Physiological And Molecular Mechanisms Of Aluminum Tolerance In Tibetan Annual Wild Barley

Aluminum (Al) toxicity is a major factor limiting crop production in acid soils. It is estimated that30-40%of arable soils and up to70%of potentially arable soils in the world are acidic and may be prone to Al toxicity. The strategies for maintaining production on acid soils include lime application to raise soil pH and use of plants with high tolerance to acid soils. Development and planting of Al tolerant cultivars is a cost-effective and practically acceptable approach for full utilization of acid soil. Barley (Hordeum vulgare L.), the fourth most important cereal crop in the world, is more sensitive to Al than other small grain cereals such as rye, oats, wheat and triticale. Wild barley offers a great source of useful genes and genetic variation for crop improvement. Annual wild barley from Qinghai-Tibet Plateau is regarded as one of the progenitors of cultivated barley and is rich in genetic diversity. Accordingly, the present study was carried out to elucidate the physiological and molecular mechanism of Al tolerance/resistance in Tibetan wild barley, and to identify specific proteins and relevant genes for Al tolerance through proteome and genome analysis, and the Al tolerant barley cultivar Dayton was used as a reference. In addition, the application of citrate and phosphorus on the alleviation of Al toxicity in the selected barley genotypes were also studied. The main results were summarized as follows:1. Difference in response to Al stress among Tibetan wild barley genotypesWe carried out a comprehensive program combining short-term hydroponic screening via hematoxylin-staining-root-regrowth procedure and filter-paper-based evaluation of diverse germplasm in response to Al/acid stress using105annual Tibetan wild barley and45cultivated barley genotypes. Root elongation among the105Tibetan wild barley genotypes varied significantly after Al exposure, ranging from62.9-80.0%in variation coefficients and4.35-4.45in diversity index. These genotypic differences to Al stress were fairly consistent in both the hydroponic and filter-paper-based evaluations—XZ16, XZ166, XZ113were selected as Al-tolerant/resistant genotypes and XZ61, XZ45, XZ98as Al-sensitive wild genotypes. Furthermore, significantly lower Al concentrations in roots/shoots were detected in the3selected Al-tolerant genotypes than in the3sensitive genotypes in the filter-paper-based experiment. Meanwhile, XZ16was the least affected by Al toxicity in regard to reduced SPAD value (chlorophyll meter readings), plant height, root length, dry biomass and tillers per plant in the long-term hydroponic experiment compared with the Al-tolerant cultivated barley cv. Dayton, while XZ61had the severest stress symptoms.2. Characteristics of photosynthetic performance, chlorophyll fluorescence and Al uptake of Tibetan wild barley in response to aluminum stressGreenhouse hydroponic dose-response and time-course experiments were conducted to investigate genotypic differences in response to Al stress among two contrasting Tibetan wild barley genotypes XZ16(Al tolerant) and XZ61(Al sensitive) and cv. Dayton (Al tolerant). The results showed that Tibetan wild barley XZ16exhibited superior Al-resistantce/tolerance reflected by significant low Al uptake and high chlorophyll fluorescence (Fv/Fm), and an enhancement in net photosynthetic rate (Pn), stomatal conductance (Gs) and transpiration rate (Tr), and maintaining relatively lower intercellular CO2concentration (Ci) under100μM Al in contrast to XZ61and Dayton.3. Characteristics of antioxidant capacity, ATPase activity and nutrient concentration of Tibetan wild barley in response to aluminum stressAl-stress-hydroponic-experiments were conducted to investigate genotypic differences of nutrient element contents, ATPase activity and antioxidatants enzymes in response to Al stress among XZ16and XZ61and cv. Dayton, and as affected by anion inhibitor of niflumic acid (NIF). The results showed that Tibetan wild barley XZ16exhibited superior Al-resistantce/tolerance reflected by significant low Al uptake and malondialdehyde (MDA) accumulation, and exhibited higher P, S, Ca and Mg contents in roots and shoots under Al stress compared with XZ61and Dayton. Moreover, activities of catalase (CAT) and ascorbate peroxidase (APX) in ROS scavenging systems play an important role in alleviating Al induced oxidative stress in XZ16. Moreover, under25-200μM Al concentration or15d of Al exposure, XZ16maintained higher H+-, Ca2+Mg2+-and total-ATPase activities over XZ61. Thus, it may be beneficial to improve Al tolerance in XZ16to maintain its high ATPase activity, high nutrient elements contents and some antioxidant enzyme activities under Al stress.4. Characteristics of organic acid secretion