Download Emerging Themes of Plant Signal Transduction

The Plant Cell, Vol. 6, 1529-1541, November 1994 0 1994 American Society of Plant Physiologists
REVIEW ARTICLE
Emerging Themes of Plant Signal Transduction
Chris Bowler a and Nam-Hai Chua bi'
a
Stazione Zoologica, Villa Comunale 1, 80121 Naples, ltaly
Laboratory of Plant Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, New York 10021-6399
INTRODUCTION
The successful existence of all higher organisms is dependent upon their ability to coordinate complex developmental
changes and to sense and respond to fluctuations in their surroundings. Responses to developmental and environmental
cues occur by stimulus-response coupling: a stimulus is perceived by the cell, a signal is generated and transmitted (signal
transduction), and a biochemical change is instigated (the response). This process most often requires the recognition of
the stimulus by a receptor and the subsequent use of chemical second messengers and/or effector proteins to transmit
a signal that will then trigger the appropriate response. Key
properties of signal transduction are speed, sensitivity
(achieved by amplification), and specificity, all of which are
controlled by a network of positively and negatively acting elements. Whereas the positively acting intermediates are
essential to drive signal transduction, the negative elements
are responsible for ensuring a response that is quantitatively
appropriate, correctly timed, and highly coordinated with other
activities of the cell. Specifically, negative control can (1) allow changes in sensitivity to a particular stimulus, permitting
a signaling pathway to work over a broad dynamic range of
stimulus intensities; (2) terminate a response when it is completed, even if the stimulus may remain (sometimes referred
to as desensitization or adaptation); and (3) allow a signaling
pathway to be reused.
In animal cells, the signal transduction events that couple
avariety of stimuli with their responseshave been well characterized, as have the mechanisms for their negative control.
Plants, however, are challenged in different ways from animals
because their developmentalprograms are generally more flexible and because they are unable to escape at will from
unfavorable environmental situations. These differences indicate the increased adaptability of plants over animals and
lead us to speculate about the nature and control of the sensing systems plants employ to regulate their development and
behavior. Have these different constraints necessitatedthe evolution of mechanisms altogether different from those found
in animals? To some extent the answer appears to be yes; both
To whom correspondence should be addressed.
genetic and biochemical approaches have now yielded sufficient information for us to realize that, whereas the basic
mechanisms may often be conserved (e.g., the use of the same
second messengers and similar protein phosphorylationl
dephosphorylation reactions), there are often very real differences in the components of the signaling machinery and in
their regulation. In some cases, plant signaling appears more
analogous to bacterial signaling systems (Hughes, 1994),
whereas in others it may be altogether novel.
Studies of signal transduction pathways are facilitated by
some knowledge about the specificity of the stimulus, the biochemical nature of the receptor, and the specificity of the
responses. Specificity of the response for the stimulus is particularly important for genetic studies because in many cases
it is desirable to generate mutants that are constitutive for a
response in the absence of the stimulus, for example, the cop
and det mutants, which are constitutive for lighi responses in
darkness, and the cif mutants, which display constitutive ethylene responses in the absence of exogenous ethylene (see
below). In these cases, to be sure that the mutant is blocked
in the signal transduction pathway of interest, it is important
to be able to show that all of the phenotypes of the mutant
are consistent with all or at least a subset of the normally observed phenotype of wild-type plants exposed to the stimulus.
This is particularly difficult to demonstrate in plants because
many responses can be mediated by different stimuli. For
example, the effects of light on plant morphology (photomorphogenesis) and gene expression can be recapitulated to a
large extent by cytokinin, and the response to pests and pathogens may involve the actions of (at least) ethylene, jasmonic
acid, salicylic acid, systemin, and electrical signals.
Furthermore, genetic screens for constitutive mutants are
largely dependent on the assumption that there are negative
regulators that normally repress signal transduction in the absence of the stimulus and that the stimulus acts (at least at
this step) by overcoming the repression. For some kinds of
signal transduction pathways, for example, in which the negative regulator is one step in a linear pathway, loss of function
mutations in such a negative regulator would produce a constitutive phenotype in the absence of any stimulus. More
frequently, however, individual steps in signal transduction (at
1530
The Plant Cell
least those upstream of transcription factors) are controlled
by counteracting positive and negative regulators. In such
cases, loss of function mutationsin a negative regulator would
not result in a constitutive phenotype unless the signal transduction pathway had a basal level flux passing through it in
the absence of the stimulus. This flux could be a result of a
basal level activity of the signaling intermediates. For example, although a kinase may be highly stimulated in the presence
of inducer, it may also have a low constitutive activity in its
absence, as may the “complementary”phosphatasethat counteracts the kinase activity. In the case of second messenger
pathways, the flux is known as a ”current” and relates to a nearly
equal hydrolysis and synthesis of the second messenger (e.g.,
cGMP) in the absence of inducer. Stimulation of the pathway
then disturbs this cycle to result in a net production of the active signal transducing molecule (see Koch, 1992).
Whereas genetic approaches can yield information about
the role of particular molecules during plant growth, biochemical methods can allow the activities of signal transducing
intermediates to be identified. However, as with genetic approaches, biochemicalstudies of plant signal transduction can
be hampered by insufficient knowledge of stimulus-response
specificity. Consequently, although several signal transducing molecules (both second messengers and proteins) have
been identifiedbiochemically, their roles in specific responses
are often obscure.
Notwithstanding the limitations of genetic and biochemical
approaches, intensive research is now yielding information
about several signal transduction pathways in plants, and some
consistem features have emerged. In this review, we attempt
to highlight these themes by discussing recently published
work on signaling mediatedby phytochrome, ethylene, abscisic
acid (ABA), and auxin. Particular emphasis is given to the negative control of these pathways, because it is one recurring
theme that has emerged. We have illustrateda variety of ideas
by presenting models of signal transduction that are consistent with the currently available experimental data. These
schemes are intended to be thought provoking and may not
necessarily be correct. This review is by no means exhaustive, and the reader is referred to the original articles as well
as to other recent reviews on various other aspects of plant
signaling involved in these and other responses(Trewavas and
Gilroy, 1991; Estelle, 1992; Bush, 1993; Deng, 1994; Jones,
1994; Lamb, 1994).
PHYTOCHROME
Phytochrome is the only plant protein that has been unequivocally identified as a receptor (for review, see Quail, 1991; Furuya,
1993). Phytochromeapoproteins are usually encoded by small
multigene families-for example, five phytochrome genes
(PHYA-PHYE) have been identified in Arabidopsis (Sharrock
and Qyail, 1989; Clack et al., 1994)-and each phytochrome
is thought to have a different physiological role (Whitelam and
Harberd, 1994). Holophytochromeis a dimer containing a linear, covalently bound chromophore that mediates light
perception. The photoreceptor is synthesized in the redabsorbing Pr form, and absorption of red light alters its conformation to the far-red-absorbing form, Pfr, which is generally
considered to be the physiologicallyactive form. Pfr responses
can be reversed by far-red light, which converts Pfr back to
Pr. Of all the photoreceptors so far characterized, this property, that is, to behave as a molecular light switch, is unique
to phytochrome. Its molecular structure is also unique; although
we know that it is a receptor, it does not look like any other
receptor so far characterized. Most conspicuously, phytochrome is cytoplasmic, and as yet there is no convincing
evidence that it ever uses membranes as a platform to mount
its biochemical reactions (Quail, 1991).
How the phytochrome molecule transduces the light stimulus is still unknown. Structure-function studies have not
answered many of the crucial questions about phytochrome
activity, largely because the studies have been confined to
overexpression of mutant phytochrome gene sequences in
wild-type plants, which also contain the endogenous phytochromes. Although much of the N-terminal region, which
contains the chromophore binding domain, is involved in light
detection, it appears that the C-terminal region is more likely
to be involved in mediating biological activity, that is, signal
transduction (Cherry et al., 1993; Boylan et al., 1994). For example, removal of the C-terminal 35 residues from oat PHYA
results in a protein that is physicochemically normal in vitro
but has no activity in vivo (Cherry et al., 1993). Nonetheless,
the first N-terminal 69 residues are probably also important
in this regard (Cherry et al., 1992; Stockhaus et al., 1992).
In addition to genetic approaches (see below), phytochrome
signal transduction has been studied by a variety of biochemical approaches: (1) using pharmacological agonists and
antagonists to interferewith phytochrome-mediatedevents, (2)
studying phosphorylation reactions or examining the effects
of antibodies that bind to signal-transducingcomponents of
animal cells, and most recently, (3) using a microinjection-based
approach to deliver putative signaling intermediates directly
into the cells of a phytochrome-deficient tomato mutant. These
approaches (reviewed in Millar et al., 1994) have identifiedheterotrimeric Ggroteins as the most upstreamcomponent of the
phytochrome signaling machinery, although this has not been
definitively proven (because the G-protein(s)has not yet been
purified and characterized). If true, this would indicate that
phytochromeeither localizes transiently to membranes or acts
through an intermediarytransducer to activate this membranebound complex. Alternatively, it is possible that the component with which phytochrome interacts does not have all the
characteristics of animal heterotrimeric G-proteins, which at
any rate are not integral membrane proteins, being localized
to membranes via lipid tags (Casey, 1994).
