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This is to certify that the
dissertation entitled

CHARACTERIZATION OF A FAMILY OF VACUOLAR
SORTING RECEPTORS IN ARABIDOPSIS THALIANA

presented by

EMILY AVlLA-TEEGUARDEN

has been accepted towards fulfillment
of the requirements for the

Doctoral degree in Cell and Molecular Biologx

 

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CHARACTERIZATION OF A FAMILY OF VACUOLAR SORTING RECEPTORS IN
ARABIDOPSIS THALIANA

By

Emily Avila-Teeguarden

A DISSERTATION

Submitted to
Michigan State University
In partial fulfillment of the requirements
For the degree of

DOCTOR OF PHILOSOPHY
Cell and Molecular Biology Program

2004

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ABSTRACT

CHARACTERIZATION OF THE FAMILY OF VACUOLAR SORTING RECEPTORS
IN ARABIDOPSIS THALIANA

By

Emily Avila-Teeguarden

In plant cells, soluble proteins delivered to the vacuole via the endomernbrane
system contain a vacuolar sorting signal (V SS). Several types of VSSs have been
identified in plants. One class of VSS is the N-terminal propeptide (NTPP) that is
cleaved from the mature protein and contains a conserved peptide motif required for
vacuolar sorting. A putative vacuolar sorting receptor (V SR) for NTPP-containing
proteins is the Arabidopsis vacuolar sorting receptor 1 (AtVSRl; formerly AtELP).
AtVSRl is a type I transmembrane protein with a protease-associated domain and three
cysteine-rich EGF repeats. Plant VSRs interact with NTPP—containing proteins in a
sequence-specific manner to direct their delivery to lytic vacuoles via a prevacuolar
compartment. AtVSRI has six homologues in the Arabidopsis genome. Expression of all
members of this gene family was detected in various plant tissues. To understand the
specific roles played by each of these proteins, I transformed promoter::GUS fusion
constructs into Arabidopsis to determine the cell type-specific expression pattern of each
gene. From this approach, I determined that many of the AtVSRs were expressed in cell
type specific expression patterns. For example, the AtVSR3 gene was specifically
expressed in the guard cells of true leaves. Other genes, such as AtVSRI, were expressed
throughout the vascular tissue as well as in developing and mature embryos. Two genes,

AtVSR2 and AtVSR5, showed much broader expression patterns than RT-PCR results

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indicated. These results are likely due to the presence of negative regulatory elements in
the introns or 3’UTR of the genes that were absent from the promoter: :GUS fusions.

Reverse genetics and biochemical approaches were also used to understand the
collective and individual functions of these proteins. An antisense approach was used to
post-transcriptionally silence the entire AtVSR gene family. No plants were obtained that
silenced expression from all the AtVSRs. Furthermore, there was very low heritability of
the AtVSR silencing. These results indicated that at least some amount of AtVSR protein
is essential to plant growth and development. Transgenic plants that had the lowest
levels of AtVSR expression showed severe defects in root and shoot gravitropism,
defects in leaf and flower development, and produced very few seeds. The pleiotropic
effects of silencing the AtVSR gene family indicated that these genes play numerous and
varied roles in plant development. To determine the functions of individual AtVSRs, we
took advantage of other reverse genetic strategies. Specifically, RNA interference of the
AtVSR3 gene produced plants that accumulated anthocyanins in the cotyledons and were
smaller than wildtype seedlings. Preliminary results also suggested that the stomata may
not respond to signal transduction pathways that cause stomata to close. Two
independent knockout lines of AtVSR7 produced very small plants. Overall, these results
demonstrated that plant VSRs function in very specific pathways which has not been
shown for other eukaryotes. Other projects presented here relate to vacuolar biogenesis.
Specifically, I initiated a high-throughput confocal microscopy screen for mutants that
did not form vacuoles properly and a proteomics survey of plant cell vacuoles. These
projects have helped Dr. Raikhel’s lab as well as the scientific community at large move

into a high-throughput systems biology approach to plant science.

To The Memory of Alberto Luis Avila (1931-1987)

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ACKNOWLEDGMENTS

I must first thank my advisor and mentor, Dr. Natasha Raikhel. Natasha has been
more of a mother figure to me than anyone in my life. She has taught me by example
how to acknowledge and deal with the challenges in my life. I will always be grateful to
her for giving me the strength I needed to change my life for the better.

I am very appreciative of my committee members, Dr. Ken Keegstra, Dr. Joanne
Whallon, Dr. Gregg Howe, and Dr. Jack Preiss. Their patience and guidance has been a
blessing.

I must also thank Dr. Marci Surpin for all of her help and support both
professionally and personally. Also, I specially thank Dr. Marci Surpin, Dr. Glenn Hicks,
and Dr. Clay Carter for critical reading of this thesis. All of the past and present
members of the Raikhel lab have been excellent teachers, colleagues, and fi'iends. I
would like to specifically thank Dr. Sharif Ahmed, who helped me continue his work on
vacuolar sorting receptors. I would also like to thank Dr. Thomas Girke and Dr. Curt
Wilkerson for their help with bioinforrnatics. Much of this work could not have been
done without them. Dr. Natasha Raikhel, Dr. Shirley Owen, Dr. Joanne Whallon, Dr.
David Carter, Cathy Ecker and Dr. Marguerite (Rita) Varagona all encouraged my
interest in microscopy. On a personal level, I am grateful to Rita for taking me under her
wing and encouraging me.

I would also like to thank my collaborators and colleagues on specific projects.
Dr. Jen Sheen graciously shared her pRJGZ3 vector for the promoterzzGUS studies. Dr.

Patti Springer, Dr. Rob Martienssen, and Dr. Ueli Grossniklaus generously shared their

 

 

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Gene Trap and Enhancer Trap lines with me as well as their knowledge about them. I
also thank the Salk Institute Genomic Analysis Laboratory for providing the Sequence-
. indexed Arabidopsis T-DNA Insertion Mutants. Dr. Vicki Chandler and Dr. Robert
Jorgensen kindly sent the dsRNA vector. Dr. Chris Somerville generously provided the
358::GFPzA-TIP line. Dr. Jian-Kang Zhu generously allowed me to use the thermal
camera in his laboratory. Dr. Louis King helped me try to sort vacuoles by FACS.
Syngenta kindly analyzed my vacuolar protein extracts.

On a personal level, I am thankful for my dearest husband, Thomas Teeguarden.
His never-ending patience and faith in me is amazing. He is definitely my inspiration to
become a better person. I am also extremely grateful to Carmen, Scherrie, Karen, Ginny,
Cathy, Sharon, Christine, Robbie, and Margaret. As a survivor of child abuse, I was
convinced that nobody, including myself, would ever understand the long-term effects I
faced, except for this incredible group. There will always be a special place in my heart
for these brave women. In this context, I must also thank my sister, Elaine, for calmly
waiting 15 years and for being so prepared to immediately help me when I was ready to
start to heal.

Finally, this work was supported in part by the MSU-DOE Plant Research

Laboratory and a Graduate Research Fellowship from the National Science Foundation.

vi

LIST OF

LIST OF

LIST OF

CILAPTE .

 

lntroduct:

CHAPTE

ExpreSSli

 

TABLE OF CONTENTS

LIST OF TABLES ..................................................................................... x
LIST OF FIGURES .................................................................................. xi
LIST OF ABBREVIATIONS .................................................................... xiii
CHAPTER 1

Introduction: An Overview of Protein Transport through the Endomembrane System. . .l

I. Introduction .......................................................................... 2
II. Lysosomal Transport in Animal Cells ........................................... 9
III. Vacuolar Transport in S. cerevisae ............................................. 11
IV. Soluble Vacuolar Protein Transport in Plants ................................. 15
V. Thesis Overview ................................................................... 26
VI. References .......................................................................... 29

CHAPTER 2

Expression Analysis of the Vacuolar Sorting Receptor Family in Arabidopsis ................ 39
Abstract ...................................................................................... 40
Introduction .................................................................................. 41
Materials and Methods ..................................................................... 43
Results ........................................................................................ 49
Discussion ................................................................................... 69
References ................................................................................... 79

vii

 

 

C ILA}

Functi

CHAPT
Conclus

IE

 

 

CHAPTER 3

Functional Analysis of the AtVSR Family ....................................................... 84
Abstract .......................................... . ............................................ 85
Introduction .................................................................................. 86
Materials and Methods ..................................................................... 86
Results ....................................................................................... 92
Discussion .................................................................................. 107
References ................................................................................. 111

CHAPTER 4

Conclusions and Future Directions ............................................................... 115
Expression Analysis of the AtVSR Gene Family ................................................. 116
Subcellular Localization of AtVSRs ................................................................... 116
Recycling of Plant VSRs ..................................................................................... l 17
Reverse Genetics Approaches to Determine the Functions of AtVSRs .............. 119
Identification of the In Vivo Targets of AtVSRs ................................................. 120
Conclusions .......................................................................................................... 121
References .................................................................................. 122

APPENDICES

Appendix A: Supplementary Information for Chapter 2 .................................... 125
References ............................................................................................................ 132

viii

 

 

Appendix
Disrupted

Tagged C

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Appendix B: Tools to Study Plant Organelle Biogenesis. Point Mutation Lines with

Disrupted Vacuoles and High-Speed Confocal Screening of Green Fluorescent Protein-

Tagged Organelles ................................................................................. 134
Conclusions .......................................................................................................... 141
References ............................................................................................................ 144

Appendix C: Proteomic Analysis of Plant Cell Vacuoles ................................... 146
Introduction .......................................................................................................... 147
Materials and Methods ......................................................................................... 147
Results .................................................................................................................. 148
Conclusions .......................................................................................................... 1 59
References ............................................................................................................ 160

Appendix D: Immunolocalization of Ple in Wildtype, vtiI 1, and vti12 Seedlings. . ..161

Introduction .......................................................................................................... 162
Materials and Methods ......................................................................................... 164
Results .................................................................................................................. 164
Discussion ............................................................................................................ 166
References ............................................................................................................ 168
BIBLIOGRAPHY
Bibliography .................................................................................................................... 1 7O

ix

 

Table 1.1
Table 1.2
Table 1.3
Table 2.1

Table 2.2

Table A}
Table A;
Table ATI
Table A4
Table A..I
Table M

Table Cl

 

LIST OF TABLES

Table 1.1.N-Terminal Propeptides ............................................................... 19
Table 1.2.C-Terminal Propeptides ............................................................... 19
Table 1.3.Nomenclature for Arabidopsis Vacuolar Sorting Receptors ....................... 27

Table 2.1.Putative Plant VSRs . . .51

Table 2.2.Summary of the Spatial and Temporal Expression Data Obtained for the AtVSR

Genes ................................................................................. 70
Table A.1. ESTs and cDNAs for the AtVSR] Gene ........................................... 126
Table A2. Promoter Elements Found in the AtVSR] Promoter ............................. 127

Table A3. Promoter Elements That Are Present in the AtVSRZ Promoter. . . . . . . . 128

Table A4. Promoter Elements That Are Present in the AtVSR3 Promoter. . . . . . . . . 129

Table A5. Promoter Elements in the AtVSR4 Promoter ..................................... 130
Table A.6. Promoter Elements in the AtVSR6 Promoter ..................................... 131
Table C.1. Proteins Identified in Isolated Vacuoles ........................................... 154

 

 

LIST OF FIGURES

Figure 1.1.A schematic of the endomembrane system ............................................ 3
Figure 1.2.Many factors are required for vesicle formation ..................................... 4
Figure 1.3.The interaction of SNAREs drives membrane fusion between a vesicle and
its target organelle ........................................................................... 6
Figure 1.4.Structure of the S. cerevisiae Vplep vacuolar sorting receptor and a plant
vacuolar sorting receptor ................................................................. 13
Figure 1.5.Protein transport through the endomembrane system of plants. .. . ..........16
Figure 1.6.Vacuole sorting signals in plants ..................................................... 18
Figure 1.7 .The current model of plant VSR function and the localization of other VSRs
within the endomembrane system ........................................................ 25
Figure 2.1.Gene structures of the AtVSR family ................................................. 50
Figure 2.2.Phylogenetic analysis of the plant VSRs ............................................ 52
Figure 2.3.Phylogenetic analysis of the PA domain of the plant VSRs ...................... 54
Figure 2.4.RT-PCR expression analysis of AtVSR genes ....................................... 55

Figure 2.5.Expression analysis of AtVSR] by PromoterzzGUS fusions in Arabidopsis. . . .57
Figure 2.6.Expression patterns of GUS fi'om the AtVSRZ promoter ................................. 60
Figure 2.7.Expression analysis of AtVSR3 by PromoterzzGUS fiisions in Arabidopsis. . ..61

Figure 2.8.AtVSR3 expression decreases in the presence of dehydration, cold, or 250 uM
ABA .......................................................................................... 63

Figure 2.9.Expression analysis of AtVSR4 by PromoterzzGUS fusions in Arabidopsis ..... 64
Figure 2.10.Expression analysis of the AtVSR5 promoter by GUS assay. . . . . . . . . . . ..........67
Figure 2.11.Expression patterns from the At VSR? promoter ................................... 68

Figure 3.1 .Some antisense AtVSR plants accumulate little or no AtVSR protein ............ 93

xi

Figure 3.2.Antisense AtVSR plants showed defects in plant development .................. 95
Figure 3.3.AtCPY is not delivered to the vacuole in two antisense plants. . . . . ...............96

Figure 3.4.Gene trap insertion in the AtVSR3 gene .............................................. 98
Figure 3.5.Visual characterization of AtVSR3 RN Ai plants .................................. 100

Figure 3.6.The average temperature of AtVSR3 RNAi seedlings is less than the average
temperature ofwildtype seedlmgleZ

Figure 3.7 .Gerrnination frequency of wildtype and AtVSR3 RNAi seeds .................. 103
Figure 3.8.Characterization of AtVSR7 knockout lines ................................................... 105
Figure 3.9.A VSR coirnmunoprecipitated with AtCPY ................................................... 108
Figure B.1.GFP:ATIP is expressed in the tonoplast of 358::GFP: -TIP transgenic

seedlings .................................................................................... 137
Figure B.2.Examples of vacuolar mutants identified .......................................... 139
Figure B.3. Assembly of screening plates ....................................................... 142

Figure C.1. ct-TIP and y-TIP peptide antibodies do not cross-react. . . . . . ....150

Figure C.2.Vacuoles that contain GFP in the tonoplast are recognized by flow
cytometry .................................................................................. 152

Figure D.1. PIN 1 localized to the basal plasma membrane in zig/vtiI I and vti12 .......... 165

xii

 

mTH’
ITW
III?
ABA

ABC

AS

ATP
BP80

CCV
CDJIPR
cDNA
CPY
CTPP
DNA
DOF
DPBF
ctRXA,
DV
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ELP
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FAQS
GA
GFP
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IGFH

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LV

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mRNA
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y-TIP
A-TIP
ABA
ABC

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BP-80
bub
CCV
CD—MPR
cDNA
CPY
CTPP
DNA
DOF
DPBF
dsRNA
DV
EGF
ELP
EMS
ER
EST
FACS
GA
GFP
GUS
IGFII
KLH
KV
LV
M-6-P

MPR
mRNA

NASC

LIST OF ABBREVIATIONS

=Alpha Ionoplast Intrinsic Protein
=§_amma Ionoplast Intrinsic Protein
=_Delta Ionoplast Intrinsic Protein
=Afiscisic Acid

=ATP-_B_inding _C_assette

=gggregates of QF P

=A1_ka1ine Phosphatase

=Adaptor Protein

=AU-Rich Plement

=Adenosine Triphosphate

=__B_inding Protein of mkDa
=b_u_bble—_l_)_ath

=Qlathn'n-Qoated Xesicle
=Qation-I)_ependent _M_annose-6-Phosphate Receptor
=§omplementary Qeoxyribogucleic Acid
=Qarboxypeptidase X

=_C_-_T_erminal Propeptide
=Qeoxyribogucleic Acid
=QNA-Binding With One Ringer

=Qc3 Promoter-Rinding P actor
=I)_ouble-§_tranded Ribor_1ucleic Acid
=Qense Xesicle

=_E_pidennal growth P actor

=Rpidermal Growth Factor Receptor-Like Protein
=_E_thyh11_ethane Sulfonate
=Pndoplasmic Reticulum

=Rxpressed §equence Iag
=Pluorescence Assisted Qell §orter
=Qibberellic Acid

=_G_reen Pluorescent Protein
=B-ngcuronidase

=Insulin-Like growth PactorR
=Reyhole Limpet Remocyanin
=RDEL Xesicle

=L_yfic Xacuole
=M_annose—§-Phosphate

=l\_/I_ultidrug Resistance-associated Protein
=_M_annose-6-Phosphate Receptor
=Mflsenger Ribogucleic Acid
=_M_ultiyesicular Rody

=_N_ottingham Arabidopsis §tock _Centre

xiii

 

 

NTPP
NtSyrl
OSMl
PA

PAC
PBS
PCR
PIN
PPV
PrA

PrB
PSV
PTGS
PV72
PVC
RER
RNA
RNAi
RT-PCR
SEF
SGR
SH-EP
SMD
SNARE
ssVSS
SYP
TAIL-PCR
TAIR
T-DNA
T-SNARE

TGN
TMD

tVS

UTR
VCLl
V-SNARE

VPE
VPS
VSP
VSR
VSS

VTI

=_N_-Iermina1 Propeptide

=Iobacco Syntaxin Related Protein

=Qsmotic Stress-Sensitive Mutant
=Protease-Associated

=Precursor Agcumulating Vesicle

=Phosphate Suffered Saline

=Polymerase Chain Reaction

=P_in-Formed

=Precursor Protein _Y_esicle

=floteinase A

=Proteinase_R

=Protein Storage Xacuole

=Post-Iranscriptional _G_ene Silencing

=Precursor Xesicle Protein of EkDa

=Preyacuolar Qompartment

=Rough Endoplasmic Reticulum

=Ribogucleic Acid

=MA Interference

=Reverse Transcription-Polymerase Qhain Reaction
=Soybean Embryo Pactor

=Shoot Qravitropism

=SulflIydrol _Endopeptidase

=Stanford Microarray Qatabase

=Soluble fl-Ethylmaleimide-Sensitive Factor Adaptor Protein Rgceptor
=Sequence—Specific _Y_acuolar Sorting Signal
=Syntaxin of Plants

=Ihermal Asymmetric Interlaced Polymerase Qhain Reaction
=Ihe Arabidopsis Information Resource
=Iransferred _Qeoxyribogucleic Acid
=Iarget-_S_oluble R-Ethylmaleimide—Sensitive Factor Adaptor Protein
R_eceptor

=Irans-Qolgi fletwork

=_T_ransmembrane Romain

=Irans-yacuolar strands

=QnIranslated Region

=yaguoleIessl

=_\_/esicle Soluble fl-Ethylmaleimide-Sensitive Factor Adaptor Protein
R_eceptor

=Xacuolar Processing Pnzyme

=_\_/acuolar Protein Sorting

=Xegetative Storage Protein

=Xacuolar Sorting Receptor

=Xacuolar Sorting Signal

=XPS-Ien Romologue

=\_fPS-_T_en Interacting Factor

xiv

Chapter 1

Introduction

An Overview of Protein Trafficking Through the Endomembrane System

 

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I. Introduction

The eukaryotic cell is compartmentalized into numerous types of organelles that
perform specific functions. In order for organelles to perform their fimctions, proteins
must be correctly targeted to the appropriate compartment. Protein targeting to
organelles such as the nucleus, chloroplast, mitochondria, and other organelles occurs
from the cytoplasm directly to the organelle. The endoplasmic reticulum (ER), Golgi,
vacuole, and plasma membrane are connected through the endomembrane system (Figure
1.1). Soluble proteins are delivered to organelles of the endomembrane system via
vesicle trafficking (Alberts et al., 1994)(Figures 1.2 and 1.3). In general, the process of
vesicle trafficking from one organelle to another occurs in the following way: cargo
proteins are brought to a specific domain of the originating organelle by aggregation,
bulk flow, or through interaction with protein sorting receptors (Figure 1.2). Adaptor
proteins on the surface of the developing vesicle recruit coat proteins to the budding
vesicle. Coatamer is the coat protein that surrounds vesicles that emerge from the cis-
Golgi and the ER whereas clathrin coats the vesicles that bud from the trans-Golgi
Network (TGN) and the plasma membrane. The combined effects of the coat proteins
and other accessory proteins force the vesicle to bleb away from the organelle (Figure
1.2). Then, the coat proteins are shed from the vesicle afier its release from the
originating organelle (Figure 1.3).

The shedding of the vesicle coat exposes SNAREs (soluble 1_\l_-ethy1ma1eimide-
sensitive factor _adaptor protein geceptor) on the vesicle surface that help specify the

identity and appropriate destination organelle of that vesicle (Figure 1.3).

 

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Figure 1.1. A schematic of the endomembrane system. mRNAs encoding soluble
endomembrane proteins are exported from the nucleus and cotranslationally inserted into
the rough endoplasmic reticulum (RER) (colored lines). ER resident proteins remain in
the ER (red circles), whereas other proteins are delivered to the Golgi via vesicle
trafficking (green, yellow, and blue circles). Golgi-resident proteins are delivered to the
cis, medial, or trans Golgi (green circles). At the TGN, a sorting event occurs in which
secreted proteins (dark blue circles) are delivered to the plasma membrane by the default
pathway and vacuolar/endosomal proteins (yellow circles) are packaged into vesicles
(usually CCV) and are delivered to the vacuole/lysosome via an endosome/PVC.
Adapted from Alberts, et al., 1994.

 

Figure 1.2. Many factors are required for vesicle formation. The first step of vesicle
formation is that the cargo proteins are recognized by protein sorting receptors or
aggregate into a specific area of the organelle (A). v-SNARES also accumulate on the
surface of the developing vesicle for later identification of the vesicle. The cytoplasmic
tail of the receptor protein interacts with the coat forming complex to affix a coat around
the budding vesicle (B). The coat protein and other factors pinch off the membrane to
release the budding vesicle (C). Adapted from Alberts, 1994.

Organelle

 

- of Origin E22] v-SNARE _< Protein Sorting Receptor

Q Vesicle 0

cis t-SNARE .
Complex

Cargo Protein (3 Adaptor Complex

Aggregating m Coat Protein

Protein

 

Figure 1.3. The interaction of SNAREs drives membrane fusion between a vesicle
and its target organelle. The coat protein is shed from the vesicle after budding fi'om
the originating organelle (A). The v-SNAREs are thus exposed on the surface of the
vesicle and can interact with the t-SNAREs of the target organelle (B). Membrane fusion
occurs and the contents of the vesicle are deposited into the destination organelle (C). At
this step, the receptor protein releases the cargo in the destination organelle. Adapted
from Alberts, 1994.

a

 

O Cargo Protein -<Protein Sorting Receptor 0 Vesicle v-SNARE

Destination cis t-SNARE

an ' "'—
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Because vesicles are arriving and departing from multiple organelles, it is
necessary for the vesicle and target organelle to be appropriately identified. Proper
identification of compartments is achieved through the action of SNARE proteins
(Sanderfoot et al., 2000). There are vesicle (v)-SNAREs that are localized to specific
vesicles as well as target (t)-SNAREs that are localized to the membranes of
compartments receiving vesicles (Figures 1.2 and 1.3). Three t-SNAREs on the target
organelle form a cis-SNARE complex that can interact with a v-SNARE on the surface of
a vesicle destined for the organelle (Figures 1.2 and1.3) (Surpin and Raikhel, 2004). The
interaction between the v-SNARE and its cognate cis-SNARE complex allows for the
membrane fusion between the vesicle and target organelle (Figure 1.3) (Surpin and
Raikhel, 2004). The membrane fusion event allows the deposition of the vesicle contents
into the target compartment.

