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This is to certify that the
dissertation entitled

BIOCHEMICAL AND FUNCTIONAL CHARACTERIZATION
OF ARABIDOPSIS PLASTIDIC PYRUVATE KINASES

presented by

CARL ANDRE

has been accepted towards fulfillment
of the requirements for the

PhD. degree in PLANT BIOLOGY

 

 

 

Major Professor’s Signature)

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MSU is an affirmative-action, equal-opportunity employer

 

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6/07 p:/C|RCIDaleDue.indd-p.1

BIOCHEMICAL AND FUNCTIONAL CHARACTERIZATION OF ARABIDOPSIS
PLASTIDIC PYRUVATE KINASES

By

Carl Andre

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Department of Plant Biology

2007

ABSTRACT

BIOCHEMICAL AND FUNCTIONAL CHARACTERIZATION OF ARABIDOPSIS
PLASTIDIC PYRUVATE KINASES

By

Carl Andre
In plants, flux through the Embden-Meyerhoff pathway (glycolysis) is vital for supplying
precursors for many biosynthetic pathways, including fatty acid, amino acid, and
isoprenoid biosynthesis. Additionally, the production of ATP by glycolysis is expected to
be important in non-photosynthetic tissues. Pyruvate kinase (PK) catalyzes the final
reaction of glycolysis and in plants many of the upstream glycolytic enzymes are
regulated directly or indirectly by its activity. The regulation of individual PK isoforrns is
thus tailored such that glycolytic flux is appropriate for the tissue in which they reside.
The specific roles of individual PKs are further defined by compartmentation between the
cytosol and plastid. The Arabidopsis genome has fourteen putative PK-encoding genes.
Three of these encode proteins which are located in the plastid and constitute 470 kDa
plastidic PK (PKp) complexes composed of four a- and either four [51- or four Bz-subunits.
Interaction of the a-subunit with either B-subunit confers unique regulatory properties to
the enzymes which suggest specific roles in plant metabolism. Notably, the (13. enzyme
has higher specific activity and is less sensitive to allosteric regulation by metabolites.
Developing Arabidopsis embryos synthesize triacyl glycerol (TAG) to fuel seed
germination and establishment. A mutant disrupted in the [ii-encoding gene (named pkp 1)
has impaired seed-PKp activity and a 60% reduction in TAG accumulation with a
reciprocal increase in carbohydrate content. Rescue of this phenotype can be achieved by

ectopic overexpression of either B-subunit encoding gene, although [32 is not capable of

full restoration. Thus, a specific role for the GB] enzyme is in catabolizing imported sugar
to provide the precursors for high rates of fatty acid synthesis in embryos. As expected,
pka seeds do not germinate and establish as efficiently as wild type. However, this
defect is not completely attributable to a lack of TAG and it seems that PKp activity is
also necessary for proper metabolism in germinating seeds. It was found that pka
seedlings and mature plants have much in common with mutants deficient in isoprenoid
and tocopherol biosynthesis, but there was no obvious defect in fatty acid metabolism.
Apparently, the function of PKp in leaves is also to provide precursors for whichever

biosynthetic processes are in highest demand.

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ACKNOWLEDGMENTS

Firstly, I am gratefully indebted to my thesis advisor, Dr. Christoph Benning. He worked
tirelessly to insure that I had a place in his lab to develop my scientific skills, despite a
seeming lack of interest from funding agencies in supporting this project. He allowed for
creativity and exploration while still establishing clear goals and demanding work to be
performed at the best of my abilities. My thesis advisory committee, Dr. J ianping Hu, Dr.
John Ohlrogge, and Dr. Andreas Weber also deserve my thanks. They made themselves
accessible for discussion at their own inconvenience and contributed thoughtful insight
about my research. I am thankful to the Michigan State University US. Department of
Energy Plant Research Laboratory and to the Department of Plant Biology for accepting
me as a graduate student and for fostering some of the best plant science in the world. I
only hope that I can help uphold the reputation for which these departments are known. I
would also like to acknowledge the Department of Biochemistry and Molecular Biology
for hosting me as one of their own and for allowing me to use their numerous facilities.
Included are the labs of Dr. Jack Preiss and Joe Leykam where l invaded and used
equipment for extended periods of (sometimes frustrating) time. I have also benefited
from the community here at MSU where numerous discussions with graduate students
and postdocs, especially the members of the Benning lab, past and present that always
made work a pleasant place, helped guide me in the right direction. I can leave MSU
knowing that I worked and socialized with some of the best scientists and people there

are. Thanks.

iv

TABLE OF CONTENTS

LIST OF TABLES ............................................................................................................. vii
LIST OF FIGURES .......................... . ................................................................................ viii
CHAPTER 1 - Seed storage compound accumulation and related metabolisms
in plants ....................................................................................................... 1
1 .1 . Introduction ......................................................................................................... 2
1.2. Embryo development .......................................................................................... 3
1.3. Signals that control embryo maturation and storage compound accumulation .. 4
1.3.1. Genetic regulators ....................................................................................... 4
1.3.2. Small molecule regulators ........................................................................... 5
1.4. Considerations for seed oil biosynthesis ............................................................. 8
1.4.1. Competition between biochemical pathways .............................................. 8
1.4.2. Supply of energy ....................................................................................... 1 1
1.4.3. Supply of reducing equivalents ................................................................. 14
1.5. The biosynthetic pathway from sucrose to oil .................................................. 16
1.5.1. Synthesis of fatty acids and triacyl glycerol .............................................. 18
1.5.2. Supply of carbon precursors for fatty acid synthesis ................................ 20
1.5.3. Glycolysis in oil seeds .............................................................................. 25
1.6. Pyruvate kinase ................................................................................................. 28
1.6.1. Pyruvate kinase phylogeny ....................................................................... 29
1.6.2. Pyruvate kinase molecular architecture .................................................... 30
1.6.3. Mechanisms of regulation ......................................................................... 31
1.6.4. Analysis of pyruvate kinase mutants ........................................................ 33
1.6.5. Pyruvate kinase moonlighting ................................................................... 34
1.7. Rationale and outlook ....................................................................................... 34
References ..................................................................................................................... 36

CHAPTER 2 - Molecular and kinetic analysis of Arabidopsis plastidic pyruvate

kinase ....................................................................................................... 49
Abstract ......................................................................................................................... 50
Introduction ................................................................................................................... 51
Materials and Methods .................................................................................................. 53
Results ............................................................................................................................ 62
Discussion ..................................................................................................................... 82
References ..................................................................................................................... 87

CHAPTER 3 - Analysis of carbon metabolism and storage compound accumulation
in seeds of an Arabidopsis mutant deficient in a plastidic pyruvate

kinase ....................................................................................................... 9 1
Abstract ..................................................................................................................... 92
Introduction ................................................................................................................... 93

Materials and Methods .................................................................................................. 96

Results .......................................................................................................................... 101
Discussion ................................................................................................................... l 18
References ................................................................................................................... 1 24

CHAPTER 4 - Germination, establishment, and grth of Arabidopsis plants

lacking a plastidic pyruvate kinase ......................................................... 128

Abstract ....................................................................................................................... 129
Introduction ................................................................................................................. 1 30
Materials and Methods ................................................................................................ 133
Results .......................................................................................................................... 136
Discussion ................................................................................................................... 149
References ................................................................................................................... 154
CHAPTER 5 - Conclusions and perspectives ................................................................. 157
Appendix A ..................................................................................................................... 166
Appendix B ..................................................................................................................... 181
Appendix C ..................................................................................................................... 192
Appendix D ..................................................................................................................... 204

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LIST OF TABLES

Table 2.1. Primer sequences .............................................................................................. 56
Table 2.2. PKp kinetic constants ..... g ................................................................................. 80
Table 2.3. Metabolite effectors of PKp activity ............................................................. 81
Table 3.1. WT and pka seed storage compound accumulation ...................................... 105
Table 3.2. Metabolite levels in WT and pka developing seeds ..................................... 115
Table 4.1. Leaf pigments in 25 day old plants grown on soil or on agar plates .............. 145

vii

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LIST OF FIGURES

Figure 1.1. Competition for resources between seed storage compounds ........................... 9
Figure 1.2. Simplified scheme of carbon metabolism in developing seeds ....................... 17
Figure 1.3. Alternative pathways of pyruvate production ................................................. 23
Figure 1.4. The glycolytic network in green oil seeds ....................................................... 27
Figure 1.5. Pyruvate kinase centrality to plant metabolism ............................................... 28
Figure 2.1. Pyruvate kinase similarity and selected gene expression in Arabidopsis ........ 64
Figure 2.2. Phylogenetic analysis of plant PK protein sequences ..................................... 66
Figure 2.3. Subcellular localizations of pyruvate kinase subunits ..................................... 68
Figure 2.4. PKp protein purification and initial activity assay ........................................... 71
Figure 2.5. In vitro interaction of PKp subunits ................................................................. 72
Figure 2.6. In vivo co-immunoprecipitation of PKp subunits ............................................ 75
Figure 2.7. Kinetics of active PKp complex formation ...................................................... 77
Figure 2.8. PKp subunit inactivation .................................................................................. 79
Figure 3.1. Identification of a SALK T-DNA mutant in PKp-Bl ..................................... 102
Figure 3.2. Reverse transcriptase-PCR analysis of PKp gene expression ........................ 103
Figure 3.3. pka seed phenotypes .................................................................................... 104
Figure 3.4. PK activity in pkp] and wild-type seeds ....................................................... 107
Figure 3.5. Oil and protein phenotype of pkp] seeds ...................................................... 109
Figure 3.6. Rescue of the pka seed oil phenotype .......................................................... 1 11
Figure 3.7. PKp gene expression and enzyme activity in rescued pka lines .................. 1 13
Figure 3.8. Carbohydrate accumulation and phosphofi'uctolinase enzyme activity

in pkpl and wild-type seeds ............................................................................................. 116

viii

Figure 4.1. Sucrose dependent establishment and root elongation in pka ..................... 137
Figure 4.2. Delayed germination and induction of PKp activity in pkpl ......................... 138
Figure 4.3. Inhibition of pkp] germination by exogenous sucrose .................................. 140
Figure 4.4. Hypocotyl elongation and PKp activity in dark-grown seedlings ................. 141
Figure 4.5. Fatty acid composition in germinating seeds and seedlings .......................... 142
Figure 4.6. Seed germination following accelerated aging treatment ............................. 143
Figure 4.7. Plant growth and morphology of pkpl .......................................................... 144
Figure 4.8. Pyruvate kinase activity in rosette leaves during the day and night .............. 146
Figure 4.9. Lipid profile and composition of pkpl leaves ............................................... 147
Figure 4.10. Carbohydrate content of 25 day old leaves ................................................. 148
Figure A]. Cytosolic localization of G6PD5 and G6PD6 .............................................. 172
Figure A.2. Single and double mutants for G6PD5 and G6PD6 .................................... 174
Figure A.3. G6PDH isoforms and activity in developing siliques .................................. 176
Figure 3.1. Impaired seedling establishment in the wriI mutant .................................... 187
Figure 3.2. Glycolytic enzyme activities in green seedlings ........................................... 188
Figure C.l. Relative gene expression of putative Arabidopsis lipin homologues ........... 198
Figure C.2. Gene structure of the two Arabidopsis LpinI homologues ........................... 199
Figure C.3. Lipid analysis of Arabidopsis lipin single and double mutants .................... 200
Figure D.l. DGTS biosynthetic pathway and involvement of RthaAB ........................ 206
Figure D.2. BtaA-catalyzed DGHS biosynthesis from radiolabeled substrates .............. 212

ix

Chapter 1

Seed storage compound accumulation and related metabolisms in plants

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1.1. Introduction

The reproductive success, and in part evolutionary fitness, of most plants is largely
dependent on sexual reproduction involving fertilization of an ovule and subsequent
embryo development. The challenges of reproduction vary, and are greatly influenced by
the environment in which the plants take root. Regardless of such factors, the packaging
of nutrient reserves in seeds has evolved as a major strategy to insure the establishment
and survival of the next generation. Gerrninating seeds must metabolize their own storage
reserves until the seedlings establish photosynthetic rates capable of sustaining growth.
Carbohydrates, oils in the form of triacylglycerol (TAG), and proteins constitute the
reserves used for this establishment. In addition to their indispensability for plant survival,
these compounds have broad economic importance as agricultural commodities. For this
reason, we need to investigate and understand the metabolism which leads to the
accumulation of these compounds with the eventual goal of engineering plants to be more
productive. Seed oil, in particular, is of utmost interest given the current Zeitgeist. The
model plant Arabidopsis serves as an excellent organism in which to study seed oil
biosynthesis, as it is very closely related to canola (Brassica napus) which is a major oil
seed crop in the Northern hemisphere. In this chapter, I will review the metabolisms
involved in storage compound accumulation, with a focus on oil, and I will introduce
some of the current hypotheses pertaining to regulation of biosynthesis and turnover. I
will establish that the glycolytic enzyme pyruvate kinase is pivotal in these processes and
that it represents a step that needs to be researched further to fully understand the

metabolism of developing oil seed embryos.

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1.2. Embryo development

Plant embryogenesis is initiated by a fertilization event in which one male gametophyte
unites with a single female gametophyte to form a zygote. An additional fertilization
occurs between another male gametophyte and a specialized diploid cell within the
embryo sac resulting in the formation of the triploid endosperm. The function of the
endosperm is to nourish the embryo during development and germination and in some
cases (e. g. cereals) it is the site of storage product accumulation (Berger 2003, Penfield et
al. 2004). Once fertilized, the development of plant embryos generally occurs in three
distinct phases: 1) cellularization and establishment of a body plan, 2) deposition of
storage reserves and maturation, and 3) acquisition of desiccation tolerance (Goldberg et
al. 1994). Immediately following the double fertilization event, the seed rapidly grows
mostly due to syncytial development of the endosperm (Berger 1999). During this time (~
5 days in Arabidopsis) the zygote goes through many rounds of asymmetrical cell
division and establishes tissues that will eventually become the hypocotyl and cotyledons
(Laux and Jurgens 1997). Upon entering the maturation phase, cell division slows in lieu
of expansion as cotyledonary cells begin to fill with storage compounds. This phase of
development is depicted in Chapter 3, Figure 3.3A, and is associated with distinct
changes in gene expression as discussed below. During this time, the water content of the
seed steadily declines (Baud et al. 2002). The acquisition of desiccation tolerance and
seed dormancy is characterized by nearly complete loss of water content from the seed
and is associated with expression of presumably cryo-protective protein-encoding LEA
genes (Wise and Tunnacliffe 2004) and programmed cell death of maternal tissues which

will eventually become the seed coat.

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1.3. Signals that control embryo maturation and storage compound accumulation
Embryo maturation, as described above, is the phase during which storage compounds
are biosynthesized and deposited in developing seeds. The regulation of this process is at
best loosely understood. There are, however, known transcriptional and metabolic cues
which exert some control over this process. It is difficult to tease apart these two means
of regulation as metabolism is directly influenced by the genes which are active in a

given tissue, the expression of which may in turn be controlled by cellular metabolites.

1.3.1. Genetic regulators

Arabidopsis has served as the model for the study of the transcriptional control of seed
maturation. The changes in metabolic gene expression during the shift from embryo
morphogenesis to storage compound accumulation have been documented (Girke et al.
2000, Ruuska et al. 2002, White et al. 2000). Additionally, some genetic factors which
control such changes in metabolic gene expression have been elucidated. The most
upstream factor identified to date is PKL (PICKLE). The PKL gene encodes a chromatin-
remodeling factor that specifically represses embryo associated transcription and
metabolism in non-embryo tissues (Ogas et al. 1999, Rider Jr et al. 2003, Rider Jr. et al.
2004). Indeed, pkl mutants ectopically accumulate seed storage compounds in roots
(Ogas et al. 1997). Some of the specific targets of PKL-mediated repression encode the
transcription factors LECI, LEC2, and F US3. These three LEAF Y COTYLEDON-class
transcription factors are regulators of embryo identity and are so named because of the
leaf-like appearance of cotyledons of the respective mutants. Overexpression of these

genes results in spurious embryo formation in vegetative tissues (Lotan et al. 1998, Stone

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reduced amounts of storage protein and oil in mutant seeds (Meinke et a1. 1994). The
LEC-class transcription factors are considered master regulators and are hypothesized to
target a subset of transcription factors controlling embryo maturation. One potential
target of LECl is the WRII gene. Overexpression of WRII in the lac] background
rescues the desiccation intolerant phenotype and the promoter of WRII contains a
potential DNA binding site of LECl (Cemac and Benning, unpublished). The WRII gene
encodes an AP2/EREB domain transcription factor that regulates sugar metabolism (1'. e.
glycolysis) in tissues where WRI] is expressed, namely, seeds and roots (Cemac and
Benning 2004, Ruuska et al. 2002). The wriI mutant was originally identified in a screen
for mutants specifically reduced in seed oil accumulation (F ocks and Benning 1998).
Overexpression of WRII in seedlings was subsequently found to cause an embryo
identity characterized by the synthesis and accumulation of TAG and seed storage
proteins (Cemac and Benning 2004). While this is an incomplete review of all the genes
involved in regulating embryo maturation, it is clear that there is a strong relationship
between transcriptional regulation of embryo identity and seed storage compound

biosynthesis.

1.3.2. Small molecule regulators

The list of small molecules which potentially regulate seed maturation, and thus storage
compound accumulation, is growing. The phytohorrnone abscisic acid (ABA) is one well
documented regulator of these processes. There are two peaks of ABA accumulation

during seed maturation of most species (Karssen et al. 1983). The first peak potentially

regulates the transition from embryo cell division to cell enlargement in Arabidopsis as
ABA is known to induce the expression of cell cycle inhibitory factors (Wang et al. 1998).
During this time ABA also induces the expression of seed storage protein genes (Crouch
and Sussex 1981). An Arabidopsis mutant defective in the sensing of ABA, abi3-3, fails
to accumulate seed storage proteins (Koomneef et al. 1989). However, as mentioned
above, it is difficult to tease apart transcriptional and metabolite-based regulation and it
has been shown that ABI3 interacts in the signaling mediated by the LEC-class
transcription factors described earlier (Parcy et al. 1997).

Non-hormonal metabolite signals are gaining recognition as potential regulators
of seed maturation. Among these, the most attention has fallen upon sugars. Sugars,
especially glucose, have been identified as regulators of photosynthetic gene expression
and as components of signal transduction networks in Arabidopsis vegetative tissue (J ang
et a1. 1997, Jang and Sheen 1994). Developing seeds import large amounts of sugar from
maternal tissue, and it is expected that sugar signaling networks would exist here as well.
In legume seeds, the transition from cell division to storage product accumulation is
correlated with a shift from a high hexose to sucrose ratio to a high sucrose to hexose
ratio (Borisjuk et al. 2003, Weber et al. 1996). It has been proposed that the
concentrations of the sugars themselves provide signals that regulate this developmental
transition (Weber et a1. 1997). A similar switch in the ratio of hexose to sucrose is
observed in Arabidopsis; however, it comes after the onset of high rates of oil
biosynthesis and may be a result of changed metabolism rather than triggering those
changes (Baud et al. 2002, Hill et al. 2003). The apetalaZ (ap2) mutant of Arabidopsis

produces seeds with higher mass and yield (Ohto et a1. 2005). The increased cell size and

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number in ap2 embryos is correlated with an increased hexose to sucrose ratio throughout
embryo development. The modulation of sugar metabolism appears to have an effect on
embryo development in this mutant, although again, it is unclear whether the altered
sugar ratio causes the phenotype or is the result of it. Transgenic tobacco plants with
seed-specific overexpression of Invertase were used to examine importance of the switch
from hexose to sucrose in oil seed crops. The transgenic lines maintained a very high
hexose to sucrose ratio throughout embryogenesis but developed normally, suggesting
that the switch from hexose to sucrose accumulation is not a signal (Tomlinson et al.
2004). Another sugar, trehalose-6-phosphate (T6P), is involved in embryo maturation. As
opposed to sucrose and hexoses, T6P exists at very low concentrations in Arabidopsis
embryos and cannot serve as an important energy source, which buttresses the notion of it
as a signaling molecule. The importance of this molecule in regulating embryo
development is evidenced by an Arabidopsis mutant deficient in T6P-synthase which is
embryo lethal (Eastrnond et al. 2002). Furthermore, an analysis of the aborted embryos
revealed a dearth of oil accompanied by an increase in starch accumulation (Gomez et al.
2006). Components involved in the T6P signaling network remain to be identified. The
amino acid asparagine may be another metabolite regulator of embryo maturation. An
analysis of soybean seeds from lnigh- and low-seed protein lines revealed a tight
correlation between the amount of aspargine present in the embryo and the amount of
seed storage protein in the dry seed (Hemandez-Sebastia et al. 2005). It is unknown
whether this regulation is due to effects on transcription of storage protein genes or due to

substrate availability.

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1.4. Considerations for seed oil biosynthesis

The composition of seed storage compounds varies between plants, but a focus on
Arabidopsis and other oil seed crops is pertinent in the context of the research presented
in later chapters. Approximately 30% of the dry weight of an Arabidopsis seed is in the
form of TAG, while another 30% is composed of seed storage proteins (Focks and
Benning 1998). Starch transiently accumulates in developing embryos, but is not a major
product in mature Arabidopsis seeds. The timing of these processes has been studied at
the metabolic and transcriptional levels using Arabidopsis (Baud et al. 2002, Ruuska et al.
2002). At the onset of embryo cell expansion, which is 5 days after flowering (DAF),
starch is synthesized in the plastids and accumulates through 9 DAF at which point it is
degraded. Coincident with starch degradation are the synthesis of TAG and seed storage
proteins, both of which continue to accumulate linearly until about 17 DAF, when the
seed begins to desiccate and become metabolically quiescent. Figure 1.1 depicts a
simplified scheme for the biosynthesis of seed storage compounds in oil seeds. Sucrose
and some amino acids are provided by the maternal source organs through the endosperrn
liquid and are then metabolized by the embryo (Schwender and Ohlrogge 2002). The
precursors of each storage compound are derived from connected biochemical pathways,
of which fine regulation is necessary to balance the final reserve composition of the seed.

Such carbon partitioning will be discussed in more detail in the following section.

1.4.1. Competition between biochemical pathways
The biosynthetic pathways in Figure 1.1 are simplified to emphasize the potential for

competition for resources between the biosyntheses of starch, oil, and protein. Seed

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storage protein biosynthesis is the most independent pathway. Stable isotope labeling of
canola embryos revealed that 30% of the amino acids used in the synthesis of storage
proteins are imported and 70% arise from de novo biosynthesis from sugars (Schwender
and Ohlrogge 2002). Labeled amino acids were not catabolized and incorporated into
fatty acids. Moreover, there appears to be independent sugar metabolism for protein and
fatty acid synthesis, which is supported by numerous Arabidopsis mutants in which
decreases in seed oil or protein does not result in compensatory increases in the other
major storage compound. The wriI mutant, for instance, is reduced 80% in oil with no
effect on the protein content (Focks and Benning 1998). Conversely, abscisic acid
biosynthetic and signaling mutants exhibit reductions in seed storage protein content with

no affect on oil (Finkelstein and Somerville 1990).

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Sucrose and amino acids are imported from source tissue via the phloem and are
incorporated into the seed metabolic network. Images are TEM micrographs of
representative storage organelles from Arabidopsis developing embryos. Pyr, pyruvate;

TAG, triacylglycerol; TCA, tricarboxylic acid.

In oil seeds, the true competition occurs between starch and oil biosynthesis. The
transient accumulation of starch in Arabidopsis seeds is hypothesized to serve several
possible functions: 1) to maintain the sink strength of the embryonic tissue during fatty
acid biosynthesis (Da Silva et al. 1997), 2) to supply carbon precursors for fatty acid
synthesis upon its degradation (Norton and Harris 1975), and 3) to supply sugar (and thus
osmolarity) needed for the acquisition of desiccation tolerance (Leprince et al. 1990).
Two studies have directly addressed the question of the firnction of transitory starch in
Arabidopsis seeds. In one a mutant of plastidic phosphoglucomutase (png) which is
compromised in starch biosynthesis was analyzed with respect to seed storage
compounds (Periappuram et al. 2000). A 40% reduction in seed oil content was found in
pgm] . The authors, however, could not conclude whether the reduction in starch
compromised the sink strength or the carbon supply for fatty acid synthesis. Another
study utilized embryo specific reduction of ADP-glucose pyrophosphorylase to impair
transient starch accumulation in canola embryos (Vigeolas et al. 2004). While starch
accumulation was severely compromised in the transgenic lines, oil biosynthesis was
compromised only during the early stages of development. The authors concluded that
starch was not a source of sugar for the production of fatty acid precursors during the
main stages of oil biosynthesis. These two studies present no clear conclusions about the
role of transitory starch accumulation. There is additional correlative evidence, though,
that links starch accumulation to fatty acid synthesis in Arabidopsis embryos. Three
mutants defective in oil accumulation, wril, led, and shrunken seed 1 (sseI, encoding a
homolog of the peroxisome biogenesis factor Pex16p), all display compensatory

increases in starch accumulation (Focks and Benning 1998, Lin et al. 1999, Lin et al.

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2004, Meinke et al. 1994). In the case of wriI and sseI which both have observed
decreases in the rate of fatty acid synthesis, sugars (hexose and sucrose) also accumulated
to higher than wild-type levels in the developing seeds. These findings can be explained
by two hypotheses: 1) starch biosynthesis functions to lessen the osmotic potential of the
embryo by consuming excess sugar, thus maintaining the embryo status as a sink tissue,
or 2) excess sugar inhibits the degradation of starch which would normally be needed to
maintain a sugar supply to feed fatty acid synthesis. As the starchless mutants described
above are desiccation tolerant, it is unlikely that starch serves to facilitate sugar
accumulation for this purpose. Yet, no definitive conclusions can be drawn about the role

of transitory starch accumulation in oil seeds.

1.4.2. Supply of energy

Fatty acid biosynthesis is dependent on a steady supply of adenosine triphosphate (ATP).
Starch and amino acid synthesis along with the assembly of storage proteins (4 ATP or
GTP per peptide bond) occurring in the embryo no doubt compete for the same ATP'
(Regierer et al. 2002). The committed step of fatty acid synthesis catalyzed by acetyl-
CoA carboxylase (ACCase) uses ATP in a two step reaction converting acetyl-CoA into
malonyl-CoA. It is the first step that is ATP depedent, in which a carboxyl group from
bicarbonate is transferred to a biotin prosthetic group on the enzyme (Ohlrogge and
Browse 1995). The synthesis of one 18 carbon fatty acid thus requires 9 ATP for the
ACCase reaction alone. ACCase is considered the rate limiting reaction of fatty acid
synthesis and its activity is light dependent by means of redox-regulation, pH, and Mg2+

concentration'(Hunter and Ohlrogge 1998, Sasaki et a1. 1997). Indeed, fatty acid

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synthesis in canola embryos is stimulated by light (Bao et al. 1998, Goffinan et al. 2005,
Ruuska et al. 2004). Such light-dependent activation may be one means of coordinating
ACCase activity with the production of ATP by photosynthesis in green tissues. However,
not all oil seeds are green, and even then only as much as 30% of ambient light may
reach the embryo (Eastrnond et al. 1996, King et a1. 1998). Other means of producing
ATP include the lower half of glycolysis and mitochondrial respiration. The contribution
of glycolysis to ATP production has not been directly tested to date, however,
mitochondrial respiration has been studied in oil seeds. Stable isotope labeling studies
revealed that only as much as 22% of the needed ATP can be generated through oxidative
phosphorylation in canola embryos (Schwender et al. 2006). Increasing the respiration
rate through indirect activation of the mitochondrial pyruvate dehydrogenase complex in
Arabidopsis resulted in higher oil yield, suggesting that increased ATP production in
mitochondria may stimulate fatty acid synthesis (Marillia et al. 2003). Export of ATP
fi'om the mitochondria occurs through the adenine nucleotide carrier and subsequent
uptake by plastids would be through a known ATP/ADP transporter (Resier et al. 2004),
both in counter exchange with ADP.

Oxygen tension may have a role in regulating mitochondrial respiration in seeds.
In Arabidopsis siliques, the ambient oxygen concentration is low, and further reduction
brought on by growth in sub-ambient [0;] limited seed growth and oil accumulation
(Porterfield et al. 1999). Additional studies in canola revealed that oxygen concentration
in seeds is also low, and that increasing [0;] in low light conditions results in elevated
ATP and UDP concentrations, accompanied by faster lipid synthesis (Vigeolas et al.

2003). Although, when light is not limiting increasing atmospheric [02] has no

12

stimulatory effect, suggesting that in green seeds photosynthesis alone is capable of
supplying the needed ATP (Goffman et al. 2005). Glycolysis is known to be induced in
hypoxic tissues such as roots and seeds (Plaxton and Podesta 2006); however, its
contribution to ATP pools has not been investigated.

In any case, ATP must be imported into (if not generated in) the plastid for use in
the relevant biosynthetic processes. The Arabidopsis genome encodes two plastidic
ADP/ATP transporters, and a double mutant lacking both displays 40% reduced lipid and
and about 30% reduced protein accumulation in seeds, presumably due to a lack of ATP
in plastids (Reiser et al. 2004). Interestingly, mRNA levels of genes encoding subunits of
plastidic pyruvate kinase (examined in this thesis) were higher in this mutant. The authors
speculate this was a mechanism to compensate for reduced ATP import by instead
producing it in the plastid, although, pyruvate kinase enzyme activity was not measured.

The importance of ATP supply is further highlighted by an observed shift from
ATP to pyrophosphate (PPi) consuming metabolism during the phase of maximum fatty
acid biosynthesis in Arabidopsis seeds. This shifi was observed at the level of
transcription and enzyme activity (Baud and Graham 2006, Ruuska et al. 2002). In
particular, ATP-dependent phosphofructokinase and invertase (which leads to
downstream ATP consumption) activities are replaced by PPi-dependent
phosphofructokinase and sucrose synthase (for the cleavage of sucrose). The current
hypothesis is that in response to a limited oxygen supply, and thus ATP, this shift is
induced in an effort to conserve the adenylate pool for use in fatty acid synthesis. A
similar pattern is seen in potato tubers where ATP is required for starch synthesis

(Appeldoom et al. 1997).

13

1.4.3. Supply of reducing equivalents

Reducing equivalents in the form of NADH and NADPH are necessary for the synthesis,
elongation, and desaturation of fatty acids. The reductases involved in fatty acid synthesis
specifically use NADPH (3-ketoacy1-ACP reductase) and NADH (enoyl-ACP reductase)
(Caughey and Kekwick 1982, Shimakata and Stumpf 1982, Slabas et al. 1986). Synthesis
of a saturated 18 carbon fatty acid requires 8 NADH and 8 NADPH. Subsequent
desaturation of fatty acids indirectly requires the input of NAD(P)H (Shanklin and
Cahoon 1998). The very long chain fatty acids found in wax, suberin, spingolipids and
seed oil, are elongated in the cytosol via a similar mechanism as fatty acid synthesis in
the plastid, and thus also require 2 NAD(P)H for each two carbon addition (Barrett and
Harwood 1998). The NADH required for fatty acid biosynthesis could be supplied from
the plastidic pyruvate dehydrogenase complex, which generates NADH and fatty acid
precursors (acetyl-CoA) in a 1:1 ratio. In green seeds exposed to light photosynthesis
could produce the required NADPH, as is the case with ATP. As described above, light
stimulates fatty acid synthesis in embryos and this could likely be a result of not only
ATP, but also NADPH production (Bao et al. 1998, Goffinan et al. 2005, Ruuska et al.
2004). Indeed, canola embryos contain chloroplasts similar to shade grown leaves and are
capable of photosynthesis (Asokanthan et al. 1997, Eastmond et al. 1996, King et al.
1998). Calculations based on reported 02 evolution rates from canola embryos indicate
that photosynthesis could provide all of the NADPH required for fatty acid synthesis
(Ruuska et al. 2004). Indeed, carbon use efficiency for storage oil synthesis is greatly

reduced in dark-cultured canola embryos compared to those grown in even low light

14

COIII

plio1

 

I V.

OI

 

conditions (50 umol m_2 s", Goffrnan et al. 2005). This effect could be due to the role of
photosynthesis in producing reductant and ATP.

The oxidative pentose phosphate pathway (OPPP) generates 2 NADPH for each
glucose molecule metabolized, and this could help fulfill the requirements for the
reductant necessary for high rates of fatty acid synthesis (Eastmond and Rawsthome
2000). The first enzyme of the OPPP, glucose-6-phosphate dehydrogenase (G6PDH), is
feedback inhibited by NADPH and would presumably be inactivated in reducing
conditions (Scheibe and Anderson 1981, Wakao and Benning 2005). Additionally, in vivo
labeling revealed that a maximum of 25% to 45% of the reductant required for oil
biosynthesis is provided by the OPPP (Schwender et al. 2003). Furthermore, an
Arabidopsis double mutant which is defective in both cytosolic G6PDH isoforms
(resulting in a 50% decrease in total enzyme activity) has no reduction in oil content (see
Appendix A). Plastidic NADP-dependent malic enzyme (NADP-ME) also produces
NADPH and a role for this enzyme in canola oil metabolism has been proposed (Kang
and Rawsthome 1994, Singal et al. 1995). However, metabolic flux analysis of cultured
canola embryos revealed that malate is not a major contributor to fatty acid synthesis
(Schwender and Ohlrogge 2002). NADP-ME mutants have been isolated from
Arabidopsis, and loss of seed specific isoforms has no effect on oil accumulation
(Wheeler et al. 2005). Taken together, it seems that the OPPP and NADP-ME play little
or no role in green seeds, but in non-photosynthetic seeds there is evidence to the
contrary. Labeling studies with sunflower (Helianthus annuus) found that malate is the
preferred substrate of fatty acid synthesis when fed to isolated leucoplasts (Pleite et al.

