Download 10 Phytohormones and Signal Transduction Pathways in Plants

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