Conservation and Innovation in Plant Signaling Pathways

Cell, Vol. 103, 201–209, October 13, 2000, Copyright 2000 by Cell Press

Conservation and Innovation in Plant Signaling Pathways Donald R. McCarty*‡ and Joanne Chory† * Plant Molecular and Cellular Biology Program Horticultural Sciences Department University of Florida Gainesville, Florida 32611 † The Howard Hughes Medical Institute The Salk Institute for Biological Studies La Jolla, California 92037

Introduction As nature’s second grand experiment in complex development, vascular plants have strategic importance to our understanding of the mechanisms and evolution of signal transduction pathways. At least a billion years have passed since plants, animals, and fungi diverged at the base of the eukaryotic crown (Doolittle et al., 1996). While it is unclear whether multicellularity evolved independently in plant and animal lineages, these phyla have at the very least undergone major independent changes in structure and organization. The emergence of complete genome sequences from strategic eukaryotic models and recent advances in the molecular analysis of plant signaling pathways allow comparative analysis of the signal transduction pathways in plants and animals. The results offer new insight into the nature and complexity of signaling pathways that were present in a common ancestor. Moreover, we can begin to discern the sources of novelty and innovation that differentiate the plant and animal lineages particularly with respect to development. In this review, we pay particular attention to origins of novelty in the evolution of signaling pathways in plants. The three important signal transduction pathways depicted in Figure 1 illustrate an intermingling of conserved and novel mechanisms that is typical of plant signal transduction pathways. Auxin (indole acetic acid) signaling, a central pathway of plant development, is mediated by a highly conserved ubiquitin ligase complex (del Pozo et al., 1998; Gray et al., 1999). The pathway is defined by the AXR (AUXIN RESISTANT) and TIR (AUXIN TRANSPORT INHIBITOR RESISTANT) mutants of A. thaliana. AXR1 and a partner protein, ECR1, comprise a RUB (related to ubiquitin)-activating enzyme analogous to E1 of the ubiquitin pathway (del Pozo et al., 1998). These proteins together with a RUB-conjugating enzyme, RCE1, RUB-modify AtCUL, a cullin homolog (del Pozo and Estelle, 1999). AtCUL is a component of an SCF (SKP-cullin-F-box) ubiquitin ligase complex that includes TIR1, the F-box protein, and ASK1, a homolog of yeast SKP1 (Gray et al., 1999). Mutations in TIR1 and ASK1 inhibit the auxin response, suggesting that the SCFTIR1 complex regulates turnover of a repressor. Possible downstream targets of the SCFTIR1 complex include IAA domain proteins such as those defined by ‡ To whom correspondence should be addressed (e-mail: drm@

ufl.edu).

Review

the dominant auxin insensitive mutants, AXR2 and AXR3 (Rouse et al., 1998; Nagpal et al., 2000). The IAA homology domain is conserved in a large family of auxin induced proteins in plants. Dominant mutations in the IAA domain that confer insensitivity to auxin also strongly inhibit turnover of IAA proteins (Worley et al., 2000). Protein–protein interactions mediated by the IAA domains are proposed to modify activity of the ARF (auxin response factor) transcription factors bound to auxin response elements (AXRE) of auxin induced genes (Ulmasov et al., 1999). Phytochrome, the photoreceptor for red and far-red light responses, exists in two photo-interconvertible forms (Figure 1 and below). Phytochrome is a red-lightactivated serine/threonine kinase (Yeh and Lagarias, 1998; Fankhauser et al., 1999). Five isoforms of phytochrome (A, B, C, D, and E) that have different physiological functions have been identified in Arabidopsis. Upon stimulation with red light, phytochrome moves from the cytosol to the nucleus. The different isoforms of phytochrome are translocated to the nucleus with different kinetics. One identified substrate of the PHYA kinase, PKS1, is localized in the cytosol, where it acts as a negative regulator of phytochrome signaling (Fankhauser et al., 1999). Nuclear-localized PHYB interacts specifically with PIF3 (PHYTOCHROME INTERACTING FACTOR), a helix-loop-helix transcription factor that binds a light response element upstream of one class of light regulated genes (Martinez-Garcia, 2000). Thus, phytochrome signaling involves both nuclear and cytosolic interactions. Key components of the ethylene signal transduction pathway (reviewed by Johnson and Ecker, 1998) include ETR1 (ETHYLENE TRIPLE RESPONSE-1), the ethylene receptor; CTR1 (CONSTITUTIVE ETHYLENE RESPONSE-1), a raf-like protein kinase; EIN2 (ETHYLENE INSENSITIVE-2), a membrane protein related to mammalian NRAMP proteins; and EIN3 (ETHYLENE INSENSITIVE-3), a novel transcription factor. In the absence of ethylene, ETR1 and related receptors actively inhibit the ethylene response (Hua and Meyerowitz, 1998). The inhibitory action of ETR1 requires the CTR1 kinase. Hence, ethylene binding to ETR1 is proposed to cause inactivation of CTR. Inactivation of CTR1 potentiates signaling mediated by the C-terminal cytoplasmic domain of EIN2 (Alonso et al., 1999). EIN2 signaling leads to activation of the EIN3 transcription factor in the nucleus. EIN3 is a direct activator of the ETHYLENE RESPONSE FACTOR (ERF) genes. ERF transcription factors in turn bind to ethylene response elements of downstream ethylene induced genes. In order to understand the forces that have shaped the evolution of these and other signaling pathways, it is useful to place them in the broader context of plant biology. Plant evolution clearly has taken a very different course under a very different set of constraints than animal evolution. Among the more obvious features are photoautotrophic growth, absence of mobility, and the presence of a semirigid cell wall. The capture and assimilation of a bacterial progenitor of the chloroplast early

