BACKGROUND
soybean is one of the most important crops in the world.
overall yield is highly affected by water deficit stress, particularly when the stress occurs during flowering and early pod expansion  <cit> .
to overcome the water limitation and facilitate the continued expansion of soybean productivity and crop improvement, implementation of modern biotechnology such as genetic engineering of plants to produce drought-tolerant cultivars, rises as a potential solution  <cit> .
however, the achievement of such a goal is highly dependent on the elucidation of the molecular mechanisms of drought tolerance and their interaction with environmental cues.
a better understanding of these aspects would help identify candidate genes for genetic engineering of improved stress-tolerant crops  <cit> .
to better fit to the surrounding environmental conditions, such as season and light/darkness and temperature variations, it is known that plants, as sessile organisms, coordinate and regulate their metabolism and physiology through an endogenous circadian clock.
this clock drives rhythms at the molecular and cellular levels and, thus, temporally regulates plant physiology and behavior to anticipate changes in the environment  <cit> .
according to khan et al.
<cit> , the consequence of proper clock and environment synchronization is optimized fitness.
however, abiotic stresses, such as drought, change the clock synchrony, altering the circadian rhythm in response to dehydration  <cit> .
genome-wide analysis of mrna expression shows daily oscillation coordinated by circadian rhythms, which, in turn, regulate various biological processes , such as seed dormancy and germination  <cit> , hormone metabolism , and grapevine fruit ripening  <cit> , among other processes  <cit> .
gene expression regulated by the circadian rhythm also has been observed in some plant responses to abiotic stress .
in the process of cold acclimation in arabidopsis, gene expression was affected by time of day, revealing an interaction between cold and diurnal regulation that drives transcriptome changes  <cit> .
furthermore, time of day also was an important cue for triggering changes in the populus transcriptome  <cit>  and in arabidopsis plants in response to soil drying  <cit> .
in soybean, evidence also suggests that the circadian rhythm plays a role in regulating genes involved in developing seeds  <cit> .
although soybean is one of the most studied crops using molecular biology tools, little is known about how daily oscillations of gene expression are affected by drought stress during the survival or acclimation response.
the regulation of a proline-rich-protein gene, induced under drought and salt stresses in specific tissues of soybean seedlings, was demonstrated to be circadian-controlled  <cit> .
considering the dynamic changes of plant metabolism that occur to coordinate the daily variation in light and temperature, the evaluation of gene expression at different time periods of the day becomes valuable for identifying times during which key genes might be most influential in the defense response  <cit> .
in arabidospis, hormone-related genes, specifically the abscisic acid -responsive genes, are correlated with diurnal oscillations  <cit> .
the functional roles of some representatives, like the dehydrins class and the rd29a, or the cold-regulated cor15b/15a and the low temperature-induced lti <dig>  may be related to responses to water deficit and low temperature, respectively  <cit> .
recently, our research team has demonstrated how drought impacts diurnal oscillation of both drought-responsive and circadian clock genes in soybean  <cit> .
drought stress induced marked reduction in gene expression levels of several circadian clock-like components, such as gmlcl1-, gmelf4-, and gmprr-like genes.
the same conditions produced a phase advance of expression for the gmtoc1-, gmlux- and gmprr7-like genes.
similarly, the daily oscillation pattern of the soybean drought-responsive genes dreb-, bzip-, gols-, rab18- and remorin-like changed significantly following plant exposure to water deficit.
with the goal of detecting genome-wide transcriptome changes during the entire period that plants were exposed to moderate water deficit and if such variations occurred in a time-of-day-dependent manner, we analyzed multiple time points in a diel period.
here, we present a survey of soybean genes expressed under stress and their daily oscillation waveforms during a 24-h time course.
we also determined their abundance, and we suggest the putative biological roles of these differentially expressed genes.
RESULTS
genes differentially expressed in response to water deficit
the expression pattern of soybean genotype br <dig>  previously characterized as drought sensitive  <cit> , was evaluated under normal and water deficit conditions, during a 24-h time course.
to identify differentially expressed genes  in response to water deficit treatment, we applied a stringent statistical test to determine whether genes were either up- or down-regulated compared to those of plants under optimal hydration conditions.
the resulting ratio represented the fold-change  for each gene.
to avoid false positives and reliably identify the most significant changes in gene expression, only genes with fc ≤ - <dig>  and ≥  <dig>  were considered.
we also applied a stringent statistical significance cutoff  to improve confidence .
there were larger sets of differentially expressed genes in zt <dig>  and zt <dig>  compared to the other periods.
in zt <dig>  the majority of genes  were down-regulated under water deficit stress condition, this is in contrast to the other periods , in which genes were primarily up-regulated  .
in general, a moderate expression ratio was detected for most genes, but high fold-changes for some genes were observed in the zt <dig> and zt <dig> periods.
the highest differential expression was detected in zt <dig> for glyma18g <dig> , which codes for a udp-glucosyl transferase 73b <dig>  and for glyma10g <dig> , a cap <dig> protein.
in zt <dig>  the highest confidence levels were found for glyma03g <dig>  a gene related to the fatty acid hydroxylase superfamily and for glyma17g <dig>  with unknown function .fig.
<dig> volcano plots.
expression data were plotted on a log <dig> scale  versus a -log <dig> transformation of the p-value .
datasets were filtered to remove genes with low expression levels , and a significance cut off  was applied .
zt <dig> ; zt <dig> ; zt <dig> ; zt <dig> ; zt <dig> ; and zt <dig> .
arrows indicate the highest expression in zt <dig>  and the highest confidence in zt <dig>
in this study, we found  <dig> genes that were differentially expressed in soybean plants in response to moderate water deficit during a 24-h time course .
these genes represented  <dig>  % of the  <dig>  predicted genes in the glyma  <dig>  soybean genome assembly  <cit> .
however, considering that some genes  were either up- or down- regulated during multiple time periods,  <dig> genes  were uniquely expressed in response to water deficit.
the number of genes exclusively expressed in each time period and those observed in multiple ones are presented in fig.
<dig> and in additional file  <dig>  most of the degs were identified in the early and late morning periods .
approximately  <dig>  %  and 53 %  were expressed exclusively in one period, whereas others were detected in both periods  .
fewer genes were differentially expressed in zt <dig>  and zt <dig>  in response to water deficit .
however, most of these genes were exclusively detected in zt <dig>  and zt <dig> , respectively .
similarly, in zt <dig>  ,  <dig>  % of the genes  were found only in this time period .
at pre-dawn ,  <dig> genes  were identified as differentially expressed , from which  <dig> up-  and  <dig> down-regulated  genes were observed exclusively in this time period .fig.
<dig> edwards’s diagram.
number of genes that were differentially expressed in each time period or in more than one period.
zt <dig> ; zt <dig> ; zt <dig> ; zt <dig> ; zt <dig> ; and zt <dig>
in general, genes differentially expressed in zt0–zt <dig> kept their expression profiles during both time periods, either increasing  or decreasing  their differential expression levels from early to late morning .
three up-regulated genes that were detected during morning  also exhibited decreased differential expression  or changed their profiles  at the late day  .
we found  <dig> genes identified as differentially expressed in both zt <dig>  and zt <dig>  .
dynamic changes in expression were not observed for these genes, with the exception of glyma11g <dig>  which codes for an oxidative stress  <dig> protein that was up-regulated in zt <dig> but down-regulated in zt <dig> .fig.