2005). Moreover, the consumption of malate was reduced when co-fed with glucose-6-

15

phosphate (G6P), and vice versa. Labeled G6P was not incorporated into fatty acids, but
instead was entered into the OPPP. The balancing of flux between the OPPP and NADP-
ME suggests that these pathways both contribute to NADPH pools in isolated leucoplasts.
However, more recent steady-state isotopic-labeling experiments using cultured embryos
indicated very little flux of malate into oil (Alonso et al. 2007). NADP-ME has also been
implicated in the supply of reductant in castor (Ricinus communis) seeds. As with
sunflower, malate was the preferred substrate for fatty acid synthesis in isolated
leucoplasts (Smith et al. 1992) and a correlation was observed between leucoplast
NADP-ME activity and the onset of fatty acid synthesis (Shearer and Dennis 2005).
Clearly, green and non-green oil seeds differ in how they supply ATP and reducing
equivalents for fatty acid synthesis, but the unifying theme is that in either there are

multiple overlapping and interacting pathways for the production of both.

1.5. The biosynthetic pathway from sucrose to oil

The biosynthetic pathways leading to the accumulation of TAG in oil seeds have been
extensively studied in Arabidopsis and other oil seed crops. Sucrose imported from
maternal tissues is the main carbon source for these metabolisms. There is no symplasmic
connection between maternal and filial tissues and so sucrose (or hexose) is released to
the seed apoplasm and is then imported into cotyledonary cells of the embryo by
membrane localized transporters (Rosche et al. 2002). Once inside the embryo hexose is
converted eventually to fatty acids and incorporated into TAG which is deposited into oil
bodies (see Figure 1.1). Oil bodies originate from the endoplasmic reticulum (ER) and

are delineated by a phospholipid monolayer heavily embedded with proteins (Galili et al.

16

SC

 

Fig
Em.

an.

 

 

1998). Figure 1.2 is based on metabolic flux analysis and transcript profiling of green oil
seeds (Ruuska et al. 2002, Schwender et al. 2004a, Schwender et al. 2004b) and depicts
the major pathway of carbon metabolism from the loading of sucrose to the budding of
oil bodies from the ER. Emphasis is given to the supply of carbon precursors for fatty
acid synthesis, but all metabolisms will be covered in forthcoming sections.

Sucrose

PM
f " . 1
Cytosol

 

 

Sucrose—> Hexose-P 66

P .
RuBisCO
Shunt

3PGA

pep
*L *L’
ATP Free FA
fr ATP

Pyr
‘T'PY' PY' ATPxi U

NAD(P)H

   

Glycolysis
Gluco eo

 

II

 

 

 

 

 

 

 

 

 

Mitochondrion
L A

 

 

 

 

Plastid
LL Jr

 

 

Figure 1.2. Simplified scheme of carbon metabolism in developing seeds

Emphasis is on oil production. Asterisks demark the pyruvate kinase reaction. Single
arrows can indicate multiple reactions. 3PGA, 3-phosphoglycerate, ATP, adenosine
triphosphate; ER, endoplasmic reticulum; FA, fatty acid; FAS, fatty acid synthase; G6P,
glucose 6-phosphate; Gluconeo., gluconeogenesis; PEP, phosphoenolpyruvate; PM,

plasma membrane; Pyr, pyruvate; TAG, triacylglycerols; TCA, tricarboxylic acid.

17

L.

h!

1.5.1. Synthesis of fatty acids and triacylglycerol

The site of almost all de novo fatty acid synthesis in plants is the plastid (Ohlrogge et al.
1979). Fatty acids are synthesized by cycles of elongation in two carbon increments, with
malonyl-CoA acting as the carbon donor. The synthesis of malonyl-CoA in plastids is
performed by a multimeric bacterial-type ACCase and as mentioned above is subject to
regulation by light through various mechanisms. Attempts to boost seed oil yield by
increasing the activity of ACCase have had limited success to date and indicate a more
complex regulation of fatty acid synthesis (Thelen and Ohlrogge 2002). To be used as a
substrate by the fatty acid synthase complex (FAS), malonate must first be transfered to
acyl carrier protein (ACP). Malonyl-CoAzACP transacylase performs this transfer and the
activity of this enzyme may also be subject to regulation by light through redox
mechanisms (Lemaire et al. 2004). All subsequent steps in the synthesis of fatty acids in
the plastid involve ACP and a bacterial type II (multimeric) FAS. A carbon-carbon bond
and the release of one C02 from malonate are the products of condensation of malonyl-
ACP with acyl-ACP (or acetyl-CoA). Three separate condensing enzymes known as 3-
ketoacyl-ACP synthases (KAS) can perform this reaction depending on the initial acyl
chain length. The first round of condensation to form a four-carbon product, which uses
acetyl-CoA as a primer, is carried out by KASIII (J aworski et al. 1989). Elongation from
4 carbons up to 16 is done by KASI and the final addition to make an 18-carbon fatty
acid is done by KASII. Each condensation yields a 3-ketoacyl-ACP product which must
be reduced (using NADPH), dehydrated, and then reduced again (using NADH) to yield
a saturated product ready for the next round of condensation. These steps are catalyzed

by 3-ketoacyl-ACP reductase, 3-hydroxyacyl-ACP dehydratase, and enoyl-ACP

18

l'Ct

dc

EF

tht

at.

dra
of.
et 2

_V€i

Ch;

AC

CV

 

 

reductase, which together with KAS make up the multimeric F AS complex. As a final
step, a portion of the 18-carbon ACP can be desaturated by a plastid localized A9
desaturase to form oleoyl-ACP. Additional desaturation steps are carried out in both the
ER and the plastid by membrane bound desaturases. These modifications occur once fatty
acids are esterified to a glycerol backbone and are the targets of metabolic engineering as
the production of very long-chain polyunsaturated fatty acids in plants is becoming an
attractive alternative to natural sources (Truksa et al. 2006).

Fatty acid synthesis is completed by cleavage of a 16- or 18-carbon chain from
ACP by an acyl-ACP thioesterase (FAT). Two forms of FAT exist, FATA and F ATB,
which preferentially cleave unsaturated and saturated acids, respectively. Overexpression
of a lauric acid-specific FATB encoding cDNA in Arabidopsis or canola resulted in a
dramatic increase in the lauric acid content of seed oil, thus underscoring the importance
of FAT enzyme activity in regulating seed oil composition (Voelker et al. 1992, Voelker
et al. 1996). Once cleaved from ACP fatty acids are exported from the plastid in an as of
yet undetermined manner and are subsequently esterified to CoA in the cytosol. The
cytolsol is the site of fatty acid elongation which yields the 20 to 24-carbon very long-
chain fatty acids (VLCFAs) present in many seed oils. A homodimeric, eukaryotic-type
ACCase and a membrane bound acyl elongation complex participate in elongation in the
cytosol and are regulated differently than their plastidic counterparts (Bao et al. 1998).
Although, ACCase is still a bottleneck and reducing the activity of the cytosolic enzyme
resulted in reduced VLCFA content in Arabidopsis seed oil (Baud et al. 2003). Many
other modifications to fatty acids occur, such as hydroxylation, methylation, etc.., and

these unusual fatty acids are typically specific to seed oil (V oelker and Kinney 2001).

19

Theei
2003)

edhed

tutip
phosr
200i)
totht
the 52
(Ilkl
isco
padr
toI)
red;

0\ Cf

AI—l
"It

 

 

The enzymes responsible for such modifications are being elucidated (e. g. Bao et al.
2002), and progress is being made in understanding how these unusual fatty acids are
edited out of membrane lipids (Voelker and Kinney 2001).

The assembly of triacyl glycerol from acyl-CoAs and membrane lipids occurs by
two pathways: the Kennedy pathway, and the recently described
phosphatidylcholine:diacyl glycerol acyltransferase (PDAT) pathway (Dahlqvist et al.
2000). In both pathways, the initial steps are identical. Fatty acids in the ER are esterified
to the sn-1 position of a glycerol-3 phosphate backbone followed by a second transfer to
the sn-2 position. The resulting phosphatidic acid is then converted to diacylglycerol
(DAG) by the action of a phosphatidic acid phosphatase. In the Kennedy pathway, DAG
is converted to TAG by the action of DAG acyltransferase (DAGAT). In the PDAT
pathway, an acyl chain from the sn-2 position of phosphatidylcholine (PC) is transferred
to DAG to form TAG. An Arabidopsis mutant defective in a seed specific DAGAT has
reduced seed oil (Katavic et al. 1995, Routaboul et al. 1999, Zou et al. 1997). Conversely,
overexpression of the same gene results in enhanced seed oil content (J ako et al. 2001).
While no direct role in seed oil biosynthesis has been attributed to any of the six genes
encoding PDAT isoforms in Arabidopsis, one is specifically expressed in developing
seeds (Stahl et al. 2004). The PDAT pathway may be a means of discriminating TAG-
specific fatty acids out of membrane lipids. In any case, the genetic evidence for

Arabidopsis points to the DAGAT pathway as the major source of TAG.

1.5.2. Supply of carbon precursors for fatty acid synthesis

Plastidic fatty acid synthesis and subsequent elongation in the cytosol are dependent on a

20

SICE

C011

cha
C0.
Ind
nea
incl
cou

SO. U

 

 

steady supply of carbon precursors. Imported photosynthate in the form of sucrose or
hexose is the major source of carbon for a developing oil seed and must be broken down
to serve this purpose. The initial steps involve glycolysis and the newly discovered
RuBisCO shunt, and these will be discussed later. Here, I will address the direct supply of
acetyl—CoA for ACCase in the plastid and cytosol. Because acetyl-CoA is membrane
impermeable (Liedvogel 1986), its supply will be considered separately for each
compartment.

The generation of cytosolic acetyl-CoA has only recently been extensively studied
in plants. A cytosolic ATP-citrate lyase (ACL) from Arabidopsis has been identified and
characterized (Fatland et al. 2002). This enzyme is the main source of cytosolic acetyl-
CoA in animals and it was speculated that a similar situation might be occurring in plants.
Indeed, when the activity of this enzyme is ablated by expression of antisense cDNA
nearly all anabolic processes involving cytosolic acetyl-CoA pools were perturbed,
including VLCFA biosynthesis (Fatland et al. 2005). It appeared that no other pathways
could compensate for the loss of ACL activityin leaves suggesting that it is the sole
source of cytosolic acetyl-CoA in that tissue. The substrate of this enzyme, citrate, is
synthesized in the TCA cycle, and ultimately from glycolysis of imported sugars. It
should be noted, however, that the synthesis of VLCFA in seeds was relatively
unaffected in the ACL antisense plants.

Two enzymes exist in plastids which potentially supply acetyl-CoA to ACCase:
the pyruvate dehydrogenase complex (PDC), and acetyl-CoA synthetase (ACS).
Expression analysis of ACS and components of the PDC in Arabidopsis suggest that

ACS makes little contribution to acetyl-CoA production in plastids of developing seeds

21

acid

this

PD<

PD

(lo

(151:

Hi

(Ke et al. 2000). Additionally, pyruvate is preferred over acetate as a substrate for fatty
acid synthesis in isolated canola embryos (Kang and Rawsthome 1994). When combined
this evidence strongly supports the PDC as being the source of plastidic acetyl-CoA. The
PDC reaction also generates NADH at a one to one ratio with acetyl-CoA, which as
described earlier could support the demands of fatty acid synthesis. One consequence of
PDC activity is the release of C02, which results in high concentrations of C02 in
developing seeds (Goffinan et al. 2004). The concentration of C02 is potentially not as
damaging though as the fact that one third of fixed carbon in pyruvate is lost at this step.
However, a refixation shunt involving RuBisCO has been discovered which partially
makes up for the loss of carbon by PDC, at least in green seeds (Schwender et al. 2004a).
The amount of acetyl-CoA generated by PDC is entirely dependent on the amount
of pyruvate available in the plastid. Figure 1.3 depicts pathways that can contribute to the
steady state pool of plastidic pyruvate. The most direct route is the import of pyruvate
from the cytosol. A plastid localized pyruvate transporter is hypothesized to exist based
on the need for pyruvate translocation during C4 carbon fixation. However, no report has
yet been made of the transporter’s identity. This pathway would also require a supply of
cytosolic PEP and the activity of cytosolic pyruvate kinase (PKC). The activity of PKc is
high in developing embryos of castor and soybean (Glycine max) (Turner et al. 2005).
Moreover, isolated plastids from canola embryos are capable of incorporating ld’C-labeled
pyruvate into fatty acids (Eastmond and Rawsthome 2000, Kang and Rawsthome 1994).
However, combined data from metabolic flux analyses of cultured canola embryos
estimates that a maximum of 30% of the pyruvate used in fatty acid synthesis is

generated in this manner (Schwender et al. 2004b).

22

1h:

0 I

v«

 

 

 

 

 

 

 

 

 

 

 

 

 

PEP ADP
C) ATP
<> '''''''''''''''''''''''''''''''' f. Pyr
NADPH
NADP+ NADP+
NADH C)
malate NADPH
Cytosol PlastidJ
.J

 

 

 

Figure 1.3. Alternative pathways of pyruvate production

Arrows represent reactions catalyzed by single enzymes. Gray ovals are plastid envelope

transporters. The pyruvate transporter is marked with “2’. 1) cytosolic pyruvate kinase; 2)
plastidic pyruvate kinase; 3) PEP carboxylase; 4) NAD-malate dehydrogenase; 5) NADP-

malic enzyme. 0AA, oxaloacetate; PEP, phosphoenolpyruvate; Pyr, pyruvate.

Alternatively, plastidic pyruvate kinase (PKp) can generate pyruvate directly in
the plastid. In this case, plastidic or cytosolic PEP can serve as the substrate. The import
of PEP by plastids of canola embryos has been demonstrated (Kubis et al. 2004) and
microarray analysis of developing Arabidopsis embryos suggested that most PEP is
generated in the cytosol (Ruuska et al. 2002). A mutant of the seed resident PEP
transporter (chlorophyll a binding protein underexpressed, cue!) has been identified, but
unfortunately has not been analyzed with a direct focus on the role of this transporter in
oil biosynthesis (Knappe et al. 2003, Li et al. 1995, Vol] et a1. 2003). A recent steady-

state carbon flux analysis on cultured embryos of canola led to the proposal of a

23

RuBi
plast
react
to P]
Alih

PG!

alto
cari
\i'h':
PET

an'

 

 

RuBisCO shunt involving reactions of the reductive pentose phosphate pathway in the
plastid (Schwender et al. 2004a). The proposed pathway bypasses the initial glycolytic
reactions in the cytosol and generates PGA in the plastid, which could then be converted
to PEP in the plastid, which could compensate for import deficiency in the cue] mutant.
Although, the labeling studies of Schwender and others (2004a) cannot define whether
PGA is converted to PEP in the plastid or cytosol.

Figure 1.3 also contains a three enzyme pathway which bypasses pyruvate kinase
altogether. In the first step, cytosolic PEP is converted to oxaloacetate (0AA) by PEP
carboxylase (PEPC). Then, malate dehydrogenase (MDH) converts 0AA to malate,
which can be imported into the plastid and metabolized by NADP-ME to generate
pyruvate. Malate is transported across the plastid envelope by a malate/Pi translocator
and supports the highest rate of fatty acid synthesis in isolated plastids from castor
embryos (Eastmond et al. 1997, Smith et a1. 1992). However, NADP-ME mutants have
been isolated from Arabidopsis, and loss of seed specific isoforms has no effect on oil
accumulation (Wheeler et al. 2005). Generally, PEPC is thought to serve an anaplerotic
role in developing seeds, replenishing TCA intermediates consumed during storage
protein biosynthesis (Chollet et al. 1996).

The mystery of the source of pyruvate in developing seeds is further complicated
by the need for ATP generation in the cytosol or plastid versus the need for the transfer of
reducing potential to the plastid (see section 1.4). The pathways illustrated in Figure 1.3
are capable of doing both. The combined evidence leads to the hypothesis that in green
oil seeds, the pyruvate kinase pathway(s) are predominant, but that in non-green or

protein-storing seeds, the pathway is flexible and may involve PK bypasses.

24

react
glue
bjp:

Fun

 

1.5.3. Glycolysis in oil seeds

The PEP used for the synthesis of pyruvate is ultimately produced by some or all of the
reactions of glycolysis. Stable isotope labeling has been used to demonstrate that 90% of
glucose fed to developing canola embryos is converted to pyruvate by the RuBisCO
bypass and the second half of glycolysis (Schwender et al. 2002, Schwender et al. 2004a).
Furthermore, a link between glycolysis and seed oil metabolism is apparent in the wriI
mutant of Arabidopsis, in which a general reduction in glycolytic activity results in an
80% reduction in seed oil (F ocks and Benning 1998). Glycolysis in plants occurs in both
the cytosol and the plastid and both compartments are connected through plastid
membrane transporters (Plaxton 1996, Weber 2004). The glycolytic intermediates of
developing canola embryos appear to be in near equilibrium between the cytosol and
plastid (Schwender et al. 2003), and activities of the full glycolytic sequence have been
detected in both compartments in embryos (Eastrnond and Rawsthome 2000). It is
therefore likely that changes in glycolytic enzyme activities influence the transport of
related intermediates across the plastid envelope. This compartmentation raises the
question of a preferred route of glucose metabolism in developing oil seed embryos.
Expressed sequence tag (EST) analysis of developing Arabidopsis seeds indicated that
mRNAs encoding cytosolic enzymes for the entire glycolytic pathway are abundant, but
that only mRNAs encoding plastidic enzymes for the second half of the pathway
metabolizing trioses are abundant (White et al. 2000). The recently discovered RuBisCO
shunt (Figure 1.2, (Schwender et al. 2004a) bypasses the initial reactions of glycolysis in
the plastid and could explain the dearth of ESTs for these genes. Microarray data from

Arabidopsis extended these findings by detecting a shift in expression from genes

25

encoding cytosolic glycolytic enzymes to those encoding plastidic glycolytic enzymes at
the onset of storage compound accumulation (Ruuska et al. 2002).

The regulation of glycolysis in oil seeds occurs at least on the transcriptional and
metabolic levels. The WRII transcription factor has been shown to induce glycolytic
gene expression in germinating seedlings and the same trans-activation is thought to
occur in developing seeds (Cemac and Benning 2004). Specifically, the activities of PPi-
dependent phosphofructokinase (PFP), enolase, and pyruvate kinase are reduced in the
wri] seeds (F ocks and Benning 1998). Furthermore, genes encoding all three enzymes
are coordinately expressed with WRII in developing seeds (Schmid et al. 2005). These
activities represent the most highly regulated or regulatory steps of plant glycolysis and
are shown in Figure 1.4. The most highly regulated step in Figure 1.4 is the conversion of
fructose 6-phosphate to fructose 1,6-bisphosphate by the ATP- and PPi-dependent
phosphofructokinases (PFK and PFP, respectively). In Arabidopsis PFK activity is high
early during embryo morphogenesis while PF P activity is induced during the phase of
storage compound accumulation (Baud and Graham 2006). The inhibition of these
enzymes (directly or indirectly) by PEP constitutes the bottom-up regulation of glycolysis
in plants (Plaxton 1996) and, at least for PFP, this inhibition may be intensified by the
hypoxic conditions present in developing seeds (Podesta and Plaxton 2003). Furthermore,
these reactions are both reversible and may have roles in controlling the partition of
carbon between starch biosynthesis and the production of precursors for fatty acid
synthesis. The activities of enolase and PK can have great influence over the
concentration of the regulatory metabolite PEP. Little is known about the enolase present

in seed tissues, and for that matter, from plants at all. An enolase encoding gene is

26

upregulated during hypoxic conditions in rice and may suggest a mechanism by which
this enzyme is induced in seed tissues (Umeda and Uchimiya 1994). On the other hand,
much is known about pyruvate kinase and given its potential importance in seed

metabolism it will be discussed separately.

 

 

 

 

 

 

 

 

1‘
GGP G6P
l
.l.
PFK PFP H 3:113:00
A A F1,6-P2
: E H Plastid
C? C? It
PEP : 3PGA
F6P—>F2,6'-P2 I ll
F6P,2K if 2PGA
l 2PGA 551qu
l ,ENOcH PKp
. '2 PEP—>Pyr
JJ

 

 

Figure 1.4. The glycolytic network in green oil seeds

Enzymes and transporters are shown as gray ovals. Individual arrows represent single
reactions. Dashed lines represent metabolite based regulation. 2PGA, 2-
phosphoglycerate; 3PGA, 3-phosphoglycerate; ENO, enolase; F 1,6-P2, fructose 1,6-
bisphosphate; F2,6-P2, fructose 2,6-bisphosphate; F6P, fructose 6-phosphate; F6P,2K,
fructose 6-phosphate 2 kinase; G6P, glucose 6-phosphate; PEP, phosphoenolpyruvate;
PFP, pyrophosphate-dependent phosphofi'uctokinase; Pyr, pyruvate.

1.6. Pyruvate kinase

27

Pyruvate kinase catalyzes the conversion of pyruvate to PEP, coupled to the substrate
level phosphorylation of ADP to generate ATP (Figure 1.5). In most eukaryotes studied
PK is cytosolic, but in plants the enzyme also occurs in the plastid. Plant PK activities
arise from the expression of multiple isozymes with different biochemical properties that

depend on the tissue and plant source.

Oxaloacetate m PE P mm Shikimic Acid

TCA Cycle ADP Aromatic Amino Acids
Amino Acids ‘ Phenolic Compounds
PK lndole Compounds

 

 

all?

.thawwruw. Pyr-U vate II emu-“W““v‘ valine

 

Alanine

    

Leucine
\ Lysine
Acetyl CoA lsoprenoids (MEP pathway)
Fatty Acids
TCA Cycle

lsoprenoids (Mevolnate pathway)

Figure 1.5. Pyruvate kinase centrality to plant metabolism
Solid arrows represent direct metabolic conversions. Metabolic pathways are represented
by broken arrows. MEP, methyl-erythritol phosphate; PEP, phosphoenolpyruvate; PK,

pyruvate kinase.

28

ArabidOpsis, for instance, has 14 annotated PK genes that likely exhibit a large degree of
variation with respect to regulation of gene expression and enzyme activity (Arabidopsis
Genome Initiative 2000). The potential influence on the energy status, glycolytic activity,
and fatty acid precursor pool in seeds. alone makes PK an interesting enzyme to study.
However, as shown in Figure 1.5, PK lies at a crossroad and its activity may have an

impact on nearly all of plant metabolism.

1.6.1. Pyruvate kinase phylogeny

Pyruvate kinase is an ancient enzyme and occurs in all kingdoms of life. Previous
phylogenetic analyses of diverse PK nucleotide (Munoz and Ponce 2003) or amino acid
sequences (Hattori et al. 1995, Oria-Hemandez et al. 2006, Schramm et al. 2000)
revealed common clustering patterns. Two major families of PK exist; one contains
animal, fungal, some bacterial, and plant cytosolic enzymes, while the other includes
archaebacterial, cyanobacterial, and plant plastidic enzymes. This segregation suggests
distinct ancestral origins of the cytosolic and plastidic enzymes. Most notably, a
cyanobacterial enzyme appears to be the origin of plant plastidic PK, which may be a
result of the endosymbiotic event which gave rise to plastids. In mammals, two genes
encode four separate PK isoenzymes by way of alternative splicing (Noguchi et al. 1986,
Satoh et a1. 1988). The phenomenon has not been documented in other systems but raises
the possibility of much more PK enzyme diversity than can be assessed by gene sequence

alone.

29

1.6.2. Pyruvate kinase molecular architecture

In all but one of the organisms studied to date, PK exists as a protein complex. Most non-
plant PKs exist as homotetrarners with individual subunits having molecular masses of
56-60 kDa (Muirhead 1990, Munoz and Ponce 2003). The subunit structure of the plant
enzymes differs and is somewhat ambiguous. Purified cytosolic PK (PKc) from
germinating castor seeds is an (1202 heterotetramer with subunits of 56- and 57 kDa
(Plaxton 1988). Leaf PKc from castor has been found as both a homo- and heterotetramer
and the enzymes from developing seeds and cotyledons are homoteteramers (Hu and
Plaxton 1996, Plaxton 1989). Analyses of PKcs from canola are consistent with this
heterogeneity. In one study, PKcs from developing and germinating canola seeds were
isolated as heterotetramers with subunits the proportions of which differed between the
tissues (Sangwan et al. 1992). However, the enzyme from a suspension cell culture was
later shown to be a homotetramer (Smith et a1. 2000).

Plastidic PKs (PKp) are also non-unifonn in their subunit organization and in one
case independent analyses of the same enzyme have resulted in conflicting conclusions.
The PKp from developing castor endosperm is composed of a and B subunits. Studies of
recombinant versions of these proteins led to the conclusion that PKp-a and PKp-B are
distinct enzymes (Blakeley et a1. 1995, Blakeley and Dennis 1993, Wan et al. 1995).
However, others have determined that a native version of the same enzyme is a
heterohexarner composed of both subunits (Negm et al. 1995, Plaxton et al. 1990, Plaxton
1991). Analysis of leucoplast PK from canola suspension cells revealed a single
heterohexameric enzyme composed of equal amounts of a and [3 subunits (Plaxton et al.

2002). Combined, the data from canola and castor suggest PKp is normally a

30

heterohexamer of (1303 stoichiometry. However, the relative expression and actual amount
of the individual subunits varies depending on the tissue (Blakeley et al. 1995, Sangwan

et al. 1992) and may indicate variable subunit stoichiometry.

1.6.3 Mechanisms of regulation

Pyruvate kinase enzyme activity is dependent on monovalent and divalent cations , with
K+ and Mg2+ typically filling the role (Munoz and Ponce 2003). There are some K+-
independent enzymes and phylogenic analysis correlates this feature with specific amino
acid residues and suggests that K+-dependence evolved from K+-independent enzymes
(Oria-Hemandez et al. 2006). The evolution of Kl-dependence is thought to have been
driven by the fact that K+ facilitates active conformation acquisition and subsequent
binding of ADP (Oria-Hernandez et al. 2005).

Most non-plant PK’s are allosterically regulated, often involving activation by
adenosine monophosphate (AMP), fi'uctose 1,6-bisphosphate and other hexose
metabolism intermediates. The contact region between subunits is responsible for the
allostery and mutation of a single amino acid in this region is sufficient to modify the
allosteric properties of an enzyme (Ikeda et al. 1997, Valentini et al. 2000). Plant PKs are
typically insensitive to allosteric regulation by FBP but are regulated more by central
carbon metabolites and pH effects (Plaxton 1996). Plant cytosolic PKs typically have pH
optima of approximately pH 7.0, whereas plastidic PKs are most active at pH 8.0 (Hu and
Plaxton 1996, Plaxton et al. 2002, Smith et al. 2000). The most common metabolite
regulators of plant PK activity are tricarboxylic acid cycle intermediates and amino acids,

and they typically act as inhibitors (Plaxton and Podesta 2006). In general, non-plant

31

enzymes are activated by upstream metabolites, whereas plant PKs are inhibited by
downstream metabolites. This generalization reflects the role of PK in the respective
organisms. In non-plants, PK functions primarily in carbohydrate catabolism and the
production of energy. In plants energy can be produced by photosynthesis and the role of
PK is mainly to supply various metabolic pathways with carbon precursors. In summary,
non-plant enzymes are activated when energy is needed and plant enzymes are inhibited
when carbon precursors are abundant.

The multimeric structure of PK allows for additional levels of regulation.
Catalysis only occurs when individual subunits associate and an active complex is formed.
In vitro, this can be stimulated by the addition of polyethylene glycol, resulting in
enzyme activation (Podesta and Plaxton 1993). This suggests that in vivo, association and
dissociation of subunits may influence PK activity. Each PK subunit has intrinsic
characteristics that confer regulatory properties to the complexes in which they are found.
Thus, the potential for multiple subunit stoichiometries for heteromeric enzymes
introduces the possibility of multiple regulatory states. The novel regulatory properties of
PK hybrids formed by combining subunits of normally homomeric mammalian enzymes
provides proof of this concept (Dyson and Cardenas 1973, Hubbard and Cardenas 1975).
Individual subunits can be further regulated by covalent modification. Marrnnalian liver
PK is inhibited by phosphorylation on serine residues and recently a soybean PKc was
shown to be targeted for degradation by phosphorylation (Munoz and Ponce 2003, Tang
et al. 2003). The availability of a particular subunit for complex formation is also
controlled by effects on gene expression, translation, compartmentation, and protein

turnover and these (and other) factors likely help govern PK activity in any given tissue.

32

1.6.4 Analysis of pyruvate kinase mutants

Pyruvate kinase deficiency occurs or has been induced in many organisms but only one
case has been reported in plants. In humans, PK deficiency is the most common
glycolytic defect and is the leading cause of hereditary non spherocytic haemolytic
anaemia with 180 known mutations which result in the disease (Zanella et al. 2007).
Erythrocytes are entirely dependent on glycolysis for ATP production and so a severe
reduction in PK activity can result in death at infancy (Zanella et al. 2007). In yeast
(Saccharomyces cerevisiae), PK deficiency results in an inability to grow on glucose or
other fermentable sugars (Sprague, Jr. 1977). Interestingly though, and counter intuitively,
flux through the tricarboxylic acid cycle is stimulated in the absence of PK activity
(Pearce et al. 2001 ). A similar glucose non-fermentable phenotype is observed in
Escherichia coli PK mutants (Pertierra and Cooper 1977). It is clear from the above cases
that PK plays a pivotal role in regulating glycolytic flux and energy production in cells
lacking respiratory capacity.

Pyruvate kinase deficiency has been induced in tobacco (Nicotiana tabacum)
inadvertently by ectopic expression of a potato (Solanum tuberosum) tuber PKc fused to a
chloroplast transit peptide (Gottlob-McHugh et al. 1992). Instead of increasing plastid
localized PK activity, PKc was silenced. As a result, root biomass was reduced relative to
the shoot suggesting an altered sourcezsink relationship (Knowles et al. 1998). Further
analysis revealed impaired export of photosynthate from leaves at night along with a
higher rate of respiration (Grodzinski et al. 1999). Whether the reasons for higher

respiration are the same as those for yeast and bacterial PK mutants is unknown.

33

1.6.5. Pyruvate kinase moonlighting

Ancient enzymes such as PK often have roles in addition to their primary enzymatic
function. Hexokinasel from Arabidopsis, for instance, has recently been identified as a
glucose sensor and can translocate into the nucleus where it mediates expression of sugar
responsive genes (Cho et a1. 2006, Moore et a1. 2003). Another glycolytic enzyme,
enolase, acts as transcriptional regulator in response to cold stress in Arabidopsis (Lee et
al. 2002). Most recently, a mammalian PK was found to translocate into the nucleus in
response to interleukin-3 stimulation, where it promotes cell proliferation (Hoshino et al.
2007). Several studies using other systems find PK in other non-traditional roles. Using a
rat cDNA library and a KATP channel as bait in a yeast two-hybrid experiment, PK along
with glyceraldehyde-3 -phosphate dehydrogenase and triose-phosphate isomerase were
found to form a complex which regulates flux of K+ (Dhar-Chowdhury et al. 2005).
Another study identified PK as a lysophosphatidic acid (LPA) binding protein (Desmaret
et al. 2005). PK activity was inhibited upon binding LPA, implying LPA as a signaling
molecule which controls metabolism. No moonlighting functions have yet been

determined for a plant PK.

1.7. Rationale and outlook

Modeling the seed metabolic network based on transcript abundance and metabolic flux
analysis places PKp at an important node that connects catabolic and anabolic processes
(Figure 1.2, Figure 1.5). A hypothesis based on this model is that most of the carbon
precursors used in fatty acid synthesis are produced in the plastid by PKp, and that PKp

also provides energy to the developing embryo in the form of ATP. While native PKps

34

have been isolated and described from castor and canola, little has been done to directly
test the function of these enzymes in vivo. Use of the model plant Arabidopsis makes
investigating the connection between in vitro properties and in vivo function readily
doable. Embryo tissue is not abundant in Arabidopsis but the availability of the entire
genome sequence makes possible the rapid cloning of putative PK genes and
heterologous production of the respective proteins. Publicly available gene expression
data can help this process by hastening the identification of those genes which are
actively transcribed in the tissue of interest. By this method I have identified and
expressed in E. coli the genes which encode PKps involved in seed metabolism, in lieu of
purifying the native enzyme from embryo tissue. Molecular and kinetic characterizations
of the recombinant proteins indicated that they perform their annotated function and were
used to make predictions about how they function in vivo. A shortcoming of previous
studies of seed resident PKps is the lack of any genetic evidence to confirm any such
predictions. To this end, I took advantage of the Arabidopsis T-DNA insertion mutant
population. Plants that are impaired in PKID activity were used to test the hypothesis that
PKp is crucial for the production of precursors for fatty acid synthesis in green oil seeds.
Further analysis of the mutants also lead to the discovery of novel functions for PKp. By
taking such a two-prong approach, I was able to draw conclusions that unite in vitro and
in vivo data for the synthesis of a more complete picture of seed oil metabolism. Finally, I
will outline some strategies for the application of my findings towards metabolic

engineering for the production of valuable phytochemicals.

35

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48

Chapter 2

Molecular and kinetic analysis of Arabidopsis plastidic pyruvate kinase1

 

' This work has (been published in Andre, C., Froehlich, J.E., Moll, M.R., and Benning, C. (2007) A
heteromeric plastidic pyruvate kinase complex involved in seed oil biosynthesis in Arabidopsis. Plant Cell
19: 1-17. Figure 2.33 was provided by J .E. Froehlich and M.R. Moll contributed to Figure 2.5A-E

49

Abstract

Plastidic pyruvate kinase (PKp) catalyzes a highly regulated, ATP-producing reaction of
glycolysis. PKp occupies a highly branched node in the triacyl glycerol biosynthetic
network of developing seeds and is expected to be an important point for the regulation
of carbon partitioning. A detailed biochemical characterization of this enzyme will
provide a framework for future manipulations of seed metabolism. The Arabidopsis
genome encodes l4 putative isoforms of pyruvate kinases. Three genes encode subunits a,
[31, and [32 of plastidic pyruvate kinase (PKp). Recombinant protein production and
subsequent kinetic analysis was used to show that active PKp complexes are composed of
a and either [3. or [32 subunits. Enzyme activity is dependent the formation of a complex
between a and [3 subunits, although the presence of either [3. or B2 results in unique kinetic
and regulatory properties. The plastid enzyme prevalent in developing seeds likely has a

subunit composition of 4(1451, is most active at pH 8 and is inhibited by glutamate.