Downstream of G-protein activation, phytochrome utilizes
Ca2+and cGMP (Neuhaus et al., 1993; Bowler et al., 1994a).
A role for Ca2+ in phytochrome signaling was first postulated
Plant Signal Transduction
10 years ago, and circumstantial evidence has accumulated
since then to support this hypothesis (reviewed in Tretyn et
al., 1991; Millar et al., 1994). However, it is only recently that
a red/far-red-reversible Ca2+ transient was directly observed
in plant cells (Shacklock et al., 1992). Subsequently, microinjection of Ca2+ and Ca2+-activated calmodulin (Ca2+/CaM) into
phytochrome-deficient tomato cells provided direct evidence
that these molecules are involved in PHYA signal transduction, specifically in stimulating chloroplast development
(Neuhaus et al., 1993). Using the same microinjection methodology, cGMP was found to mediate PHYA-dependent
anthocyanin biosynthesis and also to participate, together with
Ca2+ and Ca2+/CaM, in coordinating chloroplast development
(Bowler et al., 1994a). The use by the plant of Ca2+ and cGMP
to mediate phototransduction has an interesting evolutionary
parallel with the photoperception mechanisms found in retinal cells, even though the photoreceptors, phytochrome in the
plant and rhodopsin in the retina, are structurally completely
unrelated (Koutalos and Yau, 1993).
Figure 1A summarizes our current biochemical knowledge
of PHYA signal transduction pathways, based on the results
of microinjection experiments in tomato and studies of endogenous gene expression in soybean cells (Neuhaus et al., 1993;
Bowler et al., 1994a, 1994b). Most interestingly, the targets of
the three different positively acting pathways are divided very
clearly along functional lines. The cGMP pathway appears,
thus far, to be dedicated to the production of the photoprotective anthocyanin pigments, whereas the Ca2+- and Ca2+/
cGMP-dependent pathways control chloroplast development
by stimulating genes encoding subunits of the photosynthetic
complexes.
Obviously, with these divisions along functional lines, it is
very important that the final responses of the different pathways be carefully coordinated to allow, for example, for
photoprotectant production before photosynthesis (known as
juvenile anthocyanin; Drumm-Herrel, 1984) and to ensure
proper stoichiometry of the different photosynthetic complexes.
We have begun to understand how coordination is achieved
by specifically manipulating signal flow through the different
pathways, either by microinjecting large or small amounts of
signal intermediates or by using pharmacological agonists and
antagonists (Bowler et al., 1994b). Coordination appears to be
controlled by negative regulatory mechanisms acting at the
level of individual pathways (e.g., desensitization of the cGMP
pathway to terminate light-induced expression of the genes
encoding anthocyanin biosynthetic enzymes, such as chalcone synthase [CHS], in continuous light) and by cross-talk
mechanisms between the pathways (Bowler et al., 1994b).
The negative interactions between the pathways (summarized in Figures 1B and 1C), which we have collectively termed
reciprocal control, are presumably important at different times.
We believe that the negative regulation of the Ca2+ and
Ca2+/cGMP pathways by high levels of cGMP (Figure 1C) may
act during the early stages of light exposure to suppress synthesis of photosynthetic complexes at a time when there are
not sufficient photoprotectants, whereas negative regulation
1531
Pfr'
Pfr'
r
iu]
* chs
Negative
IRegulation
» cab
^
Pfr'
chs
iu|
^ cab
Figure 1. Summary of Current Biochemical Models of PHYA Signal
Transduction.
(A) Design of the three basic signaling pathways dependent on cGMP
and/or Ca2+. The products of these pathways (anthocyanin pigment
formation within the vacuole and chloroplast development within the
cytoplasm) are shown in highly schematized cells. Examples of gene
targets for each pathway (chs, fnr, and cab) are also indicated. Inhibitors specifically blocking the different pathways (Gen. and Trif.) are
shown and their block points indicated by two parallel lines drawn across
the arrows within the main pathways.
(B) Negative regulation of the cGMP pathway by the Ca2+ pathway.
(C) Negative regulation of the Ca2+- and Ca2+/cGMP-dependent pathways by the cGMP pathway.
In (B) and (C), the blue lines indicate the negative interactions, and
brackets indicate the positions of the components that current information suggests are responsible for these negative interactions. In
(B), negative regulation does not affect the Ca2+/cGMP-dependent
pathway, whereas in (C) it does. Go, a subunit of heterotrimeric
G-protein; CaM, Ca2+-activated calmodulin; Gen., Genistein; Trif.,
Trifluoperazine; chs, chalcone synthase; fnr, ferredoxin NADP+ oxidoreductase; cab, chlorophyll alb binding protein.
1532
The Plant Cell
of the cGMP pathway by the Ca2+pathway (Figure 16) suppresses the production of anthocyanins when they are no
longer required. These hypotheses are generally supported
by the timing of expression of genes encoding anthocyanin
biosynthetic enzymes and chloroplast components in soybean
cells (Bowler et al., 1994b) and in seedlings exposed to red
or white light (Drumm-Herrel, 1984; Brodenfeldt and Mohr,
1988; Wenng et al., 1990; Ehmann et al., 1991). Furthermore,
because we have found that the Ca*+/cGMP-dependentpathway has a lower concentration requirement (probably by nearly
an order of magnitude) for cGMP than does the cGMPdependent pathway (Bowler et al., 1994b), it would appear that
full chloroplast development and synthesis of all the photosynthetic components can proceed in the absence of anthocyanin
pigment biosynthesis.
Such a scenario clearly highlights the importance of negative regulation for coordinating phytochrome signal transduction.
Although we can only speculate about the precise mechanisms
involved, the experimental results so far obtained (Bowler et
al., 1994b) indicate that the most obviously apparent forms
of negative regulation involve interactions betweendownstream
components of the signaling pathways (Figures 1B and 1C).
In addition to these interactions, the sheer number of examples from animal cells would tend to suggest that phytochrome
signaling will also be attenuatedat the leve1of the photoreceptor
itself. One possibility is that phytochrome is inactivated by phosphorylation, like rhodopsin (Kawamura, 1993) and many other
animal receptors. Biochemical data have indicated that phytochrome is phosphorylated in vivo (Hunt and Pratt, 1980).
Furthermore, mutation of PHYA N-terminal serine residues to
alanine residues results in a molecule that has increased biological activity (Stockhaus et al., 1992), suggesting that one
desensitization mechanismfor phytochromesignaling may normally be the phosphorylation of these residues. Wong et al.
(1986) have purified a polycation-dependent kinase that may
be responsible for phosphorylating phytochrome in vivo, and
its targets indeed appear to be N-terminal serine residues, at
least in vitro (McMichael and Lagarias, 1990). A different
phytochrome-associatedkinase has also been purified (Grimm
et al., 1989). Interestingly, unlike the polycation-stimulated kinase, this kinase has been found to be stimulated by Ca2+
and cGMP (Grimm et al., 1989). This observation suggests
that the two signaling molecules that PHYA uses for its responses may also regulate photoreceptor desensitization,
providing a mechanism for feedback regulation to control the
activity of the photoreceptor.
It may also be significant that the Pfr form of PHYA is known
to be very unstable (perhaps becauseof ubiquitination; Jabben
et al., 1989) and is rapidly sequestered into large aggregates
in the cytoplasm (Quail, 1991). Controlled removal of physiologically active PHYA from its signaling machinery by
sequestration andlor degradation would clearly constitute a
form of desensitization,and such mechanisms have now been
found in severa1other signaling systems (see below). Although
the other phytochromes are not sequestered, sequestration
may well prove to be one mechanism for attenuating PHYA
signal transduction.
The importance of negative regulation for light responses
is underlined by the types of mutant that have been isolated
in screens for mutants with defective photomorphogenesis.
In Arabidopsis, two types of mutant have been studied (Chory,
1993): (1) those that have a partially etiolated morphologywhen
grown in the light (e.g., a long hypocotyl), and (2) those that
show characteristics of light-grown plants when grown in the
dark (e.g., short hypocotyls, open cotyledons, and expression
of light-regulatedgenes). In simple terms, the former class can
be thought of as light-insensitive mutants, whereas the latter
can be thought of as mutants constitutive for light responses.