<dig> genes differentially expressed in different time periods with a diverse expression pattern.
gene expression was analyzed with edger statistical test to determine a ratio of expression  between control and drought-stressed plants.
the y-axis represents the fold-change value.
all data shown are statistically significant
eighty other genes were expressed in early and late morning  and again in zt <dig>  equivalent gene expression profiles were observed at these time periods, as exemplified by glyma03g <dig>  -related, glyma05g <dig> , glyma06g <dig> , glyma06g <dig> , glyma06g <dig>  glyma06g <dig>  glyma14g <dig> , and glyma17g <dig>  whose expression peaks reached their highest induced levels in zt <dig> .
alternatively, glyma15g <dig>  which codes for a 9-cis-epoxycarotenoid dioxygenase  <dig>  enzyme, exhibited a decreasing expression pattern between the light and dark periods , although it maintained its up-regulated profile.
glyma17g <dig> , glyma19g <dig> , and glyma02g <dig> were up-regulated at midday  but down-regulated in zt <dig> .
no genes in common were detected for all time periods; however glyma08g <dig> and glyma20g <dig> were up-regulated in five out of the six sampling times, the exception being the zt <dig> period .
interestingly, the expression levels of these two genes were higher in zt <dig> and zt4compared to the other periods .
similar expression profiles were observed for glyma01g <dig> , glyma03g <dig> , and glyma19g <dig>  in zt0 − zt4 − zt16 − zt <dig> .
during these same time periods, we found seven other genes that were up- or down-regulated.
however, no contrasting changes were observed among these periods.
some genes  were down-regulated in zt <dig> and up-regulated in zt <dig>  a transition light–dark period .
likewise, the transcript glyma04g <dig>  identified in zt0–zt8–zt <dig> was down-regulated  in the early morning, up-regulated later during the day , and down-regulated at the end of the dark period  , showing oscillating expression during the day.
in addition, an important water-deficit related gene  was dynamically up-regulated over time , showing higher differential expression levels in zt <dig> and zt <dig> periods compared to zt <dig> and zt <dig> .
a list comparing all genes identified as differentially expressed in all time periods is presented in additional file  <dig>
functional roles of differentially expressed soybean genes in response to water deficit
gene ontology  terms were associated with the degs to assess their putative biological roles.
we performed an enrichment analysis of such terms comparing the list of degs identified in each time point with the annotation of the entire soybean genome .
initially, we found a set of  <dig> go terms enriched among time periods , including biological process, molecular function and cellular component terms.
aiming to summarize the go terms obtained, the resulting lists were analyzed by revigo method  <cit>  to remove redundant go terms.
approximately 50 % of the processes expressed by soybean plants under water deficit were enriched in zt <dig>  processes including the regulation of biological processes, transcription, and transcription factor activity were up-regulated in zt <dig>  whereas, among the down-regulated genes, the enriched processes were primarily represented by translation and metabolic processes .
likewise, the same processes observed in both down- and up-regulated genes in zt <dig>  among others, were enriched in zt <dig> .
in the zt <dig> time period, there were no enriched processes for down-regulated genes, but dna binding and processes related to cellular component organization were significantly represented among up-regulated genes .
lipid metabolic processes were enriched in zt <dig>  while translation and structural molecular activity were down-regulated .
in zt <dig>  no enriched processes were detected for up-regulated genes, and only processes related to cellular-component terms were detected among down-regulated genes .
interestingly, for genes expressed in zt <dig> , we observed that transcription factor activity and dna metabolism were the only two significant processes enriched in  <dig>  % of the annotated genes, the same processes enriched during light periods .
all enriched processes observed during the 24-h time course are described in additional file  <dig> fig.
<dig> enrichment analyses of functional roles.
genes were associated with gene ontology terms  and compared to the soybean genome  using agrigo and revigo.
main enriched processes are presented for each time period.
red bars : genes present in soybean genome; blue bars : differentially expressed genes
diel oscillations in the expression of soybean genes under water deficit
expression data  from control plants and those under water deficit were collected separately to determine the putative rhythmicity of gene expression.
rhythmic waveforms were detected for  <dig>  and  <dig>  genes in control and water deficit plants , respectively, which correspond to  <dig>  % and  <dig>  % of the whole genome  .
as expected, functional classes were similar in both the control and treated plants since they shared the majority of the genes .
most were organized into protein  and rna  classes.
few differences were observed in classes including transport and signaling between the control  and drought-stressed plants  .fig.
<dig> functional classification of genes with oscillating expression in control and stressed plants.
reads per kilobase per million  values were analyzed using the jtk cycle algorithm to detect cycling waveforms of gene expression.
the number of genes detected in each class was normalized using the total number of genes of the stress dataset.
of the total number of genes,  <dig>  %  and  <dig>  %  were attributed to hypothetical pathways by associating the similarity of motifs and domains with other plant genes described in various databases .
approximately 35 % of the genes were not assigned to a pathway for both the control and stressed plants.
a total genes detected in each control  and stressed plants .
b genes found exclusively in the control  and stress conditions
we also identified groups of genes that showed expression fluctuations exclusively in control  or water deficit  conditions .
under normal water supply, genes expressed in a time-of-day-dependent manner played a role in several plant biological processes, particularly in the rna  and protein  classes.
interestingly, genes associated with stress response  were detected only in the control plants .
most of those genes are involved in the response to biotic stress  and heat stress .
similarly, expression of genes related to redox regulation metabolism  was observed in the control plants .
the functionality of genes detected only in the water deficit condition was significantly increased  in signaling , rna , and protein  categories.
additionally, genes involved in hormone , amino acid , and lipid  metabolism also were more highly represented .
after subjecting plants to water deficit, genes from the stress response class increased by  <dig>  % and were particularly associated with cold , pr-proteins , and light , among others  .
to assess the association levels of the expression datasets  between time periods, a matrix of similarity was calculated using the pearson correlation coefficient .
positive correlations were observed for all comparisons in the control and water deficit sets of genes at the  <dig>  p-value.
in the control plants, major differences were identified in the groups zt0 − zt <dig> , zt4 − zt <dig> , zt8 − zt <dig> , and zt8 − zt <dig> .
the main dissimilarities between the water deficit treated groups occurred in the zt0 − zt <dig> , zt4 − zt <dig> , zt8 − zt <dig> , and zt8 − zt <dig>  time periods.
although expression for these sets of genes varied in plants under normal water availability, these groups decreased similarity under stress, indicating that the applied water deficit influenced the expression of those genes.
additionally, in the stressed group, the zt0 − zt <dig> , zt0 − zt <dig> , zt4 − zt <dig> , zt4 − zt <dig> , and zt12 − zt <dig>  comparisons showed significant changes in the associations between different time periods .fig.
<dig> pearson’s correlation matrix.
gene expression data  from the  control and  stress groups were individually analyzed through pairwise comparisons to assess the similarities and dissimilarities among time periods.
the matrices of scatterplots indicate the association, correlation, and p-value  of each comparison
considering the  <dig>  genes shared between the control and stress conditions,  <dig> were specific for the stress response , and, of those,  <dig> were responsive to abiotic stress, primarily heat and drought/salt stresses .
this group of genes was distinct from those exclusively observed in the control or stress treatments.
although many genes specifically expressed in response to stress were detected in both control and stressed plants, it is important to emphasize that these results represent gene expression data derived from rpkm values.
since such genes appeared exclusively in control or stressed plants, the ratio of differential gene expression could not be analyzed using the software package edger; thus, it was not possible to infer about their up- or down-regulation under water deficit condition.
according to the rhythmicity analysis of the  <dig>  common genes,  <dig>  % of the genes under water deficit shortened their predicted period of expression  from  <dig> to 20 h  or from  <dig> to 22 h  .
similarly,  <dig>  % of the common genes also shifted their phase  under water deficit.
most  advanced their expression phase under stress condition, modifying their peak from  <dig> to 22 h  to 0 h  .
conversely, a set of  <dig> genes delayed the lag phase, showing their expression peak at 20 − 22 h in stress conditions, rather than at 0 h  .
under water deficit,  <dig> genes  reduced their expression amplitude , whereas  <dig>  increased it .
some genes that showed oscillating expression were plotted on graphs to observe such patterns during the day .
glyma07g <dig>  and glyma16g <dig>  were not expressed in response to water deficit , but their expression was shaped by time of day in control and stressed plants .
similarly, glyma13g <dig>  also exhibited significant oscillations in both control and stressed plants, although it was induced during zt <dig> .
for all three genes, the per was maintained in stressed plants, although the phase advanced and amp was shifted, in general .
oscillation patterns for genes differentially expressed during the light periods were observed for glyma06g <dig> , which exhibited notable increased expression from zt <dig>  to zt <dig>  , and for glyma12g <dig> , whose oscillation was observed specifically in control plants .
glyma04g <dig>  showed differential expression at midday , in zt <dig>  and in the dark period at zt <dig>  .
interestingly, the expression peaks observed in these time periods were identified as differential in response to water deficit.
however, overall, each control and stressed plant showed significant contrasting expression profiles .
glyma12g <dig>  was up-regulated during midday  and pre-dawn periods , however, the diel fluctuations of expression were significant only in control plants .
according to the rhythmicity analysis, glyma08g <dig>  and glyma15g <dig>  showed similar expression profiles, peaking in zt <dig> and zt <dig> exclusively in stress condition  with ratios from  <dig> to  <dig> times higher .
interestingly, glyma01g <dig>  showed a different profile under water deficit condition, with peak expression in zt <dig>  however, in control condition peak expression was observed in zt <dig>  levels that resulted in a ratio of  <dig>  fc .
the per  and phase  of this oscillating expression were maintained after plants became drought-stressed, but amp was increased from approximately  <dig>   to  <dig>   .fig.