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Figure 1. Key Signal Transduction Pathways in Plants Signal transduction pathways for auxin, red, and far red light and ethylene as they are currently understood are diagrammed. Components of the pathways are described in the text. Two defined activities of PHY-fr are shown. PHYA-fr phosphorylates PKS1 localized in the cytosol (Fankhauser et al., 1999). Nuclear localized PHYB-fr interacts specifically with the PIF3 transcription factor (Martinez-Garcia et al., 2000), effecting regulation of one class of light-induced genes (LIGs). The ERF transcription factors contain AP2 DNA binding domains specific for the ethylene response elements in promoters of ethylene induced genes (EIGs). AIG, auxin induced gene. Cell membranes are represented by paired horizontal lines, dashed lines represent the nuclear envelope. The pathways shown are simplified for purposes of this review.

in plant evolution had a profound influence on signaling mechanisms found in modern plants. For a sessile photosynthetic organism, sensing the external environment is possibly as important as signaling among cells within the organism. Consequently, developmental pathways in plants are more strongly coupled to light and other external environmental cues (e.g., temperature and water) than is typical in animals. In general, the architecture and life history of an individual will be strongly shaped by its environment.

ditions and times of development, the photoreceptors may also act redundantly or antagonistically. It is somewhat surprising that the most widely conserved photoreceptor in nature, rhodopsin, is conspicuously absent from land plants, although rhodopsin photoreceptors are found in the eye-spot of flagellate algae such as chlamydomonas and volvox. The literature on plant photoreceptors has been reviewed extensively and we refer the readers to some excellent recent reviews (Deng and Quail, 1999; Smith, 1999; Neff et al., 2000).

Signals from the Environment Numerous environmental factors influence plant development. Temperature, light, touch, water, and gravity are among the stimuli that serve as signals for the activation of endogenous developmental programs. Of these, light has an especially important role, not only as an energy source for photosynthesis, but also as a stimulus for many developmental processes throughout the life cycle of plants, from seed germination through flowering. Consequently, plants have the richest array of light sensing mechanisms of any group of organisms (Deng and Quail, 1999; reviewed in Neff et al., 2000). The phytochrome pathway (Figure 1) is integrated with signaling by the photolyase-related blue/UV-A light absorbing receptors called the cryptochromes (Cashmore, 1999) and the phototropin-type blue light receptors (Christie et al., 1998). In combination, these receptors enable plants to discriminate an extraordinary range of quantum fluence rates, spectral quality, and photoperiod conditions. These photoreceptors sometimes act independently of one another, but in certain growth con-

Signaling in a Biophysical Context Biophysics is seemingly never far from the surface in plant biology. Plants are famous for their expressions of mathematical symmetry at the organismal level. The arrangement of florets in the head of the sunflower in near perfect Fibonnaci spirals has fascinated mathematicians and artists for centuries. Water movement is the principle engine of plant growth by cell expansion. The direction and extent of turgor-driven growth is limited by the cell wall (Cosgrove, 1997). In addition, turgor maintains the rigidity of the plant body by placing the cell wall under constant tension. Hence, factors that affect osmotic turgor pressure and extensibility properties of the cell wall are key targets of signal transduction pathways that regulate growth. The cell wall is possibly also a physical barrier to diffusion and perception of intercellular signals within plants. The network of plasmadesmatal connections that unite the cytosolic compartments of all cells in the plant into a single symplasm provides one mechanism for circumventing that barrier. The symplasm includes

