BACKGROUND
brassinosteroids  are a group of plant steroidal hormones that regulate various aspects of plant growth and development, including cell elongation, photomorphogenesis, xylem differentiation, and seed germination  <cit> , as well as adaptation to abiotic and biotic environmental stresses  <cit> .
molecular genetic studies of br-deficient and br-insensitive mutants have established an essential role for brs in plant development and led to the identification and characterization of several br signaling components  <cit> .
a large number of br-regulated genes have been identified by microarray studies; most of the known br-regulated genes are associated with plant growth and development, such as cell wall modification, cytoskeleton formation, and hormone synthesis  <cit> .
how br regulates gene expression is currently understood for only a small proportion of genes.
in the current model of br-controlled gene expression, br binding to bri <dig>  a plasma membrane localized leucine-rich repeat receptor-like kinase , induces association of bri <dig> with its coreceptor bak <dig>  which enhances signaling output through reciprocal bri <dig> transphosphorylation  <cit> .
bri <dig> binding to br inactivates bin <dig>  a glycogen synthase kinase- <dig>  and possibly activates the phosphatase bsu <dig>  bin <dig> negatively regulates transcription factors bzr <dig> and bes <dig> by phosphorylating them, while bsu <dig> positively regulates br signaling by dephosphorylating bzr <dig> and bes <dig>  activated bzr <dig> and bes <dig> accumulate in the nucleus and directly bind to cgtgg motif in the promoters of br biosysnthesis genes cpd and dwf <dig>  <cit>  and to e box sequence  in the saur-aci promoter  <cit> , respectively, to affect gene expression.
the recent demonstrations that bes <dig> interacts with other transcription factors such as bims  <cit> , myb <dig>  which acts as a positive regulator of the hypersensitive cell death response  <cit> , and the jumonji  domain-containing proteins elf <dig> and ref <dig> that are involved in regulating flowering time  <cit> , points to recruitment of different proteins by bes <dig> as one of the ways by which br affects diverse biological processes.
the role of brs in plant stress responses has been confirmed in several studies  <cit> .
br promotes tolerance in plants to a wide range of stresses, including heat, cold, drought and salinity, and this increase is generally correlated with higher expression of stress marker genes, such as heat shock protein  genes, rd29a and erd <dig>  <cit> , indicating that increased expression of stress-responsive genes is responsible, in part, for the higher stress tolerance in br-treated plants.
the mechanisms by which br controls plant stress responses and regulates the expression of stress response genes are not known.
since different plant hormones can regulate similar physiological processes, and cross-talk between different hormones can occur at the level of hormone biosynthesis, signal transduction or gene expression  <cit> , it was proposed that br regulates plant stress responses via cross-talk with other hormones  <cit> .
the plant growth regulators with documented roles in plant adaptation to abiotic and biotic stresses are abscisic acid , ethylene , jasmonic acid  and salicylic acid .
sa, ja and et are important in defense against pathogen and pest attack  <cit> , whereas aba is a key molecule involved in salt and drought stress  <cit> .
sa, et, aba and ja have also been linked to heat stress.
studies of hormone deficient and insensitive mutants have demonstrated the involvement of sa, et and aba in acquired thermotolerance of plants  <cit> , and additionally for sa and ja, a role in basal thermotolerance of plants  <cit> .
although experimental evidence points to interactions of br with auxin  <cit> , gibberellin   <cit> , aba  <cit> , et  <cit>  and ja  <cit> , the relationship of br with these hormones has been documented primarily in plant growth regulatory processes.
furthermore, with the exception of br-auxin interaction, little is known in terms of genes how br interacts with other hormones.
recent progress made towards understanding br-auxin interaction can serve as a paradigm for how two hormones could interact at multi-levels.
auxin and br share a number of target genes, many of which are involved in growth-related processes  <cit> .
since promoter regions in br-responsive genes are enriched in auxin response factor -binding sites, and binding sites of bes <dig> are overrepresented in genes regulated by both hormones, regulatory elements in gene promoters represent a point of cross-talk between auxin and br.
recently, the br-regulated bin <dig> kinase was demonstrated to phosphorylate arf <dig>  a member of the arf family of transcriptional regulators, leading to loss of arf <dig> dna binding and repression activities  <cit> .
thus, in this model arf <dig> links br and auxin signaling pathways.
in addition to gene coregulation, br can also promote auxin transport  <cit> , and optimal auxin action is dependent on br levels  <cit> .
the role of br in plant responses to abiotic stress has become well established over the last decade, but there are very few reports indicating how br interacts with other stress-related hormones and their signaling pathways in conferring stress tolerance.
while there exists evidence to indicate that br increases et and ja levels under normal growth conditions  <cit> , there appears to be only one report linking br with increase in aba levels in the lower plant chlorella vulgaris under stress condition  <cit> .
recently it was demonstrated that aba inhibits br signaling through phosphorylation of bes <dig>  <cit> .
currently there are no studies at the genetic level as to how br interacts with other hormones under stress conditions.
here we asked the question whether one or more stress-related hormones, such as aba, et, ja or sa, have a major role in br-mediated stress tolerance.
arabidopsis mutants with either disrupted or enhanced hormone pathways were tested for phenotypes and gene expression in response to br under high temperature and high salt conditions.
our results suggest that in arabidopsis the nonexpressor of pathogenesis-related genes <dig>  is likely a critical component of br-mediated effects on thermotolerance and salt tolerance, that br exerts anti-stress effects both independently as well as through interactions with other hormones, aba inhibits br effects during heat stress, and that br shares transcriptional targets with other hormones.
RESULTS
ebr effects on basal thermotolerance in different hormone genotypes
we have previously demonstrated that ebr enhances the basic thermotolerance of brassica napus, tomato  <cit>  and arabidopsis seedlings  <cit> .
since the effects of ebr on stress tolerance are most pronounced when seedlings are grown in the presence of ebr , we postulated the involvement of other phytohormones in this process  <cit> .
several hormone pathways, such as of aba, et, sa and ja, have been linked with one or more environmental stresses, including heat stress .
we therefore evaluated the effects of ebr on thermotolerance in a subset of arabidopsis hormone mutants altered in either biosynthesis or signaling of these phytohormones .
we first studied the effects of ebr on basal thermotolerance in sa genotypes npr1- <dig> , but not all sa responses), eds5- <dig> , and cpr5- <dig>  by exposing 21-day-old seedlings to 43°c for  <dig> h, allowing them to recover at 22°c for  <dig> days and then scoring for dead and surviving seedlings.
wt, eds5- <dig>  npr1- <dig> and cpr5- <dig> seedlings grown in the absence of ebr had average survival rates of  <dig> ,  <dig> ,  <dig>  and  <dig> %, respectively, and those grown in the presence of ebr had survival rates of  <dig> ,  <dig> ,  <dig>  and 83%, respectively .
from these results it is clear that the survival rates of wt and different sa genotypes were significantly increased by ebr treatment, but the increase was considerably less in case of npr1- <dig> as compared to other genotypes within this group.
photographs of all genotypes grown in the presence or absence of ebr under no-stress conditions can be seen in additional file 1: pictures of 21-day-old seedlings.
sa synthesis
key regulator of sa-mediated systemic acquired
resistance  pathway;
mutants are sa-insensitive and hypersusceptible
participates in signal transduction pathways involved in plant defense;
catalyzes dehydration of the hydroperoxide to an unstable allene oxide in the ja biosynthetic pathway;
for genotypes related to ethylene, ebr increased survival rates of wt, ein <dig> , and eto1- <dig>  seedlings to significant levels as compared to seedlings with no treatment .
