An Analysis of ERA 1 Functlon in Arabidopsis

by

Dario Bonetta

A thesis eubmitted in confomity with the requirements

for the degree of Doctor of Philosophy 8 Graduate DeparbmMt of Botany University of Toronto

@ Copyright by Dario Bonetta 2000 uisitions and Acquisitions et 7-Bib qiephic Services rM#N bibliographiques

The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant B la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distn'bute or sell reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/nlm, de reproduction sur papier ou sur format électronique.

The author retains ownership of the L'auteur conseme la propriété du copyright in ththesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permi*ssion. autorisation. ABSTRACT The elaboration of multicellular st~duresis a complex balance between cell division, growth and differentiation. At the molecular level, this balance is maintained by a dynamic array of regulatory mechanisrns which are tightly coordinated. This coordination is partly achieved through the pst-translational modification of proteins by famesyl lipid attachment. Since its initial description, protein famesylation has figured an important rnechanism for modifying protein function either by facilitating protein interaction with cell membranes or by mediating protein-protein interaction. Although a great deal of information has accumulated on the characteristics of the famesyltransferases which catalyze the attachment of farnesyl to various proteins, the divenity of proteins that are post-translationally famesylated has made it difficult to assess the utility of this type of modification in regulating cellular events. The cornmon thread linking many famesylated proteins, however, is their involvement in cell division and growth. ln this regard, this study describes the isolation of an Ambidopsis nucleosome assembly protein (NAP1) which is famesylated and which interacts with Arabidopsis cyclin 61. In addition, the analysis of era al of Arabidopsis, which is cuventl y the only famesylt ransferase loss-of- function mutant of any multicellular organism, has afforded the unique opportunity of assessing the role that farnesylation plays in regulating various processes during the development of a multicellular organism. This characterization indicates that famesyltransferase activity is required for proper coordination of cell division and differentiation during Arabidopsis vegetative and reproductive development. Loss of famesylation affects both apical and axillary meristem development and these phenotypes are contingent on plant growth conditions. Farnesylation appears to negatively regulate cell-cycle gene expression in the lower central core of apical meristems. This region has been identified previously by both genetic and molecular studies to be essential for overall menstem organization. It appears famesylation also plays a role in the organization of this center. UST OF ABBREVIATIONS BAC bacterhl artifcial chromosome bp, kb base pair, kilobase (pair) BSA bovine senirn albumin CAMP cyclic adenosine-3'-5'monophosphate cDNA complernentary DNA

OC degrees Centigrade d deoxy Da Dalton Dex dextrose DNA deoxyribonucleic acid DNAse deoxyribonuclease dNTP deoxynucleotide DTT 1 ,&dithiothreitol E Einstein E. coli Eschen'chia coli EMS ethylenediaminesulfonic acid EDTA ethylenediaminetetra-acetic acid EGTA ethyleneglycd-bis@-aminoethyl ether) N,N, N',N'-tetraacetic acid EST expressed sequence tag Fig . figure FPP famesyl pyrophosphate Fiase famesy ltransferase 9 sravny G protein guanine nucleotide-binding protein Ga1 galactose G@PP geranylgeranyl pyrophosphate GGTase geranylgeranyltmnsfetase GTP guanosine 5'-triphosphate GTPase guanosine 5'-triphosphatase h houi(s) Hepes N-[2-hydroxyethyl]pipe~ine-N'-[2.eaianesulfonicacid] IPTG isop ropyl $-D-thiogalacbpy mnoside Km aMty constant LB Luria-Bertani broth M molar mA milliAmpere(s) min rninute(s) iii mol mole(s) MPa megaPascals N. number NCBl National Center for Blotedinology Information 00 optical density PAGE polyacryiamide gel electropho resis PBS phosphate buffered saline probability ioselectric point 3-hydroxy-e(2-sulfO-e(4-sulfophenyIazo) ph- napthalenedisulfonic acid PMSF phenyl melhyl solfonyl fluoride Raf raffinose RNA ribonucleic acid s.d. standard deviation SOS sodium dodecylsuHate TCA trich loroacetic acid Tris N, N, N', Nt-tetramethylethylethylenediamine v volt(s) vol volume volume per volume weight per volume 5-b romo4-~hlor~-indolylB-D-galactopyranoside 5-bromo-4-chlore3-indolyl p-Dglucopyranoside TABLE OF CONTENTS

Chapter 1 Plant Development and CeII Signaling...... -.

Special features of plant development and structure ...... ee.e.e...... ss...... 0s...2 The role of hormones in plant development...... e...e...... 4

ABA as a mediator of plant water SWUS...... e~e.e.o...... s.....s.e.e.,...... *...*...... *e.9

Genetic analysis of ABA synthesis and signal transduction ...... ee...e..oe...... ~....e.. .0...14 ABA biosynthetic-mutant phenotypes...... 19

ABA response mutants ...... ~o.....e.e...e....e..e...... e.e.o...... es... .s.....o...o....21 More genetic ~alysis...... s.o...e...... oe..~.o~~~...... e.....ee.....ss...... e...... ~..e.31

Research 0bj8Ctk8~.e....~..~~~**eo.m~e~oeo*~~~***e****m*~~~~~e~*~o.e*~~-m~~o~~~..~~ee~~~~~~o~~oœ.~~~~~.~~~~~~~o.~~~~~~~~~~.32 Yeast interaction trap cloning ...... *..41 GST-NAP1 overexpression plasmid constnidon...... ***44 Afïinity chromatogmphy...... *...... *...... *.45 Affinity Purification of Arabidopsis NAP1 binding proteins ...... *.**.48 Generation and affinity purification of anti-NAPI antibodies ...... *.49 Phenotypic characterization of wild-type and era 7 plants ...... s...... C**.m50 Cyclin-GUS Staining ...... s...... 50 ln sihr Hybridization...... 51 pEGCYC and pEGABl construction ...... 52

Chapter 3 Farnesylation Intlwnter Reproductive Development in Arabldopsk ...... *...... *58 lntroduct ion...... *...... *...... *59 Rmb...... ~~~~~t~sC~~~~~m~~**~C*~~~~~~e~~~~~~~~~~~~~~~~~~e~~*~~~~~~~~~~~~~~~**~~~~~******~~~~~~~~~~s~~~*~e~m*~s~~~~62 era 1 mutants show subtle morphological phenotypes in constant light ...... 64 era 1mutants have reduced branching in short day conditions ...... 64 era 1 affects reproductive apical meristems development in short day ...... 68 era 7 mutants affect axillary meristem development in short day conditions ...... 69

ERAI is involved in meiosis ...... CC.**~.*C.*...**.*...... *....***.*.....*.....*..~e...... **m.... *.....74 era 7 mutants have altered cyclin gene expression in short dey ...... *.76 ERAl mRNA expression patterns are conelated with short day grown ml phenotypes ...... *.....*.....m.**m*s*e**D*bm****m79 Suppressors and enhancers of era 7 ...... *...... *.*.80 Discussion...... 85 Famesylation and cellular dflemntiatim...... *...... *...... mm85

Famesylation and the cell cycle ***e*~~*m**e*~**m~mm*m~~~**~me~~~~~c**n**~*mm*mmee~*cm**m*s*~~c~c~*c~cso~o~om~*~~e*m86 Famesyiation and ABk...... C*C~CC*m89 Chapter 4 Napl ir Çarnesylated and Interacts with CYC1At ~e.*e~.~m.~e.em*m~emm~eemm~mmemm~memmem91 Introduction...... 92

Re8utb...... *.W....***...... m...... e...... m...... 9Q Yeast interaction trap cloning ...... 99 NAPl analysis ...... ~...... s...... 05 Recombinant NAPl protein characteristics...... 108 NAPl is famesylated in vitro ...... 110 NAP1 interacts with a cydin B...... 1 12 NAPl protein expression ...... 115 Discu8sion...... ~...... m...... e...... 118 €RA1 interacting proteins ...... e.m...... e...m...... 118 NAP1 in vivo function ...... 20

Chapter 5 GemlConclusions ...... m...... 124

Model for ERA1 function in the meristem .*..**.*..* .. **.*.*...**.*..*..*....*..*....**.**.*...... * 25 Future prospects...... 1 26 A mode1 for signal integration ...... 3 1

APPENDIX 1 ...... 1 37 BIBLIOGR APHY ...... 1 42

vii Statement of Publications The research presented in this thesis has appeared or will be submitted as a series of original publications in referred joumals: Chaptw 3: Bonetta, D., Bayliss, P., Sage, T. and McCourt, P. (1 999) Famesylation is involved meristem organization in ArabMopds. Submitted to Planta.

Chapter 4: Bonetta, 0. and McCourt, P. (1999) NAPl famesylation and its interaction with cyclin B1 of Arabidopsis. ln pre paration.

viii LIST OF FIGURES Fig. 1.1 The structure and some of the functions of plant hormones ...... 6 Fig. 1.2 Schematic showing the ABA biosynthetic pathway in higher plants...... 16 Fig. 1.3 Possible role for famesylation in ABA-rnediated signaling ...... 30 Ftg. 2.1 pEG202 and pJG4-5 ...... n...... ~...... 53 Fig . 2.2 pSH18.34 ...... e.e...*...*...... -54 Fig .2.3 pSH17-4 and pRFHM 1...... ~...... em...~...... 55 Fig. 2.4 pGEXNAP.....~...... ~...... 56 Fig . 2.5 pBC-ERA used for generating RNA probes for in situ hybridization...... 57 Fig. 3.1 Famesylation assay on flower bud tissue of wild-type (WT) and of era 1 mutants...... 63 Fig. 3.2 Whole plant morphology of wild-type and ma1 plants grown in continuous light or short day ...... 66 Fig. 3.3 Meristem development in wild-type and era 1 in short days ...... 70 Fig. 3.4 Organ number frequencies par whori in wild-type and era 1flowers ..n.n...... m...... 72 Fig. 3.5 Characteristics of morphology of reproductive structures in era 7 in short day ...... 73 Fig . 3.6 Pollen development in wild-type and era 1...... 75 Fig. 3.7 Distribution of CYC1At::GUS expressing cells in root and vegetative meristems of wild-type and enil ...... m..m.nn..m...78 Fig. 3.8 ln situ hybridization of wild-type tissue fram SD grown plants

usirg ER41 DIGRNA p~be.*en.*œm..bB*.~D~****m.**B~~B.*B~~.*~m~m~~~~~O~~~~~m~eB*~*O~emb~~~~se~~ ~.~~nnmm~~~~nmm**~om82 Fig. 4.1 Cartoon showing the potential fates of famesylated pmteins...... 98 Fig. 4.2 Assay to detemine that the pEGERA product does not activate transcription of the LexAopLEU2 reporter...... m.me....lO1 Fig. 4.3 Scheme showing the set-up for a mating assay used to test interacting clones obtained in the screen...... 103 Fig. 4.4 Norlhem blot analysis using the NAPl cDNA to probe total seedling mRNA ...... 106 Fig. 4.5 A schematic showing the structure of aie Arebidopsis NAPl gene ...... 107 Fig. 4.6 GST-NAPI fusion protein expression in E. coN...... 109 Fig. 4.8 Famesylation and geranylgeranylation assays using wiicl-?yy and era 1 protein extracts ...... 111 Fig. 4.8 NAP1 interading proteins...... 113 Fig .4.9 Yeast interaction assay showhg the specific interaction of NAP1 with CYCAL ...... 114 Fig . 4.10 Western blot probed with antCNAP1 antibodies ...... 117 Fig. 5.1 Model showing the possible relationship between €RA1 and ABA eff ects in meristem function...... 135 Fig. 5.2 Schematic showing a simple neural net and its possible analogy to factors affecting germination ...... 136 LIST OF TABLES Table 1.1 Coneletion between the effects of abscisic acid (ABA) and water CHAPTER 1 PLANT DEVELOPMENT AND CELL SlGNALlNQ Spial Ibsturm of plant &vel~mnntand sûuctum The complexity of tissue differentiation in higher animals requires that the most critical phases of development occur in a more or less protected environment that is free from variation or extemal perturbation. In contrast, the ecological niche occupied by plants requires a potentially different strategy of development. Rather than a hierarchical mode of development, which is Rnely tuned mostly by endogenous factors, plant development occurs mostly after embryqenesis in an indeteminate way, and seems to be influenced mostly by extemal factors. Continual organogenesis in plants might mean that plants are capable of reorganizing their growth to best exploit their physical environment and of exhibiting a range of phenotypes from a single genotype. In plants, each organ would be essentially the outcome of the influence of the environment on the genotype. Changes in photoperbd or light intensity, nutrient and water status, or pathogens can have profound effects on the overall architecture of plants. A possible underlying consequence of this is that the number of tissue types in higher plants is greatly reduced compared to animals. In flowering plants for example, there are no more than 40 cell types which are generally only distinguishable by their anatomical context (Lyndon, 1990). In addition, there is a certain degree of functional redundancy between cell types in a plant and most are not teminally differentiated; retaining the capacity to redifferentiate into other cell types often independently of neighboring cells. The classic example of cellular totipotency is the ability of tobacco leaf protoplasts to fom an embryonic calus in tissue culture. Cells can dedifferentiate, divide, organize a polarized embryo and grow into a fertile plant that is phenotypically indistinguishable from a sexually derived individual, if supplied with the appropriate chernical information (Walbot, 1996). Although tissue regeneration in higher animals also exists, it is limited to only certain tissues under specific circumstances, such as limb regeneration in newts. In contrast plants continually modify their development in response to their sumwndings. In a sense, it would seem that the degree of tissue-type complexity is relinquished for an equal degree of complexity in developmental potential. Therefon, complexity in plants might exist at the cellular level rather than at the tissue or organ level, since individual plant cells retain such a high degree of developemntal potential. Plant structure is largely the repetition of metameric units originating from specialized meristems; primary apical mot and shoot meristems and secondary axillary meristems. The underiying developmental plastisticity of plants is largely a result of the unique properties of these meristems. For example, microsurgical experiments where apical meristems were divided into a?leas? six parts (Sussex, 1952) demonstrated that these cm then fom autonomous apical meristems, and indicated that meristems are self-regulating with the ability to reorganize themselves intemally. Moreover, meristems are capable of self-repliceting via axillaiy buds or pericycle cells, and shoot apical meristems can become rnodified to fonn floral meristems. Environmental information greatly influences the number of meristems and thus overall plant architecture. An example of this is the variability in branching patterns within the same species depending on the growth conditions (Suzuki et al., 1981). In addition, meristems can be rendered quiescent by adverse growth conditions and reactivated by favorable conditions. Unlike animals, plants are organized such that there is no central controlling tissue, like a nervous system, and the various tissues retain some degree of autonomy. In this way different component parts can act somewhat independently of one another. Indeed, plants have even been likened to a colonial organism (White, 1979) in that they may be considered as a collection of individual meristem members. Although this concept may be somewhat of an overstatement, it is useful in thinking about plant development, since it implies a high level of developmental plasticity that can accommodate envimnrnental signals through the individual meristems without strong reference to other meristems in the plant. It also implies an indeterminate and modular type of development which affords plants the ability to best exploit their local environment and maximize resource capture. Consequently, a certain degree of cornpetition for resources woufd exist between the various meristems. An example of this might be apical dominance, whem the apical shoot meristem exerts an inhibitory effect on axillary meristems and removal of the apical rneristem can induce the nonnally quiescent axillary meristems to grow. At some level, however, integration between meristems must exist otherwise the apical rneristem couM not exert its dominance over the axillary meristems.

The roks of hormones In plant dswIopment Although cellular communication is not unique to muîticellular organisms (Shapiro, 1998), multicellularity is accompanied by a greater dependence on signaling mechanisms that allow cells to specialize and coordinat0 their metabolisrn and growth for the benefit of the organism as a whoie. The mechanisms for cellular communication in animals are dependent on the transmittance of bioelectric signals by the nervous system and on a wide range of signaling molecules or Rrst messmgers which include proteins, amino acids, nucleotides, steroids, fatty acM derivatives, and dissolved gases (Hardie, 1991). Most of these molecules differ from each other by the medium through which they diffuse; the circulatory systern or extracellular fluid. For example, hormones are synthesized by specialized endocrine glands and carried to their target cells by the ciculartoiy systern where they have an effect. In contrast, local mediators are released by many unspecialized cell types and diffuse through the extracellular fluid to affect cells in the same local area (Hardie, 1991). However, the distinction between signaling molecules as either hormone or local mediator is sometimes difficult. Indeed, these signaling molecules are not fundamentally different in that their mode of action is very similar. The common feature of cell signaling systems is that the target cell perceives a signaling molecule by way of a receptor. The binding of the signaling molecules to the receptor then initiates a response in the target cell. The receptor can be either a transmembrane protein, and the signaling molecule is perceived at the plasma membrane, or the receptor can be intracellular, and the signaling molecule has to

4 travene aie plasma membrane to have an effect inside the cell. In the latter case the signaling molecule has to be wfficiently small and lipophilic enough to cross the plasma membrane, as is the case wiai most steroM hormones. Since these molecules are hydrophobic they are transportecl in aqueous solutions, such as the bloodstream, by carrier proteiris from which they dissociate befon enterbig the target cell (Norman and Litwack, 1987). Once perceived, the initiation of signal transduction cascades by signaling molecules is often mediated by second messmgers, which amplify the initial signal. This is often achieved by reoeptor linked enzyme systerns which genemte the second messenger or modify other downstream components of the signaling pathway (Hardie, 1991). - - -. Oenerally, animal signaling molecules are comrned with maintainhg a constant intemal environment, or homeostasis, which is required for the optimal functkning d the cells. This Is Men achieved by molecules which work in antagonistïc pairs, such as insulin anâ gfucagon (Hardie, 1991). Similarly, a comparativeiy smaller number of plant signaling molecules can be koadly cîassifled into growth- promoting and growth-inhibitory regulators (Fig. 1.1). The origin of plant signaling molecules is reflecteû in Wrstructure; ail of than are simple rnodYicatiom d common metabdites such as amino adds, fatty adds, and caratenoids, which have now been

Cooped to serin, a different hrmüon. Plant sipling mdecules are not al1 strküy chmical in nature, however, since signaling peptides have been identifiecl in a number of specieg (Fnnssen, 1998). The smaller number of plant slgnaling molecules compared to animals may be a lieflection of either r;iedUCBC( complexity or of a more generalized, less spedalized regulatory system. The legs s~*alitednature of plant signaling molecufes is indifecüy rdlecbed by their functknal redundancy where any one of them may paiaelly substitute for amther to alter plant giowai and dewlopment (Laopold, 1972). i=bwewr,tfmre is Sall no cîear indicatidn O( whether Fig. 1.1 The structure and some of the kinctions of plant hormones. Their major site of biosynthesis (s.b.) and the possible mode of transport (t.) are indicated. Based on information from Davies (1995). fundamental systems that control the synthesis andlor translocation of other factors, and the sensitivity of target tissues to them. Based on animal systems, hormonal regulation requires an emitting tissue and a receptor, as well as a system designed to control hormone synthesis, transport and inactivation. Plant signaling molecules have been likened to animal hormones in that they appear to exert an effect over cellular processes in locations that are remote frorn their apparent site of synthesis, and at very low concentrations. Some plant signaling molecules seem to fit al1 these criteria, while others meet only sorne. For example, ethylene acts as local mediator and affects either the cell that produces it or neighboring cells. Indeed, it seems that most plant cells have the capacity to synthesize al1 the various plant signaling molecules, so they could potentially act as local mediators. This duality has caused much controversy (Trewavas, 1981) about whether plant signaling molecules should be considered hormones at all. However, if al1 plant signaling molecules were to behave solely as local mediators they would contribute little to cell communication and other kinds of coordination would have to be evoked. Regardless, this duality might be a reflection of the less specialized nature of plant signaling molecules. A higher level of cornplexity might be achieved by the individual cells, with different cells within a tissue exhibiting different degrees of sensitivity to a given molecule or a combination of molecules. Since in some instances plant signaling molecules can act at distance, the term plant hormone is still the best functional definition. For instance, there is now substantial genetic and molecular evidence that awin is transported from the apex toward the roots by a family of auxin efflux camer proteins (Leyser, 1999). There is also evidence that ABA is transported from the mots to the shoot via the xylern, to cause stomatal closure when roots experience reduced soi1 moisture (Davies et al., 1987). It also appears that thee are medianisms that are analogous to animal hormone perception and signal transduction in plants. For exemple, the- is hicreasing evidence that each of the various classes of plant hormones may be perceived by membrane receptors. For example, the ethylene receptor is a transmembrane protein with homology to histidine-kinase two component response regulators of bacteria (8leeker and Schaller, 1996). Similady, cytokinins are thought to be perceiveci by receptonr with this structure (Kakimoto, 1998; Plakidou-Dymock et al., 1998). The putative brassinosteroid receptor is a transmembrane receptor kinase containing long leucine- rich repeats (LRR) and a cytoplasmic serine-threonine kinase domain (Li and Chory, 1997). On the basis of membranaimpeneable auxin analog studies, auxin is thought to act at the cell surface (Venis and Napier, 1990). Although a receptor has not been identified for gibberellins, the molecule is thought to be perceived at the plasma membrane (Gilroy and Jones, 1994; Hooley et el., 1991). A similar scenario appean to be tnie for ABA with perception of the molecule at the plasma membrane (Anderson et a/., 1994; Gilroy and Jones, 1994; Hornberg and Weiler, 1984 123). However, there is also evidence for its intracellular perception (Allan et a/., 1994; Pedron et al., 1998). Common components of hormone signal transduction are second messengers. These include molecules such as cyclic nucleotides, rnolecular calcium, and calcium- mobilizing second messengers, which include phosphoinosidite breakdown products such as inositol-1,4,54riphosphate (IP,) and diacylglycerol (DAO) (Hardie, 1991). Evidence is now accumulating that many plant hormone rnediated pathways invohre these second rnessengers (Trewavas, 1997; Walden, 1998) with calcium being a common denominator. In addition, signal transduction involving trimeric G protein signaling have been implicated in various plant hormone signaling pathways (Hooley, 1998). Based on these obsenrations it seems that, at least at this level, plant homones are perceived and transduced by mechanisms similar to those employed in animals. Perhaps the main difference between plant hormones and animal signaling mechanisms is that plant hormones are appear to be generally concemed with integrating responses to various environmental inputs. The evidence to support this is the following: the synthesis of some hormones can increase in response to environmental stimuli, and the sensitivity to various hormones can increase if plants are exposed to a particular environmental condition. For exemple, ABA levels increase upon drought stress in both leaves and mots (Davies et al., 1987; Hartung and Davies, 1991), and high temperature can increase auxin production in hypocotyls, which then stimulates hypocotyl elongation (Gray et al., 1998). The sensitivity to ABA can be increased if plants are droughted for a period of time prior to ABA exposure (Trewavas and Jones, 199 1) . Ethylene cm differentially regulate hypocotyl elongation depending on growth conditions: in the dark, dongetion is inhibited by ethylene, and in the light, promoted by ethylene (Smalle et a/., 1997). In this way, plant hormones, in various combinations, could act to integrate multiple environmental inputs to minimize any serious adverse efFects resulting in poor resource avaitability and rnaximize plant growth.

ABA au a redator of pknt waiw statu8 The water status of any organism cm profoundly affect the physiology of that organism. Normal growth and function in plants depends on a relatively high cellular water content. Consequently plants experiencing drought stress also experience a diverse set of physiological, metabolic and developmental changes. These responses are often dependent on the developrnental stage of the plant and on the species. Cellular responses to other environmental changes, such as increased salinity and cold temperatures, are dehydration related and can evoke responses in plants similar to drought stress. For instance, saline conditions cause water stress due to low water potential in roots. Dehydration can also be caused by ice crystals present in the extracellular spaces at low temperahires. The survival of plants is contingent on the ability to slow or stop growth and develop resistence to stress at critical stages of life. Fast or short-temi responses to water stress include changes that reduce water loss through transpiration, white skwer or long-terni responses invohre changes where growth is suspended or slowed, such as domancy. Domancy is a particularly impoitant phenornenon since it provides a means for plants to escape conditions that could othennrise be potentîally lethal to actively growing plants. In annual plants domancy is characteristic in seeds, while in biennial or perennial species, donnancy is also common in buds, seeds and storage organs such as bulbs and tubers, Apait from simple growth inhibition, dehydration can have profound long-term effects on the development and morphology of plants (Table 1.1). Generally, a differential sensitivity of roots and shoots (with root growth being less sensitive) to water deficit leads to large increases in the root to shoot ratio (Saab et a!., 1990). Water deflcits can also came the abscission of baves and fruits. Leaf size is generally reduced because of reduced cell expansion and cell division, and in some instances there is a reduction in stomata and an increase in the number of trichornes (Quarrie and Jones, 1977). Reproductive developrnent can also be sensitive to water stress, and flowering can be advanced in annuals and delayed in perennials (Jones, 1992). Many aspects of cellular metabolism are also affected by water stress. General tendencies are an increase in degradation processes; decreases in protein synthesis; decreases in photosynthetic rates; accumulation of free amino acids (such as praline), glycine-betaine, polyamines, and sugars, al1 of which could act as osrnoprotectants (Tabaeizadeh, 1998). Although rnany of these changes can be considered adaptive responses, these processes can also be assumed to be mechanisms aimed at reducing cell and tissue damage. For example, rince water deficits, Iike other forms of stress, can contribute to the accumulation of free radicals in cells, conesponding increases in neutralizing enzymes such as superoxide dismutase are induced (Tabaeizadeh, 1998). It is still not clear, however, how even small changes in water status can elicit such profound metabolic and developmental effects. One hypothesis is that changes in fable 1.1 Cornlalion between the dftsof abaisic (AM) and watw stress on short- and long- term respons8s baseci on the similarity of responses to water stress and exogenous AM(adapted from Jones (1Q921j. The strength of the eorrdatian ranges ftom weak (+) to strong (+++).