<dig> oscillations of expression of genes observed in soybean response to water deficit stress.
waveforms were detected through rhythmicity analysis using the jtk cycle algorithm.
a
glyma07g <dig>  coding for germin-like protein 1; b
glyma16g <dig>  germin 3; c
glyma13g <dig>  germin-like protein 1; d
glyma06g <dig>  burp domain-containing protein; e
glyma12g <dig>  burp domain-containing protein; f
glyma04g <dig>  burp domain-containing protein; g
glyma12g <dig>  burp domain-containing protein; h
glyma08g <dig>  9-cis-epoxycarotenoid dioxygenase 5; i
glyma15g <dig>  9-cis-epoxycarotenoid dioxygenase 3; j
glyma01g <dig>  9-cis-epoxycarotenoid dioxygenase 4
in accordance with our goals, we compared the set of genes that were differentially expressed  in response to water deficit  to genes with putative cycling expression in either control or drought-stressed plants .
among the  <dig> degs in response to water deficit,  <dig>  were observed with oscillating expression in at least one analyzed condition.
in zt <dig>  a large set of differentially expressed genes was composed of more down-  than up-regulated genes  .
in zt <dig>  we observed the opposite expression pattern, with  <dig> up- and  <dig> down-regulated genes .
during the zt <dig>  and zt <dig> , oscillations in gene expression occurred predominantly for up-regulated genes .
in the midnight period , the pattern was similar to the pattern from early morning , showing more down-  than up-regulated genes  .
conversely, there was a balanced set of genes in the pre-dawn  period, with  <dig> up- and  <dig> down-regulated genes .
regarding the zt0 − zt <dig> periods,  <dig> genes overlapped within zt0 − zt <dig>  however, approximately  <dig>  % of the genes from zt <dig> were uniquely expressed.
few genes exhibited overlapped expression within zt12 − zt <dig>  and zt16 − zt <dig> .
no genes were found between zt <dig> and zt <dig>  of the  <dig> expressed genes identified in common to both the light and dark periods,  <dig> were expressed in the early morning  and also detected at the pre-daw period .
all degs that exhibited oscillating expression  were mapped to the main pathways involved in plant response to stress .
genes expressed in zt <dig> were included in many pathways, including signaling and cell wall metabolism, that were repressed in response to water deficit during this time period.
conversely, some genes from the abiotic stress class were highly induced in a similar manner to hormone  signaling pathways and heat-shock proteins  .
the jasmonate pathway was also induced, as indicated by the expression of three genes related to lipoxygenase , allene oxide synthase , and 12-oxo-pda-reductase .
the jasmonate pathway remained induced in zt <dig> but was represented by two other genes encoding allene oxide synthase  and 12-oxo-pda-reductase .
glyma07g <dig>  which was moderately expressed in the early morning , also was highly up-regulated during pre-dawn  .
according to these results, some genes detected in a specific pathway were not expressed during the entire diel period, but genes encoding different proteins seem to be involved in the same stress-related pathways.
the expression of genes related to pr-proteins is another example of how different genes are involved in maintaining increased or decreased metabolic activity, since most genes were distinct in each time period, and only one down-regulated gene at dawn   was positively regulated in zt <dig> .
all other genes related to pr-proteins in zt <dig> were exclusively detected at this time period .
in the abiotic stress class , genes were predominantly up-regulated .
similar profiles were observed for redox-state metabolism; hsps; aba and other genes involved in hormone signaling; the transcription factors myb, wrky, dof, and erf; and for the class of secondary metabolites.
because the sets of degs were smaller in the zt <dig> and zt <dig> time periods, the profiles observed for the metabolic pathways were under-represented.
in zt <dig>  few genes related to signaling and hormone signaling  were up-regulated.
conversely, such signaling and hormone signaling  processes became repressed in zt <dig>  although many genes were down-regulated during pre-dawn , up-regulated ones were identified as involved in the processes of secondary metabolism, transcription factors, aba and ethylene hormone signaling, and redox-state metabolism .fig.
<dig> functional roles triggered in soybean plants under water deficit stress.
genes that were differentially expressed in response to water deficit and that exhibited oscillations during the time periods analyzed were mapped to specific stress-related pathways.
the color scale shows the log <dig> fold change: red = up-regulated and blue = down-regulated
validation of gene expression
expression of genes related to proteins involved in plants’ responses to water deficit stress as the remorin , gols , dreb <dig> , rab <dig>  and bzip  were analyzed by the method 2^-.
a ratio of expression  was calculated by dividing the expression detected in drought-stressed plants by the one observed in control .
the gene-coding for rab <dig> presented the highest fold-change in both zt <dig> and zt <dig> time-periods .
in general, linear equation demonstrated a good correlation between both experiments.
genes used in this analysis represent a subset from those evaluated in marcolino’s et al.
<cit>  study.fig.
<dig> validation of gene expression.
relative expression of the glyma19g <dig> , glyma19g <dig> , glyma14g <dig> , glyma09g <dig>  and glyma02g <dig>  was measured using the method 2^- in  control and drought-stressed soybean plants at specific time-periods.
glyma13g <dig>  and glyma15g <dig>  were used as endogenous genes.
b a correlation between rna-seq and qpcr data is shown
discussion
the biological processes of plants are coordinated with physical and biochemical reactions, such as water intake, gene expression control, protein synthesis, and post-translational modification.
receptors in cell membranes communicate adverse signals from the surrounding environment to synchronize metabolic processes  <cit> .
to protect themselves, plants under water deficit change their normal cellular activities, such as movement, secretion, enzyme activity, and gene expression.
in arabidopsis, transcriptome reconfiguration in response to drought is associated with distinct hormonal and stress response pathways induced at different times of the day  <cit> .
soybean genes modulating responses to water deficit stress were expressed in different time periods during the course of 24 h .
whereas many genes involved in translation and bioenergetic processes were repressed in most time periods, enriched processes, such as gene expression and transcription factor activity/dna binding, were predominantly up-regulated until late day  and were detected again during the pre-dawn period.
most of these transcription factors  are directly involved in plant responses to water deficit.
studies have demonstrated the circadian rhythm control of gene expression for different plant species .
in arabidopsis, the number of genes under circadian regulation has been estimated to be hundreds  <cit>  to thousands regulating the control of auxin signaling  <cit> .
covington et al.
