BACKGROUND
in order to acclimate and protect themselves, plants translate environmental challenges such as drought, waterlogging, extreme temperatures, soil salinity, wounding, and pathogen attack into internal signals through hormones, second messengers, and transcription factors .
the phytohormone abscisic acid  regulates abiotic stress responses and other vital processes in plant growth and development, especially during seed maturation reviewed in  <cit> .
mutant plants with altered aba biosynthesis, perception or response have been crucial in deciphering the various components involved in aba responses.
recently, a family of  <dig> novel start domain proteins, named as pyr/pyl/rcars  has been identified as intracellular aba receptors that interact with and inhibit several protein phosphatase 2cs  including aba insensitive <dig> and  <dig> , homology to abi <dig> , and pp2ca  <cit> .
such phosphatases are negative regulators of aba signaling.
a recent model for aba signaling, based on several independent crystallographic studies for example  <cit> , proposes that in the presence of aba, receptors of the pyr/pyl/rcar family bind to pp2cs which in turn release inhibition on a subfamily of snf1-related protein kinase <dig>  kinases.
these kinases then phosphorylate and subsequently activate transcription factors including aba responsive elements-binding factor / aba responsive elements-binding protein -type bzip tfs.
abfs in turn bind to aba-responsive promoter elements  to activate aba-responsive gene expression.
the molecular basis of adaptive responses to abiotic stresses such as low temperature, desiccation and salinity, has been elucidated by identifying genes such as responsive to dehydration , kykna-indusoitu , responsive to aba , cold-regulated , low-temperature-induced , and drought-induced .
manipulation of many of these genes resulted in plants with improved tolerance to drought, salt, cold and freezing reviewed in  <cit> .
molecular and genetic studies suggest that aba-dependent and –independent pathways operate in abiotic stresses and aba-dependent pathways are predominant in drought stress responses  <cit> .
environmental factors such as drought, extreme temperature and pathogen infection as well as endogenous factors including age affect the onset and progression of leaf senescence reviewed in  <cit> .
unlike abscission and dehiscence, leaf senescence is a specialized form of programmed cell death, which is a genetically regulated process of slow cell death of the entire leaf and is preceded by the reallocation of nitrogen, phosphorus, and metals to other parts of the plant.
an early manifestation of senescence in leaves is loss of chlorophyll.
leaf mesophyll cells start to senesce first, followed by other cell types, and exhibit an incoherent pattern of localized cell death, which eventually spreads to the whole leaf.
senescence is accelerated by aba, ethylene, jasmonates , and salicylic acid , and is delayed by cytokinins  and auxin reviewed in  <cit> .
however, extensive cross talk among these signaling pathways during senescence complicates understanding of the initiation and progression of senescence.
therefore, key components in senescence signaling remain largely unknown.
senescence is an important aspect of drought responses.
accelerated leaf senescence followed by leaf abscission is triggered by prolonged stress to reduce water loss, remobilize nutrients to young leaves and to enable survival of the plant  <cit> .
the myb family tfs comprises around  <dig> genes in arabidopsis and is the largest tf gene family reviewed in  <cit> .
myb proteins contain a conserved dna-binding myb domain of about  <dig> amino acids, and are classified into three subfamilies based on the presence of one, two or three myb domains reviewed in  <cit> .
the plant specific and largest myb family consists of r2r3-type factors which contain two repeats and comprise  <dig> genes in arabidopsis.
r2r3-myb genes are involved in various plant-specific processes such as regulation of secondary metabolism, modulation of development, determination of cell fate and identity and responses to environmental factors and hormone.
the gene further characterized in this paper, atmybr1/myb <dig>  was weakly induced by 24 h treatment with aba but strongly induced by the hyperactive aba analog -8′ acetylene aba   <cit> .
most aba-regulated genes are similarly regulated by water stress, however mybr <dig> was selected for further functional characterization because its expression was paradoxically repressed by drought and elevated by re-watering  <cit>  suggesting a novel role in aba signaling.
jung et al.
<cit>  reported that over-expression of mybr <dig> increased stress tolerance but unexpectedly repressed many known stress-related genes.
subsequent studies have revealed that this gene is regulated by a mitogen-activated protein kinase  cascade.
following stress treatment, mitogen-activated protein kinase  is activated and phosphorylates the bzip tf vire2-interacting protein  <dig> , which then rapidly activates the expression of mybr <dig> and other stress genes through promoter binding  <cit> .
further studies have shown that mybr <dig> interacts directly with, and is phosphorylated by, mpk <dig> at ser <dig>  <cit>  and possibly ser <dig>  <cit>  and that the ser <dig> phosphorylation is required for mybr <dig> function  <cit> .
in this study, we functionally characterized the atmybr <dig> tf by studying an arabidopsis t-dna insertion mutant mybr <dig> and overexpression lines of atmybr <dig> .
we show that mybr <dig> down regulates many aba responsive genes including those involved in abiotic stresses and negatively regulates drought responses and senescence.
moreover, direct involvement of mybr <dig> in early aba signaling is suggested by our observation that mybr <dig> protein interacts with pyl <dig>  an aba receptor.
RESULTS
atmybr <dig> represses genes induced by a hyperactive aba analog
we showed in a previous study that atmybr <dig> was induced weakly by -aba and more strongly by 24 h treatment with a hyperactive aba analog pbi <dig> -8′ acetylene aba) indicating mybr <dig> is likely a component of the aba signaling pathway  <cit> .
it has been shown previously that pbi <dig> induces aba responsive genes almost identically to the natural enantiomer s--aba.
however, because pbi <dig> is catabolized much less rapidly than -aba and accumulates to higher levels in plant tissue  <cit>  it is an effective tool to study weak and transiently expressed aba-responsive genes such as abi <dig>  abi <dig>  lti <dig>  knat <dig> and mybr <dig> itself  <cit> .
therefore we used pbi <dig> to define the role of mybr <dig> in aba signaling.
in addition to using pbi <dig> to study the function of atmybr <dig>  we generated transgenic arabidopsis 35spro:mybr <dig> plants .
after kanamycin selection, three lines with single inserts were selected and their homozygous progeny plants were grown as experimental materials.
the level of overexpression of mybr <dig> was 23-fold in gain-of-function oxmybr <dig> line 42– <dig>  12-fold in line 31– <dig> and 11-fold in line 1– <dig> and was undetectable by qpcr in loss-of-function mybr <dig> <cit> .
to reduce the likelihood of identifying phenotypic artifacts due to mis-expression, the phenotypes of all the overexpression lines were compared for qualitative consistency throughout the experiments.
we compared gene expression in different genotypes using arabidopsis oligoarrays representing a comprehensive set of approximately  <dig>  expressed genes.
the comparisons were:  genotype comparisons of untreated plants: oxmybr <dig>  or mybr <dig> versus wt,  genotype comparisons after pbi <dig> treatment: oxmybr <dig>  or mybr <dig> versus wt, and  effect of pbi <dig> treatments on each genotype: oxmybr <dig>  mybr <dig> and wt treated with pbi <dig> versus the same genotype without the treatment.
the experimental design is illustrated in additional file 1: figure s <dig> online.
samples were treated with pbi <dig> for 24 h on the basis that the accumulation and effects of pbi <dig> on gene expression was maximum at 24 h  <cit> .
the total numbers of differentially expressed genes are listed in table  <dig> and the gene lists and data may be found in additional file 2: table s <dig>  there were a total of  <dig> differentially regulated genes from all comparisons.
in the absence of pbi <dig> treatment, comparisons of oxmybr <dig> or mybr <dig> vs. wt yielded a very small number of differentially regulated genes .
treatment with pbi <dig> greatly increased numbers of differentially expressed genes and revealed differences between genotypes.
analysis of the direct effect of pbi <dig> on gene expression showed that mybr <dig> represses expression of many genes induced by pbi <dig> in wt  in terms of both total numbers  and expression ratios .
it is noteworthy that there were very few differentially regulated genes from comparisons of mybr <dig> versus wt both with  and without  pbi <dig> treatment.
this suggests the likelihood that mybr <dig> is functionally redundant with at least one other closely related gene.
mybr <dig>  is reported as the closest homolog of mybr <dig> based on sequence similarities in the c-terminal regions of the respective proteins and lack of homology with other myb-type proteins  <cit> .
mybr <dig> has been reported to be involved in auxin signal transduction.
mybr <dig> over-expression results in reduced root and shoot growth, and root phenotypes in loss-function mybr <dig> lines varied with application of auxin  <cit> .
however, its role in aba responses and stress signaling or whether it acts cooperatively with mybr <dig> remains unclear.
