Hormones in plants. Molecular Endocrinology, 2006: ISBN: Editor: Patricia Joseph-Bravo

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1 Research Signpost 37/661 (2), Fort P.O., Trivandrum , Kerala, India Molecular Endocrinology, 2006: ISBN: Editor: Patricia Joseph-Bravo 10 Hormones in plants Alejandra A. Covarrubias, Gladys I. Cassab * and Federico Sánchez # Departamento de Biología Molecular de Plantas, Instituto de Biotecnología Universidad Nacional Autonoma de México (UNAM), Mexico * gladys@ibt.unam.mx; # federico@ibt.unam.mx Abstract Plants and animals are multicellular organisms that evolved from two different and successful life strategies; however, this does not preclude that, as all eukaryotic organisms, plants need to process the huge amount of information that continuously receive and store for making adaptive decisions. An efficient chemical communication system has been selected in plants that have allowed them to accomplish normal development and adaptation to the changing environment. These chemicals act as signaling molecules that coordinate the perception and the responses to the different stimuli. Some of these signaling molecules Correspondence/Reprint request: Dr. Alejandra A. Covarrubias, Departamento de Biología Molecular de Plantas, Instituto de Biotecnología, Universidad Nacional Autonoma de México (UNAM), Apdo. Postal Cuernavaca, Mor , México. crobles@ibt.unam.mx

2 194 Alejandra A. Covarrubias et al. belong to the group of endogenous plant growth regulators also named phytohormones. The original use of the term hormone in plant physiology comes from the concept established in animal physiology of a hormone. This implies a localized site for its synthesis, its transport to a target tissue, and the eliciting at low concentrations of a response in the target tissue. However, the many differences found in the mode of action between mammalian and plant hormones have led to the conclusion that the definition of a hormone in the mammalian sense does not apply to plant hormones. In this review we will provide a general view of the concept of hormones in plants and summarize the information regarding the mode of action of the best-characterized phytohormones, including recent results that suggest an intimate cross-talk between the signal transduction pathways of different plant hormones. Introduction Plants and animals represent two different life strategies to successfully face the existence of multiple cells in an organism. These plans expose two distinctive types of body structures, on one side; animals are motile organisms, whereas on the other side, plants are rooted to the soil. The plant s plan was the result of a decision made several billion years ago to gather energy via photosynthesis. These evolutionary decisions are the consequence of different cellular structures, cellular organization and cellular communication, which does not exclude the possibility of using common elements to attain similar results. The sessile nature of plants imposed the evolution of a differentiated modular structure that allowed them to tolerate predation and environmental injure because some modules will be able to survive to further regenerate the individual. In contrast to animals, plants present a reduced functional specialization of cells and tissues, even though, they have to contend against the very different physical environments to which their organs are exposed; roots are below ground whereas shoots are exposed to light. Hence there are differences between stimuli and signals perceived by the root compared with the shoot. As all eukaryotic organisms, plants need to process the huge amount of information that continuously receive and store for making adaptive decisions. Among these decisions, plants are able to generate many phenotypes from a single genotype (phenotypic plasticity), they are capable of modifying their developmental programs to cope with an environment of enormous variability, in order to counteract the restrictions imposed by their immobility. An efficient chemical communication system has been selected in plants that have allowed them to accomplish normal development and adaptation to the changing environment. These chemicals act as signaling molecules that coordinate the perception and the responses to the different stimuli. Some of these signaling molecules belong to the group of endogenous plant growth regulators also named phytohormones (Fig. 1).

3 Hormones in plants 195 Figure 1. The chemical structure of some phytohormones. The original use of the term hormone in plant physiology comes from the concept established in animal physiology of a hormone. This implies a localized site for its synthesis, its transport to a target tissue, and the eliciting at low concentrations of a response in the target tissue. Now it is clear that the definition of a hormone in the mammalian sense does not apply to plant hormones. Although the synthesis of phytohormones may be localized to a particular organ, it may also occur in diverse tissues, or cells within tissues. Phytohormones are able to exert their action locally or at a distance (e.g. some are transported from roots to leaves to produce their physiological effect, and some others bring about changes in the same tissue, or within the same cell where they are synthesized). These characteristics have led to consider that transport is not an essential property of a plant hormone. Moreover a debate exists around the idea that the control carried out by phytohormones is not only by concentration but also by a change in sensitivity of the target tissues or cells to these compounds. Hence, we suggest to the reader to take these considerations in mind, and do not expect a phytohormone to behave as an animal hormone, plant hormones are a distinctive group of natural compounds with unique metabolism and properties that have the ability to act as chemical messengers to induce a physiological adjustment in response to particular stimuli. The growth of a coleoptile towards light is known as phototropism and is a common behavior among plants. The experiments of Darwin on the phototropism of coleoptiles were the first to suggest the presence of a longdistance signal, which functions in the recognition of a light stimulus to the

4 196 Alejandra A. Covarrubias et al. coleoptile s tip. Further studies revealed that the growth towards light is caused by an elongation of the cells at the side that is shielded from the light. The phototropic reaction does not happen if the coleoptile s tip is removed, though it can be induced again by the replacement of the tip. This indicates the existence of a substance that is spread from tip to bottom (basipetal direction) and that causes the elongation [1]. F. Went, a Dutch plant physiologist, called this effector auxin or growth regulating substance [2-3]. Subsequently, other investigations have led to the discovery of other hormones such as gibberellins (GA), cytokinins (CK), abscisic acid (ABA) and ethylene. More recently, other compounds have been added to the list such as jasmonates (JA) and brassinosteroids (BR). The structural simplicity of plant hormones as well as their apparent low variety do not seem to match with the multiple types of cells that perceived them and the so different nature of the responses they provoked. This may be because in plants the variability is provided by the activity of the different concentrations, the different sensitivity in the various tissues or cell types, and the interactions between the different phytohormones. The introduction of molecular genetics and the use of the model genetic system in the study of plant hormones have unraveled the molecular basis of how this simple compounds turn on or off a cellular response. At this point some components of phytohormones signal transduction pathways have been identified, such as receptors, signaling intermediates (kinases, phosphatases, etc.) and downstream transcription factors. However, still there are puzzles to be solved, which come from reiterated questions such as how many different hormones can affect the same process and, at the same time, how a single hormone can influence so many dissimilar responses. The answers to these questions will lead us to understand how hormones coordinate overall plant growth, development and adaptation to the changing environment. The purpose of this review is to summarize the information regarding the mode of action of the best-characterized phytohormones, including recent results that suggest an intimate cross-talk between the signal transduction pathways of different plant hormones. Auxins Auxin is one of the most important molecules regulating plant growth and morphogenesis. As a critical plant hormone, auxin modulates such diverse processes as tropic responses to light and gravity, general root and shoot architecture, organ patterning, vascular development and growth in tissue culture [4]. The importance of auxin for human sustenance is both vital and readily apparent: auxin is required for plant growth. Anthropogenic manipulation of auxin physiology has assisted plant propagation, and, through, the blind pressure of artificial selection, the development of modern crop varieties [5].

5 Hormones in plants 197 Auxin biology is among the oldest fields of experimental plant research. Charles Darwin performed early auxin experiments, observing the effects of a hypothetical substance modulating plant shoot or root elongation to allow tropic growth toward light or toward the gravity vector [1]. Since one of the earliest noted auxin effects was in phototropism, the curvature of stems toward a light source was used as a bioassay for the identification of auxin. Application of auxin to decapitated shoots can induce bending in the absence of a light stimulus [3]. Rapidly, it became obvious that auxin moved in a polar fashion from cell-to-cell along the apical-basal axis. Nevertheless, molecules and the mechanisms driving this transport remained unknown for the next seventy years. Auxin polar transport In the 1970s, the chemiosmotic hypothesis was postulated proposing that cellular accumulation of auxin is driven by electrical and ph gradients maintained across cell membranes [6], while secretion of auxin was hypothesized to be performed by elusive auxin carriers. After entering cells in its protonated form, or through the action of a saturable uptake carrier, auxin becomes rapidly deprotonated due to the more basic ph values of the cytoplasm. As a result, auxin is effectively trapped inside cells or within vesicular compartments. Consequently, export of auxin from cells requires the action of auxin efflux carriers. In 1996, the identity of the first auxin influx carrier AUX1, was revealed by Malcom Bennett and coworkers [7]. Two years later, four groups simultaneously isolated genes encoding putative auxin efflux carriers, among which PIN1 [8] was the first characterized as a member of a large multigene family. The question is then, do PIN1 and AUX1 transport auxin across the plasma membrane or are they vesicular transporters? In order to achieve the polar cell-to-cell transport of auxin, cells must be able not only to accumulate auxin but also to export auxin in a polar fashion. Thus, auxin polar transport depends upon the polar localization of the efflux carriers PIN. PIN proteins cycle very quickly and constitutively between the plasma membrane (PM) and endosomes. Furthermore, auxin transport inhibitors do not affect auxin transport per se but rather inhibit the recycling of PIN1. Recently, it has been proposed that auxin inhibits endocytosis, by inhibiting the internalization step of PIN constitute cycling and thereby increasing levels of PINs at the PM. Concomitantly, auxin promotes its own efflux from cells by a vesicle trafficking dependent mechanism. Therefore, by modulating PIN protein trafficking auxin regulates PIN abundance and activity at the cell surface, providing a mechanism for the feedback regulation of auxin transport. In animals, constitutive cycling is an entry point for multiple regulations, including signaling molecules. Through this mechanism, hormones such as insulin or vasopressin can control the relative rates of endocytosis and

