ISSN 0026-8933, Molecular Biology, 2007, Vol. 41, No. 3, pp. 427–437. © Pleiades Publishing, Inc., 2007.
Original Russian Text © G.V. Novikova, I.E. Moshkov, D.A. Los, 2007, published in Molekulyarnaya Biologiya, 2007, Vol. 41, No. 3, pp. 478–490.
TO THE 40th ANNIVERSARY OF MOLEKULYARNAYA BIOLOGIYA
MOLECULAR ADAPTATION
UDC 579.23''315
Protein Sensors and Transducers of Cold and Osmotic Stress
in Cyanobacteria and Plants
G. V. Novikova, I. E. Moshkov, and D. A. Los
Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Moscow, 127276 Russia; e-mail: [email protected]
Received and accepted for publication December 26, 2006
Abstract—Genome-wide analysis of gene expression at the transcriptional level with DNA microarrays identified almost all genes induced by particular stress in cyanobacteria and plants. Adaptation to stress conditions
starts with the perception and transduction of the stress signal. A combination of systematic mutagenesis of
potential sensors and transducers with genome transcription profiling allowed significant progress in understanding the mechanisms responsible for the perception of stress signals in photosynthesizing cells. The review
considers the recent data on the cyanobacterial and plant signaling systems perceiving and transmitting the cold,
hyperosmotic, and salt stress signals.
DOI: 10.1134/S0026893307030089
Key words: cold stress, histidine kinases, hyperosmotic stress, response regulators, salt stress, cold stress, sensors
INTRODUCTION
Abiotic stress factors affecting photosynthesizing
organisms suppress many biochemical reactions and
physiological responses at various levels. When stress
exposure is nonlethal, organisms activate the expression of various genes and synthesize proteins and specific metabolites essential for the survival under the
new conditions.
The first step of acclimation is the perception of the
stress signal and its transmission to the regulatory
regions of relevant genes. Organisms and cells have
specific protein sensors to perceive environmental
changes and proteins to transmit these signals.
As a result of their sessile life style, plants have
acquired multiple mechanisms allowing their adaptation and acclimation to particular climatic conditions
of the habitat. Studies of the molecular mechanisms of
plant adaptation to unfavorable factors is of both theoretical and applied interest, because the capability to
withstand unfavorable climatic conditions directly
affects the diversity of plant species and the productivity of crops, influencing the human food supply.
Modern biochemical and molecular-biological
techniques make it possible to study the mechanisms
of plant adaptation and acclimation at the molecular
level. To date, the genome has been sequenced completely for Arabidopsis and rice and, partly, for
tobacco, lotus, and about a dozen of other plants and
cyanobacteria. DNA microarrays have been developed to study the changes in genome expression in
response to various stress factors. Many earlier
unknown genes have been found to respond to
changes in ambient temperature. Yet the functions are
still unknown for most of these genes, and the molecular mechanisms responsible for the perception of
temperature signals and the plant response to lower
temperatures are poorly understood. This review summarizes the data on sensor proteins of photosynthesizing organisms. These proteins perceive environmental
changes and trigger the expression of genes essential
for adaptation to stress conditions.
TWO-COMPONENT SYSTEMS FOR SENSING
AND TRANSMISSION OF STRESS SIGNALS
A typical two-component system of signal perception and transduction includes a His kinase (Hik) and
the corresponding response regulator (Rre). Pairs of
hik and rre regulatory genes are close in the genome
and form operons in most eubacteria, such as Escherichia coli and Bacillus subtilis. Hik perceives the
environmental changes via its sensor domain, which
leads to autophosphorylation of His in the conserved
His kinase domain [1]. Then, the phosphoryl group is
transferred from Hik to Asp in a conserved domain of
the corresponding Rre. Phosphorylated Rre changes
its conformation and binds to the promoter region of
target genes, activating (positive regulation) or
repressing (negative regulation) their transcription.
Two-component regulatory systems are found in
prokaryotes (including cyanobacteria), fungi (including yeasts), plants, and primitive animals [2] but not in
higher animals and humans. Ser/Thr and Tyr protein
427
428
NOVIKOVA et al.
Cold stress
Hik33
?
Rre26
ndhD2
hliA
hliB
hliC
fus
ycf39
sigD
feoB
crtP
slr1544
sll1483
sll1541
sll0086
slr0616
slr1747
ssr2016
slr0400
sll1911
slr0401
sll0815
sll1770
Total: 21
crhR
rlpA
rbp1
cbiO
mutS
desB
slr0082
slr1927
sll1611
slr0955
slr0236
sll0494
slr1436
slr1974
Total: 15
Plasma membrane
Fig. 1 Perception of cold stress by cyanobacterium
Synechocystis. The relevant two-component regulatory system includes His kinase Hik33 and response regulator
Rre26; its target genes are shown. ?, an unidentified system
of cold-dependent transcriptional induction.
kinases mostly act as sensors in eukaryotes, in particular, in higher animals and higher plants [3].
The chromosome of freshwater unicellular cyanobacterium Synechocystis harbors 44 hik genes and 42
rre genes [4]. In addition, three hik and three rre genes
are in Synechocystis plasmids [5]. Unlike in E. coli
and B. subtilis, the hik and corresponding rre genes do
not form operons in the Synechocysts genome. Hence,
libraries of specific knockouts, including 42 hik
mutants and 41 rre mutants, were obtained to study
the specific functions of individual regulatory genes
(http://kazusa.or.jp/cyano/Synechocystis/mutants/index.html). Complete segregation of
mutant chromosomes was not achieved for hik2,
hik11, hik26, rre23, rre25, and rre26.
