荫蔽胁迫下生长素调控大豆苗期叶片生长的机理研究

Soybean is widely relay-intercropped with taller crops in southern China, the seedling of soybean often grew under shade stress in relay intercropping. Leaf traits are closely associated with shade tolerance, as the leaf area determines light interception and photosynthesis in leaf mesophyll determines light conversion. It is important to know mechanism of shade tolerance in laminar of soybean seedling for improving light use utilization in shade in relay intercropping system. Our previous studies found that soybean laminar became smaller and thinner when grew under shade in relay intercropping, and many differentially expressed genes involved in responses to stimulus of auxin. Therefore, we hypothesized that the auxin was induced by shade in soybean laminar, and further regulated the cell proliferation and cell expansion, finally inhibited the leaf expansion growth. To validate the hypothesis, we plan to use inter-disciplinary researching methods by setting different light environment and exogenous auxin treatments on two soybean cultivars with different leaflet area. We proposed to emphatically analyze the production and polar transporation of endogenous auxin, cell cycle, plasma membrane H+-ATPase activity, and EXPANSIN expression level in laminar of soybean seedling under shade. The results could confirm the effects of shade on auxin content, cell proliferation and cell expansion, and the responses of cell proliferation and cell expansion to application of exogenous auxin and inhibitor of auxin polar transport in soybean laminar, so as to explore the regulatory mechanism of auxin on laminar expansion growth in soybean seedling. This study will provide a theoretical support for regulations on growth of soybean seedlings in relay intercropping system.

大豆在南方地区广泛与高秆作物套作，苗期遭受荫蔽胁迫。叶片性状决定了光能截获与光能转化，与耐荫性密切相关。明确套作大豆苗期叶片的耐荫机理对提高荫蔽下的光能利用至关重要。我们前期研究发现：套作荫蔽下大豆叶片变小变薄，且大量差异表达基因参与了响应生长素刺激。因此申请者假设荫蔽胁迫改变了大豆叶片生长过程中的生长素含量，进而调控了细胞增殖与（或）细胞扩展，最终抑制了叶片扩张生长。为验证这一假设，本研究拟以不同小叶面积大豆品种为材料，综合利用植物生理、细胞生物、作物栽培等学科研究手段，设置不同光照和外源生长素调控处理，着重从叶片生长素合成量和极性运输、细胞周期、质膜H+泵活力、扩展蛋白基因表达等角度开展分析，明确荫蔽对大豆苗期叶片生长素含量、细胞增殖和细胞扩展的影响，以及叶片细胞增殖与细胞扩展对外源生长素调控的响应，阐明生长素调控大豆苗期叶片生长的生理机制，为套作大豆苗期生长调控提供理论基础。

在玉米-大豆套作模式下，大豆苗期处于荫蔽环境，光能利用受到极大影响，迫切需要提高大豆耐荫性。叶片性状与耐荫性密切相关，叶片面积直接决定了光能截获。适宜套作的耐荫品种通常叶面积较大，有利于光能截获。前期研究发现，套作荫蔽下大豆叶片变小变薄，且大量差异表达基因的生物信息学信息指示参与了响应生长素刺激。但目前尚不清楚荫蔽下生长素如何调控大豆叶片生长。因此探明荫蔽胁迫下生长素调控大豆苗期叶片扩张生长的生理机制，对套作大豆苗期生长调控，提高其耐荫性具有重要意义。本项目以不同小叶面积大豆品种为材料，设置不同光照环境和外源生长素调控处理，从大豆苗期叶片的内源激素含量、细胞增殖与细胞扩展、基因表达等层面开展分析，明确荫蔽对大豆苗期叶片生长过程中激素含量、细胞增殖与细胞扩展的影响，以及大豆叶片细胞增殖与细胞扩展对外源生长素调控的响应机制，并在大田环境对生长素抑制剂的调控效果进行了验证。研究发现，荫蔽下大豆顶叶小叶面积均发生下降，叶片解剖结构观察显示荫蔽下叶片细胞数量显著降低。利用流式细胞术检测细胞周期发现，荫蔽和生长素抑制剂NPA处理均造成细胞有丝分裂强度下降。在叶片扩张相关基因表达层面也表明叶片扩张相关基因在荫蔽和NPA处理下均以下调表达为主。因此荫蔽下大豆叶片面积下降的主因在于细胞数量下降，NPA处理进一步加剧了细胞数量下降和叶片面积下降。叶片生长素和细胞分裂素的含量随着叶片的扩张生长均呈现逐渐下降的趋势。但在茎尖的叶片扩张早期，荫蔽处理后叶片IAA显著上升，而叶片玉米素（Zeatin）和反式玉米素核苷（tZR）含量的含量则发生下降。NPA处理后叶片IAA含量发生下降，而玉米素和反式玉米素核苷的作用不显著。结合叶片表型形态性状和细胞学特点，细胞分裂素及其调控的细胞增殖分裂，是决定荫蔽下大豆叶片面积扩张的关键点。因此，耐荫大豆的选择与调控目标明确为内源细胞分裂素CTKs参与的大叶型大豆品种筛选。