Analysis of the Function of Cathepsin B As a Regulator of Plant Programmed Cell Death

ANALYSIS OF THE FUNCTION OF CATHEPSIN B AS A REGULATOR OF PLANT PROGRAMMED CELL DEATH

A thesis submitted to The University of Manchester for the degree of PhD in the Faculty of Life Science

2013

Yao-Min Cai Faculty of Life Science

DECLARATION

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ABSTRACT

Thesis title: Analysis of the function of cathepsin B as a regulator of plant programmed cell death.

Candidate’s Name: Yao-Min Cai

Degree Title: Degree of Doctor of Philosophy in Faculty of Life Science, The University of Manchester

Date: 2/7/2013

Programmed cell death (PCD) plays critical roles not only when plants are challenged with biotic or abiotic stresses, but also in developmental processes. The molecular mechanism for plant PCD has not been fully identified. A conserved activity, caspase-3- like activity, has been reported in several cases of plant PCD. In this thesis, I present research on the role of cathepsin B, a cysteine protease possessing caspase-3-like activity in Arabidopsis, in PCD. The investigation of the function of cathepsin B in ER stress- induced PCD, using cathepsin B mutant Arabidopsis lines, suggested it positively regulates ER stress-induced PCD. Surprisingly, another protease possessing caspase-3- like activity, PBA1, exhibited a suppression role in ER stress-induced PCD. Several further investigations on the molecular mechanisms in ER stress-induced PCD indicated that lacking cathepsin B reduced misfolded protein accumulation by increasing Unfolded Protein Response (UPR) gene expression. I also analysed which PCD type was mediated by cathepsin B by examining several morphological and biochemical markers, and found that cathepsin B mediated vacuolar PCD in ER stress-induced PCD, while necrotic PCD was induced in the absence of cathepsin B. In order to position cathepsin B in the ER stress-induced PCD pathway, I analysed cathepsin B in mutant lines for several genes. A putative pathway was formed in which Mitogen activated protein kinase 6 (MPK6) positively and Arabidopsis BAX inhibitor-1 (AtBI-1) negatively regulated cathepsin B. In addition to ER stress-induced PCD, I also examined the function of cathepsin B in developmental PCD. Cathepsin B exhibited a positive role in a PCD occurring in cells of seed inner integument layers but not in the PCD during xylem formation. Moreover, a novel PCD was discovered in micropylar endosperm during germination. The micropylar endosperm PCD implicated cathepsin B. Finally, a positive role of cathepsin B in KOD- induced PCD was confirmed, and how cathepsin B could be involved in KOD-induced PCD was analysed via DNA microarray.

In summary, cathepsin B had a central role in regulating ER stress-induced PCD and development-induced PCD.

TABLE OF CONTENTS

DECLARATION ...... 2 ABSTRACT ...... 3 TABLE OF CONTENTS ...... 4 FIGURE LIST ...... 7 TABLE LIST ...... 12 ABBREVIATION ...... 13 CHAPTER 1 GENERAL INTRODUCTION ...... 15

1.1 WHAT IS PROGRAMMED CELL DEATH? ...... 15 1.2 CLASSIFICATION OF PROGRAMMED CELL DEATH ...... 15 1.2.1 Animal apoptosis ...... 15 1.2.2 Animal Autophagic Cell Death ...... 15 1.2.3 Animal Programmed necrosis ...... 16 1.2.4 Plant PCD classification ...... 17 1.3 FUNCTION OF PROGRAMMED CELL DEATH IN PLANTS ...... 18 1.3.1 PCD in plant development processes ...... 18 1.3.2 PCD in stress responses...... 20 1.4 ER STRESS-INDUCED PCD...... 21 1.5 PLANT "CASPASE" CANDIDATES AND THEIR ROLES IN PROGRAMMED CELL DEATH ...... 24 1.5.1 Metacaspase ...... 25 1.5.2 Vacuolar processing enzyme (VPE) ...... 28 1.5.3 Saspase ...... 29 1.5.4 Phytaspase ...... 30 1.5.5 Proteasome subunit β1 ...... 30 1.5.6 Introduction to cathepsin B ...... 30 1.6 PROJECT AIM AND OBJECTIVES ...... 32 1.6.1 Aim...... 32 1.6.2 Objectives ...... 32 CHAPTER 2 MATERIAL AND METHOD ...... 34

2.1 MATERIAL ...... 34 2.1.1 Plant material ...... 34 2.1.2 Plasmid material ...... 34 2.2 PLANT BASED EXPERIMENTS ...... 36 2.2.1 Plant growth condition ...... 36 2.2.2 Genotyping ...... 36 2.2.3 Comparison of seed germination rates...... 37 2.2.4 Protoplasts isolation ...... 37 2.2.5 Yariv reagent treatment ...... 38 2.2.6 Tunicamycin treatment ...... 38 2.2.7 Measuring Ion leakage ...... 38 2.3 DNA AND RNA BASED EXPERIMENTS ...... 39 2.3.1 Fusion-PCR ...... 39 2.3.2 DNA digestion and ligation ...... 40 2.3.3 DNA sequencing ...... 41 2.3.4 RNA isolation ...... 41 2.3.5 Quantitative Real Time-PCR (QRT-PCR) ...... 42 2.3.6 Semi-quantitative RT-PCR ...... 43 2.3.7 Agarose Gel electrophoresis ...... 43 4

2.4 E. COLI TRANSFORMATION ...... 44 2.5 PLASMID AMPLIFICATION AND PURIFICATION ...... 44 2.6 PLANT TRANSFORMATION AND TRANSFECTION ...... 45 2.6.1Preparing Agrobacterium tumefaciens (GV3101 pMP90RK) competent cells ...... 45 2.6.2 Plasmid transformation into Agrobacterium tumefaciens ...... 45 2.6.3 Agrobacterium-mediated transformation of Arabidopsis ...... 46 2.6.4 Arabidopsis seedling transfection ...... 46 2.6.5 Transfection of tobacco leaves ...... 47 2.6.6 Biolistic particle bombardment ...... 48 2.7 BIOCHEMISTRY EXPERIMENTS ...... 48 2.7.1 Analysis of caspase-3-like activity ...... 48 2.7.2 Analysis of 20S proteasome activity ...... 49 2.7.3 Activity labelling by Biotin-DEVD-FMK...... 49 2.7.4 Measuring ATP level ...... 50 2.7.5 Separation of mitochondria for cytochrome c release analysis ...... 50 2.7.6 SDS-PAGE and western blot ...... 50 2.8 HISTOLOGY EXPERIMENTS ...... 51 2.8.1 Detection of PCD by dye exclusion staining ...... 51 2.8.2 Staining the vacuole with BCECF ...... 52 2.8.3 Detection of ROS accumulation ...... 52 2.8.4 Microscopy ...... 52 2.8.5 GUS staining ...... 53 2.8.6 Toluidine blue staining Arabidopsis xylem section ...... 53 2.8.7 Measurement of integument layer thickness ...... 53 2.9 BIOINFORMATICS ANALYSIS ...... 54 2.9.1 Microarray analysis ...... 54 2.9.2 Orthologue idetification using blast ...... 54 CHAPTER 3 ANALYSIS OF THE ROLE OF CATHEPSIN B IN ER STRESS- INDUCED PCD ...... 55

3.1 INTRODUCTION ...... 55 3.2 RESULTS ...... 56 3.2.1 is Cathepsin B involved in ER stress-induced PCD? ...... 56 3.2.2 Analysis of cathepsin B paralogues redundancy ...... 60 3.2.3 Analysis of Cathepsin B expression in ER stress ...... 62 3.2.4 Analysis of the role of PBA1 in ER stress-induced PCD ...... 64 3.2.5 The function of proteasome α subunit in ER stress-induced PCD ...... 68 3.2.6 Effect of Blocking proteasome activity on cathepsin B activity ...... 70 3.3 DISCUSSION ...... 71 CHAPTER 4 MODE OF ACTION OF CATHEPSIN B IN ER STRESS-INDUCED PCD ...... 74

4.1 INTRODUCTION ...... 74 4.2 RESULTS ...... 75 4.2.1 Which PCD type is mediated by cathepsin B? ...... 75 4.2.2 Does cathepsin B control tonoplast rupture during vacuolar PCD? ...... 79 4.2.3 Analysis of the subcellular localisation of cathepsin B ...... 82 4.2.4 Does VPE control the activation of cathepsin B? ...... 87 4.3 DISCUSSION ...... 90 CHAPTER 5 FITTING CATHEPSIN B INTO THE ER STRESS-INDUCED PCD PATHWAY ...... 92 5

5.1 INTRODUCTION ...... 92 5.2 RESULTS ...... 94 5.2.1 Does cathepsin B deficiency affect the UPR? ...... 94 5.2.2 Analysis of the signal cascade order between BAX inhibitor and cathepsin B ...... 98 5.2.3 Analysis of the signal cascade order between Mitogen-activated protein kinase 6 and cathepsin B ...... 102 5.2.4 Role of NAC transcription factor in ER stress-induced PCD ...... 105 5.2.5 Investigation of the role of Arabidopsis G protein β subunit (AGB1) in regulating cathepsin B activity in ER STRESS-induced PCD...... 109 5.2.6 Cytochrome c behavior in cathepsin B triple mutant background in ER stress-induced PCD .... 110 5.3 DISCUSSION ...... 112 CHAPTER 6. CATHEPSIN B AND DEVELOPMENTAL PCD...... 115

6.1 INTRODUCTION ...... 115 6.2 RESULTS ...... 117 6.2.1 Effect of cathepsin B mutant on xylem formation ...... 117 6.2.2 Effect of cathepsin B mutant on PCD of inner integument cells ...... 118 6.2.3 Effect of the cathepsin B mutant on seed germination ...... 119 6.2.4 Programmed cell death occurred before micropylar endosperm rupture ...... 121 6.3 DISCUSSION ...... 130 CHAPTER 7 ANALYSIS OF CATHEPSIN B AND KOD-INDUCED PCD PATHWAY ...... 132

7.1 INTRODUCTION ...... 132 7.2 RESULTS ...... 134 7.2.1 Function of cathepsin B in KOD-induced PCD ...... 134 7.2.2 Test of DEX-KOD inducible system ...... 137 7.2.3 Microarray analysis after three hour DEX induction ...... 140 7.2.4 Microarray analysis after five hour DEX induction ...... 144 7.3 DISCUSSION ...... 155 CHAPTER 8 GENERAL CONCLUSION AND DISCUSSION ...... 157

8.1 CONTRASTING ROLE OF CATHEPSIN B AND PBA1 ...... 157 8.2 DO CATHEPSIN B AND THE PROTEASOME INTERACT WITH EACH OTHER? ...... 158 8.3 ARE THERE TWO PCD TYPES INDUCED BY ER STRESS-INDUCED PCD? ...... 159 8.4 CATHEPSIN B IS PART OF THE ER STRESS-INDUCED PCD SIGNAL PATHWAY ...... 160 8.5 CATHEPSIN B MEDIATES THE KOD-INDUCED PCD PATHWAY ...... 162 8.6 CATHEPSIN B IN DEVELOPMENTAL PCD ...... 163 8.7 FUTURE WORK ...... 163 REFERENCE ...... 166 APPENDIX GENE LIST OF FIVE HOUR KOD INDUCTION ...... 176

Word count: 56,854 words

FIGURE LIST

FIGURE 1.1 ER STRESS SIGNAL PATHWAY IN PLANTS ...... 22 FIGURE 1.2 ER STRESS-INDUCED PCD PATHWAYS IN PLANTS ...... 24 FIGURE 1.3. DIAGRAM OF METACASPASES AND CASPASES...... 25 FIGURE 2.1. PXCS- ATCATHB3ΔC::MRFP-HASTREP PLASMID DIAGRAM. 35 FIGURE 3.1 ION LEAKAGES OF ER STRESS-INDUCED PCD IN COL-0 AND ATCATHB#62 ...... 57 FIGURE 3.2 YELLOWING OF LEAVES AND ER STRESS-INDUCED PCD IN COL-0 AND ATCATHB#62...... 57 FIGURE 3.3 COMPARISON OF ER STRESS-INDUCED PCD IN COL-0 AND ATCATHB#62 PROTOPLASTS ...... 58 FIGURE 3.4. DEFINING SEEDLING GROWTH STATUSES DURING ER STRESS...... 58 FIGURE 3.5. COMPARISON OF COL-0 AND CATHEPSIN B TRIPLE MUTANT SEEDLINGS GROWN UNDER ER STRESS...... 59 FIGURE 3.6 TM INDUCED ION LEAKAGES ON ATCATHB3 OVEREXPRESSION LINES...... 60 FIGURE 3.7 CATHEPSIN B GENE REDUNDANCY IN ARABIDOPSIS SEEDLINGS UNDER ER STRESS...... 61 FIGURE 3.8 TM INDUCED ION LEAKAGES IN LEAVES OF COL-0 AND DOUBLE KNOCK OUT CATHEPSIN B LINES...... 62 FIGURE 3.9 CATHEPSIN B ACTIVITY LABELLING AFTER TM TREATMENT IN WILD TYPE AND CATHEPSIN B TRIPLE MUTANT LINE...... 63 FIGURE 3.10. INDUCTION OF CATHEPSIN B TRANSCRIPTS IN ER STRESS...... 64 FIGURE 3.11 CATHEPSIN B AND PBA1 CONTRIBUTION TO CASPASE-3- LIKE ACTIVITY DURING ER STRESS-INDUCED PCD THREE DAYS AFTER INFILTRATION...... 65 FIGURE 3.12. EFFECT OF ADDING THE INHIBITOR Β-LACTONE TO TM INDUCED ION LEAKAGES...... 66 FIGURE 3.13. Β-LACTONE EFFICIENCY OF INHIBITION OF PROTEASOME ACTIVITY IN VIVO...... 66 FIGURE 3.14. INCREASED ION LEAKAGES AFTER TM INFILTRATION OF PBA1 SILENCING LINES...... 68 FIGURE 3.15 COMPARISON TM INDUCED ION LEAKAGES FROM COL-0 AND PAF2-6...... 69

FIGURE 3.16. TM INDUCED CASPASE-3-LIKE ACTIVITY IN COL-0 AND PAF2-6 LEAVES AT THREE DAYS POST INFILTRATION ...... 69 FIGURE 3.17 EFFECT OF THE INHIBITOR Β-LACTONE ON BIOTIN-DEVD- FMK ACTIVITY LABELLING ...... 70 FIGURE 4.1. MORPHOLOGY OF COL-0 AND ATCATHB#62 DEAD CELLS AFTER TM TREATMENT...... 76 FIGURE 4.2. ATP LEVEL IN COL-0 AND ATCATHB#62 CELLS DURING ER STRESS-INDUCED PCD...... 77 FIGURE 4.3. ROS ACCUMULATION IN COL-0 AND ATCATHB#62 ARABIDOPSIS LEAVES DURING ER STRESS-INDUCED PCD...... 78 FIGURE 4.4. YARIV-INDUCED NECROTIC PCD IN LEAVES...... 78 FIGURE 4.5. YARIV REAGENT-INDUCED ION LEAKAGE IN COL-0 AND ATCATHB#62 LEAVES ON DAY THREE...... 79 FIGURE 4.6. TONOPLAST RUPTURE IN COL-0 AND ATCATHB#62 CELLS DURING ER STRESS-INDUCED PCD...... 80 FIGURE 4.7. VACUOLE INTEGRITY IN COL-0 AND ATCATHB#62 USING BCECF STAINING AFTER TM TREATMENT...... 82 FIGURE 4.8. A DIAGRAM THE ATCATHB3::MRFP CONSTRUCTS USED...... 83 FIGURE 4.9. SUBCELLULAR LOCALISATION OF ATCATHB3::MRFP IN ARABIDOPSIS...... 84 FIGURE 4.10. SUBCELLULAR LOCALISATION OF ATCATHB3::MRFP IN NICOTIANA BENTHAMIANA...... 84 FIGURE 4.11 (LEFT PANEL). DETECTION OF ATCATHB3::MRFP BY WESTERN BLOT...... 85 FIGURE 4.11 (RIGHT PANEL). DETECTION OF ATCATHB3ΔC::MRFP BY WESTERN BLOT ...... 85 FIGURE 4.12. SUBCELLULAR LOCALISATION OF ATCATHB3ΔC::MRFP IN ARABIDOPSIS...... 86 FIGURE 4.13. SUBCELLULAR LOCALISATION OF ATCATHB3ΔC::MRFP IN NICOTIANA BENTHAMIANA...... 87 FIGURE 4.14. CASPASE-3-LIKE ACTIVITY IN VPE NULL DURING ER STRESS-INDUCED PCD...... 88 FIGURE 4.15. ACTIVITY LABELLING OF CATHEPSIN B IN COL-0 AND VPE NULL...... 88 FIGURE 4.16. ION LEAKAGES OF COL-0 AND VPE NULL LINE DURING ER STRESS-INDUCED PCD...... 89 FIGURE 5.1. UBIQUITINATED PROTEIN ACCUMULATION IN COL-0 AND ATCATHB#62 DURING ER STRESS...... 95

FIGURE 5.2. 20S PROTEASOME ACTIVITY IN COL-0 AND ATCATHB#62 UNDER ER STRESS...... 96 FIGURE 5.3. TRANSCRIPT LEVELS OF UPR MARKER GENES AT EARLY STAGES OF ER STRESS IN COL-0 AND ATCATHB#62...... 97 FIGURE 5.4. BZIP60 SPLICING PROFILE BETWEEN COL-0 AND ATCATHB#62 DURING ER STRESS...... 98 FIGURE 5.5. ION LEAKAGES OF COL-0 AND ATBI-1 OX2 LINE IN ER STRESS-INDUCED PCD...... 99 FIGURE 5.6. CASPASE-3-LIKE ACTIVITIES COMPOSITION IN COL-0 AND ATBI-1 OX BACKGROUNDS DURING ER STRESS-INDUCED PCD...... 100 FIGURE 5.7. AFFINITY LABELLING OF CATHEPSIN B IN COL-0 AND ATBI-1 OX UNDER ER-STRESS...... 101 FIGURE 5.8. CATHEPSIN B TRANSCRIPT LEVEL IN COL-0 AND ATBI-1 OX IN ER STRESS-INDUCED PCD...... 102 FIGURE 5.9. ER STRESS-INDUCED CASPASE-3-LIKE ACTIVITY IN WS AND MPK6-1BACKGROUNDS...... 103 FIGURE 5.10. CATHEPSIN B ACTIVITY LABELLING IN WS AND MPK6-1 DURING ER STRESS-INDUCED PCD...... 104 FIGURE 5.11. ION LEAKAGES OF WS AND MPK6-1 DURING ER STRESS- INDUCED PCD...... 105 FIGURE 5.12. SEQUENCES ALIGNMENT BETWEEN ATNAC036 AND GMNAC6...... 106 FIGURE 5.13. CASPASE-3-LIKE ACTIVITIES IN COL-0 AND NAC TRANSCRIPTION FACTORS MUTANT LINES AFTER TM TREATMENT. .. 107 FIGURE 5.14 ION LEAKAGES OF COL-0 AND DIFFERENT NAC TRANSCRIPTION FACTORS MUTANT LINES UNDER TM TREATMENT. .. 108 FIGURE 5.15. CASPASE-3-LIKE ACTIVITY IN AGB1 BACKGROUND DURING ER STRESS-INDUCED PCD...... 109 FIGURE 5.16. CATHEPSIN B ACTIVITY LABELLING IN COL-0 AND AGB1 DURING ER-STRESS INDUCED PCD...... 110 FIGURE 5.17. CYTOCHROME C RELEASE FROM MITOCHONDRIA IN COL- 0 AND ATCATHB#62 PROTOPLASTS DURING ER STRESS-INDUCED PCD. 111 FIGURE 6.1. STEM SECTIONS OF COL-0 AND ATCATHB#62...... 117 FIGURE 6.2. INNER INTEGUMENT LAYER THICKNESS OF COL-0 AND ATCATHB#62 SEEDS...... 118 FIGURE 6.3. COMPARISON OF INTEGUMENT CELL LAYER THICKNESS BETWEEN COL-0 AND ATCATHB#62 SEEDS...... 119

FIGURE 6.4. ILLUSTRATION OF FOUR ARABIDOPSIS SEEDS GERMINATION STAGES...... 120 FIGURE 6.5. COMPARISON OF GERMINATION RATES BETWEEN COL-0 AND ATCATHB#62...... 121 FIGURE 6.6. PROGRAMMED CELL DEATH IN MICROPYLAR ENDOSPERM CELLS DURING GERMINATION...... 122 FIGURE 6.7. ROS ACCUMULATION IN MICROPYLAR ENDOSPERM DURING GERMINATION...... 123 FIGURE 6.8. COMPARISON OF MICROPYLAR ENDOSPERM PCD BETWEEN COL-0 AND ATCATHB#62 SEEDS...... 124 FIGURE 6.9. PCD OF COL-0 AND ATCATHB#62 SEEDS MICROPYLAR ENDOSPERM CELLS AT STAGE III...... 125 FIGURE 6.10. RATIOS OF MICROPYLAR ENDOSPERM PCD IN COL-0 AND ATCATHB#62 SEEDS AT STAGE III...... 125 FIGURE 6.11. ABA REDUCED COL-0 SEED GERMINATION RATE...... 126 FIGURE 6.12. PCD OF ABA TREATED COL-0 SEEDS MICROPYLAR ENDOSPERM CELLS AT STAGE II AND III...... 127 FIGURE 6.13. NOVEL CAP LIKE STRUCTURE ON ABA TREATED SEEDLING RADICLE ...... 128 FIGURE 6.14. COMPARISON OF THE PERCENTAGE OF ROOT TIPS WITH OR WITHOUT CAP-LIKE STRUCTURE IN ABA TREATED AND UNTREATED SEEDS...... 128 FIGURE 6.15. COMPARISON OF GERMINATION RATES BETWEEN COL-0 AND VPE NULL SEEDS...... 129 FIGURE 6.16. COMPARISON OF ME PCD IN COL-0 AND VPE NULL SEEDS...... 129 FIGURE 7.1. KOD PEPTIDE INDUCED PCD IN ARABIDOPSIS PROTOPLASTS...... 135 FIGURE 7.2. EFFECT OF THE PROTEASE INHIBITORS DEVD-FMK AND CA074ME ON KOD- INDUCED PCD...... 135 FIGURE 7.3. KOD PEPTIDE INDUCED PCD IN CATHEPSIN B DEFICIENT PROTOPLASTS...... 136 FIGURE 7.4. CATHEPSIN B TRANSCRIPT LEVEL DURING KOD PEPTIDE INDUCED PCD...... 137 FIGURE 7.5. A DIAGRAM OF THE DEX-KOD INDUCIBLE SYSTEM...... 138 FIGURE 7.6 DEX INDUCTION OF KOD EXPRESSION ACTIVATED PCD IN SEEDLINGS...... 138

FIGURE 7.7. GUS EXPRESSION PROFILE UNDER THE CONTROL OF THE DEX INDUCIBLE SYSTEM...... 139 FIGURE 7.8. GO ANALYSIS OF GENES INDUCED BY THREE HOURS DEX TREATMENT...... 142 FIGURE 7.9. THE INTERACTION NETWORK OF RAV1 AND TGA1 (BZIP47)...... 144 FIGURE 7.10. GENE ONTOLOGY ANALYSIS OF THE GENE LIST FOR THE FIVE-HOUR DEX TREATMENT...... 148 FIGURE 7.11 VENN DIAGRAM OF THE KOD INDUCED GENES WITH PSEUDOMONAS SYRINGAE DC3000 INDUCED GENES ...... 148 FIGURE 7.12 VENN DIAGRAM OF KOD INDUCED GENES AND FLG22 INDUCED GENES...... 149 FIGURE 7.13. INTERACTIONS SUGGESTED AMONG THE KOD-INDUCED GENES AFTER FIVE HOURS DEX TREATMENT...... 151 TABLE 7.5. POTENTIAL METABOLISM PATHWAY IN KOD INDUCED GENES AT FIVE HOURS...... 151 FIGURE 7.14 GENE ONTOLOGY ANALYSIS FOR THE GENES DOWN- REGULATION AT FIVE HOURS (NEXT PAGE)...... 152 FIGURE 8.1 POTENTIAL CATHEPSIN B MEDIATING SIGNAL PATHWAY OF ER STRESS-INDUCED PCD...... 161 FIGURE 8.2. PUTATIVE KOD-INDUCED PCD SIGNAL PATHWAY...... 162

TABLE LIST

TABLE 1.1. CRITERION OF DIFFERENT TYPES OF PCD (REDRAWN FROM VAN DOORN 2011) ...... 18 TABLE 2.1 ARABIDOPSIS MUTANT LINES PURCHASED FROM NASC ...... 34 TABLE 2.2. PRIMERS FOR MAKING ATCATHB3ΔC::MRFP FUSION SEQUENCE...... 35 TABLE 2.3 PRIMERS USED FOR QRT-PCR ...... 42 TABLE 2.4 PRIMERS USED FOR SEMI-QUANTITATIVE RT-PCR ...... 43 TABLE 7.1 GENE LIST AT THE TWO-FOLD CHANGE THRESHOLD AT THREE HOURS DEX TREATMENT...... 140 TABLE 7.2 TRANSCRIPTION FACTORS (TFS) PREDICTION FOR THE REGULATION OF THE UP-REGULATED GENES IN THREE HOUR DEX TREATMENT ...... 143 TABLE 7.3 GO OF GENES INDUCED BY KOD AND AVRRPM1 INDUCED GENE LISTS...... 149 TABLE 7.4. GO OF GENES COMMON TO THE DEX-KOD AND FLG22 GENE LIST...... 150 TABLE 7.6. POTENTIAL METABOLISM PATHWAY IDENTIFIED IN KOD REPRESSED GENES AT FIVE HOURS...... 152

ABBREVIATION

Agro agrobacterium

AGB1 arabidopsis G protein β subunit 1

BAX BCL-2-associated protein X

BCECF-AM 2′,7′-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester

BCL B cell pymphoma

BSA bovine serum albumin

CPA cyclopiazonic acid

DCFDA 2’,7’ –dichlorofluorescein diacetate

DEVD Asp-Glu-Val-Asp

E. coli Escherichia coli

EDTA ethylenediamine tetraacetic acid

ER endosplamic reticulum

FR Phe-Arg

GFP Green fluorescent protein

GO gene ontology

GUS β-glucronidase

Hsc heat shock cognate

IETD Ile-Glu-Thr-Asp

KOD Kiss-of-Death peptide

MAPK mitogen activated protein kinase

MEFs mouse embryonic fibrablasts

NAC NAM, ATAF, CUC domain

NRP N-Rich Protein

ORF open reading frame

PAF6 20 S proteasome subunit α 6

PAGE poly acrylamide gel electrophoresis

PARP poly(ADP-ribose) polymerase 13

PBA1 20S proteasome subunit β 1

PCD programmed cell death

PCR polymerase chain reaction

PEMs pro-embryogenic masses

PMSF phenymethanesulfonylfluoride

RFP red fluorescent protein

RIP Receptor-Interaction Protein

ROS reactive oxygen species

RTase reverse transcriptase

SDS sodium dodecyl sulfate

T-DNA transfer DNA

TMV tobacco mosaic virus

TNF tumour necrotic factor

TUNEL terminal deoxynucleotidyl transferase mediated dutp nick end labelling

UPR unfolded protein response

UV ultraviolet

VEID Val-Glu-Ile-Asp

VPE vacuolar processing enzyme

YVAD Try-Val-Ala-Asp

Chapter 1 General introduction

1.1 What is Programmed Cell Death?

Programmed Cell Death (PCD) is usually considered as a self-destruction process in cells. In contrast to the physical destruction of cells, the essence of PCD is to be genetically controlled. PCD was used as early as 1964 in a paper written by Lockshin and Williams (Lockshin and Williams, 1964). Decades after that research on programmed cell death, the mechanisms of its regulation have been widely investigated in both animals and plants.

1.2 Classification of Programmed Cell Death

Various morphological and biochemical alterations at the cellular level have been observed during programmed cell death. Several authors have tried to define different types of programmed cell death, according to their morphological and biochemical features. To avoid confusion and miscommunication, three major types of programmed cell death were originally defined in animal biology, they are: apoptosis, autophagic cell death and programmed necrosis (Galluzzi et al., 2012; Kroemer et al., 2009).

1.2.1 Animal apoptosis

Apoptosis is the classical cell death type which is often described in the literature. It is usually accompanied by several morphological changes, such as chromatin condensation, nuclear fragmentation, cell shrinkage and plasma membrane blebbing (reviewed in Kroemer et al., 2009). In terms of biochemical markers, activation of caspases, a group of cysteine proteases which exhibit specific substrate cleavage activities, has long been considered as a marker of apoptosis (Yuan and Horvitz, 2004; Danial and Korsmeyer, 2004). 1.2.2 Animal Autophagic Cell Death

Autophagic cell death is a second type of programmed cell death where the cell is capable of degradation of its own cellular components using lysosomes (Tsujimoto and Shimizu, 2005). People sometimes confuse autophagic cell death and autophagy. The latter is often

15 referred to as a protein and organelle turnover process in eukaryotic cells. Autophagy includes three major forms: macroautophagy, microautophagy and chaperone-mediated autophagy. In macroautophagy, unwanted proteins or organelles are wrapped by a double membrane vesicle to form an autophagosome. The autophagosome will dock onto the lysosome and release its content into it for degradation. In microautophagy, proteins or organelles are imported into the lysosome without forming autophagosome. In chaperone- mediated autophagy, proteins or organelles can be recognized by the Heat shock cognate 71 kDa protein (Hsc70) and form a complex. This complex will go to the lysosome and release its cargo into it (Stromhaug and Klionsky, 2001; Bandyopadhyay et al., 2008). Autophagy does not necessarily end up with cell death (Tsujimoto and Shimizu, 2005; Kroemer et al., 2009). In many situations, autophagy acts as a pro-survival process. For example in mammalian cells, withdrawal of the cell growth factor IL-3 was able to induce autophagosome (characterised as a double membrane vesicle) formation which promoted nutrient recycling within the cell and maintained cell viability (Lum et al., 2005). In autophagic cell death, the formation of autophagosome is also induced. This is observed in Bax/Bak-deficient mouse embryonic fibroblasts (MEFs) cells. When these cells were treated with cytotoxic agents, such as etoposide, autophagosomes emerged followed by PCD (Shimizu et al., 2004). Taken together, autophagy is both involved in cell survival and cell death. But a question is how to tell apart the cell survival effect of autophagy from its cell death effect. Shimizu and colleagues proposed that the protein expression level of autophagy proteins ATG5 and ATG6 could be used as markers to distinguish pro- survival autophagy from autophagic cell death. This was because ATG5 and ATG6 maintained at low level when autophagy did not lead to cell death, whereas ATG5 and ATG6 increased during autophagic cell death in mouse embryonic fibroblasts cells (Shimizu et al., 2004).

1.2.3 Animal Programmed necrosis

Programmed necrosis is a new proposed concept. Necrotic PCD or necrosis has long been considered as an uncontrolled process. This prevalent idea was not challenged until recent discoveries suggested that Tumour Necrotic Factor (TNF)-induced necrosis was also genetically regulated (Moquin and Chan, 2010; Zhang et al., 2009; He et al., 2009). A key regulator of programmed necrosis, Receptor-Interaction Protein 3 (RIP3), was also discovered (He et al., 2009). It was shown that TNF-induced apoptosis was not affected in RIP3-/- mutant of MEFs, but RIP3-/- MEFs cells were resistant to TNF-induced programmed necrosis (He et al., 2009). In contrast to apoptosis, programmed necrosis is 16 independent of caspase activation. Indeed it has been shown that Fas, a member of the TNF family, could induce programmed necrosis in primary T cells without activating caspases (Holler et al., 2000).

1.2.4 Plant PCD classification

The classification of plant PCD in different types lags behind its counterpart in animal model. A Consensus was not established until year 2011, although dispute over some details is still going on (Van Doorn et al., 2011; van Doorn, 2011). So far, apoptosis has not been observed in plants (though "apoptotic-like programmed cell death" has been used) and it could be due to the fact that plants have a cell wall that prevents a dead cell to be engulfed by phagocyte (which does not exist in plants either). Based on recent reports, plant PCD can be sub-divided into two major types: vacuolar PCD and necrotic PCD (Van Doorn et al., 2011). In vacuolar PCD, vacuole membrane rupture is followed by the vanishing of most of the cell content (Van Doorn et al., 2011). Although "vacuole membrane rupture" is included in the definition of vacuolar PCD, this collapse of the tonoplast cannot be used as a single marker for vacuolar PCD, because in some situations, tonoplast rupture is not equal to cell death. For example, the disappearance of the vacuole membrane in sieve elements did not lead sieve elements cells to death (Wang et al., 2008). Rapid cleaning of cells contents should always be observed in vacuolar PCD, and considered as a major morphological feature to designate a PCD type as vacuolar PCD (van Doorn, 2011).

In necrotic PCD, some cell constituents are still left in cells after PCD is completed (Van Doorn et al., 2011). Usually, protoplast shrinkage occurred during this type of PCD (Van Doorn, 2011).

In addition a few biochemical features are employed to distinguish different cell death types. For instance, accumulation of Reactive Oxygen Species (ROS) and drop of the intracellular ATP level are thought to be markers for necrotic PCD (Van Doorn et al., 2011). Table 1.1 lists some morphological and biochemical features to distinguish vacuolar PCD and necrotic PCD.

Table 1.1. Criterion of different types of PCD (redrawn from Van Doorn 2011)

In practice, PCD in given conditions can sometimes show mixed features of both vacuolar PCD and necrotic PCD. For example, in the PCD detected in incompatible pollen, the PCD exhibited some features of vacuolar PCD, such as rearrangement of the actin cytoskeleton and the rupture of tonoplast (Bosch and Franklin-Tong, 2008). Meanwhile, cells organelle swelling, which belongs to the necrotic PCD markers, was also observed (Geitmann et al., 2004; Bosch and Franklin-Tong, 2008).

1.3 Function of programmed cell death in plants

Generally speaking, PCD takes part in two major aspects of plant physiology processes: plant development processes and stress responses. PCD brings various consequences to plants in different situations. Several examples of PCD in plants, which are relevant to this thesis, are presented in the following section.

1.3.1 PCD in plant development processes

PCD can be observed at various stages throughout the whole life of a plant. At an early stage of the life of a plant example includes somatic embryogenesis where PCD is involved in somatic embryogenesis transition and suspensor cells degeneration in Norway spruce cells (Filonova et al., 2000). The somatic embryogenesis of Norway spruce cells is characterized by two major phases: a proliferation of pro-embryogenic masses (PEMs)

18 phase and a somatic embryo phase (Filonova et al., 2000). PEMs were composed of two types of cells, small dense cytoplasmic cells and enlarged highly vacuolated cells (Filonova et al., 2000). The second type of PEMs cells were permeable to Evan’s blue, which is a dye for detecting dead cells (Filonova et al., 2000). PCD of PEMs was shown to correlate to the emergence of somatic embryos. An increased DNA fragmentation in PEMs detected by TUNEL assay, a classical cell death assay, was accompanied by increased somatic embryo formation (Filonova et al., 2000). Moreover, the inhibition of VEIDase activity, a protease activity involved in PCD, blocked embryo/suspensor differentiation in Norway spruce cells (Bozhkov et al., 2004). Linked to this, PCD in Arabidopsis suspensor cells has already been reported for a long time (Jones and Dangl, 1996). A recent report supported the idea that PCD occurred in Arabidopsis suspensor cells (Blanvillain et al., 2011). Blanvillain and colleagues used a promoter trap approach to identify a peptide, Kiss-of-Death (KOD expressed in suspensor cells during embryogenesis that was an inducer of PCD (Blanvillain et al., 2011).

Following embryogenesis, a next important stage where PCD also participates is seed development and germination. In Arabidopsis seed development, the nuclei of Arabidopsis seed coat inner integument layer 2 (ii2) and 3 (ii3) were degraded between the globular stage and the heart stage of embryogenesis. This nucleus degradation is considered as a sign of PCD occurring in inner integument layer cells (Nakaune et al., 2005). PCD in ii2 and ii3 results in thin integument layers whereas blocking ii2 and ii3 PCD by knocking out its regulator δ Vacuolar Processing Enzyme (VPE) maintained integument layer thickness (Nakaune et al., 2005). Another instance of PCD was reported in seed germination. During the germination of cereal grains, some hydrolytic enzymes are believed to be released out of the aleurone cells via PCD, to break down endosperm reserves for embryo growth (Young and Gallie, 2000; Fath et al., 2000).

One remarkable example of PCD involvement in post-embryo stages is in xylem formation. Xylem is a critical tissue for plants that confers plants with "woody" characteristic and is responsible for fluid conducting. Xylem consists of three different tissues: xylem parenchyma, xylem fibres and tracheary elements (TEs). TE differentiation exhibits several hallmarks of PCD. For instance, DNA fragmentation was detected in the xylem of Pisum sativum using TUNEL assay (Mittler and Lam, 1995). Moreover, it has been observed that xylem cell content was largely cleared after tonoplast rupture in Zinnia TEs (Kuriyama, 1999). In xylem fibres, cells content was cleaned up during transition

19 from cambium cells to xylem fibre, which indicated that PCD occurred in xylem fibre as well (Courtois-Moreau et al., 2009; Bollhöner et al., 2012).

In the last stage of plant life, PCD manifests itself by participating in senescence. Senescence is a critical physiological process for plants. It takes place in fruit ripening, leaf ageing and petal withering. A study of senescence in leaves of several species, including Philodendron hastatum, Epipremnum aureum, Bauhinia purpurea, Delonix regia, and Butea monosperma, showed that DNA fragmentation, detected via gel electrophoresis and TUNEL assay, occurred during senescence (Yen and Yang, 1998). This strongly indicated that PCD was involved in senescence. The function of PCD in senescence is believed to participate to the remobilisation of nutrient from old organs or tissues to new organs or tissues (Uauy et al., 2006; Van Doorn and Woltering, 2004).

1.3.2 PCD in stress responses

Besides plant developmental processes, PCD also participates in plant responses to biotic and abiotic stresses. When plants are challenged with pathogen, plants can activate the Hypersensitive Response (HR) to defend against the pathogen invasion (Mur et al., 2008; Coll et al., 2011). HR includes the activation of a localised PCD. For example, Arabidopsis Col-0 leaves inoculated with Pseudomonas syringae DC3000 rpm1 stained positive for trypan blue, indicating that Arabidopsis leaves infected with this pathogen elicited PCD (McLellan et al., 2009). PCD has long been considered as a strategy to restrict pathogen growth and spreading during HR. However, several recent reports indicated that blocking PCD during HR did not promote pathogen growth suggesting it was not strictly required for confining pathogen spreading (Király et al., 1972; Coll et al., 2010).

In abiotic stresses, PCD also was shown to play a role. For instance, during drought stress, Arabidopsis seedling roots were shown to stop growing. These roots stained positive for trypan blue (Duan et al., 2010). Similarly in barley roots, salt stress was able to induce DNA degradation in meristematic cells (Katsuhara and Kawasaki, 1996). These are two examples where PCD may play a role in modifying root architecture in response to stress. In both cases, it was suggested that cells experienced a prolonged ER stress that was the trigger to PCD.

1.4 ER stress-induced PCD.

The Endoplasmic Reticulum (ER) is an essential organelle within the cell. Major functions of ER are protein translation, folding, modification and secretion. During translation at the surface of the ER, nascent proteins are modified by the addition of oligosaccharides to their Asparagine (N) residues. This process is called N-glycosylation. N-glycosylation is key to protein fates. Improper glycosylation results in misfolded proteins, which are subject to ER associated degradation (ERAD). However, if the bulk of unfolded proteins remain in the ER lumen, it can cause ER stress and a prolonged ER stress eventually results in PCD in animal cells (Hetz, 2012). A similar situation was also observed in plants (Liu and Howell, 2010).

The signal transduction from sensing misfolded protein accumulation to executing PCD is a complex network. Based on current knowledge, scientists are able to outline an ER stress signal pathway in plants, although not all components have been identified (Iwata and Koizumi, 2012; Liu and Howell, 2010). ER stress signal transduction starts from sensing the aggregation of misfolded proteins in the ER lumen. In Arabidopsis, three ER membrane resident transmembrane proteins, Inositol-Requiring Enzyme 1 (IRE1), basic leucine zipper 28 (bZIP28) transcription factor and basic leucine zipper 17 (bZIP17) transcription factor are reported to sense aggregation of misfolded proteins in the ER lumen (Liu et al., 2007a, 2007b; Liu and Howell, 2010; Iwata and Koizumi, 2012). IRE1 spans the ER membrane with its luminal sensing domain inside the ER lumen and its phosphorylation kinase and ribonuclease domains on the cytoplasm side (Iwata and Koizumi, 2012). The molecular mechanism, by which IRE1 senses misfolded proteins in plants, has not been reported as far as I know. But research on plant IRE1 counterpart in yeast may help understand how IRE1 senses misfolded protein accumulation. At low level of misfolded proteins accumulation, a chaperone protein, BIP, binds to IRE1 and inactivates it. When the level of misfolded protein increases, BIP dissociates from IRE1 to bind misfolded proteins and this results in IRE1 activation. Active IRE1 forms a dimer and activates its ribonuclease activity (Pincus et al., 2010; Shamu and Walter, 1996), which then activates downstream signals. In Arabidopsis, the active IRE1 splices the bZIP60 mRNA. Spliced bZIP60 mRNA does not code for a transmembrane domain anymore. This results in a spliced form of the bZIP60 protein, which translocates to the nucleus, promoting expression of Unfolded Protein Response (UPR) genes (Deng et al., 2011b). A second ER stress sensor in Arabidopsis is bZIP28. bZIP28 resides on the ER

21 membrane. Upon sensing misfolded protein accumulation in the ER lumen, the C- terminal of bZIP28 is removed via proteolysis, which result in the translocation of bZIP28ΔC to the nucleus where it forms a complex with Nuclear Factor-Y (NF-Y) subunits (Liu et al., 2007; Liu and Howell, 2010). This complex subsequently binds to ER stress-responsive elements (ERSE) to promote expressions of some of the UPR genes (Liu and Howell, 2010). The third potential ER stress sensor in Arabidopsis is bZIP17. The activation of bZIP17 is similar to that of bZIP28. bZIP17 localises on the ER membrane and can be processed by a subtilisin-like serine protease (AtS1P). The processed bZIP17 translocates to the nucleus to activate downstream genes (Liu et al. 2007). Although the mode of action of bZIP17 was discovered in a salt stress context, two clues correlate the bZIP17 signal pathway to the ER stress signal pathway. First, The subcellular localisation and mode of activation of bZIP17 resemble that of bZIP28; Second, bZIP17 is processed by Arabidopsis serine-1-protease (AtSIP) whose counterpart in animal cells is regulating the ER stress signal pathway by cleaving Activating Transcription Factor 6 (ATF6) (Walter and Ron, 2011; Liu and Howell, 2010). Figure 1.1 presents a current model of signal transduction from sensing stress to inducing UPR in Arabidopsis.

Figure 1.1 ER stress signal pathway in plants A model of the signal transduction pathway of ER stress in Arabidopsis is drawn here. Three transmembrane proteins IRE1 (blue domain and red domain), bZIP28 and bZIP17 (green domain and yellow domain) are localized on the ER membrane. During ER stress, the RNase domain of IRE1 (red) is activated and cleaves the bZIP60 mRNA (bZIP60u) to give a spliced form mRNA (bZIP60s). bZIP60s translates to a protein which subsequently translocates to the nucleus to activate UPR-related genes. bZIP17 is processed by AtS1P (brown-orange) to remove its C- terminus (yellow) and then relocates to the nucleus. Whether bZIP17 activates UPR genes is not clear (indicated by dash line). bZIP28 c-terminal (yellow) is also removed during ER stress causing relocation to the nucleus to promote UPR gene expression. The protease responsible for 22 bZIP28 cleavage is unclear. It is assumed that AtS1P and AtS2P (brown-orange) may be responsible for bZIP28 processing.

At the early stage of ER stress, cells elicit UPR to rescue cells from ER stress. However, when ER stress persists, cells may enter a PCD program. More and more researches imply that ER stress-induced PCD is not only a simple response to the aggregation of misfolded proteins in plants at the cellular level, but is also a part of important plant physiology processes. For instance, ER stress triggered PCD can mediate the colonization of Piriformospora indica in Arabidopsis roots (Qiang et al., 2012). Colonization by Piriformospora indica induced ER stress as indicated by the ER swelling observed and in addition PCD was detected in cells in contact with Piriformospora indica. A close link between ER stress and PCD is supported by the increased colonisation of Piriformospora indica onto an ER stress hypersensitive Arabidopsis line (bip2 mutant line) (Qiang et al., 2012). Compared to the signal transduction from sensing ER-stress to UPR induction, the signal transduction from prolonged ER stress to PCD is not so clear. So far, several regulators have been found to modulate ER stress-induced PCD in plants (Figure 1.2). Arabidopsis Bax inhibitor-1 (AtBI-1) is a conserved PCD repressor in both animal and plant cells (Shimizu et al., 2004; Yue et al., 2012). The role of AtBI-1 in ER stress-induced PCD was reported in Arabidopsis. Overexpression of AtBI-1 reduced ER stress-induced PCD in Arabidopsis, while the AtBI-1 knock-out line was more sensitive to ER stress-induced PCD (Watanabe and Lam, 2008, 2006). A second ER stress-induced PCD regulator belongs to the N-Rich Proteins (NRPs) and was found in Glycine max (Costa et al., 2008). Treating Glycine max with tunicamycin (Tm), an ER stress inducer, induced NRPs transcript level. Overexpression of NRP-A and B promoted PCD in Glycine max protoplast and Nicotiana tabacum leaves (Costa et al., 2008). Thirdly, a recent report identified a NAC (NAM, ATAF1, ATAF2 and CUC2) domain protein, GmNAC6, downstream of NRPs in the ER stress-induced PCD pathway in Glycine max (Faria et al., 2011). NRP-A and B were found to activate the GmNAC6 promoter in Glycine max (Faria et al., 2011). In turn, overexpression of GmNAC6 induced PCD in Nicotiana tabacum leaves and Glycine max cells. A final regulator is the G protein β subunit 1 (AGB1) in Arabidopsis (Wang et al., 2007). Wang et al (2007) reported that Arabidopsis AGB1 knock out (agb1) seedlings survived better under ER stress than wild type. By contrast, Chen and Brandizzi (2012) observed that agb1 seedlings were however more sensitive to ER stress when grown on MS medium containing Tm (despite same condition as that in Wang et al., 2007 report) (Chen and Brandizzi, 2012; Wang et al., 2007). Whether AGB1

23 plays a positive or a negative role in PCD is therefore unclear now.

