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Functional characterization of a gain-of-function mutant of AtMKK9 in Arabidopsis thaliana Cluis, Corinne Pamela 2006

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F U N C T I O N A L C H A R A C T E R I Z A T I O N OF A GAIN-OF-FUNCTION M U T A N T OF A T M K K 9 IN ARABIDOPSIS T H A L I A N A  By Corinne Pamela Cluis B.Sc., M c G i l l University, 2003  A THESIS S U B M I T T E D IN P A R T I A L F U L F I L M E N T OF THE REQUIREMENTS FOR T H E D E G R E E OF M A S T E R OF SCIENCE in T H E F A C U L T Y OF G R A D U A T E STUDIES (Botany)  T H E U N I V E R S I T Y OF BRITISH C O L U M B I A December 2005 © Corinne Pamela Cluis, 2005  Abstract The relatively small number o f M A P K K s encoded in the Arabidopsis genome suggests that this particular class o f kinases acts as a point o f convergence within the plant's 'integration o f external stimuli and their transduction to elicit biological responses. In an effort to gain information about the function o f the M A P K K , A t M K K 9 , in Arabidopsis, I have  characterized  several  aspects o f the  phenotype  of  DEX:CA-MKK9-FLAG  transgenic plants, which express an inducible constitutively active version o f A t M K K 9 , C A - M K K 9 . I have found that C A - M K K 9 expression can control the production o f ethylene by activating a downstream M A P K , A t M P K 6 , which is known to promote the stabilization o f ethylene biosynthesis enzymes. C A - M K K 9 induction was correlated with an increase i n A t M P K 6 activity  in planta, and was rapidly followed by production of a  burst o f ethylene in the induced plant tissues. I hypothesized that C A - M K K 9 directly activates A t M P K 6 , and demonstrated that a recombinant version o f C A - M K K 9 was capable of phosphorylating A t M P K 6  in vitro. In addition, the production o f the hormone  was abolished when C A - M K K 9 was expressed in a  mpk6 knock-out background, thus  proving that A t M P K 6 is required for C A - M K K 9 - m e d i a t e d ethylene biosynthesis. I have also confirmed preliminary data from our laboratory suggesting that C A - M K K 9 plays a role in oxidative programmed cell death. The necrotic lesions induced by C A - M K K 9 were still observed in the  mpk6 background, indicating that programmed cell death was  triggered by C A - M K K 9 activity independently o f A t M P K 6 activity and o f ethylene overproduction. In addition, in order to investigate short-term transcriptional events triggered by C A - M K K 9 , I attempted to capture the transcriptional profile of D E X : C A M K K 9 - F L A G plants using two-channel oligonucleotide microarrays. The C A - M K K 9 affected genes included a number o f genes involved i n the octadecanoid pathway, and their promoters were enriched i n A B R E - l i k e elements. However, my attempts to validate the microarray results using additional biological replicates and quantitative real-time PCR  revealed that the majority o f these early-response  microarray results  were  apparently false positives, indicating that the microarray experiment was probably inappropriately constructed for capturing early transcriptional responses to C A - M K K 9 using the dexamethasone-inducible system.  ii  Table of contents Abstract  ii  Table of contents  iii  List of Tables  vii  List of Figures  viii  Abbreviations  ix  Acknowledgments  x  Research contributions  1.  .  x  Material contributions  x  Support and guidance  xi  General Introduction 1.1.  Programmed cell death in plants  1 1  1.1.1.  Morphological and biochemical characteristics o f P C D  1  1.1.2.  P C D in plant development  2  1.1.3.  P C D in stress responses  3  Signaling events controlling P C D  4  1.2.  1.2.1.  Experimental models in the study o f P C D signaling pathways  4  1.2.2.  Reactive oxygen species and nitric oxide i n P C D  5  1.2.3.  Salicylic acid: a positive regulator o f P C D  7  1.2.4.  Jasmonates: negative regulators o f P C D  8  1.3.  Ethylene in stress responses and P C D  9  1.3.1.  Ethylene is a major signaling molecule in stress responses  1.3.2.  Ethylene biosynthesis and its regulation during stress responses  10  1.3.3.  Ethylene crosstalk with other hormones during P C D  12  1.4.  Mitogen-activated protein kinase cascades and signal transduction  9  13 iii  1.4.1.  M A P K cascades in plants  15  1.4.2.  M A P K signaling in plant stress responses  18  1.4.3.  M A P kinase substrates  22  1.4.4.  Genetic approaches to the study o f plant M A P K cascades  23  1.5. 2.  Problem statement and thesis objectives  26  C A - M K K 9 modulates ethylene biosynthesis  28  2.1.  Introduction  28  2.2.  Material and methods  31  2.2.1.  Plant material  31  2.2.2.  R T - P C R analysis  31  2.2.3.  Ethylene measurements  32  2.2.4.  Hydrogen peroxide detection  33  2.2.5.  In vitro phosphorylation assay  2.2.6.  Protein extraction and western blot analysis  34  2.2.7.  Generation o f D E X : C A - M K K 9 - F L A G / m p M transgenic plants  35  2.3.  33  Results  36  2.3.1.  The C A - M K K 9 transgene is rapidly activated following D E X induction 36  2.3.2.  C A - M K K 9 activity results in the formation o f lesions  2.3.3.  C A - M K K 9 - m e d i a t e d cell death is associated with  2.3.4.  C A - M K K 9 causes a rapid increase in ethylene biosynthesis  H  2  O  41 2  accumulation... 42 45  2.3.5. C A - M K K 9 is similar to C A - M K K 4 in acting as a mediator of ethylene biosynthesis 46 2.3.6.  C A - M K K 9 induction correlates with increased activity of A t M P K 6  in vivo 48  2.3.7.  A t M K K 9 activates A t M P K 6 in vitro  49  2.3.8.  A t M P K 6 is necessary for C A - M K K 9 - m e d i a t e d ethylene production  51 iv  2.3.9. 2.4.  3.  A t M P K 6 is not required for C A - M K K 9 - m e d i a t e d P C D  53  Discussion  54  Investigation of short-term transcriptional events induced by CA-MKK9  63  3.1.  Introduction  63  3.2.  Material and methods  65  3.2.1.  Plant material and treatments  65  3.2.2.  Microarrays  66  3.2.3.  Functional and promoter analysis  69  3.2.4.  Quantitative Real-time R T - P C R  69  3.3.  Results  71  3.3.1. Experimental design for the study o f C A - M K K 9 - m e d i a t e d transcriptional changes 71 3.3.2.  C A - M K K 9 mediates significant short-term transcriptional changes  3.3.3. activity  Functional analysis o f transcriptional changes resulting from C A - M K K 9 76  3.3.4.  Promoter analysis o f C A - M K K 9 - a f f e c t e d genes  3.3.5. CA-MKK9 biosynthesis genes 3.3.6. 3.4.  4.  induction results  in the down-regulation  Validation o f the most robust C A - M K K 9 - a f f e c t e d genes  Discussion  Future directions  75  78 o f jasmonate 81 87 89  94  4.1.  Role of A t M K K 9 in ethylene biosynthesis in vivo  94  4.2.  Investigation into the C A - M K K 9 - a c t i v a t e d 42 k D a M A P K  95  4.3.  Signaling events i n C A - M K K 9 - m e d i a t e d P C D  96  4.4.  Mechanisms o f C A - M K K 9 regulation o f W R K Y 3 0  97  4.5.  Concluding remarks  98  5.  Bibliography  6.  Appendix  99 110  6.1.  Microarray data analysis  110  6.2.  C A - M K K 9 - a c f f e c t e d genes  114  vi  List of Tables Table 3.1. Primers used for Q R T - P C R  70  Table 3.2. Gene ontology summaries o f C A - M K K 9 - a f f e c t e d genes  76  Table 3.3. Over-represented regulated genes  cw-acting elements among C A - M K K 9 - u p - and down79  Table 3.4. Octadecanoid pathway genes significantly altered by C A - M K K 9 activity  82  Table 3.5. Most significant C A - M K K 9 - a f f e c t e d genes  87  Table 6.1. Factorial design o f the microarray experiment  110  Table 6.2. C A - M K K 9 - a f f e c t e d genes  114  List of Figures Figure 1.1. Proposed M A P K cascades in Arabidopsis and tobacco  17  Figure 2.1. Construction of C A - M K K 9 and insertion into the D E X - i n d u c i b l e vector to produce p T A 7 0 0 2 - C A - M K K 9 - F L A G 37 Figure 2.2. Time-course o f C A - M K K 9 activation following D E X induction  40  Figure 2.3. Time-course o f cell death progression following C A - M K K 9 activation  42  Figure 2.4. C A - M K K 9 activity results in H2O2 accumulation  44  Figure 2.5. Time-course of ethylene production in D E X : C A - M K K 9 - F L A G plants  46  Figure 2.6. Ethylene production in D E X : C A - M K K 9 - F L A G and D E X : C A - M K K 4 - F L A G plants 47 Figure 2.7. A t M P K 6 protein and activity levels following C A - M K K 9 activation  49  Figure 2.8. C A - M K K 9 kinase activity on A t M P K 6 and A t M P K 3 in vitro  51  Figure 2.9. Time-course plants  of C A - M K K 9  induction in D E X : C A - M K K 9 - F L A G / m / ? & ( 5 52  Figure 2.10. A t M P K 6 is required for C A - M K K 9 - m e d i a t e d ethylene production  53  Figure 2.11. Lesion formation in D E X : C A - M K K 9 - F L A G / m p k 6 plant  54  Figure 2.12. M o d e l o f C A - M K K 9 participation in ethylene biosynthesis  57  Figure 3.1. G V G transcript levels in D E X : C A - M K K 9 - F L A G and E V transgenic lines. 73 Figure 3.2. Microarray experimental design  75  Figure 3.3. Putative M A P K phosphorylation sites in A B R E - b i n d i n g factors  80  Figure 3.4. The jasmonate biosynthesis pathway  81  Figure 3.5. Technical validation of J A biosynthesis genes  84  Figure 3.6. Q R T - P C R validation of down-regulated jasmonate biosynthesis predicted by microarray to be down-regulated by C A - M K K 9 Figure 3.7. Q R T - P C R validation of the most significant C A - M K K 9 - a f f e c t e d genes  genes 86 88  Figure 6.1. Effect of dye type, transgenic lines and time-point factors on gene expression differentials 113 Vlll  Abbreviations ABRE ACC ACO ACS AGI ANOVA ATP BLASTn bp  abscissic acid response element 1 -aminocyclopropane-1 -carboxy lie acid 1 -aminocyclopropane-1 -carboxy lie acid oxidase 1 -aminocyclopropane-1 -carboxy lie acid synthase Arabidopsis genome initiative analysis of variance Adenosine triphosphate basic local alignment sequence tool (nucleotide) base pair  BSA  bovine serum albumine  C(T)  Threshold cycle  CA cDNA  constitutively active Complementary deoxyribonucleic acid  MAPKK MAPKKK MeJA mRNA MS NADPH NahG  mitogen-activated protein kinase kinase mitogen-activated protein kinase kinase kinase methyljasmonate messenger ribonucleic acid Murashige and Skoog nicotamide adenine dinucleotide phosphate salicylate hydroxylase  NO  nitric oxide  0?"  superoxide anion  ONOO"  peroxynitrite  PCD  programmed cell death  PCR  polymerase chain reaction  ppm  part per million  Pro  proline  Cy3  cyanine 3 bihexanoic acid dye  p-value  Cy5  cyanine 5 bihexanoic acid dye  PVDF  polyvinylidene fluoride  DAB  3,3'-diaminobenzidine  PVPP  polyvinylpolypyrrolidone  DEPC  Diethylpyrocarbonate  QRT  quantitative real-time  DEX  Dexamethasone  RNA  ribonucleic acid  DNA  deoxyribonucleic acid  ROS  reactive oxygen species  dNTP  deoxynucleotide triphosphate  probability value  rpm  revolution per minute  Dithiothreitol  RT  reverse transcription  dTTP  deoxythymidine triphosphate  SA  salicylic acid  dUTP  deoxyuracil triphosphate  DTT  S-AdoMet  EDTA  ethylenediaminetetraacetic acid  SAR  EGTA  ethyleneglycol-6w(P-aminoethyl)N,N,N',N'-tetraacetic Acid  SDS  GO GST H 0 2  2  HEPES HR JA  gene ontology glutathione s-transferase Hydrogen peroxide N-(2-hydroxyethyl)piperazine-N;-(2ethanesulfonic acid) hypersensitive response  SDSPAGE Ser  S-adenosyl-methionine systemic acquired resistance sodium dodecyl sulfate sodium dodecyl sulfate polyacrylamide gel electrophoresis serine  SOD  superoxide dismutase  SSC  sodium chloride-sodium citrate solution  TBST  tris-buffered saline/Tween-20  jasmonic acid, jasmonate  Thr  threonine  kDa  kiloDalton  Tris  tris hydroxymethylaminoethane  log  Logarithm  Tyr  tyrosine  MAPK  mitogen-activated protein kinase  VSN  variance stabilization normalization  ix  Acknowledgments  Research contributions  The  research presented here was greatly enhanced by the contributions o f several  colleagues. In particular, Marcus Samuel has made this whole project possible by cloning A t M K K 9 , and by generating the D E X : C A - M K K 9 - F L A G plants. Marcus first identified the cell death and oxidative burst phenotypes induced by C A - M K K 9 , which I then explored and characterized in this thesis (Chapter 2, Figure 2.3). Marcus also generated the  original  DEX:CA-MKK4-FLAG  plants  (Chapter  2, Figure 2.6).  I wish  to  acknowledge the work o f Somrudee Sritubtim, who first screened and maintained the transgenic D E X : C A - M K K 9 - F L A G and E V lines used in this study, and whose careful observations first pointed to a possible role o f A t M K K 9 in ethylene biosynthesis (Chapter 2, Figure 2.5). In addition, I thank Jin Suk Lee, who was entirely responsible for the cloning and conversion into kinase-inactive form o f M P K 3  1  and M P K 6 , and for the 1  purification o f all the recombinant proteins used for my in vitro kinase assays (Chapter 2, Figure 2.8). Finally, R i c k White, a statistical consultant for Genome B C , designed and conducted the four-way A N O V A used for the analysis o f my microarray results (Chapter 3).  Material contributions  I would like to thank D r Y u e l i n Zhang (Botany Department, U B C ) for the generous gift of the  mpk6 knock-out seeds, which allowed me to generate the D E X : C A - M K K 9 -  ¥LAG/mpk6 double mutant (Chapter 2). I would also like to thank the Genome B C funded  Treenomix project for allowing me to use their Arabidopsis full genome 70-mer  oligo microarrays, and their facilities for array hybridization and analysis. Mattheus provided expert  advice and technical support  throughout  Nathalie  those  array  experiments.  x  Support and guidance  This thesis, which represents by far my greatest scientific achievement, would not be as it is today without the encouragement, guidance and inspiration offered by many people. In particular, sincere thanks are due to Brian Ellis, my research supervisor, for sharing his knowledge and passion for biology, and for giving more resources and freedom than most Masters students get to have. I am indebted as well to my colleagues o f the Ellis lab, Hardy H a l l , Greg Lampard, A l e x Lane, Jin Suk Lee, Marcus Samuel, Somrudee Sritubtim and A n k i t Walia, whose technical expertise and critical inputs have greatly improved my work. I would also like to acknowledge the Botany Department, which, through the approachability and kindness of its members, and the quality and originality o f its curriculum, has opened m y mind to new fields and ideas i n science. O n that same note, I wish to thank all the good friends I have made among the Botany graduate students and postdocs, without whom my time in Vancouver would certainly not have been so much fun. M a n y thanks as well to my close family and friends, who have accompanied and supported me throughout my Master's program: Vincent, my life partner, for always being there for me, and whose support and care in the difficult times were invaluable. I also thank my father, Daniel, for teaching me curiosity, perseverance and for passing on the science bug. Finally, I wish to dedicate this thesis to the memory of my mother, Paulette, who was an inspiration for me throughout my life, and whom I miss everyday.  xi  1.  General Introduction  1.1.  Programmed cell death in plants  1.1.1. Morphological and biochemical characteristics o f P C D  Programmed cell death ( P C D ) is a genetically determined process that functions to remove harmful or redundant cells during development or in response to environmental cues. P C D is most well studied in animal systems, but is now well established as playing major roles in the plant life cycle as well.  Plant P C D , both development- and stress-induced, shares physiological and metabolic features with apoptosis, a well-studied form o f P C D in animals. A t the cellular level, apoptosis is characterized by condensation o f the chromatin, fragmentation o f the D N A , shrinkage o f the cytoplasm, and blebbing o f the plasma membrane into so calledapoptotic bodies. Likewise, plant P C D has been found to involve the condensation and shrinkage o f the cytoplasm, as well as the degradation o f genomic D N A by nucleases and the formation o f DNA-containing bodies (Hoeberichts and Woltering, 2003). Moreover, in animals, apoptosis is mainly associated with the activation o f highly specific proteases called caspases, which are responsible for the ordered dismantlement o f the metabolism that eventually kills the cell (Lam et al., 2001; Hoeberichts and Woltering, 2003). Inhibitor studies have suggested that caspase activity may also be important for plant P C D (Hoeberichts and Woltering, 2003), and one recent study described the purification and characterization o f a caspase-like protein involved in P C D in tobacco (Chichkova et  1  al., 2004). Finally, metazoan apoptosis is also associated with the release o f so-called cell-death activators, triggered by disruption o f mitochondrial outer membrane integrity, a process which is hypothesized to occur in plants as well, since plant P C D has been linked  to changes  in mitochondrial membrane  permeability (Lam et al., 2001;  Hoeberichts and Woltering, 2003).  1.1.2. P C D in plant development  Several plant developmental processes involve the participation o f P C D . For example, P C D is essential to the formation o f the endosperm, the development o f both phloem and xylem tissue, and the formation o f epidermal trichomes, and is also involved in the processes o f pollen tube elongation and plant senescence (Greenberg, 1996; Rao and Davis, 2001). Senescence is the most extensively studied form o f developmental P C D . Defined as the final stage o f leaf development, senescence is an active process where nutrients from the leaves are relocated to other actively growing parts o f the plant, such as the reproductive organs (Lohman et al., 1994; Weaver et al., 1998). Senescence is characterized by a decline in photosynthetic activity in the affected tissue, and is accompanied by the loss o f chlorophyll, and increased protein degradation and lipid peroxidation (Miller et al., 1999; Quirino et al., 2000). Under normal conditions, senescence is a constant and predictable process that occurs in an age-dependent manner. However, senescence can also be accelerated by a number o f stimuli, including drought, darkness and acute ozone treatment (Weaver et a l , 1998; M i l l e r et al., 1999).  2  1.1.3. P C D in stress responses  P C D can occur during plant responses to both abiotic and biotic stresses. For example, P C D has been reported in plants challenged with cold, U V , and ozone (Koukalova et al., 1997; Danon and Gallois, 1998; Rao and Davis, 1999) and is proposed to serve as a means o f restricting the spread o f pathogens such as bacteria, fungi and viruses (Greenberg, 1996).  In particular, P C D is an important part o f the hypersensitive response ( H R ) , a form o f rapidly induced disease resistance triggered by specific pathogens on a gene-for-gene basis. It involves the expression o f a range of defense molecules, as well as the death o f cells at and surrounding the infection site (Heath, 2000). The cell death component o f the H R aims at preventing the pathogen from acquiring nutrients and water, thus resulting in its containment and subsequent death (Beers and M c D o w e l l , 2001). P C D during the H R also serves a signaling purpose, triggering defense responses in neighboring tissue and activating systemic acquired resistance in the whole plant (Heath, 2000). The H R occurs during  a plant's  interaction  with  an  incompatible or  avirulent  pathogen.  This  incompatibility usually implies that the plant possesses a dominant R gene whose activity allows the plant cell to recognize and respond to the presence o f an avr gene in the pathogen. This recognition event is most well studied in plants responding to biotrophic fungal pathogens, or bacterial pathogens, and these studies have shown that there is considerable variation i n the timing, the signaling events and the phenotype o f the H R , both among plant species, and depending on the pathogen or elicitor involved (Heath, 2000). 3  1.2.  Signaling events controlling PCD  1.2.1. Experimental models i n the study o f P C D signaling pathways  The onset and spread o f P C D is controlled by complex signaling networks. In recent years, these signaling events have mainly been studied in the context o f either senescence or the H R , where P C D is known to bear structural and genetic similarities in both processes (Quirino et al., 2000; Rao and Davis, 2001). Moreover, research in the P C D field has been facilitated by the use o f simple experimental models, which combine the  use o f model plants such as Arabidopsis thaliana (Arabidopsis), Lycopersicon  esculentum (tomato) and Nicotiana tabacum (tobacco), either as whole plants or as cell cultures, with simplified P C D contexts. Stresses commonly employed i n these studies are ozone, fungal toxins and bacterial elicitors. In particular, ozone stress has emerged as a simple and efficient tool for studying P C D . It is believed that ozone reacts with various molecules in the cell's extracellular space, generating an oxidative burst which mimics the initial events o f pathogen infection leading to the H R (Rao and Davis, 2001; Overmyer et al., 2003). Moreover, it has been suggested that l o w levels o f ozone trigger premature and accelerated senescence (Pell et al., 1997). Altogether, research on these models has defined many o f the signaling events regulating P C D . It appears that reactive oxygen species, salicylic acid, jasmonates and ethylene, are particularly important global regulators i n the complex network o f events that orchestrate the onset, propagation and containment o f P C D .  4  1.2.2. Reactive oxygen species and nitric oxide in P C D  Although some exceptions exist, the inter- and intra-cellular production o f reactive oxygen species (ROS), called the oxidative burst, is considered as a hallmark o f plant P C D (Heath, 2000). During plant defense, the oxidative burst is typically biphasic. The first phase, which occurs early and transiently, and is believed to be nonspecific as to the type o f pathogen, forms part o f a general stress response o f the plant. The second phase of R O S accumulation, on the other hand, occurs later, is long-lasting, and appears to be specific to the H R response (Lamb and Dixon, 1997).  The most common forms o f R O S associated with the stress-induced oxidative burst include hydrogen peroxide (H2O2) and superoxide anion radicals (O2""). The production of R O S during the oxidative burst is thought to arise from the activity o f several enzymes,  including plasma  membrane  NADPH-oxidases,  cell  wall  peroxidases,  apoplastic amine oxidase, as well as from the mitochondrial electron transport chain (Lam et al., 2001; Rao and Davis, 2001; Overmyer et al., 2003). In particular, the role o f a membrane-bound N A D P H oxidase complex sharing homology to mammalian enzymes, is well established to participate in the conversion o f O2 into O2'" during plant defense (Torres et al., 2002; Yoshioka et al., 2003). The expression o f plant genes coding for respiratory burst oxidase homologues ( R B O H ) is induced by HR-inducing elicitors. Moreover, both i n tobacco and Arabidopsis, the phenotype o f plants that have reduced expression o f R B O H genes reveals that these enzymes play crucial roles in the H R triggered by pathogens (Torres et al., 2002; Yoshioka et al., 2003). In Arabidopsis, an  atrbohD/F double-mutant displayed a major reduction in H2O2 accumulation following 5  infection by avirulent bacteria, and reduced P C D (Torres et al., 2002). Interestingly, knocking out either AtrbohD or AtrbohF alone does not have the same effect on pathogen-induced H R , suggesting that these enzymes interact in a complex way to modulate the onset o f P C D (Torres et al., 2002). A recent study indicates that AtrbohD can also negatively regulate P C D , as the of the runaway cell death mutant,  atrbohD mutant accentuates the severity o f P C D  Isdl (Torres et al., 2005).  