The actions of vesicle budding and fusion occur at all points in the
endomembrane system in order to deliver newly synthesized proteins to the appropriate
organelle for their firnctions (Figure 1.1). Soluble endomembrane proteins have an
amino-terminal signal peptide that is recognized by the ribosomes so that the nascent
polypeptide is transported to the rough ER (RER) membrane. The protein is then
cotranslationally inserted into the lumen of the ER and the signal peptide is cleaved from
the protein. Most proteins that belong in downstream compartments of the
endomembrane system are delivered to the cis-Golgi and onward to the TGN. At the
TGN, a sorting event occurs in which proteins that have positive sorting information are
separated away fiom secreted proteins. Proteins without any intracellular sorting

information are secreted to the plasma membrane by the default pathway. Vacuolar

 

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proteins carry sorting signals that direct their delivery to vacuoles and will be discussed
in more detail later (Vitale and Raikhel, 1999).

Plants have multiple types of vacuoles and, based on genome sequencing projects,
encode many more genes than other organisms to carry out trafficking through the
endomembrane system (Paris etal., 1996; Sanderfoot et al., 2000; Park et al., 2004). For
example, one vacuolar sorting receptor, vacuolar protein sorting 10 (V ple), has been
characterized in S. cerevisae (Marcusson et al., 1994) and two other putative vacuolar
sorting receptors have been identified in the S. cerevisae (Cooper and Stevens, 1996;
Westphal et al., 1996). Likewise, two receptors that deliver proteins fiom the TGN to the
lysosome have been characterized in humans (Dahms and Hancock, 2002). By sequence
homology, Arabidopsis encodes seven genes that may function as vacuolar sorting
receptors (VSR) (Hadlington and Denecke, 2000; Shimada et al., 2003). It is still not
clear why higher plants encode so many more vacuolar sorting receptors than yeast or
mammals. The focus of my research has been to understand the expression patterns
and/or functions of a family of putative vacuolar sorting receptors in Arabidopsis. A
background of vacuolar/lysosomal transport that has been characterized in other systems
is useful to my study since many of the models and ideas about vacuolar transport in

plants stem from what has been learned in animals and yeast.

11. Lysosomal Transport in Animal Cells
The lysosome is the animal cell equivalent of the plant cell lytic vacuole. The
lysosomes are acidic compartments that degrade proteins. Protein trafficking to the lytic

vacuole is essential. The absence of vacuolar trafficking in humans leads to I-Cell

 

 

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Disease, which kills patients during childhood (Dahms and Hancock, 2002). Proteins
that are targeted to the lysosome, such as proteases, are modified in the Golgi with a
mannose-6-phosphate (M-6-P) moiety that serves as a lysosomal sorting signal (von
Figura and Hasilik, 1986).

Two mannose-6-phosphate receptors (MPR) have been isolated and characterized,
the 46kDa cation-dependent MPR (CD-MPR) and the ~300kDa insulin-like growth factor
II/MPR (IGFII/MPR) (Sahagian et al., 1981; Hoflack and Komfeld, 1985a, b; Dahms and
Hancock, 2002). MPRs are type I transmembrane proteins that contain an N-terminal
signal peptide, a long extracytoplasmic domain and a short cytoplasmic tail (Dahms and
Hancock, 2002). MPR localizes to the Golgi, clathrin-coated vesicles (CCV), plasma
membrane, and endosomes, but not in lysosomes (Fischer et al., 1980; Sahagian et al.,
1981; Campbell and Rome, 1983; Geuze et al., 1984; Klumperman et al., 1993; Le
Borgne and Hoflack, 1997). Lysosomal proteins carrying the M-6-P signal also localize
to the late Golgi, TGN, and CCVs (Campbell and Rome, 1983; Geuze et al., 1984; Geuze
et al., 1985; von Figura and Hasilik, 1986). Therefore, receptor and cargo proteins are
colocalized upstream of the lysosome. Furthermore, MPRs interact with the M-6-P
moiety of lysosomal enzymes with high affinity and dissociate in pH conditions that
resemble an intermediate endosomal compartment (Sahagian et al., 1981; Fischer et al.,
1982; Tong et al., 1989; Tong and Komfeld, 1989; Dahms and Hancock, 2002). The
cytoplasmic tail of MPR has a tyrosine motif and other motifs that are responsible for its
subcellular localization (Dell'Angelica and Payne, 2001). From these observations, a
model of MPR function in lysosomal trafficking has developed (Dahms and Hancock,

2002). The M-6-P moiety of the cargo protein interacts with MPR in the in the neutral

10

. "5 funk“:

 

I

adaptor

pH em

CCV b

the moi

lysoson

III. Va

 

 

pH environment of the TGN. The cytoplasmic tail of MPR interacts with clathrin coat
adaptor proteins and other factors to package the cargo and receptor into a CCV. The
CCV buds from the TGN and is delivered to the endosome. MPR releases the cargo in
the more acidic environment of the endosome. Finally, the cargo is delivered to the

lysosome while MPR is recycled either to the TGN or to the plasma membrane.

111. Vacuolar Transport in S. cerevisiae

In yeast, as in animals, the primary function of the vacuole is protein degradation.
However, the vacuole is not an essential organelle in yeast (Horazdovsky et al., 1995).
The dispensability of the vacuole has made yeast a useful system for studying trafficking
through the endomembrane system (Horazdovsky et al., 1995). There are multiple
pathways to the vacuole in S. cerevisiae that include vesicle trafficking from the TGN,
cytoplasm-to-vacuole traffic, endocytosis, as well as autophagy. Two of the vesicle-
mediated pathways from the TGN to the vacuole are the carboxypeptidase Y (CPY)
pathway which delivers proteins to the vacuole via a prevacuolar compartment and the
alkaline phosphatase (ALP) pathway that bypasses the prevacuolar compartment (PVC)
(Horazdovsky et al., 1995; Cowles etal., 1997; Piper et al., 1997).

Unlike animals, plant and fimgal vacuolar proteins have peptide-based vacuolar
sorting signals (VSSs) that are part of the translated protein, rather than a sugar
modification to the protein. For example, mutations of single amino acids within the [Q]-
[R]-[P]-[L] motif of CPY results in secretion of the protein (V alls et al., 1987; Valls et

al., 1990). This motif is also sufficient to redirect normally secreted proteins to the

11

 

\'3CU(

TICCBS

 

I'DSIO

rec.VCIt'r;

 

 

vacuole (Johnson et al., 1987). Therefore, the QRPL motif of the CPY precursor is
necessary and sufficient to direct a protein to the yeast vacuole.

In S. cerevisiae, a vacuolar sorting receptor was identified in a mutant screen to
find lines that secrete CPY, rather than deliver it to the vacuole (Marcusson et al., 1994).
Vplep (vacuolar protein sorting 10) is a type I transmembrane protein with a large
lumenal domain (1373 amino acids), a transmembrane domain of 20 amino acids, and a
160-amino acid cytoplasmic tail (Figure 1.4) (Horazdovsky et al., 1995; Jorgensen et al.,
1999). The lumenal domain begins with an amino terminal signal peptide for insertion
into the ER membrane. The remainder of the lumenal domain is composed of domain 1
and domain 2 (Figure 1.4) (Horazdovsky et al., 1995; Jorgensen et al., 1999). Each
domain has a cysteine-rich region (Jorgensen et al., 1999). The cytosolic domain has a
tyrosine motif (Cooper and Stevens, 1996). Thus, Vple is very similar in structure to
MPR.

Biochemical and microscopy approaches indicate that both CPY and Vple
localize to the late Golgi, CCVs, and a prevacuolar compartment (Vida et al., 1993;
Marcusson et al., 1994; Cooper and Stevens, 1996; Seaman et al., 1997; Deloche et al.,
2001). Furthermore, CPY interacts with Vps10 in the late Golgi (Marcusson et al., 1994;
Cooper and Stevens, 1996). These data indicate that vacuolar proteins interact with the
Vple receptor in the late Golgi and both proteins are delivered to the PVC via CCVs.
This pathway is analogous to the MPR pathway of animals. The stoichiometry of the
Vple-CPY interaction is 1:1, however, CPY is expressed 20 times higher than the
Vple (Cooper and Stevens, 1996). This suggests that the Vple receptor must be

recycled for multiple rounds of vacuolar sorting (Cooper and Stevens, 1996).

12

 

 

   

Figure ‘
plant V;
(A) (cc,

:1
13.
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. m
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A Domain 1 Domain 2 1393

 

 

 

 

 

   

 

 

 

 

"HI

 

 

 

1373 1553
B
F KKK!
1
Signal Transmembrane g . . Cysteine-Rich
I Peptide El Domain Tyrosrne Motif E Repeats

m PA Domain

Figure 1.4. Structure of the S. cerevisiae Vplep vacuolar sorting receptor and a
plant vacuolar sorting receptor. A schematic representing the structure of Vplep
(A) (Cooper & Stevens, 1996; Westphal, et al., 1996; Jorgensen, et al., 1990) and a
typical plant VSR (B) (Ahmed, et al., 1997; Shimada, et al., 2003; Paris, et al., 1997).
The lumenal region of Vple is divided into domain 1 and domain 2 with each domain
carrying a cysteine-rich motif. The cytoplasmic tail has a Tyrosine motif. Plant VSRs
also have cysteine-rich repeats in the lumenal domain and a tyrosine motif in the c-
terminal tail. They also have a PA domain in the lumenal region of the protein. The
schematic is not drawn to scale.

13

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The C-terminal tail contains a tyrosine-based motif that is thought to be responsible for
the recycling of Vps10p to the Golgi (Deloche et al., 2001). Mutation of the tyrosine in
the signal leads to degradation of Vplep in the vacuole and secretion of CPY (Deloche
et al., 2001). Candidates for proteins that interact with the C-terminal tail of Vplep to
direct its subcellular localization include adaptor protein-1 (AP-1), sorting nexins such as
VpsSp, Vp329p, Vps30p, and Vps35p (Horazdovsky et al., 1997; Seaman et al., 1997).
Therefore, it is likely that Vplep relies on these proteins to be recycled for further
rounds of lysosomal trafiicking.

From the accumulated data, a model of Vplep function is proposed that is
analogous to lysosomal sorting in animal cells (Horazdovsky et al., 1995). The QRPL
motif of precursor CPY interacts with the second domain of Vplep in the late Golgi
(Marcusson et al., 1994; Cooper and Stevens, 1996). The receptor-CPY complex is
packaged into CCVs and delivered to a PVC (Vida et al., 1993; Deloche et al., 2001).
The CPY precursor dissociates from Vplep in the PVC (Cooper and Stevens, 1996).
CPY is then delivered to the vacuole and Vplep is recycled to the Golgi (Horazdovsky
et al., 1995; Horazdovsky et al., 1997).

Vplep is not the receptor for all soluble vacuolar proteins, nor is the CCV
pathway the only pathway for protein transport to the vacuole. For example, the vacuolar
localization of the proteinase A (PrA), proteinase B (PrB), and ALP are not significantly
affected in the Avps10 mutant (Marcusson et al., 1994). Therefore, PrA and PrB must be
delivered to the vacuole by a Vple-independent pathway (Marcusson et al., 1994). The
S. cerevisiae genome encodes two more genes, VTHl and VTH2 (yps Ien Romologue)

that show significant similarity to VPS10 (Cooper and Stevens, 1996; Westphal et al.,

14

 

1996).

is 50° 0
al.. 19¢
studied

recepto

IV. So

 

 

 

1996). The proteins are 70% identical to Vps10 over the whole protein, specifically, there
is 50% identity in the first domain and 88% identity in the second domain (W estphal et
al., 1996). However, they are not expressed to detectable levels under the conditions
studied (W estphal et al., 1996). So, it is not known whether these are firnctional

receptors.

IV. Soluble Vacuolar Protein Transport in Plants

The plant cell vacuole is an essential organelle (Rojo et al., 2001). Arabidopsis
embryos that cannot form a vacuole, such as the vacuolessl mutant accumulate
autophagosome structures and die in the torpedo stage (Rojo et al., 2001). Transport
through the endomembrane system is important to a plant’s ability to respond to its
environment (Surpin and Raikhel, 2004). For example, the Arabidopsis VTI (Vple-
interacting factor) SNAREs mediate the gravitropic and starvation responses (Surpin et
al., 2003), whereas NtSyrl mediates stress response pathways in tobacco (Leyman et al.,
2000). Other components of vesicle trafficking in plants also play important roles in
signal transduction, and solute homeostasis (Weintraub, 1952; MacRobbie, 1999; Kato et
al., 2002; Zhu et al., 2002). Plants encode more genes whose proteins function in the
endomembrane system than other organisms and direct proteins to multiple types of
vacuoles (Figure 1.5) (Paris et al., 1996; Sanderfoot et al., 2000; Park et al., 2004).

The lytic vacuole is an acidic organelle equivalent to the yeast vacuole and animal
lysosome. The lytic vacuole is responsible for ion homeostasis, storage of secondary

metabolites such as anthocyanins, as well as protein turnover (Surpin and Raikhel, 2004).

15

aa “a
PAC/KV/ERV”
. ® ——»
«E/
. o
O.
O

  
  
  

 

Figure 1.5. Protein transport through the endomembrane system of plants. Proteins
are cotranslationally inserted into the rough endoplasmic reticulum (RER). Some
proteins (red circles) aggregate into precursor accumulating vesicles (PAC), KDEL
vesicles (KV), or ER bodies that bleb off the ER and either fuse with multivesicular
bodies (MVB) or are delivered to the protein storage vacuole (PSV), marked by dI‘IP in
developing seeds or by ATIP in vegetative cells (3). Other proteins are delivered to the
Golgi via vesicle trafficking (red, blue, and yellow circles). Storage proteins (red
circles) are packaged into dense vesicles that can fuse with MVBs or be delivered
directly to the PSV (2). Soluble proteins intended for the lytic vacuole (yellow circles)
are packaged into clathrin coated vesicles (CCV) and delivered to the lytic vacuole (LV),
marked by 11' IP and sometimes rxT 1?, via the prevacuolar compartment (PVC). Secreted
proteins (blue circles) are also packaged into CCVs that are delivered to the plasma
membrane (PM) and cell wall (CW). The separate pathways to the vacuole are
maintained even in cell types in which the PSV and LV fuse to form the large central
vacuole (CV).

 

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The tonoplast of the lytic vacuole is marked by the y-TIP aquaporin (Figure 1.5) (Paris et
al., 1996; Jauh et al., 1999). Proteases are abundant in the lytic vacuole. Some proteases,
such as Aleurain, are now common markers for the lytic vacuole (Ahmed et al., 2000).

The protein storage vacuole (PSV) is a neutral pH organelle that is unique to
plants (Figure 1.5) (Vitale and Raikhel, 1999). The role of the PSV is to store proteins
such as lectins, chitinases, legumins (V itale and Raikhel, 1999). The PSV is most
commonly associated with developing seeds but can also form in vegetative tissues under
specific conditions (Paris et al., 1996; Jauh et al., 1999). The PSV tonoplast is
characterized by the presence of a-TIP in seeds and with or-TIP or A-TIP in vegetative
tissues (Figure 1.5) (Paris et al., 1996; Jauh et al., 1999). In many cell types, the lytic
vacuole and the PSV fuse to form a large, central vacuole (Paris et al., 1996). As a result
of the fusion, the central vacuole tonoplast contains a-TIP and y-TIP (Figure 1.5) (V itale
and Raikhel, 1999). The central vacuole contains both storage and lytic vacuolar soluble
proteins.

Positive sorting information is required for protein sorting to the vacuole and, like
S. cerevisae, plants use peptide based VSSs that are part of the translated protein. Three
types of vacuolar sorting signals have been identified in plants (Figure 1.6) (V itale and
Raikhel, 1999). They include the N-terminal propeptide (NTPP; Table 1.1), the C-
terrninal propeptide (CTPP; Table 1.2), and an internal signal. It should also be
mentioned that the C-terminal ER retention signal, KDEL, can also function as a VSS
under certain conditions (V itale and Raikhel, 1999; Schmid et al., 2001; Okamoto et al.,

2003).

17

 

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1999),”

 

Amino Terminal Propeptide (NTPP) C-Terminal Propeptide (CTPP)
i l I I

. ~c 1 . 'T—c
N‘Tlll[::Wn~"_p-mw' N—fiz:I ,:_wwr

The Internal Signal The KDEL Signal

L1 wrists

 

Figure 1.6. Vacuole sorting signals in plants. The schematic depicts three types of
vacuolar sorting signals (VSS) that have been identified in plants and the KDEL signal.
All of the proteins have a signal peptide (blue box) for cotranslational insertion in into
the ER. The signal peptide is cleaved from the protein after insertion into the ER
(arrow after blue box). The NTPP (red box) is a sequence specific VSS. The signal is
cleaved from the mature protein after delivery to the vacuole (arrow afier red box).
The CTPP (grey box) is a C-terminal VSS that is not sequence specific, but may form a
distinctive tertiary structure. The CTPP is also cleaved from the mature protein afier
delivery to the vacuole (arrow before grey box). The internal signal (green box) is
necessary for delivery to the vacuole and is part of the mature protein. The KDEL
signal (purple box) normally functions as an ER retention signal, however, can act as a
vacuolar sorting signal under specific conditions. (Reviewed in Vitale & Raikhel,
1999).

"I

" g L‘- man-r.

 

protl

to di

 

 

 

 

 

The NTPP and CTPP signals are amino acid sequences that are cleaved from the mature
protein after delivery to the vacuole (Figure 1.6). However, each type of VSS is thought

to direct proteins to different vacuoles by different pathways.

Table 1.1. N-Terminal Propeptides

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Protein Amino Acid Sequence of Signal Reference
(amino acid position)

N-terminal N T PP

Sweet Potato Sporamin (22)HSRFNPIRLPTTHEPA... (Matsuoka and Nakamura,
1991)

Barley Aleurain (22)SSSFADS&_IRPVTDRAAS. .. (Holwerda et al., 1992)

Arabidopsis Aleurain (22)ANIGFDES_Nfl{MVSDGLR. .. (Ahmed et al., 2000)

Potato PTZO (19)STFTSKNPI_N:_LPSDA. .. (Koide et al., 1999)

Internal NTPP

Castor Bean Ricin (303)8LLIRPWPNFN. . .(576) (Frigerio et al., 2001)

C-terminal N T PP

Brazil Nut 2S Albumin (l30)NL_P_SMRCPMGGSIAGF(C-terrninus) (Kirsch et al., 1996)

Arabidopsis ZS Albumin (150)VCP_N;S_FPS(C-terminus) (D'Hondt et al., 1993)

Table 1.2. C-Terminal Propeptides

Protein Amino Acid Sequence of Signal Reference

Barley Lectin VFAEAIAANSTLVAE (Bednarek et al., 1990)

Tobacco Chitinase GLLVDTM (Neuhaus et al., 1991)

Phaeseolin AFVY (Frigerio et al., 1998;
Holkeri and Vitale, 2001)

a’-Soybean B.Conglycinin PLSSILRAFY (Nishizawa et al., 2003)

Tobacco Proteinase Inhibitor SEYASKVDEYVGENDLQKSKVAVS (Miller et al., 1999)

Tobacco AP24 QAHPNFPLEMPGSDEVAK (Melchers et al., 1993)

Horseradish Peroxidase Cla LLHDMVEVVDFVSSM (Matsui et al., 2003)

B-l,3—Glucanase VSGGVWDSSVETNATASLVSEM (Melchers et al., 1993)

 

 

 

 

Just as multiple pathways to the vacuole exist in other organisms, plants are
thought to deliver proteins to the vacuole by a dense vesicle pathway (Figure 1.5,
pathway 2), a precursor protein vesicle (PPV) pathway (Figure 1.5, pathway 3), and a

CCV pathway (Figure 1.5, pathway 1) (Hayashi et al., 2001; Schmid et al., 2001;

19

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cam:
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Okamoto et al., 2003; Rojo et al., 2003a). It is likely that there is cross-talk between
these pathways (Surpin and Raikhel, 2004).

The evidence for the presence of multiple pathways to vacuoles in plants first
came from the observation that the trafficking of NTPP and CTPP proteins have different
sensitivities to wortrnannin, a PI-3-kinase inhibitor (Matsuoka et al., 1995). PSV proteins
localize to dense vesicles and not to CCVs (Hohl et al., 1996). Therefore, soluble PSV
proteins are packaged into dense vesicles at the TGN and are delivered to the protein
storage vacuole (Hohl et al., 1996; Paris et al., 1996). Proteins with CTPP signals are
thought to travel by the dense vesicle pathway (Matsuoka et al., 1995; Hohl et al., 1996).
While there is no conserved sequence motif, characterized CTPPs tend to be hydrophobic
and are strictly located at the C-terminus of a protein (Bednarek et al., 1990; Dombrowski
et al., 1993; Neuhaus et al., 1994). A receptor for CTPP proteins has not been identified.
Some researchers have speculated that aggregation of these proteins in the Golgi is the
mechanism of their trafficking from the TGN (V itale and Raikhel, 1999). This model is
analogous to a regulated secretion model that has been described in animals (V itale and
Denecke, 1999). However, the vacuolar sorting of CTPP proteins is saturable, suggesting
that a receptor is involved in the process (N euhaus et al., 1994).

Some proteins that reside in the storage vacuole actually aggregate in the ER into
vesicles that bleb from the ER (Okamoto et al., 1994; Hayashi et al., 2001; Schmid et al.,
2001; Okamoto et al., 2003; Rojo et al., 2003b). These vesicles are called ER bodies,
precursor accumulating vesicles (PAC), PPVs, and KDEL vesicles (KV) (Shimada et al.,
1997; Hayashi et al., 2001; Tsuru-Furuno et al., 2001; Rojo et al., 2003b). The PPVs can

fuse with dense vesicles to form multivesicular bodies (MVBs) which are delivered to the

20

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‘ win-awn
. ,._.

