Drought stress is the most prevalent and prominent abiotic stress influencing plants growth and development [2]. In cereals, water deficit affects many morphological, biochemical and physiological parameters. Leaf and root growth are inhibited under moderate and severe stress [35]. Drought stress affects photosynthesis, water relations, nutrients uptake, oxidative status, osmotic balance and hormonal balance which impact the yield [3],[18]. Therefore, to ensure food security, the selection of tolerant varieties becomes indispensable. In this study, Moroccan varieties of barley (Hordeum vulgare L.) were screened for physiological and biochemical characteristics to select varieties able to cope with drought stress.
The results showed a significant intraspecific variability regarding the adaptation to drought conditions, as reported in many papers studying barley growth under drought stress [36],[12],[20],[37]. RWC is a critical physiological criterion for determining the degree of tissue and cell hydration required for optimal physiological growth and functioning in plants [2],[38]. Preservation of high value of RWC under drought stress indicates an important drought tolerance [39],[40]. In our results, a significant reduction of RWC was noticed under drought stress in leaves of barley varieties. Similar results were reported in other studies when barley plants were exposed to water deficit [10], [11], [12], [13]. As shown in Fig. 1A, RWC of ADRAR, AMALOU, AMIRA, FIRDAWS and TAMELLALT are the most influenced by drought stress. This means that these varieties are less able to keep cell turgor in their leaves under drought stress. However, LAANACEUR, MASSINE, OUSSAMA, and TAFFA are less influenced, meaning high capability of these varieties to keep cells turgor in their leaves under drought conditions.
Drought stress induced a significant decrease of chlorophyll content (SPAD value) as shown in Fig. 1C. Such results were also reported by other studies dealing with cereals [2], [41].Chlorophyll reduction may be due to electrolytes leakage from thylakoïds membrane and lipids peroxidation [2], protoplasm dehydration, and less photo-assimilation level [9]. The increase of electrolytes leakage in leaf tissues is considered as an index of membrane damage and deterioration [42] and as the parameter indicating membrane steadiness [43]. The increase of EL percentage under stress conditions is always associated with sensitivity of plant to oxidative stress [44]. In our findings, different levels of increase of EL% were noted, which is in agreement with the literature [9], [14]. As presented in Fig. 1B, TAMELLALT variety was the most influenced by water deficit regarding electrolytes leakage, followed by FIDRAWS, TAFFA, AMIRA and ADRAR. This indicates membrane cell damage in the tissues of those varieties under drought stress. However, for the other varieties, there was no significant impact of drought stress on their EL% indicating an important cell membrane steadiness under drought stress and high tolerance.
Under drought conditions as other abiotic stresses, the accumulation of proline is common in most of cereals [9] [18], [45], [46]. This molecule is an important osmoregulator for membrane stability, buffering cellular redox potential, and scavenging free radicals [18]. Proline can also play an important role in activation of detoxification pathway [47], which makes varieties with ability to produce more proline under stress, to be more tolerant. This was the case in FIRDAWS, LAANACEUR, MASSINE, and OUSSAMA. These varieties accumulated the highest levels of proline under drought stress as shown in Fig. 2A, elucidating that these varieties are more resistant to drought stress. The accumulation of proline is widely reported as a known response in stress tolerant plants. Many roles have been attributed to this molecule regarding its involvement in plant tolerance to abiotic stress, but the accurate mode of its action is still unclear. It is also important to keep in mind that high levels of proline could have negative impact on plant cells. The ability to maintain the intracellular proline content in balance seems to be essential for plant tolerance and survival [48]. Actually, Proline levels in the cell are determined by the balance between biosynthesis and catabolism. The proline is synthesized from ornithine and glutamate, the glutamate pathway being more predominant in the plant cell [48]. The glutamate is converted to pyrroline 5-carboxylate (P5C) by the action of the pyrroline-5-carboxylate synthase (P5CS). The intermediate P5C is then reduced to proline by the P5C reductase (PC5R). Under stress conditions, transcriptional activation of P5CS and PCR genes was reported to increase while less impact was recorded in the ornithine route [49]. P5CS accumulates in the chloroplasts, leading to enhanced proline biosynthesis [50]. Two forms of P5CS exist in plant cell. P5CS1 which is chloroplastic and involved in the stress induced proline biosynthesis and P5CS2 is responsible for development processes [51]. In the other hand, the level of proline content in the cell is regulated by the action of proline dehydrogenase (proDH) and pyrroline 5-carboxylate dehydrogenase (P5CDH). These enzymes are transcriptionally regulated by developmental and environmental signals and proline catabolism is enhanced during recovery period from stress [49]. The level of accumulation of proline is reported to vary markedly between species and between genotypes within species [52], [53]. This could be linked to the complexity of the signaling networks and the multigenic processes involved in the regulation of the proline biosnynthesis pathway and its accumulation in plant cells under environmental constraints. In Medicago trancatula, Helianthus annuus, and Sesamum indicum plants, it has clearly been reported that the extent of oxidative stress varies among genotypes within a species, when submitted to stress conditions [54].
