Summary Nitrate (NO3−) serves as the primary inorganic nitrogen source assimilated by most terrestrial plants. The acquisition of nitrate from the soil is facilitated by NITRATE TRANSPORTERS (NRTs), with NRT2.1 being the key high‐affinity nitrate transporter. The activity of NRT2.1, which has multiple potential phosphorylation sites, is intricately regulated under various physiological conditions. Here, we discovered that CALCIUM‐DEPENDENT PROTEIN KINASE 28 (CPK28) positively regulates nitrate uptake under nitrogen deprivation conditions. We found CPK28 as the kinase targeted by immunoprecipitation followed by mass spectrometry and examined the in‐planta phosphorylation status of NRT2.1 in cpk28 mutant plants by employing quantitative MS‐based phosphoproteomics. Through a combination of in vitro phosphorylation experiment and immunoblotting using phospho‐specific antibody, we successfully demonstrated that CPK28 specifically phosphorylates NRT2.1 at Ser21. Functional analysis conducted in Xenopus oocytes revealed that co‐expression of CPK28 significantly enhanced high‐affinity nitrate uptake of NRT2.1. Further investigation using transgenic plants showed that the phosphomimic variant NRT2.1S21E, but not the nonphosphorylatable variant NRT2.1S21A, fully restored high‐affinity 15NO3− uptake ability in both nrt2.1 and cpk28 mutant backgrounds. This study clarifies that the kinase activity of CPK28 is promoted during nitrogen deprivation conditions. These significant findings provide valuable insights into the intricate regulatory mechanisms that govern nitrate‐demand adaptation.

中文翻译：

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CPK28 介导的磷酸化增强了 NRT2.1 在氮剥夺过程中的硝酸盐转运活性


摘要 硝酸盐 （NO3−） 是大多数陆生植物吸收的主要无机氮源。硝酸盐转运蛋白 （NRT） 促进了从土壤中获取硝酸盐，其中 NRT2.1 是关键的高亲和力硝酸盐转运蛋白。NRT2.1 具有多个潜在的磷酸化位点，其活性在各种生理条件下受到错综复杂的调节。在这里，我们发现钙依赖性蛋白激酶 28 （CPK28） 在缺氮条件下正向调节硝酸盐摄取。我们发现 CPK28 是免疫沉淀和质谱法靶向的激酶，并通过采用基于定量 MS 的磷酸化蛋白质组学检测了 CPK28 突变植物中 NRT2.1 的植物内磷酸化状态。通过体外磷酸化实验和使用磷酸化特异性抗体的免疫印迹相结合，我们成功证明 CPK28 在 Ser21 位点特异性磷酸化 NRT2.1。在非洲爪蟾卵母细胞中进行的功能分析显示，CPK28 的共表达显着增强了 NRT2.1 的高亲和力硝酸盐摄取。使用转基因植物的进一步研究表明，磷酸化变体 NRT2.1S21E，而不是非磷酸化变体 NRT2.1S21A，在 nrt2.1 和 cpk28 突变体背景中完全恢复了高亲和力 15NO3− 摄取能力。本研究阐明了 CPK28 的激酶活性在缺氮条件下得到促进。这些重要的发现为控制硝酸盐需求适应的复杂调节机制提供了有价值的见解。