 

“Hue
Subch

munf

 

Ibhk
as A1

”SUI

 

PSV (Tse et al., 2004). It has even been suggested that MVBs may be a PVC for the
PSV (Tse et al., 2004). Proteins that have KDEL signals and/or novel, uncharacterized
signals use this pathway. The KDEL signal normally functions as an ER—retention signal
when present at the C-termini of proteins. However, some proteins that have C-terminal
KDEL signals, such as the papain type cysteine proteinase, SH-EP, are delivered to the
vacuole during senescence and programmed cell death via ER-derived vesicles (V itale
and Denecke, 1999; Tsuru-Furuno et al., 2001). What is still unclear is how the KDEL
signal can function as an ER retention signal and as a VSS. The context of the protein
and the environmental conditions likely regulate this process although the mechanisms of
such regulation are still unclear. The KDEL signal can confer ER and vacuolar
localization to green fluorescent protein (GFP) when KDEL is fused to the C-terrninus of
GFP and stably expressed fi'om a constitutive promoter in Arabidopsis (Di Sansebastiano
et al., 1998; Hayashi et al., 2001). Further insight into the dual functions of the KDEL
signal may come from hi gh-throughput proteomics and protein localization projects.
Proteins that have NTPP VSSs are thought to be delivered to the lytic vacuole by
a CCV pathway that is analogous to what has been described in yeast and animals
(Ahmed et al., 2000). The NTPP has been extensively characterized in plants and has a
conserved sequence motif. Site-directed mutagenesis of the prosporamin propeptide and
subsequent transient expression in Tobacco BY-2 cells revealed that the consensus NTPP
motif is: [preferably Asn]-[not acidic]-[Ile or Leu]-[X]-[Large and hydrophobic]
(Matsuoka and Nakamura, 1999). Although the NTPP of known vacuolar proteins, such
as Aleurain, is at the amino terminus, the placement of a functional NPIR motif is not

restricted to the amino-terminus (Kirsch et al., 1996; Koide et al., 1997; Frigerio et al.,

21

 

200 1

etal

prote

Luca.

Pump
(Arab
Iike pr
80 wa
the N“
Precu:

Arab;

 

Chile: ‘

199::

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Clem

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2001). An interesting example of this comes from studies of the ricin precursor (Frigerio
et al., 2001). The ricin precursor was previously thought to have an internal sorting
signal (Frigerio et al., 2001). However, it is now clear that the internal signal of ricin has
an NPIR motif that directs ricin to the vacuole (Frigerio et al., 2001). Similar results
have been observed for 2S albumin which also has an NPIR motif in its C-terminus
(Kirsch et al., 1996; Shimada et al., 2002). What is curious about these precursor

proteins is that these are proteins that are localized to the PSV, not to the lytic vacuole.

Vacuolar Sorting Receptors of Plants

Receptors for NTPP-containing proteins include pea binding protein-80 (BF-80),
pumpkin PV72 (precursor yesicle protein of flkDa), and Arabidopsis AtVSRl/AtELP
(Arabidopsis vacuolar sorting receptor l/Arabidopsis gpidermal growth factor receptor-
Iike protein) (Kirsch et al., 1994; Ahmed et al., 1997 ; Shimada et al., 2002). The pea BP-
80 was identified by affinity chromatography of CCVs to identify proteins which bind to
the NTPP of Barley Aleurain (Kirsch et al., 1994). The pumpkin PV72 was isolated from
precursor accumulating (PAC) vesicles of developing seeds (Shimada et al., 1997). The
Arabidopsis AtVSRl/AtELP was identified by a bioinformatics approach to identify
cysteine-rich repeats that are present in receptor proteins of other systems (Ahmed et al.,
1997)

Like MPR and Vple, plant VSRs are type I transmembrane proteins with long
lumenal domains followed by short transmembrane domains and short cytoplasmic
domains (Figure 1.4) (Ahmed et al., 1997). The amino terminus of the plant VSR has a

signal peptide for cotranslational insertion into the ER membrane (Ahmed et al., 1997).

22

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1997;
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A protease-associated (PA) domain follows the signal peptide (Mahon and Bateman,
2000). There are also three cysteine-rich epidermal growth factor (EGF) repeats within
the lumenal domain, one of which is a calcium-dependent EGF repeat (Watanabe et al.,
2002). The cytoplasmic domain has a tyrosine motif that interacts with the clathrin coat
adaptor proteins at the TGN (Sanderfoot et al., 1998; Happel et al., 2004). It also
contains a diacidic motif for export from the ER (Matsuoka and Bednarek, 1998).

AtVSRl and BP-80 have been localized to the TGN, CCVs, the PVC, and
colocalize with proteins destined for the lytic vacuole (Kirsch et al., 1994; Ahmed et al.,
1997; Sanderfoot et al., 1998; Ahmed et al., 2000; Li et al., 2002). These locations are
consistent with what is known about the vacuolar/lysosomal sorting receptors of animals
and yeast, MPR and Vple. On the other hand, PV72 localizes with PSV proteins in
PAC vesicles (Shimada et al., 1997). BP-80 and AtVSRl interact with NTPP signals in
vitro in a sequence-specific and pH-dependent manner (Kirsch et al., 1994; Ahmed et al.,
1997; Paris et al., 1997; Ahmed et al., 2000). These interactions are also similar to the
CCV pathways of animals and yeast. However, PV72 interacts with 2S Albumin in a
calcium-dependent manner (Shimada et al., 2002; Watanabe et al., 2002). Fluorescence-
based ligand binding assays of deletion mutants of BP-80 indicate that while the region
responsible for binding the ligand is N-terminal to the EGF repeats, the EGF repeats
enhance the stability of the interaction (Cao etal., 2000). Similarly, PV72 interacts with
28 Albumin in a region outside of the EGF repeats in a calcium-dependent manner
(Shimada et al., 2002; Watanabe et al., 2002). The third EGF repeat mediates the
calcium-dependency of the interaction (Shimada et al., 2002; Watanabe et al., 2002).

Thus, despite having different localization patterns and ligand dissociation requirements,

23

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the n
PV72
repea
ergo
The l
identi
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intera

(Sand
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the mechanisms and domains required for the interaction are quite similar. BP-80 and
PV72 both interact with cargo proteins in a region that is amino terminal to the EGF
repeats and use the EGF repeats to stabilize the interaction between the VSR and the
cargo protein. It is possible that the PA domain is the site of cargo protein interaction.
The PA domain is a region upstream of the EGF repeats. The PA domain has been
identified in diverse proteins (Mahon and Bateman, 2000). While the function of the PA
domain is not known, it has been proposed that the PA domain is a site of ligand
interactions (Mahon and Bateman, 2000).

The cytoplasmic tail of the plant VSR has a tyrosine motif like Vple and MPR
(Sanderfoot etal., 1998; Bonifacino and Dell'Angelica, 1999; Shimada et al., 2002). The
tyrosine motif is a short amino acid sequence composed of: [Y]-[X]-[X]-[Bulky
hydrophobic] (Bonifacino and Dell'Angelica, 1999). The Tyrosine motif interacts with
clathrin coat adaptor proteins either at the plasma membrane or at the TGN (Sanderfoot et
al., 1998; Bonifacino and Dell'Angelica, 1999; Happel et al., 2004). The tyrosine motif
of AtVSRl and BP-80 specifically interacts with the TGN-specific adaptor proteins and
does not interact with the plasma membrane specific adaptor protein (Sanderfoot et al.,
1998; Happel et al., 2004). These. results suggest that the cytoplasmic tail functions
similarly to the C-terminal tail of Vple and MPR.

The current model of how this protein functions in the context of the CCV
pathway is that the receptor interacts with an NTPP-containing protein at the TGN and
both proteins are packaged into CCVs that are delivered to the PVC (Figure 1.7) (Ahmed
et al., 2000). The receptor releases the cargo in the more acidic pH environment of the

PVC and is presumably recycled to the TGN by vesicle trafficking whereas the cargo is

24

      
 

PPV/PAC/KV/ER Body

 

WWWNN XMW

Figure 1.7. The current model of plant VSR function and the localization of other
VSRs within the endomembrane system. Soluble proteins intended for the lytic
vacuole (yellow circles) are recognized by plant VSRs (green hooks) and both are
packaged into clathrin coated vesicles (CCV) and delivered to the PVC. At the PVC, the
VSR dissociates from the cargo protein. The cargo protein is delivered to the lytic
vacuole, whereas the VSR is recycled back to the TGN. VSRs have also been detected
in PPVs as well as in MVBs (purple hooks) (Shimada, et al., 1997; Tsuru-Furuno, et al.,
2001).

25

delivered to the lytic vacuole by either vesicle trafficking or fusion of the PVC with the
vacuole. However, two observations indicate that this model is over-simplified. First,
the Arabidopsis genome encodes multiple genes that show high similarity to
AtVSRl/AtELP and thus may also function as VSRs. Multiple homologues of BP-80
and AtELP have since been identified in bean, pumpkin, wheat, and rice (Paris and
Neuhaus, 2002). The Arabidopsis genome encodes seven genes that show at least 50%
identity at the amino acid level to AtVSRl/AtELP. Rice encodes nine genes that are
homologous with AtVSRl/AtELP and BP-80 (unpublished results). Second, despite
sharing high amino acid identity, plant VSRs are found in different types of vesicles and
have been implicated in different types of vacuolar transport pathways. Recently, data
from a reverse genetics approach to understand plant VSRs demonstrated that AtVSR]
mediates the delivery of 2S Albumin to PSVs in developing Arabidopsis seeds (Shimada
et al., 2003). These results indicate that AtVSRl may also function like PV72 (Shimada
et al., 2003). Furthermore, an AtVSRl homolog was isolated from KV vesicles from
developing seeds of Vigna mun (Tsuru-Furuno et al., 2001). While cross-talk between
the vacuolar trafficking pathways may account for some of these results, it is now
probable that the same family of receptors mediates vacuolar protein traffic by different

pathways.

V. Thesis Overview

As mentioned above, the Arabidopsis family of VSRs contains seven members.

These proteins have been called by different names in the past (Table 1.3). The current

26

 

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200.

__-'-I
.1”
J. 3'

LIET:T

lyILILT

1
Di

 

nomenclature proposed is that by Dr. Hara-Nishimura and coworkers (Shimada et al.,

2003).

Table 1.3. Nomenclature for Arabidopsis Vacuolar Sorting Receptors

 

 

 

 

 

 

 

 

 

 

 

AGI Accession Nomenclature I Previously Used Nomenclature From Shimada et al.,
2003

At3g52850 AtELP AtELP/AtVSR]

At2g30290 AtVSR3 AtVSR2

At2g14740 AtVSRl AtVSR3

At2gl4720 AtVSR2 AtVSR4

At2g34940 AtVSR4 AtVSRS

Atl g3 0900 AtVSR6 AtVSR6

At4g201 10 AtVSRS AtVSR7

 

 

For my dissertation, I have adopted the nomenclature from Shimada, et al., 2003.
An important goal in this field is to identify the individual and overlapping functions of
the AtVSRs. A keen understanding of this issue will help us understand why plant VSRs
occur in such large families. My goals for this study were to determine the function(s) of
these genes. One possibility is that each gene performs a similar function and the
expression of each is restricted to specific cell types. The second possibility is that each
protein performs a distinct function and thus there are multiple VSRs expressed in most
plant cells. The third possibility is that the VSRs have completely redundant functions. It
is also possible that a combination of these scenarios exists.

To address this question, I determined the expression patterns of the individual
AtVSR genes and took advantage of the many bioinformatics tools available. To address
the function of each gene at the protein level, I attempted to isolate knockouts of each
gene, post-transcriptionally silenced individual members as well as the entire family of
AtVSRs, and used biochemical means to identify the receptor of specific soluble vacuolar

proteins. These approaches could lead to a greater understanding of the expression

27

 

pattc‘

tunc

 

 

 

 

patterns of members of the VSR family as well as an understanding of the physiological

function of at least one of the VSR proteins.

28

Alb

Bed

Bani

Cam

Cao

 

C00;

 

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37

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38

Chapter 2

Expression Analysis of the Vacuolar Sorting Receptor Family in Arabidopsis

39

Abstract

In plant cells, soluble proteins that are delivered to the vacuole via the
endomembrane system contain a vacuolar sorting signal (VSS). Three types of VSSs
have been identified in plants. One class of VSS is the N-terrninal propeptide (NTPP)
that is cleaved from the mature protein and contains a conserved peptide motif required
for vacuolar sorting. A putative vacuolar sorting receptor (VSR) for NTPP-containing
proteins is the Arabidopsis VSRl, formerly known as AtELP. AtVSRl is a type I
transmembrane protein with three cysteine-rich EGF repeats. Homologues of AtVSRl
have been identified in numerous plant species. The current model of VSR function is
that the VSR interacts with NTPP-containing proteins at the trans-Golgi Network (TGN)
and recruits them into clathrin-coated vesicles that are delivered to the prevacuolar
compartment (PVC). At the PVC, the VSR releases the cargo and is returned to the TGN
while the cargo proteins are sent to the vacuole. Phylogenetic analysis of all known plant
VSRs indicated that the VSRs are subdivided into three groups. AtVSR] has six
homologues in the Arabidopsis genome with at least one homolog in each of the three
groups. Expression of all members of this gene family has been detected in various plant
tissues by RT-PCR. The cell type-specific expression pattern of each gene was
determined by promoterzzGUS fusion and indicated that the AtVSRs have cell-type
specific expression patterns. The overall results indicated that the seven members of the

AtVSR gene family are not completely redundant.

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Introduction

The plant cell vacuole is a multifunctional organelle that is essential to plant
growth and development (Rojo et al., 2001; Surpin and Raikhel, 2004). However, the
identity and function of the vacuole is dependent on the proper targeting of vacuolar
proteins. Soluble proteins are delivered to the vacuole via vesicle trafficking of the
endomembrane system. These proteins are first cotranslationally inserted into the rough
endoplasmic reticulum (RER). In the ER, some vacuolar proteins will segregate into ER
bodies that bleb off the ER membrane and are delivered to the vacuole (Shimada et al.,
1997; Tsuru-Furuno et al., 2001; Rojo et al., 2003). Most vacuolar proteins will be
delivered to the Golgi. Proteins destined for the vacuole are sorted fi'om secreted proteins
at the TGN (V itale and Raikhel, 1999). Vacuolar proteins are distinguished from
secreted proteins by amino-acid sequence-based sorting signals.

Three types of vacuolar sorting signals (VSSs) have been identified in plants. All
three signals are peptide sequences present in the protein. The amino-terminal
propeptides (NTPP) and carboxy-terminal propeptides (CTPP) are amino acid sequences
present at either end of the protein. Both signals are cleaved from the mature protein.
There are also internal signals that are necessary for vacuolar delivery, but these are
poorly characterized and not removed from the mature protein (V itale and Raikhel,
1999). The type of vacuolar sorting signal appears to determine the pathway by which
the protein is delivered to the vacuole (Matsuoka et al., 1995). Proteins that have CTPPs
are packaged into dense vesicles and are delivered to protein storage vacuoles (PSVs). A

CTPP receptor has not yet been identified. On the other hand, proteins that have an

41

 

 

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NTPP are recognized by a vacuolar sorting receptor (VSR) in a sequence specific manner
and are delivered to the lytic vacuole via the PVC (Surpin and Raikhel, 2004).

VSRs have been identified in numerous plants by many methods. The VSR from
pea, BP-80 (binding protein of 80kDa), was isolated from pea clathrin-coated vesicles
(CCVs) by its affinity for the NTPP of the soluble vacuolar protein, barley aleurain
(Kirsch et al., 1994). The first VSR characterized from Arabidopsis, AtELP/AtVSRl
(Arabidopsis EGF receptor-like protein/Arabidopsis vacuolar sorting receptor 1), was
identified by a bioinformatics approach based on the observation that receptor proteins in
other systems have several conserved motifs (Ahmed et al., 1997). The VSR from
pumpkin was identified by a protein analysis of precursor accumulating (PAC) vesicles
(Shimada et al., 2002). Likewise, a VSR from Vigna mango was identified from a
protein analysis of KDEL-vesicles that travel fiom the ER to the vacuole (Tsuru-Furuno
et al., 2001).

All of the plant VSRs share at least 50% sequence identity throughout the whole
protein. The VSRs are type-1 transmembrane proteins that have a large lumenal domain
and a short cytoplasmic domain (Ahmed et al., 1997). The lumenal domain has a signal
peptide for insertion into the ER, followed by a protease-associated (PA) domain (Mahon
and Bateman, 2000). Researchers have speculated that the PA domain may be the site at
which the VSR interacts with protein cargo in the TGN (Cao et al., 2000; Mahon and
Bateman, 2000; Watanabe et al., 2002). The lumenal domain also contains three
epidermal growth factor (EGF) repeats that may regulate the protein-protein interactions

with ligands (Ahmed et al., 1997; Watanabe et al., 2002). The cytoplasmic tail mediates

42

 

the

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CC

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the

 

 

 

 

 

the interactions of the VSR with vesicle-forming machinery and contains sequences that
pertain to the sorting of the VSR itself (Sanderfoot et al., 1998; Happel et al., 2004).

The model of VSR function in plants is that the VSR interacts with a soluble
vacuolar protein in the near neutral pH of the TGN. Both proteins are packaged into
CCVs and are delivered to the PVC. Then, the VSR releases the cargo into the more
acidic environment of the PVC and is recycled back to the TGN while the cargo is sent to
the vacuole (Ahmed et al., 2000). With the identification of VSRs in vesicles other than
CCVs (Shimada et al., 1997; Tsuru-Furuno et al., 2001), and the observation that the
Arabidopsis genome encodes seven putative VSRs (Hadlington and Denecke, 2000;
Shimada et al., 2003), it is now clear that this model of VSR function is not necessarily
wrong, but should be expanded. In order to develop a more comprehensive model of
VSR function in plants, I addressed the functions of the individual AtVSR genes by
analyzing the phylogenetic relationship between all the identified plant VSRs, and by
determining the expression pattern for each member of the Arabidopsis gene family
through multiple experimental approaches. Towards this end, I determined the
expression patterns of each gene by RT-PCR and promoterzzGUS fusions. I also used
bioinformatics to Team more about the VSR expression patterns and to identify regulatory

elements within each promoter.

Materials and Methods

Images in this dissertation are presented in color.

R T -PCR of the AtVSR Genes

43

 

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Gene-specific primers were designed for each gene and synthesized by the MSU
Macromolecular Structural Facility or Fisher Scientific: ELF/VSR] Forward = CTT
GGG CTT TTC ACT CTC TCG TTT C; ELP/VSRI Reverse = TCC AAC TTT GCC
TGA ACC TAT GC; VSR2 Forward = TCC CAA T AC TCA ACT TTC TTC TCA AC;
VSR2 Reverse = GTT TTC ACT ATT TGG TTG TGT GTA C; VSR3 Forward = CCT
TGT CCT TCG AAT TTG TTC TTT G; VSR3 Reverse = TCT AGA GTC CTT CCC
GGG GAA TAA ATA GAT G; VSR4 Forward = CTA TTG ACT AGC TTG TCC AGT
TCT CCG TA; VSR4 Reverse = AGT GCA AAG AGA AGA CAT GTC AGT GC; VSR5
Forward = ATG GCT CGT GTG GGG TTG TAT TTG ACT ACC; VSR5 Reverse; AGT
CTT GGT TAA TGC TTT GGC TAT C; VSR6 Forward = ATG TCT TTG ATT CAT
AAA GGA GCC AC; VSR6 Reverse = CAG AAG TTA ATC TCA GCT GTT GGT G;
VSR7 Forward = GAG ATG GGT TTA GTC AAC GGG AGA G; VSR7 Reverse = GTA
AAA GGC TCG GCT TCT GAT GGA AC. Total RNA was extracted from the
following Arabidopsis tissues with the RNeasy Kit (Qiagen): dry seeds, seven-day old
seedlings, three-weekold roots, young rosette leaves, mature rosette leaves, bolts, flowers,
and green siliques. RT-PCR was canied out for each gene using the One-Step RT-PCR
Kit (Qiagen) with 500 ng total RNA template and 20-25 cycles of RT-PCR, unless
otherwise indicated. The PCR products were electrophoresed on a 1% agarose gel with
ethidium bromide and photographed by a gel documentation system (BioRad). The DNA
of all PCR products was sequenced by the University of California, Riverside (UCR)

Center for Genomics DNA Sequencing Facility to confirm their identities.

Preparation of Constructs: PromoterxGFPsGUS Fusion Constructs

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All PCR products used to generate the promoter fusion constructs were amplified fi'om
wild type Columbia genomic DNA using Takara Ex-Taq DNA Polymerase and following
the manufacturer’s protocol. The PCR products were purified using a PCR purification
kit (Qiagen) and cloned into pGEM-T Easy (Promega). DNA sequencing of the PCR
products and the constructs was done by the UCR DNA Sequencing Facility. After each
construct was prepared as described below, they were transformed into wild type
Arabidopsis (Columbia ecotype) by Agrobacterium-mediated stable transformation.
Seeds collected from the transformed plants were germinated on agar containing
Murashige Minimal Organics Medium, 30 mg/L hygromycin (Sigma), and 25mg/L
Carbenecillin (Sigma).

35S::GFP:GUS

We received the pRJG23 construct as a generous gift from Dr. Jen Sheen. For
ease of cloning into pCAMBIA, the GFP::GUS fragment of pRJGZ3 was cloned into pBS
SK+ with an EcoRI/HindIII double digest. The GFP::GUS fragment was then cloned
into pCAMBIA 1300MCS with a KpnI/Sacl double digest and ligation to make
pCAMBIA::GFP:GUS.

VSR] promoter: .' GFP:G US

The 2520 bp region upstream of the AtVSR] start codon was amplified with the
following primers: forward = AAG GTA AGA AAT ATT CTC TCA T'TT TC; reverse =
GAT ATC GAA ACG AGA GAG TGA AAA GAA GAA G. An EcoRV restriction
enzyme site (underlined) was added to the reverse primer for ease of cloning. The VSR7
promoter was cloned into the pCAMBIA vector via an SphI digest of pGEM:: VSR]

promoter and pCAMBIA followed by ligation and transformation into DHSa. The 35S

45

 

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promoter was then replaced with the GFP:GUS fragment of pBS:.-GFP:GUS by a
ClaI/PstI digest and ligation to make pCAMBIA : : VSRI promoter: : GFP:GUS.

VSR2promoter::GFP:GUS

The 2340 bp region upstream of the AtVSR2 start codon was amplified with the
following primers: Forward = ATA CTC ATC AAT GGA TAG CCA GC; Reverse =
AAG CTT TCT TAT CAA GAA ACT TAT GTT TAG. A HindIII restriction enzyme
site (underlined) was added to the reverse primer for cloning reasons.
pGEM:: VSRZpromoter and pCAMBM::GFP:GUS were digested with Sacl (New
England Biolabs) and ClaI (New England Biolabs) to replace the 358 promoter with the
VSR2 promoter to make VSR2promoter::GFP:GUS.

VSR3promoter: .' GF P.'G US

The 2160 bp region upstream of the AtVSR3 start codon was amplified by PCR
with the following primers: Forward = ATA AGC TTA ACG ACT ACT GCG TAT
TGG AGA GC; Reverse = ATA AGC TTT GGA AGG TAA CAC AGA AGC TGC.
HindIII restriction enzyme sites (underlined) were added for cloning purposes.
35S::GFP:GUS and pGEM::VSR3p were digested with HindIII (New England Biolabs)
and ligated to replace the 35S promoter with the VSR3 promoter.