the ja mutants aos  and jar1- <dig>  belong to different backgrounds; jar1- <dig> is in arabidopsis ecotype columbia , whereas aos is in col- <dig> background.
ebr increased survival rates of aos and jar1- <dig> seedlings to amounts similar to corresponding wt seedlings .
the aba mutants aba1- <dig>  and abi1- <dig>  are from the landsberg erecta  background; hence, wt ler was used for comparison with these mutants.
under the conditions used, ebr was less effective in wt ler as compared to wt col  .
since aba has been linked with heat tolerance  <cit> , we expected aba mutants to be less thermotolerant than wt even in the presence of ebr.
contrary to our expectation we found that the effect of ebr was most distinct in aba1- <dig>  as compared to wt  and abi1- <dig>  seedlings .
these results suggest that aba masks br effects on the hs response pathway of wt arabidopsis seedlings.
from the survival data represented in figure  <dig> it is clear that when ebr effect in any hormone genotype is viewed in reference to the effect on the corresponding wt, ebr could increase the basal thermotolerance of all hormone genotypes, but its effect was minimal in npr1- <dig>  since the sa-deficient nahg transgenic line  <cit>  and the ja response defective coi <dig> mutant  <cit>  could not be obtained for this study, we cannot yet conclude that sa and ja are dispensable for br-mediated increase in thermotolerance.
however, from the collection of mutants used here it would appear that br can exert anti-stress effects that are independent of aba, et, ja and sa, at least to some extent.
the dependency of br on npr <dig> in mediating stress tolerance is a first time observation made in this study.
whether br modulates npr <dig> activity via sa or br pathway or both remains to be determined.
ebr effects on oxidative damage in hormone mutants
heat stress produces oxidative damage, which as a result of lipid peroxidation leads to the production of thiobarbituric acid reactive substances   <cit> .
to complement the results of the hs phenotype of seedlings, oxidative damage levels were assessed in untreated and ebr-treated seedlings.
measurements of tbars in time course experiments determined that maximum oxidative damage occurred during recovery from hs .
therefore, seedlings exposed to hs and then allowed to recover for  <dig> days at 22°c were used for tbars analysis.
with the exception of npr1- <dig> and cpr5- <dig>  ebr treatment reduced the levels of oxidative damage in wt and other mutant seedlings as compared to their untreated counterparts .
wt col and ler exhibited 45% and 60% reduction in tbars production, respectively, while the mutant seedlings showed 25-50% less tbars, in response to ebr treatment.
consistent with its lower survival rate , the npr1- <dig> mutant showed an insignificant  <dig> % reduction in tbars production in response to ebr treatment .
however, in contrast to the relatively higher survival of cpr5- <dig> seedlings, the reduction in oxidative damage in these seedlings in response to ebr measured only 25% relative to no treatment.
overall, these results demonstrated that ebr treatment can reduce oxidative damage during hs and that this effect is not critically dependent on any one hormone in question, although a functional npr <dig> protein appears to be required for a complete effect of ebr on thermotolerance of seedlings.
ebr induces higher accumulation of hsp <dig> in aba1-1
we have found that ebr treatment leads to significant increases in the levels of hsps during hs in b. napus  <cit> , but the effect of ebr on hsp levels in arabidopsis is subtle  <cit> .
we wished to see how ebr would affect the accumulation of hsp <dig> in various mutant seedlings in the absence of hs  and in response to hs .
with the exception of aba1- <dig> , no significant differences in the steady state levels of hsp <dig> were observed between ebr-treated and untreated mutant seedlings, including npr1- <dig> .
ebr-treated aba1- <dig> seedlings accumulated approximately 3- and  <dig> -fold higher levels of hsp <dig> at  <dig> and  <dig> h of hs, respectively, as compared to untreated aba1- <dig> seedlings .
by contrast, ebr-treated wt seedlings showed a maximum of  <dig> -fold increase in hsp <dig> levels at  <dig> h of hs as compared to untreated seedlings.
the fold-change values are average of three different experiments, which consistently produced the same pattern of protein accumulation.
thus, with respect to higher survival following hs and greater accumulation of hsp <dig> during hs, ebr produced most distinct effects in the aba-deficient aba1- <dig> mutant.
these results reinforce the idea that aba suppresses br effects in wt seedlings.
although convincing evidence for antagonism between br and aba in plant growth regulatory processes, such as germination has been provided before  <cit> , the demonstration at the genetic and molecular levels of an antagonistic relationship between the two hormones in plant stress response is new.
ebr upregulates the expression of sa, ja/et and aba-response genes in both wt and corresponding mutants
plant hormone responses in arabidopsis have been correlated with the expression of hormone-specific marker genes.
we studied the expression of few such genes that are well documented to be induced by sa, ja/et or aba, both before and after treatment with ebr.
pr- <dig>  transcription factor wrky <dig> and wak <dig> are known to be regulated primarily by sa  <cit> ; pdf <dig> , lox <dig> and hel by ja/et  <cit> ; and rd <dig> and ltp <dig> by aba  <cit> .
the steady-state levels of pr- <dig>  wrky <dig> and wak <dig> transcripts were elevated by ebr in wt and sa-related genotypes, including npr1- <dig>  albeit at different levels .
the npr1- <dig> genotype is defective in the expression of pr genes in response to sa  <cit> .
full-scale induction of wrky <dig> and wak <dig> by sa also requires a functional npr <dig>  <cit> .
our results clearly indicate that ebr can mediate induction of pr- <dig>  wrky <dig> and wak <dig> to levels seen in figure 3a in an npr1-independent manner.
ebr treatment also enhanced the expression of the ja/et marker gene pdf <dig>  in wt, aos, jar1- <dig> and eto1- <dig> backgrounds, but not to the same extent in ein <dig> .
the effect of ebr on lox <dig> expression was distinct in col and jar1- <dig> backgrounds, but not in col- <dig> and aos backgrounds.
increase in the expression of the hel gene by ebr was only slight.
the aba-responsive ltp <dig> showed dramatic induction by ebr in wt and abi1- <dig> background , but not in aba1- <dig>  the transcript levels of the aba-marker gene rd <dig> were upregulated by ebr only slightly in aba1- <dig> and abi1- <dig> mutant seedlings, but significantly in wt , indicating interaction between aba and br in affecting gene expression.
to determine the interaction of ebr with sa, ja/et and aba in the regulation of gst <dig>  a gene common to abiotic stress and defense pathways  <cit> , we compared its transcript levels in untreated and ebr-treated wt and mutant seedlings.
ebr enhanced gst <dig> transcript levels in sa , as well as in all other hormone genotypes studied .
as would be expected, cpr5- <dig> had the highest expression of gst <dig> even in the absence of ebr.
exogenous br negatively regulates the br biosynthetic gene dwf <dig>  <cit> .
to ensure that transport, perception and signaling of br is intact in npr1- <dig>  which was most inert to ebr effects, dwf <dig> expression was determined in wt and npr1- <dig>  the fact that dwf <dig> levels were reduced in ebr-treated wt and npr1- <dig> seedlings as compared to untreated seedlings  suggests that the br pathway is intact in npr1- <dig>
taken together, these results demonstrate that most, if not all, of the sa, ja/et and aba-responsive genes tested in the present study are also upregulated by br both in wt and mutant backgrounds, albeit to different levels.