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Figure 2. Structural Families of Receptor S/T Protein Kinases in Plants Five major families of plant receptor kinases are classified by their putative extracellular domains. Approximate gene numbers for each family in Arabidopsis are indicated. Where known, genetically defined functions for members of each family are listed below. The LRR-, CR4- and S-type receptors are referenced in the text; WAK type (Wall-Associated Kinase; He et al., 1998); lectin type (Herve et al., 1996).

the phloem sieve tube complex that forms the active component of the plant vascular system. Recent studies suggest that mRNA, transcription factors, and other proteins may be selectively transported long distances through this network (Lucas et al., 1995; XoconostleCazares et al., 1999). As in animals and fungi, the cortical cytoskeleton has a major role in directing deposition of the extracellular matrix and organizing cell expansion in plants (reviewed in Fowler and Quatrano, 1997). At least some aspects of that mechanism are conserved across eukaryotes. Recent studies have illuminated the role Rho GTPases play in organizing tip growth of the pollen tube (Kost et al., 1999), suggesting a conserved function of Rho signaling in polarized cell enlargement in eukaryotic cells. An interesting point of divergence is that plants evidently lack integrins, proteins that provide transmembrane coupling between the cytoskeleton and the extracellular matrix in animal cells (Fowler and Quatrano, 1997). The wall associated kinases of plants (He et al., 1998) (Figure 2), a novel class of receptor-kinase-like proteins that have a cell-wall-bound extracellular domain, are candidates for this role. While the mechanisms that transduce physical forces exerted by and against the cell wall are not yet understood in plants, it is likely that strain transmitted through the semirigid cell wall can be perceived over long distances in plant organs. A variety of specific responses to touch and other external physical manipulations (e.g., wind) have been described in plants (Trewavas and Knight, 1994). The touch response is mediated in part by a class of touch-induced calmodulin genes (Braam and Davis, 1990). Responses to external forces modify plant architecture and stature. Individual plant cells probably respond continuously to complex physical fields throughout development. In contrast to conven-

tional molecular signals, the shear, pressure, and tension forces transmitted through the cell wall arise from collective activities of many cells and do not have a discrete biochemical source. In that sense, the cell wall provides a uniquely integrative signaling medium. Are these information-rich physical fields actually used in development? The spiral pattern of the sunflower inflorescence is an elaborate example of phyllotaxy, the radial pattern of organ placement around the plant stem. A favored model for radial positioning of organ primordia in the meristem invokes a lateral inhibition mechanism (reviewed by Veit, 1998). Each newly formed organ primordium is the source of an inhibitory field that prevents initiation of another organ in its immediate vicinity. While the nature of this field is not known, physical strain is one candidate. The late Paul Green proposed that phyllotaxy patterns correspond to surface folds that would in theory minimize strain in the growing surface of the meristem (Green et al., 1996). In this elegant physical model, a relaxation mechanism that minimizes surface strain combined with boundary conditions set by the inelastic meristem perimeter is sufficient to generate the observed patterns. Whether or not it survives the tests of time and experiment, Green’s hypothesis draws appropriate attention to the biophysical context of plant development. Conceivably, the radial positioning mechanism might have evolved as a solution to the physical problem rather than as a mechanism for organizing the plant body plan, per se. Signaling in Development At the level of biological function, there is substantial evidence that key elements of pathways related to stress (Mizoguchi et al., 1997), defense (Bergey et al., 1996; Durner et al., 1999), sugar (Koch, 1996; Jang et al., 1997), and osmotic responses (Kutz and Burg, 1998) are at