50

Introduction

The biochemical reactions leading to the accumulation of seed oil are well characterized
(Ohlrogge and Browse 1995). Furthermore, the use of stable isotope labeling and forward
genetics has linked the supply of precursors for fatty acid biosynthesis in embryos to
glycolysis (F ocks and Benning 1998, Schwender and Ohlrogge 2002). A key regulatory
step in plant glycolysis is pyruvate kinase, as its products inhibit the upstream glycolytic
reaction catalyzed by phosphofructokinase (Plaxton 1996). Pyruvate kinase (EC 2.7.1.40)
occurs as both cytosolic and plastidic isoforms and catalyzes the ADP-dependent
conversion of phosphoenolpyruvate (PEP) to pyruvate while producing ATP. With
respect to seed oil, plastidic pyruvate kinase (PKp) activity and concentration have been
shown to correlate with the most active stage of lipid biosynthesis in developing Brassica
napus embryos (Sangwan et al. 1992). Microarray data of developing Arabidopsis seeds
show that the transcript level of a putative PKp encoding gene coincides with the most
active period of TAG synthesis (Ruuska et al. 2002, Schmid et a1. 2005). In addition,
embryo PKp from B. napus is activated by 6-phosphogluconate, an intermediate of the
OPPP, suggesting a coordination between the production of precursors and reducing
equivalents for fatty acid synthesis (Plaxton et al. 2002).

Plant PK activities arise from the expression of multiple isozymes with different
biochemical properties that depend on the tissue and plant source. Arabidopsis, for
instance, has 14 annotated PK genes that likely exhibit a large degree of variation with
respect to regulation of gene expression and enzyme activity (Arabidopsis Genome
Initiative 2000). Plastidic pyruvate kinase has been purified and characterized from castor

(Ricinus communis) endosperm and B. napus suspension cell cultures Wegm et al. 1995,

51

Plaxton et a1. 1990, Plaxton et al. 2002). Both enzymes consist of a. and [3 subunits and
exist as 3a3B heterohexamers. Both are regulated by metabolites of central carbon
metabolism and have pH optima of approximately 8.0. In the case of the B. napus, a
scheme was formulated in which the (kinetic properties of PKp were used to infer a role
for the enzyme in regulating the supply of precursors for fatty acid synthesis. The
characterization of orthologous PKp(s) from the model plant Arabidopsis is pertinent as
such a similarly derived model could then be tested using available genetic tools. Here,
potential PKp s from Arabidopsis were identified and heterologously produced using E.
coli. The resulting recombinant proteins were then characterized at the molecular and

biochemical levels to gain insight into this enzyme’s role in seed oil biosynthesis.

52

Materials and Methods

Bioinformatics

Pyruvate kinase genes highly expressed in seeds were initially identified in the seed EST
Database (White et al. 2000). The 14 putative Arabidopsis PK-annotated sequences from
the TAIR website (www.arabidopsis.org) were used for the current work. Other
annotated PK sequences were down loaded from NCBI. Predicted full-length protein
sequences were aligned using ClustalW (Li 2003) available from the Biology Workbench
(San Diego Supercomputer Center, University of California, San Diego;
http2//workbench.sdsc.edu/). A phylogenetic tree was generated in Phylip format and
bootstrapped using a random number generator seed of 111 and 1000 bootstrap trials. The
phylogram was visualized using the TreeView program (version 1.6.6; Page 1996). No
manual adjustments were made to the initial alignment. Global gene expression data was

mined from the AtGenExpress developmental database (Schmid et al. 2005).

GFP fusion localization

The cDNAs for the three PK subunits were amplified using primers specific for GFP
fusion construct generation as listed in Table 2.1. Fully sequenced products were then
inserted into the T-DNA vector pCAMBIA1302 (CAMBIA, Canberra, Australia), which
contains a CaMV 358 promoter and a C-terrninal GF P encoding sequence. The resulting
constructs were electroporated into Agrobacterium tumefaciens as described below and
were then transiently expressed in Nicotiana benthamiana using a published protocol
(Voinnet et al. 2003). After 3-4 days, leaf samples were mounted in water on slides and

were directly examined using a Zeiss LSMS confocal microscope. Excitation light was

53

provided by an argon laser at 488 nm. GFP fluorescence was observed with a band-pass
filter of 505 to 530 nm and chlorophyll fluorescence with a 650-nm long-pass filter.
Enhanced-quality images were acquired with the LSMS imaging system software, and
post acquisition image processing was performed with the LSMS image browser and
Adobe Photoshop software. This work was performed at the Center for Advanced

Microscopy (Michigan State University).

Pea chloroplast import assays

The cDNAs encoding PKp-a, PKp-BI, and PKp-B2 inserted into pBluescript II (Stratagene)
were used in this study. These genes were transcribed/translated, and proteins were
subsequently labeled with [3SS]-Met using the TNT-coupled wheat germ extract system
according to the manufacturer's recommendations (Promega, Madison, WI, USA). The
PKp plasmids were linearized prior to translation with the T3 or T7 RNA Polymerase
TNT-coupled wheat germ extract system. The plasmid containing the gene encoding the
RuBisCO small subunit used for control purposes has been described (Olsen and
Keegstra 1992). Pea plants (Pisum sativum var Little Marvel; Olds Seed Co., Madison,
WI, USA) were grown under natural light in the greenhouse at 18 to 20°C. Chloroplasts
were isolated from 8- to 12-d-old plants as described previously (Bruce et al., 1994).
Binding or import reactions were performed according to published protocols (Tranel et
al. 1995). Post-treatrnents of import reactions with either thermolysin or trypsin were
performed as described previously (Jackson et al. 1998). All fractions were analyzed by

SDS-PAGE (Laemmli 1970) and fluorography (Tranel et al. 1995).

54

cDNA cloning and recombinant protein production

The cDNAs corresponding to At3g22960, At5g52920, and Atl g32440 were generated
from total silique RNA isolated as previously described (V erwoerd et al. 1989). Reverse
transcription was done with the Qiagen (Valencia, CA, USA) Omniscript RT kit and 600
ng of total RNA. Primers listed in Table 2.1 were used for PCR amplification of cDNAs
to generate products with and without predicted chloroplast transit peptides (cTPs) and
with or without epitope tags. All cDNAs were inserted into pBluescript II (Stratagene,
LaJolla, CA, USA) and sequenced at the MSU Research Technology Support Facility.
The vector pET-15b (Novagen, San Diego, CA, USA) was used for recombinant protein
expression in E. coli strain BL21 (DE3) pLysS (Novagen). The PKp-a encoding fragment
was inserted into the BamHI and Klenow-filled NdeI sites of pET-l 5b by digesting with
KasI, filling in with Klenow, and then by digesting with BamHI and ligating. The open
reading frame for PKp-Bi was inserted into the BamHI and Klenow-filled NdeI sites of
pET-l 5b by digesting with BglII, filling in with Klenow, and then by digesting with
BamHI and ligating. The PKp-B2 encoding fragment was inserted into the XhoI and
Klenow-filled NdeI sites of pET-15b by first digesting with SpeI, filling in with Klenow,
and then by digesting with XhoI and ligating. Proteins were expressed at 28°C by
inducing at an OD600 of 0.6 with 0.5 mM IPTG and allowing the cultures to grow for 4
more hours. His-tagged proteins were recovered over Ni-NTA resin using standard
protocols. Purified proteins were exchanged into a buffer of 50 mM NaxHxPO4 pH 7.9,
150 mM NaCl, 5 mM MgCl2, and 10% glycerol. The 6X-His tags were cleaved using a
Thrombin cleavage capture kit available from Novagen. Proteins were quantified with the

Bradford method using reagent from Sigrna-Aldrich (St. Louis, Mo, USA).

55

Table 2.1 Primer sequences

 

Gene or SALK
Line

Primer

 

PKP'Bz

SALK_013574
SALK_142845

PKp-a

SALK_096141

SALK_024870

PKP'BI

P,PE(t) 5 ’-ACTAGTATTAAAATCTCCGAAGATAG-3 ’

P(r) 5 ’ -CTCGAGTCATCCACCTATCTTTATCT-3 ’

PE(r) 5 ’-CTCGAGTCATCCCTTGTCATCGTCATCCTT
ATAATCTCCACCTATCTTTATCTT-3 ’

0,0E(f) 5 ’-GGTACCCCTCAGGTTTCTCTGCTCAT-3 ’

0(r) 5 ’ -GGTACCACTGTGAGTGATTCAAAAAA-3 ’

0E(r) 5’- GGTACCTCATCCCTTGTCATCGTCATCCTT
ATAATCTCCACCTATCTTTATCTT-3 ’

G(f) 5 ’ -GATATCGCTGCTTATGGTCAAATCTC-3 ’

G(r) 5 ’-GATATCTCCACCTATCTTTATCTTAC-3 ’

RT(f) 5 ’-GGGGATGTACCGCAGCCGATA-3 ’

RT(r) 5 ’-GGATGCCGAGGTTCTGACAGG-3 ’

RP 5’-TTTCACACAACAAATTCGTTCATT-3 ’

LP 5’-CAGCTTCCGCGAGTTTCCAAATCA-3 ’

Same as SALK_013574

P,PE(t) 5’-GGCGCCTCCTCGTCATCATCTCC-3’

P(r)-5’-GGATCCT1‘ACGGGACGTTCATI‘ACCT-3’

PE(r) 5’-GGATCCTTACAAATCCTCCTCACTAATCAA
CTTTTGCTCCGGGACG'ITCATTACCTG-3’

0E(r) 5’-GGTACCAGCCAACTGTCCTGAGATTT-3’

013(1) 5’-GGTACCTTACAAATCCTCCTCACTAATCAA
CTT'I‘TGCTCCGGGACGTTCATTACCTG-B’

G(t) 5’-ACATGTCTCAGTCTAT1"CAATTCTCC-3’

G(r) 5’-ACTAGTCGGGACGTTCAT'I‘ACCTGGA-3’

LP 5’-CCAAATTCAACACTCTCACACTTCG-3’

RP 5’-CCATCCCACCATCAACCAAAA-3’

RT(t) 5’-GCTGCTCGTTCCCGTGGAGG-3’

RT(r) 5’-TTGAAGCGGTACAGACTCAT‘-3’

LP 5’-TCTCGGACATGCTGCAATCAA-3’

RP 5’-TTCGCATCAGTCATCTTCGTCTTC-3’

P,PE(f) 5 ’-AGATCTGCTCGTGTTGAGACTGA-3 ’

P(r) 5 ’ -GGATCCTTAAACCTTGCGGACTTGGA-3 ’

PE(r) 5 ’-GGATCCTTATGCATAATCGGGAACATCATA
GGGATAAACCTTGCGGACTTGGAT-3 ’

0,0E(f) 5 ’ -GGTACCCTTCACTACTCTGTCTCAGC-3 ’

0(r) 5 ’-GGTACCCAAAAACGAGGTTCTACATA-3 ’

0E(r) 5 ’-GGTACCTTATGCATAATCGGGAACATCATA
GGGATAAACCTTGCGGACTTGGAT-3 ’

G(f) 5 ’ -CCATGGCTCAAGTGGTTGCTACCAGG-3 ’

G(r) 3 ’-ACTAGTAACCTTGCGGACTTGGATGT-3 ’

 

56

Table 2.1 Continued
SALK_04293 8 LP 5 ’-TGAGATAGCA' I " I “ I CAA I“I"I'GATGCG-3 ’

RP 5 ’ -GGCAAATCATTCACTTAGGATGGA-3 ’

RTI (f) 5 ’-CTGGGATGAATGTTGCTAGG-3 ’

RT2(r) 5 ’ -GTCAACTTTGTTCTCCACTCC-3 ’
SALK_042681 LP 5’-ACAGCCAATCTGGCGATCTCA-3’

RF 5 ’-TAT'TACAGGTCTATTTCTTTCGG-3 ’

 

T-DNA LB 5’-GTTCACGTAGTGGGCCATCG-3 ’

RT(f) 5'-AACAATCGATGGACCTGACTCG-3'
RT(r) 5'-TGCGACAATGGAACTGGAATGG-3'

ACTIN-l

 

Primers used for cloning of recombinant proteins without predicted cTP with (PE)
or without (P) eptiope tags, for cloning of full length proteins for overexpression in
plants with (OE) or without (0) epitope tags, for GFP fusion construct making
(G), for genotyping of SALK_KO lines (LP, RP) and for expression analysis (RT)
Native-PAGE analysis and gel filtration chromatography
Native-polyacrylamide gel electrophoresis (PAGE) was performed using 7.5%
acrylamide Ready-Gels from Bio-Rad (Hercules, CA, USA). Freshly prepared protein
was used and 5 pmols of each PK subunit were loaded per well. The gels were run at 4°C
at 140 V. PK activity staining was done as previously described, except that 50 mM
HEPES-KOH pH 8.0 buffer was used (Rivoal et al. 2002). Irnmunoblotting was done
using standard protocols and monoclonal anti-c-myc, anti-FLAG, and anti-HA antibodies
from Sigma-Aldrich. Antibodies were tested for specificity against epitope tagged and
untagged versions of all three PK subunits. Gel filtration was accomplished using a
Superdex 200 HR10/30 column with a flow rate of 0.4 mL min'l and a buffer of 50 mM

HEPES-KOH pH 8.0, 10% glycerol, 50 mM KCl, 5 mM MgCl2, 1 mM EDTA, and

0.04% NaN3. A standard curve was generated with the HMW gel filtration calibration kit

57

fi'om GE Healthcare (Piscataway, NJ, USA). For the samples, 300 uL of 0.5 mg mL'1

(per subunit) solution was injected and 0.35 mL fractions were collected.

Co-immunopreciptation analysis 7
Full length epitope tagged versions of PKp-a, PKp-BI, and PKp-I32 were produced by PCR
with primers listed in Table 2.1. The respective DNA fragments were then inserted into
the KpnI site of a modified pCAMBIAl 300 vector (CAMBIA), which contained the
EcoRI/HindIII expression cassette from pBIN 121 (Clontech, Palo Alto, CA, USA).
Arabidopsis was stably transformed with these constructs and protein expression was
monitored by immunoblotting. Arabidopsis plants were prepared for transformation as
previously described (Cemac and Benning 2004). When ready, plants were transformed
using the floral dip method (Clough and Bent 1998). Competent cells of Agrobacterium
tumefaciens strain C5 8C1 GV3101 pMP90 (Koncz and Schell 1986) were prepared and
transformed as previously described (Shen and F orde 1989).

Silique tissue was ground in 3 volumes (w/v) of extraction buffer containing 50
mM HEPES-KOH pH 8.0, 5 mM MgCl2, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, lmM
DTT, 0.1% Triton-X100, 10% glycerol, 2 mM benzamidine, 2 mM e-arnino-n-caproic
acid, 1 mM PMSF, and 1 mM PEP. Debris was removed by centrifugation at 16,000g for
10 min and the supernatant was used for SDS-PAGE or co-IP. For co-IP, 250 uL of
supernatant was pre-cleared by incubation for 1 hour at 4°C with 30 uL of a 50% slurry
of Protein-A sepharose. The supernatant was kept and mixed with 5 pg of the appropriate
antibody and was then nutated at 4°C. After 1 hour, 30 uL of Protein-A sepharose slurry

was added and the mixture rocked for an additional hour. After this time, the Protein-A

58

sepharose with bound antibody and proteins was washed 4 times in extraction buffer and
then mixed with SDS-PAGE sample buffer, heated, and the supernatant loaded on gels.
Excised bands were submitted to the MSU Research Technology Support Facility for
tryptic digest and LC/MS/MS. The generated data was then compared against the

Arabidopsis proteome using MASCOT software (Matrix Science, Boston, MA, USA).

PK enzyme assays and kinetic analysis
All chemicals were from Si grna-Aldrich. Pyruvate kinase activity was detected by
coupling the production of pyruvate to the conversion of NADH to NAD+ by lactate
dehydrogenase unless otherwise noted. Reactions were kept at 25°C, were started by the
addition of enzyme mix, and were linear for at least 5 minutes. Absorbance at 340 nm
was monitored using a F LUOstar Optima 96-well plate reader (BMG Labtech, Offenburg,
Germany). Standard PKp reaction mixtures contained 50 mM HEPES-KOH pH 8.0, 5%
PEG-8000, 50 mM KCI, 15 mM MgCl2, 1 mM DTT, 2 mM PEP, 1 mM ADP, 0.2 mM
NADH, and 2 U ml‘1 desalted rabbit muscle lactate dehydrogenase. PEP phosphatase
activity was corrected for by omitting ADP from the reaction. Reactions at pH 7.0 were
done using 50 mM MOPS pH 7.0 instead of HEPES.

For kinetic analysis 2.5 pmol of each subunit were mixed and used per reaction.
S05 and Vmax values were determined by fitting the Hill equation to plots of initial
velocity versus substrate concentration using origin 7.0 (OriginLab Corporation,
Northampton, MA, USA). pH optimum curves were generated using a 25 mM MES, 25
mM Bis-Tris-propane buffer over a range of pH’s. For inhibitor/activator studies

metabolite stocks were made equimolar with MgC12 and were pH adjusted to 8.0.

59

Metabolites were tested at pH 8.0 with 100 uM PEP and 150 uM ADP for GB] and 150
pM PEP and 300 uM ADP for (102. The metabolites tested were: glucose, fructose, 6GP,
GlP, F6P, F16bP, DHAP, G3P, 2PG, acetate, 0AA, citrate, iso-citrate, 2-oxoglutarate,
succinate, fumerate, malate, gro3P, 2P-glycolate, glycolate, Ala, Arg, Asn, Asp, Cys, Gln,
Glu, Gly, His, Leu, Lys, Met, Ser, AMP, ADP, ATP, ADP-Glc, UDP-Glc, UDP-Gal, R5P,
6PG, KPi, NaNO3, and NH4C1, all at 10 mM; MgPPi, NADH, NADPH, NAD+, NADP+,
oxalate, Ile, Phe, Pro, Thr, Trp, Tyr, Val, CoA, Mal-CoA, Ac-CoA, and shikimate, all at
0.5 mM; F26bP, Oleoyl-CoA, Oleate, and LPA all at 0.05 mM. Oxalate, glyoxylate, and
0AA were found to inhibit the LDH reaction so PK activity was measured by coupling to
the ATP dependent conversion of glucose to glucose-6-phosphate by hexokinase
followed by the NAD+-dependent conversion of glucose-6-phosphate to 6-
phosphogluconate by glucose-6-phosphate dehydrogenase. The reaction mix was
adjusted to contain no NADH or LDH, but instead to have 1 mM NAD+, 5 mM glucose,
2 U mL'1 hexokinase, and 2 U mL'l G6PDH. 150 and Ka values are the concentration of a
metabolite required for 50% maximum inhibition or activation, respectively. They were
calculated by fitting a modified Hill equation to plots of initial velocity versus effector
concentration as previously described (Ballicora et al. 2005).

Site-directed mutagenesis of PK subunits was done using the QuikChange-XL
Site-Directed Mutagenesis Kit using primers designed to the manufacturer’s
specifications (Qiagen). An absolutely conserved lysine residue in the PK active site was
mutated to leucine for each subunit (PKp-a, K344L; PKp-Bl, K325L; PKp-B2, K314L).
This mutation has previously been shown to abolish PK activity (Sakai 2005). Chemical

inactivation was achieved by incubating 5 uM solutions of purified subunits with either

60

water or 100-fold molar excess of 2,4,6-trinitrobenzenesulfonic (TNBS) acid in the dark
at room temperature for 1 hour. This treatment has been shown to inactivate PK subunits
by covalent modification of lysine residues without abolishing protein interactions
(Hollenberg et al. 1971) Excess TNBS was quenched with an equal volume of 100 mM
Tris-Cl pH 7.5 for 20 minutes on ice and the proteins were used directly in enzyme

assays.

Accession numbers

Arabidopsis Genome Initiative locus identifiers (www.mabidopsisprg) used in this study
are as follows: At3g22960 (encoding PKp-a), At5g52920 (encoding PKp-Bl), Atl g32440
(encoding PKp-le, At2g36580, At3g04050, At3g25960, At3g49160, At3g52990,
At3g55650, At3g55810, At4g26390, At5g08570, At5g56350, At5g63680. Genbank
accession numbers for non-Arabidopsis protein sequences used are as follows: Nt PKp-A,
Q40545; Os PKp-A, NP_001059042; Rc PKp-A, Q43117; NtPKp-G, 040546; Se
PCC630] PK, YP_172116; Se WH8102 PK, NP_897391; Pfu PK, NP_578917; Sa PK,
YP_256251; Ec PK-l, AAA24392; Hs PK-L, BAA02515; An PK, Q12669; Sc PYKl,

NP_009362; Os PKG, BAD81116; Nt PKc, Q42954; St PKC, P22200; Gm PKc, Q42806

61

Results

Identification of plastid localized and seed resident pyruvate kinases

The Arabidopsis genome encodes 14 putative PK isoforms (Arabidopsis Genome
Initiative 2000). All but one (encoded by At3g49160) of the predicted PKs contain a fully
conserved PK active site of [LIVAC]-x-[LIVM]-[LIVM]-[SAPCV]-K-[LIV]-E-
[NKRST]-x-[DEQHS]-[GSTA]-[LIVM] (listed at European Bioinformatics Institute,

. http://www.ebi.ac.uL0 and are presumably active enzymes. Several cross-kingdom PK
phylogenies have been published (e. g. Hattori et al. 1995, Munoz and Ponce 2003,
Schramm et a1. 2000), but only one putative PKc from Arabidopsis was included in these
studies. When the 14 Arabidopsis PK amino acid sequences are aligned with bonafide
PKs from other organisms they segregate into cytosolic and plastidic clades (Figure
2.1A). This amino acid sequence similarity-based segregation is supported by the
exclusive prediction of chloroplast transit peptides at the N-terrnini of the four predicted
PKps using ChloroP and TargetP (Emanuelsson et al. 1999, Emanuelsson et al. 2000).
The PKps in Figure 2.1A are further divided between a and [3 subunits. One Arabidopsis
PKp subunit (encoded by At3g22960) is most similar to described PKp-as. Two others,
which show 63% amino acid identity to each other (encoded by At5g52920 ,PKp-BI;
At1g32440, PKp-B2), are most similar to known PKp-Bs (Figure 2.1A).

Three of the four predicted Arabidopsis PKp genes were identified as seed
expressed by EST analysis of developing seeds (White et al. 2000). Two, PKp-a and PK,,-
[31, represent the highest level of induction of any PK gene in seed and are coordinately
expressed in all tissues while the PKp-B2 encoding gene has very low transcript

accumulation in any tissue (Figure 2. 1 B, Schmid et al. 2005).

62

Figure 2.1. Pyruvate kinase similarity and selected gene expression in Arabidopsis

(A) Pyruvate kinase phylogeny. Amino acid sequences of Arabidopsis (gene loci in bold)
and other bonafide pyruvate kinases were used. Bootstrap values are indicted at
branches; or and [3 represent plastidic PK subunit families. The scale represents 10%
difference. An, Aspergillus niger; Ec, Eschericia coli; Gm, Glycine max; Hs, Homo
sapiens; Nt, Nicotiana tabacum; Os, 0ryza sativa; Pfu, Pyrococcusfuriosus; Rc, Ricinus
communus; Sa, Sulfolobus acidocaldarius; Se, Synechocystis sp.; Sc, Saccharomyces
cerevisiae; St, Solanum tuberosum

(B) Relative gene expression of putative Arabidopsis PKp-a (At3g22960), PKp-BI
(At5g52920), and PKp-B2 (At1g32440) encoding genes (derived from published
microarray data (Schmid et al., 2005)). DAF, days after flowering. Values are the mean i

so (n=3).

63

 

A At3g49160
0

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

      

.2
79
‘03
3
CL
Se PCC6301 PK
1000 Se WH8102 PK
Pfu PK
Ec PK-1
325 Hs PK-L
1000 SC PYK1
OS PKG
o NtPKc
% _ Atsgsssso
m 9 At4g2ssso
£633 St ch
O Gm PKG
Athessso
1°00 At5908570
N3904050
_ At3925960
813 10010000 At3g55810
1000 M3955650
J—-—At3952990
01 1WN2936580
B A
a?
g 7000
E5 6000
g 5000
B 4000
8 3000
g; 2000
9 1000 I
E o I I . I. . , . I . . I I I
w eaaaaagecm U-U- LLLL
a a222§25$§535§§355
FEE-Eat“? “"3 3°88
8 g g
5 .5
(I)

Figure 2.1. Pyruvate kinase similarity and selected gene expression in Arabidopsis

64

Figure 2.2. Phylogenetic analysis of plant PK protein sequences.

Amino acid sequence-based phylogeny of annotated PKs from plant and algal sources.
Some sequences are the same as used in Figure 2.1. The rice (Os), corn (Z. mays),
Chlamydomonas (C. reinhardtii), Cyanidioschyzon merolae (C. merolae), and
Physcomitrella patens (P. patens) sequences are new and accession numbers are listed

after the genus identifier.

65

P. patens 12219237
— P. patens 12159402
01 P. patens 12109480
Os12905110
0511905110
At3952990
A12936580
P. patens 1 :134756
2. mays 5_91860

 

0511910980
C. reinhardtii C_600010
. ‘P ”1 98597

 

    

 

 

 

 

 

 

 

 

Se WH8102 PK
Se PCC6301 PK

  

P. patens 1:147287

 

 

 

 
  

P. patens1z147118
At3g431

60
C. rein. C_550090

 

 

 

 

C. mero/ae CMP260C
__+ C. reinhardtii C_2590004
C. reinhardtii C_670023

 

 

 

C. reinhardtii C_910058
P. patens 12188818
P. patens 1:172921
P. patens12186171
P. patens 12131669
P. patens 12169084
Z. mays 5_3070
Z. mays 5_3071
0504958110
SI PKc
Gm PKc
A15963680
At5908570
0501916960
Nt PKc
At5956350
At4926390
At3904050
At3955810
At3955650
At3925960

 
 
  
  
  
  
   
  
  
 
  

Figure 2.2. Phylogenetic analysis of plant PK protein sequences

66

An additional phylogenetic analysis was performed using amino acid sequences of
annotated plant and algal PKs to understand the evolutionary history of the PKp-a and
PKp-B subfamilies (Figure 2.2). Both the 0t- and B-subunit subfamilies are populated by
proteins from algae, moss, and higher plants. Furthermore, both subfamilies originate
from a branch containing cyanobacterial PKs. When combined, these observations
suggest that the evolution of separate PKp subunits occurred after the secondary

endosymbiotic event which gave rise to photosynthetic eukaryotes.

Predicted PKp subunits are plastid localized

To study the subcellular localization of the putative PKps, C-terminal green fluorescent
protein (GFP) fusion constructs were generated and transiently expressed in Nicotiana
benthamiana under the control of the CaMV 35$ promoter. Figur2.3A shows that for all
three putative PKps a punctate GFP signal was observed at the periphery of chloroplasts.
No GFP signal was associated with structures other than chloroplasts. All three proteins
in question are predicted to be soluble and stromal. The localized patterns observed in
Figure 2.3A could be due to the abundance of the fusion proteins, caused by
overexpression and resulting in possible overloading of the import apparatus. In vitro
chloroplast import followed by protease protection assays were conducted to obtain
independent evidence for plastid localization of the PKp subunits. When in vitro produced
3SS-labeled PKp subunits were incubated with isolated pea chloroplasts, the proteins were
imported and processed to their presumably mature forms (Figure 2.3B). All three were

resistant to treatment with thermolysin and trypsin and were found in the soluble fraction.

67

- - + Trypsin
- + - Thermolysin
P S P S P S
4— pPKp-O
“I n "K mPKp-o

‘“ PPKp'131

«PPKirflz
~~ ~~~- ----+—mPKp-i32

 

. . -o—mSS

Figure 2.3. Subcellular localizations of pyruvate kinase subunits

(A) Transient expression of GFP fusion constructs in N. benthamiana. GF P, green
fluorescent protein fluorescence; CAF, chlorophyll autofluorescence; the bar is 5 uM
long.

(B) In vitro import of PKp subunits into isolated pea chloroplasts. After import,
chloroplasts were subjected to either no treatment (-) or to post-treatment (+) with either
Thermolysin or Trypsin. Intact chloroplasts were subsequently recovered by
centrifugation through 40% Percoll cushion and fractionated into a total membrane (P)
and a supernatant (S) fraction. A11 fractions were analyzed by SDS-PAGE and
fluorography. MM, molecular masses of precursor and mature proteins based on Rf
analysis; TP, represents 10% of translation reaction added; p, precursor or m, mature

form; pSS, precursor of the small subunit of RuBisCO included as control.

68

Based on the data, the three putative PKp subunits are localized to chloroplasts and are
imported and processed into mature, soluble, stromal proteins as predicted by the analysis
of their amino acid sequence. The molecular masses indicated in Figure 2.3B were
derived from Rf analysis and are in agreement with the transit peptide cleavage site
predictions made by ChloroP. The full length precursor proteins were calculated to be
65.1 kDa, 63.5 kDa, and 62.6 kDa with predicted transit peptides of 47, 63, and 55 amino
acids for PKp-a, PKp'BI, and PKp'BZ, respectively. The mobility shifts of all three mature
proteins reveal no major discrepancies between the predicted and observed transit peptide

cleavage sites.

Heteromeric subunit composition of recombinant PKps

A recombinant approach was taken to study Arabidopsis seed PKp subunit composition
due to the scarcity of embryo tissue for native protein purification. The cDNAs encoding
the three putative PKp subunits lacking the predicted transit peptides were isolated by
reverse transcription and PCR and were inserted into an E. coli expression vector with an
N-terminal 6X-His tag and a thrombin site for removal of the tag after purification. Gel
electrophoresis (SDS-PAGE) and subsequent immunoblotting were used to confirm
purification of single proteins and complete cleavage of the tag (Figure 2.4A). Initial tests
indicated that in liquid assays none of the subunits had PK activity on their own and that
only GB] and (102 combinations were active (Figure 2.4B). Epitope-tagged versions of the
proteins were also generated that in addition to the N-terrninal His-tag had short C-
tenninal epitope tags. PKp-a was fused with c-myc (EQKLISEEDL), PKp'BI With HA

(YPYDVPDYA), and PKp-B2 with FLAG (DYKDDDDKG). The antibodies used for

69

detection of the epitopes were shown to be lacking of any cross reactivity. Native-PAGE
with epitope-tagged proteins was used to explore this subunit requirement in more detail.
Figure 2.5, panels A-E show five identical native-PAGE gels developed in different ways.
The gel in Figure 2.5A was stained for PK activity. Only the (113. and (1132 mixtures were
active. Moreover, the activities coincided with less mobile bands as shown by the
Coomassie brilliant blue (CBB) stained gel (Figure 2.58). In the case of (1131, detection of
the individual u-myc and Bl-HA fusion proteins with specific antibodies revealed a

higher molecular mass complex coinciding in mobility with the active complex in Figure
2.5A, suggesting that the active complex is composed of both a and BI subunits (Figure

2.5c, D).

70

A uncut thrombin cut
0 I32 I31 0 I32 I31
82 kDa—

64 kDa— -*‘-- .. W = -- _ m coomassie
49 kDa — '

 

  

64 kDa —-. i’ “-mu -; Anti His

A

 

OD

PK Activity (pmol/sec x 103) w
.a N

 

 

O

0 Br B2 031 0132 131132 051 ClI32
(+)ADP (-)ADP

 

Figure 2.4. PKp protein purification and initial activity assay

(A) SDS-PAGE and anti-His immunblot of purified PKp subunits. Individual subunits
were subjected to no treatment or treatment with thrombin to remove the His tag. 500 ng
protein was loaded per well. Immunoblot shows His tag has been removed after treatment
with thrombin.

(B) Pyruvate kinase activity of various subunit mixtures. Equal volumes of purified
subunits were assayed alone or in combination. Approximately 0.1 ng of each subunit
was used in each assay with saturating substrate concentrations. No PEP phosphatase

activity was observed in control reactions without ADP.

 

 

 

 

   

. 5 6°
w (i .1 (9 a"
$2 52 £3 3 x- o #0
~z~ A A .2- A
A :S f x, .; §§
A 92 Q2 Q2 0 F :2
*. * 2
PKActivity 3
.669 kD °\° 100 +031
. M a V80 +0
Coomassre. . , Y 5 , . - {—232 kDa "5 ‘
ir'“ g.."" <1:40.
i ‘ E 20
CA * 01* I i a; 0‘
"t'myc I II - I “3'6 0 1o 20 30 40750 60 7060
D(PKp-0) “ ' I g Fraction
Anti HA 0
E(PKp-B1) L‘ I I I G Fractionfl'ZTEIIB'
32 kD 2“ .
64 Kile-jg? ;.:s.::.~.;_«..... .
Anti KFLAGW L4. 49 k0 - , ‘ ~~
PKp—Bz)

Figure 2.5. In vitro interaction of PKp subunits

(A-E) Identically loaded native-PAGE gels showing in vitro interaction of PKp subunits.
10 pmol (~0.6 pg) of each subunit was used per lane (A) PK activity stained gel. (B)
Coomassie stained gel. (C) Immunological detection of a-myc with anti c-myc (D)
Immunological detection of 131-HA with anti HA (E) Immunological detection of B2-
FLAG with anti FLAG. * is used to denote bands corresponding to the active PK
complexes.

