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Mutation in a chlorophyll binding motif of Brassica ferrochelatase enhances both heme and chlorophyll biosynthesis

ABSTRACT: The heme branch of tetrapyrrole biosynthesis contributes to the regulation of chlorophyll levels. However, the molecular mechanism underlying the balance between chlorophyll and heme synthesis remains elusive. Here, we identified a dark green leaf mutant dg, which contains high levels of chlorophyll b, from an ethyl methanesulfonate (EMS) -induced mutant library of Chinese cabbage. The dg phenotype is attributed to a nonsynonymous homozygous mutation in ferrochelatase 2 (BrFC2), leading to an arginine (R) to lysine (K) amino acid substitution in the conserved chlorophyll a/b-binding motif (CAB) of BrFC2. Overexpression of mutated BrFC2 (dBrFC2) in Chinese cabbage results in a pronounced phenotype associated with the dark green leaves, in contrast to transgenic lines overexpressing wild-type BrFC2 with a low-chlorophyll phenotype. Increased dBrFC2 homodimer formation stimulates heme production in dg compared with wildtype BrFC2. Moreover, we found that dBrFC2 interacts with protochlorophyllide oxidoreductase B1 and B2 (BrPORB1 and BrPORB2) to enhance their binding ability to substrate protochlorophyllide (Pchlide), thereby promoting BrPORBs-catalyzed production of chlorophyllide (Chlide) that can be directly converted into chlorophyll. Our results reveal that dBrFC2 is a strong gain-of-function mutation contributing to balancing heme and chlorophyll synthesis via a novel regulatory mechanism in which dBrFC2 promotes BrPORB enzymatic reaction to enhance chlorophyll synthesis.

ORGANISM(S): Brassica Rapa Subsp. Pekinensis

SUBMITTER: Jianjun Zhao

PROVIDER: PXD037223 | iProX | Sun Oct 09 00:00:00 BST 2022

REPOSITORIES: iProX

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Project description:The leaf of Chinese cabbage is the major place of photosynthesis, the mutation of leaf may directly affect the rate of plant growth and development and the formation of leafy head, and ultimately influence the yield and quality of Chinese cabbage. We identified a developmentally retarded mutant (drm) exhibiting stable inheritance, which was derived from Chinese cabbage DH line âFTâ using a combination of isolated microspore culture and radiation treatment (60Co Î³-rays). The drm exhibited slow growth and development at the seedling and heading stages, leading to the production of a tiny, leafy head, as well as chlorophyll-deficient leaves, especially in seedlings. Genetic analysis indicated that the phenotype of drm was controlled by a single recessive nuclear gene. Compared with wild-type line âFTâ, the drmâs chlorophyll content was significantly reduced and its chloroplast structure was abnormal. Moreover, the photosynthetic efficiency and chlorophyll fluorescence parameters were significantly decreased. The changes in leaf color, combined with these altered physiological characters may influence the growth and development of plant, ultimately resulting in the developmentally retarded phenotype of drm. To further understand the molecular regulatory mechanisms of phenotypic differences between âFTâ and drm, comparative transcriptome analysis were performed using RNA-Seq, a total of 338 differentially expressed genes (DEGs) were detected between âFTâ and drm. According to GO and KEGG pathway analysis, a number of DEGs which involved in the chlorophyll degradation and photosynthesis were identified, such as chlorophyllase and ribulose-1,5-bisphosphate carboxylase/oxygenase. In addition, the expression patterns of 12 DEGs, including three chlorophyll degradation- and photosynthesis-related genes and nine randomly selected genes, were confirmed by qRT-PCR. Numerous single nucleotide polymorphisms were also identified, providing a valuable resource for research and molecular marker-assistant breeding in Chinese cabbage. These results contribute to our understanding of the molecular regulatory mechanisms underlying growth and development and lay the foundation for future genetic and functional genomics studies in Chinese cabbage. The RNA from the third true leaves (day 15 to day 24 after the appearance of the third true leaves) of a developmentally retarded mutant (drm) and its wild type âFTâ in Chinese cabbage were sequenced by RNA-Seq, in triplicate.

Project description:The leaf of Chinese cabbage is the major place of photosynthesis, the mutation of leaf may directly affect the rate of plant growth and development and the formation of leafy head, and ultimately influence the yield and quality of Chinese cabbage. We identified a developmentally retarded mutant (drm) exhibiting stable inheritance, which was derived from Chinese cabbage DH line ‘FT’ using a combination of isolated microspore culture and radiation treatment (60Co γ-rays). The drm exhibited slow growth and development at the seedling and heading stages, leading to the production of a tiny, leafy head, as well as chlorophyll-deficient leaves, especially in seedlings. Genetic analysis indicated that the phenotype of drm was controlled by a single recessive nuclear gene. Compared with wild-type line ‘FT’, the drm’s chlorophyll content was significantly reduced and its chloroplast structure was abnormal. Moreover, the photosynthetic efficiency and chlorophyll fluorescence parameters were significantly decreased. The changes in leaf color, combined with these altered physiological characters may influence the growth and development of plant, ultimately resulting in the developmentally retarded phenotype of drm. To further understand the molecular regulatory mechanisms of phenotypic differences between ‘FT’ and drm, comparative transcriptome analysis were performed using RNA-Seq, a total of 338 differentially expressed genes (DEGs) were detected between ‘FT’ and drm. According to GO and KEGG pathway analysis, a number of DEGs which involved in the chlorophyll degradation and photosynthesis were identified, such as chlorophyllase and ribulose-1,5-bisphosphate carboxylase/oxygenase. In addition, the expression patterns of 12 DEGs, including three chlorophyll degradation- and photosynthesis-related genes and nine randomly selected genes, were confirmed by qRT-PCR. Numerous single nucleotide polymorphisms were also identified, providing a valuable resource for research and molecular marker-assistant breeding in Chinese cabbage. These results contribute to our understanding of the molecular regulatory mechanisms underlying growth and development and lay the foundation for future genetic and functional genomics studies in Chinese cabbage.

Dataset Information

Mutation in a chlorophyll binding motif of Brassica ferrochelatase enhances both heme and chlorophyll biosynthesis

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