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Chapter 10 / Signal Transduction Pathways in Plants 137 10 Phytohormones and Signal Transduction Pathways in Plants William Teale, PhD, Ivan Paponov, PhD, Olaf Tietz, PhD, and Klaus Palme, PhD CONTENTS INTRODUCTION SIGNAL TRANSDUCTION PATHWAYS OF PLANTS ROLE OF PHYTOHORMONES AUXINS AUXIN PERCEPTION EFFECT OF AUXIN SIGNALING ON GENE EXPRESSION GIBBERELLINS CYTOKININS BRASSINOSTEROIDS ABSCISIC ACID ETHYLENE PERSPECTIVE INTRODUCTION Since the divergence of plants and animals about 1.5 billion yr ago, the signal transduction pathways in both kingdoms have been subjected to very different selection pressures. These fundamental differences have influenced the evolution of both the signaling molecules themselves and the mechanisms by which signals are relayed. Among these differences, a plant’s ability to continuously form new organs during its postembryonic development, the increased frequency of high degrees of both ploidy and gene duplication in many higher plants, and the multicellular haploid gametophytes of more primitive plants could be particularly significant. Particular developmental processes, such as totipotency (the ability of a plant to regenerate itself from vegetative tissue), have enabled plants to increase their reproductive potential and are consequences of the idiosyncrasies of a plant’s cellular signaling mechanisms. 2. SIGNAL TRANSDUCTION PATHWAYS OF PLANTS The emergence of complete genome sequences from strategic eukaryotic models has allowed the comparative analysis of plant and animal signal transduction pathways. In both cases, this analysis has offered insight into the features of specific signaling pathways that were not achievable at the time the previous edition of this book was published. It is now hoped that by looking closely at the emerging differences between analogous signaling pathways in plants and animals, it will be possible to identify their relationship to the divergence From: Endocrinology: Basic and Clinical Principles, Second Edition (S. Melmed and P. M. Conn, eds.) © Humana Press Inc., Totowa, NJ of the two lineages. Excellent recent reviews on this 137 138 topic have been published over the past 5 yr (Cock et al., 2002; Wendehenne et al., 2001). Here we give a brief overview of selected examples in order to illustrate some interesting features of plant signaling pathways and then discuss these pathways in the context of novel developments in plant evolution. As a result of photoautotrophism, the evolution of plants has been constrained by the absence of mobility and the presence of relatively rigid cell walls. The capture and integration of chloroplasts from bacterial progenitors profoundly influenced the signaling mechanisms of modern plants. Not surprisingly, for sessile photosynthetic organisms able to sense carefully their fluctuating environment, the developmental pathways of plants are irrevocably and necessarily linked to the perception of external cues. Temperature, light, touch, water, and gravity can all activate endogenous developmental programs. Of these, light has an especially important role, not only as the energy source for photosynthesis, but also as a stimulus for many developmental processes throughout the life cycle of plants, from seed germination to flowering. Consequently, plants have the richest array of light-sensing mechanisms of any group of organisms. These photoreceptors are able to measure not only the intensity but also the quality of light available to the plant. Phytochromes, e.g., are the photoreceptors for red and far-red light responses (Nagy and Schäfer, 2002). They are red-light-activated serine/ threonine kinases that exist in two photointerconvertible forms. On stimulation with red light, they move from the cytosol to the nucleus, where they interact with proteins such as the helix-loop-helix transcription factor phytochrome-interacting factor (PIF3; Martinez-Garcia et al., 2000). These proteins then bind to light-responsive promoter elements leading to transcription, thereby achieving light-regulated gene activation (Tyagi and Gaur, 2003). Thus, phytochrome signaling involves both nuclear and cytosolic interactions. Comparative genomic analysis of plant genomes from species such as Arabidopsis thaliana (thale cress) and Oryza sativa (rice) has revealed many signaling compounds that are highly conserved between animals and plants. The reiteration of core signaling mechanisms in plants and animals suggests that overall differences between the two kingdoms evolved via the modification of basic ancestral pathways. However, this basic similarity is found in combination with many novel elements or motifs. Overall organizational principles are shared among plants and animals, indicating that a core of conserved signaling