6 198 Alejandra A. Covarrubias et al. exocytosis and thereby regulate the concentrations, and thus, the activity of surface-localized proteins, including ion and water channels, transporters and receptors [9]. In plants, only auxins had an effect on trafficking, including brefeldin A (BFA)-induced internalization. Even high concentrations of several phytohormones including abscisic acid, brassinosteroids, cytokinins, ethylene and gibberellins had no detectable effect on various trafficking processes. This indicates that auxin, as some animal hormones, modulate the activity of proteins by regulating their intracellular traffic. Vesicle-mediated polar auxin transport would closely resemble, at least in some aspects, neurotransmitter-bases cell-to-cell communication at neuronal synapses [10-13]. Similar to auxin, the neurotransmitter glutamate is a derivative of amino acid metabolism and is actively loaded into endocytic/recycling synaptic vesicles and secreted into the synaptic cleft trough regulated exocytosis [14]. Interestingly, both PIN1 and AUX1 carriers are members of the amino acid transporter superfamily, which includes families related to neurotransmitter transporters. Specifically, PIN1 is a member of the TC2.A69 family, which is distantly related to Na + symporters from animals (TC2.28), while AUX1 shows limited homology with VIAAT/VGAT neurotransmitters [15]. Moreover, the ANT1 amino acid transporter can also transport auxin and shows similarity to GABA neurotransmitter transporters. As it is the case with auxin carriers, glutamate transporters were also originally considered to be active at the PM, although physiological studies failed to prove this notion. Only later was it shown that glutamate transporters are vesicular transporters responsible for loading of cytoplasmic glutamate into synaptic vesicles. Similarly to PIN1 and AUX1, vesicular neurotransmitter transporters recycle via vesicular trafficking pathways [14-15]. The universal force behind the loading of neurotransmitter molecules into synaptic vesicles comes from the proton gradient generated by vacuolar (vesicular) ATPases, which is also true for auxin loading into PMderived vesicles. There are several further similarities between polar cell-to-cell transport of auxin in plants and synaptic transmission of signals in animal excitatory tissues. Polar auxin transport requires BIG, a large protein with similarity to calossin [12], which controls vesicle recycling during synaptic transmission at neuromuscular junctions [16]. Henceforth, higher plants show neuronal-like features in that the end-poles of elongating plant cells resemble chemical synapses [13]. Moreover, immunofluorescence localization of the actin cytoskeleton has revealed that the non-growing cross-walls involved in the intercellular transport of auxin are enriched in F-actin and plant-specific unconventional class VIII myosin. Because the cross-walls at the end-poles represent unique actin- and pectin-based adhesive domains, Baluska et al. [17] proposed the concept of plant developmental synapse, in which auxin and pectin-derived signaling molecules act as plant-specific transmitters for

7 Hormones in plants 199 cell-to-cell communication. In conclusion, besides its hormone-like properties, the vesicle-based cell-to-cell transport implicates neurotransmitter-like features of auxin too, making this small but extremely powerful molecule unique in the whole eukaryotic super-kingdom. Auxin signaling IAA biosynthesis, metabolism, and transport together ensure that appropriate auxin levels are in place to orchestrate plant development. We will now discuss how the signaling between auxin and downstream effectors might occur. Auxin rapidly and transiently induces accumulation of at least three families of transcripts; SMALL AUXIN-UP RNAs (SAURs), GH3-related transcripts, and AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) family members. Many genes with auxin-induced expression, including most SAUR, GH3, Aux/IAA genes, share a common sequence in their upstream regulatory regions, TGTCTC or variants. Regions including this sequence, known as the Auxin- Responsive Element, or AuxRE, confer auxin-induced gene expression in synthetic constructs. Identification of the AuxRE led to isolation of ARF1, the founding member of the AuxRE-binding protein family. ARF proteins can either activate or repress target gene transcription, depending on the nature of a central domain [reviewed in 18]. Mutations in several Arabidopsis ARF genes confer gene-specific developmental defects. Mutations in ETTIN/SRF3 (ETT) lead to floral abnormalities. Defects in MONOPTEROS/ARF5 (MP), a transcriptionally activating ARF, result in aberrant seedling morphology, often with a single cotyledon and a loss of roots. Mutations in a second activating ARF, NON- PHOTOTROPIC HYPOCOTYL4 (NPH4/TIR5/MSG/ARF7), confer deficient shoot phototropism, an auxin-mediated process. The diversity of arf mutant phenotypes makes it clear that the rules governing the interactions between ARFs and AuxRE will be complex; the fact that only a few arf mutants have been reported indicates that much of this complexity remains to be uncovered [reviewed in 18]. Gain-of-function Aux/IAA mutations generally reduce auxin sensitivity in root elongation assays and confer dramatic auxin-related developmental defects, including altered gravitropism and apical dominance in axr2/iaa7, axr3/iaa17 and axr5/iaa1; severe lateral root defects in iaa28 and slr/iaa14, photomorphogenetic defects in shy2/iaa3, hypocotyls tropism defects in msg2/iaa19, and embryonic patterning defects in bdl/iaa12. Remarkably, these dominant missense mutations all map to a small region of domain II and several have been shown to stabilize the encoded Aux/IAA proteins. In contrast to the dramatic defects conferred by stabilizing Aux/IAA proteins, the few reported loss-of-function Aux/IAA alleles confer only subtle phenotypes [reviewed in 22].

8 200 Alejandra A. Covarrubias et al. The Aux/IAA proteins, which inhibit auxin responses, are unstable even in the absence of a stimulus. Auxin application further destabilizes Aux/IAA proteins, which is presumed to free activating ARF proteins from repression and thus allow auxin-induced gene expression. Aux/IAA proteins are unstable because they are targets of ubiquitin-mediated degradation, catalyzed by an SCF-type ubiquitin protein ligase [19]. SCFs have a characteristic subunit structure, consisting of a SKP1 protein, a Cullin, an F-box protein, and RBX1. For Arabidopsis Aux/IAA, the relevant F-box protein is the leucine-rich repeat TIR1 protein. The interaction between Aux/IAA and SCF TIR1 is central to auxin biology. Mutations in Aux/IAAs that severely dampen down their interaction with SCF TIR1 also increase their stability and confer defects in auxin-induced gene expression, causing a wide range of auxin-related morphological phenotypes. A similar suite of effects result from mutations that disrupt SCF TIR1 function. Among these, mutations in TIR1 have relatively mild phenotypes, presumably because of redundancy with its close homologues [19]. Auxin influences the interaction by affecting SCF TIR1 rather than Aux/IAAs, and this interaction implies a direct binding of auxin with TIR1 (Fig. 2). Thereby, TIR1 has been proposed to be the auxin receptor, which mediates transcriptional responses to auxin [19-20]. Figure 2. Auxin response through the TIR1 auxin receptor pathway. Transcriptionally activating ARFs are bound to auxin responsive promoter elements but are counteracted by Aux/IAA transcriptional repressors. Auxin binds to TIR1. TIR1 mediates the degradation of the Aux/IAA protein by the proteasome (via the ubiquitin ligase SCFTIR). Increased Aux/IAA degradation in response to auxin frees the activating ARF proteins from repression, allowing auxin-responsive transcription. Among the auxin-induced transcripts are those encoding the Aux/IAA repressors themselves, creating a negative feedback regulatory system.

9 Hormones in plants 201 If TIR1 is an auxin receptor, then why is tir1 phenotype so weak? It appears that genetic redundancy is the answer. TIR1 is one of six closely related F-box proteins, and a quadruple tir1 afb1 aafb2 afb3 has less auxin binding capacity than tir1 alone [21]. Progressive inactivation of the four genes progressively lessens auxin responses; the quadruple mutant displays a range of phenotypes indicative of reduced auxin responses. Growth of the most severely affected individuals is arrested after germination with a single cotyledon and no root [27], phenocopying monopteros, a loss of function ARF mutant, and bodenlos, a gain-of-function Aux/IAA mutant. Thus, TIR1, AFB1, AFB2, and AFB3 appear to function as auxin receptors that together are necessary for many Arabidopsis auxin responses. Of course, TIR1 and the AFB proteins may not be the only auxin receptors. Other auxin binding proteins, such as ABP1, might mediate certain cellular responses to auxin. Some changes occur too quickly following auxin exposure to result from a transcriptional mechanism such as that manipulated by SCF TIR1. The discovery that TIR1 is the auxin receptor reveal us a new kind of receptor, a protein with F-box, which do not use kinases in its signaling cascade as other receptors do and allows an appealingly short signal transduction chain from stimulus to response. Auxin interaction with other hormones Auxin, of course, does not act alone but rather in the environment of other regulators of plant growth and development. One of the most-told stories in plant biology is the relationship between auxin and cytokinin, which can be employed in vitro to induce root and shoot development, respectively [22]. Auxin and cytokinin levels are inversely correlated in vivo and auxin treatment can rapidly inhibit cytokinin biosynthesis. Auxin and the gaseous hormone ethylene are also intimately linked. Exogenous auxin exposure stimulates ethylene production through induction of a gene encoding the rate-limiting enzyme in ethylene biosynthesis. Conversely, ethylene inhibits lateral and basipetal auxin transport. As with ethylene, auxin elicits increased gibberelic acid (GA) production, and basipetally transported auxin is necessary for the production of the active gibberellins GA1 and GA3 in barley. GAs act, at least in part, through promoting degradation of DELLA repressors disrupting auxin transport precludes GA-mediated DELLA protein degradation. Auxin response is also connected to brassinosteroids (BRs), which act in concert with auxin to promote root gravitropic curvature in maize. BR and auxin treatments induce accumulation of many of the same transcripts. Exposure to the hormone abscisic acid (ABA) decreases free IAA levels while increasing esterified IAA conjugates in muskmelon ovaries. Antagonistic to auxin, exogenous ABA inhibits lateral root formation [reviewed in 23].