The light-insensitive mutants are recessive, indicating that
the mutations most likely affect positively acting components
of the light signaling machinery. For example, one would expect that some are deficient in photoperception and may
contain mutated photoreceptors. This has been borne out by
the characterization of the genetic lesions of mutants within
this class, denoted hy for long hypocotyl. hyl, hy2, and hy6
mutants are believed to have defects in phytochrome chromophore biosynthesis or attachment (Parks and Quail, 1991;
Nagatani et al., 1993); hy3 mutants (now denoted phyB; Quail
et al., 1994) have mutations in their PHYB genes (Reed et al.,
1993); hy8 mutants (now phyA; Quail et al., 1994) have defective PHYA genes (Dehesh et al., 1993; Nagatani et al., 1993;
Whitelam et al., 1993); and hy4 mutants are probably blue-light
photoreceptor mutants (Ahmad and Cashmore, 1993). One
would also expect some hy mutants to be defective in positively acting light signal transduction intermediates; however,
with the possible exceptions of hy5 (Chory, 1992), and fhyl
and fhy3 (Whitelam et al., 1993), such mutants have not been
found.
The paucityof mutants that are likely to bedefective in positive elements downstream of phytochrome suggests that such
mutants are extremely difficult to find in these screens. If signa1 transduction from each of the phytochromes were to
converge in the same pathway, such mutations might even be
lethal, because too many processes would be affected. Observations that PHYA and PHYB do have some overlapping
functions (Reed et al., 1994) would argue that at least some
signaling pathways are indeed shared. However, the fhyl and
fhy3 mutants do appear to affect PHYA responses specifically
(Whitelam et al., 1993; Johnson et al., 1994). Whether the
cGMP and Ca2+ pathways that have been defined biochemically (Figure 1) are used specifically by PHYA or are shared
by different phytochromesis an interesting question. Although
these pathways have been linked to PHYA (Neuhaus et al.,
1993; Bowler et al., 1994a), it is clearly possible that they may
also be used by the other phytochromes or even by the
bluelUVA photoreceptors.
The “constitutive” mutants, called der (for deetiolated) and
cop (for constitutive photomorphogenic), were isolated in
Plant Signal Transduction
screens for seedlings showing light-grown morphologies(e.g.,
short hypocotyls and open cotyledons) in darkness (discussed
in Chory, 1993; Deng, 1994). The recessive nature of these
mutantssuggests that the mutations lie within negative regulatory molecules. Epistasis tests using the hy mutants have
indicated that the gene products function downstream of both
the phytochromes and the bluelUVA photoreceptors (Chory,
1993; Deng, 1994; but see Millar et al., 1994). It is difficult,
however, to conclude that they are light perception and phototransduction mutants because their phenotypes are not
restricted to those we would expect if photoreceptor signaling
was specificallyderegulated (discussed in Millar et al., 1994).
The problem lies in the complexity of the photomorphogenic
developmentalprogram, because photomorphogenictraits are
influenced not only by light but also by (at least) cytokinin and
ABA (Millar et al., 1994). For example, it has been shown that
der7 can be phenocopied in wild-type seedlings by addition
of cytokinin (Chory et al., 1991, 1994). Furthermore, the fact
that six of the 11 constitutive mutants characterized to date
(deu, cop7, cop8, cop9, cop70, and cop77) are allelic to previously isolated seedling lethal mutants known as fusca (fus)
mutants (Castle and Meinke, 1994; McNellis et al., 1994; Misbra
et al., 1994; Wei et al., 1994a) also indicates that the mutant
phenotypesare more pleiotropic than was originally suspected.
With the realization that morphological screens do not allow the specific isolation of mutants with altered phytochrome
responses (except for the elegant utilization of responses to
constant red or far-red light irradiationto discriminate between
different phytochromes; Parks and Quail, 1993), other screens
have been developed in an attempt to improve specificity.
These screens have been based on the aberrant expression
of a light-regulatedhygromycinresistancegene (Susek et al.,
1993; Li et al., 1994). Although the mutants isolated have not
yet been characterized to determine whether they are defective specifically in the response to phytochrome, the type of
screens used (i.e., designed to isolate mutants constitutivefor
a response in the absence of the normal stirnulus) and the
fact that all but one of the mutations are recessive indicate
that these mutants are blocked in some aspect of negative
regulation.
Although genetic screens for constitutive mutants select predominantlyfor mutantswith defects in negative regulators, the
number of mutants now isolated indicates that negative regulation may be a common mechanism for controlling plant
photomorphogenesisand is perhaps as important as regulation by positive elements. Even though the cop and der mutants
do not appear to have defects specific to phytochromesignaling, their phenotypes imply that the mutations lie within
negativelyacting global regulatorsfor plant development that
are able to receive signals from multiple stimuli and integrate
them into a morphological (i.e., developmental) response. Because many of the cop and der mutants are allelic to seedling
lethal mutants, it has been suggested that this class of molecule is critically important for controlling certain aspects of plant
1533
development (Castle and Meinke, 1994). It is clearly of great
importance, therefore, to understand how such molecules work.
We now have knowledge of the structure of four of these
negative regulators: FUSG (COPll), COP9 (FUS7), DETl
(FUS2), and COPl (FUS1). FUSG (Castle and Meinke, 1994)
and COP9 (Wei et al., 1994b) have entirely novel sequences
but are probably cytoplasmically localized. COP9 exists as a
large complex whose formation or stability requiresCOP8 and
COP11, because it is absent in cop8 and cop77 mutants (Wei
et al., 1994b). This is some of the first evidence for molecular
interactions among different COPlDETlFUS gene products and
indicates that at least COP8, COP9, and COP11 may act together to control a particular biochemical process. The
sequence of DETl is also novel, although the protein is nuclear (Pepper et al., 1994). Unlike these novel proteins, COPl
contains a combinationof recognizablestructural motifs: a zine
finger domain at the N terminus (similar to a newly characterized zinc binding domain called a RlNG finger; Boddy et al.,
1994), a putative coiled-coilregion, and a domain at the C terminus with multiple WD-40 repeats homologous to the p
subunits of heterotrimeric G-proteins (Deng et al., 1992;
McNellis et al., 1994).
In spite of an extensive analysis of the COP7 gene in an allelic series of cOp7 mutants, no domain has clearly emerged
as being more important than the others (McNellis et al., 1994).
However, the homology of COPl (particularly the Gghomologous domain) with the Drosophila TAF1180 protein, a
subunit of the TFllD complex that is required for RNA polymerase II transcription initiation, may suggest a role for COPl
in repressing general transcription (McNellis et al., 1994), which
would be consistent with its expected function as a negatively
acting global regulator of plant responses. If so, COPl would
be expected to be a nuclear protein. The nuclear localization
of DETl may hint that its function is also to repress transcription. Although DETl does not appear to bind DNA directly
(Pepper et al., 1994), its repressor activity may be manifested
ata much more general level, for example, by remodeling the
nucleosomes to organize repressive regions of chromatin
(Cooper et al., 1994; lmbalzano et al., 1994; Kwon et al., 1994).
COPl may have a similar function, because Gp-homologous
domains are present within at least one such global repressor, yeast TUPl (Deng et al., 1992; Cooper et al., 1994).
Biochemical experiments are clearly required to examine this
possibility.
The current lack of overlap between the biochemical (Figure 1) and the genetic (Chory, 1992; Ang and Deng, 1994)
models of phytochrome signaling deserves some comment
and is a major challenge to address in the future. Severa1points
are relevant. First, the biochemical pathways have been defined mainly by their positive elements, whereas all the gene
products inferred from mutant studies are likely to be repressors
(with the possible exceptions of hy5, fhy7, and fhy3). This is
simply a result of the different experimental approaches: the
microinjection experimentswere designed to identify positively
1534
The Plant Cell
acting signal intermediates, whereas the genetic screens were
designed to yield predominantly negative regulators.
Recent biochemicalstudies of the negative regulatory mechanisms involved in phytochromesignaling (Bowler et al., 1994b)
can, however, allow us to predict the phenotypes of mutants
altered in specific negative regulatory aspects of phytochrome
signaling, for example, in CHSdesensitization or in reciproca1
control. Recessive mutants of this kind might not be expected
to be constitutive for a light response in darkness (as are cop
and detmutants) because at this leve1 in the signal transduction
pathway, there does not appear to be a “dark current” (i.e., turnover; see Introduction) of Ca2+ or cGMP (Bowler et al., 199413).
With respect to their responses in the light (a condition under
which we do believethere is a turnover of second messengers;
Bowler et al., 1994b), phenotypes would be more apparent.
For example, mutant seedlings unable to repress the Ca2+
and Ca2+/cGMP-dependentpathways (Figure 1C) might be
expected to display a light fluence-dependent bleaching due
to chloroplast development before sufficient photoprotectants
have been synthesized. Because the screens used so far to
isolate photomorphogenic mutants have been based largely
on aberrant phenotypes in darkness rather than aberrant
phenotypes in dynamic responses to light signals, such mutants have not yet been studied.
