Phosphorylation of WHIRLY1 by CIPK14 Shifts Its Localization and Dual Functions in Arabidopsis

Plastid-to-nucleus retrograde signaling is critical for normal growth and development in plants. The dual-function and dual-located ssDNA binding protein WHIRLY1 (WHY1) has been proposed to coordinate the retrograde signaling from plastids to the nucleus. However, the regulatory mechanism governing the functional switch of WHY1 for mediating plastid-to-nucleus retrograde signaling remains unknown. Here, we report that the Calcineurin B-Like-Interacting Protein Kinase14 (CIPK14) interacts with and phosphorylates WHY1 in Arabidopsis. Phosphorylation of WHY1 results in increased accumulation in the nucleus and enhanced binding with the promoter of WRKY53, which encodes a key transcription factor regulating leaf senescence in Arabidopsis. Transgenic plants overexpressing CIPK14 showed an increased nuclear isoform but decreased plastid isoform of WHY1, among which 95% of transgenic lines showed the stay-green phenotype and 5% of lines showed the variegated pale-green phenotype. Interestingly, the phenotypes of both types of transgenic plants could be recovered by overexpression of plastid-form WHY1. In contrast, knockdown of CIPK14 caused early senescence and even seedling-lethal phenotypes along with elevated expression of senescence-related genes such as WRKY53, SAG12, and NDHF but decreased expression of MER11, RAD50, and POR genes, which could be rescued by overexpression of CIPK14 but not by overexpressing plastid-form or nuclear-form WHY1; the stay-green plants overexpressing CIPK14 showed reduced expression of WRKY53, SAG12, NDHF, and large plastid rRNA. Consistently, the accumulation of nuclear-form WHY1 was significantly reduced in the CIPK14 knockdown lines, resulting in a low ratio of nuclear-/plastid- form WHY1. Taken together, our results demonstrate that CIPK14 regulates the phosphorylation and organellar distributions of WHY1 and pinpoint that CIPK14 may function as a cellular switch between leaf senescence and plastid development for coordinating the intercellular signaling in Arabidopsis.