10 202 Alejandra A. Covarrubias et al. Brassinosteroids Brassinosteroids (BRs), the polyhydroxylated steroid hormones of plants, regulate the growth and differentiation of plants throughout their life cycle. Over the past several years, genetic and biochemical approaches have yielded great progress in understanding BR signaling. In contrast to animal steroids signals, BRs are perceived by a PM-localized receptor kinase. This kinase is encoded by the BRI1 gene, which was initially identified as brassinolide (BL)- insensitive mutant [24]. bri1 mutants display a light-grown morphology in the dark, show extremely dwarfed growth in the light, and have numerous other phenotypes, all of which are also seen in strong BR biosynthetic mutants. BRI1 is part of a large, plant-specific family of Serine/Threonine leucine-rich repeat receptor-like kinase (S/T LRR RLKs), consisting of more than 20 members in Arabidopsis. By its overall structure, BRI1 is an archetypal receptor kinase (with 24 LRRs, an intracellular region with a S/T kinase and a short C-terminal extension), and several lines of evidence established BRI1 as the single most important BR binding activity in Arabidopsis [25]. Recently, the BRI1 ortholog in tomato was shown to act as the receptor for systemin as well as for BRs. However, systemin, a small peptide signal involved in plant defense, is present only in a subgroup of higher plants, not including Arabidopsis. Why BRI1 was co-opted for this dual role is not known [26]. The direct targets of BRI1 in vivo are not known, but several candidates exist. BAK1 and its homologs may be the main direct targets that initiate signaling events that ultimately inactive the downstream kinase BIN2. BIN2 encodes a S/T kinase, 70% similar in its catalytic domain to the mammalian GSK3, which have been involved in numerous signaling pathways and controlling metabolism, cell fate determination, and tissue patterning in various organisms. Several lines of evidence suggest that increased activity of BIN2 negatively regulates BR signal transduction. BRs are perceived by the BRI1 receptor kinase, which is located in the cell membrane and is thought to act in concert with BAK1, a second receptor kinase. The BR signal is transduced via nuclear-localized proteins BZR1 and BES1. BIN2, a negative regulator of the pathway, controls this process by phosphorylating BZR and BES1, which are then degraded by the proteasome. BRs, appear to control the pathway, at least in part, by negatively regulating BIN2 and consequently relieving the repression of BZR1 and BES activity (Fig. 3). BES and BZR1 are positive regulators, and the drastic deletion of the central region of BES1, gives rise to constitutive BR response [25]. Genomic effects of BRs Three independent laboratories have analyzed the genomic short-term effects of BRs [25]. Surprisingly, the findings from these groups showed little overlap in the genes identified, although similarities in the broad functional

11 Hormones in plants 203 categories represented by each group s gene list could be observed. One important result common to all three reports was the modest nature of the BR response. Whereas studies on other plant hormones, such as auxin, have reported transcript-induction in excess of 10-fold, few BR-regulated genes were induced by more than 2-fold. In fact, more than 80% of consistently detected BR-regulated genes show estimated expression changes of less than twofold. Determining whether such modest effects are biologically relevant will be a critical question for future studies of the BR response. Several alternative explanations have been proposed, including larger changes in a small subset of cells, highly responsive pathways, and modest expression changes being coupled with large changes in protein stability or activity. Which pathways are clearly influenced by BRs, as assayed by the genomic response? A large proportion of the genes identified by this analysis have no known function. However, a well-known function of BRs is the loosening of the cell wall and biogenesis of new cell wall material. One of the first genes identified, as BR induced was BRU1 in soybean, encoding a xyloglucan endotransglusylases/hydrolases (XTHs). Consistent with their role in cell growth, many cell wall components and the enzymes that produce them are BR regulated. Interestingly, a number of genes involved in the production and secretion of very-long chain fatty acids are also up-regulated following BR treatment. This may reveal an increased requirement for waxy cuticle to cover promptly elongating epidermal cells and may contribute to the biotic and abiotic stress protective effects of BR treatment. The cytoskeleton is also a target of BR regulation. In particular, two tubulin-encoding genes are upregulated by BRs. Some studies suggest that one aspect of the dwarfing phenotype observed in BR mutants results from a defect in microtubule organization and concomitant loss of cellulose microfibrils. BR treatment of the BR-deficient mutant induces correct orientation of cortical microtubules. As it was mentioned earlier, the connections with other hormones are plentiful, including component of both biosynthesis and signaling pathways. A large number of genes previously identified as auxin-responsive has been observed by many groups, which reveals the close association of the BR and auxin genomic responses. Several Aux/IAA transcriptional repressors are upregulated by BRs and three ARF transcription factors are down-regulated. Genes implicated in IAA homeostasis have also been found, and a number of genes involved in auxin transport are down-regulated. Together, these effects might serve to reinforce local peaks in auxin concentration, perhaps as part of a canalization process. Nonetheless, multiple studies using Arabidopsis mutants with perturbed BR or auxin signaling have demonstrated the interdependence of these two hormones in the control of hypocotyl elongation and gene expression [27-28] (Fig. 3). Furthermore, effects of BRs on ethylene biosynthesis enzymes, such as ACC synthase, were observed in mung bean. BRs also repress

12 204 Alejandra A. Covarrubias et al. Figure 3. Brassinosteroid and auxin signaling in Arabidopsis. BR signaling: BR perception by the BRI1-BAK1 receptor complex is ensued by dephosphorylation and nuclear localization of BZR1 and BES1, resulting in the regulation of target genes. BIN2 phosphorylates BZR1 and BES1, heading to their degradation by the proteasome. This process is negatively regulated by BRs. Auxin signaling: (see Fig.1). Auxin perception by TIR is followed by the degradation of the Aux/IAAs via the ubiquitin ligase SCF TIR to the proteasome. This relieves the repressive effects of Aux/IAAs on ARF action and transcriptional regulation can resume. BR and auxin crosstalk: the BR and auxin signaling pathways converge at the level of transcriptional regulation of target genes with common regulatory elements in the control of hypocotyl elongation. Adapted from [28]. transcriptional regulators induced by citokinins. On the other hand, there is a clear antagonistic relationship with the light response and the genomic responses of BRs. Three photoreceptors, phototropin 1 and phytochromes B and E, are down regulated by BRs. In conclusion, there is more to be learned regarding BR signaling, particularly about the determinants of BR homeostasis. Where and when BRs are synthesized and degraded, how they are transported out of the cell, and to what extent they are distributed in the plant. Furthermore, it remains to be determined the integration of BRs with other key signals, such as auxin and light, in order to gain insight into the complexity of plant development.

13 Hormones in plants 205 Cytokinins Cytokinins are adenine derivatives that have diverse effects on important physiological functions in plants. For instance, they induce cell division and de novo shoot formation, delay senescence, activate dormant lateral buds, and increase sink strength. There are two types of active cytokinins: zeatin and isopentenyladenine. Cytokinins are inactivated by glycosylation, and some glycosylated cytokinins are regarded as a storage form. Because cytokinins exist in apoplasm as well as in the cytoplasm, specific transmembrane transportes for cytokinins may exist. Cytokinin oxidase/dehydrogenase degrades cytokinins by cleaving the side chain [reviewed in 29]. Cytokinins receptors are histidine kinases It is now apparent that cytokinins are perceived by histidine kinases (HKs) and transduced by a two-component signaling system. Signal-induced phosphorylation of proteins is an often-used regulatory mechanism to transduce intracellular or extracellular signals. In animals, phosphorylation on a hydroxyl group of Ser, Thr, or Tyr residues is predominantly used. By contrast, in bacteria, phosphorylation in His and Asp is mainly used. This later mode has been referred to as the two-component system. Until 1993, it was thought that the two-component system was present only in prokaryotes; then two HKs, the ethylene receptor ETR1 of Arabidopsis [30] and the osmosensor Sln1 of Saccharomyces cerevisiae [31] were found. However, no homologous proteins for this system are known in animals, including Drosophila melanogaster, Caenorhabditis elegans and Homo sapiens, for which entire genomes have been sequenced. As the name indicates, prototypical two-component systems consist of two proteins, the HK and the response regulator (RR). Most HKs are transmembrane receptors with signal-sensing domain (input domain) positioned in the extracellular space and a signal-transducing domain (transmitter domain) positioned in the cytoplasm. RRs are characterized by the presence of a receiver domain, and an output (effect) domain that regulates downstream events. Although plant responses to cytokinins have been studied since the discovery of kinetin in 1956 [32], the first and long-sought-after cytokinin receptor (CRE1) was identified only about fours years ago. Cytokinin receptors were identified using forward and reverse genetics. In one screen, Arabidopsis calli resistant to cytokinin in tissue culture yield a mutant named cytokinin response 1-1 (cre1-1) [33]. The responsible gene CRE1 is identical to WOL and AHK4, and codes for a HK. To determine the molecular of function of CRE1, a mutant of S. cerevisiae was used, in which the only HK gene, Sln1, was disrupted. Disruption of Sln1 is lethal to yeast, owing to the lack of phosphotransfer. When introduce into a sln1 mutant, CRE1 rescued the lethality only in the presence of cytokinins. These results, coupled with the insensitivity to cytokinins of the Arabidopsis mutants of cre1, provided evidence the CRE1

