| Drought is one of major meteorological disaster constraining crop productivity worldwide. One of the cost-effective and efficient approaches is to develop drought resistant/tolerant crop cultivars to ensure agricultural production. To breed crop cultivars tolerant to drought stress, it is important to identify genetic resources in potential of drought tolerance and understand its mechanism. Tibetan annual wild barley (H. vulgare ssp. spontaneum and ssp.agriocrithum) posses rich in genetic diversity and specific genes associated with abiotic-tolerance for the improvement of modern barley cultivars. The present study was carried out, using drought-tolerant Tibetan wild barley genotype (XZ5) identified in our previous research, to identify stress-specific proteins for drought tolerance in wild barley via two-dimensional gel electrophoresis and mass spectrometry and bio-informatics. According to the data of proteomic analyses, coding sequences (CDS) of drought tolerance relevant genes were cloned from different wild barley geneotypes (XZ5and drought-sensitive XZ54) and cv. ZAU3by degenerate PCR, followed by databases homologous searches. Meanwhile, we investigated the protective effect of foliar-application of betaine against drought-induced growth and photosynthesis inhibition, oxidative stress and damage in ultrastructure in barley seedlings. The main results were summarized as follows:1. Comparative proteomic analysis of drought tolerance in the two contrasting Tibetan wild barley genotypes and cv ZAU3sA greenhouse pot experiment was performed to compare proteomic characteristics of two contrasting Tibetan wild barley genotypes (drought-tolerant XZ5and drought-sensitive XZ54) and cv. ZAU3under drought stress at soil moisture content10%(SMC10) and4%(SMC4) and subsequently2days (R1) and5days (R2) of recovery. More than1700protein spots were identified in each gel. We identified38drought-tolerance-associated proteins, including Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) and Thioredoxin M (Trx-M), through mass spectrometry and data bank analysis. These proteins were mainly categorized into photosynthesis, stress response, metabolic process, energy and amino-acid biosynthesis. Specifically,6protein spots were exclusively expressed or up-regulated under drought stress in XZ5but not in XZ54, including melanoma-associated antigen p97, type I chlorophyll a/b-binding protein b, glutathione S-transferase1, ribulosebisphosphate carboxylase large chain. Moreover, type I chlorophyll a/b-binding protein b was specifically expressed in XZ5but not in either XZ54or ZAU3. These proteins may play crucial roles in drought-tolerance inXZ5.2. Comparing the CDS sequence of rbcL and Trx-M genes from XZ5, XZ54and ZAU3and sequence analysisCoding Sequences (CDS) of rbcL and Trx-M genes were cloned from different wild barley geneotypes (XZ5and XZ54) and cv. ZAU3by degenerate PCR. CDS length of rbcL and Trx-M was1401bp (the partial-length CDS region, coding467amino acids) and528bp (full-length CDS region, coding176amino acids), respectively, and both contained no introns.The nucleotide and amino acid sequences showed high identity with other plant rbcL and Trx-M genes.Comparison of gene sequences among XZ5, XZ54and ZAU3revealed5and2SNPs in rbcL and Trx-M, with two2SNPs of missense mutation in the both genes. SNPs in sequencesmay resultin different protein structurewhich can affect its function. Protein structure was predicted by different methods. The results of primary structure analysis show that two genes of molecular weight (MV) were5.17and1.92KDa, respectively. rbcL with a pI (isoelecric point) of6.44; Trx-M with a pI of8.57. Moreover, the SignalP solfttware predicted that rbcL and Trx-M genes were not containing a signal peptide, for non secretory proteins. BLASTP searches of the rbcL and Trx-M amino acid sequences obtained in this study were performed to determine homology between the sequences with those previously reported. The results of sequence alignment showed that the amino acid sequence from XZ5, XZ54and ZAU3had high homology with that of other plants. Secondary structure analysis indicated that the alpha helix, β-sheet, turn and coil of rbcL and Trx-M by ANHEPROT software were37%,19%,24%,20%;31%,29%,26%,14%, respectively.3D structure was constructed by Phyre2. In addition, conserved domain prediction by SMART (http://smart.embl-heidelberg.de/) indicates that rbcL have two conserved domains:RuBisco_large_N domain and RuBisco_large_domain, their functions are ribulose-bisphosphate carboxylase activity and magnesium ion binding. For RuBisco_large_N domain the interval from XZ5, XZ54and ZAU3was21-146, for RuBisco_large_domain the intervals from XZ5, XZ54and ZAU3were154-462,154-433,154-462, respectively. And69-172conserved Thioredoxin domain interval belonged to Trx-M from XZ5, XZ54and ZAU3, their function is cell redox homeostasis. 3. Alleviation of drought stress adverse effects by foliar-application of betaineThe hydroponic experiment was performed to study the alleviation effects of exogenous betaine on drought stress. There were4treatments:basal nutrient solution (control, BNS), BNS+500μM betaine (betaine), BNS+20%PEG6000(drought), and BNS+20%PEG6000+500uM betaine (drought+betaine). Results showed that foliar spraying500μM betaine (drought+betaine) significantly alleviated drought stress in barley seedlings of cv. ZJU9(H. vulgare). The plant height, root length and dry weight of seedlings under drought+betaine were significantly higher than that of drought alone treatment. Meanwhile, exogenous betaine significantly increased root/leaf POD, CAT, APX activities (c.f.15.5%/19.8%,13.5%/7.9%,10.0%/31.9%,10.45%/11.3%higher than drought alone treatment). Betaine meliorated drought-induced damages on leaf and root ultrastructures, effectively improved the stability and integrity of nuclear membrane of root meristem, and increased the cell viability in roots tips. In addition, exogenous betaine effectively increased photosynthetic rate of barley under drought stress. |
PDF Full Text Request