genes and pathways is used repeatedly in many different developmental contexts. RAS genes are a good example to illustrate this argument. RAS genes belong to the small guanosine Part III / Insects / Plants / Comparative 5´-triphosphatase protein family. They are master regulators of numerous cellular processes including signaling, cargo transport, and nuclear transport. They are regarded as molecular switches that alternate between an active and an inactive state, thereby ensuring the flow of information at the expense of guanosine 5´triphosphate. This molecular switch appears to have been developed early, and throughout evolution, it has been adapted to a variety of tasks. Small G proteins are classified in five families: the RAS (according to the oncogene Ras from rat sarcoma virus), the RAB (Ras of brain), the ARF (ADP ribosylation factor), the RAN (Ras-related nuclear protein), and the RHO (Ras homologous) family. They interact with partner proteins (effectors) to form dynamic complexes regulating a plethora of crucial cellular processes. In plants, however, no RAS genes, but only members of the RAB, ARF, and the RAN families have been found. An additional plant-specific family of small G proteins is named ROP, for RHO of plants (Vernoud et al., 2003). Apparently, only members of those families that play intricate roles in metabolite transport and cell polarity control have been conserved in plants. It is conceivable that the sessile nature of plants demands tight control over secretory pathways to enable and precisely adjust the cell elongation processes. In this case, homeostatic control of cellular membrane compartments, transport of macromolecules between intracellular compartments and the extracellular space, and nuclear transport would have added importance for the evolutionary success of plants. Despite conservation of the basic secretory machinery between plants and other eukaryotes, several recent findings suggest distinct structural and functional differences in plants. It is therefore expected that the systematic functional analysis of key players of plant secretion will uncover novel insights into the processes by which the formation of transport vesicles and intracellular trafficking by internal and external cues are controlled, and by which vesicles are delivered to target membranes. Ultimately, from analysis of these processes, researchers will learn important lessons on how plant cells control apical and basal cell polarity. Moreover, such approaches will not only uncover important aspects of the organizational blueprint of the plant secretory pathway, but also reveal fundamental functional differences between plants and other eukaryotes and indicate how these differences relate specifically to the relationship between form and function in plants. Analysis of the plant cargo delivery system provides privileged views not just into unique aspects of secretion control, but also into many other plant-specific processes, such as hor- Chapter 10 / Signal Transduction Pathways in Plants 139 Fig. 1. Phytohormones: chemical structure and properties. monal control of growth, gravitropic and phototropic responses, establishment and maintenance of cell polarity, cell differentiation, mediation of disease resistance, and fruit ripening. In the long term, insight into these fundamental processes will be important for many biotechnological applications. 3. ROLE OF PHYTOHORMONES Auxin, cytokinin, abscisic acid (ABA), gibberellin (GA), and ethylene are the five classic hormone pathways that appear very early in plant evolution and have been adapted to functional uses in many contexts of plant development (Fig. 1). Brassinosteroids are a relatively recent addition to this list, but must also be considered as potent plant growth regulators. These phytohormones are secondary metabolites that play physiological roles at specific stages of a plant’s lifecycle. They are typically considered in terms of three sequential events: their biosynthesis, their perception, and the signals that are subsequently initiated as a consequence. The effects of a phytohormone are commonly demonstrated either by their exogenous application to a growing plant, or by the inhibition or exaggeration of their influence in mutant plants. Such plants may be affected in the rate of biosynthesis of a particular hormone, in the sensing of a hormone’s presence, or in the subsequent transduction of a downstream signaling cascade. Phytohormones represent integral components of the mechanisms by which a plant regulates both its own development and its response to the wide variety of stimuli it receives from its environment. Since Charles and Francis Darwin first attributed the bending of a grass coleoptile toward light to the action of a growth mediator, research into the biosynthesis