(F) PK activity elution profile after F PLC over Superdex-200

(G) SDS-PAGE gel of most active fractions from (F). 75mg of protein was loaded per

lane.

72

The result is less clear for the (1132 complex or and I31 subunits (Figure 2.5C, D). The result
is less clear for the (1132 complex because the B2 subunit alone forms a higher molecular
mass complex with the same mobility as the active enzyme (Figure 2.5A, B, E). However,
the a-myc protein is present in the 0132 higher molecular complex (Figure 2.5C). Thus, it
is likely that the higher molecular mass active complex in the 01132 mixture is also
composed of both subunits.

Gel-filtration chromatography was used to estimate the molecular masses of the
active PKp heteromers. Figure 2.5F shows that for both 043. and (102 PK activity eluted as
a single peak. The molecular masses of these active complexes were calculated to be 463
i 10 kDa for (1131 and 476 i: 10 kDa for (1132, which is consistent with octomeric
complexes of 60 kDa (1 subunits and 57 kDa [3 subunits. Based on SDS-PAGE analysis,
the most active F PLC fractions contained equal amounts of or and [3 subunits (Figure
2.5G). Thus, the active PKs appear to be heterooctomers composed of 4 0. and 4 [3
subunits.

The in vivo interaction of the PKp subunits was tested using co-
irmnunoprecipitation (co-1P). Three constructs containing full-length, epitope-tagged
cDNAs encoding the 0., BI, and B2 subunits driven by the CaMV 35S promoter were
introduced into Arabidopsis. Only the a-myc fusion protein could be directly
immunoprecipitated from plant tissue. One possible explanation is that the epitope tags in
the [3 subunits were not accessible in the native complex. The SDS-PAGE gel in Figure
2.6A show the result of co-IP experiments with silique tissue from wild type and three

35S:0t-myc plants.

73

Figure 2.6 In vivo co-immunoprecipitation of PKp subunits

(A) Coomassie Brilliant Blue stained gel and anti myc immunoblot of co-
immunoprecipitated (Co-1P) proteins. Total proteins were extracted from wild-type and
3SS:a-myc silique tissue and were subjected to Co-IP. About 37.5 pg of crude protein
and half of the total eluate were loaded per lane. Bands unique to the 35S20t-myc co-IP
lanes (in brackets) were excised and identified. IgG heavy chain is indicated on
immunoblot for reference. WT, wild type.

(B) Protein sequences, predicted transit peptides, and proteomics coverage of PK,-

0 ,PKp-Bl ,and PKp-B2. Gene Loci, encoded subunit, and percent coverage by proteomics
are listed. ChloroP predicted chloroplast transit peptides are in bold. Peptide fragments

identified by proteomics are highlighted in gray.

74

A Co-I P Crude

 

 

BSSzo-myc WT 355:0-myc WT

 

coomaSSIe
64 kDa
49kDa
37 kDa
64kDa _ ,» m _ ___ \Anti myc
49 kDa — ...... .- ..., .2...- o-myc

“IgG heavy chain

At3922960, PKP-a, 67% coverage

 

  
 
 
 

 

 

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a'VIv'ytlsghmasIvsrcrpdcpIfafttttsvrrrlnlqwgllpfrlsfsddmasnlnktffillksrgmIksgdlvr
avsdmlqsiqvmnvp

 

At5952920, PKp-B1, 21% coverage

IIICH' I“ I “a r V C VaVlallvvuIsIJIou

 

 

 

 

 

r. ' .1 I n x _‘L I 1 I u - A. 1
I l' l. r' "vale 2‘1 9 r 5,. IUUIIIIV _ .
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qsgtqpimsqsthmqwkv

 

At1g32440, PKp'Bz. 10% coverage

 

 

 

 

 

 

 

 

:01 v r‘l v a a _‘1"""' npna
l |_l l .1 .L J I Al ' L' J' I J L JL
1 1 ar .» a
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'I I A A .l .1 I A L I I II LA. I
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_. . 1 u .l L 1 ll_ - n 11.. u ._ 1 l 1. -
IIGGOIPVI. .. J a 9‘ ,_ a I ,_ 1 Illlql
I I ' I .IJ _IA II II I II L .‘I ' LLI I 'I '
J ‘Ia r a a ‘l a-I '- a —-r ‘I as

Figure 2.6 In vivo co-immunoprecipitation of PKp subunits

75

A small amount of a-myc protein was detected by an anti myc immunoblot in crude
extracts from 3SSza-myc plants. After immunoprecipitation, the a-myc protein was
enriched and became visible on CBB-stained gels. In addition, another protein running
slightly faster than a-myc was visible on the CBB-stained gel. The CBB-stained and
immuno-reactive bands running at 49 kDa and at a slightly less molecular mass were also
present in the wild-type control. These were presumably products of the degradation of
the anti-myc IgG during elution from the Protein-A sepharose. All three PKp subunits are
very close in size and could co-migrate during SDS-PAGE. Therefore, a gel slice
including a section above and below the a-myc protein from the co-IP reaction (indicated
by brackets in Figure 2.6A) was excised and the contained proteins were subjected to
tryptic digest and mass spectrometry (LC/MS/MS). Analysis of the mass spectrometry
data identified peptides of all three PKp subunits with significant individual ion scores
(Mowse scores >31, p<0.05): PKp-u (encoded by At3g22960), PKp-Bl (encoded by
At5g52920), and PKp-Bz (encoded by At1g32440), with 29, 7, and 3 non-redundant
peptides representing 67%, 21%, and 10% coverage of the predicted mature proteins,

respectively (Figure 2.68).

Kinetic characterization of PK complexes

Enzyme activity analysis was performed using reconstituted PKp complexes. The
maximum PK activity for the (1B; and 0432 heteromers was reached within 1 minute of
mixing the subunits (Figure 2.7A). Reciprocal titrations in saturating substrate conditions
showed that plots of PK activity versus subunit equivalents follow hyperbolic curves

when one subunit is held constant and the other titrated (Figure 2.78).

76

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.2
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o 1 2 3 4 5 6

pm ol/subunit

Figure 2.7 Kinetics of active PKp complex formation

(A) Time course of PK activity after mixing subunits. Numbers above bars represent the
time (minutes) incubated prior to assay. 2 pmol of each subunit was used per assay under
saturating substrate conditions. Activity is expressed relative to the 60 minute sample.
(B) Subunit titration curves with PKp subunits. For Cor-VB], the a subunit was held
constant and the [3; subunit was variable. For CBl-Va, the B1 subunit was held constant
and the a subunit was variable. The same notation applies to the [32 titrations. 2.5 pmol of
the constant subunit was used per reaction in saturating substrate conditions. Equivalents
refers to the molar ratio of the variable subunit to the fixed one.

(C) Pyruvate kinase activity relative to protein concentration. Equal amounts of either a

and B. or a and [32 were mixed and assayed under sub-saturating substrate conditions.

77

Furthermore, when equal molar ratios of subunits were used, enzyme activity increased
linearly with increasing protein concentration (Figure 2.7C). These results suggest that
the association (and thus activity) of the subunits is dependent only on protein
concentration and that there is little or no cooperativity of subunit association.
Site-directed mutagenesis and chemical inactivation were performed to explore
the subunit requirement in more detail (Figure 2.8). Both treatments were directed at a
lysine residue in the PK active site required for phosphoryl group transfer. In the case of
the site-directed mutant proteins (Figure 2.8A,B), PK activity was only observed when
both a wild-type or and a wild-type [3 subunit were present. No activity was seen when
only one subunit had a wild-type active site. The inclusion of a mutant protein extract did
not inhibit the activity of wild-type complexes, indicating the contaminating bands seen
in the SDM lanes of the SDS-PAGE gel of Figure 2.8A did not interfere with PK activity.
Thus, the loss of PK activity was due only to mutation of the active site of either subunit
of the PKp complex. Treatment with 2,4,6-trinitrobenzenesulfonic acid was employed to
chemically inactivate the PK subunits. Inactivation was incomplete as determined by
mixing TNBS-treated a and [3 subunits. Nonetheless, maximum PK activity was only
observed with mixtures of mock-treated proteins. Activity was reduced 60-80% when
either subunit was chemically inactivated. Addition of an inactivated subunit to an active
mixture did not affect PK activity, meaning carryover of a component of the TNBS
treatment was not responsible for reductions in PK activity. Therefore, the observed
reductions in PK activity when using one TNBS-treated subunit in combination with a
mock-treated one were due only to modification of lysine residues on the protein.

Unmodified active sites in both subunits were required for PK catalytic activity.

78

A SDM WT
a I31 I32 0 B1 82

 

 

 

CBB
82 kDa-—
64kDa—- * *W """" *——*--
49 kDa-
82 kDa— ~ Anti His
64 kDa— a“
49 kDa— - ‘4“
B 14 C 12
c12 C10
E10 35 3
o 9
5.8 0.6
m6 ‘3’
E E 4
5 4 3
2 2
0 0
N N N W I", N'} \ \ W W \
a a.» a; . a WWW. as
We: saw é 5" $0.00
g2 a s s°

Figure 2.8. PKp subunit inactivation

(A) SDS-PAGE and anti-His immunoblot of affinity purified site-directed mutant (SDM)
and wild-type (WT) PKp subunits. Approximately 15 pmol of each subunit was loaded
per well. Conditions for protein purification were the same for all six proteins, but
contaminating protein bands can be seen in the mutant protein lanes. The immunblot
reveals which bands are the actual his-tagged PKp subunits (marked with * on CBB gel).
(B) PK activity using wild-type and site-directed mutant (denoted with K—->L) proteins.
Assays were done at pH 8.0 with saturating substrates and 2 pmol of each subunit.

(C) PK activity using wild-type and chemically inactivated (denoted with i) proteins.

Assays were done at pH 8.0 with saturating substrates and 2 pmol of each subunit.

79

Further kinetic experiments were done using equal molar ratios of 0431 or (132
mixtures. The enzymes had a strict requirement for Mg2+ and K+ and were completely
inactivated after 3 min incubation at 50-55°C. The pH optima were found to be pH 7.8-
8.0 (Table 2.2) and subsequent reactions were conducted at pH 8.0. The V.,“,m of 01(3) was
approximately 2-fold higher than that of uBz and the S0_5(s) for ADP and PEP were 1.5
and 3-fold lower, respectively, for 01131 (Table 2.2). Both complexes displayed sigrnoidal
saturation kinetics for ADP and PEP, with 01B. having greater Hill coefficients (Table 2.2).
Both enzymes were capable of catalyzing phosphoryl group transfer from PEP to NDPs
other then ADP. Activities detected using 10 mM CDP, GDP, or UDP were 85, 57, and
67% and 13, 24, and 19% for 0431 and 0B2, respectively, when compared to an ADP

control. Clearly, the (1132 enzyme preferred ADP while (1B; was less discriminatory.

Table 2.2 PKp kinetic constants

 

 

 

Enzyme
Constant 11B. (1132
pH Optimum 8.0 7.8
Vmax (U/mg) 13.5 :t 0.3 7.6 :1: 0.4

PEP 80.5 (11M) 75.1 i 7.0 (2.0) 118.6 fl: 11.5 (1.2)
ADP $0.5 (11M) 113.9 i 9.1 (1.9) 303.6 :I: 27.8 (1.2)
K...t (s") 9.5 x 104 5.3 x104

 

Hill coefficients for PEP and ADP are given in parentheses.
Values are the mean :1: SD (n=4). PEP,
phosphoenolpyruvate; ADP, adenosine diphosphate.

80

Numerous metabolites and signaling compounds (fully listed in Materials and
Methods) were tested as effectors of Arabidopsis PKp activity at subsaturating
concentrations of PEP and ADP. Those compounds which had a significant effect are
listed in Table 2.3. Only one activator, 6-phosphogluconate, was identified and it only
activates 01132. The rest of the effectors acted as inhibitors with the most effective being
glutamate and oxalate (Table 2.3). Values for the constants 150 and Ka were calculated
(see Materials and Methods for definitions) for effectors capable of 50% relative
activation or inhibition and they clearly show that (1B2 is the more sensitive enzyme with
respect to these compounds. Neither enzyme was sensitive to treatment with dithiothreitol
(DTT) or sodium tetrathionate (N aTT) suggesting a lack of redox regulation contrary to
what was previously observed for plastidic glucose 6-phosphate dehydrogenase isoforms

of Arabidopsis (Wakao and Benning 2005).

Table 2.3 Metabolite effectors of PKp activity

 

 

 

Concentration Relative
Tested (mM) Activity (%) 150 (mM) K“ (mM)
Metabolite 0101 (102 a0. 0102 uBl 0102
6PG 0.05 96 192 - - - 0.02
Glutamate 5 59 30 6.2 2.1 - -
Oxalate 0.2 71 50 0.41 0.21 - -
ISO-Citrate 10 70 75 ND. ND. - -
AMP 1 77 96 ND. ND. - -
ATP 1 77 87 ND. ND. - -
Glyoxylate 5 83 79 ND. ND. - -
OAA 2 82 88 ND. ND. - -

 

Values represent the mean of at least four repeats. Activity is percent
relative to a no effector control (set at 100). 6-PG, 6-phoshpogluconate;
AMP, adenosine monophosphate; ATP, adenosine triphosphate; OAA,
oxaloacetate; ND, not determined

81

Discussion

Arabidopsis has two heterooctomeric PKps

Previous kinetic analyses of plant PKs have been limited to enzymes which can be
purified in the native state from dissected tissue samples (e. g. Hu and Plaxton 1996,
Plaxton et al. 2002, Smith et al. 2000, Turner et al. 2005). The results of such studies
have revealed that PK isoforms vary depending on the tissue and subcellular
compartment in question. Arabidopsis tissues, and especially developing seed, represent a
challenge to the traditional enzyme purification approach due to the difficulty in
amassing the quantities of tissue required. However, this work is facilitated by the
available resources and model characteristics of Arabidopsis and the findings are crucial
if we want to achieve a complete understanding of the biology of this plant. A
bioinformatics approach was taken to identify the candidate seed expressed PKp encoding
genes in the Arabidopsis genome sequence. Four potential plastid targeted, seed-resident
PKs were identified based on phylogenetics and gene expression data (Figure 2.1). The
prediction of plastid localization of these proteins was subsequently confirmed using GFP
fusion protein localization and pea chloroplast in viva import assays (Figure 2.3). Indeed,
PKp-a and PKp-Bl have been found in the stromal fraction of chloroplasts using a
proteomics approach (Friso et al. 2004). PKp-B. was also previously identified in a
mitochondrial proteomics study, but plastid contamination could not be ruled out (Giege
et al. 2003). Previous research on PKp-u orthologs from B. napus and R. communis
detailed the proteins’ transit peptide cleavage sites (Plaxton et a1. 2002, Wan et a1. 1995).
The N-terminal sequence of the mature B. napus protein aligns with a region about 40

amino acids c-terminal to the predicted Arabidopsis cleavage site, while the R. communis

82

protein has a transit peptide of 83 amino acids. These data suggest that the transit peptide
of PKp-a may be longer than the predicted 55 amino acids. However, analysis of the
observed molecular masses of the precursor and mature proteins afier pea chloroplast
import support the predictions made byTargetP and ChloroP (Fig 3). Moreover, the
Arabidopsis PKp-a protein contains a domain (79% amino acid identity to R. communis
PKp-A) which is responsible for altered import characteristics of R. communis PKp-A
(Wan et al. 1995). Our results indicate that this sequence does not affect the ability of
Arabidopsis PKp-u to be imported into and processed in pea chloroplasts.

It was determined that the Arabidopsis PKp enzymes are most likely ~460 kDa
heterooctomers of 60 and 57 kDa subunits with 41140 stoichiometry (Figure 2.5). The
reconstitution of active PKps from individually purified inactive subunits in this study as
well as the in vivo Co-IP results leave little ambiguity as to the heteromeric structure of
the Arabidopsis PKps. Some studies of recombinant PKp polypeptides from developing R.
communis endosperm concluded that the PKp-u and PKp-B homologs are distinct enzymes
(Blakeley and Dennis 1993, Blakeley et al. 1995, Wan et al. 1995), while others have
determined that the same proteins are actually subunits of a single heteromeric PKp
(N egm et al. 1995, Plaxton 1991, Plaxton et a1. 1990). The data presented here support

the conclusion that PKp is a complex composed of two different subunits.

Arabidopsis PKP activity is determined by two [3 subunits
Kinetic analysis revealed that the reconstituted Arabidopsis enzymes behave much like

previously documented PKps (pH optima of approximately 8.0, Mg2+ and K+ requirement,

and $0.5 values-for PEP and ADP in the 100-300 11M range). A pH optimum of 8.0 is

83

potentially important for these enzymes. Such a pH is generated in the plastid stroma in
response to light. As the plastids of Arabidopsis seeds are green and presumably
photosynthetic, it is possible that in vivo PKp is light-activated via alkalinization of the
stroma. Such regulation of PKp could contribute to the light-induced stimulation of fatty
acid synthesis in green seeds (Goffman et al. 2005). What else was discovered were
distinct differences between the two isoforms in Arabidopsis. The 01131 form is a more
efficient enzyme with a higher specific activity and lower So. 5 values for both PEP (75
uM vs 120 11M) and ADP (Table 2.2). The concentration of PEP in potato tuber tissue has
been estimated to be around 50-100 11M (F arre et al. 2001) and assuming the same is true
for metabolically active Arabidopsis tissues, (1131 would be expected to be more active in
vivo. In addition, (113. is 3-5 times more efficient at utilizing alternative nucleoside
diphosphates. The 0102 enzyme, on the other hand, is more responsive to the strong
metabolite effectors glutamate, oxalate, and 6-PG (Table 2.3). In fact, 031 is completely
insensitive to the activating effect of 6-PG. This is interesting since what has been
described as the major PKp from B. napus is activated by 6-PG (Plaxton et al. 2002).
Apparently, Arabidopsis and B. napus have diverged in this regard as the gene for the [32
subunit present in the the 6-PG—regulated PK, (1102 enzyme) from Arabidopsis is hardly
expressed in any tissue (Figure 2.18). A distinct feature of the Arabidopsis PKps is their
inhibition by glutamate (Table 2.2). Regulation by this effector has been reported for
PKcs from other plants but not PKps (Hu and Plaxton 1996, Smith et al. 2000). It should
be noted that while the presence of either [31 or [32 in the Arabidopsis PKp complex results
in different regulatory properties, these subunits should not be considered purely

regulatory. They both contain fully conserved PK active sites in which chemical or

84

genetic modification results in an inactive PK complex (Figure 2.8). These experiments,
in combination with the fact that the 01 subunit has no activity when assayed alone (Figure
2.48), support a model in which the both a and [3. or [32 subunits are required for enzyme
activity and the specific interactions between the subunits result in differential kinetic
properties.

The primary structures of the recombinant PKps may not be the same as the native
enzymes and thus the kinetic data must be considered with caution. However, as the
transit peptide cleavage site predictions agree well with the observed molecular masses of
precursor and mature PK subunits (Figure 2.38), the differences are likely to be minimal
between the native and recombinant proteins. It should also be noted that specific post-
translational modifications could result in changes to the enzyme structure and or
function, and these possibilities were not explored. Soybean PKC, for instance, can be
partially degraded at the c-terminus in vivo, which results in altered regulatory properties
(Tang et al. 2003).

Perhaps most significant is the realization that PKp from Arabidopsis exists as
differentially regulated heteromers resulting from the interaction of the 01 subunit with
one or the other [3 subunit. The two PKp-B subunits of Arabidopsis likely arose through
gene duplication as indicated by the similarity of their amino acid sequence and gene
structure and subsequently evolved unique regulatory features. The 043. enzyme is likely
to be dominant in most tissues based on gene expression (Figure 2.1), but it is possible
that under conditions when glycolysis needs to be more regulated the 0102 enzyme is
produced at higher rates. The regulatory properties of the 01[3. enzyme, notably the lower

805 for ADP and PEP, higher Vmax, lower sensitivity to metabolic regulation, and ability

85

to utilize other NDPs more efficiently, make it a better enzyme for processing carbon at
high rates, independent of the metabolic status of the tissue. The 0t[32 enzyme, however, is
less active and more susceptible to metabolite-based regulation and may serve more
specialized roles. It will be interesting to study the in vivo relationship of these subunits
and whether or not one enzyme complex can contain a mixture of [3 subunits that would

further fine tune PK activity for specific metabolic demands.

86

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Verwoerd, T.C., Dekker, B.M.M., and Hoekema, A. (1989) A small-scale procedure
for the rapid isolation of plant RNAs. Nucleic Acids Res. 17:2362.

Wan, J., Blakeley, S.D., Dennis, D.T., K0, K. (1995) Import characteristics of a
leucoplast pyruvate kinase are influenced by a 19-amino acid domain within the protein. J.
Biol. Chem. 270:16731-16739.

Voinnet, O., Rivas, S., Mestre, P., and Baulcombe, D. (2003) An enhanced transient
expression system in plants based on suppression of gene silencing by the p19 protein of
tomato bushy stunt virus. Plant J. 33:949-956.

Wakao, S. and Benning, C. (2005) Genome-wide analysis of glucose-6-phosphate
dehydrogenases in Arabidopsis. Plant J. 41:243-256.

White, J .A., Todd, J ., Newman, T., Focks, N., Girke, T., de Ilarduya, O.M.,
Jaworski, J .G., Ohlrogge, J.B., and Benning, C. (2000) A new set of Arabidopsis
expressed sequence tags from developing seeds. The metabolic pathway from
carbohydrates to seed oil. Plant Physiol. 124:1582-1594.

9O

Chapter 3
Analysis of carbon metabolism and storage compound accumulation in seeds of an

Arabidopsis mutant deficient in a plastidic pyruvate kinase2

 

2 This work has been published in Andre, C., Froehlich, J .E., Moll, M.R., and Benning, C. (2007) A
heteromeric plastidic pyruvate kinase complex involved in seed oil biosynthesis in Arabidopsis. Plant Cell
1921-17. [performed all of the experiments shown here.

91

Abstract

Glycolysis is a ubiquitous pathway thought to be essential for the production of oil in
developing seeds of Arabidopsis and oil crops. Compartmentation of primary metabolism
in developing embryos poses a significant challenge towards testing this hypothesis and
for the engineering of seed biomass production. It also raises the question whether there
is a preferred route of carbon from imported photosynthate to seed oil in the embryo.
Disruption of the gene encoding the [31 subunit of plastidic pyruvate kinase (PKp) causes
a 75% reduction in enzyme activity and a 60% reduction in seed oil content. The seed oil
phenotype is fully restored by expression of the [3. subunit-encoding cDNA, and partially
by the [32 subunit-encoding cDNA. Additionally, carbohydrates accumulate in the mutant
seeds, possibly due to reduced activity of upstream glycolytic enzymes. Therefore, the
identified pyruvate kinase catalyzes a crucial step in the conversion of photosynthate into
oil suggesting a preferred plastid route from its substrate phosphoenolpyruvate to fatty

acids.

92

Introduction

An important metabolic function of a developing Arabidopsis (Arabidopsis thaliana)
seed is the deposition of storage reserves: oil in the form of triacylglycerols (TAG), but
also proteins, oligo- and polysaccharides (Baud et al. 2002). As sucrose is the major
photosynthetic product transported in the phloem the embryo is required to catabolize
incoming sucrose and convert it into the more efficient storage compounds mentioned
above. Glycolysis is central to this process as it converts sugars into precursors for
protein and fatty acid synthesis while concomitantly producing ATP by substrate level
phosphorylation. In fact, stable isotope labeling has been used to demonstrate that 90% of
glucose fed to developing canola (Brassica napus) (a close relative of Arabidopsis and
oilseed crop) embryos is converted to pyruvate by the RuBisCO bypass and the lower
half of glycolysis (Schwender and Ohlrogge 2002, Schwender et al. 2004a). Furthermore,
a clear link between glycolysis and seed metabolism is apparent in the wrinkled] (wriI)
mutant of Arabidopsis, in which a general reduction in glycolytic activity results in an
80% reduction in seed oil (Focks and Benning 1998).

Glycolysis in plants occurs in both the cytosol and the plastid and both
compartments are connected through plastid membrane transporters (Plaxton 1996,
Weber 2004). The intermediates of upper glycolysis of developing B. napus embryos
appear to be in near equilibrium between the cytosol and plastid (Schwender et al. 2003),
and enzymes of the full glycolytic sequence have been detected in both compartments in
embryos (Eastmond and Rawsthome 2000). It is therefore likely that changes in
glycolytic enzyme activities influence the transport of related intermediates across the

plastid envelope. This compartmentation raises the question of a preferred route of

93

glucose metabolism in developing oil seed embryos. Expressed sequence tag (EST)
analysis of developing Arabidopsis seeds indicated that mRNAs encoding cytosolic
enzymes for the entire glycolytic pathway are abundant, but that only mRNAs encoding
plastidic enzymes for the second half of the pathway metabolizing trioses are abundant
(White et al. 2000). Microarray experiments extended these findings by detecting a shift
in expression from genes encoding cytosolic glycolytic enzymes to those encoding
plastidic glycolytic enzymes at the onset of storage compound accumulation. While gene
expression does not necessary reflect enzyme activity or metabolite flux, the microarray
data led to the hypothesis that glucose is broken down in the cytosol to
phosphoenolpyruvate (PEP), which is then imported into the plastid and metabolized by
plastidic pyruvate kinase (PKp) (Figure 1.2, Ruuska et al. 2002). Revisions to this scheme
were introduced based on more recent experiments (2'. e. RuBisCO bypass, see Schwender
et al. 2004a) but in the model the lower half of glycolysis metabolizing PEP remains
unaltered. While the import of PEP by plastids of B. napus embryos has been
demonstrated (Kubis et al. 2004), the chlorophyll a binding protein underexpressed
(cue!) mutant of Arabidopsis lacking a seed-expressed PEP transporter has no reported
seed reserve phenotype (Li et al. 1995). A recent steady-state carbon flux analysis on
cultured embryos of B. napus led to the proposal of a RuBisCO shunt involving reactions
of the reductive pentose phosphate pathway in the plastid (Schwender et al. 2004a). The
proposed pathway bypasses the initial glycolytic reactions in the cytosol and generates
PEP in the plastid, which could compensate for the PEP import deficiency in the cue]
mutant. Alternatively, pyruvate generated by cytosolic pyruvate kinase (PKC) could be

imported and uSed directly for fatty acid biosynthesis. Isolated plastids from B. napus

94

embryos are capable of incorporating l4C labeled pyruvate into fatty acids (Eastmond and
Rawsthome 2000, Kang and Rawsthome 1994), but no plastidic pyruvate transporter has
been reported. A current model of primary metabolism in developing Arabidopsis seeds
is shown in Figure 1.2. Direct molecular or genetic corroboration of this scheme is
generally lacking and the focusing on PKp in developing Arabidopsis seeds should be

highly informative in the testing of this hypothesis.

95

Materials and Methods

Plant growth and transformation

All Arabidopsis plants were of the Col-2 ecotype, except for the SALK T-DNA lines
which were Col-0. All seeds were first sterilized in 20% bleach, 0.05% TritonX-lOO for
20 min and were then rinsed 5 times with water and plated on half-strength MS medium,
pH 5.9, 0.9% agar, and 2% sucrose. When appropriate, kanamycin or hygromycin B were
included in the medium at 50 pg mL'1 and 25 pg mL", respectively. Seeds were stratified
at 4°C for 3 days prior to being germinated in an incubator (AR-75; Percival Scientific,
Boone, IA, USA) at a photon flux density of 60—80 pmol m_2 sec-I and a light period of
16 h (22°C), and a dark period of 8 h (18°C). Seedlings were transferred to 3.5 inch
square pots and were grown in a soil mix as described previously (Xu et al. 2002) and
were grown under a 16-h photoperiod with a day temperature of 22°C and a night
temperature of 20°C at a photon flux density of 100—120 umol m—2 sec‘l. The plants
were fertilized with half-strength Miracle-Gro (Scotts, Marysville, OH, USA) plant food
every fifteen days. Wild-type and mutant Arabidopsis plants were prepared for
transformation as previously described (Cemac and Benning 2004). When ready, plants
were transformed using the floral dip method (Clough and Bent 1998). Competent cells
ongrobacterium tumefaciens strain C58Cl GV3101 pMP90 (Koncz and Schell 1986)

were prepared and transformed as previously described (Shen and Forde 1989).
T-DNA mutant isolation and characterization

T-DNA insertion lines were obtained from the SALK T-DNA insertion population

(Alonso et al. 2003). Mutants were selected on growth medium containing kanamycin

96

and T-DNA insertions were confirmed using PCR primers specific for gene sequences
and the T-DNA left border. Primers were designed using the i-sect tool
(http://signal.salk.edu/tdnaprimers.html). Insertion sites were confirmed by sequencing
the PCR product of the left border primer and a gene specific primer. Expression of the
target gene was analyzed with RT-PCR using primers listed in Table 2.1. Total RNA was
isolated from seedlings using the Qiagen RNeasy kit. 600 ng of total RNA was used for
reverse transcription using the Qiagen Omniscript RT kit. PCR was done using 5% of the
RT product. Actinl (At2g37620) was used for control purposes. The PCR consisted of 25
cycles of 95°C for 30 sec, 60°C for 45 sec, and 72°C for 1 min, followed by a 10 min
72°C extension. Complementation of the mutant was done using full length cDNAs
inserted into the Kpnl site of the CaMV 353 containing pCAMBIAl300 derivative
mentioned above. An antisense construct was generated using the same full length cDNA
for PKp-B, inserted in the antisense orientation into the Kpnl site of the binary vector
pBinAR-Hyg (Dormann and Benning, 1998) containing the 12S seed storage protein
promoter from Arabidopsis (Ohlrogge J, Benning C, Gao H, Girke T, and White J,
Inventors; Plant seed specific promoters. US patent 7,081,565. 2006 July 25). Analysis of
gene expression in the rescued lines was done using RNA gel blots. Total silique RNA
was extracted using a previously described protocol (Verwoerd et al. 1989) followed by
DNase treatment and cleanup using the Qiagen RNeasy kit. Northern analysis (5 pg total
RNA) was performed as previously described (D6rmann and Benning 1998). The blots
were analyzed using a phosphor imager (Molecular Dynamics, Amersham, Piscataway,

NJ, USA).

97

Analysis of chlorophyll content

Chlorophyll was quantified in seeds as previously described (Lichtenthaler 1987). Seeds
were imaged using a Leica M2 12.5 dissecting microscope (Leica Microsystems, Wetzlar,
Germany) equipped with a Spot Insight color camera (Diagnostic Instruments, Sterling

Heights, MI, USA).

Transmission electron microscopy

For electron microscopy, seeds were dissected out of staged silques and soaked in water
for 1 hour. Embryos were then expelled from their seed coats by pressing the soaked
seeds between two glass microscope slides and were then embedded in 2% agarose. The
embryos were fixed for 2 hours at room temperature with 2.5% glutaraldehyde, 2.5%
paraformaldehyde in 0.1 M cacodylate buffer and then post-fixed in 1% (w/v) osmium
tetrachloride in 0.1 M cacodylate buffer. The samples were then dehydrated in a graded
series of acetone, embedded in Poly BD 812 resin, and sectioned. The thin sections (~70
to 100 nm) were stained with uranyl acetate and lead citrate prior to examination in a

J EOL 100CX electron microscope (J EOL, Japan).

Developing seed enzyme assays

Enzyme activities were measured from developing seed proteins extracted in a buffer of
50 mM Tris-Cl pH 7.5, 5 mM MgC12, 1 mM EDTA, 1 mM EGTA, lmM DTT, 0.1%
Triton-X100, 10% glycerol, 2 mM benzamidine, 2 mM e-amino-n-caproic acid, and 1
mM PMSF. Pyruvate kinase activity was detected by coupling the production of pyruvate

to the conversion of NADH to NAD+ by lactate dehydrogenase. Reactions were kept at

98

25°C, were started by the addition of enzyme mix, and were linear for at least 5 minutes.
Absorbance at 340 nm was monitored using a FLUOstar Optima 96-well plate reader
(BMG Labtech, Offenburg, Germany). Standard PKp reaction mixtures contained 50 mM
HEPES-KOH pH 8.0, 5% PEG-8000, 50 mM KCl, 15 mM MgC12, 1 mM DTT, 2 mM
PEP, 1 mM ADP, 0.2 mM NADH, and 2 U ml'l desalted rabbit muscle lactate
dehydrogenase. PEP phosphatase activity was corrected for by omitting ADP from the
reaction. Reactions at pH 7.0 were done using 50 mM MOPS pH 7.0 instead of HEPES.
ATP-dependent phosphofructokinase (PFK) and pyrophosphate-dependent
phosphofructokinase (PFP) activities were measured as previously described (Burrell et

al. 1994).

Seed metabolite analysis

Seed oil quantification by fatty acid methyl ester analysis was done as previously
described (Focks and Benning 1998). Seed storage proteins were extracted from 50 mg of
dry seeds by first grinding in a mortar and pestle followed by 2 extractions of the tissue
with 30 volumes of hexane. The delipidated seed material was pelleted by centrifugation
at 13,000 g for 10 min. The pellet was then dried in a speed-vac and extracted twice for
15 min with 0.5 volumes of 50 mM Tris-HCl pH 8, 200 mM NaCl, 5 mM EDTA, 0.1%
TWeen-20, 2 mM benzamidine, 2 mM s-amino-n-caproic acid, 1 mM PMSF. Water bath
sonication was used to resuspend the pellets. The supernatants from the extractions were
combined and a 1:5 dilution was used to quantify protein using the Bio-Rad DC Protein
Assay Kit. For SDS-PAGE, the equivalent 3 seeds worth of total protein was loaded per

lane. Mature seed free amino acids were extracted from 25 mg of seed tissue 3 times with

99

400 uL of 70% methanol. The supematants were combined and extracted 2 times with an
equal volume of chloroform to remove the lipids. The remaining aqueous phase was dried
under vacuum and then resuspended in 20 mM HCL. Extracts (20 uL) were then loaded
and run on over a strong anion exchange column using a Hitachi Amino Acid Analyzer
(Hitachi High Technologies America, Inc., San Jose, CA, USA) at the MSU
Macromolecular Structure, Sequencing, and Synthesis Facility. Glucose, fructose,
sucrose, and starch were extracted from developing seeds and quantified as previously
described (Focks and Benning 1998). PEP and pyruvate were extracted from developing
seeds with perchloric acid and quantified using a NADH fluorescence assay as previously
described (Hausler et al. 2000). ADP and ATP were measured in the same extracts with
an ATP Bioluminescence Assay Kit (Sigma) using a previously described protocol

(Ruuska et a1, 2000).

