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This is to certify that the

dissertation entitled

VACUOLAR TRANSPORT 0F ctVSS-BEARING PROTEINS: A
GENETIC APPROACH

presented by

Sridhar Venkataraman

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Biochemistry and

 

 

Molecular Biology

Major professor

1L/11459l

Date

 

MS U i: an Affirmative Action/Equal Opportunity Institution 0-12771

PLACE IN RETURN BOX to remove this checkout from your record.
To AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

6’01 c-JCIFlC/DateDue.p65-p.15

 

VACUOLAR TRANSPORT OF ctVSS-BEARING PROTEINS: A GENETIC
APPROACH
By

Sridhar Venkataraman

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

2001

ABSTRACT

VACUOLAR TRANSPORT OF ctVSS-BEARING PROTEINS: A GENETIC
APPROACH

By

Sridhar Venkataraman

Plants respond to stress and pathogen attack by synthesizing and storing defense-
related proteins in the vacuole. Many of these proteins are targeted to the vacuole by
virtue of sorting information residing in a C-terminal vacuolar sorting signal (ctVSS).
While many studies have been conducted to identify the nature of these sorting signals,
very little is known about the factors they interact with or the factors involved in the
transport mechanism that delivers these proteins to the vacuole. A genetic approach has
been used in Arabidopsis thaliana (I...) Heynh, to identify loci involved in the ctVSS-
bearing protein transport pathway. Mutants were screened from a collection of EMS-
treated Arabidopsis thaliana ecotype Columbia seeds expressing rat-preputial {5-D
glucuronidase with a ctVSS from tobacco chitinase and barley lectin. Mutants cvsI and
cst (for ctVSS vacuolar sorting) were identified and characterized by imrnunoelectron
microscopy. In these mutants, barley lectin was found to be partially mislocalized in the
apOplast along the cell wall and in intercellular spaces of root sections. Such results were
not observed in leaf sections, indicating the identification of root specific mutants. The
mutant locus of cvsl was mapped to a contig of BACs between AtSOl9l and DFR on
Chromosome V. Further analyses will have to be carried out to clone the cvsI mutant
locus. However, the research presented describes identification of a factor potentially

involved in transporting ctVSS bearing proteins to the vacuole.

To my parents Vasantha and R. Venkataraman and my wife Meera

iii

ACKNOWLEDGMENTS

My graduate studies have involved many colleagues and faculty members who
have been part of my development as a student. First and foremost, I would like to thank
my advisor Natasha for having supported me all these years and helped me conduct my
research. She has been instrumental in teaching me how to be critical in reviewing
scientific work. I would also like to thank Dr Kenneth Keegstra for helping me join the
PRL. I owe a lot to him. My committee members, Dr Kroos, Dr Preiss and Dr Smith,
have also helped me through these years as a PhD student.

A number of colleagues have taught me a lot in these years: Ahmed Faik, John
Froehlich, Brett Brzobohaty, Philipp Kapranov, Krzysztof Szczyglowski and Sergei
Mekhedov. Coming to the Raikhel Lab: Past - Jim “Jim is always right” Dombrowski,
Maor “Bapeti” Bar-Peled, Sharif “Jim 11” Ahmed; and present — Diane Bassham, Enrique
Rojo, Valya Kovaleva, Rodrigo Sarria-Millan, Haiyan Zheng, Ivan Delgado, Emily
Avila-Teegarden, Curtis Wilkerson and Sybil Myers.

I am especially grateful to Jim Dombrowski to have initiated my project. These
past few months would have been impossible without the help of Tony Sanderfoot (the
Universal Thesis Master) and John Scott-Craig (Seminario Oratorio Major).

Finally my family members: my wife Meera, my parents Vasantha and R.
Venkataraman, Latha, Zoltan and now Milan. I must not forget some of my friends who
have been around when I needed them: Mundath Narayanan Menon, Penmetsa

Ramachandra Varma, Hemant Varma and Satyashayee Misra.

iv

 

 

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TABLE OF CONTENTS

List of tables ....................................................................................... viii
List of figures ......................................................................................... x
Abbreviations ....................................................................................... xii
CHAPTER 1
Introduction: Protein targeting to the vacuole: the ctVSS story ................................. 1
Plant Vacuole ................................................................................. 2
Storage proteins and the PSV ............................................................... 2
Protein targeting to the vacuole in plants .................................................. 4
The endoplasmic reticulum ................................................................ 6
Golgi and the plant secretory pathway .................................................... 7
Sorting determinants ........................................................................ 8
ssVSS ............................................................................... 9
ctVSS .............................................................................. l 1
Sorting machinery ......................................................................... 12
Statement of purpose ............................................................................... 14
CHAPTER 2

Development of a reporter line for use in a screen to identify mutants impaired in ctVSS-

sorting in Arabidopsis ............................................................................... 15
Introduction ................................................................................. 16
Results and Discussion ................................................................... 18
Conclusion ................................................................................. 32

Materials and Methods .................................................................... 33

 

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EMS mutagenesis and characterization of ctVSS-missorting plants ........................ 39
Introduction ................................................................................. 40
Results and Discussion .................................................................... 41
Conclusion .................................................................................. 54
Materials and Methods ..................................................................... 54

CHAPTER 4

Characterization of mutants and selection of cvsI ............................................. 56
Introduction ................................................................................. 57
Results and Discussion ................................................................... 57
Conclusion ................................................................................. 63

Materials and Methods ................................................................... 64

CHAPTER 5

Mapping cvsI to a contig of BACs ................................................................ 67
Introduction .................................................................................. 68
Results and Discussion .................................................................... 68
Conclusion ................................................................................. 80

Materials and Methods ................................................................... 82

CHAPTER 6

Identification of ORFs and complementation of cvsl .......................................... 86
Introduction ................................................................................. 87
Results and Discussion ................................................................... 88
Conclusion ................................................................................. 93

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Materials and Methods .................................................................... 94

CHAPTER 7
Conclusions and Future .................................................................... 95
Conclusions ......................................................................... 96
The Future ........................................................................ 100
REFERENCES .................................................................................... 104
APPENDIX A
Mapping and Linkage analysis made simple: A web-based approach ..................... 115
Abstract .................................................................................... 1 16'
Introduction ............................................................................... 1 17
Results ...................................................................................... 117
References ................................................................................. l 22
HTML code ............................................................................... 123
APPENDIX B
Bradford analysis made simple: A web-based approach .................................... 130
Introduction ............................................................................... 131
Results and Conclusion .................................................................. 131
References ................................................................................. l3 1
HTML Code ............................................................................... 133
APPENDIX C ...................................................................................... 137

Tables: Genes present on BACs MBB18, MKDlO, K15E6, MXF12, K3K3 and MUL8s
and the proteins encoded

vii

Alec.

 

LIST OF TABLES

Table 1.1 List of representative vacuolar sorting signals ....................................... 9
Table 2.1 B-glucuronidase activity in protein extract from primary transforrnant .........23
Table 2.2 Frequency of T2 progeny surviving kanamycin selection ............................ 24
Table 2.3 Reporter segregation in 20 T3 progeny pools of line CRBT3-6 .................. 26
Table 2.4 Reporter segregation in 20 T3 progeny pools of line CRBT3-9 .................. 27
Table 2.5 Evaluation of reporter levels in tertiary transformants ............................. 28
Table 2.6 Kanamycin resistance in quaternary transformants ................................ 29
Table 2.7 Reporter level estimation in quaternary transformants ............................ 30
Table 3.1 Pool-wise summary of number of mutants identified .............................. 4-4
Table 3.2 List of mutants identified and the pools they were identified from .............. 45
Table 3.3 Evaluation of M3 lines by Rat-GUS secretion assay and EM ..................... 46
Table 5.1 lO-Sample mapping analysis .......................................................... 70
Table 5.2 46-Sample mapping analysis .......................................................... 72
Table 5.3 Fine map between AthPHYC and LFY3 ............................................ 73
Table 5.4 Markers used to map cvsI within between ATSOl91 and DF R ................. 79
Table 5.5 Fine mapping analysis of cvsI between AtSOl9l and DFR ..................... 81
Table 6.1 List of BACs that map close to locus cvsl .......................................... 88
Table 6.2 List of genes neighboring cvsI ....................................................... 90
Table C.l Genes present on the BAC MBB18 and the proteins encoded ................. 138
Table C.2 Genes present on the BAC MKDlO and the proteins encoded ................. 139
Table C.3 Genes present on the BAC K15E6 and the proteins encoded .................. 140
Table C.4 Genes present on the BAC MXF12 and the proteins encoded .................. 141

viii

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Table C.5 Genes present on the BAC K3K3 and the proteins encoded ................... 142

Table C.6 Genes present on the BAC MUL8 and the proteins encoded .................. 143

ix

LIST OF FIGURES

Figure 1.1 Model of the plant secretory pathway .............................................. 5

Figure 1.2 Vacuolar soluble proteins and their targeting signals ............................ 8

Figure 2.1 Verification of Rat-GUS activity in primary transformants ..................... 21
Figure 2.2 B-glucuronidase activity in protein extract from primary transformant ........ 22
Figure 2.3 Western blot to verify the expression of the barley lectin (BL) ................. 23
Figure 2.4 Verification of BL in CRBT4 seedlings by western analysis .................... 30
Figure 2.5 Western analysis of protein extracts from different seedling tissues ........... 33
Figure 2.6 Diagram of plasmid pMOGCRB ................................................... 34
Figure 2.7 Diagram of plasmid pMOGRGD ................................................... 35
Figure 3.1 Mutant Screen on vertical plates .................................................... 42
Figure 3.2 Immunolocalization of BL in section of root of mutant E1 1C .................. 48
Figure 3.3 Immunolocalization of BL in section of root of mutant El 1C .................. 49
Figure 3.4 Immunolocalization of BL in section of root of mutant El 1C .................. 50
Figure 3.5 Immunolocalization of BL in section of leaf of mutant El 1C .................. 51
Figure 3.6 Non-immune control: section of root of mutant E11C ........................... 52
Figure 4.1 Northern analysis of root RNA from mature plants .............................. 58
Figure 4.2 Western analysis of barley lectin (BL) in mutant and control tissues .......... 60
Figure 4.3 Examination of floral phenotype of cvsI ........................................... 62

Figure 5.] Alignment of BAC contig between AtSOl91 and DFR on chromosome V ...74
Figure 5.2 Alignment of BAC MBB18 (Col, 28295 — 28460) with Clone co954 (Ler) ..75
Figure 5.3 Sequence alignment of a section of BAC MKM21 (C01) and cl 103 (Ler) ...76

Figure 5.4 Sequence alignment of a section of BAC K15E6 (C01) and c951 (Ler) ....... 77

Figure 5.5 Alignment of BAC MUL8 (Col, 48930 — 49068) with Clone c1765 (Ler) ...78
Figure 5.6 BAC contig spanning cvs] locus .................................................... 82

Figure 7.1 Model of the ctVSS sorting pathway ................................................ 99

xi

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ABBREVIATIONS

AP-l
BAC
BCIP
BL
BP80
BSA
CAPS
CCV
cDNA
cM
Col
COPI
COPII
Cotyl
CTAB
ctVSS
CVS
CW
Da
DEPC
DMSO
DNA
dNTPs
E. coli
EDTA
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F2

F V
GTP
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MES
MOPS
mRNA
MU

Clathrin-associated adaptor complex 1
Bacterial artificial chromosome
5-Bromo-4-chloro-3-indolyl phosphate p-Toluidine Salt
Barley lectin

Binding Protein 80

Bovine serum albumin (fraction V)

Cleaved amplified polymorphic sequences
Clathrin-coated vesicles

copy deoxyribo nucleic acid

centi Morgan

Arabidopsis thaliana ecotype Columbia
Coat-protein (I) coated vesicles

Coat-protein (II) coated vesicles

Cotyledon

Cetyltn'methylammonium bromide

C-terminal vacuolar sorting signal

ctVSS vacuolar sorting (gene or mutant)

Cell wall

Dalton

Diethyl pyrocarbonate

Dirnethyl sulfoxide

Deoxyribo nucleic acid

Deoxynucleotide triphosphate

Escherichia coli

Ethylenediaminetetraacetic acid disodium salt
Ethyl methane sulfonate

Endoplasmic reticulum

First filial generation

Second filial generation (usually self-fertilized progeny of F 1)
Fused vacuole

Guanosine 5'-triphosphate Sodium salt
Heterozygous

ltercellular spaces

Arabidopsis thaliana ecotype landsberg erecta
Lytic vacuole

Primary mutagenized generation

Secondary mutagenized generation (usually self-fertilized progeny
Of Ml)

Tertiary mutagenized generation (usually self-fertilized progeny
0f M2)

2-(N-Morpholino)ethanesulfonic acid
3-(N-Morpholino)propanesulfonic acid
Messenger ribonucleic acid

Methyl umbelliferone

xii

a.

 

ORF
PAC
PBS
PCR
PHA
PMSF
PSV
PsVSS
PT

PVC
Rat-GUS
Rat-GUS-ctVSS

Rat-GUS-Delta
RF

RFLP

RNA

RR Buffer
SDS
SDS-PAGE
SDV
SNARE

SP

SSLP
SsVSS

Open reading frame

Precursor accumulating vesicles
Phosphate-buffered saline

Polymerase chain reaction
Phytohemagglutinin
Phenylmethylsulfonyl fluoride

Protein storage vacuole

Physical structure vacuolar sorting signal
Potato inhibitor terminator

Prevacuolar compartment

Rat preputial B-glucuronidase

Rat preputial B-glucuronidase fused to tobacco basic chitinase
ctVSS

Secreted Rat preputial B-glucuronidase
Recombination frequency

Restriction fragment length polymorphism
Ribonucleic acid

RNA resuspension buffer

Sodium dodecyl sulfate

Sodium dodecyl sulfate poly acrylamide gel electrophoresis
Smooth dense vesicles

soluble N-ethyl maleirnide sensitive factor adaptor protein receptor
Signal peptide

Simple sequence length polymorphism
Sequence specific vacuolar sorting signal
Secretory vesicle

Primary transformants

Secondary transformants

Tertiary transformants

Quaternary transformants

Fifth generation transformants

Tris Borate EDTA

Tobacco basic chitinase

Transfer DNA

Tris EDTA

Trans-Golgi network

Tonoplast intrinsic protein

Vacuole

Volume/volume

Vacuolar protein aggregates

Vacuolar protein sorting

weight/volume

Wheat germ agglutinin

Wild type

5-Bromo-4-chloro-3-indolyl beta-D-glucuronide Sodium salt

xiii

 

 

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YAC Yeast artificial chromosome

xiv

Chapter 1

Protein targeting to the plant vacuole: the ctVSS story

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INTRODUCTION

Plant vacuoles

Plant vacuoles were first observed to be large spaces devoid of cytoplasmic matter
and hence termed vacuole. This large organelle occupies 30 to 90 % of the plant cell
depending on the type of cell under observation. Functionally the vacuole is a large
repository of inorganic ions, organic acids, sugars, secondary metabolites, plant defense
compounds, stress-related solutes, detoxified substances, enzymes and storage proteins
enclosed by the vacuole membrane (tonoplast) (Marty, 1999). Recent evidence from
barley root cells, barley aleurone cells and tobacco cell (Paris et a1, 1996, Swanson et a1,
1998 and Di Sansebastiano et al, 1998) indicates that two or more types of vacuoles may
exist in a plant cell, each characterized by different pH optima (Di Sansebastiano et a1,
1998) and different aquaporins in the respective tonoplasts (J auh et a1, 1998). The low pH
vacuole is termed the lytic vacuole (LV) due to the presence of low pH optima hydrolytic
enzymes, whereas the near-neutral vacuole, the protein storage vacuole (PSV) (Marty,
1999). The LV is analogous to the yeast vacuole and the mammalian lysosome while the
PSV is unique to plants (Marty, 1999). The lytic vacuole has been reviewed in detail by

Marty (1999) and will not be discussed.

Storage proteins and the PSV

The protein storage vacuole has been found to have a tonoplast decorated with Ot-
TIP, an aquaporin found in seeds and root tips. It differs from the lytic vacuole in its
inability to be stained with neutral red, a dye that accumulates in acidic compartments,

indicating a neutral pH (Di Sansebastiano et a1, 1998). This compartment accumulates

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storage proteins such as globulins and prolamins, proteins characterized by their relative
solubility in aqueous or ethanolic buffers. Globulins are found in all dicots and many
monocots. Globulins are of the vicilin type (78 globulins) or the legumin type (11S
globulins). Common 78 globulins include vicillin (from Pisum sativum), phaseolin (from
Phaseon vulgaris) and conglycinin (from Glycine max). The legumin category of
proteins includes glycinin from Glycine max. Prolamins are predominantly found in the
endosperm of monocots such as wheat and rice. Apart from storage proteins, the PSV
also contains many ancillary storage proteins such as barley lectin and bean
phytohemagglutinin as well as hydrolase inhibitors such as tat-amylase inhibitors (Marty,
1999). These proteins may serve a defense role in plants against herbivory, insect and
pathogen attack (reviewed by Marty, 1999; Herman and Larkins, 1999).

The ability of plants to synthesize and store proteins has made them very
important as a primary source of protein for animal species. In modern times, these
aspects of plants have been utilized in developing engineered proteins in plants to
improve the nutritive value of plants and their products (Lawrence et a1, 1994). These
aspects have also been exploited in developing plants with therapeutic properties. The
Escherichia coli enterotoxin B protein has been expressed in potatoes as an oral vaccine
against E. coli-induced diarrhea (Mason et a1, 1998). Similarly, the hepatitis B surface
antigen has been expressed in potatoes as an oral vaccine against hepatitis B (Richter et
al, 2000) and the Guy 13 monoclonal antibody against Streptococcus mutans (agent of
dental caries) was expressed in tobacco (Cabanes-Macheteau et al, 1999). While most of
these developments are still in their infancy, the prospects of plant based therapies for

common ailments are exciting.

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Protein targeting to the vacuole in plants

Plant proteins destined for the vacuole are synthesized as precursors by ribosomes
associated with the endoplasmic reticulum (ER), and are co-translationally inserted into
the ER lumen (see Figure 1.1). In the ER lumen, they undergo several modifications such
as the proteolytic processing of the signal peptide, N-linked glycosylation, disulfide bond
formation, and protein folding. These steps are regulated by a “quality control” step
following which these proteins are thought to be packaged into COPII vesicles bound for
the Golgi complex (see Pirnpl et a1, 2000). Certain proteins such as cereal prolamins and
pumpkin ZS albumin are packaged into protein bodies or precursor accumulating (PAC)
vesicles bound for the protein storage vacuole. Afier the COPII vesicles deliver their
cargo to the cis-Golgi, proteins further undergo maturation processes such as complex
mannosylation of the glycan as they move through the medial and trans-Golgi via a COPI
vesicle mediated manner (see Pirnpl et al, 2000). Eventually proteins arrive at the trans-
Golgi network (TGN) where they are sorted according to localization signals residing on
the protein. Proteins with vacuolar targeting signals are sent to the vacuole via the
prevacuolar compartment (PVC) in clathrin-coated vesicles or smooth dense vesicles
(depending on the targeting signal). Proteins, which lack a vacuolar targeting signal or a
Golgi retention signal, are delivered to the extracellular space via the default pathway of

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Figure 1.1 Model of the plant secretory pathway

Soluble proteins are synthesized by ribosomes associated with the Endoplasmic Reticulum (ER).
Once in the lumen of the ER they are processed and eventually packaged into a) COPII vesicles
containing cargo such as barley lectin or sporamin (yellow) or b) Precursor Accumulating
Vesicles (PAC) as in the case of pumpkin ZS albumin (red). COPII vesicles deliver their cargo to
the Golgi complex where they are processed as they proceed by a COPI vesicle mediated process
(dark green). PAC vesicles, in contrast, deliver their cargo (red) to the Protein Storage Vacuole
(PSV). Proteins processed in the Golgi network reach the TGN where they are sorted.

l) ssVSS bearing proteins (blue) are packaged into Clathrin-Coated Vesicles (CCV) en route to
the prevacuolar compartment (PVC). These proteins ultimately reach the Lytic Vacuole (LV).

2) Proteins bearing a ctVSS such as barley lectin or a psVSS such as legumin (brown) are
packaged into Smooth Dense Vesicles (SDV) en route to the PSV. In vegetative cells, the PSV
and LV oflen fuse to form one large vacuole (FV).

3) Proteins which lack a Golgi retention signal or a vacuolar targeting signal are packaged into
secretory vesicles and their cargo (green) are secreted into the apoplast.

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The Endoplasmic reticulum

Storage proteins are synthesized on the rough endoplasmic reticulum (ER) and are
co-translationally inserted into the ER lumen. This entry into the ER is specified by a
signal peptide residing at the N-tenninus of the nascent polypeptide and consists of 16 to
30 residues organized in three domains: a positively charged amino terminus, followed by
a hydrophobic region and a polar region terminated by a cleavage site (von Heijne, 1985).
The signal peptide is cleaved upon entry into the ER. Once in the ER lumen, proteins
undergo various modifications such as N-linked glycosylation, disulphide bond
formation, oxidation of prolines to hydroxyprolines, trimming of the glycan and protein
folding (reviewed in Galili et a1, 1998). There is significant evidence to indicate the
existence of a quality control step in the ER resulting in the reverse translocation of
misfolded proteins to the cytosol (reviewed in Vitale and Denecke, 1999). Proteins such
as pumpkin 2S albuminand monocot prolamins exit the ER via PAC vesicles or protein
bodies respectively and are transported directly to the vacuole (Okita and Rogers, l996;
Galili et a1, 1998; Hara-Nishimura et all, 1998; Choi et a1, 2000).

The transport of soluble proteins from the ER to the Golgi has been found to
occur via COPII vesicles in yeast (for review see Barlowe, 1998). This mechanism
involves Sec12p (GTP-exchange protein), Sarlp (a GTPase), Se023p, Sec24p, Sec13p
and Sec31p (for review see Barlowe, 1998). Similarly, in plants the ER to Golgi traffic is
attributed to a homologous COPII system. This hypothesis was supported by the
identification of plant homologues of the yeast proteins. The homologues identified are
AtSARl (Bar-Peled and Raikhel, 1997; Takeuchi et a1, 2000), AtSEClZ (Bar-Peled and

Raikhel, 1997), and AtSEC23 (Movafeghi et a1, 1999).

 

 

 

 

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Golgi and the plant secretory pathway

The plant Golgi body can be discerned as a stack of membrane-bound
compartments (cistemae) associated with a large network of tubules and vesicle buds
called the trans-Golgi network or TGN. The number of Golgi stacks varies depending on
the plant species and can range from one stack per Chlamydomonas cell to thousands in
giant fiber cells in cotton (for review Andreeva et a1, 1998). Plant Golgi bodies carry out
a number of functions including protein glycosylation, lipid biosynthesis and
polysaccharide synthesis. BaSed on the localization of Golgi enzymes, polysaccharides,
and various modifications that proteins undergo in the Golgi stacks, the Golgi cistemae
are classified into cis-, medial- and trans-Golgi (Dupree and Sherrier, 1998).

Typically, proteins are delivered from the ER to the cis-Golgi, proceed through
the medial- and trans-Golgi undergoing various modifications. The modifications include
protein glycan al,3-fucosylation (Lerouge et al, 1998), protein glycan Bl,2-xylosy1ation
(Lerouge et a1, 1998), proteolytic processing (Jiang and Rogers, 1999) and complex
mannosylations of the glycan (Lerouge et a1, 1998). Intra-Golgi traffic in plants has been
proposed to occur via a COPI vesicle-mediated manner (Andreeva et a1, 2000, Pirnpl et
a1, 2000) or via cistemal maturation as in the case of scales in algae (Brown, 1969) and
procollagen in mammalian cells (Bonfanti et a1, 1998).

Eventually, proteins arrive at the TGN en route for two broad destinations: the
extracellular space via the default pathway of exocytosis, or the vacuole via a sorting
determinant-mediated pathway. It can be said that in the absence of a positive sorting
determinant, soluble proteins are secreted via the default pathway. Currently, very little is

known about the vesicular machinery of exocytosis. However it is clear that the process is

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mediated by calcium ions and annexins and that these vesicles are delivered to the plasma
membrane by microfilaments (Carroll et a1, 1998, Battey et a1, 1999, Sutter et al. 2000).
The presence of a positive sorting determinant directs proteins to the vacuole (V itale and

Raikhel, 1999).

