Drought tolerance is a very complex plant trait reflecting the intricacy of the abiotic stress response [28]. When confronted with water deficit, plants alter the transcript abundance of a large number of genes with diverse functions and complex interactions [29]. In roots, the drought cues are perceived by unknown sensors, and passed down through several signal transduction pathways, resulting in the expression of drought-responsive genes including those conferring drought tolerance [30]. The convolution of drought response and adaptation is apparent in the present study as measured by the number of driven genes that depict the wider molecular response of SC56 than Tx7000 to water deprivation. Beyond the response to drought, the results suggest that the expanded response to water status includes also optimal water conditions.
SC56 biologically outperforms Tx7000 in wet and drought conditions
In contrast to Tx7000_cont > SC56_cont, which showed no significantly enriched biological processes, SC56_cont > Tx7000_cont exhibited the highest count of enriched biological processes as well as DEGs. The mechanisms upregulated by the drought-tolerant SC56 line compared to TX7000 under wet conditions are all drivers of growth. Anabolism was the principal branch of metabolism that has been enhanced as indicated by more than a hundred DEGs involved in the overall biosynthesis machinery with many contributing into the all-important translation of proteins as well as the biogenesis of oligosaccharides. Also, metabolic processes related to amino acids (‘amino acids and derivatives,’ ‘oxoacid’ and ‘carboxylic acid’) were particularly enhanced in SC56; besides being the building blocks of protein, amino acids are also involved in a plethora of cellular reactions influencing plant growth and development, generation of metabolic energy or redox power, and resistance to stress [31]. Furthermore, under optimal conditions, SC56 compared to Tx7000 exhibited unambiguous superior redox homeostasis (‘cell homeostasis,’ ‘cell redox homeostasis’ and ‘regulation of biological quality’) compared to Tx7000. In plants, ROS are continuously produced as a result of oxygen metabolism; if accumulated, ROS can lead to cell damage, but at certain levels have vital roles in cell signaling. Redox homeostasis is also crucial for proper cell functioning since various cellular signaling pathways regulating cell division and stress reaction systems are sensitive to redox imbalance [32]. The above results point to the superior biological status of SC56 that possibly predisposes this genotype to mount a more efficient response to drought stress. In particular, ‘cell redox homeostasis’ was also overexpressed in another stay-green genotype (B35) versus a senescent genotype under wet conditions [33], hinting at the possible importance of redox balance before stress onset in the of stay-green drought tolerance.
The outcomes of differential expression between SC56 and Tx7000 genotypes under drought also confirm a more efficient adaptation of SC56 to stress conditions that is marked by growth metabolism. While Tx7000_treat > SC56_treat showed no significantly enriched biological processes, SC56_treat > Tx7000_treat exhibited significantly enriched processes similar to those of SC56_cont > Tx7000_cont (‘biosynthesis,’ ‘translation,’ ‘amino acid and derivative metabolism,’ ‘ncRNA,’ ‘small molecule,’ and ‘tRNA’). Moreover, in conditions of water deprivation, SC56 displayed enhanced ‘transmembrane transport’ with the overexpression of 19 proteins compared to Tx7000. This trait of the drought-resistant genotype appears to be significant in the light of the increasing evidence that plant membrane transport systems play a substantial role in adaptation to drought [33-37]. Notably, the transmembrane transporter, Zinc-Induced Facilitator-Like 1 (ZIFL1) was overexpressed in SC56. A splicing isoform ZIFL1.3 of this gene was shown to mediates drought tolerance by regulating stomatal closure [38].
SC56 relies on a plethora of stress tolerance genes in response to drought
To decipher the molecular basis of the drought resilience of SC56, gene expression of SC56 plants was compared to the drought-sensitive Tx7000 plants in dry conditions. As discussed, SC56_treat > Tx7000_treat exhibited a significantly larger number of DEGs than Tx7000_treat > SC56_treat. The examination of these DEGs relative to the known Arabidopsis drought-responsive genes uncovered that the ampler response of SC56 to water shortage also comprised a greater count of such genes since 57 were found in S_treat > Tx7000 versus only 18 in Tx_treat > S_treat. Relative to other stress resistances, resistance to drought is very challenging to evaluate [39] since it is associated with a number of physiological, morphological, and molecular events. According to their function, drought-inducible genes can be classified into two groups. The first group encodes proteins that likely operate in stress tolerance and are referred to as ‘functional proteins’ while the second group referred to as ‘regulatory proteins’ [40] encodes factors involved in regulation of signal transduction and expression of genes putatively acting in stress response and are termed. With functional genomics advances, it has become evident that genes of both groups can confer stress tolerance.
