Directory UMM :Data Elmu:jurnal:P:PlantScience:Plant Science_BioMedNet:241-260:
trends in plant science
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Early signal transduction pathways in
plant–pathogen interactions
Eduardo Blumwald, Gilad S. Aharon and Bernard C-H. Lam
Disease resistance depends on the ability of the plant to recognize a pathogen early in the
infection process. Molecules that indicate the presence of the pathogen (elicitors) activate
host receptors and these rapidly generate an internal signal that triggers early defense
responses. Several transduction pathways that relay the initial recognition signal through a
series of cytosolic and membrane-delimited pathways have been proposed. Activation of
these signal transduction pathways ensures an elicitor-induced response that is quantitative,
timely and coordinated with other activities of the host cells.
R
ecognition of microorganisms by the plant cell depends on
the generation of elicitors by the pathogen1. These can be
non-race-specific elicitors, such as fungal or plant cell
wall fragments released during the infection process, which elicit
a defense response that helps to minimize disease. Race-specific
elicitors are molecules that are encoded by avirulence (Avr) genes
in the pathogen2. Resistance involves the specific recognition of
the invading pathogen by a dominant or semi-dominant plant resistance gene product (R gene). This type of interaction is termed
gene for gene, where for each gene that confers resistance in the
host there is a corresponding gene in the pathogen that confers its
virulence3. The cloning of plant resistance (R) genes from host–
pathogen model systems has facilitated a leap in understanding
the molecular basis of host resistance. The proposed model suggests that the direct or indirect interaction of the AVR and R polypeptides triggers resistance4. Based on sequence analysis of known
R genes, it has been hypothesized that R gene products encode receptors capable of binding AVR products as ligands5. Expression
of these gene products in susceptible plants resulted in specific
resistance3, demonstrating that even susceptible plants possess the
underlying biochemical machinery required for defense. Thus, the
difference between resistance and susceptibility appears to lie in
the proper recognition of the AVR products.
The amino acid sequences of R gene products have homology
to a number of structural protein–protein interaction domains,
suggesting that they play a role as signaling intermediates in a
reaction cascade2. In addition, it appears that multiple products,
some of which may be closely linked to the original R gene, are
required for the manifestation of resistance. One example of
this involves the functional interaction between the tomato Pto
and Prf gene products for expression of resistance to Pseudomonas syringae pv. tomato. The Pto gene product is a serine/
threonine kinase6, whereas the Prf gene product contains leucinerich regions (LRRs), nucleotide binding sites (NBS) and leucine
zippers (LZ) domains7. It is likely that other R genes, such as those
of the Cf family, may interact with other gene products to confer
resistance. This notion is also supported by biochemical evidence. For example, binding of the AVR9 peptide to plant membranes could not be correlated with the presence of the Cf9 gene8,
suggesting that the R gene product might not be exclusively involved in recognition, but also in the coordination of membrane
signaling.
Signaling
Immediately downstream of the initial elicitor–receptor recognition, the activation of ion fluxes and the production of H2O2 are
the initial responses detected in plant cells. Biochemical evidence
suggests that these processes, which occur prior to the transcriptional activation of defense-related genes, appear to be mediated
through the regulation of plasma membrane-bound enzymes. These
include changes in Ca2+-ATPase (Ref. 9) and H+-ATPase (Ref.
10) activities, the activation of plasma membrane-bound ion channels11,12 and the induction of a plasma membrane-bound NADPH oxidase13,14. These
AVR
biophysical and biochemical processes can
Cf2
Cf9
induce changes in the electrical potential
NH2
NH2
difference (membrane potential) and pH
gradient across the host plasma membrane
Leucine-rich
Leucine-rich
that may affect the activity of ion channels
repeats
repeats
7TMS
and other ion transporters. Changes in the
Defense
plasma membrane-bound oxidase electron
responses
transport chain will mediate the production
?
of H2O2 and active oxygen species (termed
α
α β γ
the oxidative burst), which may affect the
COOH COOH
GTP
attacking pathogen and the host cell at the
GDP GTP
site of infection.
A number of signal transduction pathFig. 1. Putative mechanism for the involvement of membrane-associated G proteins in host–
ways have been proposed to mediate these
pathogen interactions. Receptor proteins, encoded by the Cf genes, are predicted to consist
early responses in host cells, ensuring an
predominantly of leucine-rich repeats (LRR). Binding of the avirulence (AVR) peptide to the
receptor results in the activation of a G protein-coupled receptor (7TMS), probably via extraelicitor-induced response that is quanticellular protein–protein interactions through the conserved leucine-rich repeats. The questatively appropriate, correctly timed and
tion mark indicates a putative membrane-localized intermediate in the signaling pathway.
highly coordinated with other activities of
the host plant cells. These pathways might
342
September 1998, Vol. 3, No. 9
Copyright © 1998 Elsevier Science Ltd. All rights reserved. 1360 - 1385/98/$19.00 PII: S1360-1385(98)01289-8
trends in plant science
reviews
comprise G proteins (to relay the initial elicitor–receptor recognition through a series of membrane-delimited pathways), changes
in cytosolic Ca2+ concentrations and protein kinases/phosphatases,
that affect the activity of key enzymes.
G proteins
G proteins act as molecular signal transducers whose active or inactive states depend on the binding of GTP or GDP, respectively.