Sorting determinants
Sorting determinants can be broadly classified into two categories, propeptide

mediated sorting and mature protein domain-mediated (psVSS) sorting (Figure 1.2). In

 

 

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Figure 1.2 Vacuolar soluble proteins and their targeting signals

SP = Signal peptide, ssVSS = sequence specific vacuolar sorting signal, ctVSS = C-
terrninal vacuolar sorting signal, psVSS = protein structure vacuolar sorting signal.

the case of propeptide-bearing proteins, the propeptide is eventually cleaved and is absent
fi'om the mature protein in the vacuole. The study of a number of vacuolar hydrolases,

lectins, seed storage proteins and protease inhibitors indicates that vacuolar sorting is

mediated by the presence of a propeptide signal at the N-terminus (ssVSS) of the protein

 

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(adjacent to the signal peptide) as in the case of sweet potato sporamin and barley
aleurain (Figure 1.2) or at the C-terminus (ctVSS) of the protein (Figure 1.2) as in barley
lectin (BL), tobacco chitinase (TC), kiwi actinidin (Paul et al, 1995), bean phaseolin and
brazil nut ZS albumin (reviewed by Venkataraman and Raikhel, 1998; Matsuoka and
Neuhaus, 1999; Vitale and Raikhel, 1999). The analysis of the psVSS has been reviewed

in detail by Neuhaus and Rogers (1998) and will not be covered in this review.

ssVSS

Recently Matsuoka (2000) summarized the properties of the ssVSS as to consist
of an Xl-Xz-I/L-X3-X4 motif where X1 lacks a small hydrophobic side chain and Asn is
preferred, X2 is a non acidic amino acid, X3 is any amino acid and X4 is a large and

preferably hydrophobic amino acid (Table 1.1). Although these signals were originally

Table 1.1 List of representative vacuolar sorting signals

 

 

 

Protein ssVSS

Sweet potato sporaminl . . RFNPIRLPTTHEPA . .

Barley aleurain' . .SSSSFADSNPIRPVDTDRAAST. .

Arabidopsis aleura' 2 . .ANIGFDESNPIRMVSDGLREVC. .

Protein ctVSS

Barley lectinl . .VFAEAIAANSTLVAE

Tobacco chitinasel - - GLLVDTM

Bean phaseollinl . .AFVY

Tobacco osmotinI . . CPYGSAHNETTNFPLEMPTSSTHEVAK

Kiwi actinidin3 . . QNHPKPYSSLINPPAFSMSKDGPVGVDDGQRYSA

 

ssVSS = sequence specific vacuolar sorting signal; ctVSS = C-terminal vacuolar sorting
signal. Letters in bold face represent amino acids forming the conserved motif. 1 =
reviewed by Matsuoka and Neuhaus (1999). 2 = Ahmed et a1 (2000). 3 = Paul et al (1995)

 

tder.
Sig:
tine.
nut I

NAB.

 

that 1
601g:

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5.00m:
51mm"
lira f
imam

lll Son

identified at the N-terminus of cargo proteins, Koide et a1 (1997) demonstrated that the
signal is also functional if artificially placed at the C-terminus of a cargo protein. This
finding may explain the properties of the C-terminally located targeting signals of Brazil
nut ZS albumin (Kirsh et al, 1996), the pumpkin ZS albumin (Shimada et a1, 1997) and
NaPI (Miller et al, 1999). These C-terminally located signals have a XIXZI/LX3X4 motif
and bind the ssVSS-specific vacuolar sorting receptor BP80 or its orthologs (Matsuoka,
2000). Jiang and Rogers (1998) have demonstrated that the cleavage of the ssVSS occurs
in the PVC and that the mature protein is then transported to the vacuole.

To differentiate between proteins with a vacuolar sorting determinant and those
that lack a sorting determinant, the existence of receptors at the trans-Golgi or trans-
Golgi network was postulated. Using synthetic peptides corresponding to the ssVSS of
barley aleurain and the non-functional mutant ssVSS, Kirsch et al (1994) purified from
pea clathrin coated vesicles, an 80 kDa protein (BP80). This protein was found to interact
with the aleurain ssVSS with a kd of 37 nM in a pH dependent manner. Later the Raikhel
group cloned AtELP (Ahmed et al, 1997), the Arabidopsis ssVSS receptor, and
demonstrated that this protein is localized in the TGN and interacts with synthetic
peptides representing ssVSSs in vitro (Ahmed et al, 2000). The role of AtELP as an
ssVSS receptor was further strengthened by observing the co-localization of AtELP and
sporamin in Arabidopsis cells. However AtELP did not co-localize with BL in
Arabidopsis cells indicating that the receptor was specific for ssVSS bearing proteins in
viva. Synthetic peptides representing the cytosolic domain of AtELP were found to
interact with mammalian AP-l adaptor complexes in-vitro confirming the role of AtELP

in sorting ssVSS-bearing proteins and delivering them into clathrin-coated vesicles

10

 

IV '5

 

 

 

mlSSL‘
secret
Greer
filncti.
rest»;

Vacuo

(Sanderfoot et al, 1998). AtELP was also co-localized with AtSYPZl (a syntaxin found
on the PVC) indicating that this receptor shuttles between the TGN and the PVC

(Sanderfoot et al, 1998).

ctVSS

While a consensus motif has been identified for the ssVSS sorting determinants,
ctVSS signals do not possess a common motif (Table 1.1) at the level of their primary
structure. This type of sorting determinant is found in a number of lectins, storage
proteins, hydrolases and defense related proteins. To date ctVSSs from barley lectin
(Dombrowski et al, 1993), tobacco chitinase (Neuhaus et al, 1994), kiwi actinidin (Paul et
al, 1995), bean phaseolin (Frigerio et a1, 1998) and tobacco osmotin (Sato et a1, 1995)
have been characterized. The deletion of this determinant from the protein results in the
missorting to the apoplast, and conversely the addition of this signal to heterologous
secreted proteins such as cucumber chitinase (Neuhaus et al, 1991), hen egg white
lysozyme (Neuhaus et al, 1995), rat preputial B-glucuronidase (Neuhaus et al, 1995),
Green Fluorescent Protein (Di Sansebastiano et a1, 1998) and sporamin with a non-
functional ssVSS (Matsuoka et al, 1995) resulted in the vacuolar localization of the
respective chimeric proteins. It has been postulated that the ctVSS may be cleaved in the
vacuole by an aspartic proteinase (Amidon et a1, 1999). Extensive site directed mutation
analyses have been performed on the tobacco chitinase ctVSS: 'GLLVDTM' (Neuhaus et
a1, 1994) and the BL ctVSS 'VFAEAIAANSTLVAE' (Dombrowski et al, 1993). The
terminal methionine of the TC-ctVSS could be deleted without effect, while all other

residues were required. Replacing 2LLV4 with SSS or 4VDTM7 with LLLL did not reduce

11

I
he

 

b}?

THC L

 

 

simn
Secret
sauna
50m.

bten t

 

Sonin

ssl'gg.

the sorting efficiency while replacing the terminal M with a G reduced vacuolar sorting
by 50%. In the case of the BL-ctVSS as little as lVFA3 was shown to be functional in
vacuolar targeting, while the addition of two G residues at the C-terminus of the motif
abrogated vacuolar targeting. Although glycosylation is not necessary for vacuolar
targeting in plants, translocating the glycosylation site from a -7 position to a -1 position
in the ctVSS of BL resulted in secretion of the protein (for review Dombrowski and
Raikhel, 1996). This is attributed to a steric hindrance rather than a role for the glycan.
The lack of a consensus motif within the ctVSSs indicates that a structural feature of the
ctVSS might be responsible for the selectivity. It has been suggested that the ctVSS of
barley lectin adopts an amphipathic alpha helix structure (Dombrowski and Raikhel,
1996). However, such a structural feature has not been demonstrated to be necessary for
ctVSS-mediated vacuolar sorting.

Currently, no receptor has been identified for the ctVSS pathway. It has been
shown that if ctVSS proteins are expressed to high levels, then the proteins can be
secreted (Neuhaus et a1, 1994; Frigerio et al, 1998) indicating that the pathway is
saturable. Frigerio et a1 (1998) suggest the existence of a specific machinery involved in
sorting ctVSS bearing proteins onward to the vacuole. However, such proteins have not

been identified.

Sorting machinery
While a large body of evidence has been accumulated regarding the transport of
ssVSS-bearing proteins, very little is known about the pathway transporting ctVSS-

bearing proteins. However, there are several differences between the two pathways.

12

 

ct\'S

 

(163110
C0352
\BTIO‘

Sét‘fel

obser
VBC 110
Chara.
Other 1

1‘.
I

B0163.

Matsuoka et a1 (1995) demonstrated that both barley lectin and tobacco chitinase are
secreted from tobacco protoplasts when treated with 33 M of wortmannin, whereas
sporamin is correctly targeted to the vacuole. Though wortmannin is an inhibitor of
phosphatidyl inositol-3 kinase in mammalian cells, its specific role in the plant secretory
pathway has not been elucidated. It is also known that ctVSS-bearing proteins have not
been found in clathrin-coated vesicles that contain aleurain (an ssVSS-bearing protein).
Finally, the final destinations for these proteins are different as the ssVSS-bearing
proteins are delivered to the lytic vacuole (decorated with 'y-TIP, an aquaporin), while
ctVSS-bearing proteins are delivered to the PSV (decorated with OL-TIP). These evidences
demonstrate that the ctVSS and ssVSS-mediated pathways are different. The lack of a
consensus motif in the ctVSS and the variation in the length of the ctVSS among the
various known ctVSS sequences has hampered the understanding of this part of the
secretory pathway.

In summary, the study of vacuolar protein transport has come a long way since
observing spaces in the cell that were devoid of cytoplasmic contents. A number of
vacuolar proteins have been identified and their targeting motifs dissected. The
characterization of these sorting signals led to the identification of sorting receptors and
other proteins required for this machinery. However, the lack of a consensus sequence has
hampered the understanding of the machinery involved in sorting ctVSS-bearing soluble

proteins to the vacuole.

l3

 

.‘lrait
to 6.
mute:
defee

throw

 

 

 

"Statement of purpose"

The research presented aims at identifying proteins involved in ctVSS-mediated
protein transport. So far it is known that a class of soluble proteins reach the vacuole by
virtue of their ctVSSs. In view of the general features of the various secretory pathway
models proposed, it is conceivable to expect a variety of proteins involved in the
pathway. Some of these might be receptors recognizing ctVSS-bearing cargo and sorting
them into vesicles bound for the vacuole. This pathway may also involve a specialized
class of vesicles decorated with a complement of proteins. The fate of these vesicles may
be regulated by certain factors.

To characterize the pathway, a genetic approach has been undertaken in
Arabdopsis thaliana to identify factors involved in the pathway, described in Chapters 2
to 6. Through a mutant screen cm] and cst were identified as ctVSS vacuolar sorting
mutants. cm] was found to be a single gene recessive mutation causing a leaky sorting
defect in root cells. This mutation was mapped to a contig of bacterial artificial

chromosomes between AtSOl9l and DFR on chromosome V.

14

Chapter 2

Development of a reporter line for use in a screen to identify mutants impaired in
ctVSS-sorting in Arabidopsis

15

 

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INTRODUCTION

The study of the secretory pathway in yeast has advanced due to the large
collection of vps mutants impaired in vacuolar protein transport (Raymond et a1, 1992).
More than 50 genes have been identified whose products play important roles in the
pathway. The identification of the plant homologues of yeast and mammalian secretory
pathway proteins has advanced the understanding of this mechanism in plants. This
approach was initiated by complementing the yeast pep12 mutant with an Arabidopsis
cDNA library resulting in the identification of AtPEPlZ (later renamed SYPZl) (Bassham
et al, 1995), the first plant syntaxin to be identified. Since then AtSYP4l and AtSYP42
(Bassham et al, 2000), AtVPS45 (Bassham et al, 2000), AtVTIla and b (Zheng et a1, 1999),
have been identified and characterized. It is likely that these proteins are involved in the
ssVSS pathway. On the other hand, wortmannin sensitivity studies (Matsuoka et a1, 1995)
and ctVSS mutation studies (Dombrowski et al, 1993, Neuhaus et al, 1994) have shown that
the ctVSS sorting pathway may be different from that of the ssVSS-sorting pathway. The
ctVSS mediated pathway appears to be unique to plants as Gal and Raikhel (1994) showed
that the yeast vacuolar targeting machinery does not transport barley lectin to the yeast
vacuole. Taken together the ctVSS-mediated pathway may be unique to plants and may
involve proteins hitherto unknown in other systems.

To identify proteins involved in targeting ctVSS-bearing proteins to the vacuole, a
plant mutant screen was envisaged. A number of candidate ctVSS-bearing proteins have
been identified in Arabidopsis such as AtOsmotin (Capelli et a1, 1997), myrosinase-binding
protein (Takechi et al, 1999), vacuolar invertases such as AtFruct3 and AtFruct4

(Haouazine-Takvorian et al, 1997), and a vacuolar chitinase, AtChib (Samac et a1, 1990).

16

Ht.

let:

an;

 

 

 

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tmr'

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”1110:.
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However, the role of the putative ctVSS in these proteins has not been characterized. Barley
lectin (BL), a root cap and developing embryo protein has been expressed in Arabidopsis
and extensively characterized (Dombrowski, 1995; Ahmed et al, 2000). In Arabidopsis
thaliana ecotype RLD, constitutively-expressed BL was localized in the vacuole by
immunoelectron microscopy and the precursor protein was shown by pulse-chase analysis
to be proteolytically cleaved to the mature size. It was also demonstrated that BL lacking
a ctVSS was secreted and localized in the apoplast. Another reporter active in plant
vacuoles is rat preputial B-glucuronidase (Rat-GUS, Nishimura et al, 1986) when fused to
the ctVSS of tobacco chitinase (Neuhaus et a1, 1995). Rat-GUS is a homotetrameric
protein consisting of 75 kDa (648 amino acids) subunits, synthesized in rat cells as a
precursor with a 15 amino acid C-terminal extension. This extension bears a
glycosylation site for the phosphomannosyl glycan required for targeting to the lysosome
via the mannose-6 phosphate receptor in animal cells (Johnson et al, 1990). The deletion
of the C-terminal extension (Rat-GUS-Delta) resulted in the secretion of the protein in
tobacco cell suspension protoplasts and a reduction in activity. The level of expression,
however, was not affected. With the addition of the ctVSS from tobacco chitinase (Rat-
GUS-ctVSS), the protein was efficiently directed to tobacco protoplast vacuoles
(Neuhaus et al, 1995).

To identify mutants impaired in the ctVSS pathway in the absence of known
endogenous reporter, a transgenic approach was used whereby mutants would be
identified which mislocalized heterologous ctVSS-bearing proteins. As the mutant screen
involved chemical mutagenesis (see Chapter 3 for details), two reporters were used, one

as a primary reporter to screen mutants visually and the other as a secondary reporter to

17

\‘Cl‘f.

mur.

 

53D"

“‘1 ,

BL

 

' -‘DV
JAM

map:
I l

 

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due

verify the mutants by electron microscopy. Such an approach would ensure that cis-
mutants (defective only in the targeting signal of one reporter) would be eliminated by the
secondary screen. To screen a large collection of mutants, a visual screen was devised
with Rat-GUS-ctVSS as the primary reporter as its enzymatic activity is easily detected in
vivo. To perform the secondary screen, BL was chosen as a reporter. The localization of
BL could be performed by immunoelectron microscopy. Since this screen required a
stable line expressing both reporters, it was necessary to develop a line with a single
insertion locus that was homozygous for the reporter construct. This would facilitate
mapping and positional cloning at a later stage. The development of a transgenic

homozygous single locus seed line for EMS mutagenesis is presented.

RESULTS AND DISCUSSION

While important results have been obtained describing protein sequences necessary
for sorting soluble proteins to the vacuole, little is known of the machinery that carries out
the sorting in plants. The goal of this study was to develop a line of Arabidopsis plants
stably transformed with a vacuolar reporter construct for creating ctVSS-sorting mutants.
Mutants with aberrant sorting pathways would then be expected to mis-direct their cargo to
the apoplast via the default pathway of exocytosis. In order to screen for mutants defective
in the transport machinery, a reporter construct was developed containing cDNAs encoding
two reporter proteins: Rat-GUS-ctVSS and BL. Rat-GUS-ctVSS served as the primary
rePorter in a visual screen and BL as a secondary reporter to verify the trans nature of the
mutation. Two reporters were used in the screen to eliminate false positives that would arise

due to chemically mutagenized vacuolar targeting signals (cis—mutations). As a control, a

18

 

 

 

U37:

1101..

 

Rat-

VCClt .

C0111.“

 

 

transgenic line was developed (Rat-GUS-Delta) which lacks a vacuolar targeting signal and

would be secreted into the apoplast.

Rat-GUS-ctVSS/BL Reporter construct and Rat-GUS-Delta construct

A binary vector construct was created in pMOG800 (Mogen International, The
Netherlands), with Rat-GUS-ctVSS driven by a double 358 promoter and BL driven by a
second double 35S promoter resulting in pMOGCRB. Agrobacterium tumefaciens
(GV3101 pMP90) cells were transformed with this construct and used to infect Arabidopsis
thaliana ecotype Columbia plants by vacuum infiltration (Bent et al, 1994). In parallel, Rat-
GUS-Delta (Neuhaus et al, 1995) driven by a 35S promoter was cloned into a pMOG800
vector and used to similarly transform Arabidopsis thaliana ecotype Columbia plants. This

control line helped establish the screening assay.

Primary transformants and evaluations

Eleven Arabidopsis thaliana ecotype Columbia plants were independently
infiltrated with a suspension of Agrobacterium cells carrying the pMOGCRB construct
according to Bent et al (1994). About 3000 seeds per parent were screened on kanamycin
media and an average of 15 plants per parent were found resistant to the antibiotic after 2
weeks of treatment. Since the insertion of the T-DNA is random, the activity of the
transgene is known to vary from plant to plant depending on the site of insertion. Also,
Within a single independently transformed plant, several transformation events could occur
giving rise to different levels of expression of the transgene. Leaves of kanamycin resistant

seedlings were assayed for Rat-GUS activity. Briefly, leaves were removed from three

19

seedlings per transformed parent and wounded to allow the entry of the Rat-GUS substrate,
5-bromo-4~chloro-3-indolyl B-D-glucuronide (X-GlcU), into the cell. The activity was
evaluated visually. The results were compared with wild type plants (Figure 2.1a, b, c and
d). Although wild type Arabidopsis has been reported to contain B-glucuronidase activity
(Wozniak and Owens, 1994), wild type Arabidopsis plants did not pick up any stain after 2
hours of staining at 37 0C. In contrast, pMOGCRB plants stained very strongly. Activity
was detected in trichomes and leaf veins. This confirmed that Rat-GUS was active when
expressed in plants as previously shown by Neuhaus et al (1995). Among independent
transformants, seedlings were selected visually for high B—glucuronidase activity. These
plants were subsequently transferred to soil in growth chambers. It is likely that some of the
lines have multiple insertions of the T-DNA and therefore exhibit higher levels of activity.
At this stage however, it was difficult to evaluate the number of insertions based solely on a
visual comparison of the level of activity.

The histochemical assay was complemented by a quantitative fluorometric assay
(adapted from Jefferson, 1987). B-Glucuronidase activity could be detected only in
pMOGCRB plants (Figure 2.2 and Table 2.1) using 10 11g total protein per assay. The
reaction appeared to proceed in a linear manner with respect to time. To confirm the
presence of the secondary reporter, protein extracts were analyzed for the presence of BL.
BL was detected by western blotting using anti-WGA sera (Ahmed et a1, 2000). At a
dilution of 1:1000, the antibody cross-reacted with a single product of the expected size

(Figure 2.3).

20

 

Panel A Panel C

 

  

 

 

 

Panel B Panel D

Figure 2.1 Verification of Rat-GUS activity in primary transformants

A single leaf was harvested from each of 3 kanamycin resistant seedlings per independent
transformant and wounded with a sterile micropipet tip (to allow the dye to enter the cells) in a
microtiter plate. The leaf was then incubated overnight in 100 ml of assay buffer (0.1 M
sodium acetate pH 4.8, 1 mg/ml BSA, 2 mM X-GlcU, 0.5 mM potassium ferrocyanide and
0.5 mM potassium ferricyanide) at 37 0C (modified from Guerineau and Mullineaux, 1993).
Panel A) Leaf samples fi'orn kanamycin resistant primary transformant seedlings of 8
independent Columbia plants transformed with pMOBCRB stained for Rat-GUS activity
(blue). Panel B) Leaf samples from wild type Columbia seedlings stained for Rat-GUS
activity. Panel C) Close up of one microtiter well with a pMOGCRB primary
transformant leaf stained for Rat-GUS activity (blue). Panel D) Close up of one microtiter
well with a wild type leaf stained for Rat-GUS activity. The comparison of panels C and D
indicate the strong blue stain in pMOGCRB transformants unlike wild type leaf samples.
Images in this dissertation are presented in color.

21

16

 

 

 

 

 

 

TE

:9 14 -~

0

b

n 12 +

E

\ MOGCRB
: 10 T P .
E

2 a ~~ .
2

.2 WI'
= 6 -tr-—

3

.0

E

a

'5.

5

O

2

 

 

 

 

_. ._.</L

-0-—
i: A A
v V
0 I l l
0 20 40 60

Tlmc (minutes)

Figure 2.2 B-glucuronidase activity in protein extract from primary transformant

Specific Activity of 10 ug of leaf protein from a) pMOGCRB plants (blue) and b) WT
plants (green) measured at 20-minute intervals by estimating the concentration of methyl
umbelliferone released from methyl umbelliferyl B-D glucuronide. This assay
demonstrated that pMOGCRB transformants expressed an active Rat-GUS as seen by the
linear activity of a one-hour time course. This activity was absent in wild type tissue

samples.

22

Table 2.1 B-glucuronidase activity in protein extract from primary transformant

 

 

 

 

 

 

Time (minutes) Buffer MOGCRB ild Type
0 0.56 0.56 0.56
20 0.56 3.3 0.56
40 0.56 8.06 0.56
60 0.56 13.68 0.56

 

 

 

 

 

Concentration of Methyl Umbelliferone (MU) released by B-glucuronidase activity in
samples measured in mM of MU released per mg total protein per minute. Samples include
Buffer (Extraction buffer), pMOGCRB transformants and Wild Type (Wild type
Arabidopsis Ecotype Columbia). This assay demonstrated that pMOGCRB transformants
expressed an active Rat-GUS as seen by the linear activity of a one-hour time course. This
activity was absent in wild type tissue samples.

 

 

WGA lwr [pMOGCRB
H w

A

 

 

 

Figure 2.3 Western blot to verify the expression of the barley lectin (BL)

BL was detected using rabbit anti-wheat germ agglutinin (WGA), Lane WGA (100 ng
wheat germ agglutinin) was used as a size marker, Lane WT (60 ug total wild type
Columbia leaf protein), pMOGCRB (60 ug total pMOGCRB primary transformant leaf
protein). Proteins were detected using rabbit anti-WGA (1: 1000) as a primary antibody.
This analysis demonstrated that pMOGCRB transformants expressed BL unlike wild type
tissues.

23

 

 

 

Tal'

 

 

Secondary transformants

Lines with a single reporter construct are expected to be hemizygous primary
transformants and their progeny after self-fertilization (secondary transformants) segregate
so that lA:(25 %) of the progeny are homozygous for the reporter, l/2(50 %) are hemizygous
for the reporter and l/:(25 %) lack the reporter. Since resistance to kanamycin selection is a
dominant trait, 3% (75 %) of the secondary transformants with a single reporter locus should
be resistant to kanamycin. To identify single insertion secondary transformants, secondary
transformants were germinated on kanamycin media and the ratio of kanamycin resistant

seedlings to the total number of seedlings was determined (see Table 2.2). To identify lines

Table 2.2 Frequency of T3 progeny surviving kanamycin selection

 

 

 

 

 

 

 

 

 

 

 

 

Line Resistant Susceptible Level of resistance x2 p-value
(°/o)

CRB T3 —1 49 1 98 14.1 1 x 10“
CRB T2 —2 60 0 100 20 7.7 x 10:6
CRB r2 —3 73 0 100 24.3 8.1 x 10'7
CRB r2 —5 86 0 100 28.6 8.5 x 10‘8
CRB r2 —6 65 20 765* 0.098 0.75
CRB r2 —9 64 12 84* 3.43 0.063
CRB Tz-ll 56 3 94.9 13.44 2x104

 

 

 

 

Lines of transgenic lines and level of resistance to kanamycin are displayed. * At a 0.05
level of significance, the frequency fits a 3:1 ratio indicating a single gene insertion into
the genome (using the chi-square test of goodness of fit). Lines CRB T2-6 and CRB T2-9
appeared to have a single insertion locus.

with a single reporter locus, a chi-square test of goodness of fit was performed on the ratios
obtained against an expected ratio of 75 % (3: 1) and the p-value was estimated for each

line. These were tested against a critical p-value of 0.05. Lines, that had a p-value smaller

than 0.05 were discarded, as their resistance ratio did not fit the required ratio of 75 %.

24

‘JJ
_.

Tert

COB.