14 206 Alejandra A. Covarrubias et al. is a cytokinin receptor. The extracellular domains at the N-terminal regions of the cytokinin receptors are the Cyclase/Histidine kinase-associated Sensing Extracellular (CHASE) domain, which is found in diverse receptors of prokaryotes, plants and Dictyostelium discoideum. The wooden leg (wol) mutant carries a mutation in the CHASE domain and is cytokinin resistant. The wol mutation disrupts the cytokinin-binding activity of CRE1/WOL/AHK4 and disrupts the ability of CRE1/WOL/AHK4 to confer cytokinin dependency on the sln1 mutant yeast, suggesting that the CHASE domain senses cytokinins. Roles of response regulators in cytokinin signaling The immediate downstream partners of the AHKs are a set of five HISTIDINE-CONTAINING PHOSPHOTRANSMITTERS (AHPs). Further downstream, Arabidopsis has a large number ARRs. It is assumed that these varied components form a complex phosphorelay network (AHK AHPt ARR) that is likely to be involved in signaling pathways. The common feature of RRs is that they contain the so-called receiver domains. Arabidopsis ARRs are classified into two major sub-groups on the basis of structural designs (type-a RRs, and type-b RRs). The proposed cytokinin signaling mechanism involves four principal steps: first, cytokinin receptor AHKs sense the signal and then phosphorylate AHPs; second, phospho-ahps move into the nucleus and donate a phosphoryl group to type-b ARRs; third, phosphorylated type-b ARRs serve as transcriptional activators, resulting in the rapid induction of type-a ARR genes; and finally, accumulated type-a ARRs somehow act as repressors that mediate a negative feedback loop in the circuitry (Fig. 4). Thus, cytokinins activate type-b ARRs, which are required for most or all cytokinin responses, and that type-b ARRs directly activate type-a ARR genes. This architecture is strikingly similar to that of auxin signaling (Fig. 2), although their components belong to completely different protein families, since type-b ARRs are transcriptional activators and their genes are not influenced by cytokinins, while type-a ARRs are transcriptional repressors and their genes are induced by cytokinins [29]. The role of cytokinins in whole plants How well do we know the role of cytokinins? Probably not very well. The role of cytokinins has been deduced mostly from the effects of cytokinin application, in other words, from the effects of extra cytokinins added to internal cytokinins. This shortcoming has recently begun to be addressed through utilizing genes for cytokinin oxidases to lower endogenous cytokin levels in tobacco [34]. In these transgenic lines, growth of aerial parts was severely retarded caused by a reduced rate of cell division: cell size increased while cell number decreased. By contrast, total root mass increased, which resulted from the increased size of the cell division zone. It was thus proposed

15 Hormones in plants 207 that cytokinins are positive regulators of cell division in the shoot apical meristem and negative regulators of cell division in the root apical meristem. Cytokinins are involved in the regulation of both G 1 -S and G 2 -M transitions. Their involvement in G1-S regulation is supported by the observation that cytokinins increase the G1 cyclin, cyclin D3, and that constitutive expression of cyclin D3 caused cytokinin-independent growth of Arabidopsis calli [35]. Another issue concerning the role of cytokinins is whether they are systemic (endocrine) mediators or local (paracrine) mediators. There are experimental results that support either conclusion. Therefore, the role and mechanisms of long-distance, of short-distance, and transmembrane transport of cytokinins are important issues yet to be solved. Figure 4. A model of cytokinin signaling in Arabidopsis. The three cytokinin receptors CRE1/WOL/AHK4/AHK2, and AHK3 bind to cytokinins and initiate phosphotransfer. The phosphoryl group, indicated by encircled letter P, is then transferred to the HPt domain proteins, AHPs. Phosphorylated AHPs are translocated to the nuclei and probably transfer the phosphoryl group to type-a ARRs and/or type-b ARRs. Unphosphorylated type-a ARRs act negatively on type-b mediated transcription, and phosphorylation of type-a ARRs may relieve their negative action. Cytokinins thus activate type-b ARRs, which are required for most or all cytokinin responses, and type-b ARRs directly activate type-a ARR genes. Adapted from [29]. PM, plasma membrane; NM, nuclear membrane.

16 208 Alejandra A. Covarrubias et al. Abscisic acid Abscisic acid (ABA) is a naturally occurring compound in plants. It is a sesquiterpenoid (15-carbon) (see Fig. 1), which is partially produced via the mevalonic pathway in chloroplasts and other plastids. In contrast to auxins, gibberellins, and cytokinins, which are represented by various active derivatives, ABA is a single compound. Several groups discovered ABA independently in the early 1960s. These groups believed that this compound was involved in the abscission of fruit and dormancy of woody plants, which led them to call it "abscisin II" or "dormin". The name abscisic acid was coined by a compromise between the two groups [36]. Up to date, the role of ABA in these processes is still not clear. However, it is well known that ABA regulates many important aspects of plant development, including the synthesis of seed storage proteins and lipids, the promotion of seed desiccation tolerance and dormancy, and the inhibition of the phase transitions from embryonic to germinative growth and from vegetative to reproductive growth [reviewed by 37, 38, 39]. In addition, ABA mediates some aspects of physiological responses to environmental stresses such as water deficit - induced stomatal closure, the induction of tolerance to dehydration and water deficit, salt, hypoxic, and cold stress, and wound or pathogen response [37, 38, 40]. All this information regarding ABA roles indicates that this phytohormone influences most aspects of plant growth and development to some extent, which is partly due to interactions with other phytohormones. That is the case of the antagonistic interactions between ABA and gibberellic acid (GA), cytokinins, or auxins, as well as the recently showed connections between signaling by ABA and ethylene, brassinosteroid, light, or sugars [reviewed by 38, 41, 42, 43, 44, 45, 46]. Actually, an attractive idea to explain the complex processes involved in plant growth and development is to think that they are the result of the crosstalk between different signaling pathways that share signaling nodes, which interact with a variety of components conforming a complex signaling web. Since ABA discovery in the early 1960s [36], different approaches by several research groups have led to the identification of regulatory factors that control ABA response; also, genetic screenings of large mutagenized populations or transgenic approaches, in which a target gene is overexpressed or disrupted, have conducted to the discovery of ABA functions and of the various elements implicated not only in the ABA signaling web but also in its synthesis pathway and the corresponding regulatory elements. In addition, biochemical studies have identified an assortment of gene promoter elements, kinases, kinase inhibitors, phosphatases, phospholipases, and transcription factors involved in ABA responses, and for some processes, the roles of secondary messengers and signaling intermediates in the regulation of cellular responses to ABA have been demonstrated [reviewed in 43].

17 Hormones in plants 209 ABA signaling The first step in a hormone response requires some kind of recognition event. Circumstantial and indirect evidence suggest that there are multiple ABA receptor types, which may be both intracellular and/or extracellular [37, 48, 49, 50, 51, 52]. Recently, Razem and colleagues, using a biochemical approach, have been able to isolate a barley protein that has ABA-binding activity, named ABAP1 [53]. They showed that an RNA-binding protein called FCA binds to ABA and is regulated by it, and that FCA is involved in the inhibition of flowering, a less-studied function of ABA. Also, they found that the barley ABAP1 is homologous to the Arabidopsis FCA protein in its carboxy-terminal half. Arabidopsis FCA protein also showed a high affinity for active ABA analogues [53]. The use of ABA analogs in germination and gene expression bioassays has suggested the existence of multiple ABA receptors with different structural requirements for activity in different response pathways [54]. In contrast to studies of GA, ethylene and cytokinin signaling, a genetic approach has failed to identify any putative ABA receptor(s), to date. Consequently, the greatest progress has been made using biochemical and cell biological approaches. However, these strategies only have led to the identification of ABA binding proteins [55] and carrier-mediated uptake of ABA, which have not been linked to the physiological effects of ABA [44]. Interestingly, an additional set of data suggests that ABA has direct effects on membrane fluidity and thermal behavior [56] raising the possibility that ABA activity does not require interaction with a receptor. These results lead to consider the fact that ABA is analogous to lipophilic vitamins such as β- tocopherol (vitamin E) or vitamin K, low-molecular-weight compounds that are required in animals for fertility and blood clotting, respectively [57]. In the case of vitamin E, it has been shown that it can modulate transcription, although, the molecular mechanism of action is not known [58]. Furthermore, attention should be paid to the fact that ABA in plants and retinoic acid in animals are synthesized from β-carotene by oxidative cleavage catalyzed by a carotenoid dioxygenase, an evolutionarily conserved enzyme [59, 60]. Considering this analogy, ABA could bind to an intracellular receptor capable of acting as a transcription factor. Different selection approaches have been followed to isolate mutants affected in the ABA response [61]. Most of them have been applied in Arabidopsis thaliana because its advantages as experimental model. For example, production of seeds exhibiting precocious or defective germination [62, 63], germination under NaCl containing medium [64], loss or gain of sensitivity to ABA at germination [63, 65], screening for suppressors of ABA- INSENSITIVE (ABI) lines [6], increased transpiration rates resulting in lower leaf temperatures under standard or water limited conditions [67, 68], altered