The G proteins include two major subfamilies, the heterotrimeric
G proteins and the small G proteins. Whereas the heterotrimeric
G proteins contain a, b and g subunits, the small G proteins appear to be similar to free a subunits, operating without the bg
heterodimer. Generally, it is the a subunit of the heterotrimeric
G protein that has the receptor-binding region and possesses a
guanosine nucleotide binding site and GTPase activity15. Both
classes of G proteins use the GTP/GDP cycle as a molecular
switch for signal transduction. Interaction of the G protein with
the activated receptor promotes the exchange of GDP, bound to
the a subunit, for GTP and the subsequent dissociation of the
a–GTP complex from the bg heterodimer.
A variety of evidence suggests a role for heterotrimeric G proteins in the plant defense response pathway. Most of the research
has been based on the use of non-hydrolyzable GTP analogs16,17,
toxins such as CTX (Ref. 18), mastoparan11,16,19, and recombinant
G proteins20. Although much of the evidence is correlative, some
interesting hypotheses can be proposed. For example, it could be
argued that the activation of defense responses by nonhydrolyzable GTP analogs is a consequence of receptor activation caused
by the interaction of these analogs with the NBS domain of the R
gene product. However, this possibility is unlikely given the low
affinity of the NBS for the nucleotides, which is several orders
of magnitude lower than that of the G proteins20. A more plausible explanation is that G proteins are activated downstream of
elicitor–receptor recognition. The Cf family of receptors consists
of extracellular LRRs. Although the N-terminal LRRs appear to
be different, the C-terminal LRRs are conserved among the different genes cloned to date. It is conceivable that the conserved
LRRs are involved in extracellular protein interactions while the
N-terminal LRRs are involved in ligand binding. In such a model,
rather than the receptor protein interacting with an additional molecule intracellularly, it might interact with a G protein-coupled
receptor (Fig. 1). Data showing the direct activation of a plasma
membrane Ca2+ channel by a recombinant a-subunit suggests that
the activation of defense responses could be G protein mediated
through plasma membrane-delimited pathways20.
Protein kinases
The phosphorylation of proteins, probably initiated by the receptor, is thought to relay the defense signal to different downstream
effectors. In some cases, the receptor contains a kinase domain
that may trigger the phosphorylation cascade, whereas in others a
secondary messenger such as Ca2+ may trigger the protein kinases.
The R gene class containing rice Xa-21 (Ref. 21) and wheat Lr10
(Ref. 22) encodes receptor-like kinases with an extracellular domain,
a membrane-spanning domain and a cytosolic serine/threonine
kinase domain. It might be expected that following perception of a
signal from the pathogen (such as an elicitor), the kinase domain of
these proteins would trigger a cascade of phosphorylation. Unfortunately, no downstream-interacting proteins for these receptorlike kinases have been identified to date. The tomato Pto gene is a
unique class of R gene that encodes a cytosolic protein with serine/
threonine kinase activity. Several proteins that interact with the Pto
kinase have been characterized23. The Pti1 (‘Pto-interacting 1’) gene
encodes a serine/threonine kinase that is probably a downstream
substrate of Pto; three other proteins, encoded by the genes Pti4,
Pti5 and Pti6, were also found to interact with the Pto kinase.
Each of the three proteins was shown to be a transcription factor
that binds to the promoter region of a large number of pathogenesisrelated (PR) genes24. In parsley, recognition of a nonspecific elicitor by the host cell also triggers a signaling pathway mediated by a
mitogen-activated protein (MAP) kinase25. This elicitor-responsive
MAP kinase (ERM kinase) is likely to be involved in transcriptional activation as the kinase translocates into the nucleus upon
elicitor treatment25.
Fungal elicitors induce changes in the phosphorylation status of
proteins in tomato cells in suspension culture26, and these changes
correlate with an increase in cytosolic free Ca2+ concentrations.
The dephosphorylation of the plasma membrane H+-ATPase was
evident soon after treatment with elicitors from incompatible races
of Cladosporium fulvum26. Distinct peaks of protein kinase C
(PKC)-like and Ca2+/calmodulin-dependent protein kinase activities were observed 40 min and 90 min, respectively, after treatment, and this increase in cytosolic kinase activity was associated
with the rephosphorylation of the plasma membrane H+-ATPase.
Among the plant calcium-dependent protein kinases, calmodulinlike domain protein kinases (CDPKs) have attracted great attention27. Although CDPKs have been implicated in general stress
responses28, specific evidence for their participation in signal transduction pathways during plant–pathogen interaction is still lacking.
However, increasing evidence suggests that the elevated cytosolic
calcium concentrations is a common consequence of pathogen perception11,24,29,30, and it is likely that CDPKs are involved in these
signaling processes.
The biophysical and biochemical response
Plasma membrane H+-ATPase
The plasma membrane H+-ATPase is essential for the growth of
cells. Proton extrusion provides the electrochemical gradient of
protons across the plasma membrane that drives the different H+coupled (antiport and symport) and membrane potential-coupled
(uniport) transport mechanisms for the uptake and extrusion of
solutes. In addition, the membrane potential regulates a number
of plasma membrane-bound ion channels, and acidification of
the extracellular medium regulates the physical and biochemical
properties of the cell wall. Changes in the host plasma membrane
H+-ATPase activity (with the associated changes in ion fluxes across
the plasma membrane) are among the earliest events associated
with elicitation. In some cases, treatment with elicitors results in an
inhibition of H+-ATPase activity and a concomitant depolarization
of the plasma membrane potential10,31. In other cases, treatment with
elicitors results in activation of plasma membrane H+-ATPase, with
consequent acidification of the extracellular milieu and hyperpolarization of the membrane potential10. In tomato, activation of
host plasma membrane H+-ATPase by elicitors from incompatible
races of C. fulvum appears to be mediated by a heterotrimeric G
protein that activates a membrane-bound phosphatase to induce
the dephosphorylation of the H+-pump17,26. It has been proposed
that the differential effect of elicitors on the plasma membrane H+ATPase and the resultant acidification or alkalinization of the extracellular medium is in response to the difference between specific
and nonspecific elicitors32.