100

Results

Disrupting the PKp-Bl encoding gene causes a reduction of PKp activity and seed oil
content

Multiple independent SALK T-DNA Insertion lines were obtained to study the in vivo
function of the Arabidopsis PKps (Alonso et a1. 2003). Six lines were reanalyzed for exact
insertion site location (PKp-a; SALK_096141, SALK_024870, PKp-Br; SALK_042938,
SALK_042681, PKp-Bz; SALK_013574, SALK_142845), but only one (SALK_042938
in PKp-Bl) carried an insertion in a translated portion of the gene (Figure 3.1A). The line
SALK_096141 has an insertion in an intron of the PKp-a encoding gene, but RNA levels
were unaffected in these plants. Genotyping by PCR revealed homozygous
SALK_042938 mutants completely lacking transcripts from the PKp-B. encoding gene as
detected by RT-PCR (Figure 3. l B, C). Transcript amounts for the PKp-a and PKp-Bz
encoding genes were unchanged (Figure 3.2). The mutant will be referred to as pka .
Notably, of the six analyzed mutants, only 19ka had any visible seed phenotype. The
mature seeds of pka were wrinkled when observed under a dissecting microscope and
developing mutant embryos contained less chlorophyll (Figure 3.3A, B). Rescue of pka
with ectopic expression of the PKp-B. encoding gene restored chlorophyll content to
nearly wild-type levels, however, overexpressing the PKp—Bz encoding gene was much
less effective. Transmission electron microscopy was done to assess any ultrastructural
perturbations in the pkpl mutant. The 5k-magnification (Figure 3.3C, upper panels)
micrographs show representative cotyledonary cells from 13 DAF embryos. Wild-type
cells are full of oil bodies (ob) by this time and still contain some starch (st) in the

plastids. The pka mutant has much smaller oil bodies than wild type, but has much

101

larger starch granules. The 67k-magnification images (Figure 3.3C, lower panels) show
in more detail the thylakoid structure of the mutant. While organized similarly, the pkp]

thylakoids are less extensive than wild type.

A SALK_096141 SALK_024870

 

SALK_013574
SALK_142845
no .

   

SALK_042938 SALK_042681
TDNA

      

LP RTi RP RT2

Actin1 RT1/RT2
1Kb

Figure 3.]. Identification of a SALK T-DNA mutant in PKp-Bl

(A) Gene structure of the three PK subunits and locations of T-DNA insertions. Black
box on T—DNA is the left border. LP and RP depict locations of primers used for
genotyping in (B). RT] and RT2 depict locations of primers used for RT-PCR in (C).
ATG, start codon; STOP, stop codon.

(B) PCR based genotyping of SALK_042938. LP and RP refer to At5g52920 specific
primers shown in (A) and in Table 2.1. LB refers to the T-DNA lefi border primer in
Table 4. WT, wild type.

(C) RT-PCR to measure PKP-Bl encoding gene expression in SALK_042938. Actinl
(At2g3 7620) is the control. RTl and RT2 refer to At5g52920 specific primers shown in

(A) and in Table 2.1. WT, wild type

102

Actin
WT pkp1

1200bp
800bp

400bp

m
. 74......
w
~—
—_
_
—
a——.
m

~ ”on

   

Figure 3.2. Reverse transcriptase-PCR analysis of PKp gene expression

PKp-a and PKp-Bz gene expression in 11 days afler flowering seeds of the pka and WT

background. Actinl (At2g37620) is the control. WT, wild type.

103

ADAF5 7 13 15]dry

mlmflflfifl .
manual 5

 

   

ED

 

—* N N (A)
01 O 01 o
r r r

10'
5-

ng chlorophyll/seed

 

 

 

.5 '7 9 1'1 1'3 1'5 17
Days After Flowering

Figure 3.3. pkpl seed phenotypes

(A) Seed phenotypes of pka and wild type (WT). Embryos were dissected out of
developing seeds at the time (DAF, days after flowering) indicated. Fully dessicated
mature seeds are shown as well. The bar represents 0.2 mm.

(B) Total chlorophyll content in developing seeds of pka, wild type (WT), and lines
rescued with CaMV 3SS-driven expression of either PKp-B. (RBI-23) or PKp-Bz (RB2-3).
Forty seeds were measured per sample. Values are the mean i SD (n=6).

(C) Electron micrographs of cells from 13 DAF wild-type (WT) and pkp] cotyledons.
Starch granules (st) and oil bodies (0b) are marked with arrows in the upper panels.
Higher magnification in the lower panels reveals thylakoid membranes (th) inside
plastids. 5k and 67k denote magnification used. Asterisks (*) protein bodies. Bars in
upper panels represent 2 pm. Bars in lower panels represent 0.5 pm. Lefi panels, wild

type; right panels, pkpl mutant.

104

The smaller oil bodies, reduced thylakoid membranes, and wrinkled seeds of pka
suggest a reduction in lipid biosynthesis and possibly storage compound accumulation.
Therefore, oil and protein were quantified in the mature pka seeds (Table 3.1). The
mutant accumulated only 40% as much oil as wild type, yet there was only a 15%
reduction in protein. Test crosses and analysis of the F1 progeny indicated that the low oil
phenotype of pkpl is a recessive trait largely dependent on the genotype of the embryo. A
homozygous pkpl sporophyte did however result in a 15% reduction in seed oil when

pollinated with wild-type pollen (Table 3.1) indicating a small maternal effect.

Table 3.1 WT and [7ka seed storage compound accumulation

 

 

WT .0ka 2;)? pkg/469x

Wag/”261$ 6.77 i 0.7 2.71 i 0.2 5.68 :t 0.8 6.9 i 0.2
(“Z/13:23 5.23 i 0.6 4.39 :t 0.5 ND. ND.
SEE/$23; 19.2 :1: 0.9 14.4 i 1.1 ND. ND.

 

Values are the mean of three repeats 3: standard deviation. Seed mass
determined by measuring the weight of 500 seeds three times. ND, not
determined

Enzyme activities in developing seeds were measured to determine the extent of
the PKp defect in pka . The presence of a potentially large number of different cytosolic
and plastidic PK isoforms complicates the interpretation of activity assays using crude
extracts. However, two factors aide in validating seed PKID activity in crude extracts: 1. all
14 PK encoding genes are not highly expressed in any given tissue at the same time
(Schmid et al. 2005), 2. cytosolic PKs typically have pH optima of approximately pH 7.0,

whereas plastidic PKs have pH optima of approximately pH 8.0 (Hu and Plaxton 1996,

105

Plaxton et al. 2002, Smith et al. 2000, this work). Figure 3.4A shows the time course of
PK specific activity using protein extracts of seed dissected from staged siliques. At pH
8.0 wild-type PK specific activity is greatest at 7 days after flowering (DAF) and steadily
declines throughout seed development. This pattern agrees with the expression profiles of
PKp-u and PKp-Bl encoding genes as shown in Figure 2.1, but is shifted to later DAF
possibly due to differences in growth conditions. The pkp] mutant at pH 8.0, however,
has 3-fold reduced PK specific activity at 7 DAF and does not change within
experimental limitations throughout the rest of the time course. Pyruvate kinase specific
activity was not reduced in the mutant at pH 7.0 but was actually increased at 11 and 15
DAF. Mutant and wild-type protein extracts were also assayed in the presence of 5 mM
glutamate or 0.2 mM 6-phosphogluconate (Figure 3.48). Recall that glutamate at 5 mM
inhibited recombinant (1B1 by about 40%, while 01132 was inhibited by 70% (Table 2.2).
Native PK specific activity in 9-11 DAF wild-type seed extract was inhibited only about
25% by 5 mM glutamate while that from pka was unaffected. As shown in Chapter 2, 6-
phosphogluconate is a potent activator of 01132 (Table 2.2), yet had no effect on wild-type

or pka seed PK specific activity.

106

>

 

 

 

     

 

 

 

 

 

 

 

 

500
3: 300‘ 350 None
0
g 200‘ .E 300‘ lg'U
E 1001 .g 250, -6PG
a
0 pH 7' i ' T ' 3’ 200‘ .
c 400‘ 5 1501
g E 1001 . ,
a 300‘ 50 1 7;,
:5) 1001 WT9-11DAF pkp19-11DAF
o - - - . .
5 7 9 11 13 15 17

Days After Flowering

Figure 3.4. PK specific activity in pkp1 and wild-type seeds

(A) Total PK specific activity measured at pH 8 and pH 7 in saturating substrate
conditions. WT, wild type. One mU is defined a 1 nmol pyruvate formed per minute.
Values are the mean i SD (n=4).

(B) Native seed PK specific activity at 9-11 DAF in response to metabolite effectors.
Activity was measured at pH 8.0 with subsaturating substrate concentrations. WT, wild
type. None, no effectors; glu, 5 mM glutamate; 6-PG, 0.2 mM 6-phosphogluconate. One

mU is defined a 1 nmol pyruvate formed per minute. Values are the mean :I: SD (n=4).

107

Seed fatty acid and protein profiles in pkp1

Fatty acid methylester (FAME) analysis of developing seeds revealed that pkp1
accumulates oil in the same temporal pattern as wild type, but at a much lower rate
(Figure 3.5A). The fatty acid composition of the oil in mature seeds was also analyzed.
The pkp1 mutant had a decrease in stearic (18:0), oleic (18:1), and linoleic (18:2) acids
along with an increase in linolenic (18:3). The proportion of very long chain (20 and 22
carbons) to long chain (16 and 18 carbons) fatty acids was also increased in pkp1 (Figure
3.5B). Total protein extracts were made from mature wild-type and pkp1 seeds and were
run on an SDS-PAGE gel to analyze the storage protein profile. Apparently, there are no
differences in the major storage protein profiles between wild type and pkp1 (Figure
3.5C) and the 15% reduction in protein content seen in pkp1 (Table 3.1) is not specific to
any one protein. Free amino acid content of mature seeds was also compared between
wild type and the mutant. Analysis of amino acid extracts by HPLC revealed increases in
the proportions of glycine, arginine, and glutarnine in pkp1 (Figure 3.5D). On the other
hand, aspartate, aspargine, glutamate, and valine were in greater proportion in the wild-
type seeds. Statistically, however, the differences were very small between wild type and

pkp1 for any of the listed amino acids.

Restoration of seed oil content in pkp1 expressing B-subunit-encoding cDNAs
Dealing with a single T-DNA insertion line, which could potentially harbor secondary
mutations that cannot be detected by genotyping, required additional precautions to
unambiguously link the target genotype with the observed phenotype, in this case the

reduction in oil content.

108

 

 

 

 

 

    
  

 

 

A 7 C WT pkp1
3 +WT ——
g6 +pkp1
B15 ,._. M
3
m4‘
E3 “wwfi‘m‘fi’flj—QS-a
52‘ “3--“‘5330—128-5
o 1,
.—
5 7 9 11 13 15 '7
Days After Flowering
835
.WT
30 kap1
25
°\° .
—020
E15
10 I
5
0 I I I E a
‘P. 0 ‘.'. 9‘. 9. 0" ‘.'. 9 ‘.'.
:2 <2 2 e a e a a a.

 

Figure 3.5. Oil and protein phenotype of pkp1 seeds

(A) Fatty acid accumulation in developing seeds. DAF, days after flowering; FAME,
fatty acid methyl ester; WT, wild type. Values are the mean 5: SD (n=6).

(B) Fatty acid profile of desiccated mature seeds. Values obtained from FAME analysis
of dry seeds. WT, wild type. Values are the mean :t SD (n=6).

(C) CBB stained SDS-PAGE gel of total protein extracts from wild-type (WT) and pkp1
seeds. Major storage protein bands are denoted in the margin.

(D) Free amino acid profiles of wild-type (WT) and pkp1 mature seeds. Values are the

mean :t SD (n=3).

109

To address this issue we constructed transgenic lines in the pkp1 background expressing
cDNAs that encode either PKp-Bl or PKp-Bl subunits and included these transgenic lines
in the analysis. Expression of these cDNAs was expected to restore the oil content if this
phenotype was due to the disruption in the PKp-B. encoding gene in the pkp1 line thereby
confirming the link between genotype and phenotype.

A T-DNA construct containing a CaMV 35S driven cDNA encoding PKp-fil was
used to rescue the lipid phenotype of pkp1 . Antibiotic resistance was used to select
transformants and rescued lines were identified by scoring for visual rescue of the
wrinkled seed phenotype. Of 43 independent transformants, 8 appeared to be rescued
based on seed morphology. Homozygous T3 seeds from individual rescued lines were
subjected to FAME analysis (Figure 3.6A). Overexpression of the PKp-Bl encoding
cDNA rescues the lipid phenotype of the mutant, but does not result in an increase in oil
amount in excess of wild-type seed oil content in any of the lines. A PKp-Bz encoding
cDNA was similarly overexpressed in pkp1 and transformants were selected. Twenty
independent transformants were identified and 6 of these had rescued seed morphology.
FAME analysis was performed on homozygous T3 seeds from these lines and showed
rescue of the low-oil phenotype of pkp1 (Figure 3.6B). However, the restoration of oil
content was not as complete as was achieved by overexpressing the PKp-Bl encoding
cDNA. Overexpression of the cDNA encoding PKp-Bl was able to restore the fatty acid
profile of pkp1 to wild type, while the PKp-Bz encoding cDNA overexpressors had the
same fatty acid profile as the pkp1 mutant, despite being almost completely rescued in

terms of oil accumulation (Figure 3.6C).

110

>

 

 

o—xmcohwmxr
CU

Total FAME (pg/seed)

O-INOJAUIO'DNCD

Total FAME (pg/seed)

 

 

MW :

RB1-7 :
RB1-22 :—_—+
RB1-23 :3
RB1-24 :21~
RB1-26 :
RB1-31 :
RB1-34 :1
R9143 :9

WT

 

mol %

   

 

    

 
   

 

I
I
‘5‘.
w
1—

Figure 3.6. Rescue of the pkp1 seed oil phenotype

I
‘3’. ‘7. 9
co 0 N
v— N N

I I I
9. 9 9.
CD CD 0
\— 1— N

(A) Oil amounts in mature seeds of pkp1 overexpressing the PKp-B. encoding cDNA. The
8 individual rescued lines are denoted with R131 and a number. WT, wild type. Values are

the mean i SD (n=3).

(B) Oil amounts in mature seeds of pkp1 overexpressing the PKp-Bz encoding cDNA. The
6 individual rescued lines are denoted with R132 and a number. WT, wild type. Values are

the mean d: SD (n=3).

(D) Average fatty acid profiles of the 8 RBI and 6 R132 lines compared to wild type (WT)

and pkp1. Values are the mean at SD (n=6).

111

The complementation data strongly suggest that the seed oil phenotype of pkp1 is
caused by the T-DNA insertion located in the PKp-Bl encoding gene. To fiirther exclude
the possibility of second site mutations causing the phenotype, an antisense construct was
generated to reduce the transcript amount of the PKp-Bl encoding gene specifically in
seeds. The Arabidopsis l2S seed storage protein promoter was used for this purpose.

Of twenty wild-type transformants identified, three had seeds with phenotypes
reminiscent of the pkp1 mutant. None of the empty vector control lines displayed altered
seed morphology (wrinkledness). Analysis of FAMEs was performed on the wrinkled
seeds and the three lines contained 3.92 :t 0.6, 2.73 i 0.3, and 2.22 i 0.2 pg of oil per
seed. The recapitulation of the pkp1 seed phenotype using antisense repression
independently corroborates that the low oil phenotype can be caused by reduction or

abolishment of expression of the PKPI gene encoding PKp-Bl.

Rescued lines have subunit-specific restoration of PK activity

PK gene expression and enzyme activity in the rescued lines were explored. Figure 3.7A
shows RNA gel blots of 9-11 DAF silique tissue probed with PKp gene specific probes.
The top panel shows no accumulation of PKp-13. transcript in pkp1 (as demonstrated by
RT-PCR in Figure 3.1C) and restoration only in the R131 lines. In the middle panel, very
little PKp-132 transcript is present in wild type, pkp1, or the R131 lines. However, the R132
lines clearly have increased amounts of the transcript. Based on gene expression, the R131
and R132 lines appear to have 01131 or (1132, respectively, as the dominant PK in silique tissue
and thus provided an opportunity to further test the metabolite regulation observed for the

recombinant 01131 and 01132 complexes.

112

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a...

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"I PKp-B2

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120 120
100 100
.S c
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340 540
E E
20 20
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Figure 3.7. PKp gene expression and enzyme activity in rescued pkp1 lines

(A) Expression of PKp encoding genes in siliques of wild type (WT), pkp1, and rescued
lines. R131 is overexpression of PKp-13. and R132 denotes overexpression of PKp-132. Actin1
probe was used for loading control.

(B) Native PK specific activity of 9-1 1 DAF siliques in response to metabolite effectors.
Lefi panel includes wild type (WT), pka , and pkp1 lines rescued with overexpression of
PKp-13. (R131). Right panel includes wild type (WT), pkp1, and pka lines rescued with
overexpression of PKp-132 (R132). Activity was measured at pH 8.0 with subsaturating
substrate concentrations. None, no effectors; glu, 5 mM glutamate; 6-PG, 0.2 mM 6-
phosphogluconate. One mU is defined a 1 nmol pyruvate formed per minute. Values are

the mean 1 SD (n=4).

113

PK specific activity was first measured at pH 8.0 from 9-11 DAF silique material in the
absence of effectors. The left and right panels of Figure 3.7B show that PK specific
activity was restored in the R13. and R132 lines. The inclusion of 5 mM glutamate in the
assay mixture resulted in a moderate inhibition of native PK specific activity in wild type
and all of the rescued lines, as was observed for wild type in Figure 3.48. The PK
specific activity from the R132 lines, however, responded differently from wild type and
the R131 lines to the presence of 0.2 mM 6-phosphogluconate. PK was activated by 6-
phosphogluconate in the R132 lines, which provides needed correlation between the

observed metabolite regulation of recombinant and native PK specific activity.

Altered substrate/product ratios and accumulation of glycolytic precursors in the
pka mutant

The effects of the pkp1 mutation on the pools of the substrates (PEP and ADP) and
products (Pyr and ATP) of PK in developing seeds were analyzed. Metabolite
measurements were made at 5-7 DAF and at 11-13 DAF, when PK specific activity and
chlorophyll content, respectively, are most affected in the mutant seeds. Table 3.2 shows
the results of these analyses. At 5-7 DAF, the amount of Pyr in pkp1 is decreased,
resulting in a 40% reduction in the Pyr/PEP ratio. At 11-13 DAF, there is no difference in
the ratio of Pyr/PEP between wild type and pkp1. Instead, in pkp1, the absolute amounts
of Pyr and PEP are proportionally increased. The PEP and Pyr data show that PK specific
activity positively correlates with the Pyr/PEP ratio, due mostly to effects on the steady
state levels of Pyr. The ATP/ADP ratio is increased almost 2 fold in pkp1 when both PK

specific activity (5-7 DAF) and chlorophyll content (1 l-13 DAF) are the most reduced.

114

Table 3.2 Metabolite levels in WT and pkp1 developing seeds

 

 

PEP Pyr ADP ATP
DAF nmol/ g FW nmol/g FW Pyr/PEP nmng FW nmol/g FW ATP/ADP
WT 5-7 17.5 i 2.5 89.1i 7.9 5.1 15.8 :1: 1.6 53.7 d: 3.7 3.4
11-13 13.3 11.6 36.5 i 2.7 2.7 12.8 i1.2 38.8 i 3.7 3.0
k 1 5-7 18.6 i1.5 58.1 :1: 6.1 3.1 13.0 :1: 1.3 66.4 :t 3.4 5.1
p p 11-13 22.2 i 4.1 59.1 i 10.4 2.7 10.9 d: 2.6 57.3 i 7.0 5.3

 

Values represent the mean i SD of at least three repeats. DAF, days after flowering; PEP,
phosphoenolpyruvate; Pyr, pyruvate; ADP, adenosine diphosphate; ATP, adenosine triphosphate; FW,
fresh weight
It seems that the metabolic perturbations in pkp1 actually result in increased energy status
in the developing seeds. This is somewhat surprising as PK activity and photosynthesis
are expected to contribute to the ATP pool.

Pyruvate kinase is a control point for glycolysis as its activity has a direct impact
on ATP and PEP, the latter of which is an inhibitor of phosphofructokinase in anoxic
tissues such as seeds (Plaxton 1996). It is reasonable that a reduction in PK activity
would result in an inhibition of glycolytic flux and an accumulation of carbohydrate
precursors. Thus, hexose, sucrose, and starch were also measured in developing wild-type
and pkp1 seed. Figure 3.8A shows that in wild type hexoses accumulated early during
development and steadily decreased, while sucrose followed the opposite trend. The pkp1
mutant seeds followed the same trends, but contained twice as much of both compounds
during peak accumulation times. Starch in the wild type showed the same transient
accumulation pattern as previously documented (Focks and Benning 1998, Baud et al.

2002). However, the pkp1 mutant continued to store starch throughout embryo

development (Figure 3.8A).

115

‘\

IL

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‘ Hexoses _._ WT

-- pkp1
g 200.

 

PF W
K :pkp1

 

600 Sucrose

2’ 300
100

N

o

O
mU/mg protein

 

Starch 50 .

 

 

 

 

9 13 9
Days After Flowering

9 0.4

 

 

 

5 7 9 11 13 15 17
Days After Flowering

Figure 3.8. Carbohydrate accumulation and phosphofi'uctokinase enzyme activity in
pkp1 and wild—type seeds

(A) Hexoses (glucose plus fructose), sucrose, and starch levels in developing seeds. WT,
wild type. Values are the mean 1: SD (n=4).

(B) ATP-dependent phosphofructokinase (PFK) and pyrophosphate-dependent
phosphofructokinase (PFP) activity in developing seeds. One mU is defined a 1 nmol

pyruvate formed per minute. WT, wild type.

116

ATP-dependent and PPi-dependent phosphofructokinase (PF K and PF P,
respectively) activities were measured in developing seeds at 9 and 13 DAF (Figure
3.8B), when pkp1 has increased hexoses only, or increased sucrose and starch,
respectively. PF K activity decreases in the wild type between the two timepoints while
PFP activity increases. In the pkp1 mutant, the PF K and PFP activities change as in wild
type, but are reduced by 25% and 40%, respectively. Taken together, the data in Figure
3.8 suggest that a reduction in PKp activity impairs the catabolism of carbohydrates in

developing seeds, possibly due to a reduction in upstream glycolytic activities.

117

Discussion
The (1131 form of PKp is dominant in seeds

A reverse genetic approach was taken to directly test the metabolic function of
PKp in developing Arabidopsis seeds. Mutants in the PKp-a gene, which should result in
complete inactivation of both PKps, could not be isolated. Only one mutant, in the PKp-131
encoding gene, was identified which had no transcript accumulation and a seed
phenotype (Figure 3.1, Table 3.1). Methods to separate the native Arabidopsis PK
isoforms (isoelectric focusing, zymograms, native-PAGE) were unsuccessful and so PKp
activity was assayed at pH 8.0 to minimize the background from cytosolic enzymes. The
PK specific activity profile at pH 8.0 in wild type very closely matches the expression of
the 01 and 13. subunit encoding genes (Figure 2. 1 B), while in pkp1 the activity is greatly
reduced and does not change (Figure 3.4A). In addition, wild-type and pkp1 seed PK
activities were insensitive to activation by 6PG (Figure 3.4B). In contrast, seed PKp
activity in pkp1 lines that have been rescued with overexpression of the PKp-132 encoding
cDNA (R132) are activated in the presence of 6PG (Figure 3.7B) All these results agree
with the in vitro enzyme characterization presented in Chapter 2 and corroborate a
specific inactivation of the 11131 enzyme in the pkp1 mutant. Furthermore, the pkp1 mutant
supports the initial hypothesis that the influx of photosynthate into embryo tissue and the
high demand for lipid and amino acid precursors requires high PKp activity. The
regulatory properties of the 01131 enzyme mentioned above make it a prime candidate for
this role. Taken together with the lack of transcript accumulation for the gene encoding
PKp-132 (Figure 2. 1 B), these data indicate that the 0113. enzyme is the major PKp isoform

present in developing Arabidopsis seeds. The increase in PK specific activity at pH 7.0 in

118

pkp1 suggests a compensatory mechanism in the mutant (Figure 3.4A). It is possible that
more pyruvate is being generated in the cytosol by another isoform of PK. A preliminary
flux map of carbon metabolism in B. napus embryos shows that 30% of the pyruvate used
for fatty acid synthesis is cytosolic in origin (Schwender et al. 2003, Schwender et al.
2004b, Schwender and Ohlrogge 2002). If the same is true for Arabidopsis, the increase
in PK specific activity at pH 7.0 only marginally compensates, as pkp1 still has a 60%
reduction in seed oil. Based on gene expression data (Figure 2.18, Figure 3.2), a small
amount of the PKp-132 subunit could also be present in developing seed. The presence of
this subunit could explain the incomplete loss of PK activity and seed oil in the pkp1

mutant.

Seed metabolism is dependent on proper PKp function

The pkp1 mutant seeds are smaller and less green than wild-type seeds (Figure 3.3). The
reduction in chlorophyll in pkp1 developing seeds could be the result of sugar
accumulation (Figure 3.8A) as sugars are known to repress chlorophyll accumulation and
photosynthetic gene expression (J ang et al. 1997, J ang and Sheen 1994). It is also
possible that a pleiotropic effect of the pkp1 mutation is reduced chlorophyll biosynthesis.
Dark treatment has been shown to decrease fatty acid synthesis by 23% in B. napus
embryos (Ruuska et al. 2004). However, all light-activated processes are affected by this
treatment and the reduced biosynthetic capability was linked to a lack of light induced
activation of certain enzymes. In the case of pkp], light still penetrates the seed and is

capable of inducing enzyme activities and other processes. Thus, the primary metabolic

119

defect brought on by a reduction in PKp activity is likely the major factor contributing to
the low oil phenotype of pkp1 .

In both wild-type and pkp1 seeds, fatty acids accumulate in a linear time course
from about 5 DAF until at least 15 DAF (Figure 3.5A). The rate of accumulation, though,
is reduced by about 60% in the mutant, which correlates with the reduction in seed oil
and the reduction in total PK specific activity at pH 8.0. The fatty acid profile of mature
pkp1 seeds (Figure 3.5B) is very similar to that of the wriI mutant and plants with altered
biotin carboxyl carrier protein gene expression (Focks and Benning 1998, Thelen and
Ohlrogge 2002). These mutations impair fatty acid synthesis either by reducing the
supply of precursors or by inhibiting the ACCase reaction, respectively. It is likely that in
the pkp1 mutant a reduction in precursors for fatty acid synthesis is the cause of the
altered fatty acid profile. Indeed, the steady state level of pyruvate is reduced in pkp1
seeds compared to wild type at 5-7 DAF, which correlates with the onset of fatty acid
biosynthesis (Table 3.2). It should be noted that these measurements do not distinguish
the subcellular compartmentation of the metabolites in question. Interestingly, the
amounts of PEP and pyruvate, but not the ratio of the two, are increased in pkp1 at 11-13
DAF (Table 3.2). The idea of a reduction in the supply of precursors for fatty acid
biosynthesis is further supported by the accumulation of carbohydrates in the mutant seed,
a phenotype similar to that of wriI (Figure 3.8A). It is also possible that the reduction in
the rate of fatty acid synthesis is brought on by a lack of ATP. Seeds of B. napus (and
likely Arabidopsis) are a low oxygen environment (Vigeolas et al. 2003) and even with
photosynthesis PK could have an important role in the production of ATP. A mutant

defective in a plastidic ATP/ADP transporter with reduced ATP import capacity into

120

plastids has reduced oil content in its seeds (Reiser et al. 2004). Moreover, this mutant
was shown to compensate for reduced ATP import by increasing the expression of the
genes encoding PKp-B. and PKp-132. A similar increase in transcript level for any of the
PKp subunits is not seen in pkp1 seeds (Figure 3.2). Additionally, steady state levels of
ATP in the mutant seeds were actually increased relative to wild type (Table 3.2),
possibly as a result of increased cytosolic PK (PKC) activity in the mutant (Figure 3.4A).
Elevated PKc activity in pkp1 might be an indicator of enhanced cytosolic glycolytic flux
into mitochondrial respiration, which could also elevate the ATP/ADP ratio. Based on the
pkp1 mutant phenotype it is unlikely that ATP production is a major function of PKp in
developing Arabidopsis seeds. The pkp1 mutant is rescued by ectopic overexpression of
the PKp-Bl and PKp-132 encoding cDNAs except that oil accumulation is recovered less
fully in the R132 lines (Figure 3.6). A similar pattern of rescue was observed for seed
chlorophyll accumulation (Figure 3.3B) and fatty acid profile (Figure 3.6C), in which the
R132 lines were not rescued as completely as the R131 lines. The maximum activity
observed in seed extracts was similar in all of the rescued lines (Figure 3.7B). Thus, it is
likely that distinct regulatory features determined for the two PKp complexes (detailed in
Chapter 2) account for the observed physiological differences between the R131 and R132
rescued plants. No transgenics were observed that had an increase in seed oil content or
in PK activity, despite higher than wild-type expression levels of the PKp-BI or PKp-132
encoding genes (Fig 3.7A). This might indicate that in these lines the amount of PKp-u is
limiting as it is required for activity.

Concomitant with the reduction in seed oil in pkp1 is the accumulation of

increased amounts of hexoses, sucrose, and starch (Figure 3.8A). It seems that there is a

121

redirection of carbon partitioning in pkp1 in which less hexose and sucrose are
catabolized via glycolysis, but are instead incorporated into starch. However, the starch
accumulated in pkp1 seeds at 15 DAF only accounts for approximately 20% of the carbon
not incorporated into fatty acids (Table 3.2, Figure 3.8A). One potential mechanism for
the observed accumulation of carbohydrates is that elevated PEP (Table 3.2), which is a
potent inhibitor of plant phosphofructokinases (Plaxton and Podesta 2006), could slow
the entry of hexose—phosphates into glycolysis, resulting in a misregulation of metabolism.
The reduction of PFK and PFP activities in pkp1 support this hypothesis (Figure 3.8B). It
should be noted that the assays for PFK and PF P are set up in such a way as to maximally
activate the enzymes, and thus measure activity as it relates to total protein amounts. The
observed reductions in activities therefore do not reflect any potential additional
regulation of the enzymes caused by altered metabolite levels. The mechanisms that
down-regulate these activities in pkp1 are unknown, but one could speculate that sugar
signaling is somehow involved. It is interesting that in pkp1 excess carbon is not
redirected into storage protein synthesis. Instead, protein levels are slightly decreased
(Table 3.1), which could be a result of decreased glycolytic flux and reduced substrate
availability. Two plastid-localized enzymes of branched chain amino acid biosynthesis,
acetolactate synthase (ALS) and dihydrodipicolinate synthase (DHPS), use pyruvate as a
substrate and have Km values of 1 614 mM for ALS and about 1.7 mM for DHPS
(Dumer and Boger 1990, Dereppe et al. 1992). The plastidic pyruvate dehydrogenase
(ptPDC) complex has a Km for pyruvate of about 300 11M (Camp et al. 1988). Thus,
amino acid synthesis could be out competed by ptPDC in situations of limiting pyruvate

availability. However, based on these properties alone, one would expect a decrease in

122

protein content greater than that of oil in pkp1. One possible explanation for the observed
phenotype is that branched chain amino acids are only a minor component of total seed
protein (Figure 3.5D) and restriction of their synthesis has little effect on protein content.
Increased cytosolic PK specific activity in pkp1 (PK at pH 7.0, Figure 3.4A) could also
help maintain almost wild-type protein levels by feeding into the TCA cycle and
increasing the supply of carbon skeletons available for the synthesis of other amino acids.
The results of this study are in support of the metabolic model depicted in Figure
1.2 in which PEP metabolized by PKp in the plastid is the main source of pyruvate for
fatty acid and amino acid syntheses. The compartmentation of metabolism apparently
serves to isolate metabolic pathways such that specific products can be generated from
distinct pools of substrates. In the case of seed oil metabolism, pyruvate generated in the
plastid is used mainly for fatty acid synthesis and cannot be fully replaced by cytosolic
pools. It seems that Arabidopsis seeds are programmed to make oil from plastidic
pyruvate and if that pathway is perturbed, as is the case in the pkp1 mutant, some of the
carbon (20% in pkp1) is stored in a different form, e. g. starch. As such the pkp1 mutant

provides an example for a plant with altered carbon partitioning in developing seeds.

123

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127

Chapter 4
Germination, establishment, and growth of Arabidopsis plants lacking a plastidic

pyruvate kinase

128

Abstract

Catabolism of storage reserves is essential for seed germination and establishment. An
Arabidopsis mutant (pkp1) deficient in plastidic pyruvate kinase (PKp) which is unable to
amass storage oil to the same extent as wild type is abnormal in these processes.
Germination is delayed in the mutant and seedling establishment is dependent on an
exogenous sugar supply. It appears, however, as though these phenotypes are not entirely
caused specifically by a lack of seed oil and may be related to reduced PKp activity.
Gerrninating seeds of pkp1 are unable to metabolize storage oil and cannot utilize applied
sucrose for hypocotyl elongation in the dark. Additionally, seed longevity is greatly
reduced in pkp1 indicating a potential lack of seed tocopherols. Mature pkp1 plants are
slightly chlorotic and contain less glucose and fructose. Thus, it appears as though PKp is

necessary for proper metabolic function during all aspects of plant growth.

