PBJ：浙大张国平丨HvAKT2和HvHAK1通过增强叶肉H+稳态提升耐旱能力

NMT是基因功能的活体检测技术，已被103位诺贝尔奖得主所在单位，及北大、清华、中科院使用。

期刊：Plant Biotechnology Journal
主题：HvAKT2和HvHAK1通过增强叶肉H+稳态提升耐旱能力

标题：HvAKT2 and HvHAK1 Confer Drought Tolerance in Barley through Enhanced Leaf Mesophyll H+ Homeostasis

影响因子：6.840
检测指标：K+、H+、Ca2+流速

检测样品：大麦叶肉细胞

K+、H+、Ca2+流实验处理方法：

1）20% PEG瞬时处理大麦叶片
制备叶肉组织，测试液中平衡2h后，用20％ PEG瞬时处理
2）20%PEG预处理
制备叶肉组织，用20% PEG处理0、1、12、24h
K+、H+、Ca2+流实验测试液成份：
0.1 mMCaCl2，0.5 mM KCl，pH 5.8

作者：浙江大学作物科学研究所张国平、邬飞波、陈仲华、冯雪

英文摘要

Plant K+ uptake typically consists low‐affinity mechanisms mediated by Shaker K+ channels (AKT/KAT/KC) and high‐affinity mechanisms regulated by HAK/KUP/KT transporters, which are extensively studied. However, the evolutionary and genetic roles of both K+ uptake mechanisms for drought tolerance are not fully explored in crops adapted to dryland agriculture.

Here, we employed evolutionary bioinformatics, biotechnological and electrophysiological approaches to determine the role of two important K+ transporters HvAKT2 and HvHAK1 in drought tolerance in barley. HvAKT2 and HvHAK1 were cloned and functionally characterized using barley stripe mosaic virus‐induced gene silencing (BSMV‐VIGS) in drought‐tolerant wild barley XZ5 and agrobacterium‐mediated gene transfer in the barley cultivar Golden Promise. The hallmarks of the K+ selective filters of AKT2 and HAK1 are both found in homologues from strepotophyte algae, and they are evolutionarily conserved in strepotophyte algae and land plants. HvAKT2 and HvHAK1 are both localized to the plasma membrane and have high selectivity to K+ and Rb+ over other tested cations.

Overexpression of HvAKT2 and HvHAK1 enhanced K+ uptake and H+ homeostasis leading to drought tolerance in these transgenic lines. Moreover, HvAKT2‐ and HvHAK1‐overexpressing lines showed distinct response of K+, H+, and Ca2+ fluxes across plasma membrane and production of nitric oxide and hydrogen peroxide in leaves as compared to the wild type and silenced lines. High‐ and low‐affinity K+ uptake mechanisms and their coordination with H+ homeostasis play essential roles in drought adaptation of wild barley.

These findings can potentially facilitate future breeding programs for resilient cereal crops in a changing global climate.

中文摘要（谷歌机翻）

植物对钾的吸收通常包括由摇床K+通道（AKT / KAT / KC）介导的低亲和力机制和受HAK / KUP / KT转运蛋白调控的高亲和力机制，对此进行了广泛的研究。但是，在适应旱地农业的农作物中，两种K +吸收机制对干旱的耐受性的进化和遗传作用尚未得到充分研究。

在这里，我们采用了进化生物信息学，生物技术和电生理方法来确定两个重要的K+转运蛋白HvAKT2和HvHAK1在大麦的耐旱性中的作用。使用耐旱的野生大麦XZ5中的大麦条纹花叶病毒诱导的基因沉默（BSMV-VIGS）克隆了HvAKT2和HvHAK1，并在大麦品种Golden Promise中进行了农杆菌介导的基因转移。AKT2和HAK1的K+选择性过滤器的标志都存在于链藻科藻类的同系物中，并且在链藻科藻类和陆地植物中是进化保守的。HvAKT2和HvHAK1都位于质膜上，并且对K+和Rb+的选择性比其他测试阳离子高。

在这些转基因品系中，HvAKT2和HvHAK1的过表达增强了K+吸收和H+稳态，从而导致了干旱耐受性。此外，与野生型和沉默株系相比，HvAKT2和HvHAK1过表达株系在整个质膜上表现出不同的K+，H+和Ca2+通量响应以及叶片中一氧化氮和过氧化氢的产生。高亲和力和低亲和力的K+吸收机制及其与H+稳态的协调在野生大麦的干旱适应中起着重要作用。

这些发现有可能在未来全球气候变化的情况下促进未来谷物抗逆性作物的育种计划。

图1植物和藻类中AKT2和HAK1进化分析

图2 大麦叶片HvHA1表达量、H+-ATP酶活性和离子流变化