these results point to overlapping gene targets of aba, ja/et or sa and br, as well as to hormone interactions controlling the final output.
upregulation of a subset of genes by ebr is likely via a br-regulated pathway
since the effect of ebr on thermotolerance is best seen when plants are grown in the presence of ebr for 2- <dig> weeks, the gene expression studies leading to figure  <dig> were also conducted in plants receiving long-term exposure to ebr.
to determine whether the ja/et and aba-responsive genes also respond to a short-term ebr treatment, we studied the expression of a subset of genes by qrt-pcr in response to a  <dig> h treatment with ebr.
it is to be noted that br effects on gene expression can take as long as 18- <dig> hrs  <cit> .
similar to the results of long-term treatment, the  <dig> h short-term treatment with ebr enhanced the transcript levels of pr- <dig> , pdf <dig>  , rd <dig>  and gst <dig> , suggesting that changes in the expression of these genes likely occur via a br-regulated pathway .
further studies are required to confirm this idea.
downregulation of dwf <dig> by exogenous br was used as an experimental control .
ebr rescues hypersensitivity of ein <dig> to inhibition of germination by salt stress
we have previously shown that ebr helps to overcome salt stress-induced inhibition of seed germination in b. napus  <cit> .
more recently it was demonstrated that br-deficient and br signaling-defective mutants are more inhibited in germination on salt while a bri <dig> overexpressing transgenic line is more resistant to salt than wt  <cit> .
in this study, we first screened npr1- <dig>  ein <dig>  eto1- <dig>  jar1- <dig> and abi1- <dig> mutants for their germination efficiency on  <dig> mm nacl.
as noted before with ein2- <dig>  <cit> , ein <dig> was more sensitive to nacl-mediated inhibition of germination as compared to wt .
by contrast, eto1- <dig>  npr1- <dig>  jar1- <dig> and abi1- <dig> were less sensitive than wt .
thus, to study the effects of ebr on salt stress we selected ein <dig> because of its hypersensitivity to salt stress, and eto1- <dig> and npr1- <dig> because of their relatively higher susceptibility to hs even after ebr treatment.
seeds were allowed to germinate on  <dig> mm nacl in the presence or absence of ebr, and seedlings with emerged cotyledons were scored after  <dig> days.
inhibition of germination of ein <dig> seeds by salt was significantly reduced in the presence of ebr .
wt seeds showed germination rates of 55% and > 80% on  <dig> mm nacl in the absence and presence of ebr, respectively, while ein <dig> seeds had germination rates of ~10% and 80% under the same conditions .
seeds of eto1- <dig> and npr1- <dig> germinated at similar rates  on  <dig> mm nacl in the absence or presence of ebr .
however, despite the good germination efficiency on salt, the average survival rates of eto1- <dig> and npr1- <dig> on salt were only  <dig> and 22%, respectively , but unlike wt, survival of the mutant seedlings could not be rescued by ebr treatment .
these results indicate that npr <dig> has a role in salt stress and that br effects on seedling survival under salt stress require a functional npr <dig>  phenotypes of wt, ein <dig>  eto1- <dig> and npr1- <dig> seedlings grown in the presence or absence of ebr under no-salt conditions can be seen in additional file 2: pictures of 3-day-old seedlings.
discussion
feeding br through roots  not only rescues growth defects of br-deficient mutants  <cit> , but also leads to increased stress tolerance in wt seedlings  <cit> .
furthermore, overexpression of br biosynthesis genes leads to increased vegetative and seed yield in transgenic seedlings  <cit> .
in each case it is rationalized that br-mediated outcomes likely involve both direct and indirect effects of br.
in the present study we focused on understanding br interactions with other stress hormones in mediating increase in stress tolerance mainly because 1) br is known to interact with other plant hormones in regulating plant developmental processes, and 2) multiple hormone signaling pathways play a role in acquisition of stress tolerance.
we evaluated a subset of signaling, biosynthetic and constitutively active mutants of aba, et, ja and sa for thermo and salt tolerance in untreated and br-treated states to assess the importance of these hormones in br-mediated increase in stress tolerance of arabidopsis seedlings.
here we demonstrate that npr <dig>  a protein well recognized for its role in sa-mediated sar and cross-talk inhibition of ja-mediated defense responses, also has a role in br-mediated stress tolerance.
thermotolerance defects of hormone mutants and br effects
sa, and more recently ja, have been linked with thermotolerance.
in case of sa genotypes, it is known that npr1- <dig> is compromised in basal thermotolerance, while cpr5- <dig> has greater thermotolerance than wt  <cit> .
recently it was demonstrated that a jar1-dependent pathway is also required for basal thermotolerance  <cit> .
using a collection of genotypes with basal thermotolerance either lower or higher than wt, we found that ebr treatment could significantly increase the basic thermotolerance of these genotypes and that this increase was comparable to the increase in wt.
an exception to this result within the sa, ja and et genotypes was npr1- <dig>  indicating that a functional npr <dig> is required for full manifestation of br's effects.
although collectively our data seems to suggest that br is not critically dependent on sa levels and ja signaling for its antistress effects, further confirmation is required with genotypes such as nahg and coi <dig> to make an unequivocal claim.
even if br works to some extent independently of other hormones in conferring heat tolerance, the mere fact that aba, br, et, ja and sa all play a role in thermotolerance of arabidopsis plants suggests that there must be some redundant and some specific events in the mechanisms by which these hormones produce their effects.
with the exception of br where some information has been obtained  <cit> , molecular changes mediated by aba, et, ja and sa that lead to thermotolerance are largely unknown.
in case of ethylene, the ethylene response factor protein, jerf <dig>  has been demonstrated to activate the expression of oxidative genes, resulting in decreased accumulation of ros and, in turn, enhanced adaptation to drought, freezing, and salt in tobacco  <cit> .
a similar role for jerf <dig> can be envisioned in response to hs.
a number of aba-regulated genes have been implicated in drought tolerance  <cit> .
recent functional characterization of the aba-regulated erd <dig> and erd <dig> indicated that these proteins could prevent the heat-induced aggregation and/or inactivation of various enzyme substrates  <cit> .
thus, induction of genes functionally similar to molecular chaperones by aba during hs may help combat the denaturing stress effects of hs.
previous studies involving treatment with exogenous aba  <cit> , aba and abi mutants  <cit> , and high aba producing lines  <cit> , have demonstrated the positive effects of aba on thermotolerance.
under our experimental conditions the differences in the survival rates of untreated wt and aba1- <dig> and abi1- <dig> mutant seedlings were not striking, but the most pronounced effects of ebr with respect to survival within this set of plants was seen in aba1- <dig> seedlings , indicating that endogenous aba levels suppress br effects.
this notion is supported further by higher accumulation of hsp <dig>  a representative of the hsp families of proteins that are known markers of thermotolerance, in aba1- <dig> seedlings as compared to wt .
it should be noted that neither sa, nor aba or br mutants are compromised in hsp accumulation  <cit>  .
we have previously demonstrated that treatment with exogenous ebr can significantly increase hsp accumulation in b. napus  <cit> , but that this effect in arabidopsis is subtle  <cit> .
thus, the clear enhancement of hsp accumulation in response to ebr during hs in aba1- <dig>  but not in wt , confirms that endogenous aba levels suppress br effects in wt even under stress conditions.
while this work was under preparation, a study describing aba inhibition of br signaling was reported whereby it was shown that aba increases the expression levels of br biosynthesis gene dwf <dig> and cpd to a greater extent in aba <dig> than in wt, reinforcing the idea that aba inhibits br signaling in wt and that this inhibition is relieved in the aba <dig> background  <cit> .