 

Lines CRBT2-6 (p-value = 0.75) and CRBT2-9 (p-value = 0.063) were selected, as their p-
values were greater than 0.05 indicating a low deviation from the expected ratio of 75%.
Data from the progeny of lines CRBT2-4, 7, 8 and 10 were not consistent with an expected

3:1 ratio and these lines were not pursued.

Tertiary transformants

Once a single locus line is identified, it is necessary to identify homozygous
progeny of this line. This was accomplished by scoring the progeny of twenty individual
T2 plants from each progeny pool of lines CRBT26 and CRBT29. Seeds from these lines
(T3) were germinated on kanamycin media and the ratio of kanamycin resistant seedling to
the total number of seedlings was determined (see Tables 2.3 and 2.4). Ratios thus obtained
were categorized in classes of 0 % (reporter-less segregants), 75 % (progeny of
heterozygous secondary transformants) and 100 % (progeny of homozygous secondary
transformants) by performing a chi-square goodness of fit test. As none of the T3 pools of
line CRBT2-9 were completely susceptible to kanamycin, these were discarded. Among the
T3 pools of line CRBT2-6, lines CRBT36-18 and CRBT36-Z4 were identified to be
homozygous and were analyzed firrther.

Leaves were harvested from 10 individual T3 plants from these lines and total
protein extracts prepared. These extracts were analyzed for BL by western analysis and for
Rat-GUS by fluorometric activity assays (Table 2.5). Significant activity and protein levels

could not be detected from the progeny of line CRBT26.Z4 and these lines were

25

Table 2.3 Reporter segregation in 20 T3 progeny pools of line CRBT3-6

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

CRBT3-6 Total % Resistance Category
16 45 71 75*
17 119 92 100
18 86 99 100**
19 120 97 100
20 96 nd nd
21 107 82 75*
22 127 98 100
23 129 89 100
24 90 100 100**
25 72 86 75*
26 72 100 100
27 126 97 100
28 66 100 100
29 52 92 100
30 84 76 75*
31 47 91 100
32 48 77 75*
33 36 0 0
34 101 92 100
35 32 7 0

 

 

 

 

 

The segregation of the reporter construct in the progeny of line CRBT3-6 was evaluated
by estimating the resistance to kanamycin in 20 progeny pools of this line. Lines of T3
(CRBT3-6), the total number of seedlings sampled, the percentage that survived the
selection and the category they were classified in. * 3:1 segregation of reporter construct
** Lines selected for further studies. Lines CRBT3-6.18 and CRBT3-6.24 were selected for
further studies.

26

 

 

Ta

 

 

 

 

Table 2.4 Reporter segregation in 20 T3 progeny pools of line CRBT3-9

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

CRBT3-9 Total % Resistance Category
1 69 96 100
2 48 73 75*
3 44 82 75*
4 104 98 100
5 196 99 100
6 69 78 75*
7 219 97 100
8 91 100 100
9 62 73 75*
10 55 96 100
11 73 95 100
12 44 77 75*
13 73 97 100
14 53 91 100
15 69 93 100
16 98 100 100
17 76 96 100
18 37 89 75*
19 47 100 100
20 109 89 100

 

 

 

The segregation of the reporter construct in the progeny of line CRBT3-9 was evaluated
by estimating the resistance to kanamycin in 20 progeny pools of this line. Lines of T3
(CRBT3-9), the total number of seedlings sampled, the percentage that survived the
selection and the category they were classified in. * 3:1 segregation of reporter construct.
This line was discarded as kanamycin susceptible progeny could not be detected (see
text).

27

 

 

 

Ta

 

 

 

Table 2.5 Evaluation of reporter levels in tertiary transformants

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sample Activity (mM MU BL
lmgprotein/min)

Ext Buffer 0.00 -
Wt 0.31 -
CRBT36-l8-l 5.19 -
CRBT36-18-2 0.92 +
CRBT36-l 8-3 2.14 -
CRBT36-l8-4 8.25 +
CRBT36-18-5 3.36 +
CRBT36-18-6 2.75 +
CRBT36-18-7 3.36 -
CRBT36-l8-8 1.83 -
CRBT36-18-9 1.83 -

CRBT36-l8-10 0.92 -/+
CRBT36-24-1 0.00 -
CRBT36-24-2 0.00 -
CRBT36-24-3 0.00 -
CRBT36-Z4-4 0.00 -
CRBT36-24-5 0.00 -
CRBT36-24-6 0.00 -
CRBT36-24-7 0.3 l -
CRBT36-Z4-8 0.00 -
CRBT36-24-9 0.00 -
CRBT36-24-10 0.00 -

 

 

The levels of reporters in tertiary transformants were evaluated by measuring Rat-GUS
activity and BL by western analysis. B-Glucuronidase activity (mM methyl
umbelliferone/mg protein/min). Samples include Buffer (Extraction buffer), leaf protein
from each of ten plants selected from CRBT26-l8 and CRBT26-24 (transgenic for a single
insertion locus of pMOGCRB) and Wild Type (Wild type Arabidopsis ecotype Columbia).
BL was detected by western analysis. + = High levels of BL, -/+ = moderate levels of
protein and - = no protein. Lines CRBT36-18-l, CRBT36-18-4 and CRBT36-18-5 were
selected for further analysis.

28

pit

Ta

 

 

 

discarded. Lines CRBT36.18.01, 6.18.04 and 6.18.05 (see Table 2.5) appeared to have
significant GUS activities and BL levels. Seeds from three plants (CRBT36.18.01,

6.18.04 and 6.18.05) were harvested for further analysis.

Quaternary transformants
To confirm that homozygous tertiary transformants yielded stable progeny, the
progeny of T3 lines (CRBT36.18.01, 6.18.04 and 6.18.05) were analyzed by plating on

kanamycin media and scoring for survival (Table 2.6). BL analyses by western blotting

Table 2.6 Kanamycin resistance in quaternary transformants

 

 

 

 

Genotype Total # of % Kanamycin
Seeds Resistance
CRBT46.18.1 81 96.3
CRBT46.18.4 79 88.6
CRBT46.18.5 80 100‘

 

 

 

 

 

Progeny of lines CRBT46.18.1, CRBT46.18.4 and CRBT46.18.5 were germinated on
kanamycin media and the survival ratio was determined. Genotype = Lines of T4 (progeny
of CRBT46-18). Total # of Seeds = total number of seedlings sampled. % Kanamycin
Resistance = the percentage of seedlings that survived the selection. . Complete resistance
to kanamycin (indicating homozygosity). Line CRBT46.18.5 was found to have a 100 %
kanamycin resistance indicating a stable homozygous reporter and was thus chosen for
further studies.

(Figure 2.4) and B-glucuronidase activity assays were performed on leaf tissue
(summarized in Table 2.7). Line CRBT46.18.05 was found to have a 100 % survival on
kanamycin and detectable amounts of BL protein and Rat-GUS activity. This line was

bulked for EMS mutagenesis (yielding line CRBT56.18.05x). Qualitatively, it was observed

that the levels of both reporters were lower in CRBT56. 18.05x than in primary

Z9

 

 

Ta

 

 

El;
BL
1116
bu:
CR
We
CR

 

 

 

 

 

 

 

Figure 2.4 Verification of BL in CRBT4 seedlings by western analysis

Total leaf protein from 3 CRBT4 seedlings (40 ug) was resolved by SDS-PAGE and
immunoblotted. Resolved proteins were visualized by a primary antibody treatment with
rabbit anti-WGA sera, followed by treatment with goat anti-rabbit coupled to alkaline
phosphatase. Blots were developed according to Harlow and Lane, 1988 (see Materials
and Methods). Lane 1 = CRBT4-6.l8-1, Lane 2 = CRBT4-6.l8-4 and Lane 3 = CRBT4-
6.18-5. All 3 lines tested expressed BL.

Table 2.7 Reporter level estimation in quaternary transformants

Buffer .00
.05

46.18.] 1.73
46.184 1.36
46.185 .23

 

Evaluation of reporter levels in tertiary transformants by measuring Rat-GUS activity and
BL by western analysis (see Figure 2.4 for western analysis). B-Glucuronidase activity is
measured in mM methyl umbelliferone/mg protein/min. Samples include Buffer (Extraction
buffer), leaf protein from quaternary transformant lines CRBT46.18-1, CRBT46.18-4 and
CRBT46.18-5 and WT (Wild type Arabidopsis ecotype Columbia). BL was detected by
western analysis (see Figure 2.4). + = High levels of BL and - = no protein. Line
CRBT46. 18.5 performed well with respect to BL and Rat-GUS.

30

 

 

 

 

 

if.»

 

transformants. In order to determine if this line was suitable for a mutant screen a “screen

test” was performed.

Screen test

To screen for mutants missorting Rat-GUS and BL to the apoplast (see Chapter 3
for details), it is necessary to detect Rat-GUS activity specifically in the apoplast. To
achieve this, an assay has been designed in which the substrate for Rat-GUS [5-bromo-4-
chloro-3-indolyl a-D-glucuronide (X-GlcU)] is localized in the apoplast. X-GlcU is a stain
that requires 2-5 % detergent to penetrate the plasma membrane. By applying X-GlcU to
seedlings without any detergent, the dye diffuses into the apoplast but does not cross the
plasma membrane (T ml] and Deikrnan, 1998). However, upon the application of X-GlcU in
the presence of Z % (v/v) Triton X-100, the cell membranes are perrneabilized and the
substrate diffuses into the cell.

After obtaining seeds homozygous for a single reporter locus expressing BL and
Rat-GUS (line CRBT56.18.05x), a control screen was performed to test the line for
suitability in a mutant screen. As a control seeds expressing a secreted Rat-GUS (Rat-GUS—
Delta, Rat-GUS lacking a vacuolar targeting signal) were used. In this assay it was expected
that Rat-GUS-ctVSS would be localized in the vacuole in the absence of detergent. Seeds of
wild type Arabidopsis, CRBT56.18.05x and Rat-GUS-Delta were germinated on media
plates vertically and stained with X-GlcU. While roots of Rat—GUS-Delta turned blue
(indicating Rat-GUS activity in the apoplast) within 2 hours at room temperature, roots of
wild type seedlings and CRBT56.18.05x seedlings did not pick up any stain. To verify that

the CRBT56.18.05x seedlings were white due to a vacuole-localized Rat-GUS, seedlings of

31

 

 

It

er;

3H

I
Gt
\.

C0

 

 

wild type Arabidopsis, CRBT56.18.05x and Rat-GUS-Delta were germinated on media
plates vertically and stained with X-GlcU in the presence of 2 % (v/v) Triton X-100. It was
observed that only the wild type seedlings remained white while both Rat-GUS-Delta roots
and CRBT56.18.05x root turned blue. This confirmed that CRBT56.18.05x seedlings were
expressing Rat-GUS but it was localized intracellularly, while Rat-GUS-Delta was secreted
and hydrolyzed X-GlcU resulting in blue roots. Upon the application of Triton X-100, the
cell membrane was permeabilized and the dye was accessible to the enzyme. Thus both Rat-
GUS-Delta seedlings and CRBT56.18.05x seedlings turned blue.

Since the secondary screen was based on the localization of BL, the expression of
BL protein in CRBT36.18.05x seedlings was investigated. Ten day-old CRBT56.18.05x
seedlings were analyzed for the presence of BL by germinating parental seeds on vertical
media plates for 10 days and harvesting the tissues in 3 categories: root tips, lateral root
zone and cotyledons. Total protein was extracted and analyzed by western blotting
(Figure 2.5). BL was detected in the lateral root and in the root tip but not in cotyledonary

leaves.

CONCLUSION

Using an Agrobacterium-mediated transformation approach pMOGCRB, a reporter
construct encoding Rat-GUS-ctVSS and BL was introduced into Arabidopsis thaliana
ecotype Columbia. Following each generation by kanamycin selection and measuring

activities of Rat-GUS and protein levels of BL, a line (CRBT56.18.05x)

32

 

 

ho:
det
COS

tip

.111.

De

 

 

 

 

 

 

 

Figure 2.5 Western analysis of protein extracts from different seedling tissues

Total protein fi'om cotyledonary leaves, lateral roots and root tips (~40 11g) was resolved
by SDS-PAGE and immunoblotted. Resolved proteins were visualized by a primary
antibody treatment with rabbit anti WGA, followed by treatment with goat anti-rabbit
coupled to alkaline phosphatase. Blots were developed according to Harlow and Lane,
1988 (see Materials and Methods). Lane 1 = Cotyledonary leaf protein (“Cotyl” in
diagram), Lane 2 = Lateral root protein (“Lateral” in diagram) and Lane 3 = Root tip
protein (“Tip” in diagram). While BL was not detected in cotyledonary tissues, it was
more abundant in lateral roots rather than root tips.

homozygous for the reporter construct was developed. Although both reporters could be
detected in line CRBT56.18.05x, the activity levels were not very high possibly due to
cossuppression of the reporters. By performing a screen test for Rat-GUS secretion and root

tip protein analysis for BL, the line was found to be suitable for EMS mutagenesis.

MATERIALS AND METHODS
Development of the reporter construct pMOGCRB

All chemicals, unless otherwise indicated, were purchased from Sigma Chemical Co
(St. Louis, MO, USA). The Kpnl site of pMOG843 (Mogen Intemational, The Netherlands)
was removed by blunt-ending and religation. The plasmid was then cleaved with XbaI. The

cauliflower mosaic virus 3SS promoter (35S)/potato inhibitor terminator (PT) fragment was

33

 

 

pl
111
st:

Int:

 

 

Flgt
Rag
Pot;
pro:
Ftp}

l

 

.0:

 

introduced into the Xbal site of pBluescript (modified by eliminating the HindIII site). Two
clones were isolated (in both orientations) designated 10pBS843 (orientation: Sacll, 35S,
PT and Kan and 14 pBS843 (orientation: KpnI, 358, PT and SacII). The coding region of
BL was excised from pTAW50 (Matsuoka et al, 1995) with EcoRI and introduced into the
HindIII site of 10pBS843 by blunt end cloning, yielding p10BL4. The coding region of Rat-
GUS-ctVSS was excised from pRatGUSTail (Dr J-M. Neuhaus, Universite de Neuchatel,
Switzerland) with BamHI and Pstl and inserted into the BglII-PstI sites of pIC19H (Marsh
et a1, 1984) yielding p19HRGT. The coding region of Rat-GUS-ctVSS was excised from
p19HRGT with HindIII and inserted into the HindIII site of l4pBSS43 yielding p14RGT2.
The coding region of p10BL4 was excised as an XbaI fiagment and inserted into the SpeI
site of p14RGT2 yielding pBLRT9. A 2.1 kbp Sacll fragment from pMOG800 (Mogen
International, The Netherlands) containing the multiple cloning site of pMOG800 was
inserted into the Sacll site of pBLRT9, yielding pSMlO. The BL—Rat-GUS-ctVSS coding
region of pSMlO was excised as an X1101 fragment and inserted into the X1101 site of

pMOG800, yielding pMOGCRB (Figure 2.6).

Rat-GUS BL

_ ori RKZ

7

NPTH NPTIII

Figure 2.6 Diagram of plasmid pMOGCRB

Rat-GUS = Rat-GUS-ctVSS (driven by a double 35S promoter and terminated with a
potato protease inhibitor terminator). BL = Barley lectin (driven by a double 35$
promoter and terminated with a potato protease inhibitor terminator). Ori RKZ = origin of
replication. NPTII = Neomycin phosphotransferase for kanamycin selection in plants.
NPTIII = Neomycin phosphotransferase for kanamycin selection in bacteria.

34

 

 

 

Sta

Slit

Tie

13

terr
phi; i
phi.

P111

1171:

liar;

 

{ll/if

 

88:

 

Development of the control construct: pMOGRGD

Clone pJMNRGDelta was obtained from Dr J -M Neuhaus (Universite de Neuchatel,
Switzerland) and digested with Ele. A 2722 bp EcoRI fragment consisting of Rat-GUS-
Delta driven by a 358 promoter and with a 35S terminator was subcloned into the EcoRI
site of pMOG800 (Mogen International, The Netherlands) yielding pMOGRGD (Figure

2.7).

Rat-GUS-Delta

ori RKZ

~

 

7

NPTII NPTIII

Figure 2.7 Diagram of plasmid pMOGRGD
Rat-GUS-Delta = Rat-GUS-Delta (secreted version, driven by a 35S promoter and
terminated with a 358 terminator). Ori RKZ = origin of replication. NPTII = Neomycin
phosphotransferase for kanamycin selection in plants. NPTIII = Neomycin
phosphotransferase for kanamycin selection in bacteria.
Plants, Bacteria

All DNA constructions were carried out in Escherichia coli strain DHSa. All plant
transformations were carried out in Arabidopsis thaliana, ecotype Columbia. All plant
transformations were carried out by vacuum infiltrating plants with Agrobacterium
tumefaciens GV3101 pMP90 electroporated with pMOGCRB or pMOGRGD. Vacuum
infiltration was carried according to Bent et al, (1994). Seeds (T1) were harvested and

germinated on media containing 40 mg/l kanamycin sulfate and 250 mg/l vancomycin.

35

Kanamycin resistant seedlings were transferred to 'hard selection media' containing 1.5 %

(w/v) phytoagar for 1 week and then transplanted into soil.

Leaf activity assay

A single leaf was harvested from kanamycin resistant seedlings and wounded with a
sterile micropipet tip in a microtiter plate. The leaf was then incubated overnight in 100 11.1
of assay buffer [0.1 M sodium acetate pH 4.8, 1 mg/ml bovine serum albumin (BSA), 2
mM 5-bromo-4»chloro-3-indolyl B-D-glucuronide (X-GlcU), 0.5 mM potassium
ferrocyanide and 0.5 mM potassium ferricyanide] at 37 oC (modified from Guerineau and

Mullineaux, 1993). The level of expression of Rat-GUS was evaluated visually.

Quantitative B-glucuronidase Assay

Tissue (0.2 g) was harvested from seedlings in soil and homogenized with liquid
nitrogen, 0.5 g purified sand (J.T. Baker Chemical Co. Phillipsburg, NJ, USA), 100 mg
polyvinyl polypyrrolidone using a mortar and a pestle. The powder was resuspended in 2.5
ml Rat-GUS extraction buffer [phosphate-buffered saline (PBS), 0.1% (v/v) Triton X-100,
0.1 % (w/v) sodium-N-lauryl sarcosine, 1 mM phenylmethylsulfonyl fluoride (PMSF)], and
then centrifuged twice at 4 0C for 10 minutes at 15000 g. Protein contents were estimated by
Bradford's assay (Bio-Rad Laboratories, Hercules, CA) using the Bradford analysis
package, bradfordhsa.htm (available at http://www.msu.edu/~venkata1/bradfordhsa.htm,
see Appendix B). Protein extracts were diluted to a concentration of 0.4 rig/1.11 with 0.1 M
sodium acetate pH 4.8 containing 1 mg/ml BSA. The reaction was initiated by adding assay

buffer (0.1 M sodium acetate pH 4.8, 1 mg/ml BSA, 2 mM methyl umbelliferone B-D

36

 

 

Cfl

 

glucuronide) to 25 [.11 of diluted protein extracts and incubated at 37 oC. Every 20 minutes
10 pl was sampled and the reaction was stopped with 1.5 ml 0.25 M sodium carbonate.
Fluorescence was measured by exciting at 365 nm and measuring the emission at 455 nm

(adapted from Jefferson, 1987).

“Roots, shoots and tips” Assay

Seeds of CRBT5-6.l8.5x were sterilized and germinated vertically on media plates
[Gamborg’s-BS basal medium with minimal organics (Sigma) 0.32 % (w/v), 2-(N-
morpholino) ethanesulfonic acid (MES) 0.05% (w/v) and sucrose 2% (w/v), pH 5.7] for 10
days. This was achieved by setting the plates upright to allow the roots to germinate on the
surface of the medium. Tissue was harvested from seedlings in three categories: root tips,
lateral roots and cotyledonary leaves and homogenized with liquid nitrogen, using a Kontes
motorized pestle. The powder was resuspended in protein extraction buffer [PBS, 0.1%
(v/v) Triton X-100, 0.1 % (w/v) sodium-N-lauryl sarcosine, 1 mM PMSF], and then
centrifuged twice at 4 0C for 10 minutes at 15000 g. Protein contents were estimated by
Bradford's assay (Bio-Rad Laboratories, Hercules, CA) using the Bradford analysis

package, bradfordhsahtm (see Appendix B).

SDS-PAGE and Western blotting

Protein extracts were diluted with half a volume of 3X Laemmli buffer (Harlow and
Lane, 1988). The diluted samples (25 111) were boiled, treated with 5 ul acetarrride solution
(5 1.11 1X Laemmli buffer, 2 mg Tris base, 1.33 mg iodoacetamide) and the mixture was

incubated at 37 0C for 30 minutes prior to SDS-PAGE. Proteins were irnmunoblotted onto

37

 

 

 

 

)1

bar:

by

Sta

Sltl
d4}

(0'

 

 

 

nitrocellulose filters, transfer-verified by staining with Ponceau S, blocked and incubated
with primary antibodies (rabbit anti-WGA, Ahmed et al, 2000) at 1:1000 dilution and with
secondary antibodies (goat anti-rabbit coupled to alkaline phosphatase) at 1:1000 dilution
according to Harlow and Lane (1988). Protein bands were visualized using 50 ml of
alkaline phosphatase buffer (100 mM sodium chloride, 5 mM magnesium chloride, 100
mM Tris, pH 9.5) containing 0.16 mg/ml 5-bromo-4-chloro-3-indolyl phosphate and 0.35
mg/ml nitro blue tetrazolirun chloride. After the development of bands, the blots were

rinsed with distilled water and photographed.

Visual test Screen

Seeds were sterilized and germinated vertically on media plates [Gamborg’s-BS
basal medium with minimal organics (Sigma Chemical Company, St Louis MO, USA) 0.32
% (w/v), MES 0.05% (w/v) and sucrose 2% (w/v), pH 5.7) for one week. This was achieved
by setting the plates upright to allow the roots to germinate on the surface of the medium.
Staining was canied out with X-GlcU solution [X-GlcU (0.1 mg/ml of final suspension),
dissolved in DMSO (0.1 % of the final suspension) and the solution dispersed in 0.05 %
sterile agarose]. Seedlings were overlaid with 10 ml of X-GlcU solution and scored after 5
days. Detergent test were carried as above except that staining was carried out with X-GlcU
(0.1 mg/ml of final suspension), dissolved in DMSO [0.1 % (v/v) of the final suspension]

and the solution dispersed in 0.05 % (v/v) sterile agarose containing 2 % (v/v) Triton X-

100.

38

Chapter 3

EMS mutagenesis and characterization of ctVSS-missorting plants

39

 

13...; a. I

 

 

 

 

 

INTRODUCTION

The current understanding of the various proteins involved in the secretory
pathway has been largely aided by the development of yeast mutants, aberrant for a
variety of morphological characters or in the transport of cargo proteins such as
carboxypeptidase Y (for review Klionsky et al, 1990). Mutants can be developed in plants
using several approaches such as insertion mutagenesis, physical mutagenesis and
chemical mutagenesis. Insertion mutagens include transposons (reviewed by Coupland,
1992) and T-DNA (reviewed by Koncz et al, 1992). Both of these mutagens are inserted
into the genome at random and this aspect as been used to generate large collections of
insertion mutants. This technique makes cloning the locus of interest relatively easy as the
sequence of the insertion is known and several molecular techniques are available to
clone and sequence regions flanking the insertion. Physical mutagens such as fast neutron
bombardment, X-ray and y-ray irradiation cause deletions in the genome. Both insertion
mutagenesis and physical mutagenesis result in null mutants as the gene of interest is
either interrupted by an insertion or deleted by a physical mutagen. Chemical mutagenesis
using ethyl methane sulphonate (EMS) has been a common approach in Arabidopsis
thaliana due to the generation of a large number of point mutations in a single step (Redei
and Koncz, 1992). Point mutations provide a convenient approach in studying essential
events as they do not necessarily result in the loss of the gene function but often result in
alterations in the gene product. Many of these mutations are prominent enough to result in
a measurable phenotype but not deleterious enough to be lethal to the plant. A
mutagenesis scheme resulting in null mutants would thus not be appropriate to study

protein transport to the vacuole, as the vacuole is an essential organelle and a complete

40

loss of a particular gene product may cause lethality. Point mutants however would be
more appropriate to study vacuolar protein sorting, as the mutants could be leaky. In this

chapter the identification of mutants aberrant in vacuolar protein transport is presented.

RESULTS AND DISCUSSION

Seeds of line CRBT56.18.05x (see Chapter 2), homozygous for BL and Rat-GUS-
ctVSS were mutagenized with 0.3 % EMS and germinated in soil (according to Haughn and
Somerville, 1986). They were allowed to self-fertilize and set seeds (M2 seeds). Each pool
thus represented a large family of mutants (M2).

