Comparative Proteomic Analysis of Coregulation of CIPK14 and WHIRLY1/3 Mediated Pale Yellowing of Leaves in Arabidopsis

Pale yellowing of leaf variegation is observed in the mutant Arabidopsis lines Calcineurin B-Like-Interacting Protein Kinase14 (CIPK14) overexpression (oeCIPK14) and double-knockout WHIRLY1/WHIRLY3 (why1/3). Further, the relative distribution of WHIRLY1 (WHY1) protein between plastids and the nucleus is affected by the phosphorylation of WHY1 by CIPK14. To elucidate the coregulation of CIPK14 and WHIRLY1/WHIRLY3-mediated pale yellowing of leaves, a differential proteomic analysis was conducted between the oeCIPK14 variegated (oeCIPK14-var) line, why1/3 variegated (why1/3-var) line, and wild type (WT). More than 800 protein spots were resolved on each gel, and 67 differentially abundant proteins (DAPs) were identified by matrix-assisted laser desorption ionization-time of flight/time of flight mass spectrometry (MALDI-TOF/TOF-MS). Of these 67 proteins, 34 DAPs were in the oeCIPK14-var line and 33 DAPs were in the why1/3-var line compared to the WT. Five overlapping proteins were differentially expressed in both the oeCIPK14-var and why1/3-var lines: ATP-dependent Clp protease proteolytic subunit-related protein 3 (ClpR3), Ribulose bisphosphate carboxylase large chain (RBCL), Beta-amylase 3 (BAM3), Ribosome-recycling factor (RRF), and Ribulose bisphosphate carboxylase small chain (RBCS). Bioinformatics analysis showed that most of the DAPs are involved in photosynthesis, defense and antioxidation pathways, protein metabolism, amino acid metabolism, energy metabolism, malate biosynthesis, lipid metabolism, and transcription. Thus, in the why1/3-var and oeCIPK14-var lines, there was a decrease in the photosystem parameters, including the content of chlorophyll, the photochemical efficiency of photosystem (PS II) (F(v)/F(m)), and electron transport rates (ETRs), but there was an increase in non-photochemical quenching (NPQ). Both mutants showed high sensitivity to intense light. Based on the annotation of the DAPs from both why1/3-var and oeCIPK14-var lines, we conclude that the CIPK14 phosphorylation-mediated WHY1 deficiency in plastids is related to the impairment of protein metabolism, leading to chloroplast dysfunction.