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Plant Physiol, January 2001, Vol. 125, pp. 81-84 Hormone Response Mutants. A Plethora of SurprisesMichigan State University-Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824-1312
Plant hormone mutants can be
classified into two categories: mutants that are impaired in hormone
biosynthesis and mutants that are impaired in their response to
hormones. Twenty-five years ago, the most frequently encountered
hormone mutants were gibberellin (GA) biosynthetic mutants. They
exhibited a dwarf phenotype that could be restored to wild type by
application of GA, and they were often used for GA bioassays. Using
mutants to elucidate the pathway of hormone biosynthesis is
conceptually straightforward and proved to be a fruitful approach in
the case of GA (9), abscisic acid (5), and brassinosteroids (BRs; 4, 15). The genetic approach played a minor role, if any, in the
elucidation of auxin, cytokinin, and ethylene biosynthesis.
Twenty-five years ago, the nature of plant hormone receptors and of
hormonal signal transduction pathways was an enigma. Efforts to
identify hormone receptors by conventional hormone-binding experiments
generated far more frustrations than results. The advent of molecular
genetics with Arabidopsis as a model organism opened the door to
successful experimentation on plant hormone receptors and signal
transduction pathways, and some of the results turned out to be quite
unexpected. Space limitations restrict me to three case histories,
those of ethylene, GA, and BRs.
Etiolated dicot seedlings respond to applied ethylene by a
reduction in stem elongation, thickening of the stem, and an
exaggerated closure of the apical hook. This so-called "triple
response" was first used by Bleecker et al. (1) and Guzmán and
Ecker (6) as a screen for the identification of ethylene-insensitive
mutants. The rationale was, of course, that such mutants might be
impaired in ethylene sensing or in downstream reactions of the signal
transduction pathway. A screen of 75,000 M2 seedlings derived from mutagenized seeds yielded three mutants with heritable insensitivity to ethylene (1). Of these, the etr mutant was investigated further (Fig.
1). The
mutation was dominant, all ethylene responses examined were blocked,
and crude binding experiments indicated that the function of the
ethylene receptor might be impaired. Jump 5 years forward in time: The
ETR1 gene has now been cloned, and its product found to be
related to bacterial two-component receptor systems (3). Two-component
regulators are typically composed of a sensor protein with an input
domain that receives signals and a catalytic transmitter domain that
autophosphorylates on an internal His residue. The second component, a
response regulator protein, is composed of a receiver domain that
receives phosphate from the transmitter on an Asp residue, and an
output domain that mediates responses depending on the phosphorylation
state of the receiver. The ETR1 protein has a receiver domain fused to
the C terminus of the His kinase domain. Another fast-forward in time:
There is not one but five ETR1-like genes in Arabidopsis and
also in tomatoes; there appears to be functional redundancy between the
ETR-like proteins; and the ethylene-binding properties of ETR1 are
consistent with its role as receptor (for review, see 2). These results
were quite unexpected and raised a number of important
questions.
First, why are mutations in the ETR gene family that confer ethylene insensitivity dominant? There are two models that can explain dominance. If proteins of the ETR family form multimers, a mutation in one component could inactivate the complex. Alternatively, mutations such as etr1 may lead to a gain of function. According to this model, the ethylene receptors would be "on" or would signal in the absence and would be "off" in the presence of ethylene. The etr1 mutation and similar mutations in other members of the ETR family eliminate binding of ethylene to the receptor (2). According to the gain-of-function model, mutated receptors would continue signaling in the presence of ethylene. The gain-of-function alternative was shown to be the correct one, and the consequences are quite counterintuitive (2). For example, an increase in the number of receptors would lower rather than raise sensitivity to ethylene because more ethylene would be required to reduce the output of the receptors. Second, why are there multiple ethylene receptors? This redundancy maintains ethylene responsiveness, even when the function of one receptor is lost by mutation. Furthermore, there may be subtle differences in the properties of receptor isoforms. They may, for example, have different affinities to ethylene, thereby extending the dynamic range of ethylene action (2). Third, how does a prokaryotic sensing system such as ETR fit into the signal transduction pathway of eukaryotes? The first component of this pathway downstream from the ethylene receptor turned out to be a typical eukaryotic signal transduction element, namely a Raf-like protein kinase called CTR1 (11). Because the ctr1 mutant has the phenotype of an ethylene-treated plant, the CTR1 protein negatively regulates the ethylene transduction pathway. Although it is not yet known whether CTR1 is part of an MAP-kinase cascade, there is precedence for the coupling of a prokaryotic two-component sensing system to an MAP-kinase cascade in yeast (14). Mutant analysis resulted in the identification of further downstream elements of the ethylene signal transduction pathway, which leads, via EIN3, to transcriptional regulation of ethylene-responsive genes (for review, see 10). What important questions are to be solved in the future? There are two signal transduction pathways that need to be elucidated with respect to ethylene action. Molecular genetics led to the identification of elements in the ethylene response pathway. Characterization of additional ethylene response mutants, coupled with biochemical approaches, may complete the picture. Of course, one still will have to explain by what mechanisms the multitude of diverse ethylene responses are controlled. What determines the specificity of ethylene responses in one cell type versus another, or even within the same cells? Ethylene-regulated processes are mostly initiated by an increase in ethylene synthesis. Ethylene synthesis is under the control of environmental and/or endogenous signals. Thus, to understand ethylene responses, it will be necessary to identify the exogenous and endogenous factors that control ethylene synthesis and to elucidate the signal transduction pathways that lead to an induction of ethylene biosynthesis.
