Assessing genetic variability and physiological traits conferring drought stress tolerance in cereals

Drought is the most devastating environmental stress affecting plant growth and productivity. Almost half of the terrestrial land surface in the world i.e., 6.45 billion hectares, is composed of dry land. Global warming is expected to further exacerbates the intensity of drought and could result in significant (over 75%) losses in agricultural production worldwide. Wheat and barley are amongst most important cereal crops in the world. Wheat is moderately drought tolerant while barley is classified as relatively drought tolerant. Given the extent of dry land in the world and predicted population growth to 9.3 billion by 2050, creating drought tolerant barley and wheat germplasm is the ultimate priority of the plant breeders. However, the progress in breeding for drought tolerance is significantly handicapped by the lack of convenient and reliable phenotyping methods to screen a large germplasm. Drought tolerance is a complex trait. As a result of a large genetic diversity of barley and wheat, plants show a plethora of morphological, physiological and biochemical responses to drought stress and rely on different adaptive mechanisms. These adaptive mechanisms include mainly stomatal regulations, signalling pathways, root related traits and osmoregulation. Different plant species cope with drought stress either by closing their stomata to prevent water loss or maintaining relative water content by osmotic adjustment by increased accumulation of organic and inorganic osmolytes. Taken together, it remains still elusive which trait should be targeted for drought tolerance in barley and wheat. Addressing the above issues, thirty varieties of barley (Hordeum vulgare L.), 18 bread wheat (Triticum aestivum L.) and two durum wheat (Triticum durum) varieties were collected from different geographical locations for the present study. Two different type of glasshouse experiments were performed. The first experiment was conducted in large tanks applying drought treatment to plants at three to four leaf stage by withholding irrigation for seven weeks. The plants were evaluated based on the visual damage at 3rd, 5th and 7th week of drought imposed and a visual score (0-10, 0= no visual symptoms to stress and 10= all plants are dead) was given to the plants by counting the number of chlorotic and necrotic leaves. The second experiment was performed in the pots and at two to three leaf stage, when seedlings were subjected to three irrigation regimes: control (100% field capacity) and two stress treatments (25% and 12% of full field capacity). After six weeks of drought imposed, plant agronomical (shoot fresh weight, shoot dry weight) and physiological (chlorophyll content, chlorophyll fluorescence, stomatal conductance and relative water content) characteristics were measured. We also assessed the suitability of different screening techniques to determine drought tolerance. Visual evaluation based on drought damage index provided a simple and feasible approach to measure the tolerance to water stress as it does not require any special equipmental expertise and can be used for screening on a large scale. SPAD and Fv/Fm measurements were quick and non-invasive and deemed as suitable indices for measuring drought tolerance. However, maintaining field capacity on daily basis in the pot experiment was labour-intensive and time-consuming job and not recommended for a large-scale screening. Based on drought damage index (DDI), barley genotypes were divided into four groups i.e., tolerant, moderately tolerant, moderately sensitive and sensitive. In the second experiment, biomass and physiological traits were evaluated. Stomatal conductance, chlorophyll content (SPAD), chlorophyll fluorescence (Fv/Fm), relative water content (RWC) were significantly reduced for the plants grown under 25% and 12% of full field capacity. The genotypes showed similar trend for drought tolerance and susceptibility in both the experiments. Cultivars Numar, Flagship, ZUG293 and X026 were referred as highly drought tolerant (DDI <6.5 and accumulated high biomass) and Franklin and Gairdner (DDI >9 and accumulated low biomass) as highly drought sensitive. A significant correlation was found between shoot dry weight of plants grown under 12% field capacity and chlorophyll content (SPAD), chlorophyll fluorescence (Fv/Fm), stomatal conductance (Gs), relative water content (RWC) and fresh weight (FW) of plants grown under 25% and 12% field capacity irrigation regime. In wheat, significant genotypic differences were observed among all genotypes under drought stress. Based on both screening experiments, genotypes Albidum24, Tainong292 and Mahon Demias were classified as drought tolerant with DDI<6.5, and relatively high biomass, SPAD and Fv/Fm values. Genotypes Onohoiskaja4, Kord Cl Plus (DD1>7.5) and Zhemgmai9023 (DDI>9) had lowest biomass and relative water content under severe stress (12% of full field capacity) and