VSR4promoter:: GFP:G US

The 2100 bp region upstream of the AtVSR4 start codon was amplified with the
following primers: Forward = GCA TGC AAC CAC AAT TCA CGA AAC CCT AAT
TTC; reverse = GGT ACC AAC AAA CAC CAA ATT CAA ACG GAT CAA C. SphI
and KpnI restriction enzyme sites (underlined) were added to the ends of the primers for

cloning purposes. pCAMBIA 1300MCS and pGEM::VSR4promoter were digested with

46

 

 

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SphI (New England Biolabs). The VSR4 promoter fragment was then ligated into
pCAMBIA. The resulting construct and pBS::GFP:GUS were then both digested with
SacI/Kpnl (New England Biolabs) in order to replace the 358 promoter with the
GFP:GUS fragment to make VSR4promoter::GFP:GUS.

VSR5promoter::GFP:GUS

The 2400 bp region upstream of the AtVSR5 start codon was amplified with the
following primers: forward = GCA TGC AGA CTT CAC ATA GAG ACG ATG GGA
TG; reverse = GAT ATC TGA ACC TTA ATG TAT ACG GAA GAG ACG. SphI and
EcoRV restriction enzyme sites (underlined) were added in the primers for cloning
purposes. To make VSR5promoter::GFP:GUS, pGEM::VSR5promoter was digested
with Sphl and SalI; pBS:::GFP:GUS was digested with SalI/Sacl (New England
Biolabs); and pCAMBIA I300MCS was digested with SphI/Sacl (New England Biolabs) .
The relevant fragments from each digest were joined in a 3-body ligation and
transformed into DHSa.

VSR 7promoter::GFP:GUS

The 2340 bp region upstream of the AtVSR7 gene was amplified with the
following primers: forward = GCA TGC AAG AAC ACT GTC AAT ACA CAA CAT
G; reverse = AAG AAG AGT TTG ATC GAT GAT AAC C. An SphI restriction
enzyme site (underlined) was added to the forward primer for cloning purposes. To make
the VSR 7promoter: .' GFP:G US, pGEM:: VSR 7promoter was digested with PstI/Apal
(New England Biolabs); pBS::GFP:GUS was digested with ApaI/Sacl (New England
Biolabs); and pCAMBIA 1300MCS was digested with PstI/SacI. The relevant fragments

from each digest were ligated in a 3-body ligation and transformed into DHSa.

47

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Histochemical Staining of GUS

Plant tissue was stained following the protocol of Jefferson (Jefferson et al., 1987)
with modifications. The stained tissues were cleared in 70% Ethanol and visualized by
either the MZIII Dissection Microscope (Leica) or the Insight Confocal Microscope
(Meridian). Micrographs were taken with a retail grade digital camera on the Leica or the

Hamamatsu CCD camera on the Insight.

Expression Analysis of AtVSR3 Under Stress Conditions

Wildtype Columbia Arabidopsis plants were grown on agar containing Murashige
Minimal Organics Medium (Invitrogen/Gibco) under constant light at 22°C for four
weeks. The plants were then subjected to various stresses as indicated below following
the published protocols (Y amaguchi-Shinozaki and Shinozaki, 1994). For cold
treatment, the plants were transferred to fresh agar plates and placed in 23°C or in 4°C for
the indicated time points.

For drought stress, the plants were transferred to either a fresh agar plate as a
control or transferred to a piece of Whatrnann filter paper in a Petri dish. Both plates
were incubated at 23°C under low light conditions for the indicated time points.

For salt stress, the plants were transferred to liquid media containing Murashige
Minimal Organics Medium with 250mM NaCl or to liquid media with an equal volume
of sterile water as a control. For ABA treatment, the plants were transferred to liquid
media with 250uM ABA or an equal volume of ethanol as a control. Flasks for both salt

and ABA treatments were incubated on a rotary shaker for the indicated time points.

48

RNA was extracted fi'om the indicated time points for all of the samples using the
RNeasy Extraction Kit (Qiagen). 500ng-lug of RNA was used as a template in One-Step
RT-PCR (Qiagen) with gene-specific primers for AtVSR3 (described above) or ubiquitin
(forward = GAT CTT TGC CGG AAA ACA ATT GGA GGA TGG T; reverse = CGA
CTT GTC ATT AGA AAG AAA GAG ATA ACA AGG). The PCR products were
electrophoresed in a 1% agarose gel containing ethidium bromide and photographed with
a Biorad Gel Documentation system (Biorad). Densitometry analysis of the PCR

products was accomplished with the MCID Elite 6.0 software.

Results
Phylogenetic Analysis of the Plant VSR Gene Family Reveals Three Groups of VSRs

The Arabidopsis genome encodes seven putative VSRs that share high sequence
identity (Hadlington and Denecke, 2000; Shimada et al., 2003). A gene family is a group
of genes that share a common splicing pattern and likely arose from gene duplication
(Sanderfoot et al., 2000). To determine whether the Arabidopsis putative VSR genes are
members of the same gene family, I compared the splicing patterns of the AtVSR genes
(Figure 2.1). AtVSRs 1,3,4,5, and 6 have identical patterns; AtVSR2 has one extra intron
and AtVSR7 lacks what is the fifth intron in the other genes. Therefore, these are
remarkably similar, indicating that the At VSRs arose from a single progenitor gene and
thus are members of the same gene family. VSRs have been experimentally identified in
Arabidopsis, pea, pumpkin, and Vigna mungo (Kirsch et al., 1994; Ahmed et al., 1997;

Shimada et al., 1997; Tsuru-Furuno et al., 2001). VSRs were also found through

49

AtVSR] W

At VSR2

Figure 2.]. Gene structures of the At VSR family. The schematic is a depiction of
the cDNA and intron position for each gene. The black lines represent the cDNA
for each gene. The white triangles represent the positions of introns that are found
in the same location of each gene. The black triangles represent introns that are not
found in all At VSR genes.

50

 

 

 

 

bioinformatics in almond, maize, rice, sunflower, wheat, and Physcomitrella patiens

(Table 2.1).

Table 2.1. Putative Plant VSRs

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Organism Accession or AGI Full Length
Number/Name Sequence Known?
Arabidopsis thaliana At3g52850/AtVSRl Yes
At2g30290/AtVSR2 Yes
At2g14740/AtVSR3 Yes
At2g14720/AtVSR4 Yes
At2g34940/AtVSR5 Yes
Atl g3 0900/AtVSR6 Yes
At4g201 10/AtVSR7 Yes
Pisum sativum (Garden Pea) P93484/BP-80 Yes
Cucurbita (Pumpkin) 048662/PV72 Yes
PV82 No
Oryza sativa (Rice) 83 52.m04840 Yes
8362.m01486 Yes
8360.m02012 Yes
8354.m04241 No
8354.m04240 No
8355.m04617 No
Physcomitrella patens Q9AWB4 No
Helianthus annuus (Sunflower) Q94IN3 No
Q94IN4 No
Q9ARG7 No
Prunus dulcis (Almond) Q9SDR8 No
Zea mays (Maize) P93645 No
Vigna mun (Black gram) Q93X09 Yes
T riticum aestivum (Wheat) Q9LLR3 Yes

 

 

 

To understand how the VSRs are related, 1

relationship between these proteins based on their amino acid sequences.

determined the phylogenetic

The

phylogenetic relationship of these proteins for which full length sequence was known was

calculated with PHYLIP, a software package that determines the relatedness of a group of

proteins and uses a midpoint method to determine the root of the tree (Felsenstein, 1988).

The results indicated that the VSRs separated into three groups (Figure 2.2)

51

Rice EST 8352.m04840 l

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

— 98 Arabidopsis At4g20110/ AtVSR7
_i 100 3
Arabidopsis At1g30900/ AtVSR6
100
Arabidopsis At2g34940/ AtVSR5 I
Rice EST 8362.m01486 '
—*1°°—— Pea BP-80
2
._ 88
At2g14740/ AtVSR3
At2g14720/ AtVSR4 ,
Wheat VSR ‘
Rice EST 8360.m02012
—» 99 , Arabidopsis At2g30290/ AtVSR2
41 1
Arabidopsis At3g52850/AtVSR1
100
Vigna mungo VSR
59

 

Pumpkin PV72 _/

Figure 2.2. Phylogenetic analysis of the plant VSRs. Phylogenetic analysis
indicated that the family of VSRs clusters into three groups. The firll length amino acid
sequences were analyzed to determine their relationships to one another. The tree was
calculated with the PHYLIP package (F elsenstein 1989) using the PROTDIST program
for calculating the distance matrix (categories model as distance matrix), the
NEIGHBOR-JOINING method for tree construction and the midpoint method in
RETREE for defining the root of the trees. The internal bootstrap values were obtained
from 100 alignment re-sampling replicates.

 

0.1

52

(Paris and Neuhaus, 2002). The first group contained AtVSRl and PV72, among other
proteins. The second group was composed of pea BP-80 as well as other proteins. The
third group was composed of three uncharacterized putative AtVSRs and two putative
rice VSRs.

All putative plant VSRs have a PA domain that is upstream of the cysteine-rich
EGF repeats (Mahon and Bateman, 2000; Shimada et al., 2003). The PA domain is
thought to be the site of protein-protein interaction for proteins (Mahon and Bateman,
2000). Moreover, the NTPP-interaction domains of BP-80 and PV72 are N-terminal to
the cysteine-rich repeats (Cao et al., 2000; Watanabe et al., 2002). Therefore, it is
possible that the PA domain is the site of cargo protein interaction (Mahon and Bateman,
2000). I examined the phylogenetic relationship of the VSRs using only the amino acid
sequence from the PA domain of each VSR for which the full length protein sequence
was known (Figure 2.3). The phylogeny of the PA domain of the VSRs was identical to
the phylogeny of the entire VSRs. This information suggested that the relatedness of the
plant VSRs could be indicative of their function. To address the functions of the

AtVSRs, I looked at the expression patterns for each Arabidopsis VSR gene.

Expression Patterns of AtVSR] and AtVSR, Members of the Group 1 VSRs

AtVSR] and AtVSR2 proteins are members of the first group of plant VSRs
(Figure 2.2). To determine the expression patterns of AtVSR] and AtVSR2, RT-PCR was
carried out with gene-specific primers using total RNA from various Arabidopsis tissues

as a template. Each PCR was limited to less than 28 cycles to stay within a linear range

53

 

Rice EST 8352.m04840 I
__ 97 . .
Arabrdopsrs At4g201 10/ AtVSR7

93 3
ArabidopsisAtl g30900/AtVSR6

—l:_
Arabidopsis At2g34940/ AtVSR5

Rice EST 8362.m01486

 

 

 

 

 

 

_ 88
—— Pea BP-80

 

_ 62
I ArabidopsisAt2g14740/ AtVSR3

 

99

 

Arabidopsis At2g14720/ AtVSR4

 

 

_/
Wheat VSR I

98
Rice EST 8360.m02012

 

_J 73 ArabidopsisAt2g30290/AtVSR2

| 39 l
Arabidopsis At3g52850/ AtELP/AtVSRl
55
' Vigna mango VSR
l 43
Pumpkin PV72 _/

Figure 2.3. Phylogenetic analysis of the PA domain of the plant VSRs. Phylogenetic
analysis of the PA domain of plant VSRs indicated that the putative ligand binding
domain of the family of VSRs clusters into three separate goups. The amino acid
sequence of the PA domains of the plant VSRs were compared to determine their
relationships to one another. The tree was calculated as described in Figure 2.2.

 

 

 

 

 

0.1

54

of amplification and thus obtain a more accurate picture of the relative levels of gene
expression in the various tissues (Figure 2.4). AtVSR] expression was detected in all
tissues examined (Figure 2.4). However, the expression of AtVSR2 was restricted mostly
to the roots plus a barely detectable amount present in young leaves (Figure 2.4). The
difference in expression patterns between At VSRI and AtVSR2 suggested that the
functions of these two genes were not redundant.

The widespread expression pattern of AtVSR] led to the question of whether
AtVSR] was simply a ubiquitously expressed housekeeping gene or whether it was
expressed in specific cell types throughout the plant. I was also interested in confirming
the root localization of AtVSR2. To answer these questions, I made DNA constructs that
contained the putative promoter region for each gene fused to the coding region of ,B-
glucuronidase (GUS). The transcriptional fusions were transferred to a binary vector for
stable transformation in Arabidopsis. The constructs were transformed into Arabidopsis,
and putative transforrnants were selected for resistance to hygromycin and propagated.
The T2 and T3 generations were screened for GUS activity.

Five transgenic lines were selected for analysis by GUS assay for the AtVSR]
promoter. GUS activity showed that AtVSR] was expressed in the mature embryo of
imbibed seeds, as well as the meristematic region and vasculature of seedlings (Figure
2.5A-D). In mature plants, GUS protein was detected throughout the vascular system of
roots, leaves, and inflorescences (Figure 2.5E-G). GUS was also detected in the flower
carpel as well as in the maturing embryo from an immature silique (Figure 2.5H,I).
These results were consistent with the results obtained by RT-PCR (Figure 2.4), but

suggested that AtVSRl may have a more specific function than a house-keeping protein.

55

 

 

 

AtELP/AtVSR]

AtVSR2

AtVSR3

AtVSR4

AtVSR5

AtVSR6

AtVSR 7

 

Figure 2.4. RT-PCR expression analysis of AtVSR genes. RNA was extracted from
various Arabidopsis tissues as indicated in the figure. 500 ng to 1 ug of RNA was used
as a template in RT-PCR with gene-specific primers and 20-25 cycles of PCR after
reverse transcription cDNA synthesis.

56

Figure 2.5. Expression analysis of AtVSR] by Promoter::GUS fusions in Arabidopsis.
At VSR] promoter::GUS transgenic plants were histochemically stained with the X-
Glucuronide substrate to visualize the tissue and cell-type specific expression pattern of
AtELP/AtVSR]. GUS activity is seen the mature embryo (A). GUS activity is also
detected in the meristematic region of 7-day old seedlings (B and C). 2-week old plants
and 4-week old plants express GUS throughout the vascular tissue (D and E,
respectively). Closer examination of the leaves and the roots confirmed that the GUS
activity is localized to the vascular tissue (F, G). In flowers, GUS is detected in the
carpel (H). GUS is also detected in maturing embryos from green siliques (I).

57

 

58

Eleven AtVSR2 promoter: :GUS lines from three independent transformations
were analyzed to gain more insight into the expression pattern of AtVSR2. In agreement
with the RT-PCR results, GUS activity was detected in the roots (Figure 2.6E).
However, GUS was also detected in the cotyledons of mature embryos (Figure 2.6A),
throughout seven-day old seedlings (Figure 2.6B), in the vasculature of four-week old
plants (Figure 2.6C,D), as well as in the flowers of transgenic plants (Figure 2.6E). The
discrepancy in the expression patterns obtained by RT-PCR and promoter::GUS fusion

could be due to the absence of a repression element(s) in the promoter: :GUS fusion.

Expression Patterns of AtVSR3 and AtVSR4, Members of the Group 2 VSRs

AtVSR3 and AtVSR4 proteins are part of the second group of the AtVSR
phylogenetic tree (Figure 2.2). We first determined the expression patterns of AtVSR3
and AtVSR4 by RT-PCR (Figure 2.4). The expression of AtVSR3 was highest in young
leaves and flowers although still detected in all other tissues examined (Figure 2.4).
AtVSR3 expression pattern was detected in seeds with at least 28 cycles of PCR (data not
shown). The AtVSR4 transcript was detected at low levels throughout the plant (Figure
2.4).

To determine the cell type-specific expression patterns of AtVSR3 and AtVSR4,
we made reporter fusions to the predicted promoter regions of each gene. Fifteen
transgenic lines from three independent pools of plants carrying the AtVSR3 promoter
fused to GUS were analyzed. The GUS activity of these plants indicated that AtVSR3
was expressed in the cotyledons of imbibed seeds, as well as the roots, hypocotyls, and

cotyledons of seven-day old seedlings (Figure 2.7A-C). Surprisingly, expression from

59

 

Figure 2.6. Expression patterns of GUS from the AtVSR2 promoter. AtVSR2
promoter::GUS transgenic plants were histochemically stained with the X-Glucuronide
substrate to visualize the tissue and cell-type specific expression pattern of AtVSR3.
GUS activity was detected in the cotyledons of mature embryos (A) and throughout a
seven-day old seedling (B). However, four-week old plant only showed GUS activity in
the vasculature of the true leaves (C, D) and roots (E). GUS activity was also detected in
the sepals, stamens, and carpel of the flower (F).

60

 

Figure 2.7. Expression analysis of AtVSR3 by Promoter::GUS fusions in
Arabidopsis. AtVSR3 promoterzzGUS transgenic plants were histochemically stained
with the X-Glucuronide substrate to visualize the tissue and cell-type specific expression
pattern of At VSR3. GUS activity was detected in the cotyledons of mature embryos (A).
7-day old seedlings (B) express GUS throughout the cotyledons, hypocotyl, and primary
root (C), although restricted to the margins of the true leaf. 4-week old plants (D) show
similar results of GUS expression throughout the cotyledons that is primarily restricted
to the hydathodes in the true leaves (D, E). Upon closer examination of the rosette leaf
(40X magnification) revealed GUS activity specifically in the guard cells (F). In the
flowers, GUS is detected primarily in the carpel, but can be faintly seen in the anthers as
well (G). GUS was not detected in the maturing embryos of green siliques (H), although
it was present throughout the maternal tissues of the silique.

61

the AtVSR3 promoter was restricted to the guard cells and hydathodes of true leaves
(Figure 2.7B,D-F). In the flower of the transgenic plants, GUS activity was detected in
the anthers and carpel (Figure 2.7G). Finally, GUS was detected in immature siliques
although it appeared to be excluded from the developing seed (Figure 2.7H).

Guard cell function is regulated by various stresses and by the plant hormone
ABA (Abscisic Acid) (Blatt, 2000). Therefore, we determined whether the expression of
AtVSR3 was regulated by signal transduction pathways that are initiated by stress or
hormones. Four-week old wild type Columbia plants were subjected to cold, high salt,
dehydration, and ABA for various time points. RNA was extracted from the plants and
used as a template for RT-PCR with primers specific for AtVSR3 cDNA. The expression
of AtVSR3 was repressed under cold, dehydration, and in ABA (Figure 2.8A-F).
However, AtVSR3 expression was not affected by high salt (Figure 2.8G,H). These
results indicated that AtVSR3 is affected by certain stresses related to the ABA response
pathways, but not by all of them.

Eight transgenic lines carrying the AtVSR4 promoter fused to GUS were analyzed
to determine the expression pattern from the AtVSR4 promoter. No GUS activity was
detected in any of the transgenic plants (Figure 2.9). Most likely, the discrepancy is that
the promoterzzGUS fusion construct does not encode the complete AtVSR4 promoter or

the construct is missing intron-encoded regulatory elements.

Expression Patterns of AtVSR5, AtVSR6, and AtVSR 7, Members of the Group 3 VSRs

AtVSR5, AtVSR6, and AtVSR7 belong to the third group of VSRs. The third group

was composed of putative VSRs that have not been characterized at the protein level. We

62

 

Figure 2.8 AtVSR3 Expression decreases in the presence of dehydration, cold, or 250
uM ABA. Arabidopsis plants were grown on agar for four weeks. The plants were then
transferred to either fresh agar plates or petri dishes with filter paper for the indicated
time points (A, B); transferred to fresh plates and kept at 4°c or 23°c for the indicated
time points (C, D); or were transferred to liquid media in the presence or absence of
250uM ABA for the indicated time points (E, F). RNA was extracted and 1 ug total
RNA template was used in 25 cycles of RT-PCR with primers specific to AtVSR3 or to
ubiquitin. The intensity of each band was measured for the treatment and the control.
Within each experiment, the subtracted pixel intensity of the treated sample was divided
by the subtracted pixel intensity of the untreated sample for the same time point. The
average of three independent experiments and their relative standard deviations were
graphed in the chart.

63

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64

9.33.5453:

 

Figure 2.9. Expression analysis of VSR4 by Promoter::GUS fusions in Arabidopsis.
GUS activity was not detected in any cells examined of hygromycin resistant plants,
including mature embryos (A), 7-day old seedlings (B), roots (C), 4-week old plants (D),
rosette leaves (E), flowers (F), and immature siliques (G).

65

first examined the expression patterns of these genes by RT-PCR. At VSR5 expression
was only detected in the roots by RT-PCR (Figure 2.4). AtVSR6 and AtVSR7 mRNA
transcripts were detected in most tissues examined except seeds and young leaves (Figure
2.4).

As with the other Arabidopsis VSRs, we expressed promoterxGUSt fusions of
each putative promoter in Arabidopsis. Eleven transgenic lines fi'om three independent
pools were studied for the AtVSR5 promoter. Rather than observing GUS activity only in
the roots, we observed very strong GUS staining throughout the vegetative tissues of the
plant (Figure 2.10). The effect was seen in several independent lines, ruling out the
possibility that the construct inserted into the genome near an enhancer for one line.
Constructs containing the putative AtVSR6 promoter fused to GUS were prepared and
transformed into Arabidopsis. We were unable to obtain any transforrnants from this
construct. The likely explanation is that a point mutation occurred in the binary vector of
the construct that interfered with the function of the vector in plants. Seven lines were
analyzed to study GUS expression from the AtVSR7 promoter (Figure 2.11). Consistent
with the RT-PCR results, GUS was detected at the distal ends of leaves and in the roots.
GUS was not detected in other parts of the plants. The RT-PCR results were very faint
for the other tissues and RT-PCR is more sensitive than a GUS assay. Therefore, in other
tissues, GUS might have been present at levels too low to detect by eye or by

microscopy.

66

  

Pin. ,
Figure 2.10. Expression analysis of theAtVSR5 Promoter by GUS Assay. GUS activity
was strongly detected in the mature embryo (A), the seven-day old seedling (B), and the
rosette leaf (C). GUS was detected in the distal end of cauline leaves (D), the sepals,
petals, filament and carpel of the flower (E), but only at the base of the silique (F).

67

 

Figure 2.11. Expression patterns from the AtVSR7 Promoter. GUS activity was
detected at the distal ends of cotyledons and the root-shoot transition zone of seedlings
(A). GUS was faintly detected in the roots and root hairs (B). GUS was detected in
the distal ends of true leaves of the rosette plant (C).

 

fii,.b§ul u .1; K...

Discussion

Three approaches were taken to determine the expression patterns of members of
the AtVSR gene family, including bioinformatics, RT-PCR, and promoter: :reporter
fusions. Expressed Sequence Tags (EST), the Stanford Microarray Database (SMD),
Alfymetrix Chip, and predicted promoter element data are available to the public through
The Arabidopsis Information Resource (TAIR) at http://www.arabidopsis.org/, the
Nottingham Arabidopsis Stock Centre (NASC) Affymetrix Database at
http://www.ssbd'Lc2.nottingham.ac.uk/; and the Plant cis-acting regulatory DNA elements
(PLACE) database at htIp://www.dna.affic.go.ip/htdocs/PLACE/sggnalscanhtml/ (Higo
et al., 1999). We mined these databases for information relating to the expression
patterns of each gene.

