BACKGROUND
drought is the most important abiotic stress in terms of limiting crop productivity worldwide.
water availability is, therefore, of primary importance for a non-limiting crop production in the current changing global climate scenario.
the slogan “more crop per drop”  <cit>  was the track for crop improvement in water limited environments aiming to address the growing demand for water, food and commodities  of the growing world population  <cit> .
among the c <dig> cereals, sorghum bicolor is the species most suited to environments that are prone to drought.
its tolerance to drought is a consequence of morphological and anatomical characteristics  and physiological responses   <cit> .
the high genetic variability among sorghum genotypes and the relatively small size of its genome make this cereal a good model for the identification of drought related genomic regions and genes valuable to unravel the high complexity of drought tolerance related traits  <cit> .
several sorghum linkage maps, including high density maps  <cit> , have been built using different types of dna markers  <cit> .
different genomic regions related to drought tolerance at pre-flowering and post-flowering stage were identified  <cit>  but it was the availability of the sorghum genome sequence  <cit>  that has enabled the monitoring of the genome-wide gene expression profile at a single time in response to several abiotic stresses through microarray or rna-seq analysis .
these studies resulted in the identification of drought stress responsive genes and their regulatory elements.
several transcriptomics studies were carried out on sorghum using rna-seq analysis to monitor gene expression in response to osmotic stress and abscisic acid  <cit> , to provide a s. bicolor expression atlas on the dynamic genotype-specific expression profiles  <cit> , or to identify genome-wide snps that can potentially enhance genetic analysis and the application of molecular markers in sorghum genomics and breeding  <cit> .
in addition to physiologic or agronomic approaches, genomics offer new opportunities for dissecting quantitative traits into their single determinants  paving the way to marker-assisted selection  or direct gene editing via genetic engineering  <cit> .
drought stress elicits a wide range of responses in plants  <cit> .
it increases oxidative damage in chloroplasts  <cit> , reduces photosynthesis , limits metabolic reactions  <cit> , triggers sugar catabolism, in order to provide osmotically active compound and signal molecules , and modifies cellular lipid composition  <cit> .
to cope with drought stress, plants have developed various strategies, such as generation of larger and deeper root systems  <cit> , regulation of stomatal closure to reduce water loss  <cit> , accumulation of compatible solutes and protective proteins  <cit> , and an increase in the level of antioxidants  <cit> .
identification of drought resistant traits was frequently labelled as “complex” although we already know the results of all the modifications adopted by plants to cope with drought stress  <cit> .
in this study we have furthered extended the knowledge on the drought response of two sorghum genotypes through transcriptomic analysis  <cit> .
a massive parallel sequencing of rna  on the illumina platform was used to provide a thorough scenario on the whole sorghum transcriptome in response to drought stress.
several categories of key genes involved in drought response have been identified.
RESULTS
physiological responses to drought stress
twenty sorghum plants  were subjected to severe drought stress by withholding water from  <dig> dae  until  <dig> dae when  <dig>  ftsw  was reached in all the stressed plants .
subsequently the stressed plants were kept at  <dig>  ftsw by irrigating daily for nine days, while the control plants were kept at ftsw values higher than  <dig>  for the entire duration of the experiment .
the daily transpired water  was under  <dig> gr for the stressed plant, while it was up to  <dig> gr for the control plants .fig.
<dig> trend of ftsw and daily transpired water during the dry-down experiment.
on the left axis with circles symbols the trend of ftsw during the dry-down: with full circles the ww plants and with the empty circles the ds ones.
on the right axis with triangles the daily transpired water: full triangles for the ww plants and empty triangles for the ds ones.
dae = days after emergence.
mean of  <dig> plants ± se
leaf area, chlorophyll fluorescence parameters  and gas exchange measurements  were quantified for the entire duration of the experiment .
the decreased ftsw led to a reduction in rwc  values and these changes were greater in the sensitive genotype is <dig> than in the tolerant genotype is <dig> .
drought stress also dramatically reduced chlorophyll fluorescence and photosynthetic rate.
under stress conditions the tolerant genotype is <dig> showed a significantly higher value of fv/fm than the sensitive genotype is <dig> .
the same trend was observed for Φpsii:  <dig>  and  <dig>  for the tolerant and the sensitive genotype, respectively.
in contrast, the qnp under drought stress was higher in the sensitive genotype is <dig> than in the tolerant genotype is <dig> .table  <dig> physiological responses of sorghum genotypes to drought stress
lsd
analysis of relative water content , chlorophyll fluorescence , gas exchange , intrinsic  and agronomic wue  in sorghum plants in well-watered  and drought stress  conditions at vegetative stage of 9th leaf.
values followed by the same letter are not statistically significant at lsd test p <  <dig>  performed on the interaction genotype× irrigation.
drought stress affected pn in both the genotypes differently; the sensitive genotype is <dig> had a greater reduction in pn  while the tolerant genotype is <dig> showed a pn reduction of  <dig>  %.
transpiration  did not differ between the ww  and ds  plants of the tolerant genotype is <dig>  while there was a statistically significant difference between the ww and ds plants of the sensitive genotype is <dig>  the intrinsic water use efficiency  decreased linearly for the ds plants of both genotypes from the beginning of the experiment  until harvest , while the ww plants kept their wuei close to 6 μmol mmol− <dig> .
wuei of ds plants of the tolerant genotype is <dig> was significantly higher than that of ds plants belonging to the sensitive genotype is <dig> during the stress period  .
the agronomic water use efficiency , calculated at harvest, was higher for the tolerant genotype is <dig>  than for the sensitive genotype is <dig> , thereby confirming the trend highlighted by wuei.fig.
<dig> trend of wuei calculated during the dry down experiment.
circles represent the sensitive genotype is <dig> and triangles the tolerant is <dig>  for both the genotypes the full symbols represents the ww plants whilst the empty symbols represent the ds ones.
mean of  <dig> plants ± se
drought stress reveals different intergenic transcripts and novel splice sites
transcription profiles of is <dig> and is <dig> under well-watered  and drought-stressed  conditions were explored using the illumina genome analyzer deep sequencing.
three biological replicates were analysed for each condition, resulting in twelve samples.
in total,  <dig>  billion clean reads, each  <dig> nucleotides long, were generated, with approximately  <dig> million clean reads from each sample.
the reads mapping to the reference genome were categorised into two classes: uniquely mapped reads, that are reads that map to only one position in the reference genome, and multi-position match, that are reads mapping to more than one position in the reference genome .
the assembled transcripts were mapped on the genome: on average 72 % were known transcripts, 10 % were novel transcripts and 18 % were intergenic transcripts .table  <dig> number of reads sequenced and mapped with soapaligner/soap2
the numbers of unique mapped reads plus the multi-position match equals the total number of mapped reads in well-watered  and drought stress  conditions
percentage of total mapped reads on the reference genome, percentage of match with known transcripts, with intergenic transcripts and novel transcripts identified, and alternative splicing events identified
drought stress induced alternative splicing events  in the two genotypes : in the sensitive genotype is <dig> no difference in ase were found, while in the tolerant genotype is <dig> the ase were increased by 18 %.
drought stress triggers differential expression of particular genes and go classes
each condition was represented by three biological replicates, resulting in eighteen pairwise comparisons between control and stressed plants of the two genotypes.
the transcript abundance of each gene was calculated as reads per kilobase transcriptome per million mapped reads  .
this value was used to determine the differential expression analysis as log <dig> ratio between ds and ww plants per genotype and between the two genotypes under ww and ds conditions.
four comparisons were analysed in this study: i) the genotypes is <dig> and is <dig> under ww conditions , ii) the genotypes is <dig> and is <dig> under ds conditions , iii) the genotype is <dig> in response to ds conditions , iv) the genotype is <dig> in response to ds conditions .fig.
