Physiological And Molecular Mechanisms Of Exogenous Application Of IAA Increasing Aluminum Resistance In Black Soybean

Aluminum (Al) toxicity is a major limiting factor for plant growth and crop production in acid soils, which cover more than 30% of the world’s arable land. Al firstly inhibited the growth and development of plant roots, even at micromolar concentrations. Consequently, the water and nutrient uptake by plant roots were decreased, which results in poor growth and biomass production.In the long-term evolutionary process, plants have evolved diverse mechanisms to resist to Al stress. And the Al-activated release of organic acids from root tips, intracellular compartmentalization of Al and enhancement of the activities of the anti-oxidative enzymes have been characterized as the most common Al resistant mechanisms in plants. Auxin (indole-3-acetic acid, IAA), the earliest discovered plant hormone, not only plays important roles in plant normal growth and development, but also plays regulatory roles in plant resistance to various stresses. In this study, an acid and Al-resistant black soybean (RB) and an acid and Al-sensitive black soybean (SB) were used as the plant materials, the physiological and molecular mechanisms of exogenous application of IAA enhancing these two cultivars resist to Al stress were investigated. The main results were as follows:1. Exogenous application of IAA can not only alleviate the Al-induced inhibition of primary root growth, but also promote the lateral root development and increase the biomass in both RB and SB. After application different concentrations of IAA (0.5,1, 25 and 50 μmol·L-1) in 50 μmol·L-1 Al treatment solution, primary root length, lateral root number and length, root/shoot ratio and fresh weight of RB were increased in a concentration-dependent manner, compared with Al treatment. Adding 50 μmol·L-1 IAA in the Al treatment solution has the best promotion effect on primary root length, lateral root density and length, root/shoot ratio and fresh weight of RB, and increased approximately 26.72,110.69,12.89,58.76 and 51.94%, respectively, compared with Al treatment. Similarly, SB has been effectively increased. Whereas, exogenous applying 25 μmol·L-1 IAA in 50 μmol·L-1 Al treatment solution has the best promotion effect on SB primary root length, lateral root density and length, root/shoot ratio and fresh weight, and increased approximately 41.02,63.26,16.01,28.87 and 39.43%, respectively, compared with only Al treatment. Taken together, these results suggested that exogenous applying IAA can efficiently alleviate Al toxicity to plant.2. The physiological and molecular mechanism of exogenous application of IAA increasing Al resistance were further analyzed in the Al resistance black soybean cultivar (Tamba black soybean, RB). The results showed that after exposure to 25,50 and 200 μM AICl3 for 24 h, the contents of endogenous IAA, citrate exudation and the activities of the PM H+-ATPase in RB roots were promoted first, but then decreased. However, the contents of H2O2 and MDA were increased significantly with Al concentrations increasing. Additionally, the contents of Al, MDA and H2O2 in RB roots were significantly decreased while 50μM IAA was added to 25,50 and 200 μM Al (A25, A50 and A200) treatment solutions. Furthermore, the citrate exudation increased approximately 2.39,1.73 and 6.85-fold and the activities of the PM H+-ATPase enhanced approximately 1.09,1.74 and 1.45-fold than that of only Al treatment in RB roots, respectively. These results indicated that exogenous application of IAA significantly enhanced Al resistance of RB by enhancement of citrate exudation and the PM H+-ATPase activity under Al stress. Thereby, the expression profiles of genes related to citrate exduation (MATE and STOP1) and the PM H+-ATPase activity (GHA2 and 14-3-3 protein) were further analyzed. The results showed that although exogenous application of 50 μM IAA obviously increased the mRNA abundance of MATE and STOP1, the expression of GHA2 and 14-3-3 protein was only significantly induced Al. Whereas, immunoprecipitation and western-blot analyses showed that the phosphorylation levels of the PM H+-ATPase and its interaction with the 14-3-3 protein were enhanced after 50 μM IAA were added in the Al treatment solution. These results suggested that exogenous application of IAA in Al treatment solution not only stimulated the expression of the genes (MATE and STOP1) related to citrate exudation, but also enhanced the phosphorylation levels and interaction with the 14-3-3 protein of the PM H+ -ATPase under Al stress, which resulted in increase in the Al-induced citrate exudation and Al resistance. And this mechanism were further confirmed in the transgentic tobacco that overexpression of △GHA2 (without C-terminal self-suppression domain) or inhibition the expression of PM H+-ATPase (pma4) through RNA interference (RNAi).3. It is well known that the plant Al resistance capacity is regulated by stimulating multiple genes expression. To further and comprehensive understand the molecular mechanism of exogenous application of IAA increasing aluminum resistance in RB, the digital gene expression profiling technology (DGE) was used to identify the IAA responsive genes in RB roots under Al stress. Of the 1104 genes that were significantly induced by Al stress, there were 207 genes were significantly up-regulated or down-regulated by exogenous application of IAA or TIBA under Al stress, including a series of Al resistance genes such as MATE, ABC channel protein and oxidative stress related genes. Furthermore, genes involved in regulation of IAA contents (GH3 and PIN), Ca2+ signal transduction (calcium binding protein PBP1), bZIP and WRKY transcription factor were also significantly up-regulated or down-regulated by exogenous application of IAA or TIBA under Al stress. Similarly, the gene encoding PM H+-ATPase was also only induced by Al stress. Of the 467 genes that were down-regulated by Al stress, there were 103 genes, including the metabolism related genes such as sterol reductase, fatty acid desaturase and protein synthesis and degradation related genes were up-regulated or down-regulated by exogenous application of IAA or TIBA under Al stress. Thereby, these results indicated that exogenous application of IAA not only further enhanced a series of Al resistance and signal transduction genes expression, but also effectively reversed the expression of genes related to RB root normal growth and development, which increased RB resistance to Al stress.