Currently, the simplest way to reconcile the apparent absence of a “dark current” with the constitutive phenotypes of
cop and det mutants is if COP and DET act downstream of
the second messengers. For example, they could be general
transcriptional repressors normally inactivated by Ca2+and
cGMP, among other things. In their absence (i.e., in mutants),
light-responsiveand other genes would no longer be repressed
in the dark and constitutive transcription would ensue, even
though Ca2+and cGMP levels would remain unaltered. Current information about the structure of COP1 and DET1 does
indeed suggest that they are somehow involved in regulating
transcription (see above). The possibility that COP and DET
act downstream of Ca2+and cGMP can be tested by examining whether or not the pharmacological inhibitors of the
phytochrome pathways (Bowler et al., 1994b) are able to reverse the constitutive phenotypes of the mutants in the dark.
A second important difference betweenthe biochemicaland
genetic data is that the biochemical pathways have defined
very specific cellular reactions (leading to chloroplast development and anthocyanin biosynthesis) and have not even
begun to address the developmental inputs that may affect
the stimulation of these responses. Anthocyanin biosynthesis, for example, is known to be under exquisite photobiological
control during development (Drumm-Herrel, 1984; Frohnmeyer
et al., 1992; Nick et al., 1993). The majority of mutants have,
by contrast, been isolated based on their developmental aberrations. Most likely, therefore, the gene products affected in
these mutants are involved in coordinating global developmental switches to a wide range of different signals, including
light, rather than being specific to some aspect of the phytochrome cellular signaling system that has so far been defined
biochemically.
ETHYLENE
Gaseous ethylene is an important growth regulator, influencing a variety of plant responses that include germination, leaf
abscission, and fruit ripening, as well as responsesto environmental stresses such as wounding, chilling, and pathogen
attack. In contrast to phytochrome, biochemical approaches
have contributed little to our current knowledge of ethylene
signal transduction, and recent interest has arisen largely as
a result of genetic approaches that have identified gene
products involved in regulating ethylene responses. These
approaches have exploited a morphological response to ethylene observed in etiolated seedlings known as the “triple
response:’which comprises three distinct changes in seedling
morphology: inhibition of hypocotyl elongation, hypocotyl thickening, and an exaggerated tightening of the apical hook. As
previously described for the photomorphogenesis mutants,
both “insensitive” and ‘%onstitutive” mutants have been
screened for and isolated from Arabidopsis (Guzmán and
Ecker, 1990). The success of these morphological screens is
a result of the fact that the simultaneous appearance of these
three responses is highly specific for ethylene; hence, the
mutants isolated are very likely to have defects in the same
signal transduction pathway. This greatly facilitates the interpretation of results from epistasis tests and contrasts with
the mutants so far isolated in screens for photomorphogenic
phenotypes, which may have defects in any one of severa1
signaling pathways.
For the ethylene-insensitive mutants, three complementation groups have been reported, designated etrl (Bleecker et
al., 1988) and ein2 and ein3 (Guzmán and Ecker, 1990; Kieber
et al., 1993). etrl mutations are dominant and are allelic to the
dominant einl mutants isolated by Guzmdn and Ecker (1990;
Chang et al., 1993). ein2 and ein3, on the other hand, are recessive mutations. From screens for constitutive triple response
mutants, two mutant classes have been identified that can be
distinguished based on whether or not they overproduce ethylene (Kieber et al., 1993). Ethylene overproducersare not likely
to be mutated in ethylene perception or signal transduction,
whereas those that have normal levels of ethylene are. Of this
latter class, only one complementation group has currently
been reported, designated ctr7. The recessive nature of the
constitutive phenotype indicates that CTRl is a negative regulator of ethylene responses. Epistatic relationships among
these mutants have been examined, and the results suggest
the order of action as being ETRl, CTRl, and EIN3 (Kieber
et al., 1993).
ETR7 has now been cloned, and the strong similarity of the
C terminus to domains of proteins involved in the twocomponent signal-transducing mechanisms of bacteria allows
some speculation about its possible mode of action (Chang
et al., 1993). The two-component pathway of prokaryotes, as
its name suggests, usually contains two proteins (Parkinson,
1993). One is called the “sensor” and contains both an “input”
domain (which senses the stimulus) and a “transmitter.” The
Plant Signal Transduction
1535
other, called the "response regulator," consists of both a "receiver" module (which receives the signal from the transmitter,
via phosphorylation) and an "output" domain, which triggers
the response. ETR1 contains a "transmitter-homologous domain and a "received-homologous domain downstream of an
N-terminal region of 313 amino acids whose sequence is novel.
It has been postulated that this N-terminal domain may be the
ethylene-sensing region, whereas the C terminus may be involved in signal transmission (perhaps like phytochrome). ETR1
may therefore be an ethylene receptor, as is also supported
by the fact that etrl mutants have a lower capacity to bind ethylene compared with wild-type plants (Bleecker et al., 1988).
The N-terminal region of ETR1 also contains three potential
membrane-spanning domains, indicating that the protein is
membrane localized, even though ethylene is able to diffuse
through membranes. All the etrl mutations so far characterized lie within these putative membrane-spanning domains
(Chang et al., 1993).
Based on the homology of the C terminus of ETR1 with the
prokaryotic proteins, it is possible that perception is signaled
via phosphorylation reactions. The prokaryotic transmitters are
phosphorylated on a histidine residue, and the conservation
of this residue in ETR1 suggests that histidine may also be
phosphorylated in ETRL Histidine phosphorylation was once
thought to be restricted to prokaryotes, but with the isolation
of not only Arabidopsis ETR1, but also yeast SLN1, an osmolarity sensor that also shows homology with the "transmitter" and
"receiver" domains of bacterial two-component systems (Ota
and Varshavsky, 1993), the interesting possibility now arises
that phosphorylation of histidine residues may prove to be an
important regulatory mechanism in eukaryotic cells.
CTR1 has also been cloned recently (Kieber et al., 1993).
It contains no obvious membrane-spanning domains, but its
C terminus has considerable homology with protein kinases,
particularly with members of the Raf family of serine/threonine kinases, which activate MAP kinase cascade pathways
(Blumer and Johnson, 1994). All five mutations sequenced are
in the putative kinase catalytic domain. The utilization by the
plant of ETR1 and CTR1 in ethylene signal transduction therefore appears to be an interesting combination of a prokaryotic
signaling system with a typical eukaryotic one. A similar system has also been found in yeast for controlling adaptation
to osmotic stress (Maeda et al., 1994).
How does ethylene signal transduction work? One possible model, based on current information, is presented in Figure
2. It is reasonable to speculate that ETR1 is an ethylene receptor and that it controls ethylene responses via a phosphorylation
cascade that involves CTR1 and EIN3. Because CTR1 appears
to be a kinase, it is unlikely that it acts by itself, and the phosphorylation status of its substrate probably is also controlled
by a "complementary" protein phosphatase. However, the nature of the phenotype conferred by Ctrl and the fact that the
mutations are recessive suggest that CTR1 is a negative regulator of ethylene responses. Furthermore, the fact that the
phenotype is manifested in the absence of exogenous ethylene would therefore indicate that there is always a flux through
Response
Figure 2. Possible Model for Ethylene Signal Transduction for Controlling the Triple Response.
ETR1 is shown as a dimeric plasma membrane-localized ethylene
receptor whose phosphorylation status, indicated by 0 , is altered
by ethylene binding. Possible interactions with the CTR1-regulated step
are indicated by gray arrows. CTR1 is shown as being activated by
phosphorylation and as repressing the activity of its target by phosphorylating it. A phosphatase is proposed to work together with CTR1
to control the phosphorylation status of the CTR1 target. EIN3 is shown
as subsequently being activated by phosphorylation mediated by the
target of CTRL It is also possible that EIN3 is the direct downstream
target of CTRL A thick arrow indicates the unknown remainder of the
pathway leading to the triple response.
the pathway (perhaps as a result of low constitutive activities
of the phosphatases and kinases; see Introduction) and that
one role of CTR1 is to attenuate this signal flow in the absence
of ethylene by phosphorylation of its target substrate(s). Mutation of CTR1 would then prevent phosphorylation of its target,
allowing the basal level signal to pass this regulatory step unhindered, thus leading to the triple response in the absence
of ethylene. In this model, dephosphorylation would be the
forward reaction, at least for the CTR1-controlled step.
The triple response elicited by exogenous ethylene probably occurs as a result of increased flux through the pathway
initiated by a phosphorylation cascade from ethylene-stimulated ETRL If ETR1 were to interact directly with the
CTR1-regulated step in the presence of ethylene, it could overcome CTR1 repression by inactivating CTR1, by activating the
phosphatase partner of CTRL or by both (Figure 2). In either
case, the ethylene-insensitive phenotype of etrl mutants can
be most simply explained if the mutations were to result in an
inactive protein that can no longer carry out its subsequent
interaction(s). Dominance could then be explained if ETR1
1536
The Plant Cell
functions as a multisubunit complex, because the presence
of mutant subunits would disrupt its activity (Chang et al., 1993).
