DnaJ-type Zinc Finger Protein OsDJA10 Positively Regulates Chloroplast Development in Rice

doi:10.13560/j.cnki.biotech.bull.1985.2026-0023

Abstract

Abstract:

Objective The function of DnaJ-type zinc finger protein molecular chaperones in rice remains incompletely understood. Using the OsDJA10 gene (LOC_Os08g36140) as a model, this study investigates the molecular mechanisms underlying the albino phenotype in its knockout (KO) lines and the yellowing phenotype in its RNA interference (RNAi) lines. This study also evaluates the high-light tolerance of its overexpression (OE) lines, providing a theoretical reference for breeding stress-resistant rice varieties. Method CRISPR/Cas9 gene editing, RNAi, and Agrobacterium-mediated transformation were employed to generate knockout, knockdown, and overexpression lines, respectively. The biological functions of the OsDJA10 gene were elucidated by measuring photosynthetic pigments, chlorophyll fluorescence, chloroplast ultrastructure, antioxidant enzyme activities, relative water content and relative electrical conductivity of leaves and roots, and growth traits, combined with subcellular localization, RT-qPCR, and transcriptomic analysis. Result OsDJA10 knockout seedlings exhibited a white-leaf lethal phenotype, with near-complete absence of plant pigments, mature chloroplasts, and thylakoid stacks. Expression of genes related to chloroplast structure and development was significantly downregulated, and differentially expressed genes were significantly enriched in photosynthetic pathways. RNAi-treated plants exhibited leaf chlorosis, significantly reduced photosynthetic pigment content compared to wild-type Zhonghua 11, and markedly downregulated F_v/F_m and NPQ. Chloroplast number decreased significantly, volume shrank markedly, thylakoid stacks were remodeled, and expression of chloroplast structure and development-related genes was downregulated. OsDJA10 -overexpressing lines showed significantly reduced plant height, panicle length, and flag leaf length, with no positive effect on yield; however, they exhibited significantly higher tolerance to high-light stress compared to wild-type Zhonghua 11. Conclusion The OsDJA10 gene positively regulates chloroplast development in rice by influencing photosynthetic gene transcription and photosynthetic organ stability, while enhancing tolerance to high-light stress.

Key words: rice, DnaJ-type protein, chloroplast, transcription, high light stress, OsDJA10, molecular chaperone, photosynthesis

REN Qiong, ZHONG Jiao, DUAN Yu, QIN Tong, KANG Zhen-hui. DnaJ-type Zinc Finger Protein OsDJA10 Positively Regulates Chloroplast Development in Rice[J]. Biotechnology Bulletin, 2026, 42(9): 93-106.

Figures/Tables 10

Fig. 1 Identification of OsDJA10A: The coding sequence and amino acid sequence of LOC_Os08g36140. The red box indicates the conserved motif of the DnaJ-type “CxxCxGxG” zinc finger domain. B: Chromosomal localization of the rice DnaJ gene. The red letters denote the chromosomal position of OsDJA10. C: Phylogenetic tree of the DnaJ family in rice and Arabidopsis

Fig. 2 Expression pattern and subcellular localization of OsDJA10A: Expression of OsDJA10 in different tissues of rice. B: Circadian expression of OsDJA10. C: Expression analysis of OsDJA10 under different stress conditions. Cold stress: 4 ℃; high temperature: 42 ℃. D: Expression of OsDJA10 at different time points under high light stress. E: Subcellular localization of OsDJA10 in rice protoplasts. Green fluorescence indicates GFP signal, red fluorescence indicates chloroplast autofluorescence. Bright: Bright field. Data are presented as mean ± SD. n=3. Scale bar = 10 μm. Data were analyzed using t-tests. **** P < 0.000 1. The same below

Fig. 3 Analysis and identification of OsDJA10 knockout mutantsA: sgRNA target site location for OsDJA10. The blue boxes represent exons, the green boxes represent start codons, and the red boxes represent stop codons. B: Summary of CRISPR/Cas9 genome editing frequencies across strains. C: Sequence alignment of CRISPR/Cas9-edited T0 generation. D: Sequence alignment of CRISPR/Cas9-edited T1 generation. Underlined sequences denote sgRNA and PAM sequences; blue letters indicate base substitutions, dashed lines represent deleted bases, and red letters indicate inserted nucleotide sequences