and mode of action of phytohormones has developed into one of the most widely studied aspects of plant biology (Davies, 1995). We now give an overview of the current understanding of both how higher (seed-bearing) plants perceive phytohormones and how this perception is translated into a physiological response. Plants, owing to their sessile nature, cannot move autonomously in response to environmental stimuli in the same way as many animals can. As already inferred, this restraint has been overcome, at least in part, by the extension of the role of hormones from that of regulator (either metabolic or developmental) into the means by which a response to environmental stimuli are elicited. For example, Darwin’s first experiments on coleoptile bending represent the attempt of a young grass shoot to increase its photosynthetic capacity. It was subsequently demonstrated that the response is mediated by production of indole-acetic acid (IAA) (a member of the auxin class of phytohormones) in the shoot tip, followed by asymmetrical redistribution throughout the growing plant. Cells respond to the concentration of IAA by elongating in a dose-responsive manner, producing a physiologic response. 140 Part III / Insects / Plants / Comparative 4. AUXINS Auxins are vital mediators of developmental and physiological responses in plants and a paradigm for plant growth regulators. They regulate apical-basal polarity in embryonic development; apical dominance in shoots; induction of lateral and adventitious roots; vascular tissue differentiation; and cell growth in both stems and coleoptiles, including the asymmetric growth associated with phototropic and gravitropic responses (Davies, 1995). Concentration, perception, and the effect that signaling has on gene expression are central issues when considering the phytohormone signaling pathways that affect growth and regulation. In relation to auxin, it has been suggested that efflux-mediated gradients are the underlying driving force for the formation of all plant organs, regardless of their developmental origin and fate. An attractive theory is therefore that the relative concentration of auxin is particularly important in plant development. Both the concentration of auxin in any one cell and the steepness of the auxin concentration gradient over a group of cells are determined by the rate of auxin synthesis in source cells, the rate of its transport through a tissue, and the overall rate of its degradation or conjugation (the majority of auxin present in any one cell exists as biologically inactive conjugate). Auxin is transported from the shoot downward. However, the prevailing model of the initiation of auxin gradients in the apical meristem has been questioned by the demonstration that all parts of young plants can synthesize IAA, thus potentially diminishing the importance of polar auxin transport (Ljung et al., 2001). In the 1920s, Cholodny and Went independently suggested the chemiosmotic hypothesis of auxin transport, which was later refined by Rubery and Sheldrake (1974) and Raven (1975). The theory predicts the existence of an auxin efflux carrier that actively and asymmetrically redistributes auxin in root and stem tissue on gravitropic or phototropic stimulation. Auxin movement both into and out of cells requires specialized carriers (Friml and Palme, 2002). Several Arabidopsis genes encoding putative auxin carriers have been identified during the past decade. The amino acid permease-like gene AUX1 and the family of bacterial transporter-like PIN genes encode putative auxin influx (Bennett et al., 1996) and efflux carriers, respectively. Characterization of the first putative auxin efflux carrier PIN1 (Gälweiler et al., 1998) gave context to auxin’s asymmetric localization. PIN1 encodes a 622amino-acid protein with 12 predicted transmembranespanning segments (Fig. 2). It shares similarity with a group of transporters from bacteria of the major facilitator class, evidence supporting a transport function (Gälweiler et al., 1998; Pao et al., 1998). A search of the Arabidopsis genome for genes with homology to PIN1 revealed another seven genes belonging to the same family. Similar sequences have been found in all other plants now sequenced, but not in animals, indicating that PIN proteins have evolved exclusively in plants. Based on genetic evidence, PIN proteins are strong candidates for either the auxin efflux carrier itself or an important regulatory component of the efflux machinery (Palme and Gälweiler, 1999). More important, the distributions of PIN1 and other PIN proteins in the plasma membrane of auxin-transporting cells of stems and roots were found to be dynamic and asymmetric according to the direction of auxin flux (Friml et al., 2002b; Gälweiler et al., 1998; Geldner et al., 2001; Müller