18 210 Alejandra A. Covarrubias et al. embryonic development [69], modified expression patterns of reporter genes [70], defects in root growth [71], screening for suppressors or enhancers of non-germinating lines deficient in GA or [72, 73], defects in seedling growth under particular growth conditions [74], etc. These experimental strategies not only strengthen the participation of ABA in processes such as germination, seed development, stress responses and transpiration, but also allowed the identification of different factors or elements implicated in the corresponding signaling pathways, such as a variety of transcription factors, RNA binding proteins, protein kinases and phosphatases, phosphoinositide metabolism enzymes, etc. [reviewed in 43, 75, 76, 77]. Cross-talk in ABA signaling The involvement of ABA in different processes as well as the link between multiple signaling pathways was clearly shown by the identification of ABA related loci in the course of detection of mutants by selection of sugar-resistant seedlings [78, 79, 80, 81, 82], or that showed defects in their responses to different hormones such as auxin, ethylene or brassinosteroids [45,83, 84, 85,]. Since ABA inhibits seed germination at physiological concentrations, it appeared that germination was a suitable assay for genetic analysis of the ABA pathway; however, because germination efficiency depends on many internal and external factors, the assumption that some of the ABA response genes identified by these means were not involved directly in the ABA signaling pathway is viable. To date, over 50 loci have been shown to affect ABA responsiveness in mutant screens, yet there is a low probability that all these genes are required for primary ABA signaling. In fact, some of these genes are known to be involved in other hormone responses such as ethylene and GA synthesis and signaling [6, 72, 73]. The experiences obtained in the various mutant screen carried out to date indicate that many of the identified ABA response genes function in many pathways and it has been difficult to interpret their role in ABA signaling. This has been the case of mutations in the Arabidopsis ERA1 gene, which originally was identified as a farnesyl transferase that worked as a negative regulator of ABA signaling [86]. Further phenotypic characterization of era1 mutants showed that many of the phenotypes showed by these mutants are not directly regulated by ABA, including alterations in floral morphology, inflorescence branching, and pollen development [87, 88]. However, these pleiotropic effects could be explained because the analysis of the information from Arabidopsis genome [89] revealed many potential targets for this enzyme, none of which can be assigned to the ABA signaling pathway. Additional interactions between ABA and ethylene signaling pathways have been detected in different development stages such post-germinative growth. Although these hormones act antagonistically at germination, both inhibit root growth. The fact that

19 Hormones in plants 211 chemical or genetic disruption of ethylene signaling results in reduced sensitivity of root growth to inhibition by ABA is consistent with an scenario where they act in the same parallel pathways controlling root growth [72, 73] (Fig. 5). Further interactions are evident by the ABA effects on germination and the antagonistic effects exerted by GAs, ethylene, and brassinosteroids (BRs). That is why, initially, ABA-deficient mutants were isolated as suppressors of nongermination caused by GA deficiency [90] and the GA response mutants sleepy (sly) was isolated as a suppressor of abi1-1 mutant [6]. Recently, it has been shown that the non-germination phenotype of the sly mutant can be rescued by BR treatment, whereas BR-deficient or BR-insensitive lines are hypersensitive to ABA inhibition of germination [91]. Figure 5. ABA Signaling in Arabidopsis. Some of the components that participate in the ABA signaling network are considered to illustrate the different kind of molecules involved to transmit to the cell the presence of ABA, which results in the cell response to this phytohormone. This figure also illustrates the different levels of regulation that are influenced in these signaling pathways, as well as the interconnections between ABA, ethylene and sugar signaling. Adapted from [223]. FCA corresponds to the molecule identified as one of the ABA receptors (see the text). The existence of multiple ABA receptors is an open possibility.

20 212 Alejandra A. Covarrubias et al. Many of the elements that constitute the ABA response as well as its signaling pathways have been identified as ABA regulated genes. Several of these genes are involved in response to water deficit stress conditions in vegetative tissues; some others expressed in maturing seeds are required for the synthesis of storage proteins or for the acquisition of desiccation tolerance [reviewed by 40, 92, 93]. The molecular and biochemical characterisation of these genes led to the identification of those cis-acting sequences required or sufficient for ABA mediated regulation of gene expression: the so-called ABA-response elements (ABREs), the coupling element-like sequences (CE3), the RY/Sph elements and the sequences recognize by the MYB- and MYCclass transcription factors. Some of the corresponding trans-acting factors have been identified. That is the case of proteins containing basic Leu zipper domains (bzips) and those with B3 domains that bind to the ABREs and RY elements, respectively. Also, MYB- and MYC-class transcription factors regulated by ABA have been characterized, and it has been shown that the gene expression response mediated by them is slower than that transmitted by the bzip-abre system [40]. The modular nature of promoters and the existence of different classes of regulators exhibit a scenario on ABAregulated gene expression, in which redundancy and combinatorial controls are evident, in agreement with a complex signaling web responsible of plant growth and development. ABA signaling in guard cells One of the best-characterised ABA mediated processes is that concerned to the opening and closing of stomata, which is the result of the modulation of the turgor and shape of guard cells [94, 95]. This has been possible because this cell type provides a relatively simple biological system that allows testing the roles of candidate secondary messengers and signaling intermediates in regulating single-cell responses. The primary role of stomata is to optimize leaf gas exchange (the uptake of CO 2 for photosynthesis and the loss of water vapor during transpiration) under changing environmental conditions, this is carried out through the response to stimuli such as light, atmospheric CO 2 concentration and atmospheric humidity [96, 97]. Hence, when the environment imposes a condition that requires fully turgid guard cells, the stomatal pore gates open, permitting gas exchange or transpiration. In contrast, under conditions of turgor loss (e.g. low humidity), the pore closes. These changes in guard cell turgor are, largely, but not exclusively driven by the influx and efflux of K +, with either Cl - or malate, across the plasma and tonoplast (vacuolar) membranes [98]. Now, we know that ABA brings about reductions in stomatal aperture by promoting not only stomatal closure but also by inhibiting stomatal opening. These are two separate turgor-driven processes and involve the coordinated activation and inhibition of inwardly and

21 Hormones in plants 213 outwardly directed cation and anion channels present in the plasma and tonoplast membranes. Some of the elements involved in those events elicited by ABA in order to obtain the cell behavior described above have been identified. These include protein kinases and phosphatases, an increase in cytosolic ph, slow anion channels, K + channels, activation of phospholipase D, and an increase in the concentration of free calcium ions in the guard cell + cytosol via activation of several Ca 2 channel types. Several pathways downstream of ABA, such as plasma membrane calcium permeable ion channel, phospholipase C/InsP3, cyclic ADP ribose, and possibly inositol hexakisphosphate (IP6), have as target the increment of Ca + 2 in the cytosol, which then controls some of the ion channels whose activity regulates guard cell turgor changes. However, not all the channels are regulated by increases in the cytosolic Ca + 2, so it is likely that the overall control of guard cell turgor requires additional calcium independent components or pathways [75, 77, 95, 98]. At present, it is not clear how these components and pathways might interact with each other. In the last years, new intermediates involved in ABA signaling have been discovered including reactive oxygen species [68] a heterotrimeric G-protein α-subunit [49] and sphingosine-1-phosphate, a mobilizing compound of cytosolic Ca + 2 [99]. Furthermore, ABH1, a nuclear mrna cap binding protein, which is a component of the cap binding complex, has been implicated in the control of the abundance of transcript(s), whose product(s) are involved in controlling guard cell sensitivity to ABA [100]. Apparently, ABH1 influences ABA signaling pathways upstream of those factors implicated in the increase of Ca + 2 in the cytosol [75, 95]. The role of ABA in seed maturation and dormancy The role of ABA in seed maturation is well recognized. The increase in ABA content in the seed correlates with the embryogenesis transition stage, in which the developing embryo stops cell division and starts growing by cell enlargement. Interestingly, this is consistent with the ability of ABA to induce the expression of a cyclin dependent kinase inhibitor (ICK1) [101] that would lead to cell cycle arrest at the G1/S transition phase. In many species two peaks of ABA accumulation have been detected, which in some cases correlate with low levels of germination of isolated embryos [102]. The ABA that accumulates in the seed is essential for the induction of dormancy; however, this quiescent stage of the seed is maintained regardless a substantial decrease in ABA by seed maturity, suggesting that the endogenous ABA is not the only signal for dormancy maintenance in mature seeds. Consistent with this, it has been suggested that GAs are required to overcome the germination constraints imposed by the ABA-mediated embryo dormancy. This hypothesis is supported by the hyperdormant phenotype of the comatose (cts) mutant, which shows a seed-specific defect in GA response [103], contrasting with the

22 214 Alejandra A. Covarrubias et al. pleiotropic effects on growth of mutants affected in ABA response loci. The diversity of results obtained along the study of the control of dormancy indicate that the timing of the decision of whether or not germinate differ within and among species and something similar happens with some of the regulatory factors involved in this process [104]. ABA biosynthesis As in many other cases, the concentration of ABA will depend on its rate of synthesis and catabolism and on its rate of import and export from the cell. ABA constitutes an example of a phytohormone, whose levels rise and fall dramatically in several kinds of tissues in response to developmental and environmental cues. Since the discovery of ABA, much effort has been devoted to understand how ABA levels are regulated by biosynthesis and metabolism. Genetic and biochemical studies have allowed the identification of those enzymes involved, particularly, in the ABA biosynthesis in higher plants. In fact, many aspects of this pathway are now understood in great detail. In higher plants ABA is synthesized from an indirect pathway through the cleavage of a C 40 carotenoid precursor, steps that occur in plastids until the formation of the intermediary compound xanthoxin, which is exported to the cytosol, where it is converted to ABA through two-step reaction via ABAaldehyde [105, 106]. The regulation of ABA biosynthesis is clearly of great importance in controlling ABA levels and, consequently, in modulating plant stress responses and development. Indirect evidence indicates that it is the first committed step in the ABA biosynthesis pathway, leading to the production of xanthoxin, the only one involved in the regulation of the overall rate of ABA production. Therefore, the characterisation of this control point will provide insights into the regulation of many ABA regulated processes. Aditionally, circumstantial evidence has suggested that the signal transduction pathways for stress-induced ABA biosynthesis may involve redox signals, Ca 2 + signaling, and protein phosphorylation and dephosphorylation events [76, 107]. ABA is synthesized in leaves and transported both to the root and throughout to the shoot system, primarily via the phloem; although its transport can also occur via xylem. In contrast to auxins, the ABA transport does not exhibit polarity. ABA can also be translocated through parenchyma cells. Roots can also synthesize ABA and transport the hormone into the shoot in response to water limitation [96, 108]. The integration of the information obtained to date regarding ABA function and signaling evokes an unfinished picture, where even though nearly 50 genes have been implicated in the regulation of ABA responses in Arabidopsis alone, by molecular and genetic studies still there are gaps to be filled. Many interactions with different pathways that mediate responses to other signals have been described but it is unclear whether these interactions

