CHARACTERIZATION OF THE PEROXISOMAL UBIQUITIN-CONJUGATING
ENZYME 22 PROTEIN IN ARABIDOPSIS THALIANA
By
Ye Xu

A THESIS
Submitted to
Michigan State University
in partial fulfillment of requirements
for the degree of
Plant Biology – Master of Sciences
2014

ABSTRACT
CHARACTERIZATION OF THE PEROXISOMAL UBIQUITIN-CONJUGATING
ENZYME 22 PROTEIN IN ARABIDOPSIS THALIANA
By
Ye Xu
Peroxisomes are small yet critical organelles that are present in nearly all eukaryotes.
Peroxisomes house a broad range of metabolic and biochemical pathways that are vital
for organismal development and metabolism. Unlike mitochondria and chloroplasts,
which possess DNA transcription and protein translation machineries, almost all
peroxisome matrix proteins are post-translationally targeted to peroxisomes after they
are translated on free polyribosomes in the cytosol. Therefore, it is important to
understand peroxisome protein import as well as degradation mechanisms. A number of
proteins involved in peroxisome matrix protein import have been identified. However,
our knowledge towards matrix protein degradation is still scarce. In this research, I
explored the function of a newly identified component of the peroxisomal localized
ubiquitin-proteasome system, i.e. ubiquitin-conjugating enzyme 22 (UBC22) in
Arabidopsis. I provide evidence that AtUBC22 targets to peroxisomes through its Cterminal tri-peptide KRL>. Mutant analysis shows that UBC22 might act as a negative
regulator of peroxisome IBA metabolism. UBC22 also has an effect on seed formation
and seedling development. Homologous sequences of AtUBC22 that contain
peroxisomal targeting signal type 1 are present in other plant species, indicating a
plant-specific role of UBC22 in the peroxisome.

Copyright by
YE XU
2014

ACKNOWLEDGEMENTS

I would like to first give special thanks to my adviser Dr. Jianping Hu for letting
me join this great lab and participating in the amazing world of peroxisome research.
She has been super supportive and patient, and has constantly encouraged me to keep
thinking, trying and exploring further. Without her, I would not have been able to finish
this thesis.
I am also very grateful to have Dr. John Ohlrogge and Dr. Eva Farré as my
committee members during the two years of study at MSU. Dr. Ohlrogge is a role model
for me and has inspired me a lot, both in science and in life. He has always been so
supportive and helpful even before he became my committee and I learnt a lot from him.
Dr. Farré provided me with new perspectives in thinking about this project and gave me
many critical suggestions on writing this thesis. Their support and advice have made me
a better researcher.
I thank all Hu lab members, Dr. Navneet Kaur, Dr. Gaëlle Cassin, Ronghui Pan
and Jiying Li. I would like to especially thank Navneet for her selfless help on my
research and studies; she is a talented ‘teacher’ and great companion for every late
night and weekends in the lab. Thanks also go to Ronghui Pan for being a good
neighbor and helping me take care of my plants when I was on vacation.
I would like to thank many people in plant biology department and in PRL.
Thanks to Dr. Alan Prather and Dr. Shin-Han Shiu for giving me the warmest help
during the times I was feeling low. I sincere thank Dr. Weili Yang from Dr. Ohlrogge’s

iii

lab for kindly helping me generate and analyze the fatty acid data. I also thank Dr.
Giovanni Stefano on interesting discussion regarding confocal imaging.
I especially thank Yashiuan Lai for her company and support at every important
moment during the past two years.
Lastly, I would like give my thanks to my parents and grandparents for their
endless love and support.

iv

TABLE OF CONTENTS

LIST OF TABLE………………………………………………………………………………vii
LIST OF FIGURES……………………………………………………………………………viii
KEY TO ABBREVIATIONS…………………………………………………………………ix
CHAPTER 1: LITERATURE REVIEW Peroxisomal Function, Biogenesis and
Peroxisome-associated Proteolysis…………………………………………………………1
1.1 Introduction…………………………………………………………………………2
1.2 Function of plant peroxisomes……………………………………………………4
1.2.1 Peroxisomal ß-oxidation…………………………………………………4
1.2.1.1 Fatty acid ß-oxidation………………………………………5
1.2.1.2 Conversion of IBA to IAA…………………………………6
1.2.1.3 JA biosynthesis……………………………………………7
1.2.2 Peroxisome and photorespiration………………………………………9
1.3 Peroxisomal protein remodeling and proteases………………………………10
1.3.1 Peroxisomal matrix protein import……………………………………10
1.3.2 Peroxisomal protein degradation……………………………………17
1.4 Aims of the thesis research………………………………………………………19
REFERENCES………………………………………………………………………23
C H AP T E R 2 : C h a r a c t e r i za t i o n o f t h e A ra b i d o p s i s p e ro xi s o m a l p ro t e in
UBIQUITIN-CONJUGATING ENZYME 22…………………………………………………35
Abstract……………………………………………………………………………36
2.1 Introduction………………………………………………………………………37
2.2 Results……………………………………………………………………………40
2.2.1 AtUBC22 is an E2 enzyme predicted to be peroxisomal…………40
2.2.2 UBC22 amino acid sequence analysis……………………………42
2.2.3 AtUBC22 is localized to Arabidopsis peroxisomes and its C-terminal
tripeptide KRL> is required for proper peroxisome targeting……………43
2.2.4 UBC22 is ubiquitously expressed in plants…………………………44
2.4.5 Null mutants of UBC22 are more sensitive to exogenous IBA……45
2.4.6 Null ubc22 mutants produce bigger plants and larger and heavier
seeds that store a higher amount of protein and fatty acids……………46
2.4.7 Null ubc22 mutants have reduced seed yield………………………48
2.3 Discussion…………………………………………………………………………50
2.4 Methods......……………………………………………………………….………78
2.4.1 Plant material, growth conditions, and plant transformation………78
2.4.2 RT-PCR…………………………………………………………………79
2.4.3 Gene cloning and plasmid construction……………………………79
2.4.4 Confocal microscopy analyses………………………………………80
2.4.5 Sucrose dependence assay…………………………………………80

v

2.4.6 IBA resistance assay…………………………………………………81
2.4.7 Sequence alignment and phylogenetic analysis……………………81
2.4.8 Pollen viability assay…………………………………………………82
Acknowledgements........……………………....…………………………………….82
REFERENCES………………….……………………..………………………………84

vi

LIST OF TABLES

Table 2.1. Arabidopsis UBCc domain-containing proteins………………………………54

vii

LIST OF FIGURES

Figure 1.1. Model for peroxisome matrix protein import machinery……………………21
Figure 2.1. Sequence and structural analysis of UBC22…………………………………56
Figure 2.2. Phylogenetic analysis of Arabidopsis UBC22………………………………57
Figure 2.3. Amino acid sequence alignment of Arabidopsis UBC22 and homologous
sequences………………………………………………………………………………………58
Figure 2.4. UBC22 localizes to the peroxisome through a C-terminal PTS1 (KRL>)…59
Figure 2.5. Expression patterns of UBC22 in Arabidopsis………………………………60
Figure 2.6. Expression level of UBC22 in Arabidopsis……………………………………62
Figure 2.7. Sucrose-dependence assays on the ubc22 mutants………………………63
Figure 2.8. Quantification of root (A) and hypocotyl (B) lengths of seedlings in the
sucrose dependence assay…………………………………………………………………64
Figure 2.9. ubc22 mutants are hypersensitive to IBA……………………………………65
Figure 2.10. Arabidopsis ubc22 have larger first true leaves and cotyledons…………66
Figure 2.11. Arabidopsis ubc22 have similar primary root growth rate to Col-0………67
Figure 2.12. Comparison of root cell size between ubc22 and Col-0……………………69
Figure 2.13 Morphological analysis of ubc22 seeds………………………………………70
Figure 2.14. Analysis of the weight, protein content and fatty acids of ubc22
mutants…………………………………………………………………………………………72
Figure 2.15. Silique analysis of ubc22 mutants……………………………………………74
Figure 2.16. Unfertilized ovules of ubc22 mutants in the silique…………………………75
Figure 2.17. in vitro pollen activity test by Alexander staining……………………………76
Figure 2.18. Amino acid sequence alignment of Arabidopsis UBC22 and its putative
homologous in other species…………………………………………………………………77

viii

KEY TO ABBREBIATIONS
β-gal

Beta-galactosidase

Δ

Deletion

35S

Cauliflower Mosaic Virus 35S promoter

AA

Amino acid

ABC

ATP binding cassette

ABRC

Arabidopsis Biological Resource Center

Acetyl-CoA

Acetyl coenzyme A

AD

Activation domain

Ala

Alanine

ATP

Adenosin-5’-Triphosphate

BD

DNA binding domain

BLAST

Basic Local Alignment Search Tool

C

Celsius

CAT

Catalase

CFP

Cyan fluorescent protein

Col-0

Columbia ecotype 0

C-terminal

Carboxy terminal

Cys

Cysteine

DsRed

Discosoma species red fluorescent protein

DUB

Deubiquitinase

ER

Endoplasmic reticulum

FA

Fatty acid
ix

GFP

Green fluorescent protein

GOX

glycolate oxidase

H2O2

Hydrogen peroxide

IAA

Indole-3-acetic acid

IBA

Indole 3-butyric acid

IPTG

Isopropylthio-β-galactoside

JA

Jasmonic acid

JATI

Jasmonic acid triggered immunity

KDa

Kilo Dalton

Lys

Lysine

NM

Nanometer

N-terminal

Amino terminal

OPDA

12-oxo-phytodienoic acid

PBDs

Peroxisome biogenesis disorders

PEX

Peroxin

PIM

Peroxisome import machinery

PMPs

Peroxisome membrane proteins

PTMs

Post-translational modifications

PTS1

Peroxisomal targeting signal 1

PTS2

Peroxisomal targeting signal 2

RING

Really interesting new gene

RNS

Reactive nitrogen species

ROS

Reactive oxygen species

x

RuBisCO

Ribulose-1, 5-bisphosphate carboxylase/oxygenase

RuBP

Ribulose-1,5-bisphosphate

SD

Synthetic dropout

SD/-HUTL

SD media lacking histidine, uracil, tryptophan, and leucine

Ser

Serine

TAG

Triacylglycerols

Thr

Threonine

Tyr

Tyrosine

T-DNA

Transfer DNA

Ub

Ubiquitin

UBC

Ubiquitin conjugating enzyme

UBQ

Ubiquitin

UPS

Ubiquitin (Ub)-proteasome system

VLCFA

Very long chain fatty acids

WT

Wild-type

YFP

Yellow fluorescent protein

xi

CHAPER 1
LITERATURE REVIEW
Peroxisomal Function, Biogenesis and Peroxisome-associated Proteolysis