et al., 1998; Steinmann et al., 1999) (Fig. 3). Auxin gradients in plant tissue appear to be sink driven; gradient formation seems to be regulated by auxin transport (rather than degradation) machinery. For example, the formation of a maximum auxin concentration at the Arabidopsis root apex depends on the activity of PIN4 (Friml et al., 2002a). It is likely that the activity of the efflux complex is regulated by phosphorylation (Delbarre et al., 1998). Auxin efflux was found to be more sensitive to the specific transport inhibitor N-1-naphthylphthalamic acid (NPA) in seedlings of an Arabidopsis mutant named rcn1 (“root curl in NPA”) than in the wild type. The RCN1 gene encodes a subunit of protein phosphatase 2A (Garbers et al., 1996). Furthermore, the mutant can be phenocopied with a phosphatase inhibitor (Deruere et al., 1999). The protein kinase PINOID enhances polar auxin transport (Benjamins et al., 2001) and is another potential component of the hypothetical auxin-efflux complex. 5. AUXIN PERCEPTION According to the widely accepted theory, phytohormone signaling begins with the perception of free hormone by a specific receptor. In the case of auxin, there is evidence for multiple sites of auxin perception. It therefore appears that, at least initially, the auxin signal can transduce through more than one signaling pathway. To date, the best-characterized auxin-binding protein is ABP1 (Napier et al., 2002), which was originally identified, purified, and cloned from maize (Hesse et al., 1989; Löbler and Klämbt, 1985). The high binding constant of auxin and ABP1 has inspired much research, however, the protein has no homology to any other known receptor family, and it is ubiquitous in vascular plants, including the pteridophytes and bryophytes (Napier et al., 2002). A KDEL retention motif at the C-terminus of ABP1 ensures an ER loca- Chapter 10 / Signal Transduction Pathways in Plants 141 Fig. 2. Predicted AtPIN1 protein structure. tion (Henderson et al., 1997; Tian et al., 1995); however, some ABP1 does pass along the constitutive secretion pathway to the plasma membrane and cell surface (Diekmann et al., 1995; Henderson et al., 1997). The ER location makes the characterization of ABP1 more complex because most of the physiological data demonstrate activity of ABP1 on the plasma membrane. Here, auxin is able to control several cellular responses, including tobacco mesophyll protoplast hyperpolarization (Leblanc et al., 1999a, 1999b), tobacco mesophyll protoplast division (Fellner et al., 1996), expansion of tobacco and maize cells in culture (Jones et al., 1998), and maize protoplast swelling (Steffens et al., 2001). These effects can be inhibited by the application of anti-ABP1 antibodies, which are unable to enter the cell. It has therefore been concluded that ABP1 is able to elicit a physiological response in the presence of auxin at the surface of the plasma membrane. A functional role of ABP1 inside the ER has not been shown; these data may be reconsidered, however, because auxin efflux carriers are now known to cycle continuously in membrane vesicles between the plasma membrane and the endosome (Geldner et al., 2001). There is considerable speculation about the possible role of auxin transporters in auxin signaling. It is possible that measurement of the flux of auxin through either influx or efflux carriers (or both) monitors auxin level in the cell. It is also possible that specific transporter family members no longer act as transporters but have evolved a receptor function (Friml and Palme, 2002). Sugar sensing is important for plants and yeast to report the carbohydrate status within cells and outside of cells. It has been demonstrated that some proteins that show transporter topology do not transport sugars but sense sugar outside of cells and control transcription of sugar transporters that control the sugar homeostasis in cells (Lalonde et al., 1999). A similar mechanism for auxin perception is conceivable. Fig. 3. AtPIN1 (inner arrows) and AtPIN2 (outer arrows) in Arabidopsis root tip. Arrows indicate the direction of Auxin fluxes in marked cell files. 