水稻OsNPF4.x响应丛枝菌根共生信号的生理和分子机制研究

Arbuscular mycorrhizal (AM) symbiosis between plant roots and arbuscular mycorrhizal fungi is one of the important evolutionary mechanisms of terrestrial plants to increase fitness to nutrient stresses. Several types of mycorrhiza-induced transporters responsible for translocating nutrients, such as Pi and NH4+, across the intraradical symbiotic interface have been isolated and well studied. However, no genes associated with transporting NO3- across the symbiotic interface have been evidenced to be present in plants. In our previous work, we have identified a mycorrhiza-induced gene, OsNPF4.x, from the rice NPF gene family, within which members are always characterized to have the ability of transporting NO3-. Expression analysis using GUS reporter revealed that the expression of OsNPF4.x was mainly confined to distinct cells containing arbuscules in rice mycorrhizal roots. OsNPF4.x was also observed to show response to NO3-, and its transcripts were significantly downregulated in response to ABA and SA applications. Given that NO3- has been repeatedly documented to be a signaling molecule that is able to modulate plant root development, it is tempting to make a hypothesis that OsNPF4.x could modulate the establishment of AM symbiosis, probably through mediating NO3- and/or ABA/SA (or other phytohormones) signaling pathways. In this study, we will functionally characterize this mycorrhiza-induced NPF gene, by enhancing or silencing its expression in transgenic plants, to testify its potential roles in transporting NO3- across the symbiotic interface and regulating mycorrhizal symbiosis and the related signaling pathways. The transgenic plants carrying mutated promoters (a series of truncations and deletions) fusing GUS reporter gene will also be employed to explore the putative cis-elements conferring AM-induced expression in OsNPF4.x promoter. In addition, EMSA (Electrophoretic Mobility Shift Assay) and Yeast One-Hybridization system will be performed to screen and testify the potential transcription factor(s) recognizing the AM-responsive cis-element(s). Accomplishing these studies, it will allow us to elucidate the molecular mechanisms underlying the response regulation and physiological roles of OsNPF4.x during the establishment of AM symbiosis in rice.

丛枝菌根是土壤中的菌根真菌与植物根系间建立形成的互惠共生体。研究表明植物中存在着受菌根共生诱导、负责共生界面养分（如Pi、NH4+）吸收的转运体。但至今尚未有直接证据证明菌根共生界面上存在运输NO3-的转运体。前期我们从水稻菌根中鉴定到一个被强烈诱导表达的NPF家族（该家族大部分成员都可以转运NO3-）新成员OsNPF4.x。该基因主要在含有丛枝的细胞中表达，对NO3-有响应，ABA和SA处理显著下调其表达。鉴于NO3-可以作为信号分子调控根系发育，我们提出了该假说：OsNPF4.x能够通过NO3-或ABA/SA介导的信号途径调控水稻丛枝菌根共生。本项目将利用超表达和突变体材料研究该基因对水稻菌根途径NO3-吸收和菌根共生的影响。项目还将利用启动子功能分析和酵母单杂交系统筛选启动子中响应菌根共生信号的元件和与之作用的转录因子，以揭示OsNPF4.x响应和可能调控水稻丛枝菌根共生的作用机制。

自然界中很多植物都能够与丛枝菌根真菌形成菌根共生体系，以增加对矿质养分的吸收。以前普遍认为菌根共生主要是促进植物吸收土壤中的磷素营养，但最近越来越多的研究显示，菌根植物中氮素营养也能得到显著改善，但其中的分子机制还不明确。本项目以水稻为研究对象，证实了水稻中存在一条以OsNPF4.5硝酸盐转运蛋白为介导的菌根氮吸收途径。在硝态氮作为N源的培养条件下，接种AM真菌Rhizophagus irregularis能够显著促进水稻生长和氮素吸收利用。通过分室接种实验证实，在硝态氮培养条件下，水稻通过菌根途径吸收的氮可达水稻总吸收氮的42%左右。RNA sequencing转录组测序和荧光定量RT-PCR分析发现，在水稻菌根中存在多个被显著诱导特异/增强表达的NRT1/NPF家族的硝酸盐转运蛋白基因，其中OsNPF4.5（在项目申请书中命名为OsNPF4.x）为上调最高的硝酸盐转运蛋白。组织定位分析显示，OsNPF4.5几乎只在被AM真菌侵染的根系强烈表达，且表达部位主要集中在被菌丝侵染形成丛枝的皮层细胞中，而在其它组织器中几乎都不表达。亚细胞定位分析显示，在组成型启动子驱动下，OsNPF4.5-GFP融合蛋白能够定位到细胞质膜上。对启动子区功能分析显示，OsNPF4.5响应菌根共生的表达模式受到OsNPF4.5核心启动子上游-177和-154bp的作用区段调控。在蛙卵中异源表达OsNPF4.5证实，在pH为5.5的培养条件下，OsNPF4.5在蛙卵中具有低亲和性的硝酸盐转运活性，但在pH为7.4的培养条件下，OsNPF4.5在蛙卵中不具备硝酸盐转运活性，暗示了OsNPF4.5可能编码一个依赖于pH的低亲和性硝酸盐转运蛋白。在水稻中组成型超表达OsNPF4.5能够显著促进根系对硝酸盐吸收和植株生长，但并不能促进NH4+的吸收。osnpf4.5突变体植株在接种AM真菌条件下，与野生型相比，突变体地上部生物量和氮浓度显著降低，侵染率和丛枝形成丰度也受到显著抑制。菌-根分室培养实验证实，在硝酸盐培养条件下，突变OSNPF4.5可降低大约45%的菌根途径氮吸收。