离子通道蛋白 SLAH3 调控植物对高铵和低 pH 胁迫响应的分子机理研究

Ammonium and nitrate are essential inorganic nitrogen source for plants. However, when ammonium becomes the sole or dominant inorganic nitrogen source, it inhibits the growth of plants, known as ammonium toxicity. Nitrate can effectively alleviate ammonium toxicity, the molecular mechanism of which is unknown. Our previous work indicated that ammonium toxicity is likely caused by rapid acidification of rhizosphere upon high ammonium condition. We also found a gene SLAH3, encoding a nitrate efflux channel in Arabidopsis root, is involved in regulating high ammonium and low pH stresses. Our current study identified a protein kinase SNRK1.1 that interacts with and strongly phosphorylates the C terminal of SLAH3. Genetic results suggested that SNRK1.1 negatively regulates SLAH3 in dealing with high ammonium and low pH conditions. We also found that the genome of Medicago truncatula contains homologs of AtSLAH3. The expression of several MtSLAH3 was able to rescue the phenotype of Arabidopsis slah3 mutant, suggesting the function of SLAH3 is conserved in Medicago and Arabidopsis. Based on our preliminary genetic and biochemical results, the current project proposes to reveal how SNRK1.1 regulates SLAH3 in regulating adaption of high ammonium and low pH, and ultimately to elucidate the molecular mechanisms of SLAH3 in Arabidopsis as well as in Medicago truncatula.

铵根离子和硝酸根离子是植物重要无机氮源。铵根离子作为唯一或优势无机氮源时通常会对植物生长产生抑制，称为铵毒。硝酸根离子可以有效地缓解铵毒，其分子机理未知。我们前期的工作发现铵毒的产生与高铵条件下植物对根际的迅速酸化有关；同时揭示了拟南芥根中的一个硝酸根离子通道蛋白SLAH3，参与了抗高铵与适应酸性环境过程。目前的工作鉴定到一个蛋白激酶SnRK1.1，结合并磷酸化SLAH3的C端。初步的遗传学实验结果表明SnRK1.1可能通过负调节SLAH3参与抗铵毒、低pH的过程。同时发现豆科植物蒺藜苜蓿中存在SLAH3的同源基因，其中部分基因的表达可以恢复拟南芥slah3突变体表型，说明SLAH3在拟南芥和苜蓿中功能具有保守性。本项目旨在已获得的研究基础上，揭示SnRK1.1如何调控SLAH3，参与抗高铵、低pH的过程，揭示SLAH3相关调控网络的详细分子机理；同时揭示蒺藜苜蓿中SLAH3的生物学功能。

铵态氮（NH4+）和硝态氮（NO3-）是植物的主要无机氮(N)供应来源，但NH4+作为唯一或主要的氮源的时候，会对许多植物造成生长抑制，因此被称为铵毒。有研究表明少量NO3-可以显著减轻铵毒，团队早期研究发现阴离子通道SLAC1同系物SLAH3（anion channel SLAC1 homologue 3，SLAH3）参与了这一过程，但SLAH3如何调控硝酸盐依赖的铵毒缓解机制仍不清楚。.本项目针对离子通道蛋白SLAH3调控植物对高铵和低 pH 胁迫响应的分子机理展开，首先项目团队通过酵母双杂交筛选构建的蛋白激酶库，发现SnRK1.1与SLAH3存在相互作用，蛋白激酶SnRK1.1是一个参与能量平衡和各种逆境胁迫应答的关键调节因子。当植物受到高铵胁迫时，定位于细胞质的SnRK1.1迁移到细胞核。SnRK1.1的易位减弱了它对SLAH3的抑制作用，进而允许未知激酶介导的SLAH3激活。同时，高NH4+/低pH胁迫可能导致严重的能量缺乏，导致核定位的SnRK1.1被强烈激活，从而使SnRK1.1调节下游组分，如负责能量/胁迫相关基因表达的转录因子。SLAH3介导的硝酸盐外流最终导致硝酸盐在根际积累，从而使高铵毒害得到缓解。与先前发现的将SLAH3的N末端磷酸化并作为SLAH3激活剂的SLAH3调节因子不同，该项目揭示了SnRK1.1使SLAH3的C末端磷酸化，并作为SLAH3的抑制子。.总之，该项目的主要研究结果表明激酶SnRK1.1负向调节硝酸盐通道SLAH3，从而介导硝酸盐依赖的铵毒性缓解机制。为构建抗高铵、低pH的苜蓿材料提供理论依据，对绿色农业发展具有举足轻重的作用。