1

1.1 Introduction
Peroxisomes are small, highly dynamic, and single-membrane-bounded
organelles present in nearly all eukaryotes with the exception of mature erythrocytes
and spermatozoa (Novikoff et al., 1973). They are typically spherical, ranging from 0.1
to 1.5 micrometer in diameter. The number, morphology and function of peroxisomes,
i.e. regular peroxisomes, glyoxysomes of plant seedlings and some fungi, glycosomes
of trypanosomes, and Woronin bodies of filamentous fungi, vary dramatically among
different cell types, tissues and species (Baker and Sparkes, 2005). Peroxisomes are
involved in a wide variety of biological processes, and their metabolic and biochemical
functions are critical in many aspects of eukaryotic development.
Some peroxisomal functions, such as the detoxification of hydrogen peroxide
(H2O2) and fatty acid β-oxidation, are well conserved from yeast to man. As a versatile
organelle, peroxisomes also contribute to a variety of other metabolic functions, some of
which are lineage-specific. In mammals, peroxisomes carry enzymes involved in the
synthesis of plasmalogens, cholesterol and bile acids as well as the oxidation of
alcohols. Studies have shown that Alzheimer's disease might be associated
with reduced level of brain plasmalogens (Rucktäschel et al., 2011; Grimm et al., 2011).
Mutations in peroxins (PEX), i.e. proteins required for peroxisome biogenesis, lead to
peroxisome biogenesis disorders (PBDs), the most severe of which (such as the
Zellweger syndrome (ZS)) causes infant fatality (Fujiki et al., 2012; Faust et al., 2014).
In plants, peroxisomes harbor diversified metabolic pathways and display various
morphologies at different developmental stages and under various environmental
conditions. In addition to lipid metabolism, the core enzymes of the -oxidation pathway
2

are also involved in the synthesis of the plant hormone jasmonic acid (JA) and
conversion of the protoauxin indole-3-butyric acid (IBA) to the active auxin indole-3acetic acid (IAA). Furthermore, plant peroxisomes carry some unique functions, such as
the glyoxylate cycle, photorespiration as well as nitrogen metabolism (Hu et al., 2012).
Fungal peroxisomes contain enzymes involved in the catabolism of unusual carbon and
nitrogen sources, such as methanol, glycolate and spermidine. Yeast peroxisomes
house important steps of lysine biosynthesis, and penicillin filamentous fungi generate
penicillin in these organelles (Sibirny, 2012; Meijer et al., 2010). Over the past decade,
a growing amount of evidence indicates that defects in peroxisome biogenesis and
metabolic functions cause fatality in early life of human and lethality to plant embryos,
implicating the essential role of peroxisomes in these eukaryotes (Sparkes et al., 2003;
Lee and Raymond, 2013). Recent studies revealed novel functions of peroxisomes,
such as their role in mammalian cell calcium homeostasis (Lasorsa et al., 2008) and
plant Vitamin K biosynthesis (Babujee et al., 2010; Widhalm et al., 2012).
Due to their sessile nature, plants need to deal with various environmental and
developmental cues. Peroxisomes are key components of this process due to their
functions such as degradation of very long chain fatty acids (VLCFA) to support
seedling establishment of oilseed plants like Arabidopsis, biosynthesis of JA in
response to wounding and pathogens, detoxification of harmful by-products such as
reactive oxidative and nitrogen species (ROS and RNS, respectively), leaf senescence,
and fruit maturation (Hu et al., 2012; Linka et al., 2008). Unlike mitochondria and
chloroplasts that contain DNA transcription and protein translation machineries, almost
all peroxisome proteins are post-translationally targeted to peroxisomes after they are
3

synthesized on free polyribosomes in the cytosol, with the exception of some
peroxisome membrane proteins (PMPs) that are originated from the endoplasmic
reticulum (ER) (Rucktäschel et al., 2011). Proteins are imported into peroxisomes with
the assistance of their targeting signals and peroxin (PEX) proteins. However, far less is
known about how matrix proteins are degraded. There are many questions waiting for
answers. For example, how and when do peroxisomes choose to degrade certain
proteins? Does degradation of matrix proteins occur in peroxisomes or in the cytosol
and what is the role of ubiquitination in this process?
1.2 Function of plant peroxisomes
The chief metabolic processes in peroxisomes that are crucial to plant
development are fatty acid (FA) -oxidation, generation of phytohormones JA and IAA,
glyoxylate cycle in seedlings, and photorespiration. In addition, plant peroxisomes also
contribute to other metabolic pathways, for instance biosynthesis of salicylic acid, biotin
and isoprenoids, and metabolism of urate, polyamines, sulfite, and branched-chain
amino acids (Kaur and Hu, 2009). Here, we will focus on the two major functions of
peroxisome: ß-oxidation and photorespiration.
1.2.1 Peroxisomal ß-oxidation
Plant ß-oxidation solely occurs in peroxisomes, which contain a complete set of
enzymes for the metabolism of fatty acids to acetyl coenzyme A (acetyl-CoA), as well as
the conversion of IBA to active auxin IAA and 12-oxo-phytodienoic acid (OPDA) to JA
(Hu et al., 2012; Brown et al., 2013).

4

1.2.1.1 Fatty acid ß-oxidation
Plant fatty acid ß-oxidation provides energy and carbon for post-germinative
growth before plants establish photosynthesis, and it is also active throughout the life
cycle of a plant. As the major components of plant oilseed reserve lipids, triacylglycerols
(TAGs) are mobilized from oil bodies and imported into peroxisomes, where they are
degraded to acyl-CoA and release acetyl-CoA during -oxidation. The resulting acetylCoA pool is then further metabolized in the TCA cycle in the mitochondria to succinate
and malate for gluconeogenesis or to citrate (Pracharoenwattana and Smith, 2008;
Kunze et al., 2006). Mutants that fail to break down storage fatty acids are unable to
supply the necessary energy and carbon for the seedling to grow into a photosynthetic
plant, and therefore typically show short primary roots and hypocotyls. This phenotype
can be rescued by providing external sucrose. Recent studies show that peroxisome ßoxidation

also

breaks

down

lipids

during

leaf

senescence;

in

Arabidopsis,

Brachypodium and switchgrass at least 80% of leaf fatty acids are degraded during
senescence (Troncoso-Ponce et al., 2013; Yang and Ohlrogge, 2009). However,
Arabidopsis mutants of the core β-oxidation pathway enzymes, acx1 acx2, lacs6 lacs7,
and kat2, which are defective in the breakdown of reserve oil in seeds during seed
germination, do not show substantial deficiencies in the degradation of fatty acids during
leaf senescence.
In Arabidopsis, fatty acids are transported into peroxisomes from oil bodies by
the peroxisomal ATP binding cassette (ABC) transporter protein CTS/PXA1/PED3. After
being imported into peroxisomes, saturated fatty acids (C18- C20) are activated to acylCoA by long chain acyl-CoA synthases 6 and 7 (LAC6 and LAC7) (Fulda et al., 2002;
5

Hu et al., 2012). Bioinformatics and genome analysis revealed 63 genes that encode
ATP-dependent acyl-activating enzymes (AAEs) in Arabidopsis, and 17 members of this
superfamily (including LAC6 and LAC7) carry a putative peroxisome targeting signal
(PTS) (Shockey et al., 2002; Sigrun Reumann et al., 2004). In the ß-oxidation pathway,
activated acyl-CoA are metabolized sequentially by acyl- CoA oxidase (ACX),

the

multifunctional proteins MFP2 or AIM1, and a thiolase PED1/KAT2 to produce acetylCoA and an acyl-CoA (C16 - C18) that is two Cs shorter. The shortened acyl-CoA is
then further degraded through the same catalytic cycle in peroxisomes (Hu et al., 2012).
The activity of enzymes that are involved in fatty acid degradation varies depending on
their affinity to substrates of various lengths, and therefore different enzymes participate
at different stages in the pathway. For instances, AIM1 prefers short-chain substrates
while MFP2 has higher activity towards long-chain acyl-CoAs (Arent et al., 2010; Rylott
et al., 2006). In Arabidopsis and other plants, acetyl-CoA can be converted to succinate
and malate, which will be later used for gluconeogenesis, or it can also be converted to
citrate which then is exported to mitochondria (Kunze et al., 2006; Pracharoenwattana
and Smith, 2008).
1.2.1.2 Conversion of IBA to IAA
IAA is the major form of plant auxin. As a critical plant hormone, IAA regulates a
variety of developmental events and environmental responses through the control of cell
division and expansion in lateral root initiation, root and stem elongation, and leaf
expansion, etc. (Davies, 2010). IBA is also a natural auxin, which is structurally similar
to IAA but has two additional methylene groups in the side chain. Like IAA, exogenous
IBA can inhibit primary root elongation and induces lateral root formation (Zolman et al.,
6

2000). IBA can be converted to IAA in plant peroxisomes, and IAA can also be
converted back to IBA as a storage form to maintain IAA homeostasis (Ludwig-Muller et
al., 1994).
IBA fulfills its bioactivity mainly through its conversion to IAA. Mutants that are
defective in this event fail or partially fail to convert IBA into IAA and thus their seedlings
are resistant to exogenous IBA – i.e. showing reduced response to IBA’s inhibition on
primary root elongation and induction of lateral root formation. Several mutants
defective in fatty acids ß-oxidation show IBA resistant phenotypes. Some IBA resistant
mutants are sucrose dependent during early developmental stages, indicating that the
four-carbon side chain in IBA can be modified to the two-carbon side chain in IAA in a
ß-oxidation pathway parallel to fatty acid ß-oxidation in peroxisomes (Zolman and
Bartel, 2004; Zolman et al., 2001). IBA is imported into peroxisomes, activated to IBACoA, and further converted to IAA-CoA through a pathway similar to fatty acid ßoxidation. The pxa1 mutant shows both sucrose dependent and IBA resistant
phenotypes, indicating that other than importing fatty acids, PXA1 might also contribute
to IBA import. Some fatty acid ß-oxidation pathway mutants like acxs, aim1 and
ped1/kat2 are also resistant to exogenous IBA, while ibr1, ibr3, ibr10 only show IBA
resistant phenotypes, suggesting that the latter mutants might only be defective in IBACoA ß-oxidation and therefore the corresponding protein products are specific for the
IBA pathway.
1.2.1.3 JA biosynthesis
Plants are the nutritional sources for a variety of living things including herbivores,
7

microorganisms and pathogens. In order to survive, plants synthesize JA to trigger
downstream chemical defense compounds to repel plant consumers. JA is an important
plant hormone that regulates plant growth and development including nectar
accumulation, leaf senescence and abscission, flower development, etc. It is also
involved in biotic and abiotic stress responses, like the pathogen- or wound-induced
multistage process called JA-triggered immunity (JATI) (Campos et al., 2014; He and
Gan, 2002; Radhika et al., 2010).
The production of active JA is initiated in plastids, where the polyunsaturated
fatty acid C18 precursor linolenic acid (LA) is converted to the lipid intermediate 12-oxophytodienoic acid (OPDA), which is further transported to peroxisomes and converted to
JA through ß-oxidation. JA is then released to the cytosol and conjugated with Ile to
form JA-Ile, which presumably diffuses into the nucleus and binds to the JAZ
(JAsmonate ZIM domain) repressors and targets them to degradation via the ubiquitindependent protein degradation pathway. Degradation of JAZ proteins releases the
repression on transcription factors (TFs) for JA-response genes (Campos et al., 2014).
In Arabidopsis, the peroxisomal steps of this pathway start from the import of OPDA by
PXA1. OPDA is then reduced by a flavin-dependent 12-oxophytodienoate reductase
(OPR), OPR3, to 3-oxo-2-(20-[Z]-pentenyl)-cyclopentane-1-octanoic acid (OPC-8)
(Breithaupt et al., 2009). After OPC-8 is activated by an OPC-8:0 CoA ligase 1 (OPCL1)
and forms OPC8:0-CoA (Koo et al., 2006), OPC8:0-CoA goes through three rounds of
ß-oxidation with each round removing two carbon units, to finally form JA-CoA. Similar
to fatty acid ß-oxidation, this process involves ACX (ACX1 and ACX5), AIM1 and KAT2
(Castillo et al., 2004; Afitlhile et al., 2005; Vrebalov et al., 2005; Schilmiller et al., 2007).
8