Phylogenetic analysis indicated that the plant AtVSR gene family can be sub-
divided characterized into three groups (Figure 2.2) (Paris and Neuhaus, 2002).
Furthermore, phylogenetic analysis of a region predicted to be the domain responsible for
cargo protein interaction that is present in all the plant VSRs gave similar results (Figure
2.3). From this information, we hypothesized that there were three functional groups
within the VSR family. A summary of the observations made in this chapter is presented

in Table 2.2.

Group 1 VSRs

The first group is composed of PV72, AtVSRl, a VSR identified in the ER bodies

of Vigna mango, and proteins from Arabidopsis, rice, and wheat. Previous reports

69

EST/MA/ACC

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Promoter Elements

 

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70

suggest that PV72, AtVSRl, and the Vigna mungo VSR function, at least in part, as
VSRs for soluble proteins that are destined for the PSV (Shimada et al., 1997; Tsuru-
Furuno et al., 2001; Shimada et al., 2003). Therefore, it is possible that the first group of
VSRs primarily function in the delivery of storage proteins to the PSV. Expression
analysis of AtVSR] indicated that it was expressed in developing and mature seeds as
well as throughout the vasculature of the mature plant. ESTs for AtELP/AtVSR] were
found in developing seeds, green siliques, 3-day old seedling hypocotyls, and dehydrated
rosette plants (Table A.1). The presence of seed and seedling ESTs for AtVSR] was
consistent with our RT-PCR and GUS results. Five ESTs for AtVSR] were also
identified in dehydrated plants (Table A.1). Furthermore, microarray data suggested that
AtELP/AtVSR] expression was also regulated by ABA (SMD experiments 11895,
11757)

An analysis of the AtVSR] promoter by the PLACE Signal Scan Database (Higo
et al., 1999) revealed promoter elements for expression in endosperrn, embryo, and
mature seeds (Table A2). In particular, the AtVSR] promoter contains a combination of
the GCN4 motif, the AACA motif, and the ACGT motif in close proximity to each other
(Table A2). The combination of these three motifs in a short region is necessary to
activate expression in endosperrn (Wu et al., 2000). A second combination of cis-
elements found in the AtVSR] promoter that enhances expression in seeds was the
combination of the SEF3 (soybean embryo factor) element, the SEF4 element, and the
RY element (Table A2) (Lessard et al., 1991; Fujiwara and Beachy, 1994). A number
of other elements that activate or enhance expression in seeds were also found in the

AtVSR] promoter such as the -300 element (one copy at -10), the TAT CCA motif (four

71

copies), the DPBF (Dc3 promoter-binding factor) consensus motif (two copies), and the
E-Box (10 copies) (Table A.2). The -300 element is a promoter element that is
consistently found within the first 300 bases upstream of the start codon of seed storage
genes (Thomas and Flavell, 1990). The DPBF consensus motif and the E-Box enhance
expression in seeds (Kim et al., 1997). The possibility that these are functional elements
in the AtVSR] promoter is supported by the RT-PCR and GUS data that indicated that
AtVSR] is expressed in developing and mature embryos. Taken together, these
observations further suggested that AtVSRl plays a role in processes related to seed
development and certain stresses. PSVs accumulate during both of these processes (Paris
et al., 1996; Jauh et al., 1999; Park et al., 2004). Therefore, the expression data obtained
for AtVSR] were consistent with the model that AtVSRl functions in the transport of
proteins to the PSV and that AtVSRl is important for seed development.

RT-PCR and promoter: :reporter studies demonstrated that the At VSR2 gene is
expressed in the roots. However, the RT-PCR data indicated that AtVSR2 was only
expressed in the roots, whereas analysis of transgenic plants containing the putative
AtVSR2 promoter fused to GUS suggested that AtVSR2 is expressed in other tissues as
well. The promoter::reporter transgenic plants were generated by transforming three
separate pots of wildtype Arabidopsis plants with multiple plants per pot. While it was
possible that the construct entered into the genome next to an expression enhancer for one
line, it was unlikely that a similar event would occur for at least three independent lines.
Restriction digest and DNA sequencing analysis confirmed that the construct did not
have any part of the 35S promoter and did have the putative At VSR2 promoter (Data not

shown). Promoter analysis through the PLACE Database (Higo et al., 1999) revealed 26

72

copies of a promoter element that activates expression in the roots (Table A.3) (Elmayan
and Tepfer, 1995). Therefore, it is possible that the high expression in roots is due to the
26 copies of this root expression promoter element.

The AtVSR2::GUS plants also showed weak GUS activity in the cotyledons of
seeds and in seedlings. In agreement with this data, the AtVSR2 promoter encodes many
seed expression elements (Table A.3). One of these elements is a version of the RY
element that is less able to suppress expression in leaves than another version of the RY
element (Fujiwara and Beachy, 1994). Perhaps this is why there is GUS activity in the
cotyledons and the vasculature of the leaves of the transgenic plants that was not
accounted for by RT-PCR. Finally, Affymetrix studies of the pollen transcriptome
indicate that AtVSR2 expression in the pollen is 2.8 times higher than in leaves, roots,
seedlings, and siliques (Becker et al., 2003). I also saw AtVSR2 expression in the pollen
in the promoter::GUS fusion studies. The vacuole is a dynamic and important
compartment in pollen (Hicks et al., 2004). Therefore, it is possible that AtVSR2
participates in pollen growth. Overall, these results were not strong indicators as to
whether AtVSR2 is also a receptor for storage proteins. However, a-TIP is detected in
roots and leaves, suggesting that PSVs are also present in those tissues (Paris et al., 1996;
Park et al., 2004). While it is possible that AtVSR2 is the VSR for storage proteins, this

hypothesis must be addressed by a biochemical or reverse genetics approach.

Group 2 VSRs
The second group of VSRs was composed of the previously characterized BP-80

from pea, two homologues from Arabidopsis (AtVSR3 and AtVSR4), and a putative VSR

73

from rice. BP-8O was identified as a VSR for the CCV pathway and is strictly localized
to CCVs relative to dense vesicles (Kirsch et al., 1994). Thus, members of this group
may be responsible for soluble proteins that are delivered to the lytic vacuole by the CCV
pathway.

The expression pattern of AtVSR3 is perhaps the most intriguing of the AtVSRs.
AtVSR3 is expressed in mature embryos and ubiquitously expressed in seven-day old
seedlings. However, beginning with the first true leaves, expression is restricted to the
guard cells and the hydathodes until flowering, when GUS activity is also detected in the
anthers and the carpels. Further RT-PCR analysis indicated that AtVSR3 is repressed by
ABA, dehydration, and cold treatment. Affymetrix data also indicated that AtVSR3 is
specifically expressed in guard cells and is repressed by ABA (Leonhardt et al., 2004).
An analysis of the putative AtVSR3 promoter revealed that the AtVSR3 promoter has 25
copies of the DOF (DNA-binding with one finger) motif that has been observed in the
promoters of other guard cell-specific genes and promoter elements for ABA-mediated
repression (Table A4) (Higo et al., 1999; Plesch et al., 2001; Mena et al., 2002). The
endomembrane system has many components that have been implicated in regulation of
stomata] movement (Blatt, 2002; Geelen et al., 2002; Zhu et al., 2002). It is exciting to
speculate that AtVSR3 is the VSR for a vacuolar protein that functions in keeping the
stomata open through some function in the vacuole. When the plant is exposed to cold or
drought stress, the stomata close, and the ABA signal transduction pathway represses the
expression of AtVSR3 and presumably represses the expression of the specific cargo

protein as well.

74

AtVSR4 expression was detected in all tissues by RT-PCR. However, GUS assays
of transgenic promoter: :reporter plants did not reveal any GUS activity at all. The region
of DNA between AtVSR4 and its immediately adjacent gene, At2g14 730, is only 500
bases and only this region was used to prepare the promoter::GUS construct. At2g14 730
is a hypothetical protein of unknown function for which no ESTs have been identified. It
is possible that the promoter of the AtVSR4 gene actually extends farther into the
At2g14 730 gene. Therefore, the likely possibility is that the promoter for AtVSR4 is
longer than predicted and therefore, only part of the promoter is present in the reporter
fusion construct. ESTs for AtVSR4 have been identified in developing seeds (Accession
BE25715), roots (AV545281), and dehydrated rosette plants (AV795948), confirming
that AtVSR4 is expressed. Some promoter elements for seed expression are present in the
1500 bases upstream of the AtVSR4 start codon (Table A5). Thus, the expression pattern
of AtVSR4 is similar to AtVSR]. Based on the phylogenetic tree of the AtVSR gene
family, it is possible that AtVSR4 is performing a distinct function from AtVSRl in the

same tissues.

Group 3 VSRs

The third group of VSRs was composed of proteins that have not been studied
previously. RT-PCR demonstrated that AtVSR5 expression only occurs in the roots.
Contradictory to this data, the GUS assays showed ubiquitous expression of GUS from
the predicted promoter of AtVSR5 . Transcriptional and post-transcriptional regulatory
cis-elements can exist in the introns as well as in the 3’UTR (untranslated region) of

genes. Therefore, it is likely that suppressor elements of AtVSR5 exist outside of the

75

promoter region. The effects of these elements would be seen in the RT-PCR results, but
would be masked in the GUS assays since only the upstream sequences were used in the
reporter firsion. An analysis of the introns and 3’UTR of AtVSR5 manually and by the
PLACE database did not reveal any characterized repression elements. The 3’UTR has
two copies of the AU-Rich Element (ARE) that confers mRNA instability to a gene.
However, the ARE is usually present much more abundantly in previously characterized
mRNAs (Gutierrez et al., 1999). Therefore, the most likely explanation is that there was
negative cis-regulatory information present in the introns or the 3’UTR of the At VSR5
gene. The absence of the negative regulators would allow expression from the AtVSR5
promoter to occur uninhibited. To this end, it would be interesting to examine the
expression pattern of different portions of the entire AtVSR5 gene by reporter fusions to
identify such elements. Bioinforrnatic data was very limited for AtVSR5. However, in
agreement with the RT-PCR results, the only ESTs that have been identified were both
from root tissue (Accessions AU235566 and AU226294).

RT-PCR of AtVSR6 indicated that it is expressed in most vegetative tissues with
the exception of young leaves. I was not able to recover any transgenic plants for
promoter analysis. One microarray indicated that AtVSR6 may be more highly expressed
in response to starvation (D. Bassham, personal communication). Consistent with this,
the promoter of AtVSR6 contains five promoter elements that are thought to be starvation
and/or Gibberellic Acid (GA) response elements in the a—amylase promoter (Table A6).
Perhaps one of the roles of AtVSR6 is to help the plant to adapt to starvation conditions.

Other endomembrane trafficking proteins are involved in plant adaptation to starvation,

76

such at AtVT112 (Surpin et al., 2003). Therefore, it would be interesting to examine the
possible interactions between AtVT112 and AtVSR6.

RT-PCR indicated that AtVSR 7 was expressed in seedlings, roots, and mature
leaves. This was confirmed by promoter::reporter analysis. Furthermore, the GUS
assays indicated that AtVSR7 expression in leaves was restricted to the distal end.
Microarray data indicated that AtVSR7 expression increased in response to phosphate
starvation (Hammond et al., 2003). We did not find any promoter elements that
corresponded with these expression patterns. AtVSR7 may have a very specific role in
plant adaptation to phosphate starvation. Therefore, the third group of VSRs may help
the plant adapt to changing environmental conditions.

With these and previously published results in mind, we can hypothesize that the
VSRs cluster into three functional groups. The first group is responsible for protein
transport to PSVs. The second group is responsible for CCV trafficking to the lytic
vacuole. The third group helps the plant adapt to specific environmental conditions.
From the hypothesis we can predict that the subcellular localization of the AtVSR protein
should be indicative of its function. For example, if AtVSRl is implicated in the
transport of storage proteins to vacuoles by the dense vesicle pathway, then AtVSRl
protein should primarily be localized to dense and /or PAC vesicles. Likewise, the BP-80
group of AtVSRs should primarily localize to CCVs. However, we can not expect that a
specific VSR will only be found in a particular type of vesicle because there is often
crosstalk among the different pathways to the vacuole (Hoh et al., 1995). Another
prediction would be that mutants of an individual VSR that did not produce functional

protein of a specific AtVSR would have a phenotype indicative of its specific function.

77

For example, if a member of the third group of VSRs is involved in helping the plant to
cope with a specific stress such as starvation, then a null mutant of that VSR would be
less able to cope with starvation than a wildtype plant. The next steps are to use reverse
genetic and biochemical approaches to test these predictions. Overall, the expression
patterns of the individual AtVSRs were consistent with the predicted function of each

group based on previous reports and our own phylogenetic analysis.

78

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Shimada, T., Fuji, K., Tamura, K., Kondo, M., N ishimura, M., and Hara-
N ishimura, I. (2003). Vacuolar sorting receptor for seed storage proteins in
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Surpin, M., and Raikhel, N. (2004). Traffic jams affect plant development and signal
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Surpin, M., Zheng, H., Morita, M.T., Saito, C., Avila, E., Blakeslee, J .J.,
Bandyopadhyay, A., Kovaleva, V., Carter, D., Murphy, A., Tasaka, M., and
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2899.

82

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83

Chapter 3

Functional Analysis of the AtVSR Family

84

Abstract

Soluble vacuolar proteins are delivered to the vacuole by vesicle trafficking
through the endomembrane system. First, the proteins are cotranslationally inserted into
the endoplasmic reticulum and transported to the Golgi. At the trans-Golgi Network
(TGN), a vacuolar sorting signal (V SS) is recognized by the vacuolar sorting machinery
and the protein is delivered to the vacuole. Three types of VSSs have been identified in
plants. One type of VSS, the amino-terminal propeptide (NTPP), is an amino acid
sequence that is cleaved from the mature protein after delivery to the vacuole. The NTPP
has a conserved motif that is recognized by a vacuolar sorting receptor (V SR) in a
sequence-specific manner. The VSR is a type I transmembrane protein with a large
lumenal domain and a short cytoplasmic tail. Previous studies have indicated that VSRs
interact with the NTPP signal at the TGN. Then, both proteins are packaged into
clathrin-coated vesicles and are delivered to the PVC. The cargo and receptor dissociate
in the acidic environment of the PVC and the receptor is recycled to the TGN whereas the
cargo is delivered to the vacuole. However, other members of the VSR family have been
identified in endomembrane vesicles outside of this pathway. Therefore, it is likely that
the model of VSR function needs to be expanded to accomodate other possible vacuolar
trafficking pathways. The Arabidopsis genome encodes seven VSRs, all of which are all
expressed in diverse tissues at various points in the plant life cycle. I took a reverse
genetics approach to determine the overlapping and individual functions of the AtVSRs by
using antisense technologies and T-DNA insertion lines. Determining the firnctions of
the AtVSR family will lead to a greater overall understanding of protein trafficking

through the endomembrane system.

85

Introduction

Protein targeting to the vacuole occurs via vesicle trafficking through the
endomembrane system. Three types of vacuolar sorting signals have been identified in
plants: 1') the N-terminal propeptide (NTPP), ii) the C-terminal propeptide (CTPP), and
iii) the internal signal (Surpin and Raikhel, 2004). The NTPP is recognized by a vacuolar
sorting receptor (VSR) that mediates its delivery to the vacuole via the prevacuolar
compartment (PVC) (Ahmed et al., 1997; Paris et al., 1997). The Arabidopsis genome
encodes seven VSRs. All seven genes are expressed, and thus probably produce
functional proteins (Chapter 2). Phylogenetic analysis indicated that all plant VSRs
could be subdivided into three functional groups (Chapter 2). There are at least two
A rabidopsis members represented in each group. Recent reports suggest that AtVSRl is
involved in the vacuolar trafficking of 2S albumin (Shimada et al., 2003). However,
specific functions have not been assigned to any other AtVSRs. In this chapter, I
describe the reverse genetic and biochemical approaches I used to determine the
overlapping and unique functions of the AtVSRs. First, I took an antisense approach to
get an overview of the function of the entire AtVSR family. Second, I used T-DNA
insertion lines and RNAi to determine the functions of individual At VSRs. Finally, to
identify the VSR that functionally interacts with a known vacuolar protein, I used
antibodies against known vacuolar proteins to coimmunoprecipitate AtVSRs. Once the
procedure is optimized, the immunoprecipitated protein will be analyzed by proteomic

techniques to identify the specific AtVSR that interacts with the vacuolar protein in viva.

Materials and Methods

86

Plant Growth Conditions and Transformation Protocols

Arabidopsis (Columbia, unless otherwise indicated) seeds were sterilized in 50%
bleach/0.05% Tween-20 (Sigma) and rinsed six times with sterile water. After cold
treatment in 4°C for 24 hours, the seeds were sown on phytagar containing Murashige
Minimal Organics Medium (Invitrogen/Gibco), and selection if needed, and grown under
constant light conditions at approximately 23°C. After two weeks, the seedlings were
transferred to a commercial soil mixture containing slow-release fertilizer pellets
(Osmocote) and a fungicide (Marathon). The plants were grown under long day

conditions (16 hours light/8 hours dark) at approximately 23°C.

Plant Transformation

The DNA constructs were transformed into Agrobacterium tumefaciens by a
freeze-thaw protocol fi'om Dr. Steve Farrand’s lab. The GV3101 strain of Agrobacteriurn
was grown in liquid media overnight at 28°C. Two milliliters of culture were transferred
to an eppendorf tube on ice. The cells were washed in 10 mM Tris pH 7.5 and
resuspended in cold luria broth. Two micrograms of plasmid DNA were added to the
cells and the mixture was frozen in liquid nitrogen for five minutes. The mixture was
transferred to 37°C for five minutes. Then, fresh luria broth was added and the cells were
incubated in a 28°C shaker for three hours and transferred to selection agar. The plates
were incubated at 28°C for two days. Antibiotic resistant colonies were screened for the
presence of the construct by PCR and restriction digests. Then, the constructs were
transformed into Arabidopsis using the floral dip protocol fi'om Dr. Andrew Bent (Bent et

al., 1994).

87

Photography and Microscopy

Seedlings were examined and photographed with a Leica MZIII Dissection
Microscope with a Canon G4 digital camera attached. Microscopy of guard cells was
accomplished on an Insight Confocal Microscope (Meridian) with a 20X and a 40X

objective and micrographs were taken with a CCD camera (Hamamatsu).

Nucleic Acid Extraction from Plants and Subsequent Analysis

Genomic DNA was extracted fiom two small rosette leaves of an individual plant
following a published protocol (Weigel and Glazebrook, 2002). PCR was carried out
with Takara Ex-Taq (Takara Shuzo) following the manufacturer’s instructions. Total
RNA was extracted from rosette leaves using the RNeasy Plant Mini Kit (Qiagen). RT-
PCR was carried out using 500ng total RNA, unless otherwise indicated, with the One-

Step RT-PCR Kit (Qiagen) following the manufacturer’s protocol.

Antisense AtVSR plants

The coding region for AtVSRl/AtELP was cloned into the PGA748 binary vector
in the opposite orientation of a 35S promoter by Dr. Sharif Ahmed. The construct was
transformed into Arabidopsis. To detect the presence of AtVSR protein, rosette leaves
were excised from each plant and homogenized on ice in the presence of 3X Laemmli’s
buffer. The protein was separated by SDS-PAGE on a 10% polyacrylarnide gel, blotted
to nitrocellulose membrane, and probed with anti-VSR antibodies or anti-AtCPY

antibodies followed by phosphatase-labelled anti-rabbit secondary antibodies. The blot

88

was developed in Tris buffer, pH 9.5 with Nitro Blue Tetrazolium (NET) and Bovine

Calf Intestinal Phosphatase (BCIP).

Characterization of the AtVSR3 gene trap line
The seeds were generously provided by Dr. Ueli Grossniklaus. Primers used in the
genotyping PCRs were: VSR3 forward — CCT TGT CCT TCG AAT TTG TTC TTT G;
GUS Reverse — GCT CTA GAT CGG CGA ACT GAT CGT TAA AAC; VSR3 Reverse
— GTC CTT CCC GGG GAA TAA ATA GAT G. The PCR products were purified with
a PCR purification kit (Qiagen) and sequenced by the Michigan State University DNA
Sequencing Facility and the University of California, Riverside DNA Sequencing
Facility.
Post-Transcriptional Silencing of the AtVSR3 Gene

The pFGC5941 dsRNA vector was a generous gift from Dr. Richard Jorgensen
and Dr. Vicki Chandler. I followed the cloning strategy recommended by the ChromDB
web page (http://ag.arizona.edu/chromatin/strategy.html). The primers to amplify the
coding region for the last 10 amino acids and 3’UTR of AtVSR3 were the following:
Forward — TCT AGA GGC GCG CCT CCC GAA CCA CGT TGA AT G ATG AAC G;
Reverse — GGA TCC ATT TAA ATA ACA ACA TGA ACT CTA AAA CAA GTA AC.
The forward primer added an Xba I and Asc I restriction enzyme site (underlined) for
cloning purposes. The reverse primer added a BamHI and a Swa I restriction enzyme
site (underlined) to the 3’ end for cloning purposes. The PCR product was cloned into
pGEM-T Easy (Promega) and sequenced by the UCR DNA Sequencing Facility. To add

the 3’ end of AtVSR3 in the forward orientation, this construct and the vector were

89

digested with A801 and SwaI. The relevant digestion products were purified from a 1%
low melt agarose gel and the DNA was purified from the gel using a kit (Qiagen). The
products were ligated with T4 Ligase (Roche) and transformed into DHSu. To add the
same AtVSR3 fragment in the opposite orientation, the new construct and the pGEM
construct were digested with BamHI and XbaI, ligated, and transformed as described
above. The integrity of the final construct was confirmed by triple restriction digest with
NotI, EcoRI, and PstI. The construct was transformed into Arabidopsis. Transgenic
seedlings were obtained by germinating the seeds on selection media. RT-PCR was
canied out for each putative transformant with primers corresponding to regions of the
AtVSR3 gene outside of the domain used in the dsRNA construct: forward — CCT TGT
CCT TCG AAT TI‘G TTC TTT G; reverse — TCT AGA GTC CTT CCC GGG GAA

TAA ATA GAT G.

Germination Assay

Approximately 300 wildtype and mutant seeds were sterilized and transferred to
Murashige Minimal Organics Medium (Gibco/Invitrogen) that contained either 3 uM
ABA in methanol or 500 pl methanol as a control. The plates were stored at 4°C in the
dark for three days. Then, the plates were transferred to room temperature conditions
with constant light. Germinated and ungerminated seeds were counted for five days.
Each seed was examined by a dissection microscope and a positive germination was

considered to be a seed for which the radicle had emerged fiom the seed coat.

