A missense mutation of plastid RPS4 is associated with chlorophyll deficiency in Chinese cabbage (Brassica campestris ssp. pekinensis)

Xiaoyan Tang, Yiheng Wang, Yun Zhang, Shengnan Huang, Zhiyong Liu, Danli Fei, Hui Feng

Plastome mutants are ideal resources for elucidating the functions of plastid genes. Numerous studies have been conducted for the function of plastid genes in barley and tobacco; however, related information is limited in Chinese cabbage. A chlorophyll-deficient mutant of Chinese cabbage that was derived by ethyl methanesulfonate treatment on isolated microspores showed uniformly pale green inner leaves and slow growth compared with that shown by the wild type "Fukuda 50' ('FT'). Genetic analysis revealed that cdm was cytoplasmically inherited. Physiological and ultrastructural analyses of cdm showed impaired photosynthesis and abnormal chloroplast development. Utilizing next generation sequencing, the complete plastomes of cdm and 'FT' were respectively re-mapped to the reference genome of Chinese cabbage, and an A-to-C base substitution with a mutation ratio higher than 99% was detected. The missense mutation of plastid ribosomal protein S4 led to valine substitution for glycine at residue 193. The expression level of rps4 was analyzed using quantitative real-time PCR and found lower in than in 'FT'. RNA gel-blot assays showed that the abundance of mature 23S rRNA, 16S rRNA, 5S rRNA, and 4.5S rRNA significantly decreased and that the processing of 23S, 16S rRNA, and 4.5S rRNA was seriously impaired, affecting the ribosomal function in cdm. These findings indicated that cdm was a plastome mutant and that chlorophyll deficiency might be due to an A-to-C base substitution of the plastome-encoded rps4 that impaired the rRNA processing and affected the ribosomal function.