Recent studies revealed that the Synechocystis Hik
proteins are involved in perceiving and transmitting
the signals of cold stress [6, 7], phosphorus deficiency
[8], high ocmolarity [9, 10], nickel deficiency [11],
manganese deficiency [12], and high NaCl concentrations [13, 14]. In this review, we focus on the gene
expression regulation by two-component systems in
cold and hyperosmotic stress.
Hik33–Rre26 Two-Component System Regulates
the Expression of Genes Induced
at Low Temperatures in Synechocystis
The cold stress sensor is Hik33 in Synechocystis
[6] and its analog DesK in B. subtilis [15]. Gene
expression profiling with DNA microarrays in the
∆hik33 mutant showed that Hik33 regulates 21 out of
35 genes induced at low temperatures (Fig. 1). The
other 14 cold-inducible genes are probably controlled
by some other regulatory systems. Screening of the
rre mutants indicated that Rre26 mediates the effect of
Hik33 on gene induction at lower temperatures. Mutations of the two corresponding genes block the induction of the same gene sets at low temperatures, suggesting a Hik33–Rre26 two-component system for
cold stress perception and cold signal transduction
[16].
Two-Component Systems Involved in Perception
and Signal Transduction in Hyperosmotic
and Salt Stress in Synechocystis
Although hyperosmotic and salt stress exposures
are often considered the same, they are far from being
so. Hyperosmotic stress quickly expels water from the
cytoplasm; as a result, the cytoplasm volume is
reduced and the ion concentration within the cell
increases. Salt stress similarly reduces the cytoplasm
volume at the first step [17, 18], which is followed by
a rapid increase in Na+ and Cl– concentrations within
the cell [19] owing to activation of Na+/K+–Cl– channels.
Systematic analysis of gene expression via hik and
rre mutagenesis combined with transcription profiling
with DNA microarrays identified five two-component
Hik–Rre systems involved in regulating gene expression in response to hyperosmotic stress: Hik33–
Rre31, Hik34–Rre1, Hik16–Hik41–Rre17, Hik10–
Rre3, and Hik2–Rre1 [10]. The signal transduction
pathways and target genes controlled by individual
regulatory systems are shown in Fig. 2.
The Hik33–Rre31 system is responsible for the
induction of 11 genes. The Hik10–Rre3 system regulates the expression of only one gene, htrA, which
codes for Ser protease. In the system of three proteins,
Hik16 and Hik41 probably act as a sensor and an
intermediate signal transducers and are His kinases,
while Rre17 is responsible for the transcriptional
induction of two genes with unknown functions,
sll0939 and slr0967. The Hik34–Rre1 system regulates 19 genes, including many genes for heat shock
proteins. The Hik2–Rre1 system is responsible for the
induction of sigB, which codes for an alternative σ
subunit of RNA polymerase, and four other genes
with unknown functions. In addition, 14 out of 48
genes induced by hyperosmotic stress are not regulated by any of the five Hik–Rre systems identified.
The mechanisms controlling stress-inducible transcription of these genes remain obscure [10].
Studies of the systems involved in the perception
and signal transduction in salt stress (0.5 M NaCl)
implicated the above five two-component systems in
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SENSORS AND TRANSDUCERS OF STRESS SIGNALS
regulating the salt stress response [14]. The systems
mostly regulate the expression of the same genes, but
to a different extent. However, there are examples
where individual Hik–Rre systems induce different
genes in salt and hyperosmotic stress (Fig. 2), which
is difficult to explain in terms of the existing concept
of the function of two-component regulatory systems.
For instance, Hik33 senses both salt and hyperosmotic
stress and transmits the signal to Rre31 in either case.
Yet Rre31 controls different gene sets in different
stress conditions; the mechanism of such differentiated regulation is unclear. Another open question is
the mechanism whereby one sensor kinase transmits
the signal to different response regulators, as is the
case with Hik33, which interacts with Rre26 in cold
stress and with Rre31 in salt and hyperosmotic stress.
It is likely that some additional factors interact with
the identified regulatory components to determine signal specificity. This assumption needs experimental
verification.
Hyperosmotic stress
(‡)
Hik33
Rre31
fabG
hliA
hliB
hliC
gloA
sigD
sll1483
slr1544
ssr2016
ssl3446
sll1541
Total: 11
Hik16
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No. 3
2007
?
Hik2
Hik41
Rre1
Rre1
Rre17
Rre3
sll0939
slr0967
htrA
Total: 2
Total: 1
hspA sll0846
clpB1 slr1963
sodB slr0959
htpG sll1884
dnak2 slr1603
spkH slr1945
groES ssl2971
groEL1slr1413
groEL2
dnaJ
Total: 19
sigB
sll0528
slr1119
slr0852
ssr3188
Total: 5
rlpA
repA
glpD
sll1863
sll1862
sll1772
slr0581
ssr1853
slr0112
sll0294
slr0895
slr1501
sll0293
sll0470
Total: 14
Plasma membrane
Salt stress
(b)
OSMOTIC STRESS IN PLANTS
MOLECULAR BIOLOGY
Hik10
Hik34
Hik16
Hik33
Water deficiency (drought), extreme temperature,
and/or high salinity of soil are the severe abiotic factors that limit the growth and development of plants
and may cause a complete elimination of susceptible
species. The extraordinary capability of plants to survive osmotic stress is related to their ability to alter
growth and development (duration of the life cycle,
inhibited growth of above-ground organs, activated
growth of roots), the osmotic and turgor pressures
(changes in transport of potentially toxic ions: uptake,
secretion, and vacuolar compartmentalization), and
metabolism (synthesis of compatible osmolites).