The screen consisted of identifying seedlings whose roots acquired a blue color due
to the hydrolysis of 5-bromo—4-chloro-3-indolyl B-D-glucuronide (X-GlcU) by mislocalized
Rat-GUS in the apoplast. To screen mutants, M2 seeds were germinated vertically on
media for one week and then treated with X-GlcU (without any detergent). Within 48
hours, roots of seedlings could be seen to stain clearly (Figure 3.1). As a standard control,
wild type Columbia seeds, Rat-GUS-Delta seeds (a control construct lacking the vacuolar
targeting signal, see Chapter 2) and CRBT56.18.05x seeds were treated the same way and
analyzed. Trull and Deikman (1998) have demonstrated that secreted acid phosphatase can
be detected by staining seedlings with 5-bromo-4-chloro-3-indolyl phosphate (BCIP),
indicating that BCIP is localized in the apoplast and not membrane permeable. This screen

was based on similar properties of X-GlcU. X-GlcU is a stain that requires 2-5 % detergent

to penetrate the plasma membrane.

41

   

Figure 3.1 Mutant Screen on vertical plates

Seedlings were germinated on media plates (see Materials and methods) for five days. To
obtain roots developing on the surface of the medium, the plates were set upright. To
visualize the secreted Rat-GUS activity, seedlings were stained with 5-bromo-4-chloro-3-
indolyl B-D-glucuronide (X-GlcU) in 0.1 % agarose. Subsequently plates were maintained
horizontally for the stain solution to spread on the plate. Seedlings that turned blue due to
secreted Rat-GUS activity were subsequently transferred to media plates lacking X-GlcU
for one week and then transferred to soil. Panel A: one blue-root seedling among several
white seedlings. Panel B: close up of blue seedling.

42

In all cases, wild type and parental seedlings did not stain blue, while Rat-GUS-
Delta seedlings picked up the stain within 2 hours. M2 seedlings, however, displayed a
variety of phenotypes, as seen by the intensity of staining and the location of the stain.
About 25000 seedlings have been screened out of 47 pools (Table 3.1) and 86 mutants
identified. Of these about 25 survived the transfer to soil (Table 3.2) and 14 of these were

selfed to obtain M3 seeds (Table 3.3).

Analysis of M3 seeds

Often a number of candidate mutants are identified in a preliminary screen of M2
seeds. Some of these may not be determined by a genetic defect. This can be verified by
evaluating the M3 generation. In order to determine if the phenotype was heritable, the M3
seeds were plated and stained (Table 3.3). It was observed that certain M3 lines such as
e5a, e10a, e10b, e11c, e12-1, e13-1, e19-1, e4-I and e21a secreted Rat-GUS activity as
seen by the visual assay. In contrast, M3 lines e6a and e19-10 did not secrete Rat-GUS
activity. This indicated that the defects in lines e5a, e10a, e10b, e11c, e12-l, eI3-1, e19-
1, e4—l and e21 a were heritable and passed on to the next generation while lines e6a and
e19-10 did not have a heritable defect.

To investigate whether the secretion of Rat-GUS was due to a cis-mutation or a
trans-mutation, the localization of BL in lines found to secrete Rat-GUS in a heritable
manner, was investigated by immunoelectron microscopy. Blue seedlings were sectioned

and immunoelectron microscopy was carried out using antisera specific for BL. All

43

Table 3.1 Pool-wise summary of number of mutants identified

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Pool-wise # of # screened Pool-wise # of # screened
summary mutants summary mutants
l 1 600 26 3 400
2 2 600 28 1 ' 200
3 l 550 29 O 250
4 1 600 30 5 950
5 2 400 31 O 300
6 5 200 32 O 600
7 3 350 33 0 800
8 O 500 34 0 1500
9 6 950 35 7 900
10 6 300 36 0 1350
ll 7 400 37 6 750
12 6 600 38 3 700
13 1 250 39 2 400
14 l 400 40 3 1000
15 0 300 41 4 1100
16 1 200 42 0 950
17 Z 300 43 3 350
18 1 450 44 0 900
19 1 200 45 0 500
20 0 200 46 1 400
21 1 650 47 0 950
25 O 150
Total 86 24400
Controls
CRBT5-6. 18-5x 1 800
Rat-GUS-Delta 500 500
Wild Type 0 900

 

To screen the mutagenized CRBT5-6.l8-5x M2 seeds, samples from each pool were
germinated and set upright for roots to develop on the surface of the medium. Seedlings
were stained for secreted Rat-GUS activity. This table shows a pool-wise summary of the
number of mutants obtained and the number of seedlings screened. Control assays were
performed with CRBT5-6.l8-5x, Rat-GUS-Delta lines and Wild Type Columbia lines. A
total of 24400 M2 seedlings were scored out of 47 pools of M2 families.

 

Table 3.2 List of mutants identified and the pools they were identified from

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Mutants Pool
e01-1 1
e04-1 4
e05a 5
e06a 6
e09a 9
e10—1 10
e10-3 10

e100 10
e10b 10
e100 10
e] la 1 1
e11b l 1
e11c 11
e12-I 12
e12-2 12
e13-I 13
el4a l4
e19-01 l9
el9-02 19
e1 9-1 0 19
e21a 21
33 7a 37
e40a 40
e40b 40
e410 41
Total 25 15

 

To screen the mutagenized CRBT5-6.l8-5x M2 seeds, samples from each pool were
germinated and set upright for roots to develop on the surface of the medium. Seedlings
were stained for secreted Rat-GUS activity. This lists 25 mutants belonging to 15 families
(M2 pools) that survived the transfer to soil.

45

Table 3.3 Evaluation of M3 lines by Rat-GUS secretion assay and EM

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

M3 Line Blue: White Secretion of BL by electron
microscopy (EM)
e5a 41 :0 Yes
e9a ND Yes
e1 0a 89:0 Yes
e1 0b 104: 1 Yes
E1 1c* 79:0 Yes
e12-1 37:15 Yes
E13-1** 48:0 Yes
e1 9-1 3 :2 No
e4-I 51 :0 EM data not available
e6a 0:63 EM data not available
e10—3 ND EM data not available
e10c ND EM data not available
E19—10 0:56 EM data not available
e21a 3:0 EM data not available

 

To determine the heritability of the secretion phenotype observed in the M2 seedlings,
their progeny were scored for secretion of Rat-GUS by performing the root-staining
assay. To investigate if the secretion of Rat-GUS was due to a cis-mutation, root and leaf
sections were prepared from some of the secreting mutants. The localization of barley
lectin (BL, the secondary reporter) was determined by immunoelectron microscopy (see
Materials and Methods). This table summarizes the M3 lines verified by listing the ratio
of blue root-seedlings to white root seedlings and whether nus-localization (secretion) of
BL in the apoplast was detected by electron microscopy. In certain cases exact counts
could not be obtained (ND). In certain case electron microscopy was not performed and
such situations have been described as “EM data not available”. * Mutant e1 1 c has been
renamed cvsl. ** Mutant e13—I has been renamed cvs2

46

mutants analyzed (e5a, e9a, e10a, e10b, e11c, e12-1, e13-1 and e19-1) showed the
presence of BL in both the vacuole and the apoplast except for 219-1 where BL was
localized only in the vacuole. This suggested that the screen was robust and that the
majority of mutants identified had defects in trans rather than a cis-mutation. It is
possible that mutant e19-1 had a mutation in the ctVSS of Rat-GUS causing it to be
secreted while leaving BL unaffected and correctly localized in the vacuole.

In all the mutants that appeared to missort BL, BL was also found in the vacuole
indicating that no “null” mutant was observed. Vacuolar BL was found in osmiophyllic
vacuolar protein aggregates (VPA, Figure 3.2, 3.4 and 3.5). In contrast, apoplastic BL was
either found as a “layer” along the cell wall (Figure 3.3 and 3.4) or in osmiophyllic
protein aggregates found in the intercellular spaces (Figure 3.2 and 3.4) at the junction of
two or more cells. Sections of leaves were also exarrrined but no missorting was detected
as all the BL was localized in the vacuole (Figure 3.5). As a control, sections were treated
with non-immune sera (Figure 3.6) and no specific labeling was detected. Moreover the
VPA in the non-immune treatment was not labeled with irnmunogold particles, indicating
that the labeling was specific for BL. However, BL protein has not been detected by
electron microscopy in the parental root tips.

Partial mislocalization of BL can be attributed to a leaky mutation in the secretory
machinery causing some portion of the protein to be transported to the vacuole and the
other portion secreted to the apoplast. Considering that the vacuole is an essential
organelle of the plant cell, null mutants could have been lethal and therefore been
eliminated from the screen. It was also interesting that the mutations only affected protein

transport in the root and not in the leaf. This bias may have occurred as the visual

47

 

 

Figure 3.2 Immunolocalization of BL in section of root of mutant eIIc

Blue roots were sectioned and prepared for immunoelectron microscopy (see Material
and Methods) by Dr. V. Kovaleva. Immunodetection of BL was performed using rabbit
anti-WGA antibodies. Arrow points to protein aggregates labeled with irnmunogold (10
nm) detecting the presence of BL. V = vacuole, VPA = vacuolar protein aggregate, CW =
cell wall and ICS = intercellular space. Size bar = 0.5 pm.

48

 

 

Figure 3.3 Immunolocalization of BL in section of root of mutant e11c

Blue roots were sectioned and prepared for immunoelectron microscopy (see Material
and Methods) by Dr. V. Kovaleva. Immunodetecu'on of BL was performed using rabbit
anti-WGA antibodies. Arrow points to protein aggregates labeled with irnmunogold (10
nm) detecting the presence of BL. V = vacuole and CW = cell wall. Size bar = 0.25 pm.

49

 

Figure 3.4 Immunolocalization of BL in section of root of mutant e11c

Blue roots were sectioned and prepared for immunoelectron microscopy (see Material
and Methods) by Dr. V. Kovaleva. Immunodetection of BL was performed using rabbit
anti-WGA antibodies. Arrow points to protein aggregates labeled with irnmunogold (10
nm) detecting the presence of BL. V = vacuole, VPA = vacuolar protein aggregate, CW =
cell wall and ICS = intercellular space. Size bar = 0.25 pm.

50

 

Figure 3.5 Immunolocalization of BL in section of leaf of mutant eIIc

Leaves from seedlings with blue roots were sectioned and prepared for immunoelectron
microscopy (see Material and Methods) by Dr. V. Kovaleva. Immunodetection of BL was
performed using rabbit anti-WGA antibodies. Arrow points to protein aggregates labeled
with irnmunogold (10 nm) detecting the presence of BL. V = vacuole, VPA = vacuolar
protein aggregate and CW = cell wall. Size bar = 0.25 um.

51

 

 

Figure 3.6 Non-immune control: section of root of mutant eIIc
Blue roots were sectioned and prepared for immunoelectron microscopy (see Material
and Methods) by Dr. V. Kovaleva. Control micrography was performed using non-
immune sera. Broken arrow points to irnmunogold particles (10 nm). V = vacuole, VPA =
vacuolar protein aggregate and CW = cell wall. Size bar = 0.5 pm.

52

screen was performed on roots. Since roots of Arabidopsis are white, upon the hydrolysis
of X-GlcU, blue color could be visualized in vivo without damaging the seedling, unlike
leaf staining which required the treatment of the tissue with solvents such as methanol
and detergents to remove chlorophyll and other pigments that interfere with the
visualization of the indigo dye. Such treatments would also damage the plasma membrane
and the tonoplast potentially causing artefactual mislocalizations of Rat-GUS.

It is now apparent that higher organisms such as plants have genes that can be
classified into gene families based on their function and sequence similarity. Clark and
Roux (1999) have shown that the expression pattern of homologous genes or members of
the same family can vary in a tissue specific manner. This phenomenon would explain
why a specific mutation affecting the transport of ctVSS-bearing soluble proteins in roots
does not affect that in leaves. Previous models based on components of the yeast
secretory pathway did not account for tissue specificity and gene families as a unicellular
model was under investigation. Kj emtrup et a1 (1995) demonstrated the transport of bean
phytohemagglutinin (PHA) can vary in various tissues. In the meristem of the primary
root, PHA is localized in the vacuole, whereas in elongated root cells, PHA is secreted.
This indicates a tissue- and cell type-dependent variation in the properties of the secretory
pathway. It is difficult, at this stage to determine the reason for obtaining root specific
mutants. The positional cloning of the mutant loci will eventually shed some light on this

phenomenon.

53

CONCLUSION

By screening a mutagenized a collection of seeds expressing Rat-GUS-ctVSS and
BL, several mutants have been identified secreting Rat-GUS to the apoplast. Electron-
microscopy revealed that the mutant phenotype is leaky as BL is observed both in the
vacuole and in the apoplast. The lack of a mutant phenotype in leaf sections indicates the
identification of root specific mutants that appear impaired in protein transport to the

vacuole.

MATERIAL AND METHODS
EMS Mutagenesis

EMS mutagenesis was performed according to Haughn and Sommerville (1986).
Rat-GUS-BL (CRBT5 6.18.5x) A. thaliana seeds (1 g) were soaked for 16 hours in 100 ml
of 0.3 % (v/v) ethyl methane sulfonate (Sigma Chemical Company, St Louis MO, USA).
They were subsequently washed with water 20 times, and sown in 12 flats (30 cm x 55 cm x
5 cm). They were grown under conditions of 22 0C, 110 uEinsteins of light at a 24-hour

light duration for 2 months. Seeds were harvested in 47 pools, threshed and stored at 4 °C.

Visual Screen

Seeds (~5 mg) were sterilized and germinated vertically on media plates
[Gamborg’s-BS basal medium with minimal organics (Sigma) 0.32 % (w/v), 2-(N-
morpholino)ethanesulfonic acid (MES) 0.05% (w/v) and sucrose 2% (w/v), pH 5.7] for
one week. Staining was carried out with X-GlcU (0.1 mg/ml of final suspension), dissolved

in DMSO (0.1 % (v/v) of the final suspension) and the solution dispersed in 0.05 % (w/v)

54

sterile agarose. Seedlings were overlaid with 10 ml of X-GlcU solution and scored after 5
days. Seedlings with blue roots were identified and transferred to media plates (lacking X-

GlcU) and grown until lateral roots developed and transferred to soil for recovery.

lmmunoelectron microscopy

The root or leaf segments of mutant Arabidopsis seedlings were fixed in a mixture
of 1.5% (v/v) formaldehyde, 0.5% (v/v) glutaraldehyde in 0.01 M sodium phosphate, pH
7.4, for 2.5 hours at room temperature on a rotator. The specimens were rinsed in the
same buffer and postfixed in 0.5% (w/v) 0804 for 1 hour at room temperature.
Dehydrated specimens were embedded in London Resin White (Polysciences,
Warrington, PA). Ultrathin sections were made with an Ultracut S microtome (Reichert-
Jung, Vienna, Austria) with a diamond knife and collected on nickel grids pre-coated
with Formvar. Irnmunolabeling carried out according to Sanderfoot et a1 (1998) using
primary rabbit polyclonal antibodies against wheat germ agglutinin (rabbit anti-WGA)
and goat anti-rabbit IgG coupled directly to 10 nm colloidal gold particles. Control grids
were treated with non-immune sera. The sections were examined with a Philips CM-lO
transmission electron microscope. All labeling experiments were conducted several times
each on independent sections. Tissue preparation and microscopy was carried out by Dr

V. Kovaleva.

55

Chapter 4

Characterization of mutants and selection of cvsI

56

INTRODUCTION

Mutant screens often yield a large number of putative mutants that have to be
characterized further prior to mapping the mutation. To identify mutations that affect the
fate of reporter proteins, it is imperative to ensure that the reporter itself is not affected by
the mutation. Transgenic reporters can also be affected by mutations that interfere with
the expression of the transgene. Since a transgenic reporter construct was used to identify
mutants impaired in the transport of ctVSS-bearing proteins, it was necessary to further
analyze the performance of the reporters. Once a collection of mutants which display the

desired phenotype is obtained, one can proceed with mapping and positional cloning.

RESULTS AND DISCUSSION
Identification and characterization of mutants

As the ctVSS-dependent sorting pathway is saturable (Frigerio et al, 1998 and
Neuhaus et al, 1994), the possibility of secretion due to the over-expression of reporter
proteins was investigated. The expression profile of Rat-GUS and BL in mutants e5a,
e11c, e12-I, eI3-1, CRBT5-6.l8.5x (referred to in this chapter as “parental”) was
compared to that of a line of Arabidopsis plants expressing BL (referred to in this chapter
as “control BL” line), in which BL is localized in the vacuole (Ahmed et al, 2000). Root
tissue was harvested from plants grown in liquid culture. RNA was extracted from these
tissues and analyzed by northern blotting. Filters were hybridized with randomly primed
32P-labeled probes using fragments of cDNAs of Rat-GUS, BL or the 17S ribosomal
RNA from rice as a loading control (Zarembinski and Theologis, 1993). Northern

analysis (Figure 4.1) revealed that mutants could be categorized into two classes: low

57

 

Barley
Lectin

Rat-GUS

17S
rRNA

 

 

Figure 4.1 Northern analysis of root RNA from mature plants

Seedlings of mutant, CRBT5-6.18.5x and control barley lectin transformant were grown in
liquid culture to obtain a large root mass (see Materials and Methods). RNA was
extracted and 10 pg total RNA from each culture was resolved by MOPS-agarose
electrophoresis. The RNA was then transferred to nitrocellulose and hybridized with
randomly primed 32P labeled probes. Probes used in the analysis were barley lectin, Rat-
GUS and 178 ribosomal rDNA (see Materials and Methods) as a loading control.

Lane 1 = Barley lectin control, Lane 2 = CRBT5-6.18.5x, Lane 3 = mutant e5a, Lane 4 =
mutant e13-1, Lane 5 = mutant e12-1 and Lane 6 = mutant e11c.

58

expressers (e110 and e13-1) and over-expressers (e5a and e12-1). The levels of BL
message in the two ‘over-expressing’ mutants (850 and 812-1) matched with that of the
control BL line. In contrast, mutants e] [C and e13-1 and parental lines had very low
levels of BL message. By probing the northern blot with a probe for Rat-GUS, it was
observed that levels of Rat-GUS message mimicked levels of BL message in all lines
tested in that lines expressing high levels of BL also expressed high levels of Rat-GUS.
Similarly, lines expressing low levels of BL had low levels of Rat-GUS. This indicated
that the same mechanism determined the level of expression of both reporters.

The mRNA profile analysis was complemented with a western analysis using
protein from the same tissues to determine the pattern of protein levels in mutants (e5a,
e11c, e12—1, e13-1), parental and the control BL line. Total root protein was extracted
from mature plants grown in liquid culture and the level of BL was detected by western
analysis (Figure 4.2). It was observed that mutants could be categorized into two classes:
low expressers (e! [C and e13-1) and over-expressers (e50 and e12-1). The amount of BL
in the low expresser was similar to that of the parental line indicating that the phenotype
of secretion seen in these lines was not due to a saturation of the pathway but was perhaps
a defect in the sorting machinery. This observation was further strengthened by results
which demonstrated that the level of BL in the low expressing mutants was much lower
than the control BL line, where BL is properly sorted to the vacuole. Taken together these
results indicate that in mutants e] lo and e13-1, the apoplastic localization of BL was not
due to saturation of the transport machinery but may be due to a defect in the transport

machinery caused by a trans-mutation.

59

 

 

 

 

 

 

Figure 4.2 Western analysis of barley lectin (BL) in mutant and control tissues
Seedlings of mutant, CRBT5-6.18.5x and control barley lectin transformant were grown in
liquid culture to obtain a large root mass (see Materials and Methods). Protein was
extracted and 10 pg total protein fi'om each culture was resolved by SDS-PAGE and
immunoblotted. Resolved proteins were visualized by a primary antibody treatment with
rabbit anti-WGA, followed by treatment with goat anti-rabbit coupled to alkaline
phosphatase. Blots were developed according to Harlow and Lane, 1988 (see Materials
and Methods). Lane 1 = Barley lectin control, Lane 2 = CRBT5-6.18.5x, Lane 3 = mutant
e12-1, Lane 4 = mutant e13-1, Lane 5 = mutant e11c and Lane 6 = mutant e5a.

A comparison of barley lectin protein levels in young parental tissues and mature
tissues from liquid cultures revealed that while the protein can be detected in root tips
(see Figure 2.5), it is absent in mature tissues. This was indicative of cosuppression, a
phenomenon commonly seen in plants transformed with constitutive promoters such as
the cauliflower mosaic 358. These lines demonstrated a reduction of BL and Rat-GUS in
mature tissues, such as those obtained from liquid cultures. This is very similar to results
shown by Park et al (1996), where promoter homology was shown to cause the
cossupression of transgenes driven by identical promoters. By using the same promoter
(double 358 promoter) to drive BL and Rat-GUS, it is conceivable to suggest that
promoter-homology may have induced silencing of the transgenes. Van Houdt et a1
(1997) showed that aging reinforced the silencing of transgenic neomycin
phosphotransferase in tobacco plants. Palauqui et a1 (1996) showed that in tobacco lines,
cosuppression can be induced during a period between 15 days after germination and

flowering. Further Balandin and Castresana (1997) observed that silencing in tobacco

occurs a few weeks after germination and is maintained throughout the vegetative phase

60

and flowering. However, silencing is reversed in developing seeds and fruits derived from
meiotically divided cells (Balandin and Castresana, 1997). At this stage, it is difficult to
determine if the silencing of BL and Rat-GUS are due to promoter homology or the
presence of multiple insertions (tandem repeats) at the reporter locus.

The cossupressed condition in mutants e11c and e13-1 would suggest that in these
mutants, the ctVSS transport pathway may not be saturated and hence the partial
mislocalization of BL in root tips as detected by electron microscopy, is due to a defect in
the transport machinery. Unfortunately BL has not been detected in parental roots by
electron microscopy although it is detectable in root tips by western analysis (see Figure
2.5). However, the identification of mutant e19-1 (see Table 3.3) where Rat-GUS is
secreted but BL is not mislocalized, indicates that transgenic BL is localized in the
vacuole. This is also evident in leaf sections where BL was found exclusively in the
vacuole. Taken together it can be implied that mutants e11c and e13-1 partially missort
BL to the apoplast due to a root-specific defect in the transport machinery and not
saturation of the pathway.

It was also observed that in mutant e11c, the terminal inflorescence was
“compressed” (Figure 4.3a and b) in comparison with the parental terminal inflorescence
(Figure 4.3a and c), a phenotype that is consistent with a poor elongation of intemodes in
floral tissues. At this stage, it is not clear if the secretion phenotype and the inflorescence
phenotype are linked. Mutant e11c has now been renamed cvsI for ctVSS vacuolar

sorting defect and mutant e13-1 has been renamed cvs2.

61

 

 

 

 

 

 

Figure 4.3 Examination of floral phenotype of MI
Mature inflorescences of CRBT5-6.l8.5x and mutant cvsI were compared. Panel A =
Inflorescence of mutant cvsI (left) and CRBT5-6.l8.5x (right). Panel B = Close up of
inflorescence of mutant cvs] with “compressed” phenotype. Panel C = Close up of
inflorescence of CRBT5-6. 18.5,,
Genetics of mutant cvsl

In order to understand the nature of the mutation and eventually clone the gene
whose mutation resulted in the phenotype, the mutant cvsI was backcrossed to the
parental line (F1 cross) (according to Raventos et al, 2000). This progeny was germinated
in plates set vertically so that the roots develop on the surface of the medium. Upon
staining with X-GlcU, it was found that none of the roots turned blue. This indicated a
recessive phenotype, as mutant cvsI was homozygous (see Table 3.3). To confirm this
finding, the F1 seedlings were transferred to soil and their selfed progeny harvested. This
progeny (F2) was scored for segregation of the phenotype by performing the Rat-GUS

secretion assay. Results indicated a 94 white seedlings (vacuolar) to 33 blue seedlings

(secretion) ratio. Applying chi-square analysis to this information against an expected

62

segregation ratio of 3:1 (vacuolar: secreted) for a recessive mutation, a chi-square value
of 0.066 and a p-value of 0.797 were obtained. (Note the smaller the chi-square the better
and the higher the p-value the better so as to "fail to reject a null hypothesis of equality of
ratios"). The large p-value strongly indicates the lack of a significant deviation in the
sample from an expected ratio of 3:1. These findings confirmed the results of secretion
assays performed on the F 1 generation indicating that mutant cvsl had a single gene

recessive mutation.