to investigate possible redundant functions of mybr <dig>  and mybr <dig> , gene expression analysis was performed using agilent microarrays containing  <dig>  arabidopsis thaliana reporter sequences.
gene expression was compared between pairs of genotypes treated with pbi <dig> i) mybr <dig> versus mybr <dig> and ii) mybr <dig> versus mybr1xmybr <dig>  the number of differentially expressed genes was  <dig> for mybr <dig> vs mybr <dig> and  <dig> for mybr <dig> vs mybr <dig> x mybr <dig> .
the increase in differentially expressed genes in the double mutant comparison suggests that mybr <dig> and mybr <dig> act in a synergistic manner however, only six out of  <dig> genes in the first comparison and  <dig> out of  <dig> genes in the second comparison were present in the above mentioned list of  <dig> genes differentially expressed in all comparisons.
atmybr <dig> represses many aba inducible stress genes
many stress genes that are highly induced by aba, are repressed by mybr <dig> .
however, since mybr <dig> did not appear to repress all pbi <dig> induced genes, we examined more closely the gene expression patterns affected by mybr <dig> and pbi <dig>  in this analysis, we added  <dig> statistically significant genes to the total gene list of table  <dig> for detailed analysis and interpretation.
these  <dig> genes were not listed in table  <dig> since their fold change was below the  <dig>  ratio threshold.
however, changes in expression of these genes were either verified by qpcr and direct spot visualization in base or were present in our comparative analysis of our microarray data with data published by van der graaff et al.
<cit> .
several distinct gene regulation patterns  were identified by comparing pbi <dig> treatment and mybr <dig> overexpression  and the gene lists and expression data may be found in additional file 3: table s <dig>  we excluded the comparison of mybr <dig> vs. wt from table  <dig> because of the small number of differentially expressed genes.
similarly, we also excluded microarray data of mybr <dig> from comparisons mybr <dig> versus mybr <dig> and mybr <dig> versus mybr1xmybr <dig>  many well-characterized abiotic stress responsive genes were grouped in classes a, f and g, of which  <dig> genes  were repressed by mybr <dig>   <dig> genes  were activated by mybr <dig> and  <dig> genes  were unaffected by mybr <dig>  therefore, mybr <dig> represses a subset of about 43% of aba-responsive genes.
it has been suggested that stress responsive genes are under regulation by both aba-dependent and aba-independent pathways for a review see  <cit> .
there are four dehydration responsive element binding protein <dig> /c-repeat binding factor  transcription factor genes that have been suggested to mediate aba independent cold stress responses.
here, dreb1a/cbf <dig> was induced by pbi <dig> and repressed by mybr <dig> suggesting that dreb1a/cbf <dig> is aba-dependent.
mybr <dig>
overexpression on aba-related gene expression
o
mybr <dig>
mybr <dig>
mybr <dig>
bk: background; mybr1: loss-of-function of mybr1; ox: overexpressed; gene regulation symbols-- ↑: upregulation; ↓: downregulation; —: either ‘not differentially expressed’ or ‘undetectable’.
many senescence associated genes including sag12/13/21/29/ <dig>  early responsive to dehydration <dig>  and apg8a are also grouped in class a .
there were a total of  <dig> genes that were either upregulated  or down regulated  by mybr <dig> in the same direction as aba .
among the downregulated genes were many associated with photosynthesis and biotic stresses.
a total of  <dig> genes  were regulated by mybr <dig>  but unaffected by pbi <dig>  among these, several genes involved in jasmonate and auxin action were repressed.
promoters of many genes associated with drought, cold stress and salinity contain aba-responsive element , abre binding factor  and dehydration responsive element   <cit> .
a search for statistically over-represented cis-acting motifs present in the promoters of classified genes sets in table  <dig> was carried out against agris and place databases using the analysis tools in athena  <cit>  and the results are summarized in additional file 1: table s <dig>  these promoter motifs mainly consisted of four groups.
the majority of motifs are associated with aba.
other motifs are related to stress, light regulation, gibberellins  and circadian clock, suggesting cross talk between these pathways as was also observed previously  <cit> .
there was a general similarity in the presence of aba and stress motifs in genes that were regulated specifically by aba and those that were regulated in the opposite direction by mybr <dig>  no significant enrichment was detected in genes that were regulated specifically by mybr <dig>
atmybr <dig> reduces drought tolerance
to define the function of mybr <dig> during drought stress, plants were treated with 10% and 15% polyethylene glycol  for  <dig> d. consistent with the down regulation of the stress responsive genes , oxmybr <dig> plants showed the highest degree of stress  following peg treatment and homozygous mybr1xmybr <dig> plants showed the least damage .
subsequently, we found that oxmybr <dig> rosettes lost water and chlorophyll faster than wt and loss-of-function mutants .
therefore, consistent with the down regulation of stress responsive genes, the data suggests that oxmybr <dig> plants transpired water faster and are consequently less drought tolerant than wt plants.
drought stress was also imposed by withholding water for  <dig> d on seedlings from all available genotypes.
surprisingly, we recorded a 100% survival in all  <dig> replicates of oxmybr <dig>  .
results obtained when drought stress was imposed by withholding water were opposite to what was observed following peg mediated drought stress.
when performing the soil drying experiments above it was observed that, when plants from each genotype were provided with equal volumes of water, the oxmybr <dig> soil dried more slowly than other genotypes.
therefore to investigate the contradictory results obtained by peg and soil drying experiments, we conducted transpirational water loss assays on whole plants .
in these experiments, soil water loss by evaporation was prevented so that plant water use could be monitored.
the results showed that oxmybr <dig> plants lost water slower than wt and mutants, which is contrary to what was observed in detached whole rosette leaves .
in other words, oxmybr <dig> plants extracted and/or used less water from the soil than other genotypes, even though the transpirational capacity of the detached leaves was relatively higher.
therefore, we investigated the water conservation characteristic of oxmybr <dig> further.
we measured the soil water content after imposing an  <dig> d drought.
results confirmed that water uptake was less in oxmybr <dig> than in wt and loss-of-function mutants during drought .
under normal  growth conditions, oxmybr <dig> plants grew more slowly than wt and loss-of-function mutants  and we show later that oxmybr <dig> lines have shorter primary roots than other genotypes.
therefore, with respect to mybr <dig> function, we conclude that reduced water use in whole oxmybr <dig> plants in drying soil is not due to a genetically determined reduction in transpiration but is rather a consequence of lower biomass leading to slower depletion of soil moisture.
the reduced growth rate of oxmybr <dig> lines was also noted by jung et al.
<cit>  and in soybean by seo et al.
<cit> .
as an aside, we note that the residual water content of mybr <dig> material was slightly higher than the other loss-of-function lines .
this may be an indication that the functions of mybr <dig> are not identical with those of mybr <dig>  as discussed later.
mybr1pro:gus is expressed under abiotic stress and during senescence, mechanical wounding and floral organ abscission
a  <dig>  kb promoter fragment of mybr <dig> including the 5′utr was fused to the β-glucuronidase  reporter gene  and the expression of mybr <dig> was examined histochemically.
gus staining was performed on homozygous t <dig> and t <dig> plants.
in  <dig> d old seedlings , gus expression driven by the mybr <dig> promoter was observed in cotyledons and true leaves.
in contrast to very high gus expression in cotyledons, gus expression was lower in younger true leaves relative to older leaves and was absent in the newly emerged leaves.
intriguingly, gus expression was observed in patches in younger leaves and was absent around the vascular regions of both older and younger leaves hinting that mybr <dig> could be involved in senescence since this pattern was reminiscent of the development of visible senescence in leaves reviewed in  <cit> .
under normal conditions, expression of gus was also observed in hydathodes of all leaf margins as well as embryo, suspensor, endosperm, root, stigma, sepal, petal and anther filament but was absent in stem, cauline leaf, anther, silique and testa .
gus expression was observed in embryo and endosperm dissected from siliques at developmental stages from 6– <dig> dpa as well as from dry and imbibed  mature seeds .
the intensity of gus staining increased with development in embryos but remained constant in endosperm except at  <dig> dpa when the gus expression was lower.
gus expression was high and remained constant in embryos collected from dry seeds and seeds imbibed up to 24 h but declined subsequently.
gus expression in endosperm of dry and imbibed seeds remained high.
water stress significantly reduced gus expression driven by the mybr <dig> promoter in leaves but not in roots relative to control .
drought induced reduction of mybr <dig> expression is consistent with the reduced expression of mybr <dig> under drought treatment observed by huang et al.