Some of the two-component regulatorsof bacteria are known
to function as dimers (Parkinson, 1993).
If ETRl were to interactwith the CTR1-controlled step in the
absence of ethylene (e.g., to activate CTRl), other, more complicated schemes could be proposed to account for the
phenotypes of the mutants (discussed in Chang et al., 1993).
However, with the current absence of any biochemical information about an interaction between ETRl and CTRl, all
models remain highly speculative. First and foremost, kinase
activity must be demonstratedfor each protein. Subsequently,
important questions that can be answered include whether
ETRl works as a multimer, how the activities of ETRl and CTR1
change in response to ethylene, and how their activities are
altered in etrl and ctrl mutants.
If there is a basal leve1 flux through the pathway in the absence of ethylene, ectopic expression of CTRl would be
predicted to result in ethylene insensitivity, because the equilibrium of the phosphorylation reaction would have shifted
toward the production of the inactive form of the target of CTRl.
However, ectopic expression of a modified (i.e., deregulated)
CTRl molecule should be even more effective in causing ethylene insensitivity.
Overexpression of the gene that encodes the protein phosphatase partner of CTRl (Figure 2) would cause a constitutive
triple response unless its activity were also regulated by ethylene, in which case only a deregulated form might show a
constitutivetriple response. In analogy with these predictions,
pharmacological inhibitors of CTRl activity might cause a triple response, whereas an inhibitor of the phosphatase might
prevent it. Some experiments detailing the effects of protein
kinase and phosphatase inhibitorson ethylene responses have
been reported, although these have been studied with respect
to their effects on the ethylene-mediatedinduction of pathogenesis-related genes (Raz and Fluhr, 1993). The results
indicate that protein kinase inhibitorscan block the response,
whereas protein phosphatase inhibitors can stimulate pathogenesis-related gene induction in the absence of ethylene, the
opposite of what we would predict if CTRl and its phosphatase partner were, respectively, the targets of these inhibitors.
In similar experiments, Ca2+ has also been implicated as a
second messenger in ethylene-mediated pathogenesis responses (Raz and Fluhr, 1992), although its potentialto regulate
the phosphorylationcascade involved in the triple response
remains to be critically evaluated.
ABSClSlC AClD
Among other things, ABA regulates embryo maturation and
seed dormancy and inhibits mitotic activity in root meristems.
During stress conditions such as water shortage, it also induces
stomatal closure (Zeevaart and Creelman, 1988). Mutants
defective in these responses have been isolated in screens
for seedlings that grow on normally inhibitory concentrations
of ABA. Much like the hy mutants, these mutants can therefore be thought of as “insensitives,”and they have been named
abi mutants, for abscisic acid insensitive (Koornneef et al.,
1984). These mutants are severely impaired in a wide spectrum of ABA responses, and they show phenotypes including
excessive water loss (causedby an inabilityto regulate stomatal
function) and reduced seed dormancy. These phenotypes
would therefore suggest that the mutations affect ABA responses specifically. One such mutant, abil, which is dominant,
has recently been cloned (Leung et al., 1994; Meyer et al.,
1994). The gene product, ABI1, has a nove1 structure containing a domain highly homologousto the serineAhreonine protein
phosphatase type 2C, together with an N-terminalCa2+binding site. Functionally,therefore, it may be the plant equivalent
of calcineurin, the Ca2+-dependentphosphatase present in
animal and yeast cells, even though it is structurallyvery different: calcineurin is a heterodimeric protein composed of a Ca2+
binding regulatory domain and a catalytic subunit that has no
homology with protein phosphatase type 2C (Cohen, 1989).
There is very good evidence for the involvement of Ca2+in
ABA responses, at least in controlling stomatal closure (Gilroy
et al., 1991). Logically, therefore, the structure of ABI1 would
indicate that it is a downstream target of ABA-stimulated Ca2+.
This can be tested by examining ABA-mediated Ca2+ increases in wild-type and mutant cells and by examining whether
responses to artificially increased Ca2+(e.g., stomatal closure
induced by release of caged Ca2+;Gilroy et al., 1990) are affected in the mutant.
Based on current information, models of ABA signaling
should incorporate a plasma membrane-localized receptor
(Anderson et al., 1994; Gilroy and Jones, 1994), Ca2+, and
ABI1 and should account for the dominant nature of the abil
mutation (Koornneef et al., 1984). In simple terms, two functional
possibilities exist, dependent upon whether phosphorylation
or dephosphorylation is the forward reaction for the ABI1regulated step in ABA responses (Figure 3). If dephosphorylation is the forward reaction, then ABA-mediated increases
in cytosolic Ca2+ may activate ABI1, which would then
dephosphorylate its target (Figure 3A). If phosphorylation is
the forward reaction, ABll would be predicted to function as
a negative regulator, constitutively suppressing forward flow
through the signal transductionpathway in the absence of ABA
(Figure 36). In the first scenario (Figure3A), dominance could
be explained most simply if ABll were part of a multisubunit
complex, because the mutant subunits would disrupt the activity of the complex. In the latter scheme (Figure 3B), the
dominance of the abil mutation could be explained if the mutation were a result of a loss of sensitivity to Ca2+, resulting
in phosphataseactivity that was no longer repressible by Ca2+
and hence causing a permanent switching off of the pathway.
Once phosphatase activity of ABll has been demonstrated,
Plant Signal Transduction
ABA—^H -
Response
B
-I-
Response
Figure 3. Two Possible Models for the Role of ABU in ABA Signaling.
The ABA receptor is shown to be localized to the plasma membrane
and to receive the ABA signal externally (see Anderson et al., 1994;
Gilroy and Jones, 1994). In both models, an increase in cytosolic Ca2+
is shown to precede ABU action, and the putative protein kinase partner of ABU is shown as being regulated by Ca2+ in the opposite
manner to ABU.
(A) A model in which dephosphorylation, mediated by Ca2+-activated
ABU, leads to activation of the target of ABU and consequently to ABA
responses.
(B) An alternative model in which phosphorylation of the ABU target
leads to its activation and to subsequent ABA responses. In this case,
Ca2+ would be predicted to repress ABU activity.
biochemical experiments should be able to resolve these different possibilities.
AUXIN
Auxin has been implicated in regulating almost every aspect
of plant development. Although we now have some understanding of its biosynthesis, biochemical responses, and physiology,
molecular studies of its action have proven very difficult (Estelle,
1992). As may also be the case with other hormones, one likely
way that auxin responses are regulated is by controlling the
amount of free hormone that is available for signal transduction (Szerszen et al., 1994). The conjugation of free hormone
to form inactive pools can potentially attenuate signal transduction, whereas its release from these conjugates could
activate it. In addition to this type of control, two recent articles have now provided other clues about auxin signaling
mechanisms (Leyser et al., 1993; Abel et al., 1994).
The first article details the cloning of the auxin resistance
gene AXR1 from Arabidopsis (Leyser et al., 1993). To date, 20
axrl mutants have been isolated in screens for mutants able
1537
to grow on normally inhibitory concentrations of auxin, and
all are recessive (Estelle and Somerville, 1987; Lincoln et al.,
1990). Other phenotypes of these mutants include decreases
in height, in apical dominance, in root gravitropism, in hypocotyl
length, and in fertility. Cloning and sequencing of the AXR1
gene revealed significant similarity to the ubiquitin-activating
enzyme E1 (Leyser et at., 1993), although there are sufficient
important differences between the sequences to make it clear
thatAXRI is not an E1 homolog. Nonetheless, the similarities
do suggest that the ubiquitination pathway for protein degradation may somehow play an important role in auxin responses.
This possibility has also been inferred from an independent
series of experiments. Abel et al. (1994) have found that a family
of conserved genes induced rapidly and strongly by auxin in
excised stem sections encodes nuclear proteins that have very
short half-lives. Each of these proteins contains a motif similar to that found in the DNA binding domain of prokaryotic
represser proteins such as Arc, suggesting that they are DNA
binding proteins that may regulate auxin responses. Their short
half-lives have been proposed to be a result of selective degradation by ubiquitination pathways that possibly involve the
AXR1 gene product (Abel et al., 1994), a possibility that can
now be tested by examining the half-lives of these proteins
in axrl mutants. Overall, this would provide a mechanism for
executing rapid responses to auxin that could be equally rapidly attenuated.
