Genome-Wide Identification of Calcium-Dependent Protein Kinases in Chlamydomonas reinhardtii and Functional Analyses in Nitrogen Deficiency-Induced Oil Accumulation.

Yajun Li,Xiaowen Fei,Xiaodong Deng,Weiju Zhu,Jiangyue Li,Haofu Dai

doi:10.3389/fpls.2019.01147

Abstract

Calcium-dependent protein kinases (CDPKs) are recognized as important calcium (Ca2+) sensors in signal transduction and play multiple roles in plant growth and developmental processes, as well as in response to various environmental stresses. However, little information is available about the CDPK family in the green microalga Chlamydomonas reinhardtii. In this study, 15 CrCDPK genes were identified in C. reinhardtii genome, and their functions in nitrogen (N) deficiency-induced oil accumulation were analyzed. Our results showed that all CrCDPK proteins harbored the typical elongation factor (EF)-hand Ca2+-binding and protein kinase domains. Phylogenetic analysis revealed that these CrCDPKs were clustered into one group together with a subclade of several CPKs from Arabidopsis and rice, clearly separating from the remaining AtCPKs and OsCPKs. These genes were located in 10 chromosomes and one scaffold of C. reinhardtii and contained 6–17 exons. RNA sequencing and quantitative reverse transcription (qRT)-PCR assays indicated that most of these CrCDPKs were significantly induced by N deficiency and salt stress. Lanthanum chloride (LaCl3), a plasma membrane Ca2+ channel blocker, limited oil accumulation in C. reinhardtii under N-deficient conditions, suggesting that Ca2+ was involved in N deficiency-induced oil accumulation. Furthermore, RNA interference (RNAi) silencing analyses demonstrated that six CrCDPKs played positive roles and three CrCDPKs played negative roles in N deficiency-induced oil accumulation in C. reinhardtii.

Highlights

Calcium ion (Ca2+), a ubiquitous second messenger in cell signaling, plays essential roles in many physiological processes, such as plant growth, development, and abiotic and biotic stresses (Hetherington and Brownlee, 2004)
Ca2+ binding to elongation factor (EF)-hands induces a conformational change in Calcium-dependent protein kinases (CDPKs), resulting in the refolding and exposure of the active site of the kinase domain and activating the CDPK to phosphorylate an array of substrates
We identified 15 CDPK genes on the basis of a systematic genome-wide analysis in C. reinhardtii and analyzed their protein motifs, phylogenetic relationship, chromosome localization, gene structure, expression profiles under various abiotic stresses, and the functions of CrCDPKs in N deficiency-induced oil accumulation

Summary

Introduction

Calcium ion (Ca2+), a ubiquitous second messenger in cell signaling, plays essential roles in many physiological processes, such as plant growth, development, and abiotic and biotic stresses (Hetherington and Brownlee, 2004). The CDPK family harbors four functional domains, namely, the variable N-terminal domain, highly conserved Ser/Thr kinase domain, autoinhibitory junction domain, and CaM-like domain. The N-terminal domain contains potential myristoylation or palmitoylation sites for membrane association, and the Ser/Thr kinase domain is required for substrate phosphorylation. The autoinhibitory junction domain modulates the kinase activity of CDPKs via a pseudosubstrate mechanism, and the CaM-like domain contains four elongation factor (EF)-hand motifs for binding to Ca2+ (Liese and Romeis, 2013; Schulz et al, 2013; Liu et al, 2018). Ca2+ binding to EF-hands induces a conformational change in CDPK, resulting in the refolding and exposure of the active site of the kinase domain and activating the CDPK to phosphorylate an array of substrates

Methods

Results

Conclusion