灯盏乙素促进根伸长的类激素效应及其机制研究

Plants produce abundant secondary metabolites, but the biological functions of most these metabolites are still unknown. Scutellarin is a flavonoid, extracted from Erigeron breviscapus(vant.) Hand. -Mazz., and is of great pharmaceutical interests. Our previous studies showed that low concentration scutellarin significantly promote root elongation and that the promoting effect can be dose-dependently antagonized by two typical hormones, methyl jasmonate and 2, 4-dichlorophenoxyacetic acid (2, 4-D). These results indicate scutellarin play a hormone-like role in regulating root elongation. Moreover, scutellarin enhance the transport and accumulation of auxin which is the crucial phytohormone regulating root development. According to the above findings, we propose a hypotheses that scutellarin play its hormone-like stimulus effect on root elongation by crosstalk with auxin. Therefore, this project is design to delve further into the below three aspects in Arabidopsis thaliana. We will detect 1) the relationship between the scutellarin effect and auxin transport as well as auxin signal response, 2) whether scutellarin could activate the pathway that flavonol biosynthesis regulate auxin transport, 3) the transcriptional responses at the global genome level under scutellarin treatment. Based on these results we will select the candidate pathways and genes with great regulatory function for the stimulus effect of scutellarin and validate them by experiments. In summary, our goal is to identify the targets of scutellarin effect and to understand relevant mechanisms. These efforts will promote the use of scutellarin in plant biotechnology and will provide new knowledge for the biological effects of secondary metabolites and functions of phytohormone.

植物有大量次生代谢物，但人们对其生物学功能知之甚少。灯盏乙素(S)是从短亭飞蓬中提取的黄酮类次生代谢物，有重要药用。我们前期研究发现：S在低浓度下即能极大地促进拟南芥根的伸长（平均增长3倍），此促进作用呈剂量依赖性并可被激素茉莉酸甲酯和生长素2,4-D拮抗，显示其以类似激素的作用调控根伸长的过程；并且S能增强调控根发育的核心激素生长素的运输和积累。由此我们提出假说，S通过与生长素协同调控来发挥其类似植物激素的根伸长促进效应。本项目拟以拟南芥为研究材料，分析1)S效应与生长素极性运输和信号响应的关系、2) S能否激活黄酮醇调控生长素的信号通路、3)S诱导全基因组的转录响应，在此基础上筛选出对S根伸长促进效应发挥关键作用的调控通路及基因并实验验证，进而探索S这一全新生物学效应的作用靶点和分子机制，发掘它在植物生物技术中的应用价值，为探索植物次生代谢物的生物学效应和植物激素的作用提供新的知识。

黄酮类化合物是广泛存在于植物中的一类具有重要生物活性的次生代谢产物，此前有报道多种黄酮类化合物都具有抑制植物根部生长素极性运输（PAT）并抑制根伸长的功能，而本项目首次发现了一个具有相反效应即促进PAT和根伸长的黄酮类化合物灯盏乙素。经过三年研究，我们：(1) 发现灯盏乙素与已报道黄酮类PAT抑制剂的主要结构区别在于6-OH，并应用另一种6-OH化合物黄芩素证明6-OH是决定黄酮类化合物对根伸长不同效应的关键因子；(2) 应用PAT抑制剂TIBA证明有6-OH的黄酮类化合物通过促进PAT促进根伸长；(3) 分析灯盏乙素处理后拟南芥的转录组响应，发现响应生长素刺激是差异基因显著富集的生物学过程，并检测关键基因表达，从转录组水平验证灯盏乙素促进PAT；(4) 检测黄酮醇合成途径关键基因表达，同时原位检测根部不同种类黄酮醇积累，证明灯盏乙素促进植物根部黄酮醇积累，并提高槲皮素/山奈酚比值以响应其促进PAT的效应；(5) 检测差异基因共表达网络中关键基因的表达，并借助基因缺失突变株，证实灯盏乙素促进根伸长的作用通过转录因子NUTCRACKER（NUC）基因实现，而NUC是细胞不对称分裂（ACD）的调控因子之一；(6) 检测对根伸长效应相反的灯盏乙素和黄芩素及柚皮素对ACD调控通路中NUC基因上游调控蛋白的作用，发现灯盏乙素和黄芩素显著促进SHR和SCR结合，而柚皮素显著促进RBR和SCR结合并抑制SHR和SCR结合，证明黄酮类有无6-OH对RBR-SCR-SHR调控网络作用方式不同，有6-OH的灯盏乙素和黄芩素促进SHR-SCR活性复合体形成、诱导NUC基因表达、引发ACD过程、促进根发育，而无6-OH的柚皮素则促进RBR-SCR非活性复合体形成、不能诱导NUC基因表达。.项目顺利完成，协助培养硕士一人，发表SCI论文两篇，申报专利两项，还有一篇论文正在投稿。