To analyze the differences in the drought tolerance of the G. herbaceum accessions at physiological and molecular levels, the plants were exposed to moderate and severe drought stress. It was observed that Vagad and RAHS-14 showed substantial differences in several physiological parameters and relative gene expression in response to drought. Vagad responded to moderate and severe drought by a gradual decline in A, gs, and E and, thus, has better WUE (Figure 2). In contrast, RAHS-14 had higher A and gs and, thus, lower WUE, which continued under moderate drought and then declined under severe stress (Figure 2a-d). In Vagad, earlier stomatal closure (Figure 2b) provides a large safety margin against embolism formation as observed in several species [20–22]; rather, plants are capable of pre-empting the water stress-induced xylem cavitation by closing their stomata [23–25]. Further, a reduction in A and a higher NPQ in Vagad accession under drought stress suggested that stress had been imposed and hypothesized that antioxidant defense systems and secondary metabolic pathways would have been enhanced in response. Indeed, our microarray expression analysis results showed that in Vagad, various primary and secondary metabolic pathways were enhanced (Figure 5). Flavonoid biosynthesis pathways especially lead to the formation of xanthophyll and finally convert it into anthocyanin, antheraxanthin, and zeaxanthin, which allows the quenching of excess energy from chlorophyll before it reaches reaction centers ; the transcript representing flavonoid biosynthesis pathways was found to be enriched in Vagad (Figure 5). In contrast, the xanthophyll-cycle conversion state was lower in RAHS-14 (Figure 2e and Additional file 7). Concurrent to physiological data, transcriptome analysis showed a higher expression of the vitamins metabolic process, such as α-tocopherol in Vagad (Additional file 13; worksheet 1). The α-tocopherol is a powerful antioxidant that scavenges and prevents the formation of free radicals and prevents lipid peroxidation, thereby resulting in damage to thylakoid and chloroplast membranes . Besides flavonoid pathways in Vagad, various other metabolic pathways, including polyketide biosynthesis, phenyl propanoid biosynthesis, and shikimate pathways, synthesize protective molecules such as stilbene, coumarins, and ligninis and are enriched in Vagad, which might help in the detoxification of free radicals and give an advantage to Vagad for surviving in drought stress (Figure 4). These secondary metabolites, particularly phenylpropanoid, were widely reported for their multiple function in response to various forms of environmental stress . The genes related to the shikimate and phenylpropanoid pathways have been reported to express a higher level in drought-tolerant tomato cultivars (Solanum lycopersicum L.) as compared with drought-sensitive tomato cultivars . In RAHS-14, the lipid metabolism processes were significantly higher (Figure 5). In water-deficit conditions, the membranes are the main targets of the degradative process, resulting in the formation of polar and non-polar lipid molecules . Thus, the higher lipid metabolic processes in RAHS-14 probably reflect higher membrane degeneration and, hence, the necessity of higher lipid metabolism for membrane homeostasis correlated with their susceptibility of drought stress. Vagad, in contrast to RAHS-14, maintained lipid metabolism and membrane integrity to resist the drought stress. RAHS-14 showed a higher expression of genes related to nucleic acid (DNA/RNA) metabolism, whereas the expression of these genes remains unchanged in Vagad (Additional file 5 and Figure 6). Higher nucleic acid metabolism in RAHS-14 might show an interesting mechanism related to the energy state of the cell that is represented by the salvage pathways . Phosphoribosyl-1-pyrophosphate, a key intermediate component for the synthesis of ribose-5-phosphate in nucleic acid metabolism, showed higher expression in RAHS-14 as representing the operation mechanism of salvage pathways and helps RAHS-14 keep the energy pools from being used up too quickly (Additional file 13; worksheet 2). It indicated that RAHS-14 responds to drought by inducing energy-consuming processes, whereas Vagad has various inherent primary and secondary metabolic processes that maintain growth, albeit slow even under drought. This is further evident from dark respiration (R) data which show that R declined in Vagad and increased in RAHS-14 during drought (Figure 2h). Many genes that were up-regulated in response to drought stress in Vagad are reported to be involved in multiple mechanisms that may contribute to drought tolerance (Additional files 4 and 15, worksheet 1). For example, Aquaporins (AQP), a water-selective channel protein, known to mediate and regulate rapid transmembrane water flow during a wide range of stress response, stomatal movement, and water channel movements, was expressed at a higher level in Vagad (Additional file 4) . Similarly, many tonoplastic intrinsic proteins (TIPs) from maize, Arabidopsis, and radish are known to control water exchange between cytosol and vacuole in salt stress and drought stress  and these genes were expressed at a higher level in Vagad (Additional file 4). DnaJ heat shock protein/chaperone and Delta 1-pyrroline-5-carboxylate synthase 2 (P5CS2), which enhance root biomass, flowering, and seed setting during abiotic stress , were expressed at a higher level in Vagad (Additional files 2 and 3). Yet another significantly induced gene in Vagad was the