<cit> .
gus reporter activity was rapidly and strongly induced immediately after mechanical wounding of leaves .
similar high mybr <dig> expression was also observed at the abscission zone  on the pedicel following sepal, petal, and anther filament abscission .
prior to the abscission of floral organs , no gus expression was visible on the pedicel.
however, we did not observe either accelerated- or delayed/abolished floral organ shedding in mybr <dig> loss-and gain-of-function mutants, suggesting that the high gus expression at the az is due to the wounding response.
atmybr <dig> delays leaf senescence
early in leaf senescence chloroplasts disassemble with subsequent degradation of chlorophyll and visible leaf yellowing.
to further investigate the role of mybr <dig> in leaf longevity, detached rosette true leaves numbers 3– <dig> , from  <dig> d old soil grown plants were incubated in buffer as described  <cit>  in two different sets.
leaves were photographed and the chlorophyll content was quantified on  <dig> d for one set and after 6– <dig> d of dark-induced senescence treatment for the other set.
in freshly harvested leaves, the chlorophyll content was higher in one line of oxmybr <dig>  and two reciprocal double mutants of mybr <dig> and mybr <dig> than the rest of the genotypes .
following dark-induced senescence, oxmybr <dig> lines showed increased leaf longevity and slowed chlorophyll degradation relative to wt leaves .
interestingly, increased leaf longevity in oxmybr <dig> lines was in contrast to that in mybr <dig> and reciprocal mybr <dig> & mybr <dig> mutants which exhibited early leaf senescence and accelerated chlorophyll degradation relative to wt  although the chlorophyll content before treatment was relatively high in mybr <dig> and mybr <dig> double mutants .
indeed, among the eight genotypes, leaves of mybr <dig> & mybr <dig> were least green following senescence treatment and showed the fastest chlorophyll breakdown .
to further investigate the role of mybr <dig> in leaf senescence, excised leaves of  <dig> d old transgenic mybr1pro:gus were stained for gus before and after dark induced senescence .
in untreated plants, gus staining was strong in older leaves and was absent in younger leaves and in vascular region.
gus staining became weaker overall in dark-treated leaves relative to fresh leaves but among the dark treated leaves, there was more gus staining in senescent, yellow leaves  than green ones, further suggesting that mybr <dig> plays a role during leaf senescence.
atmybr <dig> regulates the expression of senescence genes
to investigate whether mybr <dig> regulates the expression of senescence genes, we compared the differentially expressed gene lists of table  <dig> with microarray data obtained by van der graaff et al.
<cit>  on various stages and types of leaf senescence .
the number of common genes between the two microarray analyses is  <dig>  which covers 52% of our differential gene list.
the increase of mybr <dig> induction with the progression of senescence is high in ns, low in dis and det whereas mybr <dig> induction is slightly increased in sink-to-source transition  stage of ns  <cit> .
the regulation of these  <dig> common genes by aba, mybr <dig> and senescence revealed the interaction between aba and mybr <dig> during senescence .
out of  <dig> genes in class a , 88% were induced by both aba and ns but repressed by mybr <dig>  clearly demonstrating that mybr <dig> is a suppressor and aba is an activator of leaf senescence.
furthermore, out of  <dig> genes in class c , 95-96% genes are downregulated by ns, dis and det and from  <dig> genes in class g , 86% are upregulated by ns in the same direction as by aba showing a significant role of aba in leaf senescence.
of a large number of common genes , 89-93% are downregulated by aba, mybr <dig> and senescence and many of these genes are involved in protein synthesis , photosynthesis , auxin responses  and biotic stress .
furthermore, we performed qrt-pcr on senescence marker genes in rosette leaves numbers  <dig> and  <dig> in all the genotypes .
consistent with our detached leaf senescence analysis , accelerated leaf senescence of mybr1xmybr <dig> was associated with upregulated expression of sag <dig>  sag <dig> and senescence <dig>  relative to all other genotypes .
sag <dig> expression was  <dig>  fold higher in mybr1xmybr <dig> relative to wt.
the expression of sag <dig>  sag <dig>  sen <dig> and sen <dig> were downregulated in oxmybr <dig> relative to wt, consistent with the senescence-suppressing effect of mybr <dig>  changes in expression of these genes in the single mutants mybr <dig> and mybr <dig> were generally small.
however, expression of sag <dig> and sen <dig> did not show a reciprocal relationship between gain and loss of function genotypes.
nevertheless, it is apparent that mybr <dig> negatively regulates senescence based on  <dig> of the  <dig> marker genes.
protein degradation occurs during senescence via different pathways such as autophagy  and the 26s proteosome and components of both pathways were transcriptionally activated during senescence  <cit> .
our differential gene list contains  <dig> genes involved in protein degradation pathways .
here we report the repression of  <dig> genes by oxmybr <dig>  where  <dig> genes encode components of e <dig> ), e <dig> ubiquitin-ligase complex and 20s core particle of 26s proteosome and three genes are apg8a/8f/8e.
interestingly, only three genes in the ubiquitin pathway - phd finger family, early-responsive to dehydration 16/ubiquitin etension protein  <dig>  and suppresor of nim1- <dig>  were upregulated by oxmybr <dig>  son <dig> is an f-box protein component of e3-ubiquitin ligase complex which negatively regulates, through the ubiquitin-proteosome pathway, a novel defense response that is independent of systemic acquired resistance  <cit> .
on the other hand,  <dig> genes involved in protein degradation were activated by aba in both wt and mybr <dig>  and many of them are also upregulated by ns, dis and det  <cit> .
content of endogenous cytokinins and jasmonic acid
to investigate the role of mybr <dig> in relation to hormonal pathways, endogenous hormone levels were measured quantitatively by lc-ms/ms in rosette leaves numbers  <dig> and  <dig> of three weeks old plants .
trans-zeatin  and n6- adenine  and their sugar derivatives are the major cytokinins  in arabidopsis <cit> .
levels of several cks were significantly increased in oxmybr <dig> relative to other genotypes.
on the other hand, ja was significantly higher in mybr <dig> x mybr <dig> relative to other genotypes.
this suggests that suppression of leaf senescence by mybr <dig> is associated with increases in cks and conversely that increased senescence is associated with higher ja.
in our microarray data, oxmybr <dig>  downregulated arr <dig> , a transcriptional repressor of ck signaling  <cit> .
however, the down regulation of arr <dig> by aba  and senescence  <cit>  is contradictory and may be due to a feedback effect.
furthermore, we did not detect differential expression of genes involved in ck metabolism but a posttranscriptional regulatory effect of mybr <dig> on expression of these genes cannot be ruled out.
oxmybr <dig> leaves contained similar levels of aba and its metabolite dihydrophaseic acid to those measured in wt/mybr <dig> and mybr1xmybr <dig>  levels of sa and iaa were also not significantly altered.
it is surprising that aba levels remained constant despite the strong effect of atmybr <dig> overexpression on aba responses.
to investigate further, we performed qrt-pcr  on six aba downstream effectors; abi <dig>  eel and abf1/2/3/ <dig> in plants of oxmybr <dig> , wt, mybr1/ <dig> and mybr <dig> x mybr <dig> .
no significant differences of expression of these genes were evident among the genotypes tested.
mybr <dig> mis-expression affects leaf and root morphology
we examined the roots of gain-and loss of mybr <dig> function genotypes .
primary roots of oxmybr <dig> lines were drastically shorter, whereas those of mybr <dig> were notably longer relative to wt.
this shorter root phenotype of oxmybr <dig> may contribute toward reduced water uptake in the oxmybr <dig> lines as noted earlier, and may help explain the differences between results of peg treatments and soil drying experiments described above.
the arabidopsis mutant amp <dig>  with a high level of endogenous cks, had increased numbers of rosette leaves  <cit> .
conversely, plants overexpressing catabolic ck oxidases had fewer leaves than wt plants  <cit> .
we counted rosette leaves in seedlings of two lines of oxmybr <dig>  wt and mybr <dig>  seedlings of oxmybr <dig> lines had consistently more rosette leaves relative to other genotypes and double mutant mybr1xmybr <dig> had fewer leaves relative to all other genotypes .
differences between genotypes are slightly enhanced by acc treatment.
using two way anova, there is a consistent, statistically significant difference in leaf number between mybr <dig> x mybr <dig> and the oxmybr <dig> lines but no significant difference between control and acc treatments.
mybr <dig> physically interacts with pyl <dig> and ino
further information on the mechanistic role of mybr <dig> in signaling was obtained by identifying protein-protein interactions using the yeast 2-hybrid system.
initially pyr1-like <dig>  and inner no outer  proteins were identified as interacting with mybr <dig> by screening an arabidopsis cdna library made from different stages of vegetative and floral tissues with a full length mybr <dig> fused to the dna-binding  domain of the yeast gal <dig> protein.