Rmrponao Watar otroa8 ABA Corrdrtion Short torm Stomatal conductance decrease decrease Photosynthesis decf8858 decrease Membrane permeability/ increaseî increw ion transport decrease decrease Long tom: bfochamlcrl and phyrlologic#l Specifii mRNA end increase increase protein synt hesis Proline and betaine increase increase aecumulaüon Osrnotic adaptaîbn Y= Y- Photosynthetic decrease decrease enzyme activity Dessication increase increase tolerance Saiinity and cou induces induces tolerance Wax production increase increase Long tum: growth Geoefal growth Yes inhibition Cell division decrease decrease Cell expansion decrease decrease Germination inhibits inhibis Root growth increasei increW decrease decrease Root:shoot ratio increase Long torm: morphology Production of increase increase trichornes Stomatal index dectease decrease Induction of Y= Y= dormamy ,on@tom: raproductivo Fbwering in annuals often advanced often advanceâ Flower induction inhibital inhiôited in perenniais Flower abscission increased Pollen viability decreased decteased Seed set decreased decreased €mûryu maturation 3 cell turgor or size are perceived by membrane stretch sensors (Tomos and Pritchard, 1994). Altematively, osmosenson, analogous to those employed in microorganisrns, may be the phary mode of perception for water status changes. Indeed, based on similarity to osmosensors found in yeast, candidate plant osrnosensors have been isolated (Bray, 1997; Shinozaki and Yamaguchi-Shinozaki, 1997). Most of the changes induced by water deficit, however, are probably secondaty and are the result of the operation of plant regulatory systems. A mechanisrn to integrate water sensory signals with regulatoiy responses is through plant hormones. Although other hormones may be involved, the clearest evidence is that ABA performs the major role in the integration of responses to water stress and of other environmental stresses such as salinity and temperature (Mansfield and Davies, 1983). This correlation was originally made by observations that ABA concentrations increase rapidly upon water stress and by the close correspondence between the responses to water deficits and to exogenous ABA over a range of short- and long-term responses (Table 1-1)- The best characterized short-tem or fast response to exogenous ABA is stomatal closure (MacRobbie, 1998). It is now known that ABA stimulates the activation of slow (S-type) anion channels, resulting in the sustanied efflux of anions from the guard cell, causing depolarization of the guard cell plasma membrane. This depolarization activates a rapid efflux of K+ ions out of the guard cells, which is enhanced by cytosolic alkalization and Ca2+. The resulting simultaneous efflux of K+ and anions lowers the turgor and the volume of guard cells, resulting in stomatal closure. There is evidence that ABA initiates the release of Ca2+ from intemal stores; however, how this is achieved is still a matter for speculation (Grabov and Blatt, 1998). Current models implicate the activation of phospholipase C, which hydrolyses phosphoinositides to fom inositol-3- phosphate (IP3) and diecylglyceid (DAG) (Trewavas and Malho, 1998). The IP3 would elevate the concentration of free Ca2+ in the cytosol and DAO would activate protein kinase C. The exact sequence of events in guaid cell signal transduction is not known (MacRobbie, 1998). Most long-temi responses to ABA involve changes in gene expression. After embryonic pattern formation is completed, ABA concentrations rise at the same time that domancy and storage reserves are accumulated in the seed (Rock and Quatrano, 1995). Many of the genes expressed duri-2 !hi8 revelopmental phase can be induced by exogenous ABA in the embiyo and in vegetative tissues (Ingram and Bartels, 1996). In addition, several of these genes are also induced by dehydration through drought, sait or cold (Ishitani et al, 1997). lndeed the majority of ABA induced genes have been identified through studies aimed at defining dehydration regulated genes (Bray, 1997; lngram and Bartels, 1996; Shinozaki and Yamaguchi-Shinozaki, 1996; Shinozaki and Yamaguchi-Shinozaki, 1997; Tabaeizadeh, 1998). The gene products of these can be classified into at least two broad categories. One class includes proteins required for reducing cellular damage such as water channels, enzymes required for the synthesis of osrnoprotectants or proteins that are thought to protect macromolecules (8.g.. LEA proteins, osmotin, antifreeze proteins), proteinases, and detoxification enzymes (9.g.. catalase, superoxide dismutase, ascorbate peroxidase) (Shinozaki and Yamaguchi- Shinozaki, 1996). The second class consists of regulatory proteins involved in either signal transduction and gene activation. Some of these include MA? kinases, calcium dependent kinases, phospholipase C, and a nurnber of transcription factors (Shinozaki and Yamaguchi-Shinozaki, 1996). Based on gene induction by dehydration in either ABA-deficient or ABA- insensitive mutant backgrounds, t has becorne clear that both ABA-dependent and -independent responses to dehydration exkt (Bray, 1997; Shinozaki and Yamaguchi- Shinozaki, 1997; Tabaeizadeh, 1998). In this respect, distinct ABA and drought responsive cieregulatocy elements have been defined (Busk and Montserrat, 1998) and some of the transacting factors that bind these efements identified (Guiltinan et al., 1990; Kusano et al., 1985; Oeda et al., 1991). It is evident from these studies that overlapping signal transduction cascades are involved in response to dehydration; with A6A mediating or potentiating some branches of these pathways. The signaling pathways that transduce fast and slow responses to ABA are slowly emerging. However, there is no reason to believe that fast and slow responses are transduced by the same mechanisms, as is sometimes assumed. That is, that stimulation of stomatal closing is transduced by the same factors as long-tenn dehydration responses. By way of comparison, it is becoming evident in animal systems that for some steroid hormones slow or long-term responses are achieved through different transduction mechanisms from fast or short-ten responses (Wehling, 1997). A relevant analogy with respect to ABA is aldosterone, which regulates ion pumps (particularly sodium and potassium) by both fast and slow responses. The fast responses are achieved through the binding of the hormone to membrane localized receptors, which could elicit a signal transduction cascade involving second messengers to affect ion pumps. The slow responses are achieved by the binding of the homone to intracellular receptors, which affect gene transcription (Wehling, 1997). Since no ABA receptors are currently available, such a scenario is only speculative, but the analogy might provide insight into how ABA exerts it effects, and might explain the observation of both extracellular and intracellular ABA perception (Allan et al., 1994; Anderson et al., 1994; Gilroy and Jones, 1994).

Genetic 8nrty.s of ABA synthesk and signal tmnsduction ABA biosynthesis and catabolism An integral part of hormone regulation and hrnction requires insight into its biosynthesis and breakdown. A full understanding of the metabolism of ABA in relation to its signaling function can provide insight into its mode of action. This can be illustrated again by enaiogy to signalhg molecules in animals. For example, retinoic acid synthesis and breakdown is tightîy regulated, and its rnetabolic enzymes are specifically localiied to regions in embryos where mtinoic acid exelts its effects on differentiation and tissue

14 maintenance (Holleman et el., 19Q8). If a similar regionalization of ABA synthesis and breakdown exk, then questions about tissue responsiveness and regulation of signaling can be addressed. The endogenous levels of ABA can be significantly reduced through mutation. Mutation affecting ABA biosynthesis have been isolated in a variety of species including Atabidopsis thaliena (aba), Nicotiana plumbaginifola (aba), Zea mays (vivipamus), Lycopersicon esculentum (Ilacca, sitiens, notabiw, Pisum sativum (wifty), and Solanum tuberosum (droopji)(Taylor, 1991). These mutants have been essential in dissecting the ABA biosynthetic pathway. It is now evident that ABA is synthesized primarily through the breakdown of xanthophyll carotenoids (Walton and Li, 1995). Mutations in genes which encode enzymes in the early steps of carotenoid synthesis have been isolated in maire (vp2, vp5, vp7, or vpg. These cause reductions carotenoids required for photosystem protection; resulting in plant photobleaching and ABA deficiency (Neill et aL, 1986). Mutations causing defects in downstream steps of carotenoid synthesis, however, do not show photobleaching. For example, in Arabidopsis and in N .plumbaginMolia, the aba 1 and aba2 mutations inhibit the epoxidation of zeaxanthin and do not cause photobleaching (Hurry et al., 1997; Marin et al., 1996; Rock et al., 1992; Rock and Zeevaart, 1991 ; Tardy and Havaux, 1996). The N. plumbaginiolia ABA2 gene has been cloned and shown to encode zeaxanthin epoxidase, which is imported into the chloroplast (Marin et al, 1998). This protein catalyzes the conversion of zeaxanthin to antheraxanthin and then to violaxanthin (Fig. 1.2). In maize, the vp14 mutation causes vivipary in seeds. The recent cloning of the VP14 gene has shown that the VP14 protein catalyzes the first committed step of ABA synthesis by the oxidative cleavage of 9-ciisxanthophylls to xanthoxin (Schwaitr et al., 1997b; Tan et al., 1997). In addition, based on low stringency southern analysis, it appeanr that VP14 is a member of a multigene family (wiai 4 to 6 members) (Tan et al., 1997). Fig. 1.2 Schematic showing the ABA biosynthetic pathway in higher plants. The metabolic blocks in various auxotrophic mutants are indicated. Also shown are the major ABA breakdown products. Adapted frorn Finkelstein and teevaart (1 994).

Mutations affecting later steps of ABA synthesis have been isolated in a nurnber of species (Taylor, 1991). For example, the ah2 mutation of Arabidopsis causes a defect in the conversion of xanthoxin to ABA-aklehyde, and the &a3 mutation causes a defect in the synthesis of the molybdenum cofactor required for the activity of ABA- aldehyde oxidase (see Fig. 1.2) (Leon-Kloosterziel et a/., 1996; Schwartz et al., 1997a). The corresponding mutation in Neplurnbaginifolia, aba 1, causes a sirnilar defect (see Fig. 1.2). The cloning of genes required for ABA synthesis, especially those required for downstream metabolic steps, has allowed researchers to assess, at least at a crude level, the tissue and developmental regulation of ABA synthesis. For instance, based on northern blot analysis, the steady state levels of Neplumbaginifolia ABA2 mRNA are higher in leaves than roots (Audran et al., 1998). In addition, the A BA2 mRNA levels in roots, but not leaves, are increased in response to drought stress, corresponding to a concomitant ABA accumulation in this tissue, The peak levels in ABAZ expression, however, are during seed development. High expression of the maize VP14 transcript is also coincident with embryo development and is upregulated upon water stress in the root. However, VP14 mRNA is also upregulated in leaves upon water stress (Tan et aL, 1997). This difference in regulation between ABA2 and VP14 could reflect the specific step along the biosynthetic pathway at which the two genes are required. The regulation of AM2 may minor the regulation of photosynthetic genes, since it may be required for proper photosystem function. In leaves, ABA2 expression would not be regulated by water stress, since in this tissue the relative pools of violaxanthin would be kept constitutively high. In roots, and possibly other non-photosynthetic tissues, ABA2 regulation would be more responsive to stress signals. On the other hand, since VP14 is fuither downstream of AB& its regulation may more accurately reflect where and when ABA is synthesized de novo in rasponse to various stresses. The exprestsion patterns of ABAZ and VPf4 mise some impoitant points about ABA regulation. Stress induced ABA production in the mots has been interpreted to mean that the roots an, the primary sensor of water deficit, with ABA being transported through the xylem to leaves to effect stomatal closure (Davies et al., 1987). Attematively, ABA could be synthesized locally in leaves in cells near stomata (Comish and Zeevaart, 1985). The expression of VP74 in both leaves and roots in response to dehydration suggest that both scenarios are possible. Although, this does not exclude the possibility that ABA produced in roots could act as a primary signal for the induction of ABA synthesis in leaves. Moreover, the suggestion that VP14 is part of a srnaIl family of genes implies that ABA biosynthesis could be tigMly controlled if these VPlOlike genes are differentially regulated. The steady state levels of ABA in plants are controlled by a balance between synthesis and degradation. The primary pathway for ABA inactivation in leaves, developing seeds and seedlings, and in tissues recovering from water stress, is thought to be through its conversion to 8'-hydroxy-ABA (ABA-OH) and the conversion of ABA- OH to phaseic acid (PA) (Creelrnan and Zeevaart, 1984; Walton and Li, 1995), and in some tissues to dihydroxyphaseic acid (DPA) (Parry, 1993; Walton and Li, 1995; Zeevaart and Creelman, 1988). In general, the biological activity of ABA-OH, PA and DPA are lower than ABA (Walker-Simmons et al., 1997). The enzyme responsible for the Rrst conversion step, 8'-hydroxylase, is a cytochrome P450 monoxygenase (Krochco et al., 1998). The activity of the enzyme is inducible by its substrate, (+)-ABA, in some tissues and is possibly regulated by osrnotic stress (Cutler et el., 1997). The concerted synthesis and breakdown of ABA could provide an added level of regulation to mediate ABA responses. The continued isolation of genes involved in ABA metabolism, will therefore allow the possible regionalization of ABA synthesis to be assessed at the cellular level by in situ hybridization and immunolocalization experiments. This information will provide additional information conceming which cellular and developmental processes are mediated by ABA. In addition, it will allow researchers to determine which tissues are the primary source of ABA and what role ABA transport plays in stress signaling. ABA biosynthetic-mutdnt phnoîypes In general, experiments which depend on exogenously applied ABA do not give unambiguous results. One problern associated with the application of ABA is that the distribution of applied ABA rnay be very different from endogenous ABA distribution. Genetic perturbation of endogenous ABA content is the most poweiful tool for studying the role of ABA in physiological processes and plant development, and by using the appropriate genotypes can give unambiguous results. In addition, the similarities arnong the mutant phenotypes are an indication that the effects of ABA are similar in most plant species. The conselvation of ABA effects means that, regardless of species, the most probable cause is ABA deficiency and that results obtained in one species can be extrapolated to other species.

Stomatal closure All ABA auxotrophs have a tendency to wilt (Donkin et al., 1983; Koomneef et al., 1982; Tal, 1966; Waggoner and Simmonds, 1966). This effect is caused by disniptions In stomatal regulation that normally reduces water loss through transpiration. Responses of stomata of tomato flacca mutants (Bradford et al., 1983) or wilty pea mutants (Donkin et al., 1983) to other stimuli however are normal. For example, stomata close in response to elevated CO2 or decreesed light intensities. However, in these cases stomata rarely close completely. Similady, stomata of tomato tlacca and sitiens fail to close completely even when guard cells were plasmolysed (Quanie, 1Q82a; Tal, 1966) or when treated with stimuli that effect wild-type stomatal closure (Tal, 1966). These results have been interpreted to indicate that guard cell wall extensability is reduced in flecca and sitiens (Tal and Imber, 1972), aithough there is no direct evidence for this. In addition, stomatal aperture can be reduced if fiacca guard cells are treated with ABA a few hours prior to plasmolysis (Tal et al., 1974). From these observations, it is char that a necessary controllhg factor in stornatai closure is ABA. GrowUl inhibition The growth inhibitory effects of ABA have been documented for a number of biological systems (Bomman, 1983). Foc instance, plants treated with ABA have smaller leaves and shorter stems (Qua-, 1982b). However, the leaf size of ABA auxotrophs seems to be reduced compared with the corresponding wild-types (Bradford, 1983; Koomneef et al., 1982; Taylor, 1987; Taylor and Tarr, 1984). This phenotype seems to apply reganlless of whether plants are grown without humidity contro! or under nearly saturating humidity (Jones et a/., 1987). However, no data are available on the leaf turgor pressures for auxotrophs grown under high humidity; therefore it is not possible to assess whether the smaller leaf size is due to poorer leaf water relations during expansion or whether ABA is responsible for the reduced leaf size. When comparing the height of aba mutants ni Arabidopsis with wild-type, the heights of the mutants were reduced (Koomneef et al., 1982). In contrast, when auxotrophs in tomato were grown at greater than 90% relative hunidity, they could grow up to 50% taller than wild-type, with thinner stems and with more rapid leaf production (Jones et al, 1987). The generally unhealthy status of ABA auxotrophs, however, limits the interpretation of some of these observations. Perhaps a better way of assessing the role of ABA in cell growth, would be to generate cell cultures from sorne of the ABA auxotrophs, so that water limitations are not an issue. In addition, an important aspect of growth where ABA migM be involved, that has not been tested in ABA auxotrophs, is in branching patterns. The repmion of axillary shoot outgrowth by ABA application and the decline in ABA content in axillary buds upon apical shoot decapitation implies that ABA could limit branching. Moreover, in non-branching torosa, blind and lateml suppmssorrnutants of tomato, the ABA content in axillary buds is increased cornpared to the branching wild-type line (Mapelli and Kinet, 1992; Mapelli and Rocchi, 1985; Tucker, 1978). The involvement of ABA in this aspect of growth couM easiîy be tested by using the appropriate auxotmphic backgrounds.

20 Seed donnancy Treatment of seeds wRh ABA can render them dormant for the duration of the ABA treatment, and once the ABA is removed the donnancy disappears (Schopfer and Plachy, 1984). An allelic series of ABA auxotrophic mutations in Arabidopsis has demonstrated that seed dormancy correlates directly with the amount of ABA synthesized by aie embryo (Karssen et al., 1983). Reduced seed dormancy caused by ABA-auxotrophy Is exemplified by the high percentage of germination of freshly hawested seeds and of seeds in the darùness (Koomneef et a/., 1982). Moreover, the gibberellin (GA) requirement for Arabidopsis seeds to geminate is circumvented in ABA- deficient mutants, which permits seeds to geminate in the presence of GA biosynthetic inhibiton such as uniconizol and paclobutrazol. Similady, GA-auxotrophs that normally require exogenous GA to geminate will germinate if they are also ABA-auxotrophic. In fact, these features have benthe basis for the selection of ABA biosynthesis mutations at the ABA 1, ABA2, and ABA3 loci in Arabidopsis (Koomneef et al., 1982; Leon- Kloosterziel et a/., 1996).

A working hypothesis is that ABA acts through a standard signal transduction pathway, in which the binding of the hormone to a receptor elicits a transduction cascade. Accordingly, the identification of specib genes or gene families that are expresseci in response to ABA would represent the end point of the signaling pathway. Experiments that focus on ABA-dependent gene expression are mostly designed to identify proteins that respond to ABA aftei the signal has been transduced. In contrast, genetic analysis has been an invaluable tool for the isolation of genes involved in the eailier steps of ABA signal transduction. Auxotrophic mutations are useful since they define the processes that are affected by ABA. Ideally, one can expect that mutations in genes that disrupt any one of aie positively acting components of a signal transduction pathway should have phenotypes similar to aiose of the biosynthetic auxotroph. In tum it is possible to distinguish between response and auxotrophic mutants because the former is not rescued by homeapplication. Mutational analysis has been confined to a few species in which mutants can be easily identified (Reid and Howell, 1995). Since, exogenous ABA can inhibit the precocious germination of immature embryos (Rodc and Quatrano, 1995), genetic screens to distinguish genes in ABA responses in ArabidoNs, ussally entail identifying seeds that genninate in the presence of ABA concentrations that normally inhibit wild-type germination. The populadty of screening for defective ABA responses at the level of seed germination reflects the relative ease with which screens and selections can be perfoned on large numben of mutagenized individuals. To date, five distinct mutations, designated abi ('MA insensitive'), have been identified (Finkelstein, 1994; Koomneef et al., 1984). fhe abil and abi2 mutations are semi-dominant and appear to alter both vegetative and seed ABA-regulated functions. For example, seeds defective at these loci show reduced domancy and mature plants wilt excessively under mild water deficit because of a loss of stomatal regulation. The other abi mutations are recessive and their phenotypes appear to be restricted to late seed development. A second class of mutations that cause seeds to show an enhanced Lesponse to ABA (efa) at the level of seed germination have also been identified (Cutler et al., 1996). The en mutations are recessive, indicating that the genes involved might encode negative regulators of ABA action. Although molecular analysis of al1 of these ABA mutants is not complete, their phenotypes imply that there are at least two partially redundant pathways for ABA signaling: one spedfic for seed development and the other for both seed and vegetative responses. However, this interpretation is probably an oversimplification, since mutations that affect sensitivity to ABA have been fortuitously isolated through screens for mutations confemng response phenotypes to other plant hormones (Table 1.2), indicating that a certain degree of cross-talk between different hormone response pathways also exits. Table 1.2 Chamterisücs of mutations atfecting ABA ssnsitivity. Mapted fiom Lamg and OlieudPt (1998). Sensitivity: in swds is the sensia'vity to exogerious AB.tha! inhibits grninafion compared to wildt-type; in stomata is aie secisitivity of -mata to exogerw,us ABA that elfectively causes stomatai dosure compared to wiid-type; in mots is the senJitlvity of swdling root gtowth to e>cogenous ABA compared to wiH-typa R/D refm to whether mutant alleles am recesslve (R), dominant (D) or mi- dominant (SD) with respect to the wild-type allele. ------Spacio8 Mutation DIR Phanotypa ~mnrproduct tunction

ABA insenslthrity Transcription factor in seds ABA insensitivity ? in seeds ABA insensitivity Protein phosphabse 2C in seeds 8 stornata ABA lnsensitivity Protein phoeptiatase 2C in seeds & stomata ABA insensrtivity Transcription factor in seeds ABA insensitivity Transcription factor in seeds ABA insensltivity ? in seeds

ABA supersensitivity in seeds 8 stomata ABA supersensitivity in seeûs ABA supersensithty Membrane protein in seeds, fnserisitivity In roots; ethyîene insensitivity Resistam to auxin, ethylene and ABA in mots

superwnsaivlty~ awin and ABA in rools &stoITmta Notwithstanding this potentially broad hormone response netwoik, the genetic studies with abi and era mutations are compelling because some of the response mutant phenotypes overlap those of ABA-auxotrophic mutants. The majority of abi mutants have varying degrees of diminished domiancy, while era mutants have prolonged domiancy. Many of the response mutant phenotypes, however, do not overlap with those of the auxotrophs and are discussed below. This lack of conespondence might imply that many of the response mutants isolated so far are not defective in molecules that are restricted to ABA signal transduction pathways.

AB13 function The developmental phenotypes resulting from different alleles of the same locus often Vary widely depending on the severity of the allele in question. For example, aside from reduced dormancy and seed ABA sensitivity, early phenotypic cornparison showed little difference between a weak allele of ebi3 (abi3-1) and a wild-type allele at the level of seed development (Koornneef et al., 1984). The limited phenotypes of abi3-l mutants suggest that the AB13 gene product is restricted to seed ABA signaling. However, nuIl alleles of abi3 (abi3-6) resul in highly non-dormant, underdeveloped seeds that are desiccation intolerant (Nambara et aL, 1995; Ooms et al., 1993). Processes such as the breakdown of chlorophyll, accumulation of storage resewes and premature activation of the germination program, ail processes not normally observed in ABA auxotrophs or abil and abi2 mutants, suggest that AB13 might have functions broader than just ABA signal transduction. It is interesting that seveie alleles of abi3 share many phenotypic similarities with the vivIpamus (pl)mutation of maize and that the molecular identity of these two genes suggests that they encode evolutionarily conserved seed-specific transcriptional activatom (Giraudat et al., 1992; McCarty et al., 1991). The evidence that AB13 specifms seed developmental programs, apart from the nul1 mutant phenotype, is that iîs ectopie expression causes an ABA-dependent, seed- specific mRNA accumulation in leaves (Parcy et al., 1994). However, these AB13- controlled expression patterns require a functional AM1 protein because they are lost in an abil mutant background (Parcy and Giraudat, 1997). These results indicate that AB13 is suffcient to change ABA responsiveness in vegetative tissues and that A811 is required for these ABlSregulated processes. It is difncult to assign genetic relationships based on ectopie expression studies, however, since overexpression can distort iegulatory circuits. For example, loss of stomatal control in abil mutants is suppressed by AB13 overexpression (Parcy and Giraudat, 1997). Since AB13 is a transcription factor, this result is surprising because guard cell closure in response to ABA application is too rapid to involve gene expression. Possibly, these studies, and the expansive role of AB13 beyond ABA-established seed maturation, might mean that this gene is actually a global regulator of cell fate rather than an ABA signaling rnolecule perse. If AB13 h~san instructive role, the responding cells should develop along one pathway in the presence of the AB13 signal, and along another pathway in its absence. Alternatively, if AB13 has a permissive function, then cells should be dependent on an AB13 signal to complete their differentiation. For example, if embryonic cells are more sensitive to ABA than other cells in the plant then conversion of cells to a more embryonic state might make them more sensitive to ABA. Ectopic expression of AB13 suggests that it has an instructive role, because the domain of embryo-specific gene expression is expanded. If AB13 is instructive then it would not be directly involved in ABA signal transduction but would 'select' cells to become sensitive to the hormone. Thus, loss of AB13 function resuits in cells that are unable to respond to ABA appropriately. Another possibility is that AB13 is a shared component of both ABA signal transduction and seed maturation pathways, thereby allowing coordinate regulation of ABA-dependent and ABA-independent processes. A coordinate model of AB13 action is supported by the demonstration that maize VPI participates in both seed-maturation specific programs and the repression of germinative programs (Haecker et al., 1995). Thus, mutually exclusive developmental piograms in main seed development are regulated by the AB13 homolog. Alternatively, ABA could be directly invohred in ABA signaling. Support for this possibility cornes from ment experiments where tobacco seed ABA concentrations were dramatically reduced by the transgenic expression of an ABA-specific antibody under the control of a seed specific promoter, which is active from the late phase of development to the first part of late embryogenesis (Phillips et al., 1997). lt was calculated that the recombinant antibody bound most of the available free ABA until dey 20 of tobacco development. The advantage of antibody ABA-rnodulation is that the rnethod blocks the ABA molecule itself In a seed specific manner, bypassing potentially lethal vegetative effects. The anti-ABA single chah Fv (scFv) antibody caused a developmental switch in the germination program, which was characterized by the formation of chloroplasts containing photosynthetic pigments, and a significant reduction in the ernbryo of abundant storage protein and oil bodies. These seed phenotype was most similar to abi3 nul1 mutants (Nambara et el., l995), suggesting that available ABA auxotrophic mutations in Arebidopîs are still leaky enough to allow seed maturation to proceed unhindered. In addition, ultrastructural analysis of scFv- expressing seeds indicated an alteration in cotyledon development that was more reminiscent of fus3 and lecl mutants of Ambidopsis (Keith et al., 1994; Meinke, 1992; West et al., 1994). The FUS3 and LEC1 genes encode proteins that are important in specifying cotyledon identity, since loss of these gene functions resuits in a leaf-like development of the cotyledons. Originally, these genes were not considered to have a direct role in ABA signal transduction, because the fus3 and lecl mutant seeds ntain normal sensitivity to exogenous ABA (Keith et al., 1994; Meinke, 1992). Recent molecular studies, however, indicate that different combinations of abi3, -3, and lecl alleles impact on seed ABA sensitivity, with FUS3 and LEC1 regulating the abundance of the AB13 protein (Parcy et al., 1997). In addition, the FUS3 gene is releted to the VPI/AB13 gene family, but is of reduced length (312 amino acids versus 720 residues for ABI3), and high homology in the protein is restricted to a stretch of et least 100 amino acids defined as the 83 domain (Luerssen et al., 1998). This domain can mediate sequence- specific DNA-binding in vitro (Suzuki et el., 1997) and is critical for gene activation in an ABA independent manner (Carson et al., 1997). The FUS3 protein lacks an amino- terminal A-domain which mediates the ABA response in VP1 (Carson et al., 1997), which is consistent with the ABA independence of fus3 mutants. Thus, the action of AB13 might be modulateci by both ABA and developmental regulators such as FUS3 and LECI. Because FUS3 and LEC1 are essential components of cotyledon identity, their interaction with AB13, an ABA-associated ernbryogenesis regulator, may allow the coordinate control of ABA-regulated seed functions with morphological development. The seed specific phenotypes of abi3 and abi4 mutations were originally interpreted to mean that these genes are seed specific and do not have a function during vegetative development. However, the cloning of the AB14 gene indicates that its expression is not limited to seeds (Finkelstein et al., 1998). This seems to be true for AB13 as well (Rohde et ai., 1999). On the basis of reporter gene and in situ hybridization studies, AB13 is induced in apical meristems in response to extended periods of darkness or to ABA treatments which inhibit growth. AB13 expression is not evident in deetioIated (del) mutants which fomr leaves in the darkness. These results suggests that AB13, apart from preventing precocious germination, might also be required in the maintenance of a quiescent state in apical meristems in response to growth inhibitory conditions (Rohde et al., 1999).