Some of these responses are directly induced by the
primary stress signal, while some others result from
secondary signals, triggered by primary stress. Such
secondary signals include phytohormones, abscisic
acid (ABA), ethylene, reactive oxygen species (especially H2O2), and intracellular secondary messengers
(Ca2+). Because of the complex nature of osmotic
stress, studies of the perception and transduction of
the primary stress signal are hindered by the fact that
plants are exposed to stress through several developmental stages. Stress factors, such as drought or
hyperosmosis, do not arise abruptly. These factors act
gradually, and experience in hormone studies shows
that the observed pattern depends on the rate and duration of stress development rather than on the stress
severity. Hence, the question arises as to whether laboratory simulation of stress conditions adequately
reflects the actual events. Since experiments are performed with intact plants or organs, it is clear that the
stress response is determined by the programs realized
in cells of different types. For instance, cells of different types may have specific transcription factors or
differ in the relative contents of particular sensors.
429
Hik10
Hik34
Hik2
Hik41
Rre31
Rre1
Rre1
Rre17
Rre3
hliA
hliB
hliC
sigD
sll1483
slr1544
ssr2016
hspA slr1915
hik34 slr1916
clpB1 sll1844
pbp
ssl2971
sodB slr0095
dnaK2 slr1192
groEL2 sll1022
sll0846 slr1413
slr1603 sll1107
slr1963
sigB
dnaJ
riml
sll0528
slr0959
slr1686
slr0852
ssr3188
sll0939
slr0967
sll0938
htrA
ndhR sll1722 sll2194
glpD slr1687 sll1620
ycf21 slr0895 ssr2153
ggtB slr1704 slr1894
ggpS ssl3044 slr1501
menG sll1652 slr0082
suhB slr0581 slr1932
sll1863 slr1738 sll1236
sll1862 sll1621
ÇÒ„Ó:
Total: 88
ÇÒ„Ó:
Total: 33
ÇÒ„Ó:
Total: 11
Total: 26
Total: 7
Total: 19
?
Plasma membrane
Fig. 2. Synechocystis two-component systems perceiving
and transmitting the signals of (a) hyperosmotic and (b) salt
stress.
When considering the perception and transduction of
stress signals in plant cells, it is necessary to take
account of multiple cross-talks of signaling pathways
[20].
With this in mind, below we discuss the data on
sensors perceiving osmotic stress and components
involved in the corresponding signal transduction
pathways.
Plant Osmosensors
As all living organisms, plant cells perceive and
process information with the use of various receptors
exposed on the cell surface. Membrane protein
kinases of two classes—receptor-like Ser/Thr kinases
(RLKs) [21] and receptor His kinases (HKs) [22]—
act as receptors in osmotic stress.
It became clear in the late 1990s that plant cells
have stress sensors. Arabidopsis thaliana was found
430
NOVIKOVA et al.
to possess AtHK1, which is homologous to Saccharomyces cerevisiae SLN1. In yeast cells, SLN1 senses
the changes in osmolarity and triggers a two-component system, which activates MAPK (mitogen-activated protein kinase) pathways.
Although direct evidence for the osmosensor role
of AtHK1 in A. thaliana is still lacking, AtHK1
expression is known to increase in salt stress (250 mM
NaCl) or a decrease in temperature to 4°ë [23]. In the
sln1∆sho1∆ double mutant (the two affected proteins
act as osmosensors in yeasts), AtHK1 expression
inhibited the salt-sensitive sln1∆sho1∆ phenotype
[23]. It is important to note that AtHK1 did not interact with any of the five A. thaliana response regulators
examined [22]. Yeast SHO1, which acts as an osmosensor at a high osmolarity, contains four transmembrane domains and the SH3 domain exposed into the
cytoplasm and lacks enzymatic activity. SHO1 initiates a signaling pathway typical of higher eukaryotic
cells [24, 25].
At a high osmolarity, HK CRE1, which acts as a
membrane cytokinin receptor in A. thaliana, substitutes HK SLN1 in sln1∆ yeast cells when activated
with its ligand zeatin [26]. Such CRE1 activity was
also observed when osmotic stress was simulated with
sorbitol and, consequently, the turgor pressure and
cell volume decreased [27]. CRE1 and SLN1 are similar in domain organization but are highly homologous
only in the cytoplasmic kinase and sensor domains.
The questions arise as to how CRE1 perceives the
changes in turgor pressure and which domains are
indispensable for the osmosensor function of activated CRE1. Reiser et al. [27] assumed that CRE1
mediates a physical contact of the cell wall with the
plasma membrane. The osmosensor function requires
a unique combination of the periplasmatic and transmembrane domains of CRE1, as well as the integrity
of the total periplasmatic domain. The above property
of CRE1 is of immense importance, suggesting that
plants have an osmosensing system that is structurally
and functionally similar to its yeast analog. This
assumption needs verification with plant cells.
