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. Overall design: 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.

组成型光形态建成1（CONSTITUTIVELY PHOTOMORPHOGENIC 1, COP1）通过靶向大量底物进行泛素化与降解，介导植物体内多种细胞生理过程。本研究发现，在COP1抑制子5（COP1 SUPPRESSOR 5, CSU5，又名WRKY32）的第409位氨基酸位点处，亮氨酸（Leu）替换脯氨酸（Pro）可显著抑制cop1-6突变体的短下胚轴与子叶扩张表型。CSU5P409L可促进植物下胚轴生长，并抑制子叶张开。CSU5功能缺失突变体幼苗表现为下胚轴伸长，而过表达CSU5则会导致下胚轴缩短。CSU5可直接结合HY5的启动子区域以激活其转录。尽管COP1可与CSU5及CSU5P409L均发生互作，但COP1仅负调控CSU5的稳定性，而对CSU5P409L无此作用。相较于CSU5，CSU5P409L展现出更强的DNA结合能力，并可影响更多基因的表达。本研究不仅揭示了CSU5在促进光形态建成中的分子功能，同时也为通过改造光信号通路关键组分来调控植物生长提供了新思路。整体实验设计：从在持续白光（14.59 μmol·m⁻²·s⁻¹）条件下生长5天的哥伦比亚野生型（Col）、csu5-1及csu5-3突变体幼苗中提取总RNA。采用DNase I处理mRNA，并通过安捷伦2100生物分析仪（Agilent 2100 Bioanalyzer）完成质控。构建cDNA文库后，由诺禾致源（Novogene）按照厂商操作指南（HiSequation 2500用户手册），使用Illumina HiSeq 2500测序平台完成测序。测序读段采用Trimmomatic进行预处理，以去除低质量读段与接头污染（Bolger等，2014）。将高质量读段比对至拟南芥基因组（TAIR 10），比对软件为HISAT2（版本2.05；Kim等，2015），参数使用默认设置。随后通过HTseq（Anders等，2015）计算每个基因的原始读段数，再进行差异基因表达分析。将倍数变化（fold change）>1.5且错误发现率（FDR）<0.01的基因定义为差异表达基因。