A proposed role for AXR1 must account for the hormoneinsensitive, recessive nature of axrl mutants. Most simply, there
are two formal possibilities: (1) that it degrades a represser
(i.e., that it negatively regulates a negative regulator) or (2) that
it is involved in positively regulating auxin responses. The second possibility is difficult to reconcile with a role in protein
degradation, although auxin responses may involve the repression of other pathways. AXR1 could achieve this by degrading
key positive regulators within these pathways. Further genetic
and biochemical characterization is necessary to examine
these possibilities. Unfortunately, a major obstacle for assigning
an auxin-specific function to AXR1 will be the known pleiotropy
of auxin responses.
Although the function of AXR1 in auxin signal transduction
is unclear, its identification as a possible participant in ubiquitinmediated protein degradation is interesting because selective
protein degradation is an emerging theme for desensitizing
some signal transduction pathways in animal cells. Examples
include regulation of cyclins, the human growth regulator p53,
Drosophila Ftz, and yeast MATa2 (Gottesman and Maurizi,
1992). Compared with phosphorylation and other reversible
modifications, proteolysis is a drastic way to inactivate a protein, but it is especially important for controlling proteins that
are required either for only limited periods of time or under
specific developmental or metabolic conditions and whose
presence may be detrimental under other conditions. For example, cyclin acts to stimulate cell division, but its degradation
is an essential regulatory mechanism for allowing the cell to
advance to the next phase of the cell cycle.
1538
The Plant Cell
PERSPECTIVES
This brief overview of recent advances in the understanding
of plant signal transduction mechanisms serves to illUStrate
severa1 emerging features: (1) the involvement of nove1 proteins containing more than one functional domain, (2) the
concerted utilization of signaling systems previously thought
to be specific for prokaryotes or eukaryotes, (3) the presence
of basal leve1fluxes through signaling pathwaysin the absence
of a stimulus, (4) the central importance of negative regulation, and (5) the significance of cross-talk between pathways.
The mechanism by which hybrid molecules such as COP1,
ETR1, and A811 receiveand transduce signals will be important
to understand, because it is possible that they are central
players in transmitting cross-talk information, for example, between different hormones. Cross-talk is likely to be of extreme
importance in plants because of their developmental plasticity. The importance of cross-talk has indeed been inferred from
the phenotypes of many of the mutants isolated in screens
designed initially to be specific for one stimulus. Witness, for
example, the pleiotropy of the cop and det mutants and the
resistanceof auxin-resistantmutantsto other hormones (Pickett
et al., 1990; Wilson et al., 1990). Such mechanisms are likely
to be elucidated largely by genetic approaches; in fact, the
information already available gives us some clues about possible mechanisms. For example, A611 may be regulated by
Ca2+signals generated not only from ABA, but also from other
hormones or from phytochrome, because all are known to
stimulate increases in cytosolic Ca2+ (see Bush, 1993, and
referencestherein). Similarly, the COP and DET proteins may
be targets for more than one stimulus, thus allowing access
by different stimuli to the same signal transduction pathway
or at least the same target. In addition, it is possible that different signals activate the same target in different cell types or
that the same protein can behave as either an activator or a
repressor, depending on the context (Lehming et al., 1994).
The emergence of negative regulation as a central theme
in plant signaling may to some extent be a biased interpretation of the true situation because of the type of genetic screens
used to date. Nonetheless, there are clearly negative regulatory molecules of extreme importance in plant development,
of which the COR DET, and CTR1 proteins are obvious examples. Recessive mutations in some of the ACD and LSD genes
result in plants that spontaneously form necrotic lesions on
leaves even in the absence of pathogens (Dietrich et al., 1994;
Greenberg et al., 1994); these genes are also likely to encode
negative regulators, implying that the defense response to
pathogens may be mediated, to some extent, by release of
repression mechanisms. The presence of signaling pathways
primed to respond but blocked by repressors (such as the ethylene pathway and the defense response pathway) is a
conceptually simple as well as an efficient mechanism for permitting cross-talk. Moreover, the lack of rigid developmental
constraints during plant development may indicate that such
negative regulatory mechanisms will be found to be prevalent
in plant signal transduction.
Future work must confirm or identify by biochemicalmethods
the functions of gene products identified in genetic screens.
Activities proposed for different proteins-e.g., ETRI, CTR1,
and A811-must be demonstrated, their regulation understood,
and their substrates defined. The development of new methods
may also be required to open up a signal transduction pathway biochemically. For example, the extension of microinjection
and related methodologies to Arabidopsis should prove to be
extremely powerful, as has been recently shown for tomato
(Neuhaus et al., 1993; 8owler et al., 1994a).
Combined with biochemical approaches, we believe new
genetic screens should be designed to isolate mutants more
likely to be affected in specific signaling pathways. Rather than
designing screens aimed at isolating mutants that are constitutively switched on or off, it is now both feasible (Millar et al.,
1992) and worthwhile to design screens for isolating mutants
that are affected in their quantitative responses to different
stimulus strengths. By screening for aberrant phenotypes in
dynamic responses to a particular stimulus, mutants with
defects specific to the stimulus are more likely to be obtained.
Furthermore, the nature of the phenotype (e.g., a hypersensitive or an amplitude response) can give an indication of whether
the mutation affects the activity of the receptor or downstream
signal-transducing steps within the signaling pathway.
In summary, both the genetic and biochemical approaches
that have been applied to the study of plant signal transduction are clearly beginning to reveal the details of a variety of
signaling networks. The union of these approaches should now
be seen as the next major objective, because it will allow both
the elucidation of the underlying mechanisms and the identification of the molecules responsible. Ultimately, it should
permit a thorough understanding of how plants perceive,
respond, and adapt to changing developmental and environmental stimuli.
ACKNOWLEDGMENTS
We would like to thank our colleagues in the laboratory for many
stimulating discussions and especially Drs. Diana Horvath, Raphael
Mayer, and Diane Shevell for their comments on the manuscript.We
are also grateful to Dr. Joe Ecker for sharing his insightsinto ethylene
responseswith us. C.B. was supported in New York by a Science and
Engineering Research Council (NorthAtlantic Treaty Organization) postdoctoral fellowship and later by afellowshipfrom the Normanand Rosita
Winston Foundation. Research in New York was supported by National
lnstitutes of Health Grant No. 44640 to N.-H.C.
Received July 15, 1994; accepted September 16,1994.
REFERENCES
Abel, S., Oeller, P.W., and Theologis, A. (1994). Early auxin-induced
genes encode short-livednuclear proteins. Proc. Natl. Acad. Sci.
USA 91, 326-330.
Plant Signal Transduction
1539
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.
Chory, J., Reinecke, D., Sim, S.,Washburn, T., and Brenner, M.
(1994).A role for cytokinins in deetiolation in Arabidopsis. Plant Physiol. 104, 339-347.
Anderson, B.E., Ward, J.M., and Schroeder, J.I. (1994). Evidence
for an extracellular reception site for abscisic acid in Commelina
guard cells. Plant Physiol. 104, 1177-1183.
Clack, T., Mathews, S., and Sharrock, R.A. (1994). The phytochrome
apoprotein family in Arabidopsis is encoded by five genes: The sequences and expression of PHYD and PHYE. Plant MOI. Biol. 25,
413-427.
Ang, L.-H., and Deng, X.-W. (1994). Regulatory hierarchyof photomorphogenic loci: Allele-specific and light-dependentinteraction between
the HY5 and COP7 loci. Plant Cell 6, 613-628.
Bleecker, A.B., Estelle, M.A., Somenrille, C., and Kende, H. (1988).
lnsensitivity to ethylene conferred by a dominant mutation in
Arabidopsis thaliana. Science 241, 1086-1089.
Blumer, K.J., and Johnson, G.L. (1994). Diversity in function and regulation of MAP kinase pathways. Trends Biol. Sci. 19, 236-240.
Boddy, M.N., Fleemont, P.S.,and Borden, K.L.B. (1994). The p53associated protein MDM2 contains a newly characterized zincbinding domain called the RlNG finger. Trends Biol. Sci. 19,198-199.
Bowler, C., Neuhaus, G., Yamagata, H., and Chua, N.-H. (1994a).
Cyclic GMP and calcium mediate phytochrome phototransduction.
Cell 77, 73-81.
Bowler, C., Yamagata, H., Neuhaus, G., and Chua, N.-H. (1994b).
Phytochrome signal transduction pathways are regulated by reciprocal control mechanisms. Genes Dev. 8, 2188-2202.
Boylan, M., Douglas, N., and Quail, P.H. (1994). Dominant negative
suppression of Arabidopsis photoresponses by mutant phytochrome
A sequences identifies spatially discrete regulatory domains in the
photoreceptor. Plant Cell 6, 449-460.
Brijdenfeldt, R., and Mohr, H. (1988). Time courses for phytochromeinduced enzyme levels in phenylpropanoid metabolism (phenylalanine ammonia-lyase, naringenin-chalconesynthase) compared with
time courses for phytochrome-mediatedend-product accumulation
(anthocyanin, que rcetin). PIanta 176, 383-390.