6. EFFECT OF AUXIN SIGNALING ON GENE EXPRESSION Auxin-dependent transcriptional activation can occur within minutes of a signal being perceived (Abel and Theologis, 1996). In the nucleus, the regulation of gene expression by auxin can be mediated by the action of two families of auxin-induced proteins: the Aux/IAA proteins and the auxin response factors (ARFs) (Hagen and Guilfoyle, 2002). ARFs bind to auxin response promoter elements upstream of genes and activate or repress their transcription. Aux/IAA proteins can dimerize with ARF proteins, thus inhibiting their activity (Tiwari et al., 142 Part III / Insects / Plants / Comparative 2003). However, they have very short half-lives, ranging from a few minutes to a few hours (Abel et al., 1994; Gray et al., 2001). A normal auxin response is dependent on this rapid turnover of Aux/IAA proteins, as it lowers the concentration of the inhibitory ARF-Aux/IAA dimer (Ulmasov et al., 1997; Worley et al., 2000). Aux/IAA proteins are found in all higher plants and are characterized by four highly conserved domains (Abel and Theologis, 1996; Guilfoyle et al., 1998). In yeast two-hybrid assays, their dimerization with ARF proteins has been shown to involve two of these domains (which are similar to those of the ARF proteins) (Ulmasov et al., 1997). Another domain, domain II, is crucial for Aux/IAA function, with many lines of evidence demonstrating that it is the target for Aux/IAA protein destabilization, ensuring the rapid turnover required for a normal auxin response. The fusion of Aux/ IAA proteins to reporter proteins, such as luciferase or β-glucuronase (GUS), results in the destabilization of the reporter protein (Gray et al. 2001;Worley et al., 2000). This indicates that Aux/IAA proteins contain a transferable destabilization sequence. A nonfunctional domain II, as found in the auxin-resistant mutants axr3-1, axr21, and shy2, dramatically increases the protein’s half-life and prevents the ARF proteins from functioning (Gray et al., 2001; Ouellet et al., 2001; Worley et al., 2000). The stabilization of an Aux/IAA-reporter fusion by inhibitors of the 26S proteasome indicates that auxin signaling requires SCFTIR1-mediated turnover of Aux/IAA proteins. 7. GIBBERELLINS GAs are a large group of diterpenes comprising well over a hundred members. However, only a handful can elicit a physiological response, the others being representative of a large and complicated web of biosynthetic pathways from ent-kaurene, the product of the first dedicated step of GA biosynthesis. These biosynthetic pathways are now well understood. GAs regulate a wide range of physiological processes, including cell division and cell elongation, and are crucial to the control of processes as diverse as germination, stem elongation, flowering, fruit ripening, and senescence. The last 5 yr have seen a dramatic increase in our understanding of the processes involved in GA signal transduction (Gomi and Matsuoka, 2003), however, the exact mechanisms by which a plant’s response to GA is brought about remain unclear. A class of transcription factors, the DELLA proteins, has emerged as a central mediator of many GA responses, although they probably do not bind DNA directly (Dill et al., 2001). It was decided that these proteins (named after a five-amino-acid N-terminal motif) are important components of the GA signal transduction pathway after the analysis of a number of dwarfed mutants from a range of species (Sun, 2000). An important example (and the mutant from which the first member of the family was cloned) is the gai1-1 mutant of Arabidopsis. Plants with a dominant mutation at the GAI allele display a semidwarf phenotype, insensitive to the exogenous application of GA (Peng and Harberd, 1993). All results indicate that the DELLA proteins are negative regulators of GA signaling. Altogether there are five DELLA proteins in Arabidopsis (GAI, RGA, RGL1, RGL2, and RGL3), but only one in rice (SLR1) and barley (SLN1). They share a high degree of sequence homology and belong to a wider group of plant transcription factors called the GRAS family. All of these proteins share the same basic structure, with an N-terminal GA-signal-perception domain, a serine/threonine-rich domain, a leucine zipper, and a C-terminal regulatory domain conserved among GRAS proteins. This structure has been used to suggest a model for the mode of action of SLR1 where the protein exists as a dimer, the subunits linked by the leucine zipper (Itoh et al., 2002). The active form (receiving no GA signal) represses the GA response via the C-terminus, which is deactivated when a GA signal is bound by the DELLA domain. As is the case for auxin, the GA receptor is still unknown. However, there is evidence that the transduction of the GA signal from the plasma membrane to the nucleus involves G proteins (Ueguchi-Tanaka et al., 2000). A range of secondary messengers have also been shown to be involved in this process, but the exact role of many remains unclear (Sun, 2000). As in animals, it has recently emerged that the post-translational modification of proteins by the addition of Olinked N-acetylglucosamine could be involved in signaling processes (Thornton et al., 1999). Analysis of two mutants of Arabidopsis, partially rescued by the application of exogenous GA, has revealed two putative O-GlcNAc transferases (by homology with known enzymatic sequences) thought to be involved in the GA signaling pathway (Hartweck et al., 2002). In animals, the transferase ability has been shown to compete with phosphorylation of serine and threonine residues, suggesting a possible mechanism for their mode of action in plants, and a link to DELLA protein function. 