Analysis of Seedlings with Thermal Camera

90

Seedlings were grown as described for the germination assay without ABA for
two weeks. The plates were photographed with a retail-grade digital camera and

analyzed with a ThermaCAM 560 from F LIR Systems.

Reverse Genetics of AtVSR7

ET2539 seeds were generously provided by Dr. Rob Martienssen. The plants
were genotyped by PCR with the following primers: GUS — GCT CTA GAT CGG CGA
ACT GAT CGT TAA AAC; AtVSR7 Forward - GAG ATG GGT TTA GTC AAC GGG
AGA G. RT-PCR was carried out with the AtVSR7 forward primer described above and
the following primer: AtVSR7 Reverse — GTA AAA GGC TCG GCT TCT GAT GGA
AC. Western blot analysis of the AtCPY in mutant and wildtype plants was carried out
as described in the Antisense materials and methods. SALK 005814 seeds were obtained
from the Arabidopsis Biological Resource Center (ABRC). PCR genotyping was
accomplished with the following primers: VSR 7exon8forward — TAT CTA CTG GCT
GCA AAT GTC CTG AAG GTT TCC, VSR73’UTRreverse — CAA GAA GCT TTC

ATA GCT CAG TTG GTT AG, SalkLBbl — GCG TGG ACC GCT TGC AAC T.

Immunoprecipitation

Seeds were germinated and grown for seven days in liquid media as described
above. Golgi were extracted from approximately 5g of whole seedlings following the
protocol described in Munoz et a1. (1996). Immunoprecipitations were carried out using
a protocol from Dr. Alessandro Vitale’s laboratory (D'Amico et al., 1992) with AtCPY

antibodies or the corresponding preimmune antisera (Rojo et al., 2003a). The fractions

91

were separated by 10% SDS-PAGE, blotted to nitrocellulose membrane, and probed with

anti-VSR antibodies (Ahmed et al., 1997).

Results
Antisense Silencing of the AtVSR Gene Family

The functions of the AtVSR gene family were examined by transforming
Arabidopsis with a large, conserved region of the gene family in the antisense orientation
relative to the constitutive 35$ promoter. Expression of the transgene should result in
post-transcriptional gene silencing (PTGS) of the endogenous gene family. A previous
graduate student, Dr. Sharif Ahmed, attempted to express antisense AtELP/AtVSR] fiom
the 358 constitutive promoter in Arabidopsis (Sharif Ahmed and Natasha Raikhel,
unpublished results). Together, Dr. Ahmed and I were able to obtain only three
transformants. The three plants set very few seeds and the germination rate of those seeds
was very low.

I characterized progeny of the three transformants. Antibodies against the
AtVSRs could not detect protein in three of the eight plants that germinated (Figure 3.1).
The three plants that did not have detectable AtVSR protein also did not accumulate RT-
PCR-detectable levels of mRNA for AtVSR4, AtVSR6 and AtVSR7 (data not shown). In
addition, plant number three (Figure 3.1) did not accumulate At VSRI transcript; plant
number seven (Figure 3.1) did not accumulate AtVSR5 transcript; and plant number eight

(Figure 3.1) did not accumulate mRNA for AtVSR2 or AtVSR3 (data not shown).

92

WT l 2 3 4 5 .6 7 8 M kDa

- m... .. .- . ' ‘ —.101
AtVSR_ 69
l
r-TIPj -’ ...— ~ .- i “128
. - . «~ - _. .: -M'zls

Figure 3.1. Some antisense AtVSR plants accumulate little or no AtVSR protein
Protein was extracted from the leaves of eight antisense plants. The protein was
separated by SDS-PAGE, blotted onto a nitrocellulose membrane, and probed with
antibodies against the AtVSRs (Ahmed et al., 1997) or against the vegetative
aquaporin, 'y-TIP (28kDa). The y-TIP antibodies are described in detail in Appendix
C. Plants 3, 7, and 8 accumulated less AtVSR protein (~80kDa) than WT or other
antisense plants.

93

The absence of a viable plant that silenced the entire AtVSR family indicated that a plant
can not survive without some amount of VSR protein.

The three plants with reduced AtVSR family protein showed. defects in
gravitropism, leaf morphology, and flower morphology (Figure 3.2). Specifically, the
roots of the seedlings consistently grew towards the plate lid (opposite the gravity vector)
and pushed the cotyledons and leaves into the agar (Figure 3.2A). Furthermore, the stem
of the bolting plant did not grow upward; it curled around inside the pot (compare
Figures 3.2 C and D). The leaves formed “cups” and “umbrellas” or would roll under
themselves (Figure 3.2B). There were also severe defects in flower development (Figure
3.2B). This phenotype was not observed in plants that accumulated wildtype levels or
higher levels of AtVSR protein. Very few seeds were set by the antisense plants and
again there was very low germination of the few seeds that were obtained. All of the
progeny plants that germinated showed no suppression of AtVSR genes.

While it was clear that the reduction of AtVSR protein caused a pleiotropic
phenotype, it was not obvious how protein trafficking to the vacuole was affected in the
antisense plants. Therefore, I determined whether the vacuolar trafficking of AtCPY was
affected in the antisense plants. AtCPY is an Arabidopsis soluble vacuolar protein that is
often used as a marker for transport to the vacuole (Rojo et al., 2003b). The intermediate
form of AtCPY is 43 kDa, and is processed to a 24 kDa protein after it is delivered to the
vacuole (Rojo et al., 2003a). Therefore, the absence of the 24 kDa band would indicate
that AtCPY vacuolar trafficking is blocked in the antisense plants. Two of these plants (3
and 8) lacked the 24 kDa form of CPY, indicating that at least one pathway to the vacuole

is dependent on the expression of members of the AtVSR family (Figure 3.3).

94

 

Figure 3.2. Antisense AtVSR plants showed defects in plant development. The
seedlings showed defects in root and shoot gravitropism (A). The rosette leaves
formed “cup” and “umbrella” shapes (B). While a wildtype plant bolts upward (C),
the bolts of the antisense plants coil around the rosette (D). There are also severe
defects in the flowers (E).

95

iCPY'>

mCPY-p

Figure 3.3. AtCPY is not delivered to the vacuole in two antisense plants.
Vacuolar transport of AtCPY is determined by the processing of AtCPY from an
intermediate form of ~43kDa to a mature 24kDa form. In wildtype plants, the
intermediate form of CPY (iCPY) is delivered to the vacuole and processed to
the mature form of CPY (mCPY; see WT lane). Under normal conditions, the
transport and/or processing of AtCPY is slow enough that both forms are
detected in wildtype by western blot analysis (Rojo et al., 2003). Thus, both
forms of AtCPY are present in the lanes containing protein extracted from
wildtype and sample 7, which accumulated a small amount of AtVSR protein.
However, the mature form of AtCPY was not detected in protein extracted from
plants 3 and 8, which did not accumulate any detectable AtVSR protein.

96

While it was not possible to obtain viable progeny from the AtVSR antisense
plants and learn more from this approach, the results indicated that the AtVSR genes are
important to many aspects of plant development. Furthermore, it is likely that at least
some amount of AtVSR protein is essential to the plant since there was such a strong

selection against the transmission of silencing to the progeny.

Functional Analysis of AtVSR3

RT-PCR and promoter::reporter fusion studies indicated that AtVSR3 was
expressed in guard cells and was regulated by ABA (Chapter 2). Similar results have
been reported for other components of the endomembrane system, such as SYP121 and
SYP61 (Leyman et al., 2000; Zhu et al., 2002). Therefore, I was very interested in
determining whether AtVSR3 had a functional role in the ABA signal transduction
pathway, and I took a reverse genetics approach to determine the function of AtVSR3. A
line carrying an insertion in the AtVSR3 gene was found in the Gene Trap collection fiom
Dr. Rob Martienssen (Springer et al., 1995). The Gene Trap collection is a collection of
Arabidopsis lines that were transformed with a non-autonomous transposon (Springer et
al., 1995). TAIL-PCR was used to determine the location of each T-DNA in the
collection and the information was stored in a BLAST-searchable database in Dr.
Martienssen’s laboratory. A BLAST search of this database with the coding region of
AtVSR3 revealed a gene trap line (GT3281) containing an insertion in the antisense
orientation at 182 basepairs after the start codon of AtVSR3 (Figure 3.4). I was unable to
obtain homozygous mutants of this line, suggesting that the lack of functional AtVSR3

caused garnetophytic, embryonic, or germination lethality.

97

i 2 345 67 89101112

 

V
wA

-WT1234 5 67 891011121314

W W...

3'33.=8’w Insertion (1+2)

 

Figure 3.4. Gene trap insertion in the AtVSR3 gene. The schematic depicts the
AtVSR3 gene with the number of each exon (black box) indicated above the exon
(A). The insertion (white triangle) was in the first exon of the AtVSR3 gene.
Primers (black arowheads) were designed to genotype these plants. PCR
genotyping of the plants indicated that I could not obtain a homozygous mutant
(B). The top gel shows the products of PCR reactions using primers 1 and 3,
demonstrating that all of the plants have at least one copy of the AtVSR3 gene
without an insertion. The bottom gel shows the products of PCRs using primers 1
and 2, demonstrating that one copy of the insertion is present in many of the
plants.

98

I determined whether there was only one insertion in these plants by TAIL-PCR (Liu et
al., 1995; Liu and Whittier, 1995). TAIL-PCR results for GT3281 indicated that at least
two other insertions were present. This data, in conjuction with the potential lethal
phenotype of the AtVSR3 insertion, made GT3281 a difficult line to study.

Thus, I addressed the firnction of AtVSR3 by using an RNAi approach to post-
transcriptionally silence or at least reduce the accmnulation of AtVSR3 transcript. DNA
encoding the last 10 amino acids of AtVSR3 and the 3’UTR of AtVSR3 were cloned into a
RNA interference (RNAi) expression vector in which the AtVSR3 DNA is in both the
forward orientation and the reverse orientation and the two orientations are separated by a
GUS intron (Chuang and Meyerowitz, 2000). The 3’ end of AtVSR3 was chosen because
this region is not as well conserved among the AtVSR genes. Wildtype Arabidopsis
plants were transformed with this construct. Seventy-one putative transformants were
obtained from three independent transformations. RNA was extracted from each plant
and 500ng of each sample was used as a template in semi-quantitative RT-PCR with
AtVSR3-specific primers. An example is shown for fourteen plants in Figure 3.5A. The
pixel intensity of each band was determined with the MCID Elite 6.0 image analysis
software to objectively compare the amount of VSR3 transcript accumulated in wildtype
to that of the transforrnants (data not shown). The RNA extraction and RT-PCR
experiments were repeated to ensure that the results were real. Three of the
transforrnants accumulated reduced amounts of AtVSR3 RNA and one transformant did
not accumulate any AtVSR3 transcript (Figure 3.5A). The seedlings that had little or no
AtVSR3 mRNA were small and accumulated more anthocyanins than wildtype seedlings

and the true leaves showed slight defects in leaf shape (Figure 3.5B-D). An analysis of

99

 

 

 

 

 

 

 

 

 

 

Total Number of Guard Cells

    

’5 ‘3’, ’c ‘9‘? a;

Figure 3.5. Visual characterization of VSR3 RNAi plants. RT-PCR of wildtype
(WT) and 14 putative transformants with AtVSR3-specific primers (A, top panel) or
with ubiquitin-specific primers (A,bottom panel). Three transforrnants had reduced
amounts of AtVSR3 mRNA (A-1.11, 1.18, and 2.6) and one line that did not accumulate
any AtVSR3 mRNA (A-3.l), while the levels of ubiquitin were at normal levels.
Seedling 3.1 is shown as a representative sample of the phenotype observed for 1.11,
1.18, 2.6, and 3.1 (CD) compared to wildtype (B). The mutant seedlings were smaller
than wildtype (B) with mishapen cotyledons that accumulated anthocyanins (C). The
true leaves of the silenced lines had slightly aberrant leaf structure as shown in panels C
and D. The abaxial surface of mature rosette leaves from four-week old plants
produced similar amounts of guard cells as a wildype plant (WT) and a putative
transformant (2.11) that accumulated normal levels of AtVSR3 transcript (E). A closer
look at the guard cells suggested that AtVSR3 dsRNA plants did not have as many
closed stomata as their wildtype counterparts under the same conditions (F). Closed
stomata were considered to be those which had a diameter less than 2.5um.

   

   

100

A“

 

gieihgeb h . Iran. .
. .. 5a.... . .

the abaxial surface of mature rosette leaves revealed that the RNAi plants produced
similar amounts of guard cells as their wildtype counterparts (Figure 3.5E). This
suggested that AtVSR3 does not play a significant role in stomatal development.
However, more guard cells in the mutants were open than in the wildtype plants under the
same conditions (Figure 3.5F), suggesting that AtVSR3 may participate in guard cell
opening and closing. If stomata of the mutants did not close, then the plants would have
higher rates of transpiration, and thus remain cooler than wildtype plants (Merlot et al.,
2002). Therefore, I compared the temperatures of wildtype and RNAi plants (line 3.1)
for differences in temperature using a thermal camera (Figure 3.6). The analysis
demonstrated that the mutants were an average 0.8°C (P<0.0001) cooler than their
wildtype counterparts (Figure 3.6). These results indicated that AtVSR3 may play a role

in stomatal movement.

The lack of VSR3 protein causes an ABA-insensitive phenotype in seeds

Guard cell movement is regulated by the phytohormone, ABA. In chapter two, I
reported that AtVSR3 expression was down-regulated by ABA. These results, combined
with the phenotype of the RNAi lines suggested that AtVSR3 is part of the ABA signal
transduction pathway. To confirm this, I asked whether AtVSR3 RNAi seeds were
insensitive to ABA in a germination assay. Normally, ABA inhibits seed germination in
wildtype seeds. However, mutants that do not respond to ABA will germinate in the
presence of ABA. Wildtype and RNAi line 3.1 germination rates were compared for five

days in the presence or absence of ABA (Figure 3.7). While wildtype seeds had a lower

10]

27 .3 'C

20

24

 

Figure 3.6. The average temperature of AtVSR3 RNAi seedlings is less
than the average temperature of wildtype seedlings. Wildtype and RNAi
mutant line 3.1 seeds were sown on agar plates and grown for two weeks (A —
wildtype on lefi and 3.1 on right. The plates were imaged with a thermal
camera (B — wildtype on left and 3.1 on right) and the results were presented in
a color format with a corresponding look up table. The temperatures of random
spots on the plate were collected and compared between the wildtype and
mutant plates. The average temperature over 33 random spots for two plates
was 0.8% higher in wildtype with a significance of P<0.0001.

102

 

% Germination

 

 

Figure 3.7 Germination frequency 01' wildtype and AtVSR3 RNAi
seeds. The germination frequency of wildtype seeds in the absence of
ABA (blue bar) is significantly higher than in the presence of ABA
(purple bar). However, RNAi 3.1 seeds did not show a significant
germination decrease in the presence of ABA. The y-axis is
measuring the percentage of germination.

103

germination frequency in the presence of ABA, there was no significant difference
between the germination rates of line 3.1 in the presence or absence of ABA (Figure 3.7).
These results further indicated that AtVSR3 functions in the ABA signal transduction

pathway.

Reverse Genetics of the AtVSR7 Gene

Two lines carrying an insertion in the AtVSR7 gene were found in the enhancer
trap collection (ET2539) (Sundaresan et al., 1995) and the SALK collection (SALK
005814) (Alonso et al., 2003). The enhancer trap line was found using the same
techniques as described for finding the AtVSR3 gene trap line. I found the SALK
insertion line by querying the TAIR database for information about AtVSR7
(http://www.arabidopsis.org). The locations of the insertions for the SALK lines were
also determined by TAIL-PCR (http://signa1.salk.edu). 1 confirmed the location of each
insert by TAIL-PCR. Both lines appeared to have a single insert based on TAIL-PCR
and segregation analysis of the resistance gene (data not shown). The insertion in the
enhancer trap line was located in the fourth intron of AtVSR 7, while the insertion in the
SALK line was located immediately after the stop codon of the gene. The homozygous
plants did not produce mRNA of AtVSR7 (Figure 3.8A). The plants had very stunted
growth throughout their lifecycle (Figure 3.8B, C). This phenotype could be due to the
production of less cells or due to the production of smaller cells. To address this issue, I
compared the sizes of leaf epidermal cells (data not shown) and leaf mesophyll cells

(Figure 3.8D) by microscopy of rosette leaves of the same age that had been cleared in

104

 

    
 

        

J n :1! n n mm w 3.“!
7" °t
Q "3
s9 o"
- é§ Q,
AtVSR]
WT ET2539

 

Figure 3.8. Characterization of AtVSR7 knockout lines. A schematic of the
AtVSR7 gene is depicted in panel A. The exons are depicted as a black line whereas
the introns are depicted as white triangles. The ET2539 insertion was located in the
fourth intron (large, black triangle) and the Salk insertion was located immediately
after the stop codon (large, grey triangle). Homozygous ET2539 plants do not
accumulate AtVSR7 mRNA, but do accumulate normal levels of other AtVSRs such
as AtVSR] (A). The seedlings are very small (B), as were the mature plants (C).
Micrographs of rosette leaf mesophyll cells are shown to demonstrate that the small
plants had smaller cells (D, bottom panel) than their wildtype counterparts (D, top
panel).

105

70% ethanol. The results indicated that the mutants produced smaller plants because
their cells were smaller (Figure 3.8D).

Based on sequence homology, the putative function of AtVSR7 is a vacuolar
sorting receptor. From this hypothesis, I predicted that an atvsr7 mutant would be unable
to deliver some proteins to the vacuole. This was a difficult hypothesis to test because
very few soluble vacuolar proteins have been well characterized in Arabidopsis.
However, antibodies for two Arabidopsis vacuolar markers are available. These proteins
are AtAleurain and AtCPY (Ahmed et al., 2000; Rojo et al., 2003a). I checked whether
the lack of AtVSR7 protein prevented the proper localization of AtAleurain or AtCPY.
However, both proteins were delivered to the vacuole with the same efficiency as
wildtype plants (data not shown), indicating that either AtVSR7 is not the VSR for these
proteins or that another member of the AtVSR family can take over the function of

AtVSR7 in its absence.

Immunoprecipitation of Vacuolar Sorting Receptors

A major challenge in determining the function of a plant VSR is that the cargo
proteins for a specific VSR are not known. Thus, there is no straightforward assay that
can be used to identify the in vivo function of each AtVSR. To address this issue, I took
a biochemical approach to identify the specific AtVSRs that interact with a known
vacuolar protein. Antibodies are available for characterized vacuolar proteins, such as
AtCPY and AtAleurain. These antibodies could be tools to identify the functions of the
AtVSRs. Seedlings were grown in liquid media for seven days in constant light and

fractions enriched for Golgi were extracted from the tissue. The Golgi-enriched fraction

106

was solubilized in detergent and incubated with AtCPY antibodies. Then, the mixture
was incubated with protein A sepharose beads, washed, and resuspended in Laemmli’s
buffer (Laemmli, 1970). Proteins from each of the steps were analyzed by western blot
analysis with anti-VSR antibodies to determine whether a VSR was co-
immunoprecipitated with the CPY antibody (Figure 3.9). An 80kDa band that reacted
with anti-VSR antibodies was present in the AtCPY immunoprecipitation that was not
present in the preimmune immunoprecipitation (Figure 3.9). A large amount of IgG
heavy chain was also present, and this impeded the identification of the AtVSR by mass
spectrometry techniques. I am now trying column chromatography to obtain a cleaner
elution of the coimmunoprecipitation so that it will be possible to identify which member

of the AtVSR family interacted with AtCPY in seven-day old seedlings.

Discussion

The overall function of the AtVSR gene family was studied by antisense
technology. The germination rate of the transforrnants was very low and most of the
seedlings did not suppress AtVSR expression. In chapter 2, I reported that many AtVSRs
were expressed in seeds. In particular, AtVSR] expression was detected in seeds by RT-
PCR, and promoter::GUS fusions, and seed ESTs for AtVSR] were identified.
Furthermore, a knockout line of AtVSR] indicates that it may function in the vacuolar
sorting of ZS Albumin in Arabidopsis seeds (Shimada et al., 2003). However, the
knockout line itself is not lethal (Shimada et al., 2003). The low germination rate and
low heritability of the antisense phenomena observed for the antisense plants in

combination with the viable, null mutant of AtVSR] reported from Dr. Hara-Nishimura’s

107

Washes

Pre

CPY

 

Figure 3.9 A VSR coimmunoprecipitated with AtCPY. Golgi-enriched
fractions were immunoprecipitated (IP) with preimmune antibodies or anti-AtCPY
antibodies. After three washes, the immunoprecipitations were resuspended in 3X
Laemmli’s buffer to release the proteins from the protein A sepharose beads. The
protein from 40ul of each fraction was separated by SDS-PAGE and transferred to
nitrocellulose membrane. The membranes were probed with antibodies against
AtVSR protein. AtVSR protein (80 kDa) is present in the Golgi sample, the flow-
through of the preimmune immunoprecipitation, and the elution of the CPY IP.

108

lab (Shimada et al., 2003) indicated that: i) another AtVSR has limited functional
redundancy with AtVSRl , or ii) a different AtVSR serves an essential function in seeds.
The antisense VSR seedlings showed defects in shoot and root gravitropism.
Shoot gravitropism is regulated by the plant hormone auxin and the endomembrane
system is involved in auxin signal transduction (Kato et al., 2002; Surpin et al., 2003).
Specifically, absence of functional AtVTIll v-SNARE results in defects in shoot
gravitropism (Kato et al., 2002; Surpin et al., 2003). Also, AtVTlll colocalizes with
AtVSRs (Zheng et al., 1999). The vsrl knockout mutant does not appear to have any
obvious gravitropic defects (Shimada et al., 2003). Therefore, it is possible that at least
one or more AtVSRs, other than AtVSRl, plays a role in gravitropism. However, this

remains to be seen directly.

AtVSR3 plays a role in guard cell function

Results from the AtVSR3 RNAi lines suggested that the little or no AtVSR3
protein results in plants that may have a decreased ability to close stomata relative to
wildtype plants. I demonstrated that AtVSR3 is ubiquitously expressed in seedlings and is
only expressed in the guard cells of true leaves (Chapter 2). An ATP-binding cassette
(ABC) transporter, AtMRP5 (multidrug resistance-associated protein), has a very similar
expression pattern by promoter::GUS fusions (Gaedeke et al., 2001). Furthermore, the
guard cells of a knockout line for AtMRP5 do not close in response to ABA (Klein et al.,
2003). Therefore, AtVSR3 and AtMRP5 exhibited similar expression patterns and
mutants that down regulate or lack the expression of these genes had similar phenotypes

(Gaedeke et al., 2001; Klein et al., 2003). This suggests that the two proteins may

109

function in the same pathway. The MRP5 protein consists of 1501 amino acids. An
NPIR motif (exactly N-P-I-R) occurs at amino acids 865-869, directly in the middle of
the protein. The NPIR motif does fimction as a VSS from the internal sequence of the
protein, ricin (Frigerio et al., 2001). However, MRP5 is an integral membrane protein
and very little is known about the vacuolar trafficking of integral membrane proteins
(Brandizzi et al., 2002). Clearly, the potential interactions of these two proteins should

be examined further.