it has been shown by many groups that the  <dig> members of the pyr/pyl/rcar family are intracellular aba receptors that interact with and inhibit several pp2c-type protein phosphatases including abi <dig>  abi <dig>  hab <dig> and pp2ca  <cit> .
ino encodes a yabby-type tf and is required for both polarity determination and outer integument initiation in ovule development  <cit> .
mybr <dig> interaction with pyl <dig> encouraged us to subsequently fuse all  <dig> pyr/pyl/rcar genes as well as ino and mybr <dig> to the transcription-activation  domain of the yeast gal <dig> protein.
interestingly, only pyl <dig>  ino and mybr <dig> interacted with mybr <dig> .
interaction of mybr <dig> and pyl <dig> was confirmed by three independent experiments using a more stringent screening of positive clones on four drop out media  in the presence of antibiotic aureobasidin a. furthermore, we also fused the full length mybr <dig> to the bd-domain and found that it also interacts only with pyl <dig> out of the  <dig> pyl family members.
but mybr <dig> showed no interaction with ino which suggests that, despite their shared roles in stress response and senescence, mybr <dig> and mybr <dig> have some non-redundant functions.
in figure  <dig>  yeast colonies resulting from interaction of ad-pgadt <dig>  -mybr <dig>  -pyl <dig>  and -ino with bd-mybr <dig> were smaller compared to those with bd-mybr <dig> and bd-pgbt <dig>  it should be noted that we observed slight autoactivation and notable toxicity/reduced cell growth from high expression of mybr <dig> using the pgbkt <dig> plasmid and hence used the lower expressing plasmid pgbt <dig>
next, we examined whether the protein-protein interactions described above are affected by phytohormones .
in addition, mybr <dig> is reported to modulate auxin signaling  <cit>  and therefore we also tested inhibitors of auxin signaling  and transport .
however, the above interactions were constitutive and not affected by phytohormone additives.
we performed bimolecular fluorescence complementation  assays in nicotiana benthamiana leaf epidermal cells to independently verify the interactions of pyl <dig> with mybr <dig> and mybr <dig> and of mybr <dig> with mybr <dig>  it has been shown previously that pyl <dig> and mybr <dig> are localized in the nucleus  <cit>  and interaction between mybr <dig> and arf <dig> occurs in the nucleus  <cit> .
all interactions of pyl <dig> with mybr <dig> and mybr <dig> and of mybr <dig> with mybr <dig> are high with consistent fluorescent signal .
yeast two-hybrid and bifc approaches confirmed the interactions of pyl <dig> with mybr <dig> and mybr <dig> and between mybr <dig> and mybr <dig> and showed that pyl <dig> may modulate the binding of mybr <dig> and mybr <dig> to dna and/or that both mybr <dig> and mybr <dig> may modulate pyl <dig> function.
the interaction of mybr <dig>   <dig> with only pyl <dig> but not with other members of pyr/pyl/rcar family suggests that these interactions define very specific functional roles.
discussion
we previously identified mybr <dig> as a weakly aba responsive gene  <cit>  and here we provide evidence that it is a repressor of aba signaling during seedling growth, drought and senescence.
it is now clear that mybr <dig> is part of the aba/abiotic stress response and wounding/ abscission response networks, both of which involve senescence responses.
mybr <dig> acts as a negative regulator  of responses to stress, wounding and abscission in favor of normal growth and development.
mybr <dig> is by no means unique in its ability to negatively regulate aba and stress responses.
other examples include the ap <dig> domain tfs aba repressor <dig>   <cit>  and ethylene response factor <dig>   <cit>  and the homeodomain protein hb <dig> <cit> .
our original observations that mybr <dig> was induced by pbi <dig>  induced weakly by aba, repressed by drought and paradoxically induced by rewatering after drought stress  <cit>  have been confirmed and can now be rationalized.
under non-stressed conditions, aba treatment produces unnecessary stress responses and mybr <dig> induction blocks these responses to restore normal patterns of gene expression.
under water stress conditions mybr <dig> is not expressed, allowing the full effects of aba to be manifested and allows adaptive responses to be maintained during drought stress.
on recovery from stress, mybr <dig> expression leads to repression of aba responses that are no longer required.
the regulation and effects of mybr <dig> are summarized in figure  <dig>
although induced senescence during prolonged drought has survival value by conserving water and nutrients  <cit> , there are clearly finely balanced advantages and disadvantages to irreversible loss of vegetative matter.
in fact, by introducing a novel feedback mechanism to suppress drought induced senescence in tobacco, rivero et al.
<cit>  demonstrated striking beneficial effects, suggesting that, in a crop plant context, induced senescence can be disadvantageous.
therefore, it seems that mybr <dig> is a component of an endogenous homoeostatic mechanism to balance growth, high seed production and risk of death versus senescence, survival and minimal seed production.
given that senescence of older leaves is a normal stage of leaf development, mybr <dig> appears to also play a role in determining the normal length of the leaf adult phase.
senescence induces protein degradation pathways  <cit>  and the effects of mybr <dig> are associated with reduced/delayed expression of ubiquitin- and autophagy mediated protein degradation and increased production of cks.
previous studies have associated drought-induced leaf senescence with reduced cks  <cit>  and increased ck biosynthesis blocks leaf senescence  <cit> .
higher levels of cks, reduced primary root growth and more adult leaves in oxmybr <dig> lines are also consistent with increased ck effects.
however, there are other hormonal interactions.
mybr <dig> appears to repress jasmonate effects – which likely also contributes to suppression of wounding responses.
jung et al.
<cit>  demonstrated that mybr <dig> was induced by jasmonate and also showed that jasmonate responses were repressed.
more recently shim et al.
<cit>  show that mybr <dig> represses ja defense responses and activates salicylic acid-mediated defenses via wrk <dig> leading to enhanced responses to biotrophic pathogens and attenuated responses to necrotrophic pathogens.
we propose a model of mybr1-repression of aba signaling during drought and senescence .
it has been shown previously that pyl <dig> is localized in both cytoplasm and nucleus and the interaction between pp2c <dig> and pyl <dig> takes place in the nucleus  <cit> .
in addition, mybr <dig> is also localized in the nucleus  <cit> .
therefore, the interaction of mybr <dig> with pyl <dig> suggests a direct role of mybr <dig> in modulating aba perception.
the uniqueness of the interaction with pyl <dig>  provides an example of receptor specificity - an aba receptor mediating a specific sub-network of responses.
the existence of such effects was suggested by comparison of the effects of aba analogs in huang et al.
<cit> .
previous papers have noted that binding of pyl <dig> to pp2cs does not appear to be dependent on aba, so the regulatory significance of the pyl8-aba complex is not clear.
increased drought tolerance and aba hypersensitivity in seed of 35spro:pyl <dig> lines showed that pyl <dig> is an overall positive regulator of aba signaling  <cit> .
binding of mybr <dig> to pyl <dig> may block interaction with and inhibition of pp2cs.
alternatively, pyl <dig> may regulate mybr <dig> binding to dna.
since pyl8-pp2c binding is independent of aba, pyl <dig> may be responsible for constitutive aba signaling that is independent of aba itself or aba may be required to fully potentiate pyl8-pp2c interaction.
future studies will further explore the mybr1-pyl <dig> interaction in relation to mybr <dig> function.
the weak phenotypes of the mybr <dig> and mybr <dig> mutants and the enhanced effects in the double mybr <dig> x mybr <dig> mutant strongly suggest that mybr <dig> and mybr <dig> are partially redundant and the yeast two hybrid data indicates that they may form heterodimers .
however, mybr <dig> has mainly been associated with auxin signaling and root development  <cit> , shows differing mybr2pro::gus expression patterns compared to mybr1pro::gus  <cit> , and has not been distinctly associated with aba or jasmonate response as our data and others suggest for mybr <dig> <cit> .the specific interaction of mybr <dig>  with ino suggests that there are at least some unique functions of mybr <dig> not shared by mybr <dig>  however, the significance of the mybr1-ino interaction is unknown at this time.
ino encodes a yabby-type transcription factor and is only known to be involved in ovule development  <cit>  and there is no specific mybr <dig> phenotype associated with flowers.
the effects of mybr <dig> overexpression in arabidopsis were also studied by jung et al.
<cit> , but some of their results were significantly different to those reported here.
jung et al.