Besides the proteins mentioned above, an induction of caspase-3-like activity has been observed at least twice in the context of ER stress-induced PCD. In Glycine max, overexpression of NRP-A/B and NAC could induce caspase-3-like activity (Costa et la., 2008; Faria et al., 2011). Treatment of soybean suspension cells with an ER stress inducer, cyclopiazonic acid (CPA), increased caspase-3-like activity (Zuppini et al., 2004). These reports implied that proteases responsible for caspase-3-like activity are regulators of ER stress-induced PCD.

Figure 1.2 ER stress-induced PCD pathways in plants A current model of ER stress-induced PCD pathways in plants is presented above. Three major signal streams are found and marked with different colours (green, yellow and blue). Arabidopsis Bax inhibitor-1 represses ER stress-induced PCD by inhibiting an unidentified component. ER stress induces expression of NRP-A and B, which subsequently induce GmNAC6 expression. This signal transduction can activate caspase-3-like activity. AGB1 function during ER stress-induced PCD is not determined, so this pathway is marked with dash line.

1.5 Plant "caspase" candidates and their roles in 24 programmed cell death

Caspase-like activity have been reported to participate in plant PCD (see above section). Studies have successfully identified several plants proteases responsible for various caspases activities in the past decade.

1.5.1 Metacaspase

Many efforts went into the search for plants caspases homologues before year 2000. Unfortunately, these searches were not fruitful. Clues were not found until Uren and his colleagues identified metacaspases, which were picked up in database searches using re- iterative PSI-BLAST (Uren et al., 2000). Metacaspases share some features with caspases. For example, two important amino acids residues, a histidine (H) and a cysteine (C) are also found in metacaspases. A diagram of metacaspases and caspases structures is shown in Figure 1.3.

Figure 1.3. Diagram of metacaspases and caspases. A diagram of metacaspases and caspases structure is shown. Two caspases in animal cells are shown, caspase 1 and caspase 3 as representatives of two types of caspases. Two types of metacaspases are also presented. Features of caspases and metacaspases are shown in different colour: caspases pro-domain (Dark blue), zinc finger motif (yellow), caspases large subunit (orange), caspases small subunit (pink), metacaspases pro-domain (green), metacaspases p20 domain (blue) and metacaspases p10 domain (red). Two conserved amino acids residues are marked as H (Histidine) and C (Cysteine).

The Arabidopsis metacaspase family has 9 members (AtMC1-9) and is divided into two types according to their structures (Figure 1.3). Type I metacaspases harbour an N- terminal pro-domain (contains proline-rich motif and zinc-finger motif). Type II metacaspases do not have the N-terminal pro-domain (Tsiatsiani et al., 2011). The main

25 parts of metacaspases are consisted of a large subunit P20 and a small subunit P10 (Vercammen et al., 2004). Both types of metacaspases possess a His-Cys catalytic diad which is similar to the one in caspases. This elicited the question of whether metacaspases have caspase-like activity. Initially, a metacaspase in Norway spruce was shown to regulate VEIDase activity (a caspase-6 like activity) (Bozhkov et al., 2005). This finding leads to a preliminary conclusion that metacaspases possess caspase-like activity. However, studies on AtMC4 and AtMC9 enzymatic activities showed clearly that metacaspases do not have caspase-like activity (Vercamen et al., 2004). Purified recombinant AtMC4 and AtMC9 from bacterial cells and several caspase specific substrates were used in the study and surprisingly, AtMC4 and AtMC9 cleaved less than 5% of the DEVD substrate compared to nearly 95% of DEVD cleaved by human caspase- 7. Instead, AtMC4 and AtMC9 showed a strong preference for substrates with arginine (R) at P1 position (Vercammen et al., 2004). More evidence was provided by He and colleagues’ report (He et al., 2008). The authors tested AtMC8 activity against several enzymatic substrates including DEVD and VEID. In consistent with the former report, AtMC8 has a strong enzymatic activity with GRR rather than DEVD or VEID (He et al., 2008). Type I metacaspases in Arabidopsis were also tested for enzymatic activity using AtMC1. In line with other metacaspases, which have been shown above, the favourite cleavage P1 site for AtMC1 was arginine or lysine as well (Watanabe, 2005).

Although metacaspases do not have caspase-like activity, they are essential for regulating PCD in plants. Some preliminary data implied that metacaspases might be involved in PCD. For example, a metacaspase gene in tomato was induced in response to Botrytic cinerea infection (Hoeberichts et al., 2003). Studies on Norway spruce metacaspase function during embryogenesis unveiled a role for metacaspases in PCD. Loss-of-function of metacaspases (mcII-pa) in Norway spruce suppressed suspensor PCD during embryogenesis and caused developmental arrest (Bozhkov et al., 2005). From then, other metacaspases were investigated under different conditions, such as oxidative-stress induced PCD. AtMC8 transcript level in Arabidopsis subjected to UV-C and H2O2 increased dramatically. AtMC8 knock out lines had a reduced PCD triggered by H2O2 and UV-C in protoplast. Moreover, overexpression of AtMC8 made protoplast more sensitive to UV-C and H2O2 induced PCD (He et al., 2008). Recent reports indicated that AtMC4 was also a positive regulator in oxidative stress induced PCD. Loss-of-function of AtMC4 rendered Arabidopsis more resistant to the mycotoxin Fumonisin B1. In addition, overexpression of AtMC4 led to more PCD induced by methyl viologen (MV) or

26 acifluorfen (AF) (Watanabe and Lam, 2011). Apart from abiotic stress-induced PCD, metacaspases also regulate biotic stress-induced PCD, such as Pseudomonas infection. Knocking out AtMC4 reduced PCD when plants were subjected to P. syringae syringae avrrpt2 (Watanabe and Lam, 2011). In another good experimental system, mutants for LESION SIMULATING DISEASE RESISTANCE 1 (LSD1) were used. LSD1 gene encodes a zinc-finger protein and functions as a negative regulator of plants PCD (Dietrich et al., 1997). Treating lsd1 Arabidopsis with the salicylic acid analogue benzo (1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH) can induce PCD. This PCD was suppressed by knocking out AtMC1. Intriguingly, knocking out of AtMC2 accelerated BTH induced PCD (Coll et al., 2010). This suggested that metacaspases have both promoting and inhibiting roles in plant PCD.

Controlling of metacaspases activities is a way of regulation of metacaspases function in plant PCD. Metacaspases activity can be regulated in several ways, presumably to fit in different signal pathways in different contexts. Like caspases, metacaspases need to be processed for activation (maturation). Metacaspases undergo autoprocessing during maturation (Vercaman et al., 2004). For instance, recombinant AtMC9 purified from E.coli gradually removed its pro-domain and self-processed into two major fragments: the P20 domain and P10 domain (Vercamen et al., 2004). Metacaspase-like activity can also be affected by zinc. Adding extra zinc to Norway spruce embryo suppressed metacaspases-like activity in vivo and perturbed embryo suspensor cells PCD (Helmersson et al., 2008). As a cysteine protease, a specific cysteine residue is essential for metacaspase catalytic activity; modification on this cysteine residue can affect activity. For example, S-nitrosylation on AtMC9 Cys-147 blocked its maturation in vitro (Belenghi et al., 2007). Another strategy to inhibit metacaspases activity is via protease inhibitors. AtSerpin1 was found to be a suicide inhibitor to AtMC9. Incubating AtMC9 with AtSerpin1 repressed AtMC9 activity (Vercammen et al., 2006).

Seeking in vivo substrates for metacaspases is important to annotate metacaspases function. The first substrate for metacaspases was identified by Sundström and colleagues in Norway spruce. Tudor staphylococcal nuclease (TSN) was found to be a substrate of a metacaspase in Norway spruce, mcII-pa (Sundström et al., 2009). TSN is a substrate of caspases-3 and cleavage of TSN participate in the PCD pathway in animal cells (Tong et al., 1995). This finding supports the idea that metacaspases are plants caspases, however, not all people are convinced and controversy is still going on (Carmona-Gutierrez et al.,

2010; Enoksson and Salvesen, 2010).

1.5.2 Vacuolar processing enzyme (VPE)

VPE is a member of plant cysteine proteases. One major function for VPE is to process protein precursors, which are localized inside vacuole, to their mature forms (Kinoshita et al., 1995). The VPE gene family has four members in the Arabidopsis genome, which are designated as alpha (α), beta (β), gamma (γ) and delta (δ) VPE. These four VPEs have different tissue expression profiles. α and γVPE are expressed only in vegetative organs such as leaves, while β and δVPE are expressed in seeds (Kinoshita et al., 1995; Nakaune et al., 2005).

A unique characteristic of VPE is that it possesses caspase-1-like activity. This was found by Hatsugai and colleagues (2004). Activity labelling by biotin-YVAD-FMK, an inhibitor binding caspase-1, was absent in VPE down-regulated leaf protein extracts (Hatsugai et al., 2004). In addition, seed protein extract of δVPE knock out in Arabidopsis also had an abolished biotin-YVAD-FMK labelling. Moreover, incubating recombinant VPE with Ac- YVAD-FMK repressed its activity (Nakuane et al., 2005). In another experimental system, Arabidopsis VPE knock-out had a reduced caspase-1-like activity during colonization by P. indica (Qiang et al., 2012). These discoveries have firmly established that VPE is a plant caspase-1-like protease.

Many studies have pointed out that VPEs are indispensable for PCD in plants. Silencing of VPE in tobacco leaves hugely reduced tobacco mosaic virus (TMV) induced PCD. The function of VPE during TMV-induced PCD was proposed to govern tonoplast rupture (Hatsugai et al., 2004). The tonoplast rupture was seen during TMV-induced PCD, but blocked by loss-of-function of VPE (Hatsugai et al., 2004). Besides virus-induced PCD, VPE is also crucial for elicitor-triggered immunity (ETI): silencing of tobacco VPE blocked harpin (a protein produced by Erwinia amylovora)-induced HR PCD. Meanwhile, knockdown of VPE impaired stomata closure triggered by infiltration of harpin (Zhang et al., 2010). In the context of mycotoxin fumonisin B1 induced PCD, VPE also played an important role. Infiltration of fumonisin B1 to Arabidopsis leaves elicited PCD which was characterized by vacuole rupture, VPE deficiency alleviated fumonisin B1 induced PCD (Kuroyanagi et al., 2005). The authors also showed that γVPE had more influence on fumonisin induced PCD among four VPE paralogues, as knock out of γVPE reduced more PCD than other VPEs. 28

VPE was also shown to participate in PCD caused by plant-microbe interaction (Qiang et al., 2012). Piriformospora indica is a fungus that colonized to plants roots. The colonization process required PCD of the colonized cells. Knocking out of VPE blocked PCD in colonized cells and subsequent growth of Piriformospora indica (Qiang et al., 2012).

VPEs are also involved in developmental PCD. During the late stage of embryogenesis, seed inner integument cells (ii2 and ii3) shrink and die. Loss-of-function of δVPE prevented PCD to occur in inner integument cells during Arabidopsis seeds maturation and maintain integument thickness (Nakaune et al., 2005). In addition, a recent finding indicated that VPE was involved in apical dominance (AD) in potato. Cold treatment was shown to induce PCD in apical bud meristem and caused the loss of AD in potato tuber buds. However, application of a VPE inhibitor, Ac-YVAD-CHO, to apical bud accelerated its development and maintain apical dominance (Teper-Bamnolker et al., 2012). In rice,

OsVPE2 and OsVPE3 transcript levels were induced when treated with H2O2. This was repressed by overexpression of BCL2, a human PCD repressor (Deng et al., 2011a). This shows that VPE also had a role in PCD in rice

1.5.3 Saspase

A study on Avena sativa revealed two novel proteases which harbour caspase-like activity. In contrast to other caspase-like proteases, these two proteases belong to the serine proteases family; therefore, they were named as "saspase" (SAS-1 and SAS-2). SAS-1 and SAS-2 were initially identified as 84KD proteases which were labelled by Biotin- YVAD-CMK. Adding Ac-YVAD-CMK compromised the activity labelling by Biotin- YVAD-CMK, this suggested the binding of Biotin-YVAD-CMK to saspases was specific (Coffeen and Wolpert, 2004). Purification of saspases from plants established a good system for investigating their protease substrate profile. It was found that saspases were prone to cleave caspase-6, caspase-8 and caspase-1 specific substrate, such as VKMD, IETD and YVAD (Coffeen and Wolpert, 2004). Saspases had null activity on caspase-3 substrate (DEVD). Analysis of the effect of pH on saspases activity indicated that the optimum pH was between pH5.5 and pH6.5. Observation of saspases in extracellular fraction after victorin treatment supported the idea that saspases executed their function in an acidic environment (Coffeen and Wolpert, 2004). Saspases exhibited auto proteolytic

29 activation. Native saspases presented three bands in western blot analysis whereas denatured saspases only had one band. However, whether this auto proteolysis was associated with their maturation or not is unclear (Coffeen and Wolpert, 2004).

1.5.4 Phytaspase

As works on looking for plant caspases are still going on, new candidates emerge. Phytaspase is a newly found aspartate-specific protease that exhibited caspase-like activity. Phytaspase was discovered in tobacco and rice (Chichkova et al., 2010). Inhibition of phytaspase activity by different caspase inhibitors indicated that phytaspase was more likely to bind VEID (caspase-6 substrate). Phytaspase was shown to regulate TMV-triggered HR PCD. Silencing of phytaspase resulted in less PCD in TMV infected area, whereas overexpression of phytaspases caused more severe PCD (Chikova et al., 2010). Moreover, phytaspase were involved in oxidative stress-induced PCD. Overproducing of phytaspases made tobacco leaves discs more sensitive to MV-induced PCD, down-regulation of phytaspases rendered them more resistance to MV (Chikova et al., 2010). A substrate for phytaspase was also proposed. Phytaspase was found to cleave Agrobacterium tumefaciens VirD2 protein in vitro at a TATD motif. This protein could be cleaved by human caspase-3 at the same motif (Chichkova et al., 2004). This findings support the idea that phytaspases are plant caspase candidates.

1.5.5 Proteasome subunit β1

Proteases which are responsible for caspase-3-like activity in plants have puzzled plant scientists for long. Proteasome subunit β1 (PBA1) was reported to possess caspase-3-like activity recently. Applying proteasome specific inhibitors, like β-lactone or aPnLd, to leaf extracts could reduce caspase-3-like activity. Purification and activity labelling of PBA1 with Biotin-DEVD-FMK also support that PBA1 had caspase-3-like activity. PBA1 was capable of mediating HR PCD induced by pathogen bacteria (P. syringae DC3000/avrrpm1) in Arabidopsis leaves. A novel tonoplast-cytoplasm membrane fusion was involved in this process, and controlled by PBA1. Down-regulation of PBA1 blocked the membrane fusion and HR PCD (Hatsugai et al., 2009).

1.5.6 Introduction to cathepsin B

Cathepsin B is a member of cysteine proteases found in lysosomes in animal cells (Mort and Buttle, 1997). In animal models, cathepsin B was found to be associated with many diseases, for example, cathepsin B may facilitate tumour progression (Podgorski and Sloane, 2003). Cathepsin B was also found to mediate PCD. Activation of death receptors, such as TNF receptor, leads cells to death and inhibition of cathepsin B reduced this PCD. During TNF induced apoptosis, cathepsin B translocated/leaked out from the lysosome to the cytoplasm where it is believed to execute a PCD function (Foghsgaard et al., 2001).

Research on plants cathepsin B is less intensive compared with animal models. But plants cathepsin B is also found to be an important component of PCD. Cathepsin B transcript level was hugely induced in barley aleurone layers in response to incubation with gibberellic acid (GA3). This induction was suppressed by adding ABA to aleurone cells (Martínez et al., 2003). PCD was found to occur in aleurone cells during seed development (Young and Gallie, 2000; Fath et al., 2000). In addition, GA3 and ABA antagonistically regulate PCD in aleurone cells (Lovegrove and Hooley, 2000). The combination of these findings implies that cathepsin B is able to regulate PCD.

More reports providing direct evidence for cathepsin B regulation of PCD have been published from then on. The apple pathogen Erwinia amylovora (Eam) was used to induce non-host HR PCD in tobacco leaves. It was observed that cathepsin B transcript level and activity were induced as early as six hours after inoculation with Eam. Cathepsin B inhibitors, such as CA074me, suppressed Eam-induced HR PCD. To rule out the possibility that cathepsin B inhibitors may interfere with other components for PCD, virus induced gene silencing (VIGS) was used to down regulate Nicotiana benthamiana cathepsin B transcript level. Measuring of PCD by trypan blue (TB) staining showed that cell death was suppressed in cathepsin B silencing plants 18 hours after inoculation with Eam and Pseudomonas syringae pv. Tomato (Pst) (Gilroy et al., 2007). Cathepsin B was found to regulate PCD not only in Nicotiana benthamiana, but also in Arabidopsis. Arabidopsis has three cathepsin B paralogues, which seem to have redundant function in pathogen defence. A triple cathepsin B mutant line showed a decrease in TB positive staining; while cathepsin B single or double knockout lines do not show significant difference in HR-induced PCD. Meanwhile, the three cathepsin B genes in Arabidopsis showed different expression level after Pseudomonas syringae DC3000 inoculation

(McLellan et al., 2009). All these reports pointed out that cathepsin B positively regulated HR-induced PCD.

During dark-induced senescence of Arabidopsis leaves, all three cathepsin B paralogues transcript level increased. Down-regulation of cathepsin B in the triple knockout line had a significant impact on reducing chlorophyll content loss, which was an indicator of reduced senescence. Furthermore, reduction of the Senescence-Associated-Gene-12 (SAG12) transcript level was observed in the cathepsin B triple knockout background during senescence (Mclellan et al., 2009). All these firmly established cathepsin B as a mediator of PCD.

1.6 Project aim and objectives

1.6.1 Aim

Several reports have implicated caspase-3-like activity in plant PCD, and ER stress- induced PCD is a good example of this. Previous data in our lab showed that cathepsin B possesses caspase-3-like activity. It is therefore worth investigating cathepsin B’s role in plant PCD as a caspase-3 candidate. Meanwhile, PBA1 was also reported to possess caspase-3-like activity. This raises the fundamental question of whether cathepsin B and PBA1 have similar or different roles in plant PCD, as both of them have caspase-3-like activity.

1.6.2 Objectives

Analysis of cathepsin B and PBA1 roles in ER stress-induced cell death As both cathepsin B and PBA1 have caspase-3-like activity, it is important to know whether they are functional synergically or antagonistically. I carried out this research in context of ER-stress induced PCD which has been shown to be regulated by caspase-3- like activity.

Investigation of how cathepsin B regulates ER stress induced PCD Cathepsin B triple mutant will be used to study its possible role in UPR and which type of PCD is mediated by cathepsin B. Meanwhile, subcellular localisation of cathepsin B will be investigated.

Fitting cathepsin B in ER stress induced PCD regulatory network. Different mutant lines for genes involved in UPR and PCD will be used to investigate their interactions with cathepsin B in ER stress induced PCD.

Investigation of the roles of cathepsin B in developmental PCD I will carry out research on cathepsin B's possible function in various plants development processes, including seeds germination, xylem formation, and inner integument cells destruction.

Investigation of the function of cathepsin B in KOD induced PCD KOD-induced PCD has been shown to increase caspase-3-like activity, it will be interesting to know whether cathepsin B is involved in KOD-induced PCD.

Chapter 2 Material and Method

2.1 Material

2.1.1 Plant material

Vpe null, ipba1-8, ipba1-11 and ipba1-23 Arabidopsis mutant seeds were kindly gifts from Professor Ikuko Hara-Nishimura. atcathb#62, in which AtCathB1 and AtCathB3 were knocked out while AtCathB2 was silenced by RNAi, was given by Professor Paul Birch. Cathepsin B double knockout lines were obtained by crossing two single knockout lines. Columbia-0 (Col-0) and WASSILEWSKIJA (Ws) were used as wild type Arabidopsis in different experiments. Seeds purchased from NASC are listed in Table 2.1.

Table 2.1 Arabidopsis mutant lines purchased from NASC

2.1.2 Plasmid material

AtCathB3::mRFP plasmid was obtained from Dr. Yuan Ge. AtCathB3ΔC::mRFP plasmid was generated using fusion PCR (see below). AtCathB3ΔC-F and AtCathB3ΔC-R primers were used for cloning AtCathB3ΔC and mRFP-FW and mRFP-RW primers were used for cloning mRFP. Sequences of these primers can be found in Table 2.2. A diagram of AtCathB3ΔC::mRFP construct is also shown in Figure 2.1.

Table 2.2. Primers for making AtCathB3ΔC::mRFP fusion sequence.

Figure 2.1. pXCS- AtCathB3ΔC::mRFP-HAStrep plasmid diagram. A diagram of pXCS-AtCathB3ΔC::mRFP-HAStrep plasmid was generated using Serial Cloner software. Features of this plasmid are marked with different colours. Features include: AtCathB3ΔC (light green), mRFP (red), 35S promoter (orange), HA and Strep tag (dark green), selection markers for screening phosphinothricin acetyltransferase (Pat, pale green), beta-lactamase gene (AmpR, pale green), replication origin (blue), E. coli replication origin (ColE1, blue), Nopaline synthase promoter (NOS-P, blue).

2.2 Plant based experiments

2.2.1 Plant growth condition

MS medium Murashige & Skoog medium (MS Duchefa) 4.4 g/L Glucose 20 g/L MES 0.5 g/L Adjust pH to 5.6-5.8 Phytagel 4 g/L Autoclave

Growth of Arabidopsis thaliana was on MS medium. Seeds were surface sterilized by immersing seeds in 70% ethanol for 10 minutes. Seeds were dried in a flow bench. After ethanol was evaporated, seeds were resuspended in 0.1% agar and sown on MS medium plates. Seeds were put in 4 ℃ for at least two days before growing in 16 h light, 8 h dark, 22 ℃. Plants growing in soil were either directly sown on soil or transferred from MS medium plate to soil at designated stages.

2.2.2 Genotyping

PCR reaction PCR program 2×terra PCR direct buffer 10 µL Denature 98 ℃ 2 min

Primer 1(10 µM) 0.5 µL Cycle starts 98 ℃ 10 s Primer 2(10 µM) 0.5 µL Annealing 60 ℃ 15 s

Tissue sample Elongation 72 ℃ 2 min Terra PCR Direct Cycle ends 30 cycles

Polymerase Mix (1.25 U/µL ) 0.5 µL

Milli-Q H2O up to 20 µL

Arabidopsis insertion verification primers were designed using the software T-DNA Primer Design (http://signal.salk.edu/tdnaprimers.2.html). PCR was carried out using Terra PCR DNA polymerase mix genotyping kit (Clontech). Each reaction was loaded in a 200 µL PCR tube with left/right border primers (LP/RP) for amplifying a wild type gene or the LBb1.3 primer and the RP primer for amplifying an inserted sequence in Salk T- DNA insertion lines. A small leaf disc (diameter < 1 mm) was added into the reaction 36 mix. Amplified fragments were separated using gel electrophoresis and visualized using Safeview.

2.2.3 Comparison of seed germination rates

Arabidopsis seeds, harvested at the same time, were sterilized by submerging the seeds in 70% ethanol for 10 minutes at room temperature before sowing on MS plates. More than 100 seeds were sown on three plates for each type of seed. Seeds were treated with cold (4℃) before germinated at 22 ℃. Germination rates were scored. T-Test was applied to statistically compare germination rates differences.

2.2.4 Protoplasts isolation

Leaf digestion medium 1×MS Sucrose 0.4 M Cellulose R-10 (Yakult) 0.96% Macerozyme (Yakult) 0.64% Adjust pH to 5.8. Sterilized with 0.22μm filter

W5 wash medium NaCl 154 mM KCl 5 mM Glucose 5 mM

CaCl2 125 mM pH 5.6-5.8

Protoplast culture medium 1×MS Sucrose 0.4M Mannitol 0.4M MES 0.5g/L Adjust pH to 5.8. Sterilized by autoclaving

Protoplasts isolation followed the description in Wu et al. 2009 with slight modifications (Wu et al., 2009). Detached leaves were laid on black floor marking tape (SLS, TAP1052)

37 with abaxial surface facing up. Cuticles and epidermis were removed using Magic tape (Scotch, 3M). 10 mL leaf digestion medium was added into a petri dish. Leaves were placed in digestion medium with abaxial surface facing towards digestion medium and incubated at room temperature with mild agitation for an hour. Digestion medium was transferred to a 15ml falcon tube. Isolated protoplasts and tissue debris were separated by centrifuging at 90 g for five minutes. Protoplasts floating at the top of medium were transferred into a new tube and washed protoplasts with W5 medium for three times. Protoplasts were resuspended in protoplast culture medium for further testing.

2.2.5 Yariv reagent treatment

Yariv reagent (3 mg/mL, gift of Prof Simon Turner, Manchester) was infiltrated into one half of an Arabidopsis leaf. The other half was infiltrated with water. Infiltrated plants grew in growth cabinet (16 h light, 22 ℃) for further three days. Images were taken by Nikon D3100 camera.

2.2.6 Tunicamycin treatment

15 µg/mL tunicamycin was composed by diluting 3 µL 5 mg/mL tunicamycin into 990 µL water plus 7 µL DMSO. Tunicamycin was infiltrated into one half of an Arabidopsis leaf. The other half was infiltrated with 1% (v/v) DMSO. Infiltrated plants grew in growth cabinet (16 h light, 22 ℃) for further analysis at designated days. For comparing seedlings growth on tunicamycin MS medium, 15 µg/mL tunicamycin was incorporated in MS medium. Arabidopsis seeds were germinated and grew on the plate. Seedlings in each category were scored.

2.2.7 Measuring Ion leakage

Leaves with various treatments were detached. Three equal size leaf discs were punched off from leaves by a cork borer (5 mm diameter). Three leaf discs were floated in 800μl deionized water in a 24-well plate at room temperature for an hour. The conductivity of each well was measured using a conductivity meter (Horiba B-173). The conductivity meter was calibrated with the standard KCL solution (1.417 mS/cm) each time before reading. For every sample, 150 μL liquid was loaded in conductivity meter to measure its conductivity.

2.3 DNA and RNA based experiments

2.3.1 Fusion-PCR

PCR reaction PCR program

5×Hi-Fi buffer 10 µL Denature 98℃ 5 min Forward Primer (10 µM) 1 µL Cycle starts 98℃ 30 s Reverse Primer (10 µM) 1 µL 5 mM dNTP 1 µL Annealing 60℃ 30 s Template 0.5 µL Elongation 72℃ 1 min Velocity DNA Cycle ends 32 cycles Polymerase (2 U/µL) 1 µL Elongation 72℃ 5 min H2O 35.5 µL Hold 8℃ Total 50 µL

Fusion PCR reaction Fusion PCR program

5×Hi-Fi buffer 10 µL Denature 98℃ 5 min Forward primer (10 µM) 1 µL Cycle starts 98℃ 30 s Reverse primer (10 µM) 1 µL Annealing 63℃ 30 s 5 mM dNTP 2 µL Template 1 µL Elongation 72℃ 5 min (Purified DNA fragments) Cycle ends 30 cycles Velocity DNA (2 U/µL) 1 µL Elongation 72℃ 5 min Polymerase Hold 8℃ H2O 34µL Total 50 µL

Fusion PCR with mRFP was carried out essent ially as described in Atanassov et al. 2009 (Atanassov et al., 2009). Briefly, the 3’ primer of the preceding sequence and the 5’ primer of the following sequence were design to have an overlap sequence. In this thesis, the 3’ primer of the preceding sequence AtCathB3ΔC-F. The 5’ primer of the following sequence was mRFP-FW. The preceding and following sequences were cloned independently from plasmid template in the first round of PCR (See above for the reaction system and program). Then the amplicons of were purified and mixed together as a new template to obtain the fusion sequence. In the fusion PCR, the 5’ primer of the preceding sequence and the 3’ primer of the following sequence were used to amplify the fused sequence.

2.3.2 DNA digestion and ligation

Restriction enzyme digestion Ligation reaction Buffer H 10 µL 2×T4 DNA ligation Buffer 10 µL EcoR1 (20 U/µL) 1 µL Dilution buffer 1 µL Xho1 (20 U/µL) 1 µL Vector ~100 ng DNA fragment Insert ~300 ng or plasmid more than 500 ng T4 ligase (5 U/µL) 1 µL Add H2O to total 20 µL Add H2O to total 20 µL

Both a DNA fragment and a vector plasmid were digested using restriction enzymes. In this thesis, EcoR1 and Xho1 were used. The digested DNA fragment and the digested vector plasmid were purified using the DNA purification kit (NucleoSpin® Extract II, Macherey-Nagel) according to the manufacturer instructions. In brief, two volumes NT buffer was added to one volume of PCR sample. If DNA was purified from an agarose gel, 200 µL NT buffer was added to 100 mg gel and the mixture was incubated at 50 ℃ for five to ten minutes. After that, the mixture was loaded into NucleoSpin® Extract II colum, and centrifuged at 11,000 g for one minute. Flow-through was discarded and 600 µL NT3 buffer was added, followed by centrifuging at 11,000 g for one minute. Flow- through was discarded and the column was centrifuged at 11,000 g for further two minutes. To elute DNA, 15-50 µL NE buffer was added into column and incubated at room temperature for one minute. The purified DNA was finally eluted down by centrifuging at 11,000 g for one minute. Digested and purified DNA fragments and plasmid were mixed as described below for ligation using Rapid DNA Ligation Kit (Roche). The ligation reaction mix was shown (see above). Incubate the ligation reaction mix at room temperature (15-25 ℃) for one hour. The reaction was ready for transformation directly after incubation.

2.3.3 DNA sequencing

Sequencing reaction Sequencing reaction program 5×SBS buffer 4 µL Denature 95℃ 5 min Sequencing terminator 2 µL Cycle starts 95℃ 30 s Primer 10 µM 1 µL Annealing 55℃ 30 s DNA template more than 500 ng Elongation 60℃ 3 min Milli-Q H2O up to 20 µL Cycle ends 36 cycles Hold 8℃

DNA sequencing reaction was carried out using BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and a thermo cycler. The reaction is as described above. PCR products were transferred to a new 1.5 mL Eppendorf tube. 20 µL Milli-Q

H2O was added into the tube, 1 µL glycol blue, 4 µL NaOAc and 80 µL absolute ethanol were into the tube sequentially. The mixture was incubated at room temperature for 10 minutes. DNA was precipitated and spun down by centrifuging at 11,000 g for 20 minutes. DNA pellet was washed with 70% ethanol. DNA sample was dried at room temperature before sending to sequencing unit for further processing. Sequencing results were analysed with the software Chromas.

2.3.4 RNA isolation

DNase treatment reaction cDNA synthesis RNA 1 μg DNase-treated RNA 5μL 10×DNase RQ1 buffer 1μL oligo-dT 10 μM 1μL RNase free DNase I (1U/μL) 2μL DTT 100 mM 1 μL Add Milli-Q H2O up to total 10 μL dNTP 2 μL 5×Reverse transcriptase buffer 4 μL RTase (u/µL) 1 μL Milli-Q H2O 6 μL

Total RNA was isolated using NucleoSpin® RNA II kit (Macherey-Nagel) according to the manufacturer’s instructions. The purified RNA was treated with RNase free DNase I (Promega) at 37 ℃ for 30 minutes and inactivated at 65℃ for 20 minutes; the reaction is shown above. The DNase-treated RNA sample was sub-divided into two halves. One half was subjected to cDNA synthesis using a MMLV cDNA synthesis kit (Promega). The DNase-treated RNA was incubated with 10 μM oligo-dT at 70 ℃ for 10 minutes to avoid secondary structure before adding other components for the cDNA synthesis reaction. The sample was incubated at 42 ℃ for one hour and inactivated at 70 ℃ for 10 minutes. The other half was mixed with the same buffer but without reverse transcriptase as a control for genomic DNA contamination. Both half samples were diluted to 200μl final volume. 41

2.3.5 Quantitative Real Time-PCR (QRT-PCR)

QRT-PCR reaction 2×MEZA blue 12.5 μL Forward primer 10 μM 0.75 μL Reverse primer 10 μM 0.75 μL cDNA 5 μL from 200 μL Milli-Q H2O 6 μL cDNA samples were mixed with 2×MEZA blue QRT-PCR kit (Eurogentec) in a 96-well plate (Bio-Rad). The reaction ingredients are shown above. Raw fluorescence intensity data from each reaction was recorded using an ABI7000 sequence detection system (Applied Biosystems). The SYBR detector was selected to detect amplification. Four standard genomic DNA samples were also used to compose a standard curve. DNA quantity, in terms of copy number, of a sample was calculated by the ABI7000 sequence software (Applied Biosystems). Samples were normalized using reference genes copy number. Two reference genes were used in this thesis, 18S and Actin2. Primers for QRT- PCR were designed using the software Primer 3, and primers sequences were listed in Table 2.3. Table 2.3 Primers used for QRT-PCR

2.3.6 Semi-quantitative RT-PCR

RT-PCR Reaction RT-PCR program 2×Ready mix 10 μL Denature 95 ℃ 5 min Forward primer 10 μM 1 μL Cycle starts 95 ℃ 30 s

Reverse primer10 μM 1 μL Annealing X ℃ 30 s

cDNA 5 μL Elongation 72 ℃ 5 min

H2O 3 μL Cycle ends X cycles

Total 20 μL Elongation 72 ℃ 5 min Hold 8 ℃

Annealing temperature was set according to each primer sequence. To avoid amplifying DNA fragments to a saturated stage, cycle number was determined by preliminary experimental tests. Primers used for semi-quantitative RT-PCR are listed in Table 2.4. Table 2.4 primers used for semi-quantitative RT-PCR

2.3.7 Agarose Gel electrophoresis

0.5×TBE buffer Tris base 5.4 g Boric acid 2.75 g EDTA 0.5 M pH 8.0 2 mL

H2O up to 1 L

The required amount of agarose was added into 50 mL 0.5×TBE buffer to a final concentration between 0.8%-2% （according to experiment requirements）. Agarose was melted by heating the mixture in microwave for one minute, followed by cooling the mixture at room temperature to 60 ℃. 12.5 μL Safeview (NBS) was added to 50 μL melted agarose and the mixture was poured it into a gel casting tray. DNA samples were separated with 100 V/cm for 25 minutes using a minigel. DNA bands were visualized using Gel Doc XR+ (Bio-Rad). The excitation wavelength was 290 nm; the emission wavelength was 515 nm.

2.4 E. coli transformation

Luria-Bertani (LB) medium LB broth powder 20 g/L H2O up to 1 L Adjust pH to 7.0 Medium was sterilized by autoclaving

1-3 µL plasmids were added to E. coli competent cells and the mixture of competent cells and plasmids were incubated on ice for 30 minutes. After that, the mixture was incubated at 42℃ for 50 s then put back on ice for further incubation for two minutes. One millilitre LB medium was added into the E. coli and plasmid mixture, and then incubated the mixture at 37 ℃ for one hour. E. coli was finally plated on LB agar plate (1.5% Agar, Sigma-Aldrich) with the appropriate antibiotic (75 μg/mL carbenicillin was used in this experiment). Transformed E. coli grew on LB plate in 37 ℃ incubator over-night.

2.5 Plasmid amplification and purification

To purify plasmid from E. coli, E. coli was cultured in LB medium including appropriate antibiotics at 37 ℃ for over-night. The culture was harvested by centrifuging at 11,000 g for 30 s. Plasmid purification was carried out using the plasmid purification kit (NucleoSpin® Plasmid, Macherey-Nagel) according to the manufacturer instructions. 500 μL A1 buffer was added to resuspend E. coli. 500 μL A2 buffer was added and incubated at room temperature for five minutes. A3 buffer was added after incubation at room temperature followed by centrifuging at 11,000 g for 10 minutes. Supernatant was loaded into NucleoSpin® plasmid column and centrifuged at 11,000 g for one minute. 600 μL A4

44 buffer was added to column and centrifuged at 11,000 g for one minute. The flow-through was discarded and the column was centrifuged for further two minutes at 11,000 g. AE buffer was preheated at 70 ℃ and added to column to elute the plasmid DNA by centrifuging at 11,000 g for one minute.

2.6 Plant transformation and transfection

2.6.1Preparing Agrobacterium tumefaciens (GV3101 pMP90RK) competent cells

TE buffer Tris-Cl 10 mM EDTA 1 mM Adjust pH to 7.5

Preparing Agrobacterium competent cells are described as following: the appropriate Agrobacterium strain was inoculated into 3 mL LB medium and incubated at 30 ℃ overnight. On the next day, 1 mL overnight culture was inoculated to 200 mL LB medium. The culture grew till OD600 reached 0.5, then the culture was harvested by centrifuging at 4,500 g for 10 minutes. The pellet was resuspended and washed with sterile TE buffer followed by spinning down at 4,500 g for 10 minutes. The pellet was resuspended in 20 mL LB medium. Resuspended Agrobacterium was aliquoted to Eppendorf tubes with 250 μL in each tube and instant froze in liquid nitrogen.

2.6.2 Plasmid transformation into Agrobacterium tumefaciens

To introduce plasmids into Agrobacterium, a freeze-thaw method was used. Frozen Agrobacterium competent cells were thawed on ice. 10μl of purified plasmid was added to Agrobacterium competent cells and incubated on ice for five minutes. The mixture was then instantly frozen in liquid nitrogen for five minutes followed by five minutes incubation in 37 ℃ water bath. After water bath, 1 mL LB medium was added into the mixture and incubated at room temperature for two to four hours. Agrobacterium was finally placed on LB plate with appropriate antibiotics. To verify the transformants, colonies growing on LB plate were re-streaked on a new LB plate with same antibiotics. Agrobacterium growing on new LB plate was picked out and resuspended in 50μL TE

45 buffer. The TE buffer with Agrobacterium was boiled at 90 ℃ for five minutes. One μL of this boiled mixture was used as template to amplify the transformed plasmid. Positive colonies would be identified if the transformed plasmid DNA sequences could be amplified.

2.6.3 Agrobacterium-mediated transformation of Arabidopsis

YEP medium Yeast extract 10 g Bacto-peptone 10 g NaCl 5 g Add water to 1 L Adjust pH to 7 and autoclave for 30 minutes

Floral dipping buffer Sucrose 5% Silwet L-77 (OSI) 0.005%

Agrobacterium containing the selected plasmid was inoculated in a small culture (less than 5 mL) of YEP medium for overnight with the appropriate antibiotics. The small culture was transferred into a big culture and incubated for overnight. The Agrobacterium culture was harvested by centrifuging at 4,500 g for 15 minutes on the next day, and resuspended in floral dipping buffer. Flower buds were submerged in dipping buffer containing Agrobacterium and 1 bar vacuum was applied for one minute. Arabidopsis was returned to the growth chamber and was covered with a propagator for 24 h to keep humidity. Descendent seeds were harvested and the transformed seeds were selected with appropriate antibiotics or herbicides on MS plate.

2.6.4 Arabidopsis seedling transfection

2-YT medium Bacto-peptone 16 g/mL Yeast extract 10 g/mL NaCl 5 g/mL 46

Adjust pH to 7

Seedling infiltration buffer Sucrose 5% Acetosyringone (Sigma-Aldrich) 200 μM

Plasmids transfection into Arabidopsis seedlings were carried out as described in Marion et al. 2008. Seeds were germinated on sterile mesh (Saatitech reference PA500/38, pore size 500 μm) in a six-well plate. Each well contained 3 mL MS medium which was underneath the sterile mesh. Transfection was started on the third day after germination. Agrobacterium containing an appropriate plasmid was inoculated into 3 mL 2-YT liquid medium overnight. On the next day, 1 mL overnight pre-culture was added into 4 mL new 2-YT medium, and grew overnight. Agrobacterium was harvested by centrifuging at 4,500 g for 10 minutes on the next day. The Agrobacterium pellet was resuspended in seedling infiltration buffer. The resuspended culture was added into wells in such a way that it submerged the seedlings. Then seedlings and Agrobacterium culture were subjected to vacuum of 1 atm for one minute. The Agrobacterium culture was removed after the vacuum step, and seedlings grew for further three days before analysis.

2.6.5 Transfection of tobacco leaves

Infiltration buffer MES (Sigma-Aldrich) pH5.6 50mM

Na3PO4 2mM Glucose 0.5% Acetosyringone 100 μM

For transfection of tobacco leaves mediated by Agrobacterium-infiltration, Agrobacterium was inoculated in 5 mL YEP and incubated overnight. The culture was harvested by a centrifuging at 2,200 g for five minutes, and the pellet was resuspended in the infiltration buffer, followed by diluting the culture to OD600=0.1. Dilute culture was incubated at room temperature with mild agitation for four hours before infiltration. Agrobacterium was infiltrated into tobacco leaves with a 1 mL syringe.

2.6.6 Biolistic particle bombardment

2-5 μg of purified plasmids were mixed with 100 μL 2.5M CaCl2 and 20 μL 0.1 M spermine (Sigma-Aldrich). This mixture was mixed with 30 μL gold particles (Bio-Rad). The mixture was blended using vortex for three minutes and incubated on ice for 15 minutes. The supernatant was removed after incubation and particles were resuspended in 500 μL 100% ethanol. The mixture was incubated on ice for another 15 minutes. The supernatant was removed. Gold particles were resuspended in 200 μL 100% ethanol, and incubated for 20 minutes. The supernatant was replace with 38 μL 100% ethanol. Gold particles were loaded onto macroprojectiles. Gold particles bound with plasmid were delivered into tobacco leaf discs using a Biolistic PDS-1000/HE Particle delivery system (Bio-Rad) with 1,100 psi rupture discs and 1 atm vacuum at the distance of nine centimetres.

2.7 Biochemistry experiments

2.7.1 Analysis of caspase-3-like activity

Protease extraction buffer for activity measurement Dithiothreitol (DTT) 3 mM Phenylmethanesulfonylfluoride (PMSF) 100 μM ddH2O 2×enzymatic assay buffer NaOAc 100 mM EDTA 2 mM

NaN3 0.04%

CaCl2 2 mM DTT 6 mM Adjust pH to 5.5

To assess caspase-3-like activity, plant leaf samples with various treatments were homogenized by grinding in the sample isolation buffer. Protein extraction was carried out in 4 ℃ cold room to possess most of proteases activity. The homogenized sample was rotated in the cold room for 10 minutes to release most of the protein contents. Soluble proteins and plant tissue debris were separated by centrifuging at 4 ℃ 13,000 g for 10 minutes. The supernatant was transferred to a new tube. Protein concentration was

48 determined using the Quick Start™ Bradford Protein Assay (Bio-Rad). 5 μL soluble proteins were mixed with 200 μL water and 795 μL Bradford reagent in a cuvette. Protein concentrations were measured by a photospectrometer at 595 nm wavelength (Eppendorf). Protein samples were adjusted to the same concentration. For enzymatic assay, a 50 μL protein sample was mixed with 50 μL 2×enzymatic assay buffer, including 50μM Ac-DEVD-Rh110 as substrate. The mixture was loaded into a 96-well plate. In the case of protease activity inhibition studies, designated concentrations of inhibitors were added into the mixture and incubated at 30 ℃ for 20 minutes before measurement. The fluorescence emitted from the release of Rh110 was measured using a Microtiter Plate Fluorometer (Labsystem, DYNEX Technologies) with the excitation wavelength of 485 nm and the emission wavelength of 530 nm. Raw data was converted to enzymatic activity by the Fluoroskan Ascent software and presented as relative fluorescence unit per minute. To compare activities among different samples, enzymatic activities were normalized over the protein concentration.

2.7.2 Analysis of 20S proteasome activity

20S proteasome activity assay buffer Tris-HCl pH7.5 50 mM KCl 25 mM NaCl 10 mM

MgCl2 1mM

For 20S proteasome activity, soluble protein was extracted with 20S proteasome activity assay buffer. Extracted protein was mixed with 20S proteasome assay buffer containing 50 μM Ac-LLVY-AMC. Enzymatic activity measurement was carried out as described above, except the excitation wavelength for AMC was 360 nm and the emission wavelength was 460 nm.

2.7.3 Activity labelling by Biotin-DEVD-FMK

Protein extraction was as described in the section of analysis of caspase-3-like activity. 50 μL of soluble protein extract were mixed with 50 μL enzymatic assay buffer including 60 μM Biotin-DEVD-FMK. The mixture was incubated in 37 ℃ water bath for an hour. Labelled proteases were visualized using western blot.

2.7.4 Measuring ATP level

The ATP level was determined using ENLITEN® ATP Assay kit (Promega). This method relied on measuring the light generated via ATP-dependent oxidation of luciferin by luciferase. Leaves with different treatments were detached from plants. Total ATP was extracted with 3% trichloroacetic acid (TCA) from leaves. pH of the extract was then adjusted to neutral by adding NaOH for further measuring. Each extract was loaded into a 96-well plate. Meanwhile, four ATP standards were also prepared and load into the same 96-well plate. A Luciferase reaction reagent was prepared separately and the ATP level in each well was determined by multimode microplate reader (Mithras LB 940). ATP level was normalized with individual leaf fresh weight.

2.7.5 Separation of mitochondria for cytochrome c release analysis

To avoid massive destruction of mitochondria, protoplast disruption was carried out with a mild method. Protoplasts after various treatments were resuspended in W5 wash buffer and forced to pass through a nylon mesh with 30 μm pores. As protoplasts average diameter was about 50 μm, when protoplasts passed through the nylon mesh, they were disrupted without disturbing mitochondria integrity. Passed-through liquid was centrifuged at 18,000 g, 4 ℃ for 15 minutes. The supernatant was considered as cytosolic fraction. Mitochondria were retrieved in the pellet. The resuspended pellet with 2×sample buffer was boiled at 95 ℃ for five minutes to release cytochrome c.

