过氧化物酶体定位的NAD激酶TaNADK4参与小麦干旱应答的分子调控机制

NAD kinase (NADK) is the sole enzyme that catalyzes NAD(H) and ATP to produce NADP(H) in almost all living organisms and therefore plays vital roles in many important metabolic pathways and stress responses of plants. In our previous studies, we found that there are 4 NADK isoforms in wheat cells and among them TaNADK4 is located in peroxisome. Further studies showed that the expression of TaNADK4 was significantly stimulated by plant hormones such as abscisic acid (ABA) and jasmonic acid (JA), and two homeobox (HOX) transcription factors, TaHOX1 and TaHD-Zipl-1, were found to bind to the promoter of TaNADK4 gene. However, the functions of TaNADK4 in peroxisome and the molecular mechanisms in drought stress tolerance are still under investigation. In the present project, the functions of TaNADK4 and its molecular mechanism in drought stress responses will be comprehensively investigated by employing a number of biological approaches including transgenic plant creation, protein interaction analysis and stress response network clarification. By this study, we will focus on (1) understand the mode of the localization of TaNADK4 to peroxisome and its biological role in the organelle; (2) clarify the molecular mechanisms of the two HOX transcription factors in the expression control of TaNADK4; (3) clarify the interactions between TaNADK4 and ABA and JA signaling; (4) demonstrate the functional mechanism of TaNADK4 in drought stress response. The results obtained here will provide new insights for the functions of peroxisome in plant stress tolerance and therefore benefits to the improvement of stress tolerance for wheat by biotechnological ways in the future.

NAD激酶（NAD kinase, NADK）是生物体内唯一催化NAD(H)和ATP合成NADP(H)的酶，在植物代谢调控中具重要功能。我们前期研究表明，小麦中存在4种NADK，其中TaNADK4定位于过氧化物酶体，其表达受脱落酸（abscisic acid, ABA）和茉莉酸（jasmonic acid, JA）及干旱等的强烈诱导，且两个HOX（homeobox）家族转录因子TaHOX1和TaHD-ZipI-1可与其启动子结合，但TaNADK4过氧化物酶体定位并参与干旱等逆境应答的机制不清。本项目拟在此基础上，通过遗传转化、蛋白互作及转录调控分析等，深入解析TaNADK4过氧化物酶体定位的调控方式，揭示两个HOX转录因子调控其表达及与ABA和JA等激素的互作关系，最终结合转基因抗性分析，阐明TaNADK4参与小麦干旱应答的分子调控机制，为过氧化酶体抗逆功能及小麦耐旱机制的阐明提供新依据。

NAD激酶（NADK）是细胞内唯一从NAD合成还原力NADPH的酶，在调节细胞内氧还平衡及依赖NAD(H)/NADP(H)的代谢反应及抗逆中起重要的作用。NADK具有区室化分布特点，在植物细胞中前期研究证明其存在于细胞质、叶绿体和过氧化物酶体，但线粒体中是否存在NADK一直没有明确报道。本项目研究中，我们以小麦NADK为研究对象，重点揭示了过氧化物酶体定位的TaNADK4的功能及其转录调控机制，发现TaNADK4的表达受ABA激素及其他多种环境因素的影响，异源过表达小麦TaNADK4的水稻植株表现出较强的抗旱能力。进一步通过酵母单杂交筛库发现两个bZIP类转录因子TaHDZIP I-1 和TaHDZIP I-4能够与TaNADK4的启动子结合，随后通过EMSA、酵母单杂、双荧光报告系统和CHIP-qPCR实验进一步证实这两个转录因子与TaNADK4启动子结合并上调TaNADK4的表达；同时，我们采用酵母双杂交、荧光素酶互补成像和免疫共沉淀等实验发现这两个转录因子能够形成同源或异源二聚体，参与对TaNADK4的转录调控。结合相关文献报道我们推测，在干旱胁迫或者ABA的诱导下，HDZIP I-1/I-4通过正向调控TaNADK4的表达来抵御干旱胁迫。此外，在本研究中，我们还首次发现了一个线粒体定位的NADK，称为TaNADK5，并发现TaNADK3在小麦中双定位于叶绿体和线粒体。该蛋白只在小麦族的物种中存在，具有NADK激酶活性。为进一步解析TaNADK4的功能，我们在全基因组的水平分析了可能与TaNADK4互作的CML家族蛋白，共鉴定到185个TaCMLs和18个TaCaMs，这些TaCMLs在细胞内有着复杂的定位，其中TaCML46-5D定位于过氧化物酶体酶体，是一个与TaNADK4互作的蛋白。本项目研究取得的结果对明确小麦TaNADK4的功能及其调控机制，以及全面解析小麦NADK家族蛋白的区室化分布特点及生物学功能，具有重要参考意义。