Calcium homeostasis
Many cellular processes, including plant responses to pathogens,
are regulated by changes in cytosolic Ca2+ concentrations, where
free Ca2+ can serve to transduce a particular stimulus to target proteins that guide the cellular response. Many of the biochemical responses associated with the defense mechanisms directly correlate
September 1998, Vol. 3, No. 9
343
trends in plant science
reviews
Ca
Elicitor
2+
+
H
Stimulated
Receptor
PK
α β γ
α
ATP
ADP
Inhibited
GTP
PP
α
GTP
Ca2+
ATP
ADP
Cytosol
Stimulated
GTP
Increase in cytosolic Ca2+ concentrations, triggering Ca2+-dependent signaling
Ca2+
Inhibited
ADP
ATP
Cytosol
Stimulated
of downstream biochemical pathways (Fig.
2). Although there is increasing evidence
for a role for Ca2+ signaling in this process, a
fundamental question remains unanswered:
how does the cell use these calcium signals
to control downstream targets and the activation of a number of Ca2+-dependent protein kinases (i.e. CDPKs, PKC and Ca2+/
CaM-dependent protein kinase)? A model
recently proposed by De Koninck and
Schulman33 may explain the concerted action of these kinases. They demonstrated
that CaM KII kinases were primed by Ca2+
bursts of a given frequency and maintained
their activity with Ca2+ signals of substantially lower frequency. This may allow the
cells to distinguish betweem specific calcium signals (intracellular calcium spikes)
and nonspecific changes in steady-state
cytosolic Ca2+ concentrations. Plant cells
could also reinforce intracellular calcium
signals (due to the influx of Ca2+ from the
extracellular space) by coupling these Ca2+
bursts with Ca2+ release from vacuoles34.
NADPH oxidase
H+
Among plant defense responses to pathogen attack, the rapid production of hydrogen
Fig. 2. Signal events leading to the increase in cytosolic Ca concentrations. The bindperoxide (H2O2) and its probable precursor,
ing of elicitors to receptors at the host plasma membrane triggers the activation of
superoxide (O22), plays an important role.
heterotrimeric G proteins. These, in turn, transduce the signal by activating phosphatases
The release of these active oxygen species
(PP) that stimulate the plasma membrane H+-ATPase. The concomitant hyperpolarization
(AOS) in the oxidative burst may affect the
of the membrane potential induces the opening of a Ca2+ channel. Further activation of the
attacking pathogen and the host cells at the
channel by the heterotrimeric G protein, modulated by a membrane-bound protein kinase
(PK), together with inhibition of the Ca2+-ATPase, results in a transient increase in cytoinfection site, thereby limiting the spread
solic Ca2+ concentrations. Alternatively, inhibition of plasma membrane H+-ATPase by
of the pathogen35. Evidence supporting the
nonspecific elicitors, with the concomitant depolarization of the membrane potential,
role of a plasma membrane-bound NADPH
induces the opening of a depolarized-activated Ca2+ channel that also results in a transient
oxidase in the oxidative burst and the simi2+
increase in cytosolic Ca concentrations. Broken lines indicate activation of heterotrimeric
larity between the NADPH oxidase in plant
G proteins.
cells and phagocytic animal cells is emerging. In phagocyte cells, the oxidase electron transport chain, located in the plasma
with an increase in cytosolic free Ca2+ concentration. Measure- membrane, comprises a cytochrome b heterodimer (gp91-phox
ments of external Ca2+ with ion-selective electrodes and of Ca2+ and p22-phox). It is nonfunctional until at least three proteins, p47fluxes using radiometric techniques have revealed a large and tran- phox, p67-phox and Rac (a monomeric G protein), move from the
sient Ca2+ influx with concomitant acidification of the extracellu- cytosol to dock on cytochrome b. The docking process involves
lar medium. This suggests a correlation between fungal elicitor the interaction of Src-homology-3 domains (SH3) on p47-phox
activity, hyperpolarization of the host cell plasma membrane, and (activated via a PKC-mediated phosphorylation) with p22-phox.
Ca2+ influx1. The activation of plasma membrane Ca2+ channels by After the docking process, the electron transporting component is
specific11 and nonspecific elicitors12 provides a direct demonstration able to transfer electrons from NADPH to oxygen36. Elicitor inof a pathway by which cytosolic free Ca2+ concentrations increase duction of the oxidative burst in soybean, parsley and Arabidopsis
to levels that can initiate various defense responses, including the is inhibited by diphenylene iodonium, an inhibitor of mammalian
production of active oxygen species, callose and phytoalexins. NADPH oxidase13,35,37. Antibodies raised against human neutroStimulation of Ca2+ influx, without a corresponding control of the phil p47-phox and p67-phox cross-react with proteins of the same
Ca2+ efflux is unlikely. The host cell might face two disadvantages: molecular mass in extracts from soybean, cotton, Arabidopsis and
• An inability to sustain the high cytosolic Ca2+ levels that are tomato13,14,37, and genes coding for plant homologs of neutrophil
responsible for subsequent biochemical changes
NADPH oxidase gp91-phox have recently been cloned38,39. As
• The inefficient utilization of energy for maintaining the func- in phagocytes, immunocytochemistry has shown that treatment
tion of the plasma membrane-bound Ca2+-ATPase.