CONCLUSION

By screening a population of EMS-mutagenized seeds expressing Rat-GUS-
ctVSS and BL, several candidate mutants were identified using the Rat-GUS secretion
assay (see Chapter 3). Irnmunoelectron microscopy revealed that in several mutants, BL
was mislocalized in the apoplast in a “leaky” manner. The evaluation of reporter message
levels and protein levels in these mutants allowed the identification of two mutants (cvsI
and cst) potentially impaired in protein transport to the vacuole, resulting in the partial
secretion of the reporter proteins. Based on the segregation ratio of the mutant phenotype
after back crossing into the parental line, it appears that the phenotype of cvsI is
determined by a single gene recessive mutation. It can be inferred that our] is a mutant
with a single gene defect resulting in the partial secretion of ctVSS-bearing vacuolar

reporter proteins.

63

MATERIALS AND METHODS
Arabidopsis Liquid Culture

Seeds (5-10) were surface-sterilized and cultured in sterile liquid culture medium
[0.43 % Murashige and Skoog salts, 2-(N-morpholino)ethanesulfonic acid (MES) 0.05%
(w/v), 1 % (w/v) sucrose at pH 5.7, supplemented with 0.1 mg/ml myo-inositol, 1 ug/ml
nicotinic acid, 1 ug/ml pyridoxine and 1 ug/ml thiamine] with agitation at 250 rpm, 120
“Einstein light for 16 hours per day for 4 weeks. Root and shoot tissue was then frozen in

liquid nitrogen and stored at —80 °C until needed.

Northern blotting

RNA for northern analysis was extracted using the approach of Houdebine and
Puissant (1990) with certain modifications. Liquid cultured root tissue from mutant and
wild type plants were harvested, frozen in liquid nitrogen and stored at —80 °C for
extraction. One gram of tissue was homogenized with a mortar and pestle in liquid
nitrogen, and the powder was extracted with 4 ml GTC solution [4 M guanidinium
thiocyanate, 25 mM sodium citrate pH 7.0, 0.5 % (w/v) sodium lauryl sarkosinate and 10
mM B-mercaptoethanol]. The suspension was mixed with 650 u] of sodium acetate pH
4.0 by vortexing, followed by vortexing with 5 ml of phenol equilibrated with TE (10
mM Tris-HCl pH 8.0, 1 mM EDTA). Finally 1 ml of chloroform-isoamyl alcohol (24:1)
was added and the suspension centrifuged at 10000 g for 10 minutes at 4 °C. The aqueous
phase (supernatant) was precipitated with 5 ml isopropyl alcohol by storing at 4 °C
overnight. The suspension was then centrifuged at 10000 g for 10 minutes at 4 °C. The

supernatant was discarded and the pellet of RNA dislodged with 1 ml of 4 M lithium

64

chloride. The precipitate was dispersed by vortexing and the RNA was precipitated by
centrifugation. The lithium chloride step was repeated. The RNA pellet was then
resuspended in 600 [.11 RR buffer [10 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.5 % (w/v)
SDS in DEPC-treated water]. The suspension was re-extracted with phenol-chloroform-
isoamyl alcohol and finally RNA was precipitated with isopropanol. The yield of RNA
was measured by absorption at 260 nm.

RNA (10 pg) was resolved by MOPS agarose gel electrophoresis as per Bar-Peled
and Raikhel (1997) and transferred to nitrocellulose membranes. Membranes were
hybridized with 32P-labeled probes as per Bar-Peled and Raikhel (1997) synthesized from
cDNA fragments encoding Rat-GUS (EcoRV fragment comprising of nucleotides 1516 to
1881) or barley lectin (ApaI-BamHI fragment comprising of nucleotides 40 to 546) or
17S ribosomal RNA from rice (see Zarembinski and Theologis, 1993). Hybridized blots

were visualized using a Molecular Dynamics phophorimager.

Western Analysis of barley lectin

Liquid cultured root tissue from mutants, parental and control BL plants were
harvested, frozen in liquid nitrogen and stored at —80 °C for extraction. One gram of
tissue was homogenized with a mortar and pestle in liquid nitrogen, and the powder was
resuspended in protein extraction buffer [PBS, 0.1% (v/v) Triton X-100, 0.1 % (w/v)
sodium-N-lauryl sarcosine, 1 mM PMSF], and then centrifirged twice at 4 0C for 10
minutes at 15000 g. Protein contents were estimated by Bradford's assay (Bio-Rad
Laboratories, Hercules, CA) using the Bradford analysis package, bradfordhsa.htm (see

Appendix B).

65

Protein extracts were diluted with half a volume of 3X Laemmli buffer (Harlow and
Lane, 1988). The diluted samples (25 ul) were boiled, treated with 5 pl acetamide solution
(5 [.11 1X Laemmli buffer, 2 mg Tris base, 1.33 mg iodoacetamide) and the mixture was
incubated at 37 0C for 30 minutes prior to resolving by SDS-PAGE. Proteins were
immunoblotted onto nitrocellulose filters, transfer confirmed by staining with Ponceau S,
blocked and incubated with primary antibodies (rabbit anti-WGA) at 1:1000 dilution and
with secondary antibodies (goat anti-rabbit linked to alkaline phosphatase) at 1:1000
dilution according to Harlow and Lane (1988). Protein bands were visualized using 50 ml of
alkaline phosphatase buffer (100 mM sodium chloride, 5 mM magnesium chloride, 100
mM Tris, pH 9.5) containing 0.16 mg/rnl 5-bromo-4-chloro-3-indolyl phosphate and 0.35
mg/ml nitro blue tetrazolium chloride. After the development of bands, the blots were

rinsed with distilled water and photographed.

Crosses

Seedlings were grown under controlled conditions of 22 °C, 110 uEinsteins of light
at an 18-hour light duration for 4 weeks until 3-4 siliques had fiuited. Stamens fiom pollen
donor flowers were dissected and used to pollinate emasculated flowers. The pollinated
flowers were covered with plastic film for 4 days until the silique started developing and

then the film was removed. Seeds were harvested and stored at 4 °C for further use.

66

Chapter 5

Mapping ml to a contig of BACs

67

INTRODUCTION

Upon the identification and characterization of a mutant, it is necessary to identify
the mutant locus and map it as well as possible to identify the gene encoding the protein
involved in the transport pathway. In Arabidopsis and other self-fertile species, this can
be achieved by crossing the mutant into a different ecotype that offers a number of
polymorphic characters for mapping. It is then possible to measure the linkage between
the mutant locus and defined polymorphic loci in a segregating mapping population. This
approach assumes that the phenotype is not ecotype-dependent. In Arabidopsis, a large
number of polymorphic loci have been identified on all five chromosomes between

ecotypes Columbia (C01) and Landsberg erecta (Ler) enabling a fine mapping analysis.

RESULTS AND DISCUSSION

The backcrossed and self-fertilized line of cvsI (hereafter called ACWS) was
crossed into Landsberg erecta (Ler). The progeny from this cross was allowed to self-
fertilize to generate a segregating population of mutants (l lBC2FlLerF2). This
population was scored for secretion of Rat-GUS by germination of seedlings on vertical
media plates and staining the seedlings with 5-bromo-4-chloro-3-indolyl B-D-glucuronide
(X-GlcU). The identification of seedlings secreting Rat-GUS activity confirmed that the
phenotype under investigation was not affected by crossing into the mapping ecotype. All
the individuals with blue roots were transferred to soil and genomic DNA was extracted
from leaves. Two types of PCR-based codominant markers were used in the mapping
analyses: Cleaved Amplified Polymorphic Sequences (CAPS) markers and Simple

Sequence Length Polymorphisms (SSLP) markers. CAPS amplify fragments of the same

68

size in both Landsberg erecta and in Columbia (Col) but have a different restriction
pattern (Konieczny and Ausubel 1993) while SSLP markers generate PCR products of
different sizes without any further treatment (Bell and Ecker, 1994).

The analysis was initially performed on 10 DNA (Table 5.1) samples from
segregating lines expressing the mutant phenotype, with markers representing various loci
on the five chromosomes. Linkage analysis was carried out by evaluating the
recombination frequency (RF) and calculating a chi-square for deviation from an
expected ratio of 122:1 (Homozygous Col: Heterozygous: Homozygous Ler). An unlinked
locus would then have a recombination frequency of 0.5 (50 %) and a low chi-square
value (high p-value), indicating a low deviation of the sample from the expected ratio of
1:2:1. Conversely a large deviation from a 1:2:1 ratio as indicated by a large chi-square
value and a low p-value would indicate that the marker locus and the mutant locus are
linked. These calculations were performed by developing an HTML-based code available
at http://www.msu.edu/~venkata1/sslpfull.htm (see Appendix A). Recombination
frequencies on chromosomes I to IV ranged between 0.4 and 0.6 indicating a lack of
linkage between the markers and the mutant locus. However markers on chromosome V
showed a linkage to the mutation as the RF ranged around 0.37 at marker ATSOZ62.
Though this was not a close linkage, it was indicative of linkage considering the small
sample size (N= 10). It was observed that the chi-square value was not very high in this

small sample.

69

Table 5.1 10-Sample mapping analysis

 

Chromosome Position Marker Total RF Linkage (cM) chi-square p-value

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

I 9.4 nga63 10 0.50 No Linkage 0.40 0.82
I 40.1 nga248 7 10.57 No Linkage 0.43 0.81
I 81.4 nga280 10 0.45 115.13 0.20 0.90
I 111.4 ngalll 10 0.60 No Linkage 4.40 0.11
1 118.3 nga692 8 0.63 No Linkage 1.00 0.61
II 62 nga361 10 0.55 No Linkage 1.80 0.4]
II 73 ngal68 10 0.50 No Linkage 0.40 0.82
111 21 ngal62 20 0.38 69.31 2.70 0.26
111 85.3 nga6 10 0.70 No Linkage 3.60 0.17
IV 19.8 ngalZ 6 0.42 89.59 3.00 0.22
IV 24.2 nga8 19 0.58 No Linkage 1.00 0.61
N 102 nga1107 10 0.55 No Linkage 1.80 0.41
V 23.1 nga249 10 0.40 80.47 1.20 0.55
V 33 nga106 10 0.35 60.20 3.40 0.18
V 51.1 nga139 26 0.37 65.61 3.77 0.15
V 65.2 Ath80262 17 0.38 72.35 2.41 0.30
V 71.13 AthPHY-C 16 0.44 103.97 0.50 0.78
V 79 AthSOl9l 16 0.44 103.97 1.50 0.47

 

 

 

 

 

Summary of a 10-sample mapping analysis covering 5 chromosomes with various SSLP
and CAPS markers. Position = Position of marker locus on chromosome (cM). Total =
Number of segregants analyzed. RF = Recombination Frequency. Linkage = Distance
(cM) between marker locus and cvsI estimated from the RF using Haldane’s mapping
function (for review Koorneef and Stam, 1992). Chi-square = chi-square calculated to
estimate deviation of observed frequency from expected frequency in case of no linkage
(Col:Het:Ler =l:2: l)

70

A similar analysis was performed on a larger sample (n = 45) shown in Table 5.2.
Previously observed results were confirmed and a linkage to markers on chromosome V
was evident with recombination frequencies of 0.28 at marker ATSOl9l and higher RFs
on either side of the marker. This is also evident from the analysis of chi-squares where a
large deviation from the expected 1:2:1 ratio for an unlinked scenario was observed. As
observed in the small sample analysis, the RF values were not as small as expected when
approaching the mutant locus. This is likely due to the nus-identification of secreting
mutants in the segregating population. Such a result is often obtained when phenotypes
have to be evaluated subjectively (personal communication from Dr. Maarten Koornneef,
Wageningen Agricultural University, Wageningen, The Netherlands). Further
optimization of the RF by incorporating the secondary reporter, BL, could not be carried
out as secreted BL in roots can only be detected by electron microscopy. The inclusion of
non-mutants in the mapping analysis would lead to a larger recombination frequency than
expected had there been no such individuals in the sample. To circumvent this problem,
a reduction in the recombination frequency and a corresponding increase in chi-square
value were used as indicators of linkage. A large-scale analysis using 135 DNA samples
from secreting segregants was canied out using markers AthPHYC, ATSOl91, DF R and
LFY 3 (Table 5.3). In this analysis, the RF was lowest around DFR. The trend of chi-
square values reflected the trend of the RF values with marker DFR having the largest
chi-square value (80.18). This suggested that mutation cvsI lay between AtSOl91 and
DFR.

As commercially available markers did not cover this region, new markers were

developed. The region between AtSOl9l and DFR is spanned by a set of 32 overlapping

71

Table 5.2 46-Sample mapping analysis

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Chromosome Position Marker Total RF Linkage (c chi-square value
I 40.6 nga248 46 0.58 No Linkage 3.05 0.22
I 81.71 nga280 46 0.50 No Linkage 0.22 0.90
I 111.4 ngalll 46 0.46 122.84 5.34 0.07
I] 50.56 nga1126 46 0.52 No Linkage 0.21 0.90
111 16.2 nga126 46 0.51 No Linkage 0.07 0.96
HI 83.5 nga6 46 0.54 No Linkage 1.48 0.48

IV 24.1 nga8 46 0.63 No Linkage 10.52 0.01
IV 60.35 Aga 46 0.41 87.15 3.35 0.19
[V 102 nga1107 46 0.43 101.84 2.35 0.31
V 33.3 ngalO6 45 0.41 86.36 3.04 0.22
V 50.48 ngal 39 46 0.38 71.54 5.26 0.07
V 65.2 AthSOZ62 46 0.33 54.93 10.02 0.01
V 71.13 AthPHYC 45 0.30 45.81 16.40 0.00
V 79 AthSOl9l 46 0.28 39.93 17.80 0.00
V 87.63 DFR 46 0.33 52.49 9.80 0.01
V 115.01 LFY3 46 0.36 62.09 7.53 0.02

 

 

 

 

 

 

 

 

 

 

Summary of a 46-sample mapping analysis covering 5 chromosomes with various SSLP
and CAPS markers. Position = Position of marker locus on chromosome (cM). Total =
Number of segregants analyzed. RF = Recombination Frequency. Linkage = Distance
(cM) between marker locus and cvsI estimated from the RF using Haldane’s mapping
function (for review Koomeef and Stam, 1992). Chi-square = Chi-square calculated to
estimate deviation of observed frequency from expected frequency in case of no linkage
(Col:Het:Ler =1:2:1)

72

Table 5.3 Fine map between AthPHYC and LFY 3

 

 

 

 

 

 

 

 

 

[Marker AthPHYC AthSOl9l DFR LFY3
[Chromosome V V V V
[Position 71.13 79.00 87.63 115.01
Total 135 135 135 135
[RF 0.27 0.25 0.23 0.37
inkage (cM) 39.04 34.66 31.72 67.06
hi-square 59.73 73.76 80.18 17.82
value 0.00 0.00 0.00 0.00

 

 

 

 

 

 

 

Fine map of cvsI between AthPHY C and LFY3 on chromosome V. Position = Position of
marker locus on chromosome V (cM). Total = Number of segregants analyzed. RF =
Recombination Frequency. Linkage = Distance (cM) between marker locus and cvs1.
Chi-square = chi-square calculated to estimate deviation of observed frequency from
expected frequency in case of no linkage (Col: Het: Ler = 1:2: 1)

bacterial artificial chromosomes (BAC) shown in Figure 5.1. New markers were
developed for BACs MBBl8, MKM21, K15E6 and MUL8. These were developed by
performing multiple FASTA analyses (Pearson and Lipman, 1988) using do_Fasta.pl (Dr.
Joe White, Department of Biochemistry, Michigan State University, East Lansing, MI,
USA). The database of Ler genomic sequences (available at
http://www.tigr.org/tdb/at/atgenome/Ler.html) was searched for identities using the
sequence of the 32 BACs. Within high identity regions, diverging domains were
identified for developing polymorphism markers. Thus markers for BACs MBB18
(Figure 5.2), MKM21 (Figure 5.3), K15E6 (Figure 5.4) and MUL8 (Figure 5.5) were
developed. Markers for BACs MBB18 and MUL8 were developed as SSLPs (Bell and
Ecker, 1994), while markers for BACs MKM21 and K15E6 were developed as CAPS

(Konieczny and Ausubel, 1993) markers (see Table 5.4). In addition CAPS markers for

73

 

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artificial chromosomes (Y ACs) spanning the same region. Available from the Kazusa

BACs are represented by blue bands and labeled. Black solid lines represent yeast
DNA Research Institute, Chiba, Japan

Figure 5.1 Alignment of BAC contig between AtSOl91 and DFR on chromosome V

74

C0954

mbb18

c0954

mbb18

co954

mbb18

C0954

mbb18.

Figure 5.2 Alignment of BAC MBBl8 (Col, 28295 — 28460) with Clone co954 (Ler)

This alignment was used to identify polymorphic regions. Letters in bold indicate forward
and reverse primers. Polymorphic stretch between 33 and 68 Ler was utilized to generate
an SSLP. Columbia DNA sequence was obtained from the Kazusa DNA Research
Institute, Chiba, Japan and Landsberg erecta sequence from The Institute for Genomic

.seq

.seq

.seq

.seq

.seq

.seq

.seq

seq

10 20 30 40 49
TAAGATCACCAAITGCRGAAACGGAACTACTACCATTGATGTAATAAC

IIIIIIIIIIIIIHIIIIIIIIIIIIIIII
TMGATCACCAM‘TGCAGAMCGGAACTACT .................
28300 28310 28320

59 69 79 89
ACCATTGATGTAATAACATAAGGGGATATTTAAAATTCTACAC --AC
|||| |||||| ||||| | l I II II
------------------ TAAGAGGATATCTAAAACTATGCTCATTAC
28330 28340 28350

99 109 119 129 139 149
TTTAAAACTATATGCTCATTACGTTCACATAA—ATAATAGATTTTGATAAGACCTTACTT
II | I II II I II II ||l| || || | I III II ll 1
GTTCACA—TAAAT----AATAGATTTCGATAAGATCTTATTTAAGGTTAAAACATTGGT-

28360 28370 28380 28390 28400

159 169 179 189 199 209
AAGGTTAATAAAAAGTAAGCATGCTTATGATGGAAAGAGAAAAGAGCTcaccrcrc
l lllll |||||||||||||||l|| |||||||||||||||||||||||ll
~—-GCGAATAAGAAGTAAGCATGCTTATGAGGGAAAGAGAAAAGAGCTGACCTCTG
28410 28420 28430 28440 28450 28460

Research, Rockville, MD, USA (see Materials and Methods).

75

c1103.seq

mkm21.seq

c1103.seq

mkm21.seq

c1103.seq

mkm21.8eq

Figure 5.3 Sequence alignment of a section of BAC MKM21 (Col) and c1103 (Ler)

This alignment was used to identify polymorphic regions. Letters in bold indicate forward
and reverse primers. Shaded box on Col sequence at 5475-5478 = polymorphic NlaIII site
Bold and underlined sequence indicates common NlaIH site in both sequences. Columbia
DNA sequence was obtained from the Kazusa DNA Research Institute, Chiba, Japan and
Landsberg erecta sequence from The Institute for Genomic Research, Rockville, MD,

40 50 6o 70 80 90
TTGAICTTGAICTCGAATGAIAAITCTCTCTGTTTTTTAGATCTAAGGTTTAATGCCGAT
||||||||||||||||||||llllllllllllllllllllll|||||||l||||||||||

MTTGAICTCGAA MTTCTCTCT TTTTTTAGA flAAGGTTTAATGCCGAT
5370 5380 5390 5400 5410 5420

100 110 120 130 140 150
TTTGGGTTGTTTTCAGTAAGAGATTTAGGCTTAAAGGACGTTTGTATCCTTGTATTATTC

II|||llll||l|Illlll||||lllll|||||||l||||||l||||| |||||| l
TTTGGGTTGTTTTCAGTAAGAGATTTAGGCTTAAAGGACGTTTGTAT ATTATCC
5430 5440 5450 5460 5470 5480

160 170 180 190 200
CCTCTGAGAATGGAGAAGTTCATGGAAA

ccrrrcarrrcrreacam
||||||||||||||||IIIIHIIII|l|||||||||||||||||||||||||l
ccrrrcarrrcrwcacam

CCTCTGAGAATGGAGAAGTTCATGGAAA
S490 5500 5510 5520 5530 5540

USA (see Materials and Methods).

76

c951.seq

k15e6.seq

c951.seq

k15e6.seq

c951.seq

k15e6.seq

c951.seq

k15e6.seq

c951.seq

k15e6.seq

c951.seq

k15e6.seq

c951.seq

k15e6.seq

c951.seq

k15e6.seq

c951.seq

k15e6.seq

38480

38540

38600

38660

489 479 469
TTGTGAATTGTGATAATATTTGTTTATTCA
|||l||||||||||||||||l|| ||||

TGGTTTTAAATcTTTTTTGTGAcGAAGAAGTTGTGAATTGTGATAATATTTGT---TTCA

38430 38440 38450 38460 38470

459 449 439 429 419 409
TTGGACAATTTAAATTGACCAACTCTTTAAAGAAGACAAGACTTTCTACAATGTAAATTG
Ill|l|l||||||||ll||||||||||||||||||||||||||l||||l|||||||||||
TTGGACAATTTAAATTGACCAACTCTTTAAAGAAGACAAGACTTTCTACAATGTAAATTG
38490 38500 38510 38520 38530

399 389 379 369 359 349
ACTACGCCCATGAGTATCATCATGATATAATCATATACATGATCTCATATATATGTGCGT

IllIIIllllllllllllllllllllllllll||||||||||||||||||||||l|||||

ACTACGCCCATGAGTATCATCATGATATAATCATATACATGATCTCATATATATGTGCGT

38550 38560 38570 38580 38590
339 329 319 309 299 289
TTGATGCGTTTTcCTATTTTGTGGTTGCTTAGTTAATCTCGTTGACTTTTGATAAAAACA
Ill||||||||||l||||IIIIIIIIIIIIIIIIIIIIIllllIIIIIIIIIIIIIIIII
TTGATGCGTTTTCCTATTTTGTGGTTGCTTAGTTAATCTCGTTGACTTTTGM MAAAAA

38610 38620 38630 38640 38650
279 269 259 249 239 229
TAGCAT‘I'ACCAAGTACTAGA'I'I‘TTAACCCGCGGTATA-GGGACGATTTA‘I'I‘TTT'I‘AA
||||| llllllllllllllllllll I |||||l| lllllllllllllllllll
TAGCA---—CAAGTACTAGATTTTAACcCTCACTATACCGCGGGACGATTTm

38670 38680 38690 38700 38710

219 209 199 189 179 169

AGATAATATATATTAAAATTTGTAAATTTTATTTTTATAAAATATTTATATTTTATAGTT

Illlll||||||l|||||l|||||||l|||||||||l||||||||l|l||||||||||||
MAAAA WTTTTAm

AGATAATATATA ATAAAATATTTATATTTTATAGTT
38720 38730 38740 38750 38760 38770
159 149 139 129 119 109

TATAATTGTTATTAAATAACGTTATACTACGAATACATGAGACAATTGTTAAAAAACTGA

TATAATTGTTATTAAGTAACGTTATACGACGAATACATGAGACAATTGTTAAAAAACTGA

38780 38790 38800 38810 38820 38830
99 89 79 69 59 49
AGTTTTAATcCTTATTAAAACAACATATTAATAATATTTTAAAATTTTATAAATATTGAT
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
MAATC NAAAAC MCA TATTAAm NAAAATTTTATAAATATTGAT
A38840 38850 38860 38870TT 38880 38890
39 29 19 9

TCAAATACATCTACCAIAIAACCCAATTCCAAAATAAAATT

Il||l||llllllllllllllllllllllIllllllllll

TCAAATACATCTACCAEATAACCCAATTCCAAAAIAAAATC
38900 38910 38920 38930

Figure 5.4 Sequence alignment of a section of BAC K15E6 (C01) and c951 (Ler)

This alignment was used to identify polymorphic regions. Letters in bold indicate forward
and reverse primers. Shaded box on Ler sequence at 239-242
polymorphism. Columbia DNA sequence was obtained from the Kazusa DNA Research
Institute, Chiba, Japan and Landsberg erecta sequence from The Institute for Genomic

Research, Rockville, MD, USA (see Materials and Methods).

77

HpaII site of

520 530 540 550
c1765 . seq mmmmAflAACTCAAGAAGAA ---------------------

mulB.seq WAAAGTAAAGAGTCATTGTTGT TCAAGAAGAATTTGTCTGAGTTTCTCTACTA
48930 48940 48950 48960 48970 48980
560 570 578
c1765.seq ------------------------------ GACGGTAAGATGTAAATAATTTTGAGTATT
lllllllllllllllllllllllll|||||
mu18.seq TTCTCTACTAAAAGTCGGAAGCAAGAAGAAGACGGTAAGATGTAAATAATTTTGAGTATT
48980 48990 49000 49010 49020 49030
580 590 600
c1765.seq TATATAAGGTTAATAGACCTGTCGGTTTM
|||||||l||||||||||||||||l|||||
mu18.seq TATATAAGGm MAGACCTGTCGGTTT
49040 49050 49060 49068

Figure 5.5 Alignment of BAC MUL8 (Col, 48930 - 49068) with Clone c1765 (Ler)

This alignment was used to identify polymorphic regions. Letters in bold indicate forward
and reverse primers. Polymorphic stretch between 48968 and 49008 in C01 was utilized to
generate an SSLP. Columbia DNA sequence was obtained from the Kazusa DNA
Research Institute, Chiba, Japan and Landsberg erecta sequence from The Institute for

Genomic Research, Rockville, MD, USA (see Materials and Methods).