8. CYTOKININS Cytokinins play a major role in many different developmental and physiological processes in plants, such as cell division, regulation of root and shoot growth and branching, chloroplast development, leaf senes- Chapter 10 / Signal Transduction Pathways in Plants cence, nutrient mobilization, biomass distribution, stress response, and pathogen resistance. In contrast to our understanding of auxin and GA signal transduction, proteins have been identified that function as cytokinin receptors. Activation tagging experiments in Arabidopsis identified CKI1, a gene encoding a receptor histidine kinase, whose overexpression was seen to induce typical cytokinin responses (Kakimoto, 1996). Although CKI1 is able to activate the cytokinin signaling pathway, it does not bind cytokinins directly (Hwang and Sheen, 2001). Nevertheless, the discovery of CKI1 suggested that the cytokinin transduction pathway in higher plants could be similar to the prokaryotic two-component system. This hypothesis was proved by the identification of CRE1/AHK4, another histidine kinase, as the first cytokinin receptor (Inoue et al., 2001; Suzuki et al., 2001; Ueguchi et al., 2001). The availability of the Arabidopsis genomic sequence led to the identification of two further cytokinin receptors (AHK2 and AHK3). After cytokinin perception, the resulting signal is transmitted by a multistep phosphorelay system through a complex form of the two-component signaling pathways that has been well characterized in prokaryotes and lower eukaryotes. Functional evidence for cytokinin sensing by the receptor CRE1/AHK4 was obtained in elegant complementation experiments in yeast and Escherichia coli, which rendered these heterologous hosts cytokinin sensitive. Two other histidine kinases, AHK2 and AHK3, have been shown to be active in the same complementation test system and to give protoplasts cytokinin sensitivity (Hwang and Sheen, 2001; Yamada et al., 2001), indicating that these two proteins are also cytokinin receptors. Each receptor comprises an N-terminal extracellular domain, a membrane anchor, and a C-terminal transmitter domain, capable of autophosphorylation. The three cytokinin receptor genes differ in their expression pattern. CRE1/AHK4 is mainly expressed in the roots, whereas AHK2 and AHK3 are present in all major organs (Inoue et al., 2001; Ueguchi et al., 2001). This tissue-specific expression of cytokinin receptors could be an additional layer of control to the perception of cytokinin. Considering the large number of response-regulator genes associated with the two-component signaling sytem (22 in Arabidopsis), it has been suggested that they could both have different functions, using different targets in addition to participating in cross talk with other hormones. There are accumulating data demonstrating that in order to understand the growth responses to cytokinins, it is important to understand such cross talk between cytokinins and nutrients as well as cytokinins and other phytohormones. Moore et al. (2003) 143 showed that application of cytokinins as well as the use of transgenic Arabidopsis lines with constitutive cytokinin signaling could overcome the glucose repression response. The insensitivity of Arabidopsis glucose insensitive2 (gin2) to auxin and hypersensitivity to cytokinin could be the clue to understanding the antagonistic interaction between cytokinins and auxin and its dependency on the glucose status of tissues. 9. BRASSINOSTEROIDS Steroids are important signaling molecules in plants as well as in animals. Since the discovery of brassinolide in 1979, brassinosteroids (BRs) have been shown to be important at a number of stages of a plant’s development, including stem elongation, germination and senescence. BRs retain the basic four-ring structure of many steroid hormones; like animal steroid hormones, they are synthesized from cholesterol. Two mutants deficient in the biosynthesis of BRs, DET2 and CPD, develop as if grown under light when grown in the dark (Chory et al., 1991; Li and Chory, 1997). This demonstrates that, in addition to these other crucial processes, BRs play an important role in photomorphogenesis. The identification of a BR receptor has been a recent significant advance in phytohormone biology; this section focuses on the mechanism by which BRs are initially sensed by the cell, before highlighting some interesting similarities between the perception and mode of action of BRs and the regulation of development in animals. In animals, steroids receptors are nuclear located (Marcinkowska and Wiedlocha, 2002); in plants, no nuclear