A811 and AB12 function The molecular characteriration of ABll and AB12 suggests that protein phosphorylation and dephosphorylation are involved in ABA action (Leung et al., 1994; Leung et al., 1997; Meyer et al., 1994). Both genes encode homologous protein type 2C phosphatases, suggesting that they have pavtially redundant functions. This redundancy could explain why only dominant mutations in these two genes can be identified in ABA- insensitive screens. In addition, dephosphorylation assays wit h abil mutant protein show decreased enzyme activity, suggesting îhat aiis mutation might act in a dominant negative manner (Bertauche et al., 1996). In this case, the abil mutant protein could interfere with a signaling cornplex of proteins that might include the AB12 protein. However, since loss-of-hrnction alleles of A811 and AB12 do not exist, the modeling of these proteins in ABA signaling is dimcult. Neveitheless, the recent achievements of patch clamping Ambidopsis guard cells has opened up the possibility of analyzing mutant cells with altered ABA response by electrophysiology (Pei et al., 1997). These studies indicate that both AB1 1 and AB12 function in stomatal guard cells by facilitating the ABA-induced activation of slow anion channels to effect stomatal closure. Furthemiore, protein kinase inhibitors can partially rescue the defects in anion channel activation of abil but not abi2. The simplest interpretation of these results is that a protein kinase(s) acts as a negative regulator of anion channel activation and that ABH reverses the effects of this kinase by inhibiting the kinase itself or by dephosphorylating the kinase substrate. Since kinase inhibitors cannot rescue the anion channel activation defect in abi2-mutant guard cells, it is possible that AB12 acts further upstream in ABA-mediated stomatal closure. However, since the substrates of neither ABll nor AB12 are known it is difficult to speculate what their roles in ABA signaling are.

ERAI functr'on Aside from phosphorylation as a mechanism for the transduction of the ABA signal, the molecular characterizetion of one of the era loci (era 1) indicates that protein lipid modification could be another mechanism for modulating ABA signal transduction in plants (Cutler et a', 1996). The €RA 1 gene encodes the fbsubunit of a heterodimeric protein famesyltransferase, an enzyme that catalyzes the attachment of a 15-carbon famesyl to specific proteins. Although little is known about famesyîtransferase protein targets in plants, this covalent modification plays a critical role in the subcellular localization and function of eukaryotic signaling molecules such as trimeric O-proteins and small GTP-binding proteins of the Ras superfamily in yeast and mammalian cells (Schafer and Rine, lSQ2). The phenotypes caused by ERAI mutation are supersensitivity to ABA at the level of seed germination and guard cell anion channel activation (Cutler et al., 1996; Pei et al., 1998a). Moreover, double mutant analysis of enlabil indicates that eni 1 can suppress ebil ABA-insensitive phenotypes in guard cells. The recessive nature of era l mutations and the ABA-supersensitivity phenotypes caused by enil suggest th& ERAl nomally functions by affecting the activity of a negative regulator of ABA signaling. A possible mechanism of how this can be achieved is exemplified by the negative regulation of G-protein-coupled receptors in visual signal transduction. In this case, after stimulus mediated receptor activation, a cytoplasmic kinase called hodopsin kinase, is translocated to the plasma membrane to specifically inliate the inactivation of the receptor (Inglese et al., 1992b). When cells are exposed to high levels of stimulatory signal the receptor becomes phosphorylated, which leads to its dissociation from downstrearn effectors. The way that hodopsiin kinase is shuttled to the receptor is by the addition of a famesyl group at its carboxy terminus (Inglese et al., 1992b). A similar scenario could be evoked for ERAI function, where it would act to target a negative regulator of an ABA-responsive receptor to downregulate the ABA signal (Fig. 1.3). Altematively, as with AB13 function, ERAI might rnodify proteins that are only periphetally associated with ABA signaling. Therefore, the eral ABA and seed domiancy defects could be a reflection of developmental and ABA pathways that converge to affect similar processes. Hopefully, once aie targets for €RA1 are known then questions of specificity cm be addressed. It is possible that a greater number of checks and balances exist in the seed since the cornmitment to germinate, unlike many othet developmental decisions in plants, cannot be reversed or reiterated. Thus the rote of ABA in the establishment of seed domancy might be buffered by other developmental pathways. Ostensibly, al1 the Ftg. 1.3 Possible role for famesylation in ABA-mediated signaling. Mutations in the ERAI gene result in loss of famesylation activity and increased ABA sensitivity. Loss-of-function mutations cause increased sensitivity, suggesting that a negative regulator of ABA signal transduction needs to be famesylated to function. (A) In the wild-type, after ABA perception by a hypothetical receptor, the signal is transduced by a receptor-coupled effector. At some point after ABA perception, a famesylated protein rnodulator (M) causes the inactivation of receptor-effector association, downregulating the ABA signal and attenuating the ABA response. (B) In the eral mutant, M is not famesylated and is not shuttled to the receptor to cause its inactivation. This lack of interference of effector function prevents attenuation of the ABA response. proteins identified by ABA sensitivity mutant screens are potential components of signaling pathways. Based on the molecular identity of these proteins that, for example, a phosphatase or a famesyltransferase would be predicted to act upstream of transcription factor.

Expanding the current collection of ABA response mutants by traditional screens will help to increase our knowledge of ABA signaling. Mutations affecting ABA sensitivity to root growth inhibition, for example, have recently been reported (Hirnmelbach et al., 1998). Moreover, since mutants already exist, it should be possible to design genetic screens to address problems of pleiotropy. One method that is widely used to identify new loci in signaling pathways is a suppressor or enhancer scteen (Karim et el., 1996). Either suppressor or enhancer mutations can identify genes located downstream of a known gene. This approach is based on the premise that such second-site mutations can cause imbalances in the flux of a pathway, either by reducing it or by exaggerating it, and consequently produce a detectable phenotype. In the case of phenotypically complex mutations such as enil, specific phenotypes can be targeted for suppression or enhancement. The success of such screens, however, is largely dependent on the initial mutant allele. There is no guarantee that suppressor or enhancer mutations will be limiteci to the same signaling pathway or to parallel redundant pathways. Apart from the isolation of mutations that affect gene interactions, mutations identified by screens that do not utilize ABA directly have been mostly set aside (0.g.. FUS3). This is because of the likelihood that these screens do not identify mutations that diredy affect ABA signaling. However, this standard is based on the assurnption that ABA provides an instructive signal. In many physiological experiments, however, ABA seems to act as a relay between the environment and the plant (Chandler and Robertson, 1994). If this is tnie, then there would be no need for specific pathways, solely for ABA signaling. The alternative is that ABA provides a permissive signal to modulate multiple pathways, thereby either ampliîying or dampening the strength of an environmental or developmental signal. This may explain why ABA and perhaps other plant hormones seem to have a role in apparently distinct pathways. Based on what is currently known about ABA signaling, however, it is difficult to distinguish between the two possibitities.

The isolation of ERA 1 loss-of-function mutants suggests that some ABA responses, such as seed germination and stornatal closing, are dependent functional farnesyltransferase activity in plants. The famesylated proteins that mediate these responses are currently not known. A clear understanding of how these proteins effect these responses will also require a sense of how famesylation affects their activity. The overall objectives of this work was to identify proteins that can be modified by ERAI and to provide a comprehensive characterization of the consequences of ÇRA 1 deletion at the whole plant level. The following was achieved: 1) The evaluation of a yeast interaction trap a method for identifying potential €RA1 target proteins. 2) The identification of a famesylated protein that is potentially involved in ceIl cycle progression. 3) The phenotypic characterization of ere 1mutant plants to better define the processes that are dependent on famesylated intemediates and to provide aie necessary background for the subsequent analysis of famesylated proteins in an in vivo context. 4) Provide a tink of the eral devekpmental phenotypes to ABA sensitivity by isolating second site suppressor and enhancet mutations. CHAPTER 2

MATERIALS AND METHODS Phtmaferia/ Deletion alleles of era 1 (era 1-2 and 1-3) are in a Meyerowitz Columbia (MCol) background; eml-7 is in a WS background. Plants refened to as wild-type are of the MCol ecotype, except where noted. For germination assays, seeds were imbibed on a solid medium consisting of 1.1 glL MS basal salts (Sigma) and 0.8% agat and supplemented with ABA when needed. Seeds were chilled on plates at 4°C for 4 days prior to being transfened to giowth shelves at rom temperature under 200 p~rn2/s light. Germination was scored after 4 days as radicle emergence. Otherwise, seeds were sown directly ont0 autaclaved soi1 (venniculite:perlite:sphagnum, 1:1:1) moistened with 20-20-20 fertilizer. Plants were grown under constant light (CL) or shoitaay (SD) conditions, which had a 14 h dark period. In al1 cases illumination was at 200 p~/m2/s and chamber temperature kept at 20°C.

Suppressor and enhancer rcreening Seeds for suppressor and enhancer mutant isolation were generated by mutagenizing either 15 000 era 1-2 seeds with 0.25% EMS or 20 000 en1-3 seeds with 0.2% EMS for 16 h at room temperature. After mutagenesis, seeds were washed extensively with distilled water over a period of 8 h. Ml seeds were transfeired to soi1 in pools of 300-350 seeds for a total of 27 pools for ere 1-2 and 30 pools for eral-3. M2 seeds were harvested from each pool, and 1000 seeds pet pool were screened on solid media containing ABA. Suppressors of eral were isolated by identifying individuals that geminated on 0.3 pM ABA. Enhancers were isolated by identifying individuals that did not germinate on 0.03 pM ABA. These were transferred to soil, and M3 seeds were retested in a secondary screen on various concentrations of ABA. Isolates that retested were grown for further analysis. All standard molecular biology techniques were performed according to Sambrook et al. (1989). These included media for propagating E. coli (LB and NZ), plasmid DNA purification techniques by the alkali method, DNA electrophoresis, noithem blot analysis, and random-primed *P-labeled probe generation. E. coli transformations were performed according to the CaCI, method. DNA fragments for subcloning were gel- purified with low-melting temperature agarose and extracted by phenollchlorofoim extraction, followed by precipitation with ethanol (Sambrook et al., 1989). Most restriction enzymes used were from PhamacialAmersham Biotech and restriction enzyme digestion was carried out according to the manufacturer's recommendations. Ligation reactions were also carried out according to the manufacturer's recommendations (New England Biolabs). First pass sequencing was petfonned at the York University DNA sequencing facility.

E. coli strains used:

JA300 FDthr leu66 BI trpC117 thy8 hsdR hsdM rspL t strfl

Veast strains used: JBY575 Mat a ura3-52 leu2-3 1 12 his3D taZOO trp 7Ma63 ad82 Gal+ EGY48 Mat a his3 trp l ura3-52 leu2::pLEUZ--LenAop6 PCR primers used (custom made by GIBC08RL):

El 01 5' GCG AGA TAA GGA AITCGATTA TCT G 3' Ml3 5' GTA AAA CGA CGG CCA G 3' LEX1 5' CGT GAG CAG AGC TTC ACC ATT G 3' BCO1 5' CCA GCC TCT TGC TGA GTG GAG ATG 3' BC02 5' GAC AAG CCG ACA ACC TTG ATT GCA G 3' CYCF 5' CTA CTA CAA ACC TGA GAA TTC AGT CTG AGA G 3' CYCR 5' CCG GTC AAC AAA GCT GCG GCC GCA GGG ATC AMGC 3' RNA Isolation hmdlings Approximately 100 mg (fresh weight) of one week old seedlings was used for RNA sample isolation. These were collected in microcentrifuge tubes, frozen in liquid nitrogen, and ground to a fine powder using a fiid plastic eppendorf pestle. After grinding, 500 pl of 80°C extraction buffer (phenol: 0.1 M LiCI, 100 mM Tris pH 8.0, 10 mM EDTA, 1% SOS; 1:1) was added. The mixture was hornogenized by vortexing for 15 min. Then, 250 pl of chlorofom:butanol(4:1) was added and the mixture vortexed for an additional 10 min. After centrifugation for 5 min, the aqueous phase was removed and the RNA precipitated by adding an equal volume of 4 M LiCl and incubating on ice over- night. The RNA was pelleted by centrifugation and pellets were dissolved in 250 pl of sterile water and reprecipitated by adding 0.1 vol of 3M sodium acetate pH 5.2 and 2 vol ethanol. After centrifugation the RNA pellets were resuspended in 50 pl of sterile water and used for northem analysis. Proteh techniques W SDS-sample buffei: 120 mM Tris-HCl pH 6.8,4% SDS, 10% 2-mercaptoethanol, 20 % glycerol (w/v), 0.025% bromophenol blue.

SOS poîyacryIamide gel electrophoresis (SDS-PAGE) Generally, 2X SOS-sample buffer was added to protein sarnples at 1:1 ratio , which were then heated to 95OC for 2 min. Protein was separated on 10 or 12% (wlv) SDS- polyacrylamide gels after Laemmli (1970) using the BioRad mini-protean II system. After separation, gels were stained with Coomassie Brilliant Blue R (0.1% wh), in 10% acetic acid, 20% methanol and destained with 10% acetic acid. For western blottirtg gels wem transfened directly to transfer buffer (see below) wHhout pnor staining.

Silver staining of poljtacryiamide geb Gels were washed for 5 min in deionized water aiter electrophoresis. These were then placed in a solution containing 10% ethanol (viv), 5% acetic acid (vlv) for 3 h to ovemight. The gels were then washed with deionized water for 5 min and then soaked for 30 min in a 10% solution (v/v) of glutaraldehyde. The unreacted glutaraldehyde was removed by five 30 min washes with deionized water. These glutaraldehydetreated gels were then soaked in an ammoniacal silver nitrate solution for 10 min. The ammoniacal silver nitrate solution was prepaied by slowly adding, with stimng, 30 ml of 1.2 M silver nitrate solution to a solution containing 10 ml of concentrated ammonium hydroxide and 1.5 ml of 10 N sodium hydroxlde in 160 ml of deion ized water. After the silver nitrate was dissolved, the final volume was adjusted to 750 ml. The gels were removed from the ammoniacal silver nitrate and treated wHh three 30 min water washes. The image was developed with a solution containing 0.1 g of citric acid and 1 ml of formaldehyde (37% commercial formaldehyde) per litet of deionized water. When the image was sufficiently developed, the reaction was stopped by placing gels in a solution of 5% acetic acid and gels were stored in the same solution.

Two-dimensional (2-0)gel electrophoresis The BioRad mini-protean II 2-D gel system was used for this analysis. Protein samples that were separated by electrophoresis in two-dimensions were solubilized in a buffer containing 9.5 M urea, 2% Triton X-100, 5% Fmercaptoethanol, 2% Ampholytes (Phamacia). Approximately 5-1 0 pg of protein was loaded on capillary tubes containing a gel monomer solution composed of 9.2 M urea, 4% acrylamide, 2% Triton X-100,2% Ampholytes, which had ben prefocused et 200 V for 10 min, 300 V for 15 min, and 400 V for 15 min, using an upper chamber buffer of 20 mM NaOH and a lower chamber buffer of 10 mM Ham,. Proteins were resolved under the same conditions at 750 V for 3 h. After firstaimension rasolution, tube gels were extnided from the capillary tubes and incubated for 15 min in 1X SDS-sample buffer and laid horizontally on the surface of a 12% SDS-polyacrylamlde gel. The second dimension was resolved following standard SDS-PAGE. Western blotting Samples were mixed with an equal volume of 2X SOS-sample bufier and boiled for 90 seconds. The 10 pl of sample was loaded per 5 mm-wide well for gels of 1 mm thickness. Molecular weight markers were loaded according to manufacturer's instructions (Biorad Labs.). Electrophoresis was carried out at 20 mA constant current per gel. Proteins were transferred to nitrocellulose membranes using a semi-dry electroblotter apparatus according to Vie rnanuf~cturer'sprotocol (Phamacia LKB) using a buffer containing 25 mM Tris, 190 mM glycine, 20% methanol. The transferred proteins were visualized by staining the membranes for 2 min with Ponceau red stain and then washed with PBS (14 mM Na,HPO, , 1.8 mM KH,PO,, pH 7.2,138 mM NaCI, 2.7 mM KCI) for 15 min at mmtemperature. To blodc nonspecific binding, membranes were soaked in PBS containing 5% (w/v) skim milk, and with 0.05% Tween-20 for at hast 30 min at rom temperature. The membranes were then incubated with primary antibody at 0.1 -1 .O pglml in the same buffer for at least 1 h at room temperature. Excess antibody was removed by washing membranes three times for 5 min each with PBS, with 0.05% Tween-20. The membranes were then incubated for 30-60 min with peroxidasetonjugated secondary antibody diluted 1 :500 to 15000. The membranes were subsequently washed three times for 5 min each with PBS, 0.05% Tween-20 and one additional time for 5 min with PBS (no Tween). Detection was achieved by incubating blots with chemiluminescent substrate for 1 min at room temperature (Amenham). Excess substrate was removed and blots were wrapped in plastic and exposed to Xiay film.

FamesyItrrins~aeumays Cnide plant extracts for famesylation assays were isoleted by grinding approximately 1 g of fresh tissue (usually flower buds) in 1 ml of extraction buffer (50 mM Hepes pH 7.5, 1 mM EDTA, 1 mM DTT. 1 mM MgCl,. 1 mM PMSF) in a mortar with a pestle on ice. Acid washed sand was included to improve shearing of the tissue. Homogenates were centrifuged a 3000 g at 4OC for 10 min to remove cell debris and sand. The supematant was then transferred to a microcentrifuge tube and centrifuged at full speed in a microcentrifuge. The supematant was then recentrifuged at 40 000 g to remove insoluble proteins. Protein concentration was detennined by the Bradford assay (Bradford, 1976). The yield was typically 10 mg/ml soluble protein. FTase activity was dete imined by assay ing for t ransfer of rH-famesyl pyrophosphate (Fpp) to a recombinant target protein. The standard reaction mixture contained the following components in a final volume of 25-50 CI:50 mM Tris-HCI, pH 7.7, 5 mM MgCl,, 5 pM ZnCI,, 2 mM DTT, 0.5 pM 3H-Fpp (typically at 8 CVmmol; American Radiochemicals), target protein at 3-5 pg, and cell extract at 100 lg. The same reaction conditions were used for geranylgeranylation assays except 3H- geranylgeranyl pyrophosphate (GGpp) was used at 0.5 FM, in place of 3H-Fpp. After incubation for 30-60 min at 30°C, reactions were stopped by adding EDTA to 20 mM. An equal volume of W SDS-sarnple buffer was added and proteins separated by SOS- PAGE. Gels were stained with Coomassie Brilliant blue R and destained with 10% acetic acid (vlv). Gels were subsequently incubated in Enhance (Amersham) for 30 min and dried at 70°C under vacuum for 1 h using a Biorad gel dryer. Dried gels were exp0~8ddirectly to prefiashed X-ray film (Arnenham) et -70°C for up to one week. When peptides were used, famesylation reaction mixtures contained 50 mM Hepes pH 7.5,s mM MgCh, 5mM OTT, 1 mM ZnCl,, 1 pg peptide and 10 prnol 3H-FPP in a final volume of 25 pl. After incubation for 30-60 min at 30°C, the reaction was stopped by adding EDTA to 20 mM. 20 pl of the samples were spotted ont0 Silica gel 60 thin layer chromatography plates and the plates developed with propanokwater (7:3)for 5 h. After development, plates were dried at room temperature and sprayed with an atomizer containing Enhance. Plates were exposed directly to X-ray film for up to one week at -70°C. To confimi that radioactive spots corresponded to peptides plates were subsequently sprayed with 0.1% ninhydih in 50% ethanol and incubated at 120°C until peptide spots appeand. Peptides used in these assays were made to order by Genemed Biotechnologies Inc., San Francisco, CA.

Y.ut growth and manlpuIotions YPD (conpiete mediwn): 1% Difco yeast extract, 2% Bacto-peptone, 2% dextrose (D- glucose). Minimal drop-out media: 0.001 5% (wtv) yeast nitrogen base without amino acids, 0.005% ammonium sulfate, 2% dextrose, and 0.002% of the appropriate amino acid drop-out powder. Amino acid combinations were made by combining 2 g each of arginine, asparagine, histidine, isoleucine, lysine, methionine, phenylalanine, proline, serine, threonine, tiyptophan, tyrosine, uracil, and valine in a mortar and homogenized by grhding with a pestle. Leucine was added to 4 g, adenine to 0.1 g, and para-amino benzoic acid to 0.2 g. Where appropriate, individual amino acids or combinations of amino acids were omitted from the powder stocks. Media designations reflect the sugar and amino acid content: e.g.. Dex his* trp'. Galactose and raffinose were added at 2% and 1% respectively. Designation of galactose/raffinose containing media are GaVRaf, followed by th8 amino acids that are omitted. For al1 solid media 2% Bacto-agar was added. X-Ga/ plates: Drop-out media supplemented with 1X BU salts and 0.008% X-Gal. 10X BU salts consist of 70 g Na2HP0,.7HP, 30 g NaH,PO, per ILwater (pH 7.0).

Yeast transformation Yeast cells were grown in the appropriate liquid media at 30°C to approximately 1-2 x 107 celldml. Cells were hawested by centrifugation (1500 g, 5 min) and washed with sterile water. fhe cells were recentrifuged and resuspended in filter sterilized 0.1 M lithium acetate (LiAc) to a titer of 2 x 100 cells/ml and transferred to a microcentrifuge tube. These were incubated et 30°C for 15 min. 50 VIof the cell suspension was then combined with 50 pg of sheared, kat-denatured salmon spem DNA and 100 ng of plasmid DNA by brief vortexing. 10 this mixture, 3ûû pl of a solution contahing 40% polyethylene glycol (PEG), 0.1 M LiAc was added. Transformation mixtures were incubated at 30°C for 30 min and subsequently heat shocked at 42OC for 20 min. The mixture was cooled to room temperature and centrifuged to pellet the cells. Cells were resuspended in 1 ml of stefle water and dilutions of these were plated ont0 selective media.

Yeest DNA mini-preparatim A large mass of yeast cells (10 mg) were scraped from a plate and resuspended in 1 ml of water in a microcentrifuge tube (OD- at 2-5). The cells were pelleted by centrifugation and resuspended in 0.5 ml of S buffer (10 mM K,HPO, pH 7.2, 10 mM EDTA, 50 mM Pmercaptoethanol, 50 pg zymolase). Tubes were incubated at 37OC for 30 min. To the cell suspension 0.1 ml of lysis solution (25 mM f ris-HCI pH 7.5, 25 mM EDTA, 2.5% SDS) was added and tubes were transferred to 65°C for 30 min. Then 166 pl of 3 M potassium acetate was added and the tubes transferred to ice for 10 min. Cellular debris and SOS were removed by centrifugation for 10 min at top speed in a microcentrifuge. The supernatant was transferred to a fresh tube and the DNA precipitated by adding 0.8 ml of cold ethanol and incubating on ice for 10 min prior to centrifugation. The DNA was washed with 70% ethanol and resuspended in 40 pl of water. 1-2 pl of this was used to transfonn E. colior for PCR.

Yeast interaction tmp donlng Interaction trap cloning in yeast was performed essentially as described in Finley and Brent (1995). All relevant maps for plasmids used in interaction trap cloning are at the end of the chapter. pEGERA eansttuctiion To constnict a pEG202-ERAI bait plasmid, a primer was designed to introduce an EooRl site at 5'-poslion 101 of the ERAI cDNA wquence (Genbank Accession N. U46574); this primer was used to PCR amplify a DNA fragment from plasmid DNA containing the ERAI cDNA, by using universal Ml3 primer as a reverse primer (M13). PCR was carried out using Vent DNA polymerase (New England Biolabs), which has proofreading activity and produces blunt ended products. A reaction profile (94OC, 30 sec; 55OC, 30 sec; 72OC, 1 min; 25 cycles) that amplified a single product of 1.2 kb was gel-purified. The PCR product (€101) was cloned into pBCSK+ (Stratagene) by digesting El01 and the pBCSK+ vector wlth €CORI and Nofl, followed by ligation at le°C for 12 h. The ligation product was transformed into DH5a and recombinant plasmids isolated from individual colonies by a plasmid DNA mini-preparation. A clone containing El01 was then used to generate an El01 insert for subcloning into pEG202 (Fig. 2.1) by digestion with EcoRl and Nofl. Positive clones were isolated and the fusion junction at the EcoRl site was confimed by sequencing using the LEXI primer. The 'bait' plasmid designated pEGERA for library screening was then transformed into the yeast strain €GY48 and colonies selected on Oex hi$* media. To confirm that LexA-ERAt fusions were translated appropriately, protein frorn his+ yeast transfomants were resuspended in 1X SOS-sample buffer, separated by SDS-PAGE, followed by western blot analysis using LexA antibody (provided by Brent lab, Harvard University). These EGY48 derivatives were then transformed with pSH18-34 (Fig. 2.2) and selected on Dex his- ura' plates. EGY48-pSH18-WpEGERA derivatives were grown in liquid Dex hiso uraœmedia and transformed with a pJG4-5 (Fig. 2.1) AlabMopsis cDNA library provided by Hong Zhang (Texas Tech, Lubbodc TX). Ten EGY48 transfomations using 100 ng of library DNA eech were perfomed in total. Transformants were selected on Dex his- ura- trp* solid media. Approxirnately 400 000 inckpendent transfonnants grew on this media. Colonies from these plates were scraped off using a sterile microscope slide and resuspended in sterile watet. These cells were subsequently used to isolate putative €RA1 interactors by replating thern on GaURaf his- ura- trp- leuœsolld media. Approximately 30 O00 coknies grew on these plates. These colonies were then transfened onto nitrocellulose filters and placed on Dex his' ura* trp' X-gai media. After 2 days growth approximately, 30% of the colonies were blue in color, indicating transcriptional activation of the pSH 18-34 reporter. 1000 of these colonies were patched onto secondary plates of the same medium and kept for further analysis.