<dig> comparison under study.
a number of degs  in each pairwise comparison.
blue and red bar are up- an down-regulated genes respectively expressed in well-watered  and drought stressed  conditions in the genotypes is <dig>  and is <dig> .
b total number of degs that passed the cut-off of log <dig> fc > <dig> in each comparison.
in yellow the number of degs resulting from the comparison between is <dig> and is <dig> in well-watered  conditions, in green the number of degs resulting from the comparison between the two genotypes under drought stress  conditions; in blue the numbers of degs in response to drought stress in is <dig> and in red the number of degs in response to drought stress in is <dig>  c venn diagram showing the numbers of up- and down- regulated genes resulted from the four comparison performed.
the number of up- or down- regulated genes shared among the four comparison is represented by overlapping circles
after applying a stringent cut-off , the comparison of genotypes is <dig> and is <dig> under ww conditions identified  <dig> differentially expressed genes , and the comparison of genotypes is <dig> and is <dig> under ds conditions identified  <dig> degs.
<dig> genes were differentially expressed in is <dig> in response to drought stress, whilst only  <dig> were differentially expressed in is <dig> .
venn diagrams highlight the overlap of degs between each pairwise comparison .
comparison between is <dig> and is <dig> under ww conditions  resulted in  <dig> up-regulated genes and  <dig> down-regulated genes.
only  <dig> genes were uniquely up- and  <dig> genes down-regulated in is <dig> in these conditions.
the singular enrichment analysis , carried out with agrigo software  on the  <dig> up-regulated genes, highlighted  <dig> go terms significantly enriched: “aromatic compound biosynthetic process” , “secondary metabolic process” , and “flavonoid biosynthetic process”  in the cellular processes category; “glutathione transferase activity” , “oxygen binding” , “udp-glucosyltransferase activity”  in molecular functions category .
“apoptosis”  and “oxidoreductase activity”  were the most enriched go terms in the biological processes and molecular function categories among the  <dig> uniquely down-regulated genes expressed in ww conditions in is <dig> .
the comparison between the two genotypes under ds conditions resulted in  <dig> up- and  <dig> down-regulated genes.
among these genes, only  <dig> and  <dig> were uniquely up- and down- regulated in the genotype is <dig> in comparison to is <dig>  “regulation of dna replication” , “cell death” , “regulation of cell growth by extracellular stimulus” , “secondary metabolic processes”  including “terpenoids biosynthetic process” , “glutathione transferase activity”  and “pre-replicative complex”   were the most enriched go terms among the  <dig> identified after sea of the  <dig> up-regulated genes.
among the  <dig> down-regulated genes  <dig> go terms were significantly enriched: “lipid localization” , “apoptosis” , “flavonol biosynthetic process” , “electron carrier activity”  and “heme binding”  .
the main difference between the two genotypes was in the total number of genes differentially expressed in response to drought stress:  <dig> for the sensitive is <dig> and  <dig> for the tolerant is <dig>  the sea analysis, performed on all the  <dig> and  <dig> degs expressed in response to drought in the genotypes is <dig> and is <dig>  showed  <dig> significantly enriched go terms  in the sensitive genotype is <dig> while  <dig> in the tolerant is <dig>  twenty go terms were enriched in both the genotypes in response to drought stress and are represented in the heat map .
“response to heat”, “rna modification”, “cytosolic part” and “ribosomal subunit” go terms were enriched with the same extent in both the genotypes.
different go enrichment was recorded between is <dig> and is <dig> for “oxidation-reduction process”, “response to abiotic stimulus”, “oxidoreductase activity”, “response to chemical stimulus”, “small molecule metabolic process”, “response to stress”, “chloroplast”, “single-organism metabolic process” and “cytoplasm component”.
all these go terms were more enriched in is <dig> than in is <dig> fig.
<dig> heat map showing the  <dig> common go terms enriched under drought stress in sorghum leaves of is <dig> and is <dig>  the cluster frequency was used as a parameter for the parametric analysis of gene enrichment analysis.
the figure was generated using r software, limma package
between the two genotypes there were  <dig> common up-regulated genes in response to drought stress and  <dig> common down-regulated genes .
the sea performed on these common degs highlighted  <dig> enriched go terms belonging to biological processes: “response to abscisic acid stimulus” , “response to water deprivation” , “photosynthesis, light reaction”  were the most enriched go .
the sea analysis performed with agrigo on the unique up-regulated genes of is20351and is <dig>  highlighted  <dig> enriched go terms in is <dig> and and  <dig> enriched go terms is <dig>  the cross comparison of sea  highlighted  <dig> common go terms .
the sea analysis performed on the unique down-regulated genes  highlighted  <dig> and  <dig> significantly enriched go terms in is <dig> and is <dig> respectively; after the cross comparison of sea only  <dig> resulted as being common to both genotypes .
drought stress affects different pathways
the kegg pathway analysis was performed to assign the related biological pathways in which degs were involved.
one-hundred and seventy-one genes, uniquely expressed in response to drought stress in both the genotypes, were assigned to  <dig> different kegg pathways belonging to  <dig> clades under five major kegg categories including ‘organismal system’ , ‘cellular process’ , ‘environmental information processing’ , ‘genetic information processing’ , and ‘metabolism’  .
gene-set enrichment analysis showed that translation, signal transduction and carbon metabolism were the top three up-regulated pathways represented by the genes uniquely expressed in response to drought stress; metabolism pathways  and signal transduction were, on the other hand, the most enriched down-regulated pathways .fig.
<dig> number of up- and down-regulated genes in each clade of the kegg pathway maps.
the  <dig> unigenes were assigned  <dig> kegg pathways within  <dig> clades under five major categories: “organismal systems” , “cellular processes” , “environmental information processing” , “genetic information processing” , “metabolism” .
per each clades are shown the up-  and the down-  regulated genes
kegg pathway analysis was also performed on the genes that were uniquely up- and down-regulated in response to drought stress in both genotypes .
transcription factors, ‘environmental information processing’ pathways, and pathways related to ‘cellular processes’ and ‘organismal system’ remained unchanged among the uniquely up-regulated genes .
the most striking differences in the transcriptomic profiles of the two genotypes in response to drought were mainly in the ‘metabolism’ pathways , in the ‘genetic information processing’ pathway  and in the number of genes not assigned to pathways .
focusing on the up-regulated ‘metabolism’ pathways, the tolerant genotype is <dig> showed a two-fold  enrichment in the metabolism of other amino acids, the nucleotide metabolism, the glycan biosynthesis metabolism and the lipid metabolism compared to the sensitive genotypes is <dig> .
amino acid metabolism, carbohydrate metabolism and energy metabolism were more enriched in the sensitive genotype is <dig> than in the tolerant genotype is <dig> .fig.
<dig> distribution in kegg pathways of the unique up- and down-regulated genes in response to drought for the genotype is <dig> and is <dig>  pie charts showing the percentage of genes up-  and down-  regulated in response to drought stress for the genotypes is <dig>  and is <dig>
the ‘metabolism’ pathways of is <dig> and is <dig> were down-regulated to the same degree in response to drought stress .
‘cellular processes’ pathways represented 4 % of the down-regulated genes in is <dig> and 2 % in is <dig> .
‘organismal system’ pathways, ‘genetic information processing’ pathways and transcription factors were down-regulated to a greater extent in the tolerant genotype is <dig> .
among the down-regulated ‘metabolism’ pathways, energy metabolism, nucleotide, cofactors and vitamins metabolism, glycan biosynthesis and metabolism, and carbohydrate metabolism pathways were down-regulated with a higher frequency in the sensitive genotype is <dig> than in the tolerant is <dig> .
drought stress response of sorghum transcriptome
the mapman software   <cit>  was used to show a pathway overview of  <dig> and  <dig> degs expressed in response to drought stress and it was selected for its capacity to show statistically significant drought mediated gene expression data for the sensitive genotype is <dig>  and the tolerant genotype is <dig> .
three main aspects were selected for a deeper evaluation of drought tolerant traits: the antioxidant and secondary metabolism pathways, light reaction and carbon fixation pathways, lipid and carbon metabolism.fig.