<cit>  reported downregulation of stress genes but increased stress tolerance and reduced water loss from detached shoots in over-expression lines and obtained similar results in soybean transgenics  <cit> .
similarly, persak and pitzschke  <cit>  reported delayed mortality of an oxmybr <dig> line relative to wild type when exposed to toxic levels of salt.
for this reason, we focused carefully on identifying the most appropriate approach to measuring drought and water loss.
we believe that our results demonstrate that the reduced size of oxmybr <dig> lines – due to slower growth of above-ground tissues and shorter primary roots – is associated with reduced water use and slower depletion of soil moisture.
this phenomenon produced an apparent increase in drought tolerance because the differential size and water use of the mybr <dig> genotypes were not taken into account.
to circumvent this issue, peg treatment  was used to reveal the increased sensitivity of oxmybr <dig> lines to water stress .
furthermore our microarray results are consistent with reduced stress responses in oxmybr <dig> lines and careful analysis of microarray results in table  <dig> in jung et al.
<cit>  suggests that many well-known positive effectors or regulators of stress responses , cor <dig>  rd29b, delta1-pyrroline-5-carboylate synthase <dig> , dreb2a) were similarly down-regulated in overexpressing atmybr <dig> plants relative to wt plants.
however, jung et al.
did not perform experiments that showed the effects of mybr <dig> overexpression on repressing aba/pbi425-induced genes .
the differences between our results and jung et al.
<cit>  in measuring drought tolerance provides a cautionary example of the complexities and subtleties of performing and interpreting drought and water use experiments.
unlike jung et al.
<cit>  and persak and pitzschke  <cit> , we did not investigate salt-stress related phenotypes related to mybr <dig> expression.
more recently, jung et al.
<cit>  suggested that mybr <dig> was induced non-specifically by phytohormones and suppressed jasmonate responses.
our data also suggest an effect of mybr <dig> on repressing ja responses, but show a direct and unambiguous link to aba signaling as described above.
CONCLUSIONS
in the last few years, considerable information has accumulated on the involvement of mybr <dig> in stress-related mapk signaling.
however, the function of the gene in relation to stress responses has remained unclear.
this study reveals that mybr <dig> is a component of aba signaling and appears to be involved in feedback maintenance of adult, pre-senescent growth, especially under conditions of stress and wounding.
as such it provides an example of a transcription factor that integrates, balances and co-ordinates hormonal, developmental and environmental signals.

 in order to acclimate and protect themselves, plants translate environmental challenges such as drought, waterlogging, extreme temperatures, soil salinity, wounding, and pathogen attack into internal signals through hormones, second messengers, and transcription factors. the phytohormone abscisic acid regulates abiotic stress responses and other vital processes in plant growth and development, especially during seed maturation reviewed in. mutant plants with altered aba biosynthesis, perception or response have been crucial in deciphering the various components involved in aba responses. recently, a family of novel start domain proteins, named as pyr/pyl/rcars has been identified as intracellular aba receptors that interact with and inhibit several protein phosphatase 2cs including aba insensitive and, homology to abi, and pp2ca. such phosphatases are negative regulators of aba signaling. a recent model for aba signaling, based on several independent crystallographic studies for example, proposes that in the presence of aba, receptors of the pyr/pyl/rcar family bind to pp2cs which in turn release inhibition on a subfamily of snf1-related protein kinase kinases. these kinases then phosphorylate and subsequently activate transcription factors including aba responsive elements-binding factor / aba responsive elements-binding protein -type bzip tfs. abfs in turn bind to aba-responsive promoter elements to activate aba-responsive gene expression. the molecular basis of adaptive responses to abiotic stresses such as low temperature, desiccation and salinity, has been elucidated by identifying genes such as responsive to dehydration, kykna-indusoitu, responsive to aba, cold-regulated, low-temperature-induced, and drought-induced. manipulation of many of these genes resulted in plants with improved tolerance to drought, salt, cold and freezing reviewed in. molecular and genetic studies suggest that aba-dependent and –independent pathways operate in abiotic stresses and aba-dependent pathways are predominant in drought stress responses. environmental factors such as drought, extreme temperature and pathogen infection as well as endogenous factors including age affect the onset and progression of leaf senescence reviewed in. unlike abscission and dehiscence, leaf senescence is a specialized form of programmed cell death, which is a genetically regulated process of slow cell death of the entire leaf and is preceded by the reallocation of nitrogen, phosphorus, and metals to other parts of the plant. an early manifestation of senescence in leaves is loss of chlorophyll. leaf mesophyll cells start to senesce first, followed by other cell types, and exhibit an incoherent pattern of localized cell death, which eventually spreads to the whole leaf. senescence is accelerated by aba, ethylene, jasmonates, and salicylic acid, and is delayed by cytokinins and auxin reviewed in. however, extensive cross talk among these signaling pathways during senescence complicates understanding of the initiation and progression of senescence. therefore, key components in senescence signaling remain largely unknown. senescence is an important aspect of drought responses. accelerated leaf senescence followed by leaf abscission is triggered by prolonged stress to reduce water loss, remobilize nutrients to young leaves and to enable survival of the plant. the myb family tfs comprises around genes in arabidopsis and is the largest tf gene family reviewed in. myb proteins contain a conserved dna-binding myb domain of about amino acids, and are classified into three subfamilies based on the presence of one, two or three myb domains reviewed in. the plant specific and largest myb family consists of r2r3-type factors which contain two repeats and comprise genes in arabidopsis. r2r3-myb genes are involved in various plant-specific processes such as regulation of secondary metabolism, modulation of development, determination of cell fate and identity and responses to environmental factors and hormone. the gene further characterized in this paper, atmybr1/myb was weakly induced by 24 h treatment with aba but strongly induced by the hyperactive aba analog -8′ acetylene aba. most aba-regulated genes are similarly regulated by water stress, however mybr was selected for further functional characterization because its expression was paradoxically repressed by drought and elevated by re-watering suggesting a novel role in aba signaling. jung et al. reported that over-expression of mybr increased stress tolerance but unexpectedly repressed many known stress-related genes. subsequent studies have revealed that this gene is regulated by a mitogen-activated protein kinase cascade. following stress treatment, mitogen-activated protein kinase is activated and phosphorylates the bzip tf vire2-interacting protein, which then rapidly activates the expression of mybr and other stress genes through promoter binding. further studies have shown that mybr interacts directly with, and is phosphorylated by, mpk at ser and possibly ser and that the ser phosphorylation is required for mybr function. in this study, we functionally characterized the atmybr tf by studying an arabidopsis t-dna insertion mutant mybr and overexpression lines of atmybr. we show that mybr down regulates many aba responsive genes including those involved in abiotic stresses and negatively regulates drought responses and senescence. moreover, direct involvement of mybr in early aba signaling is suggested by our observation that mybr protein interacts with pyl an aba receptor.  atmybr represses genes induced by a hyperactive aba analog we showed in a previous study that atmybr was induced weakly by -aba and more strongly by 24 h treatment with a hyperactive aba analog pbi -8′ acetylene aba) indicating mybr is likely a component of the aba signaling pathway. it has been shown previously that pbi induces aba responsive genes almost identically to the natural enantiomer s--aba. however, because pbi is catabolized much less rapidly than -aba and accumulates to higher levels in plant tissue it is an effective tool to study weak and transiently expressed aba-responsive genes such as abi abi lti knat and mybr itself. therefore we used pbi to define the role of mybr in aba signaling. in addition to using pbi to study the function of atmybr we generated transgenic arabidopsis 35spro:mybr plants. after kanamycin selection, three lines with single inserts were selected and their homozygous progeny plants were grown as experimental materials. the level of overexpression of mybr was 23-fold in gain-of-function oxmybr line 42– 12-fold in line 31– and 11-fold in line 1– and was undetectable by qpcr in loss-of-function mybr. to reduce the likelihood of identifying phenotypic artifacts due to mis-expression, the phenotypes of all the overexpression lines were compared for qualitative consistency throughout the experiments. we compared gene expression in different genotypes using arabidopsis oligoarrays representing a comprehensive set of approximately expressed genes. the comparisons were: genotype comparisons of untreated plants: oxmybr or mybr versus wt, genotype comparisons after pbi treatment: oxmybr or mybr versus wt, and effect of pbi treatments on each genotype: oxmybr mybr and wt treated with pbi versus the same genotype without the treatment. the experimental design is illustrated in additional file 1: figure s online. samples were treated with pbi for 24 h on the basis that the accumulation and effects of pbi on gene expression was maximum at 24 h. the total numbers of differentially expressed genes are listed in table  and the gene lists and data may be found in additional file 2: table s there were a total of differentially regulated genes from all comparisons. in the absence of pbi treatment, comparisons of oxmybr or mybr vs. wt yielded a very small number of differentially regulated genes. treatment with pbi greatly increased numbers of differentially