129

Introduction

Pyruvate kinase (PK) is a ubiquitous enzyme located at a major branch point in carbon
metabolism (see Figure 1.5). Native PKs have been purified and characterized fi'om a
variety of organisms and these studies have revealed a wide range of kinetic and
regulatory properties for these enzymes (Munoz and Ponce 2003). This diversity of
molecular and biochemical characteristics seem to define specific functions for individual
PK isoforms within a single organism. For instance, mammals have four PKs which are
differentially expressed depending on the metabolic demands of the tissue (Yarnada and
Noguchi 1999). Plant metabolism, however, is more complex than that of mammals and
this is reflected by the fact that the Arabidopsis genome encodes 14 putative PKs which
reside in both the cytosol (PKC) and the plastid (PKp; Arabidopsis Genome Initiative
2000). Its location in the metabolic network and sheer redundancy indicate that PK is a
critical enzyme for plants, but few studies have focused on this enzyme with regard to its
function in vivo.

Only one instance of a plant deficient in PK has been reported. An attempt to
engineer transgenic tobacco (Nicotiana tabacum) with increased PKp inadvertently
resulted in co-suppression of an endogenous PKc-encoding gene and consequent loss of
enzyme activity in leaves (Gottlob-McHugh et al. 1992). At the time the only apparent
phenotype was a shift from a high pyruvate/phosphoenolpyruvate (Pyr/PEP) ratio to a
low one. More detailed analysis led to the discovery of a root growth defect, despite the
PKc deficiency being localized exclusively to leaves (Knowles et al. 1998). This
phenotype was exacerbated in low light conditions. Later, it was observed that PKc

functions in regulating photoassimilate export at night by controlling carbon flow into

130

respiration (Grodzinski et al. 1999). This result is somewhat confusing as reduced PKc
activity actually resulted in increased respiratory CO2 release. However, a yeast
(Saccharomyces cerevisiae) mutant with a similar reduction in PK activity also has
elevated respiratory flux (Pearce et al. 2001). An induction of a PK bypass (as shown in
Figure 1.3) could explain this phenomenon.

The occurrence of plant PKs in the plastid and cytosol almost certainly reflects
the unique roles for the respective enzymes (Plaxton 1996). For example, Pyr transported
into mitochondria for entry into respiration is most likely derived from PKc, especially
when considering the lack of a reported plastidic Pyr transporter (Weber 2004). Plastid-
localized metabolisms which use PEP and Pyr are no doubt influenced by PKp activity.
Two enzymes in the Shikimate pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate
synthase, and 5-enolpyruvylshikimate 3-phosphate synthase, use PEP as a substrate
(Herrmann and Weaver 1999). The final product of this pathway, chorismate, is
metabolized into aromatic amino acids which themselves are the starting points for the
synthesis of a variety of secondary metabolites (e. g. anthocyanins). The plastidic
pyruvate dehydrogenase complex, which produces acetyl-CoA for fatty acid synthesis
acts on pyruvate. The first enzyme of the methylerythritol-4-phosphate (MEP) pathway,
l-deoxy-D-xylulose-5-phosphate synthase, uses pyruvate and glyceraldehyde-3-
phosphate to synthesize the first intermediate of plastidic isoprenoid synthesis
(Lichtenthaler 1999). Plastid-derived isoprenoids include the carotenoids and phytol used
in the biosynthesis of chlorophyll and tocopherol. In addition, pyruvate is a substrate in
the biosynthesic pathways of valine, lysine, and isoleucine, and can be directly converted

to alanine by a transaminase (AraCyc metabolic map, www.arabidopsis.org/tools/aracyc/).

131

Clearly, the ratio of PEP to Pyr must be balanced, in large part by PKp, such that these
biochemical pathways function properly.

In Chapter 3, I detailed the fatty acid biosynthetic defect (a plastid localized
metabolism) of an Arabidopsis mutant (pkp1) deficient in seed PKp activity. Additionally,
pkp1 seeds had much less chlorophyll than wild type and this could indicate substrate-
restricted flux through the MEP pathway (which produces the phytol side chain of
chlorophyll). Here, I performed experiments to determine the extent to which the lack of
seed oil and perturbation of plastidic metabolism affects germination, establishment, and

grth of pkp1 offspring.

132

Materials and Methods

Plant growth conditions

Wild-type plants were of the Col-2 ecotype while pkp1 and the respective rescued lines
were in the Col-0 background. All seeds were sterilized with 20% bleach, 0.05%
TritonX-IOO for 15 min and were rinsed 5 times in sterile water. Medium used for
germination and growth on agar plates was full strength MS, pH 5.8, 0.9% agar and
included 0, 2, or 4% sucrose when appropriate. Seeds were stratified at 4°C for 3 (1 prior
to being put into an incubator with a photon flux density of 60—80 pmol m"2 sec—1 and a
light period of 16 h (22°C), and a dark period of 8 h (18°C). After 10 d, seedlings were
either transferred to soil or to fresh agar plates. Soil grown plants were put into a 16-h
photoperiod with a day temperature of 22°C and a night temperature of 20°C at a photon
flux density of 100—120 pmol rn-2 sec—l. Plant growth measurements were on plants
transferred to soil at 10 d after sowing. The aerial portion of six individuals was used for

each time point.

Root and hypocotyl elongation assays

For these assays, seeds were sown in a straight line and the agar plates were arranged
vertically. Root lengths were measured to the nearest mm every 24 h. The same set of
agar plates was used throughout the experiment. Hypocotyl elongation assays were
performed using 7 d after sowing seedlings as previously described (Penfield et al. 2004).
Once exposed to the light hypocotyls were immediately measured and those plates were

not used again.

133

Seed germination assays

All seeds used were produced from mother plants grown in identical conditions and were
of the same age. Germination assays were routinely performed using the plant growth
procedures mentioned above. When appropriate, 0, 2, or 4% sucrose was added to the
medium prior to being autoclaved. Germination was scored as radicle emergence from
the seed coat and was determined every 24 h using the same set of horizontally grown
agar plates. Accelerated aging treatment was done as previously described (Sattler et al.

2004) except that 42°C was used instead of 40°C.

Pyruvate kinase enzyme activity measurements

Seedling and leaf crude protein extracts were prepared by grinding tissue in
approximately 10 volumes (uL/mg) of buffer containing 50 mM Tris-Cl pH 7.5, 5 mM
MgCl2, 1 mM EDTA, 1 mM EGTA, lmM DTT, 0.1% Triton-X100, 10% glycerol, and a
protease inhibitor mix (Complete mini, Roche). Pyruvate kinase activity was coupled to
the conversion of NADH to NAD+ by lactate dehydrogenase. Reactions were kept at
25°C, were started by the addition of enzyme mix, and were linear for at least 5 minutes.
Absorbance at 340 nm was measured using a FLUOstar Optima 96-well plate reader
(BMG Labtech, Offenburg, Germany). The PKp reaction mixtures contained 50 mM
HEPES-KOH pH 8.0, 5% PEG-8000, 50 mM KCI, 15 mM MgCl2, 1 mM DTT, 2 mM
PEP, 1 mM ADP, 0.2 mM NADH, and 2 U ml'l desalted rabbit muscle lactate
dehydrogenase. PEP phosphatase activity was corrected for by omitting ADP from the
reaction. Reactions at pH 7.0 were done using 50 mM MOPS pH 7.0 instead of HEPES.

Protein was quantified using Bradford reagent (Sigma).

134

Lipid and carbohydrate analysis

Total leaf lipids were extracted from pre-weighed tissue by vigorously shaking for 5 min
in 500 11L of methanol/chloroform/formate (22120.1, v/v). Then 250 11L of 1 M KCl, 0.2
M H3PO4 was added and the tubes were vortexed. The phases were separated by
centrifugation at 16,000 g for 5 min. The organic phase was loaded quantitatively onto a
treated (soaked in 0.15 M (N H4)2SO4 and dried, then heated to 120°C for 2.5 hrs) silica-
60 TLC plate (Baker). The solvent system used was acetone/toluene/water (91 :3027, v/v)
and staining was done with iodine and a-naphthol. Lipid composition was determined by
fatty acid methyl ester (FAME) analysis as described previously (Focks and Benning
1998). Ten seedlings or two whole leaves were used for each sample. Glucose, fructose,
sucrose, and starch were extracted and quantified as previously described (F ocks and
Benning 1998). Five 25 (1 old plants were homogenized together and about 50 mg (fresh
weight) of leaf tissue was used for each extraction. Soluble sugars were resuspended in
200 1.1L of water and 15 11L was used for each measurement. Insoluble carbohydrate
pellets were resuspended in 300 11L of 0.2N KOH and the remaining volumes were

adjusted proportionally. Starch assays were done with 15 11L of the final preparation.

Leaf pigment quantification
Chlorophyll was extracted from leaves and seedlings using 100 volumes (uL/mg) of 80%
acetone and was quantified as previously described (Lichtenthaler 1987). Anthocyanins

were extracted and quantified using a published protocol (Martin et al. 2002).

135

Results

Seed germination and seedling establishment are aberrant in pkp1

Chapter 3 describes the seed-specific phenotypes of wild type and the pkp1 mutant and
includes some data for pkp1 rescued by ectopic overexpression of PKp-Bl (RBI-23) and
pkp1 rescued by ectopic overexpression of PKp- 132 (R132-3). During routine grth of
these plants it was observed that pkp1 seedlings do not establish (defined as development
of true leaves and root elongation) when sown directly on soil. In a single repetition,
100% of wild-type seedlings and 0% of pkp1 seedlings established after 12 days when
sown on soil. This defect was rescued 100% in RB1-23 but only 80% in R132-3. A
common phenotype of low oil mutants such as pkp1 is an inability to establish in the
absence of an exogenous sugar source (Cemac et al. 2006, Lu and Hills 2002). Indeed,
pkp1 will not establish unless provided with 2% sucrose in the medium (Figure 4.1A) and
even then growth is slow in the mutant. Root elongation assays were performed to
quantify this defect. As shown in Figure 4.1 B, pkp1 roots do not grow at the same rate as
wild type or either of the rescued lines. The results also indicate that the initiation of root
growth may be delayed in pkp1 as at 3 days after sowing (DAS) its roots have not
elongated past 1 mm. One possible explanation is that germination itself is inhibited in
these seedlings.

Germination assays were conducted in the light in the presence of 2% sucrose to
explore the possibility that pkp1 seeds do not germinate as fast as wild type. Images of
seedlings taken at 3 and 6 DAS qualitatively demonstrate an abnormality in pkp1
seedlings (Figure 4.2A). By 3 DAS, wild type seeds have germinated (defined by radicle

emergence‘from the seed coat) and the roots are already elongating.

136

>
CD

8 DAS WT 10 DAS pkp1 70
- Suc + 8010 - Suc + Suc

 

              

Root Length (mm)
A
o

 

 

 

 

 

2 3 4 5 6 7 8 9 10
Days After Sowing

Figure 4.1. Sucrose dependent establishment and root elongation in pkp]

(A) Germination and seedling establishment in the presence (+) or absence (-) of 2%
sucrose (Suc). Ten days after sowing (DAS) pkp1 seedlings are at the same
developmental stage as 8 DAS wild-type seedlings.

(B) Root elongation in the presence of 2% sucrose. Without sucrose pkp1 roots do not

elongate (shown in A). Values are the mean :t SD of 25 measurements.

In p190], however, only a portion of the seeds display radicle emergence at 3 DAS and not
until 6 DAS have all of the seeds germinated. Figure 4.2B shows the germination rates of
wild type, pkp1, and the rescued lines determined from independent experiments using
seeds of the same age. After 1 day almost 100% of wild type and R131-23 and about 75%
of R132-3 seeds have germinated while in pkp1 less than 20% of the radicles have emerged.
Not until 5 DAS does pkp1 reach its maximum germination percent, which is about 90%.
Pyruvate kinase (PK) activity was measured at pH 8.0 to ascertain if reductions in

enzyme activity are correlated with the delayed germination in pkp1.

137

 

 

 

 

 

 

 

 

 

 

A B
120
3 DAS C 100 ---------------
.9 4..
E 80
E F
a) :
6 DAS 9 60 ~
C
OJ
2 40~
d) +WT
20‘ +Pkp1
“I“R131-23
Days After Sowing
140
120-
g 100-
o
a 80‘
a, .
E 60 . , ..
D
E 40 . +WT -; ........ -
+pkp1
201 "'"RB1-23
0 . . . ,
0 2 4 6 8 10
Days After Sowing

Figure 41.2. Delayed germination and induction of PKp activity in pkp1

(A) Seed germination is delayed in pkp1 in the light on MS medium with 2% sucrose.
DAS, days afier sowing. Un-germinated seeds are marked with white arrows.

(B) Seed germination rates on medium with 2% sucrose. Values are the mean i SD (n=6).
(C) Pyruvate kinase activity measured at pH 8.0 using protein extracts from germinating
seeds and seedlings grown in the light. Values are the mean 3: SD (n=3). One mU is 1

nmol pyruvate formed per min.

138

lmbibed and stratified seeds served as the 0 DAS time point and at this time all of the
lines have about the same PK specific activity (Figure 4.2C). Twenty-four h later, there is
a doubling of PK specific activity in wild type and a similar, although less intense,
induction in the rescued lines. By 5 DAS, activity has returned to the starting point where
it remains for the rest of the time course. The pattern of PK induction is skewed in pkp1
and does not peak until 3 DAS, after which it steadily declines. There is indeed a
correlation between delayed induction of PK specific activity and germination in pkp1.

Developing seeds of pkp1 accumulate carbohydrates in the form of sucrose and
starch late during embryogenesis (see Figure 3.8). High sugar concentration in the
medium has been shown to delay seed germination (Dekkers et al. 2004, Zhou et al.
1998). Therefore, it is reasonable to postulate that the sugar accumulated in pkp1 seeds is
partly responsible for the observed germination defect and that the mutant will be more
sensitive to exogenous sugar in the medium. To test this hypothesis, germination assays
were performed on agar plates containing 0, 2, or 4% sucrose. A very subtle delay in
germination is observed for wild type with increasing sugar concentration (Figure 4.3A).
In all treatments though, germination of wild-type seeds reached a maximum by 2 DAS.
As predicted, pkp1 is more sensitive to sugar in the medium. In the absence of sucrose,
the maximum number of pkp1 seeds has germinated by 3 DAS (Figure 4.3B). With 2%,
this time is extended to 5 DAS. On 4% sucrose only 50% of the pkp1 seeds have

germinated at 5 DAS and not until 10 DAS is the maximum achieved.

139

>
w

 

 

 

  
  
     

 

 

 

 

 

 

 

120 120
WT pkp1
c 100‘ A I I c 100‘
.9 .9
E 89 - .2 89 1
E E
8 60 - 8 60 4
§ 40 - § 40 -
8? +01% SUC 5 +0% SUC
20 . +2% Suc ‘L 20 1 +2% Suc
+4% Suc +4% Soc
0 . . . . . O - . . . .
0 1 2 3 4 5 6 0 1 2 3 4 5 6
Days After Sowing Days After Sowing

Figure 4.3. Inhibition of pkp1 germination by exogenous sucrose
(A) Wild type germination rates in the presence of 0, 2, or 4% sucrose.

(B) Germination of pkp1 in the presence of increasing concentrations of sucrose.

Storage lipid metabolism is defective in pkp1

Hypocotyl elongation assays in the dark are a standard means of examining storage oil
metabolism in germinating seeds. When grown in the dark in the presence or absence of
sucrose wild type hypocotyls elongate (Figure 4.4A, 4.4B). pkp1 hypocotyls do not
elongate in medium without an exogenous carbon source, which is typical of oil deficient
mutants such as wrinkled] (wriI ; Cemac et al. 2006). However, even when provided with
2% sucrose pkp1 seedlings do not elongate their hypocotyls; a phenotype which is
rescued in lines overexpressing either B-subunit encoding gene (Figure 4.4A, 4.4B).
Dark-grown seedlings must generate ATP mainly by glycolysis and respiration, whether

fueled by endogenous storage reserves or by uptake of an exogenous carbon source.

140

 

 

 

 

 

 

 

 

 

 

 

 

  

 

 

 

 

 

A B 25
E 20
.5,
2% Suc % 15
C
2
2
§ 104
0.
>4
.5
5.
00/0 SUC
0 1
+ - + - + - + - Suc
WT pkp1 RB1-23 R132-3
C D
200 250
2% Suc +WT 0% Suc
175-
1501
E .5
93 1251 2
9 e
G- 1001 o.
‘e” E’
5 75 +wr 5
E 50 +pkp1 E
...... RB _23
25 ,M‘ R132-3
0 . . 9 . - . . . . .
0 2 4 6 8 10 0 2 4 6 8 10
Days After Sowing Days After Sowing

Figure 4.4. Hypocotyl elongation and PKp activity in dark-grown seedlings

(A) Dark-grown seedlings at 7 days after sowing on MS medium with 0 or 2% sucrose.
(B) Quantification of hypocotyl lengths from seedlings in (A). Values are the mean :t SD
of 25 measurments. (+), 2% sucrose; (-), 0% sucrose.

(C-D) Pyruvate kinase activity measured at pH 8.0 from dark-grown hypocotyls in the
presence (C) or absence (D) of 2% sucrose. Values are the mean :t SD (n=3). One mU is

1 nmol pyruvate formed per min.

141

In either case, PK is important for the substrate-level phosphorylative generation of ATP
and for the production of respiratory precursors. Thus, a reduction in PK activity could
help explain the hypocotyl elongation phenotype of pkp1 . Indeed, PK specific activity is
relatively low in pkp1 etiolated seedlings when grown with or without exogenous sucrose
(Figure 4.4C, 4.4D).

Seedling establishment is largely fueled by seed storage oil breakdown. Figure
4.5A illustrates this process in seedlings grown on agar plates containing 2% sucrose.
Very long chain fatty acids (VLCFAs), which in Arabidopsis are specific to seed
triacyl glycerol (TAG), were used as markers for storage oil content. In wild type and the
rescued lines storage oil begins to be metabolized at 2 DAS and is essentially depleted by

6 DAS. Interestingly, storage lipids are not used by pkp1 seedlings (Figure 4.5A).

 

 

 

   
 

 

 

 

 

 

A 2 B
50-

’g‘ 40-

8 9".

g 92 30-

" o\°

E ‘6 20 +wr

0 E

§ +pkp1
10 -----R131-23
0 .M‘RB2'3

0 1 2 3 4 6 6 7 0 1 2 3 4 5 6 7
Days After Sowing Days After Sowing

Figure 4.5. Fatty acid composition in germinating seeds and seedlings

(A) Seed oil-specific very long chain fatty acid (VLCFA; 20:0, 20:1, 21 :0, 21:1) content
in seedlings grown on 2% sucrose. Values are the mean :t SD (n=4).

(B) mol% of linolenic acid (18:3) in seedlings grown on 2% sucrose. Values are the mean

:t SD (n=4).

142

An additional marker for seedling establishment is an increase in the proportion of
linolenic acid (18:3) in membrane lipids, which is a major component of thylakoid
membranes. By 5 DAS, the proportion of 18:3 to other fatty acids has increased by about
30% in wild type and the rescued lines (Figure 4.5B). In pkp1 the increase is limited to
about 5%. It is clear that lipid metabolism is abnormal in pkp1 seedlings.

The lipid metabolism phenotype of pkp1 seedlings is reminiscent of Arabidopsis
mutants deficient in seed tocopherol, vitamin e] and vitamin e2 (vteI, vte2; Sattler et al.
2004). Seed vitamin E deficiency leads to irreversible lipid peroxidation and thus,
reduced seed longevity. Treatment with high temperature and relative humidity simulates
natural aging and was used here to determine the extent to which pkp1 resembles the vte

mutants. Figure 4.6 shows the results of this experiment.

 

 

 

 

 

+VVT
+pkp1
.5 100‘ '3' ..... I ..... I ..... I ..... I ..... I ..... ... .......... .RB1-23
E A R624,
.9 80 .
E 1 l
m J l
060 .1, .................
E .................
240-
<0
0.
20--’
0 I "L " 5 5 = = H
01234567891011

Days After Sowing
Figure 4.6. Seed germination following accelerated aging treatment
Seeds were the same as were used in previous germination assays and were produced

from plants grown in identical conditions. Values are the mean :1: SD (n=8).

143

Wild type germination was delayed relative to untreated seeds (compare to Figure 4.2B),
but still reached nearly 100%. Germination of pkp1 was almost completely arrested.
Surprisingly, R131-23 seeds were unaffected by the treatment while R132-3 showed a

phenotype intermediate between wild type and pkp1.

Altered growth and leaf metabolism in pkp1
The pkp1 seedlings that successfully establish continue to experience difficulties in
growth after being transferred to soil. Biomass production is limited and around the time

of flowering initiation (25 DAS) pkp1 aerial parts weigh approximately half compared to

 

 

 

 

 

wild type (Figure 4.7A).
A B
100
1&7 +WT
T“; 80 . +pkp1
Er .
r: 60‘ r
.C
O')
'6‘: 40 -
3
g 20 -
LL
0 . . . .
5 10 15 20 25 30
Days After Sowing WT pkp1
35 Days After Sowing

Figure 4.7. Plant growth and morphology of pkp1

(A) Plant growth curve of wild type and pkp1. Measurements were initiated at 10 days
after sowing, when seedlings were transferred from MS medium with 2% sucrose to soil.
Each value is the mean i SD (n=6).

(B) Whole plant morphology of wild type and pkp1.

144

Taking into account the delay in germination, pkp1 follows the same developmental time
course as wild type. At 35 DAS, both genotypes are well into flowering and pkp1 is
nearly equal to wild type in size (Figure 4.7B). Another noticeable morphology of pkp1 is
slight chlorosis. When grown on soil, total chlorophyll content is reduced by 30%,
accompanied by a moderate increase in anthocyanins (Table 4.1). This phenotype is
exaggerated in plants propagated on agar plates in lower light conditions (photon flux

densities of 100—120 and 60-80 umol m”2 560—1 for soil and agar growth, respectively).

Table 4.1 Leaf pigments in 25 day old plants grown on soil or on agar plates

 

 

 

 

Soil-grown plants Agar plate-grown plants
Chlorophyll Anthocyanins Chlorophyll Anthocyanins
(ug/ mg FW) (A530-A657/ g FW) (148/ mg FW) (A530'A657/g FW)
WT 1.06 i 0.05 0.69 i 0.01 1.18 i 0.01 0.22 i 0.03
pkp1 0.76 i 0.09 0.80 i: 0.01 0.42 :t 0.02 0.54 :1: 0.02

 

Values represent the mean :t SD of at least three repeats. FW, fresh weight

Pyruvate kinase enzyme assays were performed to see if the morphological
differences of pkp1 are correlated with a reduction in activity. When measured at pH 7.0,
which is more specific for cytosolic enzymes, enzyme activity is the same between wild
type and pkp1 (Figure 4.8A). Plastidic PK’s typically have a pH optimum of 8.0, and so
any differences directly related to the pkp1 mutation are expected to be seen at this pH.
The data in Figure 4.8B establishes that PKp activity in pkp1 leaves is ablated and only

reaches about 60% of wild-type levels.

145

>
u:

 

 

 

 

60 70-
pH 7.0 .WT pH 8.0 .WT
C 50 upkp1 : 60 upkp1
'5 “ 50
3, 30 S,
E E 30
2 20 2 20
10 10
0 , 0
day night day night day night day night
15 DAS 25 DAS 15 DAS 25 DAS

Figure 4.8. Pyruvate kinase activity in rosette leaves during the day and night

(A) Pyruvate kinase activity measured at pH 7.0 For each time point, 5 whole plants were
homogenized and used to prepare protein extracts. Day = 8 hours afier lights on. Night =
8 hours after lights off. Values are the mean i SD (n=4). DAS, days after sowing.

(B) Pyruvate kinase activity measured at pH 8.0 using the same protein extracts described

in (A).

Lipid metabolism is aberrant in pkp1 seeds and seedlings and so it is logical to
presume that there is also an effect in leaves of mature plants. However, limited
examination of leaf lipids revealed very little qualitative, if any, difference between wild
type and pkp1. Figure 4.9A is a thin layer-chromatogram of total leaf lipids extracted
from 25 DAS plants. It is clear that there is no defect in the membrane lipid profile of
pkp1. Analysis of the fatty acid profile revealed a very subtle, but significant decrease in
1823 content which is accompanied by an increase in the proportion of linoleic acid (18:2;
Figure 4.98). This result is not totally unexpected as the same trend is seen in seedlings,

although to a much greater extent (Figure 4.5B).

146

 

A B
70
IWT
60‘ upkp1
MGDG 50-
o 40-
DGDG i
0
E30.
20-
10-
origin

 

 

 

 

0- _
169 16fl 162 189 163 18fl 182 183

WT pkp1

Figure 4.9. Lipid profile and composition of pkp1 leaves

(A) Thin Iayer-chromatogram of total lipids extracted quantitatively from 25 days after
sowing wild type and pkp1 leaves. Plate was stained with iodine vapor and a-naphthol to
maximize the number of lipids detected. DGDG, digalactosyldiacyl glycerol; MGDG,
monogalactosyldiacyl glycerol.

(B) Fatty acid composition of total leaf lipids from 25 days after sowing plants. Values

are the mean 3: SD (n=3).

Reduced biomass production and chlorophyll content of pkp] could reflect or cause
altered carbohydrate metabolism. Therefore, soluble sugars and starch were extracted
from leaves during the day and night and were quantified. Hexose content (glucose and
fructose only) was reduced by more than half in pkp1 during both the day and night. On

the other hand, sucrose and starch accumulation were unaffected.

147

>
CD
0

 

 

 

..s
O
.b

N

UT

 

 

     

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

IWT IWT ' IWT
upkp1 3.5‘ upm‘l upkp1
A 8 i A 2 ‘
E 3 ‘ E
§6~ 825 316
e 3’ 2 . 2’
g 4‘ 8 1.5, § 1 '
2 5 .9
a: 3 1 1 m
I 2 . (I) 0.5‘
0.51
01 r . 0 1 ~ , 0 .
Day Night Day Night Day Night

Figure 4.10. Carbohydrate content of 25 day old leaves

(A) Hexose (glucose and fructose) content of rosette leaves. 5 whole plants were
homogenized and used for extraction. Day night cycle used was 16 hrs light and 8 hrs
dark. Day = 8 hours after lights on. Night = 8 hours after lights off. Values are the mean
:t SD (n=4). FW, fresh weight.

(B) Sucrose content of the samples described in (A). Values are the mean :1: SD (n=4).

(C) Starch accumulation in the same samples as detailed in (A). Values are the mean :t

SD (n=4).

148

Discussion
PKp activity is required to fuel seed germination
Seed germination in Arabidopsis is largely driven by the metabolism of storage reserves
other than lipids, while seed oil is more important for fueling subsequent seedling
establishment (Cemac et al. 2006). Breakdown of carbohydrates to produce energy
proceeds through glycolysis, with PK generating half of the ATP and providing
precursors for respiration. In germinating wild-type seeds, a rapid increase of PKp activity
is observed coincident with radicle emergence (0-1 DAS, Figure 4.2C). In pkp1 there is a
delay in this induction, which could explain or result from the impaired germination rate
(Figure 4.2A, 4.2B). The eventual increase in PKp activity in pkp1 can be explained by
two possibilities: 1) enhanced expression of the PKp-132-encoding gene in response to
unfavorable energy status, or 2) induction of a cytosolic enzyme with higher than normal
pH optimum. The first hypothesis is supported by elevated expression of the PKp-132-
encoding gene in dark-grown seedlings of a plastidic ATP/ADP transporter mutant,
supposedly to compensate for reduced ATP import into plastids (Reiser et al. 2004).
Induction of a PKc is also not unreasonable. In seeds of pkp1 PKC activity is increased
relative to wild type (Figure 3.4).

Addition of an exogenous carbon source does not rescue the germination defect of
pkp1. In fact, increasing the amount of sucrose actually inhibits germination (Figure 4.3).
It was not tested whether this is a result of osmotic potential in the medium. The oil
deficient wri] and triacylglycerol] (tagI) mutants have a similar phenotype but in those
cases it was likened to heightened sensitivity to osmolarity (Cemac et al. 2006, Lu and

Hills 2002). However, pkp1 differs from these mutants in that there is 50% reduction in

149

PKp activity at the time when nearly 100% of wild-type seeds have germinated (Figure
4.2C). Pyruvate kinase activity in germinating wri] seeds is no different from wild type
(Cemac et al. 2006). Therefore it is possible that in pkp1 an accumulation of
carbohydrates in the seedling brought on by a reduction in glycolytic flux is responsible
for increased sensitivity to sugar in the medium. The incomplete rescue of root elongation
by sugar application bolsters the idea that pkp1 is less capable of metabolizing sucrose

(Figure 4.1).

Storage compound utilization is dependent on PKp either in seeds or seedlings
Seedling establishment and hypocotyl elongation in the dark are driven by the catabolism
of seed storage oil. Arabidopsis mutants defective in 13-oxidation (Footitt et a1. 2002,
Germain et al. 2001) fail to establish unless provided with an exogenous carbon source
such as sucrose. Similar phenotypes are observed in glyoxylate cycle and
gluconeogenesis mutants which are unable to convert storage reserves into carbohydrates
(Comah et al. 2004, Eastmond et al. 2000, Penfield et al. 2004, Rylott et al. 2003). The
pkp1 mutant has 60% less seed oil than wild type and does not elongate its hypocotyls in
the absence of sucrose (Figure 4.4A, 4.4B). In contrast to the B-oxidation, glyoxylate
cycle, and gluconeo genesis mutants, exogenous sucrose does not rescue this phenotype.
Without sucrose PKp activity is only marginally reduced in pkp] and hypocotyl
elongation is likely inhibited due to a lack of storage reserves (Figure 4.4D). However, on
2% sucrose PKp activity is significantly reduced and this may contribute to the apparent

inability to utilize the supplied sugar (Figure 4.4C). It could also be that pkp1 cannot

150

synthesize the membrane lipids necessary for expansive growth, which supported by the
fact that the mol% of 1823 does not increase in pkp1 germinating seeds (Figure 4.5B).

The pkp1 mutant does not efficiently metabolize its seed oil reserves (Figure
4.5A) and this could also cause faulty seedling establishment and hypocotyl elongation.
Storage lipid metabolism is not completely restored in the RBI-23 and R132-3 rescued lines
and this is correlated with incomplete rescue of PKp activity (Figure 4.2C). Since PKp is
not directly involved in any metabolisms associated with storage lipid breakdown another
explanation for this phenotype was sought. Accelerated aging treatment followed by
germination assays revealed that pkp1 viability is severely compromised over time
(Figure 4.6). This result together with the storage lipid breakdown defect is consistent
with a reduction in seed tocopherol (Sattler et al. 2004). Tocopherol is synthesized in the
plastid and uses phytyldiphosphate (phytyl-PP) as a precursor (Collakova and DellaPenna
2001). Phytyl-PP is synthesized exclusively in the plastid as a downsteam product of the
methylerythritol-4-phosphate (MEP) pathway and as a salvage product released during
chlorophyll degradation (Ischebeck et al. 2006). Developing seeds of pkp1 expectedly
have lower flux through PKp and therefore less Pyr for entry into the MEP pathway.
Additionally, seed chlorophyll content is drastically reduced in developing seeds possibly
to do a lack of phytol (see Table 3.2). Moreover, the cloroplastos alterado (claI) mutant
of Arabidopsis, deficient in 1-deoxy-D-xylulose-5-phosphate synthase which catalyzes
the first step of the MEP pathway (using Pyr as one substrate) has retarded germination
and reduced tocopherol content at least in leaves (Estevez et al. 2001). The combined
data are in agreement with reduced flux through the MEP pathway leading to lower

tocopherol content in pkp1 seeds. Most interestingly, RBI-23 appears to be completely

151

resistant to the accelerated aging treatment employed, suggesting an increase in seed
tocopherols (Figure 4.6). However, seed PKp activity and chlorophyll content are not
higher than wild type in this line (see Chapter 3). An increase in tocopherol could
potentially be explained by altered timing of PKp activity in these seeds. Typically, PKp
activity peaks just prior the maximum rate of oil biosynthesis. Tocopherol is synthesized
later, once all of the oil has been deposited and seeds begin to degrade and recycle
chlorophyll (Valentin et al. 2006). Expression of the PKp-Bl-encoding gene in RBI-23 is
driven by the constitutive CaMV 358 promoter and it is possible that spurious PKp

activity late during development leads to elevated tocopherol biosynthesis.

PKp activity influences chlorophyll biosynthesis and sugar accumulation in leaves
The effects of the pkp1 mutation are evident in many aspects of whole plant morphology
and physiology. The establishment and growth phenotypes of pkp1 could not be rescued
by the application of aromatic amino acids, alanine, branched chain amino acids, or with
combinations of those (data not shown). Therefore, impaired amino acid biosynthesis
brought on by altered PEP and pyruvate metabolism seems not to be responsible for the
pkp1 phenotypes. Leaves of pkp1 contain 30 to 60% less chlorophyll than wild type,
depending of if they were grown on soil or on agar plates (Table 4.1). As with seeds,
reduced PKp activity (Figure 4.8B) in leaves and resultant impairment of isoprenoid
biosynthesis could explain this. Again, the cla] mutant supports this hypothesis in that it
too has reduced chlorophyll content in leaves (Mandel et al. 1996). Increased
anthocyanins in pkp1 (Table 4.1) may also be related to PKp activity as PEP is a substrate

for the Shikimic acid pathway which gives rise to precursors for anthocyanin biosynthesis.