ERF/AP2-type transcription factor (RAP2.1), which was reported to be induced in drought and cold via an ABA-independent pathway . RAHS-14 expressed more senescence-related genes, whereas we found that in Vagad, genes related to alcohol dehydrogenase and late embryogenesis protein 5 was expressed at a higher level (Additional files 5 and 15). Previous studies indicate that the accumulation of late embryogenesis abundant proteins and alcohol dehydrogenase (Adh) gene are correlated with stress tolerance [35, 36]. The significant up-regulation of LEA and Adh genes in Vagad in drought stress suggests that these genes play an important role in conferring drought tolerance, whereas RAHS-14 tends toward the senescence during drought stress. Several transcription families were significantly and differentially expressed in both the accessions. Ethylene-responsive element binding factors (ERFs) are members of a novel family of transcription factors that are specific to plants and which regulate nuclear gene expression under various stress conditions. Six different members homologous to the Arabidopsis ERF family (At1g19210; At1g28360; At4g34410; At5g44210; At5g47220; At5g47230) were expressed exclusively in RAHS-14 under drought stress. The expression of ERF in RAHS-14 in the drought condition indicates that RAHS-14 responds to drought mainly by the ethylene pathway, thereby leading to senescence. This relates well with transcriptome data showing a higher level of senescence-related transcripts in RAHS-14. In Vagad, these AP2/EREBP TFs belong to the Cytokinin Response Factors (CRF2) class (At1g78080; At4g23750). CRFs function redundantly to regulate the various metabolic functions, including transpiration, stomatal conductance, and respiration [37, 38]. Further, in RAHS-14, seven (At1g29860; At1g80840; At2g23320; At2g24570; At2g38470; At3g56400; At4g24240) and four (At1g80840; At2g47260; At4g24240; At5g49520) WRKY transcription factors were found to be uniquely expressed in drought and irrigated conditions, respectively. Previous reports showed that abscisic acid and salicylic acid have been involved in the WRKY-mediated hormone signal pathway during abiotic and biotic stress . Their precise role in the abiotic stress response regulatory network is not fully understood . In contrast, the MYC-type bHLH transcription factor, which regulates the expression of CBF3/DREB1A in abiotic stress, was enriched in Vagad, which suggests the involvement of the MYC-type bHLH transcription factors in the expression of CBF/DREB1 genes in Vagad but less involvement in RAHS-14 . Interactions between CBF/DREB1 genes and bHLH TFs and their involvement in various oxidative-mediated processes justify their presumed roles as regulators of drought response in Vagad. Other members of TFs, such as bZIP (At2g46270; At4g34590; At5g24800) and GRAS families (At5g48150; At5g52510; At5g66770), were expressed in higher numbers in Vagad; the regulatory roles of these TFS have been reported in stress responses in plants. The GO-based analysis of both the transcriptome libraries revealed many metabolic processes and responses to various forms of abiotic stress that were specific to Vagad and RAHS-14 (Figure 11). The shift in the processes toward reproductive growth and senescence in RAHS-14 clearly showed that they had crossed the threshold of stress and were proceeding toward senescence. The up-regulation of several metallothioneins, lipid transfer proteins, lea proteins, sucrose synthase, and so on in Vagad showed the induction of defense and stress-related genes to combat drought stress. In addition, the down-regulation of many photosynthesis-related genes during drought stress (Additional file 15 worksheet 2) can be attributed to the conservation of energy for the survival in drought stress. The up-regulation of genes such as aquaporins, lea, and metallothioneins may have played a major role in imparting tolerance to Vagad.
We further compared differentially expressed genes obtained by microarray and contigs obtained in transcriptomic data using Pearson uncentered correlation. A total 167 differentially expressed genes obtained in microarray showed very high Pearson correlation coefficient of 0.845 with transcriptomic contigs obtained under drought stress condition (Additional file 16). Out of these 167 differentially expressed genes, 78 and 48 genes were uniquely represented in Vagad and RAHS-14 respectively under drought stressed condition. Uniquely represented genes under drought stress in Vagad were mainly involved in synthesis of membrane and cytoskeleton associated proteins, oxidoreductases, kinases, heat shock proteins, sugar alcohols and secondary metabolites like, cumurin and stilbene. Similarly in RAHS-14, these genes were mainly associated with transcription factors (viz, WRKY, AP2, WD40, Zinc finger and ERF), senescence associated proteins, ethylene and auxin responsive elements (Additional file 17). The GO based analysis of these 167 genes revealed many processes that were specific to Vagad and RAHS-14. The upregulation of pyrroline-5- carboxylase, ATPase, inositol etc. (Additional file 17) in Vagad may have played a major role in imparting drought tolerance. In RAHS-14 higher numbers of these genes were involved in senescence and ethylene mediated signaling which clearly indicate the RAHS- 14 crossed the threshold of drought stress and are proceeding towards senescence (Additional file 15). Thus both the method in an unbiased way identifies same mechanistic model operating for drought responsiveness in Vagad and RAHS 14.