23 Hormones in plants 215 are direct or indirect, the identity of the ABA receptor or receptors remains unknown, as well as the substrates of various kinases and phosphatases and the identities of additional signaling elements linking the interacting complex web between hormones in plants. Gibberellins Gibberellins (GAs) form a large family of tetracyclic diterpenoid compounds with the ent-gebberellane ring skeleton that were discovered by Kurosawa in Japan in the course of his studies on fungal diseases of rice (Fig. 1). In particular he was investigating the mechanism by which the fungal pathogen Gibberella fujikuroi led to morphological changes in rice plants infected with this pathogen. Kurosawa showed that the abnormal growth promoted by the fungi was due to a compound produced by G. fujikuro, to which he called gibberellin [109]. The most widely available gibberellin is GA 3, or gibberellic acid, although the most important in plants is GA 1, which is the one responsible for stem elongation. Most of the different gibberellins are precursors or metabolites of GA 1. Now it is known that GAs are present in many other fungi species, as well as in some ferns, and in many gymno- and angiosperms [110]. GAs biosynthesis GAs are synthesized by the condensation of four isoprenoids subunits via the mevalonic acid pathway, common to many biological molecules, including carotenoids and steroids, from which some other phytohormones such as ABA and brassinosteroids are produced. This complex pathway involves plastids, endoplasmic reticulum and the cytosol. Approximately, 126 GAs are known and these are numbered GA 1 through GA 126. Of the 126 known GAs, 94 have been identified only in higher plants, 16 are present in Gibberella, and 16 are present in both organisms. Like in the case of Gibberella, individual angiosperms contain many different GAs; however, not all have high biological activity. Many of them are precursors or deactivation products of the active GAs. It is possible to determine which GA(s) in a plant is (are) the active ones by using a single gene dwarf mutant and chemical growth retardants, which inhibit specific metabolic reactions. All growing and differentiated tissues are potential sites of GA biosynthesis; however, where definitive evidence exists is in the case of developing fruits and seeds, as well as in elongating internodes and petioles, in expanding leaves and in stem apical regions of several plants [111, 112]. Function of GAs The ability of GAs to stimulate stem elongation seems to be a common property of these hormones, a functional characteristic that could be assigned

24 216 Alejandra A. Covarrubias et al. to auxin; however, it should be consider that although the final physiological result can be the same, they show different tissue specificities. For example, auxins regulate the elongation of coleoptile tissues; whereas, GAs are not present in these organs and their application to decapitated coleoptiles does not stimulate their growth, illustrating their distinctive specificities. The central role of GAs in the regulation of internode elongation has been shown through the characterization of dwarf mutants, which are affected in the internode elongation process. Application of GAs to these dwarfs allowed them to grow to the same height as the wild type plants. The determination of the GAs content in the dwarf and wild type plants showed that the tall plants have more GA 1 than the dwarfs. Furthermore, the application of GA 1 to dwarf plants resulted in tall plants. Now is known that GA is synthesized in the shoot apex and transported into the elongating internodes, where it regulates their growth [113, 114]. Studies of the effects of applied GAs in various plant tissues and organs suggest that these hormones regulate numerous additional aspects of plant development. More recently, GAs have been implicated in responses such as the induction of germination in seeds requiring light or cold to germinate; the breaking of dormancy of apical buds; the mobilization of stored reserves in seeds during germination; flowering and flower development; the control of fruit setting and fruit growth; the leaf expansion process; trichome development; and the reversion of juvenility in plants [110, 115, 116, 117]. GA signaling Recently, significant advance has been obtained in the identification of upstream GAs signaling components and trans and cis-acting factors that regulate downstream Ga-responsive genes in higher plants. The placement, order, and orientation of several sequences appear to be highly conserved in different cereal species. One particular sequence (TAACAAA) can act alone to induce responsiveness to GA and has been called the gibberellin response element (GARE) [118, 119]. In addition, mutagenesis of another specific sequence, TATCCAC, results in a loss in GA 3 -induced expression. The functional characterization of these sequences indicates that the TAACAAA and the TATCCAC "boxes" act cooperatively. The participation of a third sequence (C/TCTTTTC/T), referred to as the pyrimidine box, appears to be required for full gibberellin responsiveness. Additional components that seem to play a role as part of the GAs response elements are CAACTC box, and the Box1/O2S-like element. Together these sequences have been referred to as the gibberellin response complex (GARC) [113, 119, 120, 121, 122]. The identification and characterization of the GAREs has been important in the discovery of the cognate transcription factors that act as regulators of gene expression. A Myb-like factor represents one of the transcription factors

25 Hormones in plants 217 that bind to the GA response complex of various GA responsive genes such as α-amylase and hydrolase genes [122, 123, 124]. Now it is known that gibberellin acts by promoting the synthesis of the transcriptional activation factor GA-Myb, and possibly other transcription factors as well [125, 126]. There is also growing evidence for post-transcriptional regulation of GA-Myb expression and function in aleurone cells through a number of different mechanisms. The observation that the increases in GA-Myb protein are much higher than those detected for GA-Myb transcript in GA-treated aleurone layers indicates that GA-Myb translation and/or stability may be a major target for GA signaling [126, 127]. An additional transcription factor that binds to the region containing the GARE in a barley α-amy1 promoter has been denominated HRT, which corresponds to a nuclear-localized zinc-finger protein expressed in aleurone cells. This protein binds specifically to a 21-bp promoter fragment containing a GARE, and functions as repressor of GAinduced expression of α-amy1 and α-amy2 genes [128]. The binding of the so-called DNA binding with one finger (DOF) proteins to the pyrimidine boxes has been detected for some promoters. In some cases, these transcription factors appear to act as repressors and in others they function as activators [124, 129]. GAs seem to derepress their signaling pathway by inducing the degradation of GAs signaling repressors, designated DELLA domain proteins because they share a short amino-acid sequence. These proteins repress a variety of downstream targets, which include gene transcription factors that promote GAs related processes [130, 131, 132, 133]. One of these DELLA genes, Rht, turned out to be implicated in the semi-dwarf phenotype of the wheat varieties that led to the so-called green revolution [134, 135, 136, 137, 138]. Recent evidence indicates that the DELLA proteins are targeted for degradation by an E3 ubiquitin ligase SCF complex through the ubiquitin-26s proteasome pathway. Genetic analysis in both Arabidopsis and rice identified part of an enzyme, the F-box subunit of an SCF E3 ubiquitin ligase, as a gear in this degradation mechanism [124, 132]. It was not until very recently that the missing piece in this scheme, the GAs receptor, was identified. Although the DELLA protein degradation pathway needs to be activated by GAs to cause protein destruction, none of the components of this pathway bind GA, so they cannot be considered as receptors. However, the characterization of a GA insensitive mutant, unable to respond to GAs, showed that the product of the affected gene in this mutant, the GID1 protein, functions as a positive regulator of GA signaling, and that it acts at or upstream of a rice DELLA gene, SLR1, whose degradation induced by GAs depends on a functional GID1 protein. Importantly, GID1 protein can specifically bind gibberellin, which together with the above mentioned results strongly suggest that GID1 is the GA receptor [139, 140] (Fig. 6). It is interesting

26 218 Alejandra A. Covarrubias et al. Figure 6. Gibberellin signaling. In the presence of GA, this binds the GID1 protein (GA receptor), which enables GID1 to interact with the protein turnover complex (SCF). The formation of the complex GA-GID1-SCF allows SCF to degrade the DELLA repressor, which results in the release of the transcription factors that will promote the expression of the GA responsive genes to assemble the GA response. that this mechanism of GA perception and action result to be similar to that of auxin, which acts by stimulating the interaction between an F-box protein and the rest of the SCF complex in order to target proteins for destruction. So these examples seem to constitute a functional pattern, in which plants use these small organic molecules or phytohormones to modify protein protein interactions linked to the modulation of plant development. Ethylene Ethylene (C 2 H 4 ) was the first chemically identified endogenous regulator of plant growth and development. Ethylene is a very simple regulatory molecule that exists as a gas (Fig. 1). The ability of certain gases to stimulate ripening in fruits has been known for many years. Ancient Chinese knew that their picked fruits would ripen more quickly in a room with burning incense. Also, a different role of ethylene has represented a historical practice for growers of pineapple and mango in Puerto Rico and Philippines, respectively, who used to build bonfires near their crops to induce and synchronize flowering. Neljubov, a Russian physiologist, was the first to identify ethylene