After ß -oxidation, JA-CoA is hydrolyzed by a currently unknown thiosterase to release
JA, which is then exported from peroxisomes into the cytosol for further modifications.
1.2.2 Peroxisome and Photorespiration
Photorespiration is a process that enables photosynthetic organisms to recycle
carbon from the oxygenase reaction catalyzed by ribulose-1, 5-bisphosphate
carboxylase/oxygenase (Rubisco). This procedure produces an inevitable toxic reactive
oxygen species (ROS) by-product, hydrogen peroxide (H2O2), which then can be
detoxified by a peroxisomal exclusive antioxidant catalase (CAT) (Apel and Hirt, 2004).
Besides H2O2, peroxisomes also produce other superoxide-radicals and nitric oxide
(NO•), which are known cellular signals that participate in a variety of intra- and intercellular communication (Corpas et al., 2001).
In 1920, Warburg first reported that O2 can inhibit photosynthesis. Later studies
showed that lowering O2 concentration to 2% can lead to a 20-30% increase in biomass
and yield in plants, while when O2 concentration was above the atmospheric level,
photosynthesis was severely reduced (Maurino and Peterhansel, 2010). Current
explanation of how O2 inhibits photosynthesis is that Rubisco could catalyze both the
oxygenation and carboxylation reactions with Ribulose-1, 5-bisphosphate (RuBP),
depending on the [CO2]/[O2] ratio in the chloroplast and around Rubisco (Moroney et al.,
2013). Carboxylation of RuBP produces two molecules of 3-phosphoglycerate (3-PGA),
while oxygenation synthesizes one molecule of 3-PGA and one molecule of 2phosphoglycolate (2-PG). After dephosphorylation, 2-PG enters the photorespiratory
pathway in which 75% of its carbon returns as 3-PGA to the Calvin–Benson–Bassham
9

cycle (CBB cycle) while the rest 25% of the carbon is lost as CO2. When O2
concentration around Rubisco increases, Rubisco’s affinity for CO2 decreases and
reacts more with O2 to generate 2-PG, thus carbon, biomass and yield are lost in plants
(Moroney et al., 2013). Due to this property, the photorespiration pathway is viewed as
a major target for crop yield improvement (Hu et al., 2012).
To sum up the photorespiration process, 2-PG is first dephosphorylated in the
chloroplasts by phosphoglycolate phosphatase (PGLP) to glycolate, which is then
transported to peroxisomes where it is oxidized by glycolate oxidase (GOX) to
equimolar amounts of glyoxylate and H2O2. Glyoxylate is further transaminated by
glutamate–glyoxylate amino- transferase (GGAT) to glycine, which is transported to
mitochondria. In mitochondria, glycine decarboxylase decomposes two molecules of
glycine into one molecule of serine and releases one molecule each of CO2 and
ammonia (NH4+). Serine is then transferred back to peroxisomes and metabolized by a
serine-glyoxylate aminotransferase (SGT) to hydroxypyruvate, which is further
converted to glycerate by hydroxypyruvate reductase (HPR). Glycerate is transported to
chloroplasts where it is phosphorylated by glycerate kinase (GLYK) to 3-PGA, which
then reenters the CBB cycle (Hu et al., 2012; Maurino and Peterhansel, 2010; Moroney
et al., 2013).
1.3

Peroxisomal protein remodeling and proteases

1.3.1 Peroxisomal matrix protein import
Despite their diversified functions and morphologies, all types of peroxisomes
share an essentially conserved matrix protein import system. As peroxisomes contain
10

neither genetic material nor transcription/translation machineries, all of its matrix
proteins are encoded in the nucleus, translated on free polyribosomes in the cytosol and
then targeted to peroxisomes (Léon et al., 2006b). In Arabidopsis thaliana,
approximately 280 proteins were predicted to target to the peroxisomal matrix
(Reumann et al., 2004). In addition, peroxisomes are capable of importing fully folded or
even oligomeric and co-factor bound proteins, distinguishing it from other organelles in
their protein import mechanism (Léon et al., 2006a). Different from the origin of matrix
proteins, some peroxisome membrane proteins originate in the ER. Examples include
cottonseed (Gossypium hirsutum) and pumpkin (Cucurbita maxima) ascorbate
peroxidase (APX) and Arabidopsis thaliana PEX16 (Yamaguchi et al., 1995;
Bunkelmann and Trelease, 1996; Karnik and Trelease, 2005; Nito et al., 2007).
The peroxisome protein import process for matrix proteins can be divided into
four steps (Figure 1.1): 1) the matrix protein is recognized by the receptor in the cytosol;
2) the receptor-cargo complex docks to the peroxisome membrane; 3) the cargo is
imported into the peroxisome matrix through the membrane and released into the
matrix; and 4) the receptor returns to the cytosol for recycling or degradation (Hasan et
al., 2013). At present, 19 out of the 34 PEX genes discovered have been demonstrated
to be directly involved in peroxisomal matrix protein import.
Step 1: Cargo-recognition
Following translation, nascent peroxisomal matrix proteins are recognized by
receptors through the peroxisomal targeting signal (PTS), which is either located at the
C-terminus (PTS1, peroxisomal targeting signal type 1) or the N-terminus (PTS2) of
11

matrix proteins. The majority of the matrix proteins possess PTS1, a tripeptide at the Cterminal end of the protein. A typical PTS1 is usually comprised of Ser-Lys-Leu (SKL) or
variants that fit the consensus (S/A/C)-(K/R/H)-(L/A). PTS1 sequences are recognized
by a conserved cytoplasmic receptor PEX5. PEX5 contains two functional domains: a
natively unfolded N-terminal peroxisome docking domain and a C-terminal domain,
which contains tetratricopeptide repeats (TPRs) that allow it to recognize the PTS1sequence (Carvalho et al., 2006). Recent studies reported that the conformation of
unloaded PTS1-receptor changes upon binding with cargo, allowing the receptors to
transfer into a docking competent state (Shiozawa et al., 2009; Stanley et al., 2006;
Fodor et al., 2012).
The minority of matrix proteins possesses PTS2. In Arabidopsis thaliana, about
220 proteins are predicted to contain a putative PTS1, while only 60 proteins carry
PTS2 (Lingner et al., 2011). The usage of PTS2 for peroxisome matrix protein import
varies among species. In plants, around one third of peroxisome matrix proteins contain
the PTS2 motif (Reumann et al., 2009). In contrast, in mammals only a few proteins use
this pathway, in yeast Saccharomyces cerevisiae three proteins have been identified to
harbor a PTS2 sequence (Grunau et al., 2009; Jung et al., 2010), and in some species
such as Caenorhabditis elegans and Drosophila melanogaster the PTS2 pathway is
completely absent (Motley et al., 2000; Faust et al., 2012). PTS2 is a nonapeptide within
the first 20 amino acids of a matrix protein, and possesses the consensus sequence
(RK)-(LVIQ)-XX-(LVIHQ)-(LSGAK)-X-(HQ)-(LAF) that is recognized by the tryptophanaspartic acid (WD) repeats motif of the soluble receptor PEX7 (Lazarow, 2006; Petriv et
al., 2004; Rucktäschel et al., 2011).
12

Some proteins have been reported to be imported through a non-classical
pathway. The Trypanosoma brucei glycosomal enzyme triosephosphate isomerase
(TPI) and the pumpkin catalase CAT1 both contain internal PTS motifs (Oshima et al.,
2008; Galland et al., 2010). S. cerevisiae enoyl-CoA isomerases Eci1p and Dci1p and
mammalian Cu/Zn superoxide dismutase are imported by association with another
canonical PTS-containing cargo protein through a manner called “piggy-back import”
(Islinger et al., 2009; Yang et al., 2001). Some proteins are imported through a “nonPTS import” method. For example, S. cerevisiae acyl-CoA oxidase and H. polymorpha
alcohol oxidase interact directly with Pex5’s N-terminal region without possessing a PTS
sequence (Gunkel et al., 2004; Klein et al., 2002).
Step 2: Receptor-cargo complex docking
After the receptor-cargo complex is formed in the cytosol, it associates with the
peroxisomal membrane via a docking complex. This docking complex consists of
PEX13 and PEX14 in plants, and in fungi it contains the peroxin Pex17p as well (Kiel et
al., 2006). Yeast Pex13p is an integral membrane protein that exposes both its C- and
N-termini to the cytosol and binds to Pex14p via its SH3 domain and an
intraperoxisomal binding site (Schell-steven et al., 2005; Pires et al., 2003). Pex13p also
binds to Pex5p via its C-terminal Src-homology-3 (SH3) domain in yeasts S. cerevisiae
and P. pastoris (Williams and Distel, 2006) and with Pex7p via its N-terminal domain in
S. cerevisiae (Stein et al., 2002). Pex14p forms a complex with Pex13p through its
proline-rich segment and it binds to both PTS receptors (Niederhoff et al., 2005). PEX14
was described as an integral membrane protein in Arabidopsis thaliana, while in some
other species, it was shown to be a peripheral protein (Azevedo and Schliebs, 2006).
13

Yeast Pex17p is a peripheral membrane protein, and its function in the docking complex
is still unknown (Huhse et al., 1998).
Unlike PEX5, PEX7 itself is unable to complete all steps for matrix protein import,
so it needs auxiliary proteins. In plants, PEX5 is required for PEX7 to correctly bind to
the docking complex. In mammals and rice, a long isoform (Pex5L) serves as the PEX7
co-receptor, in most yeasts and fungi the co-receptor is Pex20p, and in S. cerevisiae
the redundant proteins Pex18p and Pex21p act as the co-receptor (Schliebs and
Kunau, 2006).
Step 3: Cargo translocation and releasing
The receptor-cargo complex is translocated into peroxisomes in a folded or even
oligomerized form and then released into the peroxisomal matrix. However, the
mechanism behind this process is still unclear. Currently several hypotheses have been
proposed. In yeast S. cerevisiae, Pex5p and Pex14p are able to form a dynamic
translocation pore for the receptor-cargo complex to pass through. Evidence show that
this pore can open up to 9 nm in diameter, and even gold particles fused with a PTS
can pass through the membrane (Walton, 1996; Meinecke et al., 2010). Besides, this
process might also be facilitated by a post docking protein complex, the RING (really
interesting new gene)-finger complex that contains the peroxisomal membrane proteins
Pex2p, Pex10p and Pex12p (Platta and Erdmann, 2007). Recent studies demonstrate
that RING proteins have E3 ligase activities in S. cerevisiae and Arabidopsis thaliana.
Lack or reduction of the expression of any of these RING peroxins remarkably
decreases the import efficiency for both PTS1- and PTS2-containing proteins even after
14

docking, and knocking out PEX2, PEX10 or PEX12 in Arabidopsis thaliana causes
embryo lethality (Kaur et al., 2013; Hu et al., 2002; Embryogenesis et al., 2014;
Schumann et al., 2003; Fan et al., 2005).
After the receptor-cargo complex is translocated into the peroxisomal lumen, the
cargo is unloaded through a still unknown mechanism. In plants and mammals, PTS2
on the imported cargo is proteolytically removed after import. In mammalian cells,
Tysnd1 was demonstrated to be responsible for removing the sequence peptides from
PTS2 proteins involved in -oxidation of fatty acids (Okumoto et al., 2011). Pex8p of H.
polymorpha, an intra-peroxisomal peripheral membrane protein that contains both PTS1
and PTS2, has been proposed to play a role in cargo releasing by facilitating the
disassociation of PTS1 peptides from Pex5p in vitro (Wang et al., 2003). A recent study
in P. pastoris shows that Pex8p aids in cargo release by assisting Pex5p transfer from a
redox-regulated oligomer to a dimer (Ma et al., 2013). Furthermore, Pex8p is necessary
for the correct binding of the docking complex (Pex13p, Pex14p and Pex17p) to the
peroxisomal RING-finger complex (Pex2p, Pex10p and Pex12p) to form the
“importomer” (Agne et al., 2003). However, Pex8p seems to be exclusively present in
yeast and absent in all higher eukaryotes (Kiel et al., 2006). Another peroxin,
mammalian PEX14, has been shown to be involved in this process by helping to
release the cargo from PEX5 (Freitas et al., 2011).
Final step: Receptor recycling and degradation.
Following cargo release, receptors are dislocated from the peroxisomal matrix
and back to the cytosol for either degradation or recycling through an ubiquitin15