AtVSR7

The vsr7 mutants were very small. AtVSR7 expression was detected mostly in
roots and at the distal ends of leaves under normal conditions (Chapter 2). AtVSR7 is also
upregulated in response to phosphate starvation (Hammond et al., 2003). Numerous
vacuolar proteins are also upregulated in response to phosphate starvation (Hammond et
al., 2003). Therefore, it is possible that AtVSR7 is the receptor for one or more of these

precursor proteins.

Conclusions

The AtVSR family is thought to direct soluble VSS-containing proteins to the
vacuole. I took reverse genetics and biochemical approaches to determine their specific
functions. The overall results indicated that the AtVSRs play roles at multiple levels of
plant development and are potentially involved in hormone-related signal transduction

pathways.

110

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114

Chapter 4

Conclusions and Future Directions

115

EXpression Analysis of the AtVSR Gene Family
The AtVSR gene family is composed of seven members. In this thesis, I present

TESults showing the expression patterns of the AtVSR genes, as well as reverse genetics,
and biochemical approaches to determine the functions of the AtVSR genes. The
expression patterns of each gene indicated that the AtVSRs have tissue- and cell-type
specific expression patterns. This result implies that the AtVSR genes are not completely
redundant. Specifically, I found that none of the AtVSRs were expressed ubiquitously,
which is the expression pattern of vacuolar sorting receptors in other eukaryotes. Also,
one of the AtVSRs, AtVSR3, was specifically expressed in guard cells in an ABA-
dependent manner. This result indicated that plants encode VSRs for specific functions
in specific signal transduction pathways. This has not been reported for any other

eukaryotes, and thus is an important scientific contribution.

Subcellular Localization of AtVSRs

Homologues of the AtVSRs in other plants have been localized to organelles
outside of the CCV pathway, such as ER bodies and PAC vesicles. With this in mind,
studies of the AtVSRs should be extended to the subcellular localization of each protein.
This can be accomplished by transforming Arabidopsis plants with tagged-versions of the
individual AtVSR genes. Towards this end, we have transformed Arabidopsis with a
construct that encodes the AtVSR] promoter and the AtVSR] gene fused to the coding
region of YFP (C. Sambojou and N. Raikhel, unpublished data). A similar construct for
AtVSR3 has also been transformed into Arabidopsis. The YFP marker can be used to

determine the subcellular localization of an individual AtVSR by microscopy and

116

biochemical techniques. Confocal microscopy would give some indication of the
subcellular localization of the AtVSRs. However, immuno-electron microscopy with
anti-YFP antibodies would give a higher resolution image, and thus a more conclusive
localization of the protein. It should also be possible to fractionate the organelles by
sucrose density gradient and look for the YF P protein by western blot analysis with anti-
YFP antibodies. A comparison of the YFP fractionation pattern with markers for other
endomembrane organelles and vesicles would determine the localization of the chimeric
protein. These experiments will determine where each AtVSR participates in vacuolar

protein targeting.

Recycling of Plant VSRs

Regardless of an individual VSR’s position in the vacuolar targeting pathway,
current models predict that all VSRs are recycled from the recipient compartment to the
donor compartment. While some aspects of the recycling mechanism have been pursued,
the actual process has not been demonstrated in plants (Sanderfoot et al., 1998; Happel et
al., 2004). The plant AtVSRs, yeast Vple, and mammalian MPR all have a tyrosine
motif in the cytoplasmic tail that interacts with AP-l for clathrin-coat formation around
the budding vesicle (Sanderfoot et al., 1998; Bonifacino and Dell'Angelica, 1999;
Deloche et al., 2001) In addition to AP-l, a family of sorting nexin (SNX) proteins may
also share the responsibility of receptor protein localization (Kurten et al., 1996). A yeast
2-hybrid assay using the C-terminus of the epidermal growth factor receptor (EGFR)
identified a sorting nexin protein (SNXI) that interacted with the C-terminus of EGF R

(Kurten et al., 1996). A YLVI motif in the C-terminus of EGFR is necessary for the

117

interaction between EGFR and SNXl (Kurten et al., 1996). SNXl is a hydrophilic
peripheral membrane protein that contains a p40 phox (PX) domain that binds
phosphotidylinositol 3-phosphate (Ponting, 1996). The C-termini of SNXl has a coiled-
coil domain that is important for dimerization between SNX proteins (Seaman and
Williams, 2002). SNXs have been shown to interact with various receptors that are
transported through the endomembrane system (Haft et al., 1998).

Three proteins in yeast, Mvplp, VpsSp, and Vpsl7p, share significant homology
with human SNX] (Horazdovsky et al., 1997). Mvplp also shares significant homology
to dynamin and thus may perform a function in vacuolar sorting that is distinct from
SNX]. However, there is evidence that VpsSp and Vpsl7p function together to perform
a role in receptor recycling that is orthologous to SNXl (Horazdovsky et al., 1997).
vps5p and vpsl 7p mutants accumulate numerous small vacuoles and secrete CPY. The
defect in CPY sorting to the vacuole is due to the mislocalization of the CPY sorting
receptor, Vple. VpsSp and Vpsl7p dimerize through their coiled-coil domains to form
part of the retromer complex in yeast. The retromer complex also consists of Vp326p,
Vp829p, and Vps35p. Together, this complex is thought to mediate the recycling of
receptor proteins within the endomembrane system. Human SNXl was shown to bind a
homolog of yeast Vp327p. Therefore, Vp327p may also be a member of the retromer
complex.

A retromer complex has not yet been characterized in plants. However, there are
genes in the Arabidopsis genome whose deduced amino acid sequences share significant
sequence homology with human SNX] and yeast VpsSp. Likewise, the C-termini of

AtVSRs also have YLVI motifs. Therefore, Arabidopsis may also utilize a retromer

118

complex to recycle vacuolar sorting receptors from the prevacuolar compartment to the
TGN. This can be further examined in two ways. First, immunoprecipitation of AtVSRs
with generic previously characterized AtVSR antibodies (Ahmed et al., 1997; Li et al.,
2002) should pull out cytosolic interacting factors that can be analyzed by protein blots
and/or mass spectrometry. Second, the localization of AtVSRs should be determined in
knockout mutants for the Arabidopsis homologues of the retromer complex. These two
approaches should determine whether the mechanism of VSR recycling is analogous to

yeast or mammalian systems.

Reverse Genetics Approaches to Determine the Functions of AtVSRs

The functions of the AtVSRs were examined by reverse genetic techniques such
as post-transcriptional gene silencing and the characterization of T-DNA insertion lines.
By attempting to silence the entire AtVSR family, we obtained plants that had very low
germination rates, defects in root and shoot gravitropism, as well as defects in leaf and
flower morphology. These processes are regulated by hormones such as ABA,
gibberellic acid and auxin. With this in mind, the promoter::GUS fusion lines for the
AtVSR promoters and RT-PCR should be used to determine if any of the AtVSR genes are
upregulated or down-regulated in response to phytohormones. For example, the plants
expressing the promoter: :GUS constructs could be treated with phytohorrnones or
subjected to different environmental conditions and subsequently stained to determine
GUS activity. Similar research was accomplished in the studies of auxin-responsive
genes such as DRS (Sabatini et al., 1999). The results of these experiments would lead to

hypotheses that could be tested in knockout lines of the AtVSRs.

119

 

Reverse Genetics to Understand the Function of AtVSR3

Examination of RNAi plants that partially or completely silenced AtVSR3
revealed that the reduction or lack of AtVSR3 causes the stomata to remain open in
greater numbers than stomata from wildtype plants. The temperature of these plants was
lower than the temperature of wildtype plants as a result of the non-responsive stomata.
Similar results were observed for the ABC transporter, AtMRP5 (Klein et al., 2003),
suggesting that AtMRP5 and AtVSR3 function in the same pathway. Surprisingly,
AtMRP5 has an NPIR motif. An exciting speculation is that AtVSR3 is a vacuolar
sorting receptor for AtMRP5. Many conventional strategies to address this issue will be
difficult because both proteins are integral membrane proteins. We already have putative
transforrnants of AtVSR3 fused to YFP. Therefore, we can prepare a construct containing
AtMRP5 fused to CFP and transform it into the AtVSR3:YFP plants. The leaves of the
double-trans gene plants can be tested for fluorescence energy resonance transfer (FRET)
activity by confocal microscopy (Huang et al., 2001; Shah et al., 2002). The YFP
fluorescence is distinguished from the CF P fluorescence by the presence of specific
emission filters in the light path. Another possibility is to make antibodies against MRP5

for use in colocalization experiments with AtVSR3.

Identification of the In Vivo Targets of AtVSRs

In order to fully characterize an AtVSR, we need to identify the putative in vivo
targets of the AtVSRs. The best method for accomplishing this is to transform
A rabidopsis with epitope-tagged versions of the AtVSRs. In this way, an individual

AtVSR can be specifically immunoprecipitated. The proteins that coimmunoprecipitate

120

with the tagged protein can be analyzed by western blot or by mass spectrometry to
determine their identities. This will determine whether each AtVSR interacts with a
specific type of VSS and whether each interaction is regulated by pH, calcium, or by
another mechanism. The approaches described above will lead to a more comprehensive
understanding of VSR-mediated trafficking in plants. An added benefit to this approach
is that we will likely pull out proteins that interact with the cytoplasmic tail of the AtVSR
as well. The identification of these proteins will be useful in determining the subcellular
localization and recycling of the AtVSRs, a question that was discussed earlier in this

chapter.

Conclusions

Studying the AtVSR gene family presents an excellent opportunity to understand
how plants differ from higher eukaryotes. The results presented in this thesis
demonstrate that the endomembrane system of plants follows the same paradigm that has
been described in other eukaryotes. However, the diversity of the plant endomembrane
system has been expanded to accommodate the unique lifestyle of plants. Our lab is now
using high throughput and systems biology approaches to describe these processes in

more detail.

121

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123

APPENDICES

124

APPENDIX A

Supplementary Information for Chapter 2

125

Table A.1. ESTs and cDNAs for the AtVSR] Gene

 

 

 

 

 

 

 

 

 

 

Source Tissue and Number of ESTs and/or cDNAs GenBank Accession(s)

Found

Deve10p_ing Seeds 5-13 daf(1) BE529517

Benngg Immature Seed cDNA Library (1) M74D05

Green Siliques (4) AV567436; Z35038; Z35039; 238123

3-Day Old Seedling Hypocotyls (l) AA042124

Rosette Plants Subject to Dehydration (5) AV794541; AV826147; AV793751;
AU238557; AV794045

A-PRLZ (4) AA650957; R30384; AA605487; R90202

 

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REFERENCES

Elmayan, T., and Tepfer, M. (1995). Evaluation in tobacco of the organ specificity and
strength of the rolD promoter, domain A of the 358 promoter and the 3582
promoter. Transgenic Res 4, 388-396.

Fujiwara, T., and Beachy, R.N. (1994). Tissue-specific and temporal regulation of a
beta-conglycinin gene: roles of the RY repeat and other cis-acting elements. Plant
Mol Biol 24, 261-272.

Hwang, Y.S., Karrer, E.E., Thomas, B.R., Chen, L., and Rodriguez, R.L. (1998).
Three cis-elements required for rice alpha-amylase Amy3D expression during
sugar starvation. Plant Mol Biol 36, 331-341.

Kim, S.Y., Chung, HJ., and Thomas, TL. (1997). Isolation of a novel class of bZIP
transcription factors that interact with ABA-responsive and embryo-specification

elements in the Dc3 promoter using a modified yeast one-hybrid system. Plant J
11,1237-1251.

Kizis, D., and Pages, M. (2002). Maize DRE-binding proteins DBFl and DBF2 are
involved in rab17 regulation through the drought-responsive element in an ABA-
dependent pathway. Plant J 30, 679-689.

Lessard, P.A., Allen, R.D., Bernier, F., Crispino, J .D., Fujiwara, T., and Beachy,
R.N. (1991). Multiple nuclear factors interact with upstream sequences of
differentially regulated beta-conglycinin genes. Plant Mol Biol 16, 397-413.

Lu, C.A., Ho, T.H., Ho, S.L., and Yu, S.M. (2002). Three novel MYB proteins with one
DNA binding repeat mediate sugar and hormone regulation of alpha-amylase
gene expression. Plant Cell 14, 1963-1980.

Mena, M., Cejudo, F.J., Isabel-Lamoneda, 1., and Carbonero, P. (2002). A role for
the DOF transcription factor BPBF in the regulation of gibberellin-responsive
genes in barley aleurone. Plant Physiol 130, 11 1-1 19.

Plesch, G., Ehrhardt, T., and Mueller-Roeber, B. (2001). Involvement of TAAAG
elements suggests a role for Dof transcription factors in guard cell-specific gene
expression. Plant J 28, 455-464.

132

Thomas, M.S., and Flavell, RB. (1990). Identification of an enhancer element for the
endosperm-specific expression of high molecular weight glutenin. Plant Cell 2,
1171-1180.

Wu, C., Washida, H., Onodera, Y., Harada, K., and Takaiwa, F. (2000). Quantitative
nature of the Prolamin-box, ACGT and AACA motifs in a rice glutelin gene

promoter: minimal cis-element requirements for endosperm-specific gene
expression. Plant J 23, 415-421.

Yamauchi, D. (2001). A TGACGT motif in the 5'-upstream region of alpha-amylase
gene from Vigna mungo is a cis-element for expression in cotyledons of
germinated seeds. Plant Cell Physiol 42, 635-641.

133

Appendix B
Avila, E.L*., Zouhar, J *., Agee, A.E., Carter, D.G., Chary, S.N., Raikhel, N .V.
(2003). Tools to Study Plant Organelle Biogenesis. Point Mutation Lines
with Disrupted Vacuoles and High-Speed Confocal Screening of Green

Fluorescent Protein-Tagged Organelles. Plant Physiology, 133(4): 1673-1676.

*E.L. Avila and J. Zouhar contributed equally to this work.

134

We have focused our studies in the past several years on understanding protein
trafficking fi'om the secretory system to the vacuole—an organelle present in all plant
cells. Here, we report an approach for generating and screening plants with defects in
vacuolar biogenesis. Plant vacuoles are commonly known to be multifunctional
organelles, and recent findings have even demonstrated a variety of new roles for
vacuoles and the vesicles that deliver cargo to them. Although it has always been
assumed that vacuoles are essential for plant survival, the recent isolation of a T-DNA-
tagged mutant called vclI (vacuoleleSSI) has unequivocally demonstrated that vacuoles
are vital organelles (Rojo et al., 2001). Mutations in the yeast (Saccharomyces cerevisiae)
ortholog of VCLl, VPSl6, also block vacuole biogenesis and affect all known vacuolar
protein transport pathways in yeast; however, in contrast to VCLl, the W816 gene
product is not essential (Horazdovsky and Emr, 1993). A major effect of VCLl
inactivation is that it blocks the formation of vacuoles, leading to embryonic lethality.
Thus, although the isolation of vclI served to emphasize the importance of plant vacuoles
to plant growth and development, it is difficult to gain additional information about
vacuole biogenesis from an embryo lethal mutant. Some important proteins that likely
mediate trafficking to the vacuole are represented by single genes in the Arabidopsis
genome. For example, each of the six members of the AtC-VPS complex for which
VACUOLELESSI is a member is encoded by a single gene (Rojo et al., 2003); thus, null
mutations in these genes would also most likely be lethal.

Similar conclusions were drawn when several knockout mutants from the SNARE
family were isolated. Although a T-DNA insertion into the SYP61/08M] syntaxin is

viable (Zhu et al., 2002), some reported null mutations of syntaxin genes are not tolerated

135

by the plant. For example, a T-DNA disruption of a single member of the SYPZ and
SYP4 gene families is gametophytic lethal (Sanderfoot et al., 2001). Another knockout
mutant, “knolle ” (sypIII), is embryo/seedling lethal (Lukowitz etal., 1996). However, a
point mutation in the SYP22/SGR3 gene is viable, and the mutant lacks the shoot
gravitropic response (Y ano et al., 2003). Thus, it becomes apparent that the isolation of
plants with point mutations would be a very valuable tool to isolate viable mutants for
studying plant vacuolar biogenesis. With this in mind, we wanted to identify mutants
with small defects in vacuolar biogenesis genes that at the same time would not be lethal
to the plant.

To allow for effective visualization of vacuolar structure, we chose Arabidopsis
lines expressing a fluorescent tonoplast marker, green fluorescent protein (GFP): A-
tonoplast intrinsic protein (TIP), under the control of the 358 promoter (Cutler et al.,
2000). The tonoplast-localized GFP fusion protein in the tonoplast of these plants is
easily visualized by confocal microscopy (Fig. B.1, A—D). Homozygous seeds from
3SS::GFP: A-TIP plants were obtained, and vacuoles from these plants were isolated
using the technique described previously by Ahmed et a1. (Ahmed et al.). Proper GFP:A-
TIP localization at the tonoplast was confirmed by microscopy (Fig. B.1E). Seeds from
homozygous plants were then treated with ethyl methanesulfonate (EMS) to induce l-bp
changes throughout the genome of this line of plants. The Meridian Insight Point
Confocal Microscope (Meridian, Okemos, MI) was used to screen 7-d-old seedlings from
the M2 generation for broken or malformed vacuoles, mistargeting of the GFPzA-TIP
chimeric protein, or other interesting phenotypes. The Meridian Insight Confocal has

real-time ocular viewing confocal capability that allowed us to rapidly screen large

136

 

 

Figure B.l GFP:-TIP is expressed in the tonoplast of 3SS::GFP: -TIP transgenic
seedlings (A) such as cotyledon epidermal cells (B), hypocotyls (C), and roots (D).

E, Example of an isolated vacuole stained with neutral red (top) and with GFP
fluorescence (bottom). Scale bar 40p.

I37

numbers of seedlings. For each seedling, three types of tissues were examined:
cotyledons, hypocotyls, and roots (Fig. B.1A).

Thus far, we have screened 9,175 M2 EMS seedlings (56 pools; approximately

160 seedlings per pool) using confocal microscopy. Seedlings (620; 7%) showed
mutations in pigment development, indicating that mutagenesis and the screening
protocol were robust (Lightner and Caspar, 1998). Originally, 211 putative mutants with
defects in vacuole biogenesis were obtained; however, 110 died before setting seed
and/or did not produce any seed (F i g. B.2, I—L), resulting in our current population of 101
putative vacuolar mutants. These mutants have been sorted into four broad categories
based on their subcellular phenotypes in the M2 generation.

The first category of mutants (bub [bubble-bath]) is characterized by increased
numbers of small vacuolar vesicles in the cell (Fig. B.2, A—C). Forty-six plants fell into
this category. Among these plants, the bub phenotype was observed either in roots (three
plants), in cotyledons (29 plants), or in hypocotyls and cotyledons (six plants). Although
bub plants appear to have a large, central vacuole, they also have increased numbers of
vesicles decorated with GFPzA-TIP relative to the parental line. The severity of the
phenotype correlated with plant lethality because plants with the highly pronounced bub
Phenotype did not survive (Fig. B.2, I-K) or did not produce seeds. The second category
0f mutants contained large aggregates of GFP fluorescence (agg, Fig. B.2, D—F). Thirty-
four plants fit into this category. Among these plants, the agg phenotype was observed
Either in roots (14 plants), in hypocotyls (nine plants), in cotyledons (one plant), in
1"flypocotyl and cotyledons (one plant), or in hypocotyls and roots (one plant). Upon

closer examination of M3 agg plants, we observed that some of the aggregates are

138

RE
' '. A'vafi-J—E :9-

 

Figure B.2. Examples of vacuolar mutants identified. bub mutants have increased
numbers of vesicles in the cotyledons (A), hypocotyls (B), or roots (C). Examples of ag
mutants with aggregates in the cotyledons (D) and hypocotyls (E). A closer look at the
aggregates (F) reveals a membrane-bound vesicular structure. G, Example of the tvs
mutant class with increased transvacuolar strands. Some of the mutants had complex
phenotypes, such as disruption of cell shape (H). I to L, Many interesting M2 vacuolar
mutants that did not survive to the M3 generation. Images A to G were collected by a
Leica TCS SP2/UV Confocal Microscope (Leica Microsystems, Wetzlar, Germany).
Images H to L were collected on a Meridian Insight Point Confocal Microscope with a
CCD-cooled camera. Scale bar 40 um.

139

 

 

 

140

membrane bound compartments containing clusters of vesicles (Fig. B.2F). The third
category contained mutants that showed vacuoles apparently transected by transvacuolar
strands (tvs; eight plants; Fig. B.2, G and L). Among these plants, the tvs phenotype was
observed either in roots (three plants), in cotyledons (two plants), in hypocotyl (one
plant), in hypocotyl and cotyledons (one plant), or in hypocotyls and roots (one plant).
Further investigation of the M3 generation of tvs plants revealed that their vacuoles were
extremely dynamic with the continuous rearrangement of the transvacuolar strands.
Additional mutants appear to have unique and more complex phenotypes (Fig. B.2H).
Thirteen viable M2 plants were clustered into this category. The unique phenotypes
included defects in the regular pattern of the cotyledon epidermal cells (seven plants).
Interestingly, approximately one-half of seedling-lethal mutants (60 plants; Fig. B.2, I—L)
showed complex or unique phenotypes throughout the seedling.

Because genetic mapping of these mutants will involve large numbers of F2
seedlings, we are working toward developing a high-throughput screening process. We
are now using an Atto Pathway HT hi gh-throughput confocal microscope system (Atto
Bioscience, Rockville, MD) with culture plates of our own design for germinating and
growing seedlings (Fig. B.3). All tissues are screened manually or by an advanced
automated imaging routine without damaging the seedlings (Fig. B.3, C and D). An
example of the wild-type and mutant (bub) images produced by the Atto Pathway
microscope can be accessed in supplemental data, available in the online version of this

article at http://www.plantphysiol.org.

Conclusions

141

 

 

 

 

Figure B.3. A, Assembly of screening plates: a standard multiwell plate lid (A) has a
silicone gasket with 48 hourglass-shaped holes (B) applied to its outer surface. The
bottom half of each opening (C) is filled with solid growth medium, and a seed is
pipetted into the neck. A sheet of cellophane (D) flattens and seals the screening wells
while allowing gas exchange. The cellophane is replaced with a coverslip for imaging.
Populated plates are stacked together, sealed with surgical tape, and then incubated in
a vertical position for 7 d. To keep the seeds hydrated, a layer of agar is deposited on
the inside of each plate before it is populated and stacked. B, Populated plates viewed
from above. Gravitropism assures vertical orientation of seedlings, which are
illuminated evenly from all sides. C, Seedling in single well. The squares indicate the
standard 5 2 6 search pattern for automatically finding cotyledon, hypocotyl, and
root, respectively. Scale bar 3 mm. D, Plate in the climate controlled imaging
chamber of the Pathway HT automated imager. All tissues are close enough to the
cover glass to be imaged by the UApo/340UV 20/0.75NA objective lens, which
moves on linear motors below the sample. For more information on microscopy
methods described above, visit http://www.cepceb.ucr.edu.

