Proteomic and functional analysis of soybean chlorophyll-deficient mutant cd1 and the underlying gene encoding the CHLI subunit of Mg-chelatase

Sufficient chlorophyll biosynthesis is vital for the growth of photoautotrophic plants. Mg-chelatase, consisting of three subunits (CHLI, CHLD, and CHLH), catalyzes the insertion of Mg2+ into protoporphyrin IX, which is the rate-limiting step of chlorophyll biosynthesis. A chlorophyll-deficient mutant (cd1) was previously identified by phenotype. This mutant shows a chlorina phenotype, abnormal chloroplasts, and lower chlorophyll content, plant height, and seed yield than the WT (Nannong 86-4). In this study, map-based cloning of cd1 revealed that a missense mutation (G1709A) was present in GmCHLI1b, leading to an amino acid substitution (D278N) in the Arg-finger domain. Yeast two-hybrid assays indicated that this substitution weakened the interaction between Gmcd1 and GmCHLI1a/b. In addition, GmCHLI1b was not a target protein of GmTrxF1/2 or GmNTRC1/2 in the Y2H system but could interact with them in a bimolecular fluorescence complementation (BiFC) assay. A proteomic analysis of leaf soluble proteins identified 31 differentially accumulated proteins, mainly involved in photosynthesis, carbon fixation in photosynthetic organisms, and carbohydrate/energy and amino acid/protein metabolism. These results indicated that cd1 possesses weaker photosynthesis, respiration and protein metabolism and that its leaf redox homeostasis is disrupted. Global transcriptome analysis of chlorophyll biosynthesis and RT-PCR demonstrated a transcriptional downregulation of photosynthesis-associated genes in cd1 during the diurnal rhythm. This study provides insights into the mechanisms of molecular regulation in the cd1 mutant and adds to the knowledge of the biological and biochemical functions of GmCHLIs in soybean.