Abscisic acid (ABA) transport plays an important role in systemic plant responses to environmental factors. However, it remains largely unclear about the precise regulation of ABA transporters in plants. In this study, we show that the C-terminally encoded peptide receptor 2 (CEPR2) directly interacts with the ABA transporter NRT1.2/NPF4.6. Genetic and phenotypic analyses revealed that NRT1.2/NPF4.6 positively regulates ABA response and that NRT1.2/NPF4.6 is epistatically and negatively regulated by CEPR2. Further biochemical assays demonstrated that CEPR2 phosphorylates NRT1.2/NPF4.6 at serine 292 to promote its degradation under normal conditions. However, ABA treatment and non-phosphorylation at serine 292 prevented the degradation of NRT1.2/NPF4.6, indicating that ABA inhibits the phosphorylation of this residue. Transport assays in yeast and Xenopus oocytes revealed that non-phosphorylated NRT1.2/NPF4.6 had high levels of ABA import activity, whereas phosphorylated NRT1.2/NPF4.6 did not import ABA. Analyses of complemented nrt1.2 mutants that mimicked non-phosphorylated and phosphorylated NRT1.2/NPF4.6 confirmed that non-phosphorylated NRT1.2^S292A had high stability and ABA import activity in planta. Additional experiments showed that NRT1.2/NPF4.6 was degraded via the 26S proteasome and vacuolar degradation pathways. Furthermore, we found that three E2 ubiquitin-conjugating enzymes, UBC32, UBC33, and UBC34, interact with NRT1.2/NPF4.6 in the endoplasmic reticulum and mediate its ubiquitination. NRT1.2/NPF4.6 is epistatically and negatively regulated by UBC32, UBC33, and UBC34 in planta. Taken together, these results suggest that the stability and ABA import activity of NRT1.2/NPF4.6 are precisely regulated by its phosphorylation and degradation in response to environmental stress.

脱落酸 (ABA) 转运在植物系统对环境因素的反应中起重要作用。然而，关于植物中 ABA 转运蛋白的精确调控仍不清楚。在这项研究中，我们表明 C 末端编码的肽受体 2 (CEPR2) 直接与 ABA 转运蛋白 NRT1.2/NPF4.6 相互作用。遗传和表型分析表明，NRT1.2/NPF4.6 正向调节 ABA 反应，而 NRT1.2/NPF4.6 受 CEPR2 的上位和负向调节。进一步的生化分析表明，CEPR2 在丝氨酸 292 处磷酸化 NRT1.2/NPF4.6，以促进其在正常条件下的降解。然而，ABA 处理和 292 位丝氨酸的非磷酸化阻止了 NRT1.2/NPF4.6 的降解，表明 ABA 抑制了该残基的磷酸化。酵母中的转运测定和非洲爪蟾卵母细胞显示非磷酸化 NRT1.2/NPF4.6 具有高水平的 ABA 输入活性，而磷酸化 NRT1.2/NPF4.6 不输入 ABA。补充nrt1的分析。2个模拟非磷酸化和磷酸化NRT1.2/NPF4.6的突变体证实非磷酸化NRT1.2 ^S292A在植物中具有高稳定性和ABA导入活性. 其他实验表明，NRT1.2/NPF4.6 通过 26S 蛋白酶体和液泡降解途径降解。此外，我们发现三种 E2 泛素结合酶 UBC32、UBC33 和 UBC34 与内质网中的 NRT1.2/NPF4.6 相互作用并介导其泛素化。NRT1.2/NPF4.6在植物中受 UBC32、UBC33 和 UBC34 的上位和负调控。总之，这些结果表明 NRT1.2/NPF4.6 的稳定性和 ABA 输入活性受到其响应环境压力的磷酸化和降解的精确调节。