142

Using a mutagenized transgenic line expressing a tonoplast-localized protein fused to
GP P, we were able to screen for vacuolar biogenesis mutants using confocal microscopy.
Although we used EMS to mutagenize plants to generate point mutations, approximately
50% of the vacuolar mutants did not survive. Nevertheless, we were able to isolate four
groups of mutants that would be useful in further analysis of vacuolar biogenesis. In our
mutant screen, we found mutants that exhibit defective or modified vacuoles throughout
the plant and mutants whose vacuolar phenotype is specific to a particular tissue of the
plant. This suggests that the endomembrane system in shoots can be uncoupled from
organization of the endomembrane system in roots and indicates that vacuolar biogenesis
has tissue-specific components. It is also important to note that we never recovered a
mutant seedling that completely lacked a large, central vacuole (Fig. B.2, A—L). This
result supports previous conclusions by Rojo et a1. (2001) that the vacuole is an essential
organelle to the plant cell. It is likely that an approach encompassing only transient
disruptions of vacuolar biogenesis components, such as chemical genetics, will be
beneficial to directly target many fundamental vacuolar biogenesis proteins.

Although the screen was originally performed using the Meridian Insight Point
Confocal Microscope, a system has been developed that grows up to 48 seedlings on a
multiwell plate lid, which can be imaged automatically. Although fully automating the
process is at an early stage of development, it will eventually increase the scope of
possible experiments, including chemical genetics screens to test for the effects of drugs
on seedling germination and tissue development. One of the challenges will be to manage
the large volume of data generated by automated screening. Similar screening approaches

could offer an excellent opportunity to study the biogenesis of other plant organelles.

143

REFERENCES

Ahmed, S.U., Rojo, E., Kovaleva, V., Venkataraman, S., Dombrowski, J.E.,
Matsuoka, K., and Raikhel, N.V. (2000). The plant vacuolar sorting receptor
AtELP is involved in transport of NH2-terminal propeptide-containing vacuolar
proteins in Arabidopsis thaliana. J Cell Biol 149, 1335-1344.

Cutler, S.R., Ehrhardt, D.W., Griffitts, J.S., and Somerville, C.R. (2000). Random
GFP :: cDNA fUSiOl’lS enable visualization of subcellular structures in cells of
Arabidopsis at a high frequency. P Natl Acad Sci USA 97, 3718-3 723.

Horazdovsky, B.F., and Emr, S.D. (1993). The VPSl6 gene product associates with a
sedimentable protein complex and is essential for vacuolar protein sorting in
yeast. J Biol Chem 268, 4953-4962.

Lightner, J., and Caspar, T. (1998). Seed mutagenesis of Arabidopsis. Methods Mol
Biol 82, 91-103.

Lukowitz, W., Mayer, U., and J urgens, G. (1996). Cytokinesis in the Arabidopsis
embryo involves the syntaxin-related KNOLLE gene product. Cell 84, 61-71.

Rojo, E., Gillmor, C.S., Kovaleva, V., Somerville, C.R., and Raikhel, N.V. (2001).
VACUOLELESSI is an essential gene required for vacuole formation and
morphogenesis in Arabidopsis. Dev Cell 1, 303-310.

Rojo, E., Zouhar, J ., Kovaleva, V., Hang, S., and Raikhel, N.V. (2003). The AtC-VPS
protein complex is localized to the tonoplast and the prevacuolar compartment in
Arabidopsis. Mol Biol Cell 14, 361-369.

Sanderfoot, A.A., Pilgrim, M., Adam, L., and Raikhel, N.V. (2001). Disruption of
individual members of Arabidopsis syntaxin gene families indicates each has
essential functions. Plant Cell 13, 659-666.

Yano, D., Sato, M., Saito, C., Sato, M.R., Morita, M.T., and Tasaka, M. (2003). A
SNARE complex containing SGR3/AtVAM3 and ZIGNTIll in gravity-sensing

cells is important for Arabidopsis shoot gravitropism. Proc Natl Acad Sci U S A
100, 8589-8594.

Zhu, J ., Gong, Z., Zhang, C., Song, C.P., Damsz, B., Inan, G., Koiwa, H., Zhu, J.K.,
Hasegawa, P.M., and Bressan, R.A. (2002). OSMl/SYP61: a syntaxin protein in

144

Arabidopsis controls abscisic acid-mediated and non-abscisic acid-mediated
responses to abiotic stress. Plant Cell 14, 3009-3028.

145

Appendix C

Identification of the Protein Contents of Plant Cell Vacuoles

146

Introduction

One possible distinctive feature of AtVSR proteins may be that each interacts
with a different type of VSS, which in turn may target the protein to a distinct vacuole
(Vitale and Raikhel, 1999). To fully explore this possibility, it is necessary to
characterize many different soluble vacuolar proteins to use as markers. A proteomics
approach was used to identify protein markers for specific types of vacuoles. I attempted
to collect distinct types of vacuoles by F luorescence-Activated Cell Sorting (FACS) and

analyze their protein contents.

Materials and Methods
Preparation and Characterization of TIP Antibodies

Synthetic peptides were prepared and conjugated with KLH by the MSU
Macromolecular Structural Facility. The peptides were then injected in rat and chicken
for antibody production by Cocalico Biologicals Company. The specificity of the
antibodies was examined by western blot analysis of seed or leaf protein with 1:500

dilutions of each antibody in the presence of each synthetic peptide.

Obtaining Vacuolesfrom Arabidopsis Leaves

Plants that were homozygous for the 35S::GFP:A-TIP transgene or wild type
plants were grown on soil under long day conditions in a temperature controlled growth
chamber. Protoplasts were isolated from leaves of 4-6 week old Arabidopsis plants using

a protocol adapted from Damm & Willmitzer(l988) with modifications. Briefly, the

147

leaves were sliced into strips and digested for four hours at room temperature in the dark
in a solution containing 1% cellulose R10, 0.5% Macerozyme R10, 30mM CaClz, 0.1%
BSA, and 5mM 2-Mercaptoethanol. The resulting protoplasts were filtered through an
80pm sieve, then washed twice in 0.4M Mannitol and IOmM MES pH5.7 and centrifuged
20 minutes at 50xg in a swinging bucket rotor. Then the protoplasts were lysed and the
vacuoles were purified according to a published protocol (Ahmed et al., 2000). The
purity and concentration of vacuoles were determined by light microscopy with neutral
red staining or fluorescent microscopy to check for GFP fluorescence.
Flow Cytometry

Fractions that contained a high concentration of vacuoles without debris were
submitted for flow cytometry through the FACS Vantage Flow Cytometer (Becton
Dickinson) at the MSU Core Flow Cytometry Facility. The 488 nm argon laser was used
to excite GFP and the detector scanned for 530nm light. The sheath fluid was changed
from PBS to a mannitol solution (0.5M Mannitol, lOmM Hepes pH 7.5, lmM EDTA,

150mM NaCl) to meet the high osmolarity needs of the vacuoles.

Results

It has been suggested that distinct vacuoles within a cell can be distinguished from one
another by the presence of specific Tonoplast Intrinsic Proteins (TIPS) on the tonoplast
(J auh et al., 1999). In order to be sure that TIPS decorate distinct vacuoles in
Arabidopsis, I had to be able to specifically detect each TIP. In Arabidopsis, a-TIP, y-
TIP, and A-TIP are very similar to one another at the amino acid level; however, the C-

termini are sufficiently divergent to be able to generate specific antibodies against each

148

TIP (J auh et al., 1999). Peptides corresponding to the C-terminal amino acid residues of
a-TIP (PPTHHAHGVHQPLAPEDY), y-TIP (HEQLPTTDY), and A-TIP
(HVPLASADF) were synthesized and coupled to Keyhole Limpet Hemocyanin (KLH)
by the MSU Macromolecular Structural Facility. The peptides coupled to KLH were
solubilized and injected into chicken and rat to raise antisera against the peptide.
Chickens and rats were chosen so that double-immnocytochemistry could be done to
ensure that multiple types of vacuoles in the same cell are distinguished by the TIPS
present on the tonoplast.

The antibodies against a-TIP and y-TIP fi'om rat and chicken Specifically
recognized a band of the expected Size (28kDa) in the seeds and leaves, respectively, and
the antibody-antigen interaction could be competed away only in the presence of the
peptide used to make the Specific antibody (Figure CI). The antisera raised against A-
TIP from both rat and chicken do not specifically recognize A-TIP. These antibodies will
allow us to determine whether distinct vacuoles in Arabidopsis can be distinguished from
one another by the TIPS present on their tonoplast. They were (and are currently) also

used in numerous other projects in Dr. Raikhel’s laboratory.

Vacuoles Isolated From 3SS::GFP:A-TIP Leaves have GFP Fluorescence in the
T onoplast

If different types of vacuoles can be distinguished by TIPS, then the TIPS will be
used as markers to isolate each type of vacuole by FACS and examine the protein
contents. The first question to address was whether isolated plant cell vacuoles could be

visualized by fluorescence if a GFP-Tagged protein was localized to the tonoplast.

149

Peptide competitor: - 6:8“ «63 8‘6

a-TIP M‘“M' “aw-W
Chicken
y-TIP
Chicken
a-TIP
Rat
. 2; ' j 1 :5: i - f.
I z u .‘ a i."
.L 5.“- _ 1. - t, it
~ ;? * A 6-
1") f. :2" I; .
"3 " '_ “i ..
.- m) r"; v‘: :3: 5:.
‘Y-TIP g. '. 3 .3. a
- * . ~ ' " .r
Rat "- g L .1 ’:- r it: I}; :1;
‘ : :3“ .' 1 . --,'
-- 3,: ~ 9 f» it?

 

. 1 if s - 2; , '-
“.15..- "In; $.31”! W yééiuj
Figure C.1. a-TIP and 'y-TIP peptide antibodies do not cross-react. Arabidopsis
seed (or-TIP) or leaf (y-TIP) protein extract was separated by SDS-PAGE and
transferred to membrane. Each blot was incubated with a 1:500 dilution of the
respective antibody that had been preincubated with the peptide competitor (1.2 ug/ml)

for 1 hour. Then, each blot was rinsed well, incubated in secondary antibody, and
visualized.

150

35S::GFP: A-TIP transgenic Arabidopsis seeds were kindly provided by Chris
Somerville’s laboratory to address this issue. The transgene in these plants is expressed
in all tissues of the plant and the protein localizes to the tonoplast (Figure B.1) (Cutler et
al., 2000). Therefore, vacuoles isolated from these plants Should be recognized by GFP
fluorescence in the tonoplast. To confirm this, we isolated vacuoles fi'om leaves of the
transgenic plants and looked at them by microscopy with Neutral Red staining and by
fluorescence to observe GFP (Figure B.1). The isolated vacuoles stained red with
Neutral Red and had GFP fluorescence, indicating that isolated vacuoles can be

recognized by the presence of a GFP-linked marker protein in the tonoplast.

Flow Cytometry of Vacuoles From Wildtype and 35S::GFP:A-TIP Plants

I confirmed that GFP fluorescence was visible by microscopy in vacuoles isolated
from 35$::GFP:A-TIP leaves. The next step was to determine whether a Fluorescence
Assisted Cell Sorter (FACS) could also detect the fluorescence from isolated vacuoles.
To accomplish this, I isolated vacuoles from wild type and 35S::GFP:A-TIP transgenic
leaves and compared them by flow cytometry in the F ACS machine. Flow cytometry
will scan the sample with the FACS detector but will not sort the sample into different
populations. The results demonstrated that the FACS detector was able to distinguish
vacuoles that contained GFP in their tonoplast from vacuoles that did not have GFP
fluorescence (Figure C2).

The next goal was to determine whether plant cell vacuoles can withstand the cell

sorting procedure.

151

50

 

50

0
4O

 

   

 

             

      

O

i” .38
go 3
ON 88
9 o
O 0
10° 101 1o2 103 104 10° 101 1o2 103 104

FL1 FL1
Wildtype 353::GFP::6-TIP

Figure C.2. Vacuoles that contain GFP in the tonoplast are recognized by flow
cytometry. Vacuoles were isolated fiom leaves of wildtype and 35S::GFP::6—TIP
transgenic plants and examined separately by flow cytometry. FL1 refers to the
intensity of flourescence detected at 530 nm and counts refers to the number of times in
which a specific intensity of fluorescence was detected.

152

The FACS was setup to separate fluorescent material away from the remaining fluid
stream and was infiltrated with a mam1itol Sheath fluid to surround the vacuoles during
the cell sorting procedure. The isolated vacuoles were placed into the FACS for sorting.
For unknown reasons, the streams of fluid were periodically and randomly shifting to the
left of the original stream and no healthy vacuoles were present in the separated fraction.
When the mannitol solution was replaced with PBS, the stream-shift problem stopped,
indicating that the problem was due to the mannitol solution. However, the isolated
vacuoles were not stable in PBS due to its low osmolarity. These results suggested that
vacuolar sorting through FACS might not be feasible with the current technology

available.

Proteomics of Isolated Vacuoles

Since flow cytometry was not a feasible option, we decided to do a more global
proteomic study of vacuoles isolated from leaf protoplasts. Protoplasts were isolated
from healthy rosette leaves. Vacuoles were isolated from the protoplasts and the protein
was extracted and analyzed by Syngenta (Table CI). The purity of the vacuoles was
determined visually by staining the sample with neutral red dye and examining the

sample by microscopy.

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158

Conclusion

Currently, FACS is not a feasible option for isolating and characterizing distinct
types of plant cell vacuoles. Members of the laboratory are in collaboration with groups
to identify the protein contents of general plant cell vacuoles by isolating vacuoles using
the methods described above and submitting the vacuoles to multiple types of protein
analysis techniques. Specifically, the goal is to identify proteins that differ between
wildtype plants and mutant plants such as the vpey mutant. This strategy will allow us to
identify potential substrates or cargo of known vacuolar transport machinery.

One possibility to separate different vacuoles that we are investigating is an
optical trapping approach. Vacuoles isolated from plants with tonoplast fluorescent
markers such as GFP would be isolated from plants and placed on a microscopic grid.
The vacuoles will be monitored as they pass through the grid. When they reach a fork in
the grid, fluorescent vacuoles will be sorted away from the non-fluorescent vacuoles by
an optical trapping system. When a laser light of a specific intensity is focused on the
vacuole, the light can actually direct its movement or stop its movement, effectively
trapping the vacuole. With this method, specific types of vacuoles can be isolated from a

general population of vacuoles and analyzed by proteomic techniques.

159

REFERENCES

Ahmed, S.U., Rojo, E., Kovaleva, V., Venkataraman, S., Dombrowski, J.E.,
Matsuoka, K., and Raikhel, N .V. (2000). The plant vacuolar sorting receptor
AtELP is involved in transport of NH2-termina1 propeptide-containing vacuolar
proteins in Arabidopsis thaliana. J Cell Biol 149, 1335-1344.

Cutler, S.R., Ehrhardt, D.W., Griffitts, J.S., and Somerville, C.R. (2000). Random
GFP :: cDNA fusions enable visualization of subcellular structures in cells of
Arabidopsis at a high fiequency. P Natl Acad Sci USA 97, 3718-3723.

Damm, B., and Willmitzer, L. (1988). Regeneration of Fertile Plants from Protoplasts
of Different Arabidopsis-Thaliana Genotypes. Mol Gen Genet 213, 15-20.

J auh, G.Y., Phillips, T.E., and Rogers, J .C. (1999). Tonoplast intrinsic protein
isoforrns as markers for vacuolar functions. Plant Cell 11, 1867-1882.

Vitale, A., and Raikhel, N.V. (1999). What do proteins need to reach different vacuoles?
Trends Plant Sci 4, 149-155.

160

Appendix D

Immunolocalization of PINl in Roots and Hypocotyls of Arabidopsis Seedlings

161

 

Introduction

Protein trafficking through the endomembrane system has been implicated in a
variety of plant responses to the environment (Surpin and Raikhel, 2004). Vesicle
trafficking through the endomembrane system requires the interaction of vesicle SNAREs
(v-SNARE) and target membrane SNAREs (t-SNARE). One family of v-SNAREs that
our lab has been studying is the AtVTI (Xps Ten-Interacting Factor) family composed of
AtVTIl 1, AtVT112, and AtVTIl3. While AtVTIII and AtVT112 transcripts were found
in a variety of tissues, AtVTII3 is not expressed at detectable levels. The possibility that
AtVTlll and AtVT112 are not redundant arose when our lab demonstrated that AVTIll
and AtVT112 complemented different aspects of the Avti vacuolar trafficking phenotype
in S. cerevisiae (Zheng et al., 1999). AtVTlll colocalizes with an AtVSR protein at the
TGN and PVC in Arabidopsis while AtVT112 did not, fiirther suggesting that AtVTIll
and AtVT112 perform different functions (Zheng et al., 1999).

A mutant of AtVTIll was independently identified in a mutant screen for
components of shoot gravitropism (Kato et al., 2002). The stem of the zig/sgr4/vti11
mutant grows in a “zig—zag” fashion, and it does not respond to a change in the gravity
vector (Kato et al., 2002). However, a vti12 mutant did not have any gravitropic
phenotype (Zheng et al., 1999). In chapter three, I presented data indicating that plants
that did not accumulate detectable levels of AtVSR protein had gravitropic defects
(Chapter 3). Thus, the colocalization of AtVTlll and AtVSR with the agravitropic
phenotypes observed for mutants of AtVTIII as well as for the AtVSR family led us to
hypothesize that AtVTIll and a member(s) of the AtVSR family function together in a

pathway that mediates gravitropism. To address this issue, I determined whether

162

AtVTIll participated in the recycling of PIN 1, a gravitropism pathway that has been
previously characterized (Steinmann et al., 1999; Geldner et al., 2001; Geldner et al.,
2003)

PINl is an auxin efflux carrier that localizes to the basal plasma membrane
(Galweiler et al., 1998). It recycles to an endomembrane compartment and this recycling
is inhibited by Brefeldin A, an inhibitor of endomembrane trafficking (Geldner et al.,
2001). Therefore, PINl is dependent upon the endomembrane system for its localization
at the basal plasma membrane and its recycling to an endosomal compartment. When
PINl is absent from the basal plasma membrane, the plant exhibits auxin-related
phenotypes, including defects in gravitropism (Geldner et al., 2001; Geldner et al., 2003).
With this in mind, we speculated that AtVTIll may play a role in the localization and
recycling of PINl. To test this hypothesis, I used confocal microscopy
immunolocalization to determine whether the trafficking of PINl was blocked in
zig/vtiI 1 roots and/or hypocotyls. Our lab published the results of many approaches taken
to address the shoot gravitropic defect in Will and determine the function of AtVT112
(Surpin et al., 2003). My contribution to this project was the demonstration that AtVTIll
does not participate in the recycling of PIN 1 between the basal plasma membrane and an
endosomal compartment (Surpin et al., 2003). Therefore, AtVTIll contributes to the
negative gravitropism of stems via a pathway that is distinct from the PIN 1-related

pathway.

163

Materials and Methods

Immunolocalization of PIN 1 in wildtype, zig/vtiII, and vti12 three-day old roots
was performed essentially as described in Geldner et a1. (2001). To immunolocalize
PIN] in three-day old hypocotyls, I followed the protocol described in Geldner et al.
(2001) with the following modifications: i) 1% sucrose was added to the fixation buffer,
ii) after adhering the hypocotyls to the slides, they were digested in 0.5% pectinase
(Sigma) and 20% Triton X-100 (Sigma) at 37° C for 90 minutes. The samples were

visualized with a Leica SP2 Confocal Microscope.

Results

In wildtype plants grown under normal conditions, the subcellular localization of
PlNl is at the basal plasma membrane in both the roots and the shoots of Arabidopsis
(Geldner et al., 2001). Neither zig/vti 11 nor vti12 mutants have root gravitropic defects
(Zheng et al., 1999; Kato et al., 2002). Therefore, PINl should be localized to the basal
plasma membrane of root cells in wildtype, zig/vtiII mutants, and in vti12 mutants. As
expected, PIN] localized to the basal plasma membrane of wildtype, zig/vtiII, and vti12
roots (Figure D.1, A-C).

The aerial tissues of zig/vtil I mutants have gravitropic defects (Kato et al., 2002).
However, the vti12 mutant does not have any gravitropic defects (Zheng et al., 1999;
Surpin et al., 2003). If VTIll participates in the recycling of PlNl to the basal plasma
membrane to mediate gravitropism in aerial tissues, then PINl should be mislocalized in

the zig/vtiI 1 mutant hypocotyls. Likewise, the vti12 mutant is not agravitropic, and thus,

164

 

Figure 0.1. PINl localized to the basal plasma membrane in zig/vtil] and vti12.
Antibodies against PIN] were used to determine its subcellular localization in three-
day old seedling roots (A-C) and hypocotyls (D-F) of wildtype (A, D), zig/vtiII (B,
E), and vti12 (C, F) plants. Arrows indicate basal localization of the PIN] signal.

165

PIN] should correctly localize to the basal plasma membrane in vti12 hypocotyls. To test
this hypothesis, I determined the localization of PIN] in wildtype, zig/vtz'II, and vti12
hypocotyls by confocal microscopy irnmunolocalization. Surprisingly, I found that PlNl
localized to the basal plasma membrane in all seedling hypocotyls (Figure D.1, D-F).
Therefore, AtVTIll is not the v-SNARE present on the vesicles that recycle PIN]

between the basal plasma membrane and an endosomal compartment.

Discussion

The goal of this experiment was to determine whether the gravitropic defect
observed in zig/vtiII plants was due to the mislocalization of PIN 1 protein. By my
observations, this was not the case. However, other experiments discussed in the paper
indicate that there is an auxin transport defect in the zig/vtiII plants (Surpin et al., 2003).
Therefore, AtVTIll is required for auxin transport, but that requirement is not linked to
the proper localization of PINl (Surpin et al., 2003). PIN3 is involved in auxin transport
in addition to PIN l (Geldner et al., 200]; Friml et al., 2002). Therefore, it is possible that
PIN3 is mislocalized in zig/vtz'II mutants. Another possibility arises from the
observation that zig/vtiI 1 plants have defects in tissue identity and organization (Surpin et
al., 2003). Gravitropism is mediated from the endodermal tissues (Surpin and Raikhel,
2004). Therefore, it is possible that the misorganization of cell types in zig/vtill plants
causes the gravitropic defect (Surpin et al., 2003). These alternative explanations have
not been tested yet. Furthermore, the link between the VSRs and gravitropism has not

been established yet either. Further inquiry into the connection between VTI]1, AtVSRs,

166

 

and shoot gravitropism is clearly necessary to obtain a complete understanding of protein

trafficking through the endomembrane system and gravitropism.

167

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