Comparative Proteomic Analysis Reveals That Chlorophyll Metabolism Contributes to Leaf Color Changes in Wucai (<i>Brassica campestris</i> L.) Responding to Cold Acclimation

Chlorophyll is a vital photosynthetic pigment that plays a key role in plant development, participating in light energy capture and energy conversion. In this study, a novel wucai (Brassica campestris L.) germplasm with green outer leaves and yellow inner leaves at the adult stage (W7–2) was used to examine chlorophyll metabolism response to cold acclimation. A green leaf wucai genotype without leaf color changes named W7–1 was selected as the control to evaluate the chlorophyll metabolism changes of W7–2. Compared to W7–1, the contents of chlorophyll a (Chl a) and chlorophyll b (Chl b) in W7–2 were significantly reduced at five developmental stages (13, 21, 29, 37, and 45 days after planting (DAP)). An iTRAQ-based quantitative proteomic analysis was carried out at 21 and 29 DAP according to the leaf color changes in both of genotypes. 1409 proteins were identified, while 218 of them displayed differential accumulations between W7–2 and W7–1 during the two developmental stages. The differentially expressed proteins (DEPs) mainly assigned to chlorophyll biosynthesis, photosynthesis, carbohydrate metabolism, ribosome metabolism and posttranslational modification. Among these DEPs, NADPH-protochlorophyllide oxidoreductase (PORB) and Mg-protoporphyrin IX chelatase 1 (CHLI1) were the key enzymes participating in chlorophyll (Chl) biosynthesis, which was down-regulated at 21 DAP and up-regulated at 29 DAP in W7–2 compared with W7–1, respectively. The expression analysis of genes of three subunits of Mg-chelatase (CHLI1, CHLD, and CHLH), Genomes Uncoupled 4 (GUN4), and Thioredoxin (TRX3) associated with chlorophyll metabolism also displayed significant down-regulation in W7–2. In particular, PORB showed significant up-regulation in W7–2, significantly affecting chlorophyll biosynthesis. Additionally, differences in chlorophyll metabolism between W7–2 and W7–1 were in terms of altered photosynthesis, carbohydrate, and energy metabolism. We found that the transcription levels of most photosynthesis proteins showed significantly lower levels, and the genes expression level, associated with carbohydrate and energy metabolism, were lower in W7–2 than in W7–1. Therefore, the present study results help understand the physiological and molecular mechanisms underlying leaf coloring responding to cold acclimation