There is controversy regarding the relative importance o f CV" and H2O2 in plant P C D . H2O2 is the R O S most commonly found associated with plant cell responses to pathogens, while ozone studies rather point towards CV" as the key signal for induction o f P C D (Levine et al., 1994; Overmyer et a l , 2003). C V " does not diffuse  across  membranes, so it is likely to accumulate only briefly in the apoplast before it is converted to the more stable and diffusible H2O2 (Lamb and D i x o n , 1997). The conversion o f CV" into H2O2 can either occur spontaneously or be catalyzed by a superoxide dismutase (SOD) (Torres et al., 2002). It is possible that differences in the detected levels o f these R O S among plant species reflect the rate at which O2'" is dismutated into H2O2 (Lamb and Dixon, 1997).  The biological effects o f R O S can be potentiated by nitric oxide ( N O ) , which also plays a crucial role i n plant P C D (Heath, 2000). It has been suggested that P C D is driven not so much by the concentration o f individual R O S , as by a fine balance between O2'", H2O2, and nitric oxide (NO) levels (Delledonne et al., 2001). According to this model, O2'" is formed first, and then dismutated spontaneously or catalytically into H2O2. N O , also  6  produced upon pathogen infection, can either react with O2" to form ONOO", which unlike in animals is harmless to plants, or it can interact with H2O2 to activate P C D . In this model, i f the N O / O2'" /H2O2 ratio favours either o f the first two molecules, the formation o f O N O O " is facilitated. However, the onset o f H R is associated with an increase in S O D transcription, resulting in the increased conversion o f superoxide anion to H2O2. This enhanced accumulation o f H2O2 would promote the synergistic action o f H 0 2  2  and N O on stimulating P C D (Delledonne et a l , 2001).  The regulation o f the oxidative burst itself involves multiple signaling events, including G-protein signaling, kinase activity and ion fluxes across the plasma membrane (Lamb and Dixon, 1997). In particular, C a  influxes are suggested to mediate the activity o f the  N A D P H oxidase complex, perhaps through the activity o f a calcium-dependent protein kinase (Xing et al., 1997). In fact, kinase inhibitor studies have also revealed that kinase activity is necessary both for H2O2 accumulation and for the P C D subsequently induced during plant defense (Lamb and Dixon, 1997). In particular, the oxidative burst triggered by ozone is associated with increased activity o f mitogen-activated protein kinases ( M A P K s ) (Samuel et al., 2000).  1.2.3. Salicylic acid: a positive regulator o f P C D  The phenylpropanoid metabolite, salicylic acid ( S A ) , is a well-established player in plant defense against pathogens, and particularly during the H R . S A is considered a positive regulator o f P C D , since a number o f 'lesion mimic' mutants, which display spontaneous lesion formation, can be rescued when cellular S A is depleted by ectopic expression of 7  the bacterial enzyme, salicylate hydroxylase (NahG) (Lorrain et al., 2003). The Arabidopsis ecotype, C v i - 0 , which is hypersensitive to ozone relative to the 'Columbia' ecotype, also hyperaccumulates S A (Rao and Davis, 1999). Exogenous S A application has been shown to be associated with increased H2O2 production, possibly by stimulating S O D activity (Rao et a l , 1997). Therefore S A is thought to cooperate with R O S , perhaps in a positive feedback loop, to induce the onset o f P C D . Furthermore, mutating the isochorismate synthase gene required for S A biosynthesis, EDS 16, suppresses the runaway cell death phenotype o f the  Isdl mutant, indicating that S A is also required for  the propagation o f P C D (Torres et al., 2005).  1.2.4. Jasmonates: negative regulators o f P C D  Jasmonic acid (JA) and its methyl-ester (MeJA) are the final products o f the cyclization branch o f the octadecanoid pathway. The jasmonates, as well other long-chain fatty acidderived intermediates and derivatives, commonly referred to as octadecanoids, are wellcharacterized signals operating within plant responses to biotic and abiotic stresses, including wounding, insect herbivory, pathogen attack and elevated ozone levels (Devoto and Turner, 2005). Jasmonates are thought to act as negative regulators o f P C D (Rao and Davis, 2001; Overmyer et al., 2003), since treatment with M e J A preceding ozone exposure blocks the spread o f P C D lesions in both tobacco and Arabidopsis Cvi-0 plants (Orvar et al., 1997; Rao et al., 2000). Moreover, the JA-insensitive mutant jar-1 displays aberrant lesion spreading in response to ozone, consistent with a role for J A in the containment o f P C D to a limited number of cells (Overmyer et al., 2000).  8  1.3.  Ethylene in stress responses and PCD  1.3.1.  Ethylene is a major signaling molecule in stress responses  Ethylene  is a  gaseous  two-carbon  molecule that plays  central  roles  in many  developmental processes such seed germination, root hair formation, abscission, and senescence.  Ethylene also participates in responses to abiotic stresses that include  wounding and hypoxia, and in plant defenses against pathogens (Beers and M c D o w e l l , 2001; Wang et al., 2002), and not surprisingly, is the hormone the most ubiquitously involved in plant P C D (Greenberg, 1996; Beers and M c D o w e l l , 2001).  In Arabidopsis, the ethylene-overproducing mutants,  etol and eto3, show increased  lesion formation in response to ozone, and this effect can be blocked by inhibitors o f ethylene biosynthesis (Rao et al., 2002). Furthermore, inhibitor studies in tomato have shown that both ethylene  biosynthesis and ethylene perception are required for  development o f an ozone-induced oxidative burst and subsequent P C D (Moeder et al., 2002).  Since ethylene  production co-localizes with,  and  is required  for, H2O2  accumulation, ethylene might be involved in directly regulating R O S production (Moeder et al., 2002). Our understanding o f the role o f ethylene in P C D has been further refined by the results o f crossing an ozone-sensitive mutant,  radical-induced cell death 1 (rcdl),  with the ethylene-insensitive mutant, einl. The phenotype o f the double mutant suggests that, while ethylene signaling is not required for the initiation o f lesions during ozone treatment, it is necessary for the subsequent amplification o f P C D once the ozone treatment ends (Overmyer et al., 2000). Altogether, those data indicate that ethylene is a  9  positive regulator o f P C D and that it participates, along with R O S , in the propagation o f P C D (Overmyer et al., 2003).  1.3.2. Ethylene biosynthesis and its regulation during stress responses  The biochemical reactions leading to ethylene biosynthesis are well understood, as'are the enzymes catalyzing these reactions (Wang et al., 2002). The biosynthesis o f ethylene starts  with  the  conversion  of  S-adenosyl-methionine  (S-AdoMet)  into  1-  aminocyclopropane-1-carboxylie acid ( A C C ) by the enzyme A C C synthase ( A C S ) . A C C is then oxidized by a second enzyme, A C C oxidase ( A C O ) , to form ethylene.  The  conversion o f S-Ado-Met to A C C by A C S is considered the rate-limiting step in ethylene biosynthesis, due to the short half-life o f the A C S protein and tight regulation o f A C S activity (Tatsuki and M o r i , 2001; Wang et al., 2002; Tsuchisaka and Theologis, 2004). In Arabidopsis, the A C S gene family codes for nine A C S proteins, eight o f which are functional. The A C S holoenzyme is also dimeric, which provides additional opportunities for functional diversity (Chae and Kieber, 2005). The A C O gene family, on the other hand, has only three putative members in Arabidopsis. The expression o f the A C S genes in Arabidopsis is differentially regulated by developmental and stress stimuli (Tsuchisaka and Theologis, 2004). In tobacco, ozone treatment induces a biphasic increase in both A C S and A C O transcripts, which is correlated with a burst o f ethylene (Moeder et al., 2002). Moreover, supporting the hypothesis that ethylene is closely involved i n the production o f R O S , the expression o f A C O in tomato, as revealed in A C O promoter-P-glucuronidase fusion plants, co-localizes with the sites o f accumulation o f H2O2 (Moeder et al., 2002). In Arabidopsis, lesion 10  formation in the rcdl mutant is preceded by increased levels o f A C S and A C O transcripts ( A C S 6 and A C O l , respectively) (Overmyer et al., 2000), a pattern that was also observed in tomato, where ozone treatment triggered a rapid increase i n A C O m R N A , followed by a smaller increase in A C S 2 transcription (Tuomainen et al., 1997).  However, the up-regulation o f A C S and A C O genes cannot always fully account for the rapid ethylene burst triggered in response to certain stimuli ( K i m et al., 2003). The posttranslational control o f A C S enzymes, in particular, appears to also play an important role in stress-induced ethylene production. Several indirect lines o f evidence have pointed specifically to a role for protein phosphorylation in modulating A C S activity. For instance, the addition o f a protein kinase inhibitor rapidly blocked the increased activity of A C S triggered by fungal elicitors and by ozone in tomato, whereas the addition o f a phosphatase inhibitor could, by itself, stimulate A C S activity (Spanu et al., 1994; Tuomainen et al., 1997). Interestingly, the phosphatase inhibitor had no effect on A C S activity when added simultaneously with a protein synthesis inhibitor, suggesting that protein phosphorylation and dephosphorylation influence the turnover o f A C S rather than its catalytic activity (Spanu et al., 1994). A subsequent study directly demonstrated that tomato A C S 2 is phosphorylated at its C-terminus by a wound-induced calcium-dependent protein kinase (Tatsuki and M o r i , 2001). Further support for the idea that phosphorylation helps regulate A C S turnover came from the discovery that a protein called E T O l interacts with the A C S 5 C-terminus, thus blocking its modification and presumably promoting the degradation o f A C S 5 by a proteasome-dependent  pathway (Wang et al.,  2004). Bringing these pieces o f evidence together, L i u et al. (2004) established that a  11  specific M A P K , A t M P K 6 , could increase the stability, and thereby the activity, o f two Arabidopsis A C S isoforms, A C S 2 and A C S 6 , by phosphorylating three conserved serine residues at their C-terminus. Based on these results, the authors proposed a model whereby biotic and abiotic stresses trigger the activation o f a M A P K cascade composed of an as yet unknown M A P K K K , which acts upon A t M K K 4 and/or A t M K K 5 , which in turn act upon A t M P K 6 .  Activated A t M P K 6  then phosphorylates  ACS6,  thereby  preventing it from being targeted for degradation, and allowing increased conversion o f S-AdoMet to A C C . The A C C would be further converted into ethylene by A C O , resulting in the onset o f the ethylene response ( L i u and Zhang, 2004).  1.3.3. Ethylene crosstalk with other hormones during P C D  There is evidence for both synergistic and antagonistic interactions between ethylene and S A . For instance, the Arabidopsis mutants,  etol and eto3, hyperaccumulate ethylene and  have a higher S A content than the wild-type plants (Rao et al., 2002). Moreover, analyses of the S A signaling mutant,  npr-1, as well as of the SA-depleted transgenic NahG plants,  both revealed that the ethylene burst induced by ozone is S A signaling-dependent (Rao et al., 2002). This suggests that S A plays a role in both the initiation o f lesions and also in their propagation, perhaps by controlling ethylene production (Rao et al., 2002). In contrast with this, ozone-treated transgenic tobacco plants over-expressing the A t M P K 6 orthologue, N t S I P K , were found to be blocked in ozone-induced S A accumulation, but to produce elevated amounts o f ethylene (Samuel et al., 2005). These conflicting pieces o f evidence may reflect species differences, or additional levels o f complexity in the crosstalk between the two hormones. 12  Jasmonates also modulate the ethylene pathway. Analyses o f  jarl and ein2 mutants in  Arabidopsis have revealed that the J A pathway negatively regulates ethylene signaling during ozone-induced P C D . Blocking ethylene perception in the  ein2 mutant background,  or by addition o f the ethylene-receptor agonist norbornadiene, both reduced ozoneinduced lesion formation in the jarl mutant (Tuominen et al., 2004). O n the other hand, the  jarl mutant did not display increased ethylene production i n response to ozone,  although it did show increased ethylene-mediated jasmonates  suppress  the  (Tuominen et al., 2004).  ethylene  pathway  gene expression, indicating that  downstream  o f ethylene  biosynthesis  Since J A treatment induces the transcription o f ethylene  receptors, which themselves act as negative regulators until they bind the hormone, it has been suggested that J A could negatively regulate ethylene by decreasing the cell's sensitivity to the latter (Overmyer et al., 2003). It is thus possible that the lesion containment role o f J A is executed by reducing the ethylene signal and the ethylenedependent R O S production required for the propagation o f P C D (Overmyer et al., 2003).  1.4.  Mitogen-activated protein kinase cascades and signal transduction  Eukaryotes have developed sophisticated signal transduction strategies to quickly and precisely transmit external stimuli to the interior o f cells. One o f the most efficient signal transduction methods involves the phosphorylation o f proteins, mediated by kinases, and their dephosphorylation by phosphatases.  13  Mitogen-activated protein kinases ( M A P K ) cascades are ubiquitous players in eukaryotic signal transduction pathways, but they are best characterized in mammalian and yeast models. Typically, M A P K cascades are composed o f a M A P K kinase kinase ( M A P K K K ) phosphorylating a M A P K kinase ( M A P K K ) , which in turn phosphorylates a M A P K . M A P K K K s are activated either by M A P K K K kinases ( M A P K K K K ) or through an interaction with GTP-binding proteins (Widmann et al., 1999). They activate M A P K K s by  phosphorylating two serine/threonine  residues  in the M A P K K  activation loop  (Ser/Thr-(X)3. -Ser/Thr). M A P K K s , in turn, are dual-specificity kinases that recognize 5  and dually phosphorylate  a conserved threonine/tyrosine  motif (Thr-X-Tyr) in the  activation loop o f M A P K s (Widmann et al., 1999; Zhang and Klessig, 2001). Finally, MAPKs  are proline-directed serine/threonine  kinases that target one or more sites  distinguished by the consensus sequence Ser/Thr-Pro i n their target proteins (Treisman, 1996; Widmann et al., 1999).  In mammalian cells, M A P K cascades are involved in various cell processes, including differentiation, proliferation, stress responses and apoptosis. Mammalian M A P K s have been divided into three families: the E R K family, activated by growth factors such as E G F , the J N K / S A P K  (Jun N-terminal kinase/ stress-activated protein kinase) family,  which responds to stress and inflammatory cytokines, and the p38/Hog family, activated by osmotic stress, endotoxins and cytokines (Morris, 2001). These M A P K s activate a wide range o f targets, which include transcription factors, protein kinases, phospholipases and cytoskeletal proteins (Widmann et al., 1999).  14  With multiple M A P K cascade components that each can interact with multiple targets, it is striking that M A P K cascades can achieve specificity i n their downstream targets following activation by a given stimulus. In mammalian and yeast systems, specificity of M A P K modules is achieved through the formation o f 'signalosomes', protein complexes that include the kinases themselves, as well as scaffold proteins (Morris, 2001).  1.4.1. M A P K cascades in plants  Although they are far from being as well characterized as in yeast and mammalian systems, increasing numbers o f studies indicate that M A P K signaling cascades also play critical roles in plants. For example, M A P K  cascades have been implicated in  cytokinesis, cell death, differentiation and stress responses (Tena et al., 2001; Jonak et al., 2002).  In Arabidopsis, genomic analyses reveal that there are 20 M A P K s , 10 M A P K K s and 80 putative M A P K K K s . Based on structural motifs and sequence similarities, the plant M A P K s , which all belong to the E R K family, have been divided into four groups ( A - D ) . Except for members  o f the most distant group D , which carry a Thr-Asp-Tyr  phosphorylation motif, all plant M A P K s are activated on a Thr-Glu-Tyr motif. Plant M A P K K s have also been divided into four groups ( A - D ) . In contrast to mammalian M A P K K s where only three amino acids separate the conserved serine/threonine residues of the activation motif, plant M A P K K s have the consensus sequence Ser/Thr. Plant M A P K K K s  Ser/Thr-XXXXX-  are divided into three main groups. Group A M A P K K K s are  15  most similar to characterized yeast and animal M A P K K K s , while groups B and C are more closely related to R A F kinases (Ichimura et al., 2002).  Although the high numbers, as well as the diversity o f plant M A P kinase cascade components suggest that they may be extensively involved in plant signal transduction, there is relatively little concrete information about functional interactions between the various M A P K K K s , M A P K K s and M A P K s .  The difficulty in unravelling biologically  relevant interactions between M A P K cascades components,  in planta has a number o f  origins, including the transient nature o f M A P K K K / M A P K K / M A P K interactions, as well as functional redundancy and crosstalk between M A P K modules. Evidence so far for plant M A P K K K / M A P K K / M A P K interactions comes mainly from in vitro, yeast twohybrid and transgenic approaches. For example, interactions were detected between the Arabidopsis M A P K K K , A t M E K K l , the M A P K K , A t M K K 2 , and the M A P K , A t M P K 4 , using yeast two-hybrid and yeast M A P K mutant complementation (Ichimura et al., 1998; Mizoguchi et al., 1998). A similar approach was used in tobacco to demonstrate that the M A P K K K , N t N P K l , can activate the M A P K K , N t M E K l , which in turns phosphorylates the M A P K , N f N T F 6 . The validity o f these predicted interactions was further supported by  in planta evidence for the co-activation o f these M A P K cascade components, as well  as by the analysis o f immuno-complexes following the over-expression o f N t M E K l - and NtNTF6-fusion proteins in B Y - 2 tobacco cells (Nishihama et al., 2001; Soyano et al., 2003). The latter approach, based on the expression o f epitope-tagged proteins, was used to show that, in Arabidopsis protoplasts, A t M E K K l phosphorylates A t M K K 5 , and that A t M K K 4 and A t M K K 5 could both activate A t M P K 3 and A t M P K 6 (Asai et al., 2002).  16  The role for A t M K K 4 / A t M K K 5 as upstream activators o f A t M P K 3 / A t M P K 6 is further supported by studies on their tobacco orthologues, the M A P K K , N f M E K 2 , and the M A P K s , NtSEPK and N t W T P K , respectively. N t M E K 2 was identified as acting upstream o f N t S I P K and NtWTPK, based on the co-activation o f N t M E K 2 and N t S J P K / N t W T P K in suspension cells treated with a fungal elicitor, and the activation o f N t S I P K / N t W T P K in  planta  by a transiently expressed constitutively active N t M E K 2 (Yang et al., 2001b).  M A P K cascades characterized so far in Arabidopsis and tobacco are summarized i n Figure 1.1. j Arabidopsis  tobacco  ^ Arabidopsis  tobacco  Arabidopsis  tobacco (^NtNPK?)  AtMKK6 ) ( NtMEKl  MAPKK  Target  ^tMPM^  (NtMPKj)  MAPK  NtWIF  ACS6/ ACS2  (NtNTR^  WRKY1  Figure 1.1. Proposed MAPK cascades in Arabidopsis and tobacco. Each column corresponds to a characterized Arabidopsis MAPK cascade (black), and to the orthologous cascade in tobacco (grey). Full arrows indicate interactions supported by in planta evidence. Dotted arrows indicate interactions supported by in vitro and/or yeast expression evidence.  17  1.4.2. M A P K signaling in plant stress responses  Inhibitor studies provided the earliest evidence o f a role for kinase activity during plant stress responses (Viard et al., 1994; Suzuki and Shinshi, 1995). Numerous studies have since reported changes in the m R N A levels and/or in enzymatic activity o f M A P K s in response to various stresses (Seo et al., 1995; Jonak et al., 1996; Bogre et al., 1997; Zhang and Klessig, 1998b). In the past few years, the identity o f several o f these M A P K s has been established, and the study o f loss- and gain-of-function mutants o f specific M A P K cascade components has increased our understanding o f their roles in stress responses.  Given the estimated number o f M A P K s in Arabidopsis, it is striking that only a small number o f M A P K s appear ubiquitously involved in plant stress responses.  Three  Arabidopsis M A P K s , as well as their respective tobacco homologues (Figure 1.1), appear to play particularly important roles in stress signaling: A t M P K 6 (NtSIPK), and A t M P K 3 ( N t W I P K ) , which belong to the Group A M A P K s , and A t M P K 4 ( N t M P K 4 ) , which is a member o f the Group B M A P K s (Ichimura et al., 2002).  In Arabidopsis, A t M P K 6 was found to be activated by bacterial and fungal elicitors (Nuhse et al., 2000).  Furthermore,, loss-of-function studies indicate that this particular  M A P K is essential for resistance to virulent and avirulent pathogens (Menke et al., 2004). A s discussed above, there is now convincing evidence that one o f the functions o f A t M P K 6 is to modulate ethylene production by stabilizing A C S isoforms during stress responses ( L i u and Zhang, 2004).  Interestingly, A t M P K 6 is often co-activated with  18  A t M P K 3 . For instance, oxidative and hyperosmotic stresses activate both A t M P K 6 and A t M P K 3 (Kovtun et al., 2000; Droillard et al., 2002; Rentel et al., 2004). A t M P K 6 and A t M P K 3 are also co-activated during ozone treatment, with A t M P K 6 displaying transient activity, while A t M P K 3 shows delayed but sustained activity (Ahlfors et al., 2004). Interestingly, ozone exposure results in the translocation o f A t M P K 3 and A t M P K 6 into the nucleus, suggesting that they might modulate the activity o f transcription factors (Ahlfors et al., 2004). The M A P K K s , A t M K K 4 and A t M K K 5 , can both activate A t M P K 3 and A t M P K 6 , suggesting a role for these M A P K K s in co-regulating the two kinases (Figure 1.1) (Asai et al., 2002).  A t M P K 4 plays a negative role in the regulation o f systemic acquired resistance ( S A R ) , perhaps by impacting S A and J A signaling. This model is supported by the phenotype o f a knock-out mutant o f the kinase, which displays constitutive S A R and resistance to virulent pathogens, as well as elevated S A levels (Petersen et al., 2000).  AtMPK4,  A t M P K 6 and A t M P K 3 are all reported to be co-activated by hypoosmolarity, as well as by the bacterial elicitor, flagellin (Droillard et al., 2004). There is also evidence for activation o f A t M P K 4 solely with A t M P K 6 . For instance, both these M A P K s are transiently activated by the bacterial elicitor hairpin, independently o f the subsequent oxidative burst (Desikan et al., 2001). Interestingly, the same study suggested that A t M P K 4 plays a particularly important role in hairpin-induced P C D . In a similar manner, another study demonstrated that these two same M A P K s are also activated by a number of abiotic stresses including low temperature, dehydration, touch, hyperosmolarity and wounding (Ichimura et al., 1998). A role for A t M P K 6 and A t M P K 4 in cold  and salt  19  stress response is further supported by a more recent paper showing that their activation by A t M K K 2 results in increased freezing and salt tolerance (Teige et al., 2004), although the underlying mechanism remains unknown.  