2.7.6 SDS-PAGE and western blot

2×Sample buffer Tris-HCL pH6.8 65mM Glycerol 26% Sodium dodecyl sulfate (SDS Melford) 2% Bromophenol blue 0.01%

Coomassie blue G-250 (Sigma-Aldrich) 0.5% v/v in 50% methanol and 10% acetic acid

PBST buffer NaCl 137 mM Phosphate 12 mM KCl pH 7.4 2.7 mM Tween-20 0.1% v/v

Blocking buffer 3% BSA (Sigma-Aldrich) in PBST buffer

Ponceau S (Sigma-Aldrich) 0.1% v/v in 5% acetic acid

Extracted proteins were boiled with 2×Sample buffer at 95 ℃ for five minutes. Boiled protein samples were loaded into a commercial acrylamide gel at various % (Bio-Rad). Proteins were separated using constant electric voltage at 100 V/cm for an hour. The acrylamide gel was either stained directly with Coomassie blue G-250 or subjected to western blot. For western blot, proteins on the acrylamide gel were transferred onto a PVDF membrane (GE) using a Bio-Rad transfer cell at 50V/cm for 2 h at 4 ℃. After transfer, the PVDF membrane was incubated with the blocking buffer at room temperature for an hour or over-night at 4 ℃. The primary antibody at designated dilution was added to the membrane for further incubation at room temperature for one or two hours. The membrane was then washed with PBST buffer three times before adding the secondary antibody. To detect the protein of interest on the membrane, the membrane was incubated with an ECL solution (pico:femto 4:1, Pierce) for less than one minute before detecting bands using X-ray film (Thermo Scientific). To visualize the large subunit of Rubisco as a loading control, the PVDF membrane was stained with ponceau S.

2.8 Histology experiments

2.8.1 Detection of PCD by dye exclusion staining

Tissue samples were resuspended in culture medium (for protoplast) or distilled water (for seeds). Tissue samples were incubated with 5 μM sytox green (Invitrogen) at room temperature for 10-15 minutes. Tissue sample was rinsed with water twice before mounting onto a glass slide. Sytox green fluorescence was observed under fluorescence

51 microscope by using GFP filter.

2.8.2 Staining the vacuole with BCECF

5 mm diameter leaf discs were punctured out and incubated with 5 μM 2′,7′-bis-(2- carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) (Invitrogen) at room temperature for 10-15 minutes. After incubation, leaf discs were washed with water twice. Leaf discs were mounted onto a glass slide for further observation under fluorescence microscope. GFP filter was used to observe green fluorescence of BCECF.

2.8.3 Detection of ROS accumulation

Diaminobenzidine tetrahydrochloride (DAB) solution DAB (Sigma-Aldrich) 1 mg/mL Dissolve 1mg/mL DAB in acidic water then adjust pH back to neutral

Two methods were employed to detect ROS accumulation. For seeds, 5 μM 2’,7’ – dichlorofluorescein diacetate (DCFDA) was incubated with seeds at room temperature for 10-15 minutes. Seeds were washed with water twice and subjected to fluorescent microscope observation with a GFP filter. For leaf samples, DAB was used. Leaves with various treatments were cut and submerged in DAB staining solution (fresh prepared) in the dark overnight. On the next day, the DAB solution was removed. Pigments in the leaves were removed using 2 mg/mL chlorohydrate.

2.8.4 Microscopy

To observe fluorescent components in the cells, a Leica DM 5500B fluorescence microscope was used in this thesis. The software for capturing images and processing was SPOT imaging software. Leica TSC SP5 confocal microscope was used to get section images. Filter cubes used in this thesis were: GFP (excitation 470 nm, emission 525 nm), TX2 (excitation 560 nm, emission 645 nm). For transmission electronic microscopy (TEM), leaf discs (about 1 mm2) were cut and floated on paraformaldehyde (PFA, obtained from TEM unit) fixative buffer in a glass vial. Vacuum was employed to facilitate leaf discs absorbing fixative buffer. Once leaf discs sunk down in the fixative buffer, the whole sample was sent to TEM unit for further processing.

2.8.5 GUS staining

Staining buffer Sodium phosphate buffer 0.5 M pH7.2 1 mL Triton X-100 0.2 mL Potassium ferrocyanide 100 mM 0.2 mL Potassium ferricyanide 100 mM 0.2 mL

X-Gluc (Sigma-Aldrich) 100 mM stock in DMF, stored in the dark at -20 ℃

Tissue samples were collected and washed with staining buffer twice. X-Gluc was added to fresh staining buffer to a final concentration at 2 mM just before staining. Tissue samples were incubated with staining buffer plus X-Gluc at 37 ℃ for more than 16 h. Tissue samples were removed from staining buffer to a series different concentration ethanol (20%, 50%, 70%) to remove background colour pigments in the tissues.

2.8.6 Toluidine blue staining Arabidopsis xylem section

Stems of Arabidopsis plants were detached. Thin tissue sections from similar stem position were generated using a razor blade and incubated with 0.05% toluidine blue for two to five minutes at room temperature. Tissues were washed with water for three times and mounted onto glass slides. Stem sections were observed by bright field microscopy filter.

2.8.7 Measurement of integument layer thickness

Embryos were removed from siliques using sharp needles at designated time points. Isolated embryos were submerged in 2.5 g/L chlorohydrate (Sigma-Aldrich) for three days to remove colour. Clear embryos were mounted on glass slides and observed under a microscope. All images were taken with same magnification. The relative thickness of integument layers was arbitrary determined using the software image J expressed as pixel unit. Statistics analysis was carried out using T-test in Excel.

2.9 Bioinformatics analysis

2.9.1 Microarray analysis

RNA samples purified from Arabidopsis seedlings were sent to the Bioinformatics unit (The University of Manchester) for further processing. Microarray data were retrieved and submitted to online tools for analysis of microarray data as listed below. GO analysis AgriGO http://bioinfo.cau.edu.cn/agriGO/analysis.php BioProfiling http://bioprofiling.de/ Promoter analysis AtCisDB http://arabidopsis.med.ohio-state.edu/AtcisDB/ PlantPromoteDB http://ppdb.agr.gifu-u.ac.jp/ppdb/cgi-bin/index.cgi AthaMap http://www.athamap.de/index.php Interaction and Pathway analysis GRG-X database http://arabidopsis.med.ohio-state.edu/grgx/. MetNet http://metnetonline.org/index.php N-Browse http://www.arabidopsis.org/tools/nbrowse.jsp Tissue expression analysis ePLANT http://bar.utoronto.ca/eplant/

2.9.2 Orthologue idetification using blast

Amino acids sequences of proteins of interest were obtain from either NCBI or TAIR. To find out gene orthologues in Arabidopsis genome, amino acids sequences were input in Blast tool in TAIR website http://www.arabidopsis.org/Blast/. The TAIR10 Proteins was used as a dataset to identify gene orthologues in Arabidopsis.

Chapter 3 Analysis of the role of cathepsin B in ER stress-induced PCD

3.1 Introduction

Prolonged ER stress can induce PCD in both animal and plants cells (see introduction). However, the mechanism of ER stress-induced PCD is still elusive in plants. Caspase-3- like activity, an important PCD regulator in both animal and plants PCD, has been observed during ER stress-induced PCD in soybean suspension cells (Zuppini et al., 2004). Previous data in our lab have shown that cathepsin B, a plant cysteine protease, possesses caspase-3-like activity. This implied that cathepsin B may be a regulator in plants ER stress-induced PCD. However, a research group in Japan identified the 20S proteasome subunit β1 (PBA1) as a plant protease responsible for caspase-3-like activity (Hatsugai et al., 2009). This raises the question of which protease, cathepsin B or PBA1, is responsible for the caspase-3-like activity increase in plants ER stress-induced PCD.

In this chapter, cathepsin B mutant Arabidopsis lines are used to investigate the role of cathepsin B in plants ER stress-induced PCD. In Arabidopsis genome, there are three paralogues of cathepsin B. Three cathepsin B paralogues were named as AtCathB1 (At1g02300), AtCathB2 (At1g02305) and AtCathB3 (At4g01610). Different mutants of these paralogues were used to examine the functional redundancy. To elucidate which protease contributes to the caspase-3-like activity increase in ER stress-induced PCD and the possible functional interaction between cathepsin B and PBA1, I also investigated caspase-3 activity in various mutants of cathepsin B and PBA1.

3.2 Results

3.2.1 is Cathepsin B involved in ER stress-induced PCD?

I first examined whether cathepsin B participated in ER stress-induced PCD. Mutant and over-expression lines were employed to test Cathepsin B function during ER stress- induced PCD. Ion leakage from leaves of Col-0 and the cathepsin B triple mutant line atcathb#62 were monitored for seven days after infiltration by 15μg/mL tunicamycin (Tm). Atcathb#62 mutant line was generated by knock-out of AtCathB1 and AtCathB3 and knock-down of AtCathB2 by RNAi. Ion leakages from Col-0 leaves started increasing three days after infiltration, which suggested PCD in Col-0 leaves set out as early as three days after infiltration of Tm (Figure 3.1). However, ion leakage from atcathb#62 did not show any obvious increase till five days after infiltration (Figure 3.1 left). Leaf appearances of Col-0 and atcathb#62 lines were photographed three days after infiltration. As seen from the images, a yellowish coloration developed in the Tm treated area for both lines (Figure 3.2 red dash line area). To quantitatively compare the loss of chlorophyll coloration between Col-0 and atcathb#62, I measured the yellowish area using ImageJ. Arbitrary unit, define by pixels within designated area, of Col-0 yellowish area were 18813 (left) and 21982 (right). Arbitrary area units for atcathb#62 yellowish areas were 4356 (left) and 7702 (right) (Figure 3.2). I also carried out tests using a protoplast system. Protoplasts were isolated from Col-0 and atcathb#62 leaves, 15μg/mL final Tm was added to protoplasts to induce PCD, and sytox green was used to detect dead protoplasts. Five hours after adding Tm, about 30% protoplast showed sytox green positive in Tm treated wild type. Only 9% of dead protoplasts were seen in the atcathb#62 background (Figure 3.3). These suggested that down-regulation of cathepsin B in Arabidopsis delayed cell death induced by ER stress.

Figure 3.1 Ion leakages of ER stress-induced PCD in Col-0 and atcathb#62 15μg/mL tunicamycin was infiltrated into Col-0 and atcathb#62 leaves, water with the same volume DMSO was also infiltrated as mock. Leaf discs were punched out at various days after infiltration (dpi) and floated on de-ionized water for 1 hour. Ion leakages were measured using a conductivity meter. Relative ion leakages were obtained by subtracting mock values from Tm treated samples values. Data points are means of relative ion leakages values for three biological repeats. Error bars represent standard deviation.

Figure 3.2 Yellowing of leaves and ER stress-induced PCD in Col-0 and atcathb#62. 15μg/ml of tunicamycin was infiltrated into Col-0 and atcathb#62 leaves. Water with the same volume DMSO was also infiltrated as mock. Leaves appearance was photographed three days after infiltration. To determine sizes of yellowish areas (red dash line area), ImageJ was used to measure the area in arbitrary unit. The arbitrary unit of each area was labelled on the top of the area. Scale bar is equal to 1 cm.

Figure 3.3 Comparison of ER stress-induced PCD in Col-0 and atcathb#62 protoplasts Mesophyll protoplasts were isolated from Col-0 and atcathb#62 leaves. 15μg/ml Tm final was added to protoplasts. Five hours after treatment, protoplasts were stained using sytox green. Sytox green positive protoplasts were counted under a fluorescence microscope with a GFP filter. Bars represent the mean percentage of sytox green positive protoplasts in total protoplasts. Error bar represents standard deviation. Five populations of protoplasts in each treatment were counted and more than 50 protoplasts were in each population.

To test whether a lack of cathepsin B conferred resistance to ER stress-induced PCD in seedlings, the growth of Col-0 versus atcathb#62 seedlings on Tm (0.2μg/mL) containing MS plates was compared. Three phenotypes of seedlings were observed and categorized as ‘Dead’ (bleached cotyledons, no true leaves, short root), ‘Affected’ (yellow or green cotyledons, green true leaves, short root) and ‘Unaffected’(green cotyledon and true leaves, normal root) (Figure 3.4). Seedlings of Col-0 and atcathb#62 grown on Tm free plates exhibited normal growth; almost 99% of these seedlings were unaffected. Under Tm treatment, 94% of Col-0 seedlings were dead and 6% seedlings were affected. Atcathb#62 seedlings had 66% death and 34% affected (Figure 3.5). This demonstrated that down-regulation of cathepsin B increased resistance to ER stress in seedlings.

Figure 3.4. Defining seedling growth statuses during ER stress. Surface sterilized Arabidopsis seeds were sown on MS plates with or without 0.2μg/ml tunicamycin. Seedlings were observed seven days after transferring to growth cabinet (16 h light, 22 ℃). Different seedlings phenotypes were photographed and divided into three categories: Death; Affected and Unaffected. Scale bar is equal to 4 mm.

Figure 3.5. Comparison of Col-0 and Cathepsin B triple mutant seedlings grown under ER stress. Surface sterilized Arabidopsis seeds for wild type (Col-0) and Cathepsin B triple mutant (atcathb#62) were sown on MS plates with or without 0.2μg/ml tunicamycin (Tm). Seven days after transfer to growth cabinet, seedlings were scored in three different categories. The percentage of each category in each population was calculated. Three plates with at least 40 seedlings in each plate were scored. Error bar represents standard deviation.

To test whether up-regulation of cathepsin B would increase ER stress-induced cell death or not, I overexpressed AtCathB3 (At4g01610) in the Col-0 background. AtCathB3 fused with mRFP was transformed into Arabidopsis through agrobacterium-mediated transformation. Transformants were selected on MS medium containing 20 µg/mL hygromycin. Eight transformants, that survived on selection medium, were picked out and transferred to soil. To confirm AtCathB3::mRFP was expressed, leaves of those transformants were detached and observed under a fluorescent microscope. Only three transformants showed red fluorescence, which meant other five were false positive. One line in these three positive transformants showed unhealthy growth, so the other two were used for further experiment. 15 μg/mL Tm and same volume of DMSO were infiltrated into Col-0, AtCathB3 OX 1# and AtCathB3 OX 2# lines. Ion leakages were measured at various days post infiltration (dpi). AtCathB3 OX #1 showed fairly similar ion leakages profile to Col-0. AtCathB3 OX #2 had more ion leakages during the first five days after infiltration, but had lower ion leakages on day seven (Figure 3.6). However, the difference between AtCathB3 OX #2 and Col-0 were not significant. This suggested that overexpressing one cathepsin B paralogue, AtCathB3, could not promote ER stress- induced PCD.

Figure 3.6 Tm induced ion leakages on AtCathB3 overexpression lines. 15μg/ml tunicamycin was infiltrated in leaves of Col-0 and two AtCathB3 overexpression lines. DMSO was also injected as mock. Leaves discs were punched out at various days post infiltration (dpi) and floated in de-ionized water for one hour. Ion leakages were measured using a conductivity meter. Relative ion leakages were calculated by subtracting mock values to Tm treated samples values on the same leaf. Data points are means of relative ion leakages value for three biological repeats. Error bar represent standard deviation.

3.2.2 Analysis of cathepsin B paralogues redundancy

A previous report indicated that cathepsin B paralogues played redundant roles in the PCD during Pseudomonas syringae induced HR (McLallen et al., 2009). To test whether cathepsin B paralogues are redundant in ER stress-induced PCD, double and single mutant cathepsin B lines were used. Various cathepsin B down-regulated lines and Col-0 were sown on MS plate with or without 0.2μg/mL Tm. On Tm free MS plates, most of Col-0 and cathepsin B down-regulated seedlings were "unaffected", although sporadic ‘affected’ seedlings (less than 5%) occurred in atcathb2/atcathb3 and atcathb1/atcathb3 double knock out lines (Figure 3.7). On Tm plates, 90% of Col-0 seedlings were ‘dead’ and the last 10% were ‘affected’. Cathepsin B single knock-out atcathb1, atcathb2 and atcathb3 and double mutant atcathb1/atcathb3 had 94%, 92%, 93% and 94% ‘death’ seedlings respectively (Figure 3.7). These proportions were similar to that of the Col-0. Atcathb2/atcathb3 had slightly less ‘death’ seedlings at 83% of total seedlings. However, this difference was not significant (Figure 3.7).This suggested gene redundancy in this ER stress test at the seedling level.

To test whether cathepsin B double knockout lines were resistant to ER stress-induced PCD on leaves, I measured ion leakages on cathepsin B double knock out leaves. Ion

60 leakages after Tm infiltration of leaves from Col-0, atcathb1/atcathb3 and atcathb2/atcathb3 leaves were recorded at various days. Figure 3.8 showed that Col-0 and atcathb1/atcathb3 lines had similar profile. Atcathb2/cathb3 line had less ion leakage in the first five days but caught up with Col-0 eventually at day seven. However, the difference between Col-0 and atcathb2/cathb3 was not significant (p-value>0.05) (Figure 3.8). These data illustrated further confirmed that cathepsin B genes were functionally redundant during ER stress-induced PCD.

Figure 3.7 Cathepsin B gene redundancy in Arabidopsis seedlings under ER stress. Surface sterilized Arabidopsis seeds (single knock out: atcathb1, atcathb2 and atcathb3; double knock out: atcathb1/3 and atcathb2/3) were sown on MS plates with or without 0.2 μg/ml tunicamycin (Tm). Seven days after transfer to a growth cabinet, seedling death categories were scored, and the percentage of each category in the total population was calculated. At least 40 seedlings were scored for each triplicate.

Figure 3.8 Tm induced ion leakages in leaves of Col-0 and double knock out Cathepsin B lines. 15 μg/ml tunicamycin was infiltrated into leaves of Col-0 and cathepsin B double knock out lines (atcathb1/3 and atcathb2/3), water with the same volume of DMSO was also infiltrated as mock. Leaves discs were punched out at various days after infiltration (dpi) and floated on de-ionized water for 1 hour. Ion leakages were measured using a conductivity meter. Relative ion leakages were calculated by subtracting mock values from Tm treated samples values in the same leaf. Data points are means of relative ion leakages values of three biological repeats. Error bars indicated standard deviation.

3.2.3 Analysis of Cathepsin B expression in ER stress

As I have shown above, lacking cathepsin B leads to resistance to ER stress-induced cell death in Arabidopsis. Therefore it would be reasonable to assume that cathepsin B expression level would increase during ER stress-induced PCD. To test this hypothesis, I examined cathepsin B activity and transcript level. 15μg/mL Tm was infiltrated into Col-0 and atcathb#62 leaves to induced ER stress. Proteins were extracted three days after infiltration. Native cathepsin B in extracts of Col-0 and atcathb#62 was labeled using Biotin-DEVD-FMK. Labeled cathepsin B were blotted onto a PVDF membrane and developed. Three bands with sizes between 39 kDa and 25 kDa were labeled by Biotin- DEVD-FMK in Col-0 leaves extracts (Figure 3.9). These three bands represented various forms of cathepsin B during maturation as proved by mass spectrometry and N-terminal sequencing in previous work (Ge et al., in preparation). After Tm infiltration of leaves all these three bands gave stronger signals in Col-0 background (Figure 3.9). In leaves of the atcathb#62 line the signals of these bands were strongly reduced (Figure 3.9). This demonstrated that cathepsin B activity was increased during ER stress-induced PCD. It should be noted that a band which size was above 36 kDa was left in atcathb#62 Tm extractant. This might be due to incomplete silencing of AtCathB2 by RNAi. 62

Figure 3.9 Cathepsin B activity labelling after Tm treatment in wild type and cathepsin B triple mutant line. Tm was infiltrated into leaves of Col-0 and atcathb#62 lines, and leaves extracts were isolated from Tm treated and untreated samples. Leaf extracts were incubated with the substrate Biotin-DEVD-FMK, and labelled bands were visualized by western blot analysis using streptavidin-HRP. . Bands sizes were estimated using protein markers. Rubisco at 50 kDa was visualized by ponceau S staining of the same membrane as a loading control.

The observed increase of cathepsin B activity could be due to either more transcription or reduced protein turnover rate. To test whether increase of cathepsin B is resulted from an increase of cathepsin B transcript level, I analyzed cathepsin B transcript level. 15μg/mL Tm was infiltrated into Col-0 and atcathb#62 to induce ER stress. Total mRNA was isolated on the third day after infiltration. Cathepsin B transcript levels were determined by QRT-PCR. To compare cathepsin B transcript level among different samples, 18S mRNA level was used to normalize data. Three days after Tm treatment, the three cathepsin B paralogues transcript levels, which are presented as percentage to 18S transcript level, were increased. AtCathB3 had about 12 folds induction, AtCathB2 had 3 folds and AtCathB1 had 2 folds induction (Figure 3.10). As expected, transcript levels for the cathepsin B paralogues were either absent (AtCathB1 and AtCathB3) or strongly down regulated (AtCathB2) in leaves of the atcathb#62 lines (Figure 3.10). This suggested cathepsin B transcript level was increased by ER stress-induced PCD.

Figure 3.10. Induction of cathepsin B transcripts in ER stress. Total RNA was isolated from Col-0 and atcathb#62 leaves with or without infiltrating tunicamycin (Tm). Transcript levels of the three cathepsin B paralogues (AtCathB1, AtCathB2 and AtCathB3) were measured using QRT-PCR and sybr green. All data was normalized using 18s transcript level as a reference. Each bar represents the mean of triplicates. Error bars represent standard deviation.

3.2.4 Analysis of the role of PBA1 in ER stress-induced PCD

A previous paper reported that proteasome β subunit one (PBA1) possesses caspase-3-like activity and was capable of regulating the fusion between vacuole membrane and plasma membrane, one of the first step towards PCD induced by Pseudomonas syringae DC3000/avrRpm1 (Hatsugai et al., 2009). Since cathepsin B also has caspase-3-like activity (Ge et al., in preparation), it is important to know the interactions between these two proteases in order to understand the regulation of ER stress-induced PCD.

Caspase-3-like activity has been reported to be induced during ER stress-induced PCD (Zuppini et al., 2005). Since both cathepsin B and PBA1 have caspase-3-like activity, the source of increased caspase-3-like activity during ER stress-induced PCD was investigated in this study. Col-0 leaves were infiltrated with 15μg/mL Tm or DMSO (as mock) and caspase-3-like activity assay was carried out three days after treatment. Total caspase-3-like activity increased about 200% when compared to mock samples (Figure 3.11). To inhibit cathepsin B and PBA1 activity, I used CA074 and β-lactone respectively. CA074 reduced 30% and β-lactone 60% of total caspase-3-like activity in mock samples in vitro (Figure 3.11). In Tm treated samples CA074 inhibited 35% and β-lactone 61% of

64 total caspase-3-like activity (Figure 3.11). Therefore this experiment demonstrated that both cathepsin B and PBA1 contribute to the caspase-3-like activity in ER stress-induced PCD.

Figure 3.11 Cathepsin B and PBA1 contribution to caspase-3-like activity during ER stress-induced PCD three days after infiltration. Total soluble proteins were extracted at three days from Col-0 leaves with or without 15 μg/ml Tm infiltration. Caspase-3-like activity was measured using the fluorogenic substrate Az-DEVD-Rh110. Col-0 mock activity was set as 100%. CA074 and β-lactone were added to extracts and pre-incubated for 30 minutes at 37 °C before adding Az-DEVD-Rh110. Bars are the mean of triplicates and error bars represent standard deviation.

It was expected that cathepsin B and PBA1 would have similar function during ER stress- induced PCD as they both had caspase-3-like activity. In order to test this hypothesis, I co-infiltrated 15μg/mL Tm and 50μM β-lactone in Col-0 leaves to induce ER stress and inhibit PBA1 activity. PCD was then assessed by measuring ion leakages. Surprisingly, more ions leaked out from co-infiltrated leaves when compared to samples infiltrated with Tm only in the first five days (Figure 3.12). To confirm that caspase-3-like activity was really reduced by β-lactone in vivo, enzymatic assays were carried out. Proteins from Col- 0 leaves infiltrated with Tm with or without β-lactone were extracted three days after infiltration. Caspase-3-like activity in isolated proteins was measured using the fluorogenic substrate Ac-DEVD-Rh110. Tm treatment increased caspase-3-like activity up to about 200% of the activity from mock sample (Figure 3.13). Applying β-lactone together with Tm onto leaves reduced caspase-3-like activity increase to 120% of that from mock sample (Figure 3.13). This was less than the caspase-3-like activity induced by Tm only.

Figure 3.12. Effect of adding the inhibitor β-lactone to Tm induced ion leakages. 15 μg/ml tunicamycin was infiltrated into Col-0 leaves, water with the same volume DMSO was also infiltrated as mock. 50μM β-lactone was added to inhibit PBA1 activity. Leaves discs were punched out at various days post infiltration (dpi) and floated on de-ionized water for 1 hour. Ion leakages were measured using a conductivity meter. Relative ion leakages were calculated by subtracting mock value from Tm treated samples value from the same leaf. Data points are means of relative ion leakages value of three biological repeats. Error bar indicated standard deviation.

Figure 3.13. β-lactone efficiency of inhibition of proteasome activity in vivo. Total soluble proteins were extracted from Col-0 leaves with or without 15 μg/mL Tm treatment. 50 μM β-lactone was infiltrated to leaves with Tm to inhibit PBA1 activity in vivo. Caspase-3-like activity was then measured at five days using the fluorogenic substrate Az- DEVD-Rh110. Col-0 mock samples was set as 100%. Each bar represented mean of triplicate sample activities. Error bar represents standard deviation.

β-lactone is capable of inhibiting 20S proteasome by binding to its three β subunits, and PBA1 is one of them (Fenteany and Schreiber 1998). Due to the global inhibition activity

66 of β-lactone, an argument may be raised that the ion leakages increase caused by co- infiltration of Tm and β-lactone was not due to the inhibition of PBA1, but of some other proteasome β subunits. In another word, PBA1 function in ER stress-induced PCD was not properly investigated just by using β-lactone. To rule out this possibility, Pba1 RNAi lines were obtained from Professor Nishimura and used to investigate the subunit function on ER stress-induced PCD. Three pba1 RNAi lines, ipba1-8, ipba1-11 and ipba1-23, were used here. 15μg/mL Tm was injected into Col-0 and various pba1 RNAi lines. Ion leakages of different lines were measured at various days after infiltration. Comparison between Col-0 and ipba1-8 showed that more ions leaked out of ipba1-8 leaves at das three and day five, but similar ion leakage was seen at day seven (Figure 3.14). Tm induced more ion leakages in ipba1-11 than that in Col-0, but the difference was not significant (Figure 3.14). Ipba1-23 lines showed more ion leakage at day five and day seven than Col-0 (Figure 3.14). This suggested that silencing of pba1 promoted ER stress- induced PCD. The variation among different RNAi lines might be due to different RNAi efficiency.

Figure 3.14. Increased ion leakages after Tm infiltration of PBA1 silencing lines. 15 μg/ml Tm was infiltrated into leaves of Col-0, ipba1-8 ipba1 1-11 and ipba1-23, water with the same volume DMSO was also infiltrated as mock. Leaves discs were punched out at various days post information (dpi) and floated on de-ionized water for one hour. Ion leakages were measured using a conductivity meter. Relative ion leakages were deduced by subtracting mock values from Tm treated samples values in the same leaf. Data points are means of relative ion leakages value of three biological repeats. Error bar indicated standard deviation.

3.2.5 The function of proteasome α subunit in ER stress- induced PCD

It has been reported that blocking the α subunit of proteasome in tobacco could promote PCD (Kim et al., 2003). I therefore tested genetically whether knocking out the proteasome α subunit could increase PCD during ER stress-induced PCD or not. The Proteasome α6 (PAF2) subunit knock out line (T-DNA insertion) Paf2 was obtained from NASC and a homozygous line, paf2-6, was selected via genotyping. After Tm infiltration of Col-0 and paf2-6 leaves, ion leakages were recorded at various days post infiltration. The results showed that ion leakage of paf2-6 was higher compared to the Col-0 background from one to seven days. The difference was not large but significant (Figure 3.15). In addition, I wondered whether knocking out PAF2 could also reduce caspase-3- like activity or not. Extracts from Col-0 and paf2-6 leaves infiltrated with or without 15μg/mL Tm were measured. Three days after infiltration, caspase-3-like activity increased about 2.2 folds in Col-0 Tm extract compared to Col-0 mock as previously seen (Figure 3.16). Knocking out of PAF2 still showed about 2.2 fold increase of caspase-3- like activity during ER stress-induced PCD (Figure 3.16). This suggested that impairing proteasome function without affecting its caspase-3-like activity did not block ER stress- 68 induced PCD.

Figure 3.15 Comparison Tm induced ion leakages from Col-0 and paf2-6. 15 μg/ml tunicamycin was infiltrated into Col-0 and paf2-6 leaves, water plus same volume DMSO was also infiltrated as mock. Leaf discs were punched out at various time points as shown in graph and float in de-ionized water for 1 hour. Ion leakages were measured by conductivity meter. Relative ion leakages were deduced by subtracting mock ion leakage values from Tm treated samples ion leakages value. Data points are means of relative ion leakages value of three biological repeats. Error bar indicated standard deviation. Dpi means days post infiltration.

Figure 3.16. Tm induced caspase-3-like activity in Col-0 and paf2-6 leaves at three days post infiltration. Total protein was extracted from Col-0 and paf2-6 leaves with or without Tm treatment. Caspase-3-like activity was measured by using ac-DEVD-Rh110. Col-0 mock sample was set to 100%. Each column represented mean of triplicate sample activities. Error bars represent standard deviation.

3.2.6 Effect of Blocking proteasome activity on cathepsin B activity

The differential effect on PCD between cathepsin B and PBA1 was intriguing and I investigated whether the inhibition of PBA1 affected cathepsin B activity. Protein extracts after various treatments were incubated using Biotin-DEVD-FMK to label cathepsin B with caspase-3-like activity. I observed that a Tm treatment increased the activity labeling of cathepsin B (Figure 3.17). Meanwhile, co-infiltration of Tm and β-lactone increased even more cathepsin B activity labeling by Biotin-DEVD-FMK (Figure 3.17). This showed that blocking PBA1 activity increased cathepsin B protein amount consistent with the increased ion leakages observed.

Figure 3.17 Effect of the inhibitor β-lactone on Biotin-DEVD-FMK activity labelling. 15μg/mL Tm was infiltrated into Col-0 leaves and β-lactone was added to inhibit the proteasome subunit PBA1. Leaf protein extracts were isolated and incubated with Biotin- DEVD-FMK. Labelled proteins were visualized on western blot membrane using strep-HRP. Bands sizes were estimated according to standard protein markers. Rubisco (at 50 kDa) was visualized by ponceau s staining on the same membrane and used as a loading control.

3.3 Discussion

ER stress-induced PCD in plants is involved in several important physiological processes (summarized in introduction). Unlike research of ER stress-induced PCD in animal cells, pathways underlying ER stress-induced PCD are still elusive in plants. In this chapter, I have shown that cathepsin B is a mediator of ER stress-induced PCD. Reduction of PCD in the cathepsin B triple mutant line under Tm treatment supported the idea that cathepsin B is a positive regulator of ER stress-induced PCD. The positive regulator role of cathepsin B in PCD is in line with other reports, for example, the same cathepsin B triple mutant lines as I used in this research has been shown to alleviate pathogen induced HR PCD (Mclellan et al., 2009). It should be noted that the cathepsin B triple mutant line, atcathb#62, did not totally block ER stress-induced PCD. It might be due to incompletely silencing of AtCathB2 transcripts. This triple mutant line was generated by crossing AtCathB1 and AtCathB3 T-DNA insertion lines, and silencing AtCathB2 by RNAi. Because the AtCathB2 and AtCathB1 loci are very close, it is impossible to cross AtCathB1 and AtCathB2 knock out lines. As we can see from Figure 3.10, AtCathB2 transcript level did not drop down to zero in atcathb#62 line, although it had been strongly reduced.

Regulation of cathepsin B during ER stress-induced PCD in Arabidopsis was also examined in this chapter. I found both cathepsin B activity and transcript level were induced. As a protease, cathepsin B is expected to be processed before activation/maturation. It is assumed that only activated cathepsin B possesses caspase-3- like activity. However, affinity labeling of cathepsin B by Biotin-DEVD-FMK indicated that a 39 kDa band, presumably from full length cathepsin B, which presumably attributes to non-processed form cathepsin B, also has caspase-3-like activity. This implies that the regulation of cathepsin B activity does not totally rely on processing. Regulation at transcription stage may also play a role in controlling cathepsin B activity. This can be supported by two observations: 1. Up-regulation of cathepsin B transcript level has been firmly documented in this chapter; 2. Among three labeled forms of cathepsin B, the non- processed cathepsin B increased more than the other two. More experiment is needed in the future to prove this.

Gene redundancy in a certain gene family sometimes impedes well characterization of individual gene function. Cathepsin B gene family is composed of three paralogues in

Arabidopsis genome. Previous reports have asserted that cathepsin B paralogues exhibited redundant roles in regulating HR induced PCD (Mclellan et al., 2009). Redundancy in Cathepsin B function was also analyzed here. Neither single nor double cathepsin B mutant lines were not resistant to ER stress-induced PCD. Although AtCathB2 and AtCathB1 double mutant Arabidopsis are not available, cathepsin B paralogues redundant roles during ER stress-induced PCD still can be confirmed.

To address the question whether both cathepsin B and PBA1 contribute to the caspase-3- like activity during ER stress-induced PCD or not, I analyzed constitution of the caspase- 3-like activity in Arabidopsis during ER stress-induced PCD. The inhibition of caspase-3- ike activity by cathepsin B and PBA1 inhibitors and the depletion of Biotin-DEVD-FMK labeling in cathepsin B triple mutant line confirmed that cathepsin B contributed to caspase-3-like activity during ER stress-induced PCD. This finding further supported two previous researches that both cathepsin B and PBA1 possess caspase-3-like activity (Ge et al., in preparation; Hatsugai et al., 2009). However, neither activity inhibition of PBA1 activity nor gene silencing of PBA1 was able to reduce ER stress-induced PCD. In contrast, inhibition of PBA1 accelerated PCD induced by ER stress. PBA1 was shown to mediate PCD via membrane fusion during bacteria pathogen attack (Hatsugai et al., 2009). These opposite roles of PBA1 are possible due to different PCD inducers. During bacteria attack, PCD is dependent of PBA1 caspase-3-like activity. But during ER stress, PBA1, as a critical component of 20s proteasome is involved in ERAD (Ahner and Brodsky 2004). Inhibition of PBA1 during ER stress may impair ERAD which subsequently cause more stresses to cells. In addition, two other reports also showed that blocking proteasome activity by virus-induced gene silencing of tobacco α6 subunit was able to induce PCD in tobacco leaves (Amanso et al., 2011; Lee et al., 2003). These two reports were in line with the results that proteasome was a negative regulator of PCD. Another explanation for the negative role of the proteasome in ER stress-induced PCD lies in the interaction between proteasome and cathepsin B. Inhibition of proteasome could induce cathepsin B activity. This implies that proteasome may act upstream of cathepsin B and regulate cathepsin B by degrading. So inhibition of proteasome reduces cathepsin B degradation which subsequently promotes PCD.

Ion leakage data varied when using PBA1 RNAi lines to investigate the roles of PBA1 in ER stress-induced PCD (Figure 3.14). Ipba1-8 had more ion leakage increase than the other two RNAi lines (ipba1-11 and ipba1-23). This may be because that repression

72 efficiency of PBA1 gene expression by RNAi was different in these three lines. Ipba1-8 may have less PBA1 gene expression than the others. The PBA1 expression in these three lines has been examined (Hatsugai et al., 2009). No expression of PBA1 has been detected in these three lines using semi quantitative RT-PCR. More accurate technique, like quantitative RT-PCR, needs in the future to check PBA1 expression in these three lines. I also noted that applying β-lactone accelerated ion leakage more than RNAi lines did. As the proteasome is a complex, when PBA1 subunit was silenced, other subunit may compensate its function. But β-lactone is able to block more proteasome activity by covalently binding to more β subunits. This might be the reason why infiltration of β- lactone with Tm induced more ion leakages than that resulted from PBA1 RNAi.

I have presented findings of cathepsin B genetic function during ER stress-induced PCD. How cathepsin B regulates ER stress-induced PCD at molecular level is unclear so far. Investigation of cathepsin B molecular function will be presented in next chapter.

Chapter 4 Mode of action of cathepsin B in

ER stress-induced PCD

4.1 Introduction

ER stress-induced PCD in Arabidopsis was shown to be mediated by vacuole rupture (Qiang et al., 2012). This suggested that ER stress-induced PCD was vacuolar PCD. In our study, mutant lines for cathepsin B significantly retarded ER stress-induced PCD but not totally block it. Two hypotheses may explain this result: 1. Lacking cathepsin B merely merely delayed the vacuolar PCD signal transduction. This may be due to incomplete silencing of cathepsin B; 2. Vacuolar PCD was blocked in cathepsin B triple mutant line, but another type of PCD, for instance necrotic PCD, was induced at late stage of ER stress-induced PCD. To test these hypotheses, I compared ER stress-induced PCD in Col-0 and atcathb#62 leaves. Several characteristic morphological and biochemical features of plant PCD were carefully examined to identify the PCD type activated in Col- 0 and atcathb#62 cells during ER stress-induced PCD.

Tonoplast rupture is a critical step in vacuolar PCD. VPE controls tonoplast rupture in various PCD (Hatsugai et al., 2004; Qiang et al., 2012). In addition, Nishimura and colleagues speculated that activation of cathepsin B was under control of VPE (Hara- Nishimura et al., 2005; Shirahama-Noda et al., 2003). This implies that cathepsin B is downstream of VPE in vacuolar PCD pathway. It is also possible that cathepsin B controls tonoplast rupture. Whether this hypothesis is true or not will be examined in this chapter.

The subcellular localisation of cathepsin B needs to be determined, as it is important to understand the possible correlation between cathepsin B and VPE. In addition, to execute its function, cathepsin B needs to reach its appropriate subcellular localisation. The subcellular localisation of cathepsin B in both Arabidopsis and Nicotiana benthamiana were examined in this chapter. By employing various RFP-tagged cathepsin B constructs, I tracked cathepsin B localisation in vivo. I also adopted enzymatic assays and activity labelling to understand the genetic interaction between VPE and cathepsin B.

4.2 Results

4.2.1 Which PCD type is mediated by cathepsin B?

In the previous chapter, I have shown that ion leakages in atcathb#62 lines treated with Tm did not increased until seven days after infiltration (Figure 3.1). What caused the delay of ion leakage increase was intriguing me. One possibility was that it was just simply delayed due to the only partially blocked ER stress-induced PCD signal transduction pathway. A second hypothesis was that another type of PCD was induced when the cathepsin B mediated PCD was blocked. To test the latter hypothesis, I compared several morphological and biochemical features between Col-0 and atcathb#62 according to the consensus criteria proposed to distinguish vacuolar PCD and necrotic PCD (Van Doorn et al., 2011). Leaf samples subjected to 15 µg/mL Tm treatment for seven days were fixed, processed and imaged by transmission electron microscopy (TEM). Most Col-0 leaves after seven days of Tm treatment exhibited empty cells (Figure 4.1). However, cytosolic contents, such as chloroplast, were largely unprocessed in the Tm treated cells of atcathb#62 leaves (Figure 4.1). The plasma membrane of Col-0 was still attached to the cell wall while plasma membrane in atcathb#62 detached from the cell wall and appeared disrupted in places (red arrow Figure 4.1).

Besides morphological features, biochemical features, including ATP level and ROS accumulation, are other criteria proposed for distinguishing vacuolar PCD from necrotic PCD. In the research on animal cells, a drop in ATP level and ROS accumulation due to changes in mitochondria and lysosome are markers for programmed necrosis (Galluzzi et al., 2012; Kroemer et al., 2009). It is believed that both of them also occur in plant necrotic PCD (Van Doorn et al., 2011). I first measured ATP level in Col-0 and atcathb#62. 15 µg/mL Tm was infiltrated into leaves to trigger ER stress-induced PCD. ATP was isolated from leaf samples at designated time points and measured using the ENLITEN® luciferase ATP kit. Col-0 and atcathb#62 leaves without Tm treatment showed similar ATP level. Col-0 had around 7×106 relative light units (RLU) and atcathb#62 had 6×106 RLU (Figure 4.2). ATP level did not change three days after Tm treatment but decreased to 5×106 RLU on day seven after ER stress induction in Col-0 (Figure 4.2). atcathb#62 increased its ATP level up to 9.5×106 RLU on day three, but fell back to 4×106 RLU at seven days after induction (Figure 4.2). However, the difference between Col-0 and atcathb#62 ATP level on day seven were not significant, as T-test p value was 0.06>0.05. This suggested ATP level did not drop significantly in cathepsin B

75 triple mutant Arabidopsis. An unexpected increase of ATP level on day three in cathepsin B triple mutant background was seen. This might be due to better preservation of cell organelles, such as mitochondria, in cathepsin B triple mutant line.

Figure 4.1. Morphology of Col-0 and atcathb#62 dead cells after Tm treatment. Col-0 and atcathb#62 cells were subjected to TEM analysis seven days after 15 µg/mL Tm treatment. One mm2 leaf discs were cut off and fixed before observing. Two representatives of the whole cells per genotype are shown here (top panel). Cell details (lower panel,) showing the plasma membrane (red arrow) and the cell wall. Scale bar is equal to 200nm.

Figure 4.2. ATP level in Col-0 and atcathb#62 cells during ER stress-induced PCD. Col-0 and atcathb#62 leaves subjected to 15 µg/mL Tm treatment were collected at three and seven days after infiltration, DMSO was infiltrated into leaves instead of Tm for mock samples. Total ATP was extracted. ATP amount in each sample is presented as Relative Light Unit (RLU) and normalized against leaves fresh weight. Three independent leaves samples were measured in each treatment. Error bar is standard deviation.

ROS accumulation is another recommended criterion for distinguishing vacuolar PCD from necrotic PCD. ROS accumulation was also tested here. Col-0 and atcathb#62 leaves were infiltrated with Tm to induce ER stress. In each leaf sample, only the left half was infiltrated with Tm, the other side was infiltrated with water plus DMSO as mock. Leaves were detached on day seven and incubated with DAB (pH7.0) solution in the dark overnight. DAB reacts with ROS and gives a brown precipitate in tissues. Chlorohydrate was used to discolour leaf pigment. The brownish area indicated the leaf area where ROS accumulated. ROS did not accumulate in Col-0 mock and Tm treated sample (Figure 4.3). ROS accumulation was observed in some atcathb#62 leaves but no others (Figure 4.3). The inconsistence of ROS accumulation in atcathb#62 leaves implied that ROS was not a substantial criterion for distinguishing different types of PCD. This issue will be brought up again in discussion section.

Figure 4.3. ROS accumulation in Col-0 and atcathb#62 Arabidopsis leaves during ER stress-induced PCD. ROS in Col-0 and atcathb#62 subjected to Tm treatment (left half of each leaf) were visualized by DAB staining at seven days after infiltration. Detached leaves were incubated over-night with DAB in the dark. Leaves were cleared using chlorohydrate and mounted for microscope observation. Two representative leaves are showed. Brownish areas indicate DAB stained area.

The two biochemical features examined above did not help to fit cathepsin B in vacuolar PCD pathway. This raises the question of whether cathepsin B is involved in necrotic PCD? In order to find out whether cathepsin B also participated in necrotic PCD, I examined cathepsin B’s role in necrotic PCD using Yariv reagent. Yariv reagent was reported to induce necrotic PCD in Arabidopsis (Gao and Showalter 1999). In a preliminary trial, I found that Yariv reagent at less than 3mg/mL was not able to induce any PCD phenotype (data not shown). By contrast, infiltration with 3mg/mL Yariv reagent into Arabidopsis leaves caused cell death in both Col-0 and atcathb#62 leaves at three days after infiltration (Figure 4.4). Infiltration with water only did not result in any change in the leaves’ appearance (Figure 4.4). Yariv reagent-induced PCD was further confirmed by measuring ion leakage. Ion leakages of Col-0 and atcathb#62 leaves were recorded three days after infiltration. Infiltration with Yariv reagent resulted in 35 μS/cm ion conductance in Col-0 leaves (Figure 4.5). Conductance induced by the Yariv reagent in atcathb#62 leaves was about 28 μS/cm on day three (Figure 4.5). However, the difference between the conductance values of Col-0 and atcathb#62 was not significant, as the T-test p-value was 0.189>0.05. This suggested cathepsin B down-regulation did not block Yariv reagent-induced necrotic PCD.

Figure 4.4. Yariv-induced necrotic PCD in leaves. 3 mg/mL Yariv reagent was infiltrated into Col-0 and atcathb#62 leaves. The left half of leaves was infiltrated with Yariv reagent, the right half of leaves was infiltrated with water. Leaves were detached at three days after infiltration and photographed. Two representatives of leaves are shown here. 78

Figure 4.5. Yariv reagent-induced ion leakage in Col-0 and atcathb#62 leaves on day three. 3 mg/mL Yariv reagent was infiltrated into Col-0 and atcathb#62 leaves. Ion leakage was measured at three days after infiltration. Relative ion leakage was calculated by subtracting ion leakage of mock sample from Yariv reagent treated sample in the same leaf. Values are the average of three biological replicates. Error bar is standard deviation. A T-test indicated that the difference between Col-0 and atcathb#62 ion leakage was not significant, P- value=0.189.