As shown in Fig. 2B, except AMALOU, AMIRA, and MASSINE, other varieties recorded an increase of TSS content. For proteins content, a great increase was marked under drought stress in all varieties (Fig. 2C), which is in agreement with literature results where tolerant barley varieties accumulates sugars and proteins in their leaves under drought stress [44]. The accumulation of soluble sugars and proteins is associated with osmotic regulation of cells. TSS and proteins accumulation decreases osmotic potential inside the cell, making cells able to keep a high turgor potential [44],[55] and stabilize cell membrane by reacting with lipid bilayer [20].
Based on biochemical, molecular, and genetic findings, it has been determined that soluble sugars play a critical role in web regulation of plants adaptation against biotic and abiotic stresses [56]. In last years, sugars are more studied for their hormone-like functions, as a primary messenger in signal transduction [57]. Glucose is widely known with his role as a modulator in repression of genes implicated in ABA catabolism and activation of genes implicated in ABA biosynthesis [58]. Moreover, It’s proved that high level of sugars lead to the repression of the genes implicated in photosynthesis [59],indeed, it’s well documented that the repression of Rubisco small subunit (RBCS) gene is associated with high sugars concentration in potato and maize [56], [60]. Furthermore, sugars accumulation is linked with down regulation of many genes implicated in photosynthesis such as atp-δ thylakoid ATPase (ATP- δ) gene, chlorophyll a/b binding protein (CAB) gene, pyruvate phospho dikinase (PPDK) gene, C4 malic enzyme gene (ME1) and C4 PEP carboxylase (PEPC1) gene [56]. In 2012, Hu et al, [61]showed that proline accumulation varies increasingly with glucose concentration applied on wheat plants under salt stress. This can help plant to be more tolerant to abiotic stress by increasing proline content, with proline functions cited above.
Under drought conditions increases of MDA and hydrogen peroxide contents in leaves were observed (Fig. 3). This was also the case of other studies [19], [20]. Various environmental stresses including drought constraint induce the generation of reactive oxygen species (ROS), which can lead to the oxidation of DNA and proteins, the peroxidation of membrane lipids, hydrogen peroxide accumulation and oxidative brust cell [62], [63]. In the other hand, the lipid peroxidation could be triggered by an increased lipoxygenase activity [18]. Both enzymatic and non-enzymatic processes are reported to be involved in the formation of lipid peroxidation products in plants such as MDA and jasmonates under oxidative stress conditions [64]. In cereals, low H2O2 and MDA contents are associated with high stress tolerance ability [18]. Actually, the role of aldehyde compounds (MDA) produced under stress environmental or developmental signals depends upon their accumulation levels which are controlled by the balance between the lipid peroxidation intensity and activity of aldehyde dehydrogenases (ALDHs). It is well established that the expression and activity of ALDHs are induced by H2O2, abscissic acid and MDA [36]. These molecules could serve as signals of protection processes where ALDHs contribute to maintain the cellular redox homeostasis and reducing potential NADPH required for antioxidant activity of the ascorbate-glutathione cycle and photosynthesis process [65], [66]. In our study, MASSINE, OUSSAMA and TAMELLALT varieties are characterized both by lower values of H2O2 and MDA contents compared with other varieties, which makes them less affected by ROS and/or lipoxygenase under drought conditions. The lower content of MDA in plant cells could be interpreted as a defense mechanism signaling rather than an indicator of membrane damage and protein carbonylation [64]. When MDA accumulated in the cells at high level, proteins are carbonylated, such as PSII core proteins and Rubisco, leading to disturbances in all plant cell metabolism which may trigger cell death. All previous published results converge towards the synthesis and involvement of MDA in plant metabolism under environmental stress. However, the role of this molecule remains unclear and needs more investigations.
It is widespread in the literature that drought tolerance is associated physiologically with high values of RWC and proline content [2], [18], [38], [67]. In our results, the biplot created (Fig. 5) discriminates MASSINE and OUSSAMA as the two varieties with higher percentages of RWC, and FIRDAWS is discriminated as the variety that accumulated most of proline in their leaves under drought stress. Also, the agglomerative hierarchical cluster analysis (Fig. 5) classified these varieties with LAANACEUR in the third class considered including varieties showing high physiological tolerance against drought stress. Furthermore, high scores of H2O2, MDA contents [18], [19], [20], and EL% [9], [42], [43] were always considered as signs of plants sensitivity against abiotic stresses. The biplot of our results (Fig. 5) discriminates TAMELLALT with high percentage of electrolytes leakage, ADRAR with high value of MDA content, AMALOU and AMIRA with high scores of H2O2 content. Except AMIRA, using agglomerative hierarchical cluster analysis, these varieties were classified in the first class of sensitive varieties under drought stress. AMIRA is classified alone in the second class showing more similarity with the first class (Fig. 5).