although br and aba display an antagonistic relationship in some physiological responses, we observed in a microarray experiment that several aba-responsive genes and one aba biosynthesis gene were upregulated by ebr and hs .
these results suggest that aba levels rise in response to hs in arabidopsis and that this increase may be further augmented by br treatment.
indeed, aba content has been reported to increase in pea leaves in response to hs  <cit>  and in response to br under hs in c. vulgaris  <cit> .
based on these results it can be speculated that br augments aba levels and aba-related effects during hs , but it is only when aba levels are compromised that the br effects of enhancing stress tolerance become apparent.
npr <dig>  stress tolerance and br effects
although npr1- <dig> seedlings have been shown to be defective in basal thermotolerance, the heat sensitivity of this genotype is not dramatically lower than wt  <cit> .
however, in our study the mere  <dig> -fold increase in percent survival of npr1- <dig> in response to ebr treatment as compared to the 9-fold increase in wt col following hs , nearly no change in oxidative damage levels in npr1- <dig> in response to ebr , and the lack of ebr effect in increasing survival of npr1- <dig> seedlings on salt , explicitly indicate that a functional npr <dig> is required for the manifestation of br effects on seedling stress tolerance.
two questions arise from these observations; 1) what would be the function of npr <dig> during abiotic stress conditions, and 2) how could npr <dig> integrate in the br pathway?
npr <dig> is a redox-controlled transcriptional cofactor, which is key to development of sar and critical for modulating cross-talk between sa and ja signaling  <cit> .
in the absence of stress, npr <dig> is maintained in a large complex consisting of intermolecular disulfide bonded oligomers, which upon stress are reduced to an active monomeric state  <cit> .
the monomeric form interacts with tga-bzip transcription factors and activates defense gene expression.
ros is a common signal in plant stress responses to both abiotic and biotic stresses  <cit> .
the notion that ros could be a signal for the hs response is derived from studies of activation of heat shock transcription factors .
the eukaryotic hsf <dig> multimerizes and binds to dna upon either heating or oxidation with h2o <dig>  <cit> .
more direct evidence showing that endogenous ros production is necessary for induction of the hs response comes from the observation that a dominant negative allele of rac <dig>  the small gtpase necessary for the activation of ros production by membrane-bound nadph oxidase  <cit> , inhibits the stress-induced activation of hsf <dig>  <cit> .
thus, it is highly likely that ros production during hs also activates npr <dig>  leading to gene expression changes critical for thermotolerance.
future studies directed at global gene expression analysis in wt and npr <dig> mutant in response to hs and br as separate and combined treatments should indicate npr1-dependent molecular changes and help clarify the role of npr <dig> in br-mediated increase in thermotolerance.
the mechanism for how br could function with npr <dig>  it is speculated that ros-activated npr <dig> monomers may bind a br-activated regulator to affect stress-responsive gene expression critical to the survival of seedlings under stress conditions.
it is unlikely that npr <dig> controls br signaling via bin <dig> and bzr <dig> given that downregulation of dwf <dig> in npr1- <dig> was unaffected .
oxidative stress state in cpr <dig> and br effects
the cpr  mutants are characterized with increased concentrations of sa, constitutive expression of the pr genes, and enhanced resistance to pathogens  <cit> .
the cpr mutants are in a state of high-cellular oxidative stress state as compared to wt.
for example, cpr <dig> plants exhibited greater oxidative damage than wt under both normal growth  and chilling  conditions  <cit> , and molecular changes in cpr <dig> such as increased expression of several genes in the ros gene network, including gsts, indicate that the cellular redox balance in this mutant is deregulated  <cit> .
we also observed higher levels of gst <dig> transcript in cpr5- <dig> as compared to wt .
the high-cellular oxidative stress state of the cpr mutants, combined with the constitutively activated sa and ja/et-mediated pathways  <cit> , may trigger not only defense responses against pathogens but also abiotic stress response pathways, leading to greater survival of cpr5- <dig> seedlings than wt in response to hs .
ebr treatment further enhanced the survival rates of cpr5- <dig> seedlings exposed to hs , but had negligible effect on the oxidative damage levels in this mutant , presumably due to the inherent oxidative state of the mutant.
this result does not preclude other positive effects of ebr on cpr5- <dig> physiology, leading to increased stress tolerance.
br induces expression of other hormone marker genes
upregulation of pr- <dig> by br in eds5- <dig> and npr1- <dig> backgrounds , as well as in short-term treatment , suggests that br directly mediates expression of this gene.
interestingly, we found wrky <dig>  a transcription factor acting downstream of npr <dig> and involved in the expression of sa-induced pr genes  <cit> , to also be upregulated by br in different sa genotypes .
thus, in addition to npr <dig>  wrky <dig> may be a potential point of cross-talk between sa and br via which br may induce a subset of sa-responsive genes.
hormone response data obtained using atgenexpress visualization tool  http://jsp.weigelworld.org/expviz/expviz.jsp indicate that sa and br are the two most potent inducers of wrky <dig> expression , which reinforces the idea that wrky <dig> is an overlapping transcriptional target of both sa and br.
such a scenario could explain, in part, how br enhances plant resistance against pathogen infection  <cit> .
br could increase the expression of pdf <dig>  in ja mutants, but not to the same extent in the et-insensitive mutant ein <dig> .
thus, at present we favour the possibility that br effects on pdf <dig>  expression are mediated via the et pathway.
it should be noted that pr- <dig> and pdf <dig>  genes are constitutively expressed in cpr and ssi <dig> mutants of arabidopsis in an npr1-independent, and sa, ja and et-dependent manner  <cit> , providing precedent for the notion that more than one pathway governs the expression of these genes.
the increase in the transcript levels of the aba-responsive rd <dig> gene and the stress-induced gst <dig> gene in wt and hormone mutants by ebr, including short-term treatment of wt , suggests that these genes may also be primary targets of br.
taken together, our data indicate that br has regulatory inputs into the expression of other hormone-responsive genes, which may result from the action of the br pathway either on the promoters of these genes, or on the regulation of a hormone-signaling component or the biosynthesis of another hormone .
br effects on inhibition of seed germination by salt
we have previously shown in b. napus that ebr helps to overcome inhibition of seed germination by salt  <cit> .
to study the involvement of br and other hormones in salt tolerance, we screened wt, npr1- <dig>  jar1- <dig>  abi1- <dig>  ein <dig> and eto1- <dig> seeds for germination on  <dig> mm salt.
the ein2- <dig> mutant has been found to be hypersensitive to salt  <cit>  and ein2- <dig> displayed sensitivity to heat and osmotic stress  <cit> .
clearly, ein <dig> is an important node for interaction of stress and hormonal signaling pathways.
the fact that ebr could rescue hypersensitivity of ein <dig> to salt  and increase survival rates of ein <dig> seedlings following hs  with effects paralleling those in wt, unambiguously indicates that br can bypass et signaling in conferring stress tolerance in arabidopsis.
we included npr1- <dig> and eto1- <dig> in the salt stress study due to the hypersensitivity of these genotypes to hs even in presence of br.
interestingly, while both npr1- <dig> and eto1- <dig> were insensitive to salt during germination, both genotypes had survival rates comparable with those of wt and ein <dig> in the absence of ebr, and notably lower survival rates than wt and ein <dig> in the presence of ebr .
these results further endorse a crucial requirement of functional npr <dig> in br-mediated increase in stress tolerance and suggest further explorations of the roles of npr <dig> in abiotic stress.
the possibility that npr <dig> mediates defense responses against abiotic stresses has been suggested in two recent reports  <cit> .