<cit>  estimated that one-third of the expressed arabidopsis genes are circadian clock-controlled.
in maize, 10 % of the  <dig>  analyzed transcripts showed circadian pattern expression  <cit> .
our results suggest that 35 % of the soybean genome  showed oscillating expression in a diel period in plants growing under normal availability of water .
seventy five percent of those genes also were detected in plants under water deficit, many of them shifted their per or phase in such adverse condition .
hsu and harmer  <cit>  demonstrated that differences in gene expression, even small, are associated with changes in phase and can influence expression.
additionally, dissimilarities observed in the set of expressed genes between some light/dark periods  reinforce the evidence of daily fluctuations in gene expression in soybean plants growing under normal conditions.
transcriptome changes observed for populus submitted to drought depended on the time of day at which they were measured  <cit> .
similarly, in arabidopsis plants, the interaction with diurnal regulation was predominant in modulating the transcriptome responses to cold stress  <cit> .
taken together, these data indicate that the time of day might play an important role in regulating the expression of many soybean genes in both normal and water deficit stress condition.
gene expression changes dynamically during the course of normal development and in response to other organisms, physical damage, or adverse environmental conditions.
such behavior was observed in the expression profiles of nced enzymes , both rate-limiting components in aba biosynthesis from the cleavage of carotenoids.
in addition to the role that this phytohormone plays in normal development, aba content is also increased in response to water deficit, preserving cell water content due to stomatal closure.
in arabidopsis, nced <dig> acts in association with nced <dig> to synthesize aba under normal conditions and in response to stress conditions  <cit> .
genes related to aba biosynthesis  have been linked to circadian regulation  <cit> .
interestingly, genes for nced <dig> and nced <dig>  showed higher expression levels compared to nced genes.
however, the two exhibited similar expression peaks at zt0 − zt <dig> under stress condition .
the gene for nced <dig>  also was detected in the light period, but its waveforms were distinct for control  and stress conditions .
such changes in oscillation can be attributed to delayed amplitude under stress condition since the period and phase remained unchanged .
although the diurnal fluctuation of aba levels in tobacco plants has been implicated to occur at the end of a light period  <cit> , as detected to the expression of nced <dig> gene  in plants under normal water supply, such oscillation appeared to be limited to light under stress condition , the period that stomatal closure is needed to avoid water loss by evapotranspiration process.
similarly, the gene coding for the oxidative stress  <dig>  protein  exhibited up-regulation in response to light  but not dark  .
this oxs <dig> protein is related to cadmium ion tolerance, as demonstrated in mutants of atoxs <dig> that were unable to enhance stress tolerance  <cit> .
the authors have also associated the atoxs <dig> gene with regulation by light, suggesting a putative role for this protein in protecting the cell against photooxidation  <cit> .
we found eight paralogs in the soybean genome that were related to the arabidopsis oxs <dig> ; three of them were identified in this study as differentially expressed in response to water deficit.
in addition to glyma11g <dig> reported above, glyma01g <dig> and its paralog glyma07g <dig> also were up-regulated in zt <dig> .
although we do not have information about the functional role of those proteins in the water deficit stress scenario, it is tempting to speculate on their importance in the soybean transcriptome since drought events can produce oxidative stress in the plants  <cit> .
additionally, glyma11g <dig> showed a cycling pattern when expressed in normal condition  , suggesting that its expression can be influenced by time of day.
under water deficit, it was observed that genes were predominantly down-regulated at dawn  .
the expression levels of  <dig> down-regulated genes in zt <dig> did not increase at midday  .
some of these genes that exhibited down-regulated profiles in zt <dig> are involved in basal metabolism, such as protein synthesis and dna metabolism.
in addition, the carbohydrate metabolic process , down-regulated at dawn and midday, represented a significant change in plant bioenergetics metabolism by reducing the synthesis of organic compounds and the breakdown of carbohydrates.
this suggests that during these time periods, energetically expensive processes are being partially arrested, and energy resources are being redirected to activate protective mechanisms.
according to fraire-velázquez and balderas-hernández  <cit> , the optimization of cellular energy resources during stress is essential for plant acclimation.
dhaubhadel et al.
<cit>  reported the accumulation of hsps in brassica napus seedlings under heat stress.
this significant accumulation resulted from higher hsp synthesis even when the mrna levels were lower in treated seedlings compared to controls.
such regulation mechanisms might act under post-transcriptional control, suggesting an advantageous ability of the plant machinery to save energy or drive it to maintain the translational apparatus during stress events.
in our study, the genes encoding germin and germin-like proteins are examples of genes that were repressed in early morning until midday .
germin has been associated with many processes important for plant development and defense .
in the soybean genome,  <dig> genes annotated as germin or germin-like proteins are associated with the cupin superfamily, which includes a variety of enzymes and non-enzymatic seed-storage proteins.
we detected the paralogous glyma07g <dig> and glyma16g <dig> down-regulated in both zt <dig> and zt <dig>  which are associated, by suggestive evidence, with nutrient-reservoir activity.
conversely, glyma13g <dig>  and glyma01g <dig>  were up-regulated under water deficit  .
germin-like protein genes  exhibited similar oscillation profiles with respect to control plants since they all showed expression peaks in zt <dig>  in general, under stress, genes advanced phases and shifted amplitude.
the results obtained for glyma13g <dig>  indicate that this gene is involved in the response to water deficit stress but also indicate that different germin-like protein genes expressed in soybean growth and development might oscillate levels with the time of day.
the diverse expression patterns of germin-like protein genes during soybean development also were reported by lu et al.
<cit> .
their study demonstrated that these genes are involved in enhancement of salt tolerance, and they exhibit expression fluctuations in darkness, suggesting a circadian clock feature  <cit> .
in arabidopsis, the ortholog  of glyma16g <dig> as well as glyma07g <dig>  as other germin-like proteins , are associated with the extracellular matrix, hypothetically acting in developmental processes or stress responses  <cit> .
additionally, the circadian regulation for atger <dig>  a germin-like cell wall protein from arabidopsis, seems to occur at the beginning of the night  <cit> .
in sinapis alba, a long-day plant from the same family of arabidopsis, circadian oscillation was associated with a transcript that encodes a germin-like protein that exhibits a transcription peak during dark periods   <cit> .
likewise, circadian regulation also was suggested for the germin-like protein  <dig>  in arabidopsis  <cit> .
conversely, genes that were positively regulated in response to water deficit, such as the burp domain-containing protein , were detected in various time periods .
this class of protein contains a conserved domain found in diverse plants and is putatively involved in the localization of proteins within the cell wall matrix through association with a structural domain that might target sites for intermolecular interaction  <cit> .
although the function of many burp proteins is unknown, specific elements have been characterized, and the functional role of these proteins is associated with normal plant metabolic processes, such as seed development  <cit> .
moreover, the genes encoding the burp domain-containing proteins also are involved in rice responses to abiotic stresses  <cit> .
some members of the rice burp family are responsive to cold, aba, and drought and salt stresses and can be induced by a single stress condition or combination of treatments.
this family also exhibits temporal and spatial expression pattern differences  <cit> .
in soybean plants, the burp family contains  <dig> genes that have been classified into five subfamilies .
although these genes possess no tissue specificity, they are expressed in response to stress.
in particular, genes from the rd <dig> subfamily are the most responsive to aba, peg treatments, and salt stress  <cit> .
for the gmrd <dig> protein , the burp domain seems to have an important role for determining its apoplast localization  <cit> .
furthermore, this protein-coding gene  exhibits potential responses to salt and osmotic stresses  <cit> .
in this study, we identified four up-regulated burp genes from the classes previously determined by xu et al.
<cit> : rd <dig> , usp , and glyma12g <dig> .
expression levels exhibited by these genes indicate the responsiveness of burp-domain-containing proteins to water deficit, and the oscillation patterns detected in plants under normal development show the regulation of gene expression also might be influenced by time of the day.
the degs that showed oscillating expression in control or drought-stressed conditions  played functional roles as regulatory genes in hormone signaling, cell communication, and abiotic stress-related pathways .
few genes involved in biotic stress responses  also were down- or up-regulated during this time.
genes responsive to multiple stresses are often detected in plants under adverse conditions, since a common set of biological processes triggered by genes induced in both events converge on similar downstream responses  <cit> .
our results showed that several hormone signaling genes, including genes related to jasmonate hormone metabolism, were observed in the early and late morning , and redox reactions were up-regulated in most of the time periods .
increased hormone signaling and fluctuations in the cellular redox status have been associated with plant responses to stress, and, in addition, circadian regulation also has been implicated in these processes  <cit> .
with respect to the classification of functional roles assigned to degs, a similar fraction of genes down- and up-regulated in the same class can be observed.
for instance, glyma07g <dig>  and glyma16g <dig> , both coding for the same type of protein, showed distinct expression patterns at different time periods.
evaluation of gene expression performed at a single time point during the day can only provide information about the plant’s responsiveness to drought during that specific period in which plants were sampled.
in this context, soybean gene networks induced in response to stress and modulated over a diel period appear to be a significant feature in the acclimation process, and exploring these interactions might provide novel insight into how plants respond to water deficit.