Classitjdng hteractors To identify pJG4-5 library plasmids carrying the FTase a cDNA, individual colonies were patched ont0 Dex his- ura' trp' plates. Patched yeast cells were lifted onto nRrocellulose filters and these were transferred to 3M paper saturated with a solution containing 1M sorbitol, 0.1 M sodium citrate, 50 mM EDTA, 15 mM Di7 and 2 mg/ml yeast lytic enzyme (ICN, >70 000 unitslg), and buffered to pH 7.0. The filters were incubated ovemight at 30°C. Next day the filters were transferred to 3M papet saturated with 10% SDS for 5 min at room temperature, followed by 10 min on 0.5 M NaOH. The NaOH was neutialized by washing the filters with 0.3 M NaCI, 30 mM Na citrate, 0.2 M Tris pH 7.5, three times for 5 min each. The filten were air dried and hybridized to W-labeled a subunit DNA (Genbank Accession N. H37092) using standard protocols (Sambrook et al, 1989). The remaining positive pJG4-5 library derivatives were classified according to the resctriction enzyme profiles of the cDNAs that they contained. PCR reactions consisted of the following: 2 pl 10X Taq buffer (500 mM KCI, 100 mM Tris-HCI pH 8.3, 15 mM MgC12, 1 mglml gelatin), 2 pl dNTP mix (4 mM of dATP, dGTP, dCTP, dlTP each), 100 ng BCOl primer, 100 ng BC02 primer, and 0.5 pl of Taq pdyrnerase (- 5 UIpJ) to a final volume of 20 p1 in water. The reactions were incubated in a robocycler (Stratagene) for 35 cycles of 94OC for 1 min, 65'C for 2 min, 72% for 1 min. For each PCR reaction, 8 pl was combined with 1 pl 10X Haeill restriction buffer (New England Biolabs) and 0.5 pl of enzyme. These were incubated et 37OC for 2 h. The Haelll digests were analyzed by electrophoresis on 1.5% agarose gels. Based on the restriction pattern of their PCR products, interacting clones were classified into 14 groups. To confimi the specificity of interactions, plasmids containing interacting clones were isolated from €GY48 to test them in a mating assay (Chapter 3). To do this, interacting pJG4-5 plasmid DNA was isolated away from other other ampicillin selectable plasmids by Rrst perfomiing a yeast DNA mini-preparation. This DNA was then used to transfomi a bacterial strain that is trpC deficient (JA300). The 1'1gene of yeast can complement this mutation allowing for isolation of pJG4-5 transformants by selection on ampicillin trp- bacterial minimal media. cDNA from interacting clones was PCR amplified using BCOl and BC02 and sequenced using a poly-T primer.

GST-NAPI overexpremlon plasmld construction To generate a glutathiones-transferase fusion with NAP1, the pGEX2TK vector (Pharmacia) was used. The NAPl cDNA in the pJG4-5 vector was PCR amplified using Vent polymerase (New England Biolabs) and gel-purifid. The PCR product was cloned into pGEX2TK by ligatlon to pGEX2TK digested with Aval and where the 3' recessed ends generated by Aval were filled in with the Klenow fragment of E. coli DNA polymerase (Pharmacia) to produce blunt ends. Ligation products were transformed into DH5a and selected on LB ampicillin (100 pg/ml) plates. Recombinant plasmids were identified by PCR using BCO1 and BC02 primers to detect NAPl inseitions into pGEX2TK (Fig. 2.4). Positive clones were grown in 3 ml liquid LB broth containing 100 pg/ml ampicillin at 37% to an OD, of 1.0 at which point IPTG was added to a concentration of 0.1 mM. These cultures were further incubated at 37OC for an additional 2 h. Cells were harvested by centrifugation and resuspended in 100 pl of 1X SOS- sample buffer. Proteins were separated by SDS-PAGE and electroblotted ont0 nlrocellulose. The GST-NAP1 fusion products were detecteâ by western blot analysis using anti-haemagglutinin (HA) epitope tag antibodies (Clontech). GST-NAPI expressing clones were tmnsfomed into the E. wliBU1 straon. Reagents and Stock SoIutions: PMSF, 0.1 M stock in 100% ethanol, stored at -20°C; DNAse 1, 10 mglml in 50 mM Hepes,50 mM KCI, 1 mM DTT, and 50% glycerol, stored at -20°C; lysozyme, 100 mgiml in water; stored at -20°C. Buffem Phosphate buffered saline (PBS): 14 mM Na,HPO, , 1.8 mM KH,PO,, pH 7.2,138 mM NaCl, 2.7 mM KCI. LPC Protease Inhibitor Mix: 1 mg/ml leupeptin, 1 mglml pepstatin, 1 mglml chymostatin dissolved in dimethylsulfoxide. Extract Buffer: 50 mM Hepes-KOH, pH 7.6,50 mM KCI, 1 mM MgCl,, 1 mM EGTA, 1 mM PMSF, 1/10W LPC protease inhibitor mix. Wash Buffer: 50 mM Hepes-KOH, pH 7.6,50 mM KCI, 1 mM MgCl,, 1 mM EGTA, 1 mM DTT, 10% glycerol, 111 000 LPC protease inhibitor mix. Elution Buffer: 50 mM Hepes-KOH, pH 7.6,0.35 M KCI, 1 mM MgCl,, 1 mM EGTA, 1 mM Dm, 10% glycerol, 111 O00 LPC protease inhibitor mix. Sample Buffer: 65 mM Tris-HCI, pH 6.8,3% SDS, 5% b-mercaptoethanol, 10% glyc8f'ol.

Purification of GST-NAP Fusion Protein: Bacterial cells were grown to saturation in 400 ml of LB containing 100 mgml ampicillin. This culture was used to inoculate 6 L of fresh NZ medium containing 100 mg/ml ampicillin (a 15 fold dilution). The culture was grown at room temperature in a large container by sparging air through a bubbler directly into the liquid media and by vigorous stiring with a magnetic stir bar. When aie cell concentration reached an optical density (00,) of 1.0, IPTG was added to a concentration of 0.1 mM. and the culture was incubated at rom temperature for 3-4 h. The cells were hawested by centrifugation, and washed once with 200 ml of ice cdd PBS and mpelleted. The cells were kept on ice during harvesüng and the final ce# pellet was froten on liquid nitrogen and stored et -80°C. The frozen cell pellet was thawed by the addition of 5 vol PBS containing 1 mM EGTA, 1 rnM EDTA, 1 mM PMSF, 0.1% Tween 20, and 200 mglml lysozyme. The cell pellet was then stirred foi approximately 30 min to break up the pellet and allow lysis to occur. The cells were then passed through a French pressure cell at a pressure of 4000 MPa to achieve lysis. KCI and DTT were then added to the lysate to final concentrations of 0.3 M and 15 mM, respectively. If the extract was fairly viscous at this point, DNAse-I and MgCl, were added to final concentrations of 10 pgfrnl and 2 mM, respectively, and the lysate incubated on ice for 15 min. The extract was dialyzed against 20 vol of PBS containing 0.3 M KCI for 45 min. This step was repeated for a total of three times. The extract was centrifuged at 10,000 g for 1 h and the supernatant loaded onto a glutathione-agarose column (Sigma) at a rate of 5-10 column voVh with a peristalic pump. The column was washed at a flow rate of 10 colurnn voüh with PBS containing 1 mM DTT and 0.3 M KCI until no protein could be detected in the flowthrough (- 15 colurnn volumes) by the Bradford assay. The colurnn was eluted with 0.5 ml fractions of 50 mM Hepes (pH 8.1), 0.3 M KCI, and 5 mM reduced glutathione, and the protein in the elution fractions was determined by the Bradford assay. The peak protein fractions were pooled and dialyzed extensively against buffer containing 50 mM Hepes (pH 7.6), 0.25 M KCI, and 30% glycerol. The purified protein was frozen on liquid nitrogen and stored at -80°C. Approximately 15 mg protein for 6 L statting culture were obtained.

Construction of A flnity Columns: Based on the acidic nature of NAP1, an Amgel 15 matrix was used because of its higher binding capacity for proteins with a pl below 6.5. Affinity columns were constructed by coupling 4-6 mg of purified GST-NAP fusion protein to 1 ml of Affigel 15 matk (Biorad). To do this, the Affigel 15 was washed severel thes with buffer (50 mM Hepes, pH 7.6, 0.25 M KCI) to remove the alcohol in which it is stored. The washes were canied out by iesuspending the Afïigel15 beads in 10 vol of buffer, followed by sedimenting the beads by centrifugation. After the beads were washed they were left on ice for 20 min starting from when the wash buffer was first added. This allowed partial inactivation of the beads (the activated coupling sites on Afngel 15 beads are slowly inactivated by water) and to prevent overcoupling of the protein to the beads. 4-6 mg of purified GST-NAP protein was added to the Affigel 15 beads, and the mixture was agitated gently on a rotator. The progress of the coupling readion was monitored by periodically pelleting the beads and measuring the protein concentration in the supernatant. When approximately 80% of the input protein had been removed frorn aie supernatant, the reaction was terminated by adding 1 M Tris (pH 8) to a final concentration of 25 mM. The coupling reaction required approximately 60 min. The beads were then transferred to a small column and washed with elution buffer before being used in an affmity chromatography experiment. For storage, the columns were washed with buffer containing 100 mM Hepes (pH 7.6), 1 mM EGTA, 1 mM MgCl,, 2 mM DTT, and 50% glycerol. Columns were stored at -20°C and were reused up to three times. All steps were carried out at 4OC except where noted. Columns containing BSA were made by adding 5 ml of a 10 mg/ml BSA solution to 5 ml of packed Afngel15. The BSA solution was made up in 50 mM Hepes, pH 7.6, and was dialyzed against the same buffer extensively before being used to make colurnns. The BSA coupling reaction was started immediately after the Affigel 15 had been washed, and the reaction was allowed to proceed for at least 1 h at room temperature. BSA columns were washed with 50 mM Hepes (pH 7.6),2 M urea before use and between each use. The columns were stored at 4OC in PBS that contained 0.02% aride end were reused many times. Plants were grown in constant light conditions at 200 pE/m*/s and above ground parts were harvested just after bolting. Approximately 30 g of fresh tissue was ground on ice with a mortar and pestle in 30 ml of extract buffer along with acid treated sand to improve grinding. The homogenate was then pelleted in 50-ml disposable conical tubes at 3000 g. The supematant was removed and recentrifuged in Corex tubes at 10 000 g for 10 min at 4°C. The supernatant was Vien centrifuged at 40 000 g for 1 h at 4OC and the supematant was removed with a pipette, taking care to avoid particulate matter et the bottom of the tube. DTT was then added to the supernatant at a final concentration of 1 mM. The protein concentration of the resulting supematant was - 10 mgfml.

NAPI Afinity column purMcation The plant extract was first passed through a 5-ml BSA column. The BSA column was connected in series to the GST-NAP column, such that the flow through from the BSA column flowed directly onto the GST-NA? column. The columns were pre-equilibrated with extract buffer before loading the crude extract. The GST-NAP columns were loaded at 8 colurnn voh, and were then disconnected from the BSA colurnn and washed with wash buffer at 8 column voüh until no protein could be detected in the flow through by the Bradford assay. The columns were then eluted with elution buffer and 0.5 ml fractions collected. The elution was carried out by pipetting 0.5-ml aliquots of elution buffer directly onto the surface of the column bed. The elution buffer was then allowed to flow through the column by gravity and was collected in 1.5-ml tubes. A salt gradient was made by pipetting 0.5 ml aliquots of elution buffer, with each aliquot increasing in KCI concentration by 50 mM to give a range from 300 mM to 1 M KCI. The protein in each fraction was precipitated by addition of TCA to a final concentration of 10% from a 100% (wlv) stock. After a IO-min incubation on ice, the protein solution was centrifuged for 10 min in a microfuge et maximum speed, at 4'C1 and the supematant was iemoved. The tube was recentrifuged to remove remaining liquid from the walls of the tube and the remaining supematant was removed. The precipitate was resuspended in 50 pl of SDS-sample buffer, which was neutralized with the vapor from a cotton swab soaked in ammonium hydroxide. All steps were carried out at 4OC except where noted. A 10 pl aliquot of each protein fraction wes loaded onto a 12% polyacrylamide gel and gels were stained with siiver or used for westem blatting. For western Mots, pdyclonal antibodies against the full length human cyclin 81 protein (Santa Cruz Biotechnology) were used at 0.S pglrni.

Genention and aflinity purifIc8tion of anti-NAPf antibodies Antibodies against GST-NAPI protein were generated in New Zealand white rabbits using a standard immunization schedule (Harlow, 1988) at the University of Toronto, Division of Comparative Medicine. Affinity purification of anti-NAP1 antibodies was perfonned by using an antigen colurnn. Beads with the bound antigen were washed with 10 bed-volumes of 10 mM Tris (pH 7.5), then with 10 bed-volumes of 100 mM glycine (pH 2.5), followed by 10 volumes of Tris (pH 8.8). These were then washed again with 10 mM Tris (pH 7.5) until the flow-through reached pH 7.5. The polyclonal serurn was passed through the column at least three times to bind the antibody. The column was washed with 20 bed-volumes of 10 mM Tris (pH 7.5), and then 20 bed-volumes of 500 mM NaCl, 10 mM Tris (pH 7.5). The antigen-bound antibodies were eluted by passing 10 bed-volumes of 100 mM glycine (pH 2.5) through the column. The eluate was colleded in a tube containing 1 bed-volume of 1 M Tris (pH 8.8). The purHled antibodies were dialyzed against PBS with 0.02% sodium aride. Affinity-purified antibodies were used for probing cytosolic and microsomal fractions from wild-type and etal seedlings. Cytosolic and microsomal fractions were separated by centrifugation of crude protein extracts et 100 000 g.

Branch numbers were rneasured in 12-20 plants that had reached senescence. Vegetative and mprodudive tissues were prepared for scanning micmscopy by fixing in FAA (3.7% formaldehyde, 5% acetic acid, 50% ethanol), and dehydrated through an ethanol series, critical point dried, and sputtered with gold. For serial sectioning, tissue was Rxed in FAA, dehydrated through an ethanol series, followed by a five-step ethanol- acetone series and two changes of 100% acetone. Embedding was a bstep process beginning with a 33:1 dilution of Spurr's resin (Ladd, Burîington, VT) in acetone. After four changes in 100% Spurts, the samples were placed in a dessicator over-night and the plastic was polymerized for 8 h at 7O0C. Serial sections (1.5 pm) were used to detemine morphometric characteristics of the median longitudinal section of the apical meristem dome where the dome extended from the top of the meristem to the meristem base as defined by Lauf et al (1998). Meristems of short day grown wild-type and era 1 inflorescences were measured using Noiaiem Exposure morphometric software (Empix lmaging Mississauga, ON, Canada). Parameters measu red included; (1) dome height, width and area, 2) dome cell number, and 3) cell area = dome area / dome cell number. Values for median longitudinal sections through inflorescence meristems were compared statistically using one way analysis of variance (ps0.001) where each value represents the mean of 6-1 0 samples f standard enor.

Cyclin-GUS Staining Wild-type Columbia seed transfomed with a CYC1At:GUS reporter (Arath; CYCB1 ;l) were a gift from John Celenza (Boston University). The reporter constnict consists of the promoter and the first 150 amino aciâs of CYClAt, which includes the cyclin destruction box translationally fused to the uid A gene of E. coli.. The reporter constnict was transfefened into an eml-2 background by genetic crosses. For measurements of cyclin activity in the roots, plants in which the first tnie leaves had emerged (approxirnately one week old) were used. Values in text are averages for 40-50 primary mots f s.d. Histochemical detection of GUS activity was carried out using X-gluc as substrâte. Plant tissue was placed in 90% acetone on ice for 15 min, vacuum infiltmted with a solution consisting of 100 mM NaPo* (pH 7.0), 3 mM K3F3(CN)6,10 mM EDTA, 0.1% Triton X-100, and 750 ~~g/rnlX-gluc for 1 hour, and incubated for 12 hours. Root tissue was washed with 70% ethanol, cleared in 8:2:1 (chloral hydrate: glycerol: water) and mounted in the same solution on microscope slides. Vegetative meristems from SD grown plants were dissected when the primordium of the 4oth rosette leaf was visible under a dissecting scope and incubated in X-Gluc solution. Meristem tissue was fixed in FAA after GUS detection and dehydrated in ethanol, followed by two changes of 100% acetone. To avoid dissolution of the GUS precipitate, tissue was embedded in Spurr's resin following a shoitened protocol. This was done by placing samples in a 7:3 dilution of Spurfs resin in acetone, followed by a 1:3 resin dilution, and two changes of 100% resin with each step Iasting a maximum of 30 min. Samples were placed in a dessicator over-night and the plastic polymerized for 8 h at 70° C. Tissue was compared to samples that had not been embedded to ensure that the GUS precipitate was retained during embedding. Embedded tissue was sectioned at 2 p.Sections were examined under bnght- and da&-field optics.

In situ HybridIZation ln situ hybridization with digoxigenin (DG)-labeled RNA probes in wild-type plants was perfonned according to the Cold Spring Harbor Ambidopsis Molecular Genetics Course protocol (http://genome-www.stanford.edu/Arabidopsicshl-coue/5-insituhtm) with minor modifications. For the generation of DIG labeled RNA probes, a cDNA clone of €RA t (Cutler et al., 1996; Genbank Accession N. U46574) was subcloned into the EcoRl and Nod sles of the pBC-SK+ vector frorn Stratagene (Fig. 2.5). The 3' terminal portion containing the poly-A region was rernoved by digesting with Nrul and Nod, end- filling and religating to fonn pCBAN. Sense and antisense RNA probes wen generated by in vitro transcribing off BI@ and Hindll digested pCBAN respectively. Probes were hydrolyzed to -100 nt. Approximately 80-120 ng of probe were used per microscope slide. Tissue was fixed with either FAA or 4% paraformaldehyde, dehydrated in an

ethanol-xylene series, embedded in Paraplast+ (Fisher Scientific), sectioned at 10 (un, and mounted on silane coated slides (Sigma). Prior to hybridization, slides were treated with 26.4 mM acetic anhydride in 0.1 M triethanolamine (pH 8.0) for 10 min at room temperature, washed with W. SSC and dehydrated through an ethanol series. Following color development slides were dehydrated through an ethanol series. The length of time that slides were left in ethanol affeded the color of the alkaline phophatase product, with longer incubations confering a purplsblue signal and shorter incubations confering a birck red signal. pEGCYC and -AB1 construction A CYClAt (CYCBI ;l)cDNA cloned into the EcoRl site of pBluescript II KS+ (Stratagene) was used as template to PCR amplify a cDNA insert with the appropriate restriction sites for subcloning into ~€0202.To do this, ?CR prirners CYCF and CYCR were designed such that an EcoRl site would be introduced at the 5' 53 position of the cDNA and a Noll site at the 3' 1337 position. ?CR conditions were 94*C, 1 min; 50°C, 2 min; 72%, 1 min; 25 cycles, using Vent DNA polymerase (New England Biolabs). The PCR product (-1.1 kb) was gelpurified and digested with €CORand Nol! and ligated into the EcoRl and Non sites of pEG202, to give an in frame fusion of CYCl At with the LexA moiety. pEGABl was a gift from M. Ohassemian, and is an AB11 gene LexA fusion in pEG202. Fig. 2.1 pEG2û2 and pJG4-5. pEG202 is a yeast-E .coli shuttle vector containing a yeast expression cassette that includes: the yeast ADHI gene promoter (PADHI), the sequences encoding the first 202 amino acids of the baclerial repressor protein LexA, a polylinker, end the yeast ADHl gene teminator sequences (TADHI). It also contains an E. coliorigin of replication (pBR ori), the ampicillin resistance gene (AmpR), a yeast selectable rnarker (HIS3), and a yeast origin of replication (2p on). pEG202 confers hisœyeast strains the ability to grow in the absence of histidine and directs the constitutive expression of LexA or LexA-fusion proteins. pJG4-5 is a yeast-E. coli shuttle vector that contains a yeast expression cassette that indudes the promoter from the yeast GALl gene (POAM), the sequences encoding the 106 amino acid fusion rnoiety or activation tag (AD), and the transcription teminator sequences from the ADHI gene (TADHI). cDNAs are inserted into the unique EcoRl and Xhol sites so that cDNAs are expressed as fusions with the fusion moiety at their amino terminus. The fusion rnoiety includes the nuclear localization signal from the SV40 virus large T antigen (PPKKKRKVA), the bacterial 642 transcription activation domain, and the haemagglutinin (HA) epitope tag (YPYDVPDYA). The plasmid also contains an E. coli origin of replication (pUC ori), the ampicillin resistance gene (ArnpR), a yeast selectable rnarker (TRPI), and a yeast origin of replication (2~0ri)-

Flg. 2.2 pSH18-34. pSHl8-34 is a lac2 reporter plasmid used for measuring activation of transcription of by interacting partners and is derived from a plasmid that contains the yeast GALl promoter fused to /acZ. The GALI upstream activating sequences (UAS) have been deleted and replaced with four LexA operators (LexAop). The plasmid also contains an E. coli origin of replication (pBRori), the ampicillin resistance gene (ArnpR), a yeast selectable marker (URA3), and a yeast origin of replication (2p on).

Fig. 2.3 pSH17-4 and pRFHM1. pSH174 is used as a positive control and contains a yeast expression cassette that includes the promoter fmm the ADHl gene (PADHI), the sequences encoding the 1-87 amino acids of the LexA gene (LexA(1-87)) fused to the 74-881 amino acids of the activation domain of yeast GAL4 (GAL4(74-881)), and the ADHl transcription terrninator sequences (TAOHI). pRÇHM1 expresses e LexA fusion to a transcnptionally inert fragment of the Drosophia Bicoid gene product (Bicoid) and is used as a negative control. 60th plasmids contain an E. coli origin of nplication (pBRori), the ampicillin resistance gene (AmpR), a yeast selectable marker (HIS3), and a yeast origin of replication (2p on).

Fig. 2.4 pGEXNAP. Schematic showing a derivative of pGEX2TK, pGEXNAP, used for the oveiexpression of a GST-NAP1 fusion protein. pGEXNAP contains an expression cassette that contains the prornoter sequences of the E. coli lac2 gene (Plac), the coding sequences for the E. coli glutathione-S-transferase gene (GST), a thrombin cleavage site (LVPRGS), and the recognition sequence for the catalytic subunit of CAMP dependent heart muscle protein kinase (RRASV) in frame with the coding sequence of the Arabidopsis NAP 1 gene (NAPI). The expression of the fusion product is inducible by inclusion of IPTG in the growth medium. The plasmid also contains the E. coli laqlq repressor (laqlq) which iepresses transcrition from the lac2 promoter in the absence of IPTG, the ampicillin resistance gene (ArnpR), and an E. coli origin of replication (pBR on).

Flg. 2.5 pBC-ERA used for generating RNA probes for in situ hybridization. The poly-A tail is located between the Nd and Nd sites and was removed by digesting with these enzymes, end-filling and religation (pBCAN). To generate anti-sense DIG-RNA probe, pBCAN was digested with Hindll located in the polylinker, and by using T3 RNA polymerase. To generate sense DIG-RNA probe, pBCAN was digested with Blpl, which is intemal in ERAI, and by using TiRNA polymerase. The plasmid contains an E. coli orig in of replication (ColEl ori) and the chloramphenicol resistance gene (Cm R).