<dig> distribution of up-  and down-  regulated genes in metabolic pathways in response to drought stress for is <dig> and is <dig>  drought mediated expression changes in the metabolic pathways in leaves of is <dig>  and is <dig> .
the figure was generated using mapman and shows degs that passed the cut-off of log <dig> fc >2
response of antioxidant and secondary metabolism related genes
degs related to antioxidant and secondary metabolism were analysed together because of the strong relationship between the capacity to scavenge ros through antioxidant genes and metabolites derived from the secondary metabolism.
seventeen degs were identified in the sensitive genotype is <dig> in response to drought:  <dig> were up-regulated and  <dig> down-regulated .
in the tolerant genotype is <dig>  in the same condition, only  <dig> degs were found and three of them were up-regulated.
the sb09g <dig>  gene showed a peculiar behaviour; it was up-regulated in the tolerant genotype is <dig> and dramatically down-regulated in the sensitive is <dig>  the sb06g <dig>  gene was up-regulated in the sensitive genotype is <dig> and remained unchanged in the tolerant is <dig>  in contrast, the sb09g <dig>  gene was up-regulated in the tolerant is <dig> and its expression remained unchanged in the sensitive is <dig>
drought affected the secondary metabolism in both sorghum genotypes.
fifty degs were found in the sensitive genotype is <dig> and  <dig> in the tolerant is <dig> .
in the sensitive genotype is <dig>  about the same number of genes were up- and down-regulated , whilst in the tolerant genotype is <dig> the down-regulated genes were more than the up- regulated ones;  <dig> and  <dig>  respectively .
among the down-regulated genes, the isoprenoids and phenylpropanoids metabolism was the most affected metabolism, with  <dig> genes in is <dig> and  <dig> in is <dig>  the flavonoids pathway showed a peculiar behaviour being up-regulated by drought in the sensitive genotype is <dig> and down-regulated in the tolerant genotype is <dig>  the changes in the secondary metabolism expression pattern, for example the change in the chlorophyll/carotenoids content, was reflected in the fluorescence parameters recorded.
response of light reactions and carbon fixation pathways
the photosynthetic pathway was drastically affected by drought in the sensitive genotype is <dig>  with  <dig> genes differentially expressed in response to drought:  <dig> belong to the light reaction pathway and  <dig> to the calvin cycle.
among the  <dig> degs belonging to the light reaction pathway,  <dig> genes were down-regulated in response to drought :  <dig> code for protein belonging to the light harvesting complex i or ii ,  <dig> code for protein related to photosystem i and ii  and  <dig> codes for the gamma subunit of the atp synthase.
two isoforms of psii polypeptide subunits were strongly up-regulated together with the electron carrier ferrodoxin in the sensitive genotype is <dig> in response to drought .
in the tolerant genotype is <dig> the light reaction pathway was also affected, but to a lower extent.
only three genes belonging to the light reaction pathway were up-regulated in response to drought:  <dig> implicated in psii and one in photosynthetic electron transport, the ferrodoxin .
<dig> genes related to the carbon fixation pathway  were differentially expressed in the sensitive genotype is <dig> :  <dig> were down-regulated by drought and  <dig> were up-regulated .
in the tolerant genotype is <dig> no genes were differentially expressed in response to drought .
lipid and carbon metabolism in response to drought stress
in terms of degs the lipid metabolism was more greatly affected in the sensitive genotype is <dig> .
in this genotype fatty acid synthesis, elongation and lipid degradation via beta-oxidation cycle were all up-regulated .
phospholipid and sphingolipid syntheses were down-regulated in response to drought .
in the tolerant genotype is <dig> the steroids biosynthesis and phospholipase d were up-regulated .
also the carbon metabolism was more greatly affected by drought in the sensitive genotype is <dig> than in the tolerant is <dig>  in is <dig> drought highlighted  <dig> degs:  <dig> genes belonging to the degradation of starch and sucrose were up-regulated, and  <dig> genes were down-regulated .
in the tolerant genotype is <dig> only  <dig> genes were down-regulated .
discussion
in plants exposure to drought triggers a wide range of responses, ranging from molecular expression, biochemical metabolism to ecosystem level, that involve lots of genes and pathways related to diverse mechanisms  <cit> .
in this study we evaluated these mechanisms through rna-seq analysis of two sorghum genotypes subjected to the same extent of drought stress.
the responses differed greatly between the sensitive is <dig> and the tolerant is <dig> genotypes in terms of the number of genes and pathways involved in drought stress response, but also in terms of the constitutive expression level of several pathways.
constitutive drought tolerance trait
the trend of ftsw, together with the value of the daily transpiration rate, confirmed that the ds plants of both genotypes were subjected to the same environmental conditions and to the same extent of drought stress.
in addition, transcriptomics analysis provided unequivocal evidence on rna modifications triggered by drought stress.
“response to heat”  and “rna modification”  go terms were enriched to the same extent in both genotypes.
although the drought stress level applied was equal , the two genotypes responded differently; in is <dig> a significantly higher number of differentially expressed genes  was observed than in the tolerant genotype is <dig>  resulting in a greater enrichment of go terms related to drought stress response in is <dig> than in is <dig>  the up-regulation of genes under ww conditions of “secondary metabolic process” , and related go terms, in the genotype is <dig> confirm its intrinsic tolerance, previously only characterized from a physiological point of view  <cit> .
in this genotype, the constitutive upper level of flavonoids and secondary metabolites led to increased drought tolerance traits according to winkel-shirley  <cit> .
furthermore the “glutathione transferase activity”  was up-regulated in the tolerant genotype is <dig> confirming the role of the glutathione-s transferase family in improving environmental stress resistance in crops  <cit> .
drought tolerance strategies
drought stress results in a massive production of reactive oxygen species   <cit>  that cause oxidative stress.
the sequence of events that occur in plant tissues in response to oxidative drought-induced stress was well described by mano et al.
<cit> .
the antioxidant enzymes constitute the “first line of defence” against ros and oxidative stress generated by different abiotic and biotic injuries  <cit> .
the activity of these enzymes can be enhanced or repressed depending on the species, genotype, stress duration and severity .
in the “response to abiotic stimulus” , “oxido-reductase activity”  and “response to stress”  gene ontology categories, genes were more greatly down-regulated by drought in the sensitive genotype is <dig> than in the tolerant is <dig>  enabling us to speculate that the tolerant is <dig> had a constitutively higher expression of antioxidant genes that is not affected by drought stress.
experimental evidence showed that the antioxidant enzyme activity might be depressed in excess-light conditions, especially when plants are faced with additional stresses such as drought or temperature  <cit> .
to cope with the oxidative stress caused by drought, genes coding for secondary metabolites such as phenylpropanoids, phenolic compounds and flavonoids, are overexpressed  <cit> .
phenylpropanoids have the greatest potential to reduce ros, the polyphenols act as antioxidants to protect plants against oxidative stress  <cit> , flavonoids play different molecular functions, including stress protection in plants  <cit> , and also flavanols were found to be oxidated in response to severe drought in tea plants, suggesting their involvement in plant protection  <cit> .
all these compounds are widely synthetized in response to several abiotic stresses, including drought .
in wheat and willow leaves an increase in flavonoid and phenolic acids content was observed together with an induction of genes involved in the flavonoid biosynthetic pathway in response to various stresses, including drought  <cit> .
with our study, we confirm that under drought stress the up-regulation of these genes in the sensitive genotype is <dig> was higher than in the tolerant genotype is <dig>  whilst a constitutively higher expression of these genes in the tolerant is <dig> under control conditions led to a lower synthesis of stress induced compounds.
the accumulation of these compounds and the differential expression of the above mentioned genes remains genotype dependent  <cit> .
only in the last decade was it hypothesized that flavonoids might also play a role as antioxidant in response to severe excess of light complementing the role of antioxidant enzymes .
agati et al.