CONCLUSIONS
changes in the gene expression profile of soybean leaves triggered in response to water deficit stress were dynamically modulated in the diel period.
such results demonstrated the importance of analyzing different time periods to characterize plant responses to stress.
analysis of rhythmicity indicates that many putative cycling genes are expressed in soybean leaves under normal development.
when plants became stressed, a large number of the cycling genes found in the control plants showed a different rhythmic pattern.
in addition, other genes showed fluctuations under stress conditions.
genes that were differentially expressed in more than one time period and whose expression oscillated during the course of the day provide evidence suggesting that time of day contributes to regulation of stress responses in soybean.

 soybean is one of the most important crops in the world. overall yield is highly affected by water deficit stress, particularly when the stress occurs during flowering and early pod expansion. to overcome the water limitation and facilitate the continued expansion of soybean productivity and crop improvement, implementation of modern biotechnology such as genetic engineering of plants to produce drought-tolerant cultivars, rises as a potential solution. however, the achievement of such a goal is highly dependent on the elucidation of the molecular mechanisms of drought tolerance and their interaction with environmental cues. a better understanding of these aspects would help identify candidate genes for genetic engineering of improved stress-tolerant crops. to better fit to the surrounding environmental conditions, such as season and light/darkness and temperature variations, it is known that plants, as sessile organisms, coordinate and regulate their metabolism and physiology through an endogenous circadian clock. this clock drives rhythms at the molecular and cellular levels and, thus, temporally regulates plant physiology and behavior to anticipate changes in the environment. according to khan et al., the consequence of proper clock and environment synchronization is optimized fitness. however, abiotic stresses, such as drought, change the clock synchrony, altering the circadian rhythm in response to dehydration. genome-wide analysis of mrna expression shows daily oscillation coordinated by circadian rhythms, which, in turn, regulate various biological processes, such as seed dormancy and germination, hormone metabolism, and grapevine fruit ripening, among other processes. gene expression regulated by the circadian rhythm also has been observed in some plant responses to abiotic stress. in the process of cold acclimation in arabidopsis, gene expression was affected by time of day, revealing an interaction between cold and diurnal regulation that drives transcriptome changes. furthermore, time of day also was an important cue for triggering changes in the populus transcriptome and in arabidopsis plants in response to soil drying. in soybean, evidence also suggests that the circadian rhythm plays a role in regulating genes involved in developing seeds. although soybean is one of the most studied crops using molecular biology tools, little is known about how daily oscillations of gene expression are affected by drought stress during the survival or acclimation response. the regulation of a proline-rich-protein gene, induced under drought and salt stresses in specific tissues of soybean seedlings, was demonstrated to be circadian-controlled. considering the dynamic changes of plant metabolism that occur to coordinate the daily variation in light and temperature, the evaluation of gene expression at different time periods of the day becomes valuable for identifying times during which key genes might be most influential in the defense response. in arabidospis, hormone-related genes, specifically the abscisic acid -responsive genes, are correlated with diurnal oscillations. the functional roles of some representatives, like the dehydrins class and the rd29a, or the cold-regulated cor15b/15a and the low temperature-induced lti may be related to responses to water deficit and low temperature, respectively. recently, our research team has demonstrated how drought impacts diurnal oscillation of both drought-responsive and circadian clock genes in soybean. drought stress induced marked reduction in gene expression levels of several circadian clock-like components, such as gmlcl1-, gmelf4-, and gmprr-like genes. the same conditions produced a phase advance of expression for the gmtoc1-, gmlux- and gmprr7-like genes. similarly, the daily oscillation pattern of the soybean drought-responsive genes dreb-, bzip-, gols-, rab18- and remorin-like changed significantly following plant exposure to water deficit. with the goal of detecting genome-wide transcriptome changes during the entire period that plants were exposed to moderate water deficit and if such variations occurred in a time-of-day-dependent manner, we analyzed multiple time points in a diel period. here, we present a survey of soybean genes expressed under stress and their daily oscillation waveforms during a 24-h time course. we also determined their abundance, and we suggest the putative biological roles of these differentially expressed genes.  genes differentially expressed in response to water deficit the expression pattern of soybean genotype br previously characterized as drought sensitive, was evaluated under normal and water deficit conditions, during a 24-h time course. to identify differentially expressed genes in response to water deficit treatment, we applied a stringent statistical test to determine whether genes were either up- or down-regulated compared to those of plants under optimal hydration conditions. the resulting ratio represented the fold-change for each gene. to avoid false positives and reliably identify the most significant changes in gene expression, only genes with fc ≤ - and ≥  were considered. we also applied a stringent statistical significance cutoff to improve confidence. there were larger sets of differentially expressed genes in zt and zt compared to the other periods. in zt the majority of genes were down-regulated under water deficit stress condition, this is in contrast to the other periods, in which genes were primarily up-regulated. in general, a moderate expression ratio was detected for most genes, but high fold-changes for some genes were observed in the zt and zt periods. the highest differential expression was detected in zt for glyma18g, which codes for a udp-glucosyl transferase 73b and for glyma10g, a cap protein. in zt the highest confidence levels were found for glyma03g a gene related to the fatty acid hydroxylase superfamily and for glyma17g with unknown function.fig. volcano plots. expression data were plotted on a log scale versus a -log transformation of the p-value. datasets were filtered to remove genes with low expression levels, and a significance cut off was applied. zt; zt; zt; zt; zt; and zt. arrows indicate the highest expression in zt and the highest confidence in zt in this study, we found genes that were differentially expressed in soybean plants in response to moderate water deficit during a 24-h time course. these genes represented  % of the predicted genes in the glyma soybean genome assembly. however, considering that some genes were either up- or down- regulated during multiple time periods, genes were uniquely expressed in response to water deficit. the number of genes exclusively expressed in each time period and those observed in multiple ones are presented in fig.  and in additional file most of the degs were identified in the early and late morning periods. approximately  % and 53 % were expressed exclusively in one period, whereas others were detected in both periods. fewer genes were differentially expressed in zt and zt in response to water deficit. however, most of these genes were exclusively detected in zt and zt, respectively. similarly, in zt,  % of the genes were found only in this time period. at pre-dawn, genes were identified as differentially expressed, from which up- and down-regulated genes were observed exclusively in this time period.fig. edwards’s diagram. number of genes that were differentially expressed in each time period or in more than one period. zt; zt; zt; zt; zt; and zt in general, genes differentially expressed in zt0–zt kept their expression profiles during both time periods, either increasing or decreasing their differential expression levels from early to late morning. three up-regulated genes that were detected during morning also exhibited decreased differential expression or changed their profiles at the late day. we found genes identified as differentially expressed in both zt and zt. dynamic changes in expression were not observed for these genes, with the exception of glyma11g which codes for an oxidative stress protein that was up-regulated in zt but down-regulated in zt.fig. genes differentially expressed in different time periods with a diverse expression pattern. gene expression was analyzed with edger statistical test to determine a ratio of expression between control and drought-stressed plants. the y-axis represents the fold-change value. all data shown are statistically significant eighty other genes were expressed in early and late morning and again in zt equivalent gene expression profiles were observed at these time periods, as exemplified by glyma03g -related, glyma05g, glyma06g, glyma06g, glyma06g glyma06g glyma14g, and glyma17g whose expression peaks reached their highest induced levels in zt. alternatively, glyma15g which codes for a 9-cis-epoxycarotenoid dioxygenase enzyme, exhibited a decreasing expression pattern between the light and dark periods, although it maintained its up-regulated profile. glyma17g, glyma19g, and glyma02g were up-regulated at midday but down-regulated in zt. no genes in common were detected for all time periods; however glyma08g and glyma20g were up-regulated in five out of the six sampling times, the exception being the zt period. interestingly, the expression levels of these two genes were higher in zt and zt4compared to the other periods. similar expression profiles were observed for glyma01g, glyma03g, and glyma19g in zt0 − zt4 − zt16 − zt. during these same time periods, we found seven other genes that were up- or down-regulated. however, no contrasting changes were observed among these periods. some genes were down-regulated in zt and up-regulated in zt a transition light–dark period. likewise, the transcript glyma04g identified in zt0–zt8–zt was down-regulated in the early morning, up-regulated later during the day, and down-regulated at the end of the dark period, showing oscillating expression during the day. in addition, an important