expressed genes and revealed differences between genotypes. analysis of the direct effect of pbi on gene expression showed that mybr represses expression of many genes induced by pbi in wt in terms of both total numbers and expression ratios. it is noteworthy that there were very few differentially regulated genes from comparisons of mybr versus wt both with and without pbi treatment. this suggests the likelihood that mybr is functionally redundant with at least one other closely related gene. mybr is reported as the closest homolog of mybr based on sequence similarities in the c-terminal regions of the respective proteins and lack of homology with other myb-type proteins. mybr has been reported to be involved in auxin signal transduction. mybr over-expression results in reduced root and shoot growth, and root phenotypes in loss-function mybr lines varied with application of auxin. however, its role in aba responses and stress signaling or whether it acts cooperatively with mybr remains unclear. to investigate possible redundant functions of mybr and mybr, gene expression analysis was performed using agilent microarrays containing arabidopsis thaliana reporter sequences. gene expression was compared between pairs of genotypes treated with pbi i) mybr versus mybr and ii) mybr versus mybr1xmybr the number of differentially expressed genes was for mybr vs mybr and for mybr vs mybr x mybr. the increase in differentially expressed genes in the double mutant comparison suggests that mybr and mybr act in a synergistic manner however, only six out of genes in the first comparison and out of genes in the second comparison were present in the above mentioned list of genes differentially expressed in all comparisons. atmybr represses many aba inducible stress genes many stress genes that are highly induced by aba, are repressed by mybr. however, since mybr did not appear to repress all pbi induced genes, we examined more closely the gene expression patterns affected by mybr and pbi in this analysis, we added statistically significant genes to the total gene list of table  for detailed analysis and interpretation. these genes were not listed in table  since their fold change was below the ratio threshold. however, changes in expression of these genes were either verified by qpcr and direct spot visualization in base or were present in our comparative analysis of our microarray data with data published by van der graaff et al.. several distinct gene regulation patterns were identified by comparing pbi treatment and mybr overexpression and the gene lists and expression data may be found in additional file 3: table s we excluded the comparison of mybr vs. wt from table  because of the small number of differentially expressed genes. similarly, we also excluded microarray data of mybr from comparisons mybr versus mybr and mybr versus mybr1xmybr many well-characterized abiotic stress responsive genes were grouped in classes a, f and g, of which genes were repressed by mybr genes were activated by mybr and genes were unaffected by mybr therefore, mybr represses a subset of about 43% of aba-responsive genes. it has been suggested that stress responsive genes are under regulation by both aba-dependent and aba-independent pathways for a review see. there are four dehydration responsive element binding protein /c-repeat binding factor transcription factor genes that have been suggested to mediate aba independent cold stress responses. here, dreb1a/cbf was induced by pbi and repressed by mybr suggesting that dreb1a/cbf is aba-dependent. mybr overexpression on aba-related gene expression o mybr mybr mybr bk: background; mybr1: loss-of-function of mybr1; ox: overexpressed; gene regulation symbols-- ↑: upregulation; ↓: downregulation; —: either ‘not differentially expressed’ or ‘undetectable’. many senescence associated genes including sag12/13/21/29/ early responsive to dehydration and apg8a are also grouped in class a. there were a total of genes that were either upregulated or down regulated by mybr in the same direction as aba. among the downregulated genes were many associated with photosynthesis and biotic stresses. a total of genes were regulated by mybr but unaffected by pbi among these, several genes involved in jasmonate and auxin action were repressed. promoters of many genes associated with drought, cold stress and salinity contain aba-responsive element, abre binding factor and dehydration responsive element. a search for statistically over-represented cis-acting motifs present in the promoters of classified genes sets in table  was carried out against agris and place databases using the analysis tools in athena and the results are summarized in additional file 1: table s these promoter motifs mainly consisted of four groups. the majority of motifs are associated with aba. other motifs are related to stress, light regulation, gibberellins and circadian clock, suggesting cross talk between these pathways as was also observed previously. there was a general similarity in the presence of aba and stress motifs in genes that were regulated specifically by aba and those that were regulated in the opposite direction by mybr no significant enrichment was detected in genes that were regulated specifically by mybr atmybr reduces drought tolerance to define the function of mybr during drought stress, plants were treated with 10% and 15% polyethylene glycol for d. consistent with the down regulation of the stress responsive genes, oxmybr plants showed the highest degree of stress following peg treatment and homozygous mybr1xmybr plants showed the least damage. subsequently, we found that oxmybr rosettes lost water and chlorophyll faster than wt and loss-of-function mutants. therefore, consistent with the down regulation of stress responsive genes, the data suggests that oxmybr plants transpired water faster and are consequently less drought tolerant than wt plants. drought stress was also imposed by withholding water for d on seedlings from all available genotypes. surprisingly, we recorded a 100% survival in all replicates of oxmybr. results obtained when drought stress was imposed by withholding water were opposite to what was observed following peg mediated drought stress. when performing the soil drying experiments above it was observed that, when plants from each genotype were provided with equal volumes of water, the oxmybr soil dried more slowly than other genotypes. therefore to investigate the contradictory results obtained by peg and soil drying experiments, we conducted transpirational water loss assays on whole plants. in these experiments, soil water loss by evaporation was prevented so that plant water use could be monitored. the results showed that oxmybr plants lost water slower than wt and mutants, which is contrary to what was observed in detached whole rosette leaves. in other words, oxmybr plants extracted and/or used less water from the soil than other genotypes, even though the transpirational capacity of the detached leaves was relatively higher. therefore, we investigated the water conservation characteristic of oxmybr further. we measured the soil water content after imposing an d drought. results confirmed that water uptake was less in oxmybr than in wt and loss-of-function mutants during drought. under normal growth conditions, oxmybr plants grew more slowly than wt and loss-of-function mutants and we show later that oxmybr lines have shorter primary roots than other genotypes. therefore, with respect to mybr function, we conclude that reduced water use in whole oxmybr plants in drying soil is not due to a genetically determined reduction in transpiration but is rather a consequence of lower biomass leading to slower depletion of soil moisture. the reduced growth rate of oxmybr lines was also noted by jung et al. and in soybean by seo et al.. as an aside, we note that the residual water content of mybr material was slightly higher than the other loss-of-function lines. this may be an indication that the functions of mybr are not identical with those of mybr as discussed later. mybr1pro:gus is expressed under abiotic stress and during senescence, mechanical wounding and floral organ abscission a  kb promoter fragment of mybr including the 5′utr was fused to the β-glucuronidase reporter gene and the expression of mybr was examined histochemically. gus staining was performed on homozygous t and t plants. in d old seedlings, gus expression driven by the mybr promoter was observed in cotyledons and true leaves. in contrast to very high gus expression in cotyledons, gus expression was lower in younger true leaves relative to older leaves and was absent in the newly emerged leaves. intriguingly, gus expression was observed in patches in younger leaves and was absent around the vascular regions of both older and younger leaves hinting that mybr could be involved in senescence since this pattern was reminiscent of the development of visible senescence in leaves reviewed in. under normal conditions, expression of gus was also observed in hydathodes of all leaf margins as well as embryo, suspensor, endosperm, root, stigma, sepal, petal and anther filament but was absent in stem, cauline leaf, anther, silique and testa. gus expression was observed in embryo and endosperm dissected from siliques at developmental stages from 6– dpa as well as from dry and imbibed mature seeds. the intensity of gus staining increased with development in embryos but remained constant in endosperm except at dpa when the gus expression was lower. gus expression was high and remained constant in embryos collected from dry seeds and seeds imbibed up to 24 h but declined subsequently. gus expression in endosperm of dry and imbibed seeds remained high. water stress significantly reduced gus expression driven by the mybr promoter in leaves but not in roots relative to control. drought induced reduction of mybr expression is consistent with the reduced expression of mybr under drought treatment observed by huang et al.. gus reporter activity was rapidly and strongly induced immediately after mechanical wounding of leaves. similar high mybr expression was also observed at the abscission zone on the pedicel following sepal, petal, and anther filament abscission. prior to the abscission of floral organs, no gus expression was visible on the pedicel. however, we did not observe either accelerated- or delayed/abolished floral organ shedding in mybr loss-and gain-of-function mutants, suggesting that the high gus expression at the az is due to the wounding response. atmybr delays leaf senescence early in leaf senescence chloroplasts disassemble with subsequent degradation of chlorophyll and visible leaf yellowing. to further investigate the role of mybr in