Drought stress tolerance strategies revealed by RNA-Seq in two sorghum genotypes with contrasting WUE
Drought tolerance strategies
Drought stress results in a massive production of reactive oxygen species (ROS) [17, 18] 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. [36]. The antioxidant enzymes constitute the “first line of defence” against ROS and oxidative stress generated by different abiotic and biotic injuries [37, 38]. The activity of these enzymes can be enhanced or repressed depending on the species, genotype, stress duration and severity [39–41]. In the “response to abiotic stimulus” (GO:0009628), “oxido-reductase activity” (GO:0016491) and “response to stress” (GO:0009628) gene ontology categories, genes were more greatly down-regulated by drought in the sensitive genotype IS20351 than in the tolerant IS22330, enabling us to speculate that the tolerant IS22330 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 [42].
To cope with the oxidative stress caused by drought, genes coding for secondary metabolites such as phenylpropanoids, phenolic compounds and flavonoids, are overexpressed [43]. Phenylpropanoids have the greatest potential to reduce ROS, the polyphenols act as antioxidants to protect plants against oxidative stress [44], flavonoids play different molecular functions, including stress protection in plants [34], and also flavanols were found to be oxidated in response to severe drought in tea plants, suggesting their involvement in plant protection [45]. All these compounds are widely synthetized in response to several abiotic stresses, including drought [46–50]. 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 [51, 52]. With our study, we confirm that under drought stress the up-regulation of these genes in the sensitive genotype IS20351 was higher than in the tolerant genotype IS22330, whilst a constitutively higher expression of these genes in the tolerant IS22330 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 [53].
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 [54–57]. Agati et al. [42] found that flavonoid genes were up-regulated in response to drought in the sensitive genotype IS20351 whilst they were mostly down-regulated in the tolerant IS22330. The biosynthesis of “antioxidant” flavonoids, in fact, increases more in stress sensitive species than in stress tolerant ones [42]. 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 [58, 59]. In any case, the relationship between antioxidant enzymes and flavonoids in response to abiotic and biotic stress it is not yet well clarified [42].
Drought stress induces a decrease in the chlorophyll content, a consequential change in the chlorophyll/carotenoid ratio [60] 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 [19–21]. All these stress-induced physiological modifications (qNP and Pn) were observed to a greater extent in the sensitive genotype IS20351. 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 IS20351 and not in IS22330, in agreement with the decreased concentration of some carotenoids under severe drought stress [17, 38, 61].
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 IS20351. The decreased expression pattern mainly involved the light harvesting complex I and II and polypeptide subunits of the photosystems (I and II). In particular, the light-harvesting chlorophyll a/b-binding proteins (LHCBs) were extremely down-regulated in the sensitive genotype IS20351 according to several studies in which the down-regulation of LHCBs reduces plant tolerance [62–65]. The LHCBs, complexed with chlorophyll and xanthophylls, form the antenna complex [66] and play an important role in adaptation to environmental stress [63–65]. Their expression is regulated by multiple environmental factors including light [67], oxidative stress [68, 69] and abscisic acid (ABA) [70]. Also the genes involved in the “carbon fixation” were more greatly down-regulated in the sensitive genotype IS20351 rather than in the tolerant one. The up-regulation of Sb03g040610.1 was the main exception in the expression pattern of this genotype; this gene codes for the electron carrier ferrodoxin. Comparing the Log2 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 (5.2 and 3.4 for IS20351 and IS22330, respectively). This result indicates that the tolerant genotype IS22330 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 IS20351 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 (inactive osmotically) content decreases, whilst content of soluble sugars (osmotically active) increases, assuring the maintenance of leaf water status and plant growth [23–25]. In the sensitive genotype IS20351, starch synthases were down-regulated and enzymes involved in the degradation of starch and sucrose up-regulated. According to Sturm and Tang [71] invertases play a role in several processes ranging from phloem loading to response to abiotic and biotic stresses [23, 72]. Exogenous ABA applied in soybean green beans [73] and maize leaves exposed to drought [74] showed an increase in invertase activity. Gazarrani and McCourt [75] 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 [76–78]. Furthermore, sucrose and other sugars are energy and carbon sources required for defence response and are necessary for plant survival under drought stress conditions [79]. 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 [80, 81]. The accumulation of wax has a key role in limiting water losses from plants [82]. It is widely accepted that drought stress can increase the amount of wax in several species [83–87] and that this increase is associated with an improved drought tolerance [88]. According to our results, the sensitive genotype IS20351 up-regulated these genes in response to drought; on the contrary, the drought tolerant genotype IS22330 remained unchanged. The hypothesis is that the tolerant genotype IS22330 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. [89], no changes in omega-3 desaturase expression were highlighted in response to drought in the tolerant genotype IS22330. On the contrary, the omega-3 desaturases were down-regulated in the sensitive genotype IS20351 [89].
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. [90]. Until that moment only the implication of sphingolipids in conferring stability to plant membranes, contributing to acclimation to drought stress had been hypothesized [91]. Spiegel and Milstien [92] 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 IS20351 that down-regulated sphingolipids in response to drought, except for a ceramidase (sb03g028410.1).
In cowpea leaves a massive breakdown of membrane lipids was observed in response to drought with a more severe degradation in the sensitive plants [93]. The main enzyme responsible for the drought-induced degradation of membrane phospholipids is phospholipase D (PLD) [94]. According to El Masouf et al. [95], the drought sensitive genotype IS20351 strongly up-regulated the PLD expression, whilst in the drought tolerant IS22330 the expression was only slightly up-regulated. Recently, PLD up-regulation was associated to drought and salt stress tolerance [96–99] and the product of its activity, the phosphatidic acid, is involved in ABA signalling in stomatal movement [100]. PLDa1, in particular, is the most predominant PLD in plants activated by ABA [101].
Some interesting genes provided insight into the drought tolerance of the genotypes analysed. The Sb06g014320 gene, encoding for a glycerophosphodiester phosphodiesterase, found to be up-regulated in response to drought in sorghum leaves [12], was strongly down-regulated in response to drought in the sensitive genotype IS20351. The Sb07g027910 gene, encoding for a monogalactosyl-diacylglycerol (MGDG) synthase, found to map to a stay green QTL [102] and to be overexpressed in response to drought in sorghum leaves, was down regulated in the sensitive genotype IS20351. 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 IS20351. A confirmation of the drought tolerance of IS22330 was the overexpression of genes related to the phosphatidylinositol biosynthesis, such as sb08g016610, sb08g022520 and sb05g026855.