CONCLUSIONS
in summary we have demonstrated that 1) br-mediated increase in stress tolerance is integrated with other hormone pathways, 2) npr <dig> appears to be a critical component of br-mediated effects on thermo and salt tolerance, 3) aba inhibits br effects in abiotic stress responses, 4) the effect of br in overcoming inhibition of germination by salt is independent of ein <dig>  and 5) several hormone-responsive genes are also br-responsive.
overall, these findings point to possible cross-talk of br with sa, et and aba signaling pathways in mediating stress responses, as depicted in figure  <dig>

 brassinosteroids are a group of plant steroidal hormones that regulate various aspects of plant growth and development, including cell elongation, photomorphogenesis, xylem differentiation, and seed germination, as well as adaptation to abiotic and biotic environmental stresses. molecular genetic studies of br-deficient and br-insensitive mutants have established an essential role for brs in plant development and led to the identification and characterization of several br signaling components. a large number of br-regulated genes have been identified by microarray studies; most of the known br-regulated genes are associated with plant growth and development, such as cell wall modification, cytoskeleton formation, and hormone synthesis. how br regulates gene expression is currently understood for only a small proportion of genes. in the current model of br-controlled gene expression, br binding to bri a plasma membrane localized leucine-rich repeat receptor-like kinase, induces association of bri with its coreceptor bak which enhances signaling output through reciprocal bri transphosphorylation. bri binding to br inactivates bin a glycogen synthase kinase- and possibly activates the phosphatase bsu bin negatively regulates transcription factors bzr and bes by phosphorylating them, while bsu positively regulates br signaling by dephosphorylating bzr and bes activated bzr and bes accumulate in the nucleus and directly bind to cgtgg motif in the promoters of br biosysnthesis genes cpd and dwf and to e box sequence in the saur-aci promoter, respectively, to affect gene expression. the recent demonstrations that bes interacts with other transcription factors such as bims, myb which acts as a positive regulator of the hypersensitive cell death response, and the jumonji domain-containing proteins elf and ref that are involved in regulating flowering time, points to recruitment of different proteins by bes as one of the ways by which br affects diverse biological processes. the role of brs in plant stress responses has been confirmed in several studies. br promotes tolerance in plants to a wide range of stresses, including heat, cold, drought and salinity, and this increase is generally correlated with higher expression of stress marker genes, such as heat shock protein genes, rd29a and erd, indicating that increased expression of stress-responsive genes is responsible, in part, for the higher stress tolerance in br-treated plants. the mechanisms by which br controls plant stress responses and regulates the expression of stress response genes are not known. since different plant hormones can regulate similar physiological processes, and cross-talk between different hormones can occur at the level of hormone biosynthesis, signal transduction or gene expression, it was proposed that br regulates plant stress responses via cross-talk with other hormones. the plant growth regulators with documented roles in plant adaptation to abiotic and biotic stresses are abscisic acid, ethylene, jasmonic acid and salicylic acid. sa, ja and et are important in defense against pathogen and pest attack, whereas aba is a key molecule involved in salt and drought stress. sa, et, aba and ja have also been linked to heat stress. studies of hormone deficient and insensitive mutants have demonstrated the involvement of sa, et and aba in acquired thermotolerance of plants, and additionally for sa and ja, a role in basal thermotolerance of plants. although experimental evidence points to interactions of br with auxin, gibberellin, aba, et and ja, the relationship of br with these hormones has been documented primarily in plant growth regulatory processes. furthermore, with the exception of br-auxin interaction, little is known in terms of genes how br interacts with other hormones. recent progress made towards understanding br-auxin interaction can serve as a paradigm for how two hormones could interact at multi-levels. auxin and br share a number of target genes, many of which are involved in growth-related processes. since promoter regions in br-responsive genes are enriched in auxin response factor -binding sites, and binding sites of bes are overrepresented in genes regulated by both hormones, regulatory elements in gene promoters represent a point of cross-talk between auxin and br. recently, the br-regulated bin kinase was demonstrated to phosphorylate arf a member of the arf family of transcriptional regulators, leading to loss of arf dna binding and repression activities. thus, in this model arf links br and auxin signaling pathways. in addition to gene coregulation, br can also promote auxin transport, and optimal auxin action is dependent on br levels. the role of br in plant responses to abiotic stress has become well established over the last decade, but there are very few reports indicating how br interacts with other stress-related hormones and their signaling pathways in conferring stress tolerance. while there exists evidence to indicate that br increases et and ja levels under normal growth conditions, there appears to be only one report linking br with increase in aba levels in the lower plant chlorella vulgaris under stress condition. recently it was demonstrated that aba inhibits br signaling through phosphorylation of bes. currently there are no studies at the genetic level as to how br interacts with other hormones under stress conditions. here we asked the question whether one or more stress-related hormones, such as aba, et, ja or sa, have a major role in br-mediated stress tolerance. arabidopsis mutants with either disrupted or enhanced hormone pathways were tested for phenotypes and gene expression in response to br under high temperature and high salt conditions. our results suggest that in arabidopsis the nonexpressor of pathogenesis-related genes is likely a critical component of br-mediated effects on thermotolerance and salt tolerance, that br exerts anti-stress effects both independently as well as through interactions with other hormones, aba inhibits br effects during heat stress, and that br shares transcriptional targets with other hormones.  ebr effects on basal thermotolerance in different hormone genotypes we have previously demonstrated that ebr enhances the basic thermotolerance of brassica napus, tomato and arabidopsis seedlings. since the effects of ebr on stress tolerance are most pronounced when seedlings are grown in the presence of ebr, we postulated the involvement of other phytohormones in this process. several hormone pathways, such as of aba, et, sa and ja, have been linked with one or more environmental stresses, including heat stress. we therefore evaluated the effects of ebr on thermotolerance in a subset of arabidopsis hormone mutants altered in either biosynthesis or signaling of these phytohormones. we first studied the effects of ebr on basal thermotolerance in sa genotypes npr1-, but not all sa responses), eds5-, and cpr5- by exposing 21-day-old seedlings to 43°c for h, allowing them to recover at 22°c for days and then scoring for dead and surviving seedlings. wt, eds5- npr1- and cpr5- seedlings grown in the absence of ebr had average survival rates of,, and %, respectively, and those grown in the presence of ebr had survival rates of,, and 83%, respectively. from these results it is clear that the survival rates of wt and different sa genotypes were significantly increased by ebr treatment, but the increase was considerably less in case of npr1- as compared to other genotypes within this group. photographs of all genotypes grown in the presence or absence of ebr under no-stress conditions can be seen in additional file 1: pictures of 21-day-old seedlings. sa synthesis key regulator of sa-mediated systemic acquired resistance pathway; mutants are sa-insensitive and hypersusceptible participates in signal transduction pathways involved in plant defense; catalyzes dehydration of the hydroperoxide to an unstable allene oxide in the ja biosynthetic pathway; for genotypes related to ethylene, ebr increased survival rates of wt, ein, and eto1- seedlings to significant levels as compared to seedlings with no treatment. the ja mutants aos and jar1- belong to different backgrounds; jar1- is in arabidopsis ecotype columbia, whereas aos is in col- background. ebr increased survival rates of aos and jar1- seedlings to amounts similar to corresponding wt seedlings. the aba mutants aba1- and abi1- are from the landsberg erecta background; hence, wt