CHAPTER 3

FARNESYLATION INFLUENCES REPRODUCTIVE DEVELOPMENT IN ARABIDOPSIS

Serial sections and scanning electron microscopy in this chapter were carried out by Peter ûayliss. Morphometric measuiements of menstems and micrographs in Fig.4.5 are courtesy of Tarnmy Sage. INTRODUCTION The formation of muiticellular structures is a fine balance between cell proliferation and differentiation. Cell division requires the coordination of an extraordinary number of events. Among the factors that control cell cycle progression, an inventoiy of cyclins and cyclin-dependent kinases (Cdks) has been recognized as central to this control (Mironov et a/., 1999; Norbury and Nurse, 1992). The factors that can influence cell cycle regulators are beginning to be elucidated. However, how cell cycle is arrested and how progress through differentiation is achieved is less clear. One factor which may be significant in this respect is protein prenylation. Protein prenylation involves the attachment of a famesyl (15-carbon) or geranylgeranyl (20-carbon) isoprenoid to a select group of molecules, thereby facilitating protein interaction with membrane lipids andlor other proteins (Marshall, 1993; thang and Casey, 1996). The famesyltmnsferase (FTase) and geranylgeranyltransferases (GGTase l and Il) that carry out these respective reactions have been identified in anirnals, plants and lower eukaryotes, indicating that these mechanisms of lipid modification of proteins have been evolutionarily consewed (Casey and Seabra, 1996; Orner and Gibbs, 1994; Rodriguez- Concepcion et al., 1999). Although both farnesylation and geranylgeranylation are biochemically related, the target proteins of these enzymes are usually very different. A growing roster of proteins involved in signal transduction have been identified that are famesylated in fungi and animals. These include: rnembers of the Ras superfamily of GTPases (Casey et ai., 1989; Der and Cox, 1991), fungal mating pheromones (Anderegg et al., 1988; Davey, 1996; Davey et al., 1998), protein phosphatases (Cates et a/., l9Q6),a number of proteins involved in visual signal transduction (Inglese et el., 1992a; lnglese and Premont, 1996; Lai et al., 1990), a RhoB protein involved in cytoskeletal oiganization (Du et a', 1999; Lebowb et al., 1997b), certain Rab proteins involved in vesicular transport (Joberty et al., 1993), and nuclear lamins (Bniscalupi et al., 1997; Foisner, 1997; Moir et eL, 1995) which are involved nuclear envelope disassembly and reassemb[y during the cell cyde. Although a great deal of information has accumulated on the characteristics of FTases as well as their fundional dein indiviâual signal transduction pathways, the full extent of farnesylation as a regulatoty medranisrn in higher eukaryotes is still unclear. Studies in yeast and cultured plant and animal cells using FTase inhibitors suggests that proteins associated with cell division and growth are commonly affected (Miquel et al., 1997; Sepp-Lorenzino et al., 1991; Sepplorenzino and Rosen, 1998). In plants, Rases peak activities and the growth sensitivity of tobacco cell cultures to famesy!tmsferase inhibitors are coincident, and FTase peak activities precede the onset of mitosis (Randall et aL, 1993). F~rthemiore,FTases inhibitors are effective at blocking cell progression before the onset of 01 and S phase but not at 02. Together, these studies suggest plant famesylation is essential for normal cell cycle progression (Qian et al., 1996). The diversity of famesylated proteins in fungi, plants and anhals, however, has made it difficult to detemine how specific this type of modification is in regulating cell cycle events. Fuithemore, extrapolations from cell culture to whole organisms, particularly with the use of inhibitors, is always problematic due to the inherent difficulties of application, unifomi accessibility, and continued maintenance of inhibitor concentrations ove? the life of the organism. For these reasons Rase inhibitor experiments in animals have been limited to oncology studies and little detailed developmental analysis has been repoited. Thus, for both plants and animals the neeâ for in vivo mode1 systems for studying famesylation dependent processes is essential for developmental studies. ln Arabidopsis thalliane, loss-of-function mutations in the €RA 1 gene result in plants deficient in protein famesylation (Cutler et aL, 1996). The €RA 1 gene encodes the B subunit of a protein famesyttransferase. €RA l was found in a genetic screen for mutations that cause the plant to becorne supersensitive to the plant hormone abscisic acid. Seeds of enil are inhibited at concentrations of exqenous ABA, which are five times lower than concentrations that inhibit wild-type germination. In addition, ABA stimulateci dosure of leaf pores in 0ni7 is about three times more sensitive than in wilb type, which effedively reduces water loss through tmnspiraüon (Pei et al., 1998a). The phenotypes obsenred in eral mutants are consistent with the physiological des of ABA in plants, which include fast responses such as closing of leaf pores and slow responses such as the inhibition of growth or germination. We still, however, do not have a clear idea of how losci of famesylation can confer ABA supersensitivity. Although the simplest interpretation is that famesylation acts as a negative regulator of ABA signal transduction, the lack of a molecular target for this pathway means that interpretations of en1 phenotypes must be done with caution. Although mutants deficient in geranylgeranylation exist in Drosophih (Karim et al., 1W6), the era 7 mutant is currently the only famesyltransferase loss-of-function mutant in higher eukaryotes, which afforâs the unique opportunity of assessing the role that famesylation plays in processes during the development of a multicellular organisme Here, as observed in cell culture systems, famesylation has a role in cell cycle progression in the whole plant. This requirernent appears to be in both cell proliferation and differentiation and is dependent on the grovvth conditions. RESULTS ERAI gene dknrpdlon causm loss of mase activlfjt The ERAI gene has signifcant homology to the f! subunit of yeast and mammalian FTases, and was originally isolated by virtue of Hs losssf-hinction phenotype which confers seed supersensitivity to ABA at the level of germination (Cutler et al., 1996). Severe alleles of eml are either deletions, which encompass the entire coding region of the gene (eral-2 and eral-3),or disniption of the coding region by a T-DNA insertion (eral-7). Although FTases have been biochemically assayed in a number of plants, targets foi famesylation in Arabidopsis have not yet been detemined. As a first step, the loss of FTase acthrity was tested by detemining whether era 1- 1and era 1-2 mutant extracts could catalyre the transfer of rH-Fpp to synthetic heptapeptidea. The sequence of the heptapeptides was based on consensus amino acid sequences that had been previously shown to act as high affinity famesyl acceptors in tobacco (Randall et al., 1993). The sequence of the famesyl target peptide was N-GGCCAIM-C. As a negative control, a heptapeptide that has been shown to be a specific target for geranylgeranyl addition was used (N-GGCC&C). Since northem analysis indicated that the highest €RA 1 mRNA levels were in floral buds (Cutler, 1BQ5), cytosolic extracts from both wild- type and eta 1 plants were made from this tissue. After incubation of these extracts with the relevant peptides and 3H-Fpp, peptides were separated by thin layer chromatography (TLC) and visualized by exposure of TLC plates to X-ray film. The results of this experiment are shown in Fig. 3.1. In lane 3, wild-type extracts incubated with Hg-Fpp show a -CAIM dependent radioactive spot , suggesting that Ha-Fpp was transfened to this heptapeptide. No spot is visible when the GAIL peptide (lane 4) or no peptide (lane 2) was incubated with wild-type extracts, suggesting that the Fpp transfer is the result of FTase activity. In contrast, when extracts from eml plants are used there is no measurable transfer of Fpp to the peptides under any conditions tested (lanes 5 -8). These results suggest that the Rase activity is deficent in eni 1 plants. Fig. 3.1 Famesylation assay on flower bud tissue of wild-type (WT) and of erel mutants. Pluses or minuses represent addition or absence of the famesyl (CAIM) or geranylgeranyl (CAIL) peptide target. Lane 1 is boiled (5 min) wild-type extract. FOH, famesol; Fmp, famesyl monophosphate; Fpp, famesyl pyrophosphate; CCAIM, famesylated peptide. en1 mutants show subtle morphologI~~lphen0iVp.s in constant light In constant light conditions (CL) eral plants look relatively similar to wild-type plants except for a number of subtle phenotypes (Fig. 32A, B). Unlike wild-type, the margins of eral leaf blades do not curve downward, giving the leaves a broadened appearance. When measured, the average lengths of mature leaves are similar between wild-type and eral, while the width of eral leaves is slightly increased (Nocha van Thielen, personal communication). Growth of eral plants is slowed compared to wikl-type, and bolting time and senescence are delayed (Table 3.1). In wild-type, axillary meristems typically become evident in elher the ails of cauline leaves on the primary shoot or in the axils of rosette leaves. These axillary meristems form additional flowering axes or branches; most of which arise frorn the axils of cauline leaves and to a lesser extent from the axils of rosette leaves (Schmitt and Theres, 1999). For simplicity, branches originating from rosette leaf ails will be referred to as rosette branches and those originating from cauline leaf axils, as cauline branches. In CL, wild-type and enrl plants produce on average the same number of cauline branches (Table 3.1). However, a slight reduction in rosette branches is obsenred in eral mutants grown in CL versus wild-type (Table 3.1). Finally, era 1 flowers show a stage-specific extension of carpels above sepals, which results in a protruding carpe1 in young flowers (not shown).

en1 mutants have rrduceâ bmnching In short day conditIon8 In an effort to better assess the effects of ERAI loss-of-function we determined the growth characteristics of eral under a number of conditions, such as different light intensities and day-length variations. A number of ma l phenotypes, which were not obvious in CL, are unmasked when plants were grown under short day conditions (SD). Under these condiiions the dimensions of era 1 rosette leaves are similar to the rosette leaves of wild-type plants gmin the same oondiiions and en1 baves are not wider as they are in CL (Nocha van Thielen, personal communication). However, based on rosette leaf number the onset of flowering is slightly, but not significantly delayed in enl versus wild-type (Table 3.1). In contrast to CL conditions, wild-type plants grown under SD form fewer branches from rosette axils while cauline axil branch initiation usually increases (Fig. 3.2C, D). While the nurnber of cauline branches in wild-type increases approximately three times, in eml the number of cauline branches remains constant in SD (Table 3.1). In addition, while there is almost always a branch produced wherever a cauline leaf emerges along a wild-type stem, many of the cauline leaves dong an en-l stem do not produce a branch (Fig. 3.2F). These observations suggest that there is a general shift under short day conditions to a greater contribution of cauline branches to overall branching patterns in wild-type and that this aspect is affected in enil. In most higher plants, removal of the apical meristem has the effect of inducing growth of axillary meristems by relieving the suppression that is exerted upon them by the dominant apical meristem. To detemine whether the reduction in branch formation in era-1 was due to extrerne apical dominance, em-1 plants with only one primary inflorescence were decapitated at the base of the stem shoitly after bolting. Of the 10 plants tested, only 1 plant initiated a branch from a rosette leaf axil after 15 days frorn the time of decapitation. The basis for the altered branching pattern was determined by longitudinally sectioning era 1 and wild-type rosette or cauline axils. All the era-1 plants examined showed no obvious meristematic tissue present in positions where meristems are found in wild-type axils (Fig. 3.3A, 6). Therefore, the reduction in rosette branching seen in eral grown under SD is due to an inability of the mutant to differentiate these structures rather than an imposed inhibition of already developed meristems. Fig. 3.2 Whole plant morphology of wild-type and era l plants grown in continuous light (A, B) or short days (C, D, E) conditions. wild-type (A) (C); emv) (Dl (EL

Table 3.1 Phenotypic measurements of wilâ-type and era 1 plants under dmerent

Time to boIting (days) 20.0 *O3 (20) 24.6 k 1.7 (20) 45.4 t 1.3 (20) 51-8 * 3.7 (20) Number of rosette feaves 10.4~1.0(20) 12.5k2.6 (20) 450 * 4.1 (20) 49.0 * 4.9 (20) at bolting Number of cauline branches 2.0 * 0.9 (12) 25* 1.5 (18) 7.0 I1 .O (22) 1.8 t 1.3 (29)a Numberofrosettebranches 29*0.8(12) 1-2*1.3(18) 11.1( 0.5î0.8(19)

Values reprmt averages t s.d. Values in brackets represent number of plants measured. asignificantly ditte~entfrom wild-type (p=û.05). In inflorescences, most of the flowers of SD grown era 1 plants cluster at the inflorescence apex rather than distribute along the length of stem (Fig. 3.2D). In addition, the primary inflorescence in em l plants becornes fasciated about 15% of the time and many flowers show abnomal organ numbers. While wild-type flowers almost invariably consist of 4 sepals, 4 petals, 6 stamens and 2 carpels, typically in era 1, at least one floral whorl is affected (Fig. 3.4A). Although a mutant whor! cm contain either one extra or one less organ, the average number of organs per whorl is not significantly different from wild-type (Fig. 3.48). Consequently the total number of organs per flower is not significantly different from a wild-type mean of 16. The clustered floral apex, fasciated stems and ahormal number of floral organs, raised the possibility that era 1 apical meristem organization is abnormal. Anbidopsis shoot apical meristems consists of a central zone which is surrounded by a peripheral zone and a rib zone which is located beneath the central zone (Steeves and Sussex, 1989). During the growth of the apical meristem, undifferentiated central zone cells divide, displacing older cells towards the peripheral zones where they differentiate into organ primordia. While meristems are developmentally vegetative, the peripheral zones give n'se to the leaves. When the vegetative meristem developmentally converts to e reproductive meristem this zone produces floral meristems, which fonn flowers. To determine when possible changes to eral meristems might be occumng, meristems of both mutant and wild-type genotypes grown under SD were sampled every five days up to sixty deys after planting. Fig. 3.3C and D shows representative meristems of developmentally similar eni 1 and wild-type vegetative meristems. As shown in this Fig. and for al1 other vegetative meristems samples (not shown), no obvious differences could be detected between mutant and wild-type at the level of scanning electron microscopy. Upon transition to a reproductive apical meristem, however, mutant samples did show increases in meristem size aver wild-type samples. Median longitudinal sections through inflorescence meristems of SD grown wild-type and eral were compared and the apical dome of eral meristems are qualitatively larger than wild-type (Fig. 3.3E, F). This increase in apical meristem size in enil was reflected in quantitative measurements of dome width and height and average cell area in the meristematic region Fable 3.2). Furthemore, the number of cells in the epidemal layer and below this layer down to the level where the next primordia was initiated is approximately three times higher in em l meristems (Table 3.2). ml mutants aWtui/lary meristem dsvelopment in short day cond?tions Although aberrant flowers are produced at some frequency at the inflorescence apex, floral structures below the apex appear to be more severely affected by SD conditions in eral mutants (Fig. 3.5). These defects, which consist of individual short filaments flanked by what appear to be stipules and subtended by a mound of cells, are visible shortly after inflorescences begin to elongate (Fig. 3.5A, 6, C). This organization suggests that the short filament is a vestigial axillary meristem and the mound of cells below it a presumptive cauline leaf, indeed the components of a cauline branch. These vestigial structures remain arrested in this fonn and do not differentiate into any mature structure. The defects in differentiation of reproductive structures is represented in less extreme cases by lowen which consist of only sepals or only pedicels (Fig. 3.5D, E, F). Elongation of the primary inflorescence reveals that successful production of complete flowers along the stem occurs in a stop-and-go fashion. Groups of mature flowers are separated by intewening lengths of stem where no flowers are successfully produced (Fig. 3.5G). The phenotypic severity of eral shows an acropetal decrease, and most complete flowers form at the apex when plants are nearing senescence. Mature eni l flowers appear qualitatively larger than wild-type floweis. Fig. 3.3 Meristem development in wild-type and erel in short days. Longitudinal sections of typical rosette leaf axils of wild-type (A) and eni 7 (8). Scanning electron microscopy wild-type (C) and era l (D) mutant vegetative meristems of similar developmental ages grown under SD. Median longitudinal sections of wild-type (E) and en1 (F) inflorescence meristems. Bars = 80 Pm.

Table 3.2 Morphometric characteristics of the median longitudinal sections of inflorescence meristems of wild-type and efa 1.

Dame width Oome height # celis in LI # œlls beiow LI Dome Ama Ceil Area

mi @mi (rim2) m2i

WT 82.9 f 4.1 23.3 k 1.4 20.3 f 0.7 38.3 f 5.1 1336.9 f 148 22.e 1.7

era 1 148.8 f 6.7 39.9 f 3.0 31.4f 1.3 111.3f11.4 43W.9f64ô.5 37.&2,5

Vaules î s.d, Rg. 3.4 (A) Organ number frequencies pei whorl in wild-type and eral flowers. The frequency of organ numbers for 50 mature flowers of each genotype is shown. Each bar represents a patticular whori and the pattern represents the proportion of organs found in that whorl. The numbers indicated in parentheses are the average number of organs (I s.d.) found in that whorl. (6) Frequency of organ numbers ni a wild-type or eral flower. The bar pattern represents the proportion of organs found in the flower. The numbers indicated in parentheses are the average number of organs (Is.d.) per flower.

Fig. 3.5 Characteristics of morphology of reproductive structures in era 1 in short day. In short day, undefined structures are produced in eral (A, 8) consisting of a short filament (9 subtended by small mound of cells (m) and flanked by stipules (s). The short filaments are not always present and unbianched trichornes are occasionally visible on the cell rnounds or filaments (t in B, C). Flowers fomred in eral plants are either complete (E), produce only sepals (F) or only pedicels (O). Along the length of ere 1 stems, regions where flowers are successfully formed are intervened by regions where no differentiated structures are fomed (arrow) (O). Bars = 100 Pm.

€RA 1 i8 involvd In meiosb When crossing em 1 for genetic studies it was noted that en1 pollen clumped and plants were semi-sterile. Although most aspects of pollen development in era 1 plants proceed nonnally, there is an apparent variability in the progression of meiosis between individual cells and an uncoupling of aspects of pollen development from meiotic events often occurs. Wild-type microsporogenesis is characterized by the development of enlarged microsporocytes (microspore and pollen mother cells) from sporangenous tissue. While microsporocytes are in the early-late stages of prophase, callose is deposited around each microsporocyte (Fig. 3.6A). Meiosis is synchronous and cytokinesis is simultaneous followed by additional deposition of callose around each microspore (Fig. 3.6C). Microsporogenesis results in a tetrahedral arrangement of microspores that become separated from each other following degradation of callose walls (Fig. 3.6E). Exine formation is followed by intine formation and two mitotic divisions give rise to a tricellular pollen grain (Fig. 3.66). Microsporogenesis in en-l is similar to that of wild-type up to late prophase (Fig. 3.68). However, meiosis is asynchronous, although cytokinesis appears simultaneous during formation of microspores (Fig. 3.6D). Tetrads of microspores may be ananged in either a tetrahedral configuration or in a more linear array (Fig. 3.6F). Many microspore mother cells continue to undergo meiosis and subsequently degenerate, while other microsporocytes continue to undergo rneiosis during callose degradation (Fig. 3.6F, H). In many microspores, exine and intine formation and two mitotic divisions are completed resulting in the formation of tricellular pollen grains. Wall deposition appears to occur around other microsporocytes, and subsequent degeneration of the cytoplasm results prior the mitotic division (Fig. 3.61). Partial wall formation is also observed around enlargeâ microspores or persistent microsporocytes (Fig. 3.64. Flg. 3.6 Pollen development in wild-type and en, 1. A- Wild-type anther locule containing microsporocytes (M) in early prophase. Arrow indicates callose. B- enil anther locule at equivalent stage where the microsporocytes are in late prophase. ANOW denotes highly condensed chromosotnes. C- Wild-type rnicrosporocytes undergoing synchronous anaphase of meiosis. D- eml microsporocytes in different stages of meiosis with sorne in prophase (p), some undergoing either meiosis I or II (m) and others where meiosis is completed (c) and cytokinesis is occurring. E- Wild-type tetrads (1) formed after cytokinesis. F- Arrangement of en1 microspores after cytokinesis is either tetrahedral (arrow) or linear. Note the presence of a microsporocyte that failed to undergo rneiosis (double anow) . G- Mature tricellular pollen grains (pg) in a wild-type locule. H- Arrow indicating degenerated microspores of era 1 after cytokinesis. I- Occurrence of continued meiosis(m) in microsporocytes after callose degradation in en1. J- In eral mature pollen grains (pg), pollen grains where the cytoplasm has degenerated (1) and enlargeci microspores or persistent microsporocytes (arrow) are present. Ban, = 25 pm. ml mutants have aïtemû cyclln gene axpmmion in short dry The abnonnal rneiotic divisions and asynchronous pollen formation suggests famesylation may have a role in coordinating meiotic cell divisions in Arabidopsk Furthennore, the enlarged reproductive menstems observed in era 1suggest that mitotic cell division patterns may also be different in these mutants. To explore the possibility of cell division defects in era 1 mutants, the expression of Ambidopsis 81-type cyclin (CYCI At) was analyzed in wild-type and eral mutant backgrounds. 81-type cyclins are useful for following cell division patterns for a number of reasons. In al1 organisms, including plants, the synthesis of BI type cyclin is required for entty of cells into mitosis and expression of CYCl At has been shown to be transcriptionally regulated with peak expression during G2 to M phases (Mironov et al., 1999). FurUiermore, 6-type cyclins must be degraded for cells to exit mitosis, so the rise and fall of CYClAt levels is an excellent molecular marker of entry into and exist out of mitosis. Here, a CYC1At:GUS reporter fusion, which contahed the CYClAt prornoter and its destruction box in frame with the 3' end of the UidA gene of E. coli was followed to determine cell division patterns in era 1 and wild-type backgrounds. Because root meristems are anatomically simple? to interpret venus the shoot meristems, analysis was first performed on root samples. In one set of experiments, the numbei of primary root meristem cells in one week old seedlings that showed GUS activity were counted (Fig. 3.7). en1 roots on average contained twice as many stained cells compared to developmentally similar wild-type roots, indicating CYC1At expression is altered in eral mutants (Fig. 37A, B). Furthennore, the distribution of GUS staining within root cells was different between the two genotypes. In wild-type roots, of the cells that did stain, approximately an equal nurnber contained stained cytoplasrn or stained nucleus (Fig. 3.7A). Of cells that showed nuclear staining two classes exist. One clam were cells containing single stained nuclei while the other class were cells that contained two nuclei in which the staining was around the periphery of each nuclei (Fig. 3.7C). Rie distribution of CYC1At:GUS stain repmsents where the GUS protein is localized in the ceIl, which in tum is a reflection of where the cells are in the cell cycle. In plants, just before entering mitosis, cyclin 61 complexes localize with cytoplasmic microtubules during prophase and then abruptly translocate to the nucleus upon entry into mitosis (Mironov et al., 1999). The GUS staining in the cytoplasm most likely represents the beginning of cyclin 8 synthesis just before cells enter mitosis, whereas the nuclear staining represents peak cyclin B expression and nuclear localization in cells during mitosis. Finally, when cells are in telophase and Wo nuclei are present in each cell before cytokinesis, the GUS staining pattern represents the rernnants of cyclin degradation. In contrast to wild-type, 77% of erel root stained cells were of the cytoplasmic class with the rernaining 13% containing stained nuclei. Therefore, mutant roots have not only more stained cells than wild-type, but also have a different ratio of cell cycle stages than wild-type. Although the reason for the increased CYC1At:GUS staining in eml is not clear, the fact that the CYC1At:GUS reporter contains a ubiquitin dependent cyclin 61 destruction motif may mean famesylation affects CYC1At degradation. The staining pattern rnay also mean progression through mitosis is somehow affected. Inhibition of cyclin81 degradation has been shown to anest mitosis resulting in a decrease in the overall cycling of the cell (Murray and Hunt, 1993). The preponderance of cytoplasmically localized CYC1At:GUS staining in era 1 root meristems suggests many of these cells may be in late prophase. This is consistent with the failure of many en1 microsporocytes to enter rneiosis beyond prophase. The clear differences in CYC1At:GUS staining patterns in the roots led us to do sirnilar qualitative assessment of apical meristern CYC1At expression patterns, Longitudinally sectioned vegetative eral meristems from SD grown plants also showed altered CYC1At:GUS staining compared to wild-type meristems (Fig. 3.7). The staining pattern in wild-type apical meristems is punctate and the rnajority of cells with GUS precipitate are mostly restricted to aie leaf primordia and the peripheral zone of the meristem (Fig. 3.7D). The staining pattern in wild-type plants is consistent with Rg. 3.7 Distribution of CYC1At:GUS expressing cells in root and vegetative meristems of wild-type and en 1. bots stained for GUS activity and viewed in bright field. (A) Wild-type; (B) ere 1, (C) a representative wild-type to show the different GUS localkation patterns within individual cells. The average number of cells expressing GUS activity in prhaty roots is given at the top of (A) and (6).Longitudinal sections of wild-type (D) and era 1 (E) vegetative apical meristems stained for GUS activity viewed under da&-field optics. The presence of GUS activity under bright-field is a blue precipitate and under da&-field it appean pink to red. Photographs are representative of 6-1 0 meristems analyzed. Bar = 50 Pm. expected patterns of cell division in the meristem (Evans and Barton, 1997). As obsewed in wild-type, localized CYC1At:GUS staining patterns in both the upper central and peripheml zones of eral show only a small number of cells within these zones are entering or actually in mitosis. However, unlike wild-type meristems, a large group of weakly staining eral cells was detected in the lower central region of the meristem and in the underlying rib zone (Fig. 3.7E). It is possible that this pattern is the result of diffusion of GUS activity or substrat0 piecipitate. However, it would be expected that if this were the case then diffusion should be limited to a few sunounding cells and not such a broad area. Although the reason for the misexpression of CYCI At in the central core cells is unknown, the pattern is detected in vegetative meristems that are morphologically normal. This result indicates that the increased meristem size seen in later reproductive meristems in the mutant is most likely due to aberrant gene expression that is occuning much earlier in meristem development.

ERAI mRNA expres8ion patterns are correhW.d with ahort dsy grown era1 phenowpa Since eral phenotypes implied a greater dependence for ERAI function in short day conditions, in situ hybridization of €RA1 mRNA was carried out on tissue of short day grown wild-type plants. A basal level of ERAI transcript is present in all tissues. Higher levels are present in regions of active cell division, although this could be due to the cytoplasmic density of cells that are actively dividing (Fig. 3.8A-D). Neveiaieless, the expression patterns of ERAI mRNA correlate well with many of the eral phenotypes in plants grown in SD. For example, in both vegetative and inflorescence meristems higher transcript levels are present in the peripheral region of the meristem and in primordia (Fig. 3.8C, D). In fioral tissues €RA l mRNA is mostly localized to developing tissues such as ovule primordia and mict08porocytes (Fig. 3.8E-H). In embryos a high amount of signal is observed from at least the globular stage to the torpedo stage, and then begins to decrease in mature embtyos (Fig. 3.81-M). Expression of ERAI pea ortholog was analyzed using a promoter GUS reporter system (Zhou et al., 1997) and high GUS activity was found in the shoot apex, flower receptacles, regions of axillary bud formation and in vascular tissue. Similarly, famesyitransferase activity was detectable in all tissues tested with the highest activity in the apical bud, stem and developing fruits (Schmitt et al., 1996). The studies on pea and tomato and expression data presented here for ERAI indicate that there is some level of regulation of farnesyltransferase abundance in various tissues. More irnportantly, expression patterns of ERAI RNA correlate well with many of the eral phenotypes seen in plants grown in SD indicating these patterns are reflective of ERAI function.

Suppres80rs and enhances of eal In an effort to expand our knowledge of the pathway(s) affected by the ERAI, suppressor and enhancer screens of the eral mutant were undertaken. In the case of suppressors, second site mutations in deletion lines of eral were identified that could restore ABA seed sensitivity back to wild-type sensitivity (0.3 pM in era 1 versus 1.2 pM ABA in wild-type). The isolates from the suppressor screen are summarized in Table 3.3. A striking feature of these mutations is that most not only revelt seed ABA sensitivity back to wild-type levels, but also restore adult plant phenotypes back to a more wild-type morphology. Characteristic era l phenotypes such as protniding carpels and shortened siliques are no longer evident in these suppressor lines. This might suggest that ABA seed sensitivity is somehow linked to adult plant morphology, since suppression of ABA sensitivity at the level of germination alsa seems to suppress eral defects in adult plants. It is interesting that some of the suppressor lines isolated are alleles of abi3 (Sarkar, 1999) and that they are dominant over en1. This suggests that AB13 is not seed specific and that it could potentially be acting in aie same pathway as ERAI. If AB13 function somehow affects the cornpetence of cells to respond to ABA (se8 Chapter 1) aiese results might provide an important link between ABA sensitivity and plant morphology. The identity of other suppressor mutations could b in genes required for ABA biosynthesis, or positive regulators of ABA signaling. The continued characterization of these suppressor tines should help to provide additional insights into the molecular nature undedying these possibilities. Enhancement of ABA seed sensitivity was not as successful (see Table 3.4). It was hoped that eral phenotypic enhancement could help to genetically identify €RA1 target proteins. This was based on the premise that loss-of-function of an €RA1 target would cause a more severe phenotype than ERAI deletion alone. The limit of enhancement was 0.03 pM ABA for germination (see Table 3.4). In some cases, enhancer isolates also had more severe aduît phenotypes compared to the ere 7 parent. These included plants that showed a higher degree of fasciation or almost complete infertility. Compafatively fewer enhancers were isolated than suppressors. This rnay be a reflection of the potentially lethal consequences of isolating mutations that affect seed ABA sensitivity at such low levels of exogenous ABA (0.03-0.06 )tM versus 0.3 pM). For example, F2 progeny from enhancer isolates backcrossed to the parent era 7 allele, which were scored for germination on 0.03 and 0.06 pM ABA did not give the expected ratios for simple dominant or recessive inheritance. This suggests a complex interaction between the enhancer alleles and era 1. Compounded with their extrernely reduced fertility, the characterization of the enhancer lines to any satisfactory level was not done. Fig . 9.8 In situ hykidization of wild-type tissue from SD gmplants using ERAI DIG-RNA probe. Positive signals are red-brown to purple-blue. Sedons A. C, D, E, F, O, 1, K, L and M where hybridized to anti-sense RNA probe. Longitudinal section through an inflorescence. ComparaMe section to (A) probed with sense probe. Longitudinal section through developing flowers. Longitudinal section through a vegetative meristem. Section through an anther locule containing microsporocytes. Section showing hybridizetion to microspore tetrads and tapetum in an anther locule. Section through a gynoecium where ovule prirnordia are evident. Sense control of a comparable section to O. Section through a silique showing hybfidizaüon to a globular stage embryo. Sirnilar section of an embryo probed with sense RNA. Hybridization in a torpedo and (L) late torpedo stage embryo. Positive signal decreases in mature embryos.