Functional proteins
The functional proteins that were most frequently represented in our study are chiefly involved in enhancing the antioxidant capacity of SC56. Oxidative stress commonly occurs along with drought stress, causing lipid peroxidation, protein carbonylation, and DNA damage, which impairs their function and leads to deleterious effects on the cells [41]. Plants have thus evolved a series of enzymatic and non-enzymatic antioxidant defense mechanisms to maintain the homeostasis of the intracellular redox state. In this study, there was a stronger antioxidant machinery in SC56 compared to the sensitive line Tx7000 under severe drought stress, as apparent by the overexpressed antioxidation-related genes. Glutathione S-transferases are a family of isozymes with the ability to catalyze the conjugation of the reduced form of glutathione (GSH) to xenobiotic substrates for the purpose of detoxification. In this study, GSTU18, GSTU7, GSTT1, GSTT3, GSTZ2, ERD9, DHAR2 (discussed latter) and AT1G65820 (microsomal GST) were all upregulated in SC56. Aside from GSTs, superoxide dismutases are a powerful antioxidant family involved in destroying superoxide free radicals [42]. SOD1, which was upregulated in SC56, is a member of this family that was shown to enhance stress tolerance in plants [43]. Peroxidases are a class of proteins that breaks peroxides, and RCI3 is a member of this class that specializes in detoxifying hydrogen peroxide. RCI3, which was overexpressed in SC56, confers abiotic stress tolerance in plants [44, 45]. Tocopherols are lipophilic antioxidants synthesized exclusively by photosynthetic organisms and collectively constitute vitamin E. The enzyme VTE1, which was overexpressed in SC56 under drought stress is essential in the biosynthesis of tocopherols. In plants, tocopherols are synthesized in the chloroplasts where they protect membranes from oxidative degradation by ROS. VTE1 deficiency in Arabidopsis mutants leads to increased oxidative stress [46] whereas overexpression in tobacco prompts enhanced drought tolerance and increased chlorophyll levels [47]. Moreover, the simultaneous deficiency of VTE1 and GSH1 which is involved in glutathione biosynthesis results in oxidative stress that affects the stability and the efficiency of the photosynthetic apparatus [46]. In our study, glutathione metabolism and the biosynthesis of tocopherols were enhanced in SC56 under drought, hinting at better photosynthesis efficiency at least partly due to diminished oxidative stress.
The uncoupling proteins UCP1, UCP2, and UCP3 are a subgroup of the mitochondrial anion transporter family. The uncoupling of the mitochondrial electron transport chain from the phosphorylation of ADP optimizes the efficiency of oxidative phosphorylation and prevents generation of ROS by the respiratory chain. In plants, UCP1 is involved in maintaining the redox poise of the mitochondrial electron transport chain to facilitate photosynthetic metabolism. Disruption of UCP1 is associated with reduced photosynthetic carbon assimilation rate [48]. Furthermore, plants overexpressing UCP1 have better drought and salt tolerance and exhibit increased net photosynthesis, higher stomatal conductance, higher water retention and lower oxidative stress [49]. Thus, it appears that in conditions of abiotic stress, overexpression of UCP1 benefits the plant not only by alleviating the oxidative stress, but also by enhancing carbon assimilation. In the present study, UCP1 was upregulated in the drought-resistant SC56 genotype in conditions of water deficit.
Chloroplast-type ferredoxins (FDs) are electron transfer proteins that are involved in several metabolic processes including chlorophyll biosynthesis. FDs also participate in ROS scavenging by reducing the radical monodehydroascorbate to ascorbate. The ferredoxin isoforms FD1 and FD2, upregulated in SC56_treat > Tx7000_treat, are regulated by drought stress [50], and their knockout under heat stress was correlated to decreased ascorbate and adverse reactions to heat treatment, suggesting chloroplast FDs can confer stress tolerance [51].
Other functional proteins involved in drought tolerance in plants include proteinase inhibitors [52]. Cystatin B (CYSB) is one such protein for which transcription was upregulated in SC56 compared to Tx7000 under drought treatment. CYSB overexpression in transgenic yeast and Arabidopsis plants increases the resistance to high salt, drought, oxidative, and cold stresses [53].