ler was used for comparison with these mutants. under the conditions used, ebr was less effective in wt ler as compared to wt col. since aba has been linked with heat tolerance, we expected aba mutants to be less thermotolerant than wt even in the presence of ebr. contrary to our expectation we found that the effect of ebr was most distinct in aba1- as compared to wt and abi1- seedlings. these results suggest that aba masks br effects on the hs response pathway of wt arabidopsis seedlings. from the survival data represented in figure it is clear that when ebr effect in any hormone genotype is viewed in reference to the effect on the corresponding wt, ebr could increase the basal thermotolerance of all hormone genotypes, but its effect was minimal in npr1- since the sa-deficient nahg transgenic line and the ja response defective coi mutant could not be obtained for this study, we cannot yet conclude that sa and ja are dispensable for br-mediated increase in thermotolerance. however, from the collection of mutants used here it would appear that br can exert anti-stress effects that are independent of aba, et, ja and sa, at least to some extent. the dependency of br on npr in mediating stress tolerance is a first time observation made in this study. whether br modulates npr activity via sa or br pathway or both remains to be determined. ebr effects on oxidative damage in hormone mutants heat stress produces oxidative damage, which as a result of lipid peroxidation leads to the production of thiobarbituric acid reactive substances. to complement the results of the hs phenotype of seedlings, oxidative damage levels were assessed in untreated and ebr-treated seedlings. measurements of tbars in time course experiments determined that maximum oxidative damage occurred during recovery from hs. therefore, seedlings exposed to hs and then allowed to recover for days at 22°c were used for tbars analysis. with the exception of npr1- and cpr5- ebr treatment reduced the levels of oxidative damage in wt and other mutant seedlings as compared to their untreated counterparts. wt col and ler exhibited 45% and 60% reduction in tbars production, respectively, while the mutant seedlings showed 25-50% less tbars, in response to ebr treatment. consistent with its lower survival rate, the npr1- mutant showed an insignificant % reduction in tbars production in response to ebr treatment. however, in contrast to the relatively higher survival of cpr5- seedlings, the reduction in oxidative damage in these seedlings in response to ebr measured only 25% relative to no treatment. overall, these results demonstrated that ebr treatment can reduce oxidative damage during hs and that this effect is not critically dependent on any one hormone in question, although a functional npr protein appears to be required for a complete effect of ebr on thermotolerance of seedlings. ebr induces higher accumulation of hsp in aba1-1 we have found that ebr treatment leads to significant increases in the levels of hsps during hs in b. napus, but the effect of ebr on hsp levels in arabidopsis is subtle. we wished to see how ebr would affect the accumulation of hsp in various mutant seedlings in the absence of hs and in response to hs. with the exception of aba1-, no significant differences in the steady state levels of hsp were observed between ebr-treated and untreated mutant seedlings, including npr1-. ebr-treated aba1- seedlings accumulated approximately 3- and -fold higher levels of hsp at and h of hs, respectively, as compared to untreated aba1- seedlings. by contrast, ebr-treated wt seedlings showed a maximum of -fold increase in hsp levels at h of hs as compared to untreated seedlings. the fold-change values are average of three different experiments, which consistently produced the same pattern of protein accumulation. thus, with respect to higher survival following hs and greater accumulation of hsp during hs, ebr produced most distinct effects in the aba-deficient aba1- mutant. these results reinforce the idea that aba suppresses br effects in wt seedlings. although convincing evidence for antagonism between br and aba in plant growth regulatory processes, such as germination has been provided before, the demonstration at the genetic and molecular levels of an antagonistic relationship between the two hormones in plant stress response is new. ebr upregulates the expression of sa, ja/et and aba-response genes in both wt and corresponding mutants plant hormone responses in arabidopsis have been correlated with the expression of hormone-specific marker genes. we studied the expression of few such genes that are well documented to be induced by sa, ja/et or aba, both before and after treatment with ebr. pr- transcription factor wrky and wak are known to be regulated primarily by sa; pdf, lox and hel by ja/et; and rd and ltp by aba. the steady-state levels of pr- wrky and wak transcripts were elevated by ebr in wt and sa-related genotypes, including npr1- albeit at different levels. the npr1- genotype is defective in the expression of pr genes in response to sa. full-scale induction of wrky and wak by sa also requires a functional npr. our results clearly indicate that ebr can mediate induction of pr- wrky and wak to levels seen in figure 3a in an npr1-independent manner. ebr treatment also enhanced the expression of the ja/et marker gene pdf in wt, aos, jar1- and eto1- backgrounds, but not to the same extent in ein. the effect of ebr on lox expression was distinct in col and jar1- backgrounds, but not in col- and aos backgrounds. increase in the expression of the hel gene by ebr was only slight. the aba-responsive ltp showed dramatic induction by ebr in wt and abi1- background, but not in aba1- the transcript levels of the aba-marker gene rd were upregulated by ebr only slightly in aba1- and abi1- mutant seedlings, but significantly in wt, indicating interaction between aba and br in affecting gene expression. to determine the interaction of ebr with sa, ja/et and aba in the regulation of gst a gene common to abiotic stress and defense pathways, we compared its transcript levels in untreated and ebr-treated wt and mutant seedlings. ebr enhanced gst transcript levels in sa, as well as in all other hormone genotypes studied. as would be expected, cpr5- had the highest expression of gst even in the absence of ebr. exogenous br negatively regulates the br biosynthetic gene dwf. to ensure that transport, perception and signaling of br is intact in npr1- which was most inert to ebr effects, dwf expression was determined in wt and npr1- the fact that dwf levels were reduced in ebr-treated wt and npr1- seedlings as compared to untreated seedlings suggests that the br pathway is intact in npr1- taken together, these results demonstrate that most, if not all, of the sa, ja/et and aba-responsive genes tested in the present study are also upregulated by br both in wt and mutant backgrounds, albeit to different levels. these results point to overlapping gene targets of aba, ja/et or sa and br, as well as to hormone interactions controlling the final output. upregulation of a subset of genes by ebr is likely via a br-regulated pathway since the effect of ebr on thermotolerance is best seen when plants are grown in the presence of ebr for 2- weeks, the gene expression studies leading to figure were also conducted in plants receiving long-term exposure to ebr. to determine whether the ja/et and aba-responsive genes also respond to a short-term ebr treatment, we studied the expression of a subset of genes by qrt-pcr in response to a h treatment with ebr. it is to be noted that br effects on gene expression can take as long as 18- hrs. similar to the results of long-term treatment, the h short-term treatment with ebr enhanced the transcript levels of pr-, pdf, rd and gst, suggesting that changes in the expression of these genes likely occur via a br-regulated pathway. further studies are required to confirm this idea. downregulation of dwf by exogenous br was used as an experimental control. ebr rescues hypersensitivity of ein to inhibition of germination by salt stress we have previously shown that ebr helps to overcome salt stress-induced inhibition of seed germination in b. napus. more recently it was demonstrated that br-deficient and br signaling-defective mutants are more inhibited in germination on salt while a bri overexpressing transgenic line is more resistant to salt than wt. in this study, we first screened npr1- ein eto1- jar1- and abi1- mutants for their germination efficiency on mm nacl. as noted before with ein2-, ein was more sensitive to nacl-mediated inhibition of germination as compared to wt. by contrast, eto1- npr1- jar1- and abi1- were less sensitive than wt. thus, to study the effects of ebr on salt stress we selected ein because of its hypersensitivity to salt stress, and eto1- and npr1- because of their relatively higher susceptibility to hs even after ebr treatment. seeds were allowed to germinate on mm nacl in the presence or absence of ebr, and seedlings with emerged cotyledons were scored after