Tabk 3.3 A list of eml suppmssot Iines. Indicated W the relative suppression of erel seed sensitivity to ABA (PM). Also shown is whether the line suppresses em 1 vegetative phenotypes (wt). Mersuppressor phenotypes include: rf, reduced fertility (based on silique length); ar, aria1 rosette; ib, increased branching; df, dwarfed; sd, semi-dwarfed; hy, elongated hypocotyl on ABA; era, denotes thet plant is indistinguishable from parent. Line number indicates the allele of the parent (8.g.. 13- = erel-3),followed by the pool number and the plant (e.g.. 0103 is pool number 1, plant number 3). Vegetative phenotypes are for plants grown in constant light. abi3 alleles are italicized. [AUJ Qhmaot~ga 1*2 lut 1.2 wt 1.2 wt 1.2 wt O .9 wt 1.2 wt 0.9 wt 1.2 wt 1.2 wt 1.2 wt 1.2 wt 1.2 rf 1.2 wt; hy 0.6 era 1.2 wt 1.2 wt 1.2 wt 1.2 wt 1*2 wt 1.2 rf 1 02 wt 1.2 wt 0.9 wt 1.2 wt o. 9 wt 0.9 ad 0.9 wt 1.2 wt 1.2 ut; hy 0,9 wt 1.2 we; hy 0.6 wt 1.2 df 1.2 sd 1.2 wt 1-2 ut 1.2 ut 1.3 l& 1.2 wt 1.2 id 1.2 wt 1-2 d 0.9 wt 1.2 8r 1*2 ar 1.2 ad; rf 1.2 df 1.2 ut 1.a ~t 0-9 wt o. 9 ut 0. 9 wt 1.2 wt 0-9 wt 1.2 wt 1.2 sd 1.2 wt 1.2 ut 1.2 wt 1.2 wt 1.2 wt 1-2 wt: hy 1.2 Pd 1.2 wt; rf 1.2 wt; hy Table 3.4 A list of eml enhancer ünes. lndicated is the relative enhancement of eral seed sensitivity to ABA (PM). Also shown are the enhancer phenotypes. era denotes that plant is indistinguishable from parent. Line number indicates the allele of the parent (0.g.. 13- = em 1-9,followed by the pool number and the plant (8.g.. 0103 ie pool number 1, plant number 3). Vegetative phenotypes are for plants grown in constant light. Phanotypm era dwarfed-chlorotic dwarfed-chiorotic dwarved-chlaxo tic era fasciated pin-f ormed; fasciated era era dwarf ed-chlorotic era era era era; reduced fertility era era; reduced fertility reduced fertility; chlorotic era dwarfed-chlorotic era era DlSCUÇSION Previous studies on en, 1 mutants have linked famesylation with two classically defined ABA responses in plants, se& dorrnancy and water relations (Cutler elal., 1996; Pei et al., 1998b). However, given that famesylation is a ubiquitous function and that there are potentially numemus cellular target proteins, that only subtle phenotypes are observed in these mutants under continuous light growth conditions was intriguing. Possibly the lack of phenotypes reflects functional redundancy for farnesylated processes in plants. For example, the cross-specificity of famesyltransferases with other prenyltransferases, like GGTase I (Lebowitz et al., 1997a; Trueblood et al., 1993; Yokoyama et al., 1997) means that some compensation for loss of ERA 1 function is likely to exist in era 1 nulls. In addition, it is also possible that some degree of redundancy for ERAI function is present, as we are not certain that €RA 1 is the only 8 famesyltransferase gene in Arabidopsis. ln this study, however, an array of eral mutant phenotypes that are contingent on photoperiod growth conditions are described. This suggests that the contributions of any redundant functions are not enough to compensate for loss of the ERAl Kase activity under short day conditions. The €RA1 FTase is, therefore, necessary for a number of developmental processes in plants, and this requirement is environmentally sensitive.

FammyIation and ce1luIar dfibmntiation Under short day conditions two major famesylation-dependent phenotypes are obsenred in mature eral mutant plants; the inability to initiate or maintain lateral organs on the main stem and an enlargement of the apical meristem. Two models can be used to explain these phenotypes. The fimt possibility is that aspects of axillary and apical meristem development are regulated by independent signaling pathways that both require different famesylated intemediates to function conedly. A second possibility is that a single ptocess is affecteâ in em l mutants. However, because axillary meristem initiationlmaintenance and regulation of apical meristem sire may not be developmentally equivalent, the defect is manifested dhrently in the two eml mutant meristem types. Mutations in a number of genes in Arebidopsls have been identified that preferentially affect one meristem type over another, suggesting these tissues are not developmentally equivalent. In revoluta mutants, for example, axillary meristems have a greater tendency to abort development, while the primary apical meristem is les8 affected (Talbert et ai., 1995). Similarly, pin and pinoid mutations cause a reduction in the capacity to generate mature flowers, floral organs and cauline leaves; however, primordia are initiated from the apical meristem (Bennett et a/., 1995; Okada and Shimura, 1994). In contrast, mutations in a number of genes have been identified in Arabidopsis that increase meristem size, but do not appear to affect axillary meristem initiation or maintenance (Clark et al., 1993; Laufs et a/., t998a; Laux et a/., 1996; Leyser and Fumer, 1992). Although these genetic experiments argue for discrete signaling steps for apical versus axillav meristem development, it is also possible that these phenotypes represent the sensitivity of different meristematic tissue to genetic perturbation. For example, regulators such as LEAFY and APETEiA2 are expressed at different stages of plant development in Arabidopsis, yet loss-of-function mutations in these genes preferentially affect only floral initiation and floral organ identity (Blazquez et al., 1997; Jofuku et al., 1994). It is possible that a lack of phenotypes in tissues where gene products are produced may simply reflect genetic redundancy. Resolution of the role of famesylated proteins in meristem organization will require the identification of famesylated targets and characteriration of their functions in both axillary and apical meristems.

Farnesylation and the cell cyck As already mentioned, genetic analysis has identified a number of gene produds that appear to be invoîved in controlling meristem size. davata mutations, for example, have defineci a putative receptor kinase (CL VI) and its potential ligand (CLV3) (Clark et al., 1997; Fletcher et al., 1999). It is interesthg that, although mutations in both CLA VATA genes result in meiistem enlargement, developmental analysis suggests the undeilying cellular mechanisms of therre phenotypes and other enlarged meristem mutants may be different. For exemple, clvl meristem cells appear to have increased rates of cell division, whereas the ch3 mutants show reduced cell proliferation in the apical menstem (Laufs et al., 1gg8b). Thus, paradoxically, mutations that decrease cell division can also result in enlargement of the apical meristem. A nurnber of enlarged meristem mutants with reduced ce!! divisions have been interpreted as having aberrant differentiation. Loss of functions necessary to promote the transition of slowly dividing central zone cells into actively dividing peripheral zone cells, for example, could result in an overall increase in undifferentiated central zone cells and yet, overall have fewer cell divisions (Laufs et a/., 1998b). The demonstration that mutations in another meristem regulator gene, wuschel (wus), can cause central zone cells to be mispecified and undergo differentiation further demonstrates the complex interaction between differentiation and cell proliferation (Laux et al., 1996; Mayer et al., 1998). At this time it cannot be concluded whether the increased floral meristem size seen in eral is causes by increased cell division or aberrant differentiation. However, the total number of cells in a mutant floral meristem is approximately three times higher than wild-type, which means that either an increase in celt proliferation has occuned or that a large number of undifferentiated cells have accumulated. Although the overall number of meristem cells has increased, the CYC1At:GUS repoiter data demonstrate the complexity of meristem organization. In both wild-type and eral vegetative meristems, the upper layen show a similat small number of cells with localized steining patterns indicating mitotic rates in these cell layers is probably not difiersnt between the two genotypes. By contrast, the abnomal CYC1At:GUS reporter expression pattern in the lower central and rib zone cells in eral versus wild-type suggest thb area of the apical meristem may play an important role in subsequent floral meristem organization. In this contact, CLVl and WüS expression patterns also indicate the importance of proper central zone gene expression on the control of meristem cell proliferation (Clark et al., 1997; Mayer et al., 1998). Although €RA 1 is most highly expressed in the upper layers of the wild-type apical meristem, the altered CYC1At:GUS expression in enl is not in this region but partially overlaps CL Vl and WUS localization. Perhaps famesylation dependent signals in actively growing regions result in negative regulation of genes like CYCl At in the lower central core of the meristem. However, because of the low ubiquitous expression of ERAI mRNA throughout wild-type meristems, it is possible that the misexpression of CYCIAt is due to reduced famesylation activity in this region, leading to slower turnover of CYClAt. The ubiquitous localization of ERAl message in wild-type, versus the CYCl At expression pattern in the mutant does, however, show at that in individual cells loss of mase function does not a priori lead to ectopic expression of CYC1At. Loss of ERAI function also has effects on CYClAt expression in the roots. Interestingly, as with the apical meristem. the root pattern shows increased expression behind the central organizing center of the root meristem. Elegant cell ablation experirnents in Arabidopsis roots and studies involving WUS suggest both shoot and root meristem may represent modifications of a the same basic rneristem organization (Mayer et ab, 1998; Van Den Berg et el., 1997). The sirnilarity of expression patterns of CYCl At in both era 1 root and shoot meristems supports this contention and suggests that a famesylated protein(s) is required to negatively regulate genes in this regions. Loss of €RA1 function appean to be more detrimental to axillary meristem development than the shoot apical meristem. Mutant axillary meristems appear to be defective in either cell division andfor differentiation. Mutant side shoot structures can be initiated, but these do not produce any dlfferentiated tissues. In an instructive scenario, famesylation could be required for the formation of axillary meristematic cells. Altematively, meristem formation may occur nonnally in the mutant but famesylation is required to permit further cell proliferation of an axillary meristematic cell. This permissive scenario is similar to the function of many growth factors in animals, which are required for cell division but not differentiation. The morphology of eml abnormal side shoots is reminiscent of floral phenotypes reported for ufo mutants (Levin and Meyerowitz, 1995; Wilkinson and Haughn, 1995). In these mutants, the commitment to fon side shoots is Initiated; however, this comrnitment is eithei not maintained or b insufficiently strong. Interestingly, the UFO gene encodes a protein with an F-box motif that is thought to be involved in cell cycle regulation in both Arabidopsis and Antinhinum (Ingram et al., 1997). The production of vestigial structures and the reduction of axillary meristem formation in era 1 mutants rnay be due to a decrease in the rnitotic cornpetence of cells comprising these structures. That both cauline leaves and rosette leaf axils with no axillary meristems can be produced in era 7 mutants may just reflect different degrees of severity of this phenomenon. Plant and animal cell culture experiments involving FTase inhibitors suggest plant famesylation is essential for normal cell cycle progression (Qian et al., 1996; Randall et al., 1993). If cell division is sufkiently slowed in era 1 axillaty meristems, perhaps cells are not positioned correctly to interpret differentiation signals. The establishment of en1 cell culture lines may clarify some of these ideas.

Farneyletion and ABA There is a general consensus that ABA inhibits shoot growth by inhibiting cell proliferation. The reduction of cell division or DNA synthesis by ABA application has been described in a number of studies (Bryant and Chiatante, 1997; Dudits et aL, 1998). The first indication of how ABA may interact with the cell cycle machinery was the identification of an ABA inducible cyclin-dependent kinase inhibitor (ICK1) which interacts with Arabidopsis Cdc2a and CycD3 (Wang et al., 1998). If normal €RA1 function is to promote cell division, the reversion of most eni-l phenotypes by genetically altering ABA sensitivity implies that ERAI could somehow mediate some of these events. That the identity of sorne of the eral suppressors am abi3 aileles (Serkar, 1999) and the discovery that AB13 is expressed in quiescent apices of Arabidopsis (Rohde et al., 1999), suggests that a potentially broad regulatory network for inhibiting cell division by ABA might exist, which would include AB13 and ERAl in antagonistic roles. The molecular identity of the other suppressor genes will help to define more clearly whether this scenario is correct. In conclusion, these results suggest that, aside from regulating ABA dependent plant processes such as seed domanoy and stomatal aperture size, ERAI functions in a diverse number of cellular events ranging from rneiosis and mitosis to differentiation. That mal mutants show cyclin61 misexpression in the central meristernatic zones in both roots and shoots suggests this gene might be a negative regulator of cell cycle gene expression within these cells. The production of vestigial structures and the reduction of axillary meristem formation in era l might be due to a decrease in the mitotic cornpetence of cells comprising these structures. Since cell proliferation and differentiation are essentially mutually exclusive events, one interpretation is that if cell division is sufficiently slowed then it is possible that cells become prematurely differentiated. Paradoxically constitutive expression of a dominant negat ive allele of Cdc2 in Arabidopsis results in a decrease in cell division, but development is unaffected and plants are cornposed of fewer, but larger cells (Hemeriy et al., 1995). Some degree of regulation of farnesylation activity exists (Goalstone et al., 1997) and so the famesylation status of regulatoiy proteins in cells with heightened famesyltransferase activities would follow. Besed on what is known of protein famesylation this modification can act as a switch to affect the function of target proteins. It follows that by spatially restricting famesyltransferase activity then it would be possible to coordinate cell division with differentiation. The abHity to address these questions within the context of a whole organism should reveal how the cell cycle machinery is integrated with dmerentiation to regulate overall development. CHAPTER 4

NAP1 IS FARNESVLATED IN ARABIDOPSIS AND INTERACTS WlTH CYC1AT Many cell signaling events are mediated by the post-translational modification of proteins such as phosphorylation, glycosylation, proteolytic processing and methylation. Over the past decade lipid modification of proteins has figured a prominent mode of changing the activity and suôcellular localization of many regulatory and structural proteins. These commonly include trimeric G proteins, G-proteincoupled receptors, specific receptoi kinases (GRKs) and cell surface proteins. The process of lipid modification includes the covalent attachment of a long-chain acyl group and can be classified according to the identity of the attached lipid: palrnitoylation (C-16), myristoylation (C-14), prenylation (C-15; C-20), and glycosylphosphatidylinositol (GPI) linkage (Chow et al., 1992; Turner, 1994; Bang and Casey, 1996). These modifications also differ ni the position of their attachment along the protein sequence. Myristol groups are generally attached at the amino-terminus, palmitoyl groups are attached intemally, and prenyl and GPI groups are attached at the carboxyl-terminus (Casey and Seabra, 1996; Clarke, 1992). nie demonstration that oncogenic RAS needs to be prenylated to cause cellular transformation (Casey et al., 1989) has focused a great deal of attention on this type of modification because of its potential cancer t herapeutic implications (Sebti and Hamilton, 1997). Protein prenylation entails the covalent attachment of either a 15-carbon isoprenoid, famesyl, or a 20-carbon isoprenoid, geranylgeranyl. The specificity of isoprenoid attachment is achieved in part by the carboxyl-terminal amino acid sequence of the rnodified proteins. In general, three groups of carboxyl-terminal sequences that seive as substrates for the protein prenyltransferases. The first consensus sequence is -CaaX, where C is cysteine, 'a' is usually an aliphatic amino acid, and X is any amino acid. If the X position is occupied by a leucine, the protein is a substrat8 for geranylgeranyl attachment (flnegold et al., 1991; Kinsella et aL, 1991; Moores et al., 1991). Other amino acids occupying the X poslion define protein subtrates for famesyl modification. Apart from -CaaX consensus sequences, motifs such as CC- or -CXC serve as substrates for double geranylgeranyl addition and are found mostly in the Rab family of GTPases (Casey and Seabra, 1996; Clarke, 1992). Following isoprenoid attachment the carboxyl-terminal tripeptide is often times endoproteolysed, and the isoprenyl-cysteine is carboxymethylated. These reactions are thought to be executed a the endoplasmic reticulem (ER) which is the site of enzymes known as 'aaX proteases' and catboxyl methyltransferases (Boyartchuk et el., 1997; Boyarichuk and Rine, 1998; Kim et al., 1999; Otto et al., 1999). However, not al1 GaaX temini undergo proteolytic cleavage, since the a subunit of rabbit muscle glycogen phosphorylase kinase is famesytated but not proteolyzed (Heilmeyer et a/., 1992). Carboxyl terminal processing following farnesylation is thought to increase the hydrophobicity of the modified protein, with methylation eliminating the negative charge on the C-terminal carboxylate group, which effectively causes it to associate wit h membrane cornpartments (Parish and Rando, 1996). These processing steps rnay be particularly relevant for famesylated proteins, for which membrane association is weaker than for geranylgeranylated proteins (Bhatnagar and Gordon, 1997; Rando, 1996). Although the dominant contribution to modified-protein hydrophobicity is the C- terminal famesyl and carboxyl methyl groups, they are generally not sufficient for the specific localization of some proteins to the plasma membrane, and other functional groups are often required. Indeed, in the case of N- and H-Ras, S-palmitoylation of cysteine residues in these proteins serves as a second type of modification (Dudler and Gelb, 1996; Morello and Bouvier, 1996; Willumsen et a&, 1996). A cunent hypothesis is that famesyl attachment to Ras might sewe as an initial signal for its weak association with endomembranes, so that it may serve as a substrate for further modifications such as palmitoylation (Magee and Marshall, 1999). In other cases, such as with K-Ras4B (James et al., 1995), a stretch of basic amino acids at the C-terminus of the protein is thought to facilitate electrostatic interactions of this region of the protein with the anionic head gmup of membrane phospholipids (Bhatnegar and Gordon, 1997) (see Fig. 4.1). Aithough a Wo signal hypothesis' (either polybasic regions or palmitoylation) can explain membrane association, it is insufficient to explain the specific localization of certain prenylated proteins to diverse membranes. For example, RhoB is localized to endomembranes (Piton et al, 1996; Piron et aL, 1994), Rap2 to the Golgi apparatus (Berenger et eL, 1991) and Rac2 to the plasma membrane (Wientjes et al., 1997). A third signal may be provided by features that are inherent to individual proteins and provide the necessary information for organellar localization. The presence of Ras in specialized regions of the plasma membrane called calveolae, which are enriched in the marker protein calveolin, along with receptors and other signal transducers, and the apparent ability of Ras to bind to calveolin, would argue for such a third signal (Mineo et al., 1996). Structures related to calveolae, known as lipid rafts, which are plasma membrane domains enriched in cholesterol and sphingolipids, also have Ras protein associated with them (Simons and Ikonen, 1997). Whether similar scenarios are tnie for other prenylated proteins awaits elucidation. The membrane association of famesylated proteins, however, is sornetimes the result of protein-protein interaction, where the famesylated protein associates with membrane bound receptors. An example of this is the preferential association of the a subunit and the py subunits of transducin when the y subunit is famesylated (Fukada et ab, 1990). In addition, activation of membrane free adenylate cyclase cornplex in Saccharomyces cerevisiae is much more effective with a famesylated Ras than with aie non-famesylated fonn (Kurorda et al., 1993). Mutant foms of yeast Ras2p which are farnesylated but not palmitoylated are not localized to the plasma membrane and have only minor effects on cell growth. However, this Ras2p-mutant km is unable to mediate the transient induction of intracellular CAMP levels in response to glucose (Bhattachaiya et al., 1995) suggesting that the function of the famesyl group of Ras2p is to facilitate interaction with the membrane bound adenylate cyclase. More recently, Pexl9p famesylation in yeast has been shown to be required for proper pemxisome biogenesis and that physical interaction of PexlQp with Per3p, a membrane peroxin protein, is dependent on the famesylation status of Pexl Qp (Gotte et al., 1998). The enzymes that catalyze protein isoprenylation include: protein famesy ltransferase (FTase), the type 1 protein gerany lgeranyltransferase (GGTase-1) and the type II protein geranylgeranyltransferase (GGTasell) (Manne et al., 1990; Moores et al., 199 1; Reiss et al., 1991 a; Reiss et al*, 1992; Reiss et al., 1991 b; Seabra et al.. 1991). FTase and GGTase-I catalyze the covalent attachment of either famesyl pyrophosphate (Fpp) or geranylgeranyl pyrophosphate (GGpp) to proteins with the C- terminal -CaaX and -CaaL motifs, respectively. GGTase-Il catalyzes the attachment of GGpp to proteins with C-terminal -CXC or -CC- motifs. The occurrence of mase and GGTase-l have been reported in many eukaryotic organisms including yeast (Finegold et al., 1991; He et al., 1991; Mayer et al., 1993) plants (Cutler et al., 1996; Morehead et al., 1995; Randall et al., 1993; Schmitt et al., 1996; Yang et al., 1993; Zhou et al., 1996) and mammals (Chen et al., 1991; Reiss et al., 1991a; Reiss et al., 1992; Seabra et aL, 1991; Yokoyama and Gelb, 1993; Zhang et al., 1994). 00th FTase and GGTase-l are zinc-requiring, heterodimeric enzymes with a shared a-subunit and related subunits (Casey and Seabra, 1996). In addition, the two enzymes may also be conserved with respect to their catalytic mechanisms. On the basis of kinetic studies, both enzymes preferentially bind the isoprenoid substrate before binding their protein substrate (Pompliano et al., 1992; Yokoyama et al., 1995). In both cases the release of isoprenoid substrate is slower than the initial formation of a ternary cornplex consisting of enzyme, isoprenoid and protein substrates (Furfine et al., 1995; Yokoyama et al., 1995). Based on these levels of conservation it is not surprising that these enzymes havesome degree of isoprenoid and substrete overlap in vitro and h vivo (Armstrong et a/., 1995; Caplin et al., 1994; Trueblood et al., 1993). For example, substrate proteins with C- terminal -CIIM motifs can be geranylgeranylated by both FTase and GGTas8-l (Caplin et al., 1994). In yeast mutants lacking FTase, Ras proteins are famesylated by GGTase-1, and yeast mutants lacking GGTase-I can be rescued by FTase overexpression (Tnieblood et al., 1993). Moreover, a Ras-related GTPase, RhoB, which may have a role in the organization of the actin cytoskeleton in mammals, can be modified by either famesyl or geranylgeranyl and both isoprenoids can be transferred to the protein by GGTasel (Armstrong et aL, 1995). Distinct aspects of enzyrnatic catalysis of FTase are associated with the a and subunits on the basis of mutational analysis (Andres et al, 1993; Mitsuzawa et al., 1995; Ohya et al., 1996). The stabilization of the heterodimer and some catalytic activity is associated with the a subunit (Park and Beese, 1997), while isoprenoid and peptide binding, and direct enzyrnatic activity are associated with the $ subunit (Dunten et al., 1998; Long et al., 1998). Indeed, mutagenesis of a Zn*+-coordinatingcysteine residue in the B subunit of FTase causes a loss of isoprenoid binding and enzyme activity (Fu et al., 1996). Both protein and peptide substrates can be cross-linked to the subunit of Rase and the N-terminal and C-terminal regions of the subunit are apparently involved in the CaaX preference of the enzyme (Del Villar et al., 1997; Reiss et al., 1991a; Ying et a/., 1994). The suggestion from these studies is that the FTase B subunit determines both isoprenoid and protein substrate specificities. The determination of the three dimensional structure of FTase, however, has indicated that a cleft fomied by the interface of the a and p subunits would be the -CaaX peptide binding site (Park et aL, 1997). More recently, similar crystalographic analysis has indicated that this might not be true, and that the subunit is the sole site for peptide binding (Dunten et al., 1998). Preliminary studies in plants indicate that plant protein famesylation has similar characteristics to those of yeast and animal systerns (Qian et ai., 1996; Randall et aL, 1993; Schmitt et a/., 1996). The existence of a carboxymethyltransferase activity in plants has ais0 been demonstrated (Crowell et al., 19Q8), and the existence of an aaX- protease related gene in the Arabidopsis DNA database has been reported (Nambara and McCourt, 1999), suggesting that prenyl protein modification and processing have been conserved in plants. Rase activity in plants has been linked to cell cycls regulation (Qian et al 1996; Randall et al., 1993). However, detailed studies on the potential regulatory rote of FTase has been limited by a lack of defined famesylated target proteins. A number of strategies have been used to identify pmnylated proteins. For example, labeling studies using radioactive mevalonic acid (MVA), a precursor of Fpp and GGpp, have shown that a number of chloroplast, mitochondrial and nuclear proteins are prenylated h vivo (Pannryd et a/., I997a; Parmryd et ah, 1997b; Randall et al., 1993; Shipton et al., 1995). ûther strategies have included screening k-phage cDNA expression libraries with active, cellular, plant extracts, which transfer radiolabeled Fpp ont0 plaques expressing -CaaX containing proteins (Biermann et al., 1994; Crowell et al., 1996). However, apart from demonstrating that a potentially large number of plant proteins can undergo this type of modification, these kinds of studies have provided limited information on the identity or functional role of these proteins. Cunently, only one famesylated protein involved in heat-shock responses, ANA, has been identified and shown to be dependent on famesylation for its membrane localization (Zhu et al., 1993). The isolation of a famesyltransferase loss-of-function mutant in Arabidopsis, era 7, provides a unique oppoitunity for the functional analysis of famesylation dependent events in plants. In conjunction with biochemical data, it affords a viable system for testing the relevance of famesylated proteins in an in vivo context. To this end, a screen aimed at identifying Arabidopsjs farnesylated proteins was undertaken by using a yeast interaction trap cloning system. This rnethod had limited success and its potential drawbacks as a -CaaX protein screening method are discussed. However, it did identify a protein containing a -CaaX motif which has homology to nucleosome assembly protein 1 (NAPI) of yeast and mamrnals. Ag. 4.1 Cartoon showing the potentiai fates of famesylated proteins. (1) Cytoplasmic proteins containing 4aaX motif becorne famesylated (9), (2) then becorne bound to the ER and aieir C-temiinal 'aaX' amino acids are proteoiyzed and carboxyl methylated (Me). (3) They can aien traffic to plasma membrane by the secretoty pathway or (4) through the cytoplasm. If the protein conteins a secondary signal for palmitoylation ( 1 ), il may becorne additionally modified, and eventually associates with calveola or lipid rafts. Alternatively, membrane association may be assisted by a polybasic region (m).In addition, famesylated proteins may associate with membrane bound proteins. The evidence for these pathways is based on rnammalian Ras tmfficking to the plasma membrane, where H-Ras becornes famesylated and then palmitoylated or K-Ras which contains a polybasic region