dependent manner. This process involves a set of sub-complexes. In contrast to the
import pathway, which is ATP-independent, the export procedure demands ATP for two
different steps, ubiquitination and export. In yeast, the necessary machinery is
comprised of the RING-finger complex (Pex2p, Pex10p and Pex12p), which displays
ubiquitin protein ligase (E3) activity, the ubiquitin-conjugating enzyme (E2) Pex4p
together with its membrane anchor Pex22p, and two AAA-protein family (ATPases
Associated with diverse cellular Activities family) members Pex1p and Pex6p (Grou et
al., 2009a). Ubiquitination provides the export signal for the receptor and allows it to be
recognized by the dislocation machinery, and this requirement seems to be conserved
in all eukaryotes (Okumoto et al., 2011b). Pex5p monoubiquitination depends on the
RING complex member Pex12p (E3) and Pex4p (E2). Then ubiquitinated Pex5p is
dislocated to cytosol via Pex1p and Pex6p (Grou et al., 2009a). In mammalian cells,
instead of PEX4 and PEX22, UbcH5a, UbcH5b and UbcH5c catalyze the
monoubiquitination step (Grou et al., 2008). Pex1p and Pex6p are anchored to the
membrane via an integral peroxisomal membrane protein, i.e. Pex15p in yeast, Pex26
in mammals and APEM9 in plants. Pex6p first binds to Pex15p, then recruits Pex1p
through an ATP-dependent manner (Goto et al., 2011; Matsumoto et al., 2003;
Birschmann et al., 2003). Recent evidence shows that this AAA complex provides the
driving force for the export of Pex5p (Platta et al., 2005). It has been hypothesizes that
the receptor export machinery might function as a molecular motor in both receptor
dislocation and protein translocation (Platta et al., 2014).
Once or shortly after the receptor has been exported to the cytosol, the monoubiquitin moiety on the receptor is removed through two ways. In the non-enzymatic
16

way, the nucleophilic attack of glutathione cleaves the thioester bond between Pex5p
and ubiquitin. In the enzymatic way, the mono-ubiquitin on PEX5 is hydrolysed by
USP9X in mammals and Ubp15p in yeast (Grou et al., 2009b; Debelyy et al., 2011).
PEX5 is mono-ubiquitinated on the conserved cysteine residue for recycling.
However, when the recycling pathway is impaired, Pex5p is polyubiquitinated on lysine
residues, resulting in its degradation via the 26S proteasome system (Kiel et al., 2004).
In S. cerevisiae, ubiquitin- conjugating enzyme (E2) Ubc4p, Ubc5p and Ubc1p have
been demonstrated to be involved in the polyubiquitination of Pex5p (Kragt et al., 2005;
Kiel et al., 2004). Ubiquitination also affects the PTS2 pathway. The yeast S. cerevisiae
PTS2 co-receptor Pex18p and the P. pastoris PTS2 co-receptor Pex20p are
ubiquitinated at the peroxisome membrane. In P. pastoris, Pex4p is essential for the
mono- and polyubiquitination of Pex20p (Liu and Subramani, 2013). Due to lack of a
corresponding protease, yeast is unable to cut off the PTS2 signal from the cargo
following its import into the peroxisomal lumen (Helm et al., 2007). So far, our
knowledge about the PEX7 pathway and whether PEX7? Is ubiquitinated in this process
is still lacking.
1.3.2 Peroxisomal protein degradation
For some organelles, protein export is a vital process to guarantee the normal
function of the organelle. Failure to export certain proteins from the organelle matrix at a
specific stage or blocking the export of misfolded proteins may be detrimental to the
organelle and ultimately lead to disease. ER exports its misfolded membrane and matrix
proteins to the cytosol for degradation through the endoplasmic reticulum associated
17

degradation (ERAD) pathway. Likewise, mitochondria export damaged proteins through
mitochondrial associated degradation (MAD). Dysfunction of any of these pathways
causes severe diseases. For example, in human, failure to secrete ATZ (Alpha-1
antitrypsin Z) from ER to the blood leads to chronic liver inflammation in children and
hepato-cellular carcinoma later in life (Perlmutter, 2006). Mitochondrial loss of function
mutations in protein degradation lead to parkinson disease (Heo and Rutter, 2011). All
those examples underscore the importance of protein export in maintaining organelle
functions. However, the export mechanism and the role of cysteine-ubiquitination in this
process are poorly understood.
Several peroxisomal proteases have been reported. Arabidopsis DEG15 and its
mammalian homolog trypsin containing domain 1 (TYSND1) are ATP-dependent ser
proteases, functioning as signal peptidases that are able to cut off the PTS signal from
imported matrix proteins (Kurochkin et al., 2007; Novikoff et al., 1973; Perlmutter, 2006).
Arabidopsis plants lacking DEG15 display mild IBA resistant phenotype and fail to
process several PTS2-containing proteins (Schuhmann et al., 2008). Besides, all
currently identified Tysnd1 substrates are involved in peroxisomal -oxidation,
prompting a model in which Tysnd1 promotes the assembly of peroxisomal enzymes
into a supramolecular complex thus accelerates the β-oxidation process (Kurochkin et
al., 2007).
Plant peroxisomes also possess a quality control system that involves a Long
Form Radiation Sensitive (Lon) protease family member LON2. Similar to DEG15, LON
proteases are also ATP-dependent proteins, which have been demonstrated to play a
crucial role in the degradation of damaged and oxidized proteins in human(Bota and
18

Davies, 2002). In plants, LON isoforms are targeted to chloroplasts, mitochondria, and
peroxisomes. Its peroxisomal isoform LON2 is assumed to participate in peroxisomal
quality control processes, and has been shown to facilitate the sustained import of
matrix proteins in Arabidopsis (Bartoszewska et al., 2012). This result indicates the
importance of intra-peroxisomal proteolysis in maintaining the peroxisome import
system (Lingard and Bartel, 2009). Suppressor screens of the Arabidopsis lon2 mutants
found several independent mutations that can fully suppress lon2 defects. The genes
involved include the autophagy genes ATG2, ATG3 and ATG7, indicating the possible
existence of pexophagy (peroxisomal autophagy) in plants (Farmer et al., 2013). In
addition, LON2 plays a role in matrix protein turnover. After ~4 days of germination,
Arabidopsis seedlings no longer rely on stored fatty acids to provide energy for growth
and have established into photosynthetic plants. During this transition, enzymes of the
glyoxylate cycle, such as isocitrate lyase (ICL) and malate synthase (MLS), are
degraded. A new set of proteins involved in the photorespiratory pathway, like
hydroxypyruvate reductase1 (HPR1) (Williams, 2014), arise. Surprisingly, in Arabidopsis
lon2 mutants, glyoxylate enzymes are degraded more quickly than in the wild type,
rather than more slowly as expected, indicating that pexophagy may be accelerated
when the LON2 protease level is decreased (Bartel et al., 2014).
Up to date, two models have been proposed to explain the transition of
peroxisomes from seed peroxisomes (glyoxysomes) to leaf peroxisomes. One model
suggested that glyoxysomes directly substitute protein content and become leaf
peroxisomes to meet the metabolic requirements of the plants. The other model states
that there are two types of peroxisomes, one containing enzymes for glyoxysomal
19

metabolism and the other for photorespiration. When a plant becomes photosynthetic,
glyoxysomes are degraded and seedlings start to generate the later type of
peroxisomes (Burke and Trelease, 1975). Studies on pumpkin (Cucurbita pepo),
watermelon

(Citrullis

vulgaris),

and

cucumber

(Cucumis

sativus)

via

immunocytochemistry support the first model, in which glyoxysomes are modified to
become leaf peroxisomes without de novo biogenesis of leaf peroxisomes (Sautter,
1986; Nishimura et al., 1986; Titus and Becker, 1985).
A recent study revealed the role of a cysteine protease RESPONSE TO
DROUGHT21A-LIKE1 in Arabidopsis germination, -oxidation, and growth (Quan et al.,
2013). Several peroxisomal proteases have also been discovered through proteomics
and bioinformatics, and their functions are waiting to be uncovered.
1.4 Aims of the thesis research
Our understanding of peroxisomes advanced greatly during the past twenty
years, yet many aspects about this important organelle are still largely unknown. This
thesis aims to contribute to the understanding the proteolytic processes inside
peroxisomes and the role of ubiquitination in peroxisomal metabolism and biochemical
functions in Arabidopsis. In Chapter 2, I will focus on the characterization of the
peroxisomal associated ubiquitin-proteasome system in Arabidopsis by studying a novel
peroxisomal localized protein, ubiquitin-conjugating enzyme 22 (UBC22).

20

STEP 1 & 2

STEP 3

STEP 4

Figure 1.1. Model for peroxisome matrix protein import machinery.

21

Figure 1.1 cont’d
STEP1 & 2. Cargo-recognition and docking.
In the cytosol, peroxisomal matrix proteins harboring a PTS1 or PTS2 are recognized by
the import receptors PEX5 and PEX7 respectively. Then the loaded receptors target to
the docking complex (PEX13 and PEX14, and in fungi it contains the peroxin Pex17p as
well) on the peroxisomal membrane. Unlike PEX5, PEX7 itself is unable to target to the
docking complex and needs auxiliary proteins. In plants, PEX5 is required for PEX7 to
correctly bind to the docking complex. In mammals and rice, a long isoform (Pex5L)
serves as the PEX7 co-receptor, in most yeasts and fungi the co-receptor is Pex20p,
and in S. cerevisiae the redundant proteins Pex18p and Pex21p act as the co-receptor.
STEP3. Cargo translocation and releasing.
It is assumed that the interaction between PEX5 and PEX14 forms a transient channel
in the peroxisome membrane that allows the import of the cargo-loaded receptor
complex to enter the peroxisome. In fungi, an intraperoxisomal protein Pex8p might be
involved in cargo releasing. In plants and mammals, this mechanism is unknown.
STEP4. Receptor recycling and degradation.
Receptors are dislocated to the cytosol for either degradation or recycling through an
ubiquitin-dependent manner. This process requires ATP for ubiquitination and export,
and involves a set of sub-complexes. In yeast, this machinery is comprised of the RINGfinger complex (Pex2p, Pex10p and Pex12p) that displays ubiquitin-protein ligase (E3)
activity, ubiquitin-conjugating enzyme (E2) Pex4p together with its membrane anchor
Pex22p, and two AAA-protein Pex1p and Pex6p. Ubiquitination provides the export
signal for the receptor and allows it to be recognized by the dislocation machinery.
Pex5p monoubiquitination depends on Pex12p (E3) and Pex4p (E2). Pex5p
polyubiquitination depends on Ubc4p (E2) and the RING proteins Pex2p and Pex10p.
Ubiquitinated Pex5p is dislocated to cytosol via Pex1p and Pex6p. In mammalian cells,
UbcH5a, UbcH5b and UbcH5c catalyze the monoubiquitination step. Pex1p and Pex6p
are anchored to the membrane via an integral peroxisomal membrane protein, i.e.
Pex15p in yeast, Pex26 in mammals and APEM9 in plants. Pex6p first binds to Pex15p,
then recruits Pex1p through an ATP-dependent manner. AAA complex provides the
driving force for the export of Pex5p. Once or shortly after the receptor has been
exported to the cytosol, the mono-ubiquitin moiety on the receptor is removed

22

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23

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34

CHAPTER 2
Characterization of the Arabidopsis peroxisomal protein
UBIQUITIN-CONJUGATING ENZYME 22

This project was initiated by Dr. Navneet Kaur. Confocal microscopy images (Figure
2.4A) were obtained by Dr. Navneet Kaur.
Fatty acid composition analysis data (Figure 2.13 C&D) was obtained by Dr. Weili Yang.

