HD-Zip III和木质素在柠条叶脉响应干旱向木质部分化及促导管成熟作用解析

HD-Zip III is a transcription factor leading to vascular procambium to xylem precursor cell in leaf vein. The SHR/SCR negatively regulated the HD-Zip III through miRNA165/166 and mediated downstream gene expression and improve the response to abiotic stress. Our previous RNA-seq data analysis and anatomy observation for non-pattern woody plants Caragana korshinskii predicted that drought induced HD-Zip III to strengthen the xylem differentiation of leaf vein, and induced lignin synthesis to promote vessel maturation, therefore formed developed vein network to improve the water use efficiency and the resist water stress capacity. However, the transcriptional regulation mechanism of HD-Zip III governing vein differentiation and lignin synthesis promoting the vessel maturation in the formation of developed vein network induced by drought is still unclear, need to be verified. How does HD-Zip III work as the centre of the transcriptional regulation and lignin biosynthesis play promotional role? In this project, objective comparison of leaf vein development in normal and drought stress of C. korshinskii will be carried out through indoor drought treatment. The depth analysis of expression pattern of HD-Zip III gene and lignin synthase gene responding to drought will be done with the help of over expression and silence expression of CkHD-Zip III in tobacco as well as protein interaction and physiological response resist drought. The regulation mechanism of developed leaf vein network then will be interpreted clearly. This helps us further expand understanding on the reason of drought promote developed leaf conducting tissue.

HD-Zip III是主导叶脉维管原形成层向木质部前体细胞分化的转录因子，其上游SHR/SCR通过下调表达介导miRNA165/166对HD-Zip III的转录后负调控，可与蛋白互作调控基因表达产生逆境响应。我们前期通过对叶脉解剖结构的观察并挖掘非模式木本植物柠条RNA-seq数据，预测干旱胁迫诱导叶脉HD-Zip III加强向木质部分化，并刺激木质素合成促进导管成熟，提高水分利用效率和抵御水分胁迫能力。然而干旱所致的柠条发达叶脉网络建成受到的以HD-Zip III为核心的转录调控以及木质素合成促进导管成熟的内在机制尚不明确。本项目拟在比较正常与干旱胁迫柠条叶脉网络特征的基础上，借助柠条CkHD-Zip III基因过表达和沉默表达、蛋白互作以及抗旱性分析，解析柠条叶脉由HD-Zip III表达水平决定的粗脉向木质部分化而细脉全面发生发育的调控机制，以及木质素对导管成熟的促进作用。

柠条锦鸡儿是黄土高原退耕还林及植被恢复的先锋植物。前期研究发现柠条叶片随干旱加剧形成更加发达的叶脉木质部导管来应对干旱。这种通过构建更发达叶脉以加强水分输导的方式是自然界抗旱植物利用自身改变适应环境的有效方式，但是机理却知之甚少。决定叶片近轴面发育的因子HD-ZIP III家族的成员CkREV是响应干旱、调控叶脉更加发达的上游转录因子。我们研究发现CkREV在正常发育过程中通过正向调节CkYUC5和 CkLAX3基因的表达，有利于植物生长素的生物合成以促进植物生长，正向调控叶脉木质部导管发育相关基因CkVND6、CkVND7及木质素合成相关基因CkPAL4的表达，促进柠条叶脉木质部愈加发达。然而当植物受到干旱胁迫时，CkREV通过感知外界信号，负调控CkYUC5的表达，抑制生长素的生物合成，从而减缓植物的生长速度，降低其对水分的需求，增强植物对干旱环境的适应能力。另外CkREV还可以通过直接正调控CkWRKY53的表达、以及与同为叶脉木质部发育转录因子的CkAS1进行蛋白互作实现对CkWRKY53基因的负调控，共同形成在干旱环境下既增加水分运输又减少水分消耗和丢失的双管齐下的抗旱策略。综上所述，本项目从木质部发育和阶段性气孔开闭两个方面证实干旱地区优势灌木柠条在干旱胁迫条件下通过近轴面发育决定子HD-ZIP I转录因子家族成员CkREV这一单个基因的调控形成更发达的木质部以及相反的叶片气孔响应共同应对干旱胁迫，创新性地发现木本植物运用单一基因靶向不同器官的抗旱新机制，即柠条锦鸡儿在干旱胁迫下，CkREV定位于木质部区域以一种干旱程度依赖的方式直接调控叶脉木质部导管发育相关基因促进柠条叶脉木质部愈加发达；同时CkREV又可以直接调控生长素生物合成基因CkYUC5及气孔开闭相关基因CkWRKY53的表达，调节气孔在不同干旱程度下的开放或关闭，同时减弱植物在干旱条件下的生长速率，降低植物对水分的需求，进一步增强植物抗旱性的同时又提高了植物的产量，揭示柠条抗旱的多途径复杂机制。未来这类具有高CkREV表达的柠条无疑是具有抗旱潜能的、应对全球变化背景下以干旱高温为主要特征的西北旱区的宝贵资源。