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, 11 (1), 39

The Rice White Green Leaf 2 Gene Causes Defects in Chloroplast Development and Affects the Plastid Ribosomal Protein S9

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The Rice White Green Leaf 2 Gene Causes Defects in Chloroplast Development and Affects the Plastid Ribosomal Protein S9

Zhennan Qiu et al. Rice (N Y).

Abstract

Background: Plastid ribosomal proteins (PRPs) play important roles in the translation of key proteins involved in chloroplast development and photosynthesis. PRPs have been widely studied in many plant species; however, few studies have investigated their roles in rice.

Result: In the present study, we used ethyl methane sulfonate mutagenesis and obtained a novel rice mutant called white green leaf 2 (wgl2). The wgl2 mutants exhibited an albino phenotype from germination through the three-leaf stage, and then gradually transitioned to green through the later developmental stages. Consistent with this albino phenotype, wgl2 mutants had abnormal chloroplasts and lower levels of photosynthetic pigments. Map-based cloning and DNA sequencing analyses of wgl2 revealed a single-nucleotide substitution (G to T) in the first exon of LOC_Os03g55930, which resulted in a substitution of glycine 92 to valine (G92 V). WGL2 encodes a conserved ribosomal protein, which localizes to the chloroplast. Complementation and targeted deletion experiments confirmed that the point mutation in WGL2 is responsible for the wgl2 mutant phenotype. WGL2 is preferentially expressed in the leaf, and mutating WGL2 led to obvious changes in the expression of genes related to chlorophyll biosynthesis, photosynthesis, chloroplast development, and ribosome development compared with wild-type.

Conclusions: WGL2 encodes a conserved ribosomal protein, which localizes to the chloroplast. WGL2 is essential for early chloroplast development in rice. These results facilitate research that will further uncover the molecular mechanism of chloroplast development.

Keywords: Albino phenotype; Chloroplast development; Plastid ribosomal protein; Rice.

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These co-authors involved in the paper all consent to publish this article in Rice.

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The authors declare that they have no competing interests.

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Figures

Fig. 1
Fig. 1
Phenotypes and pigment contents of wild type and wgl2 plants. a-c Morphology of wild type (WT) and wgl2 plants at the seedling (bar = 2 cm), tillering, and heading stages, respectively, in the paddy field (bar = 10 cm). d Pigment contents of WT and wgl2 white seedlings. e Pigment contents of WT and wgl2 green plants that older than the white ones. Chl a, chlorophyll a; Chl b, chlorophyll b; Car, carotenoids. Values represent the mean ± SD of 3 biological replicates. Student’s t-test was used to generate the p values; **p < 0.01
Fig. 2
Fig. 2
Microstructures of wild type (WT) and wgl2 white seedlings chloroplasts viewed with transmission electron microscopy
Fig. 3
Fig. 3
Map-based cloning and identification of the mutation site in wgl2. a The WGL2 locus was initially mapped to Chromosome 3 (Chr. 3). b The WGL2 locus was fine-mapped to a physical interval of 99 kb using the indicated markers. c Mutation site in WGL2 at the genome and protein levels
Fig. 4
Fig. 4
Complementation and targeted deletion experiments. a From left to right: wild type (WT), wgl2 with pCAMBIA1300 (pCK/wgl2), and seedlings from three independent WGL2 complementation lines (COM-1, 2, and 3) (bar = 1 cm). b Relative WGL2 transcript levels in WT, wgl2, and complementation transgenic plants. c Sequence confirmation of the WGL2 deletion in WT plants harboring Cas9/sgRNA constructs. The 20-nt target sequence of the Cas9/sgRNA complex is underlined in blue, and the PAM site is indicated in green. For the Cas9/sgRNA-mutated DNA sequences, inserted nucleotides are shown in red, and deleted nucleotides are depicted as yellow dots. d Phenotypes of mutant seedlings harboring the WLG2 Cas9/sgRNA constructs (bar = 1 cm). e Relative transcript levels of WGL2 in WT and three independent mutant transgenic plants. The WGL2 transcript levels in WT were set to 1. Values represent the mean ± SD of 3 biological replicates
Fig. 5
Fig. 5
Subcellular localization of the WGL2-GFP protein in rice protoplasts. GFP signals show that the WGL2-GFP fusion protein produced from the p35S::WGL2-GFP construct localized to the chloroplast and the control GFP localized to the cytoplasm and nucleus. Green fluorescence shows GFP, red fluorescence shows chloroplast autofluorescence, and yellow fluorescence shows the merged fluorescence. (bar = 5 μm)
Fig. 6
Fig. 6
Phylogenic analysis of WGL2. a Amino acid sequence alignment of the 7 types of WGL2 homologs. Amino acids that were fully or partially conserved are shaded blue and green, respectively. b Phylogenic tree of OsWGL2 and its homologs. Protein sequences are Setaria italica (SiWGL2, XP_004981623.1), Sorghum bicolor (SbWGL2, XP_002463825.1), Zea mays (ZmWGL2, NP_001105916.2), Brachypodium distachyon (BdWGL2, XP_003558826.1), Brassica rapa (BrWGL2, XP_009104732.1), Arabidopsis thaliana (AtWGL2, BAD44402.1), Spinacia oleracea (SoWGL2, XP_021845391.1), Vitis vinifera (VvWGL2, XP_002283650.1), Populus trichocarpa (PtWGL2, XP_006375046.1). The tree was constructed using MEGA version 7.0. Scale represents percentage substitutions per site. Statistical support for the nodes is indicated
Fig. 7
Fig. 7
Relative WGL2 transcript levels in various organs of WT plants. The WGL2 transcript levels in the roots were set to 1. Values represent the mean ± SD of 3 biological replicates
Fig. 8
Fig. 8
Expression of genes associated with Chl biosynthesis, photosynthesis, chloroplast development, and ribosome development in the wgl2 mutant and wild type. a, c Expression of genes associated with Chl biosynthesis, photosynthesis, chloroplast development in WT and wgl2 white seedlings (a), and in WT and wgl2 green plants (c). b, d Expression of genes associated with ribosome development in WT and wgl2 white seedlings (b), and in WT and wgl2 green plants (d). The relative expression level of each gene was normalized using UBQ5 as an internal control. The expression level of each gene in WT was set to 1.0 and other samples were calculated accordingly. Values represent the mean ± SD of 3 biological replicates

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