<cit>  found that flavonoid genes were up-regulated in response to drought in the sensitive genotype is <dig> whilst they were mostly down-regulated in the tolerant is <dig>  the biosynthesis of “antioxidant” flavonoids, in fact, increases more in stress sensitive species than in stress tolerant ones  <cit> .
the reason for this lies in the fact that stress sensitive species display a less efficient “first line” of defence against ros in conditions of stress and they are therefore exposed to a more severe oxidative stress  <cit> .
in any case, the relationship between antioxidant enzymes and flavonoids in response to abiotic and biotic stress it is not yet well clarified  <cit> .
drought stress induces a decrease in the chlorophyll content, a consequential change in the chlorophyll/carotenoid ratio  <cit>  and an increase in the ratio of violaxanthin-cycle pigment.
this results in a reduction of light absorption centres, an enhancement of non-photochemical quenching in order to dissipate the excess of light, and a reduction in photosynthetic rate .
all these stress-induced physiological modifications  were observed to a greater extent in the sensitive genotype is <dig>  the physiological response is supported by the observation that a high number of genes involved in the terpenoids and carotenoids biosynthesis were down-regulated in is <dig> and not in is <dig>  in agreement with the decreased concentration of some carotenoids under severe drought stress  <cit> .
the down-regulation of genes related to carotenoids and chlorophyll biosynthetic pathways leads to the down-regulation of light reaction and carbon fixation pathways, that in fact were dramatically affected by drought in the sensitive genotype is <dig>  the decreased expression pattern mainly involved the light harvesting complex i and ii and polypeptide subunits of the photosystems .
in particular, the light-harvesting chlorophyll a/b-binding proteins  were extremely down-regulated in the sensitive genotype is <dig> according to several studies in which the down-regulation of lhcbs reduces plant tolerance .
the lhcbs, complexed with chlorophyll and xanthophylls, form the antenna complex  <cit>  and play an important role in adaptation to environmental stress .
their expression is regulated by multiple environmental factors including light  <cit> , oxidative stress  <cit>  and abscisic acid   <cit> .
also the genes involved in the “carbon fixation” were more greatly down-regulated in the sensitive genotype is <dig> rather than in the tolerant one.
the up-regulation of sb03g <dig>  was the main exception in the expression pattern of this genotype; this gene codes for the electron carrier ferrodoxin.
comparing the log <dig> values of this gene in the two genotypes, it appears that this gene was more up-regulated in the sensitive genotype than in the tolerant one .
this result indicates that the tolerant genotype is <dig> could better cope with the excess of light during drought stress.
this is further supported at a physiological level by the low qnp value recorded.
conversely, the sensitive genotype is <dig> over expressed this gene so that it can dispose the excess of electrons and consequently waste the excess of light in non-photochemical reactions.
according to literature, under drought stress starch  content decreases, whilst content of soluble sugars  increases, assuring the maintenance of leaf water status and plant growth .
in the sensitive genotype is <dig>  starch synthases were down-regulated and enzymes involved in the degradation of starch and sucrose up-regulated.
according to sturm and tang  <cit>  invertases play a role in several processes ranging from phloem loading to response to abiotic and biotic stresses  <cit> .
exogenous aba applied in soybean green beans  <cit>  and maize leaves exposed to drought  <cit>  showed an increase in invertase activity.
gazarrani and mccourt  <cit>  also highlighted that hexose-based signals originating from sucrose cleavage are implicated in the regulation of aba biosynthetic genes.
it is well known that sucrose plays a crucial role as a key molecule in energy transduction and as a regulator of cellular metabolism .
furthermore, sucrose and other sugars are energy and carbon sources required for defence response and are necessary for plant survival under drought stress conditions  <cit> .
like hormones, sucrose can act as primary messenger controlling the expression of several genes involved in sugar metabolism.
lipids are important membrane components and, under drought stress, significant modifications of the lipid membranes occur.
for this reason our investigation also focused on this metabolic pathway.
the fatty acid elongation is considered to be the rate-limiting step in cuticular wax biosynthesis  <cit> .
the accumulation of wax has a key role in limiting water losses from plants  <cit> .
it is widely accepted that drought stress can increase the amount of wax in several species  and that this increase is associated with an improved drought tolerance  <cit> .
according to our results, the sensitive genotype is <dig> up-regulated these genes in response to drought; on the contrary, the drought tolerant genotype is <dig> remained unchanged.
the hypothesis is that the tolerant genotype is <dig> has a constitutively higher expression level of genes related to drought tolerance, such as genes involved in cuticular wax synthesis and fatty acid desaturation.
this hypothesis is also confirmed by the observation that, according to torres-martin et al.
<cit> , no changes in omega- <dig> desaturase expression were highlighted in response to drought in the tolerant genotype is <dig>  on the contrary, the omega- <dig> desaturases were down-regulated in the sensitive genotype is <dig>  <cit> .
the first evidence of the involvement of sphingolipids in the signal-transduction pathways in plants, including in response to drought, was provided by ng et al.
<cit> .
until that moment only the implication of sphingolipids in conferring stability to plant membranes, contributing to acclimation to drought stress had been hypothesized  <cit> .
spiegel and milstien  <cit>  afterwards explored the link between the sphingosine-1-phosphate and the drought hormone abscisic acid in the release of calcium from the vacuole.
rna-seq results highlighted the ineffective response of the drought sensitive genotype is <dig> that down-regulated sphingolipids in response to drought, except for a ceramidase .
in cowpea leaves a massive breakdown of membrane lipids was observed in response to drought with a more severe degradation in the sensitive plants  <cit> .
the main enzyme responsible for the drought-induced degradation of membrane phospholipids is phospholipase d   <cit> .
according to el masouf et al.
<cit> , the drought sensitive genotype is <dig> strongly up-regulated the pld expression, whilst in the drought tolerant is <dig> the expression was only slightly up-regulated.
recently, pld up-regulation was associated to drought and salt stress tolerance  and the product of its activity, the phosphatidic acid, is involved in aba signalling in stomatal movement  <cit> .
plda <dig>  in particular, is the most predominant pld in plants activated by aba  <cit> .
some interesting genes provided insight into the drought tolerance of the genotypes analysed.
the sb06g <dig> gene, encoding for a glycerophosphodiester phosphodiesterase, found to be up-regulated in response to drought in sorghum leaves  <cit> , was strongly down-regulated in response to drought in the sensitive genotype is <dig>  the sb07g <dig> gene, encoding for a monogalactosyl-diacylglycerol  synthase, found to map to a stay green qtl  <cit>  and to be overexpressed in response to drought in sorghum leaves, was down regulated in the sensitive genotype is <dig>  since these genes are involved in drought tolerance related pathways, the first in choline biosynthesis and the second in phosphatidylinositol biosynthesis, a down regulation in response to drought is proof of sensitivity to drought stress for the sensitive genotype is <dig>  a confirmation of the drought tolerance of is <dig> was the overexpression of genes related to the phosphatidylinositol biosynthesis, such as sb08g <dig>  sb08g <dig> and sb05g <dig>
CONCLUSIONS
rna-seq analysis, performed in this study, proved to be a good method to investigate complex traits in different genotypes.
the sorghum transcriptome analysed in response to drought conditions revealed unequivocal traits of sensitivity and tolerance in the two sorghum genotypes studied.
the first evidence of sensitivity to drought of the genotype is <dig> was represented by the physiological measurements  that drought dramatically affected.
this evidence was confirmed at a transcriptomic level by the higher number of differentially expressed genes  observed in the sensitive genotype is <dig> and not in the tolerant genotype is <dig>  the sensitivity to drought of is <dig> was further confirmed by the lower constitutive expression level of “secondary metabolic process”  and “glutathione transferase activity”  observed under well-watered conditions in is <dig> in comparison with the tolerant genotype is <dig>  in addition, the enriched go terms analysis highlighted the differences existing between the two genotypes in coping with drought stress and the strategies adopted.
the sensitive genotype hydrolysed carbohydrates and sugars, while the tolerant is <dig> activated the synthesis of other amino acids  to cope with drought stress.
in conclusion, we can confirm that the sensitive genotype is <dig> perceived the drought stress imposed  to a greater extent than the tolerant genotype is <dig>  showing an overactive genetic response.
is <dig>  on the other hand, being generally less affected by drought in all the analysed pathways, could be used as a genetic donor to further improve the sorghum germoplasm with drought tolerance traits.