Tamura et al. [28] recently cloned NtC7 (Nicotiana
tabacum), whose expression increased rapidly (10 min)
and transiently (within 60 min) when tobacco leaves
were treated with 200 mM NaCl and/or 500 mM mannitol. Transgenic tobacco plants overexpressing NtC7
were tolerant of osmotic stress regardless of their age,
while germination of transgenic seeds and the growth
of transgenic seedlings were inhibited by NaCl and
LiCl. This finding indicates that the NtC7 function in
osmotic stress is not related to the maintenance of ion
homeostasis.
NtC7 codes for a transmembrane protein of 308
amino acid residues (33.9 kDa). Its N-terminal region
is similar to the RLK receptor domain, but NtC7 lacks
the kinase domain exposed into the cytoplasm. How
does NtC7 transmit the signal to the downstream signaling components? Note that, apart from the kinase
domain, NtC7 is homologous to tomato leaf Cf-9, a
transmembrane receptor of the Cladosporium fulvum
avr-9 elicitor. It is commonly accepted that the Cf-9
C-terminal region, which is exposed into the cytoplasm, is responsible for pathogen-induced signal
transduction. In the cytoplasm, Cf-9 probably interacts with Ser/Thr kinase Pto, which is involved in signal transduction upon a pathogen attack. It is possible
that NtC7 similarly interacts with its partners in the
cytoplasm to transmit the signal about changes in
osmolarity. Further studies will identify the NtC7
partners.
It is important that the primary receptors of abiotic
signals be searched among RLKs, which are characteristic of higher eukaryotic cells. The RLK family
harbors about 60% of all kinases and is encoded by
2.5% of genes in Arabidopsis, while there are only
11 HKs in this plant. The RLK-family proteins vary in
domain structure and have highly diverse sequences
of the extracellular domains [29], suggesting perception of various signals. In planta studies of the RLK
function will elucidate whether RLK signaling is specific for particular signals or RLKs are involved in the
cross-talk of signaling pathways triggered by many
different signals.
Plant Protein Kinases and Protein Phosphatases
Reversible phosphorylation of proteins is an
important mechanism regulating the cell response, in
particular, to osmotic stress. Among all relevant
enzymes, mitogen-activated protein kinases (MAPKs)
are a subject of intense studies. MAPKs are of special
interest partly because the well-studied S. cerevisiae
osmotic stress signaling pathway involves the HOG1
(high osmolarity glycerol response 1) MAPK cascade,
which mediates signal transduction from the receptor
to transcription factors triggering the stress response
genetic program.
There are only six different MAPK genes in the S.
cerevisiae genome and 20 MAPK genes in the A.
thaliana genome [30]. Some of these genes were
functionally characterized not only in A. thaliana, but
also in alfalfa Medicago sativa and tobacco N.
tabacum [31], although studies of A. thaliana MAPKs
are most successful.
MAPKs MPK3, MPK4, and MPK6 are activated in
response to low temperatures and osmotic and salt
stress in A. thaliana [32, 33]. The kinetic and extent of
activation vary among different MAPKs, suggesting
their independent roles in the stress response. For
instance, mutant A. thaliana seedlings with inactivated MPK4 are tolerant of hyperosmotic stress [34];
i.e., AtMPK4 can negatively regulate the response to
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SENSORS AND TRANSDUCERS OF STRESS SIGNALS
N. tabacum
A. thaliana
Signal
Hyperosmosis
Drought
NaCl
Cold
AtHK1
Sensor
Hyperosmosis
Hyposmosis
431
M. sativa
Hyperosmosis
NaCl
Cold
Drought
NtC7
åÄêäää
AtMEKK1
AtMEKK1
åÄêää
AtMKK1
AtMKK2
åÄêä
AtMPK3/6
AtMEKK1
NtNPK1
AtMKK1
NtMEK2
NtMEK2
MsSIMKK
MsPRKK
AtMPK4/6
AtMPK4/6 AtMPK4/6
NtSIPK
NtWIPK
MsSIMK
MsSAMK
AtMKP1
AtPTP1
AtMKP1
AtPTP1
AtMKK1
MsNPK1
Target
genes
Negative
regulator
MsMP2C
Fig. 3. MAPK involved in osmotic stress signal transduction in plant cells. A scheme of the signaling pathway is shown on the left.
AtHK1 and NtC7 presumably act as osmosensors. MAPK modules whose function in stress was demonstrated experimentally are
framed. Species: At, Arabidopsis thaliana; Nt, Nicotiana tabacum; Ms, Medicago sativa. Dual-specificity (AtMKP1), phosphotyrosine (AtPTP1), and PP2C (MsMP2C) MAPK-specific protein phosphatases act as negative regulators.
hyperosmicity, in contrast to the positive regulators
MPK3 and MPK6.
Of about 1000 A. thaliana protein kinase genes,
more than 100 genes code for kinases involved in
MAPK cascades (modules), including 60 MAPK
kinase kinases (MAPKKKs), 10 MAPK kinases
(MAPKKs, or MKKs), and 20 MAPKs (or MPKs)
[30]. This proportion suggests that MAPKKs act both
as a main multifunctional component integrating the
signals from upstream MAPKKKs and as a branching
point for activation of downstream MPKs.
It was demonstrated that AtMPK4/6 are involved
in transmitting the osmotic signal and MAPKK
AtMKK1 is activated in osmotic stress [35] and phosphorylates and activates AtMPK4 in vitro [36]. These
findings gave grounds to assume that A. thaliana has a
MAPK cascade that is activated in osmotic stress and
includes AtMPK4, AtMKK1, and AtMEKK1 (MAPKKK).