days. inhibition of germination of ein seeds by salt was significantly reduced in the presence of ebr. wt seeds showed germination rates of 55% and > 80% on mm nacl in the absence and presence of ebr, respectively, while ein seeds had germination rates of ~10% and 80% under the same conditions. seeds of eto1- and npr1- germinated at similar rates on mm nacl in the absence or presence of ebr. however, despite the good germination efficiency on salt, the average survival rates of eto1- and npr1- on salt were only and 22%, respectively, but unlike wt, survival of the mutant seedlings could not be rescued by ebr treatment. these results indicate that npr has a role in salt stress and that br effects on seedling survival under salt stress require a functional npr phenotypes of wt, ein eto1- and npr1- seedlings grown in the presence or absence of ebr under no-salt conditions can be seen in additional file 2: pictures of 3-day-old seedlings. discussion feeding br through roots not only rescues growth defects of br-deficient mutants, but also leads to increased stress tolerance in wt seedlings. furthermore, overexpression of br biosynthesis genes leads to increased vegetative and seed yield in transgenic seedlings. in each case it is rationalized that br-mediated outcomes likely involve both direct and indirect effects of br. in the present study we focused on understanding br interactions with other stress hormones in mediating increase in stress tolerance mainly because 1) br is known to interact with other plant hormones in regulating plant developmental processes, and 2) multiple hormone signaling pathways play a role in acquisition of stress tolerance. we evaluated a subset of signaling, biosynthetic and constitutively active mutants of aba, et, ja and sa for thermo and salt tolerance in untreated and br-treated states to assess the importance of these hormones in br-mediated increase in stress tolerance of arabidopsis seedlings. here we demonstrate that npr a protein well recognized for its role in sa-mediated sar and cross-talk inhibition of ja-mediated defense responses, also has a role in br-mediated stress tolerance. thermotolerance defects of hormone mutants and br effects sa, and more recently ja, have been linked with thermotolerance. in case of sa genotypes, it is known that npr1- is compromised in basal thermotolerance, while cpr5- has greater thermotolerance than wt. recently it was demonstrated that a jar1-dependent pathway is also required for basal thermotolerance. using a collection of genotypes with basal thermotolerance either lower or higher than wt, we found that ebr treatment could significantly increase the basic thermotolerance of these genotypes and that this increase was comparable to the increase in wt. an exception to this result within the sa, ja and et genotypes was npr1- indicating that a functional npr is required for full manifestation of br's effects. although collectively our data seems to suggest that br is not critically dependent on sa levels and ja signaling for its antistress effects, further confirmation is required with genotypes such as nahg and coi to make an unequivocal claim. even if br works to some extent independently of other hormones in conferring heat tolerance, the mere fact that aba, br, et, ja and sa all play a role in thermotolerance of arabidopsis plants suggests that there must be some redundant and some specific events in the mechanisms by which these hormones produce their effects. with the exception of br where some information has been obtained, molecular changes mediated by aba, et, ja and sa that lead to thermotolerance are largely unknown. in case of ethylene, the ethylene response factor protein, jerf has been demonstrated to activate the expression of oxidative genes, resulting in decreased accumulation of ros and, in turn, enhanced adaptation to drought, freezing, and salt in tobacco. a similar role for jerf can be envisioned in response to hs. a number of aba-regulated genes have been implicated in drought tolerance. recent functional characterization of the aba-regulated erd and erd indicated that these proteins could prevent the heat-induced aggregation and/or inactivation of various enzyme substrates. thus, induction of genes functionally similar to molecular chaperones by aba during hs may help combat the denaturing stress effects of hs. previous studies involving treatment with exogenous aba, aba and abi mutants, and high aba producing lines, have demonstrated the positive effects of aba on thermotolerance. under our experimental conditions the differences in the survival rates of untreated wt and aba1- and abi1- mutant seedlings were not striking, but the most pronounced effects of ebr with respect to survival within this set of plants was seen in aba1- seedlings, indicating that endogenous aba levels suppress br effects. this notion is supported further by higher accumulation of hsp a representative of the hsp families of proteins that are known markers of thermotolerance, in aba1- seedlings as compared to wt. it should be noted that neither sa, nor aba or br mutants are compromised in hsp accumulation. we have previously demonstrated that treatment with exogenous ebr can significantly increase hsp accumulation in b. napus, but that this effect in arabidopsis is subtle. thus, the clear enhancement of hsp accumulation in response to ebr during hs in aba1- but not in wt, confirms that endogenous aba levels suppress br effects in wt even under stress conditions. while this work was under preparation, a study describing aba inhibition of br signaling was reported whereby it was shown that aba increases the expression levels of br biosynthesis gene dwf and cpd to a greater extent in aba than in wt, reinforcing the idea that aba inhibits br signaling in wt and that this inhibition is relieved in the aba background. although br and aba display an antagonistic relationship in some physiological responses, we observed in a microarray experiment that several aba-responsive genes and one aba biosynthesis gene were upregulated by ebr and hs. these results suggest that aba levels rise in response to hs in arabidopsis and that this increase may be further augmented by br treatment. indeed, aba content has been reported to increase in pea leaves in response to hs and in response to br under hs in c. vulgaris. based on these results it can be speculated that br augments aba levels and aba-related effects during hs, but it is only when aba levels are compromised that the br effects of enhancing stress tolerance become apparent. npr stress tolerance and br effects although npr1- seedlings have been shown to be defective in basal thermotolerance, the heat sensitivity of this genotype is not dramatically lower than wt. however, in our study the mere -fold increase in percent survival of npr1- in response to ebr treatment as compared to the 9-fold increase in wt col following hs, nearly no change in oxidative damage levels in npr1- in response to ebr, and the lack of ebr effect in increasing survival of npr1- seedlings on salt, explicitly indicate that a functional npr is required for the manifestation of br effects on seedling stress tolerance. two questions arise from these observations; 1) what would be the function of npr during abiotic stress conditions, and 2) how could npr integrate in the br pathway? npr is a redox-controlled transcriptional cofactor, which is key to development of sar and critical for modulating cross-talk between sa and ja signaling. in the absence of stress, npr is maintained in a large complex consisting of intermolecular disulfide bonded oligomers, which upon stress are reduced to an active monomeric state. the monomeric form interacts with tga-bzip transcription factors and activates defense gene expression. ros is a common signal in plant stress responses to both abiotic and biotic stresses. the notion that ros could be a signal for the hs response is derived from studies of activation of heat shock transcription factors. the eukaryotic hsf multimerizes and binds to dna upon either heating or oxidation with h2o. more direct evidence showing that endogenous ros production is necessary for induction of the hs response comes from the observation that a dominant negative allele of rac the small gtpase necessary for the activation of ros production by membrane-bound nadph oxidase, inhibits the stress-induced activation of hsf. thus, it is highly likely that ros production during hs also activates npr leading to gene expression changes critical for thermotolerance. future studies directed at global gene expression analysis in wt and npr mutant in response to hs and br as separate and combined treatments should indicate npr1-dependent molecular changes and help clarify the role of npr in br-mediated increase in thermotolerance. the mechanism for how br could function with npr it is speculated that ros-activated npr monomers may bind a br-activated regulator to affect stress-responsive gene expression critical to the survival of seedlings under stress conditions. it is unlikely that npr controls