 drought is the most important abiotic stress in terms of limiting crop productivity worldwide. water availability is, therefore, of primary importance for a non-limiting crop production in the current changing global climate scenario. the slogan “more crop per drop” was the track for crop improvement in water limited environments aiming to address the growing demand for water, food and commodities of the growing world population. among the c cereals, sorghum bicolor is the species most suited to environments that are prone to drought. its tolerance to drought is a consequence of morphological and anatomical characteristics and physiological responses. the high genetic variability among sorghum genotypes and the relatively small size of its genome make this cereal a good model for the identification of drought related genomic regions and genes valuable to unravel the high complexity of drought tolerance related traits. several sorghum linkage maps, including high density maps, have been built using different types of dna markers. different genomic regions related to drought tolerance at pre-flowering and post-flowering stage were identified but it was the availability of the sorghum genome sequence that has enabled the monitoring of the genome-wide gene expression profile at a single time in response to several abiotic stresses through microarray or rna-seq analysis. these studies resulted in the identification of drought stress responsive genes and their regulatory elements. several transcriptomics studies were carried out on sorghum using rna-seq analysis to monitor gene expression in response to osmotic stress and abscisic acid, to provide a s. bicolor expression atlas on the dynamic genotype-specific expression profiles, or to identify genome-wide snps that can potentially enhance genetic analysis and the application of molecular markers in sorghum genomics and breeding. in addition to physiologic or agronomic approaches, genomics offer new opportunities for dissecting quantitative traits into their single determinants paving the way to marker-assisted selection or direct gene editing via genetic engineering. drought stress elicits a wide range of responses in plants. it increases oxidative damage in chloroplasts, reduces photosynthesis, limits metabolic reactions, triggers sugar catabolism, in order to provide osmotically active compound and signal molecules, and modifies cellular lipid composition. to cope with drought stress, plants have developed various strategies, such as generation of larger and deeper root systems, regulation of stomatal closure to reduce water loss, accumulation of compatible solutes and protective proteins, and an increase in the level of antioxidants. identification of drought resistant traits was frequently labelled as “complex” although we already know the results of all the modifications adopted by plants to cope with drought stress. in this study we have furthered extended the knowledge on the drought response of two sorghum genotypes through transcriptomic analysis. a massive parallel sequencing of rna on the illumina platform was used to provide a thorough scenario on the whole sorghum transcriptome in response to drought stress. several categories of key genes involved in drought response have been identified.  physiological responses to drought stress twenty sorghum plants were subjected to severe drought stress by withholding water from dae until dae when ftsw was reached in all the stressed plants. subsequently the stressed plants were kept at ftsw by irrigating daily for nine days, while the control plants were kept at ftsw values higher than for the entire duration of the experiment. the daily transpired water was under gr for the stressed plant, while it was up to gr for the control plants.fig. trend of ftsw and daily transpired water during the dry-down experiment. on the left axis with circles symbols the trend of ftsw during the dry-down: with full circles the ww plants and with the empty circles the ds ones. on the right axis with triangles the daily transpired water: full triangles for the ww plants and empty triangles for the ds ones. dae = days after emergence. mean of plants ± se leaf area, chlorophyll fluorescence parameters and gas exchange measurements were quantified for the entire duration of the experiment. the decreased ftsw led to a reduction in rwc values and these changes were greater in the sensitive genotype is than in the tolerant genotype is. drought stress also dramatically reduced chlorophyll fluorescence and photosynthetic rate. under stress conditions the tolerant genotype is showed a significantly higher value of fv/fm than the sensitive genotype is. the same trend was observed for Φpsii: and for the tolerant and the sensitive genotype, respectively. in contrast, the qnp under drought stress was higher in the sensitive genotype is than in the tolerant genotype is.table physiological responses of sorghum genotypes to drought stress lsd analysis of relative water content, chlorophyll fluorescence, gas exchange, intrinsic and agronomic wue in sorghum plants in well-watered and drought stress conditions at vegetative stage of 9th leaf. values followed by the same letter are not statistically significant at lsd test p < performed on the interaction genotype× irrigation. drought stress affected pn in both the genotypes differently; the sensitive genotype is had a greater reduction in pn while the tolerant genotype is showed a pn reduction of  %. transpiration did not differ between the ww and ds plants of the tolerant genotype is while there was a statistically significant difference between the ww and ds plants of the sensitive genotype is the intrinsic water use efficiency decreased linearly for the ds plants of both genotypes from the beginning of the experiment until harvest, while the ww plants kept their wuei close to 6 μmol mmol−. wuei of ds plants of the tolerant genotype is was significantly higher than that of ds plants belonging to the sensitive genotype is during the stress period. the agronomic water use efficiency, calculated at harvest, was higher for the tolerant genotype is than for the sensitive genotype is, thereby confirming the trend highlighted by wuei.fig. trend of wuei calculated during the dry down experiment. circles represent the sensitive genotype is and triangles the tolerant is for both the genotypes the full symbols represents the ww plants whilst the empty symbols represent the ds ones. mean of plants ± se drought stress reveals different intergenic transcripts and novel splice sites transcription profiles of is and is under well-watered and drought-stressed conditions were explored using the illumina genome analyzer deep sequencing. three biological replicates were analysed for each condition, resulting in twelve samples. in total, billion clean reads, each nucleotides long, were generated, with approximately million clean reads from each sample. the reads mapping to the reference genome were categorised into two classes: uniquely mapped reads, that are reads that map to only one position in the reference genome, and multi-position match, that are reads mapping to more than one position in the reference genome. the assembled transcripts were mapped on the genome: on average 72 % were known transcripts, 10 % were novel transcripts and 18 % were intergenic transcripts.table number of reads sequenced and mapped with soapaligner/soap2 the numbers of unique mapped reads plus the multi-position match equals the total number of mapped reads in well-watered and drought stress conditions percentage of total mapped reads on the reference genome, percentage of match with known transcripts, with intergenic transcripts and novel transcripts identified, and alternative splicing events identified drought stress induced alternative splicing events in the two genotypes: in the sensitive genotype is no difference in ase were found, while in the tolerant genotype is the ase were increased by 18 %. drought stress triggers differential expression of particular genes and go classes each condition was represented by three biological replicates, resulting in eighteen pairwise comparisons between control and stressed plants of the two genotypes. the transcript abundance of each gene was calculated as reads per kilobase transcriptome per million mapped reads. this value was used to determine the differential expression analysis as log ratio between ds and ww plants per genotype and between the two genotypes under ww and ds conditions. four comparisons were analysed in this study: i) the genotypes is and is under ww conditions, ii) the genotypes is and is under ds conditions, iii) the genotype is in response to ds conditions, iv) the genotype is in response to ds conditions.fig. comparison under study. a number of degs in each pairwise comparison. blue and red bar are up- an down-regulated genes respectively expressed in well-watered and drought stressed conditions in the genotypes is and is. b total number of degs that passed the cut-off of log fc > in each comparison. in yellow the number of degs resulting from the comparison between is and is in well-watered conditions, in green the number of degs resulting from the comparison between the two genotypes under drought stress conditions; in blue the numbers of degs in response to drought stress in is and in red the number of degs in response to drought stress in is c venn diagram showing the numbers of up- and down- regulated genes resulted from the four comparison performed. the number of up- or down- regulated genes shared among the four comparison is represented by overlapping circles after applying a stringent cut-off, the comparison of genotypes is and is under ww conditions identified differentially expressed genes, and the comparison of genotypes is and is under ds conditions identified degs. genes were differentially expressed in