of Tibetan wild barley in response to aluminum stressAl-stress-hydroponic-experiments were performed, and the physiochemical characteristic of two contrasting Tibetan wild barley genotypes (XZ16and XZ61) and cv. Dayton were compared.Ultrastructure of chloroplasts and root cells in XZ16was less injured than that in Dayton and XZ61. Moreover, XZ16secreted significantly more malate besides citrate over XZ61. The anion channel inhibitor NIF reduced the citrate secretion of roots in all three genotypes under Al stress. The protein synthesis inhibitor cycloheximide reduced citrate secretion from XZ16, but not from Dayton. The external of50μM citrate could alleviate the damage caused by Al on root morphological characteristics, root weight, root cell integrity and root Al accumulation, especially for Al sensitive genotype XZ61, but had no effect on injury of the chloroplasts integrity and shoot weight or Al accumulation in XZ61. In Tibetan wild barley, our findings highlight the significant correlations between Al tolerance, ATPase activity and citrate secretion, providing some insights into the physiological basis for Al-detoxification.5. Comparative proteomic analysis of aluminum tolerance in Tibetan wild and cultivated barleysThe hydroponic-experiments were performed to compare proteomic characteristics of two contrasting Tibetan wild barley genotypes XZ16and XZ61as well as cv. Dayton. Thirty-five Al-tolerance/resistance-associated proteins in roots were identified and categorized mainly in metabolism, energy, cell growth/division, protein biosynthesis, protein destination/storage, transporter, signal transduction, disease/defense, etc. Among them,30were mapped on barley genome, with16proteins being exclusively up-regulated by Al stress in XZ16, including4proteins (S-adenosylmethionine-synthase3, ATP synthase beta subunit, triosephosphate isomerase, Bp2A) specifically expressed in XZ16but not Dayton. The findings highlighted the significance of specific-proteins associated with Al tolerance, and verified Tibetan wild barley as a novel genetic resource for Al tolerance.6. Identification of specific genes for Al tolerance in Tibetan wild and cultivated barleysTo better understand the mechanisms involved in Al toxicity and tolerance in plants, microarray technology was used to evaluate changes in gene expression in roots of two contrasting Tibetan wild barley genotypes XZ16and XZ61and cv. Dayton under200μM Al stress. With the use of Affymetrix Genechip, a comparison of RNA expression profiles was made between control and Al-treated barley seedlings. A total of15genes were upregulated in XZ16but downregulated in XZ61, or682upregulated in XZ16but unchanged in XZ61,553genes unchanged in XZ16but unchanged in XZ61. These genes were identified as Al-responsive in XZ16. We found that some of the genes were associated with the function of signal factors including abscisic acid, Ca2+, salicylate, gibberellins, jasmonic acid and ethylene. These signal factors improved the expression of a series of identified genes which could be categorized mainly in detoxificants, transporters, lipid metabolism, defense, transcription and carbohydrate metabolism, etc. Furthermore, these genes in XZ16attributed the Al tolerance mechanisms consist of ROS scavenge, ion homeostasis, production of ATP, synthesis of acetyl-CoA, protein conformation and stabilization, DNA replication, glycolysis, TCA cycle, cell wall biosynthesis and elongation remodeling, etc. Thus, not only citrate secretion, the ABC transports, some types of ATPases and several genes relevant to tricarboxylic acid cycle were newly found to be associated with the Al resistance in XZ16.7. Effect of phosphorus supply on the physiological features of Tibetan wild and cultivated barleys under Al stress.Greenhouse hydroponic experiments were conducted to investigate genotypic differences in response to different concentration of phosphorus (90,180or360μM P) on Al stress among two contrasting Tibetan wild barley genotypes XZ16(Al tolerant) and XZ61(Al sensitive) and cv. Dayton (Al tolerant). The addition of the high concentration of P dissolving (360μM P) in Al solution significantly increased the biomass of root and shoots of in XZ61, however, the biomass loss of XZ61was also much higher than XZ16or Dayton. Compared to XZ61, the addition of360μM P for XZ16significantly increased the values of Gs and Tr, improved the APX and H+-ATPase activities in roots, increased the contents of nutrient elements such as P, Mg, Ca, Fe in plants, and finally induced the secretion of citrate. The content of Al in roots of the three barley genotypes, and in the shoots of XZ61and Dayton was not changed by the different levels of P addition. However, Al content in the leaves of XZ16was decreased after360μM P addition while not happened in XZ61and Dayton.