br signaling via bin and bzr given that downregulation of dwf in npr1- was unaffected. oxidative stress state in cpr and br effects the cpr mutants are characterized with increased concentrations of sa, constitutive expression of the pr genes, and enhanced resistance to pathogens. the cpr mutants are in a state of high-cellular oxidative stress state as compared to wt. for example, cpr plants exhibited greater oxidative damage than wt under both normal growth and chilling conditions, and molecular changes in cpr such as increased expression of several genes in the ros gene network, including gsts, indicate that the cellular redox balance in this mutant is deregulated. we also observed higher levels of gst transcript in cpr5- as compared to wt. the high-cellular oxidative stress state of the cpr mutants, combined with the constitutively activated sa and ja/et-mediated pathways, may trigger not only defense responses against pathogens but also abiotic stress response pathways, leading to greater survival of cpr5- seedlings than wt in response to hs. ebr treatment further enhanced the survival rates of cpr5- seedlings exposed to hs, but had negligible effect on the oxidative damage levels in this mutant, presumably due to the inherent oxidative state of the mutant. this result does not preclude other positive effects of ebr on cpr5- physiology, leading to increased stress tolerance. br induces expression of other hormone marker genes upregulation of pr- by br in eds5- and npr1- backgrounds, as well as in short-term treatment, suggests that br directly mediates expression of this gene. interestingly, we found wrky a transcription factor acting downstream of npr and involved in the expression of sa-induced pr genes, to also be upregulated by br in different sa genotypes. thus, in addition to npr wrky may be a potential point of cross-talk between sa and br via which br may induce a subset of sa-responsive genes. hormone response data obtained using atgenexpress visualization tool http://jsp.weigelworld.org/expviz/expviz.jsp indicate that sa and br are the two most potent inducers of wrky expression, which reinforces the idea that wrky is an overlapping transcriptional target of both sa and br. such a scenario could explain, in part, how br enhances plant resistance against pathogen infection. br could increase the expression of pdf in ja mutants, but not to the same extent in the et-insensitive mutant ein. thus, at present we favour the possibility that br effects on pdf expression are mediated via the et pathway. it should be noted that pr- and pdf genes are constitutively expressed in cpr and ssi mutants of arabidopsis in an npr1-independent, and sa, ja and et-dependent manner, providing precedent for the notion that more than one pathway governs the expression of these genes. the increase in the transcript levels of the aba-responsive rd gene and the stress-induced gst gene in wt and hormone mutants by ebr, including short-term treatment of wt, suggests that these genes may also be primary targets of br. taken together, our data indicate that br has regulatory inputs into the expression of other hormone-responsive genes, which may result from the action of the br pathway either on the promoters of these genes, or on the regulation of a hormone-signaling component or the biosynthesis of another hormone. br effects on inhibition of seed germination by salt we have previously shown in b. napus that ebr helps to overcome inhibition of seed germination by salt. to study the involvement of br and other hormones in salt tolerance, we screened wt, npr1- jar1- abi1- ein and eto1- seeds for germination on mm salt. the ein2- mutant has been found to be hypersensitive to salt and ein2- displayed sensitivity to heat and osmotic stress. clearly, ein is an important node for interaction of stress and hormonal signaling pathways. the fact that ebr could rescue hypersensitivity of ein to salt and increase survival rates of ein seedlings following hs with effects paralleling those in wt, unambiguously indicates that br can bypass et signaling in conferring stress tolerance in arabidopsis. we included npr1- and eto1- in the salt stress study due to the hypersensitivity of these genotypes to hs even in presence of br. interestingly, while both npr1- and eto1- were insensitive to salt during germination, both genotypes had survival rates comparable with those of wt and ein in the absence of ebr, and notably lower survival rates than wt and ein in the presence of ebr. these results further endorse a crucial requirement of functional npr in br-mediated increase in stress tolerance and suggest further explorations of the roles of npr in abiotic stress. the possibility that npr mediates defense responses against abiotic stresses has been suggested in two recent reports.  in summary we have demonstrated that 1) br-mediated increase in stress tolerance is integrated with other hormone pathways, 2) npr appears to be a critical component of br-mediated effects on thermo and salt tolerance, 3) aba inhibits br effects in abiotic stress responses, 4) the effect of br in overcoming inhibition of germination by salt is independent of ein and 5) several hormone-responsive genes are also br-responsive. overall, these findings point to possible cross-talk of br with sa, et and aba signaling pathways in mediating stress responses, as depicted in figure 
using a collection of arabidopsis mutants that are either deficient in or insensitive to abscisic acid, ethylene, jasmonic acid and salicylic acid, we studied the effects of 24-epibrassinloide on basic thermotolerance and salt tolerance of these mutants. brassinosteroids play crucial roles in plant development and also promote tolerance to a range of abiotic stresses. these results together indicate that the redox-sensitive protein npr, a master regulator of sa-mediated defense genes, is likely a critical component of ebr-mediated increase in thermotolerance and salt tolerance, but it is not required for ebr-mediated induction of pr- gene expression; that br exerts anti-stress effects independently as well as through interactions with other hormones; that aba inhibits br effects during stress; and that br shares transcriptional targets with other hormones. since br interacts with other plant hormones, it is likely that the stress tolerance conferring ability of br lies in part in its interactions with other stress hormones. ebr could rescue the et-insensitive ein mutant from its hypersensitivity to salt stress-induced inhibition of seed germination, but remained ineffective in increasing the survival of eto1- and npr1- seedlings on salt. the positive effect of ebr was significantly greater in the aba-deficient aba1- mutant as compared to wild type, indicating that aba masks br effects in plant stress responses. although much has been learned about their roles in plant development, the mechanisms by which brs control plant stress responses and regulate stress-responsive gene expression are not fully known. treatment with ebr increased expression of various hormone marker genes in both wild type and mutant seedlings, although to different levels.
 brassinosteroids play crucial roles in plant development and also promote tolerance to a range of abiotic stresses. although much has been learned about their roles in plant development, the mechanisms by which brs control plant stress responses and regulate stress-responsive gene expression are not fully known. since br interacts with other plant hormones, it is likely that the stress tolerance conferring ability of br lies in part in its interactions with other stress hormones.  using a collection of arabidopsis mutants that are either deficient in or insensitive to abscisic acid, ethylene, jasmonic acid and salicylic acid, we studied the effects of 24-epibrassinloide on basic thermotolerance and salt tolerance of these mutants. the positive impact of ebr on thermotolerance in proportion to wild type was evident in all mutants studied, with the exception of the sa-insensitive npr1- mutant. ebr could rescue the et-insensitive ein mutant from its hypersensitivity to salt stress-induced inhibition of seed germination, but remained ineffective in increasing the survival of eto1- and npr1- seedlings on salt. the positive effect of ebr was significantly greater in the aba-deficient aba1- mutant as compared to wild type, indicating that aba masks br effects in plant stress responses. treatment with ebr increased expression of various hormone marker genes in both wild type and mutant seedlings, although to different levels.  these results together indicate that the redox-sensitive protein npr, a master regulator of sa-mediated defense genes, is likely a critical component of ebr-mediated increase in thermotolerance and salt tolerance, but it is not required for ebr-mediated induction of pr- gene expression; that br exerts anti-stress effects independently as well as through interactions with other hormones; that aba inhibits br effects during stress; and that br shares transcriptional targets with other hormones. 