is in response to drought stress, whilst only were differentially expressed in is. venn diagrams highlight the overlap of degs between each pairwise comparison. comparison between is and is under ww conditions resulted in up-regulated genes and down-regulated genes. only genes were uniquely up- and genes down-regulated in is in these conditions. the singular enrichment analysis, carried out with agrigo software on the up-regulated genes, highlighted go terms significantly enriched: “aromatic compound biosynthetic process”, “secondary metabolic process”, and “flavonoid biosynthetic process” in the cellular processes category; “glutathione transferase activity”, “oxygen binding”, “udp-glucosyltransferase activity” in molecular functions category. “apoptosis” and “oxidoreductase activity” were the most enriched go terms in the biological processes and molecular function categories among the uniquely down-regulated genes expressed in ww conditions in is. the comparison between the two genotypes under ds conditions resulted in up- and down-regulated genes. among these genes, only and were uniquely up- and down- regulated in the genotype is in comparison to is “regulation of dna replication”, “cell death”, “regulation of cell growth by extracellular stimulus”, “secondary metabolic processes” including “terpenoids biosynthetic process”, “glutathione transferase activity” and “pre-replicative complex” were the most enriched go terms among the identified after sea of the up-regulated genes. among the down-regulated genes go terms were significantly enriched: “lipid localization”, “apoptosis”, “flavonol biosynthetic process”, “electron carrier activity” and “heme binding”. the main difference between the two genotypes was in the total number of genes differentially expressed in response to drought stress: for the sensitive is and for the tolerant is the sea analysis, performed on all the and degs expressed in response to drought in the genotypes is and is showed significantly enriched go terms in the sensitive genotype is while in the tolerant is twenty go terms were enriched in both the genotypes in response to drought stress and are represented in the heat map. “response to heat”, “rna modification”, “cytosolic part” and “ribosomal subunit” go terms were enriched with the same extent in both the genotypes. different go enrichment was recorded between is and is for “oxidation-reduction process”, “response to abiotic stimulus”, “oxidoreductase activity”, “response to chemical stimulus”, “small molecule metabolic process”, “response to stress”, “chloroplast”, “single-organism metabolic process” and “cytoplasm component”. all these go terms were more enriched in is than in is fig. heat map showing the common go terms enriched under drought stress in sorghum leaves of is and is the cluster frequency was used as a parameter for the parametric analysis of gene enrichment analysis. the figure was generated using r software, limma package between the two genotypes there were common up-regulated genes in response to drought stress and common down-regulated genes. the sea performed on these common degs highlighted enriched go terms belonging to biological processes: “response to abscisic acid stimulus”, “response to water deprivation”, “photosynthesis, light reaction” were the most enriched go. the sea analysis performed with agrigo on the unique up-regulated genes of is20351and is highlighted enriched go terms in is and and enriched go terms is the cross comparison of sea highlighted common go terms. the sea analysis performed on the unique down-regulated genes highlighted and significantly enriched go terms in is and is respectively; after the cross comparison of sea only resulted as being common to both genotypes. drought stress affects different pathways the kegg pathway analysis was performed to assign the related biological pathways in which degs were involved. one-hundred and seventy-one genes, uniquely expressed in response to drought stress in both the genotypes, were assigned to different kegg pathways belonging to clades under five major kegg categories including ‘organismal system’, ‘cellular process’, ‘environmental information processing’, ‘genetic information processing’, and ‘metabolism’. gene-set enrichment analysis showed that translation, signal transduction and carbon metabolism were the top three up-regulated pathways represented by the genes uniquely expressed in response to drought stress; metabolism pathways and signal transduction were, on the other hand, the most enriched down-regulated pathways.fig. number of up- and down-regulated genes in each clade of the kegg pathway maps. the unigenes were assigned kegg pathways within clades under five major categories: “organismal systems”, “cellular processes”, “environmental information processing”, “genetic information processing”, “metabolism”. per each clades are shown the up- and the down- regulated genes kegg pathway analysis was also performed on the genes that were uniquely up- and down-regulated in response to drought stress in both genotypes. transcription factors, ‘environmental information processing’ pathways, and pathways related to ‘cellular processes’ and ‘organismal system’ remained unchanged among the uniquely up-regulated genes. the most striking differences in the transcriptomic profiles of the two genotypes in response to drought were mainly in the ‘metabolism’ pathways, in the ‘genetic information processing’ pathway and in the number of genes not assigned to pathways. focusing on the up-regulated ‘metabolism’ pathways, the tolerant genotype is showed a two-fold enrichment in the metabolism of other amino acids, the nucleotide metabolism, the glycan biosynthesis metabolism and the lipid metabolism compared to the sensitive genotypes is. amino acid metabolism, carbohydrate metabolism and energy metabolism were more enriched in the sensitive genotype is than in the tolerant genotype is.fig. distribution in kegg pathways of the unique up- and down-regulated genes in response to drought for the genotype is and is pie charts showing the percentage of genes up- and down- regulated in response to drought stress for the genotypes is and is the ‘metabolism’ pathways of is and is were down-regulated to the same degree in response to drought stress. ‘cellular processes’ pathways represented 4 % of the down-regulated genes in is and 2 % in is. ‘organismal system’ pathways, ‘genetic information processing’ pathways and transcription factors were down-regulated to a greater extent in the tolerant genotype is. among the down-regulated ‘metabolism’ pathways, energy metabolism, nucleotide, cofactors and vitamins metabolism, glycan biosynthesis and metabolism, and carbohydrate metabolism pathways were down-regulated with a higher frequency in the sensitive genotype is than in the tolerant is. drought stress response of sorghum transcriptome the mapman software was used to show a pathway overview of and degs expressed in response to drought stress and it was selected for its capacity to show statistically significant drought mediated gene expression data for the sensitive genotype is and the tolerant genotype is. three main aspects were selected for a deeper evaluation of drought tolerant traits: the antioxidant and secondary metabolism pathways, light reaction and carbon fixation pathways, lipid and carbon metabolism.fig. distribution of up- and down- regulated genes in metabolic pathways in response to drought stress for is and is drought mediated expression changes in the metabolic pathways in leaves of is and is. the figure was generated using mapman and shows degs that passed the cut-off of log fc >2 response of antioxidant and secondary metabolism related genes degs related to antioxidant and secondary metabolism were analysed together because of the strong relationship between the capacity to scavenge ros through antioxidant genes and metabolites derived from the secondary metabolism. seventeen degs were identified in the sensitive genotype is in response to drought: were up-regulated and down-regulated. in the tolerant genotype is in the same condition, only degs were found and three of them were up-regulated. the sb09g gene showed a peculiar behaviour; it was up-regulated in the tolerant genotype is and dramatically down-regulated in the sensitive is the sb06g gene was up-regulated in the sensitive genotype is and remained unchanged in the tolerant is in contrast, the sb09g gene was up-regulated in the tolerant is and its expression remained unchanged in the sensitive is drought affected the secondary metabolism in both sorghum genotypes. fifty degs were found in the sensitive genotype is and in the tolerant is. in the sensitive genotype is about the same number of genes were up- and down-regulated, whilst in the tolerant genotype is the down-regulated genes were more than the up- regulated ones; and respectively. among the down-regulated genes, the isoprenoids and phenylpropanoids metabolism was the most affected metabolism, with genes in is and in is the flavonoids pathway showed a peculiar behaviour being up-regulated by drought in the sensitive genotype is and down-regulated in the tolerant genotype is the changes in the secondary metabolism expression pattern, for example the change in the chlorophyll/carotenoids content, was reflected in the fluorescence parameters recorded. response of light reactions and carbon fixation pathways the photosynthetic pathway was drastically affected by drought in the sensitive genotype is with genes differentially expressed in response to drought: belong to the light reaction pathway and to the calvin cycle. among the degs belonging to the light reaction pathway, genes were down-regulated in response to drought: code for protein belonging to the light harvesting complex i or ii, code for protein related to photosystem i and ii and codes for the gamma subunit of