磷脂酶D家族成员FaPLDα1调控高羊茅获得性耐高温的分子途径

Heat stress is one detrimental abiotic stress limiting growth of the cool-season turfgrass during summer time. Our previous study on tall fescue (Festuca arundinacea) showed that drought priming promoted the accumulation of phosphatidic acid (PA) and enhanced heat tolerance. PA has recently been identified as an important signaling molecule responding to the abiotic stresses, including drought and heat stresses. Further studies showed that drought priming induced PA accumulation was mainly contributed by phospholipase D (PLD) mediating pathway. PLD plays an important role in plants stress tolerance and PLD activity has been implicated in both signaling and/or catabolic functions. However, the role of PLD in drought-priming enhanced heat tolerance and the underlying mechanisms remain largely unknown. A PLD gene, FaPLDα1, that had relatively high expression levels under drought, was cloned from tall fescue. In addition, transferring the gene into yeast successfully enhanced its heat stress tolerance. Based on these results, in this study, we aim to 1) clarify the function of FaPLDα1 by creating its RNAi and over-expressing transgenic lines followed by analyzing the phenotype and physiological parameters under drought priming and heat stress conditions; 2) screen the interactive proteins in drought-priming enhanced heat tolerance of FaPLDα1 using Y2H, BiFC and co-IP methods; 3) investigate the effects of interactive protein on subcellular location and expression level of FaPLDα1. The results from this study could provide theoretical basis and gene resources for the improvement breeding for turfgrasses.

夏季高温是制约高羊茅等冷季型草坪草生长发育的重要逆境。申请人前期研究表明，适度干旱锻炼可显著提高高羊茅耐热性，这种获得性耐热能力与磷脂酸（PA）积累密切相关。磷脂酶D（PLD）是逆境诱导合成PA的关键酶，但逆境锻炼如何调控PLD功能促进高温耐性形成的机制尚不明确。申请人已从高羊茅中筛选到一个受干旱诱导的PLD家族基因FaPLDα1，其异源表达显著提高了酵母的高温耐性。在此基础上，本项目拟：①在高羊茅中过表达和抑制表达FaPLDα1，明确其介导获得性耐高温的功能；②采用酵母双杂、双分子荧光互补和蛋白质免疫共沉淀等技术挖掘FaPLDα1的互作蛋白，并鉴定其功能；③采用原生质体瞬时表达和原核表达技术，明确互作蛋白对FaPLDα1亚细胞定位、蛋白表达水平的影响，最终阐明FaPLDα1及其互作蛋白调控获得性耐高温的分子机制。研究结果将深化植物耐热分子机制研究，还可为草坪草耐热育种提供优异的基因资源。

高羊茅（Festuca arundinacea)是应用最为广泛的冷季型草坪草之一，高温会导致其严重枯黄死亡。随着温室效应的不断加剧，如何保障草坪常绿是草坪生产管理中的一项技术难题。“脂质第二信使”磷脂酸是植物响应高温胁迫关键的信号物质，但磷脂酸及磷脂酶D家族成员FaPLDα1在草坪草获得性高温耐性形成中的功能及其调控途径尚未见报道。本研究发现，外源喷施磷脂酸可以通过调节维持细胞膜稳定性及胁迫信号传递的脂类组分变化提高高羊茅耐热性；磷脂酸还可以通过另一信号分子H2O2的介导，激活热激转录因子及热激蛋白的表达。克隆了一个高羊茅FaPLDα1基因，FaPLDα1基因可被干旱及高温诱导表达。亚细胞定位分析显示FaPLDα1蛋白定位于细胞质及细胞膜，酵母自激活活性检测其无自激活活性；克隆了FaPLDα1的启动子序列，通过酵母单杂交系统筛选获得了3个FaPLDα1的上游转录因子（FaERF13、FaERF2及FaERF7）。异源转化结果表明转FaPLDα1提高了酵母高温、干旱及盐胁迫耐性。通过酵母双杂交系统筛选获得了8个FaPLDα1的候选互作蛋白，挑选了其中3个蛋白（FaPCAP1、FaGLN1及FaTB1），采用酵母双杂交及荧光素酶系统验证发现均与FaPLDα1存在真实互作，我们进一步在高羊茅里过表达了一个与质膜相关的植物特异性蛋白FaPCAP1基因。采用磷酸化蛋白组学的方法，研究了获得性高温耐性形成过程中蛋白磷酸化水平的变化，我们发现参与RNA剪接、转录控制、胁迫应激保护/防御和胁迫应激感知/信号传递等过程的蛋白磷酸化水平变化可能与干旱锻炼诱导的耐热性相关。此外，基序分析检测到几个不同的基序（[GS]、[DSD]和[S..E]）受干旱锻炼和高温胁迫调节，酪蛋白激酶-II和丝裂原活化蛋白激酶可能是参与干旱锻炼诱导高温耐性形成中的关键蛋白激酶。在磷酸化蛋白组分析中我们还发现FaPLDα1的互作蛋白FaPCAP1磷酸化位点T172的磷酸化水平受干旱锻炼和高温处理诱导，我们推测FaPCAP1磷酸化水平的变化可能影响其与FaPLDα1的互作，进而影响FaPLDα1蛋白活性及功能，有待进一步研究。综上，本研究结论及成果对于培育耐热性强的草坪草新品系具有重要的理论指导与现实意义。