4.2.2 Does cathepsin B control tonoplast rupture during vacuolar PCD?

The results above have shown that cathepsin B mediates vacuolar PCD but not necrotic PCD. One critical step in vacuolar PCD is tonoplast rupture. I therefore examined whether cathepsin B was required for tonoplast rupture. Tonoplast rupture has already been reported in ER stress-induced PCD (Qiang et al., 2012). In this section I examined the tonoplast behaviour during ER stress-induced PCD in a cathepsin B mutant genetic background. 15 μg/mL Tm was infiltrated into Col-0 and atcathb#62 leaves to induce ER stress. Three days after infiltration, leaves discs were cut off and subjected to TEM analysis. This observation was carried out at a different time point compared with the observation in Figure 4.1. It was because tonoplast rupture occurred before cleaning of cell contents. Tm free Col-0 and atcathb#62 leaves exhibited intact vacuole membrane (Figure 4.6 A and C red arrow). However, the Tm treatment resulted in rupture of vacuole membranes in both Col-0 and atcathb#62 cells (Figure 4.6 B and D red arrow).

Figure 4.6. Tonoplast rupture in Col-0 and atcathb#62 cells during ER stress-induced PCD. Col-0 and atcathb#62 leaves infiltrated with 15 μg/mL Tm or same volume DMSO (mock) were collected at three days after infiltration. Cells were observed under TEM. Images of cells structures were taken. Cells representative of each treatment are shown here. Red arrows are pointing at vacuole membrane. Scale bar = 200nm

To complement single cell TEM images and have a good estimate of vacuole membrane integrity in cells over the whole Tm infiltrated area, I used BCECF (2’,7’‑bis‑(2‑ carboxyethyl)‑5‑(and‑6)‑carboxy fluorescein)-AM (acetomethyl) staining of fresh leaf tissue. BCECF-AM is a cell membrane permeable chemical. BCECF-AM is non- fluorescent until its AM group is removed by cytoplasmic esterases. Moreover, the fluorescence of BCECF is dependent on pH. An acidic compartment, for example vacuole, results in stronger fluorescence. Col-0 and atcathb#62 leaves were infiltrated with 15 μg/mL Tm or same amount of DMSO as mock. Three same-size leaf discs within infiltrated area were punched out and subjected to BCECF staining. Leaf discs were incubated with BCECF at room temperature for 10 minutes. Observation using a microscope at high magnification confirmed that BCECF was localised inside the vacuoles of pavement cells (Figure 4.7 lower panel). Fluorescence images of each disc were taken under a fluorescent microscope with a GFP filter. To make sure the difference in fluorescence intensity was not due to alterations in exposure time, images were taken under the same exposure parameters. The green fluorescence emitted from BCECF was expected to diminish if the tonoplast was ruptured. Three representative leaf discs from each sample are presented in Figure 4.7. Large areas of green fluorescence were visible

80 in mock samples of both Col-0 and atcathb#62 leaves (Figure 4.7 upper panel). However, the Tm treatment strongly diminished the green fluorescence in both Col-0 and atcathb#62 (Figure 4.7 upper panel). This suggested that cathepsin B deletion did not block vacuole rupture during ER stress induced PCD. This is consistent with the TEM observation.

Figure 4.7. Vacuole integrity in Col-0 and atcathb#62 using BCECF staining after Tm treatment. Col-0 and triple mutant atcathb#62 leaves were infiltrated with 15 μg/mL Tm to induce ER stress. Same amount DMSO was infiltrated as mock. Three days after infiltration, three leaves discs were punched out and incubated with BCECF. Three representative discs of Tm treated or non-treated Col-0 and atcathb#62 leaves are shown. Images were photographed under a fluorescent microscope with GFP filter. Scale bar= 2mm. Two representative of high magnification images of Col-0 and atcathb#62 cells stained with BCECF-AM are presented in lower panel. Scale bar is equal to 15 µm.

4.2.3 Analysis of the subcellular localisation of cathepsin B

A previous report speculated that the activation of cathepsin B may be under the control of VPE, a vacuolar enzyme (Hara-Nishimura et al., 2005). In addition, VPE controls tonoplast rupture and vacuolar PCD (Van Doorn et al., 2011; Qiang et al., 2012). This implies that a functional interaction between VPE and cathepsin B exists in vacuolar PCD. Therefore, a possible genetic interaction between cathepsin B and VPE was examined here. First of all, I investigated cathepsin B subcellular localisation. Subcellular localisation is critical for most proteins to fulfil their function. For cathepsin B, the subcellular localisation may determine its possible physical interaction with VPE and its enzymatic activity due to the different pH environment in various cellular compartments. To understand the spatial distribution of cathepsin B in vivo, I carried out a subcellular localisation analysis. The full length AtCathB3 fused at C-terminus with monochrome red fluorescence protein (mRFP), AtCathB3::mRFP, was obtained from Dr. Yuan Ge (Figure 4.8). It has been shown in previous data of our lab that both the N-terminus and C- terminus of cathepsin B are cleaved during maturation (Yuan GE et al., in preparation). This feature sets an obstacle for tracking subcellular localisation of cathepsin B once it is mature. To circumvent this problem, I made another construct in which several amino acids at the C-terminus were deleted, and named it AtCathB3ΔC::mRFP (Figure 4.8). The C-terminus cleavage site was predicted according to alignment between AtCathB3 and human cathepsin B and the amino acid sequence after K380 (including K380) was removed (Figure 4.8).

Figure 4.8. A diagram the AtCathB3::mRFP constructs used. Two forms of AtCathB3 construct are presented. Each constructs was fused with mRFP (red). The Full length form construct keeps its C-terminus (green): AtCathB1610::mRFP. The truncated type of AtCathB3 lacks its C-terminus: AtCathB1610DC::mRFP. The c-terminus is removed from K380. The removed amino acid sequence is indicated in the diagram and labelled green.

AtCathB3::mRFP under the control of the 35S promoter was transformed into Arabidopsis Col-0 via agrobacterium-mediated transformation and several transgenic lines were obtained. Three high expresser lines were selected by observing mRFP fluorescence under the microscope. To observe cathepsin B subcellular localisation, epidermal cell layers were peeled off from leaves and mounted on glass slides. Fluorescence images were photographed using a fluorescence microscope. As we can see in Figure 4.9, the red fluorescence signal occupied most of the space within epidermal cells. To see clearly whether the red fluorescence signal existed in the apoplast or not, a plasmolysis was carried out to separate a protoplast and the cell wall. After plasmolysis 15 minutes with 0.5M mannitol, protoplasts had perfectly detached from the cell wall. No red signal was observed in apoplast or cell wall, all the red signal was confined to the vacuole (Figure 4.9).

Figure 4.9. Subcellular localisation of AtCathB3::mRFP in Arabidopsis. Leaf pavement cells of Arabidopsis stable transformants for 35S::AtCathB3::mRFP were visualized under fluorescence microscopy TX2 filter. Plasmolysis (0.5 M mannitol) was applied to separate protoplast from cell wall (lower panel). Cells images were photographed under fluorescence (left) and bright field (middle), and merged together (right). Scale bar is equal to 10μm.

The subcellular localisation of the AtCathB3 orthologue in tobacco has been reported to be in the cell wall (Gilroy et al., 2007). In order to know whether AtCathB3 would have a different subcellular localisation in tobacco, AtCathB3::mRFP was expressed in Nicotiana benthamiana by an agro-infiltration method. After three days incubation, leaf epidermal layers were peeled off and mounted on slides. Under a fluorescence microscope, a bright red fluorescence was observed at the edges of epidermal cell (Figure 4.10). Unfortunately, the background fluorescence emitted from chloroplasts interfered with the mRFP signal inside cell protoplasts. To get a better image, I peeled epidermal cells again carefully avoiding most of mesophyll cells, and applied plasmolysis (0.5 M mannitol). Under this condition, the red fluorescence signal was seen both inside the vacuole and the cell wall (Figure 4.10).

Figure 4.10. Subcellular localisation of AtCathB3::mRFP in Nicotiana benthamiana. 35S::AtCathB3::mRFP was transfected into Nicotiana benthamiana by agro-infiltration. Leaves pavement cells were visualized under fluorescence microscopy (filter). A 15 minutes plasmolysis (0.5 M mannitol) was applied to separate protoplast (yellow arrow) from cell wall (black arrow) (lower panel). Cells images were photographed under TX2 filter (left) and bright field (middle), and merged together (right). Scale bar is equal to 10μm.

The expression of AtCathB3::mRFP in Arabidopsis was confirmed using western blot. Soluble protein of non-transformed and transformed leaves was extracted and an anti-RFP antibody was used to detect the expression of AtCathB3::mRFP. A band whose size was

84 between 37 kDa and 28 kDa was detected in the AtCathB3::mRFP transgenic sample, whereas this band was absent in non-transformed samples (Figure 4.11 left). The size was similar to the expected mRFP (26 kDa) plus C-terminus of cathepsin B, which meant that mRFP was cleaved off from AtCathB3.

The cleavage of mRFP from AtCathB3 elicits a problem that the vacuolar localisation of cathepsin B may not be its final destination. To find out the final destination of cathepsin B, AtCathB3ΔC::mRFP was transfected into Arabidopsis seedlings. First, a western blot analysis with an mRFP antibody was carried out to check whether mRFP was still attached to AtCathB3ΔC in the leaf cells. A band of an approximate size of 50 kDa was presented in AtCathB3ΔC::mRFP sample (Figure 4.11 right). This size was similar to the expected size of a mature AtCathB3ΔC::mRFP without mRFP cleavage. It was concluded that mRFP was still attached to AtCathB3ΔC in vivo.

Figure 4.11 (left panel). Detection of AtCathB3::mRFP by western blot. Total soluble protein was extracted from leaves of a transgenic plant harbouring a 35S::AtCathB3::mRFP construct. The protein samples were separated using SDS-PAGE, transferred to a nylon membrane and incubated with an anti-RFP antibody. A single band between 28 kDa and 37 kDa was present in transformed sample only and corresponds to the expected size of mRFP. The unspecific band at 37 kDa is used as a sample loading control.

Figure 4.11 (right panel). Detection of AtCathB3ΔC::mRFP by western blot Total soluble protein was extracted from leaves of a transgenic plant harbouring a 35S::AtCathB3ΔC::mRFP construct. The protein samples were separated using SDS-PAGE, transferred to a nylon membrane and incubated with an anti-RFP antibody. Expression of AtCathB3ΔC::mRFP was detected with anti-RFP antibody in western blot. A single band at 50 kDa was presented in transformed sample only, corresponding to the full length fusion protein. The unspecific band at 37 kDa is used as a sample loading control.

I subsequently analysed the subcellular localisation of AtCathB3ΔC::mRFP in Arabidopsis by co-transfection with a subcellular marker. AtCathB3ΔC::mRFP was transiently expressed under the 35S promoter in Arabidopsis seedlings (three day old) with GFP tagged sphingosine kinase 2 (SPKH2::GFP) whose localisation had been shown 85 to be in plasma membrane (Worrall et al., 2008; Marion et al., 2008). Agrobacterium culture containing AtCathB3ΔC::mRFP and SPHK2::GFP were incubated with Arabidopsis seedlings and co-infiltrated into seedlings under one atm vacuum. The seedlings cotyledons were subjected to the confocal microscopy observation three days after infiltration. Fluorescence imaging showed that all red fluorescence signals were confined within the green fluorescence border marking the plasma membrane (Figure 4.12). This suggested that the fusion cathepsin B without C-terminus was able to localize inside the vacuole like the full length construct.

Figure 4.12. Subcellular localisation of AtCathB3ΔC::mRFP in Arabidopsis. 35S::AtCathB3ΔC::mRFP was co-expressed with 35S::SPHK2::GFP using agro-infiltration in seedlings. Leaves pavement cells were visualized under confocal fluorescence microscope. SPHK2::GFP is a marker of the plasma membrane. Red fluorescence (middle) and green fluorescence (right) images are presented with a merged image (left). Scale bar is equal to 10μm

To examine the localisation of truncated cathepsin B in tobacco cells, AtCathB3ΔC::mRFP was transfected into Nicotiana benthamiana. Expression of AtCathB3ΔC::mRFP in Nicotiana benthamiana was mediated by biolistic bombardment. A leaf disc was cut out and placed on a petri dish with agar. 35S::AtCathB3ΔC::mRFP was coated on gold particles and delivered into leaf cells using a biolistic pds-1000/he particle delivery system. Leaf cells were observed under a fluorescence microscope two days after bombardment. In the fluorescent images, red fluorescence was localized only along the periphery of pavement cell suggesting a cell wall localisation as published by Gilroy et al., 2007 (Figure 4.13). This suggested that in Nicotiana AtCathB3ΔC::mRFP does not enter the vacuole.

Figure 4.13. Subcellular localisation of AtCathB3ΔC::mRFP in Nicotiana benthamiana. 35S::AtCathB3ΔC::mRFP was transformed into Nicotiana benthamiana leaves using a biolistic method. After two days expression, leaves pavement cells were visualized under a fluorescence microscope. Photos were taken under TX2 filter cube (left) and bright field (middle). On the right is the merged image of red fluorescence (left) and bright field (middle) images. Scale bar: 5μm.

4.2.4 Does VPE control the activation of cathepsin B?

To understand whether VPE deletion may affect cathepsin B activity or not, I carried out enzymatic assays to measure caspase-3-like activity in vpe quadruple knock out line (vpe null). 15 μg/mL Tm was used to induce ER stress in both Col-0 and vpe null mutant leaves. The same volume DMSO was infiltrated into leaves as mock. Soluble protein of Col-0 and vpe null leaves were extracted and subjected to enzymatic assays. Three day treatment with Tm resulted in caspase-3-like activity increased from 100% to 250% in Col-0 (Figure 4.14). Vpe null leaves contained similar caspase-3-like activity to Col-0 in Tm free treatment (Figure 4.14). The Tm treatment also induced caspase-3-like activity in the vpe null sample, but this increase was lower than that in Col-0 and reached less than 200% of the mock treatment (Figure 4.14).

Figure 4.14. Caspase-3-like activity in vpe null during ER stress-induced PCD. 15 μg/mL Tm was used to induce ER stress in Col-0 and vpe null leaves. DMSO was infiltrated as mock. Caspase 3-like activities were determined three days after infiltration. Col-0 mock was set as 100%. Error bar = standard deviation of technical triplicates.

Since cathepsin B activity is not the only component of Arabidopsis caspase-3-like activity, the decrease in caspase-3-like activity in vpe null line could be due to a decline of cathepsin B activity or of PBA1 activity. To measure cathepsin B activity in vivo in vpe null line, I labelled cathepsin B activity with Biotin-DEVD-FMK. 15 μg/mL Tm and DMSO were infiltrated into Col-0 and vpe null leaves. Soluble proteins were isolated at three days after infiltration. Extracted proteins were subjected to activity labelling. Col-0 leaves subjected to Tm treatment showed stronger labelling of Biotin-DEVD-FMK to cathepsin B than Tm free sample (Figure 4.15). Surprisingly, vpe null showed clearly increased binding of Biotin-DEVD-FMK to cathepsin B in both samples with or without infiltrating of Tm (Figure 4.15). Clearly a Tm treatment still caused an increase in activity labelling of cathepsin B in vpe null sample as it does in Col-0 (Figure 4.15).

Figure 4.15. Activity labelling of Cathepsin B in Col-0 and vpe null. Col-0 and vpe null leaves were treated with or without 15 μg/mL Tm. Soluble proteins extracted from Col-0 and vpe null leaves were incubated with Biotin-DEVD-FMK followed by western blot. To visualise cathepsin B on blot, streptavidin-HRP was used to detect labelled cathepsin B. Bands corresponding to cathepsin B are shown. Rubisco (Rb) was visualized via ponceau S staining and used as loading control (lower panel).

To address the question of whether the increased amount of cathepsin B activity in vpe null caused more PCD or not, I measured ion leakage on Col-0 and vpe null lines in the context of ER stress-induced PCD. 15 μg/mL Tm was infiltrated into Col-0 and vpe null leaves to induce ER stress. DMSO was also injected as mock. Three leaves discs were punched out in each leaf and the ion leakage was measured using a conductivity meter at designated time points. Prolonged ER stress caused an ascending profile of ion leakage in 88

Col-0 leaves from nearly 0 μS/cm to about 70 μS/cm (Figure 4.16). Ion leakage in vpe null line only reached less than 40 μS/cm on day seven, though the overall profile of ion leakage was still ascending (Figure 4.16). This illustrated that knocking out of VPE down-regulated ER stress induced PCD but did not suppress it. At first glance, a partial reduction in PCD is consistent with the partial reduction in total caspase-3-like enzymatic activity seen in vitro but is not consistent with the activity labelling results. These observations can however be reconciled as discussed below.

Figure 4.16. Ion leakages of Col-0 and vpe null line during ER stress-induced PCD. Ion leakages of Col-0 and vpe null leaves treated with or without 15 μg/mL Tm were recorded from day one to day seven every other day. Tm and same volume DMSO were infiltrated into different halves of one leaf. Three discs were punched out in each half of a leaf. The relative ion leakage was obtained by subtracting ion leakage of mock samples from Tm treated samples. Ion leakages were measured in biological triplicates. Error bars represent standard deviation.

4.3 Discussion

The cell ultrastructure analysis of Col-0 and atcathb#62 using TEM during ER stress- induced PCD presented contrasting morphology in dead cells. Col-0 dead cells had clean and empty cell content whereas dead atcathb#62 cells were left with much cell contents unprocessed. Moreover, there was an evidence of different plasma membrane behaviour, intact plasma membrane in Col-0 and ruptured plasma membrane in atcathb#62. These observations classified ER stress-induced PCD in Col-0 as vacuolar PCD and in atcathb#62 as necrotic PCD. However, biochemical features were more variable and not conclusive in Col-0 and atcathb#62 leaves. Total ATP concentration did not separate the two types of PCD and may not be a reliable criterion to use. ROS accumulation was found to be variable. To detect ROS accumulation in leaves, I adopted DAB staining. Leaves were left in DAB solution overnight, so the penetration of DAB into leaves just relied on how efficiently leaves could uptake the DAB solution. Different soaking speeds may have caused the observed inconsistence of staining. This could be improved in the future using vacuum infiltration or adding surfactant. The implications of the important finding that only one type of cell death is cathepsin B-dependant will be further discussed in the general discussion.

The subcellular localisation of cathepsin B provides a spatial control for the execution of its function. mRFP tagged versions of the full length and C-terminus truncated cathepsin B showed that vacuole was the native localisation for cathepsin B in Arabidopsis. This is different from the subcellular localisation of one tobacco cathepsin B isoform which has been shown in the apoplast (Gilroy et al., 2007). Interestingly the same apoplast localisation of AtCathB3 in tobacco leaves was confirmed by expressing AtCathB3::mRFP in tobacco leaves. However, AtCathB3::mRFP localisation in tobacco suggested that AtCathB3 exhibited a dual localisation in apoplast and vacuole. As the C- terminal mRFP of full length cathepsin B was cleaved during maturation, the localisation of mRFP might not reflect the true localisation of mature cathepsin B in tobacco. Usually, secreted proteins can be secreted via Golgi apparatus to either the vacuole or the cell wall (Denecke et al., 1990; Hanton et al., 2007). The processing of cathepsin B might occur in the ER or the Golgi, so that the removed C-terminus plus mRFP was transported to the vacuole while the rest of cathepsin B was secreted to apoplast in tobacco. The mRFP signal from AtCathB3::mRFP in the apoplast could be due to incomplete processing of cathepsin B. The different localisations of cathepsin B in Arabidopsis and Nicotiana benthamiana may be due to species specificity of transport. Further research on the 90 transportation pathway in Arabidopsis and Nicotiana benthamiana, for example examining cathepsin B localisation in various transporter protein mutant plants, may shed light on this issue. Another possibility of different localisations of cathepsin B may be resulted from stresses brought by transformation technique, such as DNA bombardment. Investigation of translocation of cathepsin B under stress conditions in the future may be helpful to understand the localisation of cathepsin B under different conditions.

VPE has been suggested to be responsible for activation of cathepsin B (Hara-Nishimura et al., 2005). However, activity labelling of cathepsin B in vpe null Arabidopsis negated this hypothesis, because more cathepsin B activity was observed in vpe null line in the presence or absence of Tm. In addition, in the vpe null background cathepsin B was still able to undergo processing, this was consistent with a previous finding on animal cathepsin B auto-processing (Mach et al., 1993). This answers the main question I wanted to address by using the vpe null line. It should be noted that increased cathepsin B activity in vpe null line seems to be opposite of what would be expected from the ion leakage result of vpe null line. As I have shown in the previous chapter, cathepsin B acts as a positive regulator of ER stress induced PCD. So more ion leakage would be expected to match more cathepsin B activity. But in this chapter, more cathepsin B activity and less ion leakage were observed in vpe null. VPE controls tonoplast rupture, therefore the tonoplast should keep intact after Tm treatment in the absence of VPE. Cathepsin B is localised inside the vacuole and the release of cathepsin B into the cytoplasm from the vacuole may be important for executing its death function. So no matter how much more cathepsin B was in the vacuole of vpe null cells, the absence of VPE prevented cathepsin B from being released into the cytoplasm which impaired cathepsin B mediating ER stress induced PCD. The fact that PCD still did occur in the vpe null background may mean that in the absence of VPE the cell switch to necrotic PCD. This hypothesis could be further tested by the analysis of vacuole integrity associated with morphological and biochemical features of different PCD types using the vpe null line under ER stress- induced PCD.

Chapter 5 Fitting cathepsin B into the ER

stress-induced PCD pathway

5.1 Introduction

In the previous chapter, I have shown that a strong down-regulation of cathepsin B delayed ER stress-induced PCD. However, at the molecular level, it remains to explain how cathepsin B is regulated in ER stress-induced PCD. In this chapter, I am going to investigate the ER stress-induced PCD signal pathway and try to fit cathepsin B in this pathway.

I start from investigation on interaction between cathepsin B and Unfolded Protein Response (UPR). UPR is an indispensable and critical part of the regulation of ER stress- induced PCD. UPR is responsible for changing the expression of several genes which are involved in protein folding, for example Bip2, and aims at rescuing cells from ER stress- induced PCD. Therefore I examined here whether cathepsin B mutant lines could still induce UPR gene expression and reduce the level of misfolded proteins. Moreover, UPR is believed to occur at the early stage of ER stress-induced PCD (liu and howell 2010). So investigation of role of cathepsin B in UPR may help to understand whether cathepsin B functions higher up in the ER stress-induced PCD signal pathway.

Some cathepsin B potential regulators, based on research on published literatures were also examined in this chapter. Mitogen-activated protein kinase 6 (MPK6) was previous reported to regulate the activation of caspase-3-like activity in context of cadmium promoted PCD (Ye et al., 2012). This implies that MPK6 may regulate cathepsin B activity. Besides MPK6, Arabidopsis Bax inhibitor 1 (AtBI-1) is a now classic regulator of ER stress induced PCD. Overexpression of AtBI-1 in Arabidopsis alleviates ER stress- induced PCD (Watanabe and Lam, 2008). In addition, overexpression of AtBI-1 delayed leaf senescence via suppressing the activation of MPK6 (Yue et al., 2012). Although no direct evidence showed that AtBI-1 could influence caspase-3-like activity in Arabidopsis, these findings all together implied that AtBI-1 might be an indirect regulator of caspase- 3-like activity.

Other reported caspases-3-like activity regulators in plants are belonging to the family of NAC (short for NAM (no apical meristem), ATAF, CUC (cup-shaped cotyledon)) transcription factors. Overexpression of the NAC transcription factor, GmNAC6, in soybean was shown to promote caspase-3-like activity. Moreover, GmNAC6 was shown to be a downstream component of ER stress-induced PCD signal pathway (Faria et al., 2011).

I also attempted to look for down-stream components of cathepsin B in the ER stress- induced PCD signal pathway. The mitochondrion has been reported to be a component of ER stress-induced PCD in soybean (Zupinni et al., 2005) where cytochrome c was released out of mitochondria during ER stress. In animal cell models, a BH3-domain-only molecule, BID can be cleaved by cathepsin B upon perceiving an apoptosis signal (Cirman et al., 2004). Cleaved BID may help BAX to form oligomers and insert into mitochondria outer membrane, which resulted in cytochrome c release (Eskes et al., 2000). It was therefore worth investigating whether lacking cathepsin B may affect cytochrome c translocation during ER stress-induced PCD in Arabidopsis.

5.2 Results

5.2.1 Does cathepsin B deficiency affect the UPR?

During ER stress, one obvious consequence of interruption of protein homeostasis caused by ER stress is accumulation of misfolded proteins. Misfolded proteins in the ER lumen can be degraded via the ubiquitin-proteasome system. Here, I examined whether cathepsin B deficiency affects misfolded protein accumulation. I induced ER stress in Col-0 and the triple mutant line atcathb#62 leaves by infiltrating 15 µg/mL Tm into leaves. Soluble protein was isolated at designated time points (one, two and three days after infiltration). Extracted protein was separated by SDS-PAGE and blotted onto a PVDF membrane. To visualize ubiquitinated proteins, an antibody against poly-ubiquitin (P4D1) was incubated with membrane followed by ECL development. As we can see from the result, Col-0 has more ubiquitinated proteins in Tm treated samples at all three time points (Figure 5.1). In atcathb#62 samples, ubiquitinated protein level in day one sample did not showed strong induction when compared with the mock sample. However two and three days after infiltration, atcathb#62 exhibited similar ubiquitinated protein level as those in Col-0 samples (Figure 5.1). In order to make sure this result was reproducible, ubiquitinated protein accumulation on day one in Col-0 and atcathb#62 samples were measured for additional two times. This suggested that lacking cathepsin B caused less ubiquitinated protein accumulation on day one of ER stress-induced PCD.

Figure 5.1. Ubiquitinated protein accumulation in Col-0 and atcathb#62 during ER stress. Plant leaves of Col-0 and atcathb#62 were infiltrated with 15μg/mL Tm to trigger ER stressed-induced PCD, equal volume DMSO was also infiltrated as mock (M). Leaves treated with Tm were collected at one, two and three days post infiltration (dpi). Total protein was isolated and separated by SDS-PAGE. Ubiquitinated proteins were detected by western blot with an anti-ubiquitin (P4D1) antibody. The full spectrum of protein ranging from 10 kDa to 250 kDa is shown (above panel). Rubisco (Rb) was detected by ponceau S staining of the membrane and used as loading control. The result presented is representative of three repeats.

The molecular mechanism for this delay is not clear. Two hypotheses may explain the ability of cathepsin B mutant lines to delay ubiquitinated protein aggregation: 1. lacking of cathepsin B generates more proteasome activity that accelerates protein turnover rate; 2. Cathepsin B deficiency induced further UPR genes to help cells with refolding misfolded proteins. I first tested the first hypothesis. 20S proteasome catalytic activity was determined by measuring its cleavage activity against the artificial tetra peptide LLVY. I extracted soluble proteins from 15μg/mL Tm treated and non-treated Col-0 and atcathb#62 leaves at one, two and three days after infiltration. Extracts were incubated with the fluorescent 20S proteasome substrate Ac-LLVY-AMC at 37 ℃. The fluorescence intensity emitted from cleaved Ac-LLVY-AMC was recorded and the enzymatic activity rates of various treatments within 30 minutes were calculated. One day after Tm treatment, neither Col-0 nor atcathb#62 showed increased LLVY activity compared to non-treated sample (Figure 5.2). But 20S proteasome activity in atcathb#62 was only about 70% of those in Col-0 (Figure 5.2). LLVY activity started to increase two days after Tm infiltration which reached about 150% in both Col-0 and atcathb#62 lines (Figure 5.2). LLVY activity in Col-0 Tm treated sample continued going up to 200% in day three. Atcathb#62 Tm treated sample reached 180%, meanwhile, atcathb#62 Tm free sample also had 20% more activity than Col-0 Tm free sample (Figure 5.2). These data suggested that the cathepsin B mutations did not result in more 20S proteasome activity. The data is also consistent with less ubiquitinated proteins in the mutant line on day one.

Figure 5.2. 20S proteasome activity in Col-0 and atcathb#62 under ER stress. Soluble proteins were extracted from Col-0 and atcathb#62 leaves with or without Tm infiltration. 20S proteasome activity was determined by incubating the extract with ac-LLVY- AMC. Col-0 mock sample at each time point was set to 100% as reference and relative activity is presented in the chart. Error bar means standard deviation of triplicates.

The Lack of evidence supporting the idea that cathepsin B mutant line had more proteasome activity to maintain protein homeostasis during ER stress made me turn to the second hypothesis. In order to test it, I examined the transcript level of several UPR marker genes. I chose BIP2, PGS, CNX2 and PDI6 as UPR marker gene. These genes are responsible for different function during UPR. For example, BIP2, CNX2 and PDI6 are protein chaperones which help protein folding. PGS is responsible for the formation of galactinol from UDP-galactose which is involved in protein glycosylation. To quantitatively measure gene transcript levels, total RNA was extracted from leaves of various treatments. DNA free RNA was subjected to cDNA synthesis and QRT-PCR. Gene transcripts data were normalized against 18S transcript levels. In Col-0 background, BIP2, CNX2, PDI6 and PGS transcript levels increased as early as two hours after infiltration. BIP2, CNX2 and PGS peaked at two hours and then declined (Figure 5.3). PDI6 transcript level reached a small peak at 4 hours and declined as well. BIP2, CNX2 and PDI6 recovered from decline at 8 hours while PGS stayed at mock group level (Figure 5.3). By contrast, the transcript level of these genes in atcathb#62 exhibited clearly a higher level at 8 hours with in particular a 5 fold increase of PDI6, PGS and CNX2 (Figure 5.3). This suggested cathepsin B deficiency resulted in more UPR genes expression during ER stress-induced PCD.

Figure 5.3. Transcript levels of UPR marker genes at early stages of ER stress in Col-0 and atcathb#62. Transcript levels of four UPR genes, BIP2, CRT2, PDI6 and PGS, at early time points (two, four, six and eight hours after infiltration) of ER stress induced by Tm (15μg/mL) infiltration are presented here. Mock samples were infiltrated with DMSO. All copy numbers were normalized against the 18S transcript level. Error bar is standard deviation of triplicates.

The expression of UPR genes is controlled by several ER membrane-anchored signal transducer proteins. For example, bZIP60 is an ER membrane transcription factor responsible for some UPR genes activation. ER stress activates bZIP60 by splicing bZIP60 mRNA. This splicing enabled bZIP60 to translate into a non-membrane bound form that translocates to the nucleus where this truncated form of bZIP60 can trigger UPR induced expression of some genes such as PDI6 (Nagashima et al., 2011). I therefore tested whether the cathepsin B mutant line had altered bZIP60 activation or not. The spliced form and unspliced form of bZIP60 mRNA were detected via PCR amplification using different pairs of primers. Briefly, a pair of primers, bZIP60iF and bZIP60iR, which spanned a cleavage site between the sliced sequence and the unspliced sequence was used to amplify the unspliced bZIP60 mRNA. A primer pair, bZIP60sF2 and bZIP60sR2, covering the spliced sequence on both sides of the cleavage site were used to amplify the spliced form of bZIP60. A diagram of the two forms of bZIP60 with corresponding primer positions used are shown in Figure 5.4 upper panel. A small amount of spliced

97 bZIP60 was detected in the mock sample (Figure 5.4 lower panel). As expected the Tm treatment increased the spliced bZIP60 in Col-0 background while the unspliced form of bZIP60 did not change significantly (Figure 5.4 lower panel). In the atcathb#62 line, the spliced bZIP60 also increased after two-hour treatment. There was no significant difference of bZIP60 slicing profile between Col-0 and atcathb#62 (Figure 5.4 lower panel). This demonstrated that lacking cathepsin B did not change bZIP60 activation.

Figure 5.4. bZIP60 splicing profile between Col-0 and atcathb#62 during ER stress. Two forms of bZIP60 mRNA were amplified by PCR and visualized via DNA electrophoresis. bZIP60s is the spliced form and bZIP60u the unspliced form. Total RNA samples were collected from leaves during a time course of hour post infiltration (hpi) with 15μg/mL Tm. Detection was also performed in mock (M) samples infiltrated with DMSO.

5.2.2 Analysis of the signal cascade order between BAX inhibitor and cathepsin B

As an important regulator of ER stress induced PCD, Arabidopsis BAX inhibitor 1 (AtBI- 1) was examined here for a possible interaction with cathepsin B. AtBI-1 was shown to be downstream of UPR and its function is to alleviate ER stress-induced PCD in Arabidopsis (Watanabe and Lam 2008). So an AtBI-1 overexpression line (AtBi-1 OX2) was expected to show reduced ER stress-induced PCD in Arabidopsis. This possible reduction of ER stress induced PCD in AtBi-1 OX2 was investigated by measuring ion leakage. Ion leakage was triggered by infiltrating 15 μg/mL Tm into Col-0 and AtBI-1 OX2 leaves.

Ion leakages were monitored for seven days and recorded at designated time points. The ion leakage profile in Col-0 background showed a gradual increase from day three (Figure 5.5). However, ion leakage values in AtBI-1 OX2 line samples did not exhibit any obvious increase until day five (Figure 5.5). This demonstrated that overexpression of AtBI-1 reduced ER stress induced-PCD, which was consistent with the profile seen in a cathepsin B triple mutant line. Presumably overexpression prevented vacuolar PCD and not necrotic PCD.

Figure 5.5. Ion leakages of Col-0 and AtBI-1 OX2 line in ER stress-induced PCD. Ion leakages from Col-0 and AtBI-1 OX2 leaves treated with or without 15 μg/mL Tm were recorded from day one to day seven. Tm or the same volume of DMSO was infiltrated into different halves of each leaf. Three discs were punched out in each half. Relative increment of ion leakage was obtained by subtracting ion leakage of mock samples from Tm treated samples. Error bar represents standard deviation of biological triplicates.

I subsequently tested whether this reduction of ER stress-induced PCD is associated with a non-increase of cathepsin B caspase-3-like activity. 15 μg/mL Tm was infiltrated into

Col-0 and AtBI-1 OX2 lines to induced ER stress. Soluble proteins of these lines were extracted and incubated with the caspase-3 substrate Ac-DEVD-Rh110. Rates of fluorescence intensity per minute of each treatment were recorded. Caspase-3-like activity in Col-0 mock sample was set as 100% standard and relative caspase-3-like activities in other samples were calculated. The Tm treatment increased the caspase-3-like activity in Col-0 to 220%, the net increase was 120% (Figure 5.6). Increase of caspase-3-like activity in AtBI-1 OX2 line reached only about 180%, equivalent to a net increase of caspase-3-like activity of 70% (Figure 5.6). To understand which part of caspase-3-like 99 activity, activity from cathepsin B or PBA1 was repressed in AtBI-1 OX2, cathepsin B and PBA1 inhibitors were added to eliminate cathepsin B or PBA1 activity in Col-0 and AtBI-1 OX2. The rationale underlying this experiment is that if cathepsin B or PBA1 activity has already been repressed in the AtBI-1 OX2 line, adding inhibitors should not further reduced their activity. In the event, CA074 and β-lactone respectively reduced by 20% and 46% caspase-3-like activity of Tm treated Col-0 samples (Figure 5.6). In the Tm treated AtBI-1 OX2 line, CA074 and β-lactone reduced activity by 12% and 46% respectively (Figure 5.6). The difference between DEVDase activity (from 20% to 12%) repressed by CA074 in Col-0 and AtBI1 OX2 sample indicated a mild decrease of cathepsin B activity in AtBI1 OX2 Tm treated samples.

Figure 5.6. Caspase-3-like activities composition in Col-0 and AtBI-1 OX backgrounds during ER stress-induced PCD. Soluble proteins of Col-0 and AtBI-1 OX leaves after various treatments were extracted and subjected to caspase3 enzymatic assays. 15 μg/mL Tm was used to induce ER stress. The inhibitors CA074 and β-lactone were applied to suppress cathepsin B activity or proteasome activity. Col-0 activity in the mock sample was set to 100%. Error bar = standard deviation of biological triplicates.

Reduction of cathepsin B derived caspase-3-like activity in AtBI-1 OX2 line was only partial in in vitro assays. As a complement, I carried out activity labelling to check cathepsin B activity in vivo. For this, leaves were infiltrated with 15 μg/mL Tm and the amount of Biotin-DEVD-FMK bond to cathepsin B in Col-0 and AtBI-1 OX2 lines was compared on day three. As expected, labelling of cathepsin B was faint in both Col-0 and AtBI-1 OX2 mock samples. A strong increase of bound Biotin-DEVD-FMK in Col-0 was 100 observed in Tm treated sample, especially the highest molecular weight band (Figure 5.7). This increase was less in AtBI-1 samples compared to Col-0 background, consistent with the chemical inhibitor results (Figure 5.6).

Figure 5.7. Affinity labelling of Cathepsin B in Col-0 and AtBI-1 OX under ER-stress. Col-0 and AtBI-1 OX leaves were treated with or without 15 μg/mL Tm. Soluble proteins extracted from Col-0 and AtBI-1 OX leaves were incubated with Biotin-DEVD-FMK followed by western blot. Active cathepsin B was visualised using streptavidin-HRP to detect labelled cathepsin B. Three bands corresponding to cathepsin B are shown. Rubisco (Rb) was visualized via ponceau S staining and used as loading control.

In addition, I carried out QRT-PCR to know whether AtBI-1 overexpression did affect cathepsin B transcript level. Total RNA was extracted on day three from Col-0 and AtBI- 1 OX2 lines with or without induction of ER stress. The transcript level of the three cathepsin B paralogues was measured by QRT-PCR using SYBR green. Gene transcript levels were normalized against Actin2 transcript level. AtCathB2 and AtCathB3 transcript level were strongly induced by infiltration of 15 μg/mL Tm. They approached 170% and 190% of Actin2 transcript level (Figure 5.8). AtCathB1 had a relatively low expression level, but still showed an increase from 1% to 3% (Figure 5.8). AtCathB2 and AtCathB3 levels were reduced down to 75% and 46% of ACTIN2 transcript level in the AtBI-1 OX2 line (Figure 5.8). AtCathB1 transcript level went down to about null (Figure 5.8). This suggested that overexpression of AtBI-1 reduced the cathepsin B transcript level induction by Tm, but could not abolish it. This is consistent with the enzymatic results. Nevertheless this showed that the expression of the three cathepsin B genes was down- regulated indirectly by AtBI-1.

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Figure 5.8. Cathepsin B transcript level in Col-0 and AtBI-1 OX in ER stress-induced PCD. Total RNA samples were isolated from Col-0 and AtBI-1 OX leaves infiltrated with 15 μg/mL Tm or the same volume of DMSO. cDNA was synthesized from DNase treated RNA. The transcript levels of the three cathepsin B paralogues were measured by QRT-PCR. Actin2 was used as reference gene. Error bar indicates standard deviation of triplicates.

5.2.3 Analysis of the signal cascade order between Mitogen- activated protein kinase 6 and cathepsin B

MPK6 was shown to be a regulator of caspase-3-like activity in context of cadmium- induced PCD (Yue et al., 2012). In this section, I examined the relationship between MPK6 and caspase-3-like activity during ER stress-induced PCD. I used mpk6-1, a homozygous T-DNA insertion line obtained from NASC, to test how caspase-3-like activity was changed. ER stress was induced as described before. Briefly, 15 μg/mL Tm was infiltrated into Ws and mpk6-1 leaves. Leaves were collected at three days after infiltration. Soluble protein was extracted and incubated with the caspases substrate Ac- DEVD-Rh110. Caspase-3-like activity was determined by measuring the rate of fluorescence intensity increasing per minute. As we can see from Figure 5.9, three days after induction by 15 μg/mL Tm, caspase-3-like activity increased to 250% in Ws samples when compared to mock sample. However, increase of caspase-3-like activity in mpk6-1 was only about 30% (Figure 5.9). This suggested that the loss of MPK6 strongly reduced ER stress-induced caspase-3-like activity, presumably reducing both proteasome and cathepsin B activity.

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Figure 5.9. ER stress-induced caspase-3-like activity in Ws and mpk6-1backgrounds. 15 μg/mL Tm was infiltrated into leaves to induced ER stress. Total soluble proteins were isolated from Tm treated and untreated (mock) Ws and mpk6-1 leaves at three days after infiltration. Caspase-3-like activity in each sample was measured by using the fluorescent caspase-3 substrate ac-DEVD-Rh110. Caspase-3-like activity of Ws mock sample was set to 100%. Error bar represents standard deviation of triplicates.

In order to confirm that mpk6-1 is able to reduce cathepsin B activity in vivo more direct evidence was needed. Cathepsin B activity in vivo was determined by activity labelling using Biotin-DEVD-FMK. Total soluble proteins of Ws and mpk6-1 were extracted from leaves three days after designated treatments. Soluble protein extracts were incubated with Biotin-DEVD-FMK, separated by SDS-PAGE and labelled cathepsin B was visualized on western blot using streptavidin-HRP. The Tm treated Ws sample showed an increase of cathepsin B labelling by Biotin-DEVD-FMK compared to the mock sample (Figure 5.10). In mpk6-1 samples, this increased labelling did not occur, as an obvious difference existed between mock and Tm treated samples (Figure 5.10). This findings further confirmed that knocking out mpk6-1 repressed the induction of cathepsin B’s caspase-3-like activity during ER stress-induced PCD.

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Figure 5.10. Cathepsin B activity labelling in Ws and mpk6-1 during ER stress-induced PCD. WS and mpk6-1 leaves were treated with or without 15 μg/mL Tm. Total soluble proteins from leaves were isolated and incubated with Biotin-DEVD-FMK. Biotin-DEVD-FMK bound to cathepsin B was visualized by western blot. Three to four bands corresponding to cathepsin B are shown. Rubisco (Rb) was visualized via ponceau S staining and used as loading control.

I then tested whether knocking out mpk6-1 would alter ER stress-induced PCD. Ion leakage was chosen as an indicator of PCD. Ion leakage of Ws and mpk6-1 leaves was monitored within seven days after infiltrating 15 μg/mL Tm. Leaves at various time points were detached from the plants. Three discs were punched out from each treated leaf and floated on deionized water. Ion leakage of each sample was measured using a conductivity meter. Ion leakage from the mock sample was subtracted from that of Tm treated sample, so that the relative increase in ion leakage was presented in Figure 5.11. Ion leakage started increasing three days after Tm infiltration and went up to about 45 μS/cm on day seven in Ws (Figure 5.11). By contrast, mpk6-1 kept a very low level of ion leakage, ion leakage was only 10 μS/cm even on day seven (Figure 5.11). This demonstrated that the loss of MPK6-1 blocks ER stress induced PCD, although the difference in ion leakages between the genotypes from day one to five was not significant.

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Figure 5.11. Ion leakages of Ws and mpk6-1 during ER stress-induced PCD. Ion leakage from Ws and mpk6-1 leaves treated with or without 15 μg/mL Tm was recorded from day one to day seven. A half of each leaf was infiltrated with Tm and the other half was infiltrated with DMSO instead as mock. Three discs were punched out in each half leaf. Relative increment of ion leakage was obtained by subtracting ion leakage of mock samples from Tm treated samples. Error bar represent standard deviation of triplicates.

5.2.4 Role of NAC transcription factor in ER stress-induced PCD

A Glycine max NAC transcription factor (GmNAC6) has been proved to be an activator of caspase-3-like activity. The corresponding NAC transcription factor in Arabidopsis, which may regulate caspase-3-like activity, has not been reported. Here, I am trying to identify a potential Arabidopsis orthologue, which would be able to regulate caspase-3- like activity. The NAC transcript factors belong to a large family in the Arabidopsis genome. To pick up NAC orthologue candidates, I adopted two strategies: 1) To try identifying GmNAC6 orthologous sequences in Arabidopsis via BLAST; 2) To search ER stress microarray data and choose Arabidopsis NAC transcription factors, which were induced by ER stress. Three candidates were finally chosen: AtNAC036 (At2g17040), AtNAC062 (At3g49530) and AtNAC082 (At5g09330). AtNAC036 was chosen as it shared the highest similarity (67%) with GmNAC6 at the amino acid sequence level. This was based on the amino acids sequences of AtNAC036 and GmNAC6 obtained from TAIR and NCBI respectively. These sequences were aligned using EBI blast tool ClustalW2. For information, the amino acid sequences alignment is shown in Figure 5.12.

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Figure 5.12. Sequences alignment between AtNAC036 and GmNAC6. AtNAC036 (AAK32817 NCBI) and GmNAC6 (NP_001236900 NCBI) amino acids sequences were obtained from the TAIR and NCBI databases respectively. These two sequences were aligned using EBI ClustalW2. Asterisks indicate identity. Two dots indicate high similarity. One dot indicates similarity. The conserved NAM domain is labelled pink.

In an ER stress microarray data, I found that AtNAC062 and AtNAC103 were induced by ER stress (data taken from Nagashima et al., 2011). However, no appropriate T-DNA insertion line for AtNAC103 is available. So I chose an AtNAC082 mutant line, which is the closest paralogue to AtNAC103 instead. All NAC mutant Arabidopsis seeds were obtained from NASC. The atnac062 mutant line was available as homozygous in NASC. The atnac082 mutant line was generated by the Gabi-kat consortium. I screened for homozygous from fifteen T3 lines using antibiotics selection. Finally, and unfortunately, the atnac036 mutant seeds did not germinate, so I was not able to carry out further investigation on atnac036 seeds.

The possible correlation between these NAC transcription factors and caspase-3-like activity were examined individually. I started with AtNAC062. 15 μg/mL Tm was infiltrated into leaves of Col-0 and AtNAC062 knock out line (atnac062) to induce ER stress. Tm treated or untreated leaves were collected on day three and subjected to enzymatic assay. Tm induced an 80% increase of caspase-3-like activity in Col-0 leaves (Figure 5.13) and a 50% increase in atnac062 (Figure 5.13). However, the difference of caspase-3-like activity between Col-0 and atnac062 were not significant as the T-test p value was 0.06>0.05 (Figure 5.13). Caspase-3-like activity in the other NAC transcription factor mutant lines was also tested. ER stress was induced as described above. As we can see from Figure 5.13, about 100% increase of caspase-3-like activity was shown both in Col-0 and atnac082 (Figure 5.13). These findings suggested that neither AtNAC062 nor AtNAC082 affected ER stress-induced caspase-3-like activity.