of tomato cells with race-specific elicitors induces translocation
It has been shown recently that almost immediately after the treat- of p47-phox-, p67-phox- and rac-like proteins from the cytosol to
ment of tomato cells with incompatible elicitors of C. fulvum, the plasma membrane36. The assembly process also involved the
activation of plasma membrane Ca2+ channels leads to a parallel phosphorylation of these proteins36. The similarity between the
inhibition of a plasma membrane Ca2+-ATPase (Ref. 9). These re- mode of action of NADPH oxidase in tomato and in phagocytes
sults suggest a finely coordinated elicitor-dependent increase in suggests that plants and animals share common elements in this
cytosolic Ca2+ in the cell that then acts as a signal for the activation signal transduction pathway. Nevertheless, biochemical evidence
2+
344
September 1998, Vol. 3, No. 9
trends in plant science
reviews
temporal and spatial resolution of the defense response. Further
research is needed to characterize the different signaling components downstream of the initial recognition events. The molecular
characterization of the different protein kinases involved in the
signal transduction pathways and the identification of their specific targets will help to clarify the cellular network required for
activation of the defense response.
Elicitor
Receptor
β
γ α
GTP
GTP
α
PK
H2O2
Ca
2+
NADPH
O2–
CDPK
ATP
Ca2+
ADP
α
e–
GTP
PKC
NADP
+ H+
O2
GTP
ATP
CaMKII
PP
ADP
H+
Glucan
synthase
Callose
Fig. 3. A hypothetical model of the early signal transduction
events in plant–pathogen interactions. Following the events involving G proteins that lead to an increase in cytosolic Ca2+ concentrations, Ca2+ activates a calmodulin-like domain protein
kinase (CDPK). This induces NADPH oxidase activity with the
consequent production of active oxygen species (O2– ) and H2O2.
The increase in cytosolic Ca2+ concentrations will also induce b1,4 glucan synthase and the production of callose. Following these
initial events, Ca2+ activates a protein kinase C (PKC) and a Ca2+/
CaM-dependent protein kinase that rephosphorylate the H+ATPase, returning the enzyme activity to control levels. Broken
lines indicate activation of heterotrimeric G proteins.
supports the involvement of different protein kinases in the activation of p47-phox and p67-phox in phagocytes (PKC) and tomato
cells (possibly a CDPK; Ref. 14). These differences may reflect
the unique requirements for spatial and temporal distribution in
the plant cell and differences in the developmental and environmental signals to which plants must respond.
Future perspectives
Immediately downstream of pathogen recognition processes, signal transduction pathways activate key plasma membrane-bound
enzymes (Fig. 3). Evidence suggests that these pathways may involve the activation of heterotrimeric G proteins and membranebound phosphatases that subsequently mediate the activation of
plasma membrane H+-ATPase and Ca2+ channels. The increase in
cytosolic Ca2+ concentration initiates the activation of a number of
protein kinases that induce a series of defense responses (e.g. the
oxidative burst and callose synthesis) and are responsible for the
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Eduardo Blumwald*, Gilad S. Aharon and Bernard C-H. Lam are at
the Dept of Botany, University of Toronto, 25 Willcocks St,
Toronto, Ontario, Canada M5S 3B2.
*Author for correspondence (tel +1 416 978 2378;
fax +1 416 978 5878; e-mail blumwald@botany.utoronto.ca).
Regulation of nitrogen fixation in
heterocyst-forming cyanobacteria
Herbert Böhme
Some cyanobacteria are able to reduce atmospheric dinitrogen to ammonia – a process
where oxygen evolved by photosynthetic activity in the same cell is detrimental to nitrogen
fixation. Strategies to avoid oxygen range from temporal separation of nitrogen fixation and
oxygen evolution (in unicellular and filamentous, non-heterocystous strains) to spatial separation and cellular differentiation into nitrogen fixing heterocysts (in filamentous cyanobacteria).
Recent research has begun to clarify the genes involved in nitrogen fixation, the mechanisms
that regulate the expression of proteins involved in the assimilation of nitrogen from different
sources and the way in which the cell is able to sense its nitrogen status.
C
yanobacteria (blue-green algae) are a diverse group of prokaryotes. A common feature is their oxygenic photosynthesis, which is similar to that in algae and higher plants
and is the most important biological mechanism for capturing solar
energy. As sunlight is their energy source and water the reductant,
they generate oxygen in the light. Energy and reductant generated
by photosynthesis are usually used for carbon dioxide reduction.
Some strains are strict photoautotrophs, whereas others can use
exogenous carbon sources such as fructose and glucose.
Nitrogen fixation occurs only in prokaryotes and one line of
evidence for the common origin of the nitrogen fixation mechanism is the similar physical, chemical and biological characteristics of the nitrogen-fixing enzyme system in otherwise dissimilar
organisms. Many cyanobacteria are able to reduce atmospheric
346
September 1998, Vol. 3, No. 9
dinitrogen to ammonia. In some filamentous cyanobacteria nitrogen-fixing heterocysts are formed. Heterocysts are terminally differentiated cells whose interior becomes anaerobic, mainly as a
consequence of respiration, allowing the oxygen-sensitive process
of nitrogen fixation to continue. Heterocysts are spaced at semiregular intervals along the filament with approximately 7% of the
cells differentiating into heterocysts in free-living Anabaena/Nostoc
species. The regulation of dinitrogen fixation has been extensively
studied in the heterocyst system of diazotrophic cyanobacteria1.