The tobacco orthologue o f A t M P K 6 is N t S I P K , which was originally identified as a S A induced protein kinase (Zhang and Klessig, 1997). N t S I P K is rap idly activated in cell cultures and whole tobacco plants following treatment with the tobacco mosaic virus, fungal elicitor or mechanical stress (Zhang and Klessig, 1998a; Romeis et al., 1999). The orthologue o f A t M P K 3 , N t W I P K , was first characterized as a wound-inducible protein kinase  whose  transient  activity is required  for  wound-induced  J A and  MeJA  accumulation (Seo et al., 1995; Seo et a l , 1999), although N t W I P K is also activated by certain fungal elicitors (Romeis et al., 1999; Zhang et al., 2000). However, in contrast to N t S I P K , whose increased activity appears solely due to post-translational mechanisms, N t W I P K activation is typically preceded by increases in both m R N A and protein levels (Zhang and Klessig, 1998b; Zhang et al., 2000). Interestingly, N t W I P K activation by tobacco mosaic virus requires the expression o f the resistance gene N , suggesting that the kinase plays a role in the plant's defense strategy (Zhang and Klessig, 1998b). Ozone treatment, as well as external application o f R O S such as H2O2, also leads to the rapid and sustained activation o f N t S I P K , possibly along with N t W I P K (Samuel et al., 2000). A role for N t S I P K in ROS-mediated P C D is also indicated by the increased sensitivity to ozone conferred by both loss-of-function and gain-of-function mutants o f this kinase (Samuel and Ellis, 2002). In fact, the over-expression o f N t S I P K is sufficient to induce P C D (Zhang and L i u , 2001) and an increase in ethylene production ( K i m et al., 2003;  20  Samuel et al., 2005) similar to the behaviour o f its Arabidopsis orthologue, A t M P K 6 , . The over-expression o f N t M E K 2 , the upstream M A P K K o f N t S I P K and N t W I P K (Figure 1.1) (Yang et al., 2001a), leads to P C D , independently o f S A signaling (Yang et al., 2001b). Interestingly, this phenotype is dependent on the expression o f NtrbohB, a gene encoding  an  NADPH  oxidase  homologue,  suggesting  that  NtMEK2  and/or  N t S I P K / N t W I P K might play a role in inducing or controlling R O S production (Yoshioka et al., 2003).  The tobacco orthologue o f A t M P K 4 , N t M P K 4 , is activated by wounding concurrently with both N t S I P K and N t W I P K (Gomi et al., 2005). N t M P K 4 is required for wound/JAinduced gene expression and for lesion containment after ozone treatment, suggesting that N t M P K 4 plays a positive role in J A signaling. Conversely, NtMPK4-suppressed lines accumulate high amounts o f S A after ozone treatment, and N t M P K 4 expression has a negative effect on SA-induced gene expression, altogether suggesting that N t M P K 4 negatively regulates S A production. The M A P K K N t S I P K K activates N t M P K 4 , but is not able to activate N t S I P K / N t W I P K (Figure 1.1) (Gomi et al., 2005).  Overall, the ubiquitous roles o f A t M P K 3 , A t M P K 4 and A t M P K 6 , as well as o f their tobacco orthologues, in both biotic and abiotic stress responses suggest that the timing of activation o f the different kinases, and/or the co-activation o f other pathways is responsible for the final response specificity. Moreover, it appears that the co-activation of these M A P K s is due, in some cases, to the pattern o f joint activation by upstream M A P K K s , with A t M K K 4 / A t M K K 5 ( N t M E K 2 ) regulating the activation o f A t M P K 3 and  21  A t M P K 6 ( N t W I P K and N t S I P K ) , while A t M K K 2 activity results in the co-activation o f A t M P K 4 and A t M P K 6 (Figure 1.1).  1.4.3.  M A P kinase substrates  So far, only a few plant M A P K substrates have been established. One o f these is A C S 6 (and plausibly the highly related A C S 2 ) , an A C C synthase isoform discussed previously. After the phosphorylation activity o f A t M P K 6 on this protein was established  in vitro, it  was shown that the phosphorylation o f A C S 6 by A t M P K 6 increases the stability o f the enzyme and is necessary for AtMPK6-mediated ethylene accumulation  in planta (Figure  1.1) ( L i u and Zhang, 2004). Another confirmed M A P K substrate is M K S 1 , a protein o f unknown  function,  that  interacts  with  WRKY  transcription  factors  and whose  homologues in other species appear to be involved i n plant responses to pathogens. M K S 1 was shown to be phosphorylated by A t M P K 4 in vitro, a pattern which was corroborated  in planta by demonstration of physical interaction between the two proteins,  and by a marked reduction o f M K S 1 phosphorylation observed in an A f M P K 4 knock-out background (Figure 1.1) (Andreasson et al., 2005). In Ozyza sativa (rice), a transcription factor, O s E R E B P l , is activated by a M A P K , B W M K 1 . The D N A - b i n d i n g activity o f O s E R E B P l was shown to increase following phosphorylation by B W M K 1 , both in vitro and in transient expression assays i n Arabidopsis protoplasts (Cheong et al., 2003). In tobacco, a recent paper also reports that a novel transcription factor, N t W I F , is a substrate for N t W I P K  (Figure  1.1). The phosphorylating activity o f N t W I P K  on N t W I F ,  established by in vitro kinase assays, was corroborated by the increased transcription stimulating activity o f N t W I F when both proteins were transiently expressed in B Y 2 cells 22  (Yap et al., 2005). A l s o i n tobacco, the transcription factor N f W R K Y l was shown by yeast two hybrid and in vitro assays to be a putative substrate for N t S I P K (Figure 1.1) (Menke et al., 2005). Moreover, using a high-throughput microarray-based proteomic approach, several putative in vitro substrates o f A t M P K 3 and A t M P K 6 have recently been identified. They include transcription factors, transcription regulators, histones, ribosomal proteins and many others (Feilner et al., 2005). However, until these interactions are examined and confirmed  in planta, it is unclear whether they are  biologically significant.  1.4.4. Genetic approaches to the study o f plant M A P K cascades  1.4.4.1. Loss-of-function mutants The large number o f M A P K cascade components i n plants suggests that there might be redundancies in the functions o f several o f these proteins. A s a result, the loss o f a given M A P K may not lead to a visible phenotype. Nevertheless, loss-of-function approaches have, i n a few cases, provided insight into M A P K functions i n plants. For instance, examination o f a knock-out mutant of  AtMPK4, and o f an RNAi-silenced atmpk6 mutant,  revealed that both kinases play a role in disease resistance (Petersen et al., 2000; Menke et al., 2004). Moreover, loss-of-function mutants have shed light on the reciprocal influence o f certain M A P K s on each other's activity. For instance, silencing o f NtSIPK leads to the prolonged hyperactivation o f N t W I P K under ozone stress (Samuel and Ellis, 2002). Similarly, i n ozone-treated Arabidopsis plants, silencing AtMPK6 results i n the prolonged activity o f A t M P K 3 , while a knock-out mutant o f the latter displays prolonged activation o f A t M P K 6 (Miles et al., 2005). It is also noteworthy that loss-of-function 23  approaches are a powerful tool for revealing genetic interactions. Confirmation o f M A P K control over ethylene biosynthesis, for instance, relied critically on the use o f an  atmpk6  knock-out mutant ( L i u and Zhang, 2004).  1.4.4.2. Gain-of-function mutants A s M A P K cascade components are, in their native form, in an inactive state, their ectopic expression does not necessarily make it possible to study their activity in vivo. However, protein modifications by deletions or site-directed mutagenesis can mimic the activation state of certain kinases. M A P K K K s , for instance, can be made constitutively active by deletion o f their regulatory domain, which then allows investigation o f the identity o f their downstream M A P K K targets during transgenic expression in plants (Kovtun et al., 2000; Asai et al., 2002). Likewise, M A P K K s , normally activated by phosphorylation of a pair of serine/threonine residues in a conserved Ser/Thr-X(3-5)-Ser/Thr region o f their catalytic  domain,  are  routinely  made  constitutively active  by  converting  these  serine/threonine residues by site-directed mutagenesis to the acidic amino acids glutamate and aspartate, which mimics the conformation o f phosphorylated M A P K K s (Alessi et al., 1994; Mansour et a l , 1994; Wurgler-Murphy et al., 1997). This gain-of-function approach has been used in the study o f plant M A P K cascades, either to gain insight about the M A P K s activated by a M A P K K o f interest, or to study downstream and phenotypic impacts o f M A P K cascades (Desikan et al., 2001; Yang et al., 2001b; A s a i et al., 2002; K i m et al., 2003; Yoshioka et al., 2003; L i u and Zhang, 2004). Unfortunately, M A P K s themselves cannot be made constitutively active, presumably because changes in the  24  kinase three-dimensional structure brought about by phosphorylation o f the Thr-X-Tyr motif are too complex to be mimicked by simple amino acid changes.  1.4.4.3. Inducible gene expression systems The over-expression o f kinases using constitutive promoters can sometimes lead to severe growth defects, as well as cell death, making it difficult to study these proteins in a transgenic plant context (Chen et al., 2003). To overcome these difficulties, a number of regulated gene-expression systems have been developed for plant transformation. These include the dexamethasone-inducible system, the ethanol-inducible ale expression system and the estrogen receptor-based chemical-inducible system (Aoyama and Chua, 1997; Zuo et al., 2000; Roslan et al., 2001).  The dexamethasone-inducible system is based on the activity o f a chimeric transcription factor, G V G , comprising the D N A - b i n d i n g domain o f the yeast transcription factor G A L 4 , the transactivating domain o f the herpes viral protein V P 16, and the hormonebinding domain o f the rat glucocorticoid receptor, which results in nuclear translocation of the transcription factor upon binding to a glucocorticoid. When a transgenic plant expressing the  appropriate  construct  is treated with the  synthetic glucocorticoid  dexamethasone ( D E X ) , the D E X - G V G complex is transported into the nucleus. There, it binds to and activates a G A L 4 promoter. When the latter has been situated upstream o f a transgene o f interest, this activation induces transcription o f the transgene (Aoyama and Chua, 1997). The dexamethasone-inducible system is now commonly used to study the  25  activity o f M A P K s in plants ( K i m et al., 2003; Soyano et al., 2003; L i u and Zhang, 2004).  1.5.  Problem statement and thesis objectives  The relatively small number o f M A P K K s encoded in the Arabidopsis genome suggests that this particular class o f kinases acts as a point o f convergence within the plant's integration o f external stimuli and their transduction to elicit biological  responses.  Moreover, our ability to convert these enzymes to constitutively active forms through site-directed mutagenesis  makes  MAPKKs  the tools o f choice for studying the  downstream targets and phenotypes o f M A P K cascades. Use o f this gain-of-function approach as well as other strategies, has made it possible to gain insight into the biological function o f a number o f Arabidopsis M A P K K s (Asai et a l , 2002; Ren et al., 2002; Teige et  al., 2004). Nevertheless,  several other  characterized. In particular, the Group D M A P K K s ,  MAPKKs  remain to  be  which includes Arabidopsis  A t M K K 7 , A t M K O , A t M K K 9 and A t M K K l O , are largely unstudied so far (Ichimura et a l , 2002).  Our laboratory has successfully cloned A t M K K 9 and created a constitutively active ( C A ) form o f the protein. The C A version o f A t M K K 9 ( C A - M K K 9 ) , was placed under the control o f the D E X - i n d u c i b l e gene expression system, and stable transgenic Arabidopsis lines expressing D E X : C A - M K K 9 - F L A G were obtained. A preliminary analysis o f these  26  plants revealed that C A - M K K 9 could trigger lesion formation in plants, and it was thought that it might be somehow involved in ethylene biosynthesis.  Based on these preliminary results, the overall aim o f my M . S c . research was to gain a better understanding o f the function o f A t M K K 9 in Arabidopsis. More specifically, the following objectives were pursued: 1. To characterize the induction pattern o f C A - M K K 9 in D E X : C A - M K K 9 - F L A G plants, both at the m R N A and protein levels. 2.  To characterize the cell death phenotype induced by C A - M K K 9 , in the context of PCD.  3.  To investigate the role o f C A - M K K 9 in controlling ethylene production, and establish the relationship between my findings and the existing model o f M A P K control o f the ethylene biosynthesis pathway.  4.  To investigate  short-term transcriptional events resulting from  CA-MKK9  activity, using 70-mer oligomer microarrays.  27  2.  CA-MKK9 modulates ethylene biosynthesis  2.1.  Introduction  A n expression survey o f all Arabidopsis M A P K K s identified a so-far uncharacterized M A P K K , A t M K K 9 , whose expression profile was correlated with aging o f the rosette leaves, as well as with some stress responses (Ellis laboratory, unpublished data). Moreover,  the  Ellis  laboratory  recently  generated  transgenic  Arabidopsis plants  expressing an inducible gain-of-function mutant form o f A t M K K 9 ( C A - M K K 9 ) .  A  preliminary phenotypic analysis o f those transgenic plants revealed that the ectopic expression o f this kinase leads to the formation o f necrotic lesions in tobacco and Arabidopsis plants. These data have led me to explore the possible role o f A t M K K 9 programmed cell death ( P C D ) , a process playing critical roles in the plant's life cycle, including senescence and responses to abiotic and biotic stresses (Rao and Davis, 2001).  The onset o f P C D , which has been mainly studied in the context o f ozone and pathogen responses, is associated with a defined set o f genetic and metabolic events. A m o n g those, it is well established that reactive oxygen species (ROS) play a central role. The R O S most commonly associated with P C D in plants include hydrogen peroxide (H2O2) and superoxide anion radicals (O2'"), both o f which accumulate i n the apoplast in response to challenge by incompatible pathogens or by ozone.  28  Several studies have demonstrated a role for M A P K cascades in regulating P C D . In tobacco, elicitor-induced P C D is associated with the prolonged activation o f N t S I P K , as well as with the somewhat later activation o f N t W I P K (Zhang and Klessig, 1998b; Suzuki et al., 1999; Zhang et al., 2000). Ozone treatment was similarly shown to result in the activation o f N t S I P K in tobacco, and o f A t M P K 3 and A t M P K 6 in Arabidopsis (Samuel et al., 2000; Ahlfors et a l , 2004). M A P K cascades act both upstream and downstream o f R O S production. Evidence for an upstream role includes the participation of N t M E K 2 in upregulating a respiratory burst oxidase gene involved i n R O S production, as well as a role for A t M K K 4  and A t M K K 5  in inducing H2O2 accumulation and  promoting P C D in Arabidopsis (Ren et al., 2002; Yoshioka et al., 2003). O n the other hand, the fact that several M A P K are activated by oxidative stress indicate that M A P K cascades also act downstream o f R O S (Ichimura et al., 2000; Samuel et al., 2000; Desikan et al., 2001; Yuasa et al., 2001).  P C D is also tightly regulated by a complex interplay o f plant hormones, as reviewed earlier in Chapter 1. A m o n g those, ethylene is proposed to control the propagation of PCD  (Overmyer et al., 2003). Ethylene production, perception and/or signaling are  required for pathogen-induced P C D in tomato, and for both R O S accumulation and lesion formation following ozone treatment in Arabidopsis (Lund et al., 1998; Overmyer et al., 2000). Moreover, high levels o f ethylene biosynthesis are correlated with more extensive P C D following ozone treatment (Rao et al., 2002).  29  A biochemical link between M A P K signaling and ethylene biosynthesis was provided by a recent study that demonstrated that the stability o f rate-limiting enzymes i n the ethylene biosynthesis pathway is regulated by a M A P kinase cascade ( L i u and Zhang, 2004). According to this model, specific biotic and abiotic stresses trigger the activation o f a M A P K cascade composed o f an as yet unknown M A P K K K , which acts upon A t M K K 4 and/or  AtMKK5,  which  i n turn  act  upon  AtMPK6.  Activated  AtMPK6  then  phosphorylates the A C S isoform A C S 6 , thereby preventing it from being targeted for degradation, and allowing increased conversion o f S-AdoMet to A C C , which would be further converted into ethylene, resulting in the onset o f the ethylene response ( L i u and Zhang, 2004). Notably, the same group had previously reported that the inducible overexpression o f constitutively active ( C A ) versions o f A t M K K 4 and A t M K K 5 results in H2O2 accumulation and necrotic lesions in tobacco and Arabidopsis, supporting the hypothesis that high ethylene production could lead to uncontrolled propagation o f P C D (Ren et al., 2002).  In this chapter, I have characterized Arabidopsis transgenic plants expressing the gain-offunction version o f A t M K K 9 , C A - M K K 9 , under the control o f the  dexamethasone  (DEX)-inducible promoter. I have found that, as observed for C A - M K K 4 and C A - M K K 5 (Ren et al., 2002), C A - M K K 9 triggers a form o f P C D . In addition, using a combination of protein analyses, biochemical assays, and genetic approaches, I have demonstrated that C A - M K K 9 activates A t M P K 6 ,  causing a burst of ethylene release i n DEX-induced  plants. M y results also demonstrate that C A - M K K 9 - m e d i a t e d P C D occurs independently of A t M P K 6 activity, and o f the observed ethylene burst.  30  2.2.  Material and methods  2.2.1. Plant material  Arabidopsis seeds were vernalized for three days, sown on Ready-Earth planting medium, and grown under a 16:8 hours lightdark regime for nineteen to twenty-two days. DEX-inducible transgenic plants were induced by spraying the foliage uniformly with a solution 2 5 p M D E X and 0.015% Silwet-77.  2.2.2. R T - P C R analysis  R N A was extracted from frozen leaf tissue using an Rneasy Plant K i t (Quiagen). Total R N A (1 ug) was reverse-transcribed into c D N A with OligodT (Invitrogen) using R T Superscript II (Invitrogen), according to the manufacturer's instruction. The polymerase chain reaction ( P C R ) was performed in a 20pl reaction containing I X Jumpstart™ R E D T a q ™ R e a d y M i x ™ P C R Reaction M i x (Sigma), l p l c D N A (the equivalent o f 50 ng R N A ) and 0.75 u M o f each forward (F) and reverse (R) primers. A 502 bp region of the C A - M K K 9 - F L A G transcript was amplified using the following primers: I n M K K 9 F (5' CGCCGGATTCGCTAAACAGAT CTTGTCATCGTCGTCCTTGTA  3')  and  InFLAG  R  (5'  3'). A 415 bp region o f A C T 8 (Atlg49240) was  amplified using the following primers: A C T 8 F (5' A T T A A G G T C G T G G C A 3') and ACT8  R (5'  TTATCCGAGTTTGAAGAGGCTAC  3'). The P C R reactions  were  performed in a Biometra T-gradient thermocycler, using the following program: initial 31  denaturation for 2 minutes at 94°C, followed by 20 ( C A - M K K 9 - F L A G ) or 25 ( A C T 8 ) amplification cycles consisting o f denaturation (30 seconds at 94°C), annealing (30 seconds at 55°C) and extension (1 minutes at 72°C), and terminated by a final extension (5 minutes at 72°C). P C R products were analyzed by 1% agarose gel electrophoresis.  2.2.3. Ethylene measurements  Three week-old plants were induced with D E X . After the tested induction time, four or five rosettes were cut free from their root system and placed in the barrel o f a 10 m l syringe. The plunger was pushed in to leave 2 m l o f headspace i n the syringe, and the needle was sealed with parafilm. One hour later, 1 m l headspace was analyzed by gas chromatography.  Analyses presented  in Figure 2.5 were performed  using a  gas  chromatograph (Hewlett Packard Series II 5890) equipped with a C a r b o n P L O T column ( J & W Scientific) and a flame ionization detector. The samples were injected, with helium as the carrier, under the following temperature program: column temperature 275°C, injection temperature 200°C, detection temperature 250°C. Analyses presented i n Figure 2.6 and Figure 2.10 were performed using a different gas chromatograph (Varian 3400), equipped with a Porapak Q column and a flame ionization detector. The samples were injected, with helium as the carrier, under the following temperature program: column temperature 70°C, injection temperature 2°C, detection temperature 220°C. In each case the amount o f ethylene detected was quantified in terms o f peak area, and converted to parts per million (ppm) units through a standard curve established with a 100% Ethylene Standard (Alltech).  32  2.2.4. Hydrogen peroxide detection  Hydrogen peroxide was detected by 3,3'-diaminobenzidine ( D A B ) staining. A t chosen times after induction, whole rosettes were transferred to a side-armed Erlenmeyer flask containing 50 m l o f l m g / m l D A B (Sigma)-HCl, p H 4. The rosettes were vacuuminfiltrated with the solution, then transferred to wet paper towels and incubated under bright light for five hours. The tisssue was then cleared by gentle shaking i n a 3:1:1 ethanol:lactic acid:glycerol solution, in darkness, for 48 hours. Representative leaves were mounted on microscope slides and photographed. The quantification o f the D A B precipitate was done using ImageJ version 1.34s. The pictures were converted to 8-bit grayscale images, and all pixels within the leaf area with brightness values greater than 8 and smaller than 155 were quantified with help of the  Analyze Particules tool.  2.2.5. In vitro phosphorylation assay  The recombinant proteins used i n the i n vitro assays were produced i n Escherichia coli, using the Glutathione S-tran sferase (GST) Gene Fusion System, with G S T fused to their N-terminus. They had been previously purified from bacterial lysates by affinity chromatography. M P K 6 and M P K 3 had also been cleaved from G S T using the thrombin 1  1  protease, and provided to me as such by Jin Suk Lee. M P K 6 / M P K 3 (1 ug) was added to 1  0.5 pg C A - M K K 9 - G S T  i n 40 p i kinase buffer  1  [25 m M Tris pH7.5, 5 m M p-  glycerophosphate, 2 m M D T T , 0.1 m M N a V 0 , 10 m M M g C l , 200 p M A T P ] . The 3  4  2  samples were incubated for forty-five minutes at 30°C, and the reactions terminated by addition o f 5 X S D S sample buffer [0.25M T r i s - H C l p H 6.8, 9% S D S , 40% glycerol,  33  0.125% bromophenol blue and 20% v/v [3-mercaptoethanol]. After being boiled for five minutes, the proteins were separated by S D S - P A G E and analyzed by a western blot with an anti-phospho-p44/42 M A P kinase (Thr202/Tyr204) antibody (Cell Signaling).  