With the advent of the Atabidopsis genome sequencing project many potential -CaaX box containing proteins can be identified (see Appendix 1). However, at present none of these potential targets have been shown to serve as a famesylated substrate in vhOn the basis that the subunit of FTases could be responsible for -CaaX recognition and binding, I reasoned that the B subunit should then be sufficient to screen for target proteins in a yeast interaction hunt. Yeast interaction traps provide an in vivo assay for interaction between two specifically constructed proteins. Protein interaction is assayed in yeast by using transcription of yeast reporter genes to measure the protein interaction. In the system used in this study, ERAl was fused to a bacterial DNA binding domain (LexA in pEG202) to create a 'bait' protein and library cDNA encoded interacting proteins were expressed as fusions to a transcription activation domain (in pJG4-5). Proteins that interacted, activated transcription of specially designed reporter genes that carry the binding sites for LexA protein. Genes fused to the LexA domain are constitutively expressed in yeast by virtue of the ADHl promoter directing their expression. Interaction was assayed with two reporter genes containing binding sites for LexA protein. One reporter is a yeast LEU2 derivative that has its normal upstream regulatory sequences replaced with LexA operator sequences. In this study, EGY48, which has the LEU2 gene under the control of six tandem LexAsperators (LexAopLEU2),was used. Transcription from the LexAopLEU2 gene is measured by the ability of the strain to grow in the absence of leucine, which is dependent on the LEU2 gene product. The second reporter gene is lacZ(LexAop-lecZ) which provides some quantitative information about the interadion based on the level of X-Gai cieavage product produced. A library plasmid (pJG4-5) that directs the conditional expression of Anibidopsis cDNA encoded proteins fused at their amino temini to a moiety containing three domains (a nuclear localkation signal, a transcription activation dornain (842), and an epitope tag (haemagglutinin; HA)) was used. The expression of the activation domain tagged cDNA encoded protein is under the control of the yeast GAL1 promoter, which is induced by galactose and repressed by glucose. An €GY48 derivative containing a LexA-ERAI fusion (pEGERA) was transformed with a laczreporter plasmid (pSH18-34) and selected for growth on hir ura- media. To ensure that pEGERA did not activate transcription on its own, cells from independent colonies were resuspended in sterile water and spotted ont0 GaVRaf his- ura- leu' media. As a positive control pSH17-4 was used, which expresses a LexA fusion to the GAL4 activation domain which activates transcription. A negative cont rol was provided by pRFHM1, which expresses LexA fused to a transcriptionally inert fragment of the Drosophila BiCoid gene product. The results from this experirnent are shown in Fig.4.2. An additional test for transcriptional activation by the LexA-ERA fusion would be to patch the EGY48 strain containing pEGERA and pSH18-34 on Dex urawhis* X-Gal plates to check if the blue X-gal product is produced. In this case colonies remained white and no pEGERA dependent transcription was induced (not shown). This is not unexpected, however, since the LexAop-LEU2 repoiter is more sensitive than the LexAop-lac2 reporter. The EGY48 derivative containing pEGERA was transformed with a pJG4-5 Arribidopsis cDNA libraiy. Interacting proteins were identifid by their ability to confer growth on GaVRaf his' uniwtrp' leu- media. As a secondary screen, colonies that grew on these plates were lifted onto nitrocellulose filters which were subsequently placed ont0 GaVRaf hisœura- trp- X-Gal plates. 1000 colonies that induced transcription from the LexAoplacZ reporter were selected for further classification. A prîmary analysis of an additional 250 interacting pJG4-5 library plasmids had indicated that they al1 contained CONAS encoding the Ambidopsis FTase a subunit. To exclude the greater proportion of these, the 1000 pJG4-5 library derivatives were streaked ont0 Dex his' ura' trp- plates and lifted onto nitrocellulose filters, which were probed with the a- Fig. 4.2 Assay to detemine that the pEGERA product does not activate transcription of the LexAop-LEU2 reporter. Positive controls are pSH 17-4, which expresses a LexA-GAL4 fusion, and pEG202, which activates transcription to a lesser extent because of a few amino acids encoded by the polylinker. A negative control is provided by pRFHM1, which expresses a transcript ionally inert version of the Dmsophila Bicoid gene. subunit cDNA by colony hybridization. Library derivatives which did not contain a subunit DNA sequences were classified by PCR analysis. To do this, pJG4-5 library plasmids were used as template to PCR amplify interactor cDNA fragments with BCO1 and BC02 primen. BCOl primer is derived from the coding region of the B42 activation domain, 5' to cDNA inserts and BC02 from the sequence of the ADHl terminator, 3' to cDNA inserts. PCR-products were then digested with Heelll and these were analyzed by agarose gel electrophoresis. Based on their restriction enzyme profiles, pJG4-5 library PCR products were classified into 14 groups. The strength of the interaction was qualitatively assessed by plating isolates from representative groups on GaVRaf his- ura- trp' X-gal media (see Table 4.1). The specificity of the interacting isolates was tested by subjecting individual isolates from each group to a mating assay (see Fig. 4.3). Individual plG4-5 library plasmids were isolated out of EGY48 and were transformed into JBY575 (mating type a). The JBY575 derivatives were streaked onto Dex trp' media and allowed to grow. pEGERA was transformed into EGY48 (mating type a).pEG202 was independently transformed into EGY48 as a positive control for successful mating. The EGY48 derivatives were streaked onto Dex his- media and allowed to grow. The two strains were crossed by applying them to the same replica velvet and lifting their print with a YPD plate. The YPD plate was incubated at 30°C overnight to promote mating and subsequently replica plated to the following indicator plates: Dex his' trp', Dex his- trp' leu-, and GaVRaf his' trpe leu'. The hisetrpo plates contain only diploid cells. All diploid combinations grew on Dex his- trp' media and none grew on Dex his- trp' leu- media. However, only a subset of diploids grew on his- trp* leu* media in a galactose dependent way (Fig. 4.3). The pJG4-5 library plasmids that activated transcription from the Lercr\op-LEU2 reporter in a galactose dependent manner were analyzed further. cDNAs from a subset of groups were further characterized by DNA sequencing and their homology to known proteins determined by BLAST analysis (NCBI). The results of the screen are summarized in Table 4.1. Of the interadom isolated using this Fig. 4.3 Scherne showing the set-up for a mating assay used to test interacting clones obtained in the screen. The Dex his- plate (top bft) contained pEG202 and pEGERA in EGY48 streaked vertically. The Dex trpw plate (top right) contained JBV575 derivatives streaked horizontally. Each derivative contained a different TRPl library plasmid. The EGY48 derivatives were Mat a,HlS3, trpl-, and leu2-. The JBY575 derivatives were Mat a, TRPl, hisa, and leuZW.The two plates were pressed to the same replica velvet and the replica lifted onto a YPD plate (tenter). During overnight incubation at 30°C the two strains grew. At the intersections on the YPD plate the two strains mated and fonned His' Trp' diploids. The YPD plate was then replica plated to 3 plates: a Dex his- trp- plate, on which diploids grew, but neither haploid parent did; a Dex his- trpDleu' plate, on which no strain grew; a GaURaf his- trp- leu plate, on which only activation tagged cDNA encoded proteins which interacted with €RA1 grew (bottom). Different TRPl library plasmids are given a number and the group (denoted by a letter) into which they were classified based on their restriction maps. Note that not al1 TRPl library plasmids activated LEU2 expression.

Table 4.1 €RA1 interacting clones isolated in a yeast interaction trap. The homology of interacting clone cDNAs, as determined by BLAST analysis, is indicated. The strength of interaction is represented by photographs of yeast cells streaked onto GaVRaf hisDuram trpo X-Gal plates. ND = not determined. GROUP ISOLATES IDENTCrV STRENGTH OF /1 O00 l NTER ACfIO N

novei

ND

novel

LEU3

FTase a subun it

ND

NAPl

ND

ND

ND

ND

anion exchanger

ferredoxin

transketolase method, the largest class consisted of cDNAs clones of the FTase a subunit. The nexi largest class of strong interactors was a group of cDNAs encoding proteins with homology to no known proteins (Groups A and C). Additional gnwips included proteins that would not represent interactions of biological significance (e.g., LEU3). Based on their coding potential and on BLAST analysis, none of the cDNAs from these groups encode proteins containing a -CaaX motif. One group, however, of weak interacting clones did contain a putative -CeaX motif (group G in Table 4.1). This gene showed high homology to nucleosome assembly protein (NAP1) of soybean. NAP1 is a protein originally isolated in human Hela and mouse FM3A cells, shown to be involved in the nucleosome assembly process (Ishimi et al., 1984). In vitro, NAPl can transfer histone octamers from NAP1 -histone complexes to DNA, thereby inducing supercoiling of relaxed circular DNA (Ishimi and Kikuchi, 1991). In yeast, immunoprecipitation of yNAPl has revealed an in vivo association with yCLB2, a mitotic cyclin (Kellogg et al., 1995), and yNAPl is required for a subset of yCLB2 regulated mitotic events (Kellogg and Murray, 1995). In addition, cell cycle-dependent changes in the subcellular localization of NAPl suggest that it has a role in the replication dependent assembly of chromatin (Ito et al., 1996). How NAPl is regulated is still unknown and the prospect that it could be farnesylated is intriguing since this could provide some level of regulation of its activity. For these reasons, I decided to characterite the Ambidopsis NAPI to a greater extent.

NAP1 analysis To confimi that a full length or neariy full length cDNA of Ambidopsis NAPl had been isolated, seedling mRNA was probed with a PCR product derived from a group G isolate (pJGNAP), which had a cDNA insert of approximately 1.3 kb (designated NAPI) (Fig. 4.4). Besed on the site of the conesponding mRNA, the cDNA isolated from the interaction screen is approximately full length (1.1 kb). BLAST anaîysis of a paiaal cDNA sequence against Arabidopsis EST and BAC sequences provided a full length

IO5 Fig. 4.4 Northem blot analysis using the NAPl cDNA to probe total seedling mRNA (left panel). The right panel shows an autoradiogram of the hybndized NAP1 clone which is approximately 1.3 kb long. 10 kg of total RNA were loaded per lane.

Fig. 4.5 A schematic showing the structure of the Anibidopsis NAPI gene. Boxes indicate exons and the line indicates genomic sequence (A). In (B), a cornparison of NAP proteins from different organisms. Regions of highest homology are indicated by darker colors. At, Arabidopsis; Ps, garden pea; Gm, soybean: Hp, sea urchin; Mm, mouse; Sp, fission yeast; Rn, rat; Sc. budding yeast; Dm, fruit fly; NAPI, nucleosome assembly protein; NPL1, NAP-like. .L *aItt OOf ea Ott +. L md *L sequence of genomic and coding region DNA of NAPI (Fig. 4.5). Homology analysis of NAPl protein to other NAPI proteins from a variety of organisms indicates the highest homology (-72% identical) is to another Ambidopsis protein designated NAP1-like (AtNPL1) and to other plant NAPs (Fig. 3.6). Interestingly, the -CKQQ motif is absolutely conseived in al1 plant NAPs and in some animal NAPs.

Recombinant NAPI proteln characterbtior To obtain sufkient amounts of NAPI protein for carrying out basic biochemistry, NAPl was expressed as a glutathione-S-transferase (GST) fusion in E. coli, and the OST- NAPl fusion purified by virtue of its affinity for glutathione (Smith and Johnson, 1988). The purified fusion protein used in this study is shown in Fig. 4.6A. The fusion protein shows some degree of proteolytic degradation and is slightly contaminated with bacterial proteins of different molecular masses. A major breakdown product with an apparent molecular rnass of 60 kDa, is presumably NAP1 that has been clipped from GST by endogenous bacterial proteases (Fig. 4.6A). This is not unexpected since a thrombin cleavage site is engineered at the junction of the GST and NAP1 fusion. A characteristic of NAPl and NAP-related proteins is their high content of acidic amino acids, reflecting the fact that the proteins interact with highly basic histones (Ito et al., 1996). The acidic nature of the recombinant fusion protein was determined &y two- dimensional gel electrophoresis. As a marker bovine serum albumin (BSA) was used because it has an isoelectric point (pl) of 4.5. In Fig. 4.68 the rnobility of the GST-NAPI fusion is toward the acidic end of the first dimension at a position close to the BSA marker. Any contribution of the GST protein to the focusing of NAPl is assumed to be minot since the major breakdown product of aie GST-NAPI fusion is visible at a position just under the BSA spot. Based on this pattern NAP1 appears to have a pl of approximately 4.6. Fig. 4.6 GST-NAP1 hision protein expression in E. cdi. In A, lane 1-3 contain varying amounts of BSA that were included to estimate relative pmtein quantity. Lane 1 contains 1 pg of BSA, lane 2,3 M, and lane 3,5 pg. Lanes 4-6 contain varying amounts of GST-NAPI protein isolated from E. col& which runs between 97 and 66 kDA. Lane 4 contains 1 pl of protein eluted fma GST colurnn, lane 5, 3 pl, and lane 6, 5 pl. A major breakdown product which presumably results from the clipping of the GST off the NAPl protein is visible (anow).The GST-NAP1 lanes are contaminated with some bacterial protein bands. In B, two-dimensional gel electrophoresis to cietennine the apparent isoelectric point (pl) of NAP1 protein. Anow indicates NAP1.

NAM Is hmesyIMed In vitro To detemine if the GKQQ motif at the C-terminal end of NAPI couM act as an acceptor for famesyl modification, purified GST-NAP 1 was used as substrate in a famesylation assay using wild-type plant extracts. The results from this experiment are shown in Flg. 4.7A. The appearance of radioactively modified protein bands corresponding in size to OST-NAP and its breakdown product (at 60 kDa) indicate that NAP1 can be famesylated in vitro presumably via its -CKQQ motif. An additional test to conflm this, was to use eral extracts in a similar assay. The expectation was that because of the loss of FTase activity in era 1, era 7 extracts would not catalyze Fpp transfer to the GST- NAP. In addition, this would provide a measure of the specificity of the assay. While addition of rH-Fpp to GST-NA? is evident in reactions when wild-type plant extract is used, similar Fpp transfer in assays where eral extract is used is not evident (Fig. 4.78). In addition, similar assay conditions were used to test whether GST-NAP could be geranylgeranylated. Because of the possibility that the FTase deficiency in em 1 could be compensated by GGTase, assays using both wild-type and era 7 extracts were also included. rH-GGpp transfer to GST-NAP was not detectable under the assay conditions used (Fig. 4.76). This result suggests that if NAP1 is geranylgeranylated in vivo, then it is not an efficient reaction, and that lack of NAPl farnesylation in eral is not compensated by geranylgeranylation. A recent study aimed at deterrnining Fiase and GGTase-l preferences for the 'X' amino acid in CaaX peptides, has indicated that when X is a glutamine the peptide is as a good substrate for FTase but is not a good substrate for GGTase (Roskoski and Ritchie, 1998). This finding provides some indirect evidence that this may also be true in the case of NAP1. However, the ht vivo famesylation or geranylgeranylation -tus of NAP1 has not yet been detemined and the possibility that it is also geranylgeranylated cannot be excluded. Rg. 3.8 Famesylation and geranylgeranylation assays using wild-type and emt protein extracts. In al1 cases GST-NAP1 protein was used as substrate. In (A), famesylation assays using wild-type extracts. The left panel shows a Coomassie stained gel and the right panel an autoradiogram of the exposed gel. In one reaction, radiolabeled Fpp was added ('Fpp) and in a second reaction 'Fpp and 1 mM of cold Fpp was added (Fpp). Each lane contains approximately 75 pg total protein. In (B), farnesylation and geranylgeranylation teactions using wild-type (lanes 1, 2, 4) and era 1 (lanes 3, 5) extracts. Each lane contains 50 pg total protein. Lane 1 is boiled wild- type (5 min) extract with 'Fpp as substrate. The lipid substrates are indicated above the lanes. In both A and 8, proteins were separated on 12% polyacry lamide gels.

NAPl interne& wHh a cycîln 8 Additional characterization of NAPI involved detemining what potentially significant interactions NAP1 could have with other cellular proteins. As a first step, affinity chromatography was used to identify NAPl binding proteins. It was reasoned that this approach would allow the detection of proteins that bind both weakly to NAPI, as well as those that bind tightly. For these experiments, purified GST-NAPI was coupled to a column matrix. Cnide extracts from wild-type plants were loaded onto the GST-NAP1 affinity column, which was then washed with buffer and eluted with a gradient of 0.3-1 M KCI. A number of different proteins bind to NAPI (Fig. 4.8). These results are striking since the crude cell extract was made in buffer containing 0.25 M KCI, and the column washed with buffer containing the same salt concentration. That many proteins remain bound to the column under these stringent wash conditions suggest that they bind to NAPI with relatively high affinity, and are potentially specific interactions. Although these results are intriguing, at present it is not possible to identify al1 these proteins. Instead, we wanted to test whether any of the eluted proteins was a 6-type cyclin, since CL62 and CL63 interaction with yeast NAP1, as well as Xenopus NAPl with cyclin BI and 82, had already been shown to exist (Kellogg et a/., 1995). Because no antibody was available for any of the 8-type cyclins from Ambidops/s, a cornmercially available rabbit antibody against human cyclin BI was used. Western blotting with anti-cyclin BI antibodies provided evidence that one of the eluted proteins was cyclin BI-related (Fig. 4.8 bottom panel). It is noteworthy that no significant amounts of the cyclin-related protein can be detected in the input crude extracts and that it becomes enriched in fractions that are eluted from the column at high salt concentrations. This would indicate a relatively tight and specific interaction between NAPl and the cyclin related protein. To test this possible interaction in vivo and to define more clearly which of the Atabidopsis 6-type cyclins might be associated with NAPI, a yeast FIg. 4.8 NAPl interacting proteins. The top panel is a silver stained gel of protein fractions eluted from a NAPl affinity column. In lane (1), an aliquot of the wild-type crude extract that was incubated with the affinity column. In lane (WS), a sample of the flow through that initially came off the column with washes containing 250 mM KCI. Lane (WF) is the final wash with 250 mM KCI. Subsequent lanes are protein fractions that were eluted from the column at different salt concentraüons; 0.3 M, 0.4 M, 0.5 M, 0.7 M, and IM KCI. The bottom panel shows the same fractions probed with anti-cyclin 81 antibody.

Fig. 4.9 Yeast interaction assay showing the specific interaction of NAPl with cyclAt. Each horizontal panel shows the appearance of a patch of €GY48 derivatives containing different combinations of plasmids on different media (indicated on the Ieft of each panel). Each €GY48 derivative contains pSH18-34. €GY48 derivatives containing pEGABl and pJGNAP 1 or pEGCYC and pJG4-5 do not activate transcription of the LexAup-lacZor LexAop-LEU2 reporters. Photographs were taken after 3 days growth. However, €GY48 derivatives containing pEGCYC and pJGNAPI activate transcription of both reporters indicating an interaction between NAPI and the Cycl At protein. 0.x hir-un- t~4.u- interaction assay where the Anbidopsis CYCAt (CYCB1;l) gene was fused to the LexA gene (pEGCYC). pEGCYC was co-expressed in €GY48 along with the pJGNAP1 isolate and with pSH18-34. €GY48 transformants containing these plasmids grew on his- ura' trp' leu' media in a galactose dependent way and activated transcription from the LexAop-lacz reporter (Fig. 4.9). As a control to test that pJGNAP1 did not activate transcription on its own, a pEG202 derivative containing a LexA fusion to the ABH gene of Arabidopsis (pEGABI) was used. To exclude the possibility that pEGCYC could activate transcription on its own, €GY48 cells were cotransforrned with pEGCYC and pJG4-5. Control cotransformants containing either pEGCVC and pJG4-5 or pEGABl and pJGNAPl did not activate transcription of the LexAop-LEU2 or LexAop-lac2 reporters (Fig. 4.9). This suggests that the galactose dependent growth on leu' media of pEGCYC and pJGNAPl cotransformants was due to an interaction of NAPI with the cyclAt protein.

The GST-NAP1 fusion protein was used to generate rabbit polyclonal antibodies against NAPI. Affinity purified antibodies were used to probe cytosolic and microsomal protein fractions from wild-type and eral seedlings (Fig. 4.10). Since NAPl famesylation may cause it to localize to membrane fractions in cells, the rationale of this experiment was to test if any difierences between NAPl fractionation, at least at a crude level, could be detected between wild-type and era1. However, at this level there were no differences between wild-type and era 1 microsomal fractions. suggesting that NAPl fractionation with microsomal proteins is not famesylation dependent. This does not exclude the possibility, however, that famesylation is required for NAP1 localization at a subcellular level to specific organelles such as the nucleus. This possibility can be tested by immunolocalization experirnents with anti-NAP1 antibodies on wild-type and ere 1 tissues. The western blot analysis revealed an interesting feature of era 1 NAP1. Upon close inspection of the NAPl protein bands it is evident that the mobility of NAP1 in erai cytosolic and microsomal fractions is slightly increased. The fact that proteins were separated on a 12% polyacrylamide gel indicates that this change in rnobility is significant. Although this is somewhat puuling it suggests that NAPl could somehow be modified in wild-type and that this modification is absent in eral. Changes in protein rnobility of this degree could be due to protein glycosylation.. It is interesting in this regard that NAPl contains a putative glycosylation site at its N-terminal starting at the 9th amino acid position. Whether NAPl is glycosylated in vivo will require further characterization. Fig. 4.10 Western blot probed with anti-NAP1 antibodies. The top panel is a 12% polyacrylamide, Coomassie stained gel. Lanes 1 and 3 are wild-type seedling soluble protein extracts containing approximately 20 and 40 pg of total protein respectively. Lanes 2 and 4 are en1 soluble protein extracts containing similar concentrations of protein. Lanes 5 and 7 contain wild-type insoluble protein at 10 and 20 pg total protein. Lanes 6 and 8 contain eral insoluble protein at similar concentrations. The bottom panel is a western blot of to the top panel gel, which was probed with affinity purified anti-NAPl antibodies. Note the slightly increased mobility of NAPI protein in the eral extract lanes,

DISCUSSION The isolation of a farnesylation mutant in Alabidopsis raises a number of questions about the influence of this modification on plant signaling pathways. At a prirnary level, famesylation influences seed donancy and seed and stomatal sensitivity to ABA (Cutler et a/., 1996; Pei et a', 1Q98a).How this influence is exerted and through which specific proteins is still unanswered. The key to begin to address some of these questions is to identify which proteins are famesylated and how farnesyl modification can modulate their activities or functions in the context of cellular mechanisms.