leaf longevity, detached rosette true leaves numbers 3–, from d old soil grown plants were incubated in buffer as described in two different sets. leaves were photographed and the chlorophyll content was quantified on d for one set and after 6– d of dark-induced senescence treatment for the other set. in freshly harvested leaves, the chlorophyll content was higher in one line of oxmybr and two reciprocal double mutants of mybr and mybr than the rest of the genotypes. following dark-induced senescence, oxmybr lines showed increased leaf longevity and slowed chlorophyll degradation relative to wt leaves. interestingly, increased leaf longevity in oxmybr lines was in contrast to that in mybr and reciprocal mybr & mybr mutants which exhibited early leaf senescence and accelerated chlorophyll degradation relative to wt although the chlorophyll content before treatment was relatively high in mybr and mybr double mutants. indeed, among the eight genotypes, leaves of mybr & mybr were least green following senescence treatment and showed the fastest chlorophyll breakdown. to further investigate the role of mybr in leaf senescence, excised leaves of d old transgenic mybr1pro:gus were stained for gus before and after dark induced senescence. in untreated plants, gus staining was strong in older leaves and was absent in younger leaves and in vascular region. gus staining became weaker overall in dark-treated leaves relative to fresh leaves but among the dark treated leaves, there was more gus staining in senescent, yellow leaves than green ones, further suggesting that mybr plays a role during leaf senescence. atmybr regulates the expression of senescence genes to investigate whether mybr regulates the expression of senescence genes, we compared the differentially expressed gene lists of table  with microarray data obtained by van der graaff et al. on various stages and types of leaf senescence. the number of common genes between the two microarray analyses is which covers 52% of our differential gene list. the increase of mybr induction with the progression of senescence is high in ns, low in dis and det whereas mybr induction is slightly increased in sink-to-source transition stage of ns. the regulation of these common genes by aba, mybr and senescence revealed the interaction between aba and mybr during senescence. out of genes in class a, 88% were induced by both aba and ns but repressed by mybr clearly demonstrating that mybr is a suppressor and aba is an activator of leaf senescence. furthermore, out of genes in class c, 95-96% genes are downregulated by ns, dis and det and from genes in class g, 86% are upregulated by ns in the same direction as by aba showing a significant role of aba in leaf senescence. of a large number of common genes, 89-93% are downregulated by aba, mybr and senescence and many of these genes are involved in protein synthesis, photosynthesis, auxin responses and biotic stress. furthermore, we performed qrt-pcr on senescence marker genes in rosette leaves numbers and in all the genotypes. consistent with our detached leaf senescence analysis, accelerated leaf senescence of mybr1xmybr was associated with upregulated expression of sag sag and senescence relative to all other genotypes. sag expression was fold higher in mybr1xmybr relative to wt. the expression of sag sag sen and sen were downregulated in oxmybr relative to wt, consistent with the senescence-suppressing effect of mybr changes in expression of these genes in the single mutants mybr and mybr were generally small. however, expression of sag and sen did not show a reciprocal relationship between gain and loss of function genotypes. nevertheless, it is apparent that mybr negatively regulates senescence based on of the marker genes. protein degradation occurs during senescence via different pathways such as autophagy and the 26s proteosome and components of both pathways were transcriptionally activated during senescence. our differential gene list contains genes involved in protein degradation pathways. here we report the repression of genes by oxmybr where genes encode components of e ), e ubiquitin-ligase complex and 20s core particle of 26s proteosome and three genes are apg8a/8f/8e. interestingly, only three genes in the ubiquitin pathway - phd finger family, early-responsive to dehydration 16/ubiquitin etension protein and suppresor of nim1- were upregulated by oxmybr son is an f-box protein component of e3-ubiquitin ligase complex which negatively regulates, through the ubiquitin-proteosome pathway, a novel defense response that is independent of systemic acquired resistance. on the other hand, genes involved in protein degradation were activated by aba in both wt and mybr and many of them are also upregulated by ns, dis and det. content of endogenous cytokinins and jasmonic acid to investigate the role of mybr in relation to hormonal pathways, endogenous hormone levels were measured quantitatively by lc-ms/ms in rosette leaves numbers and of three weeks old plants. trans-zeatin and n6- adenine and their sugar derivatives are the major cytokinins in arabidopsis. levels of several cks were significantly increased in oxmybr relative to other genotypes. on the other hand, ja was significantly higher in mybr x mybr relative to other genotypes. this suggests that suppression of leaf senescence by mybr is associated with increases in cks and conversely that increased senescence is associated with higher ja. in our microarray data, oxmybr downregulated arr, a transcriptional repressor of ck signaling. however, the down regulation of arr by aba and senescence is contradictory and may be due to a feedback effect. furthermore, we did not detect differential expression of genes involved in ck metabolism but a posttranscriptional regulatory effect of mybr on expression of these genes cannot be ruled out. oxmybr leaves contained similar levels of aba and its metabolite dihydrophaseic acid to those measured in wt/mybr and mybr1xmybr levels of sa and iaa were also not significantly altered. it is surprising that aba levels remained constant despite the strong effect of atmybr overexpression on aba responses. to investigate further, we performed qrt-pcr on six aba downstream effectors; abi eel and abf1/2/3/ in plants of oxmybr, wt, mybr1/ and mybr x mybr. no significant differences of expression of these genes were evident among the genotypes tested. mybr mis-expression affects leaf and root morphology we examined the roots of gain-and loss of mybr function genotypes. primary roots of oxmybr lines were drastically shorter, whereas those of mybr were notably longer relative to wt. this shorter root phenotype of oxmybr may contribute toward reduced water uptake in the oxmybr lines as noted earlier, and may help explain the differences between results of peg treatments and soil drying experiments described above. the arabidopsis mutant amp with a high level of endogenous cks, had increased numbers of rosette leaves. conversely, plants overexpressing catabolic ck oxidases had fewer leaves than wt plants. we counted rosette leaves in seedlings of two lines of oxmybr wt and mybr seedlings of oxmybr lines had consistently more rosette leaves relative to other genotypes and double mutant mybr1xmybr had fewer leaves relative to all other genotypes. differences between genotypes are slightly enhanced by acc treatment. using two way anova, there is a consistent, statistically significant difference in leaf number between mybr x mybr and the oxmybr lines but no significant difference between control and acc treatments. mybr physically interacts with pyl and ino further information on the mechanistic role of mybr in signaling was obtained by identifying protein-protein interactions using the yeast 2-hybrid system. initially pyr1-like and inner no outer proteins were identified as interacting with mybr by screening an arabidopsis cdna library made from different stages of vegetative and floral tissues with a full length mybr fused to the dna-binding domain of the yeast gal protein. it has been shown by many groups that the members of the pyr/pyl/rcar family are intracellular aba receptors that interact with and inhibit several pp2c-type protein phosphatases including abi abi hab and pp2ca. ino encodes a yabby-type tf and is required for both polarity determination and outer integument initiation in ovule development. mybr interaction with pyl encouraged us to subsequently fuse all pyr/pyl/rcar genes as well as ino and mybr to the transcription-activation domain of the yeast gal protein. interestingly, only pyl ino and mybr interacted with mybr. interaction of mybr and pyl was confirmed by three independent experiments using a more stringent screening of positive clones on four drop out media in the presence of antibiotic aureobasidin a. furthermore, we also fused the full length mybr to the bd-domain and found that it also interacts only with pyl out of the pyl family members. but mybr showed no interaction with ino which suggests that, despite their shared roles in stress response and senescence, mybr and mybr have some non-redundant functions. in figure  yeast colonies resulting from interaction of ad-pgadt -mybr -pyl and -ino with bd-mybr were smaller compared to those with bd-mybr and bd-pgbt it should be noted that we observed slight autoactivation and notable toxicity/reduced cell growth from high expression of mybr using the pgbkt plasmid and hence used the lower expressing plasmid pgbt next, we examined whether the protein-protein interactions described above are affected by phytohormones. in addition, mybr is reported to modulate auxin signaling and therefore we also tested inhibitors of auxin signaling and transport. however, the above interactions were constitutive and not affected by phytohormone additives. we performed bimolecular fluorescence complementation assays in nicotiana benthamiana leaf epidermal cells to independently verify the interactions of pyl with mybr and mybr and of mybr with mybr it has been shown previously that pyl and mybr are localized in the nucleus and interaction between mybr and arf occurs in the nucleus. all interactions of pyl with mybr and mybr and of mybr with mybr are high with consistent fluorescent signal. yeast two-hybrid and bifc approaches confirmed the interactions of pyl with mybr and mybr and between mybr and mybr and showed that pyl may modulate the binding of mybr and mybr to dna and/or that both mybr and mybr may modulate pyl function. the interaction of mybr with only pyl but not with other members of pyr/pyl/rcar family suggests that these interactions define very specific functional roles. discussion we previously identified mybr as a weakly aba responsive gene and here we provide evidence that it is a repressor