Recent studies confirmed this assumption [37]. It
was shown using transient expression in A. thaliana
leaf mesophyll protoplasts and the yeast two-hybrid
system that MAPKK AtMKK2 is phosphorylated in
response to salt and cold stress and that its activated
phosphorylated form phosphorylates AtMPK4 and
AtMPK6. MAPKKK MEKK1 was identified as a
potential activator of AtMKK2. Upon simultaneous
expression of AtMKK2, AtMPK4, and AtMPK6, constitutively active AtMEKK1 significantly activated
both MAPKs in protoplasts.
The identification of the first MAPK module (Fig. 3)
activated in response to salt and cold stress is an
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important event, which would meet great enthusiasm
if not for the following. First, it is unclear which sensor perceives the stress signal and how this information is translated into a “language” understandable to
the MAPK cascade. Second, what is the mechanism
ensuring the specificity of signal transduction from
AtMEKK1 to downstream AtMKK2 and then to
AtMPK4/6? This is important to establish, because
AtMEKK1 was earlier implicated in the MAPK cascade involved in the A. thaliana response to flagellin
[38]. A well-known mechanism ensuring transduction
of individual signals involves scaffold proteins, which
are found in plants as well as in other organisms. Yet
it is unclear whether their function is similar to that of
scaffold proteins of other eukaryotes. Third, A. thaliana
plants overexpressing activated MKK2 display considerable changes in expression of 152 genes, including the marker genes of salt and cold stress [39, 40].
Hence, it is important to determine whether the transcription factors that regulate the expression of genes
involved in the response to abiotic stress act as MAPK
substrates in planta. Analysis of 1690 A. thaliana proteins with the use of protein microarrays identified
39 proteins as AtMPK6 substrates and 48 as AtMPK3
substrates, including some transcription factors [41].
To study the physiological significance of the substrates identified, it is necessary to map the phosphorylation sites and to verify the results in vivo.
Intracellular Ca2+ concentration changes in
response to osmotic, salt, and cold stress in plant cells.
Calcium-dependent protein kinases (or calmodulinlike domain protein kinases (CDPKs)) were found
only in plants and green algae. In A. thaliana, the
432
NOVIKOVA et al.
Salt
stress
ëa2+
PPI
FISL
Kinase
Ca2+
domain
SCaBP (SOS3)
Inactive
PKS (SOS2)
Ca 2
SC
+
aBP
(SO
S3)
ABI2
PPI
FISL
Kinase
domain
Activated
PKS (SOS2)
Transporter
phosphorylation
Ion homeostasis
Salt tolerance
Fig. 4. Role of SOS2, SOS3, and ABI2 in the A. thaliana
cell response to salt stress. Salt stress generates a Ca2+ signal, which is perceived by the Ca2+ sensor SCaBP (SOS3).
In the absence of stress, protein kinase S (PKS, SOS2) is
inactive owing to the intramolecular interaction between the
N-terminal kinase and C-terminal receptor domains. The
regulatory domain includes the FISL motif, which is
involved in SCaBP (SOS3) binding to PKS (SOS2), and the
PPI motif, involved in the interaction with protein phosphatase 2C (ABI2). The activation loop (black bar) is within
the kinase domain of PKS (SOS2). The Ca2+–SKBP
(SOS3) complex binds to the FISL domain and thereby activates PKS (SOS2). Activated PKS (SOS2) phosphorylates
ion transporters responsible for ion homeostasis. ABI2
either inactivates PKS (SOS2) or dephosphorylates the
transporters.
Ser/Thr-CDPKs family is one of the largest families
and includes 34 unique CDPKs and CDPK-related
protein kinases. It should be emphasized that CDPK is
a Ca2+ sensor possessing an enzymatic activity. Recent
studies focused on the specific functions of individual
CDPKs in signal transduction. Such works seem logical in view of the subcellular location of CDPKs [42]
and the diversity of their potential substrates
(enzymes of carbohydrate and nitrogen metabolism,
stress proteins, membrane transporters, ion channel
proteins, cytoskeletal proteins, and transcription factors).
A role of CDPKs as positive regulators of signal
transduction in osmotic stress can be inferred from the
induction of individual CDPK genes. It was found,
indeed, that cold and salt tolerance of rice increases
with the increasing expression of OsDCPK7, an
ortholog of AtCDPK1 [43]. Plants overexpressing
OsCDPK7 displayed induction of some genes
involved in the response to cold, but not to salt, stress.
Thus, these findings did not confirm the above
assumption. Moreover, the same cytosolic OsCDPK7
proved to play a role in two different pathways, suggesting an unknown mechanism of signaling specificity.
In leaves of facultative halophyte Mesembryanthemum crystallinum, McCPK1 expression increases
quickly and transiently in salt and osmotic stress, the
latter being more effective [44]. McCPK1 phosphorylates CSP1 (CDPK substrate protein 1) in the presence
of Ca2+ in vitro and, probably, in vivo. Note that CSP1
expression is not regulated in osmotic stress. CSP1 is
constitutively located in the nucleus and acts as a transcription factors, belonging to the family of two-component response pseudoregulators. McCPK1 is found
in the nucleus of cells exposed to salt stress and dehydration and is associated with the plasmalemma in
intact leaf cells. These findings indicate that, in hyperosmotic stress, McCPK1 phosphorylates CSP1 in the
nucleus, which changes the ability of CSP1 to regulate
the expression of genes involved in the hyperosmotic
stress response. When stress is eliminated, McCPK1
returns to the plasmalemma, allowing the cell to
respond to new stress. Thus, McCPK1 perceives the
signal when on the plasmalemma and transmits information into the nucleus to activate stress-inducible
genes.