Nitrogen fixation and heterocyst formation
During differentiation of a vegetative cell into a heterocyst, major
structural and biochemical changes occur that affect nitrogen fixation. Upon nitrogen deprivation phycobiliproteins are broken
Copyright © 1998 Elsevier Science Ltd. All rights reserved. 1360 - 1385/98/$19.00 PII: S1360-1385(98)01290-4
reviews
Early signal transduction pathways in
plant–pathogen interactions
Eduardo Blumwald, Gilad S. Aharon and Bernard C-H. Lam
Disease resistance depends on the ability of the plant to recognize a pathogen early in the
infection process. Molecules that indicate the presence of the pathogen (elicitors) activate
host receptors and these rapidly generate an internal signal that triggers early defense
responses. Several transduction pathways that relay the initial recognition signal through a
series of cytosolic and membrane-delimited pathways have been proposed. Activation of
these signal transduction pathways ensures an elicitor-induced response that is quantitative,
timely and coordinated with other activities of the host cells.
R
ecognition of microorganisms by the plant cell depends on
the generation of elicitors by the pathogen1. These can be
non-race-specific elicitors, such as fungal or plant cell
wall fragments released during the infection process, which elicit
a defense response that helps to minimize disease. Race-specific
elicitors are molecules that are encoded by avirulence (Avr) genes
in the pathogen2. Resistance involves the specific recognition of
the invading pathogen by a dominant or semi-dominant plant resistance gene product (R gene). This type of interaction is termed
gene for gene, where for each gene that confers resistance in the
host there is a corresponding gene in the pathogen that confers its
virulence3. The cloning of plant resistance (R) genes from host–
pathogen model systems has facilitated a leap in understanding
the molecular basis of host resistance. The proposed model suggests that the direct or indirect interaction of the AVR and R polypeptides triggers resistance4. Based on sequence analysis of known
R genes, it has been hypothesized that R gene products encode receptors capable of binding AVR products as ligands5. Expression
of these gene products in susceptible plants resulted in specific
resistance3, demonstrating that even susceptible plants possess the
underlying biochemical machinery required for defense. Thus, the
difference between resistance and susceptibility appears to lie in
the proper recognition of the AVR products.
The amino acid sequences of R gene products have homology
to a number of structural protein–protein interaction domains,
suggesting that they play a role as signaling intermediates in a
reaction cascade2. In addition, it appears that multiple products,
some of which may be closely linked to the original R gene, are
required for the manifestation of resistance. One example of
this involves the functional interaction between the tomato Pto
and Prf gene products for expression of resistance to Pseudomonas syringae pv. tomato. The Pto gene product is a serine/
threonine kinase6, whereas the Prf gene product contains leucinerich regions (LRRs), nucleotide binding sites (NBS) and leucine
zippers (LZ) domains7. It is likely that other R genes, such as those
of the Cf family, may interact with other gene products to confer
resistance. This notion is also supported by biochemical evidence. For example, binding of the AVR9 peptide to plant membranes could not be correlated with the presence of the Cf9 gene8,
suggesting that the R gene product might not be exclusively involved in recognition, but also in the coordination of membrane
signaling.
Signaling
Immediately downstream of the initial elicitor–receptor recognition, the activation of ion fluxes and the production of H2O2 are
the initial responses detected in plant cells. Biochemical evidence
suggests that these processes, which occur prior to the transcriptional activation of defense-related genes, appear to be mediated
through the regulation of plasma membrane-bound enzymes. These
include changes in Ca2+-ATPase (Ref. 9) and H+-ATPase (Ref.
10) activities, the activation of plasma membrane-bound ion channels11,12 and the induction of a plasma membrane-bound NADPH oxidase13,14. These
AVR
biophysical and biochemical processes can
Cf2
Cf9
induce changes in the electrical potential
NH2
NH2
difference (membrane potential) and pH
gradient across the host plasma membrane
Leucine-rich
Leucine-rich
that may affect the activity of ion channels
repeats
repeats
7TMS
and other ion transporters. Changes in the
Defense
plasma membrane-bound oxidase electron
responses
transport chain will mediate the production
?
of H2O2 and active oxygen species (termed
α
α β γ
the oxidative burst), which may affect the
COOH COOH
GTP
attacking pathogen and the host cell at the
GDP GTP
site of infection.
A number of signal transduction pathFig. 1. Putative mechanism for the involvement of membrane-associated G proteins in host–
ways have been proposed to mediate these
pathogen interactions. Receptor proteins, encoded by the Cf genes, are predicted to consist
early responses in host cells, ensuring an
predominantly of leucine-rich repeats (LRR). Binding of the avirulence (AVR) peptide to the
receptor results in the activation of a G protein-coupled receptor (7TMS), probably via extraelicitor-induced response that is quanticellular protein–protein interactions through the conserved leucine-rich repeats. The questatively appropriate, correctly timed and
tion mark indicates a putative membrane-localized intermediate in the signaling pathway.
highly coordinated with other activities of
the host plant cells. These pathways might
342
September 1998, Vol. 3, No. 9
Copyright © 1998 Elsevier Science Ltd. All rights reserved. 1360 - 1385/98/$19.00 PII: S1360-1385(98)01289-8
trends in plant science
reviews
comprise G proteins (to relay the initial elicitor–receptor recognition through a series of membrane-delimited pathways), changes
in cytosolic Ca2+ concentrations and protein kinases/phosphatases,
that affect the activity of key enzymes.
G proteins
G proteins act as molecular signal transducers whose active or inactive states depend on the binding of GTP or GDP, respectively.
