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, 145 (1), 29-40

A Chlorophyll-Deficient Rice Mutant With Impaired Chlorophyllide Esterification in Chlorophyll Biosynthesis

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A Chlorophyll-Deficient Rice Mutant With Impaired Chlorophyllide Esterification in Chlorophyll Biosynthesis

Ziming Wu et al. Plant Physiol.

Abstract

Chlorophyll (Chl) synthase catalyzes esterification of chlorophyllide to complete the last step of Chl biosynthesis. Although the Chl synthases and the corresponding genes from various organisms have been well characterized, Chl synthase mutants have not yet been reported in higher plants. In this study, a rice (Oryza Sativa) Chl-deficient mutant, yellow-green leaf1 (ygl1), was isolated, which showed yellow-green leaves in young plants with decreased Chl synthesis, increased level of tetrapyrrole intermediates, and delayed chloroplast development. Genetic analysis demonstrated that the phenotype of ygl1 was caused by a recessive mutation in a nuclear gene. The ygl1 locus was mapped to chromosome 5 and isolated by map-based cloning. Sequence analysis revealed that it encodes the Chl synthase and its identity was verified by transgenic complementation. A missense mutation was found in a highly conserved residue of YGL1 in the ygl1 mutant, resulting in reduction of the enzymatic activity. YGL1 is constitutively expressed in all tissues, and its expression is not significantly affected in the ygl1 mutant. Interestingly, the mRNA expression of the cab1R gene encoding the Chl a/b-binding protein was severely suppressed in the ygl1 mutant. Moreover, the expression of some nuclear genes associated with Chl biosynthesis or chloroplast development was also affected in ygl1 seedlings. These results indicate that the expression of nuclear genes encoding various chloroplast proteins might be feedback regulated by the level of Chl or Chl precursors.

Figures

Figure 1.
Figure 1.
Phenotypic characterization of the rice ygl1 mutants. A, Four-week-old plants. B, Ten-week-old plants. C, Fifteen-week-old plants. D, Chloroplasts of the first leaf from top to base have abundant, well-ordered stacks in 4-week-old wild type. E, Chloroplasts of the first leaf in 4-week-old ygl1 mutant have few or no membrane stacks and only occasional long, parallel, and unstacked membranes in worse order than in wild type. Examples of chloroplast (Cp), plastoglobule (Pg), and mitochondrion (Mt). Bar equals 0.5 μm.
Figure 2.
Figure 2.
Map-based cloning of the ygl1 locus. The map was constructed based on the publicly available sequence of rice chromosome 5. Seven CAPS markers (P5, P8, P11, P20, P23, P25, and P26) were produced during this study, while three SSR markers RM516, RM5454, and RM3838 were obtained from the public database, and SSR markers y1, y5, and y22 were developed in the work. A, The ygl1 locus was mapped to a region between markers RM516 and RM5454 on the long arm of rice chromosome 5 (Chr.5) with 252 recessive individuals. B, Fine mapping of the ygl1 locus between y1 and RM3838 from a segregating population of 2,741 recessive individuals. Two BAC contigs (AC144742 and AC136221) cover the ygl1 locus. C, The ygl1 gene was narrowed down to an 11-kb genomic DNA region between the CAPS markers P23 and P8, and cosegregated with P25 and P26.
Figure 3.
Figure 3.
Sequence analysis of YGL1 homologs. A, Alignment of the derived amino acid sequence of rice with published Chl and bacteriochlorophyll synthase sequences. Identical residues are boxed in black, similar residues are highlighted in gray. The Pro-198 to Ser change is indicated with an asterisk at the mutation site of ygl1. Domain II, suggested to be the binding site of the polyprenyl PP, is underlined. GenBank accession numbers for the respective protein sequences are rice (OsYGL1, ABO31092); oat (AsCHLG, AJ277210); Arabidopsis (AtCHLG, At3G51820); Synechooystis sp. PCC 6803 (SCHLG, BA000022); R. capsulacus (RcbchG, CAA77532); Rhodobacter sphaeroides (RsbchG, CP000143); Heliobacillus mobilis (HmbchG, AAC84024); and Chloroflexus aurantiacus (CabchG, AAG15227). B, A phylogenetic tree representing alignment of YGL1 proteins. The rooted tree using percentage identities is based on a multiple sequence alignment generated with the program DNAMAN. Scale represents percentage substitution per site.
Figure 4.
Figure 4.
Complementation of the ygl1 mutant by wild-type gene. The japonica rice ygl2 with ygl1 allele was used as transforming material (see “Materials and Methods”). A, Phenotypes of the wild type, the ygl2 mutant, and the transgenic plant, ygl2 (ygl1)/YGL1. Photographs were taken 2 weeks after sowing. B, Total Chl levels of wild type, ygl2 mutant, and ygl2 (ygl1)/YGL1. Chl was extracted from the second leaf of 2-week-old plants. C, Chl a/b ratio calculated from B. Error bars represent sdsd), and representative data from three independent experiments are presented.
Figure 5.
Figure 5.
Expression analysis of YGL1 by RT-PCR. Total RNA was extracted from root (R), leaf sheath (S), leaf (L), and young panicle (P) of wild type and ygl1 mutant. A, Expression patterns of YGL1 in root (R), leaf sheath (S), leaf (L), and young panicle (P) of wild type and ygl1 mutant. B, YGL1 expression in wild-type and the ygl1 mutant leaves of 2-week-old plants grown in dark or under light. C, YGL1 expression in wild-type and ygl1 mutant leaves of 4-, 10-, and 15-week-old plants. RT-PCR was repeated three times and representative results (25 cycles) are shown. Actin was amplified as a control.
Figure 6.
Figure 6.
Expression analysis of genes associated with Chl biosynthesis, photosynthesis, or chloroplast development by real-time PCR. Total RNA was extracted from leaves of 4-week-old (4w) plants. Actin was amplified as a control. Error bars represent sdsd) and representative data from three independent experiments are presented.
Figure 7.
Figure 7.
Activity of recombinant proteins and time course of Chl accumulation. A, Activity of recombinant YGL1 and ygl1 proteins in E. coli. Total enzyme activity was determined using the extracts from the induced bacterial cells with Chlide a plus PhyPP or Chlide a plus GGPP. Equal amounts of protein were used. B, Time course of Chl accumulation. C, Time course of Chl a/b ratio based on the results in B. The seedlings were grown in darkness for 1 week, Chl content and Chl a/b ratio was measured after exposure to white light for various times as indicated. Error bars represent sdsd) from three independent experiments are presented.
Figure 8.
Figure 8.
Analysis of Chl intermediates in wild-type and ygl1 mutant. Chl intermediates were measured in second leaf from 2-week-old wild-type and ygl1 mutants. A, Levels of ALA. B, Relative fluorescence of Proto IX, Mg-proto IX, Pchlide, and Chlide. Error bars represent sdsd) and representative data from three independent experiments are presented.

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