2.2.6. Protein extraction and western blot analysis  Leaves were ground i n liquid nitrogen and the powder was mixed with protein extraction buffer [lOOmM H E P E S pH7.5, 5 m M E D T A , 5 m M E G T A , I m M N a V 0 , 1 0 m M N a F , 3  4  10%) glycerol, 7.5% P V P P , 5ul [3-mercaptoethanol and one protease inhibitor tablet (Roche)]. The extract was incubated at 4°C on a reciprocal shaker for ten minutes, and centrifuged for thirty minutes at 4°C (14,000 rpm). The amount o f protein in the supernatant was quantified using the Bio-Rad protein assay kit with bovine serum albumin ( B S A ) as the standard, and the supernatant was either assayed directly or flashfrozen and stored at - 8 0 ° C . For analysis, 100 u.g total protein was concentrated by acetone precipitation. Briefly, total proteins were mixed with five volumes o f acetone, incubated ten minutes at -80°C, then overnight at -20°C. The precipitated proteins were then centrifuged at 4°C for fifteen minutes (14,000 rpm). The pellet was air-dried, resuspended in 40 p i I X S D S protein buffer [50 m M T r i s - H C l pH=6.8, 1.8 % S D S , 8% glycerol, 0.025%) bromophenol blue and 4% v/v [3-mercaptomethanol], and boiled for 5 minutes. Samples were subsequently separated on a 12% S D S - P A G E gel, and the proteins were transferred to a P V D F membrane (Millipore). For a n t i - F L A G analysis, the membrane was blocked for two hours with 5% no-fat dried milk (Carnation) in Tris-buffered saline/Tween-20 ( T B S T ) [20mM T r i s - H C l p H 7.5, 100  34  m M N a C l , 0.05% Tween-20], and then incubated for two hours in a 1:10,000 dilution o f a n t i - F L A G antibody M 2 (Sigma) in 5% no-fat dried milk and T B S T . For anti-phosphoE R K analysis, the membrane was blocked for two hours in 3% B S A , then incubated for two hours with 1:1000 phospho-p44/42 M a p kinase (Thr202/Tyr204) antibody (Cell Signaling) in 3% B S A and T B S T . For a n t i - M P K 6 analysis, the membrane was blocked two  hours in 5% B S A in T B S T , then incubated two hours in 1:5000 a n t i - M P K 6  (provided as a beta product by Sigma) in 5% B S A and T B S T . After incubation with the primary antibody, the membrane was washed four times for five minutes with T B S T , and then incubated with a 1:7500 dilution o f peroxidase-conjugated goat anti-mouse antibody (Dako) in T B S T for one hour. The membrane was washed again four times five minutes in T B S T , and then visualized by using an enhanced chemiluminescence detection kit (Amersham).  2.2.7. Generation o f D E X : C A - M K K 9 - F L A G / m / ? A : 6 ' transgenic plants  Agrobacterium tumefaciens GV3101 carrying the construct D E X : : C A M K K 9 - F L A G in the pTA7002 plasmid (Aoyama and Chua, 1997) was grown overnight in a 300 m l culture containing 50 pg/ml rifamycin, 25 pg/ml gentamycin and 50 pg/ml kanamycin. The  mpk6 knock-out mutant was transformed using the floral dip method, i.e. by dipping  the immature flowers into the  Agrobacterium cells that had been resuspended in 5%  sucrose and 0.05% Silwet-L77. T l transformants were selected by germinating the resulting seeds on M S plates containing 50 pg/ml hygromycin.  35  2.3.  Results  2.3.1. The C A - M K K 9 transgene is rapidly activated following D E X induction  In the C A - M K K 9 protein, the conserved serine residues present in the activation loop o f the kinase have been converted into glutamate residues, a modification which structurally mimics the acidic character created by post-translational phosphorylation o f serine residues, thus bypassing the requirement for a M A P K K K - d r i v e n  phosphorylation for its  activity. Furthermore, a F L A G epitope has been added to the C-terminus o f C A - M K K 9 (Figure 2.1 A ) .  This construct was put into the pTA7002 vector (Aoyama and Chua,  1997), to generate the p T A 7 0 0 2 - C A - M K K 9 - F L A G construct (Figure 2 . I B ) which, once inserted into a plant genome, allows induced expression o f C A - M K K 9 - F L A G by treating the transformed plants with the steroid hormone analogue, dexamethasone ( D E X ) (Figure 2.1C).  36  MALVRERRQLNLRLPLPPISDRRFSTSSSSATTTTVAGCNGISACDLEKLNVLGCG NGGIWKVRHKTTSEIYALKTVNGDMDPIFTRQLMREMEILRRTDSPYWKCHGIF EKPWGEVSILMEYMDGGTLESLRGGVTEQKLAGFAKQILKGLSYLHALKIVHRDIK PANLLLNSKNEVKIADFGVSKILVRELDSCNEYVGTCAYMSPERFDSESSGGSSDI YAGDIWSFGLMMLELLVGHFPLLPPGQRPDWATLMCAVCFGEPPRAPEGCSEEF RSFVECCLRKDSSKRWTAPQLLAHPFLREDLDYKDDDDK  B RB  35S)| G V G [ E 9 ^ N O ^ H P T |-|3A  NUCI  CA-MKK9FI_AG  6 X U A S GAL4  - LB  FUS  Figure 2 . 1 . Construction of CA-MKK9 and insertion into the DEX-inducible vector to produce pTA7002-CA-MKK9-FLAG. A) Amino acid sequence of CA-MKK9. In red are the serine residues converted to glutamate. In blue is the C-terminus FLAG tag B) Schematic representation of the T-DNA region of the pTA7002-CA-MKK9-FLAG construct used for transformation. RB, T-DNA right border; 35S, CaMV 35S promoter GVG, chimeric GAL4-VP16GR dexamethasone inducible transcription factor; E9, pea rbcS-E9 polyadenylation sequence; Nos, nopaline synthase promoter; HPT, hygromycin phosphotransferase; N,, nopaline synthase polyadenylation sequence, 6XUAS i4, GVG binding site; LB, left T-DNA border (Aoyama and Chua, 1997; McNellis et al., 1998) C) Schematic diagram of the DEX-inducible system. Adapted from McNellis e t a l . (1998). ga  37  The p T A 7 0 0 2 - C A - M K K 9 - F L A G construct was used to transform Arabidopsis, and stable, homozygous D E X : C A - M K K 9 - F L A G transgenic lines were obtained. In the same manner, in order to detect potential artefactual effects due to the p T A 7 0 0 2 vector alone, Arabidopsis plants were also transformed with the "empty" p T A 7 0 0 2 vector, to establish several Empty Vector ( E V ) lines.  A s groundwork for future investigation into the downstream effects o f expressing C A M K K 9 , it was important to define the time-course o f C A - M K K 9 expression after induction by D E X . Therefore, I used R T - P C R to assess the rise in C A - M K K 9 m R N A levels following the transgene induction. In order to detect solely the  CA-MKK9  transgene, and not the endogenous A t M K K 9 , primers were designed to encompass the F L A G tag fused to the C-terminus o f the C A - M K K 9 transgene. A s shown by an R T - P C R analysis (Figure 2.2A), D E X : C A - M K K 9 - F L A G plants already showed a marked rise in C A - M K K 9 m R N A only two hours after D E X treatment. Furthermore, the levels o f C A M K K 9 m R N A appeared to remain elevated for at least twelve hours after the initial induction.  I also analyzed the increase in C A - M K K 9 protein levels following D E X induction. A s previously mentioned, a F L A G tag had been fused to the C-terminus C A - M K K 9 , which should allow the translated transgene product to be detected using the a n t i - F L A G M 2 antibody. This strategy has been previously used in plant systems to detect transgenic fusion proteins (Samuel et al., 2000; Samuel and Ellis, 2002; K i m et al., 2003; L i u et al.,  38  2003). However, due to nonspecific binding o f this antibody to other proteins in the total Arabidopsis protein extracts, it proved difficult to unambiguously identify C A - M K K 9 by using the a n t i - F L A G antibody. The mass predicted for the C A - M K K 9 - F L A G protein is 36.3 k D a , but I could only detect an increase in a - 4 0 k D a protein, starting two hours after D E X induction i n the D E X : C A - M K K 9 - F L A G plants. A faint band o f similar size can also be seen in the E V plants, probably reflecting the nonspecific binding o f the antibody (Figure 2.2B). Interestingly, aberrant molecular weight o f C A - M A P K K s was also reported by Ren et al. (2002), who described up-shifts in the mobilities o f C A A t M K K 4 and C A - A t M K K 5 proteins on P A G E gels compared to wild-type proteins. Nevertheless, in order to localize more confidently the C A - M K K 9 protein, our laboratory has recently generated an a n t i - A t M K K 9 antibody. Once optimized, western blots using this new antibody should allow us to clarify the expression pattern o f the C A - M K K 9 protein.  39  Time after DEX (hrs):  D EX: CA-M KK9-F LAG L2 0  2  4  8  12  CA-MKK9FLAG  DEX:CA-MKK9-FI_AG L12 0  2  4 —  8 —  12 —  ACT8  Time after DEX (hrs):  DEX:CA-MKK9-FLAG L13 8  EVL1  12  8  12  CA-MKK9FLAG  ACT8  B DEX:CA-MKK9-FLAG L12 Time after DEX (hrs):  0  2  4  8  12  EVL1 4  8  12  CA-MKK9- J FLAG  Figure 2.2. Time-course of CA-MKK9 activation following DEX induction Three-week-old DEX:CA-MKK9-FLAG (L2, L12 and L13) and EV plants (L1) were induced with DEX and harvested at the indicated time. A) Total RNA was extracted from plant tissue, and CA-MKK9-FLAG mRNA levels were estimated by RT-PCR using AtMKK9- and FLAG-specrfic primers. Equal loading is shown by comparable ACT8 levels. B) Total proteins extracted from plant tissue (100ug) were separated by SDS-PAGE. CA-MKK9-FLAG was detected by immunoblot with an anti-FLAG antibody. Equal loading is shown by Coomassie Blue staining of the membrane.  40  Overall, the analysis o f C A - M K K 9 - F L A G expression suggests that the transgene is expressed two hours after the D E X induction, and that its expression stays high up to twelve hours, and possibly longer.  2.3.2. C A - M K K 9 activity results in the formation of lesions  Since it had been observed in a preliminary experiment that the induction o f C A - M K K 9 causes a cell death phenotype in the transgenic plants, I carried out a more detailed characterization o f the timing and pattern o f the formation o f these necrotic lesions in D E X : C A - M K K 9 - F L A G plants. A s shown in Figure 2.3, leaves began to yellow two days after D E X induction, starting from the center o f the rosette, including the meristematic region. Scattered chlorotic lesions became clearly visible on the leaves three days after induction, and by the fourth day these had taken over the entire plant (Figure 2.3). The induced D E X : C A - M K K 9 - F L A G plants did not recover, and eventually died (data not shown).  41  Days after DEX: *  CM  5  —I  Ul G  I  £5 X uUl G  1  E  ft*  Figure 2.3. Time-course of cell death progression following CA-MKK9 activation. Three-week-old DEX:CA-MKK9-FI_AG (L12 and L13) and EV (L1) plants were induced with DEX. Pictures show the same plant at the indicated time after DEX induction.  2.3.3. C A - M K K 9 - m e d i a t e d cell death is associated with H2O2 accumulation  The appearance o f the chlorotic lesions observed following C A - M K K 9  activation  suggested that they could be the result o f P C D . In plants, P C D , in particular in the context o f the H R and in response to ozone stress, is associated with the accumulation o f R O S , which include H2O2 (Lamb and Dixon, 1997). In order to assess whether the phenotype o f D E X : C A - M K K 9 - F L A G was consistent with this type o f P C D , I examined the correlation between lesion formation and accumulation o f H2O2 in the induced transgenic plants. A 3,3'-diaminobenzidine (DAB)-based histochemical assay revealed that H2O2 accumulation can be detected one day after D E X induction; i.e. immediately  42  preceding the appearance o f the first visible lesions (Figure 2.4). However, no significant H2O2 accumulation could be detected two or four hours after D E X induction, indicating that the R O S response is probably not an early consequence o f ectopic  CA-MKK9  activity (Figure 2.4). Nevertheless, the pattern and timing o f H2O2 accumulation suggest that R O S are closely associated with the death o f the leaf tissue observed in the D E X : C A - M K K 9 - F L A G transgenic plants, consistent with the hypothesis that C A - M K K 9 activity is inducing H R - l i k e P C D .  43  Hours after DEX:  0  2  4  24  Figure 2.4. CA-MKK9 activity results in H 0 accumulation. Three week-old DEX:CA-MKK9-FLAG (L12 and L13) and EV (L1) plants were induced with DEX. At the indicated time-points, H2O2 generation was assessed by infiltrating a whole plant rosette with a DAB solution. Infiltrated plants were left under light for five hours and then cleared for two days. The amount of DAB precipitate was quantified using ImageJ, and indicated on each picture as the percentage of the total leaf area. 2  2  44  2.3.4. C A - M K K 9 causes a rapid increase in ethylene biosynthesis  A s R O S production and P C D both require the action o f ethylene (Moeder et al., 2002; Rao et al., 2002), the above results suggested that that C A - M K K 9 might affect the biosynthesis o f this hormone. It has been previously reported that a M A P K  cascade  involving A t M P K 6 controls ethylene production ( L i u and Zhang, 2004). It thus seemed possible that C A - M K K 9 also triggers that signaling cascade, resulting in a rise o f ethylene production that eventually leads to cell death in the transgenic plants. A s a first step to test that hypothesis, I asked whether the activation o f the C A - M K K 9 transgene was temporally correlated with an increase in ethylene production. To answer that question, the time-course o f ethylene production was monitored in D E X : C A - M K K 9 F L A G plants after D E X induction. The induction o f C A - M K K 9 was indeed quickly followed by a rise in ethylene production (Figure 2.5). This increase was detectable four hours after the D E X treatment, and reached up to six times wild-type levels after twelve hours. The timing o f this response suggests that C A - M K K 9 rapidly modulates ethylene biosynthesis, possibly through a post-translational mechanism.  45  Figure 2.5. Time-course of ethylene production in DEX:CA-MKK9-FI_AG plants. DEX:CA-MKK9-FLAG (L2 and L12) and EV (L3 and L10) plants were induced with DEX. At the indicated time-points, four rosettes were collected and placed into a syringe. One hour after, the ethylene contained in the headspace was quantified by gas chromatography. Each time-point assay was done in triplicate. Error bars represent standard errors.  2.3.5. C A - M K K 9 is similar to C A - M K K 4 in acting as a mediator o f ethylene biosynthesis  The current model o f M A P K regulation o f ethylene production proposes that A t M K K 4 and/or A t M K K 5 act upstream o f A t M P K 6 ,  and that the latter's activity leads to  stabilization o f A C S isoforms ( L i u and Zhang, 2004). However, no experimental data has been published regarding the participation o f these specific M A P K K s in the A C S 2 / 6 stabilization process, since L i u and Zhang's (2004) model is based on the ectopic expression o f A t M K K 4 / A t M K K 5 ' s tobacco orthologue, N t M E K 2 , in Arabidopsis. Since 46  my evidence suggested that C A - M K K 9 might also participate in the same pathway, I compared the extent o f the ethylene burst in D E X : C A - M K K 9 - F L A G plants  with that  obtained in D E X : C A - M K K 4 - F L A G plants. A s shown by Figure 2.6, two D E X : C A M K K 4 - F L A G transgenic lines, L I 5 and L I 7 , produced approximately twice as much ethylene as the tested D E X : C A - M K K 9 - F L A G lines, twelve hours after induction. This confirms the role o f A t M K K 4 as acting upstream o f A t M P K 6 in the control o f ethylene biosynthesis, but also shows that A t M K K 9 can participate in an analogous fashion.  60 ,  50  g Q.  40  a.  £=  o o -o 30 o  I— Q_ CD C  a  >.  10  0  i  i I DEX:CA-MKK9-FLAG L12  i  i . i----  DEX:CA-MKK9-FLAG L13  __i  DEX:CA-MKK4-FLAG L15  t : : : J DEX:CA-MKK4-FLAG L17  i.  = EV L1  Figure 2.6. Ethylene production in DEX:CA-MKK9-FI_AG and DEX:CA-MKK4FLAG plants. Three week-old DEX:CA-MKK9-FLAG (L12 and L13), DEX:CA-MKK4-FLAG (L15 and L17) and EV (L1) were induced with DEX and collected twelve hours after. Four or five rosettes were placed into a syringe. One hour after, the ethylene contained in the headspace was quantified by gas chromatography. Each genotype was tested in triplicate. Error bars represent standard errors. 47  2.3.6.  C A - M K K 9 induction correlates with increased activity of A t M P K 6  in vivo  The rapidity with which the ethylene burst is detected following C A - M K K 9 activation implied that C A - M K K 9 must quickly activate A t M P K 6 following its own activation, i f it is to be responsible for the observed increase in ethylene production. In order to determine whether C A - M K K 9 induction correlates with increased A t M P K 6 activity in D E X : C A - M K K 9 - F L A G plants, I monitored protein and activation levels o f A t M P K 6 at different time-points after D E X induction. While protein levels were estimated using a specific a n t i - A t M P K 6  antibody, the  activity o f the kinase was assessed  with a  commercial anti-phospho-ERK antibody. This antibody was raised against the conserved phosphorylated tyrosine and threonine residues present in the activated form of the mammalian M A P K , E R K , and has been shown to also detect a number o f phosphorylated plant M A P K s , including N t S I P K / A t M P K 6 (Samuel et al., 2000; Ahlfors et al., 2004). It is well established that the phosphorylation o f the conserved - T y r - X - T h r - motif present in the activation loop o f M A P K s correlates with increased kinase activity (Ray and Sturgill, 1988), which makes the anti-phospho-ERK antibody a useful tool for assessing the presence o f activated M A P K s . I found that D E X : C A - M K K 9 - F L A G plants, following induction, displayed increased levels o f a phosphorylated - 4 5 k D a M A P K , which the a n t i - A t M P K 6 antibody confirmed to be A t M P K 6 (Figure 2.7). Activated A t M P K 6 , which was almost absent before D E X induction, was clearly detectable after two hours, and increased to a maximum between eight ( L I 3 ) and twelve ( L I 2 ) hours (Figure 2.7). This early increase in A t M P K 6 activity is concurrent with the activation o f C A - M K K 9 after D E X induction (Figure 2.2), again  48  consistent with the hypothesis that the former could be a direct target o f the latter. On the other hand, the levels o f A t M P K 6 protein remained relatively constant throughout the time-course, indicating that the increased presence o f phosphorylated A t M P K 6 is likely due to a post-translational modification rather than to an increase in the A t M P K 6 pool. Interestingly, the anti-phospho-ERK blot revealed the concurrent activation o f a second M A P K o f lower molecular weight than A t M P K 6 . W e are currently investigating the identity o f this second M A P K .  DEX:CA-MKK9-FLAG L12  DEX:CA-MKK9-FLAG L13  EV L1  Time after DEX (hrs):  0 2 4 8  12  0 2 4 8  12  0 2 4 8  12  a-p-ERK •  Figure 2.7. AtMPK6 protein and activity levels following CA-MKK9 activation. DEX:CA-MKK9-FLAG (L12 and L13) and EV (L1) plants were collected at the indicated time-points. Total proteins (100 pg) were separated by SDS-PAGE. Membranes were first blotted with anti-phospho-ERK, then stripped and re-blotted with anti-AtMPK6. Equal loading is shown by Coomassie Blue staining of the membrane.  2.3.7.  A t M K K 9 activates A t M P K 6 in vitro  T o further test the model o f an A t M K K 9 - A t M P K 6 signaling cascade, I examined the ability o f A t M K K 9 to phosphorylate recombinant A t M P K 6 in an in vitro kinase assay.  49  Moreover, in order to establish whether this assay was stringent enough to reveal specific substrate preferences, I also tested A t M K K 9 ' s ability to phosphorylate A t M P K 3 , the Arabidopsis M A P K most closely related to A t M P K 6 and also one that is known to play a role in stress responses (Kovtun et al., 2000; Asai et al., 2002; Ahlfors et al., 2004). In order to by-pass the requirement for an upstream M A P K K K for A t M K K 9 activation, I used a recombinant C A - M K K 9 - G S T protein in the assay. Because M A P K s often display auto-phosphorylating activity, the assayed recombinant M A P K s had previously been converted to kinase-inactive forms ( M A P K ) by site-directed mutagenesis, whereby the 1  lysine residue o f the kinase domain was replaced with a catalytically ineffective arginine residue. Following co-incubation o f the C A - M K K 9 with either M P K 3 ' or M P K 6 ' , the phosphorylation state o f the tested M A P K s was assessed by immunoblot using antiphospho-ERK. A s shown in Figure 2.8, C A - M K K 9 strongly increases the phosphorylation state o f M P K 6 ' . In contrast, it does not appear to have any activity against M P K 3 . Therefore, I 1  conclude that, at least  in vitro, C A - M K K 9 is capable o f activating A t M P K 6 . Together  with the rapid rise o f A t M P K 6 activity observed in D E X : C A - M K K 9 - F L A G plants, this supports my hypothesis that A t M P K 6 is activated by C A - M K K 9 in transgenic plants upon D E X induction.  50  MPK6  MPK3  j  CA-MKK9  +  -  +  +  MAPK  -  +  +  -  mm* mm a-p-ERK  Total ( proteins  +  j  + + _  CA-MKK9-GST ^-p-MPKS 1  . -CA-MKK9-GST «- —MPK6' — ~I\/IPK3'  Figure 2.8. CA-MKK9 kinase activity on AtMPK6 and AtMPK3 in vitro. Recombinant CA-MKK9-GST (0.5 ug), MPK6' (1 ug) and MPK3 (1 ug) proteins were subjected to an in vitro kinase assay. Proteins were then separated by SDSPAGE and analyzed with anti-phospho-ERK. Equal loading is shown by Coomassie Blue staining of the membrane. j  2.3.8. A t M P K 6 is necessary for C A - M K K 9 - m e d i a t e d ethylene production  M y results so far suggested that, following D E X induction, C A - M K K 9 directly activates A t M P K 6 . However, in order to establish a causal relationship between A t M P K 6 activity and the observed C A - M K K 9 - m e d i a t e d phenotypes, i.e. ethylene overproduction and P C D , I wanted to find genetic evidence o f such dependency. T o do this, I transformed a  mpk6 knock-out mutant line with the D E X : C A - M K K 9 - F L A G construct. After T3 generation plants were obtained, I screened the transgenic lines to isolate those where the level o f expression o f C A - M K K 9 following D E X induction was comparable to that observed in the original D E X : C A - M K K 9 - F L A G lines (L2, L 1 2 and L 1 3 ) (Figure 2.2A). A n R T - P C R analysis o f the time-course o f C A - M K K 9 induction in the double-mutant  51  lines revealed that three independent DEX:CA-MKK9-FLAG//w/?£r5 lines, L I , L 4 and L 7 , displayed strong induction o f C A - M K K 9 two hours after induction, with sustained transgene expression up to twelve hours after expression (Figure 2.9). A s this was very similar to what I had observed in D E X : C A - M K K 9 - F L A G plants (Figure 2.2A), it seemed reasonable to assume that these double-mutant lines would behave the same way as the single C A - M K K 9 mutant, and I therefore used these three lines in subsequent analyses.  DEX:CA-MKK9-FLAG/mpft6 L1 Time after DEX (hrs):  DEX:CA-MKK9-FLAG/mpft6 L4  DEX;CA-MKK9-FLAG/mp/f6 L7 ~""""~ ~~~~~~ 0 2 4 8 12 - —  0  2  4  8  12  0  2  4  8  12  Figure 2.9. Time-course of CA-MKK9 induction in DEX:CA-MKK9-FLAG/mp/c6 plants. Three-week-old DEX:CA-MKK9-FLAG/mp/c6 plants (L1, L4 and L7) were induced with DEX and harvested at the indicated time. Total RNA was extracted from plant tissue, and CA-MKK9-FLAG mRNA levels were estimated by RT-PCR using AtMKK9- and FLAG-specific primers. Equal loading is shown by comparable ACT8 levels.  In contrast to the pattern I observed with D E X - i n d u c e d D E X : C A - M K K 9 - F L A G plants, I found that treating the DEX:CA-MKK9-FLAG//w/?jfc<5 plants with D E X did not induce increased ethylene production following D E X treatment (Figure 2.10). This provides compelling genetic evidence that A t M P K 6  is necessary for C A - M K K 9 - m e d i a t e d  ethylene production in the transgenic plants.  52  35  DEX.CA-MKK9FLAGL12  DEX:CA-MKK9FLAGL13  DEX:CA-MKK9FLAG/mpk6 L1  DEX:CA-MKK9FLAG/mpk6 L4  DEX:CA-MKK9FLAG/mpk6 L7  EVL1  Figure 2.10. AtMPK6 is required for CA-MKK9-mediated ethylene production. DEX:CA-MKK9-FI_AG (L12 and L13), DEX:CA-MKK9-FLAG/mp/c6 (L1, L4 and L7) and EV (L1) plants were treated with DEX. Twelve hours later, four rosettes per transgenic line were placed into a syringe and the headspace was analyzed by gas chromatography. Samples were done in triplicate. Error bars represent standard error.  2.3.9. A t M P K 6 is not required for C A - M K K 9 - m e d i a t e d P C D  The development o f ROS-associated lesions in the D E X : C A - M K K 9 - F L A G (Figure 2.3, Figure 2.4) had led me to believe that AtMPK6-mediated  plants ethylene  overproduction might be the direct cause o f the C A - M K K 9 - m e d i a t e d P C D . In order to test that hypothesis, I compared the phenotype o f the  DEX:CA-MKK9-FLAG//wp&(5  plants to that o f D E X : C A - M K K 9 - F L A G plants four days after D E X induction. If the AtMPK6-mediated ethylene burst indeed caused P C D , I expected that the latter process would not be observed in the double mutant, where A t M P K 6 activity and ethylene  53  overproduction are absent (Figure 2.10). However, as shown in Figure 2.11, D E X : C A MKK9-FLAG7/wp£6 plants developed lesions comparable to those o f D E X C A - M K K 9 FLAG  plants following  D E X induction. Therefore,  C A - M K K 9 - m e d i a t e d P C D is  triggered independently o f A t M P K 6 , and is not directly caused by the overproduction o f ethylene.  DEX:CA-MKK9-FLAG L12  DEX:CA-MKK9-FLAG/mp/c6 L1  Figure 2.11. Lesion formation in DEX:CA-MKK9-FLAG/mpk6 plant. DEX:CA-MKK9-FLAG (L12) and DEX:CA-MKK9-FLAG/mpk6 (L1) plants were induced with DEX, and were photographed four days after. A comparable phenotype was observed for two additional DEX:CA-MKK9-FLAG/mp/f6 lines, L4 and L7.  