27 Hormones in plants 219 as a regulator of plant development by demonstrating that it was the active component of illuminating gas, which had previously been shown to have dramatic effects on plants in the late nineteenth century [96, 141]. He showed that ethylene causes the so-called triple response on pea seedlings, which implicates inhibition of stem elongation, increase in stem thickening, and a horizontal growth habit. In Arabidopsis, the triple response consists of an inhibition of root and hypocotyls elongation, radial swelling of the hypocotyl, and an exaggeration of the curvature of the apical hook. This response of Arabidopsis seedlings has provided a simple screen for the isolation of ethylene response mutants and has been the key in unraveling the molecular basis of ethylene signaling. The mutations isolated by this strategy affect virtually all ethylene responses in both seedlings and adult plants, suggesting that they affect central components in ethylene signaling. An additional class of mutants affect ethylene responses in only a subset of tissues, such as the root (eir1) or the apical hook (hls1) and may act late in the ethylene response pathway [30, 142, 143, 144, 145, 146, 147, 148]. Functions of ethylene The detection of ethylene by gas chromatography has established that, in general, all higher plants produce ethylene. Its production has not been detected in algae but it does occur in few bacterial species [141]. In seedlings, the shoot apex is an important site of production, which might result from the high amount of auxins in this region, because it is known that auxins stimulate ethylene formation, whereas in roots the ethylene concentration detected is relatively low. Apparently, nodes of dicot seedling stems produce much more ethylene than the amount produce by internodes, and in leaves the amounts of ethylene rise slowly until, the leaves become senescent and abscise. Ethylene is also synthesized in flowers, where it has been involved in their senescence and abscission. In many fruits little ethylene is produced until the onset of ripening, when the content of this hormone in the intercellular air spaces rises dramatically. This significant increase in ethylene stimulates ripening of many fruits. The treatment with ethylene of many different fruits triggers their ripening, whereas, increasing concentrations of carbon dioxide gas, a natural antagonist of ethylene activity, delay this maturation process. This observation is now applied in modern fruit storage methods to delay or accelerate ripening and in this way modulate the storage life of fruits. Ethylene has also been involved in the modulation responses of plants to a wide range of biotic and abiotic stresses, such as wounding and invasion by pathogenic organisms, and flooding and drought [141, 144, 148, 149]. Being a gas, ethylene moves by diffusion from its site of synthesis to its place of action. Since one of ethylene biosynthesis intermediates, 1- aminocyclopropane-1-carboxylic acid (ACC), can be transported, it has been

28 220 Alejandra A. Covarrubias et al. proposed that it may mediate the effects elicited by ethylene at a distance from the causal stimulus. Ethylene biosynthesis The biosynthetic pathway of ethylene has been extensively studied in higher plants and it has been shown that methionine is the important ethylene source because, ultimately, it is derived from the third and fourth carbon of this amino acid. Ethylene synthesis proceeds from the S-adenosyl-L-methionine (SAM) cycle. The pathway from SAM to ethylene is catalysed exclusively by the enzymes ACC synthase and ACC oxidase. Because the ACC oxidase activity is constitutive, it is considered that the rate-limiting step for ethylene production is that catalysed by the ACC synthase; however, small changes in ACC oxidase may provide fine tuning of ethylene production [149, 150, 151, 152, 153, 154]. Ethylene signaling In recent years, studies in Arabidopsis revealed that ethylene signal transduction provides an interesting example of how the cellular systems that process information have evolved in plants. This is because a family of receptors, composed of structural elements that are characteristic of signaling proteins in bacterial and fungi, perceives the ethylene signal. Even though they are able to transmit the appropriate signal by interacting with proteins that are eukaryotic in origin. This family of receptors is composed of five members, ETR1, ETR2, ERS1, ERS2, EIN4, that are bound to membranes [155, 156]. Loss-of function mutations in any single ethylene receptor have little or no effect upon seedling growth, consistent with functional overlap within the receptor family [30, 157, 158]; however, the lack of multiple receptors lead to a constitutive ethylene response, indicating the receptors are negative regulators of ethylene signaling [158]. Interestingly, whereas most receptors are localized to the plasma membrane, analysis of the ethylene receptor ETR1 supports localization to the endoplasmic reticulum (ER) [159]. Such a location is compatible with the ready diffusion of ethylene in both aqueous and lipid environments [141]. The next downstream component in the signaling pathway is CTR1, a Raf-like ser/thr kinase with similarity to a mitogen-activated protein kinase - kinase kinase (MAPKKK). These CTR1 characteristics suggested the involvement of a MAP-kinase-like signaling cascade in the regulation of ethylene signaling [160, 161]. The absence of CTR1 results in a constitutive ethylene-response, indicating that CTR1 is a negative regulator of ethylene signaling. Next in this signaling cascade is EIN2, which has similarity to members of the Nramp metal-ion transporter family [162]. The fact that the loss of EIN2 results in complete ethylene insensitivity for all ethylene responses indicates

29 Hormones in plants 221 that EIN2 plays a major role in the ethylene response as a positive regulator. Based on the similarity to Nramp, it has been proposed that EIN2 may regulate ethylene responses in part by altering ion concentrations. Although calcium has been implicated in ethylene responses [163, 164], to date there is no evidence that EIN2 could be involved neither in calcium transport nor in the transport of any other ion. In addition, although EIN2 is predicted to be membrane localized, the specific membrane system has not yet been determined. Thus the actual function of EIN2 is still a mystery. Functioning downstream of EIN2 is a small family of transcription factors that includes EIN3 and various EIN3-like (EIL) proteins [165]. Loss of function mutations in EIN3 causes partial ethylene insensitivity. This insensitivity can be rescued by expression of EIN3, EIL1 or EIL2 [165]. This EIN3/EIL family is involved in a regulatory cascade and stimulate the transcription of other transcription factors such as those in the ERF family (ethylene response factor) [162, 166], which is also known as the EREBP family for ethylene response element binding proteins [167]. These transcription factors have been shown to act as activators or repressors of downstream ethyleneresponsive genes [168]. Consistent with this, an ein3/eil1 double mutant eliminates virtually all the transcriptional response to ethylene, indicating the key role this family of transcription factors in the ethylene plant response. An important feature of the ethylene signaling pathway is that it contains both positive and negative regulators, some proteins thereby serving to induce the responses while others suppress them. The basic working model establishes that in the absence of ethylene, the ethylene receptors activate the kinase activity of CTR1, which suppresses the downstream response; hence EIN2 and the EIN3/EIL transcription factors remain inactive. In the presence of ethylene, this binds to its receptors, which no longer activate CTR1with the consequent activation of the pathway. This allows the activation of EIN2, the induction of the transcriptional cascade, and the establishment of the ethylene response. Although some differences have been observed, there is evidence that indicates that the basic elements and mechanism of the ethylene signal transduction pathway are conserved in dicots and monocots important in agriculture, such as tomato, rice, maize and wheat [169, 170, 171, 172, 173, 174]. Ethylene signaling is highly regulated at the post-transcriptional level via ubiquitin/26s proteasome-mediated degradation. Degradation is initiated by the covalent attachment of ubiquitin (Ub) moieties to the targeted protein, as regulated by the actions of Ub-conjugating and Ub-ligase enzymes. The 26S proteasome then recognizes the polyubiquitinated protein and degrades it. The clearest role for post-transcriptional regulation in the pathway for ethylene signal transduction is in regulation of the key transcription factor EIN3. In the absence of ethylene, EIN3 is continuously degraded through the proteasome mediated pathway; which preclude the activation of its transcriptional targets.

30 222 Alejandra A. Covarrubias et al. In the presence of ethylene, the EIN3 degradation is suppressed, which allows the increase in EIN3 protein levels and thus the induction of the ethylene response [175, 176, 177] (Fig. 7). Although the precise mechanisms have not yet been established, the results obtained to date make clear that varying the protein levels of EIN3 constitutes an important means to control flux through the signaling pathway. Hence, it can be considered that the EIN3 protein is in essence a bottleneck in the ethylene pathway such that slight changes in its level can have significant effects in the response. Figure 7. Model for ethylene signaling. Data indicate that the perception of ethylene occurs through receptors localized in the endoplasmic reticulum (ER) membranes. Ethylene receptors are grouped in a family of five members (ETR1, ETR2, ERS1, ERS2, EIN4), which have similarity to two-component regulators from bacteria. These receptors are negative regulators; in the presence of ethylene, this inactivates the receptors and consequently CTR1 is also inactivated. As a result, EIN2 is activated and consequently the pathway in which participate EIN3/EIL and ERF transcription factors is triggered. The end result of these actions is the cell ethylene response. In contrast, in the absence of ethylene in the air, the ethylene receptors maintain CTR1 in an active state, which then represses all downstream responses. In the absence of ethylene, the EIN3 protein levels decrease due to degradation by the ubiquitin-proteasome pathway. The possibility of a CTR1 independent pathway, in which AHP and ARR proteins participate, is considered in this illustration.

31 Hormones in plants 223 Cross-talk in ethylene signaling A number of recent studies have implicated new components in the regulation of ethylene signal transduction pathway, although their role has not been accurately established, it is possible that these components are not part of the major ethylene transduction pathway and may represent elements of interacting pathways. As it has been mentioned along this manuscript, the interactions among hormones and other physical-chemical factors is a common characteristic in plant hormone biology. An example of how multiple signals can be co-ordinated to generate effects on plant growth is apparent in the formation of the apical hook in Arabidopsis seedlings, which is proposed to occur through integration of ethylene, auxin and light signaling [178]. An additional instance is the crosstalk with sugar sensing signal transduction pathways, as evidenced by the isolation of sugar insensitive mutants that resulted to be affected in components of the ethylene signaling pathway, such as CTR1 [179]. Now it is known that cross-talk between ethylene and sugars takes place both at transcriptional and post-transcriptional levels. DNA microarray analysis demonstrated that several ethylene biosynthetic and signal transduction genes are repressed by glucose [180]. All together these data establish a clear role for ethylene-in the plant responses to sugars. The use of microarrays has allowed the global analysis of gene expression changes in response to ethylene and it has been critical to unravelling the relationship between transcriptional regulation and ethylene responses. For instance, cluster analysis of microarray data indicates that ethylene affects transcription of genes involved in many biological processes, from metabolism to signal transduction, and including, for example, protein degradation mediated by the ubiquitin-proteasome system as well as by proteases, the transport of water, peptides and ions, cell wall and lipid metabolism, etc. Although our understanding of the physiological processes regulated by ethylene are only beginning to be understood, an integrative view, shows that the data accumulated to date indicate that the ethylene response is the result of co-ordinated increases and decreases in expression of many genes and proteins, with the relative levels of these genes and proteins further regulated by inputs from other signaling pathways (162, 181, 182, 183]. The global analysis of gene expression that has been carried out to date has analysed changes in gene expression at the whole-plant level, an approach that has allowed to get an average view out the changes across multiple tissues and cell types, thereby yielding a picture of what changes in gene expression are common throughout the plant. However, it is palpable, that given the complexity of the role of ethylene in the different processes in which it participates, that a complete understanding of the function of ethylene, as well as of other plant hormones, in plant growth and development will also require