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least partially conserved in plants, animals, and fungi. These conserved pathways regulate processes that are basic to unicellular as well as multicellular organisms. In many cases, the basic functions of these pathways have been extended in significant ways. For example, sugar sensing provides a mechanism for long-distance communication between plant organs (Koch, 1996; Jang et al., 1997). In contrast, the signaling pathways that underlie much of multicellular development and patterning are, as far as we can tell, highly novel in plants. The Ras, Wnt, and hedgehog signaling pathways that are central to animal development (Scott, 2000) are not detected in plants. Although auxin signaling is mediated by a highly conserved ubiquitin mediated proteolysis apparatus (Figure 1); the downstream targets of the auxin-regulated SCFTIR1 complex are highly novel and plant specific. The generalization that developmental pathways are less conserved than responses common to unicellular organisms is consistent with the hypothesis that multicellular development evolved independently in plants and animal lineages. Although many of the signaling pathways underlying development are novel, there are shared principles. In plants, like animals, a core set of signaling pathways is used repeatedly in many different developmental contexts. The classic hormone pathways—auxin, cytokinin, abscisic acid, gibberellin, and ethylene—appear in many contexts in plant development. The reiteration of core pathways in plants and animals suggests that development evolved through duplication and innovation on basic pathways that were recruited early in evolution of the respective lineages. For example, the basic auxin/cytokinin polarity that defines the architecture of vascular plants is already established in primitive mosses that have a simple filamentous form (Schumaker and Dietrich, 1998). Another striking example of convergence is the evolution of homeotic gene action. The homeotic gene interactions underlying flower development and segmentation in plants and animals, respectively, evidently evolved according to similar principles. A series of gene duplications created a family of transcription factors capable of binding DNA as homo- and hetero-dimers. An initial state of functional redundancy undergoes relaxation as individual member genes drift and acquire altered expression patterns and new functions. The combinatorial interactions of the transcription factors in regions of overlapping expression define unique domains. The floral homeotic genes were recruited from a MADS box transcription factor family that was already large and partially redundant in the gymnosperm ancestors of the angiosperms (see Ma and dePhamphillis, 2000). Another strong theme in plant signaling networks is a prevalence of negatively regulated pathways. Loss of function mutations that cause a constitutive response in the absence of the agonist have been crucial in defining the light, gibberellin, and ethylene response pathways. In these systems, a default state is actively enforced by the pathway in the absence of agonist. Hence, the hormone independent activity of these pathways may be at least as significant physiologically as the ligand induced states. For example, loss-of-function mutations (gene knockouts) in the ethylene receptor

genes confer a constitutive ethylene response phenotype in Arabidopsis (Figure 1) (Hua and Meyerowitz, 1998). The severity of the phenotype increases as knockouts of multiple members of the receptor gene family are combined. Hence, the ethylene independent activity of the receptors is apparently necessary for normal development. It is less clear that the ethylene-induced state of the pathway is essential for development. At least one species of flowering plants, P. pectinatus, has evidently lost all capacity for ethylene biosynthesis (Summers et al., 1996). An interesting question is whether the orphaned ethylene receptors have been retained for their essential developmental function in this aquatic monocot. Sources of Innovation and Novelty in the Evolution of Plant Signal Transduction Pathways From a comparative perspective, plant signal transduction pathways display an intriguing montage of both novelty and conservation of elements with other eukaryotes. Conservation and novelty are juxtaposed at all levels: biological function, pathways, signaling molecules, transduction mechanisms, protein domains, and motifs. The emerging comparative analysis of strategic eukaryotic and prokaryotic genomes offers new insight into the origins of novelty in plant signaling pathways and how the innovation relates to the biological constraints that have shaped plant evolution. Innovation and Novelty in the Input Layer: Ligands and Receptors Plants utilize a variety of metabolites as signaling molecules, including many that have analogs in other eukaryotes. Hormones derived from aromatic amino acid, steroid, apo-carotenoid, and fatty acid derivatives mirror major classes of animal hormones. This raises the question of whether some pathways are of ancient origin. The octadecanoid (Bergey et al., 1996) and nitric oxide (Durner et al., 1999) signaling pathways that mediate plant wound and pathogen defense responses are functionally related to defense pathways in animals, suggesting that these may be ancient responses. Jasmonic acid and related octadecanoid compounds are cyclic products of lipid oxidation and are structurally related to arachidonic acid derivatives in animals. The brassinosteroid (BR) and abscisic acid (ABA) hormones are analogs of steroid and retinoid hormones of animals, respectively. Key steps in plant and animal steroid biosynthetic pathways are highly conserved. The human steroid 5␣-reductase type I or type II genes, for example, rescue the Arabidopsis det2 mutant, which is deficient in the synthesis of the steroid hormone brassinolide (Li et al., 1997). The apo-carotenoids, retinoic acid and abscisic acid, are derived from oxidative cleavage of plant carotenoids. The biochemical mechanism of apocarotenoid synthesis was illuminated by analysis of viviparous14, an ABA-deficient mutant of maize (Tan et al., 1997). VP14 defines a new class of dioxygenases that catalyze specific oxidative cleavage of carotenoids (Schwartz et al., 1997). Related genes are found in genomes of animals and bacteria that synthesize apocarotenoids (Tan et al., 1997), suggesting that this mechanism is broadly conserved in nature. A recent study (von Lintig and Vogt, 2000) confirms that the single VP14

