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Identical substitutions in magnesium chelatase paralogs result in chlorophyll-deficient soybean mutants.

ABSTRACT: The soybean [Glycine max (L.) Merr.] chlorophyll-deficient line MinnGold is a spontaneous mutant characterized by yellow foliage. Map-based cloning and transgenic complementation revealed that the mutant phenotype is caused by a nonsynonymous nucleotide substitution in the third exon of a Mg-chelatase subunit gene (ChlI1a) on chromosome 13. This gene was selected as a candidate for a different yellow foliage mutant, T219H (Y11y11), that had been previously mapped to chromosome 13. Although the phenotypes of MinnGold and T219H are clearly distinct, sequencing of ChlI1a in T219H identified a different nonsynonymous mutation in the third exon, only six base pairs from the MinnGold mutation. This information, along with previously published allelic tests, were used to identify and clone a third yellow foliage mutation, CD-5, which was previously mapped to chromosome 15. This mutation was identified in the ChlI1b gene, a paralog of ChlI1a. Sequencing of the ChlI1b allele in CD-5 identified a nonsynonymous substitution in the third exon that confers an identical amino acid change as the T219H substitution at ChlI1a. Protein sequence alignments of the two Mg-chelatase subunits indicated that the sites of amino acid modification in MinnGold, T219H, and CD-5 are highly conserved among photosynthetic species. These results suggest that amino acid alterations in this critical domain may create competitive inhibitory interactions between the mutant and wild-type ChlI1a and ChlI1b proteins.

SUBMITTER: Campbell BW

PROVIDER: S-EPMC4291463 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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Identical substitutions in magnesium chelatase paralogs result in chlorophyll-deficient soybean mutants.

Campbell Benjamin W BW Mani Dhananjay D Curtin Shaun J SJ Slattery Rebecca A RA Michno Jean-Michel JM Ort Donald R DR Schaus Philip J PJ Palmer Reid G RG Orf James H JH Stupar Robert M RM

G3 (Bethesda, Md.) 20141201 1

The soybean [Glycine max (L.) Merr.] chlorophyll-deficient line MinnGold is a spontaneous mutant characterized by yellow foliage. Map-based cloning and transgenic complementation revealed that the mutant phenotype is caused by a nonsynonymous nucleotide substitution in the third exon of a Mg-chelatase subunit gene (ChlI1a) on chromosome 13. This gene was selected as a candidate for a different yellow foliage mutant, T219H (Y11y11), that had been previously mapped to chromosome 13. Although the p ...[more]

PMID: 25452420

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Project description:Soybean (Glycine max) seed yields rely on the efficiency of photosynthesis, which is poorly understood in soybean. Chlorophyll, the major light harvesting pigment, is crucial for chloroplast biogenesis and photosynthesis. Magnesium chelatase catalyzes the insertion of Mg2+ into protoporphyrin IX in the first committed and key regulatory step of chlorophyll biosynthesis. It consists of three types of subunits, ChlI, ChlD, and ChlH. To gain a better knowledge of chlorophyll biosynthesis in soybean, we analyzed soybean Mg-chelatase subunits and their encoding genes. Soybean genome harbors 4 GmChlI genes, 2 GmChlD genes, and 3 GmChlH genes, likely evolved from two rounds of gene duplication events. The qRT-PCR analysis revealed that GmChlI, GmChlD, and GmChlH genes predominantly expressed in photosynthetic tissues, but the expression levels among paralogs are different. In silicon promoter analyses revealed these genes harbor different cis-regulatory elements in their promoter regions, suggesting they could differentially respond to various environmental and developmental signals. Subcellular localization analyses illustrated that GmChlI, GmChlD, and GmChlH isoforms are all localized in chloroplast, consistent with their functions. Yeast two hybrid and bimolecular fluorescence complementation (BiFC) assays showed each isoform has a potential to be assembled into the Mg-chelatase holocomplex. We expressed each GmChlI, GmChlD, and GmChlH isoform in Arabidopsis corresponding mutants, and results showed that 4 GmChlI and 2 GmChlD isoforms and GmChlH1 could rescue the severe phenotype of Arabidopsis mutants, indicating that they maintain normal biochemical functions in vivo. However, GmChlH2 and GmChlH3 could not completely rescue the chlorotic phenotype of Arabidopsis gun5-2 mutant, suggesting that the functions of these two proteins could be different from GmChlH1. Considering the differences shown on primary sequences, biochemical functions, and gene expression profiles, we conclude that the paralogs of each soybean Mg-chelatase subunit have diverged more or less during evolution. Soybean could have developed a complex regulatory mechanism to control chlorophyll content to adapt to different developmental and environmental situations.

Project description:Magnesium chelatase is a conserved enzyme complex responsible for the first committed step of chlorophyll biosynthesis in photosynthetic organisms, which is the addition of magnesium to the chlorophyll precursor, protoporphyrin IX. The complex is composed of the catalytic subunit ChlH, the bridging subunit ChlD, and the subunit ChlI, which serves as the motor that drives the entire complex. Although the enzyme is well-characterized functionally, high-resolution structures are available only for individual subunits. Hence, the full assembly and molecular mechanism of the enzyme complex remain unknown. Here, we used cryogenic electron microscopy, supported by biochemical analysis and mass photometry, to determine the structures of the ChlI motor subunit of magnesium chelatase under turnover conditions in the presence of ATP. Our data reveal the molecular details of ChlI oligomerization and conformational dynamics upon ATP binding and hydrolysis. These findings provide new insights into the mechanistic function of ChlI and its implications for the entire magnesium chelatase complex machinery. IMPORTANCE Photosynthesis is an essential life process that relies on chlorophyll. In photosynthetic organisms, chlorophyll synthesis involves multiple steps and depends on magnesium chelatase. This enzyme complex is responsible for inserting magnesium into the chlorophyll precursor, but the molecular mechanism of this process is not fully understood. By using cryogenic electron microscopy and conducting functional analyses, we have discovered that the motor subunit ChlI of magnesium chelatase undergoes conformational changes in the presence of ATP. Our findings offer new insights into how energy is transferred from ChlI to the other components of magnesium chelatase. This information significantly contributes to our understanding of the initial step in chlorophyll biosynthesis and lays the foundation for future studies on the entire process of chlorophyll production.

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Identical substitutions in magnesium chelatase paralogs result in chlorophyll-deficient soybean mutants.

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Identical substitutions in magnesium chelatase paralogs result in chlorophyll-deficient soybean mutants.

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