A missense mutation in COP1 SUPPRESSOR 5 causes reversed action in mediating photomorphogenic development

CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) mediates various cellular and physiological processes in plants by targeting a large number of substrates for ubiquitination and degradation. In this study, we reveal that a substitution of Pro for Leu at the site of 409 in COP1 SUPPRESSOR 5 (CSU5, also called WRKY32) largely suppresses the short hypocotyls and expanded cotyledon phenotypes of cop1-6. CSU5P409L promotes the hypocotyl growth and inhibits the opening of cotyledons in plants. Loss of CSU5 function mutant seedlings display elongated hypocotyls, whereas overexpression of CSU5 leads to shortened hypocotyls. CSU5 directly associates with the promoter regions of HY5 to activate its transcription. Although COP1 interacts with both CSU5 and CSU5P409L, it negatively controls the stability of CSU5, but not CSU5P409L. CSU5P409L exhibits enhanced DNA binding ability and affects the expression of more genes compared with CSU5. Our results not only reveal the molecular role for CSU5 in promoting photomorphogenesis, but also provide insights into manipulating plant growth by engineering key components of light signaling. Total RNA was extracted from the 5-d-old Col, csu5-1 and csu5-3 seedlings grown in constant W (14.59 μmol·m-2·s-1) light conditions. Quality control of DNase I treated mRNA was performed with an Agilent 2100 Bioanalyzer. cDNA libraries were generated, and sequencing was performed using the Illumina HiSeq 2500 platform according to the manufacturer's instruction (HiSequation 2500 user guide) by Novogene. Sequenced reads were preprocessed using Trimmomatic to remove reads in low quality and adaptor contamination (Bolger et al., 2014). The high-quality reads were aligned to Arabidopsis genome (TAIR 10) using Hisat2 (version 2.05; Kim et al., 2015) software using default parameters. Then, raw reads for each gene were calculated by HTseq (Anders et al., 2015) before calculating differential gene expression. Genes with a fold change >1.5 (FDR < 0.01) were defined as differentially expressed genes.