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homolog in the Drosophila genome is the 15,15 ␤-carotene dioxygenase responsible for retinal synthesis. Mutations in human RPE65, a homolog expressed in the retinal pigment epithelium of the eye, cause congenital retinal dystrophy (Morimura et al., 1998). There is little evidence that the conservation observed in the biosynthetic pathways extends to the signal transduction mechanisms of plant steroid and apo-carotenoid hormones. With the Arabidopsis genome sequence nearing completion, it is increasingly unlikely that genes belonging to the nuclear receptor superfamily will be found in plants. Rather, plant steroids appear to be perceived at the plasma membrane through a leucine-rich-repeat (LRR)-receptor ser/thr kinase, BRI1 (Li and Chory, 1997; Friedrichsen et al., 2000; He et al., 2000). Localization of BRI1 receptor on the plasma membrane suggests that BR signaling is initiated on the cell surface (Friedrichsen et al., 2000). Moreover, the extracellular domain of BRI1 confers BR responsiveness to heterologous cells (He et al., 2000). The possibility that membrane-bound steroid receptors exist in animals remains; however, LRR receptor S/T kinases related to BRI1 are not found in animal genomes. While the evidence favors independent evolution of steroid signaling mechanisms in plants and animals, the steroid biosynthetic pathway was evidently highly developed in the common ancestor of plants and animals. It is possible that the use of steroid signals is ancient and that the signal transduction mechanisms have diverged radically in plant and animal lineages. Perhaps the richest display of novelty in plant signal transduction mechanisms can be found in the receptors, although a few receptors such as cryptochrome (originally discovered in plants; Ahmad and Cashmore, 1993) are shared by plants, animals, and other eukaryotes. Other major families of plant receptors include receptor S/T-kinases, two component histidine kinases, LRRdomain pathogen resistance receptors and membrane channel related proteins. A few G-protein coupled receptors have been identified in Arabidopsis (PlakidouDymock et al., 1998), but the number of genes is far lower than in the C. elegans and Drosophila genomes. Membrane Channel–Related Receptors. Judging from the available genome sequence, the potential for channel type receptors is substantial in plants and still largely unexplored. One interesting example is the EIN2 gene, which encodes a 12 membrane spanning protein that is closely related to NRAMP metal transporters of animals (Alonso et al., 1999). Genetic analysis indicates that EIN2 acts downstream of the ethylene receptors (see Figure 1) and it is also implicated in jasmonic acid and abscisic acid signaling. EIN2 is the only recessive mutant known that blocks all aspects of ethylene signaling in all parts of the plant. The EIN2 protein contains a large C-terminal cytoplasmic domain that is not present in animal NRAMP proteins. When expressed by itself as a soluble protein, the C-terminal domain can partially suppress the EIN2 mutant phenotype. In this respect, the mechanism of EIN2 signaling is analogous to hexose sensors of yeast that signal through a C-terminal extension (Alonso et al., 1999). The integral membrane NRAMP domain is necessary, but not sufficient for ethylene-dependent signaling of intact EIN2. It is not known whether the function of EIN2 in ethylene signaling re-