35

Abstract
Peroxisomes are multifunctional eukaryotic organelles that mediate a broad
range of metabolic and biochemical processes critical for growth and development.
Many proteins involved in peroxisome matrix protein import, such as PEX5, PEX7 and
PEX14, have been identified. However, our knowledge of the mechanism behind matrix
protein export and degradation processes is still scarce. Here I characterized a novel
member of the peroxisomal ubiquitin-proteasome system, Arabidopsis ubiquitinconjugating enzyme 22 (AtUBC22). I demonstrated that YFP-UBC22 localizes to
peroxisomes, and this localization relies on a tripeptide KRL> located at the C-terminus
of UBC22. Null ubc22 mutants have longer primary roots and larger leaves at early
developmental stages, are hypersensitive to exogenous IBA, and yield larger seeds
despite of having reduced fertility. In summary, Arabidopsis UBC22 is a peroxisomal
protein involved in IBA metabolism and participating in plant growth and reproduction.

36

2.1 Introduction
Peroxisomes are critical organelles that are present in most eukaryotes, housing
a broad range of metabolic and biochemical processes that are vital for cells and
organisms in many aspects. Defects in peroxisomes have been linked to several severe
developmental disorders and fatal human diseases, highlighting the importance of
peroxisomes in maintaining normal cellular functions. Fatty acid -oxidation and
hydrogen peroxide (H2O2) detoxification are peroxisomal functions well conserved from
yeast to man. Proteomic analyses of peroxisomes have identified about 85 peroxisomal
genes in Homo sapiens (human) and 61 genes in Saccharomyces cerevisiae (Schrader
and Fahimi, 2008). Surprisingly, around 137 peroxisomal genes have been identified in
Arabidopsis thaliana (http://www.peroxisome.msu.edu/), indicating that compared with
yeast and human, plant peroxisomes may bear more pathways and functions than we
once thought.
In eukaryotes, organisms employ post-translational modifications (PTMs) to
increase the functional diversity of the proteome and dynamically coordinate signaling
networks. PTMs influence cells in almost all aspects through regulating protein
activities, localization, degradation, and protein interaction with other proteins, nucleic
acids, lipids, and cofactors. PTMs involve phosphorylation, ubiquitination, methylation,
acetylation, etc. Among these mechanisms, the ubiquitin (Ub)-proteasome system
(UPS) is utilized by cells to regulate the degradation of intracellular proteins and as a
type of ‘quality control’ (Varshavsky, 2012). Modification of protein by ubiquitination also
contributes to cell-cycle control, stress response, DNA repair, growth-factor signaling,
transcription, gene silencing, and other cellular processes (Hurley et al., 2006).
37

In order to destroy and recycle misfolded proteins or maintain proteinexpression
at certain levels, UPS modifies its substrates by attaching a polyubiquitin chain to it, so
that the substrate can be recognized by the 26s proteasome and be degraded. UPS can
also perform mono-ubiquitination to regulate protein activity, for instance histone H2B
mono-ubiquitination is involved in transcription regulation, differentiation as well as DNA
repair (Bonnet et al., 2014). In Arabidopsis, around 6% of the genome is dedicated to
the UPS (Hua and Vierstra, 2011).
The ubiquitination system consists of ubiquitin-activating enzymes (E1), ubiquitinconjugating enzymes (E2), and ubiquitin ligases (E3), which act sequentially in the
substrate ubiquitination reaction. Ub is a 8.5-kDa polypeptide consisting of 76 amino
acids and present in nearly all eukaryotic organisms. The initial step of this reaction is
the ATP-dependent activation of ubiquitin (Ub), in which the last residue of Ub (Gly76) is
attached to a Cys residue on the E1 enzyme via a thioester bond (Varshavsky, 2012).
E1 then transfers the “activated” Ub moiety to the active site Cys of an E2 enzyme
through a trans (thio) esterification reaction. At the final step of the ubiquitination
cascade, E2 together with the “activated” Ub moiety bind to an E3 enzyme and form E2E3 Ub ligase holoenzymes. E3 functions as the target protein recognition module that is
able to interact with both E2 and the specific substrate at the same time. After the E2E3 Ub ligase holoenzyme recognizes the target protein, E2 passes the Ub moiety to the
Lys residue of the target protein via an isopeptide bond between the C-terminal Gly of
the Ub moiety and a Lys on the substrate. Substrates are able to attach polyubiquitin
chains through the catalyzation of E2. Ubiquitination can lead to diverse effects on the
target protein depending on the position of the Lys residue that the Ub moiety is bonded
38

with, and the number and topology of the mono-ubiquitin or ubiquitin chains added to
the substrate. In eukaryotes, an E1 is able to bind to a dozen E2s, which can then bind
to hundreds of E3s. While E3s control substrate selection, E2s are involved in the
selection of the Lys residue to attach the ubiquitin chain to, thereby deciding the cellular
fate of the substrate (van Wijk and Timmers, 2010). In order to bind to Ub, a specific
domain, UBDs, which is required by enzymes that catalyze ubiquitylation or
deubiquitylation. UBDs are a class of ubiquitin-binding domains that non-covalently bind
to ubiquitin (Hicke et al., 2005). Currently there are at least 20 known UBD domains:
UBA, UIM, MIU, DUIM, CUE, GAT, VHS, NZF, A20 ZnF, UBP ZnF, PRU, UBZ, UEV,
UBCc, UBM, UBAN, GLUE, SH3, Jab1/MPN and PFU (Dikic et al., 2009; Hurley et al.,
2006).
At present, several UPS components have been identified to be involved in
peroxisome related pathways. In plants and yeasts, the peroxisomal matrix protein
receptor PEX5 is dislocated from the peroxisome matrix after delivering cargo proteins,
and moves back to the cytosol for either recycling or degradation in an ubiquitindependent manner. In yeast, and probably also in plants, this process is mediated by
the RING-finger complex (Pex2p, Pex10p and Pex12p) that displays ubiquitin-protein
ligase (E3) activity, the ubiquitin-conjugating enzyme (E2) Pex4p/ScUBC21 together
with its membrane anchor protein Pex22p, and two AAA ATPase family members
Pex1p and Pex6p (Hasan et al., 2013). Ubiquitination of Pex5p provides the export
signal for Pex5p, allowing it to be recognized by the dislocation machinery (Okumoto et
al., 2011). Pex5p depends on the RING protein Pex12p (E3) and Pex4p (E2) for monoubiquitination, after which it is dislocated to the cytosol via Pex1p and Pex6p (Grou et
39

al., 2009). In mammals, HsUBC E2D1/2/3 (UbcH5A/B/C) (E2) might be the functional
counterparts of the yeast Pex4p, as it catalyzes the mono-ubiquitination of PEX5 (Grou
et al., 2008).
Little is known about the function of the plant peroxisomal associated UPS.
Although plant peroxisomal RING proteins PEX2, PEX10 and PEX10 possess E3 Ub
ligase activity, their substrates have not been identified. The biochemical function of
plant PEX4 has not been demonstrated, either. It is also unknown whether there are
other components of the peroxisomal UPS in plants. To better understand how plant
peroxisomal function is regulated by the UPS and to identify additional components of
this system, I identified and characterized a peroxisomal localized ubiquitin-conjugating
enzyme, AtUBC22, in Arabidopsis. Through cell biological and genetic analyses, I
confirmed the peroxisomal localization of AtUBC22 and showed that it is involved in
peroxisomal IBA metabolism and plant growth and reproduction.

2.2 RESULTS
2.2.1 AtUBC22 is an E2 enzyme predicted to be peroxisomal
In order to identify additional proteins involved in the Arabidopsis peroxisome
protein proteolytic processes, we employed two criteria to search The Arabidopsis
Information Resource (TAIR) database (TAIR: https://www.arabidopsis.org/ (GarciaHernandez et al., 2002)).

First, we searched for proteins that contain a putative

ubiquitin-conjugating catalytic (UBCc) domain by consulting the TAIR Protein Domain

40

database

(ftp://ftp.arabidopsis.org/home/tair/Proteins/Domains/TAIR10_all.domains).

Several previously identified peroxisome-related ubiquitin (Ub)-proteasome system
(UPS) members share this trait in diverse species, i.e. yeast Pex4p (UBC10, PAS2),
Arabidopsis PEX4 (UBC21) and human E2D1/2/3 (UbcH5A/B/C). I identified a total of
49 Arabidopsis proteins that contain UBCc domain (Table 2.1).
Second, I searched the 49 candidates for putative peroxisome targeting signals
(PTSs), using a published database and protein targeting prediction website TargetP
(http://www.cbs.dtu.dk/services/TargetP/)

(database

available

at

http://www.plantcell.org/content/23/4/1556.full, (Lingner et al., 2011)). As a result, I was
able to narrow to a single gene, At5g05080, which encodes ubiquitin-conjugating
enzyme 22 (UBC22). UBC22 contains an UBCc domain with a conserved active-site
Cys residue as well as an experimentally verified C-terminal PTS1 tripeptide KRL>.
Previous in vitro ubiquitination assays reported that UBC22 has E3-independent
activity, implying that UBC22 is able to catalyze the formation of a poly-ubiquitin chain in
vitro without the presence of an E3 ligase. Converting the conserved active-site Cys
residue to alanine (Ala) resulted in the disability of UBC22 to bind to ubiquitin, thus
indicating that this conserved Cys is necessary to maintain the basic function of UBC22
(Takahashi et al., 2009; Kraft et al., 2005). Although UBC22 was experimentally verified
to be able to catalyze the formation of the poly-ubiquitin chain without E3, whether this
activity also occurs in vivo in Arabidopsis and its regulatory function is unknown.
Several UBC proteins that possess E3-independent activity have been reported in other
species. For example, yeast Cdc34p is believed to regulate its own levels (Skowyra et
al., 1999). In addition, unlike most Arabidopsis UBC proteins that share higher
41

similarities with other Arabidopsis UBC proteins, UBC22 displays high sequence
similarity to a human UBC protein HsUBE2S/EPF5, which has a higher expression level
in cancer tissues compared with normal tissues (Kraft et al., 2005; Welsh et al., 2001)
To further characterize UBC22. I asked the following questions: Is UBC22 a
peroxisome-localized protein? What is the possible physiological role(s) of AtUBC22 in
plants and which peroxisomal metabolic pathway is it involved in? What are its possible
substrates in plants?

2.2.2 UBC22 amino acid sequence analysis
The UBC22 gene has five exons (Figure 2.1A). Its protein product contains a
previously verified UBCc domain near the N-terminus that harbors a conserved catalytic
Cys residue required for UBC22 activity (Kraft et al., 2005; Takahashi et al., 2009), and
a non-canonical PTS1 tripeptide KRL> at its C-terminus (Figure 2.1B). Protein topology
analysis of UBC22 amino acids sequence predicted by TMHMM Server v. 2.0
(http://www.cbs.dtu.dk/services/TMHMM/ (Krogh et al., 2001)) and plant membrane
protein database (Aramemnon: http://aramemnon.botanik.uni-koeln.de/ (Schwacke et
al., 2003)) showed that UBC22 does not contain a hydrophobic region that may
constitue a transmembrane domain, indicating that AtUBC22 may not be an integral
membrane protein.
Phylogenetic analysis showed that within all known UBCs in Arabidopsis and the
yeast S. cerevisia, AtUBC22 has the highest sequence similarity to yeast

42

ScUbc10/Pex4p and Arabidopsis PEX4 (Figure 2.2). Amino acid sequence alignment of
AtUBC22 and selected known peroxisomal related E2s showed that AtUBC22 shares
35%, 41% and 36% amino acid identities with HsUBC E2D1/2/3 (UbcH5A/B/C),
ScUbc10/Pex4p and AtUBC21/PEX4, respectively. Further amino acid sequence
analysis of AtUBC22 using ClustalW2 predicted that Cys94 is the conserved E2 active
Cys residue in AtUBC22 (ClustalW2: http://www.ebi.ac.uk/Tools/msa/clustalw2/) (Figure
2.3).