were deemed as drought sensitive. Another finding of note was that barley showed rather different strategies dealing with drought as compared with wheat. In wheat, the tolerance was achieved by closing stomata (low relative Gs values- 19% to 0.6%) while in barley, the plants were able to open their stomata (high relative Gs values- 23% to 0.7%) for a longer time and maintained higher water content due to more efficient osmotic adjustment. Based on a large screening of barley genotypes for their drought tolerance, seven contrasting genotypes (Numar, ZUG293-tolerant, Commander, Fleet, X123- moderately tolerant, Franklin, Gairdner- sensitive) were selected for the third experiment. The experiment was conducted under glasshouse conditions, with genotypes grown under 100%, 25% and 12% field capacity to study various physiological traits and linking the overall drought tolerance with changes in plant water related traits, stomatal characteristics and the contribution of organic and inorganic osmolytes towards the root and shoot osmotic adjustment. The overall drought tolerance was positively correlated with root length, stomatal conductance, relative water content, stomatal density, root K\\(^+\\), root Cl\\(^-\\), leaf Cl\\(^-\\), total soluble sugars, total amino acids of plants grown under 12% field capacity irrigation regime. However, leaf water potential, leaf K\\(^+\\) and Na\\(^+\\) content of plants grown under 12% field capacity irrigation showed no correlation with drought tolerance. Taken together, these results suggest drought tolerant genotypes had higher root K\\(^+\\) and Cl\\(^-\\) and organic osmolytes content as well as high stomatal conductance (Gs), stomatal density (SD), relative water content (RWC) and root length (RL) under drought stress. The relative contribution of inorganic K\\(^+\\), Na\\(^+\\), Cl\\(^-\\)) and organic osmoles (total soluble sugars-TSS, total amino acids-TAA) towards the leaf and osmolality of plants under drought stress was in the order Cl > K > TSS > TAA > Na. The relative contribution by Cl\\(^-\\) was also the highest towards the root osmolality, followed by K\\(^+\\) and Na\\(^+\\). Abscisic acid (ABA) regulates various molecular events in response to water deficit in plants. To elucidate the abscisic acid mediated signalling in barley roots and ionic mechanisms of osmoregulation, the non-invasive ion-selective microelectrode measurements (the MIFE technique) was used. Transient ion fluxes in response to hyperosmotic stress were compared for seven barley genotypes contrasting in drought tolerance (Numar, ZUG293-tolerant, Commander, Fleet, X123-moderately tolerant, Franklin, Gairdner- sensitive). All the genotypes had uptake of K\\(^+\\) and Cl\\(^-\\) under hyperosmotic stress (200mM mannitol) in the root mature zone and net uptake of K\\(^+\\) and Cl\\(^-\\) was positively correlated this drought tolerance of the cultivar. However, there was an efflux of Na in response to hyperosmotic stress. Long term (48 hours) of hyperosmotic stress caused further increase in the uptake of K\\(^+\\) and Cl\\(^-\\) in drought tolerant genotypes. Another set of experiments was performed to measure membrane potential in the root mature zone. Drought tolerant genotypes were able to maintain more negative membrane potential values as compared to moderately tolerant and sensitive genotypes. Abscisic acid application increased Cl\\(^-\\) uptake in the roots of ZUG293 and Franklin whereas there was no significant effect of ABA on K\\(^+\\) and Na\\(^+\\) in both the genotypes suggesting the role of ABA in regulating ions in roots is opposite to the role of ABA in guard cells. Overall, this work has screened and identified barley and wheat genotypes contrasting in their drought tolerance. These genotypes are recommended for mapping double haploid (DH) population to reveal the QTLs responsible for drought tolerance in these species. We also found that barley has rather different drought tolerance mechanisms compared to wheat. This study also recommended some rapid and convenient screening methods to screen a large germplasm for drought tolerance. Inorganic osmolytes mainly Cl and K made the highest contribution to root and leaf osmolality. Leaf K\\(^+\\) and Na\\(^+\\) did not correlate with drought tolerance, however, root K\\(^+\\) and Clcorrelated with drought tolerance. Therefore, we could recommend breeders to select genotypes which are drought tolerant based on root inorganic ions such as K\\(^+\\) and Cl\\(^-\\). Hyperosmotic stress induced by mannitol caused a significant uptake of K\\(^+\\) and Cl\\(^-\\) in drought tolerant genotypes compared to moderately tolerant and sensitive suggesting the activation of HvAKT1 uptake channels and HvHAK1 (HAK/KUP/KT transporters) in tolerant genotypes, as evident from the fact that they were able to maintain more negative membrane potential under hyperosmotic stress. Both HvAKT1/HvHAK1 activated by membrane hyperpolarization brought about by increased uptake of Cl\\(^-\\). Another im...