€RA l intemctlng proteins As a first approach to identifying in vivo relevant targets, I perfomed an interaction trap screen using the FTase -subunit as bait. As expected, the mase a-subunit showed strong interaction with €RA1 in this screen. Other, strong interactions identified by the screen present some potentially exciting possibilities. Although the identity of these interacting partners (see groups A and C in Table 3.1 of results) is not known, their strong association with ERAl raises the possibility that they could be modulators of FTase action. For example, there is evidence to suggest that FTase activity is altered by phosphorylation (Goalstone et al., 1997). Additional regulation could be provided by proteins that influence its afAnity for different protein substrates. Biochemically this could be tested by detenining the effects of group A and C proteins when they are included in a standard FTase assay. in addition, it should be possible to express these interactors in an in vivo system. For example, overexpression of these proteins in plants may provide a measure of their inhibitory or stimulatory influence on mase adivity at the whole plant level. Even if these interadon represent biologically unimportant interactions, they may prove useful by virtue of their ability to bind ERAI and possibly prevent its dimerization with the a subunit. These could be expressed by using an inducible expression system and in this way they could be used to inactivate Rase activity in the plant in a contidled way. This would provide a means of inactivating ERAI at specific times during development, and the specific timing of ERAI requirement could be assessed. The identification of an Arabidopsis NAP1 homolog by interaction trap screening is intriguing. It demonstrates that the FTase B subunit is suffident for the recognlion and binding of -CaaX containing proteins, at least in the case of NAPI. Previous studies associating -CaaX recognition with the subunit were based on cross-linking studies done in vitm Here this link is shown in an in vivo contexk. However, the utility of this type of screen for isolating -CaaX containing proteins is not practical. The reasons foi this are twofold. First, a large number of positive interactions are with the a subunit of FTase and efforts to eliminate these need to be considered. Second, the strength of the interaction between the LexA-ERA1 fusion with NAPl is relatively weak. The interaction system used in this study is estimated to be capable of detecting protein interactions with dissociation constants that are in the order of greater than 10-50 pM (Brent and Finley, 1997). Protein interactions occurring at these levels include enzyme substrat0 interactions found in signaling pathways. The weak interaction between ERAI and NAPl is probably at the threshold of these levels. Such weak interactions would lead to lower plating efficiencies, leading to their under-detection in a standard screen (Estojak et a/., 1995) and many GaaX containing proteins rnay have been missed. In addition, since both interacting partners have bulky multidomain moieties fused to their amino termini, this means that amino terminal dependent interactions are blocked. For example, studies on the FTase subunit indicates that both N-terminal and C-terminal regions of the B subunit anrequired for the -CaaX specificîty of the enzyme (Del Villar et a/., 1997). Based on this information it is somewhat surprising, then, that any -CaaX containing protein was identifid at all. However, it does indicate that the C-terminal region of ERAl ie sufficient for CaaX recognition. NAPi in vivo function Genetic experiments in budding yeast indicate that NAPI is involved with the function of the cyclin CLB2 and with GIN4 kinase (Altman and Kellogg, 1997; Kellogg et al., 1995; Kellogg and Murray, 1995). Loss of NAPI function in yeast can cause defects in CL62 mediated mitotic events. One of these is a switch in the pattern of bud growth as cells enter mitosis (Kellogg and Murray, 1995). lnitially buds grow only at the tip. During mitosis. however, the CLBBCDCPB kinase complex causes bud growth to become isotropic (Lew and Reed, 1995). When NAP1 is deleted, the CLB2-CDC28 complex does not induce isotropic growth and the bud continues to grow at the tip foming elongated buds. Deletion of another gene, encoding GIN4 kinase, leads to similar defects in bud growth switching (Altman and Kellogg, 1997). Furthemore, GIN4 is specifically activated during mitosis in a NAPI dependent fashion and physically interacts with NAPl (Altman and Kellogg, 1997). GIN4 activation leads to an inhibition of polar bud growth and possibly promotes the assembly of mitotic spindles. On the basis of genetic and biochemical relationships it has been hypothesized that NAPI in yeast mediates the activation of GIN4 through the hyperphosphoryaltion of another kinase, CLA4 (Tjandra et al., 1998). The exact sequence of events in this pathway and how NAPI specifically mediates them is still unresolved. Nevertheless, it seems that, at least in yeast, NAPl is sornehow involved in conferring a certain degree of specificity ta cyclin-dependent kinase complexes. The demonstration that the Arabidopsis NAPl also interacts with a cyclin B (CYC1 At) suggests that a potentially similar modulation of cyclin dependent cell cycling events is mediated by plant NAPI. The determination of addiional Alabidopsis NAP1 protein interactions should help to resolve this to a greater extent and whether NAPl can mediate cell cycle specific phosphorylation events in plants. Apatt from its interaction with cyclin 0, NAP1 has the ability to fom histone-DNA complexes (Ishimi and Kikuchi, 1991 ). In addition, NAPl binds specifically to the amino terminal regions of corn histones and the disordered regions of linker histones in yeast (McQuibban et el., 1998). These disordered regions are subject to post-translational modification and the sites of interaction with transcriptional activator proteins (Ling et al., 1996). In this regard, NAPI has been shown to facilitate transcription factor binding to nucleosome containing target sequences (Walter et al., 1995). These results suggest that NAP1 also plays a role in modulating access to histone domains. Furthemore, changes in the distribution of DrosophiIa NAPl from the cytoplasm to the nucleus during S phase and its binding capacity for histones H2A and H28 suggests that it rnay act to shuttle histones from the cytoplasm to the chromatin assembly machinery in the nucleus during DNA replication (Ito et al., 1996). The distribution of NAPl during different phases of the cell cycle in human cultured cells, however, indicates that NAP1 is predominantly located in the cytoplasm during all phases of the cell cycle and present in the nucleus at lower levels during 01, S and M but not during 02 (Marheineke and Krude, 1998). Although the results for Orosophila and human NAPI localization are somewhat at odds, it seems that NAP1 can be differentially localized to the nucleus during different stages of cell growth. Taken together, these results imply that NAPl is involved in a complex sequence of events that are associated with cell cycle progression. Although at present it is not known if Ambidopsis NAPl is differentially localized, a potential mechanism for NAP1 localization may be dependent on its famesylation status. The demonstration that it is famesylated in vitro provides circurnstantial evidence for this possibility. However, there is no direct evidence that it is also famesylated in vivo. The demonstration that it is not famesylated in eral indicates that its famesylation may have some in vivo relevance. That significant amounts of NAPI are present in microsomal fractions of era l protein extracts, however, would argue that lack of famesylation does not mediate its membrane localization. Although a more detailed analysis of NAP1 subcellular localization in ere 1 compared to wild-type may reveal more significant differences. The apparent molecular site difference of NAP1 in wild-type and etal does suggests that a famesylation dependent modification of the protein might be occumng in vivo. The nature of this modification, if any, has not been deteminecl. Another issue that needs to be resolved is whether the -CKQQ motif found in NAPl is proteolyzed end caiboxymethylated. If it b not, it might provide some indication about the relevance of the famesyl modification and whether it seives to localize NAP1 to a membrane fraction. Furthennom, since famesylation alone is not generally sufficient for membrane localization, additional features of the NAPl protein would need to provide second signals for its membrane localization. One would be the presence of a polybasic domain, which, at a cursory level, is not present in NAPI. Another would be palmitoylation, which would be provided by a cysteine upstream of -CKQQ. Whether NAPl is palmitoylated could be easily tested in vitro. If NAP1 famesylation is not involved in its membrane localization then it may have more to do with protein-protein interaction. There are cases when protein famesylation facilitates protein-protein interaction (Marshall, 1993). For example, in yeast, PEX19p acts along with PEX3p to effect peroxisome biogenesis (Gotte et a/., 1998). The majority of PEXI 9p is cytoplasmic and its famesylation is required for its association with PEX3p, which is a membrane protein. An important issue that was not directly addressed, is the in vivo function of NAP1 in Alabidopsis. NAP1 deletion in yeast is not lethal and obvious yNAPI dependent phenotypes are only evident in a CLB2 dependent fashion (i.0. when CLB3,4 and 5 are also deleted) (Kellogg and Murray, 1995), indicating that yNAP1 requirement is CLB2 specific. Based on this result, NAPI deletion in plants rnay not be detectable in a wikl- type background. This is further complicated by the fact that a homologue of NAPl exists in Algbidopsis (NPLI), which wouki argue that simply deleting NAP1 by reverse genetic techniques would produce no observable phenotype. This could be partially circumvented, by deleting both NAPl and NPL1. An alternative, is to focus on farnesylation dependent events that are mediated by NAP1. To test this, it should be possible to overexpress NAP1 in an eral genetic background and detemine if any enal phenotypes are suppmssed. This option may be viable since ovetexpression of some farnesylated proteins can obviate their famesylation requirement (Deschenes and Broach, 1987; Trueblood et al., 1993). At a flrst approximation it seems that NAP1 in Arabidopsis may be somehow linked to the plant cell cycle via its interaction with cycl At. This is particulaify intriguing since correlations between famesylation and the cell cycle are known to exist in yeast, animals and plants (Galli et a/., 1997; Qian et al., 1996; Sepp-Lorendno et ai., 1991). The isolation of a farnesylated NAPl protein provides a potential link between famesylation and the cell cycle that had previously not been recognized. CHAPTER 5

GENERAL CONCLUSIONS Mo&/ for €RA t tunctlon fn the merbtem The reduction of cell division or DNA synthesis by ABA application (Bryant and Chiatante, 1997; Dudits et al., 1998) and the identification of an ABA inducible cyclin- dependent kinase inhibitor (ICK1) (Wang et aL, 1998), implies that cellular ABA sensitivity might affect the competence for progression through the cell cycle. The recessive nature and the ABA supersensitivity of eral mutants suggest that normal €RA1 function is to somehow required to negatively regulate ABA sensitivity. The phenotypic charcteriration of eral described in this study implies that there is also a connection between ERAI function and the competence for cells to divide. A spectulative model that atempts to address some of these observations is depicted in Fig. 5.1. In the context of wild-type meristem organization, it is possible that ERAI is required to downregulate the effects of ABA on cell proliferation in the meristem. This could be achieved by the famesylation of some modulator of ABA signal transduction which is required to attenuate the ABA signal and alleviate the inhibitory effects of ABA on cell division. In contrast, in eral mutants the famesylated modulator is not as active in attenuating the ABA signal and meristematic cells experience a chronic response to the inhibitory effects of ABA and do not divide as frequently. The consequence of the reduced cell division could cause cells not to occupy the peripheral zones of the meristem, where the frequency of cell division is normally speeded-up and oigan primordia are initiated. The increased rates of division of peripheral zone cells might indeed be a prerequisite for proper organ formation. In era 1 if cell division was significantly reduced then central zone cells would not easily make the transition to the peripheral zone. Ovei the life of the plant the meflstem might become enlarged because of an overaccumulation of cells which would have nonnally fomed side shoots or flowers. Alemaüvely, the model could be mod'ied so that ABA negatively affeds ERAI activity. In this scenario, if ERAI poshely affects proteins that are invotved in cell cycle progression then a reduction in ERA1 acüvity might be sufficient to cause an overall reduction in cell division in the meristem.

A survey of the genes that are cumntly known to affect ABA sensitivity (see Fig. 3.1) will reveal that a large number of them encode proteins that are either transcription factors or what would be assumed to be downstream components of a signal transduction pathway. Based on this mutational analysis, it would seem that an ABA receptor cannot be easily isolated through conventional screens. This has a number of implications. Mutation of a gene encoding an ABA receptor is either Iethal or there are too many receptors for mutation of only one of them to cause a detectable phenotype. An alternative view is that an ABA receptor is not, as one would initially assume. a membrane bound protein, but is rather a transcription factor, like animal steroid hormone receptors. However, considering what physiological studies have indicated, ABA does not appear to be the primary signal in most of the responses that it is involved in. By most accounts, it is a secondary rnessenger in responses that are elicited by dehydration. Dehydration-induced gene expression studies exemplify this (see chapter 1). The question then is, what does a change in ABA sensitivity mean? In the case of the ERAI, the loss-of-function phenotypes at the level of seed germination and stomatal closure would imply that it is a negative regulator of ABA signal transduction. However, it is difficult to separate whether these phenotypes are symptoms or causes of ABA sensitivity. The phenotypic analysis of era 1 implies that it is involved in cell cycle regulation. Does this mean then, that an alteration of cell division during germination sensitizes cells to ABA beyond normal levels, or does it mean that the ABA supersensitivity causes cell cycle progression to be affected? In keeping with the notion that ABA functions to integrate environmental signais, dehydration is known to cause a reduction in overall growth and cell division. Continued growth in times of water scarcity, at least in the shoot, would be detrimental at kt.The role of ABA could be to strengthen this effect by suff-iently slowing down growth, to cause cells to become semi-dormant, so that the consequences of water deprivation can be avoided. The way that this rnight be achieved would b8 to solicit molecules that are involved in cell cycle progression. Progression through the cell cycle is ultimately dependent, and integrated with, the state of a cells metabolic activity. It is difficult to imagine that a cell that is not metabolically active could build sufficient cellular components that are required by two cells. Since the metabolic activity of cells that are in a suspended state of growth would rationally be different from those that are actively growing, then is this change in metabolic activity what makes thern more sensitive to ABA? Cellular sensitivity to ABA might then hinge on the activity of proteins that effectively influence cell metabolism. In the case of AB13, for example, the genes that it controls encode proteins that are associated with the acquisition of desiccation tolerance and the induction of domancy. That is to Say, proteins that are associated with responses where cellular metabolism is presumably slowed. Deletion of AB13 would then cause cells to remain metabolically active and consequently they would no longer respond to ABA. In the case of ERA1, the opposite would be true. Its normal function seems to be to promote growth, possibly by influencing proteins required cell cycle progression (like NAPI), and its deletion causes growth to become slowed. At the whole plant level this is exemplified by long-lived enil mutants. In keeping with this notion, does the ultimate regulation of ERAI link back to molecules that gauge the metabolic activity of the ceIl, such as molecules that are involved in sugar metabolism (Zhou et ai., 1997)? The answers to some of these questions might eventually be provided once we know how ERA1 activity is regulated. The interacting proteins isolated in the yeast interaction hunt may be useful with respect to this, which at this point is a fundamental issue. Studies in mammalian cell lines indicate that insulin can stimulate FTase activity approximately 4-fold via the phosphorylation of the a subunl of FTase (Goalstone et al., 1997). It will be interesthg to see if any of aie strong ERAI interacting proteins are involved in Ambidopsib Fiase regulation. As already discussed, these could be tested in standard FTase assays to determine whether they have any stimulatory or inhibitory effects on mase activity. In addition, they could be tested in vivo by creating overexpression constnicts to detenine whether they can significantly influence FTase mediated pathways, such as the one involving NAP1. Our knowledge of ERAl regulation could also be increased by devising schemes aimed at directly identifying proteins that can change its activity. A potentially viable way of doing this could be to use yeast as an assay system. The Rarnl gene in yeast encodes the p subunit of yeast FTase, and its disruption is not lethal but causes cells to become temperature sensitive (at 37%) for growth (He et a/., 1991). Co- expression of the tomato nase a and subunits has already been shown to rescue the ram 1 temperature sensitive phenotype (Yalovsky et aL, 1997). The co-expression of €RA1 along with the a subunit of the Arabidopsis FTase should then also be able to suppress the ram 1 temperature sensitivity. If €RA1 la-subunit-coexpressing ram l derivatives are then transfomed with an Arabidopsis yeast expression library, then it should be possible to identify proteins that can cause the FTase activity to be downregulated or upregulated, which could be assessed by the growth characteristics of the mutant line at 37OC. Negative regulaton of Fiase activity would predictably cause reduced yeast growth at 37OC, while positive regulators of FTase activity would increase growth at 37OC. The era l suppressor mutations indicate that there might be a connection between ABA sensitivity and overall plant growth characteristics. fhat era 7 suppression can cause overall branching to increase (Sarkar, 19Q9), a process that implies an increase in growth, seems to support the notion that ABA sensitivity can be conelated with growth. This issue can be addressed, at least in part, by asking whether the phenotypes of the suppressor mutants, isolated away from the eral background, are characterized by increased growth rates or cell division. It will be interesting to see if any of these mutations are in genes involveci in ABA synthesis or breakdown, or possibly molecules that are negative regulators of cell cycle progression, as has already been implied for A613 (Rohde et a\., 1999). The difficulty in detemining what ABA related pathways require famesylated intemediates to function is complicated by the potentially large number of €RA1 protein targets (see Appendk 1). Afier establishing that they can be modified by famesylation, a way of dealing with this wealth of choices would be to first determine if mutation any of genes encoding these target proteins cause ABA related defects. For some potential target proteins, such as APETELAl (see Appendix l), mutant alleles already exist. WheVier apl mutants have any ABA dependent phenotypes has yet to be detennined, although this analysis could easily be done. The influence of photoperiod on vegetative and reproductive growth has been well documented and the effect of photoperiod on eral expressivity might indicate that €RA1 may function in pathways that affect inflorescence meristem Mentity. The recent finding that APETALAl (API) a meristem identity and floral transition regulator contains a CAAX box and is famesylated may explain some of the to en7 flowering phenotypes (Rodriguez-Concepcion et al., 1999). The phenotypes of apl loss of function mutants and era 1 mutants, however, share few similarÏties (Irish and Sussex, IWO; Okamuro et a!., 1997; Simon et al., 1996). This discrepancy may indicate that famesylation of AP1 may change the interaction of AP1 with other proteins involved in transition to flowering such as LEAFY (LW) and UNUSUAL FLORAL ORGANS (UFO) rather than being essential for flower formation. In this respect, in Impatiens balsamina the reversion from reproductive to vegetative growth can be effected by transferring plants from inductive to non-inductive conditions (Pouteau et al., 1997). It is interesting that the Impatiens AP1 homolog lacks a CAM box suggesting that there might be a link berneen the f~mesylationstatus of AP1 and the transition from vegetative to reproductive growth. Altematively, loss-of-function mutations in select target molecules could be created by reverse genetic techniques, and the phenotypes caused by these could be assessed with respect to ABA sensitivity. For example, the characterization of NAPI Io=-of-hinction mutants could provide a way of connechg the long standing consensus that ABA inhibits cell division. Another putative target that could function in NAPI-releted nucleosome modification, is a histone deacetylase (see Appendix 1). Its analysis could help to clarify whether famesylation is involved in coordinating the subceUular movement of these proteins at different stages of cell growth to the nucleus. Another potentially interesting proteiii is RD29A, which is transcriptionally activated by dehydration. At this point, the number potentially interesting molecules is not Iimited. The choice of which molecules to focus on will depend in part on the specific area of interest. The identity of some of the target proteins rnight also provide dues to era 1 phenotypes that have been missed. For example, one putative target protein is XTR6, which is a xyloglucan endo-l,4-b-glucanase. Based on its identity this enzyme could be involved in cell wall modification, possibly by loosening the xyloglucan matrix. We have not investigated the cell wall characteristics of eral, although this aspect of its rnorphology might be changed. lndeed a large number of genes with similarity to enzymes involved in the modification of complex carbohydrates, such as polygalacturonase, contain putative famesylation motifs. In addition, the possible role of famesylation in modulating the activity of these enzymes can be addressed. Regardless of the choice of target protein, there is cunently a lack of knowledge of what kind of cell trafficking events or protein-protein interactions are mediated by nase in plants. By focusing on one famesylated protein at a time, then it should be possible to gain sorne insight into what the common features farnesy lat ion mediated protein traff icking are. In this respect, eral mutants could provide a powerful tool for assessing their in vivo relevance.

A mokl îbr signai intrgmuon The apparent mulatude and redundancy of signals which can elicit different responses in plants is difficult to reconcite with the apparently fewer transduction pathways that cm be identified by genetic analysis. Furthermore, of the apparent hormone signaling components isolated by genetic analysis, most do not deal with primary perception (Le. receptors). The relative difficulty in isolating receptors may indicate that there is a certain degree of functional redundancy between receptors and they can only be genetically isolated if the mutations are dominant, as with ethylene receptors (Bleeker and Schaller, 1996). Altematively, mutations in genes encodlng hormone receptors may be lethal. Otherwise, it is possible that homones are not the primary source of the signa!, which would be provided by environmental stimuli and hormones would act downstream to affect signal transduction components. A way to deal with situations where multiple inputs need to be integrated has been proposed by Bray (1995), who compares signal transduction to computer-based neural networks. Briefly, a simple neural network is composed of an input layer, an intemediate hidden layer and output layer frorn which the response is obtained (Fig. 5.2A). Each layer consists of a set of neurons or switches that affect the layer below it. Connections between the layers are given a weight value. The stronger the connection, the greater the weight value. The connection weights can be adjusted during a leaming phase, where different patterns of inputs are presented. This process is repeated many times and the resulting outputs are compared to a reference optimal output. This has the effect that certain connections between each layer gain a greater weight value because they can produce the optimal output more efficiently. Eventually the network is trained to recognize multiple input pattems to give the correct output (Kartalopoulos, 1996). Once the network has leamed, then the transmission of an input through the circuit follows the connections with the highest weight values. In a biological context the rnutiple inputs would be perceived by multiple receptors (input layer), which would either positively or negatively affect cornponents in a signal transduction cascade (hidden layer), which then elicl a response (output layer). The switches of the hidden and output layer would be provided by kinases, phosphatases and other proteins involved in pst-translational modifications, since they cari change the activity of other signaling components either positively or negatively by modifying them. In this context, the connection weights would be achieved through evolution (Bray, 1995) by modifying the switch proteins so that they are more or lem responsive to the different inputs. This analogy can be extended to plant signaling processes. For example, a process such as germination is known to be either positively or negatively affected by multiple environmental and homional signals or inputs (Fig. 5.28). Moreover, some stimuli cmmore strongly affect germination and are required to a greater extent, such as light in Arabidopsis. These inputs would effect downstream signaling components or switch proteins, such as a kinases. Through its action the kinase would change the activity of components further downstream, possibiy another kinase, and so on. The sum of both positive and negative effects on the kinases will then detemine if germination is to take place or noteHormones in this scheme could act to influence the strength of environmental inputs by acting antagonistically or synergistically to these inputs. However, the perception of the hormone need not be at the same level as the enviromental inputs, and their perception could be at a level below the environmental input. Although this mode1 is speculative, it does provide a framework to think about what the phenotypes of mutants affected in any one of these component parts would be. The mode1 in Fig. 5.28 would predict that a mutation causing a defect in light perception, such as in a phytochrome, would no longer influence the downstream switch protein, in this case a hypothetical kinase. This kinase would then be under greater influence from an antagonistic signal, like ABA, which reduces its activity. This would then cause seed germination to becorne more sensitive to ABA. The mode1 would also predict that mutation of one of the kinases would cause multiple inputs, which normally affect its activity, not to be interpreted properiy. This would have the effect that the equilibrium of the pathway would be skewed in one direction, because now it is under greater influence from the alternate kinase. The mutant phenotype would then be either nontesponsiveness or hyperresponsïveness to other input signals. This is in contrast to the current view that mutations in individual receptors cause pleiotropic phenotypes. If the cmof the signaling pathway is the switch protein, however, then its mutation, and not mutation of the receptor, would cause pleiotropic phenotypes. With respect to ABA signaling, such a scenario might explain in part why mutations in genes such as ERAl and AB11 (both are switch proteins) have such pleiotropic effects, despite the fact that they do not encode receptors. Their activity would then be influenced by many input signals and not just an ABA signal. If this is tnie, then part of the reason why mutations in an ABA receptor have not been isolated, would be because its phenotypes are too subtle to be detectable. In a recent study, ABP1, an auxin binding protein, and a candidate auxin receptor based on its membrane localization, was overexpressed in tobacco (Jones et al., 1998). The expectation would be that, since auxin can prornote cell expansion, then ABPI overexpressing plants should have a dramatic alteration in their overall morphology. This was not the case, however, and transgenic plants displayed only mild increases in cell expansion, while their overall morphology was unchanged. The reason for this could be that there are multiple receptors for auxin, or that auxin is not the only signal required to increase cell expansion. The prediction from the model would be that cell expansion is under the influence of multiple receptors, both for environmental signals and for hormone signals, and simply changing one of these would not cause a dramatic change in plant morphology. Perhaps the overexpression of a switch protein, which positively affects cell expansion and whose activity is affected by environmental and auxin signals, would cause a more dramatic change in morphology. The placement of switch proteins at the center of the signaling pathway would provide a way of integrating multiple signals. The rnultiplicity of environmental signals affecting plant growth and development would require such a high level of integration. Some of the predictions made by the model could be tested with respect to germination. For example, do mutants that have a changed senslivity to a hormone also have a changed sensithrity ta environmental signals? Are they mutated in a switch protein? In the case of ERAI, one could ask, is its sensitivity to other factors, such as light, also changed? Based on the eraf germination phenotype, its normal function is to prevent or reduce an ABA signal from negatively infiuencing germination. This is like saying that its normal function is to prornote germination. Hypothetically, €RA1 could positively regulate kinase 2 in Fig.5.28, whose activity nomally promotes germination and which is also positively regulated by light. When ERA1 is rnissing it would no longer prevent the negative regulation of kinase 2 by ABA. Kinase 2 would stil! be posiüvely regulated by light, but also in a more negative way by ABA. The net result would be that germination does not proceed unhindered. The model could be modified such that €RA1 replaces kinase 2; if it is positively regulated by ABA during germination and if it positively regulates kinase 3. An essential question that arises from this is, again, how is €RA1 regulated? Is it positively or negatively regulated by ABA, and what other endogenous and exogenous signals can influence its activity? The large collection of mutants that are currently availabe in Arabidopsis , with defects in different aspects of environmental and hormone signal transduction, coupled with the biochernical analysis of ERAI, should help to test some of these ideas. Fig. 5.1 Mode1 showing the possible relationship between ERAl and ABA effects in meristem function.

Fig. 5.2 Schematic showing a simple neural net (top panel) and its possible analogy to factors affecthg germination (bottom panel). Different layers are connected by lines, indicating the strength of influence of individual components in one layer on components in the next layer. Arrows indicate a positive influence and lines indicate a negative influence. The strength of an effect is indicated by the width of the connecting line. Input Hidden Output lryet Iayer 1-t APPENDIX 1, Putative isoprenylated proteins identified by cornputer based searches for C-terminal patterns (-CXXX) in the Arebidopsis protein database.

Mmtrl biadhg protahm AAC28185.1 CAIM Metal binding protein (MBP)

metallothionein- like protein (mL) CTCK MTL CTCK

Rabl8-ras-related small GTPase

p. GTP-binding protein (GBP) SEC4 protein homolog

CTSS p-GB3 - GBP ATFP8-S. Rab1 Ras-like GBP Rabllb

Ara4-Rab Axas-Rab CCGT~ Rab8-S. Ara3 CM s RabllB from N. tabacum CLU Rac-like se D.discoideum RacE CTAA Rac GTP binding protein Arac7 CGKN Rac GTP binding protein arac8 CQW ERG protein-smali GTPase

CDFS p.glucoamylase CLAA xyloglucan endo-1,4-8- glucanase XTR6 cm xyloglucan endo trans - glycosylase related TCH4 CGAC -1,3 -glucanase CNMR p .p-1,3 -galactosyltransferase CNFR p.p-1,3-galactosyltransferase CLAQ N-acetyi-p-glucosaminidase (He-) CGSP p. polygalac turonase CLNR p.polygalacturonase CFED p.polyga1acturonase CNST p.polygalacturonase CFKT p.polygalacturonase CNHY p.polygalacturonase CQcI p.glucosyltransferase CIaW p.gl~osyltransferase CGGV* p,glucosyltransferase CMIF p. glucosylhydrolase CVGKf pxellulose synthase -1 DNA-binding pro teins AAA64789.1 CYAA p ,MADS-box protein AAB71455,l CETA p. homeobox pro tein AAC67508,1 CYAA AAC69941-1 CQTA p. homeabox protein AADlO 649-1 CKLA S. humaa Kial7-like (zincfinger pro tein) CAB36819.1 CDGA Mersitern LI (A--1) CFAA CGCF CADI s.chloroplast nucleoid DNA- binding protein CADI s.chïoroplast nucleoid DNA- binding protein CLTK p.zinc-finger (ZF) protein CLTK ZF-related protein CSIR CAWK Çcarecrow (SC) transcription factor homolog CAWR CRSS CTET p.reverse transcriptase CRLG s.RingH2 ZF CGSS Phytochrome interacting factor 3 (PIF3) basic helix- loop-helix protein

CFRA s.awin regulated gene cm s.Auxin induced (Axil-like) CNED s .Mil CNRG auxin induced gene (IAAS) cm s.auxin repressed gene CFRV s-auxin induced gene

CLLS s-membrane associated salt-inducible gene s-membrane associated salt- inducible gene CEGE s.ligand gated ionic channel weakly s.vesicle associated protein cm1 p. membrane pro tein CSAT s.&o acid transporter AATl CVNF dessication responsive protein RD29A CGEK s .peptidyl prolyl cis- transferase (cyclophilin) CMLM carbonic anhydrase-like CKYA s .cyanohyàrin lyase CYEA s.cytidine deaminase CYm s.cytidine deaminase CKFP lipase CVTF s.microtubule binding grotein Sikl cm S. pambe Isp4-like CNIF S. triacylglycerol lipase CKDD ITI6 (serine protease inhibitor)

CPAT s.strictocidine synthase cm PIG-L related (GPI synthesis) CRCY s.serine acetyltransferase CRCY sserine acetyltransferase CYMY s.phosphoribosy1 anthranilate transierase CFGV s.phosphatidylinositol/choline transfer protein p.ARI, RING finger protein

AIG1-like (disease resistance associated) CDHC RPPl (disease resistance protein) NBS/LRR (disease resistance pro tein)

CmR p.serine/threonine kinase CLCV p.serine/threonine kinase CD11 p .protein kinase

CVSN p.serine carboxypeptidase CDIG s.vesicle associated protein CAFH s-clathrin assembly protein

DNAJ isolog DNAJ isolog (AtJ3) DNJH isolog (DNJH) AAAS0234.1 mQQ nucleosome assembly protein (NAP) like AAD10147.1 cm NAPl AAB70032 CMIV histone deacetylase

AAC17618.1 CSYA s.Stigl-like (stigma associated)

CNYA S. Stigl-iike CRIY FCAl (flowering the) CKLA s nodulin ( transmembrane pro tein) AC006585.2 CAWF s early nodulin AAD21720.1 . CFCF p .MEI2-meiosis cAMP regulated AC006526. 15 CGIA chromosome segregation 1-like PIR-S55244 CFNS Polyubiquitin 4

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