2.2.3 AtUBC22 is localized to Arabidopsis peroxisomes and its C-terminal
tripeptide KRL> is required for proper peroxisome targeting
In order to confirm the predicted localization of UBC22, we co-expressed in
Arabidopsis a peroxisome marker DsRed-PTS1, and the 35Spro: Green Fluorescent
Protein (GFP)-UBC22 fusion protein. Subcellular localization of GFP-UBC22 was
examined using confocal laser scanning microscopy in Arabidopsis T1 plants by Dr.
Navneet Kaur. As shown in Figure 2.4A, GFP-UBC22 was co-localized with
fluorescence spots labeled by the peroxisome marker DsRed-PTS1, few GFP-UBC22
signal were observed in the cytosol (Figure 2.4A). However, GFP-UBC22 signals were
largely lost in T2 plants (data not shown). To confirm the localization of AtUBC22, I
generated a 35Spro: Yellow Fluorescent Protein (YFP)-UBC22 (full length UBC22 CDS)
fusion construct and transformed it into Arabidopsis. T1 transgenic seedlings are being
screened as this thesis is written. I also transiently co-expressed 35Spro: YFP-UBC22
with the peroxisomal marker DsRed-PTS1 in Nicotiana tabacum (Tobacco).
43

Fluorescence microscopy of the infiltrated tobacco leaf epidermal cells showed that, like
GFP-UBC22, which was shown to be localized to the peroxisome in Arabidopsis, YFPUBC22 also co-localized with DsRed-PTS1 in tobacco.
To determine whether the tripeptide KRL> (Lys-Arg-Leu) at the very C-terminal
end of UBC22 is required for AtUBC22’s peroxisomal targeting, I tested the localization
of YFP-UBC22KRL, in which KRL> was deleted. Fluorescence microscopy of tobacco
leaf epidermal cells showed that the fusion proteins are localized to the cytosol instead
of peroxisomes (Figure 2.4B).
The above data indicate that AtUBC22 is localized to peroxisomes, and the Cterminal tripeptide KRL> is required for the proper targeting of AtUBC22 to
peroxisomes.

2.2.4 UBC22 is ubiquitously expressed in plants
To investigate the expression pattern of UBC22 in Arabidopsis, we searched
GENEVESTIGATOR

database

(https://www.genevestigator.com/gv/plant.jsp)

for

information obtained from microarray-based analyses (Hruz et al., 2008). This resource
uses publicly available microarray data to generate expression profiling of genes.
UBC22 is highly expressed in nearly all Arabidopsis tissues throughout the life-time of a
plant (Figure 2.5 A). Further tissue specific expression profiling shows that UBC22 has
very high levels of expression in pollen and pistil (Figure 2.5B).

44

2.4.5 Null mutants of UBC22 are more sensitive to exogenous IBA
To examine the possible roles of UBC22 in peroxisome function, I characterized
two

UBC22

T-DNA

insertion

alleles:

ubc22-1

(GK-642C08)

and

ubc22-2

(SALK_011800) (Figure 2.1A) (Kleinboelting et al., 2012). Both lines are in Col-0
background and have T-DNA insertion in the fifth exon - the last exon of UBC22 (Figure
2.1A). I conducted reverse transcription PCR (RT-PCR) to analyze the transcript levels
of UBC22 in ubc22-1 and ubc22-2. Both alleles are null mutant lines, as full-length?
UBC22 transcripts were not detected in either allele (Figure 2.6).
Physiological assays were performed on the null ubc22 mutants to determine if
AtUBC22 is involved in peroxisome fatty acid β-oxidation and/or IBA metabolism.
Mutants deficient in fatty acid -oxidation fail to convert stored lipid in seeds into energy,
thus young seedlings are largely dependent on exogenous sucrose to become
established as photosynthetic plants (Hu et al., 2012). The conversion of IBA to IAA, a
phytohormone that inhibits primary root growth, also occurs in the peroxisome through
β-oxidation. Mutants defective in this process display reduced response to exogenous
IBA and therefore the primary root length of mutant seedlings is not inhibited as much
as the wild type in response to IBA (Zolman et al., 2000).
ubc22 seedlings were grown under both light and dark conditions on 1/2MS
plates with or without 0.5% (w/v) sucrose. Hypocotyl lengths of seedlings grown in the
dark and root lengths of seedlings grown in the light were compared with wild-type
seedlings grown in the same conditions (Figure 2.7). Both ubc22 mutants showed no
obvious difference from the WT plants in the sugar dependence assay (Figure 2.8),
45

indicating that peroxisomal fatty acid -oxidation is not strongly affected in the absence
of UBC22. Interestingly, the primary root lengths of 7-day old ubc22-1 and ubc22-2
seedlings were 33% and 26% respectively longer than that of Col-0 on medium with or
without sucrose (Figure 2.7A).
To test whether ubc22 mutants were compromised in IBA metabolism, we grew
plants on 1/2MS plates that contained various concentrations of IBA. By comparing the
relative root length of mutants on different IBA concentrations, we are able to test
whether the efficacy of this pathway is changed. The root lengths of ubc22-1 and
ubc22-2 lines decreased significantly in response to IBA, indicating that IBA metabolism
was not impaired in ubc22 mutants (Figure 2.9A). When compared with WT, the primary
root length of both ubc22 alleles decreased more rapidly as IBA concentration
increased (Figure 2.9B). This result shows that the efficiency of IBA metabolism seems
higher without UBC22, indicating that UBC22 might play a negative role in peroxisomal
IBA metabolism.
2.4.6 Null ubc22 mutants produce bigger plants and larger and heavier seeds that
store a higher amount of protein and fatty acids
In addition to the longer primary roots, the size of the first true leaves of ubc22-1
and ubc22-2 was about 55% larger than that of Col-0 (Figure 2.10A). To examine this
phenotype in more details, I grew mutants and Col-0 on three different media: plain
medium with agar only, 1/2 LS (Linsmaier and Skoog) medium with sucrose, and 1/2
MS (Murashige and Skoog) medium with sucrose. Primary root growth of 3d to 7d
plants was investigated. The growth rate of the primary root of the ubc22 mutants was
46

lower than Col-0 when grown on plain and 0.5x LS medium, while they grew slightly
faster than Col-0 when grown on 0.5x MS medium (Figure 2.11). From 3 to 7 days, the
difference in primary root length and cotyledon size between null UBC22 mutants and
Col-0 was very obvious. On 0.5x LS medium, primary root length of 3-day-old mutants
were about 40% longer than Col-0. This number decreased at 7 day, when ubc22-1 and
ubc22-2 seedlings were 33% and 26% respectively longer than that of Col-0. These
results suggest that the growth rate of Col-0 is faster than mutants when grown on 0.5x
LS medium at early stage whereas when plants were grown on 0.5x MS medium, the
growth rate of the primary root of the ubc22-1 was similar to Col-0, and the growth rate
of ubc22-2 was 10% faster than Col-0. Finally, ubc22 adult plants of both mutant lines
are taller than Col-0, a phenotype that will need to be analyzed in more details in the
future.
The size of plant organs is determined by both cell size and number (Hua and
Chua, 2003). To determine which factor leads to the longer primary root phenotype in
null ubc22 mutants, I performed propidium iodide (PI) staining on 5-day-old seedlings to
observe and measure the cell size and calculate cell number in roots. Cell size in the
elongation zone and division zone in both ubc22 lines was similar to Col-0 (Figure 2.12).
Since both 5-day-old ubc22 lines have longer primary root than Col-0, we conclude that
the longer primary roots of the mutants is due to the possession of higher cell numbers
in the roots.
These results prompted us to trace the phenotype back to the seeds, where the
seedlings come from. Statistically significant differences were observed with respect to
the size and weight of dry seeds between ubc22 and Col-0. Seed length and width were
47

measured using over 100 seeds each time, using seeds from independent batches. Null
ubc22 mutant seeds were statistically larger in both length and width compared to Col-0
(Figure 2.13). I further measured the weight of seeds using 1000 seeds randomly
selected from three independent batches. As shown in Figure 2.14A, null ubc22 lines
are about 45% heavier than Col-0 (Figure 2.14A).
The significant difference in size and weight between the seeds of null ubc22
mutants and WT prompted me to analyze protein and fatty acid composition of seeds.
ubc22-1 and ubc22-2 seeds contained ~36% more total protein than Col-0 (Figure
2.14B). Compared to the fatty acid composition of WT seeds, both mutant ubc22-1 and
ubc2-2 have higher amount of 18:1, 18:2, 18:3 and 20:1 fatty acids and total FAME
(fatty acid methyl esters); ubc22-1 and ubc22-2 contain 46% and 38% higher amount of
total FAME than Col-0 (Figure 2.14C). The molar percentage of each type of fatty acids
in ubc22-1 and ubc2-2 are similar to that in WT (Figure 2.14D). These data suggest
that the larger size of ubc22 mutant seeds might be due to the higher amount of stored
nutrients such as lipids and proteins in the seeds.
2.4.7 Null ubc22 mutants have reduced seed yield
To check further into seed development, siliques of the mutants were analyzed.
Siliques of both UBC22 knock out lines were about 33% shorter than Col-0 (Figure
2.15A). The number of seeds in each silique was 65% less than WT (Figure 2.15B). I
further measured the total seed weight from a single plant, and found that the total seed
weight of Col-0 per plant was about two times higher than that of the UBC22 loss-offunction mutants. I calculated the approximate number of seeds and siliques on each
48

plant for each lines, and concluded that ubc22-1 and ubc22-2 yielded on average 63%
and 66% fewer seeds, and 38% and 42% fewer siliques compared with the wild type. In
summary, although each seed of null UBC22 mutants is larger than Col-0, the total seed
production is decreased in the absence of UBC22.
In plants, seed size and number are the most direct parameters for yield (Van
Daele et al., 2012). In nature, seed number is often negatively correlated with seed size
(Alonso-Blanco et al., 1999). In this work, both ubc22 mutant lines produce substantially
fewer seeds per plant, but the seeds are larger. This might be due to less competition
between zygotes toward the limited carbon and other nutrients provided by the mother
plant.
Given the mutant phenotypes in seed formation, I checked whether there is any
pollen or pistil phenotype in the ubc22 mutants. About two thirds of the ovules in
dissected green siliques of ubc22 mutants were unfertilized, and the distribution of the
aborted ovules in the siliques was random (Figure 2.16). The total number of ovules in
both null ubc22 lines was similar to that in Col-0, indicating the low seed yield could be
ascribed to the malfunction of pollen or pistil, or both. The viability of ubc22-1 and
ubc22-2 mature pollen was investigated using Alexander’s stain, which stained the
pollen from both ubc22 lines and Col-0 at similar red purple levels, indicating that the
mutant pollen grains are as viable as those of the wild type (Figure 2.17). Further
physiological examinations of pollen and the female gametophyte in in vivo conditions
are underway.