water-deficit related gene was dynamically up-regulated over time, showing higher differential expression levels in zt and zt periods compared to zt and zt. a list comparing all genes identified as differentially expressed in all time periods is presented in additional file functional roles of differentially expressed soybean genes in response to water deficit gene ontology terms were associated with the degs to assess their putative biological roles. we performed an enrichment analysis of such terms comparing the list of degs identified in each time point with the annotation of the entire soybean genome. initially, we found a set of go terms enriched among time periods, including biological process, molecular function and cellular component terms. aiming to summarize the go terms obtained, the resulting lists were analyzed by revigo method to remove redundant go terms. approximately 50 % of the processes expressed by soybean plants under water deficit were enriched in zt processes including the regulation of biological processes, transcription, and transcription factor activity were up-regulated in zt whereas, among the down-regulated genes, the enriched processes were primarily represented by translation and metabolic processes. likewise, the same processes observed in both down- and up-regulated genes in zt among others, were enriched in zt. in the zt time period, there were no enriched processes for down-regulated genes, but dna binding and processes related to cellular component organization were significantly represented among up-regulated genes. lipid metabolic processes were enriched in zt while translation and structural molecular activity were down-regulated. in zt no enriched processes were detected for up-regulated genes, and only processes related to cellular-component terms were detected among down-regulated genes. interestingly, for genes expressed in zt, we observed that transcription factor activity and dna metabolism were the only two significant processes enriched in  % of the annotated genes, the same processes enriched during light periods. all enriched processes observed during the 24-h time course are described in additional file fig. enrichment analyses of functional roles. genes were associated with gene ontology terms and compared to the soybean genome using agrigo and revigo. main enriched processes are presented for each time period. red bars: genes present in soybean genome; blue bars: differentially expressed genes diel oscillations in the expression of soybean genes under water deficit expression data from control plants and those under water deficit were collected separately to determine the putative rhythmicity of gene expression. rhythmic waveforms were detected for and genes in control and water deficit plants, respectively, which correspond to  % and  % of the whole genome. as expected, functional classes were similar in both the control and treated plants since they shared the majority of the genes. most were organized into protein and rna classes. few differences were observed in classes including transport and signaling between the control and drought-stressed plants.fig. functional classification of genes with oscillating expression in control and stressed plants. reads per kilobase per million values were analyzed using the jtk cycle algorithm to detect cycling waveforms of gene expression. the number of genes detected in each class was normalized using the total number of genes of the stress dataset. of the total number of genes,  % and  % were attributed to hypothetical pathways by associating the similarity of motifs and domains with other plant genes described in various databases. approximately 35 % of the genes were not assigned to a pathway for both the control and stressed plants. a total genes detected in each control and stressed plants. b genes found exclusively in the control and stress conditions we also identified groups of genes that showed expression fluctuations exclusively in control or water deficit conditions. under normal water supply, genes expressed in a time-of-day-dependent manner played a role in several plant biological processes, particularly in the rna and protein classes. interestingly, genes associated with stress response were detected only in the control plants. most of those genes are involved in the response to biotic stress and heat stress. similarly, expression of genes related to redox regulation metabolism was observed in the control plants. the functionality of genes detected only in the water deficit condition was significantly increased in signaling, rna, and protein categories. additionally, genes involved in hormone, amino acid, and lipid metabolism also were more highly represented. after subjecting plants to water deficit, genes from the stress response class increased by  % and were particularly associated with cold, pr-proteins, and light, among others. to assess the association levels of the expression datasets between time periods, a matrix of similarity was calculated using the pearson correlation coefficient. positive correlations were observed for all comparisons in the control and water deficit sets of genes at the p-value. in the control plants, major differences were identified in the groups zt0 − zt, zt4 − zt, zt8 − zt, and zt8 − zt. the main dissimilarities between the water deficit treated groups occurred in the zt0 − zt, zt4 − zt, zt8 − zt, and zt8 − zt time periods. although expression for these sets of genes varied in plants under normal water availability, these groups decreased similarity under stress, indicating that the applied water deficit influenced the expression of those genes. additionally, in the stressed group, the zt0 − zt, zt0 − zt, zt4 − zt, zt4 − zt, and zt12 − zt comparisons showed significant changes in the associations between different time periods.fig. pearson’s correlation matrix. gene expression data from the control and stress groups were individually analyzed through pairwise comparisons to assess the similarities and dissimilarities among time periods. the matrices of scatterplots indicate the association, correlation, and p-value of each comparison considering the genes shared between the control and stress conditions, were specific for the stress response, and, of those, were responsive to abiotic stress, primarily heat and drought/salt stresses. this group of genes was distinct from those exclusively observed in the control or stress treatments. although many genes specifically expressed in response to stress were detected in both control and stressed plants, it is important to emphasize that these results represent gene expression data derived from rpkm values. since such genes appeared exclusively in control or stressed plants, the ratio of differential gene expression could not be analyzed using the software package edger; thus, it was not possible to infer about their up- or down-regulation under water deficit condition. according to the rhythmicity analysis of the common genes,  % of the genes under water deficit shortened their predicted period of expression from to 20 h or from to 22 h. similarly,  % of the common genes also shifted their phase under water deficit. most advanced their expression phase under stress condition, modifying their peak from to 22 h to 0 h. conversely, a set of genes delayed the lag phase, showing their expression peak at 20 − 22 h in stress conditions, rather than at 0 h. under water deficit, genes reduced their expression amplitude, whereas increased it. some genes that showed oscillating expression were plotted on graphs to observe such patterns during the day. glyma07g and glyma16g were not expressed in response to water deficit, but their expression was shaped by time of day in control and stressed plants. similarly, glyma13g also exhibited significant oscillations in both control and stressed plants, although it was induced during zt. for all three genes, the per was maintained in stressed plants, although the phase advanced and amp was shifted, in general. oscillation patterns for genes differentially expressed during the light periods were observed for glyma06g, which exhibited notable increased expression from zt to zt, and for glyma12g, whose oscillation was observed specifically in control plants. glyma04g showed differential expression at midday, in zt and in the dark period at zt. interestingly, the expression peaks observed in these time periods were identified as differential in response to water deficit. however, overall, each control and stressed plant showed significant contrasting expression profiles. glyma12g was up-regulated during midday and pre-dawn periods, however, the diel fluctuations of expression were significant only in control plants. according to the rhythmicity analysis, glyma08g and glyma15g showed similar expression profiles, peaking in zt and zt exclusively in stress condition with ratios from to times higher. interestingly, glyma01g showed a different profile under water deficit condition, with peak expression in zt however, in control condition peak expression was observed in zt levels that resulted in a ratio of  fc. the per and phase of this oscillating expression were maintained after plants became drought-stressed, but amp was increased from approximately to.fig. oscillations of expression of genes observed in soybean response to water deficit stress. waveforms were detected through rhythmicity analysis using the jtk cycle algorithm. a glyma07g coding for germin-like protein 1; b glyma16g germin 3; c glyma13g germin-like protein 1; d glyma06g burp domain-containing protein; e glyma12g burp domain-containing protein; f glyma04g burp domain-containing protein; g glyma12g burp domain-containing protein; h glyma08g 9-cis-epoxycarotenoid dioxygenase 5; i glyma15g 9-cis-epoxycarotenoid dioxygenase 3; j glyma01g 9-cis-epoxycarotenoid dioxygenase 4 in accordance with our goals, we compared the set of genes that were differentially expressed in response to water deficit to genes with putative cycling expression in either control or drought-stressed plants. among the degs in response to water deficit, were observed with oscillating expression in at least one analyzed condition. in zt a large set of differentially expressed genes was composed of more down- than up-regulated genes. in zt we observed the opposite expression pattern, with up- and down-regulated genes. during the zt and zt, oscillations in gene expression occurred predominantly for up-regulated genes. in the midnight period, the pattern was similar to the pattern from early morning, showing more down- than up-regulated genes. conversely, there was a balanced set of genes in the pre-dawn period, with up- and down-regulated genes. regarding the zt0 − zt periods, genes overlapped within zt0 − zt however, approximately  % of the genes from zt were uniquely expressed. few genes exhibited overlapped expression within zt12 − zt and zt16 − zt. no genes were found between zt and zt of the expressed genes identified in common to both the light and dark periods, were expressed in the early morning and also detected at the pre-daw period. all degs that exhibited oscillating expression were mapped to the main pathways involved in plant response to stress. genes expressed in zt were included in many pathways, including signaling and cell wall metabolism, that were repressed in response to water deficit during this time period. conversely, some genes from the abiotic stress class were highly induced in a similar manner to hormone signaling pathways and heat-shock proteins. the jasmonate pathway was also induced, as indicated by the expression of three genes related to lipoxygenase, allene oxide synthase, and 12-oxo-pda-reductase. the jasmonate pathway remained induced in zt but was represented by two other genes encoding allene oxide synthase and 12-oxo-pda-reductase. glyma07g which was moderately expressed in the early morning, also was highly up-regulated during pre-dawn. according to these results, some genes detected in a specific pathway were not expressed during the entire diel period, but genes encoding different proteins seem to be involved in the same stress-related pathways. the expression of genes related to pr-proteins is another example of how different genes are involved in maintaining increased or decreased metabolic activity, since most genes were distinct in each time period, and only one down-regulated gene at dawn was positively regulated in zt. all other genes related to pr-proteins in zt were exclusively detected at this time period. in the abiotic stress class, genes were predominantly up-regulated. similar profiles were observed for redox-state metabolism; hsps; aba and other genes involved in hormone signaling; the transcription factors myb, wrky, dof, and erf; and for the class of secondary metabolites. because the sets of degs were smaller in the zt and zt time periods, the profiles observed for the metabolic pathways were under-represented. in zt few genes related to signaling and hormone signaling were up-regulated. conversely, such signaling and hormone signaling processes became repressed in zt although many genes were down-regulated during pre-dawn, up-regulated ones were identified as involved in the processes of secondary metabolism, transcription factors, aba and ethylene hormone signaling, and redox-state metabolism.fig. functional roles triggered in soybean plants under water deficit stress. genes that were differentially expressed in response to water deficit and that exhibited oscillations during the time periods analyzed were mapped to specific stress-related pathways. the color scale shows the log fold change: red = up-regulated and blue = down-regulated validation of gene expression expression of genes related to proteins involved in plants’ responses to water deficit stress as the remorin, gols, dreb, rab and bzip were analyzed by the method 2^-. a ratio of expression was calculated by dividing the expression detected in drought-stressed plants by the one observed in control. the gene-coding for rab presented the highest fold-change in both zt and zt time-periods. in general, linear equation demonstrated a good correlation between both experiments. genes used in this analysis represent a subset from those evaluated in marcolino’s et al. study.fig. validation of gene expression. relative expression of the glyma19g, glyma19g, glyma14g, glyma09g and glyma02g was measured using the method 2^- in control and drought-stressed soybean plants at specific time-periods. glyma13g and glyma15g were used as endogenous genes. b a correlation between rna-seq and qpcr data is shown discussion the biological processes of plants are coordinated with physical and biochemical reactions, such as water intake, gene expression control, protein synthesis, and post-translational modification. receptors in cell membranes communicate adverse signals from the surrounding environment to synchronize metabolic processes. to protect themselves, plants under water deficit change their normal cellular activities, such as movement, secretion, enzyme activity, and gene expression. in arabidopsis, transcriptome reconfiguration in response to drought is associated with distinct hormonal and stress response pathways induced at different times of the day. soybean genes modulating responses to water deficit stress were expressed in different time periods during the course of 24 h. whereas many genes involved in translation and bioenergetic processes were repressed in most time periods, enriched processes, such as gene expression and transcription factor activity/dna binding, were predominantly up-regulated until late day and were detected again during the pre-dawn period. most of these transcription factors are directly involved in plant responses to water deficit. studies have demonstrated the circadian rhythm control of gene expression for different plant species. in arabidopsis, the number of genes under circadian regulation has been estimated to be hundreds to thousands regulating the control of auxin signaling. covington et al. estimated that one-third of the expressed arabidopsis genes are circadian clock-controlled. in maize, 10 % of the analyzed transcripts showed circadian pattern expression. our results suggest that 35 % of the soybean genome showed oscillating expression in a diel period in plants growing under normal availability of water. seventy five percent of those genes also were detected in plants under water deficit, many of them shifted their per or phase in such adverse condition. hsu and harmer demonstrated that differences in gene expression, even small, are associated with changes in phase and can influence expression. additionally, dissimilarities observed in the set of expressed genes between some light/dark periods reinforce the evidence of daily fluctuations in gene expression in soybean plants growing under normal conditions. transcriptome changes observed for populus submitted to drought depended on the time of day at which they were measured. similarly, in arabidopsis plants, the interaction with diurnal regulation was predominant in modulating the transcriptome responses to cold stress. taken together, these data indicate that the time of day might play an important role in regulating the expression of many soybean genes in both normal and water deficit stress condition. gene expression changes dynamically during the course of normal development and in response to other organisms, physical damage, or adverse environmental conditions. such behavior was observed in the expression profiles of nced enzymes, both rate-limiting components in aba biosynthesis from the cleavage of carotenoids. in addition to the role that this phytohormone plays in normal development, aba content is also increased in response to water deficit, preserving cell water content due to stomatal closure. in arabidopsis, nced acts in association with nced to synthesize aba under normal conditions and in response to stress conditions. genes related to aba biosynthesis have been linked to circadian regulation. interestingly, genes for nced and nced showed higher expression levels compared to nced genes. however, the two exhibited similar expression peaks at zt0 − zt under stress condition. the gene for nced also was detected in the light period, but its waveforms were distinct for control and stress conditions. such changes in oscillation can be attributed to delayed amplitude under stress condition since the period and phase remained unchanged. although the diurnal fluctuation of aba levels in tobacco plants has been implicated to occur at the end of a light period, as detected to the expression of nced gene in plants under normal water supply, such oscillation appeared to be limited to light under stress condition, the period that stomatal closure is needed to avoid water loss by evapotranspiration process. similarly, the gene coding for the oxidative stress protein exhibited up-regulation in response to light but not dark. this oxs protein is related to cadmium ion tolerance, as demonstrated in mutants of atoxs that were unable to enhance stress tolerance. the authors have also associated the atoxs gene with regulation by light, suggesting a putative role for this protein in protecting the cell against photooxidation. we found eight paralogs in the soybean genome that were related to the arabidopsis oxs; three of them were identified in this study as differentially expressed in response to water deficit. in addition to glyma11g reported above, glyma01g and its paralog glyma07g also were up-regulated in zt. although we do not have information about the functional role of those proteins in the water deficit stress scenario, it is tempting to speculate on their importance in the soybean transcriptome since drought events can produce oxidative stress in the plants. additionally, glyma11g showed a cycling pattern when expressed in normal condition, suggesting that its expression can be influenced by time of day. under water deficit, it was observed that genes were predominantly down-regulated at dawn. the expression levels of down-regulated genes in zt did not increase at midday. some of these genes that exhibited down-regulated profiles in zt are involved in basal metabolism, such as protein synthesis and dna metabolism. in addition, the carbohydrate metabolic process, down-regulated at dawn and midday, represented a significant change in plant bioenergetics metabolism by reducing the synthesis of organic compounds and the breakdown of carbohydrates. this suggests that during these time periods, energetically expensive processes are being partially arrested, and energy resources are being redirected to activate protective mechanisms. according to fraire-velázquez and balderas-hernández, the optimization of cellular energy resources during stress is essential for plant acclimation. dhaubhadel et al. reported the accumulation of hsps in brassica napus seedlings under heat stress. this significant accumulation resulted from higher hsp synthesis even when the mrna levels were lower in treated seedlings compared to controls. such regulation mechanisms might act under post-transcriptional control, suggesting an advantageous ability of the plant machinery to save energy or drive it to maintain the translational apparatus during stress events. in our study, the genes encoding germin and germin-like proteins are examples of genes that were repressed in early morning until midday. germin has been associated with many processes important for plant development and defense. in the soybean genome, genes