78

Table 5.4 Markers used to map cvs] within between ATSOl9l and DFR

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Marker Primer Primer Sequence Type Polymorphism

M8818} Forward TAAGATCACCAATI‘GCAGAAACGG SSLP Col = 169
Reverse CAGAGGTCAGCTCTI‘ITCTCITFCC Ler = 192

Kl 5136“ Forward "GTGAWGTGATMTAWGWC CAPS Col = 500
Reverse "ITITATI'ITGGAATI‘GGGTFATATGG HpaII Ler= 250, 250

MUL8& Forward CI‘AAAGTAAAGAGTCA'ITGTTGTA'IT SSLP (301:140
Reverse GTAAACCGACAGGTCTATTAACCI‘ Ler= 99

MKM218‘ Forward GATCI’FGATCWGATCTCGWGAT CAPS Col = 117, 32, 29
Reverse AAAGAATGATCI‘CAAGAAATCAAACC NlaIII Let = 149, 29

MYH19* Forward ACTAmATGoanGCAm CAPS Col=l95
Reverse ATCI‘GTAAATCTTGTATA’I'TACC Ddel Ler = 175’ 20

MSN9R“ Forward TGGCAGTAAmMTGATAMC” CAPS Col=200

MNF 13R" Forward CAGAGTCATGGTMATGCCFG CAPS Col=190
Reverse GTITCTITGAGTATI‘AGAGTCC BstNI Ler = 170, 20

 

Markers for each BAC are listed along with the sequence of the amplification primers and
the restriction enzymes (where appropriate). & = Primer sequence and polymorphism
profile developed by multiple FASTA analyses using A.t. ecotype Columbia BAC
sequences and At. ecotype Landsberg erecta random genomic clone sequences (The
Institute for Genomic Research, Rockville, MD, USA). CAPS = Cleaved Amplified
Polymorphic Sequences. SSLP = Simple Sequence Length Polymorphisms. Restriction
enzymes used in developing the polymorphism are italicized. * = Ziegelhoffer et al

(2000)

79

 

BACs MYH19, MSN9R and MNF 13R were also used (Ziegelhoffer et al, 2000). These
markers were used to map the meiotic recombination break points between AtSOl91 and
DFR (Table 5.5) indicating a recombination event between Ler and Columbia
chromosomal DNA in the neighborhood of cvsI .

Since the mutation was developed in a Columbia background, the region flanking
the mutant locus is expected to be homozygous for Columbia markers (labeled blue in
Table 5.5). The detection of a chromosomal break point on BACs MBB18 and MUL8
indicated that mutation cvsI lies “south” of the polymorphism marker for BAC MBBlS
(25231 bp on BAC MBB18) and “north” of the marker for BAC MUL8 (48929 bp on
BAC MUL8). The length of this region is about 417 kbp and is spanned by a contig of 6
overlapping BACs: MBBlS, MKDlO, K15E6, MXF 12, K3K3 and MUL8 (Figure 5.6).
As this region includes the CAPS marker for BAC K15E6, the mapping analysis
performed with this marker revealed a Columbia pattern in all samples tested. This

confirmed that the mutation lay in this region.

CONCLUSION

Using a combination of CAPS and SSLP markers the mutation in cvsI was
initially localized between ATSOl9l and DFR on chromosome V. By developing
markers specific for the BACs spanning this region along with other markers, cm] was
mapped to a 6-BAC contig including MBB18, MKDlO, K15E6, MXF 12, K3K3 and
MUL8. Further analysis by complementation using BACs and cosmid clones should

reveal the exact position of cvsI .

80

Table 5.5 Fine mapping analysis of MI between AtSOl9l and DFR

 

Markers developed for this region were used in mapping analyses and the results are
tabulated. Lines: numbers represent individual segregant DNA sample. H = Heterozygous
polymorphism (Red). C = Columbia polymorphism (Blue). L = Landsberg erecta
polymorphism (Yellow). 0 = No amplification. Blank = DNA was not sampled. Marker
= SSLP or CAPS marker used in developing the polymorphism. Position = Mbp along
chromosome V.

81

 

 

 

 

Figure 5.6 BAC contig spanning cvs] locus
Light blue boxes represent BACs and broken arrows indicate the approximate position of
the mapping markers used.

MATERIALS AND METHODS
Crosses

Line ACWS was crossed into Arabidopsis thaliana ecotype Landsberg erecta
(Ler) as follows. Seedlings were grown under controlled conditions of 22 °C, 110
uEinsteins of light at an l8-hour light duration for 4 weeks until 3-4 siliques had fruited.
Stamens from ACWS flowers were dissected and used to pollinate emasculated Ler
flowers. The pollinated flowers were covered with plastic film for 4 days until the silique
started developing and then the film was removed. Seeds (F 1) were harvested and stored at 4
°C for further use. The self-fertilized progeny from the F 1 line was harvested (F 2).

F2 seeds were sterilized in 50 % commercial bleach with a drop of Tween 20, and
rinsed several times. These seeds were plated on Arabidopsis germination media [2 %
(w/v) sucrose, Gamborg’s B5, 300 mg/L carbenicillin] for seven days and stained with 5-

bromo-4—chloro-3-indolyl B-D—glucuronide [0.1 % (w/v) in 0.05 % (w/v) agarose] for S

82

days. Blue seedlings were transferred to soil and leaves from individual seedlings

harvested.

Genomic DNA isolation

Leaves from individual blue seedlings were homogenized in liquid nitrogen using
a Kontes motorized pestle and the leaf resuspended in extraction buffer [2%
cetyltrirnethylammonium bromide (CTAB), 100 mM Tris-HCL, pH 8.0, 1.4 M NaCl, 20
mM EDTA, 0.2 % B-mercaptoethanol] and incubated at 65 0C for 1 hour. The suspension
was extracted with chloroform. The aqueous phase was then treated with ethanol to
precipitate genomic DNA. The DNA pellet was air dried at room temperature overnight
and dissolved in TE (10 mM Tris-HCl pH 7.0, 1 mM EDTA) containing 10 ug/ml bovine

pancreatic RNAse (Roche, Palo Alto, CA, USA).

PCR Amplifications: SSLP markers

SSLP analysis was performed according to Bell and Ecker, 1994. Briefly, genomic
DNA samples were amplified in a 20 111 volume of 20 mM Tris-HCl pH 8.4, 50 mM KCl,
0.2 mM dNTPs, 2 mM MgC12 containing 50 ng genomic DNA, 1 unit Taq DNA
polymerase (Gibco-BRL, Rockville, MD, USA), 5 pmoles forward primer and 5 pmoles
reverse primer. The following cycling profile was used to amplify the reactions:
denaturation at 94 °C for 2 minutes, amplification cycle (40 cycles) at 94 0C for 15
seconds, 55 °C for 15 seconds, 72 °C for 30 seconds and extension at 72 °C for 7 minutes.

SSLP primers were obtained from Research Genetics, Huntsville, AL, USA. Amplified

83

products were resolved by TEE-agarose gel electrophoresis and visualized by ethidium

bromide staining.

PCR Amplifications: CAPS markers

CAPS analysis was performed according to Konieczny and Ausubel (1993).
Briefly, genomic DNA samples were amplified in a 10 111 volume of 20 mM Tris-HCI pH
8.4, 50 mM KCl, 0.125 mM dNTPs containing 50 ng genomic DNA, 1 unit Taq DNA
polymerase (Gibco-BRL, Rockville, MD, USA), 3.2 pmoles forward primer and 3.2
pmoles reverse primer. The following cycling profile was used to amplify the reactions:
denaturation at 94 °C for 2 minutes, amplificaton cycle (50 cycles) at 94 °C for 1 minute,
56 0C for 1 minute, 72 °C for 3 minutes and extension at 72 °C for 10 minutes. CAPS
primers were obtained from Research Genetics, Huntsville, AL, USA. Amplified
products were digested with the appropriate restriction enzymes (as recommended by the
manufacturer) and resolved by TEE-agarose gel electrophoresis and visualized by

ethidium bromide staining.

Development of additional markers

BAC sequences between ATSOl91 and DFR on chromosome V (available at
http://www.kazusa.or.jp/arabi/) were compared with sequences of a Lansdberg erecta
random genomic shot gun library available at the Institute for Genomic Research, (TIGR,
Rockville, MD, USA) by multiple FASTA (Pearson and Lipman, 1988) analyses using
do_fasta.pl (kindly provided by Dr. J White, Michigan State University). Sequence

comparisons were used to identify polymorphic regions between Columbia loci and

84

Landsberg erecta loci and PCR primers were designed to amplify these regions. In
situations were a fragment length polymorphism was not available, a restriction site was
identified which would provide the required polymorphism. Using this approach,

polymorphic markers for BACs MUL8, MKM21, K15E6 and MBB18 were developed.

85

Chapter 6

Identification of ORFs and complementation of cvsl

86

INTRODUCTION

Classically mutants were mapped and then cloned by chromosome walking. This
process required the identification of several overlapping YACs (yeast artificial
chromosomes) spanning the two closest RFLP markers (Restriction Fragment Length
Polymorphism) on either side of the mutation. This was achieved by using the RF LP
marker to probe a YAC library and identify YACs that contain the RFLP marker.
Typically, the average distance between two RFLP markers was 400 kbp (Gibson and
Somerville, 1992). This could be covered by about 5 overlapping YACs depending on the
sizes of the inserts. Once the YACs bearing the RF LP markers were identified, the YAC
ends were used once again to identify overlapping YACs until the mutation was spanned.
The YAC ends would also be used as RFLP markers to identify segregants with
chromosomal break points closer to the mutation. This process was repeated until a YAC
was found which covered the mutant locus. The YAC was then subcloned into a library
of cosmids for complementation analysis. Eventually the cosmid that complemented the
mutation was sequenced and the gene identified.

With the sequencing of the Arabidopsis genome, the process of "walking YACs"
has been replaced with in silico operations that identify overlapping BACs spanning the
region that includes the mutation. Previously, mutation cvsI was mapped to a region
covered by BACs: MBB18, MKDlO, K15E6, MXF 12, K3K3 and MUL8 (see Chapter 5).

The analysis of the sequence of this region is presented.

87

RESULTS AND DISCUSSION
Mutant analysis

The mapping analysis revealed that the mutation cvs] lay between markers
ATSOl91 and DFR and more specifically on a small contig of 6 BACs: MBB18, MKDlO,
K15E6, MXF 12, K3K3 and MUL8. Since the polymorphism markers for MBB18 and
MUL8 revealed heterozygous bands in certain segregants, mutation cvsI should lie
"south" of position 25021 on BAC MBB18 and "north" of position 48901 on BAC

MUL8. The length of this region is about 417 kbp (Table 6.1).

Table 6.1 List of BACs that map close to locus cvsI

 

BACs Size (bp)
MBB18 79537
MKD 10 47460
K15E6 71736
MXF 12 66237
K3K3 68889
MUL8 83450
Total Span 417309

 

 

 

 

 

 

 

 

 

 

 

BACs spanning the region between the SSLP marker for BAC MBB18 and BAC MUL8
have been listed along with their sizes (bp).
Complementation analysis

To verify that a mutation has been mapped, it is essential to complement the
mutation by transforming the mutant with a transgene that encodes the wild type gene.
The loss of the mutant phenotype in the primary transformant (in case of a recessive
mutation) will indicate complementation. Such an event has to be verified by following

the segregation pattern in the selfed progeny (T2) to the primary transformant and ensure

88

that the loss of the mutant phenotype segregates with the complementing transgene in the
T2 lines (Donnann et al, 1999).

As BAC K15E6 (Liu et al, 1999) was available in a plant transformation vector,
mutant cvsl has been transformed with clone K15E6 using Agrobacterium-mediated
transformation (Bent et a1, 1994). Primary transformants have been selected on
hygromycin media. Results from this experiment are awaited. Complementation with
BAC K15E6 should indicate if the mutation lies on that BAC. However, this step is not
easy to accomplish. The transformation efficiency of plants is low. Once, this step has
been accomplished, the BAC will be subcloned into cosmid vectors and these will be
used for transforming mutant cvsl. Upon identifying a complementing cosmid clone, the
corresponding region will be amplified out of cvsl genomic DNA by PCR amplification
and sequenced. A comparison of the cvsl sequence to that of the wild type Columbia
sequence should indicate the site of the mutation. The gene can then be cloned from a

root specific cDNA library and this clone used to complement the mutant once again.

ORF Analysis

A parallel strategy is based on the sequence of the Arabidopsis genome. The
sequencing of the genome has revealed a lot of information about genes present in the
genome and their putative functions. Using this information, putative proteins encoded by
open reading frames (ORF) as annotated by the Kazusa DNA Research Institute and The
Institute for Genomic Research (TIGR) have been listed in Appendix C: Table C.l (BAC

MBB18), Table C.2 (BAC MKDIO), Table C.3 (BAC K15E6), Table C.4 (BAC

89

MXF12), Table C.5 (BAC K3K3) and Table C.6 (BAC MUL8). A number of candidate

genes appear to have potential roles in the secretory pathway (Table 6.2). This locus

Table 6.2 List of genes neighboring cvsl

 

 

 

 

 

 

 

 

 

 

 

Clone(s) Similarity
K15E6.14,K15E6.16,K15E6.18,
K15E6.20,K3K3.2,K3K3.4 Oxalate oxidase (germin protein)-like protein
MXF12.13,1\IIXF12.14,I\D(F12.15,
MXF12.16,MXF12.18,K3K3.6 Germin-like protein
K15E6. 19 Similar to oxalate oxidase
K15E6.6 Disease resistance protein-like
Similar to heat shock transcription factor HSF
MBB18.12,MBB18.14 30
Myrosinase binding protein-like, similar to
MBB18.7,MBB18.8 'asmonate induced protein
MKD10.5 Similar to Salt-inducible protein
MUL8.2 AHP3
MUL8.2] v-SNARE AtVTIla

 

 

 

List of genes present on the BAC contig spanning MBB18 to MUL8 encoding proteins
with a potential role in the secretory pathway. Annotation performed by the Kazusa DNA
Research Institute (Chiba, Japan) and The Institute for Genomic Research, (TIGR,
Rockville, MD, USA). See Appendix C for a detailed list of ORFs present on the BACs.

appears to encode many stress related proteins including germin-like proteins, HSF 305,
myrosinase-binding protein-like, salt-inducible proteins and signal transduction
components. The only known secretory pathway protein in this locus is the soluble N-

ethyl maleirnide sensitive factor adaptor protein receptor (SNARE) AtVTIla (Zheng et al,

19991

Germins
Germins are a family proteins typically found in monocots such as wheat and

barley. They are similar to desiccation tolerant vacuolar proteins such as vicilin and

90

legumin. They have been found to have oxalate oxidase and super oxide dismutase
activity (Gane et a1, 1998; Woo et al, 2000). They are induced during wheat seed
maturation and germination and have been implicated in pathogen defense and resistance

to abiotic stresses such as heat, salt, submergence and aluminium toxicity.

Myrosinase-binding protein

Myrosinase binding protein (MBP) is a protein that binds to myrosinase and
activates the enzyme to hydrolyze glucosinolates. Glucosinolates are a group of
thioglucosides found in crucifer species that are broken down into glucose, sulfate, and
nitriles, isothiocyanates, thiocyanates, or epithionitriles depending on the species. These
catabolites are highly toxic to insect and fungal pathogens (Geshi and Brandt, 1998).
MBP is a vacuolar protein that has a putative ctVSS and is induced by wounding and by

jasmonic acid.

Heat Shock induced factor 30 (HSF30)

HSF 30 belongs to a group of heat shock induced transcription factors first
identified in tomato (Scharf et al, 1990). These have an N-terminal DNA-binding domain
and form dimers or trimers by the interaction of their leucine zipper motifs. The C-
terrninal domains are not conserved and are implicated in specificity. Scharf et a1 (1990)
demonstrated that the DNA-binding domain interacts with the heat stress consensus

element.

91

AHP3

The sequence of ORF MUL8.2 is similar to AHP3. AHP3 is one of three proteins
identified with a histidine phosphotransfer domain (Suzuki et al, 1998). This protein is
implicated in the signal transduction pathway involving ETRI (Chang et al, 1993), a plant
sensor His kinase, and a response regulator such as ARR (Irnamura et al, 1998). AHP3 is

postulated to be a signal transducer in this pathway.

AtVTIla

While the above described proteins have a role in stress responses, disease
resistance and development, they do not have a clearly defined role in the secretory
pathway. The only gene in this BAC contig with a known role in the plant secretory
pathway is AtVTIla (Sanderfoot and Raikhel, 1998; Zheng et al, 1999; Bassham et al,
2000). This protein is a homologue of the yeast SNARE Vtilp (Fischer von Mollard and
Stevens, 1999; Tishgarten et al, 1999; Antonin et a1, 2000; Sato et a1, 2000). In plants,
AtVTIla is implicated in vesicle traffic. By subcellular fractionation studies and
immunoelectron microscopy AtVTIla has been found to be co-localized with the ssVSS
receptor, AtELP, at the TGN and with the prevacuolar compartment syntaxin AtSYP21
(Zheng et al, 1999). Its role in protein transport has not been clearly demonstrated but its
association with other proteins known to be involved in the secretory pathway such as
AtELP and AtSYP21 makes it likely to have a role in the secretory pathway.

Since cvsI is a root specific mutation, it was interesting to speculate that this

protein must be preferentially expressed in roots. Expression analyses by Zheng et a1

92

(1999) revealed that AtVTIla message was most abundant in roots. Also, the TIGR
database shows that AtVTIla is found in root cDNA libraries. Complementation analysis

and RT-PCR analysis of cvsl root mRNA should reveal if the cvsl mutation corresponds

to AtVTIla.

ORF analysis summary

Proteins possessing ctVSS are predominantly ancillary vacuolar storage proteins
(Marty, 1999). They have been found to be important against salt-stress, pathogen attack
and insect attack. It is interesting to note that a locus potentially encoding a protein
involved in the sorting of ctVSS-bearing proteins should also encode proteins involved in
defense against salt and heat stress and pathogen attack. Considering that Arabidopsis
chitinase is induced by ethylene (Samac et al, 1990), it is interesting that an ethylene
signal transduction factor, AHP3, is encoded by this locus. The most promising candidate
in this locus is AtVTIla, a plant SNARE, which has been co-localized with other

secretory pathway proteins.

CONCLUSION

The isolation of mutants impaired in ctVSS mediated protein transport to the
vacuole has generated a collection of mutants, of which cvsl was characterized and
mapped. The mutant was narrowed down to a contig of BACs that include heat and salt
stress proteins, pathogen and insect defense proteins, an ethylene signal transduction
factor and AtVTIla, a SNARE implicated in plant vesicle formation. Further studies

including complementation with BACs, followed by complementation with cosmids

93

derived from a complementing BAC should help in cloning cvsl. In parallel, sequencing
the locus in the region of AtVTIla should reveal if cvsl is really AtVTIla. If cvsl is
found to be AtVTIla this will be the first major protein identified in the ctVSS-mediated

transport pathway.

MATERIALS AND METHODS
Complementation Analysis

Clone K15E6 (Liu et al, 1999) was electroporated into Agrobacterium tumefaciens
GV3101 pMP90 and selected for transformation. This clone was used in subsequent steps
for plant transformation. Line ACWS (cvsl back-crossed and self fertilized, see Chapter 5)
was germinated on vertical media plates and stained with X-GlcU. ACWS plants were
transformed by vacuum infiltration with K15E6 (Agro) according to Bent et al., (1994).
Seeds (K15E6-Tl) were harvested and selected by germination on media containing 12.5

mg/l hygromycin and 250 mg/L carbenicillin.

94

Chapter 7

Conclusions and Future

95

CONCLUSIONS

Vacuolar proteins have been the focus of plant biology since the 1970s, as they
perform a myriad of functions essential to the plant cell. A major function of vacuolar
proteins is plant defense: defense-related macromolecules are stored in the vacuole to be
released upon wounding or pathogen attack. A number of these defense-related proteins
are ctVSS-bearing proteins. These proteins appear to be induced in the aerial tissues of
plants during wounding or pathogen attack. However, in root tissues, these are
constitutively expressed. This phenomenon might be related to the fact that roots are
continuously exposed to abiotic stresses in the course of developing in the substratum.

Vacuolar proteins are synthesized on the rough endoplasmic reticulum and are co-
translationally inserted into the ER lumen. By vesicle-mediated transport, these proteins
are thought to exit the ER and reach the cis-Golgi. Eventually, via vesicle-mediated
transport, these proteins are delivered to the trans-Golgi network. In this organelle,
vacuolar proteins are sorted away from other secretory proteins and packaged into
vesicles. The cargos of these vesicles reach the vacuole via a prevacuolar compartment
(Jiang and Rogers, 1998; Miller et al, 1999). Proteins such as barley lectin and tobacco
chitinase possess a C-terminally located vacuolar targeting signal (ctVSS). Dombrowski
et a1 (1993) and Neuhaus et a1 (1994) demonstrated that the hydrophobic residues in the
targeting signal are important and that two Gly residues added to the C-terminus of the
ctVSS could abrogate vacuolar sorting. Although glycosylation per se is not necessary for
vacuolar targeting, the addition of a glycosylation site at the C-terminus of the ctVSS
abrogates vacuolar targeting. This property of the glycan is probably due to a steric

hindrance preventing the ctVSS sorting machinery from correctly recognizing the ctVSS.

96

Roos-Runeberg et a1 (1994) have demonstrated that the ctVSS of barley lectin can be
processed by the aspartic proteinase, phytepsin, in vitro. This activity has been postulated
to occur in the vacuole. Pharmacologically, ctVSS-mediated sorting can be inhibited by
the application of 33 M of Wortmannin (an inhibitor of the mammalian phosphatidyl
inositol-3 kinase). In plants, however, the in viva activity of tobacco PI-3 kinase is not
inhibited by 33 M of wortmannin. The missorting of BL or tobacco chitinase in the
presence of wortmannin has been suggested to be due to the inhibition of phospholipid
biosynthesis by the drug.

To summarize the above, a ctVSS is a C-terminally located hydrophobic targeting
motif. This motif losses activity if two Gly residues are added at the C-terminus or a
glycosylation site is introduced at the C-terminus. This pathway is sensitive to
wortmannin. Gal and Raikhel (1994) demonstrated that the ctVSS pathway is unique to
plants in that ctVSS bearing proteins are not targeted to the yeast vacuole.

These studies have revealed the properties of the ctVSS, but very little has been
elucidated regarding proteins interacting with ctVSS-bearing proteins and the machinery
involved in transporting these proteins to the vacuole. Using classical genetics, an attempt
has been made to understand the processes involved in the transport of ctVSS-bearing
proteins by developing a screen for mutants impaired in the transport of ctVSS-bearing
proteins. This screen involved the development of a single locus line homozygous for a
reporter construct encoding barley lectin and rat preputial B-glucuronidase with a ctVSS
(Rat-GUS-ctVSS) from tobacco chitinase. This line was mutagenized and screened for
mutants with a sorting defect. Using an assay designed to visually score for secreted Rat-

GUS activity, several putative mutants were identified. These putative mutants were

97

allowed to self-fertilize and the progeny was scored for secretion. Most of the progeny
were found to have a heritable phenotype. To differentiate between cis-mutations and real
sorting defects, the localization of barley lectin was examined by immunoelectron
microscopy using anti-wheat germ agglutinin antibodies. All the mutants analyzed
accumulated barley lectin in the vacuole. In addition, the majority of the mutants analyzed
accumulated a secreted pool of barley lectin in spaces between adjacent cells and along
the wall in root sections. However in leaf sections, barley lectin was localized exclusively
in the vacuole. These results indicated the identification of root specific mutants, which
displayed a partial mislocalization of BL in the apoplast.

By studying the expression profile of Rat-GUS and BL, mutants were classified
into “over-expressers” and “low-expressers”. The over-expressers were not pursued
further as the mislocalization observed could have arisen due to a saturation of the ctVSS
transport pathway resulting in secretion. One of the low expressers, designated cvsl, was
analyzed further and found to have a single gene recessive defect. cvsl was then mapped
to a contig of BACs between AtSOl91 and DFR on chromosome V. It has been narrowed
down to a stretch of 6 overlapping BACs (MBB18, MKDlO, K15E6, MXF12, K3K3 and
MUL8) covering a span of 417 kbp. Preliminary complementation analysis has been
endeavored with BAC K15E6 and the results are awaited. This contig of BACs encodes,
salt and heat stress proteins, defense related proteins, ethylene signal transduction
proteins and the SNARE AtVTIla.