49

2.3 DISCUSSION
The biological function of peroxisomes in plant reproduction was reported
recently. The Arabidopsis PEX13/AMC gene is transiently expressed in male and
female gametophytes during fertilization (Boisson-Dernier et al., 2008). AMC functions
as a peroxin essential for protein import into peroxisomes in gametophytes. An
interesting phenomenon was discovered in the loss-of-function mutant of AMC, in which
the pollen tube reception is impaired only when an amc pollen tube reaches an amc
female gametophyte, resulting in pollen-tube overgrowth and failure to release sperm
cells. Another Arabidopsis peroxisomal gene involved in plant reproduction is DAU,
which encodes a peroxisomal membrane protein ABERRANT PEROXISOME
MORPHOLOGY9 (APEM9) that is transiently expressed from bi-cellular pollen to
mature pollen during male gametogenesis. In plants, APEM9 is an integral peroxisomal
membrane protein that functions as membrane anchor for PEX1 and PEX6 (Goto et al.,
2011). In dau pollen, peroxisome biogenesis and peroxisomal protein import are
impaired and JA level is significantly decreased (Li et al., 2014).
In this work, I discovered that UBC22 is required for some aspects of Arabidopsis
seed formation, because lack of a functional UBC22 dramatically decreases seed yield
per plant. The seed yield per plant is a vital trait for grain crop cultivars, and thus it will
be important to find out the biological mechanism of how UBC22 affects seed
production in Arabidopsis. Crop seeds are the major resources of food and nutrition for
human beings. Amino acid similarity-based searches in Zea mays (maize), Glycine max
(soybean), Oryza sativa (rice) and Triticum aestivum (wheat) databases using AtUBC22
showed that maize, rice and soybean contain putative ubiquitin-conjugating enzyme
50

family proteins that respectively share 69%, 70% and 74% amino acid identity with
AtUBC22. Interestingly, all three putative UBC22 orthologous proteins possess a KRL>
tripeptide at the C-terminal end (Figure 2.18). This result indicates that UBC22 is a
plant-specific peroxisomal protein that may carry out function that of potential values to
agriculture.
Although we have proved that Arabidopsis UBC22 is targeted to the
peroxisomes, we do not have sufficient knowledge to relate the low reproduction rate
displayed in the mutants to the dysfunction of peroxisomes. We cannot exclude the
possibility that UBC22 may also target to other organelles besides peroxisomes to fulfill
its function.
To date, our understanding about the role of peroxisomal localized E2 in
peroxisome biogenesis and metabolic functions is still scarce. In Arabidopsis, PEX4
was the only E2 known to be associated with peroxisomes before the identification of
UBC22. Yeast Pex4p binds to the peroxisomal membrane via its membrane anchor
Pex22p, and was shown in vitro to mono-ubiquitinate Pex5p during receptor export
(Platta et al., 2007). Yeast S. cerevisiae Ubc4p, Ubc5p and Ubc1p are E2 enzymes
involved in the polyubiquitination of Pex5p (Kragt et al., 2005; Kiel et al., 2004). A
previous in vitro study of AtUBC22 shows that UBC22 is able to form poly-ubiquitin
chains in the absence of an E3 (Kraft et al., 2005). However, although in vitro assays
shows UBC22 to have E3-independent polyubiquitination activity, one has to investigate
in planta to determine whether UBC22 collaborates with an E3 or directly transfers
mono- or poly-ubiquitin chains to its substrates in an E3-independent manner.

51

In peroxisomes, the four-carbon IBA is converted to the two-carbon IAA in a
mechanism similar to fatty acid ß-oxidation (Zolman and Bartel, 2004; Zolman et al.,
2001). Mutants of the fatty acid ß-oxidation pathway such as aim1 and ped1/kat2 are
also resistant to exogenous IBA, while ibr1, ibr3, ibr10 only show IBA resistant
phenotypes, suggesting that these enzymes might be specific for IBA-CoA ß-oxidation.
Our sucrose dependence experiments suggest that the peroxisomal fatty acid oxidation pathway is not strongly affected in the absence of UBC22 (Figure 2.8). In the
IBA resistant assay, IBA seems more efficient in both ubc22 mutant lines compared to
Col-0, indicating that UBC22 might act as a negative regulator in peroxisomal IBA
conversion. Since fatty acid -oxidation is unaffected in null UBC22 mutants, we
speculate that UBC22 might be involved in the regulation of IBA metabolism through
modulating the activities of IBA pathway-specific enzymes, such as IBR1, IBR3 and
IBR10.
I also report that two UBC22 knock out lines produce fewer but larger seeds. The
higher weight of null UBC22 mutant seeds might be due to less competition between
zygotes for nutrients due to the aborted ovules. Mature seeds of null ubc22 mutant lines
are equally scattered inside siliques, indicating that the length of mutant pollen tubes is
likely to be normal. Further in vivo analysis of the loss-of-function mutants, such as in
vivo pollen tube growth assay and reciprocal cross-pollinations between wild-type and
ubc22 plants, needs to be done to elucidate the mechanism behind this phenotype.
In summary, I have characterized a novel Arabidopsis peroxisomal protein,
AtUBC22, whose function might be conserved in diverse plant species such as food

52

crops maize, rice and soybean. Morphological and physiological analysis of null ubc22
mutants reveals that UBC22 has an effect on seed formation and plant growth and that
UBC22 might act as a negative regulator of peroxisomal IBA metabolism. We speculate
that UBC22 affects seed yield by exerting its function inside peroxisomes. It is possible
that certain UBC22-related peroxisomal pathways are linked to male or female
gametophytic physiological activities, which then further affect plant reproduction.
However, many questions remain to be answered. For example, which peroxisomal
pathway is UBC22 involved in? What is the physiological and molecular mechanism
behind the unfertilized ovules? How are peroxisomes linked to the decreased seed yield
phenotype?

53

Table 2.1 Arabidopsis UBCc domain-containing proteins
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39

Accession#
AT1G14400
AT1G16890
AT1G17280
AT1G36340
AT1G45050
AT1G50490
AT1G53020
AT1G53023
AT1G53025
AT1G63800
AT1G64230
AT1G75440
AT1G78870
AT2G02760
AT2G16740
AT2G16920
AT2G33770
At2G46030
AT3G08690
AT3G08700
AT3G12400
AT3G12775
AT3G13550
AT3G15355
AT3G17000
AT3G20060
AT3G24515
AT3G46460
AT3G52560
AT3G55380
AT3G57870
AT3G59410
AT3G60300
AT4G27030
AT4G27960
AT4G36410
AT4G36800
AT5G05080
AT5G13860

Protein
UBC1
UBC36,UBC13B
UBC34
UBC31
UBC15
UBC20
UBC26, PFU3
Ubiquitin-conjugating enzyme family protein
Ubiquitin-conjugating enzyme family protein
UBC5
UBC28
UBC16
UBC35,UBC13A
UBC2
UBC29
UBC23,PFU2
UBC24,PHO2
UBC6
UBC11
UBC12
ATELC, ELC
Ubiquitin-conjugating enzyme family protein
CIN4,COP10,EMB144,FUSCA 9
UBC25,PFU1
UBC32
UBC19
UBC37
UBC13
MMZ4,UEV1D,UEV1D-4
UBC14
AHUS5,ATSCE1,EMB1637,SCE1,SCE1A
ATGCN2, GCN2
RWD domain-containing protein
FAD4, FADA
UBC9
UBC17
RCE1
UBC22
ELC-LIKE, ELCH-LIKE

54

Last three a.a. at
C-terminal

TAD>
SGA>
LQL>
ANN>
DKV>
PSA>
SSR>
CSV>
SSR>
LDP>
AMG>
DKV>
SGA>
TAD>
ALF>
QQQ>
PES>
ADP>
AMG>
AMG>
LHS>
SFD>
FAK>
SSS>
DQS>
LNA>
CRP>
EMF>
TCF>
EML>
ALV>
VWS>
GDK>
NQA>
AMG>
DKV>
RCI>
KRL>
LHS>

Table 2.1 cont’d
40
41
42
43
44
45
46
47
48
49

AT5G25760
AT5G41340
AT5G41700
AT5G42990
AT5G50430
AT5G50870
AT5G53300
AT5G56150
AT5G59300
AT5G62540

UBC21,PEX4
UBC4
UBC8
UBC18
UBC33
UBC27
UBC10
UBC30
UBC7
UBC3

55

KKG>
PDP>
AMG>
DKV>
LQL>
CSA>
AMG>
AMG>
EMF>
SYV>

Figure 2.1 Sequence and structural analysis of UBC22.
(A) Genomic structure of UBC22. The T-DNA insertion sites in ubc22-1 (GK-642C08)
and ubc22-2 (SALK_011800) are indicated.
(B) Putative protein structure of UBC22. UBC domain, Ubiquitin Conjugating domain;
PTS1, Peroxisome Targeting Signal type 1.

56

Figure 2.2 Phylogenetic analysis of Arabidopsis UBC22.
Phylogenetic analysis of the relationship between Arabidopsis UBCs and the six known
yeast UBCs and three known human UBCs. Scale bar, 0.1 amino acid substitutions per
site. Bootstrap was performed from 1000 replicates. Bootstrap values are shown at the
tree nodes.

57

Figure 2.3 Amino acid sequence alignment of Arabidopsis UBC22 and
homologous sequences.
Sc, S. cerevisiae; Hs, Homo sapiens. Full amino acid regions from human UBCH5A
(HsUBC5A), Arabidopsis thaliana UBC22 (AtUBC22) and PEX4 (AtPEX4), S. cerevisiae
Ubc1p (ScUbc1p), Ubc4p (ScUbc4p), Ubc5p (ScUbc5p) were aligned using ClustalW2.
Identical residues are shaded. Conserved sequences are indicated by black boxes. Red
asterisk indicates the conserved active site Cys residue.

58

Figure 2.4 UBC22 localizes to the peroxisome through a C-terminal PTS1 (KRL>).
(A) Confocal images of 35S::GFP-UBC22 (green) co-expressed with the peroxisome
marker 35::DsRed-PTS1 (red) in leaf epidermal cells of Arabidopsis transgenic plants.
Scale bar = 10 µm. White arrow indicate co-localization of UBC22 and peroxisomes.
(B) Epifluorescence images of 35S::YFP-UBC22∆KRL (green) co-expressed with
DsRedPTS1 (red) in tobacco epidermal cells. Scale bar = 10 µm

59

Figure 2.5 Expression patterns of UBC22 in Arabidopsis

60

Figure 2.5 cont’d
(A) Expression at successive developmental stages. Y axis indicates the level of gene
expression. Data used for the analysis were retrieved from GENEVESTIGATOR.
(B) Ranking of UBC22 gene expression levels in various plant tissues.

61

Figure 2.6 Expression level of UBC22 in Arabidopsis
RT-PCR analyses of total RNA extracted from 10d seedlings of wild-type, and leaves
and flowers from 10d seedlings and 8-week loss-of-function mutants (ubc22-1 and
ubc22-2) respectively. Primers used are indicated in Fig. 2.1A. The UBQ10 transcripts
are used as loading controls.

62

Figure 2.7 Sucrose-dependence assays on the ubc22 mutants.
(A) Seven-day light-grown seedlings. Scale bar = 10 mm
(B) Seven-day dark-grown seedlings. Scale bar = 10 mm

63

Figure 2.8 Quantification of root (A) and hypocotyl (B) lengths of seedlings in the
sucrose dependence assay.
The pex4 pex22 double mutant defective in peroxisome biogenesis was used as a
positive control. Error bars represent SE. Asterisk indicates statistically significant
differences between mutants and Col-0 under the same condition (Student’s t-test,
p<0.0001).

64

Figure 2.9 ubc22 mutants are hypersensitive to IBA.
(A) Root lengths of 7-day-old seedlings grown on 1/2MS media with 15µm IBA or
200nm IAA were quantified. pex14 was used as a positive control. Error bars represent
SE (n> 40 for each genotype).
(B) Root lengths of 7-day-old seedlings grown on 1/2MS media with indicated
concentration of IBA normalized to their respective average seedling root length growth
on media without IBA. pex14 was used as a positive control.