2.4.  Discussion  The gaseous hormone ethylene is involved in several stages o f plant development, as well as in responses to biotic and abiotic stresses. The production o f ethylene is controlled by two family o f enzymes, A C C synthases ( A C S ) and A C C oxidases (Wang et al., 2002).  54  The rate o f ethylene biosynthesis can be modulated by the differential expression o f both A C S and A C O genes (Tuomainen et al., 1997; Overmyer et al., 2000; Moeder et al., 2002). Moreover, several lines o f evidence indicate that the stability o f A C S enzymes is controlled by post-translational mechanisms, allowing rapid changes i n the rate o f ethylene biosynthesis. For instance, A C S activity in tomato depends upon intact kinase activity (Spanu et al., 1994). In addition, in Arabidopsis, an ethylene regulatory protein ( E T O l ) has been shown to interact with the A C S 5 C-terminus, thus blocking its activity and presumably promoting its degradation by a proteasome-dependent pathway (Wang et al., 2004). More recently, it was demonstrated that activated A t M P K 6 phosphorylates two Arabidopsis A C S isoforms, A C S 2 and A C S 6 , thereby stabilizing them and causing increased production o f ethylene ( L i u and Zhang, 2004). The authors o f that study, using a DEX-inducible/gain-of-function  system, described a rapid increase  in  ethylene  production following the activation o f N t M E K 2 , both in tobacco and Arabidopsis ( K i m et al., 2003; L i u and Zhang, 2004).  In this Chapter, I report that another Arabidopsis M A P K K , A t M K K 9 , is also capable o f controlling ethylene biosynthesis when overexpressed in a C A form, under the control o f the DEX-inducible promoter. When I examined ethylene production in DEX-induced C A - M K K 9 plants, I found that ethylene production starts to increase only four hours after D E X induction (Figure 2.5). This was preceded by an increase i n A t M P K 6 ' s activity (Figure 2.7), which mirrored the pattern o f C A - M K K 9 induction (Figure 2.2). Since A t M P K 6 ' s activity was practically absent before D E X induction, as well as in the control line, E V L I , it appears that C A - M K K 9 ' s activity is responsible for activation o f the  55  M A P K . Nevertheless, it could be argued that the rise in A t M P K 6 activity is not due directly to C A - M K K 9 , but is an indirect effect o f the stress caused by the transgene activation. For instance, A t M P K 6 is known to be activated upon oxidative stress, and more particularly by H2O2 (Ichimura et a l , 2000; Desikan et al., 2001; Yuasa et al., 2001), and D E X : C A - M K K 9 - F L A G plants indeed show increased accumulation o f H2O2 (Figure 2.4). However, this H2O2 accumulation had not yet begun two hours after D E X induction (Figure 2.4), at which time A t M P K 6 activity was already increasing (Figure 2.7). I therefore conclude that the observed A t M P K 6 activity is not an indirect consequence o f oxidative stress. Supporting this idea, I also showed by an in vitro kinase assay that A t M P K 6 is a suitable substrate for C A - M K K 9 (Figure 2.8). In addition to these lines o f biochemical evidence, I was able to genetically demonstrate that the DEX:CA-MKK9-FLAG/mp&f5  double-mutant did not accumulate ethylene upon D E X  induction (Figure 2.10), despite comparable levels o f expression o f the  CA-MKK9  transgene (Figure 2.9). Altogether, this evidence supports my hypothesis that, upon activation in the D E X : C A - M K K 9 - F L A G plants, C A - M K K 9 rapidly activates  AtMPK6,  which in turn is known to stabilize the A C C synthases, A C S 2 and/or A C S 6 through phosphorylation o f specific sites on the A C S protein. This stabilization would then lead to the observed burst o f ethylene in induced C A - M K K 9 plants (Figure 2.12).  56  Figure 2.12. Model of CA-MKK9 participation in ethylene biosynthesis. CA-MKK9, similarly to AtMKK4/AtMKK5, activates AtMPK6, which in turn stabilized ACS6, leading to a burst of ethylene (Liu and Zhang, 2004). CA-MKK9 also activates the onset of PCD, independently of AtMPK6 and of ethylene biosynthesis.  According to Liu and Zhang (2004), the upstream MAPKKs responsible for AtMPK6 are either AtMKK4 and/or AtMKK5. However, the experimental data presented by those authors was based on the over-expression in Arabidopsis of a gain-of-function mutant of the tobacco MAPK kinase, NtMEK2, which they showed was able to activate AtMPK6 and induce ethylene production. Because the orthologues of NtMEK2 in Arabidopsis are AtMKK4 and AtMKK5, and because these two MAPKKs have also been shown to activate AtMPK6 in protoplast studies (Asai et al., 2002), Liu et al. (2004) speculated that AtMKK4 and/or AtMKK5 act upstream of AtMPK6 in controlling ethylene production in vivo. The same group had previously shown that CA-AtMKK4/AtMKK5 induces  H2O2  accumulation and PCD in Arabidopsis (Ren et al., 2002), in a manner similar to what I 57  observed in D E X : C A - M K K 9 - F L A G plants (Figure 2.3). When I compared ethylene production between D E X : C A - M K K 9 - F L A G and D E X : C A - M K K 4 - F L A G plants, both genotypes displayed a marked increase in ethylene biosynthesis twelve hours after D E X induction (Figure 2.6). This confirms L i u et al. (2004)'s model, where A t M K K 4 acts upstream o f A t M P K 6 in controlling the stability o f A C S isoforms. Although the two D E X : C A - M K K 4 transgenic lines tested produced approximately twice as much ethylene as the D E X : C A - M K K 9 - F L A G lines (Figure 2.6), it is not clear that this difference reflects the relative participation rates of the two M K K s in ethylene biosynthesis in  planta. M y experimental approach is solely based on ectopic transgene expression, and does not take into account the normal activity levels o f the endogenous M K K s , including their  spatial  or  temporal  distribution.  Nevertheless,  the  similarity  between  A t M K K 4 / A t M K K 5 - and A t M K K 9 - m e d i a t e d ethylene production and cell death raises questions about the specificity o f M A P K K s in vivo. A t M K K 4 and A t M K K 5 are part o f Group C M A P K K s , whose members are well-established upstream effectors o f Group A M A P K s (e.g. A t M P K 6 ) in stress signaling (Ichimura et al., 2002). A t M K K 9 , on the other hand, is part o f group D M A P K K s , and only bears approximately 50% amino acid sequence similarity to Group C M A P K K s . Whether the Group C and D M A P K K s are functionally redundant, or whether their specificity depends on other factors which cannot be assessed by the current gain-of-function studies, remains unclear.  The only study published so far on a Group D M A P K K describes the tomato M A P K K , L e M K K 4 . Interestingly, the over-expression o f L e M K K 4 causes cell death and correlates with the activation o f L e M P K 2 and L e M P K 3 , which are putative orthologues o f  58  A t M P K 6 and A t M P K 3 , respectively (Pedley and Martin, 2004). Moreover, the authors of that study demonstrated that L e M K K 4 strongly activates L e M P K 2 , while its capacity to activate L e M P K 3 , although detectable, is much less important (Pedley and Martin, 2004). A l l together, these data closely mirror our finding on C A - M K K 9 , suggesting that L e M K K 4 and A t M K K 9 could be functional orthologues.  Interestingly, the same study  reveals similar findings for L e M K K 2 , a Group C M A P K K , strengthening the idea that Group C and D M A P K K s may have at least partly overlapping functions in plants (Pedley and Martin, 2004).  M y results also revealed that a second M A P K o f smaller size is co-activated with A t M P K 6 following D E X induction o f the D E X : C A - M K K 9 - F L A G plants (Figure 2.7). Its apparent molecular weight, - 4 2  k D a , points toward  several Arabidopsis M A P K  candidates: A f M P K l (42.6 kDa), A t M P K 3 (42.7 kDa), A t M P K 4 (42.9 kDa), A t M P K 5 (42.9 kDa), A f M P K l 1 (42.4 kDa), A t M P K 1 3 (42.2 kDa). However, only a few o f these M A P K s are reported to be co-activated with A t M P K 6 . A t M P K 3 , i n particular, is the M A P K whose activity is the most frequently associated with that o f A t M P K 6 , under several biotic and abiotic stress conditions. However, since I found that A t M P K 3 cannot be directly activated by C A - M K K 9 (Figure 2.8), it appears unlikely that this M A P K could be activated so early after C A - M K K 9 induction. A t M P K 4 has also been reported, in a few instances, to be co-activated with A t M P K 6 (Ichimura et al., 2000; Desikan et al., 2001; Teige et al., 2004). In fact, I have obtained preliminary data suggesting that C A M K K 9 may be able to activate A t M P K 4  in vitro, although this w i l l require confirmation  59  (data not shown). Future experiments should allow me to confirm whether the 42 k D a M A P K co-activated with A t M P K 6 by C A - M K K 9 is indeed A t M P K 4 .  I have also found that expression o f C A - M K K 9 triggers a form o f P C D in Arabidopsis plants. Induced D E X : C A - M K K 9 - F L A G transgenic plants display necrotic lesions that become clearly visible two to three days after activation o f the transgene (Figure 2.3). Moreover, the appearance o f visible lesions in D E X : C A - M K K 9 - F L A G plants was preceded by the accumulation o f H2O2 (Figure 2.4). It is now well established that R O S , including H2O2, play a central role as signaling molecules orchestrating the onset and the progression o f H R - l i k e P C D (Lamb and Dixon, 1997). Moreover, a complex network o f interactions between these R O S and the hormones salicylic acid ( S A ) , ethylene and jasmonic acid (JA) regulates the initiation, progression and containment  of P C D  (Overmyer et al., 2000; Rao et al., 2002; Overmyer et a l , 2003). Ethylene has been associated with multiple forms o f P C D , and its participation in H R and ozone-induced cell death is well-established. Ethylene insensitivity is associated  with increased  resistance to ozone damage (Overmyer et al., 2000). In addition, ethylene stimulates cell death, and both its biosynthesis and perception are required for H2O2 accumulation and subsequent P C D in plant tissues in response to certain abiotic stimuli (de Jong et al., 2002; Moeder et al., 2002). It thus appeared possible that C A - M K K 9 might trigger P C D through its activity on A t M P K 6 (Figure 2.8, Figure 2.7), and the resulting overproduction of ethylene (Figure 2.5). However, I found that the D E X - i n d u c e d D E X : C A - M K K 9 ¥LAGImpk6  plants developed lesions similar to those seen on D E X : C A - M K K 9 - F L A G  plants following D E X induction (Figure 2.11), despite the wild-type levels o f ethylene,  60  and the absence o f A t M P K 6 in the double mutant genotype (Figure 2.10). O n one hand, this tells me that C A - M K K 9 - m e d i a t e d P C D is not caused by the ethylene burst observed in D E X : C A - M K K 9 - F L A G plants (Figure 2.12). In fact, although ethylene is reported to promote the spreading o f lesions during P C D , there is no evidence that it can, by itself, trigger the onset o f P C D . For example, the ethylene-overproducing mutants  etol and eto3  display increased damage in response to ozone, but do not spontaneously form lesions (Rao et al., 2002). Therefore, although I may hypothesize that D E X : C A - M K K 9 - F L A G plants should indeed be oversensitive to PCD-triggering stimuli due to high ethylene production, the latter is not sufficient to explain the spontaneous  lesion formation  observed following D E X induction. The cell death phenotype o f DEX:CA-MKK9-FLAG//w/?£f5 plants also demonstrates that C A - M K K 9 - m e d i a t e d P C D does not require the activity o f A t M P K 6 (Figure 2.12). This suggests that A t M P K 6 , although it is activated by several biotic stresses associated with P C D , plays more o f a parallel role in plant defense and is not directly responsible for activating the cell death program (Nuhse et a l , 2000; Menke et al., 2004). This conclusion contrasts with the interpretation o f studies on A t M P K 6 ' s tobacco orthologue (NtSIPK) where the authors concluded that N t S I P K overexpression is sufficient in itself to trigger P C D (Zhang and L i u , 2001). In light o f my findings, it appears that an AtMPK6-independent pathway activated by C A - M K K 9  triggers cell death in the  D E X : C A - M K K 9 - F L A G plants. Interestingly, the inhibition o f A t M P K 4 activity, which plausibly corresponds to the 42 k D a M A P K activated by C A - M K K 9 (Figure 2.7), has been correlated with the inhibition of P C D in Arabidopsis cell cultures (Desikan et al., 2001). Moreover, in tobacco, the M A P K K N t M E K 2 has been hypothesized to trigger  61  P C D by upregulating the expression of a gene coding for a respiratory burst oxidase homologue, thereby  increasing R O S production (Yoshioka  et  al., 2003).  These  observations and models provide many interesting avenues for further research into the mechanism o f C A - M K K 9 - m e d i a t e d P C D .  The gain-of-function system used in this study, as well as others (Zhang and L i u , 2001; K i m et al., 2003; L i u and Zhang, 2004; Pedley and Martin, 2004), is a useful tool for defining the M A P K s and metabolic events acting downstream o f a M A P K K o f interest. However, this system is somewhat artificial in that it presumably does not exactly reflect the usual biological context in which a M A P K K is active, and may lack modifying factors that control the specificity o f that M A P K K .  Therefore, my model, while fully  consistent with my experimental results undoubtedly presents a simplistic view of what is likely a complex signaling cascade. In order to better understand the role o f A t M K K 9 in vivo, as well as to address the issue o f overlapping function and specificity between different groups o f M A P K K s , it w i l l be necessary to monitor variation in the catalytic activity o f these M A P K K s  in planta, in their endogenous form. Such experiments would  permit us to gain a fuller understanding o f A t M K K 9 ' s role in mediating both ethylene biosynthesis and P C D , and could also provide a template for analogous studies of the roles o f other, as yet uncharacterized, M A P K K s in plant biology.  62  3.  Investigation of short-term transcriptional events induced by CA-MKK9  3.1.  Introduction  M A P K cascades have been identified as mediators of transcriptional changes in several organisms. In yeast, humans, Drosophila  melanogaster  transcription factors serve as targets of M A P K transcription factors  by  MAPKs  can modulate  and Caenorhabditis  elegans,  signaling. The phosphorylation of their  interaction  with  the  basal  transcription machinery, their D N A binding kinetics, their stability or their subcellular localization (Treisman, 1996). Although little is known about the substrates o f M A P K s in plants, there is growing evidence that transcription factors can be such phosphorylation targets. In rice, O s E R E B P l , an A P 2 / E R E B P family transcription factor, displayed enhanced D N A - b i n d i n g after phosphorylation by the nuclear-localized M A P K , B W M K 1 (Cheong et al., 2003). More recently, in tobacco, N t W I P K was found to increase the transcription modifying activity o f N t W I F , a transcription factor with homology to the A R F family (Yap et al., 2005), while N t S I P K has been reported to phosphorylate a N t W R K Y l transcription factor and thereby increase its D N A - b i n d i n g activity (Menke et a l , 2005). In addition, several studies report the translocation o f plant M A P K s into the nucleus, suggesting that they could modulate the activity o f transcription factors (Ligterink et a l , 1997; Kroj et al., 2003; Ahlfors et al., 2004; Lee et al., 2004).  63  Genome-wide expression profiling by microarrays provides an efficient way to capture the transcriptional re-modeling triggered by given stimuli. These  high-throughput  experiments can provide insight into the metabolic pathways activated or silenced by a stimulus o f interest. Moreover, they can also be used as tools to study co-regulated transcriptional modules. It is well-accepted that the transcription o f a gene is mediated by the interaction o f a transcription factor and specific D N A sequences (c/s-acting elements) in the promoter o f that gene. B y retrieving common cz's-acting elements in the upstream sequence o f co-regulated genes, one can generate hypotheses about the identity o f the transcription factors regulating the activity o f those genes (Yeung et al., 2004). This approach has been pioneered and explored mainly in the yeast model (Wyrick and Young, 2002), but appears applicable to a wide range o f organisms (Ohler and Niemann, 2001).  The inducible gain-of-function system described in Chapter 2 allowed me to trigger the activity o f A t M K K 9 , as well as o f its downstream signaling targets, i n a temporallycontrolled manner. It therefore presents an opportunity to investigate the transcriptional changes triggered by the M A P K cascade(s) to which A t M K K 9  contributes. In this  chapter, I present the results o f the transcriptional profiling o f D E X : C A - M K K 9 - F L A G plants using two-channel oligonucleotide microarrays. The goal o f this experiment was the identification o f metabolic pathways that are affected by C A - M K K 9 activity, and that might be contributing to the phenotypes o f the transgenic plants. In addition, I wished to analyze the common c/s-acting elements found in the promoters o f C A - M K K 9 - a f f e c t e d  64  genes to  create hypotheses about transcription  factors  that might  be  operating  downstream o f an A t M K K 9 / M A P K cascade.  The statistical analysis o f my microarray results showed that significant transcriptional changes could be detected two hours after C A - M K K 9 activation. The C A - M K K 9 affected genes included a number o f genes involved in the octadecanoid  pathway.  Moreover, I found that the promoters o f genes down-regulated in the short-term by C A M K K 9 were enriched i n A B R E - l i k e elements, pointing towards A B F proteins as putative targets o f M K K 9 - a c t i v a t e d M A P K s . However, my attempts to validate the microarray results using additional biological replicates and quantitative real-time ( Q R T ) - P C R revealed that the majority o f these earlyresponse microarray results were apparently false positives. I therefore conclude that the microarray experimental design I used was probably inappropriate to study early downstream targets o f C A - M K K 9 using the DEX-inducible system.  3.2.  Material and methods  3.2.1. Plant material and treatments  For each genotype and treatment, twenty to thirty plants were germinated and grown on soil. Seventeen days after germination, plants were sprayed with either a D E X solution (25 u M D E X and 0.015% Silwet-L77) or a control solution (0.015% Silwet-77). Rosettes  65  were collected and flash-frozen in liquid nitrogen, at the indicated times after the treatment.  3.2.2. Microarrays  3.2.2.1. RNA isolation Tissue (0.8-1.0 g) from the pooled frozen plants were ground in liquid nitrogen, and mixed with 15 ml T R I z o l reagent (Invitrogen). The samples were incubated for five minutes at 65°C with regular mixing, and centrifuged for twenty minutes at 4 ° C (12,000 rpm, Beckman J A 25.50). Three m l chloroform was then added and vortexed with the supernatant. The samples were spun for 30 minutes at 4 ° C (4,000 rpm, Sorvall R T H 750), and the aqueous phase transferred to new DEPC-treated Corex tubes. The extraction was repeated a second time with an equal volume o f chloroform. The samples were then precipitated with a half volume o f isopropanol and a half volume o f 0 . 8 M Na3citrate, gently mixed by inversion, and centrifuged for thirty minutes at 15°C (10,000 rpm, Beckman J A 25.50). The pellets were washed with 15 m l 70% ethanol, centrifuged for 20 minutes at 4 ° C (10,000 rpm, Beckman J A 25.50), air dried for five minutes and resuspended in 200-350 p i RNAse-free water. The R N A was quantified using a 2100 Bioanalyzer (Agilent Technologies) and re-precipitated overnight at -20°C with 0.1 volume o f 3 M N a O A c and 2.5 volumes o f 100% ethanol. Samples were then centrifuged for 15 minutes at 4 ° C (12,000g, microcentrifuge) and the pellets resuspended to a final R N A concentration o f 5 pg/pl.  66  3.2.2.2. cDNAflurorescentprobe preparation The reverse transcription reactions were performed using 70u.g total R N A into a 40 ul volume containing I X First-Strand buffer (Invitrogen), 3 . 7 5 u M Anchor-T primer, 5 0 0 u M dNTPs-dTTp, 5 0 u M d T T P , l O m M D T T and 0.025 m M C y 3 - or C y 5 - d U T P (Amersham), which were incubated for 5 minutes at 65°C and for 5 minutes at 42°C before the addition o f 40 U R N A s e O U T (Invitrogen) and o f 400 U Superscript II reverse transcriptase (Invitrogen). After incubation for two and a half hours at 4 2 ° C , the reactions were stopped with 8ul 1 M N a O H , incubated for 15 minutes at 65°, and neutralized by adding 8 ul 1 M HC1, 4 p i 1 M T r i s - H C l pH7.5 and 40 p i water. The labeled c D N A s were then purified using the Qiaquick P C R purification kit (Qiagen) according to the manufacturer's instructions and eluted twice in 50 p i . C y 3 - and Cy5-labeled samples were combined, 1 p i C y 5 - G F P was added, and the product was precipitated overnight at -20°C using 0.1 volume 3 M N a O A c and 2.5 volumes 100% ethanol.  3.2.2.3. Hybridization, scanning and image processing We used microarray slides printed with the Arabidopsis Genome Oligo Set Version 1.0 (26,090 70mer oligonucleotides). To prepare them for hybridization, the slides were shaken (80 rpm) for one hour at 48°C in prehybridization solution ( 5 X S S C , 0.1%> S D S , 0.2%) B S A ) . The slides were then rinsed twice in water, dipped five times i n isopropanol, and dried by a three minute centrifugation (2000 rpm, Haerreus) in 50 m l Falcon tubes. Meanwhile, the probes were spun down at 4°C for 30 minutes (14,000 rpm), briefly washed with 500 p i 70% ethanol and centrifuged for 15 minutes at 4 ° C (14,000 rpm), airdried, and resuspended in 3.5 p i l O m M E D T A . The probes were then denatured at 95°C 67  for two minutes, mixed with 50ul hybridization solution (Ambion) and kept at 65°C until used. The microarray slides were placed into hybridization chambers in which small slots were filled with 20 p i water to help prevent dehydration. O n each array, the probe was applied lengthwise as small drops and gently covered with an untreated glass cover slip (Fisher Scientific). The hybridization chambers were sealed and incubated in a shaking water bath (40 rpm) at 42°C for approximately eighteen hours. After the hybridization, the cover slips were removed and the slides washed for fifteen minutes once in 2 X S S C , 0.5% S D S , and twice i n 0 . 5 X S S C , 0.5% S D S , briefly rinsed in 0.1 X S S C and dried by a three minute centrifugation (2,000 rpm, Haereus) in 50ml Falcon tubes. Microarrays were scanned with a ScanArray Express (Perkin-Elmer) with laser power set at 90%) and photomultiplier tube set to 63-66%. The spots were then identified and quantified using the ImaGene software (BioDiscovery). Spot grids were placed manually and adjusted by using the Auto Adjust function three times. R a w spot and background intensities were saved as text files. Median spot and background pixel intensities were used for further analysis.  3.2.2.4. Data analysis The background was calculated as the mean of the 10 %> weakest spots for every subgrid, and subtracted from each spot signal value. The background-corrected intensities were then normalized for each channel using variance stabilization normalization ( V S N ) . This normalization method was chosen under the assumption that the variance observed between the two channels was independent o f the signal intensities (Huber et al., 2002). Therefore, for each slide, the variance was equalized across intensities.  