Taken together, a model has been proposed (Figure 7.1). Proteins bearing a ctVSS

98

 

' Plasma
Membrane

    
  

/c'vs1

4—5
CVS

 

   

Vacuole .

 

 

 

Dans-Go i Network

 

 

Figure 7.1 Model of the ctVSS sorting pathway

Proteins bearing a ctVSS (red) such as barley lectin (black) are sorted and packaged into
vesicles (magenta). These vesicles eventually deliver their cargo to the vacuole (red)
where the ctVSS is proteolytically cleaved by an enzyme such as phytepsin. The factors
involved in this pathway may include several CVS gene products. This pathway can be
blocked in mutants such as cvsl resulting in the secretion (green) of the protein to the
extracellular space.

such as barley lectin, may be sorted at the trans-Golgi network and transported to the
vacuole via a process involving several CVS gene products. In the vacuole, the ctVSS
may be proteolytically cleaved by an enzyme such as phytepsin. This pathway can be
partially blocked in mutants such as cvsl and cvs2 resulting in the secretion of the protein
to the extracellular space, or by drugs such as wortmannin. Although this model
incorporates the implied functions of the mutants characterized, it cannot be confnmed

until the genes encoding these factors are identified and analyzed.

99

THE FUTURE

To proceed finther four broad approaches can be envisaged, a biochemical
approach should be developed to identify proteins interacting with the ctVSS, a genetic
approach to identify proteins involved in the pathway, a cell biological approach to purify
intermediate compartments that accumulate precursor proteins and a bioinformatic

approach using the genomic sequence information available.

Biochemical Approach

Using synthetic peptides representing wild type and mutant ctVSS of barley lectin,
a 66 kDa ctVSS-interacting protein was identified from radish root tissues
(Venkataraman and Raikhel, unpublished). To the best of our knowledge, this was the
first demonstration of a ctVSS-specific interacting protein. Although the identity of the
protein is not known, this approach has demonstrated that in vitro methods can be
pursued to identify interacting factors. Boller and Vogeli (1984) have reported that
tobacco chitinase can accumulate in vacuoles to the extent of 1% of the total leaf protein
if the leaf is treated with ethylene. Mauch and Staehelin (1989) have demonstrated that in
ethylene-treated leaves, chitinase is exclusively localized in the vacuole. Samac et al
(1990) have demonstrated that the Arabidopsis basic chitinase is highly expressed in
ethylene-treated leaves. While the identity of the interacting factor is not known, it is
logical to assume that a tissue that over-expresses a cargo protein and does not saturate
the pathway, should over-express the receptor as well. These evidences suggest that
ethylene-treated leaves may be a suitable source of ctVSS-interacting proteins. The

specificity of the protein can be verified by applying the protein preparation to an ssVSS

100

synthetic peptide matrix or synthetic peptide matrices representing known ctVSSs such as
those of tobacco chitinase, kiwi actinidin and tobacco osmotin. Once a ctVSS-sorting
receptor is characterized, further research should attempt to identify other factors
involved in sorting, by examining factors that interact with this sorting receptor.

Based on the structure of AtELP (Ahmed et al, 1997), it can be postulated that the
ctVSS-interacting factor may have a lumenal ctVSS-interacting domain, a transmembrane
domain and a cytosolic domain for vesicle budding. One can then proceed to identify
factors that interact with the cytosolic domain and use a proteomic approach to

characterize these proteins.

Genetic Approach

The identification of proteins involved in the traffic of ctVSS-bearing proteins
through biochemical means will be limited to factors involved early in post-Golgi traffic.
To identify factors involved in later steps, a mutant approach may be useful. The
approach described in chapters 2 through 6 was helpful in identifying two mutants cvsl
and cvs2. The characterization of cvsl led to the identification of a locus potentially
involved in ctVSS-mediated protein sorting. Considering the large number of proteins
likely to be involved in protein traffic, this mutagenesis screen is clearly not saturated. By
performing a larger screen on the same collection of M2 seeds it will be possible to
identify more loci involved in the pathway. Also by crossing mutants identified, it will be
possible to determine if the mutations are allelic or novel. Considering some of the
technical drawbacks in the screen performed with respect to the use of the double 35$

promoter, it will be better to repeat a mutant screen with a population of mutagenized

101

seeds derived from a homozygous line of BL/Rat-GUS plants where each reporter is
driven by a different promoter such as a single 358 promoter or a NOS promoter. Such an
approach will limit silencing events due to promoter homology and result in a more
robust screen.

To complement a genetic screen, a battery of endogenous reporters will have to be
identified. A number of candidate ctVSS-bearing proteins have been identified in
Arabidopsis such as AtOsmotin (Capelli et al, 1997), myrosinase-binding protein
(Takechi et a1, 1999), vacuolar invertases such as AtFruct3 and AtFruct4 (Haouazine-
Takvorian et al, 1997), and a vacuolar chitinase, AtChib (Samac et al, 1990). However,
the role of the putative ctVSS in these proteins has not been characterized. The
characterization of these proteins will provide endogenous markers to follow the ctVSS
pathway in Arabidopsis thaliana. These markers will prove invaluable in characterizing

mutants identified by a genetic screen.

Cell biology

Currently, no intermediate compartment has been identified containing Golgi
modified ctVSS-bearing precursor proteins. By electron microscopy, barley lectin has
been localized in the Golgi stacks and in the vacuole. However, the intermediate
compartments through which the protein must progress have not been identified. Using a
combination of rate-zonal and isopycnic centrifugation steps, techniques will have to be
optimized to purify membrane-bound organelles which contain precursor proteins.

Certain drugs such as the ionophore, monensin, and nucleotide analogs may be useful in

102

trapping precursor in intermediate compartments or in slowing down the transport

pI'OCCSS.

Bioinformatics

With a large body of sequence information and the recent completion of the
Arabidapsis genome (Walbot, 2000), an in silica design may be used to identify potential
ctVSS pathway proteins. Currently about 30 % of the 25498 predicted gene products have
not been assigned a function (The Arabidapsis Genome Initiative, 2000). These are either
unique to plants or are homologous to unidentified gene products in other organisms.
Considering that the ctVSS pathway is unique to plants, one might start with a subset of
these unknown gene products that are unique to plants and identify proteins which appear
to have a large lumenal domain, a transmembrane domain and a cytosolic domain. Such
an analysis might lead to the identification of novel sorting receptors. Recently,
Sanderfoot et al (2000) have identified a large number of SNARE homologues in
Arabidapsis using sequence information available. Once candidate proteins are identified,
they will have to be verified by examining at their in viva localization and activity. Such
an approach may be useful in the near future when prediction programs become more

accurate .

The exploration of some of these avenues may one day complete the map that was

initiated with the identification of the ctVSS in barley lectin and tobacco chitinase.

103

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114

APPENDIX A

Mapping and Linkage Analysis made simple: A web-based approach
http://www.msu.edu/~venkatal/sslpfull.htm

115

ABSTRACT

With the establishment of molecular genetics resources to study basic
physiological and developmental fimctions in plants, mutant analysis has become
extremely important in understanding the role of a particular gene product. Such studies
involve the design of comprehensive screens to identify mutants defective in a given
function. Once identified, the gene aberrant in function is to be cloned, typically by a
map-based approach. Such an approach requires the evaluation and interpretation of
recombination frequencies between the mutant locus and linked or unlinked markers. A
JavaScript program has been developed to provide scientists all the tools required to
calculate recombination frequencies and mapping distances along with appropriate
statistics. This program is optimized to operate on any operating system and in a

networked manner.

116

INTRODUCTION

In order to determine the genes that code for properties of organisms, mutants
aberrant in a given function are convenient approaches to identify the key players in these
processes. Once a mutant is identified, approaches have to be taken to clone the gene
aberrant in the mutant. The model dicotyledonous plant, Arabidapsis thaliana has been a
model genetic system providing a convenient system to study plant biology.

Historically, mutants have been developed in Arabidapsis thaliana ecotype
Columbia (Col) or Landsberg erecta (Ler). The mutants are then crossed into Ler or Col
respectively for mapping purposes. In the case of non-lethal recessive phenotypes, the
second filial generation of the cross (F2) is screened for the mutant phenotype. Individual
plants expressing the mutant phenotype are used for mapping the mutation using

molecular markers such as CAPS or SSLPs among others.

RESULTS

In order to map mutations using the principle classical genetics with molecular
markers, one often has to calculate various parameters to evaluate the performance of a
particular molecular marker in determining the location of the mutation. Calculations of
recombination frequency (RF) are extremely important in determining the distance
between a marker and the locus of interest. The RF needs to be tested, its deviation from
an unlinked RF evaluated and finally the mapping distance calculated. In the description
that follows we have considered the case of a recessive mutation identified in a
Arabidapsis thaliana ecotype Columbia background and crossed into Arabidapsis

thaliana ecotype Landsberg erecta to develop mapping populations.

117

Recombination Frequency (RF)

The recombination frequency (RF) is the proportion of recombinants
chromosomes observed among the total number chromosomes present. This can be
equated to:
RF= (Het + 2 Ler)/(2 * (Col + Het + Ler))
where Het = number of mapping individuals heterozygous for a particular marker, Col =
number of mapping individuals homozygous for the Columbia allele for a particular
marker and Ler = number of mapping individuals homozygous for the Lansdberg erecta
allele for a particular marker
Standard Deviation of RF

The standard deviation of the RF value can be of interest to give an indication of

the spread of the results. This has been evaluated using ((RF * (1 - RF))/1‘)" 2

where T = Col + Het + Ler

Chi-Square test of goodness of fit of RF

This test allows one to determine whether the observed frequency of marker
alleles deviates from an unlinked situation of 1:2:1 (Col: Het: Ler). Since the Chi-Square
test is performed on the Observed results, in this case the frequency of individuals with
C01, Het, Ler, genotypes at a given locus and the
x2 statistic = 2‘. (O - E)2/e with n-l degrees of freedom (n = 3 in this case).
The xz statistic can be equated to (4 Col 2 + 2 Het 2 + 4 Ler 2 - T2) / T where T = Col +

Ler + Het.with 2 degrees of freedom.

118

For convenience, the observed x2 is compared to theoritical x2 at on = 0.1, 0.05 and 0.01 (
x2 values of 4.605, 5.991 and 9.210 respectively). A null hypothesis of no significant
deviation from an unlinked condition is tested under that above conditions. One can
conclude that the larger the x2 value the greater the deviation of the observed frequency
from an unlinked condition and hence greater indication of linkage between the test locus

and the mutation.

Mapping Distance

During meiosis, a large number of cross-overs occur which can be classified into
different classes such as single, 2, 3, , n cross-overs per meiosis within a particular
region. Since the number of possible cross-overs is very large over the entire chromosome
but small within the region of interest i.e. between loci considered, the probability of a
particular number of cross-overs occurring within a particular distance on a chromosome

follows a Poisson distribution. ..... (1)

Since non-zero number of cross-overs per meiosis, results in a 50 % rate of
recombinants (Stahl, 1969 for review), and a zero cross-over meiosis produces a 0 percent
rate of recombinants, it follows that the probability of obtaining at least one cross-over
within a particular distance, is the summation of the probabilities of obtaining non-zero
number of cross-overs. (2)

This is equivalent to 1- p(0) where p(0) is the probability of obtaining 0 cross-
overs within a particular distance. Since only half of those cross-overs will be

recombinant, the frequency of recombination (RF) = l/:(l-P(0))

119

 

An event following the Poisson distribution can be described by p(i) = (e"‘ xi )/ (i!)
where p(i) is the probability of occurrence of i events and x the average number of events
occurring in the given experiment.

The probability of 0 cross-overs occurring within the 2 loci follows p(i) = (e'x xi )/
(i!) where in this case i = 0 and x is the average number of Cross-overs between the 2
loci.

With i = 0, p(0) = e'x
Subsequently p(at least 1 non-zero Cross-over) = 1- e'X and p(recombinants) = 1/2x p(at
least 1 non-zero Cross-over) = 1/(1 - e").

e" = 1 - x + x2 -x3/3! + x4/41 + ......... (-x)“/n! Ad infinitum
when x << 1, all the exponents of x tend to 0 and can be neglected.
Thus when x <<1, e"= l - x

Therefore p(recombinants) = 1/(1 - e”) = 1/(1 - ( l - x)) = x/2.

If the “distance” between the two loci is defined as d, such that d is proportional
to x and equal to p(recombinants) (in the case of closely linked loci). Then
(1 = x/2 or x = 2d
Therefore p(recombinants) = l/(l - e") = 1/(1 - e'Zd).

p(recombinants) in reality is the recombination frequency or RF.

Thus RF = ‘/(1 - e'Zd)
This can be rearranged to calculate the mapping distance d

d = - ‘/2Ln ( l - 2RF) with (I expressed in Morgans.

120

A more convenient unit is the cM = 1/ 100 Morgans.
Again d = -100/2 Ln (1 - 2RF) = -50 Ln (1 - 2RF), where d is expressed in centiMorgans

(cM).

To summarize, the recombination frequency RF = 1/;1(1- e'd/SO) where d is the map
distance (in cM) between the two loci with a recombination frequency of RF and e is the
base of the natural logarithm and d = -50 Ln (l-2RF) in centiMorgans.

With a large number of samples and with large number of measurements, these
calculations can be cumbersome. We have developed a java script code to perform these
calculations and test the frequencies obtained for all the above parameters and present
them in a fashion useful to the user. Using this code it is possible analyze one’s mapping
data on any operating system (Macintosh or IBM) and using Netscape, or Internet
Explorer the two standard World Wide Web browsers.

This program allows one to go directly from raw mapping data such as counts of
occurrences of Col, Het and Ler allele profiles on electrophoresis gel as in the case of
CAPS and SSLP markers to obtain the final results such as distance of the mutant locus

from marker loci.

To complement these analytical tools and exploit the networked resources, a
number of world-wide web-based tools have also been “linked” to the program. This
program is available at http://www.msu.edu/~venkata1/sslpfull.htrn
Recently Raventos et a1 (2000) reported the use of this program for analyzing linkage

data.

121

REFERENCES
Haldane JBS (1919) J. Genet 8: 299-309.
Koopmans LH (1987) Introduction to contemporary statistical methods (2nd ed).

Koornneef M, Stam P (1992) Genetic Analysis. In Koncz C, Chua N-H and Schell J
(1992) Methods in Arabidapsis research, World Scientific Publishing.

Stahl FW (1969). The Mechanics of Inheritance. 2nd edition, Englewood Cliffs, NJ:
Prentice-Hall. A short introduction to genetics, including some advanced material

presented with a novel approach.

Suzuki DT, Griffiths AJF, Miller JH, Lewontin RC (1989) An introduction to genetic
analysis. (4th ed)

122

HTML code for sslpfull.htrn
<.HTML><.HEAD><.T1TLE>PCReactor <./TITLE>
<.SCRIPT>function PCR (Intro, NR,Prim,B10,prog)
{
if (BIO == "Sep0")

{

BlOX = NR * 2

B 10Xreal = Math.ceil (B 10X)

K20 = "10 X PCR Buffer = " + B10Xreal + " ul "
MG = NR * 0.6

MGreal = Math.ceil (MG)

K30 = "50 mM MgC12 = " + MGreal + " ul "

taq = NR * 0.4

Primer = NR " 0.25

dntp = NR "‘ 0.25

dd = (NR *19)-(BlOX + MG + taq + Primer + Primer + dntp)
ddreal = Math.ceil(dd)

}
if (BIO = "Sep")

{

BlOX = NR * 2

B l 0Xreal = Math.ceil (B 10X)

K20 = "10 X PCR Buffer == " + BlOXreal+ "111 "
MG = NR * 1.2

MGreal = Math.ceil (MG)

K30 = "50 mM MgC12 = " + MGreal + " ul "

taq = NR * 0.4

Primer = NR * 0.25

dntp = NR * 0.25

dd = (NR *19)-(B10X + MG + taq + Primer + Primer + dntp)
ddreal = Math.ceil(dd)

}
if (B10 = "Combo")

{

BlOX = NR * 3.2

B l 0Xreal = Math.ceil (B 10X)

K20 = "6.25 X PCR-Mg Buffer = " + B10Xreal + " ul "
K30 = H N

taq = NR * 0.4

Primer = NR * 0.25

dntp = NR * 0.25

dd = (NR * l9) - ( BlOX + taq + Primer + Primer + dntp)
ddreal = Math.ceil(dd)

}
if (prog == "svss1>4")

123

{

prog = " I Cycling Program = " + prog
Sl= "I Step 1:94 C for 7 min"

82: " I Step 2: 94 C for 30 sec"

S3= " I Step 3: 55 C for 15 sec"

S4= " I Step 4: 72 C for 30 sec"

SS= " I Step 5: Back to Step 2 40 times"
S6= " I Step 6: 72 C for 7 min"

S7= " I Step 7: 4 C forever"

}

if (prog == "SVSSP3")

{

prog = " I Cycling Program = " + prog
Sl= "I Step 1:94Cfor2min"

SZ= " I Step 2: 94 C for 30 sec"

S3= " I Step 3: 55 C for 15 sec"

S4= " I Step 4: 72 C for 30 sec"

SS= " I Step 5: Back to Step 2 40 times"
S6= " I Step 6: 72 C for 7 min"

S7= " I Step 7: 4 C forever"

}

if (prog == "SVSSP5")

{

prog = " I Cycling Program = " + prog
Sl= "I Step 1:94 C for 5 min"

SZ= " I Step 2: 94 C for 1 min"

S3= " I Step 3: 55 C for l min"

S4= " I Step 4: 72 C for 1 rrrin"

SS= " I Step 5: Back to Step 2 40 times"
S6= " I Step 6: 72 C for 7 min"

S7= " I Step 7: 4 C forever"

}

TexSep = Intro + "\n\nName of Primer = " + Prim +

" I Number of reactions = " + NR + "\nWater = " + ddreal
+ " 111" + prog + "\n" + K20 + 81 + "\n" + K30 + S2 +
"\ndNTPs (10 mM each) = " + dntp + " 111" + S3 + "\n" +

Prim + " Forward = " + Primer + " 111" + S4 + "\n" + Prim

+ " Reverse = " + Primer + ',' 111" + SS + "\nTaq Enzyme = "
+taq+" ul"+S6+"\n ----- > 19 ulperRXN" +S7+

"\n+ lul Template DNA"

document.PCRFonn.TextBox.value = TexSep

}
function F indMarker (marker) {

fullQuery = "httpz/lgenome-www3.stanford.edu/cgi-bin/Webdriver

124

?MIval=atdb_locus_max&name=" + escape (marker)
location.href=fullQuery

}

function MasMix () {

location.href = "http://www.msu.edu/~venkata1/sslpmas.htm"

}

function RFcM () {

location.href = "http://www.msu.edu/~venkata1/cminvort.htm"

}

function labl () {

location.href = "http://www.msu.edu/~venkata1/labsslst.htm"

}

function Eckers () {

location.href = "http://genome.bio.upenn.edu/SSLP_info/SSLP.html"

}

function EckerMaps () {

location.href = "http://genome.bio.upenn.edu/SSLP_info/SSLP_map.html"
}

function AraSer O {

location.href = "http://genome-www.stanford.edu/Arabidopsis/AT-arabgen.html"

}
function LocatMarker (Smarker)

{
fulQuery = "http://genome-www3.stanford.edu/cgi-bin/AtDB/RImap?locus=" + escape
(Smarker)
location.href=firlQuery
}
function Chisqhet(Intro,C,H,L,Al)
{
C = C * l
H = H * 1
L = L * 1
T = C + H + L
CHI=((4*C*C)+(2*H*H)+(4*L*L)-(T*T))/T
if (A1 = 0.1)
{CHITAB = 4.605}
if (A1 = 0.05)
{CHITAB = 5.991}
if (A1 = 0.01)
{CHITAB = 9.210}
if (CHI >= CHITAB)
{R = "Failed"}
if (CHI <. CHITAB)

125

{R = "Passed"}
CHISHO = (Math.floor (1000 "' CHI))/1000
document.PCRForm.Hourf.value = Intro + "\n0bserved Segregation
Ratio ofCol to Het to Ler= " + C + "z" + H + "z" + L +
"\nExpected ratio 'of Col : Het : Ler if unlinked is = " + T/4 + "z"
+ T/2 + "z" + T/4 + "\n The Chisquare value is " + CHISHO + "\nand has "
+ R + " the null hypothesis at alpha = " + Al
}
function CalculateLink (Intro, HC, Het, HL) {
N=(HC *1)+(HL*1)+(Het*l)
RF = ((Het * 1) + (HL * 2))/(2 "' N)
M1 = (Math.log(l- (2*RF))) * (-50)
Mrounded = (Math.round (100 * Ml))/100
if (Mrounded <. l)
{Mrounded = "0" + Mrounded}

if (RF >= 0.5)
{Mrounded = "Undefined & Unlinked"}
if (RF >= 0.3)
{Linko = "Not Linked"}
if (RF <. 0.3)

{ Linko = "Linked"}

SD = Math.sqrt((RF * (l - RF))lN)

R2F = RF - SD

M2 = (Math.log(l- (2*R2F))) "‘ (-50)

M2R = (Math.round (100 * M2))/ 100

if (M2R <. 1)

{M2R = "0" + M2R}

SDcM = SD

SDcMR = (Math.round (100 * SDcM))/ 100
if (SDcMR <. 1 )

{SDcMR = "0" + SDcMR}
RF = (Math.round (100 * RF))/100
if (RF <. 1 )

{RF = "O" + RF}
document.PCRForm.result0.value = RF
document.PCRForm.resultl .value = Mrounded
document.PCRForm.result2.value = Linko
document.PCRForm.result3.value = SDcMR
C = HC * 1
H=Ha*l
L = HL * 1
T = C + H + L
CHI=((4"'C*C)+(2*H*H)+(4*L*L)-(T*T))/T
if (CHI >= 9.21)

{R = "failed the null hypothesis at alpha = 0.01"}

126

if ((CHI <. 9.21) && (CHI >= 5.991))
{R = "failed the null hypothesis at alpha = 0.05"}
if ((CHI <. 5.991) && (CHI >= 4.605))
{R = "failed the null hypothesis at alpha = 0.1"}
if (CHI <. 4.605)
{R = "passed the null hypothesis at alpha = 0.1"}
CHISHOT = (Math.floor (1000 * CHI))/1000
CHISHO = CHISHOT
if (CHISHOT <. 1)
CHISHO = "0" + CHISHOT
document.PCRForm.Hourf.value = Intro + "\n\nObserved Segregation Ratio of
Col to Hetto Ler=" +C+":"+H+":"+L+
"\nExpected ratio of Col : Het : Ler if unlinked is = " + T/4 + ":"
+ T/2 + ":" + T/4 + "\nThe Chisquare value is " + CHISHO + "\nand has "
+ R + N." +
"\nThe Recombination frequency is = " + RF + " +/- " + SDcMR + "." +
"\nThe mapping distance is = " + Mrounded + " cM." +
"\nThe lower limit (RF - SD) mapping distance is = " + M2R + " cM."
}
today = new Date ()
document.write ("<.H1>SSLP Package <./Hl><.D"I>This program estimates materials
to be used for a PCR SSLP reaction and the analysis of the results<.DT>
<.F ONT SIZE = 1> A freeware product of Iyer-Venk: Developed by Meera Iyer
and Sridhar Venkataraman<./D'I><./FONT><.HR>Date: " + today)
<./SCRIP'I>
<.BODY>
<.FORM NAME = "PCRForm"><.FONT FACE = "tahoma" SIZE =2><.DT>Details of
the experiment <.IN PUT TYPE = "text" NAME = "ibox" SIZE= 50> <.DT>Number of
standard SSLP - 20 ul Reactions <.INPUT TYPE = "text" NAME = "NumR" SIZE= 3>
Name of Primer <.INPUT TYPE = "text" NAME = "Prim" SIZE= 8>
<.dt>Buffer and Mg
<.SELECT NAME= "Bufl 0Mg">
<.0PTION VALUE = "Sep0">Separate 10 X PCR buffer and MG (1.5 mM
final)<./0PT10N>
<.0PTION VALUE = "Sep">Separate 10 X PCR buffer and MG (3 mM
final)<./0PTION>
<.0PT ION VALUE = "Combo">Combo PCR buffer and MG (6.25 X and 18.75
mM)<./0PT10N>
<./SELECT>
Cyling Program
<.SELECT NAME= "Prog">
<.0PTION VALUE = "SVSSP3">SVSSP3<./0PTION>
<.0PTION VALUE = "SVSSP4">SVSSP4<./0PT10N>
<.0PTION VALUE = "SVSSPS">SVSSP5<./0PT10N>