Regulatory proteins
In the present study, several previously reported drought-response regulatory genes (MAPK1, CRK7, CRK23, HVA22, CIPK1, CRK4, and CRK23) were upregulated in SC56 compared to Tx7000 under water deficit. Some of these genes have been validated as determinants of tolerance to drought and/or other stresses in plant systems. For instance, CBL1 and CBL9 perceive the Ca2+ signaling that is triggered by drought occurrence. Both factors, then specifically interact with CBL-interacting protein kinase 1 (CIPK1) to regulate the stress response which results into drought tolerance - loss of function of either gene results into sensitivity to drought [54]. Remarkably, CIPK1 represents a convergence point for abscisic acid (ABA)-dependent and ABA-independent stress response since CLB1 and CBL9 mediate both mechanisms, respectively. In plants, receptor-like protein kinases (RLKs), of which cysteine-rich receptor-like kinases (CRKs) are a subfamily, play essential roles in signal transduction by recognizing extracellular stimuli and activating the downstream signalling pathways. In Arabidopsis, the transgenic overexpression of different CRKs (CRK5, CRK4, and CRK19) resulted into the enhancement of ABA sensitivity and drought tolerance [55]. The member of this subfamily, CRK7, that was overexpressed in SC56, was also reported to be involved in stress tolerance through a protective role against apoplastic oxidative stress [56].
SC56 triggers a negative regulator of senescence in response to drought
The stay-green trait reflects impaired or delayed chlorophyll catabolism and is divided into cosmetic stay-green, which is confined to pigment catabolism, and functional, in which the entire senescence syndrome, including chlorophyll catabolism, is delayed and/or slowed [57]. The senescence syndrome is a complex set of processes characterized by the decline of photosynthetic activity, an overall metabolic switch from anabolism to catabolism, the degradation of macromolecules, and nutrient remobilization [58]. The initiation and progression of senescence can be inhibited by the potent senescence antagonists, cytokinins, and this route was used to create cytokinin-mediated stay-greens [59,
60]. The senescence process can also be downregulated to produce stay-green phenotypes using mutated senescence-associated transcription factors [61, 62]. In the present study, the gene encoding senescence-associated E3 ubiquitin ligase 1 (SAUL1, also known as PUB44), which is involved in chlorophyll biosynthesis and catabolism, was found to be more highly expressed in SC56 compared to Tx7000 under drought. In plants, SAUL1 negatively regulates premature senescence and cell death, as mutants lacking SAUL1 display early senescence [63]. Similarly, in a more recent study, PUB12 and PUB13 which encode U-box E3 ubiquitin ligases where found to negatively regulate stress-induced leaf senescence [64]. In sorghum, the complexity of the senescence syndrome is likely reflected in the molecular basis of functional stay-green since this phenotype is a classic example of a quantitative trait with continuous variation [65]. A drought study [66] involving a sorghum population derived from SC56 x Tx7000 uncovered a total of 9 quantitative-trait-loci (QTLs) in different environments, of which 3 (Stg A, Stg G, and Stg J) overlapped with QTLs uncovered in B35 [67-69], the main source of stay-green in breeding programs. Thomas and Ougham [70] pointed out that the interactive nodes of transcriptional regulation, hormone- and ROS signaling, and sensors of environmental stresses that are associated with senescence offer massive number of junctures at which genetic modification can result in a stay-green phenotype, and constitute a rich source of variation for crop improvement. In this study, given the interconnectivity of drought tolerance networks and the high number of the obtained DEGs, SAUL1 might constitute one of several candidate genes that are significant for stay-green in SC56.
SC56 predisposed to drought tolerance under wet conditions
The overexpression of drought response genes in wet conditions before the onset of drought stress might predispose plants for a more efficient response to stress, which might be the case of the stay-green phenotype, since it was also observed in wet conditions [71-73]. SC56_cont > Tx7000_cont showed more than fifty Arabidopsis known drought-responsive genes with several shown to confer stress tolerance by transgenic overexpression or by knockdown (in Arabidopsis or tobacco), as discussed hereafter. Trehalose-6-phosphate synthase (TPS1), which is critical for the biosynthesis of the osmoprotectant trehalose was linked to dehydration tolerance [74]. Interestingly, TPS1 was also overexpressed under wet conditions in a study comparing sorghum stay-green line B35 to the senescent line R16 [33]. Similarly, the UB-like protease 1D (ULP1D) confers tolerance to different stresses [75, 76]. This protein is a deSUMOylating enzyme, which in plants is associated with developmental mechanisms and stress responses through the post-translational regulatory process of SUMOylation/deSUMOylation. Pyrophosphorylase 6 (PPa6), shown to be involved in drought tolerance [77], is part of a group of enzymes that catalyses the hydrolysis of PPi to Pi, which is central to many anabolic processes.