quires interaction with another ligand; however, the similarity to metal transporters is strongly suggestive. The Prokaryotic Heritage: Two Component Related Receptors. Two important families of receptors, phytochrome light receptors and the ethylene receptors, are derived from bacterial two-component histidine kinase systems. Direct antecedents of both receptors are found in the cyanobacteria genome, suggesting that these receptors were inherited from the progenitor of the chloroplast. However, in plants, the functions of these proteins have been extended and modified in ways that enhance integration with signaling mechanisms of the eukaryotic host cell. The plant and bacterial phytochromes are light-regulated protein kinases (see Figure 1). Bacterial phytochromes have light-dependent histidine kinase activity whereas plant phytochromes catalyze light-dependent serine/threonine phosphorylation (Yeh and Lagarias, 1998) suggesting that a serine/threonine-specific kinase evolved from an ancestral histidine kinase. A similar transformation occurred in the evolution of mitochondrial ␣-ketoacid dehydrogenase kinases (Harris et al., 1995). In both instances, ancestral histidine kinases assimilated from the respective organelle genomes apparently adapted to the dominant signaling paradigm of the eukaryotic host-serine/threonine phosphorylation. This transition may have facilitated integration of organelle and host signaling mechanisms. The biochemical mechanism of ethylene sensing also has ancient roots. The genome of Synechocystis contains a histidine kinase gene with a structure very similar to the ETR1 protein and related plant ethylene receptors, indicating that a copper containing sensor domain arose early in the evolution of photosynthetic organisms (Rodriquez et al., 1999). While the bacterial protein also binds ethylene with high affinity, cyanobacteria have no known physiological response to ethylene. While the function of the bacterial protein is still a mystery (Rodriquez et al., 1999), it is conceivable that ethylene signaling evolved through recruitment of an incidental chemical activity of the Cu(I) center that existed long before the advent of ethylene responses in plants. Curiously, key components of the ethylene pathway, EIN2 and the ETR1-like receptors, respectively, are related to eukaryotic and prokaryotic proteins that have potential metal sensing capacities. The ETR1 sensor domain is not found in other eukaryotes, and EIN2 homologs are not evident in the cyanobacteria genome. One possibility is that the ethylene pathway evolved through a convergence of eukaryotic and prokaryotic pathways related to metal homeostasis. The opportunity for integration of these pathways presumably arose during assimilation of the cyanobacterial progenitor of the chloroplast genome. Diversification of the Receptor S/T-Kinases. The receptor serine/threonine kinases comprise the largest and most diverse class of receptor proteins in plants. By contrast, receptor tyrosine kinases that are prevalent in animal signal transduction have not been detected in plants. A cursory analysis of current databases indicates that the available Arabidopsis genome sequence contains more than 300 genes structurally related to receptor serine/threonine kinases. Five structural families of plant receptor serine/threonine kinases are diagrammed

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in Figure 2, classified according to structures of their putative ligand binding domains. The leucine-rich-repeat class is the largest family, comprising more than 170 genes in Arabidopsis. A handful of plant LRR receptor kinases have genetically defined functions including meristem organization (CLV1; Clark et al., 1997), pathogen resistance (rice Xa21; Song et al., 1995), and brassinosteroid perception (BRI1; Li and Chory, 1997). Because LRR domains typically mediate protein–protein interactions (Kobe and Deisenhofer, 1995), the ligands of these receptors are expected to include peptides. Genetic and biochemical studies confirm that the ligand of CLV1 is a small secreted protein encoded by the CLV3 gene of Arabidopsis (Fletcher et al., 1999; Trotochaud et al., 2000). The CLV genes organize the apical meristem of Arabidopsis. The CLV3 peptide is expressed specifically in a small group of cells that define the central zone of the shoot apical meristem surface (Fletcher et al., 1999). This region makes contact with a separate, larger group of CLV1-expressing cells in the interior of the meristem. Mutations in either gene cause the volume of the apical meristem to increase progressively during development. The result is a “fasciated” plant phenotype. As already mentioned, the LRR receptor kinases may be involved in perception of small organic molecules as well as peptides. The BRI1 receptor is required for brassinosteroid perception in Arabidopsis (Li and Chory, 1997; He et al., 2000), suggesting that it is at least a component of the BR receptor. It is not known whether the LRR domain interacts directly with BR or through an interaction with another BR binding protein. A novel juxtaposition of domains occurs in the extracellular component of the CRINKLY4 family of receptor kinases. The CRINKLY4 mutant of maize disrupts differentiation of epidermal cells (Becraft et al., 1996). At least five related genes of unknown function exist in Arabidopsis. Although the ligand for maize CRINKLY4 has not been identified, the structure of the putative ligand binding domain suggests a capacity for peptide binding. The extracellular domain includes a CYS motif found in tumor necrosis factor receptors (TNFR) of vertebrates and their viral pathogens. The TNFR motif is notably absent from the proteomes of Drosophila, C. elegans, and yeast (Rubin et al., 2000). The presence of this domain in plants suggests that it either was present in the common ancestor of plants and animals or transferred horizontally. Transfer via a viral vector is a plausible mechanism. In plants, the evolutionary history of the TNFR domain has not yet been traced beyond the angiosperms. A second extracellular domain of CRINKLY4 is equally interesting. All CR4-like receptors contain seven copies of an ⵑ39 amino acid repeat that is distantly related to RCC1 GTPase activating proteins found in all eukaryotes (Renault et al., 1998). Molecular modeling studies support the structural homology of the CRINKLY4 domain to the RCC1 family of 7-bladed propeller proteins (D. R. M., unpublished data). If confirmed, this would be the first occurrence of the propeller fold in an extracellular context. Analysis of the self-pollen recognition mechanism of brassica indicates that at least some members of the S-locus receptor family will also have peptide ligands (Schopfer et al., 1999). In this system, the S-locus recep-