68  A four-way A N O V A was performed on the normalized data. This statistical procedure was entirely designed and carried out by R i c k White, a statistical consultant for Genome B C . A summary o f the A N O V A design and results is presented in the Appendix. For filtering, p-values for the differential effect o f C A - M K K 9 at T=2 and T=0 were considered. The fold-change between T=2 and T=0 was obtained by exponentiating (10 ) the  absolute  FLAG  T = 2  /EV  T = 2  value  of  the  difference  between  ) and l o g ( D E X : C A - M K K 9 - F L A G - F L A G 2  log (DEX:CA-MKK9-FLAG2  T = 0  /EV  T = 0  ) . Ratios smaller than  one were converted to negative ratios by raising to the power -1 (X" ). 1  3.2.3. Functional and promoter analysis  The  in silico promoter analysis was performed using the web-based software package,  "Athena" (O'Connor et al., 2005). The A G I numbers o f the down- and up-regulated genes identified in the filtered gene lists from the microarray analysis were entered in the Analysis  Suite  application  (http://www.bioinformatics2.wsu.edu/cgi-  bin/Athena/cgi/analysis_select.pl). The promoter region o f these genes was retrieved as the 1000 base-pair sequence upstream o f the transcription start, cutting off at adjacent upstream genes as necessary. The software calculated the transcription factor binding frequency and enrichment o f the tested promoters.  3.2.4.  Quantitative Real-time R T - P C R  Total R N A was extracted from new plant samples with the RNeasy Plant M i n i kit (Qiagen). Total R N A (2 pg) was used for reverse transcription with OligodT (Invitrogen) 69  using Superscript II reverse transcriptase (Invitrogen) according to the manufacturer's instructions. The resulting c D N A samples were diluted by a factor o f ten i n water. Primers  were  designed  using  'Primer3'  (http://frodo.wi.mit.edu/cgi-  bin/primer3/primer3_www.cgi) to amplify a 140 to 160 bp unique region of the tested genes, i f possible spanning an intron. The specificity o f each primer pair was tested using B L A S T ( n ) on the Arabidopsis genome.  Table 3.1. Primers used for QRT-PCR AGI number Primer name Gene Description ACT8 (QRT)F Actin (ACT8) At1g49240 ACT8 R LOX3 F At1g17420 Lipoxygenase (LOX3) LOX3 R LOX4 2 F Lipoxygenase (LOX4) At1g72520 LOX4R OPR3 F At2g06050 2-oxophytodienoate (OPR3) OPR3 R OPR1 F 2-oxophytodienoate (OPR1) At1g76680 OPR1 R JMT F Jasmonic acid carboxyl At1g 19640 JMT R methyltransferase (JMT) WRKY F WRKY transcription factor At5g24110 WRKY R (WRKY30) UPS F Universal stress protein At1g48960 UPS R Zinc finger (C2H2 type) family ZnFgF At3g45260 ZnFg R protein StP F Stress-inducible protein At1g 12270 StP R GlyH F Glycosyl hydrolase family 1 At5g28510 GlyH R proteins  Sequence 5' TCTAAGGAGGAGCAGGTTTGA 3' 5' TTATCCGAGTTTGAAGAGGCTAC 3' 5' AAGAGGTTCCTTACCCTAGACGTT 3' 5' AAGTGTCCTGCTTCGACTCTTC 3' 5' CCGGGTGTTACGTGTAGAGG 3' 5' TCGGCAAATAAACCATACTGC 3' 5' GGAGTGGTCCGTTGAGCATA 3' 5' GGCACAAGGGAACTCTAACG 3' 5' AGACGGCTTGGTATCGAAGA 3' 5' CCGTATCCTTCATGAACTGG 3' 5' GCTTATTTTGGTGAAACCTTGC 3' 5' TCCTTGACGCTCAATACAGAAA 3' 5' AACTACTCCGGCGAACTTGA 3' 5' GGGGCAATTCTGAC I I I IGA 3' 5' GCCGTCACGGATACAATCTT 3' 5' AGAAGCATCGAAGCACCAAT 3' 5' CATCATCCCCTCTCATTTCC 3' 5' CGATGACTTCTGCATCTGGA 3' 5' GGACTTTGAAACTGCTATTCAGC 3' 5' CCCTTTCCACAGCCTTGTTA 3' 5' AATGCAATGGCGATAATGGT 3' 5' GAGGAAAAATTCTTGTCCATGAG 3'  For Q R T - P C R , 2 p i o f diluted c D N A (the equivalent o f 20ng R N A ) was mixed with 0.5 u M o f each primer and I X QuantiTect S Y B R Green P C R Master M i x (Qiagen) i n a total volume o f 20 p i . The reactions were ran i n a D N A Engine O p t i c o n ™ ( M J Research), using the following program: 95°C for 15 minutes, followed by 40 cycles o f amplification (94°C for 15 seconds, 55°C for 30 seconds, 68°C for 45 seconds, and a fluorescence reading). After a final elongation step o f 5 minutes at 68°C, a melting curve was  70  performed from 60 to 90°C, with a reading followed by a 1 second hold every 0.2°C, in order to assess the specificity o f the primers. The results o f each Q R T - P C R run were analyzed using Opticon M o n i t o r ™ Analysis Software version 1.07 ( M J Research). The threshold level was adjusted manually above background fluorescence. Each P C R run included a set o f A C T 8 standards (10 to 10 molecules), used to generate a standard 1  7  curve that allowed me to convert threshold cycle (C(T)) units into molecule units. The standards were obtained by serial dilution o f a purified A C T 8 P C R product whose concentration was determined independently by spectrophotometry.  The number o f  molecules for each gene o f interest was averaged from two technical replicates and divided by the number o f A C T 8 molecules amplified from the same biological sample.  3.3.  Results  3.3.1. Experimental design for the study of C A - M K K 9 - m e d i a t e d transcriptional changes  In order to study transcriptional changes resulting from A t M K K 9 activity, I assessed the global transcriptional profile o f multiple D E X : C A - M K K 9 - F L A G transgenic lines, using Arabidopsis oligomer microarrays. Two factors were considered with particular care when choosing a time-point for monitoring C A - M K K 9 - m e d i a t e d transcriptional changes. First, I wished to choose a time-point after D E X induction when the  CA-MKK9  transgene was strongly induced. I have previously described the induction pattern o f C A M K K 9 in D E X : C A - M K K 9 - F L A G transgenic lines (L2, L I 2 and L I 3 ) and shown that the transgene is transcribed at high levels two hours after D E X induction (Chapter 2;Figure 2.2). A t this same time, I also observed increased activity o f two M A P K s , suggesting that 71  C A - M K K 9 is active in the plants (Chapter 2; Figure 2.7). Second, the time-point had to be early enough to avoid capturing gene expression changes caused by secondary signaling pathways that might be triggered by early transcriptional events resulting directly from C A - M K K 9 activity. I had observed that, following the transgene activation, DEX:CA-MKK9-FLAG  plants started overproducing ethylene four hours after D E X  treatment (Chapter2; Figure 2.5), and treating plants with ethylene is known to induce transcriptional changes (Solano et al., 1998; Guo and Ecker, 2004). A s the goal o f my experiment was not to study ethylene-mediated transcriptional changes, but rather A t M K K 9 - m e d i a t e d changes, it appeared necessary to capture the transcriptional profile of the plants before they showed detectable production o f ethylene. Taken together, these data indicated that two hours after D E X induction (T=2) was an appropriate time-point to monitor global transcriptional activity in D E X : C A - M K K 9 - F L A G plants.  There are several caveats to the use of the DEX-inducible system for a global transcriptional study. Although the DEX-inducible system has been repeatedly used in plant research, the potential influence o f the chimeric transcription factor G V G on endogenous gene expression, independently or not o f D E X treatment, remains largely uncharacterized. It has been reported that high levels o f G V G in transgenic lines expressing the empty vector are correlated with growth defects, as well as with the expression o f defense-related genes such as  PDF1.2 and PR-5 (Kang et al., 1999). This is  of particular concern i n the context o f my study, since C A - M K K 9 appears to be involved in stress responses (Chapter 2). Furthermore, as the aim o f this microarray study was to capture early changes resulting from C A - M K K 9 activity, I reasoned that the presence o f  72  undefined noise background solely due to G V G could potentially hinder my ability to successfully capture these changes. In order to minimize the impact o f that hypothetical artefactual noise on the experiment, two controls were used. First, the microarrays were performed on three independent D E X : C A - M K K 9 - F L A G transgenic lines (L2, L 1 2 and L13). Second, each o f these transgenic lines was paired with an Empty Vector ( E V ) transgenic line expressing comparable levels o f G V G . Figure 3.1 shows that D E X : C A M K K 9 - F L A G L 2 had G V G levels similar to those in E V L 3 , whereas D E X : C A - M K K 9 F L A G L 1 2 was most similar to E V L 1 0 and  D E X : C A - M K K 9 - F L A G L13 to E V L l .  Based on this information, each o f these D E X : C A - M K K 9 - F L A G lines was paired to the appropriate E V line, and both were subjected to the same treatment in the microarray experiments.  ACT8  Figure 3.1. GVG transcript levels in DEX:CA-MKK9-FLAG and EV transgenic lines. DEX: CA-MKK9-FLAG plants (L2, L12 and L13) and EV plants (L1, L3 and L10) were grown for 18 days. Total RNA was extracted and reverse-transcribed. The levels of GVG transcript were determined by RT-PCR. Equal loading in each lane is indicated by the comparable ACT8 levels detected in each sample.  73  Another point to consider i n the experimental design o f the microarray was the basal expression levels o f the C A - M K K 9 transgene before D E X induction. M y time-course study o f C A - M K K 9 induction had revealed that in D E X : C A - M K K 9 - F L A G L I 3 , low levels o f C A - M K K 9 expression could be detected even before the D E X treatment (Figure 2.2). Replicates o f this experiment, in which higher number o f P C R cycles were performed to amplify the C A - M K K 9 c D N A , showed that the transgene displays similar basal expression levels in the two other lines (L2 and L I 2 ) as well (data not shown). This indicates that G V G might have some activity in the nucleus before the D E X treatment, which,  in addition to the  basal  and  induced expression  of C A - M K K 9 ,  might  constitutively influence the transcription o f other genes. Altogether, I hypothesized that the basal activity o f G V G and o f C A - M K K 9 might create a background o f responses that would contribute an unknown component to the expression profiles o f the D E X : C A M K K 9 - F L A G / E V plants two hours after D E X induction. In order to be able to subtract that background from the two-hour post-DEX transcriptional changes,  I  therefore  assessed the transcription profile o f the same D E X : C A - M K K 9 - F L A G / E V lines pairs before the D E X treatment (T=0). The experimental design o f the microarray experiment is pictured in Figure 3.2. For the L13/L1 and the L 1 2 / L 1 2 pairs, the arrays were performed on two biological replicates. For the L 2 / L 3 pair, arrays were done on one biological replicate.  74  DEX:CA-MKK9-FLAG L2  Vs  Vs  Vs  EVL1  E V L3  EVL10  B-2  B-1  1  1 T=2  T=0  J  B-1  B-2  BE]  T-2  T-2 T-4  T-0  T=2  T=0  B-1  DEX:CA-MKK9-FLAG L12  DEX:CA-MKK9-FLAG L13  T-3  B-2  B-1  T-2 T-2  *  T-2 T- 2  Total number of slides = 27  Figure 3.2. Microarray experimental design. DEX:CA-MKK9-FLAG L13 was hybridized against EV L1, DEX:CA-MKK9-FLAG L12 against EV L10, and DEX:CA-MKK9-FLAG L2 against EV L3. Plants were treated either with DEX (T=2) or with a mock spray (T=0) and collected two hours thereafter. The number of biological replicates (B-X) and technical replicates (T-X) is indicated for each experiment. (*) indicates that for one of the technical replicates, EV L3 RNA from B-1 was used.  3.3.2.  C A - M K K 9 mediates significant short-term transcriptional changes  After background subtraction and normalization o f the data, the statistical significance o f the expression differentials was determined using a multifactorial A N O V A (Appendix). The A N O V A revealed that there were indeed a significant number o f genes whose expression was altered due to the C A - M K K 9 transgene, between T=2 and T=0, suggesting that the microarray experiment was successful in revealing short-term C A M K K 9 - m e d i a t e d transcriptional changes. I generated a list o f the most significantly altered genes by filtering the normalized data according to the following criteria: the p-value should be <0.05 for the C A - M K K 9 effect between T=2 and T=0 data, and the observed fold-change should be >2 between T=2 and 75  T=0 data. Based on these criteria, I obtained a list o f 191 genes, among which only 11 were up-regulated while 180 were down-regulated (Appendix, Table 5.2). The large number o f down-regulated genes suggested that C A - M K K 9 might generally mediate transcriptional repression. Notably, the up-regulated genes included  AtMKK9/CA-  M K K 9 , indicating that our analysis method was at least able to accurately detect the transgene induction.  3.3.3. Functional analysis o f transcriptional changes resulting from C A - M K K 9 activity  Having retrieved the 191 genes whose expression was most altered by C A - M K K 9 induction, I wished to determine whether that group shared any functional similarities. To do so, I regrouped these genes based on their common gene ontology ( G O ) terms. The relative abundance o f each G O term assignment among these genes was compared with the abundance o f the given term across a random set o f 3000 genes (Table 3.2).  GO Slim Term  CA-MKK9 Total # %  Random set %  Cellular component other membranes other cellular components cellular component unknown other intracellular components chloroplast Nucleus mitochondria other cytoplasmic components ribosome extracellular plasma membrane ER cytosol plastid Cell wall Golgi apparatus Molecular function  61 53 42 42 39 35 23 19 8 8 8 5 3 2 1 1  23.2 10.7 19.2 9.2 9.6 8.1 7.6 4.5 2.5 1.5 1.1 0.7 0.7 0.8 0.7 0.3  17.4 15.1 12.0 12.0 11.1 10.0 6.6 5.4 2.3 2.3 2.3 1.4 0.9 0.6 0.3 0.3  76  GO Slim Term Hydrolase activity other enzyme activity Molecular function unknown Nucleotide binding DNA or RNA binding protein binding kinase activity transferase activity transporter activity other binding other molecular functions structural molecule activity nucleic acid binding transcription factor activity receptor binding or activity Biological process other physiological processes other metabolic processes other cellular processes protein metabolism transport biological process unknown other biological processes cell organization and biogenesis response to stress transcription response to abiotic or biotic stimulus signal transduction DNA or RNA metabolism developmental processes Electron transport or energy pathways  Random set  CA-MKK9 Total # % 13.7 63 47 10.2 41 8.9 40 8.7 37 8.0 32 7.0 29 6.3 29 6.3 28 6.1 27 5.9 27 5.9 23 5.0 19 4.1 15 3.3 3 0.7  9.4 14.1 15.8 5.4 7.1 3.9 6.0 9.8 7.2 6.7 3.0 2.8 3.2 4.2 1.0  183 149 148 68 53 46 31 29 27 23 17 13 13 11 7  21.2 19.1 18.1 6.7 5.8 10.1 3.8 1.9 1.7 3.2 1.5 1.3 1.6 1.2 2.1  22.4 18.2 18.1 8.3 6.5 5.6 3.8 3.5 3.3 2.8 2.1 1.6 1.6 1.3 0.9  %  The "cellular component" ontology o f C A - M K K 9 - a l e r e d genes indicated that these genes contain a large proportion o f extracellular- (2.3%), plasma membrane-  (2.3%)  and  endoplasmic reticulum- (1.4%) targeted proteins, as compared to the average percentages of these proteins i n the random set (1.5%), 1.1% and 0.7%>, respectively).  A s for the "molecular function" ontology o f C A - M K K 9 - a f f e c t e d genes, it appeared that "protein binding" (7%>) and "structural molecules" (5%) were the most  overrepresented,  as compared to the average representation (3.9% and 2.8%, respectively).  77  Most importantly, the analysis o f the "biological function" ontology group revealed that 3.3% o f C A - M K K 9 - a f f e c t e d  genes were involved in "response  to stress", which  represents a 94% over-representation compared to the 1.7% o f the random set that fell into this G O category. C A - M K K 9 - a f f e c t e d genes also contained more "cell organization and biogenesis" genes (3.5%) compared to the average (1.9%). It is notable that the C A MKK9-affected  gene group contained a disproportionately low number (0.9%) o f  "electron transport and energy pathway" genes, as compared to the random group (2.1%).  3.3.4.  Promoter analysis o f C A - M K K 9 - a f f e c t e d genes  I hypothesized that genes that had a similar early expression pattern following C A M K K 9 activation would share a common set o f cw-acting elements in their promoter region. Using the web-based application 'Athena', I retrieved the promoter region o f down- and up-regulated genes o f the C A - M K K 9 - a f f e c t e d  gene list, and identified  common binding factors motifs among these promoters. The frequency o f each motif among the promoter subset was compared to its frequency i n the entire Arabidopsis genome. Motifs that were significantly overrepresented (p-value<0.05) among the up- or down-regulated genes subsets are presented in Table 3.3. The C A - M K K 9 - d o w n - r e g u l a t e d genes were mostly enriched with A B R E - l i k e motifs (pO.OOl).  The  CA-MKK9-up-regulated  genes,  on  the  other  hand,  were  most  significantly enriched with the C C A 1 binding site motif (Table 3.3). However, because the latter gene set contains only ten genes, it is unclear whether meaningful biological conclusions can be derived from its analysis.  78  Table 3.3. Over-represented c/'s-acting elements among CA-MKK9-up- and downregulated genes. Subset #  Genome  %  %  Genome #  Pvalue  C A - M K K 9 - d o w n - r e g u l a t e d genes (180) ABRE-like binding site BACGTGKM motif CCAATGT LEAFYATAG  28%  50  18%  5493  0.001  13%  24  8%  2490  0.01  MYB1 binding site motif  MTCCWACC  9%  16  4%  1438  0.01  TGTCTC  38%  68  30%  9245  0.013 0.017  Motif Name  A R F binding site motif Z-box promoter motif UPRMOTIFIIAT R A V 1 - B binding site motif D R E core motif  Consensus sequence  3  Subset  ATACGTGT  5%  9  2%  674  CC(N)i CCACG  5%  10  2%  888  0.035  CACCTG  13%  24  9%  2808  0.036  RCCGAC  24%  42  18%  5550  0.039  54%  6  23%  7075  0.025  36%  4  13%  3965  0.046  2  CA-MKK9-up-regulated genes (11) C C A 1 binding site motif L1-box promoter motif  AAMAATCT TAAATGYA  0.049 4045 4 13% 36% CACGTGMOTIF CACGTG A, adenine; C, cytosine; G, guanine ; T, thymine ; R= A or G; Y= Cor T; W = A o r T ; S= C or G; M,=A or C; K= G or T; B=C, G or T; D= A, G or T; H= A, C or T; V= A, C or G; N= A, C, G o r T. a  In order to provide groundwork for future studies on potential A t M K K 9 - d r i v e n M A P K cascades leading to transcriptional changes, I explored the hypothesis that C A - M K K 9 might alter the activity o f a A B R E - b i n d i n g factor ( A B F ) , leading to the down-regulation of the 50 genes under the bearing these A B R E motifs. There are four A B F s characterized so far in Arabidopsis. I retrieved the amino acid sequences o f the A B F s , and identified putative M A P K phosphorylation sites, as well as M A P K docking and recognition sites in those sequences. M A P K s are known to phosphorylate serine and threonine residues that are followed by a proline residue (Cohen, 1997). Moreover, transcription factors targeted by the mammalian M A P K s E R K and p38 have a docking site known as the ' D domain', which is necessary for efficient phosphorylation by M A P K s . The D domain is typically upstream o f the phosphorylation sites, composed o f a stretch o f basic amino acids, followed by a L e u - X - L e u motif and a hydrophobic region (Tanoue and Nishida, 2003). Some M A P K substrates also have a M A P K recognition site called the Phe-X-Phe-Pro  79  motif, located downstream o f the phosphorylation sites (Tanoue and Nishida, 2003). A B F 2 and A B F 3 , w h i c h contained six and four, respectively, potential phosphorylation sites, stood out as most likely targets o f a C A - M K K 9 - d r i v e n M A P K cascade (Figure 3.3), although they had neither a D domain, nor a Phe-X-Phe-Pro motif However, as P h e - X Phe-Pro motifs are present i n none o f the known plant M A P K substrates, and putative D domains are found i n some but not all o f the latter, these two transcription factors could still be considered as M A P K substrate candidates i n future investigations. ABF1 - At1g49720 MGTHIDINNLGGDTSRGNESKPU^RQSSLYSLTFDELQSTLGEPGKDFGSMNMDELU<NIWTAEDTQAFMTTTSS VAAPGF«GFVPGGNGLQRQGSLTLPRTLSQKTVDEVWKYLNSKEGSNGNTGTDALERQQTLGEMTLEDFLLRAG WKEDNTQQNENSSSGFYANNGAAGLEFGFGQPNQNSISFNGNNSSMIMNQAPGLGLKVGGTMQQQQQPHQQ QLQQPHQRLPPTIFPKQANWFAAPVNMWIRG VFGRGRRSNTGLEKWERRQKRMIKNRESAARSRARKQAYTLELEAEIESLKLVNQDLQKKQAEIMKTHNSELKEF SKQ PPLLAKRQC LR RT LTGPW ABF2 - At1g45249 MDGSMNLGNEPPGDGGGGGGLTRQGSIYSLTFDEFQSSVGKDFGSMNMDELLKNIWSAEETQAMASGWPVLG GGQEGLQLQRQGSLTLPRTLSQKTVDQVWKDLSKVGSSGVGGSNLSQVAQAQSQSQSQRQQTLGEVTLEEFLV RAGVVREEAQVAARAQIAENNKGGYFGNDANTGFSVEFQQPSPRWAAGVMGNLGAETANSLQVQGSSLPLNV NGARTTYQQSQQQQPIMPKQPGFGYGTQMGQLNSPGIRGGGLVGLGDQSLTNNVGFVQGASAAIPGALGVGAV SPVTPLSSEGIGKSNGDSSSLSPSPYMFNGGVRGRKSGTVEKWERRQRRMIKNRESAARSRARKQAYTVELEA EVAKLKEENDELQRKQARIMEMQKNQETEMRNLLQGGPKKKLRRTESGPW ABF3 - At4g34000 MGSRLNFKSFVtXBVSEQQPTVGTSIPLTRQNSWSLTFDEFQNSWGGGIGKDFGSMNMDElXKNIWTAEESHSM MGNNTSYTNISNGNSGNTVINGGGNNIGGI^VGVGGESGGFFTGGSLQRQGSLTLPRTISQKRVDDVWKELMKE DDIGNGWNGGTSGIPQRQQTLGEMTLEEFLVRAGWREEPQPVESVTNFNGGFYGFGSNGGLGTASNGFVANQ PQDLSGNGVAVRQDLLTAQTQPLQMQQPQMVQQPQMVQQPQQLIQTQERPFPKQTTIAFSNTVDWNRSQPAT QCQEVKPSILGIHNHPMNNNLLQAVDFKTGVTVAAVSPGSQMSPDLTPKSALDASLSPVPYMFGRVRKTGAVLEK VIERRQKRMIKNRESAARSRARKQAYTMELEAEIAQLKELNEELQKKQVEIMEKQKNQLLEPLRQPWGMGCKRQC LRRTLTGPW ABF4 - At3g19290 MGTHINFNNLGGGGHPGGEGSSNQMKPTGSVMPLARQSSVYSLTFDELQNTLGGPGKDFGSMNMDELLKSIWT AEEAQAMAMTSAPAATAVAQPGAGIPPPGGNLQRQGSLTLPRTISQKTVDEVWKCLITKDGNMEGSSGGGGES NVPPGRQQTLGEMTLEEFLFRAGWREDNCVQQMGQVNGNNNNGFYGNSTAAGGLGFGFGQPNQNSITFNGT NDSMILNQPPGLGLKMGGTMQQQQQQQQLLQQQQQQMQQLNQPHPQQRLPQTIFPKQANVAFSAPVNITNKG FAGAANNSINNNNGLASYGGTGVTVAATSPGTSSAENNSLSPVPYVLNRGRRSNTGLEKVIERRQRRMIKNRESA ARSRARKQAYTLELEAEIEKLKKTNQELQKKQAEMVEMQKNELKETSKRPWGSKRQCLRRTLTGPW  Figure 3.3. Putative MAPK phosphorylation sites in ABRE-binding factors. Serine or threonine residues followed by a proline are shown in bold red.  80  3.3.5. C A - M K K 9 induction results in the down-regulation o f jasmonate biosynthesis genes.  Within the C A - M K K 9 - a f f e c t e d gene group were found a number o f genes coding for enzymes o f the octadecanoid pathway, which leads to the biosynthesis o f octadecanoid signaling metabolites, including jasmonic acid (JA). The first committed step in that pathway is the conversion o f the a-linolenic acid to 13-hydroperoxylinolenic acid, catalyzed by lipoxygenase enzymes (Figure 3.4).  a-linolenic acid |  Lipoxygenase (LOX)  13-hydroperoxy linolenic acid |  Allene oxide synthase (AOS)  Allene oxide |  Allene oxide cyclase (AOC)  12-oxo-phytodienoic acid |  12-oxo-phytodienoic acid reductase (OPR)  3-oxo-2(2 '-pentenyl)-cyclopentane-1 octanoic acid  I  |  B-oxidation  i (+)-7-iso-jasmonic acid |  Jasmonic acid methyl transferase (JMT)  (+)-7-iso-methyljasmonate Figure 3.4. The jasmonate biosynthesis pathway. Adapted from Devoto and Turner (2005).  81  According to my microarray data, L O X 2 , L O X 3 and L 0 X 4 ,  which all code for  lipoxygenases, were down-regulated upon C A - M K K 9 activation. L O X 2 and L O X 4 , which displayed a -2.8 and -2.7 fold-change, respectively, between T=0 and T=2, were the most strongly down-regulated, while L O X 3 showed a -2.1 fold-change (Table 3.4). Since it appeared that this might represent a significant aspect o f C A - M K K 9 - d r i v e n transcriptional activity, I asked whether  other genes coding for enzymes o f the  octadecanoid pathway might also have been affected by C A - M K K 9 activity, but not passed the initial filtering, i.e. they might not have shown an expression difference o f more than two-fold between T=0 and T=2. Keeping the p<0.05 criterion, I found that four additional genes involved in J A biosynthesis were also modestly down-regulated in the microarray data set as a result o f C A - M K K 9 activity. L O X 5 , which also codes for a lipoxygenase, was 1.4-fold downregulated, while O P R 1 and O P R 3 , coding for O P D A reductases, were down-regulated 1.4- and 1.7-fold, respectively. Finally, J M T , coding for a  S-adenosyl-L-methionine:jasmonic  acid  carboxyl methyltransferase,  was  down-  regulated 1.3-fold (Table 3.4).  