Physiological Mechanism, Stress-Specific Proteins For The Tolerance To Combined Stress Of Drought And Salinity In Tibetan Wild Barley

Drought and salinity are the most important abiotic stresses limiting agricultural production worldwide. Meanwhile, the two abiotic stresses frequently co-occur in both natural and agricultural ecosystems. Therefore, an improvement in drought and salinity tolerance in crops is a pre-requisite for achieving economic gains. The best and most effective approach in this regard is to develop drought-and salinity-tolerant crop varieties. It is therefore important to identify the genetic resources that have high tolerances and to understand the mechanisms of drought and salinity tolerance in plants. Barley (Hordeum vulgare L), as feed, malt and human food, is the fourth important cereal crops in the world. Although barley is superior in terms of drought and salinity resistance to the most other cereal crops, its growth and production is also greatly affected by these two stresses. Annual wild barley from Qinghai-Tibet Plateau is one of the progenitors of cultivated barley and rich in genetic diversity for the use in crop improvement. Accordingly, the present study was carried out to elucidate the physiological mechanism in the tolerance to combined stress of salinity and drought in Tibetan wild barley, and to identify specific proteins for the tolerance to the combined stress through proteomics analysis. The main results were summarized as follows:1. Genotypic differences in antioxidative metabolism, water use efficiency and Na+/K+ratio in Tibetan wild and cultivated barley in response to drought and salinity combined stressGreenhouse pot experiment were conducted to investigate genotypic differences in response to individual and combined stresses of drought and salinity between Tibetan wild barley genotypes (XZ5, drought tolerant; XZ16, salinity/Al tolerant) and cv. CM72(salinity tolerant). The results showed that either drought (D) or salinity (S) alone and combination (D+S) decreased plant growth, chlorophyll content, net photosynthetic rate (Pn), stomatal conductance (gs), intercellular CO2concentration (Ci), transpiration rate (Tr) and maximal photochemical efficiency of PSII (Fv/Fm), with the largest suppression under combined stress. Under D+S stress, two wild genotypes XZ5and XZ16were less suppressed than that of CM72, with plant growth, Pn and Fv/Fm being less suppressed than in CM72. Water use efficiency (WUE) increased significantly in XZ5and XZ16on the35th and45th day after D+S treatment compared to control, but no significant change was observed in CM72. Meanwhile, XZ5and XZ16(especially XZ5) showed30.9%and12.1%higher leaf K+level and30.5%and24.1%lower Na+/K+ratio, when compared with CM72, with increased metal nutrients such as Ca, Fe and Mn under D+S. Moreover, much more increase in activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), guaiacol peroxidase (POD), ascorbate peroxidase (APX), and glutathione reductase (GR) under D+S vs control were observed in XZ5and XZ16than that of CM72, with less accumulation of malondialdehyde (MDA) and H2O2. These results suggested that high tolerance to D+S combined stress of XZ5and XZ16is closely related to lower Na+/K+ratio and enhanced WUE, and increased capacity of antioxidative performance to scavenge reactive oxygen species (ROS) and thus suppressed level of lipid peroxidation.2. Genotypic differences in ATPase, ultrastucture and some osmolytes under drought and salinity combined stress in Tibetan wild and cultivated barleyDrought and salinity alone and in combination significantly decreased water potential and osmotic potential in leaves with the less decrease in the two Tibetan wild genotypes. There is a significant difference in proline, soluble sugar and glycine-beatine between two XZ5/XZ16and CM72especially under combined stress. The highest accumulation in proline and glycine-beatine was recorded in combined stress, and soluble sugar content in drought alone with the large accumulation in two wild genotypes. In addition, XZ16showed significantly higher leaf soluble protein than XZ5and CM72under drought, salinity alone and combined stress when compared with control. Moreover, the contents of non-enzymatic antioxidants i.e reduced glutathione and ascorbate increased by20%and35%in XZ5, respectively, under combined stress, when compared with cv. CM72. Meanwhile, the activities of H+-, Ca2+/Mg2+-ATPase under combined stress in XZ16was significantly higher than in CM72but similar with XZ5. Obvious ultrastructural alterations were observed in all genotypes under drought, salinity alone and D+S treated leaf and root tip cells.3. Comparative proteomic analysis in response to combined stress of drought and salinity between Tibetan wild and cultivated barleysTwo Tibetan wild genotypes (XZ5, XZ16) and cv. CM72were subjected to drought (4%soil moisture, D), salinity (S), or a combination of both treatments (D+S) during vegatative stage. The proteomic analysis revealed that more than1200protein spots were reproducibly detected in each gel. Thirty-four proteins related to tolerance to drought and salinity alone or a combination were identified by MS. Out of