of aba signaling during seedling growth, drought and senescence. it is now clear that mybr is part of the aba/abiotic stress response and wounding/ abscission response networks, both of which involve senescence responses. mybr acts as a negative regulator of responses to stress, wounding and abscission in favor of normal growth and development. mybr is by no means unique in its ability to negatively regulate aba and stress responses. other examples include the ap domain tfs aba repressor and ethylene response factor and the homeodomain protein hb. our original observations that mybr was induced by pbi induced weakly by aba, repressed by drought and paradoxically induced by rewatering after drought stress have been confirmed and can now be rationalized. under non-stressed conditions, aba treatment produces unnecessary stress responses and mybr induction blocks these responses to restore normal patterns of gene expression. under water stress conditions mybr is not expressed, allowing the full effects of aba to be manifested and allows adaptive responses to be maintained during drought stress. on recovery from stress, mybr expression leads to repression of aba responses that are no longer required. the regulation and effects of mybr are summarized in figure  although induced senescence during prolonged drought has survival value by conserving water and nutrients, there are clearly finely balanced advantages and disadvantages to irreversible loss of vegetative matter. in fact, by introducing a novel feedback mechanism to suppress drought induced senescence in tobacco, rivero et al. demonstrated striking beneficial effects, suggesting that, in a crop plant context, induced senescence can be disadvantageous. therefore, it seems that mybr is a component of an endogenous homoeostatic mechanism to balance growth, high seed production and risk of death versus senescence, survival and minimal seed production. given that senescence of older leaves is a normal stage of leaf development, mybr appears to also play a role in determining the normal length of the leaf adult phase. senescence induces protein degradation pathways and the effects of mybr are associated with reduced/delayed expression of ubiquitin- and autophagy mediated protein degradation and increased production of cks. previous studies have associated drought-induced leaf senescence with reduced cks and increased ck biosynthesis blocks leaf senescence. higher levels of cks, reduced primary root growth and more adult leaves in oxmybr lines are also consistent with increased ck effects. however, there are other hormonal interactions. mybr appears to repress jasmonate effects – which likely also contributes to suppression of wounding responses. jung et al. demonstrated that mybr was induced by jasmonate and also showed that jasmonate responses were repressed. more recently shim et al. show that mybr represses ja defense responses and activates salicylic acid-mediated defenses via wrk leading to enhanced responses to biotrophic pathogens and attenuated responses to necrotrophic pathogens. we propose a model of mybr1-repression of aba signaling during drought and senescence. it has been shown previously that pyl is localized in both cytoplasm and nucleus and the interaction between pp2c and pyl takes place in the nucleus. in addition, mybr is also localized in the nucleus. therefore, the interaction of mybr with pyl suggests a direct role of mybr in modulating aba perception. the uniqueness of the interaction with pyl provides an example of receptor specificity - an aba receptor mediating a specific sub-network of responses. the existence of such effects was suggested by comparison of the effects of aba analogs in huang et al.. previous papers have noted that binding of pyl to pp2cs does not appear to be dependent on aba, so the regulatory significance of the pyl8-aba complex is not clear. increased drought tolerance and aba hypersensitivity in seed of 35spro:pyl lines showed that pyl is an overall positive regulator of aba signaling. binding of mybr to pyl may block interaction with and inhibition of pp2cs. alternatively, pyl may regulate mybr binding to dna. since pyl8-pp2c binding is independent of aba, pyl may be responsible for constitutive aba signaling that is independent of aba itself or aba may be required to fully potentiate pyl8-pp2c interaction. future studies will further explore the mybr1-pyl interaction in relation to mybr function. the weak phenotypes of the mybr and mybr mutants and the enhanced effects in the double mybr x mybr mutant strongly suggest that mybr and mybr are partially redundant and the yeast two hybrid data indicates that they may form heterodimers. however, mybr has mainly been associated with auxin signaling and root development, shows differing mybr2pro::gus expression patterns compared to mybr1pro::gus, and has not been distinctly associated with aba or jasmonate response as our data and others suggest for mybr.the specific interaction of mybr with ino suggests that there are at least some unique functions of mybr not shared by mybr however, the significance of the mybr1-ino interaction is unknown at this time. ino encodes a yabby-type transcription factor and is only known to be involved in ovule development and there is no specific mybr phenotype associated with flowers. the effects of mybr overexpression in arabidopsis were also studied by jung et al., but some of their results were significantly different to those reported here. jung et al. reported downregulation of stress genes but increased stress tolerance and reduced water loss from detached shoots in over-expression lines and obtained similar results in soybean transgenics. similarly, persak and pitzschke reported delayed mortality of an oxmybr line relative to wild type when exposed to toxic levels of salt. for this reason, we focused carefully on identifying the most appropriate approach to measuring drought and water loss. we believe that our results demonstrate that the reduced size of oxmybr lines – due to slower growth of above-ground tissues and shorter primary roots – is associated with reduced water use and slower depletion of soil moisture. this phenomenon produced an apparent increase in drought tolerance because the differential size and water use of the mybr genotypes were not taken into account. to circumvent this issue, peg treatment was used to reveal the increased sensitivity of oxmybr lines to water stress. furthermore our microarray results are consistent with reduced stress responses in oxmybr lines and careful analysis of microarray results in table  in jung et al. suggests that many well-known positive effectors or regulators of stress responses, cor rd29b, delta1-pyrroline-5-carboylate synthase, dreb2a) were similarly down-regulated in overexpressing atmybr plants relative to wt plants. however, jung et al. did not perform experiments that showed the effects of mybr overexpression on repressing aba/pbi425-induced genes. the differences between our results and jung et al. in measuring drought tolerance provides a cautionary example of the complexities and subtleties of performing and interpreting drought and water use experiments. unlike jung et al. and persak and pitzschke, we did not investigate salt-stress related phenotypes related to mybr expression. more recently, jung et al. suggested that mybr was induced non-specifically by phytohormones and suppressed jasmonate responses. our data also suggest an effect of mybr on repressing ja responses, but show a direct and unambiguous link to aba signaling as described above.  in the last few years, considerable information has accumulated on the involvement of mybr in stress-related mapk signaling. however, the function of the gene in relation to stress responses has remained unclear. this study reveals that mybr is a component of aba signaling and appears to be involved in feedback maintenance of adult, pre-senescent growth, especially under conditions of stress and wounding. as such it provides an example of a transcription factor that integrates, balances and co-ordinates hormonal, developmental and environmental signals. 
the transcription factor atmybr is a member of the myb family of transcription factors and is expressed throughout the plant life cycle and especially in senescing and wounded leaves. it has previously been shown to be involved in responses to abiotic stress and is regulated by phosphorylation. mybr physically interacted with aba receptor pyr1-like suggesting a direct involvement of mybr in early aba signaling. the mybr promoter strongly drove β-glucuronidase reporter gene expression in tissues immediately after wounding and many wounding/pathogenesis genes were downregulated in oxmybr oxmybr plants were more susceptible to injury under water stress than wildtype, which was correlated with suppression of many aba inducible stress genes in oxmybr conversely, mybr plants were more tolerant of water stress and exhibited reduced rates of water loss from leaves. mybr is a negative regulator of aba, stress, wounding responses and blocks senescence. when mybr was over-expressed under the control of the constitutive 35s promoter in arabidopsis thaliana, leaf senescence was delayed.
 the transcription factor atmybr is a member of the myb family of transcription factors and is expressed throughout the plant life cycle and especially in senescing and wounded leaves. it has previously been shown to be involved in responses to abiotic stress and is regulated by phosphorylation.  when mybr was over-expressed under the control of the constitutive 35s promoter in arabidopsis thaliana, leaf senescence was delayed. in contrast loss-of-function mybr plants showed more rapid chlorophyll loss and senescence. the mybr promoter strongly drove β-glucuronidase reporter gene expression in tissues immediately after wounding and many wounding/pathogenesis genes were downregulated in oxmybr oxmybr plants were more susceptible to injury under water stress than wildtype, which was correlated with suppression of many aba inducible stress genes in oxmybr conversely, mybr plants were more tolerant of water stress and exhibited reduced rates of water loss from leaves. mybr physically interacted with aba receptor pyr1-like suggesting a direct involvement of mybr in early aba signaling. mybr appears to exhibit partially redundant functions with atmybr and double mybr x mybr mutants exhibited stronger senescence and stress related phenotypes than single mybr and mybr mutants.  mybr is a negative regulator of aba, stress, wounding responses and blocks senescence. it appears to have a homeostatic function to maintain growth processes in the event of physical damage or stress. abadrought stresstranscription factorpyl8senescence 