Regulation of ion homeostasis under stress conditions is important for the formation of salt tolerance in
plants. Molecular-genetic analysis of the A. thaliana
sos mutants (salt-overlay-sensitive) identified the
components (SOS1, SOS2, and SOS3) involved in the
signal transduction pathway that transmits information about a stress-induced increase in intracellular
Ca2+ and functions to restore ion homeostasis in the
cell (Fig. 4) [45]. Studies of the Ca2+ sensor SOS3 and
its partner protein kinase SOS2 allowed the identification of the A. thaliana genes coding for eight SOS3like Ca2+ sensor/binding proteins (SCaBPs) and 23
SOS2-like protein kinases (PKSs). Like SOS3, all
other SCaBPs lack enzymatic activity and bind Ca2+,
utilizing their so-called EF hands. It is of particular
interest that SOS3 may serve as a Na+ sensor, since
Na+ binding was demonstrated for EF-hand proteins
[46].
SCaBPs and their partner PKSs are capable of
numerous interactions in vitro [47]. Phosphorylation
is probably a key event in the regulation of PKS activity, because PKS is enzymatically inactive in the
absence of SCaBP. PKS-phosphorylating kinases are
as yet unidentified, but it is known that protein phosphatase 2C (PP2C) can dephosphorylate PKS [48].
The physiological substrates of PKSs are unknown
in most cases. SOS1 (plasmalemma Na+/H+ antiMOLECULAR BIOLOGY
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porter) is an exception; its function prevents the cell
from accumulating excess Na+ in salt stress. On recent
evidence, PKS (SOS2) can regulate vacuolar transporters such as CAX1 (H+/Ca2+ antiporter) [49] and
AtNHX1 (Na+/H+ antiporter) [50], the changes in
antiporter activity being independent of the presence
or activity of SCaBP (SOS3).
Several protein phosphatases interact with PKSs
(Fig. 4). The PKS C-terminal region harbors the conserved PPI motif, which is necessary and sufficient for
the interaction with PP2C [48]. Mutations of the PPI
motif prevent the PKS (SOS2)–PP2C interaction.
PP2C has the PKI domain and utilizes it to interact
with PKSs. The PP2C–PKS interaction is impossible
when the PKI domain is altered by a mutation. The
PKS (SOS2)–PP2C (ABI2) interaction is distorted in
the abi2-1 A. thaliana mutant (ABI codes for PP2C),
but the mutant is resistant to salt stress and is insensitive to ABA [48]. It is possible that SOS2 and ABI2
control each other’s phosphorylation or regulate phosphorylation of a common substrate. Another possibility is that the ABA and SOS signaling pathways crosstalk at the ABI2 level. If so, SOS2 acts as a scaffold
protein, rendering SOS3 and ABI2 in a complex
whose specific function is associated with salt resistance in response to the Ca2+ signals arising in salt
stress. Yet this is only one of all possible scenarios.
Considerable progress has recently been made in
studies of SCaBP (SOS3) and PKS (SOS2). However,
such studies are far from being completed and many
questions are still open. What are the functions of
other members of their families and which substrates
do they affect? Which protein kinases phosphorylate
Ser/ Thr, or Tyr in PKS? What is the mechanism sustaining the function of the SCaBP–PKS signaling
module in organisms other than A. thaliana?
Increasing attention in studies of the signal transduction pathways is attracted by protein phosphatases
responsible for only transient activation of protein
kinase-involving pathways. In contrast to protein
kinases with their highly similar structures, protein
phosphatases form a less abundant family and considerably differ from each other in structure, biochemical
properties, and subcellular location. In the context of
this review, it is of special interest to consider Tyr
phosphatases (PTPs), dual specificity protein phosphatases (DsPTPs), and PP2C, since these enzymes
dephosphorylate MAPKs, including those involved in
signal transduction in osmotic stress. An A. thaliana
mutant with an increased sensitivity to short-wave UV
irradiation (UV-C) and methyl methanesulfonate
(MMS) made it possible to identify MKP1 (MAPK
phosphatase 1), which codes for DsPTP regulating the
sensitivity to DNA-damaging agents. The recessive
mkp1 mutants were hypersensitive to UV-C and MMS
but tolerant of salt stress [51]. The potential partner of
MKP1 is MPK6, while the interaction with MPK3 and
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MPK4 is weaker [51]. These findings indicate that
MKP1 controls the NaCl stress signal transduction
pathway, which involves MPK6, but it is still unclear
whether MPK6 plays a central role in A. thaliana
adaptation to salt stress and is the only target of
MKP1. On the other hand, NaCl-induced activation of
AtMPK6 and AtMPK4 does not change in the mkp1
mutant, while the mpk4 mutant displays a higher resistance to hyperosmotic stress [34]. These data make it
possible to assume that MKP1 is involved in osmotic
signal transduction, functioning in the MAPK cascade
with MAPKs other than MPK4/6. In addition to
MKP1, AtPTP1 dephosphorylates Tyr residues in
AtMPK4 and AtMPK6 in vitro [52]. It is possible that
MKP1 and/or AtPTP1 dephosphorylate AtMPK4/6 in
stress, but the conditions allowing protein phosphatases from different groups inactivate the same
substrates are a subject of further studies.