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Figure 5.13. Caspase-3-like activities in Col-0 and NAC transcription factors mutant lines after Tm treatment. Caspase-3-like activities in Col-0 and atnac062 (top panel) and atnac082 (bottom panel) leaf extracts were measured. 15 μg/mL Tm was used to induce ER stress. Soluble Proteins extracted from leaves were subjected to caspase3 enzymatic assay on day three. Col-0 mock sample was set to 100%. Error bar = standard deviation of triplicates.

Although none of the NAC transcription factors tested in this research showed insignificant influence on caspase-3-like activity induced by ER stress, it was possible that these NAC transcription factors altered ER stress-induced PCD via a caspase-3-like activity independent pathway. To test this hypothesis, 15 μg/mL Tm induced ion leakages in Col-0, atnac062 and atnac082 knock out lines were compared. Leaves were collected at designated time points. Three discs were punched out of treated leaves and floated on deionized water. Ion leakage was measured using a conductivity meter. Ion leakage in

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Col-0 was as normal, it started increasing on day three and continued going up till day seven (Figure 5.14). Atnac062 ion leakage profile closely followed Col-0 ion leakage profile. Although, on day five, more ion leakage was observed in atnac062, this increasing was not significant (Figure 5.14). Ion leakage profile of atnac082 kept slight lower than that of Col-0 all along seven days treatment, but the differences between Col-0 and atnac082 ion leakages were no more than 10 μS/cm and was not significant (Figure5.14). These data illustrated that mutation in AtNAC082 and AtNAC062 did not change ER stress-induced PCD.

Figure 5.14 Ion leakages of Col-0 and different NAC transcription factors mutant lines under Tm treatment. Atnac062 and atnac082 leaves were treated with 15μg/mL Tm to induced ER stress. A half of each leaf was infiltrated with Tm and the other half was infiltrated with DMSO instead as mock. Three discs were punched out in each half of a leaf. Relative increment of ion leakage was obtained by subtracting ion leakage of mock samples from Tm treated samples in the same leaf. . Error bar represent standard deviation of biological triplicates.

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5.2.5 Investigation of the role of Arabidopsis G protein β subunit (AGB1) in regulating cathepsin B activity in ER STRESS-induced PCD.

AGB1 is one the few identified ER stress induced PCD regulators although it has been reported separately as either a positive or a negative regulator (Wang et al., 2007; Chen et al., 2011). Functional interaction between AGB1 and cathepsin B is tested here. A homozygous AGB1 knock out line (agb1), generated by ethylmethane sulfonate mutation. Comparison of caspase-3-like activity between Col-0 and agb1 was first obtained by measuring their cleavage kinetics against Ac-DEVD-Rh110. 15 μg/mL Tm or DMSO only was infiltrated into Col-0 and agb1 leaves. Soluble Proteins were isolated three days after infiltration and incubated with Ac-DEVD-Rh110. Caspase-3-like activity increased up to about 225% of the non-stressed sample in Tm treated Col-0 sample (Figure 5.15). Caspase-3-like activity increased to similar amount, 227% in agb1 mutant sample after Tm treatment (Figure 5.15). This suggested that agb1 deletion neither increased nor reduced caspase-3-like activity induced by ER stress.

Figure 5.15. Caspase-3-like activity in agb1 background during ER stress-induced PCD. 15 μg/mL Tm was used to induce ER stress in Col-0 and agb1 leaves. DMSO was infiltrated as mock. Caspase-3-like activities were determined three days after infiltration. Col-0 mock was set as 100%. The mean of technical triplicates for each sample is shown. Error bar represents standard deviation.

To further test the influence of an AGB1 knock out on cathepsin B, I labelled cathepsin B using Biotin-DEVD-FMK. Biotin-DEVD-FMK was incubated with extracted soluble proteins from various treatment of Col-0 and agb1 leaves three days after infiltration.

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Labelled proteins were detected by streptavidin-HRP on western blot. Activity labelling of cathepsin B with Biotin-DEVD-FMK dramatically increased in Col-0 Tm treated sample (Figure 5.16). A Similar increase of labelling was observed in agb1 samples (Figure 5.16). It demonstrated that AGB1 deletion did not alter cathepsin B increased activity during ER stress induced PCD.

Figure 5.16. Cathepsin B activity labelling in Col-0 and agb1 during ER-stress induced PCD. Col-0 and agb1 leaves were treated with or without 15 μg/mL Tm. Soluble Proteins extracted from Col-0 and agb1 leaves were incubated with Biotin-DEVD-FMK followed by western blot. To visualise cathepsin B, streptavidin-HRP was used to detect labelled cathepsin B. Bands corresponding to cathepsin B are shown. Rubisco (Rb) was visualised via ponceau S staining and used as loading control (lower panel).

5.2.6 Cytochrome c behavior in cathepsin B triple mutant background in ER stress-induced PCD

During PCD of mammalian cells, cathepsin B is able to cleave BID, which subsequently helps BAX oligomers formation. Formation of BAX oligomers makes mitochondria permeable, and results in cytochrome c exportation to cytoplasm (Cirman et al., 2003). In the context of plants ER stress-induced PCD, leaking of cytochrome c out of mitochondria in Glycine max suspension cells was previous reported (Zuppini et al., 2005). This raises the question of how cytochrome c behaves in Arabidopsis ER stress- induced PCD. For this, isolation of mitochondria from leaves was tried using published protocols for suspension cells. However, it was very hard to keep the integrity of mitochondria during the extraction procedure. To better separate intact mitochondria from cytosol, a milder extraction method in protoplast was developed. Two million protoplasts were prepared from leaves of Arabidopsis plants grown in soil according to (Wu et al., 2009), treated with 15μg/mL Tm and collected at one, two, three and four hours after treatment. The same volume of DMSO was added into protoplasts as mock and the 110 sample was collected at four hours. Protoplasts were disrupted gently by passing through a 30 μm nylon mesh without breaking the mitochondria. Mitochondria were separated from other cytosolic content by centrifuging at 4 ℃ 15,000g for 15 minutes into the supernatant cytosol fraction and pellet containing isolated mitochondria. The mitochondria in the pellet were resuspended and broken up by sonication, and cytochrome c was released and collected with the supernatant after centrifugation. The cytosolic fraction and mitochondria fraction were both loaded into SDS-PAGE, cytochrome c was detected using anti-cytochrome c antibody. An antibody against the mitochondria Voltage Dependent Anion Channel 1 (VDAC1) was used as an indicator of the purity of cytosol separation. Reassuringly VDAC1 was found only in the mitochondria fraction and not in the cytosolic fraction for both Col-0 and atcathb#62 samples. This suggested no mitochondria protein contamination in the cytosolic protein extract (Figure 5.17). Unexpectedly Cytochrome c was present in both the cytosolic and mitochondria fraction in Col-0 throughout all time points (Figure 5.17). In contrast, cytochrome c was not detected in the cytosolic fraction in the atcathb#62 line (Figure 5.17). It should be noted that the amount of VDAC1 and cytochrome c in the mitochondria fraction decreased along with time after Tm infiltration, although cytochrome c in atcathb#62 appeared more stable compared to wild type (Figure 5.17). By contrast, cytochrome c in Col-0 cytosolic fraction did not decrease (Figure 5.17).

Figure 5.17. Cytochrome c release from mitochondria in Col-0 and atcathb#62 protoplasts during ER stress-induced PCD. Protoplasts were prepared from 3-week-old leaves and treated with 15μg/mL Tm. Cytosolic proteins were separated from mitochondria proteins by gentle break up of protoplasts followed by centrifugation. The two protein fractions were collected at various hours post infiltration (hpi) and submitted to western analysis. Cytochrome c was detected using an anti-Atcytochrome c antibody. VDAC1 was detected as an indicator of the presence of mitochondria in the fraction. M is DMSO only treated sample.

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5.3 Discussion

As a strategy to escape from ER stress, cells adopt various methods to sort out misfolded protein accumulation in the ER lumen. As we can clearly see from the ubiquitinated protein accumulation experiment above, the cathepsin B mutant line was capable of maintaining a low level of ubiquitinated protein at early stage of ER stress. A test on the 20S proteasome activity suggested that this is not due to faster protein turnover rate resulted from higher proteasome activity. Further investigation indicated that low level of ubiquitinated protein accumulation in cathepsin B mutant line was correlated with higher a UPR genes induction, especially for PDI6, rather than faster protein turnover rate by higher proteasome activity. However, how cathepsin B down-regulation results in a higher expression of UPR genes is unclear. I tested the activation of a well-documented plant ER-stress signal transduction by measuring the alternative splicing of the transcription factor bZIP60 mRNA. The splicing of bZIP60 mRNA was reported to result in the up-regulation of several UPR genes expression including PDI6, Comparison of bZIP60 mRNA splicing in Col-0 and atcathb#62 showed that lacking cathepsin B did not alter bZIP60 activation indicating that possibly the sensing of protein folding is not up- regulated. This result does not exclude that cathepsin B mutant may alter some other ER stress signal transducer or it may affect somehow the protein accumulation of bZIP60. There are other signal transducers that could be tested, for instance, bZIP28 which can also mediate ER stress signals (Liu and Howell 2010). The delay in the accumulation of ubiquitinated protein in the cathepsin B mutant remains to be explained.

In this chapter, I also examined the relationship between cathepsin B and several published regulators of caspase-3-like activity or ER stress. I have confirmed that MPK6 and AtBI-1 were capable of regulating caspase-3-like activity derived from cathepsin B. This is in line with a previous report which demonstrated that MPK6 regulated caspase-3- like activity in cadmium induced PCD (Ye et al., 2012). Beyond that, evidence of MPK6 in ER stress induced PCD was first presented in this chapter. Given that MPK6 is downstream of AtBI-1 in senescence (Yue et al., 2012) and overexpression of AtBI-1 did not alter UPR genes expression, including BIP2 and PDI6 (Watanabe and Lam 2008), AtBI-1, MPK6 and AtCathB are likely to form a signal cascade after UPR in ER stress-induced PCD pathway. It should be noted that overexpression of AtBI-1 only caused a slight reduction of cathepsin B activity. This might be due to an indirect regulation of cathepsin B activity of AtBI-1. AtBI-1 could regulate cathepsin B activity via controlling partially the activation of MPK6. Yue and colleagues have shown that overexpression of AtBI-1 112 caused only a mild reduction of MPK6 activation (Yue et al., 2012), consistent with the mild effect of AtBI-1 overexpression on cathepsin B activity in this study. Activity labelling of cathepsin B in mpk6-1 showed a faint double labelled-band of the cathepsin B intermediate form (Figure 5.10 bands in the middle). This was not seen in other activity labelling experiments. It may be due to different genotype background. Cathepsin B in Arabidopsis Ws may have different behaviours than that in Col-0. Mpk6 mutant line in Col-0 background should be used in the future to resolve this issue.

My search for Arabidopsis NAC transcription factors which I consider as potential regulators of cathepsin B ended up in failure. None of the NAC transcription factors tested in this chapter showed strong influence on caspase-3-like activity or ER stress induced PCD. It is may be due to the strategy used which was not suitable. For example, I picked up AtNAC062 because it was induced by ER stress. However, this induction was seen in the early time points of ER stress (within 24 hours). In my research system, where I infiltrated Tm into leaves, caspase-3-like activity is significantly increased at 72 hours. This implied that the NAC transcription factor induced in early time points may not be the one which regulates caspase-3-like activity. Choosing AtNAC082 has its own drawback. AtNAC082 was chosen as an alternative candidate for AtNAC103 based on BLAST. However, although AtNAC082 is the closest paralogue to AtNAC103, they have different subcellular localisation prediction (based on the Bar database, bar.utoronro.ca). AtNAC103 is predicted to be in cytoplasm and nucleus, while AtNAC082 is predicted in mitochondria, peroxisome and nucleus. Different spatial distribution may attribute them to different signal pathway. Testing for relevant NAC transcription factors needs more works in the future with the development of better selection parameters.

In this chapter, I also tested the effect of an agb1 knock out on cathepsin B activity in the context of ER stress-induced PCD. The Agb1 mutant did not exhibit a significant effect on the overall caspase-3-like activity or cathepsin B activity. This suggested that ER stress-induced caspase-3-like activity was not under the control of AGB1. Two previous papers showed conflicting roles for AGB1 in ER stress induced-PCD; a positive role in Wang et al., 2007, and a negative role in Chen et al., 2011. My different experiment system may be the reason why there was no effect of caspase-3-like activity in the agb1 mutant. In previous published papers, the authors treated Arabidopsis seedlings with 0.3 µg/mL Tm, which is less than the concentration I used. Low level of Tm may induce different ER stress response (Wang et al., 2007; Chen et al., 2011). This suggests that the

113 function of AGB1 in ER stress-induced PCD, if any, needs more investigation with different Tm concentration treatments.

Atcathb#62 exhibited a different patterns of cytochrome c when compared to Col-0. Cytochrome c was detected in the cytosol of Col-0 mock sample. This was unexpected and could be due to the fact that protoplasts were stressed during preparation and a small percentage of the population entered the PCD pathway. By contrast, only traces of cytochrome c signal were detected in atcathb#62 cytosolic fraction, obviously less than those in Col-0 background. This suggested protoplasts of the cathepsin B mutant line were resistant to stress and exhibited less PCD. In this experiment system, the release of cytochrome c induced by ER stress-induced PCD may be masked by other stresses. So that it cannot rule out the possibility that cathepsin B can also mediate cytochrome c release during ER stress-induced PCD. Using other systems in the future, like suspension cell culture, may help to avoid unnecessary stresses to the cell and obtain more conclusive results.

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Chapter 6. Cathepsin B and developmental

PCD.

6.1 Introduction

Many examples in the literature have emphasized that PCD is important for development. As we had shown that cathepsin B is an important regulator of PCD, the cathepsin B triple mutant line atcathb#62 offered a unique tool to investigate the role of cathepsin B and the role of PCD in various developmental pathways. Several instances of PCD occurring during Arabidopsis development were selected.

A canonical developmental process where PCD is involved is xylem formation. Several important PCD markers, for instance DNA fragmentation, have been observed during TE differentiation (Mittler and Lam 1995). As research on PCD in TE progressing, cysteine proteases, like Xylem Cysteine Protease 1/2 (XCP1/2), have already been well documented as regulators for xylem formation (Avci et al., 2008). Moreover, a recent report proposed that the proteasome was responsible for caspase-3-like activity detected during xylem formation (Han et al., 2012). This lead me to check whether cathepsin B also regulate xylem formation.

Another developmental PCD occurs during seed maturation in inner integument cells. Arabidopsis seed maturation is accompanied by inner integument layer thinning. PCD was observed to underlie thinning of inner integument layer (Nakuane et al., 2005). Whether cathepsin B, as a regulator of plant PCD, also participates in PCD in inner integument cells was examined here.

Seed germination is an important agronomic trait and PCD has been reported to occur in aleurone layers (Fath et al., 1999) and in endosperm (Fath et al., 2000). Seed germination however includes several stages and we discovered that one instance of PCD involvement has been overlooked. Micropylar endosperm rupture is considered as a rate-limited step for seed germination. Currently the accepted model suggests that micropylar endosperm “weakening” is the key process which governs the radicle penetrating through micropylar 115 endosperm at germination (Voegele et al., 2012). In this chapter, I investigated whether PCD was involved in micropylar endosperm weakening. And if so, whether cathepsin B regulates it. In previous chapter, we have seen the lack of VPE promoted cathepsin B activity during ER stress-induced PCD. In this chapter, I would like to investigate possible interaction between VPE and cathepsin B in developmental PCD as well.

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6.2 Results

6.2.1 Effect of cathepsin B mutant on xylem formation

Xylem formation is an example of cell differentiation in which PCD participates. To understand whether cathepsin B deficiency may alter xylem formation, xylem morphology and patterning were analyzed. Col-0 and atcathb#62 stems were sectioned and stained by toluidine blue. A representative of each genetic background is shown here (Figure 6.1 A and B). Xylem vessel was stained in blue (corresponding to secondary cell wall formation). Close–up images of Col-0 and atcathb#62 stem sections are also presented (Figure 6.1 C and D). Both whole-section images and close-up images did not show obvious changes in xylem morphology or patterning. This suggested that cathepsin B mutant did not alter xylem formation.

Figure 6.1. Stem sections of Col-0 and atcathb#62. Sectioned Col-0 and atcathb#62 stem tissues were subjected to toluidine blue staining. Whole section (A and B) and close-up (C and D) are shown here. A and C are from Col-0 transversal sections while B and D are from atcathb#62 sections. Vascular tissue are stained and showed in blue. Other tissues are shown as purple. Scale bar for A and B is equal to 0.25 mm. Scale bar for C and D is equal to 0.07 mm.

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6.2.2 Effect of cathepsin B mutant on PCD of inner integument cells. To check whether cathepsin B participated in inner integument cells PCD, the effect of cathepsin B deletion on inner integument cells PCD was examined. Col-0 and atcathb#62 seeds were harvested from siliques at three and seven days after pollination. Pigments were removed from harvested seeds using the chlorohydrate solution. Seeds were photographed using a microscope at low magnification and the thickness of inner integument cell layer was calculated using Image J (see Material and Methods). Inner integument cell layers were thick at three days post pollination (heart stage) in both Col-0 and atcathb#62 seeds (Figure 6.2 yellow bar). Seven days after pollination, inner integument layer cells in both Col-0 and atcathb#62 seeds became thinner (Figure 6.2 yellow bar). However, it is difficult to draw any conclusion without quantitative measurements.

Figure 6.2. Inner integument layer thickness of Col-0 and atcathb#62 seeds. Col-0 and atcathb#62 embryos at 3 and 7 days after pollination were cleared using chlorohydrate. Cleared embryos were observed under microscope. Representatives of embryos images are presented. All images were taken under the same magnification, but scaled to fit the window size. Yellow bars indicate inner integument layer thickness. Scale bar is equal to 0.2mm.

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Statistics were employed to see whether thickness of the inner integument cell layers of Col-0 and atcathb#62 seeds were significant differently or not. Col-0 and atcathb#62 seeds exhibited almost the same thickness (measured by pixel unit) on day three, when both Col-0 and atcathb#62 inner integument layers were as thick as 130 pixel unit (Figure 6.3). On day seven, the average Col-0 inner integument layer thickness was about 70 pixel unit, whereas atcathb#62 inner integument layer was about 80 pixel unit (Figure 6.3). T-Test indicated that this difference was significant as p=3.7×10-9<0.05. This suggested that deficiency of cathepsin B was able to maintain a thicker inner integument.

Figure 6.3. Comparison of integument cell layer thickness between Col-0 and atcathb#62 seeds. Col-0 and atcathb#62 embryos were detached from siliques at three and seven days after pollination. Seed pigments were removed using chlorohydrate. Images of Col-0 and atcathb#62 embryos were taken. The thicknesses of the inner integument cell layers are presented as pixel unit in Image J. More than 20 seeds of each sample were measured. Error bar is standard deviation. The difference observed at day seven is significant p=3.7×10-9<0.05.

6.2.3 Effect of the cathepsin B mutant on seed germination

Seeds germination includes several stages towards the completion of the process. First the timing of these stages was investigated prior to further investigation of gene functions during seed germination. Arabidopsis thaliana wild type Col-0 seeds imbibed water for 48 hours at 4℃ before germinating in a growth cabinet with long-day light period (22℃; 16 h light and 8 h dark). Seeds were photographed at various stages after the transfer to the growth cabinet. According to common accepted criteria, seed germination is divided

119 into four stages as shown in figure 6.4. Water uptake from the environment into dried seeds was stage I. Seeds no longer show a wrinkled surface (Figure 6.4 A). After several hours of incubation, the testa, also called seed coat, ruptured (stage II, Figure 6.4 B, yellow arrow indicates testa). But the micropylar endosperm still kept its integrity (Figure 6.4 B). Finally the micropylar endosperm ruptured and was accompanied by radicle growth through the micropylar endosperm (stage III, Figure 6.4 C and D, white arrow indicates micropylar endosperm). So only when seeds are at stage III or after, are they designated as germinated.

Figure 6.4. Illustration of four Arabidopsis seeds germination stages. Arabidopsis Col-0 seeds imbibed water in 4 ℃ for two days. After that, seeds germinated at 22 ℃, 16h light. Seeds were photographed at four different germination stages: A. seeds imbibed with water, testa had not ruptured; B. testa ruptured but micropylar endosperm was intact. Yellow arrow indicates testa; C. testa ruptured and micropylar endosperm started to rupture. White arrow indicates micropylar endosperm; D. radicle comes out of the micropylar endosperm after its rupture. T=Testa, ME=Micropylar Endosperm. Scale bar is equal to 0.1mm.

To understand whether cathepsin B is involved in the seed germination process, germination rates of Col-0 and cathepsin B mutant lines were compared. Col-0 and atcathb#62 seeds were harvested at the same time. Both seed batches were dried for two weeks before germination test. Surface sterilized Col-0 and atcathb#62 seeds were plated on MS-phytagel medium. Plates were put in 4 ℃ for two days before transfer to 16 h light/8 h dark light period at 22 ℃. The ratios of germinated vs. total seeds of Col-0 and

120 atcathb#62, determined according to the criteria described above, were scored every 24 hours till 144 hours. At the beginning of the observation, only sporadic seeds germinated. The majority Col-0 seeds germinated between 24 hours and 72 hours, when the germination rate increased from less than 5% to about 80% (Figure 6.5). After 72 hours, Col-0 seeds germination ratio increased to 90% and stop increasing (Figure 6.5). The germination rate of atcathb#62 is slower than that of Col-0, especially between 24 hours and 72 hours. For example, at 72 h, about 78% Col-0 seeds had germinated while less than 60% atcathb#62 seeds had germinated (Figure 6.5). In addition, less than 80% of atcathb#62 seeds germinated eventually, which was less than the 90% of Col-0 (Figure 6.5). This suggested that the deletion of cathepsin B not only delayed seeds germination rate, but also reduced finally seeds germination ratio.

Figure 6.5. Comparison of germination rates between Col-0 and atcathb#62. Col-0 (closed square) and atcathb#62 (closed circle) seeds were harvested and stored together. After surface sterilization with 70% ethanol, seeds were plated on MS-phytagel medium and store in the cold for two days before germination. Percentages of seeds germination were scored at various hour post imbibition. At least 40 seeds were scored in each plate. Error bar represents standard deviation of triplicates.

6.2.4 Programmed cell death occurred before micropylar endosperm rupture

I attempt in the following section to unveil how and why a deficiency in cathepsin B would delay seed germination. Micropylar endosperm rupture is a key step for seeds germination. Previous reports suggested that micropylar endosperm weakening occurred before micropylar endosperm rupture (Voegele et al., 2012). I wondered whether PCD is

121 involved in micropylar endosperm weakening or not. To check this, I carried out a cell death assay to check the viability of micropylar endosperm cells during seed germination. Col-0 seeds were germinated on wet filter paper. Col-0 seeds, which were at stage II, were collected and stained with 5 μM Sytox green. Sytox green is a chemical which enters only into dead cells and emits green fluorescence when bonding to DNA. As we can see from figure 6.6, the seed testa had ruptured but the micropylar endosperm was still intact (Figure 6.6). Under a fluorescence microscope, micropylar endosperm cells exhibited sytox green positive staining (Figure 6.6 red arrow). This suggested that PCD occurred in some micropylar endosperm cells before it ruptured.

Figure 6.6. Programmed cell death in micropylar endosperm cells during germination. Col-0 seeds were germinated on wet filter paper at 22 ℃. Seeds at stage II were stained with 5μM sytox green. Images of representative seeds were taken under fluorescence (left) or white light (right). The testa showed a brown color under bright field, whereas it also exhibited green fluorescence when observed under fluorescence. Red arrows indicate micropylar endosperm in both fluorescence and bright field images. Scale bar is equal to 0.1mm.

ROS are an important regulator in plants programmed cell death. I tested whether ROS are involved in micropylar endosperm PCD during germination. To detect ROS accumulation in micropylar endosperm, I used 2’,7’ –dichlorofluorescein diacetate (DCFDA). DCFDA is a cell permeable probe for ROS. Upon oxidization by ROS, DCFDA will transform into DCF and emits fluorescence. 5μM DCFDA was incubated with seeds at room temperature for 10 minutes, followed by washing twice in water. Micropylar endosperm fluorescence status was analyzed under a fluorescence microscope. In stage II and III seeds, strong green fluorescence localized inside micropylar endosperm cells around the radicle (Figure 6.7 red arrow). After micropylar

122 endosperm ruptured, frontier cells where micropylar endosperm ruptured showed strong fluorescence (Figure 6.7). This suggested that ROS accumulated in micropylar endosperm during micropylar endosperm PCD.

Figure 6.7. ROS accumulation in micropylar endosperm during germination. Col-0 seeds at stage II and stage III were subjected to DCFDA 5μM staining. Images of fluorescence (left panels) were photographed using a fluorescein filter. The same seeds were also photographed under bright field (right). Red arrows mark micropylar endosperm cells in both fluorescence and bright field images. Scale bar is equal to 0.2mm.

Cathepsin B was found to regulate plants PCD. Therefore I subsequently investigated whether a mutant of cathepsin B could disturb micropylar endosperm PCD during germination or not? I carried out sytox green staining on Col-0 and atcathb#62 seeds when they were at stage II. Seeds were collected and incubated with 5 μM sytox green at room temperature for more than 10min. After incubation, seeds were washed with water twice and mounted onto glass slides for observation. About 86% of Col-0 seeds showed green fluorescence in micropylar endosperm cells, whereas this ratio decreased to 40% in atcathb#62 seeds at stage II (Figure 6.8). To illustrate negative staining, a representative image of atcathb#62 negatively stained seeds is presented (Figure 6.8). The testa was still stained whereas sytox green fluorescence was completely absent in micropylar endosperm cells (Figure 6.8). This suggested that cathepsin B deletion reduced micropylar endosperm PCD during seeds germination.

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Figure 6.8. Comparison of micropylar endosperm PCD between Col-0 and atcathb#62 seeds. Col-0 and atcathb#62 seeds germinated on wet filter paper. Seeds at stage II were collected and incubated with 5μM sytox green at room temperature for about 15min. Stained seeds were observed under fluorescence microscopy. The percentages of sytox green positive seeds were scored. Seeds were scored in triplicate experiment for each line. More than 35 seeds were in each population. Error bars represent standard deviation. A representative image of negative staining in atcathb#62 seeds is also shown (lower image). Scale bar is equal to 0.1 mm.

Cathepsin B deficiency does not totally block PCD in micropylar endosperm; it implied that the influence of lacking cathepsin B on micropylar endosperm PCD was temporary. In other words, Col-0 and atcathb#62 seeds may have same percentage of micropylar endosperm PCD at a later stage of germination. To test this hypothesis, I performed sytox green staining on Col-0 and atcathb#62 seeds at stage III. Stage III seeds were collected and incubated with 5 μM sytox green at room temperature. Stained seeds were observed under a fluorescence microscope. Two representative images of Col-0 and atcathb#62 seeds are shown: sytox green could stain cells in both Col-0 and atcathb#62 seeds after micropylar endosperm rupture (Figure 6.9 red arrow). Moreover, those sytox green cells were confined to the micropylar endosperm area. I also tried and quantitatively compared micropylar endosperm PCD ratios between Col-0 and atcathb#62 seeds at stage III. Both Col-0 and atcathb#62 seeds exhibited more than 90% sytox green positive in micropylar

124 endosperm (Figure 6.10).

Figure 6.9. PCD of Col-0 and atcathb#62 seeds micropylar endosperm cells at stage III. Col-0 and atcathb#62 seeds were stained with 5μM sytox green after the micropylar endosperm ruptured (stage III). Images were taken with a fluorescence microscope under a FITC filter or bright field. Sytox green positive cells showed green fluorescent spots corresponding to the nuclei. Testas were also stained and showed green fluorescence. Red arrow marks sytox green positive micropylar endosperm cells. Yellow arrow indicates same micropylar endosperm cells in bright filed. Scale bar is equal to 0.25 mm.

Figure 6.10. Ratios of micropylar endosperm PCD in Col-0 and atcathb#62 seeds at stage III. Col-0 and atcathb#62 seeds were stained with 5μM sytox green at stage III. The percentage of seeds with sytox green positive micropylar endosperm was scored for each genotype. Triplicates were scored for each genotype. More than 30 seeds were in each replicate. Error bars represent standard deviation.

Several plant hormones, including abscisic acid (ABA), play key roles in seed germination. Previous reports have shown that ABA inhibited PCD in endosperm during seeds maturation (Young and Gallie 2000). Therefore whether ABA also inhibits micropylar endosperm PCD during germination was tested here. The inhibitory role of ABA during seed germination was confirmed in this section. As we can see from figure 6.11, Col-0 seeds sown on MS medium containing 10 μM ABA germinated very slowly 125

(Figure 6.11). Only 30% seeds germinated at 96 hours when non-treated Col-0 seeds had 80% of germinated seeds (Figure 6.11). This is in line with previous reports.

Figure 6.11. ABA reduced Col-0 seed germination rate. Col-0 seeds were germinated on MS medium with or without 10μM ABA. Germination percentage of each treatment was scored at various time points. Triplicates of at least 40 seeds in each plate were scored. Error bars represent standard deviation.

Whether the reduced germination rate of ABA treated seeds correlated with a blocked PCD in micropylar endosperm was also tested. 10 μM ABA-treated seeds, which were at stage II were subjected to sytox green staining. Micropylar endosperm cells were exposed because the testa had ruptured but no green fluorescence was observed in these cells (Figure 6.12). Moreover, ABA treated seeds at stage III or stage IV were also stained using sytox green to see whether ABA delayed rather than totally blocked micropylar endosperm PCD. None of those seeds exhibited sytox green positive cells in the ruptured micropylar endosperm in this analysis (Figure 6.12). It suggested that ABA blocked PCD in micropylar endosperm cells.

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Figure 6.12. PCD of ABA treated Col-0 seeds micropylar endosperm cells at stage II and III. Col-0 seeds treated with 10 μM ABA were subjected to sytox green staining at stage II and III. Representative images were taken using a fluorescence microscope under a GFP filter or under bright field. Sytox green positive cells showed green fluorescent spots corresponding to nuclei. Testas were stained with green fluorescence but endosperm cells were not. Red arrow marks micropylar endosperm cells. Yellow arrow indicates same micropylar endosperm cells in bright filed. Scale bar is equal to 0.2 mm.

The consequence of applying ABA to seeds during germination was not only to delay seed germination, but also caused novel morphological changes. Col-0 seeds germinated on MS medium containing 10μM ABA were photographed. Seeds at stage III and IV are presented (Figure 6.13). The micropylar endosperm layer was hard to dissociate from the root tip, even when the majority of the seedling had come out of the testa and endosperm (Figure 6.13). A layer of dark gray cells, which was named as “cap”, was left on the root tip and covered most of root tip area (Figure 6.13). This novel structure was absent in Col-0 seeds germinated on ABA free medium (Figure 6.13). To avoid the bias of being selective when taking images of seeds, I carried out counting and statistical analysis. The percentage of seedlings with a cap on the root tip was recorded. ABA free seedlings did not show any cap like structure (Figure 6.14). However, more than 80% ABA treated seeds showed a cap structure (Figure 6.14)

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Figure 6.13. Novel cap like structure on ABA treated seedling radicle Col-0 seeds germinated on ABA 10 µM (upper panel) or ABA free (lower panel) medium were photographed at stage IV. Testas show brown colour. The novel cap-like structure is indicated by a yellow arrow. Scale bar is equal to 0.2 mm.

Figure 6.14. Comparison of the percentage of root tips with or without cap-like structure in ABA treated and untreated seeds. Col-0 seeds were germinated on MS medium with or without ABA 10 µM and were observed under a dissecting microscope. The ratios of seeds with cap like structure were recorded. Four independent plates of each sample were observed with more than 25 seeds in each plate. Error bar indicates standard deviation.

Results in previous chapters implied that VPE governed tonoplast rupture, which might be a key step for releasing cathepsin B to the cytoplasm to fulfill its PCD function. So it is possible that VPE may also participate to germination in general and micropylar endosperm PCD in particular. To test this hypothesis, a vpe null line was used. Germination rates of Col-0 and vpe null lines were compared. As usually, Col-0 seeds germination rate started increased after 24 hours and reached a plateau (more than 90%) at 72 hours (Figure 6.15). vpe null seeds had only about 30% seeds germinating at 48 hours when Col-0 reached already 80% (Figure 6.15). Even after 72 hours vpe null seeds still showed a lower germination percentage than Col-0 (Figure 6.15). I then examined whether vpe null mutant reduced PCD in micropylar endosperm cells. Col-0 and vpe null seeds at stage II and after stage III, were subjected to sytox green staining followed by imaging analysis under fluorescence microscopy. More than 90% seeds in Col-0 before or after micropylar endosperm rupture showed Sytox green positive (Figure 6.16). Surprisingly vpe null mutant seeds exhibited almost the percentage as that of Col-0 seeds (Figure 6.16). It demonstrated that vpe null mutant did not block PCD in the micropylar endosperm.

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Figure 6.15. Comparison of germination rates between Col-0 and vpe null seeds. Col-0 and vpe null seeds were surface sterilized with 70% ethanol. Seeds were sown on MS- phytagel medium and put in the cold for two days before transfer to 22C, 16h light. The percentages of seed germination were scored at various hour post imbibition (hpi). Seeds were scored in triplicates of at least 40 seeds. Error bars represent standard deviation.

Figure 6.16. Comparison of ME PCD in Col-0 and vpe null seeds. Col-0 and vpe null seeds were germinated on wet filter paper. PCD in micropylar endosperm of Col-0 and vpe null seeds were detected by sytox green at stage II (Testa Rupture, TR) and stage III (Micropylar endosperm rupture, MR). The percentages of sytox green positive seeds were recorded. Each type of seeds were counted in triplicate experiments of 20 seeds in each. Error bars represent standard deviation.

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6.3 Discussion

Xylem formation is a classic developmental process, which includes PCD. A preliminary experiment in this chapter suggested that cathepsin B deficiency did not obviously alter xylem formation in Arabidopsis. However, the method used may not be enough to exclude cathepsin B from the regulation of xylem formation. As seen in previous chapters, cathepsin B deficiency did not totally block PCD. So it is possible that a different type of PCD occurred during xylem formation in the cathepsin B mutant genetic background. Some mutant and wild type analysis of xylem cells morphological features using TEM may help to answer this question.

The results in this chapter showed that PCD occurred in micropylar endosperm cells during seeds germination. This is the first time that PCD is reported in Arabidopsis micropylar endosperm during germination. Micropylar endosperm weakening is associated with radicle protrusion during seed germination. This weakening is restricted to the micropylar endosperm area and does not occur elsewhere in the remaining endosperm (Weitbrecht et al., 2011). This correlates with the local emergence of PCD in micropylar endosperm. Spatial and temporal overlap between micropylar endosperm weakening and PCD implied that PCD participates to micropylar endosperm weakening. Moreover, a novel cap-like structure was found attached to the radicle tip of ABA treated seeds. This structure, presumably, resulted from incomplete micropylar endosperm weakening was possibly due to a blocked PCD. Although cathepsin B deficiency reduced micropylar endosperm PCD, this did not totally blocked micropylar endosperm PCD in late stage. This may be the reason that the novel cap-like structure was absent in atcathb#62 seed radicles. The current model for micropylar weakening relies only on cell wall loosening by enzymes (Voegele et al., 2012). How PCD contributes to cell wall loosening is unclear at this stage. One possible consequence of PCD would be to release enzymes into the apoplast, enzyme that would change properties of the cell wall. This will be tested in the future. Cathepsin B mutant seeds exhibited a reduced germination rate compared to Col-0 seeds. However, whether this phenotype was solely due to a reduction of micropylar endosperm PCD is not for sure now. This is because cathepsin B deficiency also showed better maintenance of inner integument layer thickness, which is believed to be due to reduced PCD (Nakaune et al., 2005). Vpe null seeds, which also maintain inner tegument thickness exhibited a similar delayed seeds germination rate. In the case of VPE, the

130 reduced germination phenotype was proved to be not due to a reduction of micropylar endosperm PCD. This implies that blocking inner integument cell layer PCD without affecting micropylar endosperm PCD may delay seeds germination in itself. It cannot be excluded that some other undiscovered PCD does affect germination in the vpe and atcathb#62 mutants. Or both proteases could be required for some aspects of germination that do not involve PCD, e.g. storage protein degradation. As cathepsin B was found to have a dual role in both inner integument cell PCD and micropylar endosperm PCD, whether the delayed germination of cathepsin B mutant seeds was due to less inner integument cells PCD or less micropylar endosperm PCD needs more research in the future. For example, silencing cathepsin B under the control of tissue specific promoter may help to learn which PCD contribute more to seeds germination.

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Chapter 7 Analysis of cathepsin B and KOD-

induced PCD pathway

7.1 Introduction

A novel peptide, which was named Kiss-of-Death (KOD), has been identified as a regulator of plant PCD (Blanvillain et al., 2011). The KOD gene was discovered using a promoter trap method in Arabidopsis. The gene was identified in a line, which expressed the GUS reporter gene in suspensor cells during seeds development (Blanvillain et al., 2011). A recent report implicated an orthologue of KOD in the PCD occurring in Oryza sativa hybrid sterility (Yang et al., 2012). These findings implied that KOD might be involved in different developmental PCD processes. In addition, it was shown that KOD is required for heat induced PCD of root hair and expressed during HR, suggesting a role in stress-induced PCD (Blanvillain et al., 2011). Interestingly, KOD-induced PCD in Arabidopsis led to an increase caspase-3-like activity in transient assays (Blanvillain et al., 2011). It was therefore worth investigating whether cathepsin B could mediate KOD- induced PCD.

A functional interaction between KOD and cathepsin B can be tested genetically using KOD peptide and cathepsin B mutant lines. However, to understand how they influence on each other, if they really have a functional interaction, requires a broader analysis. As KOD was a newly identified gene in Arabidopsis, literature about its molecular function is scarce. So, it was impractical to obtain clues from the published literature for researching KOD function. To get over this obstacle, I used DNA microarrays to obtain leads on the putative function of KOD in plant PCD.

Microarray provided a dataset of gene expression changes induced by KOD. Based on the KOD-induced gene datasets, a series of bioinformatics analyses were employed to seek a mechanism by which KOD induced plant PCD. For this Gene Ontology (GO) is a commonly used method. GO analysis examines whether genes linked to a GO term are overrepresented in the microarray dataset. This method can identify which biological processes KOD-induced genes may be involved in. GO analysis may provide a profile of 132 pathways that KOD participates in. GO analysis can then be complemented by other bioinformatics approaches as more details are needed to solve how KOD-induced plant PCD. I tried three additional approaches. The first one was gene interactions analysis. Possible interactions among KOD-induced genes, based on published literatures and yeast-2-hybrid data, can be generated in silico. Putative interacting genes are clustered according to different biological processes in such a way that potential pathways involving KOD can be identified. The second approach was promoter analysis. Analysis of the promoter sequences of KOD-induced genes can identify common conserved transcription factors binding motifs. This helps to establish a signal cascade from KOD to its induced target genes. The final and third approach was an analysis of metabolic pathways in which KOD-induced genes potentially participate. Metabolic changes are important to plants when they are facing various stimuli including stress. Searching for overrepresented genes connected with specific metabolism pathways may tell us how plant cells respond to KOD in terms of metabolism changes, again giving the broad picture of a possible mode of action for the peptide. All this was carried out with the aim of generating several hypotheses for a KOD / Cathepsin B pathway that could be tested experimentally in the future.

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7.2 Results

7.2.1 Function of cathepsin B in KOD-induced PCD

Blanvillain et al., (2011) reported that KOD-induced PCD was accompanied with increased caspase-3-like activity. As cathepsin B has been shown to be responsible for caspase-3-like activity I investigated whether Cathepsin B was a part of the KOD pathway. First of all I set up a simple experimental system to induce PCD by adding synthetic KOD peptide to plant cells. For this, protoplasts were isolated from Arabidopsis Col-0 leaves and resuspended in culture medium. In order to test at which concentration KOD can effectively trigger PCD, the synthetic KOD peptide was added into protoplast culture at various final concentrations (0.5 μM, 1 μM and 5 μM). DMSO was added into protoplasts to a final concentration of 0.5% as mock treatment. PCD in KOD peptide treated protoplasts was detected using sytox green staining. Sytox green positive protoplasts, which were dead protoplasts, were scored at 0.5, 4 and 20 hours after treatment. All samples had only about 10% sytox green positive protoplasts at half an hour post treatment (Figure 7.1). As time went on, the percentages of sytox green positive protoplasts increased. 5μM KOD peptide induced a higher PCD rate than the other concentrations. 5μM KOD treated protoplasts had more than 30% dead protoplasts at four hours while the rest samples had less than 20% dead protoplasts (Figure 7.1). At 20 hours after treatment, dead protoplasts reached 50%, whereas the percentages of dead protoplasts in 0.5 μM, 1μM and mock samples were about 25% (Figure 7.1). This result suggested that at least 5 μM of KOD peptide was necessary to induce PCD in this experiment.

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Figure 7.1. KOD peptide induced PCD in Arabidopsis protoplasts. Arabidopsis Col-0 protoplasts were isolated and cultured at 106/ml. Different concentrations of KOD peptide (0.5 μM, 1 μM and 5 μM) were added in DMSO. The mock sample had DMSO only. Sytox green at 5 μM was used to detect dead protoplasts. Protoplasts were observed under fluorescence microscope at designated time points. Five populations with more than 200 protoplasts were counted in each treatment. Error bars represent standard deviation.

It has been reported that caspase-3-like activity was increased in KOD-induced PCD (Blanvillain et al., 2011). This implied that cathepsin B might be involved in KOD induced PCD. To verify this hypothesis, I used Ac-DEVD-FMK and CA074Me, which are caspase-3 and cathepsin B inhibitors, to suppress caspase-3-like activity and cathepsin B activity in KOD peptide treated protoplasts. 50 μM Ac-DEVD-FMK and 50 μM CA074Me were added to protoplasts at the same time as 5 μM KOD peptide. CA074Me was used here as it is a cell permeable inhibitor for cathepsin B. Once CA074Me enters into a cell, the methyl ester group will be cleaved by cytoplasmic esterase, and the inhibitor can interact with cathepsin B. The PCD percentages of the different treated samples were measured at four hours. 5 μM KOD peptide on its own caused about 27% protoplasts death at four hours while KOD peptide free protoplasts had only 6% death (Figure 7.2). Adding Ac-DEVD-FMK and CA074Me to protoplasts with the KOD peptide reduced death to 9% and 11% respectively (Figure 7.2). This suggested that inhibition of caspase-3-like activity or cathepsin B activity could reduce KOD-induced PCD.

Figure 7.2. Effect of the protease inhibitors DEVD-FMK and CA074Me on KOD- induced PCD. Arabidopsis Col-0 protoplasts were isolated and cultured at 106/mL. 5 μM KOD peptide was added into Col-0 protoplasts with or without 50 μM Ac-DEVD-FMK and 50 μM CA074Me. Dead protoplasts were detected using 5 μM sytox green. Protoplasts were observed under a fluorescence microscope at designated time points. Three populations with more than 200 protoplasts were counted in each treatment. Error bars represent standard deviation.

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To investigate the possibility that the reduced PCD in CA074Me treatment was due to CA074Me targeting other cathepsin proteases, for example cathepsin L, I used the cathepsin B triple mutant line, atcathb#62, to prepare protoplasts and induce PCD using KOD. 5μM KOD peptide was applied to Col-0 and atcathb#62 protoplasts. Dead protoplasts percentages were recorded at four hours after treatment. 5μM KOD peptide induced more than 30% death in Col-0 protoplasts (Figure 7.3). However, there was no KOD-induced PCD in atcathb#62 (Figure 7.3). Meanwhile, untreated Col-0 protoplasts and atcathb#62 protoplasts had about 8% death (Figure 7.3). This demonstrated that lacking cathepsin B reduced KOD-induced PCD.

Figure 7.3. KOD peptide induced PCD in cathepsin B deficient protoplasts. Arabidopsis Col-0 and triple cathepsin B mutant (atcathb#62) protoplasts were isolated and cultured at 106/mL. 5 μM synthetic KOD peptides were added. Dead protoplasts were detected using 5 μM sytox green. Protoplasts were observed under a fluorescence microscope at designated time points. Three populations with more than 200 protoplasts were counted in each treatment. Error bars represent standard deviation.

Having shown that cathepsin B was involved in KOD-induced PCD, I attempted to investigate whether cathepsin B expression could be induced by KOD peptide. 5 μM KOD peptides treated and untreated Col-0 protoplasts were harvested at four hours after treatment. Total RNA was isolated from these protoplasts, and then subjected to cDNA synthesis. Cathepsin B transcript level was visualized via semi-quantitative RT-PCR. All three cathepsin B paralogues in Arabidopsis were amplified with their gene specific primers, and actin2 was used as reference gene. As we can see from Figure 7.4, AtCathB1 had very low expression level in both KOD untreated and treated protoplast and amplification was barely detectable (Figure 7.4). AtCathB2 and AtCathB3 did not show significant change when challenged with the KOD peptide (Figure 7.4). This suggested that the KOD peptide did not alter cathepsin B transcript level at four hours after treatment.

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Figure 7.4. Cathepsin B transcript level during KOD peptide induced PCD. Protoplasts with or without 5 μM KOD peptide treatment were subjected to mRNA isolation at four hours after treatment, followed by cDNA synthesis. Cathepsin B transcript levels were detected using RT-PCR. RT-PCR was carried out with different cycles. The three paralogues of cathepsin B (B1,B2 B3) were all tested. Actin2 was used as a reference gene.