annotated as germin or germin-like proteins are associated with the cupin superfamily, which includes a variety of enzymes and non-enzymatic seed-storage proteins. we detected the paralogous glyma07g and glyma16g down-regulated in both zt and zt which are associated, by suggestive evidence, with nutrient-reservoir activity. conversely, glyma13g and glyma01g were up-regulated under water deficit. germin-like protein genes exhibited similar oscillation profiles with respect to control plants since they all showed expression peaks in zt in general, under stress, genes advanced phases and shifted amplitude. the results obtained for glyma13g indicate that this gene is involved in the response to water deficit stress but also indicate that different germin-like protein genes expressed in soybean growth and development might oscillate levels with the time of day. the diverse expression patterns of germin-like protein genes during soybean development also were reported by lu et al.. their study demonstrated that these genes are involved in enhancement of salt tolerance, and they exhibit expression fluctuations in darkness, suggesting a circadian clock feature. in arabidopsis, the ortholog of glyma16g as well as glyma07g as other germin-like proteins, are associated with the extracellular matrix, hypothetically acting in developmental processes or stress responses. additionally, the circadian regulation for atger a germin-like cell wall protein from arabidopsis, seems to occur at the beginning of the night. in sinapis alba, a long-day plant from the same family of arabidopsis, circadian oscillation was associated with a transcript that encodes a germin-like protein that exhibits a transcription peak during dark periods. likewise, circadian regulation also was suggested for the germin-like protein in arabidopsis. conversely, genes that were positively regulated in response to water deficit, such as the burp domain-containing protein, were detected in various time periods. this class of protein contains a conserved domain found in diverse plants and is putatively involved in the localization of proteins within the cell wall matrix through association with a structural domain that might target sites for intermolecular interaction. although the function of many burp proteins is unknown, specific elements have been characterized, and the functional role of these proteins is associated with normal plant metabolic processes, such as seed development. moreover, the genes encoding the burp domain-containing proteins also are involved in rice responses to abiotic stresses. some members of the rice burp family are responsive to cold, aba, and drought and salt stresses and can be induced by a single stress condition or combination of treatments. this family also exhibits temporal and spatial expression pattern differences. in soybean plants, the burp family contains genes that have been classified into five subfamilies. although these genes possess no tissue specificity, they are expressed in response to stress. in particular, genes from the rd subfamily are the most responsive to aba, peg treatments, and salt stress. for the gmrd protein, the burp domain seems to have an important role for determining its apoplast localization. furthermore, this protein-coding gene exhibits potential responses to salt and osmotic stresses. in this study, we identified four up-regulated burp genes from the classes previously determined by xu et al.: rd, usp, and glyma12g. expression levels exhibited by these genes indicate the responsiveness of burp-domain-containing proteins to water deficit, and the oscillation patterns detected in plants under normal development show the regulation of gene expression also might be influenced by time of the day. the degs that showed oscillating expression in control or drought-stressed conditions played functional roles as regulatory genes in hormone signaling, cell communication, and abiotic stress-related pathways. few genes involved in biotic stress responses also were down- or up-regulated during this time. genes responsive to multiple stresses are often detected in plants under adverse conditions, since a common set of biological processes triggered by genes induced in both events converge on similar downstream responses. our results showed that several hormone signaling genes, including genes related to jasmonate hormone metabolism, were observed in the early and late morning, and redox reactions were up-regulated in most of the time periods. increased hormone signaling and fluctuations in the cellular redox status have been associated with plant responses to stress, and, in addition, circadian regulation also has been implicated in these processes. with respect to the classification of functional roles assigned to degs, a similar fraction of genes down- and up-regulated in the same class can be observed. for instance, glyma07g and glyma16g, both coding for the same type of protein, showed distinct expression patterns at different time periods. evaluation of gene expression performed at a single time point during the day can only provide information about the plant’s responsiveness to drought during that specific period in which plants were sampled. in this context, soybean gene networks induced in response to stress and modulated over a diel period appear to be a significant feature in the acclimation process, and exploring these interactions might provide novel insight into how plants respond to water deficit.  changes in the gene expression profile of soybean leaves triggered in response to water deficit stress were dynamically modulated in the diel period. such results demonstrated the importance of analyzing different time periods to characterize plant responses to stress. analysis of rhythmicity indicates that many putative cycling genes are expressed in soybean leaves under normal development. when plants became stressed, a large number of the cycling genes found in the control plants showed a different rhythmic pattern. in addition, other genes showed fluctuations under stress conditions. genes that were differentially expressed in more than one time period and whose expression oscillated during the course of the day provide evidence suggesting that time of day contributes to regulation of stress responses in soybean. 
we have identified putative cycling genes that are expressed in soybean leaves under normal developmental conditions and genes whose expression oscillates under conditions of water deficit. major differences in gene expression were observed in the control plants from late day until predawn periods, indicating that gene expression oscillates during the course of 24 h in normal development. these results suggest that time of day, as well as light and temperature oscillations that occur considerably affect the regulation of water deficit stress response in soybean plants. additionally, genes known to be involved in secondary metabolism and hormone signaling were also expressed in the dark period. since drought can seriously affect plant growth and development and little is known about how the oscillations of gene expression during the drought stress-acclimation response in soybean is affected, we applied illumina technology to sequence cdna libraries synthesized from control and drought-stressed soybean plants to verify the dynamic changes in gene expression during a 24-h time course. cycling variables were measured from the expression data to determine the putative circadian rhythm regulation of gene expression. under water deficit, dissimilarity increased in all time-periods, indicating that the applied stress influenced gene expression. gene expression networks can be dynamically shaped to acclimate plant metabolism under environmental stressful conditions. such differences in plants under stress were primarily observed in zt to zt and also from zt to zt stress-related pathways were triggered in response to water deficit primarily during midday, when more genes were up-regulated compared to early morning. we identified genes differentially expressed in soybean plants in response to water deficit. this number increased to  % when plants were submitted to water deficit. of predicted soybean genes,  % exhibited expression oscillations in a 24 h period. of these genes, were differentially expressed during the light period, from which approximately  % were observed in both light and darkness.
 since drought can seriously affect plant growth and development and little is known about how the oscillations of gene expression during the drought stress-acclimation response in soybean is affected, we applied illumina technology to sequence cdna libraries synthesized from control and drought-stressed soybean plants to verify the dynamic changes in gene expression during a 24-h time course. cycling variables were measured from the expression data to determine the putative circadian rhythm regulation of gene expression.  we identified genes differentially expressed in soybean plants in response to water deficit. of these genes, were differentially expressed during the light period, from which approximately  % were observed in both light and darkness. we found genes that were either up- or down-regulated in different periods of the day. of predicted soybean genes,  % exhibited expression oscillations in a 24 h period. this number increased to  % when plants were submitted to water deficit. major differences in gene expression were observed in the control plants from late day until predawn periods, indicating that gene expression oscillates during the course of 24 h in normal development. under water deficit, dissimilarity increased in all time-periods, indicating that the applied stress influenced gene expression. such differences in plants under stress were primarily observed in zt to zt and also from zt to zt stress-related pathways were triggered in response to water deficit primarily during midday, when more genes were up-regulated compared to early morning. additionally, genes known to be involved in secondary metabolism and hormone signaling were also expressed in the dark period.  gene expression networks can be dynamically shaped to acclimate plant metabolism under environmental stressful conditions. we have identified putative cycling genes that are expressed in soybean leaves under normal developmental conditions and genes whose expression oscillates under conditions of water deficit. these results suggest that time of day, as well as light and temperature oscillations that occur considerably affect the regulation of water deficit stress response in soybean plants. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords abiotic stressdaily oscillationdiel regulationdroughtglycine maxplant metabolismissue-copyright-statement© the author 2015 