steroid receptors have been found, indicating that plant cells have evolved a different method to receive the BR signal. The bri mutant of Arabidopsis shows a dwarfed phenotype similar to that of mutants deficient in the biosynthesis of BRs. BRI1 was thought to be involved in signaling owing to the mutant plants’ unresponsiveness to applied brassinolide. Cloning of the bri1 gene revealed a leucine-rich repeat (LRR) receptor-like kinase, an immediate candidate for the BR receptor (Li and Chory, 1997). BRI1 was subsequently shown to be located at the plasma membrane (Friedrichsen et al., 2000), and to have a relatively high affinity for bioactive BR (Wang et al., 2001). LRR receptor kinases contain three domains: an extracellular domain comprising several leucine-rich repeating sections (in the case of BRI1, 25), a transmembrane section and a cytoplasmic kinase domain. BRI1 also contains a 70amino-acid island in the LRR domain, necessary for the protein’s function (Li and Chory, 1997). The use of chimeric proteins has demonstrated that the BRI1 extracellular domain was both necessary and sufficient 144 Part III / Insects / Plants / Comparative for the translation of a specific set of genes. When fused to the intracellular kinase domain of a similar receptor-like kinase, a protein involved triggering a plant’s defensive response to pathogens, activation of the extracellular BRI1 domain and, hence, defenserelated gene expression could be induced by BR (He et al., 2000). The LRR-receptor kinases are members of a very large class of proteins in Arabidopsis comprising 174 members with diverse function. Only a small number have been ascribed a function, including CLAVATA (involved in meristem development) and ERECTA (involved in organogenesis) (Dievart and Clark, 2003). The functions of LRR-receptor kinases are diverse. In Arabidopsis, three BRI-like proteins share high homology and are similar to protein sequences found in monocotyledonous species. Of these, BRL1 and BRL3 are able to rescue the bri1 mutation, suggesting a closely related function. The cloning of a homolog of BRI1 in tomato revealed an intriguing overlap of function of the tBRI1 (BRI of tomato) protein. Systemin is a peptide signal important in pathogen-defense responses in plants, acting by amplifying the induction of the jasmonate signaling pathway. It was discovered that the same receptor (called SR160 in the context of systemin) was responsible for both BR and systemin signaling (Montoya et al., 2002; Szekeres, 2003). This dual receptor function has also been observed in animals, with the hormone progesterone able to inhibit specifically the peptide oxytocin from binding to its receptor, a uterine G protein–coupled receptor (Grazzini et al., 1998). It has been suggested that BR could bind to its receptor while simultaneously bound to a specific protein, owing to sequence homology to animal steroid-binding proteins being found in the Arabidopsis genome (Li and Chory, 1997), and the fact that, in general, LRRs mediate protein-protein interactions rather than smaller ligand binding. 10. ABSCISIC ACID Plants control water balance with a range of strategies. For most land plants, the anatomy of leaves is centered on a balance between minimizing water loss and maximizing both exposure to the sun and the rate of diffusion of molecular oxygen away from and carbon dioxide into photosynthetic cells. This balance is essential for maintaining the flux of electrons that pass through the light-dependent reactions of photosynthesis. The leaf is an organ that is necessarily exposed to relatively high levels of sunlight and, therefore, of water loss through evaporation from pores (stomata). ABA is the phytohormone which regulates the opening and closing of stomata. It does this by controlling the turgor pressure inside the two surrounding bananashaped guard cells. Much of the work on ABA signaling has been focused on guard cells and mutants affecting their function. However, ABA influences both physiological (gene expression in response to salt stress and drought) and developmental (e.g., germination and seedling development) processes. ABA seems to affect many different signaling pathways, sometimes with a high degree of redundancy; the extent to which it mediates cross talk between environmental and developmental stimuli is currently the subject of concentrated research. The amount of free ABA able to elicit a response is thought to be dependent on many factors. These include movement of ABA through the plant, the relative rates of ABA synthesis and catabolism, and the concentration of ABA in the leaf symplast. It has become clear that the ABA signal is transduced through a number of secondary messengers, among them lipid-derived signals, H2O2, G proteins, and nitric oxide (Himmelbach et al., 2003). The