iTRAQ-based Proteomic Analysis of a Chlorophyll-Deficient Mutant Caused by Single Base Change in RPS4 of Chinese Cabbage (Brassica Campestris L. ssp. Pekinensis)

Abstract

Background: Plastids are important plant-cell organelles containing a genome and bacterial-type 70S ribosomes—primarily composed of plastid ribosomal proteins and ribosomal RNAs. In this study, a chlorophyll-deficient mutant (cdm) obtained from double-haploid Chinese cabbage ‘FT’ was identified as a plastome mutant with an A-to-C base substitution in the plastid gene encoding the ribosomal protein RPS4. To further elucidate the function and regulatory mechanisms of RPS4, a comparative proteomic analysis was conducted between cdm and ‘FT’ plants using isobaric tags and a relative and absolute quantitation by (iTRAQ)-based strategy.Results: A total of 6,245 proteins were identified, 540 of which were differentially expressed (DEPs) in the leaves of cdm as compared to those of ‘FT’—including 233 upregulated and 307 downregulated proteins. Upregulated DEPs were mainly involved in translation, organic nitrogen synthesis, ribosomes, and spliceosomes. Meanwhile, downregulated DEPs were mainly involved in photosynthesis, photosynthetic reaction centres, photosynthetic light harvesting, carbon fixation, and chlorophyll binding. Our findings indicate an important role of RPS4 in the regulation of growth and development of Chinese cabbage, possibly by regulating plastid translation activity by affecting the expression of specific photosynthesis- and cold stress-related proteins. Moreover, a multiple reaction monitoring test and quantitative real time polymerase chain reaction analysis confirmed our iTRAQ results.Conclusions: Quantitative proteomic analysis allowed us to confirm diverse changes in the metabolic pathways between cdm and ‘FT’ plants. Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed that DEPs were significantly associated with photosynthesis, chlorophyll metabolism, carbon metabolism, RNA transport, glucosinolate biosynthesis, and gene splicing. This work provides new insights into the regulation of chlorophyll biosynthesis and photosynthesis in Chinese cabbage.

Different Inhibitory Effects of Erythromycin and Chlortetracycline on Early Growth of Brassica campestris Seedlings

“…SNOWY COTYLEDON 1 (SCO1) encodes a chloroplast-localized elongation factor G that plays a role in chloroplast development in cotyledons in Arabidopsis (Arabidopsis thaliana), but not in the Chl biosynthetic pathway [33]; the knock-out mutant of this gene has pale cotyledons. Additionally, an analysis to identify differentially expressed proteins (DEPs) in the chlorophyll-deficient mutant (cdm) of B. campestris and wild-type plants found that many ribosomal proteins, such as RPL, were up-regulated in cdm; this difference in protein abundance was consistent with a qRT-PCR analysis of gene expression [34]. As transcript abundance of these genes was related to a Chl-deficient phenotype [33,34], we used qRT-PCR to analyze expression of SCO1 (Bra027021) and RPL21 (Bra036731) in B. campestris.…”

“…Additionally, an analysis to identify differentially expressed proteins (DEPs) in the chlorophyll-deficient mutant (cdm) of B. campestris and wild-type plants found that many ribosomal proteins, such as RPL, were up-regulated in cdm; this difference in protein abundance was consistent with a qRT-PCR analysis of gene expression [34]. As transcript abundance of these genes was related to a Chl-deficient phenotype [33,34], we used qRT-PCR to analyze expression of SCO1 (Bra027021) and RPL21 (Bra036731) in B. campestris. Both of these genes are encoded in the nuclear genome.…”