Flagella-Associated WDR-Containing Protein CrFAP89 Regulates Growth and Lipid Accumulation in Chlamydomonas reinhardtii.

Qiulan Luo,Yajun Li,Chaogang Wang,Wenwen Song,Zhangli Hu

doi:10.3389/fpls.2018.00691

Abstract

WD40-repeat (WDR) domain-containing proteins are subunits of multi-protein E3 ligase complexes regulating various cellular and developmental activities in eukaryotes. Chlamydomonas reinhardtii serves as a model organism to study lipid metabolism in microalgae. Under nutrition deficient conditions, C. reinhardtii accumulates lipids for survival. The proteins in C. reinhardtii flagella have diverse functions, such as controlling the motility and cell cycle, and environment sensing. Here, we characterized the function of CrFAP89, a flagella-associated WDR-containing protein, which was identified from C. reinhardtii nitrogen deficiency transcriptome analysis. Quantitative real time-PCR showed that the transcription levels of CrFAP89 were significantly enhanced upon nutrient deprivation, including nitrogen, sulfur, or iron starvation, which is considered an effective condition to promote triacylglycerol (TAG) accumulation in microalgae. Under sulfur starvation, the expression of CrFAP89 was 32.2-fold higher than the control. Furthermore, two lines of RNAi mutants of CrFAP89 were generated by transformation, with gene silencing of 24.9 and 16.4%, respectively. Inhibiting the expression of the CrFAP89 gene drastically increased cell density by 112–125% and resulted in larger cells, that more tolerant to nutrition starvation. However, the content of neutral lipids declined by 12.8–19.6%. The fatty acid content in the transgenic algae decreased by 12.4 and 13.3%, mostly decreasing the content of C16:0, C16:4, C18, and C20:1 fatty acids, while the C16:1 fatty acid in the CrFAP89 RNAi lines increased by 238.5 to 318.5%. Suppressed expression of TAG biosynthesis-related genes, such as CrDGAT1 and CrDGTTs, were detected in CrFAP89 gene silencing cells, with a reduction of 16–78%. Overall our results suggest that down-regulating of the expression of CrFAP89 in C. reinhardtii, resulting in an increase of cell growth and a decrease of fatty acid synthesis with the most significant decrease occurring in C16:0, C16:4, C18, and C20:1 fatty acid. CrFAP89 might be a regulator for lipid accumulation in C. reinhardtii.

Highlights

WD40-repeat (WDR) proteins comprise a diverse superfamily of regulatory proteins characterized by the presence of several WD motifs
The phylogenetic tree shows that the CrFAP89 from C. reinhardtii is homologous to WDR proteins from other eukaryotic species, including NOTCHLESS and Ribosome assembly protein 4 (Figure 1C), which plays a role in organ number and size, stomatal index, flowering, and seed development in higher plants and animals (Chantha et al, 2006; Stirnimann et al, 2010; Beckcormier et al, 2014)
Our results suggest that CrFAP89 functions as regulatory factor for cell growth and lipid accumulation in C. reinhardtii

Summary

Introduction

WD40-repeat (WDR) proteins comprise a diverse superfamily of regulatory proteins characterized by the presence of several WD motifs ( known as the Trp-Asp or WD40 motifs). WD40 domains are considered as the key motifs of WDR proteins, which mediate diverse protein–protein interactions, and are involved in scaffolding as well as cooperative assembly and regulation of dynamic multi-subunit complexes (Stirnimann et al, 2010). The functions of several WDR proteins in eukaryotes have been well characterized, including RNA processing, organ differentiation, and abiotic stress responses (Jesús et al, 2005; Kong et al, 2015; Chen et al, 2016). The diverse functions of WDR proteins have been characterized in plants, including abiotic stress responses, cell cycle control, signal transduction, and flowering (Zhao et al, 2007; Chuang et al, 2015; Gourlay et al, 2015); they are not well characterized in microalgae

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