Zhu Jin-Dong, Liang Yu-Ping, Yan Hong-Ru, Wu Qi-Qi, Zhang Yue-Yue, Zhou Fang-Yuan, Zhang Xiao, Zhao Xiang
State Key Laboratory of Crop Stress Adaptation and Improvement, School of Life Sciences, Henan University, Kaifeng 475004, China.
Plant Physiol. 2025 Feb 7;197(2). doi: 10.1093/plphys/kiae537.
NONPHOTOTROPIC HYPOCOTYL3 (NPH3) is a key regulator of hypocotyl phototropism under both low- and high-intensity blue light (LBL/HBL), mediating phototropin1 (phot1) and phot2 signaling. NPH3 undergoes dephosphorylation and is released from the plasma membrane (PM) upon blue light irradiation. However, how its phosphorylation status and PM localization mediate phot1 and phot2 signaling in Arabidopsis (Arabidopsis thaliana) remains elusive. In this study, we found that fusing NPH3 with GFP at its C-terminus (N3G) impaired its release from the PM, a defect exacerbated by a phosphorylation-deficient mutation, resulting in a dephosphorylated NPH3-GFP (N3AG). Unlike N3G, transgenic lines expressing N3AG exhibited defective hypocotyl phototropism under HBL, which could be rescued by myristoylation at the N-terminus of N3AG (mN3AG), indicating that NPH3 phosphorylation is not essential for HBL-induced phototropic responses when it is artificially anchored at the PM via its N-terminus. Furthermore, genetic analysis revealed that N3AG anchored to the PM by its N-terminus (as in mN3AG) only rescues phot1-mediated HBL responses, which require RPT2. However, N3AG failed to regulate phot2-mediated HBL signaling, regardless of its PM orientation. Taken together, our results revealed that NPH3 phosphorylation is essential for phot2-mediated hypocotyl phototropism under HBL, but is not required for phot1-mediated HBL signaling when the NPH3 N-terminus is PM-anchored.
非向光性下胚轴3（NONPHOTOTROPIC HYPOCOTYL3，NPH3）是低强度和高强度蓝光（LBL/HBL）下胚轴向光性的关键调节因子，介导向光素1（phot1）和向光素2（phot2）信号传导。蓝光照射后，NPH3发生去磷酸化并从质膜（PM）释放。然而，其磷酸化状态和质膜定位如何在拟南芥中介导向光素1和向光素2信号传导仍不清楚。在本研究中，我们发现将NPH3的C末端与绿色荧光蛋白（GFP）融合（N3G）会损害其从质膜的释放，磷酸化缺陷突变会加剧这种缺陷，导致去磷酸化的NPH3-GFP（N3AG）。与N3G不同，表达N3AG的转基因株系在高强度蓝光下表现出下胚轴向光性缺陷，通过在N3AG的N末端进行肉豆蔻酰化（mN3AG）可以挽救这种缺陷，这表明当通过其N末端人工锚定在质膜上时，NPH3磷酸化对于高强度蓝光诱导的向光反应不是必需的。此外，遗传分析表明，通过其N末端锚定在质膜上的N3AG（如mN3AG）仅挽救需要RPT2的向光素1介导的高强度蓝光反应。然而，无论其质膜取向如何，N3AG都无法调节向光素2介导的高强度蓝光信号传导。综上所述，我们的结果表明，NPH3磷酸化对于高强度蓝光下向光素2介导的下胚轴向光性至关重要，但当NPH3的N末端锚定在质膜上时，对于向光素1介导的高强度蓝光信号传导则不是必需的。
