H2S介导根区氧信号调控桃侧根发生的作用机制

Root zone oxygen content significantly affected the lateral root formation of peach trees. The applicant's preliminary study found that oxygen treatment can significantly increase the number of lateral root in peach trees. It was interestingly found that the expression of PpDES, a key gene encoding hydrogen sulfide synthesis in peach root under aerobic conditions, was up-regulated through high-throughput digital gene expression profiling (DGE) and exogenous hydrogen sulfide significantly increased the number of lateral peach roots and the expression of PpDES suggesting that hydrogen sulfide may be aware of changes of root zone oxygen content and involves in root zone oxygen signal regulation lateral root formation in peach trees. To verify this hypothesis, peach seedlings are selected to study the effects of different root zone oxygen content on the expression of its encoding gene，the activity of hydrogen sulfide synthesis enzyme and the content of endogenous hydrogen sulfide in peach tree root based on this project. Then RT-PCR will be carried out to determine the expression of the encoding gene of auxin synthesis and transport genes, cell cycle regulatory genes of root under the condition of different root zone oxygen content and endogenous hydrogen sulfide content in which hydrogen sulfide generator or scavenger was used to change the content of root endogenous H2S. And the content and distribution of auxin in roots was also determined. Meanwhile, the occurrence of lateral root primordia and the number of lateral roots were observed by optical microscope. These assays are used to make sure if the root zone oxygen content regulates the occurrence of lateral root in peach trees by affecting the synthesis of hydrogen sulfide which further affects the synthesis, transport and distribution of auxin in peach tree roots. This study aims to elucidate the mechanism of hydrogen sulfide involves in root zone oxygen content regulation lateral root formation in peach trees.

根区氧气含量显著影响桃侧根的发生。申请者研究发现根区增氧可显著提高桃侧根数量，通过高通量数字基因表达谱分析发现，增氧条件下桃根系H2S合成关键酶编码基因PpDES表达上调；且外源H2S处理显著提高桃实生苗根系PpDES表达量和侧根数量，表明H2S可能响应根区氧气含量变化调控桃侧根的发生。为验证该假设，本项目拟以桃实生苗为试材，进一步研究不同根区氧气含量对桃根系H2S合成关键酶编码基因表达量及其酶活性、內源H2S含量的影响；并在不同根区氧气含量条件下，运用H2S发生剂或清除剂改变根系内源H2S含量后，利用实时荧光定量技术检测根系生长素合成和转运基因、细胞周期调控基因表达量，并测定根系生长素含量；最后利用光学显微镜观测侧根原基的发生和侧根数量。探讨根系中H2S能否响应根区氧气含量变化，调控根系生长素的合成、转运和积累，从而影响侧根的发生；最终阐明H2S介导根区氧信号调控桃侧根发生的作用机制。

为明确硫化氢（H2S）介导根区氧信号调控桃侧根发生的机制，本研究以毛桃实生苗为试材，研究了根区氧信号对桃根系根系硫化氢合成关键酶编码基因表达调控作用，进一步研究了外源硫化氢对桃侧根发生的影响，利用转录组测序技术、转基因技术以及实时荧光定量PCR技术来研究H2S诱导桃侧根形成的调控网络。主要研究结果如下：.1.研究发现根区氧气含量对根系硫化氢合成关键酶编码基因表达的调控作用不明显。表明根区氧信号可能并不是主要通过硫化氢调控根系的发生和生长。.2.进一步研究表明，0.2 mM H2S供体硫氢化钠（NaHS）处理显著促进了桃苗根系的生长。在处理第5 d，NaHS使幼苗侧根数量、根系总长度和根系总表面积分别增加31.28%、14.15%和12.13%；而H2S清除剂亚牛磺酸（HT）的使用消除了H2S对侧根的诱导作用；并且，单独添加HT显著抑制了幼苗的根系生长，使侧根数量降低15.17%。.3.通过分析内源性H2S含量和生长素浓度，外源H2S在处理第1 d诱导了根系中内源性H2S的产生，而处理第5 d使根系中生长素浓度提高了44.50％。同样，添加HT消除了NaHS对内源性H2S和生长素的诱导作用。.4.NaHS及对照（清水）处理0、1和5 d的桃根系RNA-seq结果显示，在H2S处理第1 d和第5 d，分别有963个和1,113个基因响应外源H2S。通过KEGG通路分析，发现植物激素途径与H2S处理密切相关，该通路中73.68％的差异表达基因（DEG）位于生长素途径。在DEG中，有26个基因参与了生长素的合成、转运和信号转导。通过基因共表达网络分析发现，在H2S特异性基因共表达模块中，一半的生长素相关基因与生长素的极性转运相关，这表明生长素的极性转运可能在H2S诱导的根生长过程中起重要作用。.5.通过RNA-seq及RT-qPCR分析发现，在暴露于外源H2S的情况下，桃根系中有16个细胞增殖相关基因的转录也受到了外源H2S调控，其中细胞周期调控基因PpJ18（LOC18790988）和细胞壁重构相关基因PpXTH23（LOC18789215）被显著上调。在桃根系中，LBD转录因子响应外源H2S处理，其成员PpLBD16的转录水平既能响应外源H2S，也能响应外源生长素的调控而上调；过表达PpLBD16拟南芥侧根数量和桃根系过表达PpLBD16不定根数量均显著增多。