152

Unlike seeds, hexose content is reduced in pkp1 leaves (Figure 4.10A) and likely has no
role in repression of chlorophyll biosynthesis. Instead, reduced photosynthesis due to a
lack of chlorophyll might be the reason for reduced glucose and fructose. Photosynthetic
limitations could also explain the dwindled biomass of pkp1 (Figure 4.7A). Sucrose and
starch appear to be unaffected in pkp1 suggesting that any problems with carbohydrate
metabolism are localized in or around glycolysis (Figure 4.10B, 4.10C). Altered sugar
levels would be expected to result in additional pleiotropic effects as 30 to 50% of
Arabidopsis genes are at least partially transcriptionally regulated by sugar concentration
(Blasing et al. 2005). It is interesting that lipid content and composition is roughly the
same in pkp1 as in wild type. It remains to be determined if pkp1 has a lower rate of fatty
acid synthesis, as is the casein developing seeds. In conclusion, the pkp1 mutant has little
in common with previously described tobacco plants lacking PKG, thus detailing the
unique roles of plastid- and cytosolic-localized PKs. Much work remains to be done to

fully understand the role of PKp in plant growth and development.

153

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Collakova, E. and DellaPenna, D. (2001) Isolation and functional analysis of
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Cornah, J .E., Germain, V., Ward, J.L., Beale, M.H., and Smith, S.M. (2004) Lipid
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Estevez, J .M., Cantero, A., Reindl, A., Reichler, S., and Leon, P. (2001) l-Deoxy-D-
xylulose-S-phosphate synthase, a limiting enzyme for plastidic isoprenoid biosynthesis in
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Focks, N. and Benning, C. (1998) wrinkled] 2 A novel, low-seed-oil mutant of
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Plant Physiol. 118:91-101.

Footitt, S., Slocombe, S.P., Larner, V., Kurup, S., Wu, Y., Larson, T., Graham, 1.,
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Germain, V., Rylott, E.L., Larson, T.R., Sherson, S.M., Bechtold, N., Carde, J.P.,
Bryce, J.H., Graham, I.A., and Smith, S.M. (2001) Requirement for 3-ketoacyl-COA
thiolase-2 in peroxisome development, fatty acid beta-oxidation and breakdown of
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transgenic tobacco plants in the absence of cytosolic pyruvate kinase. Plant Physiol.
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Grodzinski, B., Jiao, J., Knowles, V.L., and Plaxton, W.C. (1999) Photosynthesis and
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Herrmann, KM. and Weaver L.M. (1999) The Shikimate pathway. Annu. Rev. Plant
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Ischebeck, T., Zbierzak, A.M., Kanwischer, M., and Dormann, P. (2006) A salvage
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Lu, C. and Hills, M.J. (2002) Arabidopsis mutants deficient in diacylglycerol
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(1996) CLAl, a novel gene required for chloroplast development, is highly conserved in
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Martin, T., Oswald, O., and Graham, LA. (2002) Arabidopsis seedling growth, storage
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Rylott, E.L., Gilday, A.D., and Graham, LA. (2003) The gluconeogenic enzyme
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Sattler, S.E., Gilliland, L.U., Magallanes-Lundback, M., Pollard, M., and
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Yamada, K. and Noguchi, T. (1999) Nutrient and hormonal regulation of pyruvate
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Zhou, L., Jang, J.C., Jones, T.L., and Sheen, J. (1998) Glucose and ethylene signal
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Acad. Sci. USA. 95:10294-10299.

156

Chapter 5

Conclusions and perspectives

157

Major Conclusions

This multifaceted study of Arabidopsis PKp has confirmed its central location in the seed
metabolic network (see Figure 1.2). Of two PKp complexes, the one that is most active
and least sensitive to feedback inhibition has been naturally selected for in seeds. Based
on analysis of the pkp1 mutant, one can conclude that at least 60% of the fatty acids in
Arabidopsis seed oil are ultimately derived from a single PKp (specifically 0113;).
Carbohydrates accumulated in place of 20% of the oil in pkp1 seeds. Therefore, PKp
represents a regulatory step that controls carbon partitioning in developing embryos.
Connectivity between the cytosol and plastid provided by metabolite transporters was
insufficient for increased PKC activity to fully compensate for the loss of PKp. This result
implies that pyruvate is not a major metabolite that is transported across the plastid
envelope in seed tissue. Remaining questions and directions for future research will be

discussed in the forthcoming sections.

Some additional questions

The now bonafide PKps described here account for only a fraction of the annotated PK-
encoding genes in Arabidopsis. Phylogenetic analysis suggested the presence of one other
plastid targeted PK (At3g49160; Figure 2. 1 A), but pilot experiments with recombinant
protein were unable to detect any catalytic activity. The question remains whether the
enzymes used in this study are the only PKps in Arabidopsis. If so, the presence of 10
genes encoding PKcs implies either great redundancy or the ability to very precisely
regulate cytosolic glycolysis. In any case, the lack of redundancy for PKp is surprising

compared to‘ the cytosolic enzymes.

158

The pkp1 mutant still accumulates up to 40% wild-type seed oil and the source of
carbon precursors for this residual TAG is unknown. Basal expression of PKp-flg is one
explanation. The contribution of the 01132 complex to seed oil biosynthesis could be tested
by crossing pkp1 with a null mutantpof PKp-flz. It could be that metabolic redundancy is
provided by PKc, as evidenced by induction of its activity in pkp1 seeds (Figure 3.4A). It
is also possible that the PK bypasses discussed in Chapter 1 (Figure 1.3) compensate for
the reduction in PKp activity. Preliminary experiments detected no changes in the
activities of PEP carboxylase, NAD-malate dehydrogenase, or NADP-malic enzyme in
pkp1, but more accurate measurements are needed to confirm this. There is also the
question of to what extent PKp-132 can function in place of PKp-131. The kinetic parameters
of the 01132 complex (Table 2.2) and the inability of PKp-flz overexpression to fully restore
oil content in rescued pkp1 lines (Figure 3.6B) suggests that PKp-132 is not able to
functionally replace PKp-Bl. However, other aspects of plant growth and development
seem to be restored in the 35S2PKp- ,82-rescued pkp1 lines. It seems that only in situations
of very high rates of fatty acid synthesis is it advantageous to have 01131 as the dominant
PKp.

In this work, enzyme assays at pH 8.0 were used to enrich detection of PKp
activity. This method was useful in sensing total activity, but was not able to pinpoint the
loss of a specific PKp isoform in pkp1. Procedures used to separate and detect individual
PKs were unsuccessful and so loss of a specific isoform had to be assumed. Perfection of
a zymogram technique as was used for glucose-6-phosphate dehydrogenases would be
useful here (see Appendix A). Analysis of the reconstituted enzymes also raised some

questions that deserve attention. For instance, it was not tested whether a single complex

159

can contain both 131- and 132-subunits. A mixed-composition enzyme might have
regulatory properties distinct from the 01131 and 01132 enzymes, as is the case with hybrids of
mammalian PKs (Hubbard and Cardenas 1975). The association of the individual PK
subunits is likely a dynamic process and the factors that control the strength of protein-
protein interaction were not analyzed in much detail. It was observed that K+ and PEP
were required for co-immunoprecipitation of native PKp complexes, suggesting that these
are important for subunit association. However, the effects of pH and metabolite effectors,
for example, were not examined. It could be that regulation of enzyme activity by certain
metabolites is mediated by abrogation of subunit interaction.

Several phenotypes of the plcp] mutant require additional examination. The
effects of reduced fatty acid synthesis are no doubt extended beyond TAG accumulation.
Other lipids such as those making up the hydrophobic layer on the seed coat might also
be affected. Preliminary staining of mature seeds suggested increased permeability in
pkp1, thus warranting investigation of lipids other than TAG. The reduction in
chlorophyll content in pkp1 is also intriguing. Reduced chlorophyll in seeds is correlated
with less extensive thylakoid membranes (Figure 3.3). It would be interesting to know if
the situation in leaf chloroplasts is the same. Furthermore, the cause of reduced
chlorophyll is still unknown. Is it a chlorophyll biosynthetic defect or is it the result of a
lack of thylakoid membranes brought on by impaired fatty acid synthesis? Either is
possible. Chlorophyll biosynthesis is likely not inhibited by sugar concentration in pkp1
leaves, since hexoses are actually decreased relative to wild type (Figure 4.10). Several
lines of evidence also hint at a lack of seed tocopherols in pkp1, but measurements have

not yet been'made. Both chlorophyll and tocopherol biosynthetic defects could arise from

160

reduced phytol synthesis, and so it would also be informative to measure flux through the

methylerythritol-4-phosphate (MEP) pathway in plastids.

Future Directions

In addition to answering the immediate questions listed above, future research on PKp
should be focused on systems biology and metabolic engineering. The biochemical
properties (S0, 5, Vmax, 150, K.) obtained for the PKp complexes could be incorporated into
a kinetic model of seed metabolism. The details of many other enzymes in the network
would be needed and this dissertation provides a framework for such characterizations.
Once made, a kinetic model could be used to predict the effects of specific metabolic
perturbations or enhancements. For example, the effects of having 0113. versus 01132 as the
dominant seed PKp could be predicted and then tested using pkp1 and the respective
rescued lines. A similar approach has been taken to direct the engineering of glycine
betaine metabolism in tobacco (McNeil et al. 2001). Simple metabolic profiling of pkp1
seeds and seedlings would also be informative. It is expected that the defect in central
carbon metabolism results in pleiotropic effects that would be readily elucidated with this
method. Developing Arabidopsis seeds have temporally distinct metabolic fingerprints
(Fait et al. 2006) and it would be interesting to see if PKp controls the balance of
metabolites beyond what was presented in previous chapters. Metabolic flux analysis
using stable isotopomers would also be informative for pkp1 . This method could help
determine any rerouting of metabolic fluxes that are not evident based on enzyme activity
alone. For example, alternative routes of pyruvate production may be more active in pkp1,

despite there being no changes in extractable enzyme activities. In general, a systems

161

biology approach to investigating metabolism in pkp1 would be the most efficient for
answering the obvious questions while at the same time discovering unexpected
perturbations.

Increasing oil yield in crop plants is the main driving force for this research. As
PKp is a regulatory enzyme in this process, the next logical step is to increase PKp
activity in developing seeds. This was not possible by overexpression of either 13-subunit-
encoding cDNA, probably because the amount of 01 was limiting. Therefore,
simultaneous overexpression of both 01- and 13-genes should be pursued. This can be
achieved using stacked expression constructs with seed-specific promoters. Increasing
PKp activity in non-seed tissues would also be a worthwhile endeavor. Ectopic oil
production is emerging as a means to greatly increase the yield of a single plant, as seeds
have evolved to be at or near their maximum potential. Use of tissue specific or inducible
promoters is one means of achieving this. Another way is to take advantage of
endogenous regulatory networks. The WRINKLEDI (WRll) transcription factor induces
expression of at least PK p-a and PKp-fl ,, possibly by binding directly to their promoters.
Thus, overexpression of WRII in the desired tissues could be used to increase PKp
activity, with the advantage that WRII also regulates downstream components of fatty
acid and TAG biosynthesis (Ruuska et al. 2002). A similar approach was taken to
stimulate nitrogen assimilation in maize (Zea mays). Ectopic production of the ZmDofl
transcription factor (normally involved in light response) was used to activate anaplerotic
carbon metabolism and resulted in a 30% improvement in nitrogen assimilation
(Yanagisawa et al. 2004). Increased PKp activity could also be used for the production of

useful secondary metabolites. However, the range of metabolic influence of PKp is not

162

fully known and there is (weak) precedent only for the engineering of plastid localized
isoprenoid metabolism.

Another approach to modulating PKp activity is to engineer the enzyme itself.
Targeting of specific residues for increased activity or altered regulation would be greatly
facilitated by a crystal structure for the PKp in question. The crystal structure of a
mammalian PK has been determined (Muirhead et al. 1986) and it was used to direct
mutagenesis for the conversion of a non-allosteric PK into an allosteric enzyme (Ikeda et
al. 1997). Comparison of (preferably) crystal structures or the primary sequences of 01131
and 01132 to each other and to additional plant PKs could indicate which amino acids are
good targets for modification. A non-targeted approach such as directed evolution using
error-prone PCR and DNA shuffling could also yield similarly modified enzymes. Error
prone PCR introduces random mutations and resultant protein libraries must then be
screened for the desired qualities. In DNA shuffling, PCR is used to randomly combine
domains from different genes to generate a library of chimeras which must then be
screened. These procedures have been very successful for improving the biodegradation
pathways of microorganisms (Parales and Ditty 2005). Pyruvate kinases have distinct
domains for substrate and effector binding (Munoz and Ponce 2003) and combination of
these from diverse enzymes could prove fi'uitful. A non-mutagenic approach to altering
PKp activity would be to mix whole subunits from various organisms. In this work it was
determined that the presence of either 13-subunit influences the biochemical properties of
the respective PKp complexes. It is therefore possible that hybrid enzymes with novel
kinetics and regulation could be constructed by taking advantage of natural variation.

This method has been successful for the assembly of a hybrid ADP-glucose

163

pyrophosphorylase out of Arabidopsis and potato subunits (Ventriglia et al. 2007). With
so many options for the engineering of PKp enzymes, one should carefully weigh the
benefits and pitfalls of each. Targeted mutagenesis involves the least amount of work, but
would require inferences to be drawn from crystal structures which have not yet been
determined for any plant PK. Directed evolution has a high potential for success, but the
outcomes are less predictable and are entirely dependent on the screening method used to
analyze the mutant proteins (which should number in the thousands). Combining subunits
from various organisms takes advantage of existing enzyme diversity, but one must
obtain the purified subunits to perform any experiments. In conclusion, I feel that future
research on Arabidopsis PKp should be from multiple directions and should involve
collaboration with experts in various fields. Eventually, what is learned needs to be

applied to a real crop plant so that humanity can benefit from their investment in science.

164

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Parales, RE. and Ditty, J .L. (2005) Laboratory evolution of catabolic enzymes and
pathways. Curr. Opin. Biotechnol. 16:315-325.

Ruuska, S.A., Girke, T., Benning, C., and Ohlrogge, J.B. (2002) Contrapuntal
networks of gene expression during Arabidopsis seed filling. Plant Cell 14:1191-1206.

Ventriglia, T., Ballicora, M.A., Crevillen, P., Preiss, J., and Romero, J .M. (2007)
Regulatory properties of potato-Arabidopsis hybrid ADP-glucose pyrophosphorylase.
Plant Cell Physiol. 48:875-880.

Yanagisawa, S., Akiyama, A., Kisaka, H., Uchinriya, H., and Miwa, T. (2004)
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10127833-7838.

165

Appendix A
Analysis of glucose-6-phosphate dehydrogenase activity in Arabidopsis T-DNA

insertion mutants3

 

3 This work was done in collaboration with Dr. Setsuko Wakao and is being prepared for publication in:
Wakao, S., Andre, C., and Benning C. (2007) Functional analyses of cytosolic G6PDHs and their
contribution to seed oil accumulation in Arabidopsis. Plant Physiol. I contributed Figures A. ID, A.2C, and
all of A.3.

166

Introduction

Glucose-6-phosphate dehydrogenase (G6PDH) is one of the two NADPH generating
enzymes of the oxidative pentose phosphate pathway (OPPP). All eukaryotic G6PDHs
studied are feed-back inhibited by NADPH, and together with the fact that this enzyme
catalyzes a committed step makes G6PDH the regulatory enzyme of the OPPP.

In addition to the feed-back inhibition by NADPH, the plastidic isoforms of G6PDH in
plants and algae are subject to regulation by the thioredoxin/ferredoxin system (Graeve et
al. 1994, Lendzian 1980, Scheibe and Anderson 1981, Wenderoth et al. 1997, Wendt et al.
2000). Hence, they are presumed to act as cellular redox sensors and are inactivated to
prevent unnecessary oxidation of carbon when photosynthesis is sufficient for NADPH
generation.

In plants G6PDH has been frequently described in connection to its involvement
in nitrogen assimilation. The induction of its activity or transcript has been described in
various systems including pea roots (Bowsher et al. 1992), barley roots (Wright et al.
1997), maize roots (Redinbaugh and Campbell 1998), tobacco roots and leaves (Debnam
et al. 2004) and Arabidopsis (Wang et al. 2003). G6PDH activity is necessary to supply
the reducing equivalents required for nitrogen assimilation in root cells that lack
photosynthesis (Bowsher et al. 1992, Esposito et al. 2001, Esposito et al. 2003, J in et al.
1998, Wright et al. 1997). Aside from nitrogen assimilation, G6PDH has been
hypothesized to be an important source of NADPH in other non-photosynthetic tissues
(Emes and Neuhaus 1997) and in those that synthesize large quantities of fatty acids (for
incorporation into triacylglycerol), such as pollen (N iewiadomski et al. 2005) and oil

seeds (Eastrnond and Rawsthome 1998). Green oil seeds such as those of canola

167

(Brassica napus) contain plastids similar to those of shade-adapted leaves (Asokanthan et
al. 1997) and are capable of photosynthetic NADPH production, however, only 20-30%
of ambient light penetrates the silique walls and reaches the embryo (Eastmond et al.
1996, King et al. 1998). Reported O2 evolution rates from canola embryos were used to
calculate that all of the NADPH required for fatty acid synthesis could be provided by
photosynthesis (Ruuska et al. 2004). However, in controlled experiments only a 25%
decrease in fatty acid synthesis is observed in the dark. Thus, 75% of the required
NADPH could be generated by other reactions such as those of the OPPP. In vi v0 stable
isotope labeling revealed that a maximum 25% to 45% of the reductant required for oil
biosynthesis could come from the OPPP (Schwender et al. 2003). A recent report of
G6PDHs from Arabidopsis reported the isolation of T-DNA insertion mutants for both
cytosolic isoforms (Wakao and Benning 2005). Here, I examined the effects of these

mutations on total G6PDH activity in relation to seed oil biosynthesis.

168

Materials and Methods

Plant growth conditions and transformation

All seeds were surface sterilized by incubating in 20% bleach, 0.05% Triton-X. The tubes
containing the seeds were inverted for 15 min and washed three times with water. The
seeds were suspended in 0.1% agar and plated onto MS medium (pH 5.8) (Murashige and
Skoog 1962) with 1% sucrose, 0.9% agar and were transferred to soil after 9 days. Wild-
type and mutant Arabidopsis plants were prepared for transformation as previously

described (Cemac and Benning 2004).

Transient expression of G6PDH::GF P for subcellular localization analysis

The coding region of G6PD5 and G6PD6 were amplified with the following primers; for
G6PD5, (+) 5’-GGACTAGTATGGGTI‘CTGGTCAATGGCA, (-) 5’
GGACTAGTCAATGTAGGAGGGATCCAAA, and for G6PD6, (+) 5’-
GGACTAGTATGGGATCTGGTCAATGGCA, (-) 5’-
GGACTAGTTAGTGTAGGAGGGATCCAG. The cDNAs were cloned into the Spel
site of pCAMBIAl302 (Genbank accession no. AF 234298). Onion epidermal peels were
bombarded following the methods previously described (Varagona et al. 1992) using
1100 psi (pounds per square inch) rupture discs at ~4 cm distance using a biolistic gene
delivery system (Dupont). For each construct, three peels were bombarded and incubated
overnight at 22 C in the dark. The peels were observed with a Leica DMR A2 microscope
in the fluorescence mode with the L5 filter cube (Leica Microsystems, Wetzlar,

Germany).

169

Construction of complementation vectors for G6PD5 and G6PD6

The T—DNA insertion lines were transformed with BAC clones containing the regions of
G6PD5 and G6PD6 that were isolated from a genomic library in a cosmid vector
(pBIC20) (Meyer et al. 1994). The 3’-UTR of the respective genes was used as a probe.
The T-DNA insertion lines for both GéPDS and G6PD6 have lost their kanamycin
resistance, and thus the transformants were selected by kanamycin resistance introduced

by the cosmid vector.

G6PDH activity assay

Protein extraction from various tissues and electrophoresis on cellulose acetate plates
(zymogram) were performed as described previously (Wakao and Benning 2005).
Roughly 8 volumes (v/w) of extaction buffer were used per sample for homogenization.
Liquid assay of G6PDH was performed as described previously (Wakao and Benning
2005). For identification of the middle band, intact plastids were isolated from 5 week old
wild-type Arabidopsis plants grown on soil. Five to 10 g of tissue was used per isolation.
Intact plastids were isolated from homogenized tissue using a discontinuous Percoll

gradient as previously described (Xu et al. 2002).

170

Results and Discussion

To test that G6PD5 and G6PD6 indeed encode cytosolic isoforms the subcellular
localization of the proteins was examined by transient expression of the respective
cDNAs fused to a green fluorescence protein (GFP) gene. For both constructs containing
G6PD5 and G6PD6, the green fluorescence was observed dispersed in the cytosol and
surrounding what is presumably the nucleus (Figure A. l A, Al .8). The same patterns
were observed in multiple experiments. A similar pattern was observed in cells
expressing GFP alone, which localizes to the cytosol and to the nucleus (Figure A. 1C).
This result together with the lack of a potential targeting sequence in the proteins
suggests that G6PD5 and G6PD6 are both likely to be cytosolic proteins.

There are three major active G6PDH isoforms in vivo, G6PD5, G6PD6 and an
unidentified isoform that is ubiquitous (Wakao and Benning 2005). To help rule out the
possibility that the third isoform is localized in the cytosol, we examined G6PDH activity
in isolated chloroplasts from Arabidopsis leaves. As shown in Figure A.1D, isolated
chloroplasts contain a single band on a zymogram with similar mobility as the
unidentified band detected in protein extract from buds. This result suggests that the
unidentified ubiquitous isoform is localized in the plastid and that G6PD5 and G6PD6 are
the only cytosolic isoforms with major activity in Arabidopsis.

To specifically examine the in vivo roles of the cytosolic G6PDHs T-DNA
insertion lines for the two genes were obtained from the SALK institute. Their insertion
sites were identified using PCR as previously described (Figure A28 and Wakao and

Benning 2005).

171

 

Figure A.l. Cytosolic localization of G6PD5 and G6PD6

(A-C) Onion cells were bombarded with either with pCAMBIA1302 inserted with (A)
G6PD5 or (B) G6PD6 coding sequence or (C) the vector alone.

(D) Zymogram with isolated chloroplasts shows enrichment in the G6PDH band that is
neither G6PD5 nor G6PD6. The arrow indicates origin and direction of electrophoresis.

Lm, standard G6PDH from Leuconostoc mesenteroides.

The single mutants did not have any obvious morphological phenotypes (Figure A.2A).
To address whether this was because of the redundant functions of the two G6PDH
isoforms, crosses between the two lines were performed to generate plants homozygous

for both T-DNA insertions. GéPDH activity was examined in plants of different

172

genotypes using zymograms of bud protein extracts (Figure A.2C). In each single mutant,
g6pd5 and g6pd6, a band is lost from the zymogram. In the double mutant, both G6PD5
and G6PD6 were lost. Interestingly, activity from the plastidic isoform is reduced in all of
the mutants. Residual activity of G6PD5 was observed in the double mutant, consistent
with previous observations indicating g6pd5 is not a null mutant (W akao and Benning
2005). In the g6pd6 mutant, the uppermost band on the zymogram (G6PD5) is intensified
relative to wild type. It is possible that an alternative G6PDH is induced in this mutant.
However, in the double mutant this induction is lost indicating that the upper band in the
g6pd6 zymogram is indeed G6PD5. Surprisingly the double mutant was also
indistinguishable from the WT plant, despite the loss of most of the cytosolic G6PDH
activity as observed in zymograms. Therefore we conclude that a nearly complete loss of
cytosolic G6PDH activity does not result in severe morphological phenotypes of the plant
under normal conditions. Attempts to complement the single and double mutants with a
cDNA or G6PDH fused to GFP at the N-terminal have been unsuccessful (data not
shown). Only when genomic fragments containing G6PD5 or G6PD6 were introduced
did we observe the recovery of the lost bands on zymograms (Figure A.2C). This result
together with the zymogram pattern of the double mutant proves the previously defined
zymogram bands (Wakao and Benning 2005) were indeed coded by G6PD5 and G6PD6.

Seed triacyl glycerol (TAG) was quantified to determine the effects, if any, of loss
of one or both cytosolic G6PDHs. The single mutants had no changes in seed oil content.
The double mutant had a small, albeit significant, increase in TAG (data not shown)

which was associated with an increase in seed mass and not specifically oil accumulation.

173

C

96
Lo Lm g6 rescue

 

    

“WT 6pd6 g6pd5 Double

b 500 bp
AtGGPDS SALK_045083

-p
p+ p-
Lb

AtG6PD6 SALK_016157 Y"

"pt p-

B

Figure A.2. Single and double mutants for G6PD5 and G6PD6

(A) Morphological phenotypes of the single and double mutants.

(B) Gene structure of G6PD5, G6PD6 and T-DNA insertion sites. Primers used for
genotyping PCR were designed as shown.

(C) G6PDH zymograms of single and double mutants and lines rescued by Cosmid
complementation. The arrow indicates origin and direction of electrophoresis. Lm,

standard G6PDH fi'om Leuconostoc mesenteroides.

174

To better understand the increase in seed mass for the double mutant the G6PDH
isoforms present in developing seeds were examined (Figure A.3A). In wild type, the
dominant isoforms are G6PD6 and the plastidic one. As expected, the double mutant has
no activity from any isoforms. The g6pd5 mutant is similar to wild type, except for a
reduction in the plastidic isoform. The g6pd6 mutant, on the other hand, has lost the
bands present in wild type, but shows a reciprocal increase in G6PD5 activity. This
reciprocal induction could explain why only the double mutant and not g6pd6 has a seed
mass phenotype. Liquid enzyme assays were performed with the same extracts to
quantify any changes in total G6PDH activity (Figure A.3B). It is clear that G6PDH
activity is only compromised in the double mutant and that the reciprocal induction of
G6PD5 makes up for the loss of G6PD6 in the g6pd6 mutant.

The unique attributes of plant OPPP, such as dual localization, complicate the
interpretation of G6PDHs role in whole cells. The discovery of a plastidic pentose-
phosphate transporter (Eicks et al. 2002) demonstrated a physical connection between
plastidic and cytosolic pentose phosphate pathway but how the cellular supply of
NADPH is coordinated remains unknown. Additionally, connectivity between
biochemical pathways makes interpretation difficult. Overlap of intermediates with other
metabolic pathways such as glycolysis, the TCA cycle, and amino acid and nucleotide
biosynthesis makes it difficult to discern whether the effect is primarily due to reduced
OPPP flux, NADPH supply, or something else. We speculate that in the double mutant
but not in the single mutants (because of the G6PDH activity compensation) there is

altered carbon metabolism and one of the effects is larger seeds.

175

A WT ngd5/96pd6 g6pd5 g6pd6
DAFLm’l 9 «N «'5 \‘3 Lm’l 9 «N «’5 «‘3 Lm’l e x“ «’5 «‘9 Lm’l c2, \’\ <5 <0
Lo 3:251:31 ' . 9i; i

 

 

      

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DAF
Figure A.3. G6PDH isoforms and activity in developing siliques
(A) Zymograms of protein extracts from staged siliques of the various geneotypes. An
induction of the normally inactive G6PD5 is evident for the g6pd6 mutant. The arrow
indicates origin and direction of electrophoresis. Days after flowering (DAF) is indicated
for each sample. Lm, standard G6PDH from Leuconostoc mesenteroides.
(B) Liquid enzyme assays show that only in the double mutant is G6PDH activity

reduced. DAF, days after flowering.

176

It is possible that glycolytic flux is increased in the double mutant, since glucose-6-
phosphate is not consumed by OPPP, further fueling fatty acid synthesis. Such alteration
in carbon metabolism may have occurred in non-seed tissues as well but could not be
detected. It will require metabolic profiling or metabolic flux analyses in order to study
the details of the metabolic changes in the double mutant. The fact that the single mutants
are not different from the WT again indicates the remaining G6PDH activity is

compensating for the loss of the other.

177

References

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Cernac, A. and Benning,C. (2004) WRINKLEDI encodes an AP2/EREB domain protein
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Debnam, P.M., Fernie, A.R., Leisse, A., Golding, A., Bowsher, C.G., Grimshaw, C.,
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Eastmond, P., Kolacna, L., and Rawsthome, S. (1996) Photosynthesis by developing
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Eastmond, P.J. and Rawsthome, S. (1998) Comparison of the metabolic properties of
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Eicks, M., Maurino, V., Knappe, S., Flugge, U.I., and Fischer, K. (2002) The plastidic
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Emes, M.J. and Neuhaus, H.E. (1997) Metabolism and transport in non-photosynthetic
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Esposito, S., Carfagna, S., Massaro, G., Vona, V., and Rigano, V.D. (2001) Glucose-
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Esposito, S., Massaro, G., Vona, V., Rigano, V.D., and Carfagna, S. (2003) Glutamate
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179

 

Wenderoth, 1., Scheibe, R., and von Schaewen, A. (1997) Identification of the cysteine

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180

Appendix B
Analysis of glycolytic enzyme activities in seedlings of the wrinkled] mutant of

Arabidopsis“

 

4 This work was done in collaboration with Dr. Alex Cemac and portions have been published in: Cernac,
A., Andre, C., Hoffman-Benning, S., and Benning, C. (2006)WRI1 is required for seed germination and
seedling establishment. Plant Physiol. 141:745-757. I contributed Figure B2.

181

Introduction

Seed germination in Arabidopsis initiates with the matrix-driven absorption of water,
followed by cell expansion, splitting of the seed coat, and subsequent emergence of the
radicle. The mobilization and metabolism of seed triacyl glycerol (TAG) is an intricate but
well-characterized process and has been reviewed recently (Penfield et al. 2005). Plants
with reduced TAG mobilization have been identified, including those affected in lipid
trafficking and B-oxidation (Footitt et al. 2002, Germain et al. 2001, Hayashi et al. 1998,
Lawand et al. 2002, Zolman et al. 2001). The role of the glyoxylate cycle involved in the
conversion of lipids into carbohydrates has also been examined through a series of
mutants (Comah et al. 2004, Eastmond et al. 2000, Hayashi et al. 2005), as has
gluconeogenesis itself (Penfield et al. 2004, Rylott et al. 2003). Common phenotypes,
many of which can be rescued by sugar supplementation or appropriate photosynthetic
conditions, include a reduced germination rate, arrest of development after germination,
inability to elongate the hypocotyl in the dark, and failure to elongate the root after
opening and greening of the cotyledons. Based on the analysis of these mutants a picture
has emerged suggesting that in Arabidopsis the energy for germination is derived from
stored reserves other than lipids, and that seed oil becomes vital for continued growth and
seedling establishment after the radicle has emerged.

The wrinkled] (wri]) mutant of Arabidopsis was originally isolated based on its
low seed oil content and developing wri] seeds showed reduced activity of key glycolytic
enzymes such as hexokinase (HXK) and pyruvate kinase (PK, F ocks and Benning 1998).
WRII was subsequently shown to encode an APETALA2/ethylcue-responsive element-

binding transcription factor (Cemac and Benning 2004).

182

Microarray analysis of wri]-1 and wild-type developing seeds indicated a global down-
regulation of transcripts encoding enzymes involved in carbohydrate metabolism in the
mutant (Ruuska et al. 2002) consistent with a role for WRIl in the regulation of sugar
metabolism. Further support for the function of WRII was provided when WRII was
recently identified in an activation-tagging screen targeting genes for which a strong
expression resulted in increased transcription from a known sugar-inducible promoter
(Masaki et a1. 2005). The ectopic expression of the WRII cDNA caused a sugar-inducible
accumulation of seed oil in the transgenic seedlings, which is correlated with increased
transcript amount of HXK and PK encoding genes. The general appearance of the
transgenic seedlings suggested a resumption of embryonic development following
germination. The overall conclusion was that the WRII gene product is involved in
controlling the phase of embryo maturation in which TAG accumulates, possibly by
regulating the expression of specific HXK and PK encoding genes (Cemac and Benning
2004). Seed germination and establishment are also impaired in wri] (Cemac et a1. 2006).
Establishment of wri] seedlings is dependent on a supply of sucrose and root
ultrastructure is abnormal. As the WRII gene is expressed in seedling roots, we wanted to
determine if misregulation of HXK and PK are responsible for the observed

morphological differences between wild type and wri] .

183

Materials and Methods

Growth of seedlings for root elongation and enzyme assays

Root elongation rate assays were carried out on half-strength Murashige and Skoog
medium. Seeds were sown in a straight line, 15 to 20 for wild type and rescued lines and
30 to 50 for wril-l. Seedling root length was measured every 24 h starting on the 4th (1
post incubation through the 12th (1. For RNA and protein extraction seeds were sown as
they were for root elongation except 15 x 150 mm plates were used. The seeds were
stratified as above and the plates incubated vertically for 11 d. Entire wri]-1 seedlings
were harvested for the 0 mM Suc treatment. Roots and shoots were separated by cutting
along the line of plants at the base of the hypocotyl. Harvested material was wrapped in

aluminum packets, weighed, and frozen in liquid nitrogen.