7.2.2 Test of DEX-KOD inducible system

Identifying gene expression changes during KOD-induced PCD could give clues on what links cathepsin B to KOD-induced PCD. For this I carried out a DNA microarray analysis. To induce death in a large amount of tissue, an inducible KOD expression system was used. In the system chosen, the KOD open reading frame was fused with a promoter, controlled by the glucocorticoid dexamethasone (DEX). A diagram of the DEX induction system used is presented in Figure 7.5. The corresponding construct was transformed into Arabidopsis (Col-0), and a homozygous DEX-KOD line, DEX-KOD 25#, was selected in the lab by Dr. Bennett Young. I first tested the efficiency of DEX- triggered KOD PCD in seedlings of this line. DEX-KOD 25# seeds germinated on MS medium and seedlings were transferred to a 6-well plate followed by treating with 30μM DEX at the two-cotyledon stage (three days after germination). Seedlings were then kept under a constant light environment. DEX treated DEX-KOD 25# seedlings became pale in the cotyledon tissues (Figure 7.6 the three images on the left column), whereas untreated DEX-KOD 25# still showed green cotyledons on day three (Figure 7.6, the three images on the right column). This suggested that the DEX-KOD 25# line was appropriate to induce PCD in seedlings and to harvest total RNA.

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Figure 7.5. A diagram of the DEX-KOD inducible system. A diagram of DEX-KOD inducible system is presented here. LhGR consists of a fusion between a high-affinity DNA-binding mutant of the lac repressor, and the transcription- activation-domain-II of GAL4 from Saccharomyces cerevisiae. Upon adding DEX, LhG4 dissociates from HSP90 to activate the bidirectional pOp promoter which subsequently activates the transcription of KOD::GFP and of GUS.

Figure 7.6 DEX induction of KOD expression activated PCD in seedlings. DEX-KOD 25# seeds were germinated on wet filter paper. Three days after germination, DEX at 30 μM was added (+) or not added (-) to the seedlings, and the seedlings grew with DEX for a further three days. Images of the seedlings were taken using a dissecting microscope at two days after adding DEX. Three representative images are presented for each treatment. Scale bar is equal to 5 mm.

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Expression of the KOD peptide was confirmed via GUS staining. For this, DEX-KOD 25# seedlings were germinated on MS medium and three days after germination, seedlings were treated with or without 30 μM DEX for three and five hours. Seedlings were subjected to GUS staining. Untreated DEX-KOD 25# did not exhibited any blue colour in any part of seedling (Figure 7.7). By contrast, the three-hour DEX treatment caused a strong GUS staining, but most of the expression was confined to the root (Figure 7.7). Treating DEX-KOD 25# seedlings with DEX for five hours resulted in GUS expression in most parts of the seedlings, including cotyledons, hypocotyls and roots (Figure 7.7).

Figure 7.7. GUS expression profile under the control of the DEX inducible system. Three days after germination, 30 μM DEX was added to DEX-KOD 25# seedlings. A group of DEX-KOD 25# seedlings without DEX was designated as untreated sample. Seedlings were subjected to GUS staining three or five hours after DEX treatment. After incubation in the GUS staining buffer for more than 16 h, seedlings were observed under a dissecting microscope and photographed. Two representative images of each treatment (untreated, 3 hours and 5 hours treatment) are shown above. The blue stain indicated where GUS expression occurred. GUS expression is a reporter of KOD expression in this line. Scale bar is equal to 2 mm

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7.2.3 Microarray analysis after three hour DEX induction

DEX-KOD 25# Arabidopsis seedlings (more than 30 seedlings were used in each treatment) were treated with or without DEX respectively. Samples were collected three hours after treatment for RNA extraction. Col-0 Arabidopsis seedlings were also treated with or without DEX. RNA samples of Col-0 seedlings were also harvested at the same time as the DEX-KOD 25# samples. Gene expression profiles of these samples were examined by Affymetrix ATH1 microarray. Raw microarray data were normalized by Robust Multi-array average (RMA). The threshold of significance was set at two-fold difference. Only four genes changed transcript level significantly when comparing Col-0 DEX treated sample with Col-0 untreated sample. This confirmed that DEX had very little influence on gene transcript level on Col-0 Arabidopsis (data not shown). Comparison between DEX-KOD 25# treated sample and untreated sample indicated that 19 genes were up-regulated by at least two fold and four genes were down-regulated (less than 0.5 fold). These genes are listed below (Table 7.1).

Table 7.1 Gene list at the two-fold change threshold at three hours DEX treatment. Genes, which changed transcript level more than two folds or less than 0.5 fold under three hours DEX treatment, are listed. The table is divided into three columns. Left column is the gene locus ID, middle column is the gene description, and right column shows the fold change.

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In order to learn in which biological processes the genes with differential expression participated, the genes up-regulated by two folds were subjected to gene ontology (GO) analysis. The gene list was input into AgriGO (http://bioinfo.cau.edu.cn/agriGO/analysis.php) for analysis. Overrepresented GO terms were picked out and are shown in Figure 7.8. Among 19 up-regulated genes, eight were involved in response to various stimuli (Figure 7.8). Seven of them belonged to a subclass, which was termed as response to stress (Figure 7.8). This suggested that genes responsible for response to stress mediated the early response of KOD-induced PCD.

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Figure 7.8. GO analysis of genes induced by three hours DEX treatment. The genes list for up-regulation at three hours was analyzed. The yellow blocks highlight the enriched GO terms. In each block, the GO term is shown and the P-value is in the brackets. The numbers in the bottom line shows number of gene belonging to the GO versus the total genes number in the list or in the whole genome.

The GO analysis did not identify a specific stress response, so I adopted another strategy to find out which pathway the up-regulated genes were involved in. A promoter analysis was selected as it might give some clues by identifying a single common or a set of common transcription factors potentially regulating the KOD response. Again the three hours up-regulated gene list was subjected to promoter analysis using the software AthaMap (http://www.athamap.de/index.php). The sequences of the 500 base pairs (- 500bp) upstream of the transcript starting-site (TSS) and the 50 base pairs downstream (+50bp) of the TSS were scanned. Putative transcription factor binding motifs were aligned against known conserved motif sequences. The validity of these alignments was scored based on a nucleotide substitution matrix. Motifs whose score was more than the threshold score (variable according to the matrix) were selected. Collectively the nineteen genes returned many putative transcription factor binding motifs. The binding motifs that were common across genes were selected for further analysis. A total of 59 putative binding sequences showed in more than two genes promoters (Table 7.2). To refine these results, I chose two criteria: 1. The corresponding transcription factors must be identified in Arabidopsis thaliana; 2. The corresponding transcription factors must have a high expression in the root at the two-cotyledon stage, as KOD expression was restricted within the root area at three hours DEX treatment (Figure 7.7 C,D). Using the information provided by AthaMap, the transcription factors that existed in Arabidopsis thaliana were highlighted in yellow or red in Table 7.2. The expression profile of these transcription factors in Arabidopsis tissues was examined in the Bio Array Resource (BAR). Most of these transcription factors exhibited low expression in the root at the two-cotyledon stage and these were highlighted in yellow. However, two transcription factors with high expression level in the root of two-cotyledon seedlings passed the filter and were highlighted in red. They were RAV1 (an AP2/B3 domain transcription factor, AT1G13260) and TGA1 (a bZIP transcription factor, bZIP47, AT5G65210). These two transcription factors have been reported to be involved in biotic and abiotic stress responses (Kesarwani et al., 2007, 1; Woo et al., 2010).

More effort was put into trying to fit RAV1 and TGA1 in given regulatory networks. This

142 was achieved by using the GRG-X database (http://arabidopsis.med.ohio-state.edu/grgx/). RAV1 and TGA1 were input into GRG-X. GRG-X returned a regulatory network where both RAV1 and TGA1 were involved. Note that TGA1 was named as bZIP47 in the GRG-X software according to its own nomenclature rules. In this network, several genes showed either experimentally confirmed or putative regulatory interactions with RAV1 and TGA1 (bZIP47) (Figure 7.9). The AP2 transcription factor (At4g36920) and LFY (AT5G61850) could activate RAV1. SEPALLATA3 (At1g24260) and AGL15 had unconfirmed predicted regulation function on RAV1 (Figure 7.9). A helix-loop-helix transcription factor bHLH15 (At2g20180) might be able to regulate TGA1 and SEPALLATA3 (Figure 7.9). bHLH15 might be under the regulation of AGL15 (Figure 7.9). A potential protein, which may connect RAV1 and TGA1 pathway was AGL15, however, how AGL15 would regulate TGA1 and RAV1 is unclear.

Table 7.2 Transcription factors (TFs) prediction for the regulation of the up- regulated genes in three hour DEX treatment Putative transcription factors for the 3 hours up-regulated genes are listed in the table. TFs belonging to the Arabidopsis genome are marked with yellow and red. Red items are genes of high expression at two-cotyledon stage in root.

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Figure 7.9. The interaction network of RAV1 and TGA1 (bZIP47). RAV1 and TGA1 (bZIP47) interaction network are generated by GRG-X. RAV1 and TGA1 (bZIP47) are labeled with green colour. Blue arrow means activation. Red blunt end line means inhibition. Gray ball end line (or dash line) means unknown (unconfirmed) interaction.

7.2.4 Microarray analysis after five hour DEX induction

In the three hours treatment microarray data, I did not see any cathepsin B transcript level alteration. It was possible that cathepsin B would be induced at a later stage. To check this hypothesis, I analysed KOD-induced gene change after a five hour DEX treatment. Total RNA from DEX treated DEX-KOD 25# seedlings and non-treated DEX-KOD 25# seedlings were isolated for microarray analysis. Raw data normalization was done using RMA. Overall, 445 genes exhibited more than two fold up-regulation, and 366 genes had more than two fold decrease when compared to non-treated sample (Appendix 1). However, still the cathepsin B transcript level did not change within five hours of KOD induction.

Gene ontology analysis was then carried out to understand which biological processes the KOD-induced genes are involved in. This is also helpful to find out whether KOD- induced genes may be involved in the biological processes in which cathepsin B participates, for example plant defence response. The gene loci ID from the two fold up- regulation list were input into AgriGO for gene ontology analysis. Overrepresented gene ontology terms were marked as yellow, orange and red in Figure 7.10, the colour being dependent on how significant they were. To better represent the gene ontology hierarchy, I chose a flow chart display to present GO terms. The GO chart indicated that genes that were involved in plant defence response and response to other organisms were highly

144 overrepresented (Figure 7.10 red block). In subclass GO categories, genes enriched in response to fungus, bacterium and chitin (Figure 7.10 orange block) were overrepresented. The gene response to salicylic acid stimulus was also significantly as overrepresented (p<0.05) (Figure 7.10 yellow block). Besides plant defence related GO terms, genes enriched in secondary metabolism were also identified (Figure 7.8 orange block, left part). The function of cathepsin B in plant response to pathogen has been documented (McLellan et al., 2009). This implied that cathepsin B might mediate some plant biological processes which overlapped with the KOD induced plant biological processes. This will be further discussed in the discussion section.

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Figure 7.10. Gene ontology analysis of the gene list for the five-hour DEX treatment. The gene ontology of the genes up-regulated at five hours DEX is presented. The significance of genes over-representation in certain GO terms is marked with colors. Significance increases as colour changes from yellow to red. In each block, GO number and terms are shown. The P-value is in between brackets. Numbers in the bottom line show the number of gene belonging to a certain GO versus total genes number in the list and in the whole genome.

As KOD was suggested to be a part of the plant response to bacterium and fungus (Blanvillain et al., 2011), I compared the changes in gene expression induced by DEX- KOD, DC3000 avrrpm1 and DC3000 avrrpt2. Microarray data of the Arabidopsis response to DC3000 avrrpm1 (four hour treatment) was obtained from Truman et al. (2006) report (Truman et al., 2006), and DC3000 avrrpt2 (48 hour treatment) microarray data was obtained from the database PlexDB (http://www.plexdb.org/). KOD-induced genes and DC3000 avrrpm1 and avrrpt2 induced genes were subjected to a Venn diagram analysis using BioVenn (http://www.cmbi.ru.nl/cdd/biovenn/). KOD shared 56 genes with DC3000 AvrRpm1, whereas there were only 28 in common between KOD and DC3000 AvrRpt2 induced genes (Figure 7.11). Gene ontology analysis of KOD and DC3000 AvrRpm1 common genes indicated that they belonged to three significant groups, defence response, endomembrane system and electro carrier activity (Table 7.3). Surprisingly, the 28 genes common to both KOD and DC3000 AvrRpt2 gene lists did not show any significant GO category enrichment.

Figure 7.11 Venn diagram of the KOD induced genes with pseudomonas syringae DC3000 induced genes Venn diagram representing the overlap between KOD-induced genes (five hours) and pseudomonas DC3000 avrrpm1 (four hours) and avrrpt2 (48 hours) induced genes. The total number of genes and the number of overlapping genes are shown.

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Table 7.3 GO of genes induced by KOD and AvrRpm1 induced gene lists. Gene ontology analysis of the genes common to both KOD (five hours) and pseudomonas DC3000 avrrpm1 (four hours) induced gene lists. GO terms and descriptions are presented in the table. The P-value indicates the statistical significance of the GO term detected.

KOD is a small peptide with only 25 amino acids. This reminded me that the KOD- induced signalling pathway might correlate with the flg22-induced signal pathway. Flg22 is a well-known microbe flagellin peptide, which induced plants response to pathogen attacks. Here, I compared the gene lists induced by KOD and by flg22. 515 genes showed an increased transcript level one hour after adding flg22 to Arabidopsis seedlings. 99 genes were found in both KOD and flg22 treatments (Figure 7.12). GO analysis of these 99 genes showed that three major groups were protein phosphorylation, kinase activity and response to chitin (Table 7.4).

Figure 7.12 Venn diagram of KOD induced genes and flg22 induced genes. Venn diagram representing the overlap of KOD-induced genes at five hours with flg22 induced genes at one hour. The total number of genes and the number of overlapping genes are also shown.

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Table 7.4. GO of genes common to the DEX-KOD and flg22 gene list. The results of the Gene ontology analysis for genes induced by both KOD at five hours and flg22 at one hour are displayed. GO terms and descriptions overrepresented in the overlapping gene list are presented in the table. P-value indicates the statistical significance of the enriched GO terms.

. To better understand how KOD-induced genes at five hours were involved in plant defence, I used the N-Browse online tools to analysis putative gene interactions within the gene list. The two-fold up-regulation gene list was imported into N-Browse. Several gene interactions were identified. Five clusters of genes interactions were observed and distinguished by different background colours in Figure 7.13. Gene ontology analysis indicated that these genes clusters were involved in ABA biosynthesis (green), salinity response (blue), photomorphogenesis (pink), cell fates (lilac) and xylem formation (yellow).

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Figure 7.13. Interactions suggested among the KOD-induced genes after five hours DEX treatment. KOD-induced genes at five hours, which may interact with each other were identified via N- Browse. Proposed interactions were supported by different methods. Red line means confirmed by biochemical assay. Green line means confirmed by Y2H. Five highly clustered groups, ABA biosynthesis (green), salinity response (blue), photomorphogenesis (pink), cell fates (lilac) and xylem formation (yellow), were identified.

The same gene list was also imported in MetNet for metabolite regulatory network analysis. Ten pathways were identified based on the gene overrepresentation algorithm. The top one, which was the most significantly over-represented pathway, was camalexin biosynthesis (Table 7.5). Camalexin is a type of phytoalexin, which is involved in plants defence response. Other identified pathways were related to IAA biosynthesis (Table 7.5). This might be due to the fact that camalexin derives from tryptophan via indole-3- acetaldoxime.

Table 7.5. Potential metabolism pathway in KOD induced genes at five hours. The five-hours up-regulation gene list was analysis using MetNet. Putative metabolism pathways, which are enriched in the gene list, are shown in the table. P-value indicates the statistical significance of pathway over-representation.

Genes, which were down-regulated by KOD expression at five hours were also examined. The 0.5 down-regulation gene list was subjected to gene ontology analysis and MetNet metabolism pathway analysis. Gene ontology analysis indicated that the down-regulated genes were enriched in the abiotic stress response (Figure 7.14 red block). This included the responses to cold and light (Figure 7.14 orange block). Another very highly overrepresented GO category was lipid localisation (Figure 7.14 orange block). The genes down-regulated by five hours KOD treatment were also enriched in photosynthesis related GO term (Figure 7.14 orange block). In addition, nine pathways were identified via MetNet analysis (Table 7.6). Two of them were Calvin cycle related, one was fatty

151 acid synthesis related, and one was gluconeogenesis related. This suggested that photosynthesis was repressed during KOD expression.

Table 7.6. Potential metabolism pathway identified in KOD repressed genes at five hours. Down-regulated genes at five hours were analyzed using MetNet. Putative metabolism pathways identified in the gene list are shown in the table. P-value indicates the statistical significance of genes over-representation for certain pathways.

Figure 7.14 Gene ontology analysis for the genes down-regulation at five hours (next page). Down-regulated genes at five hours were analyzed using AgriGO. The GO of these genes is represented. The significance of the over-representation of certain GO term is indicated with colors. Significance increases as color change from yellow to red. In each block, GO numbers and terms are shown. The P-value is in between brackets. The numbers in the bottom line shows the number of gene belonging to certain GO versus the total gene number in the list and the same for the whole genome.

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7.3 Discussion

The signal pathway that mediates KOD induced PCD needs investigating. In this chapter, I found that applying a cathepsin B inhibitor to protoplasts was able to block KOD- induced PCD. This finding was further supported by the result that the cathepsin B deficiency line atcathb#62 also blocked KOD-induced PCD. This demonstrated that cathepsin B was a component of the KOD-induced PCD signal pathway. However, cathepsin B may execute its function in a different way compared to its mode of action in ER stress-induced PCD, because KOD did not induce cathepsin B transcript level in our experiments using RT-PCR or microarray. Cathepsin B may be regulated via translation, activation, or translocation during KOD induced PCD rather than regulated at transcript level. This can be tested in the future by monitoring cathepsin B subcellular localisation during KOD-induced PCD.

Analysis of KOD-induced gene expression indicated that the KOD signal pathway was strongly overlapping with plant defence signal networks. Several lines of evidence supported this conclusion. 1. Gene ontology analysis of microarray data at five hours induction showed that the gene list was enriched in genes of the plant defence pathway; 2. The KOD-induced genes had a large overlap with Pseudomonas syringae DC3000 avrrpm1 and flg22 induced genes. Pseudomonas syringae DC3000 avrrpm1 and flg22 are well-documented effectors that induce plants defence responses; 3. Metabolism network analysis identified that plant phytollexin synthesis related genes were over-represented in the KOD-induced genes. It could be argued that plant cells may mistake the KOD peptide for a peptide elicitor from a foreign organism and by doing that induce effector-triggered immunity. However, the KOD peptide being encoded by the plant genome, it should not be recognized as an exogenous molecule. 4. KOD-induced genes were enriched in the GO category in response to salicylic acid stimulus. Salicylic acid is a well-documented plant hormone that regulates plant defence response (Shah, 2003). 5. Promoter analysis of KOD-induced genes after a three hours DEX treatment indicated that RAV1 and TGA1 were potential transcription factors for these genes. The roles of RAV1 and TGA1 in plant biotic and abiotic stress have been reported (Shearer et al., 2012). This is in line with the finding that KOD-induced genes overlapped with plant defence response genes. Whether RAV1 or TGA1 are real downstream components of KOD signal pathway needs more investigation in the future.

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In this chapter, we have seen that KOD induced genes had strongly overlap with plant defence genes. This was consistence with the previous results that cathepsin B mediated pseudomonas syringae induced HR PCD (Mclellan et al., 2009). Although how cathepsin B participates in KOD-induced PCD has not been resolved, these results open a new aspect for investigation of the function of cathepsin B in plant PCD. More experiments, such as analysing a microarray KOD-induced PCD at a late stage, may help to understand how cathepsin B regulates KOD-induced PCD in the future.

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Chapter 8 General conclusion and discussion

PCD has been implicated in various plant physiological processes (Greenberg 1996). However, the molecular mechanism of PCD in Arabidopsis is not fully understood. In this thesis, I investigated the function of cathepsin B, a cysteine protease, in the context of different PCD situations. These situations included ER stress-induced PCD, developmental PCD and KOD induced-PCD. Besides research on cathepsin B’s individual role in PCD, I also attempted to fit cathepsin B in a signal transduction pathway from ER stress to PCD. In this chapter, I am going to conclude and discuss major findings of my research.

8.1 Contrasting role of cathepsin B and PBA1

The role of cathepsin B in mediating pathogen-induced HR PCD has been documented (Mclellan et al., 2009; Gilroy et al., 2007). In this thesis, I reported a new role for cathepsin B in ER stress-induced PCD. Cathepsin B triple mutant in Arabidopsis (atcathb#62) had a significantly reduced ER stress-induced ion leakage. Moreover, cathepsin B triple mutant seedlings grew better than Col-0 seedlings did under ER stress treatment. These results strongly support the hypothesis that cathepsin B positively mediates ER stress-induced PCD. A previous finding in our lab indicated that Arabidopsis cathepsin B possessed caspase-3-like activity (Ge et al., in preparation). Results in this thesis further confirmed that Arabidopsis cathepsin B possessed caspase-3-like activity, as mutant of cathepsin B diminished Biotin-DEVD-FMK activity labeling. In addition, increasing activity labeling of cathepsin B by Biotin-DEVD-FMK during ER stress suggested that cathepsin B’s caspase-3-like activity contributed to around 30% of ER stress-induced caspase-3-like activity.

An unexpected finding of this thesis was that inhibiting the 20S proteasome βsubunit 1 promoted rather than blocked ER stress-induced PCD. This was confirmed by using proteasome specific inhibitors and an RNAi Arabidopsis line against PBA1, the proteasome subunit with caspase-3-like activity. Taken together these results suggested that two plant caspase-3-like activity sources, cathepsin B and PBA1, played antagonistic roles during ER stress-induced PCD. The 20S proteasome is composed of several

157 subunits. PBA1 is responsible for part of the core catalytic activity of the proteasome (Kurepa and Smalle, 2008; Parmentier et al., 1997). So it is reasonable to suggest that the down-regulation of PBA1 impairs the whole 20S proteasome function. In other words, the promotion of PCD observed in the PBA1 RNAi line was attributed to the dysfunction of whole 20S proteasome complex. This is reminiscent of the fact that inhibition of 20S proteasome by MG132 could promote ER stress-induced PCD in human cells (Lee et al., 2003; Park et al., 2011). Lee and colleagues proposed that applying proteasome inhibitors stabilized the unspliced form of the transcription factor protein XBP-1 (XBP-1u). XBP-1 transduces UPR signals via unconventional splicing of its RNA (Walter and Ron, 2011). So blocking XBP-1 splicing impeded signal transduction of UPR, and this subsequently led to more ER stress and PCD. However, a recent report showed that inhibition of the proteasome using MG132 in vascular smooth muscle cells did not block XBP-1 splicing but UPR was still repressed (Amanso et al., 2011). These findings suggested that the inhibition of proteasome would repress UPR in a XBP-1 independent fashion. Therefore inhibition of proteasome may cause more misfolded protein aggregation in ER lumen eventually enhancing the PCD. In Arabidopsis, proteasome activity may be required for the activation of bZIP60, an Arabidopsis transcription factor corresponding to animal XBP-1, although no evidence has been provided. Besides bZIP60, bZIP28 is another ER membrane bound transcription factor, which transduce ER stress signal. Activation of bZIP28 is dependent on proteolytic processing, so it is possible that the proteasome modulate ER stress signal by interfering with bZIP28 activation, if proteasome does not have an impact on bZIP60 activation. Alternatively, the 20S proteasome is responsible for degrading proteins including misfolded proteins in animal (Ahner and Brodsky, 2004; Smith et al., 2011). It is therefore possible that inhibiting the proteasome can directly reduce misfolded protein degradation, enhance ER stress and lead to enhanced PCD. Negative regulation of ER stress-induced PCD in Arabidopsis by the proteasome may be directly or indirectly due to regulating ER stress itself, independently of its caspase-3-like activity.

8.2 Do cathepsin B and the proteasome interact with each other?

Inhibiting the 20S proteasome increased Biotin-DEVD-FMK activity labeling of cathepsin B. This result seems to imply that 20S proteasome regulates cathepsin B degradation. However, further evidences may be needed to support this hypothesis. First, 158 cathepsin B must be shown ubiquitinated to attribute its degradation to the 20S proteasome. This can be examined by pull down cathepsin B and probed it with anti- ubiquitin antibody using western blot. Second, a second possibility will need to be investigated that the increased cathepsin B activity is due to more ER stress signal resulting from 20S proteasome inhibition. To test this hypothesis, a measurement of ubiquitinated protein accumulation and UPR genes transcript level in context of inhibition of proteasome are necessary.

Another pending question is whether cathepsin B regulates 20S proteasome activity? This was tested by comparing 20S proteasome activities of Col-0 and atcathb#62 leaves under ER stress treatment. The cathepsin B triple mutant had less 20S proteasome activity on day one, but did not show huge change on day two and three. This implies that cathepsin B may regulate 20S proteasome activity directly or indirectly. Research on animal cells suggested that caspase-3 activation down-regulated proteasome activity by cleaving the proteasome 19S subunit (Sun et al., 2004; Wang et al., 2010; Cohen, 2005). As cathepsin B possesses caspase-3-like activity, it is possible that cathepsin B in Arabidopsis has similar function as its counterpart in animal cells. However, experimental data in this thesis did not support this idea as we did not observe a higher proteasome activity in the presence of lower cathepsin B activity. Although some of our experimental data implied that cathepsin B and proteasome might regulate each other, more investigation needs to be done to confirm the correlation between cathepsin B and proteasome. Proteasome subunit cleavage by cathepsin B could be investigated using the cathepsin B mutant line and antibody against proteasome subunits.

8.3 Are there two PCD types induced by ER stress- induced PCD?

The cathepsin B triple mutant did not totally block ER stress-induced PCD, ion leakage still increased in atcathb#62 at a late stage. Analysis of Col-0 and atcathb#62 cells ultrastructure suggested two PCD types were involved. The morphological features of dead Col-0 leaf cells matched the criteria of vacuolar PCD, while the morphological features of dead atcathb#62 leaf cells matched criteria of necrotic PCD (Figure 4.17). In addition, mutating the cathepsin B genes did not reduce Yariv reagent-induced necrotic

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PCD. These findings are in support of cathepsin B mediating vacuolar PCD during ER stress, but not necrotic PCD. A previous report, in which cathepsin B expression was found to be induced during vacuolar PCD in maize aleurone cell, also provides indirect evidence to fit cathepsin B in the vacuolar PCD pathway (Martinez et al., 2003). I also examined two biochemical markers for distinguishing different types of PCD. Unfortunately, these markers were not consistent with the TEM criteria. For example, ATP level of Col-0 and atcathb#62 leaves did not show significant difference at day one and day seven, but atcathb#62 sample had higher ATP level at day three while I was expecting a decrease. Using ATP levels as a biochemical marker to distinguish vacuolar PCD and necrotic PCD is derived from research on animal PCD. It has been proposed that apoptosis needs ATP for packing cells debris and for degradation, so ATP level should be maintained at higher level than that in programmed necrosis (Kroemer et al., 2009; Galluzzi et al., 2012; Van Doorn et al., 2012). Based on this, ATP depletion was considered as a marker for animal programmed necrosis. However it is possible that this criterion does not apply to plant necrotic PCD.

8.4 Cathepsin B is part of the ER stress-induced PCD signal pathway

In this thesis, I also attempted to reconstruct the cathepsin B signaling pathway during ER stress-induced PCD. Several cathepsin B regulators were identified during the course of this work. MPK6 exhibited the strongest regulation on both ER stress-induced PCD and cathepsin B activity, as a MPK6 knock out line blocked ER stress-induced PCD and reduced cathepsin B caspase-3-like activity. Another regulator of cathepsin B during ER stress-induced PCD is AtBI-1. However, the effect of AtBI-1 over-expression on cathepsin B activity was not as strong as that observed in mpk6-1 line. According to some previous reports, AtBI-1 is a negative regulator of ER stress-induced PCD (Duan et al., 2010; Watanabe and Lam, 2006; Watanabe and Lam, 2008). A recent report showed that overexpression of AtBI-1 repressed activation of MPK6 (Yue et al., 2012). The extent of MPK6 repression by AtBI-1 was similar to the repression of cathepsin B by AtBI-1. These findings implied that AtBI-1 affected cathepsin B activity through regulating ER stress or MPK6 rather than having a direct influence on cathepsin B.

VPE is an important regulator for ER stress-induced PCD as a recent report suggested that lacking VPE blocked ER stress-induced PCD when the mutualistic fungus 160

Piriformospora Indica colonised Arabidopsis roots (Qiang et al., 2012). In addition, it has been proposed that cathepsin B activation might be dependent on VPE (Shirahama-Noda et al., 2003) Results in my thesis negated this hypothesis. Cathepsin B activity were not repressed in vpe null Arabidopsis no matter with or without ER stress. In contrast, cathepsin B had more activity in vpe null Arabidopsis. Interestingly, vpe null Arabidopsis still delayed Tm induced ion leakage, even though cathepsin B activity was higher than that in Col-0. One molecular function of VPE is controlling tonoplast rupture (Hatsugai et al., 2004). The subcellular localisation of cathepsin B is the vacuole. This implied that VPE loss of function blocked cathepsin B translocation from vacuole to cytoplasm where cathepsin B is considered to execute it PCD function rather than VPE loss of function reducing cathepsin B activation. A cathepsin B mediating ER stress-induced PCD signal pathway is presented in Figure 8.1 here to summarise current results.

Figure 8.1 Potential cathepsin B mediating signal pathway of ER stress-induced PCD. Above figure illustrates a potential cathepsin B mediating signal pathway of ER stress-induced PCD. This pathway was constructed based on results of this thesis and current findings in the published literature. Uncertain signal transduction is marked as dash line. ER stress is divided into two stages: initial stage (left part) and late stage (right part). At initial stage, ER stress-induced UPR aims at rescuing cells from ER stress. AtBI-1 can reduce ER stress and the proteasome is required for full UPR. If the ER stress is prolonged, MPK6 is induced and followed by activation of cathepsin B, which will leads to vacuolar PCD. AtBI-1 may reduce MPK6 activation. Proteasome possibly regulates cathepsin B activation. VPE control tonoplast rupture and doing so may control cathepsin B release from vacuole. Necrotic PCD is induced by ER stress either in parallel to vacuolar PCD or if vacuolar PCD is not activated.

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8.5 Cathepsin B mediates the KOD-induced PCD pathway

The signaling pathway of the KOD peptide, a new PCD inducer in Arabidopsis, has not been identified (Blanvillain et al., 2011). In this thesis, I discovered that the cathepsin B mutant line in Arabidopsis was resistant to KOD-induced PCD. This suggested cathepsin B was required for KOD-induced PCD pathway. The KOD-induced gene expression profiles implied that cathepsin B mediated the KOD signal pathway as part of the HR signal pathway, because genes induced by KOD were largely overlapping with genes induced by pseudomonas syringae DC3000 avrrpm1. In addition, cathepsin B has been shown to regulate pseudomonas syringae DC3000 avrrB induced HR PCD (Mclallen et al., 2009). An oxidative stress signal, ROS, may be a component of the cathepsin B- mediated KOD-induced PCD. In the microarray data, three peroxidase genes among 19 KOD induced genes at three hours treatment were identified. Moreover, cathepsin B has already been reported to be resistant to oxidative stress induced PCD (Ge et al., in preparation). A putative cathepsin B-mediated KOD-induced PCD pathway is illustrated in Figure 8.2. However, a more detailed molecular mechanism by which cathepsin B mediates KOD-induced PCD remains to be established.

Figure 8.2. Putative KOD-induced PCD signal pathway. An outline of the KOD-induced PCD signal pathway is illustrated above. The KOD peptide induces ROS accumulation. ROS induces HR where cathepsin B could mediate the PCD part of the pathway.

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8.6 Cathepsin B in developmental PCD

The findings in this thesis also explored the role of cathepsin B in developmental PCD. A Cathepsin B triple mutant showed a delayed seeds germination rate. Subsequently, Cathepsin B was shown to be involved in two PCD processes during seed maturation and seed germination. During seed maturation the decrease in integument layer thickness was shown to be a PCD process (Nakaune et al., 2005). Cathepsin B triple mutant limited the decrease of integument layer thickness. During germination, cathepsin B regulates PCD in the micropylar endosperm. The observed delay in germination rate of cathepsin B triple mutant seeds may be due principally to the reduced PCD in micropylar endosperm rather than the increased thickness of integument layers. Two reasons for this: 1) The testa, which includes the integument layers, ruptured at the same time in Col-0 and atcathb#62 seeds; 2) Although the integument layer was thicker in atcathb#62 seeds than in Col-0 seeds, no obvious difference of embryo development was observed. Cathepsin B has been shown here to be a regulator not only of stress-induced PCD, but also of at least some PCD in developmental processes. In all, this demonstrated that cathepsin B was an important and general regulator of plant PCD.

8.7 Future work

In this thesis, I have investigated the role of cathepsin B in ER stress-induced PCD. In addition, I attempted to figure out the cathepsin B signaling pathway during ER stress- induced PCD. In order to know the molecular mechanism of the cathepsin B mediated pathway, there are still several questions which need to be addressed in the future: 8.7.1 Looking for substrate. One of the molecular functions of caspase-3 is to cleave PARP during apoptosis (Boulares et al., 1999) and PARP has been shown to be cleaved during plant PCD (Sun et al., 1999). Cathepsin B possesses caspase-3-like activity. However, the substrate(s) of cathepsin B has not been identified. Without identifying at least one cathepsin B substrate, our understanding of the cathepsin B molecular function will be impeded; to the extent of undermining the attribution of a plant caspase-3 function to cathepsin B. Searching for cathepsin B in vivo substrate(s) can be achieved by pulling down cathepsin B with its interacting proteins.

8.7.2 Translocation of cathepsin B The subcellular localisation of cathepsin B underlies the execution of its function. 163

However, whether cathepsin B translocates during ER stress-induced PCD remains elusive. Some preliminary data showed that AtCathB∆C::mRFP was detected in the space between two pavement cells, which implied a cell wall localisation of cathepsin B in addition to its vacuolar localisation. More details need to be understood on how cathepsin B translocates. For example, it is unclear whether cathepsin B translocation from inside the vacuole, if it does so, is just due to tonoplast rupture or to a protein transport complex. To address such question, cathepsin B fused with mRFP can be expressed in various Arabidopsis KO lines for cellular transport-related genes. Then the translocation of cathepsin B during ER stress-induced PCD can be genetically dissected.

8.7.3 Cathepsin B upstream components The control of cathepsin B expression is also important for understanding its signalling pathway. I have shown that MPK6 can regulate cathepsin B activity. Which components connect MPK6 and cathepsin B are not known. A predicted putative phosphorylation site of cathepsin B was identified using PhosPhAT 4.0. This implied that MPK6 may regulate cathepsin B through a post-translational modification. So examining whether cathepsin B can be phosphorylated by MAP kinase may provide clues for understanding how cathepsin B activity is regulated. Analysing the cathepsin B promoter sequence and looking for transcription factors binding sites may also help to understand how cathepsin B expression is regulated.

8.7.4 What connects cathepsin B and KOD Cathepsin B and KOD-induced PCD have a strong connection. But how these two components are connected during PCD is elusive. Dissecting the KOD induced signalling pathway may help to understand how KOD and cathepsin B are connected. This can be done in the future by searching for components, which can perceive a KOD peptide signal such as e.g. a receptor.

8.7.5 Cathepsin B and xylem by TEM and micropylar endosperm weakening The cathepsin B triple mutant line in Arabidopsis did not show obvious alteration in xylem formation. It has been reported that xylem element and xylem fibril have different PCD types during their formation. It is possible that a cathepsin B mutant does not block PCD in xylem but changes the PCD program. TEM can be employed to investigate which PCD type of xylem occurs in the cathepsin B triple mutant line. On the other hand, the cathepsin B mutant displays a clearly reduced PCD of the micropylar endosperm.

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Whether this reduction of PCD is associated to changes of cell wall property is unclear. This can be investigated by examining cell wall properties of cathepsin B mutant line using atomic force microscope during germination.

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Appendix Gene list of five hour KOD induction

Up-regulation (> 2-fold)

Transcript Fold- ID Target Description Change ycf2.1 hypothetical protein 2.9 orf77.1 hypothetical protein 2.2 ndhB.1 NADH dehydrogenase ND2 2.2 rpoB RNA polymerase beta subunit 2.5 ycf6 hypothetical protein 2.0 rps14 ribosomal protein S14 2.7 similar to latex allergen from Hevea brasiliensis ;supported by full- At2g26560 length cDNA: Ceres:1999. 2.2 hypothetical protein predicted by genefinder; similar to GP|2464853|gnl|PID|e353149|Z99707;supported by full-length cDNA: At2g23270 Ceres:40943. 2.7 putative transketolase precursor ; supported by cDNA: At2g45290 gi_15982841_gb_AY057528.1_ 2.7 putative pectinesterase ; supported by cDNA: At2g45220 gi_13605695_gb_AF361829.1_AF361829 2.7 At2g47550 putative pectinesterase ;supported by full-length cDNA: Ceres:111254. 2.3 F12A21.7 hypothetical protein;supported by full-length cDNA: At1g67800 Ceres:34552. 2.1 At5g12340 putative protein similarity to predicted protein, Arabidopsis thaliana 2.6 At4g17215 Expressed protein ; supported by full-length cDNA: Ceres: 94923. 2.6 At4g14365 Expressed protein ; supported by full-length cDNA: Ceres: 37809. 2.0 At4g15120 hypothetical protein ;supported by full-length cDNA: Ceres:36129. 3.4 beta-1,3-glucanase class I precursor ;supported by full-length cDNA: At4g16260 Ceres:3176. 2.1 At4g16820 triacylglycerol lipase like protein 2.6 At4g15130 putative phosphocholine cytidylyltransferase 2.0 At4g14610 disease resistance RPS2 like protein 2.5 At1g28190 hypothetical protein predicted by genemark.hmm 2.3 hypothetical protein similar to putative heat shock protein GI:6403504 At1g44160 from [Arabidopsis thaliana] 2.1 nucellin, putative similar to nucellin GI:2290201 from [Hordeum At1g44130 vulgare] 3.5 hypothetical protein predicted by genemark.hmm;supported by full- At1g35210 length cDNA: Ceres:42217. 2.1 alcohol dehydrogenase - like protein alcohol dehydrogenase 6, Vitis vinifera, EMBL:AF195866;supported by full-length cDNA: At5g24760 Ceres:155666. 2.1 WRKY-like protein WRKY DNA binding protein - Solanum At5g13080 tuberosum, EMBL:AJ278507 3.9 At4g37010 caltractin-like protein ;supported by full-length cDNA: Ceres:26540. 2.4

176

heat shock transcription factor HSF4 ; supported by cDNA: At4g36990 gi_1619920_gb_U68017.1_ATU68017 2.7 At4g37070 patatin-like protein ;supported by full-length cDNA: Ceres:30438. 2.0 SigA binding protein ; supported by cDNA: At3g56710 gi_14596086_gb_AY042831.1_ 2.4 At1g51915 Expressed protein ; supported by full-length cDNA: Ceres: 4795. 3.5 light repressible receptor protein kinase, putative similar to light repressible receptor protein kinase GI:1321686 from [Arabidopsis At1g51890 thaliana] 3.1 At1g51920 hypothetical protein predicted by genemark.hmm 2.6 receptor-like protein kinase, putative similar to receptor-like protein At1g51860 kinase GI:9758831 from [Arabidopsis thaliana] 2.3 DNA-binding protein, putative similar to DNA-binding protein GI:19058 from [Hordeum vulgare]; supported by full-length cDNA: At1g57560 Ceres:250386. 2.6 disease resistance protein RPP1-WsB, putative similar to disease At1g57630 resistance protein RPP1-WsB GI:9279731 from [Arabidopsis thaliana] 2.8 disease resistance protein RPP1-WsA, putative similar to disease At1g57650 resistance protein RPP1-WsA GI:3860163 from [Arabidopsis thaliana] 2.0 CRK1 protein, putative similar to CRK1 protein GI:7671528 from At1g57700 [Beta vulgaris] 2.2 putative protein EXOSTOSIN-1 (PUTATIVE TUMOR At5g33290 SUPPRESSOR PROTEIN EXT1) - Homo sapiens, EMBL:S79639 2.9 RING-H2 zinc finger protein-like RING-H2 zinc finger protein ATL6 - Arabidopsis thaliana, EMBL:AF132016;supported by full-length At5g27420 cDNA: Ceres:106078. 2.4 hexose transporter - like protein hexose transporter HT2, Lycopersicon esculentum, EMBL:LES132224; supported by cDNA: At5g26340 gi_15010579_gb_AY045591.1_ 2.1 At5g25920 putative protein 2.6 receptor-like protein kinase - like receptor-like protein kinase 5, At5g25930 Arabidopsis thaliana, PIR:S27756 2.2 disease resistance protein - like disease resistance protein Hcr2-5D, At5g25910 Lycopersion esculentum, pir:T30553 2.8 cytochrome P450 71B1 - like protein cytochrome P450 71B1, Thlaspi At5g25180 arvense, SWISSPROT:C7B1_THLAR 2.1 At5g67310 cytochrome P450 3.9 At5g67340 putative protein strong similarity to unknown protein (pir||T00518) 2.5 At5g67080 protein kinase-like protein 3.4 At5g66640 putative protein similar to unknown protein (emb CAB16816.1) 2.5 At5g65600 receptor protein kinase-like protein 3.4 At5g64890 unknown protein ;supported by full-length cDNA: Ceres:9242. 2.7 At5g64900 unknown protein ; supported by full-length cDNA: Ceres: 25655. 2.2 putative protein strong similarity to unknown protein At5g64660 (emb|CAB89350.1) 2.0 peroxidase ATP3a (emb|CAA67340.1) supported by full-length At5g64100 cDNA: Ceres:3459. 2.1 At5g63970 putative protein strong similarity to unknown protein (gb|AAF01562.1) 2.4 peroxidase ATP3a homolog ;supported by full-length cDNA: At5g64110 Ceres:2577. 2.1 At5g62150 putative protein predicted protein, Arabidopsis thaliana 2.6 At5g61260 putative protein predicted proteins, Arabidopsis thaliana 2.1 anthocyanin 5-aromatic acyltransferase - like protein anthocyanin 5- aromatic acyltransferase, Gentiana triflora, EMBL:AB010708; At5g61160 supported by cDNA: gi_14335039_gb_AY037199.1_ 2.0 At5g60630 putative protein predicted protein, Arabidopsis thaliana 3.0 177

receptor like protein kinase receptor like protein kinase, Arabidopsis At5g60280 thaliana, PIR:T47484 2.2 1-aminocyclopropane-1-carboxylate oxidase - like protein 1- At5g59530 aminocyclopropane-1-carboxylate oxidase kidney bean, PIR:T10818 2.2 receptor-like protein kinase precursor - like receptor-like protein At5g58940 kinase precursor, Madagascar periwinkle, PIR:T10060 2.2 At5g57480 putative protein contains similarity to AAA-type ATPase 2.3 At5g57510 unknown protein 2.2 putative protein similar to unknown protein At5g57500 (gb|AAF08572.1);supported by full-length cDNA: Ceres:118484. 2.5 At5g57190 phosphatidylserine decarboxylase 3.0 At5g57220 cytochrome P450 2.0 At5g55560 putative protein contains similarity to NRK-related kinase 2.5 auxin-responsive-like protein ; supported by cDNA: At5g54510 gi_11041725_dbj_AB050596.1_AB050596 2.2 Myb-related transcription factor-like protein ; supported by cDNA: At5g54230 gi_5823314_gb_AF175991.1_AF175991 2.0 At5g51160 putative protein similar to unknown protein (gb|AAF43949.1) 2.0 At5g50560 putative protein similar to unknown protein (emb|CAB62355.1) 2.7 At5g48400 ligand-gated ion channel protein-like; glutamate receptor-like 3.7 At5g48410 ligand-gated ion channel protein-like; glutamate receptor-like 3.1 respiratory burst oxidase protein ; supported by cDNA: At5g47910 gi_3242788_gb_AF055357.1_AF055357 2.1 At5g47730 putative protein strong similarity to unknown protein (pir||T05949) 2.3 putative protein contains similarity to WRKY-type DNA-binding protein; supported by cDNA: At5g46350 gi_15384212_gb_AF404855.1_AF404855 2.1 At5g46080 serine/threonine protein kinase-like protein 2.1 At5g45380 urea active transporter-like protein 2.2 At5g45000 putative protein contains similarity to disease resistance protein 2.5 At5g44990 putative protein strong similarity to unknown protein (pir||T04769) 3.7 At5g44820 putative protein similar to unknown protein (pir||T04881) 2.3 putative protein contains similarity to unknown;supported by full- At5g44720 length cDNA: Ceres:4029. 2.1 At5g44380 berberine bridge enzyme-like protein 2.1 At5g44480 putative protein contains similarity to UDP-glucose 4-epimerase 2.9 At5g43590 patatin-like protein 3.5 At5g42830 N-hydroxycinnamoyl benzoyltransferase-like protein 2.2 At5g41610 Na+/H+ antiporter-like protein 2.9 At5g41680 Pto kinase interactor 1-like protein 2.1 putative protein predicted proteins, Arabidopsis thaliana;supported by At5g40690 full-length cDNA: Ceres:11539. 2.4 putative protein tail-specific thyroid hormone up-regulated - Xenopus At5g39970 laevis, EMBL:U37373 2.4 expressed protein predicted protein, Synechocystis sp., PIR:S77152; At5g39520 supported by full-length cDNA: Ceres: 5331. 2.3 frnE protein - like frnE protein, Deinococcus radiodurans, At5g38900 PIR:E75491;supported by full-length cDNA: Ceres:10982. 3.8 receptor protein kinase - like protein receptor-protein kinase-like At5g39020 protein, Arabidopsis thaliana, PIR:T45786 2.0 receptor protein kinase - like protein receptor serine/threonine kinase At5g39030 PR5K, Arabidopsis thaliana, EMBL:AT48698 2.2 proline oxidase, mitochondrial precursor -like protein PROLINE OXIDASE, MITOCHONDRIAL PRECURSOR, Arabidopsis thaliana, At5g38710 SWISSNEW:PROD 2.8