127

<./SELEC'I>

<.dt><.INPUT TYPE = "button" VALUE = "Polymerize" onClick = " PCR
(document.PCRForm.ibox.value, document.PCRForm.NumR.value,
document.PCRForm.Prim.value,document.PCRForm.Bufl 0Mg.options
[document.PCRForm.Bufl 0Mg.selectedlndex].value,document.PCRForm.Prog.options
[documentPCRForm.Prog.selectedIndex].value)"><./FONT>

<.INPUT TYPE = "button" VALUE = "Master Mixes" onClick = " MasMix 0"

target = "_new_window_"><./FON'l>

<.IN PUT TYPE = "button" VALUE = "Explain" onClick = " F indMarker
(document.PCRForm

.Prim.value)" target = "_new_window_"><./FONT><.INPUT TYPE = "button" VALUE

"Locate on RI Map" onClick = " LocatMarker (document.PCRForm.Prim.value)"
target = "_new_window_"><./FONT>
<.IN PUT TYPE = "button" VALUE = "cM <.=> RF conversions" onClick = " RFcM 0"
target = "_new_window_"><./FON'I>
<.dt><.INPUT TYPE = "button" VALUE = "GoTo Ecker's SSLP page" onClick = "
Eckers()
" target = "_new_window__"><./FONT>
<.IN PUT TYPE = "button" VALUE = "Ecker's SSLP Map page" onClick = " EckerMaps
0
" target = "_new_window_"><./FON'I>
<.INPUT TYPE = "button" VALUE = "Raikhel's SSLPs" onClick = " labl 0" target =
"_new_window__"><./FONT>
<.FONT SIZE =3><.dt><.BR><.textarea name="TextBox" rows=15
cols=70><./textarea><./BR><.D'I>
<.p><.b><.font size = 3> Analysis of Data <./font><./b>
<.dt><.FONT SIZE =3>
Details of the experiment <.INPUT TYPE = "text" NAME = "boxO" SIZE= 50><./dt>
<.DT>Number of homozygous lines in Columbia Ecotype <.INPUT TYPE = "text"
NAME = "HomCol" SIZE= 5> <./D'I>
<.DT>Number of heterozygous lines <.INPUT TYPE = "text" NAME = "Het" SIZE=
15> <./DT>
<.DT>Number of homozygous lines in Lansberg Ecotype <.INPUT TYPE = "text"
NAME =
"HomLer" SIZE= 5> <./DT>
<.dt><.INPUT TYPE = "button" VALUE = "Evaluate" onClick = "CalculateLink
(document.PCRForm.box0.value,document.PCRFonn.HomCol.value,
document.PCRForm.Het.value,
document.PCRForm.HomLer.value)">
<.P>Recombination Frequency (RF) <.INPUT TYPE = "text" NAME = "resultO"
VALUE="" SIZE=15>
<.DT>Standard Deviation in "~RF" <.INPUT TYPE = "text" NAME = "result3"
VALUE="" SIZE=15> <./D'I>
<.DT>Linked (<. 0.3 RF)/ Unlinked (> 0.3 RF) <.INPUT TYPE = "text" NAME =

128

"result2" VALUE="" SIZE=15> <./D'l>
<.DT>Mapping Distance (cM) <.INPUT TYPE = "text" NAME = "resultl" VALUE=""
SIZE=30> <./D'I>
<.BR>
<.textarea name="Hourf" rows=12 cols=70><./textarea><.BR>
<.FONT FACE="Tahoma"><.P>You are visitor no.
<.1MG SRC="http://counter.digits.corn/wc/-d/4/svsslp" ALIGN=middle

WIDTH=60 HEIGHT=20 BORDER=0 HSPACE=4 VSPACE=2>

<.A HREF="http://www.digits.com/"><.FONT
SIZE=2>http://www.digits.com<./FON'I><./A>
<.P> Should you find this program useful in your analysis,
please cite "Sridhar Venkataraman and Natasha V. Raikhel,
Arabidapsis Mapping Package using SSLP and CAPS data, 1999"
<.P><.A HREF="mailtozvenkatal@pilot.msu.edu"><.FONT FACE="Times,Times New
Roman">Please send Comments to Sridhar Venkataraman<./A><./D'I>
<.DT><.A HREF="http://www.msu.edu/~venkatal/index.htm"><.FONT FACE="Times,
Times New Roman">Back to the main page<./A><./DT><./FORM>
<.strong>A search of all messages contained in the BioSci Arabidapsis Genome
mailing or newsgroup.<.lstrong>
<.FORM ACT10N="http://genome-www.stanford.edu/cgi-bin/AtDB/AT-arabgenbiosci
search.cgi" METHOD=POST>
<.TABLE><.TR><.TD><.INPUT NAME="search" VALUE="" size=50><fTD>
<.TD><.INPUT TYPE="image" SRC="http:l/genome-www.stanford.edu/Excite/pictures/
AT-search_button.gif' NAME="searchButton" HEIGHT=20 WIDTH=75 ALT="Search"
BORDER=0><./TD><./TR><./TABLE>
<.IN PUT TYPE="hidden" NAME="sp" VALUE="sp">
<./FORM>
<.FORM NAME= "main" METHOD="POST" ACT10N="http://www.williamstone.com/
primers/calculator/calculator.cgi">
Primer Evaluation at www.williamstone.com

<.dt> <.td><.IN PUT TYPE="text" NAME="fprimer" SIZE=40><./td>

<.td><.SELECT NAME="forient"><.0PTION SELECTED VALUE="0">5' to 3'
<.0PTION VALUE="1">3' to 5'<./SELECT><./td>
<./tr><./table><./font><./td>

<.INPUT TYPE="submit" NAME="nextbutton" VALUE="Eva1uate Primer">
<.IN PUT TYPE="hidden" NAME="next" VALUE="evaluate">
<./FORM>
<./BODY>
<./HTML>

129

APPENDIX B
Bradford Analysis made simple: A web-based approach
http://www.msu.edu/~venkata1/bradfordhsa.htm

130

INTRODUCTION

A key step in protein studies is to quantify the concentration of protein present in
a given solution. A variety of assays are available for determination of protein
concentrations such as the Lowry procedure, the Biuret method, the bicinchonic acid
procedure and the Bradford assay (see Dunn, 1989 for review). The Bradford assay is a
convenient assay to measure concentrations of protein in the range of 200 11ng and
1,400 ug/ml. This assay is based on the binding of Coomassie Brilliant Blue G-250 to
primarily basic (especially arginine) and aromatic amino acid residues (Bradford, 1976).

The reaction can be detected using colorimetrically at 595 nm.

RESULTS AND CONCLUSION

To measure the concentration of a protein sample, the absorption of light at 595
nm is compared to the absorption of known concentrations of human serum albumin
(HSA). To facilitate the calculation of the concentrations an HTML code has been
written. This code carries out a regression analysis to determine the slope and y-intercept
of the standards used in the assay. These results are then used to calculate the
concentration of the test sample.

The use of this program has facilitated the evaluation of protein concentrations in

a convenient manner.

REFERENCES

Bradford MM (1976) A rapid and sensitive method for the quantitation of rnicrogram
quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:
248-54.

131

Dunn MJ (1989) Determination of total protein concentration. In Harris ELV and Angel
S (eds.) Protein purification methods: a practical approach. Oxford University Press,
Oxford, UK. pp 10-17

132

HTML code for Bradford Analysis available at
www.msu.edu/~venkata“bradfordhsa.htm

<.HTML>
<.HEAD>
<.TI'I‘LE>BradFord<./'1‘ITLE>
<.SCRIP'I>function Brad
(Intro,NS,Xl,X2,X3,X4,X5,X6,X7,X8,X9,X10,Yl,Y2,Y3,Y4,Y5,Y6,Y7,Y8,Y9,Y10,
NU,VU,AU,Nl,N2,N3,N4,N5,N6,N7,N8,N9,N10,U1,U2,U3,U4,U5,U6,U7,U8,U9,U10)
{
ARRAYNBIG = new Array(N 1,N2,N3,N4,N5,N6,N7,N8,N9,N10)
ARRAYXBIG = new Array(Xl,X2,X3,X4,X5,X6,X7,X8,X9,X10)
ARRAYYBIG = new Array(Y l,Y2,Y3,Y4,Y5,Y6,Y7,Y8,Y9,Y10)
ARRAYUBIG = new Array(U1,U2,U3,U4,U5,U6,U7,U8,U9,U10)
ARRAYUX = new Array(NU)
SIGX = 0
for (a = 0; a <. NS; a++)
{
SIGX = SIGX + (1 * ARRAYXBIG[a])
}
SIGX2 = 0
for (i=0; i <. NS; i++)
{
SIGX2 = SIGX2 + (ARRAYXBIG[i])*(ARRAYXBIG[i])
}
SIGY = 0
for (b = 0; b <. NS; b++)
{
SIGY = SIGY + (1 * ARRAYYBIG[b])
}
SIGXY = 0
for (j=0; j <. NS; j++)
{
SIGXY = SIGXY + (ARRAYXBIG[j])*(ARRAYYBIG[j])
}
B = (SIGXY - ((SIGX) * (SIGY))/NS)/ (SIGX2 - (SIGX * SIGX)/N S)
A = (SIGY/N S) - (B * SIGX/N S)
for (k=0; k <. NU; k++)
{
ARRAYUX[k] = (Math.round ((ARRAYUBIG[k] - A) * 10000/(B * VU)))/10000
} .
STDTAB = "\n\nStandard's Table" + "\n "
+ "\nIHSA (ug/ul)I OD 595 I" + "\n "
for (p=0; p <. NS; pH)
{

 

 

133

TX = ARRAYXBIG[p]
if (ARRAYXBIG[p] <. 1)
{ TX = "o" + ARRAYXBIG[p]}
TY = ARRAYYBIG[p]
if (ARRAYYBIG[p] <. 1)
{ TY = "o" + ARRAYYBIG[p]}
STDTAB = STDTAB + "\nI " + TX + " I " + TY + n I"

}
UNKTAB = "\n " + "\n\nUnknown's Table" + "\n -----------

+ "\n Sample Vol = " + VU + " 111."
+ "\nISampleI OD 595 | Cone (ug/ul)| Vol (111) for " + AU + " ug protein" + "\n --------------

 

 

 

for (CF-0; q <- NU; q++)
{
TU = ARRAYUBIG[q]
if (ARRAYUBIG[q] <. l)
{ TU = "0" + ARRAYUBIG[q]}
TUX = ARRAYUX[q]
VTUX = (Math.round (10 * AU / TUX))/10
if (ARRAYUX[q] <. l)
{ TUX = "0" + ARRAYUX[q]}
UNKTAB = UNKTAB + "\nI " + ARRAYNBIG[q] + " I " + TU + " I " + TUX + "
l H + VTUX +1! I N

}
document.ecoliForm.Brader.value = Intro + STDTAB + UNKTAB + "\n

 

 

}
today = new Date ()

document.write ("<.H1>Bradford test by Regression Analysis<./Hl><.DT>This program
evaluates protein concentrations based on 0D595<.D'I><.F ONT SIZE = 2>Protocol
<.dt> 1) Add 5 ml Bradford reagent (Bio-Rad 500-0006) to 20 ml H20. <.dt> 2) Start
the Spec and set at CD 595 nm. <.dt> 3) Aliquot 1 ml of the diluted reagent to sample
tubes, add 0,2,4,6,8 and 10 ul of <.dt> 1 mg/ml HSA (BSA can also be used but HSA is
more linear and reproducible) for standards and say 3 ul of protein extract for analysis.
<.dt>4) Quantitate and calculate below <.dt> <.F ONT SIZE = 1> A freeware product of
Iyer-Venk: Developed by Meera Iyer and Sridhar
Venkataraman<./DT><./FON'I><.HR>Date: " + today)

<./SCRIPT>

<.BODY>

<.FORM NAME = "ecoliForm">

<.P><.F ONT FACE="Tahoma" SIZE=3>Details of the experiment <.INPUT TYPE =
"text" NAME = "Intro" SIZE= 50>

<.DT>Number of Standards (including blank) <.INPUT TYPE = "text" NAME = "NoS"
SIZE= 3>

134

<.DT>Standard Blank (0 ug/ul HSA) <.INPUT TYPE = "text" NAME = "SIP" SIZE= 4>
0D <.INPUT TYPE = "text" NAME = "SOD1" SIZE= 6>

<.DT>Standard l (ug/ul HSA)<.INPUT TYPE = "text" NAME = "SZP" SIZE= 4> 0D
<.INPUT TYPE = "text" NAME = "SOD2" SIZE= 6>

<.DT>Standard 2 (ug/ul HSA)<.IN PUT TYPE = "text" NAME = "S3P" SIZE= 4> 0D
<.INPUT TYPE = "text" NAME = "SOD3" SIZE= 6>

<.DT>Standard 3 (ug/ul HSA)<.INPUT TYPE = "text" NAME = "S4P" SIZE= 4> 0D
<.INPUT TYPE = "text" NAME = "SOD4" SIZE= 6>

<.DT>Standard 4 (ug/ul HSA)<.INPUT TYPE = "text" NAME = "SSP" SIZE= 4> 0D
<.INPUT TYPE = "text" NAME = "SODS" SIZE= 6>

<.DT>Standard 5 (ug/ulHSA)<.1NPUT TYPE = "text" NAME = "S6P" SIZE= 4> 0D
<.INPUT TYPE = "text" NAME = "SOD6" SIZE= 6>

<.DT>Standard 6 (ug/ul HSA)<.INPUT TYPE = "text" NAME = "S7P" SIZE= 4> 0D
<.INPUT TYPE = "text" NAME = "SOD7" SIZE= 6>

<.DT>Standard 7 (ug/ul HSA)<.INPUT TYPE = "text" NAME = "S8P" SIZE= 4> 0D
<.INPUT TYPE = "text" NAME = "SOD8" SIZE= 6>

<.DT>Standard 8 (ug/ul HSA)<.INPUT TYPE = "text" NAME = "S9P" SIZE= 4> 0D
<.INPUT TYPE = "text" NAME = "SOD9" SIZE= 6>

<.DT>Standard 9 (ug/ul HSA)<.INPUT TYPE = "text" NAME = "S10P" SIZE= 4> 0D
<.INPUT TYPE = "text" NAME = "SODIO" SIZE= 6>

<.P>

<.DT>Number of Unknowns <.INPUT TYPE = "text" NAME = "NoU" SIZE= 3>
Volume of Unknowns (ul) <.INPUT TYPE = "text" NAME = "VoU" SIZE= 4>
Required amount of Unknowns (ug)<.INPUT TYPE = "text" NAME = "AU" SIZE= 4>
<.DT>Unknown l <.INPUT TYPE = "text" NAME = "UlP" SIZE= S> OD 595
<.INPUT TYPE = "text" NAME = "UOD1" SIZE= 6>

<.DT>Unknown 2 <.INPUT TYPE = "text" NAME = "U2P" SIZE= 5> OD 595
<.INPUT TYPE = "text" NAME = "UOD2" SIZE= 6>

<.DT>Unknown 3 <.INPUT TYPE = "text" NAME = "U3P" SIZE= 5> OD 595
<.INPUT TYPE = "text" NAME = "UOD3" SIZE= 6>

<.DT>Unknown 4 <.INPUT TYPE = "text" NAME = "U4P" SIZE= 5> OD 595
<.INPUT TYPE = "text" NAME = "UOD4" SIZE= 6>

<.DT>Unknown 5 <.INPUT TYPE = "text" NAME = "USP" SIZE= 5> OD 595
<.INPUT TYPE = "text" NAME = "UOD5" SIZE= 6>

<.DT>Unknown 6 <.INPUT TYPE = "text" NAME = "U6P" SIZE= 5> OD 595
<.INPUT TYPE = "text" NAME = "UOD6" SIZE= 6> '
<.DT>Unknown 7 <.INPUT TYPE = "text" NAME = "U7P" SIZE= 5> OD 595
<.INPUT TYPE = "text" NAME = "UOD7" SIZE= 6>

<.DT>Unknown 8 <.INPUT TYPE = "text" NAME = "U8P" SIZE= 5> OD 595
<.INPUT TYPE = "text" NAME = "UOD8" SIZE= 6>

<.DT>Unknown 9 <.INPUT TYPE = "text" NAME = "U9P" SIZE= 5> OD 595
<.INPUT TYPE = "text" NAME = "UOD9" SIZE= 6>

<.DT>Unknown 10 <.INPUT TYPE = "text" NAME = "UlOP" SIZE= 5> OD 595
<.INPUT TYPE = "text" NAME = "UOD10" SIZE= 6>

135

<.P><.INPUT TYPE = "button" VALUE = "Submit" onClick = "Brad
(document.ecoliForm.Intro.value,
document.ecoliForm.NoS.value,document.ecoliForm. S 1P.value,
document.ecoliForm.$2P.value, document.ecoliForm.S3P.value,
document.ecoliForm.S4P.value, document.ecoliForm.SSP.value,
document.ecoliForm.S6P.value, document.ecoliForm.S7P.va1ue,
document.ecoliForm.S8P.value, document.ecoliForm.S9P.value,
document.ecoliForm.S 10P.value, document.ecoliForm. SOD 1 .value,
document.ecoliForm.SOD2.value,document.ecoliForm.SOD3.value,
document.ecoliForm.SOD4.value, document.ecoliForm.SODS.value,
document.ecoliForm.SOD6.value, document.ecoliForm.SOD7.va1ue,
document.ecoliForm.SOD8.value, document.ecoliForm.SOD9.value,
document.ecoliForm. SOD l 0.value,document.ecoliForm.NoU.value,document.ecoliForm.
VoU.value,document.ecoliForm.AU.value,

document.ecoliForm.U 1 P.value, document.ecoliForm.U2P.value,
document.ecoliForm.U3P.value, document.ecoliForm.U4P.value,
document.ecoliForm.USP.value, document.ecoliForm.U6P.value,
document.ecoliForm.U7P.value, document.ecoliForm.U8P.value,
document.ecoliForm.U9P.value, document.ecoliForm.U 10P.value,
document.ecoliF orm.UOD1 .value, document.ecoliForm.UOD2.value,
document.ecoliForm.UOD3.value, document.ecoliForm.UOD4.value,
document.ecoliForm.UODS.value, document.ecoliForm.UOD6.value,
document.ecoliForm.UOD7.value, document.ecoliForm.UOD8.value,
document.ecoliForm.UOD9.value, document.ecoliForm.UOD10.value)">
<./FON"I>

<.BR>

<.textarea name="Brader" rows=50 cols=80 ><./textarea>

<.BR>

<.D'1>

<.P><.A HREF="mailtozvenkata1@pilot.msu.edu"><.FONT FACE="Times,Times New
Roman">Please send Comments to Sridhar Venkataraman<./A><./DT>
<.DT><.A HREF="http://www.msu.edu/~venkata1/index.htm"><.F ONT
FACE="Times,Times New Roman">Back to the main page<./A><./DT>
<./FORM>

<./BODY>

<./HTML>

136

APPENDIX C

137

Table C.1 Genes present on the BAC MBB18 and the proteins encoded

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Clone Similarity
'MBB18.2 [Unknown Protein
MBB 18.3 IUnknown Protein
MBB18.4 IUnknown Protein

BBl8.5 'Unknown Protein
MBB18.6 Tryptophan synthase beta chain

yrosinase binding protein-like, similar to jasmonate induced
'MBB l 8.7 rotein
yrosinase binding protein-like, similar to jasmonate induced
MBB 18.8 rotein
B 18.9 IUnknown Protein

MBB18.10 Isimilar to protein kinase
'MBB18.1 1 nknown Protein
MBBI8.12 Similar to heat shock transcription factor HSF 30
MBB 18.13 IUnknown Protein
[MBB 18.14 irnilar to heat shock transcription factor HSF 30
lMBB18.15 nknown Protein

BB 18.16 IUnknown Protein

BB18.17 DS-box protein-like
lMBB18.18 Cytochrome B-S6l
MBB18.19 Similar to guanine nucleotide exchange factor

BB 1 8.20 nknown Protein
MBB 1 8.21 [Unknown Protein
MBB18.22 IUnknown Protein
MBBl8.23 [Unknown Protein
MBBI8.24 [Unknown Protein
MBB18.25 Similar to non-LTR retroelement reverse transcriptase

 

 

 

Annotation by the Kazusa DNA Research Institute (Chiba, Japan) and The Institute for
Genomic Research, (TIGR, Rockville, MD, USA).

138

Table C.2 Genes present on the BAC MKD10 and the proteins encoded

 

 

Clone imilarity

'MKD10.2 nknown Protein

MKD10.3 IProline oxidase precursor

MKD10.4 IUnknown Protein

[MKD10.5 Similar to Salt-inducible protein

IMKD10.6 [Unknown Protein

IMKD10.8 IUnknown Protein

IMKD10.9 Similar to pollen coat protein

IM S-adenosyl methioninezsalycylic acid
KD10. 10 carboxlrnethyltranferase-like protein

IMKD10.11 [Unknown Protein

IMKD10.12 JUnknown Protein

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Annotation by the Kazusa DNA Research Institute (Chiba, Japan) and The Institute for
Genomic Research, (TIGR, Rockville, MD, USA).

139

Table C.3 Genes present on the BAC K15E6 and the proteins encoded

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

blone Similarity

K15E6.1 Similar to bZIP transcription factor

K15E6.2 Unknown Protein

K15E6.3 Amino acid transporter-like

Kl 5E6.4 Cysteine-tRN A 1i gase

K15E6.5 Unknown Protein

K15E6.6 IDisease resistance protein-like

K15E6.7 DNA-binding protein-like

K15E6.8 Unknown Protein

K15E6.9 Unknown Protein

K15E6.10 Unknown Protein

K15E6.11 Unknown Protein

K15E6. 12 FmE protein-like

K15E6.13 TRNA-IlefiAT)

K15E6. l4 xalate oxidase (gemrin proteinL-like protein
K15E6.15 Similar to non-LTR retroelement reverse transcriptase
K15E6. l6 Oxalate oxidase (gerrnin protein)-like protein
K15E6.17 Similar to retroelement pol

K15E6. 18 Oxalate oxidase (germin protein)-like protein
K15E6.19 Similar to oxalate oxidase

K15E6.20 Oxalate oxidase (germin protein)-like protein
K15E6.21 Cytochrome P-450

K15E6.22 Similar to retroelement pol

K15E6.23 nknown Protein

K15E6.24 Receptor protein kinase-like protein

 

 

Annotation by the Kazusa DNA Research Institute (Chiba, Japan) and The Institute for
Genomic Research, (TIGR, Rockville, MD, USA).

140

Table C.4 Genes present on the BAC MXF12 and the proteins encoded

12.2
12.3
12.4
12.5
12.6
12.7
12.8
12.9
12.10
12.11
12.12
12.13
12.14
12.15
12.16
12.17
12.18
12.19

 

-1ike ' kinase
nknown Protein
' kinase-like
' kinase-like
-like
ltransferease-like

imilar to Ac-like
' like
ltransferease-like
imilar to retroelement 1

like
like
nknown Protein
like '
' kinase-like

Annotation by the Kazusa DNA Research Institute (Chiba, Japan) and The Institute for
Genomic Research, (TIGR, Rockville, MD, USA).

141

Table C.5 Genes present on the BAC K3K3 and the proteins encoded

K3.2 oxidase

K33 nknown Protein
K3.4 oxidase

K3.5 ' ' to retroelement
K3.6 ' '
K3.7 Protein
K3.8 nknown Protein
K3.9 nknown Protein
K3.10 Protein

K3.] 1 Protein

K3.12 nknown Protein
K3. 1 3 nknown Protein
K3.l4 Protein

K3.15 LTR retroelement reverse
K3. 1 6 nknown Protein

K3. 1 7 ' ' '

K3. 1 8

K3 . 19 ' like

K3.20 ' like

K3.2] ' like

K3.22 ' like

K3.23 P

 

Annotation by the Kazusa DNA Research Institute (Chiba, Japan) and The Institute for
Genomic Research, (TIGR, Rockville, MD, USA).

142

Table C.6 Genes present on the BAC MUL8 and the proteins encoded

Protein

Protein
imilar to S60 S-locus
Protein
kinase-like
imilar to '
nknown Protein

bokr'os'uth'wiv;

lo

.10

.11 nknown Protein

.12 ' ll

.13 nknown Protein

.14 nknown Protein

.15 imilar to CHP-rich Zinc F '
.16 imilar to CHP-rich Zinc F'
.17 nknown Protein

.18 nknown Protein

.19 nknown Protein

.20 formation

.21 -SNARE At-VTIla

.22 '

.23 nknown

.24 P K3.23

 

Annotation by the Kazusa DNA Research Institute (Chiba, Japan) and The Institute for
Genomic Research, (TIGR, Rockville, MD, USA).

143

 

   

    

IIIIIIIIIIIIIIIIIIII

IlIllI

BIIIIIIJIZQIOM