In mammalian and yeast cells, Ser/Thr protein
phosphatase PP2C inactivates MAPK. Interestingly,
alfalfa PP2C (MP2C) produced in yeast cells proved
to suppress the lethal phenotype caused by expression
of constitutively active MAPKKK STE11, which
induces the HOG1 MAPK cascade. This gives
grounds to think that MP2C inactivates the MAPK
cascade involved in signaling in osmotic stress in
alfalfa.
It was found, indeed, that PP2C (MP2C) dephosphorylates and inactivates stress-inducible MAPK
SIMK (a homolog of AtMPK6) upon coexpression of
their genes in Petroselinum crispum protoplasts [53].
This finding is of fundamental importance, suggestive
a selective interaction of SIMK and MP2C in vivo.
Hence, it is reasonable to assume that MP2C negatively regulates the SIMK signaling pathway involved
in the salt stress response in alfalfa. The A. thaliana
genome harbors 69 PP2C genes. Among all PP2C
enzymes, ABI1 and ABI2 deserve special attention.
The role of these protein phosphatases as negative regulators of the ABA-dependent signaling pathways
leading to stomatal closure in drought has recently
been reviewed [54].
The nature of the abi1-1 mutation, identified genetically, is unclear. The abi1-1 mutant is ABA-insensitive and displays a lower activity of AtPP2C. Identification of a potential substrate of alfalfa MP2C elucidated, to a certain extent, the mechanism whereby the
abi1-1 mutation affects the PP2C function. An MP2C
mutation equivalent to abi1-1 reduced the phosphatase activity of MP2C without affecting its interaction with SIMK. Mutant MP2C was capable of inactivating SIMK [53]. It was assumed that a decrease in
phosphatase activity does not influence the PM2C–
SIMK interaction, which is mediated by the MP2C
KIM motif. In mammals and yeasts, this conserved
motif is found in proteins interacting with MAPKs
and protein phosphatases are incapable of MAPK
434
NOVIKOVA et al.
Cold
NaCl, drought
ICE1
ÄBA
ICE1
P
ICEr1
ICEr2
DREB1/CBF
?
DREB2
CBF/DREB1
CBF4
MYC/MYB
P
ABF/AREB/bZIP
MYCR/MYBR
DRE/CRT
TATA
ABRE
TATA
Fig. 5. Regulation of the expression of ABA-dependent and
ABA-independent genes involved in the cell response to
abiotic stress in A. thaliana. Abiotic factors (cold, drought,
and NaCl) activate the expression of the stress response
genes
via
stress-inducible
transcription
factors
CBF/DREB1 and DREB2 and ABA-inducible bZIP transcription factors ABF/AREB and CBF4. Transcriptional
activator ICE1 binds to cis element ICEr1, which regulates
the expression of CBF/DREB1 together with ICEr2. ABAindependent transcription factors CBF/DREB1 bind to the
cis elements of DRE/CRT to ensure the expression of the
cold stress response genes. Transcription factors are shown
with ovals; cis elements are shown with black bars. Transcription factors whose activity is regulated via phosphorylation are indicated with encircled P. Unknown posttranslational modification is indicated with encircled ?.
dephosphorylation in the absence of KIM. Indeed,
SIMK is not inactivated by PP2C ABI2, which lacks
KIM in contrast to ABI1 [53]. Although this assumption is appealing, it cannot be excluded that the catalytic domain of PP2C is involved in determining its
substrate specificity.
Transcription Factors
As plant genomes are sequenced and studies with
DNA microarrays performed, data are accumulating
concerning the changes in plant transcriptomes in
response to osmotic stress. A fundamental conclusion
based on these data is that genes induced in cold and
salt stress are similarly induced in response to water
deficiency, suggesting a cross-talk of the relevant signaling pathways, as is the case in cyanobacteria [16,
55].
In particular, signals interact and are integrated at
the level of the target gene promoters, which contain
the cis-acting dehydration-responsive element (DRE),
cold-responsive element (CRT), and ABA-responsive
element (ABRE) (Fig. 5). These molecular switches
interact with transcription factors, which ensure
expression of both genes involved in the early
response to osmotic stress and genes responsible for
stress adaptation.
A NAC-domain transcription factor family is specific to plants. Proteins of the family play diverse
roles, being involved in the responses to various factors from pathogens to abiotic stress [56]. In total, 26
NAC genes were identified in sugar cane Saccharum
officinarum. Of these, SsNAC23 is induced in cold
stress, while its expression in water deficiency is transient [57]. Rice OsNAC6 (an ortholog of SsNAC23) is
induced both in response to osmotic stress and treatment with exogenous ABA [58].
As all eukaryotes, plants have transcription factors
of the bZIP family. Rice low temperature-inducible
protein 19 (LIP19), a bZIP factor, does not bind to
DNA [58]. Like animal Fos, LIP19 binds to OsOBF1,
a potential new transcription factor of the bZIP family
[59]. In contrast to LIP19, OsOBF1 is synthesized at
an optimal temperature and binds to specific DNA
sites. When temperature decreases, OsOBF1 binds to
LIP19, and the resulting heterodimer interacts with
unidentified promoters. Since LIP19 is unstable at
normal temperatures, genes activated by the heterodimer are inactivated when plants are exposed at
higher temperatures. It is unknown whether other
bZIP factors act similarly. In A. thaliana, bZIP ABREbinding factor (ABF)/ABRE-binding protein (AREB)
activate the RD29A promoter by binding to its ABRE
(Fig. 5) and this activation is enhanced by ABAdependent phosphorylation of the transcription factor.