these34differentially expressed proteins (DEPs),15spots (drought),8(salinity) and11(D+S) DEPs were treatment-specific. Differentially regulated proteins predominantly had functions in photosynthesis, but also in detoxification, energy metabolism, and protein biosynthesis. The analysis indicated that photosynthetic electron transfers proteins, chaperone-like proteins and enzymes of ROS homeostasis play important roles in adaptation to combined stress. In addition, genetic variation identified in the proteome in response to drought and salinity represent stress adaption mechanisms in Tibetan wild barley to be exploited in future crop breeding efforts.4. Differential changes in leaf secondary metabolism between Tibetan wild and cultivated barleys under drought and salinity alone and combination and subsequent recoveryGenotypic difference in secondary metabolites and their related genes were studied in response to the separate and combined stresses of drought and salinity and subsequent recovery. The determination of callose content and the activity of chitinase, sucrose synthase (SuSy), sucrose phosphate synthase (SPS) and acid invertase (AI) showed a greater increase in XZ5and XZ16under all stress treatments than in CM72. Meanwhile, content of phenols, flavonoids, and antioxidant activity were also increased by all stress treatments in both XZ5and XZ16. In addition, those parameters still gained increase in XZ5under D+S during recovery. The expression of secondary metabolism related genes (GST, PPO, SKDH, PAL, CAD and chitinase genes) were induced by all stress conditions in wild barley and accompanied with increase in the activities of respective enzymes and with the maximum increase in XZ5. However, the contents of all enzymes, except polyphenol peroxidase (PPO) and glutathione-S-transferase (GST) were decreased in XZ5during recovery. Microscopic imaging of DNA in leaves exhibited damage with increasing tail moment under all stress treatments; however, XZ5and XZ16were less affected than CM72.5. Difference in yield and physiological features in response to drought and salinity combined stress during anthesis in Tibetan wild and cultivated barleysGreenhouse pot experiments were conducted to investigate the tolerance potential and mechanisms of Tibetan wild barley genotypes compared with salinity-tolerant cv. CM72in response to separate and combined stresses of drought and salinity during anthesis. Under salinity stress alone, plants had higher Na+concentrations in leaves than in roots and stems. Importantly, XZ5and XZ16had substantially increased leaf K+concentrations; XZ16was more efficient in restricting Na+loading in leaf and maintained a lower leaf Na+/K+ratio. Moreover, a significant decrease in cell membrane stability index (CMSI) and an increase in malondialdehyde (MDA) were accompanied by a dramatic decrease in total biomass under D+S treatment. Glycine-betaine and soluble sugars increased significantly in XZ5and XZ16under all stress conditions, along with increases in protease activity and soluble protein contents. Significant increases were observed in reduced ascorbate (ASA) and reduced glutathione (GSH) contents, and in activities of H+K+-, Na+K+-, Ca++Mg++, total-ATPase, and antioxidant enzymes under D+S treatment in XZ5and XZ16compared to CM72. Compared with control, all stress treatments significantly reduced grain yield and1000-grain weight; however, XZ5and XZ16were less affected than CM72. Our results suggest that high tolerance to D+S stress in XZ5and XZ16is closely related to the lower Na+/K+ratio, and enhanced glycine-betaine and soluble protein and sugar contents, improved protease, ATPase activities and antioxidative capacity for scavenging reactive oxygen species during anthesis.6. Differential changes in grain ultrastructure, amylase, protein and amino acid profiles between Tibetan wild and cultivated barleys under drought and salinity alone and combined stressGrain phytochemical profiles were compared in Tibetan wild barley XZ5, XZ16and cv. CM72in response to drought and salinity alone and in combination during anthesis. The total antioxidant capacity (assessed by determining ferric-reducing antioxidant potential, FRAP) in the grains increased significantly in the order as follows:D+S> drought> salinity, and XZ5> XZ16> CM72. In addition, a marked increase in the total phenol (TP) from individual and combined stresses was observed in XZ5, while a decrease occurred in CM72. Moreover, the activity of α-/β-amylase in the grains under combined stress was81.8%/16.9%in XZ5and48.6%/18.7%in XZ16higher than that of CM72. Increases in amino acids, protein content and protein fractions of albumin, globulin, hordein and glutelin were maximized under D+S, with larger values in the Tibetan wild genotypes. Observation with a scanning electron microscopy showed a distinct genotypic difference under D+S; for example, XZ5and XZ16maintained a relatively integral starch granule with a greater protein deposit/matrix, while CM72degraded by pitting. This research expands our understanding of barley drought and salt-tolerance mechanisms and provides possibility of Tibetan wild barley in developing barley cultivars with both tolerance to drought and salinity.