CPK28-mediated phosphorylation enhances nitrate transport activity of NRT2.1 during nitrogen deprivation

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 NRT2.1^S21E, but not the nonphosphorylatable variant NRT2.1^S21A, 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.