the atp synthase. two isoforms of psii polypeptide subunits were strongly up-regulated together with the electron carrier ferrodoxin in the sensitive genotype is in response to drought. in the tolerant genotype is the light reaction pathway was also affected, but to a lower extent. only three genes belonging to the light reaction pathway were up-regulated in response to drought: implicated in psii and one in photosynthetic electron transport, the ferrodoxin. genes related to the carbon fixation pathway were differentially expressed in the sensitive genotype is: were down-regulated by drought and were up-regulated. in the tolerant genotype is no genes were differentially expressed in response to drought. lipid and carbon metabolism in response to drought stress in terms of degs the lipid metabolism was more greatly affected in the sensitive genotype is. in this genotype fatty acid synthesis, elongation and lipid degradation via beta-oxidation cycle were all up-regulated. phospholipid and sphingolipid syntheses were down-regulated in response to drought. in the tolerant genotype is the steroids biosynthesis and phospholipase d were up-regulated. also the carbon metabolism was more greatly affected by drought in the sensitive genotype is than in the tolerant is in is drought highlighted degs: genes belonging to the degradation of starch and sucrose were up-regulated, and genes were down-regulated. in the tolerant genotype is only genes were down-regulated. discussion in plants exposure to drought triggers a wide range of responses, ranging from molecular expression, biochemical metabolism to ecosystem level, that involve lots of genes and pathways related to diverse mechanisms. in this study we evaluated these mechanisms through rna-seq analysis of two sorghum genotypes subjected to the same extent of drought stress. the responses differed greatly between the sensitive is and the tolerant is genotypes in terms of the number of genes and pathways involved in drought stress response, but also in terms of the constitutive expression level of several pathways. constitutive drought tolerance trait the trend of ftsw, together with the value of the daily transpiration rate, confirmed that the ds plants of both genotypes were subjected to the same environmental conditions and to the same extent of drought stress. in addition, transcriptomics analysis provided unequivocal evidence on rna modifications triggered by drought stress. “response to heat” and “rna modification” go terms were enriched to the same extent in both genotypes. although the drought stress level applied was equal, the two genotypes responded differently; in is a significantly higher number of differentially expressed genes was observed than in the tolerant genotype is resulting in a greater enrichment of go terms related to drought stress response in is than in is the up-regulation of genes under ww conditions of “secondary metabolic process”, and related go terms, in the genotype is confirm its intrinsic tolerance, previously only characterized from a physiological point of view. in this genotype, the constitutive upper level of flavonoids and secondary metabolites led to increased drought tolerance traits according to winkel-shirley. furthermore the “glutathione transferase activity” was up-regulated in the tolerant genotype is confirming the role of the glutathione-s transferase family in improving environmental stress resistance in crops. drought tolerance strategies drought stress results in a massive production of reactive oxygen species that cause oxidative stress. the sequence of events that occur in plant tissues in response to oxidative drought-induced stress was well described by mano et al.. the antioxidant enzymes constitute the “first line of defence” against ros and oxidative stress generated by different abiotic and biotic injuries. the activity of these enzymes can be enhanced or repressed depending on the species, genotype, stress duration and severity. in the “response to abiotic stimulus”, “oxido-reductase activity” and “response to stress” gene ontology categories, genes were more greatly down-regulated by drought in the sensitive genotype is than in the tolerant is enabling us to speculate that the tolerant is had a constitutively higher expression of antioxidant genes that is not affected by drought stress. experimental evidence showed that the antioxidant enzyme activity might be depressed in excess-light conditions, especially when plants are faced with additional stresses such as drought or temperature. to cope with the oxidative stress caused by drought, genes coding for secondary metabolites such as phenylpropanoids, phenolic compounds and flavonoids, are overexpressed. phenylpropanoids have the greatest potential to reduce ros, the polyphenols act as antioxidants to protect plants against oxidative stress, flavonoids play different molecular functions, including stress protection in plants, and also flavanols were found to be oxidated in response to severe drought in tea plants, suggesting their involvement in plant protection. all these compounds are widely synthetized in response to several abiotic stresses, including drought. in wheat and willow leaves an increase in flavonoid and phenolic acids content was observed together with an induction of genes involved in the flavonoid biosynthetic pathway in response to various stresses, including drought. with our study, we confirm that under drought stress the up-regulation of these genes in the sensitive genotype is was higher than in the tolerant genotype is whilst a constitutively higher expression of these genes in the tolerant is under control conditions led to a lower synthesis of stress induced compounds. the accumulation of these compounds and the differential expression of the above mentioned genes remains genotype dependent. only in the last decade was it hypothesized that flavonoids might also play a role as antioxidant in response to severe excess of light complementing the role of antioxidant enzymes. agati et al. found that flavonoid genes were up-regulated in response to drought in the sensitive genotype is whilst they were mostly down-regulated in the tolerant is the biosynthesis of “antioxidant” flavonoids, in fact, increases more in stress sensitive species than in stress tolerant ones. the reason for this lies in the fact that stress sensitive species display a less efficient “first line” of defence against ros in conditions of stress and they are therefore exposed to a more severe oxidative stress. in any case, the relationship between antioxidant enzymes and flavonoids in response to abiotic and biotic stress it is not yet well clarified. drought stress induces a decrease in the chlorophyll content, a consequential change in the chlorophyll/carotenoid ratio and an increase in the ratio of violaxanthin-cycle pigment. this results in a reduction of light absorption centres, an enhancement of non-photochemical quenching in order to dissipate the excess of light, and a reduction in photosynthetic rate. all these stress-induced physiological modifications were observed to a greater extent in the sensitive genotype is the physiological response is supported by the observation that a high number of genes involved in the terpenoids and carotenoids biosynthesis were down-regulated in is and not in is in agreement with the decreased concentration of some carotenoids under severe drought stress. the down-regulation of genes related to carotenoids and chlorophyll biosynthetic pathways leads to the down-regulation of light reaction and carbon fixation pathways, that in fact were dramatically affected by drought in the sensitive genotype is the decreased expression pattern mainly involved the light harvesting complex i and ii and polypeptide subunits of the photosystems. in particular, the light-harvesting chlorophyll a/b-binding proteins were extremely down-regulated in the sensitive genotype is according to several studies in which the down-regulation of lhcbs reduces plant tolerance. the lhcbs, complexed with chlorophyll and xanthophylls, form the antenna complex and play an important role in adaptation to environmental stress. their expression is regulated by multiple environmental factors including light, oxidative stress and abscisic acid. also the genes involved in the “carbon fixation” were more greatly down-regulated in the sensitive genotype is rather than in the tolerant one. the up-regulation of sb03g was the main exception in the expression pattern of this genotype; this gene codes for the electron carrier ferrodoxin. comparing the log values of this gene in the two genotypes, it appears that this gene was more up-regulated in the sensitive genotype than in the tolerant one. this result indicates that the tolerant genotype is could better cope with the excess of light during drought stress. this is further supported at a physiological level by the low qnp value recorded. conversely, the sensitive genotype is over expressed this gene so that it can dispose the excess of electrons and consequently waste the excess of light in non-photochemical reactions. according to literature, under drought stress starch content decreases, whilst content of soluble sugars increases, assuring the maintenance of leaf water status and plant growth. in the sensitive genotype is starch synthases were down-regulated and enzymes involved in the degradation of starch and sucrose up-regulated. according to sturm and tang invertases play a role in several processes ranging from phloem loading to response to abiotic and biotic stresses. exogenous aba applied in soybean green beans and maize leaves exposed to drought showed an increase in invertase activity. gazarrani and mccourt also highlighted that hexose-based signals originating from sucrose cleavage are implicated in the regulation of aba biosynthetic genes. it is well known that