Table 3.4. Octadecanoid pathway genes significantly altered by C A - M K K 9 activity. Oligo ID  AGI number  Name  Description  Fold-change  P-value  A009087_01  At3g45140  LOX2  Chloroplast lipoxygenase  -2.80  0.0437  A025792_01  At1g17420  LOX3  Lipoxygenase  -2.09  0.0252  A004372_01  At1g72520  LOX4  Lipoxygenase, putative  -2.73  0.0189  A011266_01  At3g22400  LOX5  Lipoxygenase, putative  -1.36  0.0188  A025313_01  At1g76680 OPR1  12-oxophytodienoic acid reductase  -1.42  0.0444  A007905_01  At2g06050  12-oxophytodienoate reductase  -1.72  0.0004  S-adenosyl-L-methionine:jasmonic acid carboxyl methyltransferase  -1.34  0.0290  A019988 01  OPR3  At1g 19640 JMT  82  Since these were not major shifts i n transcriptional intensity, and microarray data inevitably contain considerable noise, I re-evaluated the expression o f these genes by mean of an independent and more reliable method: quantitative real-time (QRT-) P C R . First, in order to see whether the microarrays results could be technically replicated, I used the same R N A samples that I had used for some o f the microarray replicates and measured the expression o f L O X 2 , L O X 3 and O P R 3 relative to the housekeeping gene A C T 8 . A s shown by Figure 3.5, Q R T - P C R analysis revealed that L O X 2 and L O X 3 were indeed down-regulated i n D E X : C A - M K K 9 - F L A G L 1 2 and L 1 3 , while O P R 3 expression was essentially unchanged. However, I also observed that these genes all appeared to be up-regulated in the E V L 1 0 line at T=2. This suggests that the down-regulation detected in the microarray analysis might be partly due to this E V - d r i v e n shift in ratio o f gene expression between D E X : C A - M K K 9 - F L A G L 1 2 and E V L10. Moreover, the presence o f gene expression changes in the E V L 1 0 genotype upon D E X induction indicates that this particular empty vector line was an inappropriate choice as a control, i n the context o f my microarray experiment.  83  L0X2 At3g45140  LOX3 At1g17420  fold change -2.80, p=0.0437  fold-change -2.1, p=0.0252 3.5  30 -  3  eculc  25  I  20 o E o 15 • >  2  5  I o  2 o * 1.5 o >  10 -  1  B  1  0.5 DEX:CA-MKK9FLAG L12  DEX:CA-MKK9FLAGL13  0  0EX:CA-MKK9FLAGL12  DEX:CA-MKK9FLAG L13  OPR3 At2g06050 fold-change-1.72, p=0.0004  T 1  ... I DEX:CA-MKK9FLAGL12  DEX:CA-MKK9FLAG L13  Figure 3.5. Technical validation of JA biosynthesis genes Total RNA of DEX:CA-MKK9-FLAG (L12 and L13) and EV (L1 and L10) used for the microarrays was used for QRT-RT analysis, to determine the abundance of LOX2, LOX3, OPR3 and ACT8 transcripts. Transcript levels are expressed as the ratio of molecules between the tested gene and ACT8 for a given samples. Values represent the average of two replicates. Error bars represent the standard error.  M y inability to validate the microarray results for O P R 3 , despite the apparent statistical robustness o f m y original results (p-value=0.004) led me to suspect that the putative down-regulation o f the suite o f J A biosynthesis genes might not be real. I therefore prepared new plant samples to more fully examine the validation o f this gene set by QRT-PCR.  Tissue samples from D E X . C A - M K K 9 - F L A G L I 2 , L 1 3 and E V L I plants  were collected i n triplicate at zero and two hours after D E X induction, i n the same manner as for the microarray experiments. Moreover, because I suspected that the twohour time-point might be so early that plant-to-plant variation i n the induction time would obscure meaningful transcriptional events, I also collected samples from additional plants  84  eight hours after the D E X induction. The E V L 1 0 control line was not tested in this experiment, as it appeared from my previous results that it displayed severe misregulation o f the J A biosynthesis genes upon D E X treatment. Figure 3.6 shows the expression pattern o f L O X 2 , L O X 3 , L O X 4 , O P R 1 , O P R 3 and J M T in the new biological replicates o f DEX-induced D E X : C A - M K K 9 - F L A G ( L I 2 and L I 3 ) and E V L I plants. Only L O X 2 and J M T appeared significantly down-regulated in both DEX:CA-MKK9-FLAG  lines, as compared to the E V line (Figure 3.6). Conversely,  L O X 4 and O P R 1 were up-regulated at T=2, while O P R 3 again appeared essentially unchanged (Figure 3.6). Most importantly, it appeared that the expression patterns o f these six genes displayed a roughly similar trend in the empty vector line ( E V L I ) as in the D E X : C A - M K K 9 - F L A G  lines (Figure 3.6). Overall, this suggests that the down-  regulation o f the octadecanoid pathway predicted by my microarray data may be more a reflection o f the effect o f D E X treatment than an effect o f C A - M K K 9 induction on the plants metabolism.  85  LOX2 At3g45140  fold-change -2.80, p=0.0437  DEX :CA-MKK9-FLAG L12 DEXCA-MKK9-FLAG L13  LOX4 At1g72520  fold-change -2.73, p=0.0189  LOX3 At1g17420 fold-change -2.09, p=0.0252  DEX:CA-MKK9-FLAG L12 DEX:CA-MKK9-FLAG L13  OPR1 At1g76680 fold-change -1.42, p=0.0444  T  DEX;CA-MKK9-FLAG L12 DEX: CA-M KK9-FIAG L13  OPR3 At2g06050 fold-change -1.72, p=0,0004  1  DEX:CA-MKK9-FLAG L12 DEXCA-MKK9-FLAG L13  EVL1  JMT At1g19640  fold-change -1.33, p=0.0290  DEX:CA-MKK9-FLAG U12  DEX:CA-MKK9-FLAG L13  DEXCA-MKK9-FLAG L12  DEXCA-MKK9-FLAG L13  Figure 3.6. QRT-PCR validation of down-regulated jasmonate biosynthesis genes predicted by microarray to be down-regulated by CA-MKK9. Independent tissue samples of DEX:CA-MKK9-FLAG (L12 and L13) and EV (L1) plants were collected 0, 2, and 8 hours after DEX treatment. The abundance of LOX2, LOX3, LOX4, OPR1, OPR3, JMT and ACT8 transcripts was determined by QRT-PCR. Transcript levels are expressed as the ratio of molecules between the tested gene and ACT8 for a given samples. Values represent the average of three independent samples, for which QRT-PCR was performed in duplicates. Error bars represent the standard error. The response of each gene as previously assessed by microarray analysis is shown in smaller font in each graph header.  86  3.3.6. Validation o f the most robust C A - M K K 9 - a f f e c t e d genes  The results described in 3.3.5 suggested that the C A - M K K 9 - a f f e c t e d gene set, as determined by the microarray experiment, contained a large proportion o f false positives. In order to determine whether this was due to insufficient stringency in our filtering strategy, or to a general problem o f the microarray results, I examined the expression pattern o f a subset o f those genes that displayed the most statistically robust expression differentials in the microarray experiments. Seeing this analysis as a potential preliminary study for further investigation, I chose to investigate only those genes for which some functional annotation was available. The five chosen genes had the lowest p-values amongst the annotated genes o f the C A - M K K 9 - a f f e c t e d gene set (Table 3.5).  Table 3.5. Most significant CA-MKK9-affected genes Oligo ID  AGI number  Description  Fold-change  P-value  A016008..01  At5g24110  WRKY 30, member of WRKY Transcription Factor; Group III  3.25  0.000054  A002005..01  At1g48960  universal stress protein (USP) family protein  3.34  0.000056  A024355..01  At3g45260  zinc finger (C2H2 type) family protein  -2.21  0.000084  A004610..01  At1g 12270  stress-inducible protein, putative  -2.05  0.000417  A019910..01  At5g28510  glycosyl hydrolase family 1 protein  -2.02  0.000487  Figure 3.7 shows the Q R T - P C R analysis o f the expression o f these five genes, in independent samples o f D E X : C A - M K K 9 - F L A G plants (L12 and L13) and E V L I plants, measured at zero and two hours after D E X induction. Despite the statistical robustness o f the expression data for these genes in the microarrays, only W R K Y 3 0 could be consistently confirmed as being up-regulated between T=0 and T=2 (Figure 3.7).  87  WRKY30 Atg24110  Universal stress protein At1g48960  fold-change 3.25, p=0.000054  fold-change 3.34, p=0.000056 0.5 0.45 0.4 0.35 0.3 0.25 0.2  0.4  0.15 0.1  0.2  0.05 0 DEX:CA-MKK9-FLAG L12  DEX:CA-MKK9-FLAG L12 DEX:CA-MKK9-FLAG L13  DEX:CA-MKK9-FLAG L13  Zinc finger protein At3g45260  Stress-inducible protein At1g12270  fold-change -2.21, p=0.000084  fold-change-2.05, p=0.000417  0.9  1.6  0.6  1.4  0.7 "5  EVL1  1.2  ao  0.6  o E 0.5 o % 0.4  1  E o 0.8 n 0.6  1  T - f -  •  stive  > 01 0.3  T — t —  S 0.4  0.2  0.2  0.1 0  0 DEX:CA-MKK9-FLAG L12 DEX:CA-MKK9-FLAG L13  DEX:CA-MKK9-FLAG L12  DEX:CA-MKK9-FLAG L13  EVL1  Glycosyl hydrolase At5g28510 fold-change -2.02, p=0.000487 0.006 0.005 8 0.004 aj o E ? 0.003 'I 0.002 E 0.001 0 D£X:CA-MKK9-FLAG L12 DEX:CA-MKK9-FLAG L13  Figure 3.7. QRT-PCR validation of the most significant CA-MKK9-affected genes. Independent tissue samples of DEX:CA-MKK9-FLAG (L12 and L13) and EV (L1) plants were collected 0, 2 hours after DEX treatment. The abundance of At5g24110, At1g48960, At3g45260, At1g12270, At5g28510 and ACT8 transcripts was determined by QRT-PCR. Transcript levels are expressed as the ratio of molecules between the tested gene and ACT8 for a given samples. Values represent the average of three independent samples, for which QRT-PCR was performed in duplicates. Error bars represent the standard error. The response of each gene as previously assessed by microarray analysis is shown in smaller font in each graph header.  88  3.4.  Discussion  In animal systems, M A P K cascades play a crucial role in relaying the information conveyed by an extracellular stimulus to the nucleus (Treisman, 1996). In plants, although the number and diversity o f M A P K modules suggests that M A P K cascades play important roles in the plant life cycle, only a few studies have explored the downstream molecular events specifically resulting from M A P K activity. Crosstalk and overlapping functions between different M A P K modules, as well as the transient nature o f their activation, can make it difficult to analyze the downstream targets o f a single M A P K . Asai et al. (2002) successfully dissected a single M A P K cascade and its downstream targets by re-constructing the cascade in Arabidopsis protoplasts, using a transient expression system. They showed that challenge with a flagellin elicitor, which is recognized by a specific membrane-bound receptor-like kinase, F L S 2 , can activate a MAPK  cascade involving A t M E K K l , A t M K K 4 / A t M K K 5  and  AtMPK3/AtMPK6,  ultimately leading to the transcription o f W R K Y 2 9 and W R K Y 1 (Asai et al., 2002).  The inducible expression  in planta o f a gain-of-function mutant o f a M A P K K , A t M K K 9 ,  should, in principle, provide another good experimental system to study the downstream targets o f a single M A P K cascade. A s shown in the previous chapter, D E X : C A - M K K 9 F L A G transgenic plants rapidly express the C A - M K K 9 transgene upon D E X induction (Chapter 2, Figure 2.2). Moreover, I have found that the transgene activation results in the activation o f A t M P K 6 . A s this M A P K can be translocated into the nucleus (Ahlfors et a l , 2004), it appeared possible that its activity would cause rapid transcriptional changes.  89  In this Chapter, I conducted a microarray experiment that aimed at capturing genomewide transcriptional events resulting from C A - M K K 9 activity. A multifactorial A N O V A allowed me to identify genes that were predicted to be significantly altered as a result o f the C A - M K K 9 transgene induction, between zero and two hours after the D E X treatment (Appendix). In order to gain clues as to the identity o f cellular processes regulated by C A - M K K 9 activity, I characterized the predicted C A - M K K 9 - a f f e c t e d gene set using gene ontology (Table 3.2). This analysis revealed that several genes o f the octadecanoid pathway appeared down-regulated i n the microarrays upon C A - M K K 9 induction (Table 3.4). Since J A and ethylene have been proposed to play antagonistic roles in-the control of pathogen- and ozone-induced programmed cell death (Overmyer et al., 2003), these results seemed especially interesting in the light o f our previous finding regarding C A M K K 9 ' s role in ethylene biosynthesis (Chapter 2). In addition, I was able to detect a significant over-representation o f A B R E - l i k e  elements in the promoter region o f the  down-regulated gene set, which suggested a link between A t M K K 9 and A B A and G A signaling, as well as A B F proteins as putative targets o f M A P K s (Skriver et al., 1991; Choi et al., 2000) (Table 3.3, Figure 3.3). However, the gene expression patterns found by the microarrays i n the C A - M K K 9 - a f f e c t e d gene set could not be validated by Q R T P C R (Figure 3.6, Figure 3.7), suggesting that the aforementioned analyses do not reflect real biological processes.  Overall, therefore,  the microarray experiment  was  not  successful in detecting early downstream transcriptional events triggered by C A - M K K 9 , despite the scale and level o f replication employed.  90  It is possible that the failure o f the microarray experiment is due to flaws in its experimental design. The hybridizations were designed to obtain a direct comparison between a given D E X : C A - M K K 9 - F L A G line and a given E V line. This model was used under the assumption that a D E X : C A - M K K 9 - F L A G line and a E V line o f comparable G V G level should behave similarly in every respect, apart from the C A - M K K 9 - m e d i a t e d transcriptional events (Figure 3.1). Moreover, I assumed that the D E X treatment would have only minor effects on the E V lines. However, I found that the E V L 1 0 line displayed marked mis-regulation o f J A biosynthesis genes upon D E X induction (Figure 3.5), making it quite plausible that this line also shows mis-regulation o f other genes. The results o f the multi-factorial A N O V A support this hypothesis, as they demonstrated that a relatively high proportion o f genes showed altered expression in the D E X : C A - M K K 9 F L A G L 1 2 / E V L 1 0 pair compared to the other two combinations o f transgenic lines (Appendix; Figure 5.1C, D ) . A re-analysis o f the G V G levels in the E V L 1 0 plants revealed much higher G V G levels than had been originally observed (data not shown), indicating that the use o f this E V line was inappropriate in the first place. Moreover, the Q R T - P C R analysis o f J A biosynthesis gene expression in another empty vector line, E V L l , showed that those genes were also down-regulated in this line, suggesting that a general pattern exists where D E X application influences the metabolism o f plants in which the G V G transcription factor is being constitutively expressed (Figure 3.6). Taken together, these observations suggest that the behavior o f pTA7002 E V lines could explain the high number o f false-positive in the microarrays. Since I was comparing gene expression between D E X : C A - M K K 9 - F L A G plants at T=2 and T=0 in an indirect manner, by looking at the ratio between D E X : C A - M K K 9 - F L A G and E V lines at both  91  time-points, any artefactual gene expression in the E V lines is likely to have been falsely interpreted as a C A - M K K 9 - d r i v e n process. Moreover, indirect comparisons have less statistical power  than  direct  ones.  Therefore,  true C A - M K K 9 - d r i v e n  expression  differential may have been missed because o f insufficient statistical power in the design. A s we were most interested i n differences between D E X . C A - M K K 9 - F L A G plants at T=2 and  T=0,  it  would  have  been  more  appropriate  to  directly co-hybridize  the  aforementioned samples together, instead of running each against a E V line.  Despite these challenges, I was able to identify and verify W R K Y 3 0 as a transcriptionally up-regulated gene responding to C A - M K K 9 activation (Figure 3.7). The W R K Y family of transcription factors is unique to the plant kingdom and contains 74 members (Ulker and Somssich, 2004). There is growing evidence that W R K Y transcription factors are downstream players i n M A P K cascade. The promoter o f W R K Y 2 9 was shown to be activated upon flagellin-dependent activation of the A t M E K K l - A t M K K 4 / 5 - A t M P K 3 / 6 cascade (Asai et al., 2002). Moreover, W R K Y 3 3 was recently identified as an in vitro substrate o f A t M P K 4 , and knock-out mutants o f both W R K Y 3 3 and A t M P K 4 showed the same SA-related phenotype (Andreasson et al., 2005). Tobacco W R K Y 1 was also recently shown to be a direct substrate for S I P K (the tobacco orthologue o f A t M P K 6 ) (Menke et al., 2005). There is only limited information available concerning the biological role o f W R K Y 3 0 in Arabidopsis. It has been reported that the transcription o f W R K Y 3 0 is activated by challenge to the plant tissue from both host and non-host types of pathogen, in a SA-independent manner (Kalde et al., 2003). It would therefore be  92  interesting to examine whether A t M K K 9 influences susceptibility to pathogen attack through its participation in the regulation of W R K Y 3 0 .  93  4.  Future directions  In an effort to gain information about the function o f the M A P K K A t M K K 9 in Arabidopsis, I have characterized several aspects o f D E X : C A - M K K 9 - F L A G transgenic plants, which express an inducible constitutively active version o f A t M K K 9 . The results of this study confirm preliminary data from our laboratory suggesting roles for A t M K K 9 in programmed cell death ( P C D ) and in regulating ethylene biosynthesis. Moreover, I have established that A t M K K 9 could act as an upstream effector o f the M A P K A t M P K 6 , and that its activity results in the up-regulation o f a specific transcription factor, W R K Y 3 0 . To conclude this thesis, I wish to propose future directions for the presented research.  4.1. Role of AtMKK9 in ethylene biosynthesis in vivo The results presented in Chapter 2 strongly suggest a role for A t M K K 9 in modulating the A t M P K 6 - c o n t r o l o f A C S stability, and thereby ethylene biosynthesis. However, as the evidence obtained so far relies solely on the ectopic expression o f C A - M K K 9 , the function o f A t M K K 9 i n its endogenous form remains purely hypothetical. In order to gain insights into the biological context i n which A t M K K 9  stimulated  ethylene  production, several approaches can be used. First, it is necessary to determine when and where A t M K K 9 is active. Publicly available gene expression databases, as well as R T P C R surveys performed i n the Ellis laboratory, suggest that the transcription o f A t M K K 9 is increased under a number o f conditions associated with ethylene production, including senescence, cold stress, and pathogen infection (data not shown). These can be used as a starting point to investigate how changes i n the activity o f A t M K K 9 might be correlated  94  with specific biological processes. Moreover, since a complex pattern o f cross-talk exists between the hormones ethylene, salicylic acid ( S A ) and jasmonates, it would also be interesting to investigate whether the exogenous application o f these hormones can affect A t M K K 9 activity. While the activity o f M A P K s i n response to different stresses can be easily assessed by subjecting proteins to in-gel kinase assays using the myelin basic protein substrate, or indirectly, as I did, using anti-phospho-ERK antibodies, measuring the activity o f MAPKKs  directly remains a technical challenge. T o do so, one might have to  immunoprecipitate either the endogenous M A P K K using a specific antibody, or an epitope-tagged version o f the M A P K K , and test its ability to activate in vitro a known cognate M A P K substrate. F o r instance, one could immunoprecipitate A t M K K 9  from  proteins extracted from tissue treated under an appropriate test condition, and measure its ability in vitro to activate recombinant A t M P K 6 , which I have already established to be an A t M K K 9 substrate. Once the biological contexts o f A t M K K 9 activity have been defined, it would be possible to explore whether reducing the activity o f A t M K K 9 , using transgenic plants expressing a D E X : A t M K K 9 - R N A i construct (which the Ellis laboratory has obtained), alters ethylene production in that particular context.  4.2. Investigation into the CA-MKK9-activated 42 kDa MAPK I have also found that the induction o f C A - M K K 9 is associated with the activation o f a 42 k D a M A P K  in vivo (Chapter 2, Figure 2.7). A s I have done for A t M P K 6 , a  combination o f crude protein extract analysis with a specific antibody, in vitro kinase assay, and epistatic analysis using a knock-out mutant o f the appropriate M A P K , would 95  allow us to characterize that second C A - M K K 9 - a c t i v a t e d M A P K cascade. A s mentioned in Chapter 2, A t M P K 4 stands out as the most likely candidate corresponding to that 42 kDa M A P K . Interestingly A t M P K 4 and A t M P K 6 are co-activated by a bacterial elicitor, harpin, independently o f R O S production. Moreover, A t M P K 4 activity is correlated with harpin-induced P C D (Desikan et al., 2001). If we can obtain data showing that C A MKK9  indeed activates A t M P K 4 , it would be interesting to investigate whether  knocking-out A t M P K 4 can suppress or reduce C A - M K K 9 - m e d i a t e d P C D . This might require the use o f an inducible R N A i suppression approach, given the dwarf phenotype o f the characterized transposon-tagged mpk4 mutant (Petersen et al., 2000),  4.3.  Signaling events in CA-MKK9-mediated PCD  The analysis o f the D E X : C A - M K K 9 - F L A G / m / ? A ; r 5 double-mutant revealed that the P C D phenotype induced by C A - M K K 9 does not require A t M P K 6 activity or increased ethylene production (Chapter 2, Figure 2.11).  In order to investigate whether C A -  MKK9-mediated P C D results from the mis-regulation o f another pathway, we could assess the status o f other major P C D regulators, S A and jasmonates. Quantitatively measuring these hormones by gas chromatography is possible, but can be technically challenging. A s a first step, or an alternative to directly measuring S A and jasmonate levels, the transcriptional activity o f marker genes for these hormones could easily be assessed by R T - P C R . These markers include, for instance, PR1 for S A and PDF 1.2 for jasmonates (Rao et al., 2002). In addition, it has been previously reported that M A P K K activity can trigger the onset o f P C D by up-regulating respiratory burst oxidase homologue genes (Yoshioka et al., 2003). 96  In Arabidopsis, the activity o f AtrbohD and AtrbohF during pathogen attack modulates R O S production and subsequent P C D (Torres et al., 2002). It would therefore  be  interesting to assess the expression o f the genes coding for these enzymes in induced D E X : C A - M K K 9 - F L A G plants. If we find that one or more Atrboh genes is indeed upregulated  following  CA-MKK9  construct with the corresponding  induction, combining the  DEX:CA-MKK9-FLAG  Atrboh knock-out mutant would allow us to assess  whether R B O H activity is required for C A - M K K 9 - m e d i a t e d P C D .  4.4. Mechanisms of CA-MKK9 regulation of WRKY30 The microarray experiment and associated Q R T - P C R presented in Chapter 3 has revealed that C A - M K K 9 induces a significant increase in the expression o f W R K Y 3 0 , a gene coding for a group III W R K Y transcription factor. Several reports have proposed a link between  WRKY  transcription factors  and M A P K  signaling (Asai  et  al., 2002;  Andreasson et al., 2005). I have established A t M P K 6 as a substrate o f A t M K K 9 . In order to test whether the up-regulation o f W R K Y 3 0 is due to A t M P K 6 activity, as opposed to another A t M K K 9 - d r i v e n pathway, we could use Q R T - P C R to compare  WRKY30  transcript levels in D E X : C A - M K K 9 - F L A G plants and the D E X : C A - M K K 9 - F L A G / m / ? & f 5 double-mutant plants. Moreover, certain W R K Y transcription factors can bind their own promoter, thereby promoting their transcription in a positive feedback loop (Asai et al., 2002). Therefore, it is possible that W R K Y 3 0 is, in fact, a direct substrate of a C A - M K K 9 - a c t i v a t e d M A P K . We could test the ability o f C A - M K K 9 - a c t i v a t e d M A P K s ,  such as A t M P K 6 ,  to  phosphorylate W R K Y 3 0 by in vitro kinase assays using P-labelled A T P . Moreover, i f 32  97  we find that W R K Y 3 0 is indeed an A t M K K 9 - M A P K substrate, we could assess changes in its ability to activate a reporter gene, such as P-glucuronidase, upon induction o f C A M K K 9 in transgenic plants.  