178

At5g23510 putative protein similar to unknown protein (gb AAF01580.1) 2.0 At5g23000 myb-related transcription factor-like 2.3 putative protein strong similarity to unknown protein At5g22540 (emb|CAB72473.1) 2.8 unknown protein ; supported by cDNA: At5g22530 gi_14532613_gb_AY039931.1_ 2.6 putative protein similar to unknown protein At5g22270 (gb|AAF02129.1);supported by full-length cDNA: Ceres:35419. 2.1 At5g22300 Nitrilase 4 (sp|P46011) ; supported by full-length cDNA: Ceres: 6220. 3.1 At5g18780 putative protein predicted proteins, Arabidopsis thaliana 2.0 anthranilate phosphoribosyltransferase, chloroplast precursor At5g17990 (sp|Q02166) ; supported by cDNA: gi_15450851_gb_AY054506.1_ 2.0 cinnamoyl CoA reductase - like protein cinnamoyl CoA reductase, Populus tremuloides, EMBL:AF217958;supported by full-length At5g14700 cDNA: Ceres:17229. 3.6 putative protein predicted proteins, Arabidopsis thaliana and At5g15120 Drosophila melanogaster 2.6 H-protein promoter binding factor-1 (gb|AAC24592.1) ; supported by At5g13820 cDNA: gi_13641339_gb_AY029195.1_ 2.5 At5g12930 putative protein predicted proteins 3.1 auxin-responsive - like protein Nt-gh3 deduced protein, Nicotiana At5g13320 tabacum, EMBL:AF123503 5.2 putative protein GLUR3 ligand-gated channel-like protein precursor, At5g11210 Arabidopsis thaliana, EMBL:AF167355 4.7 Pto kinase interactor - like protein Pto kinase interactor 1, Lycopersicon esculentum, EMBL:SL28007; supported by cDNA: At5g10520 gi_15810234_gb_AY056156.1_ 2.4 At5g09800 putative protein various predicted proteins, Arabidopsis thaliana 2.5 peptide methionine sulfoxide reductase-like protein peptide methionine sulfoxide reductase (msr) - Arabidopsis thalina, At5g07460 EMBL:AJ133753 2.8 At5g05340 peroxidase 3.9 putative protein proline-rich protein APG, Arabidopsis thaliana, At5g03610 PIR:S21961; supported by full-length cDNA: Ceres: 13022. 2.5 At5g03390 putative protein ;supported by full-length cDNA: Ceres:124173. 2.0 At5g03550 putative protein 2.8 At5g02780 putative protein In2, Zea mays, EMBL:X58573 4.3 Ca2+/H+ exchanger-like protein Ca2+/H+ exchanger - Vigna radiata, EMBL:AB012932; supported by cDNA: At5g01490 gi_15426027_gb_AF409107.1_AF409107 2.0 putative protein peroxisomal Ca-dependent solute carrier - Oryctolagus cuniculus, EMBL:AF004161;supported by full-length At5g01500 cDNA: Ceres:249772. 2.2 Ca2+-transporting ATPase -like protein Ca2+-transporting ATPase, At3g63380 calmodulin-stimulated, wild cabbage, PIR:T14453 2.7 At3g61390 putative protein several hypothetical proteins - Arabidopsis thaliana 2.0 At3g60680 putative protein various predicted Arabidopsis thaliana proteins 2.1 putative protein prib5, Ribes nigrum, EMBL:RNI7578;supported by At3g60420 full-length cDNA: Ceres:31361. 2.1 beta-glucosidase-like protein several beta-glucosidases - different At3g60120 species 8.1 At3g59660 putative protein other hypothetical proteins 2.2 At3g59700 serine/threonine-specific kinase lecRK1 precursor,lectin receptor-like 2.1 At3g57630 putative protein DOC4, Mus musculus, AF059485 2.5 putative protein metalloendopeptidase NRD2 convertase - Rattus sp, At3g57460 EMBL: X93208 2.2 179

putative protein hypothetical protein T32G6.16 - Arabidopsis thaliana, At3g57380 PIR:T00820 2.8 putative protein motif in hypothetical protein T23K23.24 - At3g56500 Arabidopsis thaliana, EMBL:AC012563 2.7 DNA-binding protein-like DNA-binding protein 4 WRKY4 - Nicotiana tabacum, EMBL:AF193771;supported by full-length cDNA: At3g56400 Ceres:34847. 4.0 Yippee-like protein Yippee protein, Homo sapiens, EMBL:AF172940; At3g55890 supported by cDNA: gi_15293034_gb_AY050951.1_ 2.5 receptor kinase - like protein receptor kinase homolog CRINKLY4, At3g55950 maize, PIR:T04108 2.3 At3g54950 putative protein patatin, Solanum tuberosum, PIR2:A29810 2.0 embryonic abundant protein -like embryonic abundant protein EMB34, Picea glauca, PIR:T09281; supported by full-length cDNA: At3g54150 Ceres: 6529. 2.8 zinc finger - like protein Zat11 zinc finger protein, Arabidopsis At3g53600 thaliana, EMBL:ATRZAT11 3.0 glucosyltransferase - like protein glucosyltransferase IS10a, salicylate- At3g53150 induced, Nicotiana tabacum, PIR:T03745 3.4 glucosyltransferase - like protein glucosyltransferase IS5a, Nicotiana At3g53160 tabacum, PIR:T03747 2.9 putative protein other hypothetical proteins in Arabidopsis thaliana; At3g52430 supported by cDNA: gi_6457330_gb_AF188329.1_AF188329 2.2 mucin -like protein hemomucin, Drosophila melanogaster, EMBL:DM42014; supported by cDNA: At3g51440 gi_13430723_gb_AF360274.1_AF360274 2.1 mucin -like protein hemomucin, Drosophila melanogaster, At3g51450 EMBL:DM42014;supported by full-length cDNA: Ceres:38956. 2.7 putative protein EREBP-3 homolog, Stylosanthes hamata, At3g50260 EMBL:U91982; supported by cDNA: gi_15010555_gb_AY045579.1_ 2.3 At3g49620 putative protein SRG1 protein - Arabidopsis thaliana, PIR:S44261 2.4 At3g49120 peroxidase ;supported by full-length cDNA: Ceres:39678. 2.0 calcium dependent protein kinase - like strong similarity to calcium At3g49370 dependent protein kinase CP4, Arabidopsis thaliana, EMBL:AF130252 2.4 putative progesterone-binding protein homolog Atmp2 ; supported by At3g48890 cDNA: gi_4960153_gb_AF153283.1_AF153283 2.2 At3g48640 hypothetical protein 3.2 At3g48650 hypothetical protein 2.1 At3g48450 hypothetical protein ;supported by full-length cDNA: Ceres:264020. 3.0 At3g48020 hypothetical protein 2.6 endochitinase-like protein BASIC ENDOCHITINASE CHB4 At3g47540 PRECURSOR - Brassica napus, SWISSPROT:CHI4_BRANA 4.7 glucosyltransferase-like protein UDP-glucose glucosyltransferase - At3g46680 Arabidopsis thaliana, EMBL:AB016819 3.0 high-affinity nitrate transporter - like protein high-affinity nitrate At3g45060 transporter ACH1, Arabidopsis thaliana, EMBL:AF019748 2.0 putative protein putative MuDR protein T22C5.27 - Arabidopsis At3g45120 thaliana, EMBL:AC012375 3.0 oxidosqualene cyclase - like protein oxidosqualene cyclase LcOSC2 - At3g45130 Luffa cylindrica, EMBL:AB033335 4.3 lipoxygenase AtLOX2 ; supported by cDNA: At3g45140 gi_431257_gb_L23968.1_ATHATLO 2.2 putative chloroplast prephenate dehydratase similar to bacterial PheA At3g44720 gene products 2.5

180

cytochrome P450 - like protein cytochrome P450, Sinapis alba, At4g39950 AF069494; supported by cDNA: gi_15028134_gb_AY046017.1_ 3.0 putative protein myosin heavy chain, Oncorhynchus mykiss, At4g40020 PIR2:S52696 2.7 putative protein DNA damage-inducible protein - Synechocystis At4g39030 sp.,PIR2:S77364 3.0 receptor protein kinase - like protein receptor protein kinase erecta, At4g39270 Arabidopsis thaliana 2.2 putative pectinesterase pectinesterase - Lycopersicon esculentum, At4g38420 PID:e312172 3.1 At4g37290 hypothetical protein ;supported by full-length cDNA: Ceres:41783. 5.3 cytochrome P450-like protein ; supported by cDNA: At4g37310 gi_15809892_gb_AY054214.1_ 2.5 putative protein amino-acid N-acetyltransferase, Escherichia coli, At4g37670 PIR1:XYECAA 2.2 putative protein predicted protein, Arabidopsis thaliana;supported by At4g37710 full-length cDNA: Ceres:207350. 2.0 phosphoserine aminotransferase ;supported by full-length cDNA: At4g35630 Ceres:34272. 2.0 At4g35060 putative protein ;supported by full-length cDNA: Ceres:265. 2.2 putative protein pEARLI 4, Arabidopsis thaliana, PATCHX:G871782; At4g35110 supported by cDNA: gi_14326526_gb_AF385717.1_AF385717 2.2 amino acid permease - like protein Lily mRNA, Lilium longiflorum, At4g35180 gb:D21814;supported by full-length cDNA: Ceres:36461. 5.4 cytochrome P450-like protein cytochrome P450 monooxygenase, At4g31950 Pisum sativum, PATCHX:G894153 2.3 putative protein BEM46 PROTEIN, Schizosaccharomyces pombe, At4g31020 PID:D1022245 2.2 At4g29140 putative protein several hypothetical proteins - Arabidopsis thaliana 2.5 At4g28550 putative protein 2.2 receptor protein kinase like protein lectin receptor-like At4g28350 serine/threonine kinase lecRK1, Arabidopsis thaliana, PIR2:S68589 2.5 Expressed protein ; supported by cDNA: At4g28085 gi_15028040_gb_AY045877.1_ 3.1 putative protein probable calcium-dependent protein kinase, Oryza At4g26470 sativa, PIR2:S56652 2.2 NPR1 like protein regulatory protein NPR1 - Arabidopsis thaliana, At4g26120 PID:g1773295 3.2 predicted protein destination factor synapse-enriched clathrin adaptor At4g25940 protein LAP - Drosophila melanogaster, PID:g4160434 2.4 possible apospory-associated like protein Pennisetum ciliare possible apospory-associated mRNA clone pSUB C, PID:g549984;supported by At4g25900 full-length cDNA: Ceres:37817. 2.1 At4g25110 putative protein extensin, Catharanthus roseus, D86853 2.2 putative Na+/H+-exchanging protein Na+/H+-exchanging protein At4g23700 slr1595 - Synechocystis sp., EMBL:D90902 4.5 serine /threonine kinase - like protein serine /threonine kinase, At4g23280 Brassica oleracea 2.0 serine/threonine kinase - like protein serine/threonine kinase, Brassica At4g23320 oleracea 2.7 myb-like protein myb-related transcription factor, Solanum tuberosum, At4g22680 PATCHX:E252796; supported by full-length cDNA: Ceres: 154343. 2.4 At4g23030 putative protein 2.9 putative protein CGI-131 protein, Homo sapiens, AF151889;supported At4g21850 by full-length cDNA: Ceres:24573. 2.3

181

peptide transporter - like protein peptide transporter (ptr1) - Hordeum At4g21680 vulgare,AF023472 3.4 serine/threonine kinase - like protein serine/threonine kinase BRLK, At4g21390 Brassica oleracea, gb:Y12531 3.5 At4g21440 myb-related protein M4 ;supported by full-length cDNA: Ceres:33333. 3.1 At4g21380 receptor-like serine/threonine protein kinase ARK3 3.6 vacuolar sorting receptor-like protein BP-80 vacuolar sorting receptor, At4g20110 Pisum sativum, PATCHX:G1737222 2.3 At5g44810 putative protein similar to unknown protein (gb|AAC79139.1) 2.9 At4g20000 hypothetical protein 2.1 putative chitinase chitinase (EC 3.2.1.14) / lysozyme (EC 3.2.1.17) PZ At4g19810 precursor, pathogenesis-related, common tobacco, PIR2:S51591 2.4 cytochrome P450 cytochrome P450, Arabidopsis thaliana; supported At4g19230 by cDNA: gi_15293092_gb_AY050980.1_ 3.8 At4g19370 hypothetical protein 2.9 putative protein various predicted proteins, Arabidopsis At4g19460 thaliana;supported by full-length cDNA: Ceres:42503. 4.2 DNA binding-like protein SPF1 protein, sweet protein, PIR2:S51529 and WRKY protein family, Petroselinum crispum, MNOS:S72443, At4g18170 MNOS:S72444, MNOS:S72445 2.1 receptor serine/threonine kinase-like protein receptor serine/threonine At4g18250 kinase PR5K, PATCHX:G1235680 4.8 membrane-bound small GTP-binding - like protein GTP-binding protein - Pisum sativum, PATCHX:D1002602 and SR1 Nt-rab11b, At4g18430 Nicotiana tabacum, PATCHX:G623578 2.6 putative transcription factor myb-related transcription factor THM16 - Lycopersicon esculentum,PID:e252796; supported by cDNA: At4g12350 gi_5823330_gb_AF175999.1_AF175999 2.2 pEARLI 1-like protein Arabidopsis thaliana pEARLI 1 mRNA, At4g12500 PID:g871780 2.9 pEARLI 1-like protein Arabidopsis thaliana pEARLI 1 mRNA, At4g12490 PID:g871780; supported by cDNA: gi_15450470_gb_AY052336.1_ 2.4 copper amine oxidase like protein (fragment2) copper amine oxidase - At4g12280 Cicer arietinum,PID:e1335964 2.3 putative phospholipase D-gamma phospholipase D-gamma - Arabidopsis thaliana,PID:g2653885; supported by cDNA: At4g11850 gi_2653884_gb_AF027408.1_AF027408 2.1 RPP1-WsA-like disease resistance protein disease resistance protein At4g11170 RPP1-WsA - Arabidopsis thaliana, PID:g3860163 5.8 RING-H2 finger protein RHA1a -like protein ;supported by full-length At4g11370 cDNA: Ceres:21591. 2.3 UDP-galactose 4-epimerase - like protein UDP-galactose 4-epimerase, Cyamopsis tetragonoloba, AJ005082;supported by full-length cDNA: At4g10960 Ceres:15608. 2.1 peroxidase C2 precursor like protein peroxidase (EC 1.11.1.7) C2 At4g08770 precursor - Armoracia rusticana,PID:d1014846 4.2 peroxidase C2 precursor like protein peroxidase (EC 1.11.1.7) C2 At4g08780 precursor - Armoracia rusticana,PID:d1014846 2.2 5-adenylylsulfate reductase ;supported by full-length cDNA: At4g04610 Ceres:40330. 3.7 At4g04490 putative receptor-like protein kinase 2.9 cyclic nucleotide gated channel (CNGC4) like protein Arabidopsis At4g01010 thaliana cyclic nucleotide gated channel (CNGC4),PID:g4378659 2.3 putative flavonol glucosyltransferase similar to Manihot esculenta flavonol 3-O-glucosyltransferase 5, GenBank accession number At4g01070 Q40287;supported by full-length cDNA: Ceres:1204. 2.5 182

hypothetical protein contains Pfam profile: PF00650 CRAL/TRIO At1g22180 domain 2.4 unknown protein similar to dimethylaniline monooxygenase (N-oxide- At1g19250 forming)-like protein GI:9759603 from [Arabidopsis thaliana] 7.9 hypothetical protein similar to hypothetical protein GI:9802750 from At1g13540 [Arabidopsis thaliana] 2.2 At1g30135 Expressed protein ; supported by full-length cDNA: Ceres: 31945. 2.4 DnaJ protein, putative contains Pfam profile: PF00226: DnaJ At1g56300 domain;supported by full-length cDNA: Ceres:25796. 2.5 glucosyl transferase, putative similar to zeatin O-xylosyltransferase At3g11340 SP:P56725 [Phaseolus vulgaris (Kidney bean) (French bean)] 3.4 trehalose-phosphatase, putative contains TIGRfam profile: TIGR00685: trehalose-phosphatase;supported by full-length cDNA: At1g35910 Ceres:255364. 5.3 lipoxygenase, putative similar to lipoxygenase GI:1407704 from [Solanum tuberosum]; supported by cDNA: At1g55020 gi_289202_gb_L04637.1_ATHLIPOXY 2.0 late embryogenesis protein, putative similar to late embryogenesis At1g54890 abundant protein (EMB7) GI:1350542 from [Picea glauca] 3.6 protein kinase, putative contains Pfam profile: PF00069: Eukaryotic At1g66880 protein kinase domain 2.2 At1g66690 unknown protein 3.9 At1g36640 unknown protein 3.1 At3g14850 hypothetical protein predicted by genemark.hmm 2.1 hypothetical protein similar to unknown protein GB:AAF14829 from At3g22260 [Arabidopsis thaliana];supported by full-length cDNA: Ceres:124466. 2.0 DNA-3-methlyadenine glycosylase (MAG) identical to DNA-3- methlyadenine glycosylase (MAG) SP:Q39147 (Arabidopsis thaliana At3g12040 (Mouse-ear cress)) 2.0 ATPase II, putative similar to GB:AAD34706 from [Homo sapiens] At3g25610 (Biochem. Biophys. Res. Commun. 257 (2), 333-339 (1999)) 2.6 dirigent protein, putative similar to dirigent protein GB:AAF25365 At3g13650 from [Thuja plicata] 2.3 unknown protein ; supported by cDNA: At3g26440 gi_16612299_gb_AF439843.1_AF439843 2.4 glutamine:fructose-6-phosphate amidotransferase, putative similar to glutamine:fructose-6-phosphate amidotransferase 2 GB:BAA74729 At3g24090 [Mus musculus] 2.0 O-acetylserine(thiol) lyase, putative similar to O-acetylserine(thiol) At3g22460 lyase GB:CAA71798 from [Brassica juncea] 2.1 unknown protein contains Pfam profile:PF00279 LTP:Plant lipid At3g22600 transfer protein family;supported by full-length cDNA: Ceres:19287. 4.6 brassinosteroid receptor kinase, putative similar to GB:AAC49810 from [Arabidospsis thaliana], contains Pfam profiles: PF00560 Leucine At3g13380 Rich Repeat (23 copies) 2.5 At3g28580 hypothetical protein predicted by genemark.hmm 6.0 longevity factor-like protein similar to LAG1Ce-1 GB:AAD16893 from [Caenorhabditis elegans] (Genome Res.(1998) 8 (12), 1259- At3g19260 1272);supported by full-length cDNA: Ceres:123372. 2.3 At3g18250 hypothetical protein predicted by genscan+ 2.6 At3g19660 unknown protein 2.1 ABC transporter, putative similar to AtMRP4 (transport of glutathione-conjugates into the vacuole) GB:CAA05625 [Arabidopsis At3g13100 thaliana]; contains Pfam profile: PF00005 ABC transporter 2.2 At2g45760 hypothetical protein predicted by genscan 2.0 At1g43000 hypothetical protein 2.0 183

At3g02800 unknown protein 2.4 At1g66480 hypothetical protein 2.6 cytochrome P450, putative contains Pfam profile: PF00067 At3g26230 cytochrome P450 2.0 cytochrome P450, putative contains Pfam profile: PF00067 cytochrome P450; supported by cDNA: At3g26200 gi_13430717_gb_AF360271.1_AF360271 2.2 DnaJ protein, putative contains Pfam profile: PF00226 DnaJ At3g13310 domain;supported by full-length cDNA: Ceres:31309. 2.3 At3g12700 hypothetical protein predicted by genemark.hmm 2.8 short-chain alcohol dehydrogenase, putative similar to short-chain alcohol dehydrogenase GB:AAF04194 [Pisum sativum]; contains At3g29250 Pfam profile: PF00106 short chain dehydrogenase 2.1 sugar transport, putative similar to D-XYLOSE-PROTON At3g18830 SYMPORTER GB:O52733 from [Lactobacillus brevis] 2.0 ethylene responsive element binding protein, putative similar to At3g23230 EREBP-4 GB:BAA07323 from [Nicotiana tabacum] 2.0 putative UDP-glucose glucosyltransferase similar to GB:Q40284 from At3g21780 [Manihot esculenta] 2.2 putative SGP1 monomeric G-protein similar to GB:CAB54517 from At3g21700 [Arabidopsis thaliana];supported by full-length cDNA: Ceres:30989. 2.0 putative serine/threonine protein kinase similar to GB:NP_035979 At3g20860 from [Mus musculus] 2.8 AIG2-like protein similar to AIG2 protein GB:P54121 from At3g28930 [Arabidopsis thaliana];supported by full-length cDNA: Ceres:28511. 3.0 putative 4-coumarate:CoA ligase 2 similar to GB:AAD47192 from At3g21230 [Arabidopsis thaliana] 4.9 putative 4-coumarate:CoA ligase 2 almost identical (1 amino acid difference) to GB:AAD47192 from [Arabidopsis thaliana]; supported At3g21240 by cDNA: gi_5702187_gb_AF106086.1_AF106086 3.2 NAM-like protein (No Apical Meristem) similar to GB:CAA63101 At3g29035 from [Petunia x hybrida] (Cell 85 (2), 159-170 (1996)) 2.0 At3g14700 hypothetical protein predicted by genscan+ 2.4 At3g13950 hypothetical protein predicted by genemark.hmm 4.6 putative cytochrome P450 similar to cytochrome P450 71B2 At3g26830 GB:O65788 [Arabidopsis thaliana] 5.1 At3g26910 unknown protein 2.4 At3g02550 unknown protein ;supported by full-length cDNA: Ceres:20907. 2.6 hypothetical protein predicted by genemark.hmm; supported by cDNA: At3g04420 gi_16974618_gb_AY060587.1_ 2.1 putative disease resistance protein similar to disease resistance protein At3g04220 RPP1-WsC GB:AAC72979 [Arabidopsis thaliana] 2.6 putative trypsin inhibitor similar to trypsin inhibitor:ISOTYPE=a GB:737210; contains Pfam profile: PF00197 trypsin and protease At3g04320 inhibitors 2.3 At3g08630 unknown protein 2.0 putative membrane protein similar to integral membrane protein, Tmp21-I (p23) GB:CAA06212 [Rattus norvegicus]; contains Pfam profile: PF01105 emp24/gp25L/p24 family; contains non-consensus At3g10780 TG acceptor splice site at exon 3 2.4 hypothetical protein predicted by genscan;supported by full-length At3g11840 cDNA: Ceres:100676. 2.1

184

hevein-like protein precursor (PR-4) identical to hevein-like protein precursor GB:P43082 [Arabidopsis thaliana], similar to wound-induced protein (WIN2) precursor GB:P09762 [Solanum tuberosum]; Pfam HMM hit: chitin_binding proteins;supported by full-length cDNA: At3g04720 Ceres:8793. 2.1 NAM-like protein (no apical meristem) similar to NAM GB:CAA63101 [Petunia x hybrida]; supported by full-length cDNA: At3g04060 Ceres: 119460. 2.5 unknown protein similar to uridylyl transferase-like proteins At3g01990 GB:AAD20075, GB:AAC00631 (Arabidopsis thaliana) 2.3 putative serine/threonine protein kinase similar to serine/threonine- specific kinase GB:S68589 [Arabidopsis thaliana]; Pfam HMM hits: putative serine/threonine protein kinase, Eukaryotic protein kinase At3g08870 domain 2.3 putative pectinacetylesterase similar to pectinacetylesterase precursor At3g09410 GB:CAA67728 [Vigna radiata] 3.8 At3g02240 unknown protein 3.8 sugar transporter, putative similar to integral membrane protein GB:U43629 from [Beta vulgaris] (Plant Physiol.(1996) 110 (2), 511- At3g05400 520) 3.6 At3g05390 hypothetical protein predicted by genscan+ 3.1 unknown protein similar to unknown protein GB:AAB84346 At3g10320 [Arabidopsis thaliana] 2.5 At3g09020 unknown protein identical to GB:AAD56318 (Arabidopsis thaliana) 3.8 putative receptor ser/thr protein kinase similar to receptor kinase GB:S70769 [Arabidopsis thaliana]; supported by full-length cDNA: At3g09010 Ceres: 124301. 5.7 putative peroxidase very similar to peroxidase GB:CAA66963 from At3g01190 [Arabidopsis thaliana];supported by full-length cDNA: Ceres:37597. 2.7 putative disease resistance protein similar to Cf-2 disease resistance At3g05370 protein GB:AAC15780 from [Lycopersicon pimpinellifolium] 2.3 At3g01175 unknown protein 3.1 hypothetical protein predicted by genscan+;supported by full-length At1g69050 cDNA: Ceres:871. 2.2 At1g17745 unknown protein ; supported by full-length cDNA: Ceres: 20582. 3.7 MAP kinase, putative similar to MAP kinase 5 GI:4239889 from [Zea At1g01560 mays] 2.3 1-aminocyclopropane-1-carboxylate synthase (ACC synthase) almost identical to 1-aminocyclopropane-1-carboxylate synthase GI:166578 from [Arabidopsis thaliana]; supported by cDNA: At1g01480 gi_166579_gb_M95595.1_ATHACSC 2.5 At1g35230 hypothetical protein ;supported by full-length cDNA: Ceres:265772. 3.2 hypothetical protein ; supported by cDNA: At1g21250 gi_14532585_gb_AY039917.1_ 2.1 At1g55210 unknown protein 2.5 disease resistance protein, putative similar to GI:1184077 from At1g47890 [Lycopersicon pimpinellifolium] (Cell 84 (3), 451-459 (1996)) 2.6 At1g67920 unknown protein ;supported by full-length cDNA: Ceres:13102. 2.9 putative S-adenosyl-L-methionine:trans-caffeoyl-Coenzyme A 3-O- methyltransferase similar to S-adenosyl-L-methionine:trans-caffeoyl- Coenzyme A 3-O-methyltransferase GB:AAA62426 [Arabidopsis thaliana] (function=disease resistance); supported by cDNA: At1g67980 gi_682727_gb_L40031.1_ATHORF 2.6 At1g30040 unknown protein 2.5 At1g68765 Expressed protein ; supported by full-length cDNA: Ceres: 39198. 2.3

185

Expressed protein ; supported by cDNA: At1g73800 gi_16648699_gb_AY058126.1_ 4.3 putative lipase/acylhydrolase contains Pfam profile: PF00657 At1g73610 lipase/acylhydrolase with GDSL-like motif 2.5 putative calmodulin-binding protein similar to calmodulin-binding At1g73805 protein GB:AAB37246 [Nicotiana tabacum] 2.8 AIG1 identical to AIG1 (exhibits RPS2- and avrRpt2-dependent induction early after infection with Pseudomonas) GB:U40856 [Arabidopsis thaliana] (Plant Cell 8 (2), 241-249 (1996)); supported by At1g33960 cDNA: gi_1127803_gb_U40856.1_ATU40856 2.7 NAM-like protein similar to NAM (no apical meristem) At1g52890 GB:CAA63101 from [Petunia x hybrida] 3.4 putative glutathione S-transferase similar to putative glutathione S- transferase GB:CAA10060 [Arabidopsis thaliana]; contains Pfam profile: PF00043 Glutathione S-transferases;supported by full-length At1g74590 cDNA: Ceres:40734. 2.6 putative receptor protein kinase similar to brassinosteroid insensitive 1 GB:AAC49810 (putative receptor protein kinase); contains Pfam profiles: PF00560 Leucine Rich Repeat (17 repeats), PF00069 Eukaryotic protein kinase domain; supported by cDNA: At1g74360 gi_15810516_gb_AY056297.1_ 3.0 At1g68450 unknown protein 3.8 unknown protein contains Pfam profile: PF00561 alpha/beta hydrolase At1g80280 fold 5.5 putative transcription factor similar to OSMYB1 GB:BAA23337 from [Oryza sativa]; supported by cDNA: At1g74080 gi_15375306_gb_AF371983.2_AF371983 4.6 hypothetical protein predicted by genscan; supported by cDNA: At1g69810 gi_15384220_gb_AF404859.1_AF404859 2.5 putative glutathione transferase similar to glutathione transferase At1g69930 GB:CAA09188 [Alopecurus myosuroides] 3.8 putative glutathione transferase similar to glutathione transferase At1g69920 GB:CAA09188 [Alopecurus myosuroides] 3.4 putative thioredoxin similar to thioredoxin GB:S58123 [Arabidopsis At1g69880 thaliana] 2.3 hypothetical protein similar to GB:AAB61488 [Arabidopsis At1g69890 thaliana];supported by full-length cDNA: Ceres:34864. 2.0 At1g10990 hypothetical protein predicted by genefinder and genscan 2.4 At2g47190 MYB transcription factor (Atmyb2) 2.5 At2g47200 hypothetical protein predicted by genefinder and genscan 2.6 At1g08050 unknown protein 2.1 oxidase, putative similar to oxidase GB:AAA32870 GI:166876 from At1g32350 (Arabidopsis thaliana);supported by full-length cDNA: Ceres:152458. 4.9 hypothetical protein predicted by genemark.hmm;supported by full- At1g78410 length cDNA: Ceres:157. 4.7 hypothetical protein similar to hypothetical protein GB:AAB67625 GI:2342727 from [Arabidopsis thaliana]; supported by full-length At1g29050 cDNA: Ceres: 30068. 2.2 ascorbate oxidase promoter-binding protein, putative similar to ascorbate oxidase promoter-binding protein GB:D45066 GI:853689 At1g29160 from [Cucurbita maxima] 2.8 maternal embryogenesis control protein (MEDEA), putative similar to At1g02580 MEDEA GB:AAC39446 GI:3089625 from [Arabidopsis thaliana] 2.9 hypothetical protein contains non-consensus splice sites.; supported by At1g02470 full-length cDNA: Ceres:29906. 2.4 At1g53620 hypothetical protein predicted by genemark.hmm 2.1 186

receptor-like serine/threonine kinase, putative similar to receptor-like serine/threonine kinase GB:AAC50043 GI:2465923 from [Arabidopsis At1g53430 thaliana] 2.1 hypothetical protein similar to reticuline oxidase-like protein At1g26420 GB:CAB45850 GI:5262224 from [Arabidopsis thaliana] 2.5 unknown protein similar to reticuline oxidase-like protein At1g26410 GB:CAB45850 GI:5262224 from [Arabidopsis thaliana] 3.1 hypothetical protein similar to reticuline oxidase-like protein GB:CAB45850 GI:5262224 from [Arabidopsis thaliana]; supported by At1g26390 cDNA: gi_15293132_gb_AY051000.1_ 4.3 hypothetical protein similar to reticuline oxidase-like protein GB:CAB45850 GI:5262224 from [Arabidopsis thaliana]; supported by At1g26380 cDNA: gi_13430839_gb_AF360332.1_AF360332 2.4 cytokinin oxidase, putative similar to GB:CAA77151 from [Zea mays] At1g75450 (Plant J. 17 (6), 615-626 (1999)) 2.2 catechol O-methyltransferase, putative similar to catechol O- At1g33030 methyltransferase GI:4808524 from [Thalictrum tuberosum] 3.8 wall-associated kinase 2, putative similar to wall-associated kinase 2 At1g79680 GI:4826399 from [Arabidopsis thaliana] 4.0 At1g21120 putative ATPase similar to GB:AAF28353 from [Fragaria x ananassa] 2.2 O-methyltransferase, putative similar to GB:AAF28353 from [Fragaria At1g21100 x ananassa]; supported by cDNA: gi_15982843_gb_AY057529.1_ 2.4 At1g07690 hypothetical protein predicted by genemark.hmm 2.0 anionic peroxidase, putative similar to anionic peroxidase GI:170202 At1g14540 from [Nicotiana sylvestris] 2.9 anionic peroxidase, putative similar to anionic peroxidase GI:170202 At1g14550 from [Nicotiana sylvestris] 4.9 wall-associated kinase 1, putative similar to wall-associated kinase 1 At1g28390 GI:3549626 from [Arabidopsis thaliana] 2.2 At1g14330 hypothetical protein predicted by genemark.hmm 2.3 hypothetical protein similar to hypothetical protein GB:AAF25996 At1g18380 GI:6714300 from [Arabidopsis thaliana] 2.2 ABC transporter, putative similar to ABC transporter GI:9279716 At1g15520 from [Arabidopsis thaliana] 3.1 leucine zipper protein, putative similar to basic leucine zipper protein At1g08325 GI:2865394 from [Zea mays] 2.1 pectin methylesterase, putative similar to pectin methylesterase At1g11370 GI:1279597 from [Nicotiana plumbaginifolia] 2.6 cinnamoyl CoA reductase, putative similar to cinnamoyl CoA reductase GB:AAF43141 GI:7239228 from [Populus tremuloides]; At1g80820 supported by full-length cDNA: Ceres: 32255. 3.0 At1g64610 hypothetical protein predicted by genemark.hmm 2.3 receptor protein kinase, putative contains Pfam profiles: PF00069: Eukaryotic protein kinase domain, multiple PF00560: Leucine Rich At1g56145 Repeat 2.4 NAC1 identical to NAC1 GB:AAF21437 GI:6649236 from At1g56010 [Arabidopsis thaliana];supported by full-length cDNA: Ceres:205648. 2.6 auxin-regulated protein GH3, putative similar to auxin-regulated At1g59500 protein GH3 GI:18590 from [Glycine max] 2.0 glutathione S-transferase, putative similar to glutathione S-transferase At1g02940 GI:860955 from [Hyoscyamus muticus] 2.2 isochorismate synthase (icsI) identical to isochorismate synthase (icsI) GB:AF078080 (catalyzes chorismic acid to isochorismic acid Plant Physiol. 118 (4), 1536 (1998)); supported by cDNA: At1g74710 gi_3348076_gb_AF078080.1_AF078080 2.0 At1g68620 unknown protein ; supported by cDNA: 2.6 187

gi_14335125_gb_AY037242.1_ serine threonine kinase, putative similar to GB:CAA73067 from (Sorghum bicolor) (Plant Mol. Biol. 36 (4), 529-539 (1998)); supported At1g48260 by cDNA: gi_14571552_gb_AY036958.1_ 2.1 At1g68630 hypothetical protein predicted by genscan+ 2.3 unknown protein contains similarity to zinc finger and C2 domain protein GI:9957238 from [Arabidopsis thaliana];supported by full- At1g70810 length cDNA: Ceres:23322. 2.1 hypothetical protein similar to hypothetical protein GB:AAF24561 At1g64170 GI:6692096 from [Arabidopsis thaliana] 2.2 hypothetical protein similar to putative serpin GB:AAD15462 At1g64020 GI:4263819 from [Arabidopsis thaliana] 2.1 hypothetical protein similar to putative serpin GB:AAD15462 At1g64010 GI:4263819 from [Arabidopsis thaliana] 2.1 unknown protein contains similarity to xenotropic and polytropic At1g14040 retrovirus receptor GB:4759334 2.7 unknown protein ; supported by cDNA: At1g75830 gi_15529223_gb_AY052236.1_ 2.2 hypothetical protein similar to gb|AF098458 latex-abundant protein At1g16420 (LAR) from Hevea brasiliensis 2.5 anthranilate N-hydroxycinnamoyl/benzoyltransferase, putative similar to anthranilate N-hydroxycinnamoyl/benzoyltransferase GB:Z84384 GI:2239084 [Dianthus caryophyllus];supported by full-length cDNA: At1g28680 Ceres:12689. 2.1 phosphoribosyl diphosphate synthase identical to phosphoribosyl diphosphate synthase GI:4902470 from (Arabidopsis thaliana); At1g10700 supported by cDNA: gi_16604385_gb_AY058091.1_ 2.4 puative calcium-transporting ATPase similar to gb|AF038007 FIC1 gene from Homo sapiens and is a member of the PF|00122 E1-E2 ATPase family. ESTs gb|T45045 and gb|AA394473 come from this At1g13210 gene 2.4 unknown protein ESTs gb|T75618 and gb|AA404816 come from this At1g10690 gene 2.0 putative cytochrome P450 strong similarity to gi|2880052 T11J7.14 putative cytochrome P450 from Arabidopsis thaliana and is a member At1g11610 of the PF|00067 Cytochrome P450 family 2.2 receptor kinase, putative similar to receptor kinase 1 [Brassica rapa] At1g65790 GB:BAA23676 2.1 hypothetical protein similar to unknown protein GI:4585976 from At1g79450 [Arabidopsis thaliana];supported by full-length cDNA: Ceres:94828. 2.3 lipase, putative similar to lipase GI:1145627 from [Arabidopsis At1g53990 thaliana] 2.7 UDP-galactose 4-epimerase-like protein similar to proteins from many bacterial species including [Bacillus subtilis] and [Methanobacterium At1g30620 thermoautotrophicum] 2.0 putative reticuline oxidase-like protein similar to GB:P30986 from [Eschscholzia californica] ( berberine bridge-forming enzyme ), ESTs gb|F19886, gb|Z30784 and gb|Z30785 come from this gene; supported At1g30700 by cDNA: gi_16930506_gb_AF419607.1_AF419607 2.1 At2g16590 unknown protein 3.3 subtilisin-like serine protease AIR3 almost identical to subtilisin-like protease AIR3 GI:4218991 from [Arabidopsis thaliana], missing 18 aa At2g04160 at C-terminus 2.3 putative C2H2-type zinc finger protein likely a nucleic acid binding At2g28710 protein 2.4

188

putative aspartate aminotransferase ;supported by full-length cDNA: At2g22250 Ceres:112880. 2.5 At2g31945 Expressed protein ; supported by full-length cDNA: Ceres: 258917. 2.1 At2g15390 unknown protein 2.7 At2g46440 putative cyclic nucleotide-regulated ion channel protein 2.8 At2g36470 unknown protein ;supported by full-length cDNA: Ceres:109103. 2.2 similar to harpin-induced protein hin1 from tobacco ;supported by full- At2g35980 length cDNA: Ceres:26418. 4.4 At2g22330 putative cytochrome P450 3.2 NAM (no apical meristem)-like protein, putative similar to NAM (no At1g02220 apical meristem)-like protein GI:9759589 from (Arabidopsis thaliana) 6.6 receptor kinase, putative similar to receptor kinase 1 GI:9294449 from At1g67520 [Arabidopsis thaliana] 3.0 putative nicotianamine synthase similar to GB:BAA74589;supported At1g09240 by full-length cDNA: Ceres:37124. 2.8 beta-glucosidase, putative similar to beta-glucosidase GI:1155254 At1g61820 from [Prunus avium] 3.0 putative IAA1 protein Match to IAA10 protein gb|U18412 from A. thaliana; supported by cDNA: At1g04100 gi_12083201_gb_AF332396.1_AF332396 2.7 Ste-20 related kinase SPAK, putative similar to Ste-20 related kinase At1g70430 SPAK GI:3851169 from [Mus musculus] 2.5 glucose-6-phosphate/phosphate-translocator precursor, putative similar to glucose-6-phosphate/phosphate-translocator precursor GI:2997591 from [Pisum sativum]; supported by cDNA: At1g61800 gi_14596172_gb_AY042874.1_ 3.5 serine/threonine protein kinase, putative similar to serine/threonine At1g61750 protein kinase GI:3080385 from [Arabidopsis thaliana] 2.2 beta-glucosidase, putative similar to beta-glucosidase GI:804655 from At1g61810 (Hordeum vulgare) 5.8 unknown protein similar to EST gb|AA598098;supported by full- At1g10140 length cDNA: Ceres:23916. 2.0 At2g17740 unknown protein 2.0 unknown protein Similar to Saccharomyces hypothetical protein YDR051c (gb|Z49209). ESTs gb|T44436,gb|42252 come from this At1g08940 gene 2.6 putative luminal binding protein Similar to Arabidopsis luminal At1g09080 binding protein (gb|D89342) 2.1 matrix metalloproteinase, putative similar to matrix metalloproteinase At1g70170 GI:7159629 from [Cucumis sativus] 2.2 unknown protein similar to putative DNA-binding protein GI:7268215 from [Arabidopsis thaliana]; supported by cDNA: At1g62300 gi_12658409_gb_AF331712.1_AF331712 2.2 receptor kinase, putative similar to receptor kinase GI:166692 from At1g61420 [Arabidopsis thaliana] 2.5 putative protein kinase similar to (Z71703), cdc2-like protein kinase; similar to ESTs gb|T20748, gb|T20464, and emb|Z17761; supported by At1g03740 cDNA: gi_14532735_gi_13430451 2.1 F-box protein family, AtFBL5 contains similarity to F-box protein FBL2 GI:6063090 from [Homo sapiens];supported by full-length At1g77000 cDNA: Ceres:3549. 2.3 Mlo protein, putative similar to Mlo protein GI:1877220 from [Hordeum vulgare]; supported by cDNA: At1g61560 gi_14091581_gb_AF369567.1_AF369567 3.0 hypothetical protein predicted by genefinder;supported by full-length At1g03850 cDNA: Ceres:271253. 3.2 189

At1g51280 hypothetical protein similar to disease resistance protein 4.0 At2g36770 putative glucosyl transferase 2.6 At2g43000 NAM (no apical meristem)-like protein 3.4 At2g15760 hypothetical protein ;supported by full-length cDNA: Ceres:5. 2.2 At2g15480 putative glucosyltransferase 2.2 At2g15490 putative glucosyltransferase 2.5 At2g19970 putative pathogenesis-related protein 2.2 At2g25440 putative disease resistance protein 2.0 At2g40110 unknown protein 2.3 At2g32140 putative disease resistance protein 3.4 At2g31990 hypothetical protein predicted by genscan 2.3 At2g35730 Expressed protein ; supported by full-length cDNA: Ceres: 5598. 3.6 At2g15040 putative disease resistance protein 2.5 putative cytochrome P450 identical to GB:D78604; supported by At2g24180 cDNA: gi_15450907_gb_AY054534.1_ 2.7 similar to axi 1 protein from Nicotiana tabacum ; supported by cDNA: At2g37980 gi_13430693_gb_AF360259.1_AF360259 2.3 unknown protein ; supported by cDNA: At2g39030 gi_15451161_gb_AY054661.1_ 7.9 hypothetical protein predicted by genscan and genefinder; supported by At2g02370 cDNA: gi_13605570_gb_AF361611.1_AF361611 2.3 At2g28760 putative nucleotide-sugar dehydratase 2.3 putative glutathione S-transferase ; supported by cDNA: At2g29460 gi_14423533_gb_AF387004.1_AF387004 2.8 putative glutathione S-transferase ; supported by cDNA: At2g29470 gi_11096003_gb_AF288185.1_AF288185 3.8 putative tropinone reductase ;supported by full-length cDNA: At2g29350 Ceres:14555. 2.8 At2g46650 putative cytochrome b5 ;supported by full-length cDNA: Ceres:3743. 2.2 Mutator-like transposase similar to MURA transposase of maize At2g32250 Mutator transposon 2.0 putative desiccation related protein ; supported by full-length cDNA: At2g46140 Ceres: 2747. 2.5 At2g29740 putative flavonol 3-O-glucosyltransferase 2.2 At2g46740 unknown protein 2.2 At2g46750 unknown protein 2.4 At2g18950 hypothetical protein predicted by genefinder 2.7 At2g39530 unknown protein 2.8 At2g39420 putative phospholipase 2.1 At2g34500 putative cytochrome P450 2.5 putative protein kinase contains a protein kinase domain profile At2g38490 (PDOC00100) 2.1 putative protein kinase contains a protein kinase domain profile (PDOC00100); supported by cDNA: At2g34180 gi_13249124_gb_AF295668.1_AF295668 2.8 At2g31030 putative oxysterol-binding protein 2.6 At2g44010 unknown protein 2.3 putative protein kinase contains a protein kinase domain profile At2g26290 (PDOC00100) 2.7 At2g44480 putative beta-glucosidase 3.3 At2g19190 putative receptor-like protein kinase 2.3 At2g30400 unknown protein 2.9 At2g30550 putative lipase ; supported by full-length cDNA: Ceres: 207043. 2.5 At2g32660 putative disease resistance protein 2.2 At2g45570 putative cytochrome P450 2.4 190

At2g30750 putative cytochrome P450 2.8 At2g30770 putative cytochrome P450 2.3

Down-regulation (<0.5)

Transcript ID Target Description Ratio putative Ca2+-ATPase ; supported by cDNA: At2g41560 gi_11493642_gb_AF200739.1_AF200739 0.2 unknown protein predicted by genscan; supported by cDNA: At2g45190 gi_3822215_gb_AF074948.1_AF074948 0.2 At1g67830 F12A21.4 similar to iEP4 gb|AAD11468.1 0.2 predicted protein ; supported by cDNA: At4g16563 gi_15809799_gb_AY054167.1_ 0.2

At4g16985 Expressed protein ; supported by full-length cDNA: Ceres: 30087. 0.2 xyloglucan endotransglycosylase-related protein XTR-7 ;supported by At4g14130 full-length cDNA: Ceres:33554. 0.2 At4g14890 ferredoxin ;supported by full-length cDNA: Ceres:22861. 0.2 At4g17600 Lil3 protein ;supported by full-length cDNA: Ceres:29150. 0.2 At4g17560 putative protein ;supported by full-length cDNA: Ceres:2161. 0.2 membrane channel like protein ;supported by full-length cDNA: At4g17340 Ceres:99796. 0.3 At4g14440 carnitine racemase like protein 0.3 ribosomal protein L10-like ribosomal protein L10- Nicotiana tabacum, EMBL:AB010879;supported by full-length cDNA: At5g13510 Ceres:29083. 0.3

