外源绿原酸缓解黑大豆SB铝毒害的机理研究

Alleviation of Al toxicity by exogenous chlorogenic acid in black soybean

关键词:  黑大豆SB, 绿原酸, 柠檬酸, 14-3-3蛋白, H⁺-ATPase, Al胁迫

Key words:  black soybean, chlorogenic acid, citric acid  14-3-3, H⁺-ATPase  Al stress

摘要:

该文研究了外源绿原酸(CGA)对Al胁迫下铝敏感型黑大豆SB根生理生化指标以及根中胁迫相关基因表达的变化,探讨外源CGA缓解SB根铝毒害的效果及分子机理。以不同浓度Al和CGA处理SB,筛选出CGA缓解Al毒害的最佳浓度,测定Al含量、抗氧化系统酶活性、14-3-3蛋白与H⁺-ATP酶的表达、H⁺泵活性。结果表明:低浓度CGA能缓解Al胁迫下黑大豆SB根伸长抑制,并促进侧根数目增加,而高浓度CGA的缓解效果下降; 0.01 g·L^-1 CGA使Al胁迫下SB根尖Al含量与MDA含量下降,促进根系柠檬酸的分泌。RT-PCR和Western Bloting分析表明0.01 g·L^-1 CGA促进Al胁迫下SB根中14-3-3b、14-3-3m、14-3-3k和GHA2基因(质膜H⁺-ATP酶)的表达,抑制MATE基因的表达。同时,0.01 g·L^-1 CGA能促进Al胁迫下质膜H⁺-ATP酶蛋白磷酸化水平以及其与14-3-3蛋白结合,且能提高质膜H⁺-ATP酶和H⁺泵活性。因此推测外源CGA可能通过增加侧根数,增强14-3-3蛋白和质膜H⁺-ATP酶基因蛋白表达水平和互作,弥补Al胁迫下MATE表达的抑制,增加柠檬酸的分泌,增强SB对铝毒害的耐受性。

Abstract:

We investigated the influences of exogenous chlorogenic acid(CGA)on the physiological parameters and Al stress-related gene expression of SB(Al-sensitive black soybean)root under Al stress. A major goal of this report was to explore the alleviating molecular mechanism of exogenous CGA on Al toxicity in SB. SB seedings were cultured in water solution and treated with 50 μmol·L^-1 Al or CGA plus 50 μmol·L^-1 Al. The optimal concentration of CGA alleviating Al toxicity was screened. And we study the effects of optimal exogenous CGA on root tip Al content, the activities of antioxidant enzyme, the expressions of 14-3-3 protein and H⁺-ATPase, and the activity of H⁺ pump under 50 μmol·L^-1 Al stress. Low concentrations of CGA alleviated inhibition of root elongation caused by Al toxicity and promoted the increase of lateral root numbers under Al stress. However, the CGA-alleviated effect was weaken with high concentration of CGA treatment. It was founded that the alleviation effect of 0.01 g·L^-1 CGA was the most significant. Exogenous 0.01 g·L^-1 CGA decreased the Al in root tips and MDA contents. And the citric acid content in root exudates of SB under Al stress was significantly increased due to the addition of exogenous 0.01 g·L^-1 CGA. Expression analysis showed that exogenous 0.01 g·L^-1 CGA enhanced the expression of three 14-3-3 isoforms(14-3-3b, 14-3-3m, and 14-3-3k)and increased the expression of GHA2 (plasma membrane H⁺-ATPase)gene, but inhibited the gene expression of MATE in SB under Al stress. The results of immunoprecipitation(IP)showed that the phosphorylation of the PM H⁺-ATPase was up regulated by the exogenous 0.01 g·L^-1 CGA, which could bind with 14-3-3 protein under Al stress. Meanwhile, plasma membrane H⁺-ATPase and H⁺ pump activities were both enhanced by exogenous 0.01 g·L^-1 CGA under Al stress. It was suggested that exogenous CGA may enhance the SB tolerance to Al stress by increasing the lateral roots number, compensating inhibition of MATE expression and interaction between them to increase citric acid exudation.