水稻缺钾响应转录因子调控钾素吸收循环利用的分子机制

One of the major molecular mechanisms for plants to adapt the limited potassium (K) supply is enhancing the transcriptional expression of HAK/KUP/KT K transporters. Up to today, the only known transcription factor in plants to positively regulate K transporters is RAP2.11 belonging to AP2/ERF family in Arabidopsis. We have cloned two RAP2.11 homologues in rice and name them as OsKTR1 and OsKTR2. Both of OsKTR1 and OsKTR2 were up-regulated by K deficiency and salt stress. Knockout of OsKTR1 in rice decreased K uptake, translocation and expression of OsHAK1, OsHAK5 and OsHAK-iv genes. OsKTR1 could directly bind to the fragments of OsHAK1/5/iv gene promoters. Meanwhile, knockout of OsHAK-iv resulted in accumulation of K in shoot and decrease of K in root. Interestingly, OsKTR1 and OsHAK1/5/iv genes showed partial overlapping in their tissue localization and responded to K deficiency, salt stress, ethylene, calcium and reactive oxygen species (ROS), while the time course of the responses to the stresses were not fully consistence, indicating that other factors (such as OsKTR2) in addition to OsKTR1 might be involved in the transcriptional regulation of K deficiency signaling pathways. .Based on these preliminary data, we propose to further characterize the function and regulation of OsKTR1 and OsKTR2 in rice adaptation to K limitation and salt stress. We will first generate the single, double and multiple knockout mutants of OsKTR1 and OsHAK1/5/iv genes, as well as the gene complementation lines. Then, these transgenic lines together with their wild type will be used to conduct the pot and field experiments with different K application levels and salt (adding NaCl) stresses. The effects of OsKTR1 and OsHAK1/5/iv expression on root acquisition of K and translocation and re-distribution of K in different organs will be analyzed in details. The possible involvement of ethylene, calcium and reactive oxygen species signaling in the transcriptional regulation of OsKTR1 will be examined in response to K deficiency and salt stress. It will be further analyzed that if the phosphorylation modification and interacting proteins of OsKTR1 controls the transcription activity of OsKTR1 in rice. Meanwhile, the commonality and difference of OsKTR1 and OsKTR2 functions and regulations in rice responses to the K deficiency and salt stress will also be conducted in parallel. The researches of this project are helpful to dissect the molecular regulatory mechanism of plants to K deficiency and to breed high K use efficient crops in future.

提高植物耐低钾(K)能力的其中一个重要的分子机制是增强高亲和K转运体HAK家族基因的表达。目前唯一明确的正向转录调控HAK基因的转录因子是属于AP2/ERF家族的拟南芥RAP2.11。在水稻中，我们克隆到与RAP2.11同源的两个转录因子基因（暂定名为OsKTR1和OsKTR2）。初步发现OsKTR1可能调控OsHAK1,OsHAK5和OsHAK-vi的表达及K的吸收与转运。本项目将构建水稻OsKTR1和OsHAK1/5/vi基因的单、双、多突变体及其基因功能回补材料，进行不同供K和盐胁迫处理，分析OsKTR1是否通过转录调控这些HAK基因表达来控制K的吸收转运及再分配，是否通过乙烯、活性氧、钙信号及磷酸化修饰和其互作蛋白调控OsKTR1的表达和转录活性，并探究OsKTR2与OsKTR1的生理功能和分子调控机制的异同。研究结果将有助于明晰作物耐钾的分子调控途径和钾高效分子育种。

植物HAK/KT/KUP（以下简称为HAK）家族钾离子转运体是根系在低钾供应环境中吸收钾的主要通路。低钾激活植物乙烯和氧化胁迫信号，增强HAK家族成员的表达，但其调控机制知之甚少。在本研究中，我们鉴定了两个高度同源、均属于AP2/ERF家族的OsKTR1和OsKTR2基因在水稻响应和调控钾吸收利用中的功能。明确了OsKTR1和OsKTR2均在细胞核内表达，具有转录调控活性。在转录水平上，OsKTR1和OsKTR2 均响应乙烯和氧化胁迫信号。体外DNA结合实验显示OsKTR1和OsKRT2均可以与多个HAK家族基因启动子中包含的GCC序列结合。.发现敲除OsKTR1或OsKRT2均抑制水稻的生长，其中OsKTR2的单突或OsKTR1/2的双突大幅降低水稻的结实率。发现OsKTR1和OsKRT2对OsHAKs 基因的表达和钾离子与重金属铯（Cs）吸收有不同的调控方式。OsKTR1突变强烈促进OsHAK1的表达，显著增加Cs的积累，而OsKTR2突变抑制OsHAK1的表达，显著降低Cs的含量。由于OsHAK1是水稻Cs吸收的关键转运体，因此，OsKTR1和OsKRT2在水稻钾的吸收利用和铯积累的阻控中具有反馈调节功能。.此外，发现OsKTR2能正调控OsHAK22的表达。OsKTR2 与OsHAK22具有相同的组织表达部位，其各自的突变均导致叶鞘中钾含量的降低，呈现容易失水和干旱胁迫症状。而OsKTR2和OsHAK22超表达均增加叶鞘中钾的含量，增强耐干旱的能力。与此同时，明确了OsHAK1和OsHAK26在水稻生长、花粉发育以及OsHAK5在调控水稻株型和产量中的功能。研究结果明晰了水稻耐低钾和阻控铯积累的分子调控途径，也为钾素高效利用与高产分子育种提供了候选基因。