32 224 Alejandra A. Covarrubias et al. more focused approaches capable of resolving changes in gene expression occurring within individual tissues and cell types. Peptide hormone signaling in plants Peptide hormone signaling in plants is a fast developing area of research. Up to now, about 20 signal peptides have been identified that regulate cell division, plant development, reproduction, root-nodule organogenesis and plant defense against herbivores and pathogens [184]. Polypeptide signaling may have had its origins in ancestral organisms that predated plants and animals, providing a foundation for the evolution of polypeptide signaling in all modern eucaryotes. In animals, polypeptide hormones classically fall into two major categories: endocrine hormones [185] and membrane-anchored growth factors [186]. Biochemical and physiological characteristics between plant and animal signaling peptides are similar: in both cases, they are derived from larger precursor proteins, act at nanomolar concentrations, and its perception is mediated by receptors. Multicellular organisms must integrate developmental and physiological processes that occur throughout their body by their vascular system. In plants, this inter-organ regulation occurs through both the xylem and the phloem (Fig. 8). The phloem comprises a cell-to-cell communication system that generates a system of sieve tubes. In Angiosperms (flowering plants) this conduit is built from file companion cell-sieve element (CC-SE) complexes [187]. Sieve elements are enucleated cells and depend on the exchange of metabolites and macromolecules through the plasmodesmata (Fig. 9) that connect them to their companion cells. Plasmodesmata are special intercellular organelles that create cytoplasmic and endomembrane continuity, allowing small molecules such as ions, metabolites and plant hormones to diffuse from neighboring cells [188, 189]. These structures facilitate the coordination of biochemical and physiological processes not only locally but systemically through the whole plant. A fundamental difference between gap junctions, intercellular communication channels found in animals, and plasmodesmata is that these later have the additional capacity to selectively mediate cell-to-cell trafficking of proteins and protein/rna complexes [187, 188, 190, 191]. In plants, development at the tissue, or organ, level is integrated by a combination of phytohormones and non-cell autonomous proteins (NCAPs). Substantial evidence indicates that plasmodesmata establish a unique regulation system by controlling the transport of a selective class of proteins such as peptide hormones and transcription factors, through several layers of cells, and the plant vascular system. Many of these small peptides and proteins appear to use the macromolecular trafficking capacity of plasmodesmata to act as NCAPs translocators, thereby regulating the biological events occurring outside the cells in which they are produced [189, 192, 193].

33 Hormones in plants 225 Figure 8. Developmental and physiological processes that occur throughout the plant body are integrated through their vascular system. In plants, inter-organ regulation occurs through both the xylem and the phloem. Specific signaling molecules induced in response to abiotic conditions (light, circadian rhythms, mineral nutrition, temperature, and water availability) and biotic challenges (viruses, bacteria, fungi, nematodes and insects) enter the phloem for long distance trafficking and are delivered to distant organs (SAM and RM). AM, auxiliary meristem; SAM, shoot apical meristem; RM, root meristem. A major difference between plant and animal development is that plant cells can not move, so that plant development depends on an exquisite balance between cell expansion and cell division. If this balance is impair then plant development will be impair or not take place at all. Peptide hormone regulatory signals include the systemin family of defense hormones found in the Solanaceae (eg, tobacco, tomato) family [184]; CLV3, a regulator of shoot apical meristem development [194]; PSK, a small polypeptide involved in mitogenesis of plant cells grown in culture [195]; ENOD40 is a highly structured plant small ORF-mRNAs involved in root nodule organogenesis [196]; RALF, a ubiquitous polypeptide in plants affecting growth and development [197]; and a insulin-like growth factor that apparently regulates protein synthesis in maize [198].

34 226 Alejandra A. Covarrubias et al. Figure 9. Plasmodesmata structure. Plasmodesmata are special intercellular organelles that create cytoplasmic and endomembrane continuity, allowing small molecules such as ions, metabolites and plant hormones to diffuse from neighboring cells. Small peptides and proteins appear to use the macromolecular trafficking capacity of plasmodesmata to act as non-cell autonomous proteins NCAPs translocators, thereby regulating the biological events occurring outside the cells in which they are produced. ER, endoplasmic reticulum. Until 1991, plants were not known to utilize polypeptides as regulatory molecules; instead they were thought to use only small organic phytohormones, including auxins, cytokinins, gibberellins, ethylene, abscisic acid [199], and more recently, brassinolides [200]. Systemin was isolated from tomato leaves in 1991 as the mobile signal for defense gene activation in the systemic wound response. The systemin polypeptide was discovered during a search for the systemic wound signal that regulates the expression of defensive genes in tomato leaves in response to insect attacks or other severe mechanical wounding [201]. Tomato and potato plants respond to insect attacks by releasing a signal or signals from the wound site that cause the synthesis and accumulation of proteinase inhibitors in both wounded leaves and distal, unwounded leaves [202]. In tomato, the polypeptide is composed of 18 amino acids with the sequence AVQSKPPSKRDPPKMQTD, and because of its association with systemic signaling, it is called systemin. This peptide hormone induces the synthesis of proteinase inhibitors in leaves of young excised tomato plants when supplied at low nanomolar levels through their cut petioles [201]. The potency is in the same range as that found for the activity of various polypeptide hormones in animals. A synthetic 14 C-labeled systemin was mobile when applied to wound sites, and its movement in the phloem correlates with the movement of the systemic signal that is produced in response to wounding, that is, 3 cm/hr [184]. The mechanism for the movement of systemin in the phloem is not understood, but for systemin to have long-range signaling effects, an amplification of the signal

35 Hormones in plants 227 most likely occurs. The presence of a 200 amino acid systemin precursor called pro-systemin in phloem parenchyma cells suggests that the interplay between the phloem and its surrounding cells may contribute to the signaling mechanism. Pro-systemin contains one copy of the systemin sequence near the C-terminus [203] indicating that systemin, like many animal and yeast polypeptide hormones, is proteolytically processed from a larger protein. Tomato pro-systemin does not exhibit a signal sequence at its N-terminus, nor is it glycosylated [204]. The lack of a signal sequence and the absence of any post-translational modification suggest that pro-systemin is not secreted through the secretory pathway. The alcalinization of tomato suspension culture cells medium, when systemin is added has been exploited as a bio-assay to search for a peptide with systemin-like properties in extracts of herbivore attacked tobacco leaves using a tobacco suspension culture. By using this assay, two peptides from tobacco were identified that matched all of the criteria for being tobacco systemin analogues and were, therefore, called TobsysI and Tob-sysII [205]. Their sequence is not homologous to the tomato systemin, but their function is homologous. From these facts, it can be inferred that structurally diverse peptides in different plant species, although belonging to the same family, can have similar functions, an observation not yet made in animals and yeast. In this case, the two tobacco systemin peptides are integral parts of a single precursor pre-pro-protein that has an N-terminal leader sequence for translocation via the secretory pathway. Recently, the tomato systemin receptor SR160 from Lycopersicon peruvianum has been characterized and its structure indicates that it belongs to a member of the plant LRR receptor kinase super family [206]. The development of higher plants is largely post-embryonic, and plant embryos contain few of the organs found in the adult plant. Plant embryos are simple in structure (Fig. 10). The apical end of the embryo contains a shoot apical meristem (SAM), which produces the above-ground organs and tissue of the plant, namely the stems, the leaves and the flowers. The basal end of the embryo contains a root meristem (RM), which gives rise to the root system. Plant organs are formed from SMs and RMs during post-embryonic development. CLAVATA 3 (CLV3) is an extracellular signaling peptide of 79 amino acids in length that is involved in the cell fate of the shoot apical meristem (SAM) of Arabidopsis. Organ position and identity of the aboveground body plan is initiated and established in the SAM [207] by an exquisite balance between the division of the stem cells found in the central zone (CZ), and the division of the differentiating cells found in the periphery of the SAM, as the plant grows. The stem cells divide slowly, while the peripheral cells do it more actively. In addition, CLAVATA 3 and CLAVATA 1, the receptor kinase of CLAVATA3, which is also expressed in some cells of the SAM of Arabidopsis [194] are both essential to maintaining the balance of cells in each

36 228 Alejandra A. Covarrubias et al. Figure 10. Structure of the Arabidopsis embryo. The apical end of the embryo contains a shoot apical meristem (SAM), which produces the above-ground organs and tissue of the plant, namely stems, leaves and flowers. The basal end of the embryo contains a root meristem (RM), which gives rise to the root system. Plant organs are formed from SMs and RMs during post-embryonic development. SAM, shoot apical meristem; RM, root meristem. zone. Mutations in both or individual genes (CLV1 and CLV3) produce identical phenotypes (larger number of cells in SAM and floral meristems, than those found in wild type meristems), suggesting that both genes are components of the same signaling pathway [207]. The CLV signaling pathway has provided an exceptional insight into how shoot apical meristems develop. A polypeptide signal and its receptor complex, which constitutes an archetypal signaling model for growth and differentiation in plants, orchestrate such developmental processes. Three novel hydroxyproline-rich glycopeptides signals (LeHypSys I, II, and III) were recently isolated from tomato (Lycopersicon esculentum) leaves that are powerful activators of the same intracellular defense-related genes that are activated by wounding and systemin, mediated by the octadecanoid pathway [208]. The three peptides are composed of 18, 20, and 15 amino acids, respectively, and are derived from a single poly-protein precursor [184],