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A Point Mutation of Magnesium Chelatase OsCHLI Gene Dampens the Interaction Between CHLI and CHLD Subunits in Rice

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Abstract

Proper chloroplast development and chlorophyll biosynthesis are essential for the photoautotrophic plants. The insertion of magnesium (Mg2+) into protoporphyrin IX (Proto), catalyzed by magnesium chelatase (Mg-chelatase), is the first committed step of chlorophyll biosynthesis. In dicot plants, a proposed model revealed that Mg-chelatase I subunit (CHLI) and Mg-chelatase D subunit (CHLD) can interact directly; however, their relation remains elusive in rice, a monocot model plant. In this study, we characterized a chlorophyll-deficiency mutant, etiolated leaf and lethal (ell), which displayed a yellow leaf in young seedlings and became lethal after three-leaf stage. Chlorophyll content in homozygous ell mutant was approximately 1 % of that in the wild type. Besides, chloroplast development in the mutant was entirely arrested and no thylakoid structure was observed. By map-based cloning, the ell locus was delimited to a 3.9-Mb interval in chromosome 3. A single-base mutation (G529C) in OsCHLI was identified, leading to an amino acid substitution (G177R) in a highly conserved region. Compared with the wild type, more Proto but less magnesium protoporphyrin IX (Mg-Proto) was measured in the ell mutant. Using protoplast transfection and callus transformation, we found that exogenous OsCHLI could consistently recover the lesion of chloroplast in the ell mutant. By subcellar localization analysis, OsCHLI was detected in the chloroplast. Despite the secondary structure of OsCHLI that was predicted to be altered in the mutant, the point mutation did not affect subcellular localization. Real-time PCR demonstrated that the ell mutation induced significantly transcriptional downregulation of the photosynthesis-associated nuclear and plastid genes. Additionally, yeast-two-hybrid experiments indicated that the single amino acid substitution blocked the intrinsic interaction between OsCHLI and OsCHLD. Moreover, OsCHLI showed physical interactions with some thioredoxins (TRXs), suggesting a similar regulatory mechanism of Mg-chelatase activity in both monocot and dicot plants.

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Abbreviations

Proto:

Protoporphyrin IX

Mg-chelatase:

Magnesium chelatase

ell :

Etiolated leaf and lethal

Mg-Proto:

Magnesium protoporphyrin IX

TRX:

Thioredoxin

EMS:

Ethyl methyl sulfonate

CHLH:

Mg-chelatase H subunit

CHLI:

Mg-chelatase I subunit

CHLD:

Mg-chelatase D subunit

Pchlide:

Protochlorophyllide

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Acknowledgments

This project was financially supported by “the Fundamental Research Funds for the Central Universities (KYY201301)”, “the Key Science and Technology Specific Projects of Guizhou Province (Grant no. 2012-6005)”, the National Science and Technology supporting program (2013BAD01B02-16), the High Technology Program from NDRC ([2012]1961), and the Jiangsu Science and Technology Development Program (BE2012303, BE2013301).

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Authors and Affiliations

  1. National Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing, 210095, China

    Huan Zhang, Linglong Liu, Maohong Cai, Jieyu Zhao, Tianhui Zheng, Xinyang Xu, Zhaoqiong Zeng, Jing Niu, Ling Jiang, Saihua Chen & Jianmin Wan

  2. Rice Research Institute, Guizhou, 550009, China

    Susong Zhu

  3. Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agriculture Sciences, Beijing, 100081, China

    Jianmin Wan

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  1. Huan Zhang
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  2. Linglong Liu
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  3. Maohong Cai
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Correspondence to Saihua Chen or Jianmin Wan.

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Supplementary Table S1

Summary of primers (DOCX 16 kb)

Supplementary Fig. S1

Abundance of Proto, Mg-Proto and Pchilide in wild type and ell mutant. Proto accumulated in ell mutant, but Mg-Proto and Pchilide were decreased compared with the wild type. WT, wild type; **p < 0.01 (GIF 15 kb)

High Resolution Image (TIFF 1872 kb)

Supplementary Fig. S2

Expression analysis of photosynthesis-associated nuclear and plastid genes in the wild type and ell mutant. rpoA (the RNA polymerase A subunit), rpoB (the RNA polymerase B subunit), Lhc (light-harvesting complex protein), psbA (a core component of photosystem II), and rps7 (ribosomal protein S7). Relative amounts were recalculated using the level of Actin transcript as internal control; Nip, Nipponbare; *p < 0.05; **p < 0.01 (GIF 20 kb)

High Resolution Image (TIFF 2672 kb)

Supplementary Fig. S3

Negative controls of BiFC assay. YFP, fluorescence of yellow fluorescent protein; Chl, chlorophyll autofluorescence; ER-mCherry, fluorescence of ER marker, mCherry ER rk CD3-959; Merged, merged image of YFP, Chl and ER-mCherry; bars = 10 μm (GIF 442 kb)

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Zhang, H., Liu, L., Cai, M. et al. A Point Mutation of Magnesium Chelatase OsCHLI Gene Dampens the Interaction Between CHLI and CHLD Subunits in Rice. Plant Mol Biol Rep 33, 1975–1987 (2015). https://doi.org/10.1007/s11105-015-0889-3

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