Root and shoot suberization in transgenic lines of grey poplar (Populus × canescens), an economically important tree species

本项目的目的是对灰杨的Suberin CRISPR-Cas9基因敲除系和35S启动子过表达系进行全面的鉴定，并与相应的野生型进行比较。除初生根外，本项目还将首次扩展杨树8-12周龄植株次生根和枝条中韧皮化周皮的形成、结构和功能的研究。这将包括以下工作包：(I)产生转基因(CRISPR-Cas9基因敲除和过表达)杨树中的Suberin关键基因。(Ii)优化生长方案，以获得处于次生发育状态的进一步发育的根和正在经历次生加厚和周皮形成的新梢。(Iii)初级根、次级加厚的根和茎周皮的详细解剖学特征，以确定与野生型和对非生物胁迫(盐分和干旱)的反应的变化相比，组织特有的Suberin的沉积或缺失。(Iv)对初生根、二次加厚根和地上部周皮中的Suberin和相关蜡量和组成进行化学分析，以确定与野生类型相比和对非生物胁迫的反应中的修饰。(V)根和地上部周皮中水分和溶质运输的特征，与野生型和对非生物胁迫的反应相比较。(Vi)与野生型对照和对非生物胁迫的响应比较，分析Suberin突变系的修饰基因表达(RNA-seq)。在上述实验的基础上，我们打算进一步阐明初生根、次生根和地上部周皮中的Suberin在适应非生物环境胁迫因子干旱和盐分中的作用。

This project aims to thoroughly characterize suberin CRISPR-Cas9 knock-out and 35S-promoter over-expression lines of grey poplar and compare them to the corresponding wild-type. Besides primary roots, this project will for the first time extend the investigations on the formation, structure, and function of suberized periderms in secondary roots and shoots of poplar forming in 8-12 week-old plants. This will include the following work packages: (i) Generation of transgenic (CRISPR-Cas9 knock-out and over-expression) poplar lines of suberin key genes. (ii) Optimization of growth protocols to attain further developed roots in their secondary developmental state and shoots, both undergoing secondary thickening and periderm formation. (iii) Detailed anatomical characterization of primary roots, secondarily thickened roots, and shoot periderms to identify tissue-specific deposition or absence of suberin in comparison to the wild-type and alterations in response to abiotic stress (salt and drought). (iv) Chemical analysis of suberin and associated wax amount and composition in primary roots, secondarily thickened roots, and shoot periderms to identify modifications in comparison to the wild-type and in response to abiotic stress. (v) Characterization of water and solute transport in roots and shoot periderms in comparison to the wild-type and in response to abiotic stress. (vi) Analysis of modified gene expression (RNA-seq) in suberin mutant lines in comparison to their wild-type reference and in response to abiotic stress. Based on these experiments described above, we intend to further elucidate the function of suberin in primary and secondary roots and shoot periderm in adaptation to the abiotic environmental stress factors drought and salt.