Bush, D.S. (1993). Regulationof cytosolic calcium in plants. Plant Physiol. 103, 7-13.
Casey, P.J. (1994). Lipid modificationsof G proteins. Curr. Opin. Cell
Biol. 6, 219-225.
Castle, L.A., and Meinke, D.W. (1994). A FUSCA gene of Arabidopsis
encodes a nove1protein essentialfor plant development. Plant Cell
6, 25-41.
Chang, C., Kwok, S.F., Bleecker, A.B., and Meyerowitz, E.M. (1993).
Arabidopsis ethylene-response gene ETR7: Similarity of product to
two-component regulators. Science 262, 539-544.
Cherry, J.R., Hondred, D., Walker, J.M., and Vierstra, R.D. (1992).
Phytochrome requires the 6-kDa N-terminaldomain for full biological activity. Proc. Natl. Acad. Sci. USA 89, 5039-5043.
Cherry, J.R., Hondred, D., Walker, J.M., Keller, J.M., Hershey, H.P.,
and Vierstra, R.D. (1993). Carboxy-terminaldeletion analysis of oat
phytochrome A reveals the presence of separate domains required
for structure and biological activity. Plant Cell 5, 565-575.
Chory, J. (1992). A genetic model for light-regulatedseedling development in Arabidopsis. Development 115, 337-354.
Cohen, P. (1989). The structure and regulation of protein phosphatases. Annu. Rev. Biochem. 58, 453-508.
Cooper, J.P., Roth, S.Y., and Simpson, R.T. (1994). The global transcriptional regulators, SSN6 and TUPI, play distinct roles in the
establishment of a repressive chromatin structure. Genes Dev. 8,
1400-1410.
Dehesh, K., Franci, C., Parks, B.M., Seeley, K.A., Short, T.W.,
Tepperman, J.M., and Quail, P.H. (1993). Arabidopsis HY8 locus
encodes phytochrome A. Plant Cell 5, 1081-1088.
Deng, X.-W. (1994). Fresh view of light signal transduction in plants.
Cell 76, 423-426.
Deng, X.-W., Matsui, M., Wei, N., Wagner, D., Chu, A.M., Feldmann,
K.A., and Quail, P.H. (1992). COP7, an Arabidopsis regulatory gene,
encodes a protein with both a zinc-binding motif and a GBhomologous domain. Cell 7 l , 791-801.
Dietrich, R.A., Delaney, T.P., Uknes, S.J., Ward, E.R., Ryals, J.A.,
and Dangl, J.L. (1994). Arabidopsis mutants simulatingdisease resistance response. Cell 77, 565-577.
Drumm-Herrel, H. (1984). Blue/UV light effects on anthocyanin synthesis. In Blue Light Effects in Biological Systems, H. Sanger, ed
(Berlin: Springer-Verlag), pp. 375-383.
Ehmann, B., Ocker, B., and Schafer, E. (1991). Development- and
light-dependent regulation of the expression of two different chalcone synthase transcripts in mustard cotyledons. Planta 183,
416-422.
Estelle, M. (1992). The plant hormone auxin: lnsight in sight. BioEssays 14, 439-444.
Estelle, M.A., and Somenrille, C. (1987). Auxin-resistant mutants of
Arabidopsis thaliana with an altered morphology. MOI.Gen. Genet.
206, 200-206.
Fmhnmeyer, H., Ehmann, B., Kretsch, T., Rocholl, M., Harter, K.,
Nagatanl, A., Furuya, M., Batschauer, A., Hahlbrock, K., and
Schgfer, E. (1992). Differential usage of photoreceptors for chalcone
synthase gene expression during plant development. Plant J. 2,
899-906.
Furuya, M. (1993). Phytochromes:Their molecular species, gene families, and functions. Annu. Rev. Plant Physiol. Plant MOI. Biol. 44,
617-645.
Gilroy, S.,and Jones, R.L. (1994). Perception of gibberellin and abscisic acid at the externa1face of the plasma membrane of barley
(Hordeum vulgare L.) aleurone protoplasts. Plant Physiol. 104,
1185-1 192.
Gilroy, S.,Read, N.D., and Trewavas, A.J. (1990). Elevation of cytoplasmic calcium by caged calcium or caged inositol trisphosphate
initiates stomatal closure. Nature 346, 769-771.
Chory, J. (1993). Out of darkness: Mutants reveal pathways controlling light-regulated development in plants. Trends Genet. 9,167-172.
Gilroy, S.,Fricker, M.D., Read, N.D., and Trewavas, A.J. (1991). Role
of calcium in signal transduction of Commelina guard cells. Plant
Cell 3, 333-344.
Chory, J., Aguilar, N., and Peto, C.A. (1991). The phenotype of
Arabidopsis thaliana detl mutants suggests a role for cytokinins in
greening. Symp. SOC.Exp. Biol. 45, 21-29.
Gottesman, S., and Maurizi, M.R. (1992). Regulation by proteolysis:
Energy-dependent proteases and their targets. Microbiol. Rev. 56,
592-621.
1540
The Plant Cell
Greenberg, J.T., Guo, A., Klessig, D.F., and Ausubel, F.M. (1994).
Programmed cell death in plants: A pathogen-triggeredresponse
activated coordinately with multiple defense functions. Cell 77,
551-563.
Grimm, R., Gast, D., and Rüdiger, W. (1989). Characterization of a
protein-kinase activity associated with phytochrome from etiolated
oat (Avena sativa L.) seedlings. Planta 178, 199-206.
Guzmln, P., and Ecker, J.R. (1990). Exploiting the triple response
of Arabidopsis to identify ethylene-related mutants. Plant Cell 2,
513-523.
Hughes, D.A. (1994). Histidine kinases hog the limelight. Nature 369,
187-188.
Hunt, R.E., and Pratt, L.H. (1980). Partia1 characterization of undegraded oat phytochrome. Biochemistry 19, 390-394.
Imbalzano, A.N., Kwon, H., Green, M.R., and Kingston, R.E. (1994).
Facilitatedbinding of TATA-binding protein to nucleosomal DNA. Nature 370, 481-485.
Jabben, M., Shanklin, J., and Vierstra, R.D. (1989). Ubiquitinphytochrome conjugates. J. Biol. Chem. 264, 4998-5005.
Johnson, E., Bradley, M., Harberd, N.P., and Whitelam, G.C. (1994).
Photoresponses of light-grownphyA mutants of Arabidopsis. Plant
Physiol. 105, 141-149.
Lincoln, C., Britton, J.H., and Estelle, M. (1990). Growth and development of the axr7 mutants of Arabidopsis. Plant Cell2,1071-1080.
Maeda, T., Wurgler-Murphy, S.M., and Saito, H. (1994). A twocomponent system that regulates an osmosensing MAP kinase cascade in yeast. Nature 369, 242-245.
McMichael, R.W.J., and Lagarias, J.C. (1990). Phosphopeptide mapping of Avena phytochrome phosphorylated by protein kinases in
vitro. Biochemistry 29, 3872-3878.
McNellis, T.W., von Arnim, A.G., Araki, T., Komeda, Y., Misbra, S.,
and Deng, X.-W. (1994). Genetic and molecular analysis of an allelic series of cOp7 mutants suggests functional roles for the multiple
protein domains. Plant Cell 6, 487-500.
Meyer, K., Leube, M.P., and Grill, E. (1994). A protein phosphatase
2C involved in ABA signal transduction in Arabidopsis thaliana.
Science 264, 1452-1455.
Millar, A.J., Short, S.R., Hiratsuka, K., Chua, N.-H., and Kay, S.A.
(1992). Firefly luciferaseas a reporter of regulatedgene expression
in higher plants. Plant MOI. Biol. Rep. 10, 324-337.
Millar, A.J., McGrath, R.B., and Chua, N.4. (1994). Phytochrome
phototransduction pathways. Annu. Rev. Genet. 28, in press.
Jones, J.D.G. (1994). Resistance crumbles? Curr. Biol. 4, 67-69.
Misbra, S., Müller, A.J., Weiland-Heidecker, U., and Jürgens, G.
(1994). The FUSCA genes of Arabidopsis: Negative regulatorsof light
responses. MOI. Gen. Genet. 244, 242-252.
Kawamura, S. (1993). Rhodopsin phosphorylation as a mechanism
of cyclic GMP phosphodiesterase regulation by S-modulin. Nature
362, 855-857.
Nagatani, A., Reed, J.W., and Chory, J. (1993). lsolation and initial
characterization of Arabidopsis mutants that are deficient in
phytochrome A. Plant Physiol. 102, 269-277.
Kieber, J.J., Rothenberg, M., Roman, G., Feldmann, K.A., and
Ecker, J.R. (1993). CTR7, a negative regulator of the ethylene response pathway in Arabidopsis, encodes a member of the Raf family
of protein kinases. Cell 72, 427-441.