Fig. 4 Homozygous mutant of OsDJA10 displays an albino phenotypeA: Phenotype of rice seeds 3 days after germination, scale bar = 1 cm; KO1, KO2, and KO3 represent positive edit lines E387-3, E387-18, and E387-20, respectively. B: Phenotype of rice seeds 9 days after germination, scale bar = 2 cm. C: Growth cycle at 21 days post-germination, scale bar = 2 cm. D: Chl a, Chl b, and Car content measurements. E: Chloroplast ultrastructure. CP: Chloroplast. Data represent the mean ± SD of six biological replicates. All data were analyzed using Student’s t-test

Fig. 5 Analysis of chloroplast-associated gene expression and protein levelsA: RT-qPCR analysis of chloroplast-encoded genes and nucleus-encoded chloroplast genes in WT and OsDJA10. Class I: PEP-dependent genes; Class II: PEP- and NEP-dependent genes; Class III: NEP-dependent genes. B: Western blot analysis of chloroplast-encoded proteins in WT and osdja10 seedlings, with β-actin as the internal control protein. Data represent the mean ± SD of three biological replicates. All data were analyzed using Student’s t-test. ***P < 0.001. The same below

Fig. 6 Knockdown of OsDJA10 confers a yellowish-green phenotype in riceA: RT-qPCR analysis of siRNAi1-i8 interference levels. Data represent the mean ± SD of three biological replicates analyzed by one-way ANOVA. B: Phenotype of WTand RNAi lines. Bar = 2 cm. C: Comparison of leaf phenotypes between WT and RNAi lines. Bar = 1 cm. D: Chla, Chlb, and Car content. E: Fresh weight. F: Root length. G: Above-ground height. H: Maximum quantum efficiency of PSII. I: Actual quantum efficiency of PSII. J: Photochemical quenching. K: Non-photochemical quenching. Data represent the mean ± SD of six biological replicates, analyzed by one-way ANOVA. L: Chloroplast ultrastructure. M: RT-qPCR analysis of chloroplast genes and nucleus-encoded chloroplast genes; class I represents PEP-dependent genes; class II represents PEP- and NEP-dependent genes; class III represents NEP-dependent genes. Data represent the mean ± SD from six biological replicates. All data were analyzed using Student’s t-test, *P <0.05,**P < 0.01. The same below

Fig. 7 Phenotypic and physiological and biochemical indicators of OsDJA10 overexpression lines under high light stressA-D: Phenotype under normal conditions (A, B) and 2 d high light treatment (C, D). Scale bar = 2 cm. E: Genomic-level molecular characterization of the OsDJA10 overexpressing line. F: Transcriptional-level molecular characterization of the OsDJA10 overexpressing line. G-R: Physiological and biochemical indicators of WT and OE lines after 8 h high light treatment. Data are presented as mean ± standard error (SE). Statistical analysis was performed using two-way ANOVA

Fig. 8 Transcriptome analysis of WT and KO plantsA: Correlation analysis of gene expression levels between WT and KO, Wt1, Wt2, and Wt3 represent three independent wild-type ZH11 lines, while Mt1, Mt2, and Mt3 represent the albino mutants KO1, KO2, and KO3 respectively. B: Statistical analysis of differentially expressed genes (DEGs) between WT and KO. C: KEGG pathway enrichment analysis. D: GO pathway enrichment analysis

Fig. 9 Agronomic traits of OsDJA10 OE linesA‒B: Maturity-stage phenotypes of WT and OE lines, bar=30 cm. C‒D: Mature spike length phenotypes of WT and OE lines, bar=2 cm. E: Grain length phenotypes of WT and OE, bar=1 cm. F: Grain width phenotypes of WT and OE, bar=1 cm. G: Plant height. H: Spike length. I: Flag leaf length. J: Flag leaf width. K: Tillering number. L: Grain length. M: Grain width. N: Thousand-grain weight. Data are presented as mean ± standard deviation; ten biological replicates. Experimental data were analyzed using Student’s t-test

Fig. 10 Model of OsDJA10 involvement in chloroplast development and albino formation

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