GTL1 - like protein GTL1, Arabidopsis thaliana, EMBL:ATAJ3215; At5g28300 supported by cDNA: gi_14423451_gb_AF386963.1_AF386963 0.3 spermine synthase (ACL5) ;supported by full-length cDNA: At5g19530 Ceres:3968. 0.3 At5g13140 putative protein ;supported by full-length cDNA: Ceres:14064. 0.3 At4g36530 putative protein ;supported by full-length cDNA: Ceres:36845. 0.3 At4g36540 putative protein ; supported by full-length cDNA: Ceres: 123997. 0.3 PPR-repeat protein, putative contains multiple PPR domains: At1g31920 PF01535: PPR repeat 0.3 cold and ABA inducible protein kin1 ;supported by full-length At5g15960 cDNA: Ceres:2270. 0.3 putative protein with poly glutamic acid stretch hypothetical protein F16B3.13 - Arabidopsis thaliana, EMBL:AC021640; supported by At5g16030 full-length cDNA: Ceres: 29745. 0.3 biotin carboxyl carrier protein precursor-like protein biotin carboxyl carrier protein (clone BP6) precursor - Brassica napus, At5g15530 EMBL:X90731;supported by full-length cDNA: Ceres:25607. 0.3 myrosinase TGG2 ; supported by cDNA: At5g25980 gi_13507564_gb_AF360348.1_AF360348 0.3 dehydration-induced protein RD22 ; supported by cDNA: At5g25610 gi_16974545_gb_AY060560.1_ 0.3 putative protein hypothetical protein - Ricinus communis, At5g25460 EMBL:Z81012;supported by full-length cDNA: Ceres:1351. 0.3 putative protein similar to unknown protein (gb|AAC18972.1); At5g67370 supported by cDNA: gi_15293188_gb_AY051028.1_ 0.3 At5g67060 putative protein similar to unknown protein (emb CAB62312.1) 0.3

191

At5g66470 GTP-binding protein-like 0.3 At5g66520 selenium-binding protein-like 0.3 At5g65640 unknown protein ;supported by full-length cDNA: Ceres:13267. 0.3 xyloglucan endo-transglycosylase-like protein ;supported by full- At5g65730 length cDNA: Ceres:12301. 0.3 asparagine synthetase (gb|AAC72837.1) ; supported by cDNA: At5g65010 gi_3859535_gb_AF095453.1_AF095453 0.3 non-phototropic hypocotyl 3 (gb|AAF05914.1) ; supported by cDNA: At5g64330 gi_6224711_gb_AF180390.1_AF180390 0.3 At5g62840 putative protein ;supported by full-length cDNA: Ceres:5589. 0.3 At5g62670 plasma membrane proton ATPase-like 0.3

ripening-related protein - like ripening-related protein, Vitis vinifera, At5g62350 EMBL:VVI237985;supported by full-length cDNA: Ceres:9669. 0.3 MYB96 transcription factor-like protein ; supported by cDNA: At5g62470 gi_5823334_gb_AF176001.1_AF176001 0.3 At5g62280 putative protein predicted proteins, Arabidopsis thaliana 0.3 At5g61130 putative protein various predicted proteins, Arabidopsis thaliana 0.3 histone H4 - like protein histone H4, Zea mays, At5g59690 PIR:HSZM4;supported by full-length cDNA: Ceres:15418. 0.3 dehydrodolichyl diphosphate - like protein dehydrodolichyl At5g58770 diphosphate, Arabidopsis thaliana, EMBL:ATH277136 0.3 similar to unknown protein (pir||S75584) ;supported by full-length At5g58260 cDNA: Ceres:3488. 0.3 non phototropic hypocotyl 1-like ; supported by cDNA: At5g58140 gi_5391441_gb_AF053941.2_AF053941 0.3 At5g57780 putative protein similar to unknown protein (emb CAB79759.1) 0.3

At5g57785 Expressed protein ; supported by full-length cDNA: Ceres: 267411. 0.3 putative protein similar to unknown protein At5g55620 (gb|AAF04428.1);supported by full-length cDNA: Ceres:27668. 0.3 At5g55740 selenium-binding protein-like 0.3 At5g55220 trigger factor-like protein 0.4 At5g53880 unknown protein ;supported by full-length cDNA: Ceres:31129. 0.4 At5g53580 aldo/keto reductase-like protein 0.4 At5g52570 beta-carotene hydroxylase 0.4

At5g51720 unknown protein ; supported by full-length cDNA: Ceres: 266744. 0.4 dihydrodipicolinate reductase-like protein ;supported by full-length At5g52100 cDNA: Ceres:107440. 0.4 At5g51540 putative protein contains similarity to endopeptidase 0.4 putative protein similar to unknown protein At5g51550 (gb|AAD25141.1);supported by full-length cDNA: Ceres:33455. 0.4

At5g50915 Expressed protein ; supported by full-length cDNA: Ceres: 36971. 0.4 putative protein similar to unknown protein (pir||S75732);supported At5g50100 by full-length cDNA: Ceres:35710. 0.4 At5g49730 FRO2-like protein; NADPH oxidase-like 0.4 putative protein similar to unknown protein (pir||S72530);supported At5g48490 by full-length cDNA: Ceres:32925. 0.4 At5g48470 unknown protein ;supported by full-length cDNA: Ceres:25275. 0.4 putative protein similar to unknown protein At5g47610 (gb|AAF16660.1);supported by full-length cDNA: Ceres:15457. 0.4 putative protein similar to unknown protein (pir||H71431);supported At5g47550 by full-length cDNA: Ceres:31680. 0.4

192

At5g46580 putative protein contains similarity to salt-inducible protein 0.4 putative protein contains similarity to FKBP-type peptidyl-prolyl cis- At5g45680 trans isomerase 0.4 unknown protein ; supported by cDNA: At5g45040 gi_13926304_gb_AF372903.1_AF372903 0.4 putative protein similar to unknown protein (gb AAD10689.1); At5g44260 supported by cDNA: gi_14334449_gb_AY034916.1_ 0.4 vegetative storage protein-like ;supported by full-length cDNA: At5g44020 Ceres:27372. 0.4 At5g43700 auxin-induced protein AUX2-11 (sp P33077) 0.4 At5g43270 squamosa promoter binding protein-like 2 (emb|CAB56576.1) 0.4 putative protein similar to unknown protein (pir||F70811);supported At5g42760 by full-length cDNA: Ceres:111265. 0.4 putative protein similar to unknown protein At5g42070 (dbj|BAA92898.1);supported by full-length cDNA: Ceres:97314. 0.4 beta-1,3-glucanase-like protein ;supported by full-length cDNA: At5g42100 Ceres:11988. 0.4 At5g41050 unknown protein ;supported by full-length cDNA: Ceres:42577. 0.4 50S ribosomal protein L27 ;supported by full-length cDNA: At5g40950 Ceres:152076. 0.4 putative protein similar to unknown protein (pir||S75227);supported At5g38520 by full-length cDNA: Ceres:22. 0.4 At5g36910 thionin Thi2.2 ;supported by full-length cDNA: Ceres:1523. 0.4 MtN3-like protein ; supported by cDNA: At5g23660 gi_3747110_gb_AF095641.1_AF095641 0.4 putative protein similar to unknown protein (emb|CAB75797.1); At5g23060 supported by cDNA: gi_15027882_gb_AY045798.1_ 0.4 At5g22340 unknown protein 0.4 putative protein 2'-hydroxyisoflavone reductase (EC 1.3.1.45) - Nicotiana tabacum, PIR:T02202;supported by full-length cDNA: At5g18660 Ceres:17121. 0.4 unknown protein ; supported by cDNA: At5g17170 gi_14334891_gb_AY035119.1_ 0.4 unknown protein ; supported by cDNA: At5g17300 gi_14190364_gb_AF378860.1_AF378860 0.4 putative protein polypeptide deformylase, Aquifex aeolicus, At5g14660 PIR:C70352;supported by full-length cDNA: Ceres:150612. 0.4 At5g14410 putative protein ;supported by full-length cDNA: Ceres:12170. 0.4 peptide transporter - like protein peptide transporter, Hordeum At5g13400 vulgare, EMBL:AF023472 0.4 putative serine rich protein predicted proteins, Arabidopsis At5g12050 thaliana;supported by full-length cDNA: Ceres:36958. 0.4

putative protein predicted proteins in castor bean, Arabidopsis At5g11420 thaliana and alfalfa.; supported by full-length cDNA: Ceres: 25522. 0.4 At5g08610 RNA helicase-like protein 0.4 At5g08050 putative protein predicted protein, Arabidopsis thaliana 0.4 putative protein SF16 protein, pollen specific - Helianthus annuus, At5g07240 PIR:T13992 0.4 cytochrome P450 90A1 (sp|Q42569) ; supported by full-length At5g05690 cDNA: Ceres: 36334. 0.4

fatty acid elongase - like protein KCS1 fatty acid elongase 3- At5g04530 ketoacyl-CoA synthase 1, Arabidopsis thaliana, EMBL:AF053345 0.4 At5g03120 putative protein ;supported by full-length cDNA: Ceres:40252. 0.4

193

At5g02710 putative protein ;supported by full-length cDNA: Ceres:158771. 0.4 protein phosphatase - like protein protein phosphatase 2C homolog, At5g02760 Mesembryanthemum crystallinum, EMBL:AF097667 0.4 one helix protein (OHP) ;supported by full-length cDNA: At5g02120 Ceres:16704. 0.4 putative protein wound-inducible protein wun1 protein - Solanum tuberosum, PIR:JQ0398;supported by full-length cDNA: At5g01740 Ceres:248967. 0.4 At5g01240 LAX1 / AUX1 -like permease 0.4 At5g01075 Expressed protein ; supported by full-length cDNA: Ceres: 1366. 0.4 putative protein outer envelope membrane protein E 6.7 - chloroplast Spinacia oleracea, PIR:A35958; supported by cDNA: At3g63160 gi_15724349_gb_AF412115.1_AF412115 0.4 putative protein chloroplast ribosome recycling factor protein - Spinacia oleracea, EMBL:AJ133751; supported by cDNA: At3g63190 gi_13926251_gb_AF372883.1_AF372883 0.4 At3g62750 beta-glucosidase-like protein several beta-glucosidases 0.4

At3g61460 RING finger protein ;supported by full-length cDNA: Ceres:25801. 0.4 GATA transcription factor 4 ; supported by cDNA: At3g60530 gi_14517394_gb_AY039532.1_ 0.4 At2g45470 unknown protein ;supported by full-length cDNA: Ceres:7709. 0.4

At3g60290 SRG1 - like protein SRG1 protein, Arabidopsis thaliana, PIR:S44261 0.4 bZIP protein ; supported by cDNA: At3g60320 gi_600854_gb_U17887.1_ATU17887 0.4 putative protein basic leucine zipper transcription activator shoot- forming PKSF1 - Paulownia kawakamii, EMBL:AF046934;supported At3g58120 by full-length cDNA: Ceres:34553. 0.4 responce reactor 4 ; supported by cDNA: At3g57040 gi_3273201_dbj_AB010918.1_AB010918 0.4 putative protein hypothetical protein - Arabidopsis thaliana, At3g57180 EMBL:AL049523 0.4 putative protein translation releasing factor RF-2 - Synechocystis sp., At3g57190 PIR:S76448 0.4 putative protein strictosidine synthase (EC 4.3.3.2) - Rauvolfia mannii At3g57030 (fragment) 0.4 At3g56290 putative protein 0.4 At3g56070 peptidylprolyl isomerase 0.4 putative protein CHLOROPLAST 30S RIBOSOMAL PROTEIN S20, SWISSPROT:RR20_GUITH; supported by cDNA: At3g56010 gi_15810456_gb_AY056267.1_ 0.4 sedoheptulose-bisphosphatase precursor ; supported by cDNA: At3g55800 gi_15451177_gb_AY054669.1_ 0.4 synaptic glycoprotein SC2-like protein synaptic glycoprotein SC2 spliced variant, Homo sapiens, EMBL:AF038958;supported by full- At3g55360 length cDNA: Ceres:6774. 0.4 Peptidase - like protein ileal peptidase, Rattus norvegicus, At3g54720 EMBL:AF009921;supported by full-length cDNA: Ceres:103193. 0.4 fructose-bisphosphatase precursor ; supported by cDNA: At3g54050 gi_14532619_gb_AY039934.1_ 0.4 nucleoid DNA-binding - like protein nucleoid DNA-binding protein cnd41, chloroplast, common tobacco, PIR:T01996; supported by full- At3g54400 length cDNA: Ceres: 8987. 0.4

194

nuclear envelope membrane protein - like LBR integral nuclear envelope inner membrane protein, Homo sapiens, EMBL:HSLBR10; At3g52940 supported by cDNA: gi_8917584_gb_AF257178.1_AF257178 0.4 beta-galactosidase precursor - like protein beta-galactosidase precursor, Carica papaya, EMBL:AF064786; supported by cDNA: At3g52840 gi_13605856_gb_AF367327.1_AF367327 0.4 mucin-like protein hemomucin, Drosophila melanogaster, At3g51420 EMBL:DM42014;supported by full-length cDNA: Ceres:38547. 0.4 non-specific lipid transfer protein ;supported by full-length cDNA: At3g51600 Ceres:8400. 0.4

putative protein steroid dehydrogenase homolog - Homo sapiens, At3g50560 EMBL:AF078850;supported by full-length cDNA: Ceres:34560. 0.4 kinesin -like protein KINESIN-LIKE PROTEIN KIF4, Homo sapiens, EMBL:AF179308; supported by cDNA: At3g50240 gi_14041828_dbj_AB061676.1_AB061676 0.4 MTN3-like protein MtN3 gene product - Medicago truncatula,PID:e1169583; supported by cDNA: At3g48740 gi_13605687_gb_AF361825.1_AF361825 0.4 putative protein hypothetical protein F12C20.9 - Arabidopsis At3g48610 thaliana, PIR:T02648 0.4 At3g48500 hypothetical protein 0.4

putative protein PMP31 protein - Candida At3g47430 boidinii,PIR2:S50280;supported by full-length cDNA: Ceres:28351. 0.4

At3g47295 Expressed protein ; supported by full-length cDNA: Ceres: 157151. 0.4 putative histone deacetylase similar to maize nucleolar histone deacetylase (U82815); supported by cDNA: At3g44750 gi_11066134_gb_AF195545.1_AF195545 0.4 lipid-transfer protein-like protein nonspecific lipid transfer protein, loblolly pine, PIR:S51816;supported by full-length cDNA: At3g43720 Ceres:8461. 0.4 hypothetical protein ; supported by cDNA: At4g39710 gi_15529203_gb_AY052226.1_ 0.4 At4g39040 putative protein 0.4 cinnamyl-alcohol dehydrogenase CAD1 ;supported by full-length At4g39330 cDNA: Ceres:34593. 0.4 putative protein phospholipase C (EC 3.1.4.3) precursor,phosphatidylinositol-specific - Listeria monocytogenes, At4g38690 PIR2:A37204 0.4 putative auxin-induced protein auxin-induced protein 10A, Glycine At4g38860 max., PIR2:JQ1099 0.4 extensin - like protein proline-rich protein, Solanum tuberosum, At4g38770 AJ000997;supported by full-length cDNA: Ceres:2315. 0.4

auxin-induced protein - like auxin-inducible SAUR gene, Raphanus At4g38840 sativus,AB000708;supported by full-length cDNA: Ceres:10140. 0.4 cinnamyl-alcohol dehydrogenase ELI3-1 ; supported by cDNA: At4g37980 gi_13430625_gb_AF360225.1_AF360225 0.4 At4g38160 putative protein 0.4 formamidase - like protein formamidase, Methylophilus methylotrophus,PIR2:S74213;supported by full-length cDNA: At4g37550 Ceres:23732. 0.4

195

subtilisin-like serine protease similar to SBT1, a subtilase from At4g34980 tomato plants GI:1771160 from [Lycopersicon esculentum] 0.4 At4g34560 putative protein 0.4 Homeodomain - like protein similaritry to homeotic protein BEL1, At4g34610 Arabidopsis thaliana, PIR2:A57632 0.4

At4g33660 Expressed protein ; supported by full-length cDNA: Ceres: 109432. 0.4

pectinesterase - like protein pectinesterase, Prunus persica, X95991; At4g33220 supported by cDNA: gi_14190428_gb_AF378892.1_AF378892 0.4

P-Protein - like protein P-Protein precursor, Solanum tuberosum, At4g33010 gb:Z99770; supported by cDNA: gi_14596024_gb_AY042800.1_ 0.4 At4g31850 putative protein crp1 protein, Zea mays, Z14393 0.4 predicted protein cation transport protein ChaC, Escherichia coli, At4g31290 PIR2:G64868;supported by full-length cDNA: Ceres:39740. 0.4 cinnamoyl-CoA reductase - like protein cinnamoyl-CoA reductase, Saccharum officinarum, gb:AJ231134; supported by full-length At4g30470 cDNA: Ceres: 150515. 0.4

photosystem II protein W - like photosystem II protein W, Porphyra At4g28660 purpurea, PIR2:S73268; supported by full-length cDNA: Ceres: 2419. 0.4 putative protein 150-kD protein, Dictyostelium discoideum, At4g28080 gb:U49332 0.4

At4g27657 Expressed protein ; supported by full-length cDNA: Ceres: 12935. 0.4 putative protein ENOD20 gene, Medicago truncatula, At4g27520 X99467;supported by full-length cDNA: Ceres:33380. 0.4 putative protein gene F19K23.12 of BAC F19K23 from Arabidopsis At4g27030 thaliana chromosome 1, PID:g2160143 0.4 fructose-bisphosphate aldolase - like protein fructose-bisphosphate aldolase, Arabidopsis thaliana, PIR1:ADMU;supported by full-length At4g26530 cDNA: Ceres:34690. 0.4 putative pathogenesis-related protein gene PR-1 protein - Medicago At4g25780 truncatula, PIR2:S47171 0.4 DRE CRT-binding protein DREB1C involved in low-temperature- responsive gene expression00; supported by cDNA: At4g25470 gi_3738227_dbj_AB007789.1_AB007789 0.4 At4g25260 putative protein pectinesterase - Citrus sinensis, PID:g2098705 0.4 At4g24510 CER2 ;supported by full-length cDNA: Ceres:33382. 0.4 At4g24700 hypothetical protein 0.4 putative major latex protein major latex protein type 1 - Arabidopsis thaliana, EMBL:X91960;supported by full-length cDNA: At4g23670 Ceres:108949. 0.4 At4g22890 putative protein ;supported by full-length cDNA: Ceres:13264. 0.4

heat shock protein - like heat shock protein 17, Triticum aestivum, At4g21870 PIR1:HHWT17;supported by full-length cDNA: Ceres:23223. 0.4 hyuC-like protein 5-substituted hydantoins to the corresponding L- At4g20070 amino acids, Pseudomonas sp.,PIR2:D42594 0.4 TMV resistance protein N - like TMV resistance protein N, Nicotiana At4g19530 glutinosa, PIR2:A54810 0.4 protein ch-42 precursor, chloroplast ;supported by full-length cDNA: At4g18480 Ceres:7501. 0.4

At4g18650 putative protein transcription factor OBF3.1, Zea mays, PIR2:S33223 0.4 196

HhoA protease precursor, putative identical to putative protease HhoA precursor [Arabidopsis thaliana] SP:Q9SEL7 GI:6690272 (unpublished, Lensch,M.H.A., Herrmann,R.G. and Sokolenko,A); At4g18370 supported by cDNA: gi:6690271 0.4 At4g12970 putative protein 0.4

putative protein pectinesterase - Citrus At4g12390 sinensis,PID:g2098711;supported by full-length cDNA: Ceres:27615. 0.4 Ribosomal protein L7Ae -like various L7Ae ribosomal proteins; At4g12600 supported by full-length cDNA: Ceres: 33381. 0.4 putative protein heat shock protein dnaJ - Bacillus At4g09350 stearothermophilus,PIR:JC4739 0.4 putative protein A. thaliana hypothetical protein F1N20.70, GenBank accession number AL022140;supported by full-length cDNA: At4g04630 Ceres:118778. 0.4 At4g04840 putative protein similar to transcriptional regulator 0.4 putative xyloglucan endotransglycosylase ;supported by full-length At4g03210 cDNA: Ceres:17748. 0.4 hypothetical protein similar to A. thaliana hypothetical protein At4g02790 T32G6.19, GenBank accession number AC002510 0.4 putative L5 ribosomal protein ;supported by full-length cDNA: At4g01310 Ceres:22919. 0.4 homeodomain protein AHDP ; supported by cDNA: At4g00730 gi_17063159_gb_AY062100.1_ 0.4 probable plasma membrane intrinsic protein 1c ; supported by cDNA: At4g00430 gi_2373400_dbj_D85192.1_D85192 0.4 At4g00360 probable cytochrome P450 0.4 At4g00400 putative protein 0.4 At4g00050 putative transcriptional regulator 0.4 putative YABBY3 axial regulator ; supported by cDNA: At4g00180 gi_14335013_gb_AY037186.1_ 0.4 phosphoribulokinase precursor identical to phosphoribulokinase precursor GB:P25697 GI:125576 from [Arabidopsis At1g32060 thaliana];supported by full-length cDNA: Ceres:11226. 0.4 fatty acid condensing enzyme CUT1, putative similar to fatty acid condensing enzyme CUT1 GI:5001734 from [Arabidopsis At1g25450 thaliana];supported by full-length cDNA: Ceres:99793. 0.4 At1g16720 unknown protein 0.4 unknown protein identical to unknown protein GI:9755444 from (Arabidopsis thaliana); supported by full-length cDNA: Ceres: At1g66940 110066. 0.4 ABC transporter, putative similar to ABC transporter GI:10280532 At1g17840 from [Homo sapiens] 0.4 tonoplast intrinsic protein, putative similar to tonoplast intrinsic protein GI:21054 from [Phaseolus vulgaris]; supported by cDNA: At1g17810 gi_2605713_gb_AF026275.1_AF026275 0.4

PSI type II chlorophyll a/b-binding protein, putative similar to PSI type II chlorophyll a/b-binding protein GI:541565 from [Arabidopsis At1g19150 thaliana];supported by full-length cDNA: Ceres:252299. 0.4 unknown protein contains similarity to alternative NADH- dehydrogenase GI:3718005 from [Yarrowia lipolytica];supported by At1g07180 full-length cDNA: Ceres:114420. 0.4 unknown protein ; supported by cDNA: At1g16880 gi_14423501_gb_AF386988.1_AF386988 0.4

197

hypothetical protein predicted by genemark.hmm;supported by full- At1g56210 length cDNA: Ceres:107869. 0.4 oxidoreductase, putative contains Pfam profile: PF01408: At1g66130 oxidoreductase, Gfo/Idh/MocA family 0.4 At1g66180 unknown protein ;supported by full-length cDNA: Ceres:99625. 0.4 thionin, putative similar to thionin [Arabidopsis thaliana] GI:1181533; supported by cDNA: At1g66100 gi_14190504_gb_AF380652.1_AF380652 0.4 At3g28220 unknown protein 0.4 cytochrome P450, putative contains Pfam profile: PF00067 cytochrome P450; supported by cDNA: At3g28740 gi_15292830_gb_AY050849.1_ 0.4 unknown protein similar to cell wall-plasma membrane linker protein At3g22120 GB:CAA64425 from [Brassica napus] 0.4 At3g13510 unknown protein ;supported by full-length cDNA: Ceres:37810. 0.4

ABC transporter, putative similar to ATP-binding cassette, sub- At3g21090 family G (WHITE), member 2 GB:NP_036050 from [Mus musculus] 0.4

phosphoribosyamidoimidazole-succinocarboxamide synthase, putative similar to PHOSPHORIBOSYLAMIDOIMIDAZOLE- SUCCINOCARBOXAMIDE SYNTHASE GB:P38025 from At3g21110 [Arabidopsis thaliana];supported by full-length cDNA: Ceres:35508. 0.4 myrosinase-associated protein, putative similar to GB:CAA71238 from [Brassica napus], conatains Pfam profile:PF00657 Lipase/Acylhydrolase with GDSL-like motif; supported by cDNA: At3g14210 gi_15450434_gb_AY052318.1_ 0.4 mrp protein, putative similar to mrp protein GB:S11948 from [Escherichia coli] (Mol. Gen. Genet. (1990) 223 (1), 121- At3g24430 133);supported by full-length cDNA: Ceres:231598. 0.5 At3g23760 unknown protein ; supported by full-length cDNA: Ceres: 5176. 0.5 hypothetical protein predicted by genemark.hmm, contains Pfam profile:F00575 S1:S1 RNA binding domain; supported by cDNA: At3g23700 gi_15146283_gb_AY049283.1_ 0.5 unknown protein contains Pfam profiles: PF00560 leucine rich At3g28040 repeat, PF00069 eukaryotic protein kinase domain 0.5 phosphate transporter, putative contains Pfam profile: PF01384 At3g26570 phosphate transporter family 0.5 glycine-rich protein, putative identical to a region of GB:JQ1064 from [Arabidopsis thaliana] ( Plant Cell 2 (5), 427-436 (1990)); At3g20470 supported by cDNA: gi_15010655_gb_AY045629.1_ 0.5 hypothetical protein predicted by genemark.hmm;supported by full- At3g17170 length cDNA: Ceres:16548. 0.5 At3g17050 unknown protein 0.5 cell elongation protein, Dwarf1 identical to GB:S71189 from [Arabidopsis thaliana]; supported by cDNA: At3g19820 gi_516042_gb_U12400.1_ATU12400 0.5 hypothetical protein similar to hypothetical protein GB:AAD50054 At3g19850 from [Arabidopsis thaliana] 0.5 unknown protein similar to NA-DAMAGE-REPAIR/TOLERATION PROTEIN DRT100 PRECURSOR GB:Q00874 from [Arabidopsis At3g20820 thaliana] 0.5 putative D-3-phosphoglycerate dehydrogenase similar to GB:O04130 from [Arabidopsis thaliana]; supported by cDNA: At3g19480 gi_14596140_gb_AY042858.1_ 0.5

198

unknown protein similar to MRP-like ABC transporter At3g21250 GB:AAC49791 from [Arabidopsis thaliana] 0.5 putative 2Fe-2S iron-sulfur cluster protein contains Pfam profile: PF00111 2Fe-2S iron-sulfur cluster binding domains; supported by At3g16250 cDNA: gi_14596158_gb_AY042867.1_ 0.5 50S ribosomal protein L15, chloroplast precursor identical to GB:P25873 from [Arabidopsis thaliana];supported by full-length At3g25920 cDNA: Ceres:10926. 0.5 putative peroxiredoxin similar to peroxiredoxin Q GB:BAA90524 from [Sedum lineare]; supported by cDNA: At3g26060 gi_15081742_gb_AY048264.1_ 0.5

nitrate transporter identical to nitrate transporter GB:CAB38706 At3g21670 [Arabidopsis thaliana];supported by full-length cDNA: Ceres:111089. 0.5

putative chlorophyll A-B binding protein similar to chlorophyll A-B binding protein 151 precursor (LHCP) GB:P27518 from [Gossypium At3g27690 hirsutum];supported by full-length cDNA: Ceres:7700. 0.5 At3g23450 hypothetical protein predicted by genemark 0.5 unknown protein predicted by genscan+;supported by full-length At3g06070 cDNA: Ceres:153279. 0.5 putative flowering-time gene CONSTANS (COL2) identical to putative flowering-time gene CONSTANS (COL2) GB:AAB67879 GI:1507699 (Arabidopsis thaliana);supported by full-length cDNA: At3g02380 Ceres:949. 0.5 At3g08030 unknown protein ;supported by full-length cDNA: Ceres:27471. 0.5 At3g05900 unknown protein 0.5 unknown protein similar to unknown protein GB:AAB67633 At3g04620 [Arabidopsis thaliana] 0.5 putative serine carboxypeptidase II similar to serine carboxypeptidase II (CP-MII) GB:CAA70815 [Hordeum vulgare];supported by full- At3g02110 length cDNA: Ceres:42069. 0.5 At3g05730 unknown protein 0.5 At3g10525 Expressed protein ; supported by full-length cDNA: Ceres: 2153. 0.5

putative aspartate kinase similar to aspartate kinase GB:CAA67376 At3g02020 (Arabidopsis thaliana); supported by full-length cDNA: Ceres: 6203. 0.5

putative APG protein similar to anter-specific proline-rich protein APG precursor SP:P40602 (Arabidopsis thaliana); contains Pfam At3g16370 profile: PF00657 Lipase Acylhydrolase with GDSL-like motif 0.5 hypothetical protein similar to unknown protein GI:3335359 from At1g01610 [Arabidopsis thaliana] 0.5 unknown protein ; supported by cDNA: At1g44000 gi_15028026_gb_AY045870.1_ 0.5 hypothetical protein ; supported by cDNA: At1g20450 gi_15081631_gb_AY048208.1_ 0.5 P-glycoprotein, putative similar to P-glycoprotein GI:6671365 At1g28010 [Gossypium hirsutum] 0.5 lipid transfer protein, putative contains Pfam profile: PF00279: Plant lipid transfer protein family; supported by full-length cDNA: Ceres: At1g27950 15027. 0.5 hypothetical protein predicted by genemark.hmm;supported by full- At1g55370 length cDNA: Ceres:32881. 0.5

199

nitrate reductase 1 (NR1) identical to nitrate reductase 1 (NR1) GB:P11832 [Arabidopsis thaliana]; supported by cDNA: At1g77760 gi_15983498_gb_AF424624.1_AF424624 0.5 AP2-containing DNA-binding protein contains Pfam profile: PF00847 At1g64380 AP2 domain 0.5 At1g49840 unknown protein 0.5 putative protein kinase similar to leucine-rich repeat transmembrane At1g80640 protein kinase 1 GB:AAC27894 from [Zea mays] 0.5 germin-like protein similar to germin precursor GB:P26759 [Triticum aestivum]; contains Pfam profile: PF01072 Germin family;supported At1g72610 by full-length cDNA: Ceres:20068. 0.5 hydroxypyruvate reductase (HPR) identical to hydroxypyruvate reductase (HPR) GB:D85339 [Arabidopsis thaliana] (Plant Cell Physiol 1997 Apr;38(4):449-55); supported by cDNA: At1g68010 gi_2055272_dbj_D85339.1_D85339 0.5 30S ribosomal protein S17, chloroplast precursor (CS17) identical to 30S ribosomal protein S17, chloroplast precursor GB:P16180 [Arabidopsis thaliana]; supported by cDNA: At1g79850 gi_14423473_gb_AF386974.1_AF386974 0.5 GAST1-like protein similar to GAST1 protein precursor GB:P27057 [Lycopersicon esculentum] (induced by gibberellins, inhibited by At1g74670 ABA Plant J 1992 Mar;2(2):153-9) 0.5 putative SET protein, phospatase 2A inhibitor similar to SET protein, phospatase 2A inhibitor GB:Q01105 [Homo sapiens] (role in the mechanism of leukemogenesis: J Biol Chem 1994 Jan 21;269(3):2258-62); contains Pfam profile: PF00956 nucleosome At1g74560 assembly protein (NAP) 0.5 very-long-chain fatty acid condensing enzyme (CUT1) identical to very-long-chain fatty acid condensing enzyme (CUT1) GB:AF129511 (required for cuticular wax biosynthesis and pollen fertility: Millar,A.A., et al., Plant Cell (1999));supported by full-length At1g68530 cDNA: Ceres:36276. 0.5 hypothetical protein predicted by genefinder;supported by full-length At1g72430 cDNA: Ceres:99920. 0.5 biotin synthase (Bio B) ;supported by full-length cDNA: At2g43360 Ceres:42038. 0.5 membrane related protein CP5, putative similar to GB:AAD28760 from [Arabidopsis thaliana]; supported by cDNA: At1g55960 gi_16604620_gb_AY059755.1_ 0.5 RING-H2 finger protein RHA3a, putative similar to RING-H2 finger protein RHA3a GI:3790573 from [Arabidopsis thaliana]; supported At1g49200 by cDNA: gi_14517431_gb_AY039551.1_ 0.5 beta-glucosidase, putative similar to beta-glucosidase GB:L41869 GI:804655 from [Hordeum vulgare];supported by full-length cDNA: At1g26560 Ceres:125606. 0.5 ribosomal protein L11, putative similar to chloroplast ribosomal protein L11 GI:21312 from [Spinacia oleracea];supported by full- At1g32990 length cDNA: Ceres:16773. 0.5

starch synthase, putative similar to starch synthase GI:21613 from At1g32900 [Solanum tuberosum];supported by full-length cDNA: Ceres:7714. 0.5 putative calcium-binding protein, calreticulin similar to SP:P12858 At1g12900 from [Arabidopsis thaliana] 0.5 subtilisin-like serine protease similar to subtilisin-type protease precursor GI:14150446 from [Glycine max];supported by full-length At1g20160 cDNA: Ceres:3907. 0.5 200

beta tubulin 1, putative similar to GB:AAD02498 from [Arabidopsis thaliana] (Plant Mol. Biol. 39 (1), 171-176 (1999)); supported by At1g20010 cDNA: gi_13605518_gb_AF361585.1_AF361585 0.5

fatty acid elongase 3-ketoacyl-CoA synthase, putative similar to GB:AAC99312 from [Arabidopsis thaliana] (Plant J. (1999) In press); At1g07720 supported by cDNA: gi_16226846_gb_AF428349.1_AF428349 0.5 phytochrome kinase substrate 1, putative similar to phytochrome kinase substrate 1 GI:5020168 from [Arabidopsis thaliana];supported At1g14280 by full-length cDNA: Ceres:97569. 0.5

At1g14345 Expressed protein ; supported by full-length cDNA: Ceres: 19170. 0.5

At1g49975 Expressed protein ; supported by full-length cDNA: Ceres: 11759. 0.5 helix-loop-helix protein homolog, putative similar to helix-loop-helix protein homolog GB:BAA87957 GI:6520231 from [Arabidopsis At1g18400 thaliana] 0.5 At1g76110 unknown protein 0.5 At1g15980 unknown protein ;supported by full-length cDNA: Ceres:122986. 0.5 RING-H2 zinc finger protein ATL5, putative similar to RING-H2 At1g22500 zinc finger protein ATL5 GI:4928401 from [Arabidopsis thaliana] 0.5 At1g64680 unknown protein ;supported by full-length cDNA: Ceres:101924. 0.5 peptide transporter, putative predicted by genemark.hmm; supported At1g64500 by cDNA: gi_15810442_gb_AY056260.1_ 0.5 TINY-like protein similar to TINY GB: CAA64359 GI:1246403 from At1g33760 [Arabidopsis thaliana] 0.5 50S ribosomal protein L21 chloroplast precursor (CL21) identical to GB:P51412 GI:1710424 from [Arabidopsis thaliana]; supported by At1g35680 cDNA: gi_16226898_gb_AF428363.1_AF428363 0.5 At1g80240 hypothetical protein predicted by genemark.hmm 0.5 late embryogenis abundant protein, putative similar to late embryogenis abundant protein 5 GI:2981167 from [Nicotiana At1g02820 tabacum];supported by full-length cDNA: Ceres:96540. 0.5 alpha-xylosidase precursor identical to alpha-xylosidase precursor GB:AAD05539 GI:4163997 from [Arabidopsis thaliana]; supported At1g68560 by cDNA: gi_15982750_gb_AY057482.1_ 0.5 ribosomal protein L18, putative similar to ribosomal protein L18 At1g48350 GI:3980238 from [Thermotoga maritima] 0.5 auxin transport protein REH1, putative similar to auxin transport protein REH1 GI:3377509 from [Oryza sativa]; supported by cDNA: At1g70940 gi_5817300_gb_AF087818.1_AF087818 0.5 unknown protein identical to residues 1 to 141 of unknown protein At1g70760 GB:AAD55491 (Arabidopsis thaliana) 0.5

At1g70985 Expressed protein ; supported by full-length cDNA: Ceres: 102374. 0.5 At1g73120 hypothetical protein predicted by genefinder 0.5 hypothetical protein predicted by genemark.hmm;supported by full- At1g49380 length cDNA: Ceres:230. 0.5 acyl CoA synthetase, putative similar to acyl CoA synthetase At1g49430 GI:1617267 from [Brassica napus] 0.5 SOUL-like protein Similar to SOUL Protein [Mus musculus] (gi|4886906) and [Homo sapiens] (gi|4886910). Location of ests PAP043 5' (gb|Z27017), PAP043 3' (gb|Z29848), 153N19T7 (gb|AA720145), and 212B8T7 (gb|N37862);supported by full-length At1g17100 cDNA: Ceres:29992. 0.5

201

unknown protein Location of est 136A23T7 (gb|T45563); supported At1g17200 by full-length cDNA: Ceres: 28177. 0.5

hypothetical protein Strong similarity to gi|4734005 F3L12.7 hypothetical protein from Arabidopsis thaliana BAC gb|AC007178; At1g15180 supported by cDNA: gi_15028308_gb_AY045957.1_ 0.5 unknown protein ESTs gb|F20110 and gb|F20109 come from this At1g15290 gene 0.5

unknown protein location of ESTs 144D22XP 3', gb|AA404877 and At1g14150 144D22T7, gb|T75757;supported by full-length cDNA: Ceres:5665. 0.5 unknown protein similar to elongation factor G SP:P34811 [Glycine max (Soybean)]; supported by cDNA: At1g62750 gi_14532623_gb_AY039936.1_ 0.5 receptor kinase (CLV1) identical to receptor kinase (CLV1) At1g75820 GB:AAB58929 GI:2160756 [Arabidopsis thaliana] 0.5 putative transport protein may be a member of the PF|00083 sugar At1g16390 transporter family 0.5 At1g75690 unknown protein 0.5 At1g65230 unknown protein 0.5 putative endoxyloglucan transferase similar to xyloglucan endotransglycosylase-related protein XTR4 (pir|IS71223);supported At1g10550 by full-length cDNA: Ceres:27813. 0.5

At2g36145 Expressed protein ; supported by full-length cDNA: Ceres: 10500. 0.5 At2g20490 unknown protein ;supported by full-length cDNA: Ceres:39169. 0.5 At2g22170 unknown protein ;supported by full-length cDNA: Ceres:15081. 0.5 putative glucosyltransferase ; supported by full-length cDNA: Ceres: At2g31750 114997. 0.5 At2g04790 hypothetical protein predicted by genefinder 0.5 RNA helicase, putative similar to RNA helicase GI:3776015 from [Arabidopsis thaliana]; supported by cDNA: At1g59990 gi_15983391_gb_AF424570.1_AF424570 0.5 unknown protein ESTs gb|T20589, gb|T04648, gb|AA597906, gb|T04111, gb|R84180, gb|R65428, gb|T44439, gb|T76570, gb|R90004, gb|T45020, gb|T42457, gb|T20921, gb|AA042762 and gb|AA720210 come from this gene;supported by full-length cDNA: At1g09310 Ceres:11073. 0.5 putative fructose bisphosphate aldolase ; supported by cDNA: At2g21330 gi_14334739_gb_AY035043.1_ 0.5 putative GDSL-motif lipase/hydrolase similar to APG proteins; pFAM domain PF00657;supported by full-length cDNA: At2g04570 Ceres:39762. 0.5 putative xyloglucan endo-transglycosylase ; supported by full-length At2g36870 cDNA: Ceres: 7831. 0.5 putative RNA polymerase sigma-70 factor ; supported by cDNA: At2g36990 gi_7209639_dbj_AB029916.1_AB029916 0.5 putative aquaporin (tonoplast intrinsic protein gamma) ;supported by At2g36830 full-length cDNA: Ceres:36633. 0.5

At2g36000 hypothetical protein ;supported by full-length cDNA: Ceres:123727. 0.5 At2g42870 unknown protein ;supported by full-length cDNA: Ceres:102453. 0.5 At2g21210 putative auxin-regulated protein 0.5 putative rubisco subunit binding-protein alpha subunit ;supported by At2g28000 full-length cDNA: Ceres:25773. 0.5

202

disease resistance protein RPS4, putative similar to disease resistance At1g65390 protein RPS4 GI:5459305 from [Arabidopsis thaliana] 0.5 putative gibberellin-regulated protein contains similarity to gibberellin-regulated protein 2 precursor (GAST1) homolog At1g22690 gb|U11765 from A. thaliana 0.5 At1g22630 unknown protein ;supported by full-length cDNA: Ceres:37537. 0.5 hypothetical protein contains similarity to lectin polypeptide At1g65400 GI:410436 from [Cucurbita maxima] 0.5

At1g12080 unknown protein ; supported by full-length cDNA: Ceres: 270281. 0.5 putative NPK1-related protein kinase 2 similar to nitrate chlorate transporter GB:Q05085 from (Arabidopsis thaliana); supported by At1g12110 cDNA: gi_166667_gb_L10357.1_ATHCHL1A 0.5 pEARLI 1-like protein may be induced when levels of Aluminum become toxic or other stresses become present in the plant;supported At1g12090 by full-length cDNA: Ceres:5712. 0.5 aminomethyltransferase-like precursor protein very strong similarity to aminomethyltransferase precursor gb|U79769 from Mesembryanthemum crystallinum. ESTs gb|T43167, gb|T21076, gb|H36999, gb|T22773, gb|N38038, gb|T13742, gb|Z26545, At1g11860 gb|T20753 and gb|W43123 come from this gene 0.5

hypothetical protein similar to lecithin:cholesterol acyltransferase At1g27480 precursor (M26268);supported by full-length cDNA: Ceres:35408. 0.5 putative pectate lyase A11 similar to GB:CAB36835;supported by At1g04680 full-length cDNA: Ceres:37952. 0.5 hypothetical protein predicted by genemark.hmm;supported by full- At1g65490 length cDNA: Ceres:2118. 0.5 hypothetical protein predicted by genscan;supported by full-length At1g09750 cDNA: Ceres:6295. 0.5 At1g09900 hypothetical protein predicted by genscan 0.5 At1g70200 unknown protein 0.5 unknown protein similar to putative glycosyl transferase GI:7268597 from [Arabidopsis thaliana]; supported by cDNA: At1g70090 gi_13878002_gb_AF370264.1_AF370264 0.5 putative protochlorophyllide reductase similar to protochlorophyllide reductase precusor; similar to ESTs gb|R30630, gb|T46162, emb|Z26728, gb|AA042736, and gb|AA042730;supported by full- At1g03630 length cDNA: Ceres:7573. 0.5 putative auxin transport protein strongly similar to auxin transport protein GB:AAD52697; supported by cDNA: At1g23080 gi_15450508_gb_AY052356.1_ 0.5 hypothetical protein predicted by genemark.hmm; supported by At1g77090 cDNA: gi_13926194_gb_AF370571.1_AF370571 0.5

Rubisco subunit binding-protein beta subunit identical to chaperonin 60 beta precursor GB:JT0901 from [Arabidopsis thaliana]; supported At1g55490 by cDNA: gi_14423415_gb_AF386945.1_AF386945 0.5 hypothetical protein predicted by genscan; supported by cDNA: At2g18300 gi_15724317_gb_AF412099.1_AF412099 0.5 At2g10940 unknown protein ;supported by full-length cDNA: Ceres:32647. 0.5 At2g16630 unknown protein 0.5 ferredoxin--nitrite reductase ; supported by cDNA: At2g15620 gi_15010613_gb_AY045608.1_ 0.5 At2g20180 unknown protein 0.5

203

putative esterase (contains an esterase/lipase/thioesterase active site serine domain (prosite: PS50187); related to plant sensitive response At2g03550 proteins 0.5 At2g03350 unknown protein ;supported by full-length cDNA: Ceres:1697. 0.5 putative serine carboxypeptidase II ;supported by full-length cDNA: At2g35780 Ceres:40628. 0.5 At2g18050 histone H1 ;supported by full-length cDNA: Ceres:112970. 0.5 At2g15050 putative lipid transfer protein 0.5 At2g15090 putative fatty acid elongase 0.5 subtilisin-like serine protease, putative contains similarity to cucumisin-like serine protease GI:3176874 from [Arabidopsis At2g05920 thaliana] 0.5 protease inhibitor II ; supported by cDNA: At2g02130 gi_15293090_gb_AY050979.1_ 0.5 protease inhibitor II identical to GB:X69139, contains a gamma- thionin family signature (PDOC00725); supported by cDNA: At2g02100 gi_13878184_gb_AF370355.1_AF370355 0.5 putative endoxyloglucan glycosyltransferase identical to At2g06850 GB:D16454;supported by full-length cDNA: Ceres:15276. 0.5 At2g29180 unknown protein 0.5 putative auxin transport protein ; supported by cDNA: At2g01420 gi_7109714_gb_AF087016.1_AF087016 0.5 unknown protein ; supported by cDNA: At2g01590 gi_14334561_gb_AY035185.1_ 0.5 putative nonspecific lipid-transfer protein ; supported by cDNA: At2g38540 gi_15146309_gb_AY049296.1_ 0.5 At2g14880 unknown protein ; supported by full-length cDNA: Ceres: 41621. 0.5 At2g46830 MYB-related transcription factor (CCA1) 0.5 ribonuclease, RNS1 identical to ribonuclease SP:P42813, GI:561998 from [Arabidopsis thaliana]; supported by full-length cDNA: At2g02990 Ceres:27242. 0.5 unknown protein ; supported by cDNA: At2g02950 gi_15450680_gb_AY052708.1_ 0.5 At2g03020 predicted by genefinder 0.5 expansin AtEx6 identical to GB U30480; supported by full-length At2g28950 cDNA: Ceres: 17914. 0.5 At2g34620 hypothetical protein predicted by genefinder 0.5 At2g39470 unknown protein ;supported by full-length cDNA: Ceres:16403. 0.5 unknown protein ; supported by cDNA: At2g41190 gi_14532707_gb_AY039978.1_ 0.5 high affinity Ca2+ antiporter identical to GB:U57411, except a possible frameshift at base 58008. Sequence has been confirmed with 5 sequencing reads.; supported by cDNA: At2g38170 gi_13937168_gb_AF372938.1_AF372938 0.5 At2g32560 unknown protein 0.5 At2g37770 putative alcohol dehydrogenase 0.5 At2g44040 unknown protein ;supported by full-length cDNA: Ceres:10293. 0.5

beta-ketoacyl-CoA synthase (FIDDLEHEAD) identical to GB:AJ010713; contains a chalcone and stilbene synthase active site At2g26250 (PF00195); supported by cDNA: gi_14517455_gb_AY039563.1_ 0.5

putative C2H2-type zinc finger protein likely a nucleic acid binding At2g41940 protein; supported by cDNA: gi_14517523_gb_AY039597.1_ 0.5 At2g32690 unknown protein 0.5

204

hypothetical protein predicted by genefinder and genscan;supported At2g32650 by full-length cDNA: Ceres:23742. 0.5

unknown protein similar to hypothetical protein PIR|S76698|S76698; At2g39670 supported by cDNA: gi_15809963_gb_AY054250.1_ 0.5 At2g33050 putative leucine-rich repeat disease resistance protein 0.5

205