The A. thaliana MYC- and MYB-family transcription factors also function as activators of the ABAdependent pathway leading to expression of dehydration-inducible (RD) genes such as RD22 and AtADH1
[60]. Transgenic plants overexpressing AtMYC and
AtMYB are hypersensitive to exogenous ABA. Such
plants display a higher tolerance of osmotic stress, but
their growth is somewhat slower than in wild-type
plants possibly because of a higher ABA content.
Apart from the transcription factors binding to the
cis elements in the promoters of stress response genes,
activation of transcription requires some extra cofactors, which determine the expression level. CRT-binding factor 1 (CBF)/DRE-binding factor 1 (DREB1)
act as transcription factors to ensure the expression of
genes responding to osmotic stress in an ABA-independent manner. A specific feature of CBF is its rapid
(15 min) and transient accumulation in plants exposed
to low temperatures. This is quite conceivable, assuming that a constitutive transcription factor is activated
at low temperatures to induce the expression of the
CBF gene [61]. Such a new regulator was recently
identified and termed the inducer of CBF expression
(ICE1). ICE1 is a MYC-like transcription factor that
is phosphorylated in plants exposed to a low positive
temperature and activates transcription of the CBF3
gene [60, 61]. CBF1/2 activate transcription of a set of
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SENSORS AND TRANSDUCERS OF STRESS SIGNALS
genes whose promoters contain CRT/DRE. The role
of transcription factors CBF and ICE1 has been
intensely studied in silico [62]. Although the ICE1dependent pathway is certainly important for the
induction of cold-responsive (COR) genes [62], it
should be noted that a substantial role can also be
played by other transcription factors. For instance, the
A. thaliana hos9-1 mutant (high expression of osmotically responsive genes) is hypersensitive to freezing.
Although its CBF expression is intact, the hos9-1
mutant is incapable of cold adaptation. It seems that A.
thaliana HOS9 controls constitutive tolerance of low
temperatures. Since the expression of CBF-controlled
genes is not altered in the hos9-1 mutant, it is possible
to assume that HOS9 is necessary for cold tolerance
and functions in a CBF-independent pathway [63].
Returning to Fig. 5, it appears that each stress signal is transmitted via a specific pathway. Yet experiments with transient expression showed that CBF and
DREB function together with ABF to increase the
RD29A expression. Hence, it is impossible to exclude
interplay of ABA-dependent and ABA-independent
pathways. Moreover, overexpression of the transcription factor genes, e.g., CBF, confers resistance not
only to low temperatures but also to changes in osmolarity, testifying again to interplay of signaling pathways.
PROSPECTS OF FURTHER STUDIES
By definition, a primary receptor recognizes its
own ligand with high specificity. A noncovalent
reversible binding of the ligand to its receptor modulates the function of a primary signal transduction
pathway, inducing a primary response. This is true for
some ligands such as phytohormones. However, the
ligand can hardly be identified in the case of abiotic
stress (cold, drought). Hence, the main problem is that
plant primary sensors of osmotic signals are as yet
unidentified. The Ca2+ signal induced in osmotic
stress is decoded and transmitted by the Ca2+ sensors,
which await further investigation. Although the role of
protein kinases and, in particular, MAPKs has been
demonstrated, in particular, for osmotic stress signal
transduction, it is still poorly understood, first, how
putative primary sensors transmit information to protein kinases. A second question is how protein kinases
are connected with transcription factors. As for protein kinases, questions again prevail over answers. In
particular, little is known on their substrates in planta.
Studies with protein microarrays have yielded data on
the potential MAPK substrates [41], but the physiological significance of these data should be verified in
vivo.
Phosphoproteomics will certainly develop as a global approach, although this needs genome sequencing
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435
in many plants, expensive high-tech equipment, and
expert evaluation.
Studies with DNA microarrays have identified
hundreds of genes whose expression changes in
response to osmotic stress. The main current problems
are to determine their regulons, identify the specific
transcription factors, and elucidate the role of the regulons in sustaining plant growth and development
under adverse conditions.
As -omics techniques will be used to study the
plant tolerance of abiotic stress in the nearest future,
important data will be obtained for the perception and
transduction of signals determining stress resistance.
It should be emphasized that an important contribution to further progress can be made by studies of
extremophilic plants such as Thellungiella halophila,
whose genome is about twice as large as the
A. thaliana genome. In contrast to A. thaliana,
T. halophila is capable of surviving extreme osmotic
stress. Studies involving T. halophila as a model in the
project “Integrating International Research of Plant
Abiotic Stress Tolerance Using Arabidopsis Relative
Model Systems (ARMS): Thellungiella halophila”
will substantially improve the understanding of the
mechanisms responsible for plant adaptation to continuously changing environmental conditions.
ACKNOWLEDGMENTS
This work was supported by the Russian Foundation for Basic Research (project nos. 06-04-48581,
05-04-50883, 05-04-49643) and the program Molecular and Cell Biology of the Presidium of the Russian
Academy of Sciences.
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