Protein extraction and enzyme assays

Approximately 100 mg of tissue was used for each protein extraction. Tissue was ground
frozen using mortar and pestle and transferred to 1.5 mL tubes. Protein for enzyme assays
was extracted as described previously except that bovine serum albumin was omitted
from the extraction buffer (Focks and Benning 1998). Protein extracts were used
immediately for enzyme assays in a double-beam spectrophotometer (Uvicon 930,
Kontron Instruments) equipped with a cell changer (model 900, Kontron). All reagents
were from Sigma-Aldrich. Pyruvate kinase was assayed in 1 mL total volume in a
reaction mix consisting of 50 mM MOPS pH 7.0, 5% PEG-8000, 50 mM KCl, 15 mM
MgC12, 1 mM dithiothreitol, 2 mM phosphoenolpyruvate, 1 mM ADP, 0.2 mM NADH,

and 2 units of desalted lactate dehydrogenase. Pyruvate kinase was also assayed at pH 8.0

184

by using 50 mM HEPES-KOH pH 8.0 instead of MOPS. The same trends were observed
using both conditions. Correction for phosphoenolpyruvate phosphatase activity was
carried out by the omission of ADP from the reaction mix. Glucokinase and Fructokinase
were assayed as previously described, but in 1 mL total volume (Wiese et al. 1999). All
reactions were done at 30°C, were initiated by the addition of protein extracts, and were
linear for at least 5 min. Total protein in each extract was determined using the DC

protein assay kit from Bio-Rad.

185

 

Results and Discussion
The poor establishment of wri]-I in medium lacking sugar is qualitatively shown in
Figure B. 1 A and was quantified by measurements of root length from day 4 through day
12 post incubation (Figure B.1B). Without any sugar supplement, the wri 1-1 seedlings
failed to establish and even after 12 (1 they did not significantly elongate their root or
form true leaves. This phenotype was similar to that observed for abi8, ntt2, and bou
mutants. The abi8 mutant is defective in ABA signaling and allelic to eldl (Brocard—
Gifford et a1. 2004, Cheng et al. 2000), bou is deficient in a mitochondrial acyl camitine
transporter (Lawand et al. 2002), and ntt2 is deficient in a plastidic ATP/ADP transporter
(Reiser et al. 2004). While Suc did not enhance the germination frequency of wri]-I it
did allow the seedlings to establish (Figure B.1A). As previously observed (Focks and
Benning 1998), wri]-1 plants grew similar to wild type on soil after they were raised on
medium containing sugar. Expression of the WRII cDNA in the transgenic lines was able
to rescue the wri]-1 root elongation defect almost to the level of wild type (Figure B.lB).
An impairment of sugar metabolism, i.e. glycolysis, in nonphotosynthetic tissues
should have a detrimental effect on seedling establishment as the root is completely
dependent upon the import and metabolism of sugar initially from the cotyledons
breaking down lipids and converting them to sugars, and later from cotyledons and true
leaves conducting photosynthesis. Thus, to better understand the physiological effects
described above, we examined the activity of hexokinase (HXK) and pyruvate kinase
(PK) in roots and shoots of 1 1-d-old developing seedlings of the different lines under

investigation.

186

 

 

Root Length (mm)
8

5°-
:1de
“m3. \
v.

4°‘ oulii1-1 }

 

Age of Seedling (days)
Figure 3.1. Impaired seedling establishment in the wri]-I mutant
(A) Appearance of 12-d-old seedlings of wild type and wri]-1 grown in the presence (+)
or absence (—) of 50 mM Suc.
(B) Root grth on medium lacking Sue. The error bars represent SD of the mean of at

least 15 root length measurements.

187

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Figure 8.2. Glycolytic enzyme activities in green seedlings
wri]-1, wild type, and WRII overexpressing seedlings in the presence or absence of

sucrose. Values are the mean i SD, n=5.

The seedlings shown in Figure Bl .A are representative of the tissue that was used
for enzyme activity measurements. The data obtained from liquid enzyme assays is
depicted in Figure B2. In wild type, PK activity in roots grown on Sue was reduced. On
the contrary, with Suc present the gluco- and fructokinase activities representing HXK

were slightly increased. There were no gross differences between the three genotypes.

188

Based on these results we concluded that glycolysis is active in ll-d-old seedlings that
have progressed through the establishment phase during which sugar supplementation
could alleviate the deficiency in the wri]-1 mutant. Therefore, WRIl does not to seem to
be directly involved in regulation of glycolysis once seedlings start growing during the
process of establishment. However, what is presumably limiting during this phase of
development in the mutant is the sparse amount of seed storage oil, which is reduced at
least 80% in wri]—1 seeds (Focks and Benning 1998). Offering Suc that can provide
energy and building blocks at this stage completely restores seedling establishment
(Figure B.1A). It seems that the role of WRIl on seedling establishment is indirect, by
way of its involvement in the up-regulation of carbohydrate metabolism in the developing

seed thereby ensuring the presence of storage oil needed for seedling establishment.

189

 

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Cernac, A., Andre, C., Hoffmann-Benning, S., and Benning, C. (2006)WR11 is
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Cernac, A. and Benning, C. (2004) WRINKLEDI encodes an AP2/EREB domain
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Footitt, S., Slocombe, S.P., Larner, V., Kurup, S., Wu, Y., Larson, T., Graham, 1.,
Baker, A., and Holdsworth, M. (2002) Control of germination and lipid mobilization by
COMATOSE, the Arabidopsis homologue of human ALDP. EMBO J. 21 :2912-2922.

Germain, V., Rylott, E.L., Larson, T.R., Sherson, S.M., Bechtold, N., Carde, J.P.,
Bryce, J.H., Graham, I.A., and Smith, S.M. (2001) Requirement for 3-ketoacyl-C0A
thiolase-2 in peroxisome development, fatty acid beta-oxidation and breakdown of
triacylglycerol in lipid bodies of Arabidopsis seedlings. Plant J. 2821-12.

Hayashi, M., Toriyama, K., Kondo, M., and Nishimura, M. (1998) 2,4-
Dichlorophenoxybutyric acid-resistant mutants of Arabidopsis have defects in
glyoxysomal fatty acid beta-oxidation. Plant Cell 10:183-195.

Hayashi, M., Yagi, M., Nito, K., Kamada, T., and Nishimura, M. (2005) Differential
contribution of two peroxisomal protein receptors to the maintenance of peroxisomal
functions in Arabidopsis. J. Biol. Chem. 280: 14829-14835.

190

 

Lawand, S., Dorne, A.J., Long, D., Coupland, G., Mache, R., and Carol, P. (2002)
Arabidopsis A BOUT DE SOUFFLE, which is homologous with mammalian camitine
acyl carrier, is required for postembryonic grth in the light. Plant Cell 14:2161-2173.

Masaki, T., Mitsui, N., Tsukagoshi, H., Nishii, T., Morikami, A., and N akamura, K.
(2005) ACTIVATOR of Spo minzzLUCl/WRINKLEDI of Arabidopsis thaliana
transactivates sugar-inducible promoters. Plant Cell Physiol. 46:547-556.

Penfield, S., Graham, S., and Graham, LA. (2005) Storage reserve mobilization in
germinating oilseeds: Arabidopsis as a model system. Biochem. Soc. Trans. 33:380-383.

Penfield, S., Rylott, E.L., Gilday, A.D., Graham, S., Larson, T.R., and Graham, LA.
(2004) Reserve mobilization in the Arabidopsis endosperm fuels hypocotyl elongation in
the dark, is independent of abscisic acid, and requires PHOSPHOENOLPYRUVATE
CARBOXYKINASEI. Plant Cell 16:2705-2718.

Reiser, J., Linka, N., Lemke, L., Jeblick, W., and Neuhaus, H.E. (2004) Molecular
physiological analysis of the two plastidic ATP/ADP transporters from Arabidopsis.
Plant Physiol. 136:3524-3536.

Ruuska, S.A., Girke, T., Benning, C., and Ohlrogge, J.B. (2002) Contrapuntal
networks of gene expression during Arabidopsis seed filling. Plant Cell 14:1191-1206.

Rylott, E.L., Gilday, A.D., and Graham, LA. (2003) The gluconeogenic enzyme
phosphoenolpyruvate carboxykinase in Arabidopsis is essential for seedling
establishment. Plant Physiol. 131:1834-1842.

Wiese, A., Groner, F., Sonnewald, U., Deppner, H., Lerchl, J., Hebbeker, U., Flugge,
U., and Weber, A. (1999) Spinach hexokinase I is located in the outer envelope
membrane of plastids. F EBS Lett. 461113-18.

Zolman, B.K., Monroe-Augustus, M., Thompson, B., Hawes, J.W., Krukenberg,
K.A., Matsuda, S.P., and Bartel, B. (2001) chyI , an Arabidopsis mutant with impaired
beta-oxidation, is defective in a peroxisomal beta-hydroxyisobutyryl-CoA hydrolase. J.
Biol. Chem. 276:31037-31046.

191

Appendix C

Leaf and seed lipid analysis of Arabidopsis mutants disrupted in Lipin genes

192

Introduction

The biochemical reactions involved in lipid homeostasis have been described, but the
regulators of these processes are more elusive. Lipodystrophy is a human metabolic
disorder which is characterized by the loss of body fat and concomitant deposition of fat
in the liver (Reitman 2005). Mice carrying mutations in the fatty liver dystrophy (fld)
gene have symptoms of lipodystrophy and serve as a model for the human disease.
Positional cloning was used to identify the gene responsible for mouse fld (Peterfy et al.
2001). The gene, called Lipin-1 (Lpinl) has homologues in all eukaryotic kingdoms. In
mice LpinI controls triacyl glycerol (TAG) accumulation in adipose tissue. LpinI knock
outs have no TAG and do not develop mature functioning adipocytes, while
overexpressors of Lpinl contain more TAG and are prone to obesity (Peterfy et al. 2005,
Phan et al. 2004). A yeast lipin mutant is also compromised in TAG accumulation (Han
et al. 2006). Combined, these phenotypes are consistent with a role for lipin in TAG
biosynthesis.

The yeast and, more recently, the mammalian lipin genes have been shown to
encode members of the Mg2+-dependent phosphatidate (PA) phosphatase (PAPl) family
(Donkor et al. 2007, Han et al. 2006). The generation of diacylglycerol from PA is a step
in the synthesis of TAG in both the Kennedy and the phosphocholinezdiacylglycerol
acyltransferase pathways (outlined in Chapter 1). While lipin is required for TAG
accumulation in yeast and mice, its contribution to TAG biosynthesis in plants has not
been investigated. In addition, PA is recognized as a secondary messenger in plant (and
other eukaryotic) signal transduction networks (Wang 2004), and an emerging role for

PA is as a membrane lipid biosynthetic intermediate which is involved in the trafficking

193

of lipids from the endoplasmic reticulum to the plastid (Awai et al. 2006, Xu et al. 2005).
The manipulation of PA levels through modulation of lipin gene expression provides an
opportunity to explore the dynamics of such lipid signaling and trafficking. Moreover, the
same mutants would provide a chance to investigate whether or not lipin plays a similar
role in seeds as in adipose tissue, with respect to TAG synthesis. The relative ease of
obtaining T-DNA insertion mutants for nearly any gene in the Arabidopsis genome

provided enough impetus to pursue these questions.

194

Materials and Methods

Gene identification

Arabidopsis lipin homologues (At3g09560 and At5g42870;LPN1 and LPLI,
respectively) were identified using. the deduced amino acid sequence of the Mus muscqu
Lpz'n] gene (accession NM_172950). Homologues were identified using the basic local
alignment search tool (BLAST) available at the TAIR website (wwwarabidopsisorg).
Global gene expression data was mined from the AtGenExpress developmental database

(Schmid et al. 2005).

T-DNA mutant isolation

Arabidopsis lipin mutants were selected from the SALK T-DNA insertion population
(Alonso et a1. 2003). Selection of mutants was on half-strength MS medium containing
25 pg mL'l Kanamycin. T-DNA insertion sites were determined by sequencing PCR
products made using primers designed with the help of the i-sect tool available on the
Salk Institute website (http://signal.salk.edu/tdnaprimers.html). Primers for LPN] were
5’-TgTTATTgTTCTCTAATTTTg-3’ and 5’-gTTTngTCAgCTCTgACTgC-3’ and
primers used for LPLI were 5’-gAATTCgCgCATAgTTngTC-3’ and 5’-
AACAAgCCCCgTATCTCCTgT-3’. The left border primer used was LBal as suggested
by the SALK Institute website (http://signal.salk.edu/). PCR conditions used standard
buffer conditions and 40 cycles of 95°C for 30 sec, 52°C for 40 sec, and 72°C for 90 sec,
followed by a 10 min 72°C extension. All subsequent genotyping was done using the

same PCR protocol.

195

Lipid analysis

Leaf samples (50-150 mg) were collected from individual plants and were weighed and
frozen at -80°C. Lipids were extracted from leaves by vigorously shaking for 5 min in
500 uL of methanol/chloroform/formate (2:1 :0.1, v/v). Then 250 pL of 1 M KCl, 0.2 M
H 3PO4 was added and the tubes were vortexed. The phases were separated by
centrifugation at 16,000 g for 5 min. The organic phase was removed and loaded
quantitatively onto a pre-treated (soaked in 0.15 M (N H4)2SO4 and dried, then heated to
120°C for 2.5 hrs) silica-60 TLC plates (Baker). Lipids were developed in a solvent
system of acetone/toluene/water (91:30:7, v/v) and were stained with iodine and a-
naphthol. Fatty acid methyl ester analysis of leaf and seed lipids was done as previously
described (F ocks and Benning 1998). One small leaf or 10 seeds were used for each

replicate.

196

 

 

Results and Discussion

Lipin homologue identification and analysis of gene expression

Arabidopsis lipin homologues were identified based on amino acid sequence similarity to
Mus musculus Lpinl. Two genes were identified which encode Lpinl homologues,
At3g09560 and At5g42870. The At3g09560 gene encodes a protein most similar to
Lpinl (23% identical, 38% similar) and was named lipin] (LPNI). The other gene,
At5g42870, encodes a protein less similar to Lpinl (22% identical, 36% similar) and thus
was named lipinI—like (LPLI). Lipin proteins contain a DEXT motif characteristic of
Mg2+-dependent phosphatases. In the Lpinl homologues from mammals, chicken, fish,
Caenorhabditis elegans, Drosophila, Ciona, and S. cerevisiae, the motif is exactly
DIDGT (Donkor et al. 2007). The Arabidopsis proteins vary only slightly and both
contain a signature sequence of DVDGT. It is therefore likely that the Arabidopsis
proteins are also Mg2+-dependent phosphatases.

Publicly available Arabidopsis gene expression data reveals the tissue specific
expression patterns for both LPN] and LPL] (Figure C.1). In general, LPN] is expressed
at the same or higher level as LPLI in any given tissue. Both genes are induced during
embryo development and have maximal expression just prior to the onset of TAG
biosynthesis and accumulation. The gene expression data suggests that both LPN] and

LPLI have roles in embryo development.

197

 

 

 

 

 

  
   

 
     

 

 

 

 

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Figure C.l. Relative gene expression of putative Arabidopsis lipin homologues
Figure is derived from published microarray data (Schmid et al., 2005). DAF, days after

flowering.

T-DNA mutant isolation and lipid analysis

The Arabidopsis T-DNA mutant population was utilized to study the in vitro function of
the Arabidopsis lipins (Alonso et al. 2003). Three lines were obtained which had inserts
in the desired genes (Figure C.2). Genotyping of putative mutants was done with PCR as
described in materials and methods and exact insertion sites were determined. As it turns
out, SALK_146637 and SALK_042850, both located in LPN], had identical insertion
sites. For all three lines, the T-DNA was inserted into a translated portion of an exon
which almost certainly causes reduced transcript abundance in homozygous lines.

However, it remains to be tested whether or not transcription is actually affected by these

198

insertions. There were no obvious morphological or developmental differences between
the single mutants and wild type. A double mutant was generated by crossing lpn] and

[pl] and it too was wild-type in appearance.

SALK_146637
SALK_042850

At3909560 - LPN 1 STOP

 

SALK_047457

At5 42870 - LPL1
ATG 9 STOP

 

 

1 Kb
Figure C.2. Gene structure of the two Arabidopsis Lpinl homologues
Locations of the T-DNA insertions are indicated. Black box on T-DNA is the left border.

ATG, start codon; STOP, stop codon.

Lipid analysis was carried out for the individual lipin mutants as well as for the
double mutant, despite not knowing the degree of mRNA reduction in any of the lines.
First, total leaf lipids were extracted quantitatively and analyzed by thin layer
chromatography (TLC). Figure C.3A shows the results of this experiment. Two staining
methods were used on the same plate to maximize the number of lipids detected.
Membrane galacto- and phospholipids dominate the stained TLC plate. It is clear that
there are no gross differences in the lipid composition between wild type and either the

single or double mutants.

199

 

 

 

 

 

 

 

 

   

 

 

  

 

    
 

 

 

  

 

 

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Figure C.3. Lipid analysis of Arabidopsis lipin single and double mutants

(A) Thin layer chromatograrn of total leaf lipids stained with iodine and a sugar-sensitive
stain. DGDG, digalactosyldiacylglycerol; MGDG, monogalactosyldiacylglycerol.

(B) Fatty acid composition of total leaf lipids. Single leaves were subjected to fatty acid
methyl ester analysis using gas chromatography. WT, wild type.

(C) Seed oil content of wild type (WT) and lipin single and double mutants as determined
by fatty acid methyl ester (FAME) analysis.

(D) Fatty acid profile from seeds used for oil content determinations in (C).

200

The fatty acid profiles of total lipids from leaves were determined as well (Figure C.3B).
Again, the mutants and wild type were remarkably similar. Seed oil content and
composition were examined in mature seeds of the lipin mutants. As shown in Figure

C .3C, there were no differences observed between any of the lines. And again, the fatty
acid profile of the lipids in questions was determined, and again, the wild type and
mutant were the same (Figure C.3D).

The lack of a lipid phenotype for these mutant lines is not totally surprising. Gene
expression levels in the mutants were not analyzed and it is possible that the T-DNA
insertions have no affect on mRNA or protein abundance. A simple RT-PCR or RNA
blot experiment would begin to address this issue. Also, there was no confirmation of the
encoded proteins’ putative catalytic fimction. Nonetheless, even if the genes in question
do encode active Mg2+-dependent phosphatidate phosphatases, and even if gene
expression is completely knocked out, the mutants might not have an easily detectable
phenotype. Phosphatidate occurs at very low concentrations in plant cells and is not
readily measured by the methods used here. Any changes in the steady state amount of
PA would have to be detected by other means. Furthermore, the fatty acid profile data in
Figures C.3B and C.3D is of total lipids and does not take into account any changes at the
level of individual lipid species (i.e. PG, MGDG, DGDG). Beyond these obvious
methodological shortcomings, the flexibility of plant metabolism could also account for
the apparent lack of aberration in the lipin mutants. For instance, certainly LPN1 and
LPLl are not the only PA phosphatases in Arabidopsis. Future studies of lipin in
Arabidopsis should be focused on mutants with documented reductions in gene

expression in combination with other lipid metabolism mutants, such as TGDl -3.

201

References

Alonso, J.M., Stepanova, A.N., Leisse, T.J., Kim, C.J., Chen, H.M., Shinn, P.,
Stevenson, D.K., Zimmerman, J ., Barajas, P., Cheuk, R., Gadrinab, C., Heller, C.,
Jeske, A., Koesema, E., Meyers, C.C., Parker, H., Prednis, L., Ansari, Y., Choy, N.,
Deen, H., Geralt, M., Hazari, N ., Hom, E., Karnes, M., Mulholland, C., Ndubaku, R.,
Schmidt, 1., Guzman, P., Aguilar-Henonin, L., Schmid, M., Weigel, D., Carter, D.E.,
Marchand, T., Risseeuw, E., Bragden, D., Zeko, A., Crosby, W.L., Berry, CC, and
Ecker, J.R. (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana.
Science 301:653-657.

Awai, K., Xu, C., Tamot, B., and Benning, C. (2006) A phosphatidic acid-binding
protein of the chloroplast inner envelope membrane involved in lipid trafficking. Proc.
Natl. Acad. Sci. U.S.A. 103:10817-10822.

Donkor, J ., Sariahmetoglu, M., Dewald, J ., Brindley, D.N., and Rene, K. (2007)
Three mammalian lipins act as phosphatidate phosphatases with distinct tissue expression
patterns. J. Biol. Chem. 282:3450-3457.

Focks, N. and Benning, C. (1998) wrinkled]: A novel, low—seed-oil mutant of
Arabidopsis with a deficiency in the seed-specific regulation of carbohydrate metabolism.
Plant Physiol. 118:91-101.

Han, C.S., Wu, W.H., and Carman, GM. (2006) The Saccharomyces cerevisiae Lipin
homolog is a M g2+-dependent phosphatidate phosphatase enzyme. 1. Biol. Chem.
281:9210-9218.

Peterfy, M., Phan, J ., and Reue, K. (2005) Alternatively spliced lipin isoforms exhibit
distinct expression pattern, subcellular localization, and role in adipogenesis. J. Biol.
Chem. 280:32883-32889.

Peterfy, M., Phan, J ., Xu, P., and Rene, K. (2001) Lipodystrophy in the fld mouse
results from mutation of a new gene encoding a nuclear protein, lipin. Nat. Genet.
27: 121-124.

lPhan, J., Peterfy, M., and Reue, K. (2004) Lipin expression preceding peroxisome
proliferator-activated receptor-gamma is critical for adipogenesis in vivo and in vitro. J.
Biol. Chem. 279:29558-29564.

Reitman, M.L. (2005) The fat and thin of lipin. Cell Metab. 1:5-6.

Schmid, M., Davison, T.S., Henz, S.R., Pape, U.J., Demar, M., Vingron, M.,
Scholkopf, B., Weigel, D., and Lohmann, J.U. (2005) A gene expression map of
Arabidopsis thaliana development. Nature Gen. 37:501-506.

Wang, X. (2004) Lipid signaling. Curr. Opin. Plant Biol. 7 2329-336.

202

 

Xu, C., Fan, J ., F roehlich, J .E., Awai, K., and Benning, C. (2005) Mutation of the
TGDl chloroplast envelope protein affects phosphatidate metabolism in Arabidopsis.
Plant Cell 17 23094-3] 10.

203

 

Appendix D
In vitro substrate specificity of the Rhodobacter sphaeroides betaine lipid

biosynthetic enzyme BtaA5

 

5 This work, was done in collaboration with Dr. Wayne Riekhof and has been published in: Riekhof, W.R..,
Andre, C., and Benning, C. (2005) Two enzymes, BtaA and BtaB, are sufficient for betaine lipid
biosynthesis in bacteria. Arch. Biochem. Biophys. 441:96-105. I contributed Figure D.2.

204

 

‘Introduction
The sessile lifestyle of many plants, bacteria, and fungi has led to the development of
complex means of coping with specific mineral deficiencies. For instance, the
replacement of abundant cellular phospholipids with alternative, non-phosphorous lipids
is one means of dealing with environmental phosphate limitation. This phenomenon has
been the most extensively documented for the a-proteobacteria Rhodobacter sphaeroides
and Sinorhizobium meliloti, which synthesize the betaine lipid diacylglyceryl-N,N,N-
trimethylhomoserine (DGTS) and omithine containing lipids to replace depleted
membrane phospholipids (Benning et al. 1995, Geiger et al. 1999).

DGTS was first discovered in a unicellular alga (Brown and Elovson 1974), but
has since been found in lower plants and fungi (Kunzler and Eichenberger 1997,
Rozentsvet et al. 2000). The structure and zwitterionic nature (Figure D.1A) of DGTS led
to the hypothesis that it replaces phosphatydylcholine in the organisms in which it occurs.
The use of radioisotope labeling in Chlamydomonas reinhardtii and in R. sphaeroides led
to the identification of methionine as the biosynthetic precursor for the homoserine
moiety and the adjoining methyl groups (Hofmann and Eichenberger 1996, Sato 1988).
Subsequently, a genetic study using R. sphaeroides resulted in the identification of a two
gene operon responsible for DGTS biosynthesis during phosphate starvation (Klug and
Benning 2001). The two genes, designated btaA and btaB, respectively encode a
proposed AdoMet/diacylglycerol 3-amino-3-carboxypropyl transferase producing the
intermediate DGHS, and a putative methyltransferase adding three methyl units to the

amino group of DGHS to form DGTS.

205

 

   

NH; Q7, Q; a?
be ~32 .5 (go
MetK 0v (on 0&7 0Q
DAG Q' Q- ‘o to
AdoMet . ml
2
O;;'/\/CSIH3
“H R1 2 < DGHS
NH3
OH OH
BtaA
5'MTA R2
DGHS R1 i w... E;§:..’.-..'L;§?'+PtdEtn
NH;
inspim (3x.
DGTS R1

Figure D.1. DGTS biosynthetic pathway and involvement of RthaAB

(A) BtaA is proposed to catalyze the transfer of the 3-amino-3-carboxypropyl group of
AdoMet to the 3-hydroxyl of DAG to form DGHS. DGHS is then N-methylated by BtaB
to form DGTS. AdoHcy, S—adenosylhomocysteine; AdoMet, S-adenosylmethionine;
DAG, diacylglycerol; DGHS, diacylglycerylhomoserine; DGTS, diacylglyceryl-(N,N,N)-
trimethylhomoserine; MetK, AdoMet synthetase; 5'MTA, 5'-methylthioadenosine.

(B) E. coli expressing btaA accumulate DGHS. The btaA expression strain (third lane)
shows a band co-migrating with authentic DGHS from a phosphate stressed R.
sphaeroides btaB KO strain (first lane). Phosphate-replete R. sphaeroides btaB K0 and

empty pQE-31 served as negative controls (second and fourth lanes, respectively).

206

 

This proposed DGTS biosynthetic scheme involving these two enzymes is depicted in
Figure D. 1 A. While these genes have been shown to be necessary for DGTS biosynthesis
in vivo, a biochemical analysis of their protein products has not been done. This work
focuses on the BtaA enzyme, due to its unusual reaction, and describes an in vitro
experiment which helped confirm its identity as an AdoMet/diacyl glycerol 3-amino-3-

carboxypropyl transferase.

207

 

Materials and Methods

In vitro activity assays of RthaA

Escherichia coli TOPIO F' harboring thaA (constructed as described in Riekhof et al.
2005) was grown in 250 ml LB-ampicillin at 37 °C to an OD of 0.7, and induced with
0.25 mM IPTG followed by an additional 4 h of growth at 28 °C. Cells were harvested by
centrifugation and the cell pellet was suspended in 10 ml of cold buffer (50 mM Hepes,

1 mM DTT, 1 mM EDTA, pH 7.3). The resuspended cells were sonicated 3—4 times, 30 3
each with a microprobe tip, and the lysate was centrifuged at 2000 g for 10 min to
remove unbroken cells and cellular debris. Aliquots (1 ml) of the cell-free extract were
then frozen in liquid N2 and stored at -80 °C prior to use. Activity under these storage
conditions did not decrease appreciably for at least 1 month.

Assays were conducted in 100 pl final volume by combining 48.75 ul of cell-fiee
extract with 48.75 pl of 100 mM Hepes, Tris—Cl, or MES, 1 mM DTT, 1 mM EDTA, at
varying initial pH to give a final pH in the range of 5.5—8.6 when mixed with the cell-free
extract (initial, pH 7.3). Reactions were initiated by addition of 25,000 dpm 1-
[I4C]AdoMet (American Radiolabeled Chemicals, 2.5 u], final concentration of AdoMet
of 2.1 uM, specific activity 7.14 MBq/nmol), or 75,000 dpm [14C]DAG (dioleoyl-rac-
glycerol, [oleoyl l-MC]; American Radiolabeled Chemicals, final concentration 6.3 uM,
specific activity 7.14 MBq/nmol). For the reaction initiated with labeled DAG, it was
essential to dry [14C]DAG, which was delivered suspended in toluene/ethanol (1 :1, v/v) at
room temperature under a stream of nitrogen. A sonicating water bath was used to
disperse the DAG at the desired concentration in a buffer of 50 mM Hepes, pH 7.8, 1 mM

EDTA, lmM EGTA, 0.1% Triton X-100. Reactions were incubated at 28 C, and

208

 

terminated by addition of 400 pl of chloroform/methanol (1 : 1, v/v) and 100 pl 0.9%
(w/v) NaCl to separate aqueous and organic phases. The organic phase was transferred to
a new tube, dried under a stream of nitrogen, and dissolved in 50 pl chloroform/methanol
(1 :1, v/v). This lipid extract was then spotted onto silica-6O TLC plates (Baker) and
developed with the solvent chloroform/acetone/methanol/acetic acid/water (10:42222:1,
v/v), followed by quantification of the signal for DGHS on a Molecular Dynamics
phosphorimager screen (Amersham, Piscataway, NJ, USA) with the ImageQuant
software package. Alternatively, TLC plates were stained with ninhydrin reagent to

visualize primary amine-containing lipids.

209

 

Results and Discussion

The btaA and btaB genes have previously been shown to be necessary for DGTS
accumulation in phosphate stressed R. sphaeroides (Klug and Benning 2001). Expression
of btaA in E. coli resulted in the accumulation of a primary amine-containing lipid band
which co-mi grated with authentic DGHS produced from phosphate stressed R.
sphaeroides btaB KO (Figure D. 1 B). We expected that E. coli expressing RthaA could
use endogenous diacyl glycerol (DAG) and S-adenosylmethionine (AdoMet) to synthesize
DGHS. Heterologous expression of RthaA confirmed the catalytic role of the encoded
protein, but did not confirm DAG or AdoMet as the substrates used for DGHS
biosynthesis.

In vitro assays of RthaA enzyme activity were conducted using 1-[I4C]AdoMet
or ['4C]DAG to follow product formation. While we were able to utilize the engineered
Hisé-tag to purify small amounts of apparently soluble RthaA protein using various
detergents in the lysis buffer, we were unable to demonstrate activity in a reconstituted
liposome system. To circumvent this problem, we developed a system to minimize the
steps between expression and enzyme assay to demonstrate the proposed reaction
catalyzed by RthaA. As we wanted to test the incorporation of label from 1-
[14C]AdoMet as well as ['4C]DAG, two sets of controlled reactions were set up keeping
all conditions the same except for the compound carrying the label. Both sets of reactions
were supplied with the two substrates, AdoMet at 2.1 pM and DAG at 6.3 pM. In the two
sets of reactions, the specific activity for one or the other substrate was kept the same. In
both sets a 3-fold higher amount of DAG was used because unlike AdoMet, DAG is

offered as a micelle suspension and a number of other reactions consume this precursor

210

 

as well (see below). This most suitable 1—3 ratio of substrates was empirically determined.

 

Given that the specific activity of both substrates and their total concentrations in the two
sets of reactions were identical, we expected that the rates for both sets of reactions were
identical if both compounds are direct substrates of the enzyme. The result is shown in
Figure D.2A. Label from AdoMetwas efficiently incorporated into DGHS by transfer of
the 3-amino-3-carboxylpropyl moiety to a lipid acceptor giving rise to a single labeled
compound in the lipid fraction of the reaction extract. This compound was not present in
the vector control consistent with it being DGHS. When labeled DAG was used, label
was observed in a number of polar lipids as DAG is a general precursor for polar lipid
biosynthesis. These labeled compounds were also present in the vector control. One
compound co-chromatographing with DGHS present in the AdoMet reaction (Figure
D.2A, box) was also present, but unlike other lipids, it was absent from the vector control
thereby suggesting that it was DGHS. Thus, labeled DAG appeared to serve as a
precursor for DGHS biosynthesis under the employed conditions. To determine the
relative rates of incorporation, the phosphor imager outputs were quantified (Figure
D28). The rates were linear for both labeled substrates (r2 = 0.9964 for the AdoMet
reaction and r2 = 0.9743 for the DAG reaction) over the incubation time suggesting that
the enzyme was present at non-saturating amounts in the reaction mixture. The rates were
very similar (29.6 relative units/min for the AdoMet reaction and 27.7 relative units/min
for the DAG reaction). Given that the specific activity for the labeled compounds and the
total substrate concentrations in both reactions were identical, one can cautiously
conclude that both compounds were incorporated into DGHS with similar efficiency as

expected for the direct substrates of the reaction.

211

[14C]-AdoMet [14C]-DAG

A thaA \fl: thaA Vic B
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6000
32.9! DAG y=29.62x+1784
. , .. .. " r - = 1—DGHS R2=o.9964
§
33000
. a: = 27.749x + 1333
.. t s Q 3 {-i 2000‘ yR2: 0.9743
I
1000*
0 . DAG
-Ori 0 2'0 40 60 80 100120140

Time (min)

Figure D.2. BtaA-catalyzed DGHS biosynthesis from radiolabeled substrates

(A) E. coli cells expressing RthaA were assayed for the ability to incorporate label from
1-[I4C]AdoMet or [”C]DAG into DGHS. Cells either contained the thaA plasmid or an
empty pQE31 vector control (Vec). Time courses are shown with incubation times (min)
indicated. Reaction products were separated by TLC and detected using a
phosphorimager. The product bands containing DGHS (missing in the vector controls)
are shown inside the box. DAG, diacylglycerol; DGHS, diacylglycerylhomoserine; Ori,
origin.

(B) Plot of DGHS synthesized from 1-[14C]AdoMet or ['4C]DAG over time. DGHS
amount (expressed as pixel density) was determined from the phosphorimage in (A). The
rates of DGHS synthesis from radiolabeled DAG or SAM are linear and have roughly the

same value.

212

The RthaA protein in a crude membrane preparation was shown to transfer the
3-amino-3-carboxypropyl moiety from AdoMet t0 DAG by demonstrating that under
identical conditions incorporation of label into DGHS from either labeled AdoMet or
DAG proceeded at the same rate (Figure D.2). This result is in agreement with the role of
RthaA that had been tentatively assigned based on mutagenesis and heterologous
expression, and rules out other contingencies for the activity of RthaA. Previous work in
a cell-free system on the cognate BtaA-type transferase activity in C. reinhardtii using
radiolabeled AdoMet to follow the reaction had given conflicting results as to the identity
of the hydrophobic substrate, indicating that DAG might not be the direct substrate
because the addition of unlabeled DAG strongly inhibited the reaction (Moore et a1.
2001). However, the point was raised that excess DAG might simply disrupt the
membrane environment in which the enzyme(s) are working, and the decrease in activity
might not be a result of enzyme inhibition, per se. The results presented here for

recombinant RthaA seemingly provide a solution to this question.

213

 

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