The GA transduction pathway is not as well understood as that of ethylene. For example, the identity of the GA receptor(s) has not yet been determined, and fewer downstream elements of the pathway have been identified. Nevertheless, interesting and surprising results have recently emerged from the analysis of GA response mutants. The first GA-insensitive mutant of Arabidopsis, gai, was described by Koornneef et al. (12). The mutation is dominant, results in severe dwarfing of the plant, and also blocks GA-mediated negative feedback inhibition of GA biosynthesis. This indicates that at least some steps in the GA signal transduction pathway are shared between diverse GA responses. GAI was found to encode a protein that has the hallmarks of a transcription factor with similarity to SCARECROW (13). It contains consensus nuclear localization signals and motifs that are necessary for the binding of transcriptional co-activators to nuclear receptors. The mutation in gai resulted in a 17-amino-acid deletion, which changed the functional properties of the protein. It can, therefore, be classified as a gain-of-function mutation. Loss-of-function mutations in GAI, such as gai-d, also resulted in a GA-insensitive phenotype. However, such mutants grew tall, even when plants were treated with an inhibitor of GA biosynthesis. The following model has been proposed by Harberd et al. (7) to explain these seemingly conflicting results: GA counteracts the action of GAI, which negatively regulates plant growth and other GA-controlled processes. Loss-of-function mutations in GAI derepress these pathways and render them GA independent. The gai mutation, on the other hand, changes the properties of GAI such that it escapes control by GA and continues to act as a suppressor of growth. A GAI-related protein, GRS [independently identified and designated as RGA by Silverstone et al. (16)], exhibits functions that are similar to and overlap with those of GAI. Mutants in the SPY locus overcome GA deficiency in Arabidopsis and resemble plants treated with GA (17). The phenotype of the spy mutant indicates that the SPY protein is also a negative regulator of GA signal transduction. Its deduced amino acid sequence shows significant similarity to O-GlcNAc transferases, leading to the conclusion that SPY may act by modifying components of the GA signal transduction pathway. Thus, unexpectedly, some parallels between the ethylene and GA transduction pathways have emerged. In both cases, the signal transduction pathway includes negative regulators whose action is relieved by the respective hormone, and, in both cases, there is redundancy built into the system. Many open questions remain, however. What is the identity of the GA receptor? What are the other components of the signal transduction pathway? Do unrelated GA responses share components of the signal transduction pathway or are, for example, GA-induced growth and GA-enhanced hydrolase activities in cereal aleurone cells mediated by different pathways?
BRs encompass a group of plant steroids that elicit a wide range of effects on growth and development when applied to plants (for review, see 4 and 15). For many years, the role of BRs as plant hormones was not widely accepted because it was difficult to assess their natural function in the absence of mutants or specific inhibitors. This skeptical view changed, however, with the discovery that two Arabidopsis mutants with de-etiolated phenotype in the dark and dwarf phenotype in the light are blocked in BR biosynthesis. Applied BR was found to repair the lesion (15). Thus, molecular genetics helped establish the role of BRs as endogenous plant hormones with well-defined functions. The one known BR-insensitive mutant, bri1, was found to encode a Leu-rich repeat (LRR) receptor kinase with a transmembrane domain (15). It has been proposed that BRI1 functions as a cell surface receptor that transduces the BR signal into the interior of the cell via protein phosphorylation. This hypothesis raises two questions: First, known steroid receptors are cytoplasmic and act through transcriptional regulation. Is BRI1 the first example of a membrane-bound steroid receptor? Second, the LRR motif is known to mediate protein-protein interactions. Does it also mediate interaction with a small molecule, such as BR? Alternatively, does BRI1 interact with another protein that binds BR? Recent results provide strong evidence that BRI1 is directly involved in BR perception (8). This conclusion was reached from experiments in which the LRR and transmembrane domains of BRI1 were fused to the protein kinase domain of XA21, a LRR receptor kinase that mediates disease resistance responses in rice. The BRI1-XA21 hybrid receptor, expressed in rice cells, elicited disease resistance responses when the cells were treated with BR. Hormone response mutants have been the key to elucidate a number of hormonal reaction mechanisms in plants. It is expected that further screens will yield new mutants that will help fill in some of the blanks in hormonal transduction pathways. However, one also has to be aware that the genetic approach has potential limitations. It has become clear that plants lacking ethylene responses can survive perfectly well, at least under laboratory conditions. Eliminating responses to other hormones may turn out to be lethal, however. Alternatively, lack-of-response mutants may indicate redundancies in the signal transduction pathways. Different types of screens, for example, activation tagging, may help overcome some of these difficulties. Also, the publication of the Arabidopsis and rice genomes may help identify candidate members of hormonal transduction pathways or may indicate heretofore unrecognized redundancies in such pathways. The next 25 years will undoubtedly bring a full understanding of hormonal reaction mechanisms, which will help us manipulate central processes in plant growth and development.
* E-mail hkende{at}pilot.msu.edu; fax 517-353-9168.
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TOP
INTRODUCTION
ETHYLENE RESPONSE MUTANTS HELP...
1 ethylene. The wild-type seedlings showed the
characteristic triple-response phenotype: reduced elongation, thickened
hypocotyl, and pronounced apical hook. The etr1 mutant
elongated normally just as wild-type seedlings would in an atmosphere
without ethylene. [Reprinted with permission from Science
241: 4869 (cover page), 1988. Copyright 1988, American
Association for the Advancement of Science. Photo by Kurt Stepnitz,
Michigan State University.]