4.5. Concluding remarks Overall, the results presented in this thesis represent essential groundwork that enables further exploration o f the biological and molecular functions o f A t M K K 9 . In particular, as shown by the proposed experiments o f this concluding chapter, I believe that we now have exciting opportunities to investigate in detail the role o f endogenous A t M K K 9 , particularly in the context o f stress responses involving ethylene. The data and resources I have developed w i l l also make it possible to define, biochemically and genetically, the pattern o f interactions between C A - M K K 9 and its downstream targets, and to place those interactions in their biological context.  98  5.  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Z u o , J., N i u , Q.W., a n d C h u a , N . H . (2000). Technical advance: A n estrogen receptorbased transactivator X V E mediates highly inducible gene expression in transgenic plants. Plant J 24, 265-273.  109  6.  Appendix  6.1.  Microarray data analysis  The data from the microarray experiment described in Chapter 3 was subjected to a fourway  A N O V A . This statistical procedure was entirely designed and conducted by a  Genome B C statistical consultant, R i c k White. The A N O V A tested the effect o f four independent factors on gene expression. Table 6.1 shows the factors tested, as well as the levels within each factor. For the sake, o f simplicity the variable numbers o f biological replicates were ignored, and all replicates were considered as technical replicates. Table 6.1. Factorial design of the microarray experiment Factor Transgene Transgenic line Dye Time-point  Levels 1. 2. 1. 2. 3. 1. 2. 1. 2.  CA-MKK9 EV DEX;CA-MKK9-FLAG L2/ EV L3 DEX;CA-MKK9-FLAG L12/EV L10 DEX;CA-MKK9-FLAG L13/ EV L1 Cy3 Cy5 T=0 T=2  This statistical test assessed importance o f the different variables accounted for in the microarrays, i.e. the two C y - dyes, the three pairs o f transgenic lines, the presence or absence o f the C A - M K K 9 transgene  and the two time-points, as well as possible  interactions between these factors (Table 6.1). B y looking at the percentage o f genes altered due to each o f the factors, it was possible to estimate whether differentials recorded by the microarray experiment were more likely to be artefactual, or to reflect changes induced by the C A - M K K 9 transgene two hours after its induction.  110  The main effect o f each factor was determined, as well as the interaction effect o f all factors combinations. M a i n and interaction effects were quantified by an estimate value and their significance by a p-value and q-value. We found that, except for the dye factor, significant gene expression differences were mainly due to interactions between factors, rather to each factor taken independently. For instance, differences due to the transgenic lines could be detected when that factor was assessed in combination with the C A - M K K 9 transgene and the T=2 time-point. Figure 6.1 presents the most relevant results o f the A N O V A , i.e. the dye effect, the transgenic line effect (with interactions with the C A M K K 9 transgene and the T=2 time-point), the T=0 time-point (with an interaction with the C A - M K K 9 transgene) and the difference between T=2 and T=0 (with an interaction with the C A - M K K 9 transgene). The percentage above each graph estimates the number of differentially expressed genes. It represents the number o f genes falling above the graph's blue bar,.i.e. above threshold o f false discovery, and therefore encompasses both some true positives (p-value <0.05) as well as some false negatives  (p-value>0.05)  (Figure 6.1). Surprisingly, more than 50% o f the observed difference can be attributed solely due to the type o f dye used to label the two c D N A preparations, which shows the importance o f 'dye balance' in microarray experiments (Figure 6.1 A ) . However, almost 45%> o f the tested genes showed differences in expression due to factors other than the dye (Figure 6 . I B ) . Although I assumed that C A - M K K 9 expression would induce a common set o f transcriptional changes in each o f the three D E X : C A - M K K 9 - F L A G transgenic lines ( L 2 , L 1 2 and L13), it was also possible that each line would display a somewhat different set o f transcriptionally altered genes. Both at T=0 (Figure 6.1C) and T=2 (Figure 6.ID), D E X : C A - M K K 9 - F L A G L 1 3 / E V L l and D E X : C A - M K K 9 - F L A G  111  L 2 / E V L 3 had only - 1 0 % genes whose differential expression could be attributed solely to the transgenic background, whereas D E X : C A - M K K 9 - F L A G L 1 2 / E V L 1 0 displayed more than 20 % o f the detected gene expressions altered because o f that factor. This indicated that either D E X : C A - M K K 9 - F L A G L 1 2 or E V L 1 0 had significantly increased levels o f background transcription variation. Ultimately, the multi-factorial A N O V A procedure permitted me to estimate the impact o f C A - M K K 9 expression after two hours induction. A s shown by Figure 6. I E , expression o f <7% o f the responding genes was altered by C A - M K K 9 before the D E X treatment (T=0), indicating that the basal expression o f the transgene was probably insignificant. B y contrast, expression o f more than 35% o f the genes was altered by C A - M K K 9 at T=2, as compared to T=0 (Figure 6.IF). Overall, this indicated that the T=2 time-point was sufficiently late to detect above-background changes.  112  B 44.63 % D i f f E x p ,  53.87 % DiffExp  t 111 Whole-model parametric p-values  Dye parametric p-values  27.17 % D i f f E x p c cu  10.6 % Diff Exp  o ti c '  s  IT  C12/P10_CA-MKK9_T= p-values  C13/P1_CA-MKK9_T=0 p-values  C2/P3_CA-MKK9_T=0 p-values  D 13.51 % D i f f E x p  13.49 % DiffExp  . Diff Exp O 8 C «? CU  CU 3  C13/P1 _CA-M KK9_T=2 p-values  C12/P10_CA-MKK9_T=2 p-values  6.89 % Diff Exp  3  s  C2/P3_CA-MKK9_T=2 p-values  36.6? % Diff Exp  cu  8  CT K  0>  cu "•  CA-MKK9_T=0 p-values  CA-MKK9_(T=2)-(T=0) p-values  Figure 6.1. Effect of dye type, transgenic lines and time-point factors on gene expression differentials. Normalized intensities were analyzed by ANOVA. A) Histogram of p-values for genes showing differential expression due to the dye factor B) Histogram of pvalues for genes showing differential expression due to factors other than the dye 113  C) Histogram of p-values for genes showing differential expression due to CAMKK9 in the L13/L1, L12/L10 and L2/L3 backgrounds at T=0 D) Histogram of pvalues for genes showing differential expression due to the L13/L1, L12/L10 and L2/L3 backgrounds at 1=2 E) Histogram of p-values for genes showing differential expression due to CA-MKK9 at T=0 F) Histogram of p-values for genes showing differential expression between T=2 and T=0 due CA-MKK9. The blue line on each graph represents the threshold for false discoveries. The percentage is the estimated the number of differentially expressed genes.  6.2.  CA-MKK9-acffected genes  From the results obtained from the A N O V A , I filtered the genes that had a p-value smaller than 0.05 for the C A - M K K 9 effect between T=2 and T=0, and a fold-change greater than 2 between T=2 and T=0. Based on these criteria, I obtained a list o f 191 genes, among which only 11 were up-regulated while 180 were down-regulated (Table 6.1). These " C A - M K K 9 - a f f e c t e d genes", were used for further analysis.  Table 6.2. CA-MKK9-affected genes AGI Annotation OligoJD number A017580 01 At5g 14370 expressed protein A022201 01 At3g60430 hypothetical protein A011628 01 At3g21320 hypothetical protein A016008 01 At5g24110 member of WRKY Transcription Factor; Group III A002005 01 At1g48960 universal stress protein (USP) family protein A024355 01 At3g45260 zinc finger (C2H2 type) family protein A020698 01 At1g73500 member of MAP Kinase Kinase At1g 14780 expressed protein A001407_01 At1g 14790 A003480 01 At1g56660 expressed protein A004610 01 At1g 12270 stress-inducible protein, putative A019910 01 At5g28510 glycosyl hydrolase family 1 protein A024327 01 AM g21730 kinesin-related protein (MKRP1) leucine-rich repeat transmembrane protein A022148_01 At3g57830 kinase, putative, several receptor-like protein kinases eukaryotic translation initiation factor 2 family A002523_01 At1g76810 protein A007441 01 At2g 16650 expressed protein A004372 01 At1g72520 lipoxygenase, putative A022092 01 At2g 12700 hypothetical protein, phosphatidylinositol-4-phosphate 5-kinase family A024399_01 At3g 14270 protein A017268 01 At5g66210 calcium dependent protein kinase  Foldchange 3.59 -2.08 -2.17 3.25 3.34 -2.21 3.62  0.00000004 0.00004081 0.00004334 0.00005380 0.00005592 0.00008371 0.00009900  -2.23  0.00022182  -2.38 -2.05 -2.02 -2.38  0.00027813 0.00041654 0.00048739 0.00055993  -2.53  0.00057055  -2.11  0.00065006  -2.37 -2.73 -2.45  0.00067833 0.00070827 0.00072686  -2.32  0.00076290  -2.07  0.00101666  P-value  114  OligoJD  AG I number  A019068_01  At5g 10570  A011361 A006305 A024172 A014851 A015780 A024227  01 01 01 01 01 01  A025843_01 A024619_01 A017280 01 A024527_01 A020705 01 A001349_01 A000484_01 A002332 01 A009035_01 A007751 01 A007745 01 A010624 01 A013282_01 A023593_01 A019768_01 A015694_01 A017036_01 A006948 01 A008938 01 A006035 01 A015257_01 A025445_01 A007228_01 A003052 01 A005267_01 A005086 01 A013882_01 A006891_01  Annotation  basic helix-loop-helix (bHLH) transcription factor family protein At3g08530 clathrin heavy chain, putative At2g34930 disease resistance family protein At5g17440 LUC7 N terminus domain-containing protein At4g34260 expressed protein At5g56420 F-box family protein At1g18620 expressed protein leucine-rich repeat family protein / protein kinase At1g53430 family protein formin homology 2 domain-containing protein / At3g25500 FH2 domain-containing protein At5g35980 expressed protein GA-responsive GAST1 protein homolog regulated by BR and GA antagonistically. At1g75750 Possibly involved in cell elongation based on expression data At5g46210 cullin, putative phosphoribulokinase (PRK) / At1g32060 phosphopentokinase tRNA synthetase class II (G, H, P and S) family At1g29870, "protein At1g21170 expressed protein SNF2 domain-containing protein / helicase At3g42670 domain-containing protein At2g34080 cysteine proteinase, putative At2g21440 RNA recognition motif (RRM)-containing protein At3g14230 AP2 domain-containing protein RAP2.2 interacts with H+-ATPase, and regulates its At4g27500 activity SWIB complex BAF60b domain-containing At2g 16480 protein / plus-3 domain-containing protein At1g70320 encodes a ubiquitin-protein ligase-like protein At1g55860 containing a HECT domain proline oxidase, putative / osmotic stressAt5g38710 responsive proline dehydrogenase 26S proteasome AAA-ATPase subunit RPT3 At5g58290 (RPT3) At2g07360 hypothetical protein At3g57410 villin 3 (VLN3) At2g22125 C2 domain-containing protein coatomer gamma-2 subunit, putative / gamma-2 At4g34450 coat protein, putative / gamma-2 COP FAT domain-containing protein / At2g 17930 phosphatidylinositol 3- and 4-kinase family protein At2g45530 zinc finger (C3HC4-type RING finger) family At2g45540 protein At1g 18040 cell division protein kinase, putative required for regulated cell expansion and normal root hair development. Encodes an evolutionarily At3g 13870 conserved protein with putative GTP-binding motifs At1g22930 T-complex protein 11 microtubule associated protein (MAP65/ASE1) At4g26760 family protein U5 small nuclear ribonucleoprotein helicase, At2g42270 putative  Foldchange  P-value  3.19  0.00117855  -2.66 -3.11 -2.02 -2.04 2.08 -2.41  0.00119467 0.00121429 0.00124474 0.00141990 0.00153535 0.00185639  -2.05  0.00190918  -2.08  0.00191832  -2.03  0.00215544  2.16  0.00222445  -2.15  0.00223091  -2.70  0.00226019  -2.53  0.00234548  -2.48  0.00240396  -2.73  0.00248381  -2.53 -2.62 -2.00  0.00260725 0.00296144 0.00302789  -2.36  0.00324237  -2.09  0.00362380  -2.39  0.00367355  -2.02  0.00376199  -2.38  0.00381498  -2.09 -2.05 -2.38  0.00406205 0.00406751 0.00410772  -2.28  0.00421507  -2.42  0.00424829  -2.05  0.00426283  -3.17  0.00433710  -2.53  0.00460739  -2.39  0.00472917  -2.22  0.00483178  -2.13  0.00484676  115  OligoJD  AGI number  A021782 01  At3g09500  A020511_01  At3g 11450  A010510_01  At3g04000  A007454_01  At2g29580  A011306_01  At3g12280  A023758_01  At1g20510  A005126 A016205 A014038 A012702 A002217 A017468 A003654  01 01 01 01 01 01 01  At5g20990 At5g05940 At4g28910 At3g22860 At1g79820 At5g25280 At1g63700  A006384_01  At2g47410  A025177 01 A006954 01 A025264 01  At3g 13470 At2g39340 At5g08680  A011866_01  At3g47900  A011688 A011639 A004615 A012703 A019449 A023293  01 01 01 01 01 01  At3g 19055 At3g13300 At1g61690 At3g52250 At1g22770 At1g73020  A024622_01  At2g 18960  A020036 A008112 A011694 A000981  01 01 01 01  At5g42220 At2g33435 At3g 18490 At1g35220  A016574_01  At5g05730  A007934 01 A018889 01 A001355 01  At2g40540 At5g65180 At1g30795  A022994_01  At5g38670  A016129 01 A025923 01  At5g45510 At4g25860  A015007_01  At4g 17330  A001250_01  At1g03090  A000336_01  At1g63830  A000052 01 A005416 01 A010358 01  At1g04810 At5g27030  Annotation 60S ribosomal protein L35 (RPL35A) DNAJ heat shock N-terminal domain-containing protein / cell division protein-related short-chain dehydrogenase/reductase (SDR) family protein zinc finger (CCCH-type) family protein / RNA recognition motif (RRM)-containing protein encodes a retinoblastoma homologue in Arabidopsis. This protein is involved in G1/S cell cycle transition in fungi, animals, and plants. 4-coumarate—CoA ligase family protein / 4coumaroyl-CoA synthase family protein homologous to E. coli MogA expressed protein expressed protein member of elF3c - eukaryotic initiation factor 3c hexose transporter, putative serine-rich protein-related member of MEKK subfamily (YODA) teransducin family protein / WD-40 repeat family protein chaperonin, putative SAC3/GANP family protein ATP synthase beta chain, mitochondrial, putative ubiquitin carboxyl-terminal hydrolase family protein hypothetical protein required for leaf development tetratricopeptide repeat (TPR)-containing protein myb family transcription factor late flowering protein expressed protein ATPase 1, plasma membrane-type, putative / proton pump 1, putative / proton-exporting ATPase, putative ubiquitin family protein RNA recognition motif (RRM)-containing protein aspartyl protease family protein expressed protein anthranilate synthase, alpha subunit, component 1-1 (ASA1) putative potassium transporter AtKT2p (AtKT2) expressed protein hydroxyproline-rich glycoprotein family protein F-box family protein, similar to SKP1 interacting partner 6 (Arabidopsis thaliana) leucine-rich repeat family protein oxysterol-binding family protein gene of unknown function expressed in seedlings, flower buds and stems MCCA is the biotinylated subunit of the dimer MCCase, which is involved in leucine degradation. proline-rich family protein, contains one predicted transmembrane domain 26S proteasome regulatory subunit WD-40 repeat family protein transcription activation domain-interacting  Foldchange  P-value  2.06  0.00489962  -2.78  0.00492631  -2.35  0.00494051  -2.59  0.00505960  -2.11  0.00510689  -2.04  0.00513408  -2.32 -2.07 -2.26 -2.74 -2.06 -2.54 -2.07  0.00517218 0.00518187 0.00519377 0.00521121 0.00522019 0.00527032 0.00528430  -2.07  0.00532912  -2.78 -2.08 -2.20  0.00544900 0.00556886 0.00567328  -2.09  0 00573976  -2.42 -2.59 -2.12 -2.34 -2.85 -2.69  0.00576834 0.00583690 0.00583922 0.00588580 0.00594350 0.00600035  -2.23  0.00641275  -2.01 -2.40 -2.10 -2.03  0.00651547 0.00666955 0.00668733 0.00679655  -2.17  0.00680408  -2.31 -2.23 -2.35  0.00712780 0.00717650 0.00729700  -2.25  0.00737730  -2.04 -3.18  0.00763283 0.00781124  -2.01  0.00783359  -2.19  0.00792468  2.05  0.00798142  -2.07 -2.32 -2.13  0.00798700 0.00817355 0.00833219  :  116  A019846 01 A017482 01  AGI number At3g21480 At2g38440 At5g65520  A002163_01  At1g 13980  A020665 A016135 A024969 A016311 A001896 A024871  01 01 01 01 01 01  At4g32330 At5g24710 At4g08410 At5g42100 At1g31730 At5g65770  A001286_01  At1g31780  A000214 A018082 A017224 A008949 A020631 A005346 A013832 A003446 A002003 A013575 A002462 A013433  01 01 01 01 01 01 01 01 01 01 01 01  At1g79280 At5g54230 At5g38370 At3g26560 At5g54720 At1g 10290 At4g29060 At1g80070 At1g21610 At4g 16660 At1g06560 At4g01530  A006925_01  At2g34210  A011741_01  At3g23430  A003159 01 A007459 01  At1g50770 At2g44480  A006978_01  At2g24050  A021895_01  At5g21274  A023935 A025950 A001320 A023141 A005998  01 01 01 01 01  At1g60030 At3g54580 At1g61210 At3g52870 At1g29790  A018921_01  At5g49920  OligoJD  A004668 01  At5g42020 At5g28540 At5g02220 At1g34410 At1g35520 At1g35540 At1g43950 At1g35240 At1g34310 At1g34390 At1g 14970  A012453_01  At3g23670  A011166 01  At3g45450  A021021_01 A019610 01  A025517_01  Foldchange  P-value  -2.37 -2.20  0.00840097 0.00840525  -2.11  0.00844582  -2.13 -2.01 -2.08 -2.69 -2.37 -2.67  0.00844932 0.00883518 0.00907410 0.00933898 0.00944185 0.00963027  -2.03  0.00985685  -2.01 -2.30 -2.92 -2.19 -2.17 -2.23 -2.13 -2.32 -3.63 -2.69 -2.01 -4.52  0.00988185 0.00991258 0.00992506 0.00993022 0.01003557 0.01007926 0.01117506 0.01121576 0.01121903 0.01156464 0.01182247 0.01216392  -2.04  0.01222729  -2.02  0.01231944  -2.10 -2.16  0.01256179 0.01319754  -2.36  0.01324092  2.26  0.01356201  -2.18 -2.03 -2.84 -2.11 -2.03  0.01360638 0.01375120 0.01385491 0.01416477 0.01446589  -2.85  0.01497564  luminal binding protein (BiP)  -2.93  0.01521725  expressed protein  -2.10  0.01624952  transcriptional factor B3 family protein / auxinresponsive factor AUX/IAA-related  -2.24  0.01658463  -2.16  0.01692409  -2.16  0.01705467  -2.11  0.01711266  Annotation protein-related putative WAVE homolog expressed protein homologous to Sec7p and YEC2 from yeast. Involved in the specification of apical-basal pattern formation. expressed protein WD-40 repeat family protein proline-rich extensin-like family protein glycosyl hydrolase family 17 protein epsilon-adaptin, putative nuclear matrix constituent protein-related conserved oligomeric Golgi complex componentrelated / COG complex component-related expressed protein putative transcription factor (MYB49) hypothetical protein ATP-dependent RNA helicase ankyrin repeat family protein dynamin-like protein 6 (ADL6) elongation factor Ts family protein wound-responsive family protein heat shock protein 70, putative / HSP70, putative NOL1/NOP2/sun family protein hypothetical protein, KOW domain-containing transcription factor family protein mutant is deficient in the transfer of phosphate from root epidermal and cortical cells to the xylem hypothetical protein glycosyl hydrolase family 1 protein MIF4G domain-containing protein / MA3 domaincontaining protein encodes a calmodulin isoform. Expressed in leaves. xanthine/uracil permease family protein proline-rich extensin-like family protein expressed protein calmodulin-binding family protein expressed protein octicosapeptide/Phox/Bemlp (PB1) domaincontaining protein  expressed protein phragmoplast-associated kinesin-related protein, putative Clp amino terminal domain-containing protein  117  OligoJD A014080 A014743 A018414 A008021 A002117  01 01 01 01 01  A001296_01 A000710 01 A001238 01  AGI number At4g00450 At4g20850 At5g09840 At2g36910 At1g79350 At1g48850 At1g48840  01 01 01 01 01  At1g48090 At1g 16270 At2g40680 At5g52065 At1g62740 At1g 15340 At1g 15770 At1g05660 At3g09840  A003280_01  At1g28060  A002139_01  At1g09770  A002953 01 A012158 01 A008463 01  At1g15130 At3g01370 At2g 16050  A017880_01  At5g67470  A020961 01  At1g21630  A019880_01  At2g42600  A014493 A025792 A015984 A020779  01 01 01 01  At4g32410 At1g 17420 At5g40450 At5g42530  A013669_01  At4g24190  A023370 01  At2g47090  A019158_01  At5g 15720  A008967 01 A017561 01  At3g54670 At5g01840  A020581_01  At1g06670  A025862 01 A001141 01 A006250 01  At1g79920 At1g06950 At2g30690  A007327_01  At2g26080  A016937 01  At5g64570  A014105_01  At4g26690  A024149 01  At2g34910  A008647_01 A003023 A020650 A000449 A025319 A024330  Foldchange -2.64 -2.25 -2.30 -2.21 -2.12  0.01730766 0.01746549 0.01754231 0.01762966 0.01810830  -2.16  0.01816726  -2.06 -2.23  0.01836100 0.01844679  hypothetical protein  -2.15  0.01890859  stress-inducible protein, putative methyl-CpG-binding domain-containing protein expressed protein polygalacturonase, putative / pectinase, putative member of AAA-type ATPases small nuclear ribonucleoprotein family protein / snRNP family protein member of MYB3R- and R2R3- type MYBencoding genes hydroxyproline-rich glycoprotein family protein expressed protein DC1 domain-containing protein formin homology 2 domain-containing protein / FH2 domain-containing protein calcium-binding EF hand family protein encodes one of four Arabidopsis phosphoenolpyruvate carboxylase proteins. cellulose synthase catalytic subunit Lipoxygenase expressed protein expressed protein encodes an ortholog of GRP94, an ER-resident HSP90-like protein and is involved in regulation of meristem size and organization. Single and double mutant analyses suggest that SHD may be required for the correct folding and/or complex formation of CLV pro zinc finger (C2H2 type) family protein GDSL-motif lipase/hydrolase family protein, similar to family II lipase EXL3 and EXL2 Cohesion ovate family protein nuclear DEIH-box helicase (NIH) encoding a putative RNA and/or DNA helicase homologous to a group of nucleic acid helicases from the DEAD/H family heat shock protein 70, putative / HSP70, putative chloroplast inner envelope protein-related expressed protein glycine dehydrogenase (decarboxylating), putative / glycine decarboxylase, putative / glycine cleavage system P-protein, putative beta-xylosidase glycerophosphoryl diester phosphodiesterase family protein expressed protein  -2.17 -2.11 -2.37 -2.30 -2.16  0.01897061 0.01925895 0.01944278 0.01955440 0.01958703  -2.41  0.02082370  -2.25  0.02108034  -2.22 -2.08 -2.25  0.02302878 0.02307892 0.02326059  -2.05  0.02346571  -2.78  0.02356713  -2.01  0.02432634  -2.10 -2.09 -2.47 2.24  0.02457772 0.02519402 0.02534816 0.02549915  -2.05  0.02582019  -2.06  0.02634684  -2.05  0.02634738  -2.20 -2.10  0.02642654 0.02734042  -2.25  0.02735415  -2.20 -2.22 -2.10  0.02756319 0.02799841 0.02946237  -2.12  0.02952559  -2.35  0.03045088  -2.17  0.03049098  -2.99  0.03110226  Annotation mutant has dwarf; late flowering phenotypes subtilase family protein expressed protein member of MDR subfamily DNA-binding protein, putative chorismate synthase, putative / 5enolpyruvylshikimate-3-phosphate phospholyase, putative C2 domain-containing protein protein kinase family protein  P-value  118  OligoJD  AGI number  A002067_01  At1g56560  A002569 01  At1g70220  A018932_01  At5g04140  A022473 01  At4g 12770  A016210_01  At5g 14780  A001488 01 A025933 01  At1g62750 At1g59610  A020824_01  At2g48080  A010371 01  At3g14010  A018064_01  At5g50920  A016207_01  At5g 13630  A005168_01  At1g53540  A009087_01  At3g45140  A013257 01  At4g35800  A025632_01  At3g61830  Annotation beta-fructofuranosidase, putative / invertase, putative / saccharase, putative / betafructosidase, putative hypothetical protein encodes a gene whose sequence is similar to ferredoxin dependent glutamate synthase (FdGOGAT). Expression in leaves is induced by light and sucrose. Proposed to be involved in photorespiration and nitrogen assimilation auxilin-related NAD-dependent formate dehydrogenase 1B (FDH1B) elongation factor Tu family protein dynamin-like protein, putative (ADL3) oxidoreductase, 20G-Fe(ll) oxygenase family protein hydroxyproline-rich glycoprotein family protein CIpC mRNA, nuclear gene encoding chloroplast protein encodes magnesium chelatase involved in plastid-to-nucleus signal transduction 17.6 kDa class I small heat shock protein (HSP17.6C-CI) chloroplast lipoxygenase required for woundinduced jasmonic acid accumulation in Arabidopsis RNA polymerase II large subunit transcriptional factor B3 family protein / auxinresponsive factor AUX/IAA-related  Foldchange  P-value  -2.07  0.03150710  -3.32  0.03158312  -2.05  0.03427149  -2.03  0.03490292  -2.13  0.03499392  -2.10 -2.27  0.03579065 0.03632609  -2.03  0.03641010  -2.05  0.03670238  -2.12  0.04055739  -2.36  0.04093862  -2.08  0.04261357  -2.80  0.04374736  -2.70  0.04459444  -2.04  0.04571103  119  

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