Neuhaus, G., Bowler, C., Kern, R., and Chua, N.-H. (1993). Calcium/calmodulin-dependent and -independent phytochrome signal
transduction pathways. Cell 73, 937-952.
Koch, K.-W. (1992). Biochemicalmechanism of light adaptation in vertebrate photoreceptors. Trends Biol. Sci. 17, 307-311.
Koornneef, M., Reuling, G., and Karssen, C.M. (1984). The isolation and characterization of abscisic acid-insensitive mutants of
Arabidopsis thaliana. Physiol. Plant. 61, 377-383.
Koutalos, Y., and Yau, K.-W. (1993). A rich complexity emerges in
phototransduction.Curr. Opin. Neurobiol. 3, 513-519.
Kwon, H., Imbalzano, A.N., Khavari, P.A., Kingston, R.E., and
Green, M.R. (1994). Nucleosome disruption and enhancement of
activator binding by a human SWllSNF complex. Nature 370,
477-481.
Lamb, C.J. (1994). Plant disease resistance genes in signal perception and transduction. Cell 76, 419-422.
Lehming, N., Thanos, D., Brickman, J.M., Ma, J., Maniatis, T., and
Ptashne, M. (1994). An HMG-like protein that can switch a transcriptional activator to a repressor. Nature 371, 175-179.
Leung, J., Bouvier-Durand, M., Morris, P.X., Guerrier, D., Chefdor,
F., and Giraudat, J. (1994). Arabidopsis ABA response gene ABl7:
Features of a calcium-modulatedprotein phosphatase. Science 264,
1448-1452.
Leyser, H.M.O., Lincoln, C.A., Timpte, C., Lammer, D., Turner, J.,
and Estelle, M. (1993). Arabidopsis auxin-resistancegene AXR7
encodes a protein related to ubiquitin-activatingenzyme El. Nature
364, 161-164.
Li, H.-m., Altschmied, L., and Chory, J. (1994). Arabidopsis mutants
define downstream branches in the phototransduction pathway.
Genes Dev. 8, 339-349.
Nick, P., Ehmann, E., Furuya, M., and Schafer, E. (1993). Cell communication, stochastic cell responses, and anthocyanin pattern in
mustard cotyledons. Plant Cell 5, 541-552.
Ota, IA., and Varshavsky, A. (1993). A yeast protein similar to bacteria1 two-component regulators. Science 262, 566-569.
Parkinson, JS(1993). Signal transduction schemes of bacteria. Cell
73, 857-871.
Parks, B.M., and Quail, P.H. (1991). Phytochrome-deficienthyl and
hy2 long hypocotyl mutants of Arabidopsis are defective in
phytochrome chromophore biosynthesis. Plant Cell 3, 1177-1186.
Parks, B.M., and Quail, P.H. (1993). hy8, a new class of Arabidopsis
long hypocotyl mutants deficient in functional phytochrome A. Plant
Cell 5, 39-48.
Pepper, A., Delaney, T., Washburn, T., b o l e , D., and Chory, J. (1994).
DE T7, a negative regulator of light-mediated development and gene
expression in Arabidopsis, encodes a nove1 nuclear-localized protein. Cell 78, 109-116.
Plckett, F.B., Wilaon, A.K., and Estelle, M. (1990). The aux7 mutation of Arabidopsis confers both auxin and ethylene resistance. Plant
Physiol. 94, 1462-1466.
Quail, P.H. (1991). Phytochrome: A light-activated molecular switch
that regulates plant gene expression. Annu. Rev. Genet. 25,389-409.
Quail, P.H., Brlggs, W.R., Chory, J., Hangarter, R.P., Harberd, N.P.,
Kendrick, R.E., Koornneef, M., Parks, E., Sharrock, R.A., Schafer,
E., Thompson, W.F., and Whitelam, G.C. (1994). Spotlight on
phytochrome nomenclature. Plant Cell 6, 468-471.
Raz, V., and Fluhr, R. (1992). Calcium requirement for ethylenedependent responses. Plant Cell 4, 1123-1130.
Plant Signal Transduction
1541
Raz, V., and Fluhr, R. (1993). Ethylene signal is transduced via protein phosphorylation events in plant cells. Plant Cell 5, 523-530.
Trewavas, A., and Gilroy, S. (1991). Signal transduction in plant cells.
Trends Genet. 7, 356-361.
Reed, J.W., Nagpal, P., Poole, D.S., Furuya, M., and Chory, J. (1993).
Mutations in the gene for the red/far-red light receptor phytochrome
B alter cell elongation and physiological responses throughout
Arabidopsis development. Plant Cell 5, 147-157.
Wel, N., Kwok, S.F., von Arnim, A.G., Lee, A., McNellis, T.W., Piekos,
B., and Deng, X.-W, (1994a). Arabidopsis COP8, COP70, and COP77
genes are involved in repressionof photomorphogenicdevelopment
in darkness. Plant Cell 6, 629-643.
Reed, J.W., Nagatani, A., Ellch, T.D., Fagan, M., and Chory, J. (1994).
Phytochrome A and phytochrome B have overlapping but distinct
functions in Arabidopis development. Plant Physiol. 104, 1139-1149.
Wei, N., Chamovitz, D.A., and Deng, X.-W. (1994b). ArabidopsisCOP9
is a component of a nove1signaling complex mediatinglight control
of development. Cell 78, 117-124.
Shacklock, P.S., Read, N.D., and Trewavas, A.J. (1992). Cytosolic
free calcium mediates red light-inducedphotomorphogenesis.Nature 358, 753-755.
Wenng, A., Batschauer, A., Ehmann, B., and Schafer, E. (1990).
Temporal pattern of gene expression in cotyledons of mustard
(Sinapis alba L.) seedlings. Bot. Acta 103, 240-243.
Sharrock, R.A., and Quail, RH. (7989). Nove1phytochrome sequences
in Ara6i&psis hdiana: Structure, evolution, and differential expression
of a plant regulatory photoreceptor family. Genes Dev. 3, 1745-1757.
Whitelam, G.C., and Harberd, N.P. (1994). Action and function of
phytochrome family members revealed through the study of mutant and transgenic plants. Plant Cell Environ. 17, 615-625.
Stockhaus, J., Nagatanl, A., Halfter, U., Kay, S., Furuya, M., and
Chua, N.-H. (1992). Serine-to-alanine substitutions at the aminoterminal region of phytochromeA result in an increase in biological
activity. Genes Dev. 6, 2364-2372.
Whitelam, G.C., Johnson, E., Peng, J., Carol, P., Andenon, M.L.,
Cowl, J.S., and Harberd, N.P. (1993). Phytochrome A null mutants
of Arabidopsis display a wild-type phenotype in white light. Plant
Cell 5, 757-768.
Susek, R.E., Ausubel, F.M., and Chory, J. (1993). Signal transduction mutants of Arabidopsis uncouple nuclear CAB and RBCS gene
expression from chloroplast development. Cell 74, 787-799.
Wilson, A.K., Pickett, F.B., Turner, J.C., and Estelle, M. (1990). A
dominant mutation in Arabidopsis confers resistanceto auxin, ethylene and abscisic acid. MOI. Gen. Genet. 222, 377-383.
Szerszen, J.B., Szczyglowski, K., and Bandunki, R.S. (1994). iaglu,
a gene from Zea mays involved in conjugation of growth hormone
indole-3-acetic acid. Science 265, 1699-1701.
Wong, Y.-S., Cheng, H.-C., Walsh, D.A., and Lagarias, J.C. (1986).
Phosphorylation of Avena phytochrome in vitro as a probe of lightinduced conformational changes. J. Biol. Chem. 261, 12089-12097.
Tretyn, A., Kendrick, R.E., and Wagner, G. (1991). The role(s) of calcium ions in phytochrome action. Photochem. Photobiol. 54,
1135-1155.
Zeevaart, J.A.D., and Creelman, R.A. (1988). Metabolismand physiology of abscisic acid. Annu. Rev. Plant Physiol. Plant MOI. Biol.
39, 439-473.
Emerging themes of plant signal transduction.
C Bowler and N H Chua
Plant Cell 1994;6;1529-1541
DOI 10.1105/tpc.6.11.1529
This information is current as of August 11, 2017
Permissions
https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X
eTOCs
Sign up for eTOCs at:
http://www.plantcell.org/cgi/alerts/ctmain
CiteTrack Alerts
Sign up for CiteTrack Alerts at:
http://www.plantcell.org/cgi/alerts/ctmain
Subscription Information
Subscription Information for The Plant Cell and Plant Physiology is available at:
http://www.aspb.org/publications/subscriptions.cfm
© American Society of Plant Biologists
ADVANCING THE SCIENCE OF PLANT BIOLOGY