sucrose plays a crucial role as a key molecule in energy transduction and as a regulator of cellular metabolism. furthermore, sucrose and other sugars are energy and carbon sources required for defence response and are necessary for plant survival under drought stress conditions. like hormones, sucrose can act as primary messenger controlling the expression of several genes involved in sugar metabolism. lipids are important membrane components and, under drought stress, significant modifications of the lipid membranes occur. for this reason our investigation also focused on this metabolic pathway. the fatty acid elongation is considered to be the rate-limiting step in cuticular wax biosynthesis. the accumulation of wax has a key role in limiting water losses from plants. it is widely accepted that drought stress can increase the amount of wax in several species and that this increase is associated with an improved drought tolerance. according to our results, the sensitive genotype is up-regulated these genes in response to drought; on the contrary, the drought tolerant genotype is remained unchanged. the hypothesis is that the tolerant genotype is has a constitutively higher expression level of genes related to drought tolerance, such as genes involved in cuticular wax synthesis and fatty acid desaturation. this hypothesis is also confirmed by the observation that, according to torres-martin et al., no changes in omega- desaturase expression were highlighted in response to drought in the tolerant genotype is on the contrary, the omega- desaturases were down-regulated in the sensitive genotype is. the first evidence of the involvement of sphingolipids in the signal-transduction pathways in plants, including in response to drought, was provided by ng et al.. until that moment only the implication of sphingolipids in conferring stability to plant membranes, contributing to acclimation to drought stress had been hypothesized. spiegel and milstien afterwards explored the link between the sphingosine-1-phosphate and the drought hormone abscisic acid in the release of calcium from the vacuole. rna-seq results highlighted the ineffective response of the drought sensitive genotype is that down-regulated sphingolipids in response to drought, except for a ceramidase. in cowpea leaves a massive breakdown of membrane lipids was observed in response to drought with a more severe degradation in the sensitive plants. the main enzyme responsible for the drought-induced degradation of membrane phospholipids is phospholipase d. according to el masouf et al., the drought sensitive genotype is strongly up-regulated the pld expression, whilst in the drought tolerant is the expression was only slightly up-regulated. recently, pld up-regulation was associated to drought and salt stress tolerance and the product of its activity, the phosphatidic acid, is involved in aba signalling in stomatal movement. plda in particular, is the most predominant pld in plants activated by aba. some interesting genes provided insight into the drought tolerance of the genotypes analysed. the sb06g gene, encoding for a glycerophosphodiester phosphodiesterase, found to be up-regulated in response to drought in sorghum leaves, was strongly down-regulated in response to drought in the sensitive genotype is the sb07g gene, encoding for a monogalactosyl-diacylglycerol synthase, found to map to a stay green qtl and to be overexpressed in response to drought in sorghum leaves, was down regulated in the sensitive genotype is since these genes are involved in drought tolerance related pathways, the first in choline biosynthesis and the second in phosphatidylinositol biosynthesis, a down regulation in response to drought is proof of sensitivity to drought stress for the sensitive genotype is a confirmation of the drought tolerance of is was the overexpression of genes related to the phosphatidylinositol biosynthesis, such as sb08g sb08g and sb05g  rna-seq analysis, performed in this study, proved to be a good method to investigate complex traits in different genotypes. the sorghum transcriptome analysed in response to drought conditions revealed unequivocal traits of sensitivity and tolerance in the two sorghum genotypes studied. the first evidence of sensitivity to drought of the genotype is was represented by the physiological measurements that drought dramatically affected. this evidence was confirmed at a transcriptomic level by the higher number of differentially expressed genes observed in the sensitive genotype is and not in the tolerant genotype is the sensitivity to drought of is was further confirmed by the lower constitutive expression level of “secondary metabolic process” and “glutathione transferase activity” observed under well-watered conditions in is in comparison with the tolerant genotype is in addition, the enriched go terms analysis highlighted the differences existing between the two genotypes in coping with drought stress and the strategies adopted. the sensitive genotype hydrolysed carbohydrates and sugars, while the tolerant is activated the synthesis of other amino acids to cope with drought stress. in conclusion, we can confirm that the sensitive genotype is perceived the drought stress imposed to a greater extent than the tolerant genotype is showing an overactive genetic response. is on the other hand, being generally less affected by drought in all the analysed pathways, could be used as a genetic donor to further improve the sorghum germoplasm with drought tolerance traits. 
the physiological measurements carried out confirmed the drought sensitivity of is and the drought tolerance of is genotypes, as previously studied. transcriptomics analysis results supported all the physiological measurements and were essential to clarify the tolerance of the two genotypes studied. genome-wide rna-seq technology was used to compare the drought response of two sorghum genotypes characterized by contrasting water use efficiency. rna-seq analysis proved to be a very useful tool to explore differences between sensitive and tolerant sorghum genotypes. at the molecular level the response to drought stress results in the differential expression of several metabolic pathways. it triggers a wide range of responses detectable at molecular, biochemical and physiological levels. drought stress is the major environmental stress that affects plant growth and productivity. the expression of drought-related genes was more abundant in the drought sensitive genotype is compared to the tolerant genotype is under drought stress gene ontology enrichment highlighted a massive increase in transcript abundance in the sensitive genotype is in “response to stress” and “abiotic stimulus”, as well as for “oxidation-reduction reaction”. the connection between differential gene expression and physiological response to drought unequivocally revealed the drought tolerance of genotype is and the strategy adopted to cope with drought stress. rna-seqdrought stresssorghum bicolorwater use efficiencydrought toleranceissue-copyright-statement© the author 2016 “antioxidant” and “secondary metabolism”, “photosynthesis and carbon fixation process”, “lipids” and “carbon metabolism” were the pathways most affected by drought in the sensitive genotype is in addition, genotype is showed a lower constitutive expression level of “secondary metabolic process” and “glutathione transferase activity” under well-watered conditions. for this reason, exploring the subtle differences in gene expression of drought sensitive and drought tolerant genotypes enables the identification of drought-related genes that could be used for selection of drought tolerance traits.
 drought stress is the major environmental stress that affects plant growth and productivity. it triggers a wide range of responses detectable at molecular, biochemical and physiological levels. at the molecular level the response to drought stress results in the differential expression of several metabolic pathways. for this reason, exploring the subtle differences in gene expression of drought sensitive and drought tolerant genotypes enables the identification of drought-related genes that could be used for selection of drought tolerance traits. genome-wide rna-seq technology was used to compare the drought response of two sorghum genotypes characterized by contrasting water use efficiency.  the physiological measurements carried out confirmed the drought sensitivity of is and the drought tolerance of is genotypes, as previously studied. the expression of drought-related genes was more abundant in the drought sensitive genotype is compared to the tolerant genotype is under drought stress gene ontology enrichment highlighted a massive increase in transcript abundance in the sensitive genotype is in “response to stress” and “abiotic stimulus”, as well as for “oxidation-reduction reaction”. “antioxidant” and “secondary metabolism”, “photosynthesis and carbon fixation process”, “lipids” and “carbon metabolism” were the pathways most affected by drought in the sensitive genotype is in addition, genotype is showed a lower constitutive expression level of “secondary metabolic process” and “glutathione transferase activity” under well-watered conditions.  rna-seq analysis proved to be a very useful tool to explore differences between sensitive and tolerant sorghum genotypes. transcriptomics analysis results supported all the physiological measurements and were essential to clarify the tolerance of the two genotypes studied. the connection between differential gene expression and physiological response to drought unequivocally revealed the drought tolerance of genotype is and the strategy adopted to cope with drought stress. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords rna-seqdrought stresssorghum bicolorwater use efficiencydrought toleranceissue-copyright-statement© the author 2016 