Table 1. DNA Binding Domains Specific to Eukaryotic Lineages Plants1 AP2 B3 EIN3 GT (trihelix) LFY

Animals2 PRD POU ETS T-box Nuclear receptors

Fungi2 APSES

1

References: AP2, Weigel, 1995; B3, Suzuki et al., 1997; EIN3, Chao et al., 1997; GT, Smalle et al., 1998; LFY, Busch et al., 1999. 2 Chervitz et al., 1998.

tor kinase is expressed in the receptive maternal tissues of the flower that make contact with incoming pollen. The presumptive ligands of this receptor are encoded by a second set of S-locus specific genes (SCR) that are tightly linked to the receptor gene (Schopfer et al., 1999). The SCR genes encode small, secreted, CYS-rich peptides that are expressed in pollen. An incompatible reaction is induced when the pollen and stigma carry the same S-locus allele. The interaction is evidently highly specific to the linked pair of receptor and ligand genes, allowing discrimination between a hundred or more different S alleles. Self-incompatibility has evolved independently in many flowering plant lineages (Charlesworth and Awadalla, 1998). Other members of the brassicaceae, including Arabidopsis, are naturally self-pollinating and have no self-incompatibility mechanism. Nevertheless, the S-locus-related receptor kinases comprise one of the largest families of receptor kinases in the Arabidopsis genome. The functions of these genes are unknown. The number of LRR, CR4-type and S-domain receptor kinase genes in Arabidopsis suggests that the potential for peptide hormones in plants is large. In an interesting contrast to animal peptide hormones, however, the putative peptide ligands identified thus far have failed to identify corresponding families of genes encoding peptide hormones. This suggests that these peptide signals are highly specialized. One other CLV3-like gene of unknown function is detected in the Arabidopsis genome. A small family of CLV3-related secreted proteins is expressed in the endosperm of maize (Opsahl-Ferstad et al., 1997) indicating conservation of the CLV3 sequence between the monocots and eudicots, but the maize homologs are evidently not involved in organizing an apical meristem. Other peptide signaling systems of plants show evidence of rapid evolution. The SCR peptides of brassica detect several divergent CYS-rich open reading frames in the genome of Arabidopsis, a self-compatible species. Systemin, the first peptide signal discovered in plants, shows wide variation in biological activity among close relatives of tomato (Constabel et al., 1998). Innovation in the Output Layer: Novel Transcription Factors An intriguing discovery stemming from genetic analysis of diverse plant signaling pathways are several novel families of DNA binding proteins (Table 1) that are unique to plants. Animal genomes encode a similar number of lineage-specific DNA binding proteins. In contrast, comparative genomics has revealed only one DNA binding protein family that is unique to fungi (Chervitz et al., 1998). The plant-specific proteins were discovered