The G proteins include two major subfamilies, the heterotrimeric
G proteins and the small G proteins. Whereas the heterotrimeric
G proteins contain a, b and g subunits, the small G proteins appear to be similar to free a subunits, operating without the bg
heterodimer. Generally, it is the a subunit of the heterotrimeric
G protein that has the receptor-binding region and possesses a
guanosine nucleotide binding site and GTPase activity15. Both
classes of G proteins use the GTP/GDP cycle as a molecular
switch for signal transduction. Interaction of the G protein with
the activated receptor promotes the exchange of GDP, bound to
the a subunit, for GTP and the subsequent dissociation of the
a–GTP complex from the bg heterodimer.
A variety of evidence suggests a role for heterotrimeric G proteins in the plant defense response pathway. Most of the research
has been based on the use of non-hydrolyzable GTP analogs16,17,
toxins such as CTX (Ref. 18), mastoparan11,16,19, and recombinant
G proteins20. Although much of the evidence is correlative, some
interesting hypotheses can be proposed. For example, it could be
argued that the activation of defense responses by nonhydrolyzable GTP analogs is a consequence of receptor activation caused
by the interaction of these analogs with the NBS domain of the R
gene product. However, this possibility is unlikely given the low
affinity of the NBS for the nucleotides, which is several orders
of magnitude lower than that of the G proteins20. A more plausible explanation is that G proteins are activated downstream of
elicitor–receptor recognition. The Cf family of receptors consists
of extracellular LRRs. Although the N-terminal LRRs appear to
be different, the C-terminal LRRs are conserved among the different genes cloned to date. It is conceivable that the conserved
LRRs are involved in extracellular protein interactions while the
N-terminal LRRs are involved in ligand binding. In such a model,
rather than the receptor protein interacting with an additional molecule intracellularly, it might interact with a G protein-coupled
receptor (Fig. 1). Data showing the direct activation of a plasma
membrane Ca2+ channel by a recombinant a-subunit suggests that
the activation of defense responses could be G protein mediated
through plasma membrane-delimited pathways20.
Protein kinases
The phosphorylation of proteins, probably initiated by the receptor, is thought to relay the defense signal to different downstream
effectors. In some cases, the receptor contains a kinase domain
that may trigger the phosphorylation cascade, whereas in others a
secondary messenger such as Ca2+ may trigger the protein kinases.
The R gene class containing rice Xa-21 (Ref. 21) and wheat Lr10
(Ref. 22) encodes receptor-like kinases with an extracellular domain,
a membrane-spanning domain and a cytosolic serine/threonine
kinase domain. It might be expected that following perception of a
signal from the pathogen (such as an elicitor), the kinase domain of
these proteins would trigger a cascade of phosphorylation. Unfortunately, no downstream-interacting proteins for these receptorlike kinases have been identified to date. The tomato Pto gene is a
unique class of R gene that encodes a cytosolic protein with serine/
threonine kinase activity. Several proteins that interact with the Pto
kinase have been characterized23. The Pti1 (‘Pto-interacting 1’) gene
encodes a serine/threonine kinase that is probably a downstream
substrate of Pto; three other proteins, encoded by the genes Pti4,
Pti5 and Pti6, were also found to interact with the Pto kinase.
Each of the three proteins was shown to be a transcription factor
that binds to the promoter region of a large number of pathogenesisrelated (PR) genes24. In parsley, recognition of a nonspecific elicitor by the host cell also triggers a signaling pathway mediated by a
mitogen-activated protein (MAP) kinase25. This elicitor-responsive
MAP kinase (ERM kinase) is likely to be involved in transcriptional activation as the kinase translocates into the nucleus upon
elicitor treatment25.
Fungal elicitors induce changes in the phosphorylation status of
proteins in tomato cells in suspension culture26, and these changes
correlate with an increase in cytosolic free Ca2+ concentrations.
The dephosphorylation of the plasma membrane H+-ATPase was
evident soon after treatment with elicitors from incompatible races
of Cladosporium fulvum26. Distinct peaks of protein kinase C
(PKC)-like and Ca2+/calmodulin-dependent protein kinase activities were observed 40 min and 90 min, respectively, after treatment, and this increase in cytosolic kinase activity was associated
with the rephosphorylation of the plasma membrane H+-ATPase.
Among the plant calcium-dependent protein kinases, calmodulinlike domain protein kinases (CDPKs) have attracted great attention27. Although CDPKs have been implicated in general stress
responses28, specific evidence for their participation in signal transduction pathways during plant–pathogen interaction is still lacking.
However, increasing evidence suggests that the elevated cytosolic
calcium concentrations is a common consequence of pathogen perception11,24,29,30, and it is likely that CDPKs are involved in these
signaling processes.
The biophysical and biochemical response
Plasma membrane H+-ATPase
The plasma membrane H+-ATPase is essential for the growth of
cells. Proton extrusion provides the electrochemical gradient of
protons across the plasma membrane that drives the different H+coupled (antiport and symport) and membrane potential-coupled
(uniport) transport mechanisms for the uptake and extrusion of
solutes. In addition, the membrane potential regulates a number
of plasma membrane-bound ion channels, and acidification of
the extracellular medium regulates the physical and biochemical
properties of the cell wall. Changes in the host plasma membrane
H+-ATPase activity (with the associated changes in ion fluxes across
the plasma membrane) are among the earliest events associated
with elicitation. In some cases, treatment with elicitors results in an
inhibition of H+-ATPase activity and a concomitant depolarization
of the plasma membrane potential10,31. In other cases, treatment with
elicitors results in activation of plasma membrane H+-ATPase, with
consequent acidification of the extracellular milieu and hyperpolarization of the membrane potential10. In tomato, activation of
host plasma membrane H+-ATPase by elicitors from incompatible
races of C. fulvum appears to be mediated by a heterotrimeric G
protein that activates a membrane-bound phosphatase to induce
the dephosphorylation of the H+-pump17,26. It has been proposed
that the differential effect of elicitors on the plasma membrane H+ATPase and the resultant acidification or alkalinization of the extracellular medium is in response to the difference between specific
and nonspecific elicitors32.