In SC56_cont > Tx7000_cont, many of the upregulated known stress tolerance genes are associated with protection against oxidative stress: the antioxidants superoxide dismutases SOD1 and SOD2 [42], vitamin C defective 1 (VTC1) [78], glyceraldehyde-3-phosphate dehydrogenase C subunit 1 (GAPC1) [79], monodehydroascorbate reductase 1 (MDAR1) [80], methionine sulfoxide reductase B 2 (MSRB2) [81] and ferritin 2 [82]. In relation to the enhanced growth capacity of SC56, possibly MSRB2 is especially relevant since, in Arabidopsis, plastidial MSRB1 and MSRB2 account for most leaf peptide MSR activity and have been shown to be essential for growth under environmental constraints due to their involvement in the preservation of the photosystem antennae [81]. In the context of the stay-green phenotype, ABC1-like kinase 1 (ABC1K1), which is upregulated in SC56 compared to Tx7000 under wet conditions, might be of particular interest since it is involved in modulating chlorophyll degradation directly by maintaining the number of Chl-binding photosynthetic thylakoid membranes and by playing a role against photooxidative stress [83]. The overexpression of numerous stress tolerance genes in non-stress conditions suggests that the transcriptional makeup of SC56 prior to the onset of stress might contribute to a strong and early stress response potentially involving a network of genes that culminate into high stress tolerance. However, despite the stress tolerance role of the abovementioned genes and despite their overexpression in SC56 under wet conditions, it is difficult to corroborate this role in this study since there was no differential upregulation in the drought treatment. Nevertheless, these genes might have been differentially expressed during an earlier time point of the drought stress that was not captured during the late drought response in these experiments (transcription levels measurement at 13 days post-drought, with an SMC of less than 10%).
Some genes that are possibly highly associated with drought resilience of SC56 would be those overexpressed under both wet and dry conditions compared to Tx7000. The scrutiny of the common DEGs in the relevant comparisons (S_cont > Tx_cont and S_treat > Tx_treat) revealed several stress response genes including glutathione transferases and heat-shock proteins. Most notably, copper/zinc superoxide dismutase 1 (SOD1), CBL-interacting protein kinase 1(CIPK1) and dehydroascorbate reductase 2 (DHAR2) were overexpressed in SC56 under both conditions and have been found to play a role in stress tolerance. As mentioned, SOD1 is a crucial ROS scavenging enzyme, and was shown to enhance oxidative stress tolerance via transgenic overexpression in tobacco [43] and in Arabidopsis [84]. Similarly, as discussed, CIPK1 is a regulatory protein that is a convergent point in ABA-dependent and ABA independent stress tolerance [55]. CIPK1 was also overexpressed in the stay-green line B35 compared to the senescent line R16 under wet conditions [33]. DHAR2 is a dehydroascorbate reductase (DHAR), which by reducing the oxidized form of ascorbic acid regulates its cellular redox state, and thus affects cell responsiveness and tolerance to environmental ROS [85]. The overexpression of DHAR2 in SC56 in comparison to Tx7000 is possibly a key element in the difference of drought tolerance between both lines. In a study using transgenic tobacco [85], suppression of DHAR caused a preferential loss of chlorophyll a, lower levels of the carbon fixing enzyme Rubisco, and a lower rate of CO2 assimilation that correlated with a slower growth and reduced foliar dry weight. In addition, premature leaf aging was observed in mature leaves as seen through an accelerated rate of loss of chlorophyll, Rubisco, light-harvesting complex II, and photosynthetic functioning. Conversely, DHAR overexpression sustained higher levels of chlorophyll, rubisco, light-harvesting complex II, and photosynthetic functioning while maintaining lower levels of lipid peroxidation, resulting in delayed leaf aging. Hence, by recycling ascorbic acid, DHAR possibly protects against ROS-mediated damage and affects the level of photosynthetic activity, thus influencing the rate of plant growth and leaf aging.