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almost exclusively in developmental pathways that are unique to plant biology. A similar generalization applies on the animal side suggesting that recruitment of new transcription factors has played a significant role in the evolution of novelty in these lineages. In plants, the B3 domain proteins are implicated in ABA (McCarty et al., 1991) and auxin response pathways (Ulmasov et al., 1999), and in the regulation of vascular patterning (Hardtke and Berleth, 1998). Other novel transcription factor families include the EIN3 family of transcription factors involved in ethylene signaling (Chao et al., 1997), and the AP2 domain proteins implicated in a variety of processes including ABA, cold, and ethylene responses, as well as floral organ identity (Weigel, 1995). In addition, the novel protein, LFY, regulates flowering (Busch et al., 1999). The superfamily of B3 domain proteins consists of four subfamilies each having a characteristic domain structure. A phylogeny of B3 sequences indicates that the ABA and auxin response factors (ARFs) separated early in plant evolution. Functional analyses suggest that B3 transcription factors are typically bifunctional. Proteins in the RAV (RELATED TO ABI3/VP1) subfamily contain a second AP2-like DNA binding domain and exhibit two distinct DNA binding specificities (Kagaya et al., 1999). In the case of the VIVIPAROUS1 (VP1) and ARF proteins, separable protein–protein interaction domains provide the key interfaces for the input of ABA and auxin signals, respectively (Carson et al., 1997; Hobo et al., 1999; Ulmasov et al., 1999). The N-terminal domain of VP1 has potent coactivator and corepressor activities in the absence of B3 (Hoecker et al., 1995; Carson et al., 1997). The coactivator domain mediates ABA-dependent activation of downstream genes through a specific interaction with a b-ZIP protein partner (Hobo et al., 1999). Auxin signaling to ARF proteins evidently occurs via protein–protein interactions mediated by the IAA homology domain (Figure 1). Other families of conserved eukaryotic transcription factors have been amplified disproportionately in plants. The MADS box and MYB gene families stand out as being particularly large compared to other eukaryotes. The Arabidopsis genome contains more than 100 R2R3 type MYB genes (Kranz et al., 1998). MYBs are implicated in a variety of functions, including regulation of secondary metabolism and cell morphology. The process of gene duplication and diversification of MYB gene function has continued in recent evolutionary times. For example, the c1 and pl genes, which are organ-specific regulators of the anthocyanin biosynthetic pathway in maize, were created by duplication of the ancestral maize genome about 12 MYA (Gaut and Doebley, 1997). Conclusions and Prospects In this brief survey, we have highlighted just a few examples where we can begin to trace the origins of novelty in the evolution of plant signaling pathways. The source materials derive from some expected sources, such as the progenitor of the chloroplast genome and genes inherited from the common ancestor of all eukaryotes, but also include a surprising number of novel genes of unknown origin. While the mechanisms that allow evolution of new functions for old genes are presumably similar in eukaryotes, there may be qualitative differ-

ences at that level as well. For example, is the creation of novelty through gene duplication and diversification more common in plants than in animals? In animals, examples of evolution through gene duplication are balanced by examples where single genes have evolved more complex regulation. That the Arabidopsis genome should have nearly twice the gene number of Drosophila, an organism that is arguably more complex, is intriguing. One possibility is that plant genomes have a higher level of functional redundancy overall. If so, a smaller fraction of gene disruptions in plants will have discernable phenotypes relative to other eukaryotes. Time will tell as we accumulate more data from gene knockout experiments in plants. Alternatively, functional complexity may be partitioned differently in plant genomes such that a larger number of less complex genes are used to span an equivalent set of functions that can be provided by a smaller number of more complex genes in animals. Plant evolution might favor a path of deploying a larger number of differentially regulated simple genes rather than a smaller number of more complex genes for a couple of reasons. First, polyploidy and aneuploidy are better tolerated in plants than in higher animals allowing more frequent wholesale duplication events. As illustrated by the history of the maize genome, polyploid genomes relax to genetic diploidy fairly rapidly, but in the process create a large number of partially redundant paralogous genes, many of which can be distinguished phenotypically (Gaut and Doebley, 1997). A second possibility is that the capacity to add regulatory complexity to individual plant genes is constrained in some way relative to animals. Upstream regulatory elements in plant genes are rarely located more than 1 kb or so from the transcription start and more often occur within the first 300 nt (roughly the span of two nucleosomes). In Arabidopsis, this compact regulatory structure is enforced by an average gene density of one gene per 4 kb (Bevan et al., 1998; Lin et al., 1999). Perhaps, in animals, the unit of duplication can be an enhancer as readily as an entire gene whereas in plants, there is a tendency to duplicate whole genes. However, the features of plant chromatin structure that would impose such a constraint are not obvious. Clearly, these are very early days in the era of comparative functional genomics, and we can look forward to many new insights into the evolution of novelty in eukaryotic signal transduction pathways. References Ahmad, M., and Cashmore, A.R. (1993). HY4 gene of A. thaliana encodes a protein with characteristics of a blue-light photoreceptor. Nature 366, 162–166. Alonso, J.M., Hirayama, T., Roman, G., Nourizadeh, S., and Ecker, J.R. (1999). EIN2, a bifunctional transducer of ethylene and stress responses in Arabidopsis. Science 284, 2148–2152. Becraft, P.W., Stinard, P.S., and McCarty, D.R. (1996). CRINKLY4: a TNFR-like receptor kinase involved in maize epidermal differentiation. Science 273, 1406–1409. Bergey, D.R., Howe, G.A., and Ryan, C.A. (1996). Polypeptide signaling for plant defensive genes exhibits analogies to defense signaling in animals. Proc. Natl. Acad. Sci. USA 93, 12053–12058. Bevan, M., Bancroft, I., Bent, E., Love, K., Goodman, H., Dean, C., Bergkamp, R., Dirkse, W., Van Staveren, M., Stiekema, W., et al.

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