Calcium homeostasis
Many cellular processes, including plant responses to pathogens,
are regulated by changes in cytosolic Ca2+ concentrations, where
free Ca2+ can serve to transduce a particular stimulus to target proteins that guide the cellular response. Many of the biochemical responses associated with the defense mechanisms directly correlate
September 1998, Vol. 3, No. 9
343
trends in plant science
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Ca
Elicitor
2+
+
H
Stimulated
Receptor
PK
α β γ
α
ATP
ADP
Inhibited
GTP
PP
α
GTP
Ca2+
ATP
ADP
Cytosol
Stimulated
GTP
Increase in cytosolic Ca2+ concentrations, triggering Ca2+-dependent signaling
Ca2+
Inhibited
ADP
ATP
Cytosol
Stimulated
of downstream biochemical pathways (Fig.
2). Although there is increasing evidence
for a role for Ca2+ signaling in this process, a
fundamental question remains unanswered:
how does the cell use these calcium signals
to control downstream targets and the activation of a number of Ca2+-dependent protein kinases (i.e. CDPKs, PKC and Ca2+/
CaM-dependent protein kinase)? A model
recently proposed by De Koninck and
Schulman33 may explain the concerted action of these kinases. They demonstrated
that CaM KII kinases were primed by Ca2+
bursts of a given frequency and maintained
their activity with Ca2+ signals of substantially lower frequency. This may allow the
cells to distinguish betweem specific calcium signals (intracellular calcium spikes)
and nonspecific changes in steady-state
cytosolic Ca2+ concentrations. Plant cells
could also reinforce intracellular calcium
signals (due to the influx of Ca2+ from the
extracellular space) by coupling these Ca2+
bursts with Ca2+ release from vacuoles34.
NADPH oxidase
H+
Among plant defense responses to pathogen attack, the rapid production of hydrogen
Fig. 2. Signal events leading to the increase in cytosolic Ca concentrations. The bindperoxide (H2O2) and its probable precursor,
ing of elicitors to receptors at the host plasma membrane triggers the activation of
superoxide (O22), plays an important role.
heterotrimeric G proteins. These, in turn, transduce the signal by activating phosphatases
The release of these active oxygen species
(PP) that stimulate the plasma membrane H+-ATPase. The concomitant hyperpolarization
(AOS) in the oxidative burst may affect the
of the membrane potential induces the opening of a Ca2+ channel. Further activation of the
attacking pathogen and the host cells at the
channel by the heterotrimeric G protein, modulated by a membrane-bound protein kinase
(PK), together with inhibition of the Ca2+-ATPase, results in a transient increase in cytoinfection site, thereby limiting the spread
solic Ca2+ concentrations. Alternatively, inhibition of plasma membrane H+-ATPase by
of the pathogen35. Evidence supporting the
nonspecific elicitors, with the concomitant depolarization of the membrane potential,
role of a plasma membrane-bound NADPH
induces the opening of a depolarized-activated Ca2+ channel that also results in a transient
oxidase in the oxidative burst and the simi2+
increase in cytosolic Ca concentrations. Broken lines indicate activation of heterotrimeric
larity between the NADPH oxidase in plant
G proteins.
cells and phagocytic animal cells is emerging. In phagocyte cells, the oxidase electron transport chain, located in the plasma
with an increase in cytosolic free Ca2+ concentration. Measure- membrane, comprises a cytochrome b heterodimer (gp91-phox
ments of external Ca2+ with ion-selective electrodes and of Ca2+ and p22-phox). It is nonfunctional until at least three proteins, p47fluxes using radiometric techniques have revealed a large and tran- phox, p67-phox and Rac (a monomeric G protein), move from the
sient Ca2+ influx with concomitant acidification of the extracellu- cytosol to dock on cytochrome b. The docking process involves
lar medium. This suggests a correlation between fungal elicitor the interaction of Src-homology-3 domains (SH3) on p47-phox
activity, hyperpolarization of the host cell plasma membrane, and (activated via a PKC-mediated phosphorylation) with p22-phox.
Ca2+ influx1. The activation of plasma membrane Ca2+ channels by After the docking process, the electron transporting component is
specific11 and nonspecific elicitors12 provides a direct demonstration able to transfer electrons from NADPH to oxygen36. Elicitor inof a pathway by which cytosolic free Ca2+ concentrations increase duction of the oxidative burst in soybean, parsley and Arabidopsis
to levels that can initiate various defense responses, including the is inhibited by diphenylene iodonium, an inhibitor of mammalian
production of active oxygen species, callose and phytoalexins. NADPH oxidase13,35,37. Antibodies raised against human neutroStimulation of Ca2+ influx, without a corresponding control of the phil p47-phox and p67-phox cross-react with proteins of the same
Ca2+ efflux is unlikely. The host cell might face two disadvantages: molecular mass in extracts from soybean, cotton, Arabidopsis and
• An inability to sustain the high cytosolic Ca2+ levels that are tomato13,14,37, and genes coding for plant homologs of neutrophil
responsible for subsequent biochemical changes
NADPH oxidase gp91-phox have recently been cloned38,39. As
• The inefficient utilization of energy for maintaining the func- in phagocytes, immunocytochemistry has shown that treatment
tion of the plasma membrane-bound Ca2+-ATPase.