65

Figure 2.10 Arabidopsis ubc22 have larger first true leaves and cotyledons
(A) True leaf size of 7-day-old seedlings grown on 1/2LS media 0.5% sucrose under
light conditions. Left panel, image of representative seedlings, scale bar = 2mm. Right
panel, average true leaves size.
(B) Four-day old seedlings of Col-0, ubc22-1and ubc22-2. Seedlings were grown on
1/2LS media with 0.5% sucrose under light conditions.
66

Figure 2.11 Arabidopsis ubc22 have similar primary root growth rate to Col-0

67

Figure 2.11 Cont.
Relative root length of 3, 5 and 7-day-old ubc22-1, ubc22-2 and Col-0 seedlings grown
on (A) plain media with agar only, (B) ½ LS media with 0.5% sucrose and (C) 1/2 MS
media with 0.5% sucrose was measured. Error bars indicate SE. Samples of each line >
30.

68

Figure 2.12 Comparison of root cell size between ubc22 and Col-0.
Roots of 5-day-old wild type, ubc22-1, and ubc22-2 were stained with propidium iodide
(PI) to visualize the cell wall. Bar = 100 m.
Green bar indicates the length of the division zone. Blue bar indicates the length of 10
cells in the division zone. Yellow bar indicates the length of one cell in the elongation
zone.

69

Figure 2.13 Morphological analysis of ubc22 seeds.

70

Figure 2.13 cont’d
(A)(B) Dry seeds observed under the light microscope. Bar = 500 m.
(C)(D) Lengths and widths of ubc22 and Col-0 seeds. >100 seeds were measured.
Asterisk indicates statistically significant differences between mutants and Col-0
(Student’s t-test, p<0.001).

71

40

C

35

nmol / seed

30
25
20

Col-0

15

ubc22-1

10

ubc22-2

5
0
16:018:018:118:218:320:020:120:220:322:022:1

total

Fatty acid

total (nmol/ seed)

mol %

D

Col-0
22.052655

ubc22-1
32.2887

ubc22-2
30.4673

30
25
20
15
10
5
0

Col-0

ubc22-1
ubc22-2
16:0 18:0 18:1 18:2 18:3 20:0 20:1 20:2 20:3 22:0 22:1

Fatty acid

Figure 2.14 Analysis of the weight, protein content and fatty acids of ubc22
mutants

72

Figure 2.14 cont’d
(A) Comparison of the average weight of 1000 seeds from ubc22 mutants and Col-0.
Seeds are from three independent batches
(B) Total protein content in 100 seeds from each genotype. Four asterisks indicate
statistically significant differences between mutants and Col-0 (Student’s t-test, p<0.01).
Error bars represent SE
(C) Total fatty acid content in seeds of WT and ubc22 mutants. Error bars represent
standard deviation.
(D) Fatty acid composition analysis in seeds. Error bars represent standard deviation.

73

B

Figure 2.15 Silique analysis of ubc22 mutants.
(A) Green siliques from 10-week plants. Silique lengths were measured using >30
siliques from each genotype. Asterisk indicates statistically significant differences
between mutants and Col-0 (Student’s t-test, p<0.0001).
(B) Comparison of seeds in the siliques of Col-0, ubc22-1and ubc22-2. The number of
seeds per silique was counted using over 40 siliques from independent batches.
Asterisk indicates statistically significant differences between mutants and Col-0
(Student’s t-test, p<0.0001). Bar = 2 mm. Pictures are from representative siliques from
each line.

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Figure 2.16 Unfertilized ovules of ubc22 mutants in the silique.
Pictures are from representative siliques from each line. White arrows indicate
unfertilized ovules. From left to right are Col-0, ubc22-1, ubc22-1.

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Figure 2.17 in vitro pollen viability test by Alexander staining
Non-aborted pollen grain stained red, aborted pollen grain were colorless or shows
pale-green color.

76

Figure 2.18 Amino acid sequence alignment of Arabidopsis UBC22 and its
putative homologues in other species.
Amino acid sequence alignment of AtUBC22 and Zea mays UBCE2s (gb|ACG34027.1),
Oryza sativa Os06g0660700 (Japonica Group, ref|NP_001058271.1), Glycine max
UBCE2S (ref|XP_006604289). Conserved sequences are indicated by black boxes.
Red asterisk indicates the conserved active site cysteine residue. Red box indicates the
C-terminal peroxisome targeting signal tri-peptide KRL>.

77

2.4 METHODS
2.4.1 Plant material, growth conditions, and plant transformation
Arabidopsis (Arabidopsis thaliana) seeds were sown on ½ Linsmairer and Skoog
medium (LS) medium containing 0.5% (w/v) sucrose. Plants were grown at 20C with
70% humidity, under white light illumination at 70 to 80 mol m-2 s-2 for 14h per day.
Homozygous

T-DNA

insertion

alleles

ubc22-1

(GK-642C08)

and

ubc22-2

(SALK_011800) were obtained from GABI-KAT and ABRC (Arabidopsis Biological
Recourse Center) respectively, both in Col-0 background. T-DNA insertion and the
homozygosity of mutants were confirmed by PCR-based screening of genomic DNA.
UBC22 transcripts level was determined by reverse transcription PCR (RT-PCR).
35Spro: DsRed-PTS1 (Zhang and Hu, 2010) was used as the peroxisomal marker
in UBC22 localization study. Transgenic plants were generated via floral-dipping
transformation of DsRed-PTS1- containing plants with Agrobacterium tumefaciens
harboring 35Spro: GFP-UBC22 fusion gene that contains the full-length CDS of UBC22.
T1 generation was screened with kanamycin and the selected plants were subjected to
confocal microscopy to observe GFP-UBC22 signals.
Nicotiana tabacum (Tobacco) were grown at 24C with 70% humidity and white
light illumination at 50 mol m-2 s-2 for 14 h per day. A. tumefaciens cells harboring
35Spro: YFP-UBC22 or 35Spro: YFP-UBC22KRL were co-infiltrated with A. tumefaciens
cells harboring 35Spro: DsRed-PTS1into mature leaves. The infiltrated tobacco plants

78

were incubated in the same growth condition for another 2 days before subjected to
confocal imaging.

2.4.2 RT-PCR
Total RNA was extracted from 2-week-old plants and adult plant flowers using
the RNeasy plant mini kit (Qiagen, USA) as instructions. 500 ng of RNA was reverse
transcribed using Omniscript RT kit (Qiagen, USA) using oligodT primers. 5 ng of cDNA
was used in PCR amplification (Promega, USA) using gene-specific primers.
PCR conditions: 1) 95°C for 2 min, 30 cycles of step 2) – 4), 2) 95°C for 30 sec,
3) 55°C for 30 sec, 4) 72°C for 1min, 5) final extension 72°C for 10 min.

2.4.3 Gene cloning and plasmid construction
CDS of UBC22 was used in localization study. Sequence was amplified from
cDNA that had been reverse transcribed from Col-0 total mRNA. PCR fragments were
cloned to a pDonorTM207 then into the pCHF3 vector. Since the GFP signals were lost
in T2 plants, I then generated YFP-UBC22 and YFP-UBC22KRL constructs using the
binary vector pEarleyGate 104 (Earley et al., 2006).YFP-UBC22KRL has the last 9
nucleotides of the gene (KRL) deleted.

79

2.4.4 Confocal microscopy analyses
Confocal images for localization were taken by Navneet. Olympus FluoView
1000 CLSM (Olympus, Tokyo, Japan) were used. For UBC22 localization image, 488
nm Argon were used for GFP excitation and 505-530 nm emission filters to acquire GFP
signal, also 543nm HeNe were used for excitation of DsRed and 560-615nm emission
filters for DsRed signal.
For root imaging, 5-day old seedlings were stained in 10 g/ml propidium iodide
(PI) for one minute, rinsed, then mounted in dH2O. PI fluorescence signals were
visualized by excitation via Kr/Ar 488-nm laser line and detection with a band-pass 570670 nm emission filter (González-García et al., 2011). Images were processed by the
Olympus FV software 4.1 and assembled by ImageJ software.

2.4.5 Sucrose dependence assay
Surface-sterilized Seeds were resuspended in sterile water and stratified at 4°C
for 48h under dark condition. Seeds were sown on 1/2MS medium without exogenous
Suc or supplemented with 0.5% (w/v) Suc and solidified with 0.8% agar. Plates were
placed in light for one hour then transferred to growth chamber and grown vertically in
the dark for seven days. pex4/pex22 were used as the positive control in this
experiment. Hypocotyl lengths were measured with a ruler or using ImageJ 1.48
(http://imagej.nih.gov/ij/). Statistical significance was calculated by the Student’s T-test

80

using GraphPad Prism 6, to determine whether there are differences between Col-0
plants and ubc22 mutants.

2.4.6 IBA resistance assay
Surface-sterilized seeds were resuspended in sterile water and stratified at 4°C
for 48h in the dark. Seeds were sown on 1/2MS medium with IBA (0M, 5M, 10M,
15M, 20M, 25M IBA), or 200nM IAA. Plates were placed vertically in growth
chamber with continuous light for 7 days. pex14 was used as the positive control in this
experiment. Primary root lengths were measured by ImageJ 1.48. Statistical
significance was calculated by the Student’s T-test using GraphPad Prism 6.

2.4.7 Sequence alignment and phylogenetic analysis
The amino acid sequences used in this study were obtained from the National
Center

for

Biotechnology

Information

website

(NCBI:

http://www.ncbi.nlm.nih.gov/protein), The Arabidopsis Information Resource (TAIR)
(www.arabidopsis.org) and UNIPROT (http://www.uniprot.org/). The sequences were
then aligned by the Clustal W method and grouped into a phylogenetic tree by MEGA6
software (http://www.megasoftware.net/) (Tamura et al., 2013). MEGA6 was used to
generate the neighbor-joining tree and perform bootstrap analysis using the distance
analysis function with 1000 replicates.

81

Amino

acid

sequences

were

aligned

by

ClustalW2

(http://www.ebi.ac.uk/Tools/msa/clustalw2/) and further shaded into graphical format by
ESPript 3 software (http://espript.ibcp.fr/ESPript/ESPript/) (Robert and Gouet, 2014).

2.4.8 Pollen viability assay
A simplified Alexander staining method as described in (Peterson et al., 2010)
was used in this study. Anthers were removed from open flowers, fixed in Carnoys
fixative (6 ethanol: 3 chloroform: 1 glacial acetic acid) for at least two hours, before they
were stained by Alexanders staining (10ml 95% ethanol, 25ml glycerol, 1ml Malachite
green (1% solution in 95% ethanol), 5ml Acid fuchsin (1% solution in water), 0.5ml
Orange G (1% solution in water), 4ml glacial acetic acid, 54.5ml distilled water) buffer
on slides. Bottom of the slides were carefully heated with a burner until the stain was
completely absorbed into pollen grains. Overheating of the samples should be avoided
in this process. Images were taken by Zeiss Axio Imager.M1 microscope (Carl Zeiss).

ACKNOWLEDGEMENTS
We thank the GABI-Kat (Germany) for the T-DNA insertion mutant ubc22-1, and
ABRC (USA) for the T-DNA insertion mutant ubc22-2. We also thank Melinda Frame
(Center of Advance Microscopy, MSU) for help with the confocal microscopy, Dr. Weili
Yang (MSU) for help with fatty acid analysis. This work was supported by grants to JH
from the Chemical Sciences, Geo-sciences and Biosciences Division, Office of Basic
82

Energy Sciences, Office of Science, U.S. Department of Energy (DE-FG0291ER20021), and National Science Foundation (MCB 1330441).

83

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