BdNRT2A and BdNRT3.2 are the major components of the High-Affinity nitrate Transport System in Brachypodium distachyon
BdNRT2A and BdNRT3.2 are the major components of the High-Affinity nitrate Transport System in Brachypodium distachyon
复制标题
BdNRT2A 和 BdNRT3.2 是二穗短柄草高亲和力硝酸盐转运系统的主要组成部分
DOI：
10.1101/2023.11.24.567652
复制
发表时间：
2023
期刊：
影响因子：
--
通讯作者：
David L
中科院分区：
文献类型：
--
作者：
David L
高效的硝态氮吸收系统有助于在低氮有效度条件下提高作物氮素利用效率。植物的高亲和力硝酸盐转运系统（HATS）在低外部硝酸盐环境下具有活性，并由一个双组分系统介导[高亲和力转运体NRT2与伴侣蛋白NRT3 （NAR2）相关]。在C3谷物的模式植物短尾草中，我们通过多种实验方法研究了bdnrt2aand bdnrt3.2的作用，包括基因表达谱、异种系统功能表征、成像细胞内定位以及基因沉默的反向遗传学。bdnrt2 . a2和bdnrt3.2基因的表达对硝酸盐有效性的响应符合HATS组分的特征。在异源表达系统中，爪蟾卵母细胞中硝酸盐的有效转运需要bdnrt2aand bdnrt3.2的共同表达。BdNRT2A-GFP和BdNRT3.2-RFP融合蛋白在拟南芥原生质体质膜上的瞬时表达实验中观察到功能相互作用。BdNRT3.2似乎对BdNRT2A的质膜定位是必要的。15用BdNRT2A突变体（两个amiRNA突变体和一个nan3诱导的NRT2A蛋白截断突变体）进行的硝酸盐内流测量证实，BdNRT2A是短枝植物中HATS的主要贡献者。为了评估其在BdNRT2A和BdNRT3.2相互作用中导致质膜靶向的潜在作用，研究人员对单子叶植物特异性的保守丝氨酸残基（S461）进行了定向诱变，以模拟非磷酸化的S461A或组成磷酸化的S461D。有趣的是，S461的磷酸化状态并没有改变这种相互作用，这表明存在更复杂的机制。综上所述，BdNRT2A和BdNRT3.2是短柄茅（Bd21-3）硝酸盐HATS活性的主要组成部分，在低氮条件下生长最佳。
An efficient nitrate uptake system contributes to the improvement of crop nitrogen use efficiency under low nitrogen availability. TheHighAffinity nitrateTransportSystem (HATS) in plants is active in low external nitrate and is mediated by a two-component system [high affinity transporters NRT2 associated to a partner protein NRT3 (NAR2)].In Brachypodium, the model plant for C3 cereals, we investigated the role ofBdNRT2AandBdNRT3.2through various experimental approaches including gene expression profiling, functional characterisation in heterologous system, intracellular localization by imaging, and reverse genetics via gene silencing.Expression ofBdNRT2.AandBdNRT3.2genes in response to nitrate availability fits with the characteristics of the HATS components. Co-expression ofBdNRT2AandBdNRT3.2is required for an effective nitrate transport in the heterologous expression system Xenopus oocytes. Functional interaction between BdNRT2A-GFP and BdNRT3.2-RFP fusion proteins has been observed at the plasma membrane in Arabidopsis protoplasts in transient expression experiments. BdNRT3.2 appeared to be necessary for the plasma membrane localization of BdNRT2A.15Nitrate influx measurements withbdnrt2amutants (two amiRNA mutants and one NaN3induced mutant with a truncated NRT2A protein), confirmed that BdNRT2A is a major contributor of the HATS in Brachypodium.Directed mutagenesis in BdNRT2A of a conserved Ser residue (S461) specific to monocotyledons has been performed to mimic a non-phosphorylated S461A or a constitutively phosphorylated S461D, in order to evaluate its potential role in the BdNRT2A and BdNRT3.2 interaction leading to plasma membrane targeting. Interestingly, the phosphorylation status of S461 did not modify the interaction, suggesting on a more complex mechanism.In conclusion, our data show that BdNRT2A and BdNRT3.2 are the main components of the nitrate HATS activity in Brachypodium (Bd21-3) and allow an optimal growth in low N conditions.