苜蓿体内H_(2)S信号与Ca^(2+)调节气孔运动的作用机制

The Mechanism of H_(2)S Signal and Ca^(2+) Regulating Stomatal Movement in Medicago sativa

为探究H_(2)S信号在苜蓿(Medicago sativa)体内调节气孔运动的作用,及在此过程中H_(2)S与Ca^(2+)的关系,以蒺藜苜蓿(Medicago truncatula)的野生型和钙离子转运体突变体为试验材料,分别从转录水平、细胞水平和生理水平开展研究。采用qRT-PCR比较相关基因的表达量变化、荧光探针显示体内Ca^(2+)含量、电极法测定H_(2)S含量、光学显微镜观察和测量气孔孔径等。结果表明:蒺藜苜蓿突变体NF3011和NF2734体内H_(2)S的含量与野生型相比极显著降低(P<0.01);H_(2)S信号在一定程度上抑制钙离子转运体编码基因MTR_6g027580的表达;外源生理浓度H_(2)S熏蒸可诱导蒺藜苜蓿气孔关闭,与Ca^(2+)通道阻断剂LaCl_(3)联合处理对野生型气孔运动未产生影响,而在突变体中的结果截然相反;利用荧光探针测定保卫细胞内的Ca^(2+)含量,所得结果与气孔孔径的变化规律完全一致。综上所述,H_(2)S信号促进叶片保卫细胞内Ca^(2+)的含量增加,最终表现为植物气孔孔径变小,在此过程中胞内Ca^(2+)含量变化主要通过Ca^(2+)转运体进行,少部分依赖Ca^(2+)离子通道。该研究结果不仅在理论上丰富了H_(2)S信号的作用机制,更具应用于苜蓿生产实践并推广于其他作物的潜力。

To explore the role of H_(2)S signal in regulating stomatal movement in alfalfa and the relationship between H_(2)S and Ca^(2+)during this process, wild type and the calcium transporter mutants of Medicago truncatula were used as experimental materials, and the transcriptional level, cellular level and physiological level were studied respectively, and the expression levels of related genes were compared by qRT-PCR, the content of Ca^(2+)in vivo was detected by fluorescent probe, the content of H_(2)S was measured by electrode method, and the stomatal aperture was observed by microscope respectively. The results showed that compared with the wild type, the content of H_(2)S in the mutant NF3011 and NF2734 decreased significantly;the H_(2)S signal inhibited the expression of calcium transporter encoding gene MTR_6g027580, the exogenous physiological concentration of H_(2)S fumigation could induce stomatal closure of Medicago truncatula, and the combination of treatment with LaCl_(3), Ca^(2+)channel blocker, had no effect on stomatal movement of wild type, but opposite in mutant;the concentration of Ca^(2+)in guard cells was measured by fluorescence probe, which were consistent with the change law of stomatal aperture. In conclusion, the content of Ca^(2+)in guard cells of leaves is induced by H_(2)S, whose stomatal aperture become smaller at the same time. In this process, the change of intracellular Ca^(2+)concentration mainly depend on Ca^(2+)transporter, and small partly depend on Ca^(2+)channel. The results of this study not only enrich the mechanism of H_(2)S signaling in theory, but also have the potential to be applied to alfalfa production and other crops.