of tomato cells with race-specific elicitors induces translocation
It has been shown recently that almost immediately after the treat- of p47-phox-, p67-phox- and rac-like proteins from the cytosol to
ment of tomato cells with incompatible elicitors of C. fulvum, the plasma membrane36. The assembly process also involved the
activation of plasma membrane Ca2+ channels leads to a parallel phosphorylation of these proteins36. The similarity between the
inhibition of a plasma membrane Ca2+-ATPase (Ref. 9). These re- mode of action of NADPH oxidase in tomato and in phagocytes
sults suggest a finely coordinated elicitor-dependent increase in suggests that plants and animals share common elements in this
cytosolic Ca2+ in the cell that then acts as a signal for the activation signal transduction pathway. Nevertheless, biochemical evidence
2+
344
September 1998, Vol. 3, No. 9
trends in plant science
reviews
temporal and spatial resolution of the defense response. Further
research is needed to characterize the different signaling components downstream of the initial recognition events. The molecular
characterization of the different protein kinases involved in the
signal transduction pathways and the identification of their specific targets will help to clarify the cellular network required for
activation of the defense response.
Elicitor
Receptor
β
γ α
GTP
GTP
α
PK
H2O2
Ca
2+
NADPH
O2–
CDPK
ATP
Ca2+
ADP
α
e–
GTP
PKC
NADP
+ H+
O2
GTP
ATP
CaMKII
PP
ADP
H+
Glucan
synthase
Callose
Fig. 3. A hypothetical model of the early signal transduction
events in plant–pathogen interactions. Following the events involving G proteins that lead to an increase in cytosolic Ca2+ concentrations, Ca2+ activates a calmodulin-like domain protein
kinase (CDPK). This induces NADPH oxidase activity with the
consequent production of active oxygen species (O2– ) and H2O2.
The increase in cytosolic Ca2+ concentrations will also induce b1,4 glucan synthase and the production of callose. Following these
initial events, Ca2+ activates a protein kinase C (PKC) and a Ca2+/
CaM-dependent protein kinase that rephosphorylate the H+ATPase, returning the enzyme activity to control levels. Broken
lines indicate activation of heterotrimeric G proteins.
supports the involvement of different protein kinases in the activation of p47-phox and p67-phox in phagocytes (PKC) and tomato
cells (possibly a CDPK; Ref. 14). These differences may reflect
the unique requirements for spatial and temporal distribution in
the plant cell and differences in the developmental and environmental signals to which plants must respond.
Future perspectives
Immediately downstream of pathogen recognition processes, signal transduction pathways activate key plasma membrane-bound
enzymes (Fig. 3). Evidence suggests that these pathways may involve the activation of heterotrimeric G proteins and membranebound phosphatases that subsequently mediate the activation of
plasma membrane H+-ATPase and Ca2+ channels. The increase in
cytosolic Ca2+ concentration initiates the activation of a number of
protein kinases that induce a series of defense responses (e.g. the
oxidative burst and callose synthesis) and are responsible for the
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Eduardo Blumwald*, Gilad S. Aharon and Bernard C-H. Lam are at
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Regulation of nitrogen fixation in
heterocyst-forming cyanobacteria
Herbert Böhme
Some cyanobacteria are able to reduce atmospheric dinitrogen to ammonia – a process
where oxygen evolved by photosynthetic activity in the same cell is detrimental to nitrogen
fixation. Strategies to avoid oxygen range from temporal separation of nitrogen fixation and
oxygen evolution (in unicellular and filamentous, non-heterocystous strains) to spatial separation and cellular differentiation into nitrogen fixing heterocysts (in filamentous cyanobacteria).
Recent research has begun to clarify the genes involved in nitrogen fixation, the mechanisms
that regulate the expression of proteins involved in the assimilation of nitrogen from different
sources and the way in which the cell is able to sense its nitrogen status.
C
yanobacteria (blue-green algae) are a diverse group of prokaryotes. A common feature is their oxygenic photosynthesis, which is similar to that in algae and higher plants
and is the most important biological mechanism for capturing solar
energy. As sunlight is their energy source and water the reductant,
they generate oxygen in the light. Energy and reductant generated
by photosynthesis are usually used for carbon dioxide reduction.
Some strains are strict photoautotrophs, whereas others can use
exogenous carbon sources such as fructose and glucose.
Nitrogen fixation occurs only in prokaryotes and one line of
evidence for the common origin of the nitrogen fixation mechanism is the similar physical, chemical and biological characteristics of the nitrogen-fixing enzyme system in otherwise dissimilar
organisms. Many cyanobacteria are able to reduce atmospheric
346
September 1998, Vol. 3, No. 9
dinitrogen to ammonia. In some filamentous cyanobacteria nitrogen-fixing heterocysts are formed. Heterocysts are terminally differentiated cells whose interior becomes anaerobic, mainly as a
consequence of respiration, allowing the oxygen-sensitive process
of nitrogen fixation to continue. Heterocysts are spaced at semiregular intervals along the filament with approximately 7% of the
cells differentiating into heterocysts in free-living Anabaena/Nostoc
species. The regulation of dinitrogen fixation has been extensively
studied in the heterocyst system of diazotrophic cyanobacteria1.
Nitrogen fixation and heterocyst formation
During differentiation of a vegetative cell into a heterocyst, major
structural and biochemical changes occur that affect nitrogen fixation. Upon nitrogen deprivation phycobiliproteins are broken
Copyright © 1998 Elsevier Science Ltd. All rights reserved. 1360 - 1385/98/$19.00 PII: S1360-1385(98)01290-4