varying cellular concentrations of these compounds unite to influence indirectly the cytosolic concentration of Ca2+, the central factor in many ABA signals (McAinsh et al., 1997). It is thought that ABA has two modes of action: the first “nongenomic” effect is able to change the turgor pressure in guard cells by altering the plasma membrane’s permeability to ions, and the second acts via changing the transcription levels of ABA-responsive genes. It is thought that both processes are reliant on alterations in the intracellular concentration of Ca2+; however, it is not yet fully understood to what extent the pathways are separated. Despite a long history of research, the nature of the initial ABA receptor remains elusive. However, it is widely believed that the initial event in the signaling cascade is the binding of ABA to either a membranebound or a cytosolic receptor. In many cases, this binding results in the activation of Ca2+-influx channels resulting in the ABA-mediated increase in intracellular Ca2+ concentration (Murata et al., 2001). Another important factor in this process is the altering permeability of the tonoplast to Ca2+; this is influenced by intracellular lipid-derived signals and cyclic adenosine 5´-diphosphate–ribose concentration, the latter dependent on the Ca2+ concentration itself (Wu et al., 2003). The overall increase in intracellular Ca2+ results first in the inhibition of K+-influx channels and, second, in the activation of K+-efflux channels and the inhibition of H+-adenosine triphosphatase. Therefore it can be seen that ABA initiates a complicated mesh of interconnecting signals, resulting in a physiologic response (Finkelstein et al., 2002). Chapter 10 / Signal Transduction Pathways in Plants 145 11. ETHYLENE 12. PERSPECTIVE It has long been known that exposure to ethylene elicits a well-characterized response in seedlings. The so-called triple response, a signature of ethylene signaling, comprises an increase in the girth of hypocotyl and root as well as the formation of an apical hook. This well-defined phenotype has been the basis of ethylene research, which is used to identify mutants in the ethylene signaling pathway. Ethylene has been shown to be important in a wide range of processes including fruit ripening, senescence, and defense response. The ethylene signaling pathway is relatively well characterized, and it has also been shown that ethylene signaling is intrinsically linked to many other phytohormonal signaling pathways. There are five ethylene receptors in Arabidopsis: ETR1, ETR2, ERS1, ERS2, and EIN4 (Stepanova and Ecker, 2000). They are all histidine kinases, the same class of two-component regulatory system as is found in the cytokinin signaling pathway. The ethylene receptors can be most easily classified in two ways. In the first, they are grouped into those with (ETR1, ETR2, and EIN4) and without (ERS1 and ERS2) a receiver domain, the domain that receives the phosphotransfer from the histidine kinase. It is thought that a signal could be transduced via a dimer of receptors (Hall et al., 2000); it has been suggested that the proteins without a receiver domain operate in a receptor complex. The second and more common classification groups the ethylene receptors according to their structure. Subfamily I (ETR1 and ERS1) has three membrane-spanning regions, whereas subfamily II (ETR2, ERS2, and EIN4) has four membrane-spanning regions and lacks conserved residues in the histidine kinase domain. Interestingly, the phenotype of receptor-deficient mutants can be rescued with an ETR1 protein with an inactivated histidine kinase domain. This work suggests that the ethylene receptor complex can transduce a signal by a mechanism other than histidine kinase–dependent phosphotransfer (Wang et al., 2003). Although it can also homodimerize (Schaller and Bleecker, 1995), ETR1 has been shown to interact directly with CTR, a protein similar to the Raf family of mitogen-activated protein kinase (MAPK) kinases . The receptor complex has been shown to be located at the endoplasmic reticular membrane (Gao et al., 2003), and negative regulation by a MAPK signaling cascade has been demonstrated in Arabidopsis and Medicago (Ouaked et al., 2003). This provide another hint as to the complex relationship between what have been traditionally regarded as discrete phytohormone signaling pathways. Understanding the significance of such integration will be a major challenge in the coming years. Over the last decade, tremendous progress has been made using genetic analysis of the model plant Arabidopsis. This has allowed researchers to dissect developmental programs as well as hormonal and environmental responses, including light regulation and plant-pathogen interactions. 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