Functional Characterization of Rice Leaf Color Gene OsClpP6

doi:10.16819/j.1001-7216.2025.240201

Abstract

Abstract:

【Objective】This study aimed to investigate the function of OsClpP6 in chloroplast development, providing new genetic resources for improving plant photosynthetic rate. 【Method】Bioinformatics analyses were conducted to characterize the OsClpP6 gene and protein. The expression pattern of OsClpP6 was assessed through real-time quantitative PCR and subcellular localization techniques. Targeted editing of the OsClpP6 gene was carried out using CRISPR/Cas9 technology to generate targeted mutations in the OsClpP6 gene. The chloroplast structure in leaf mesophyll cells of mutants was examined using a transmission electron microscopy. Additionally, RNA-seq analysis was conducted to elucidate the impact of OsClpP6 on leaf color-related pathways. 【Result】The Clp gene family plays a crucial role in the early development of chloroplast in rice and is highly conserved during plant evolution. Oryza sativa L. caseinolytic protease P6 (OsClpP6) is an important member of the rice Clp gene family. Spatial and temporal expression pattern analysis revealed that OsClpP6 is predominantly expressed in the aerial parts of seedlings and stems during the vegetative growth stage, with subcellular localization in chloroplasts confirmed through experiments in rice protoplasts. CRISPR/Cas9-mediated knockout mutants of OsClpP6, clpp6-6s-ko-1 and clpp6-6s-ko-2, were generated in the background of Huazhan. These mutants exhibited phenotypic characteristics including reduced plant height, lighter leaf color, and decreased thousand-grain weight compared to the wild type. Transmission electron microscopy observation of flag leaves during the grain-filling stage revealed significantly fewer chloroplasts, thylakoid membrane layers, and starch granules in mutant leaf mesophyll cells compared to the wild type. Transcriptome analysis showed that differentially expressed genes were mainly enriched in the photosynthetic pathway, with significant changes in the expression of key proteins involved in chloroplast development. 【Conclusion】OsClpP6 participates in chloroplast development, affecting the balance of the rice source-sink relationship and subsequently influencing thousand-grain weight.

Key words: rice, leaf color, OsClpP6, Clp complex

摘要：

【目的】探究OsClpP6在叶绿体发育过程中的功能，为提高植物光合速率提供新的基因资源。【方法】对OsClpP6基因、蛋白进行生物信息学分析，通过实时定量PCR和亚细胞定位技术分析该基因的表达模式，利用CRISPR/Cas9技术对该基因进行定点编辑，通过透射电镜观察突变体叶肉细胞叶绿体结构，利用RNA-seq分析OsClpP6影响叶色途径。【结果】Clp基因家族在水稻叶绿体早期发育中起重要作用，且在植物进化过程中十分保守。OsClpP6是水稻Clps基因家族的重要成员之一。通过研究OsClpP6的时空表达模式发现OsClpP6主要在苗期地上部分及营养生长期的茎中表达，水稻原生质体亚细胞定位结果表明，OsClpP6定位在叶绿体。在华占水稻背景下构建了OsClpP6的CRISPR/Cas9敲除突变株系clpp6-6s-ko-1、clpp6-6s-ko-2，突变体较WT株高降低、叶色变浅、千粒重减少。对WT、clpp6-6s-ko-1和clpp6-6s-ko-2灌浆期剑叶叶片进行透射电镜观察叶肉细胞的叶绿体微观结构发现，突变体叶肉细胞叶绿体结构、类囊体片层结构和淀粉粒积累明显少于WT。通过对突变体和WT进行转录组分析发现差异基因主要富集在光合作用通路，多个叶绿体发育的关键蛋白表达出现显著变化。【结论】OsClpP6通过参与叶绿体发育影响水稻源库的平衡，进而影响千粒重。

关键词: 水稻, 叶色, OsClpP6, Clp复合物

GONG Mengmeng, SONG Shufeng, QIU Mudan, DONG Hao, ZHANG Longhui, LI Lei, LI Bin, CHEN Weijun, LI Yixing, WANG Tiankang, LEI Dongyang, LI Li. Functional Characterization of Rice Leaf Color Gene OsClpP6[J]. Chinese Journal OF Rice Science, 2025, 39(2): 197-208.

龚蒙萌, 宋书锋, 邱牡丹, 董皓, 张龙辉, 李磊, 李斌, 谌伟军, 李懿星, 王天抗, 雷东阳, 李莉. 水稻叶色基因OsClpP6的功能研究[J]. 中国水稻科学, 2025, 39(2): 197-208.

Figures/Tables 9

Fig. 1. Analysis of Clp family A represents the phylogenetic tree of the Clp gene family in rice, wheat, maize, Arabidopsis thaliana, soybean, and tobacco. The outer ring displays the domains, with different colors indicating different types; B shows the sequence alignment of one gene selected from each of the five different types in A; C shows the heatmap of the spatiotemporal expression of the ClpP gene family in rice.

Table 1. Information for Clp family in rice

基因名 Gene name	登录号 Locus ID	长度 CDS length(bp)	氨基酸数 Amino acids	等电点 PI
OsClp1	LOC_Os01g16530.1	897	298	9.37
OsClp2	LOC_Os01g32350.1	945	314	8.78
OsClp3	LOC_Os02g42290.1	897	298	7.25
OsClpP5	LOC_Os03g19510.1	849	282	8.46
OsClp5	LOC_Os03g22430.1	1026	341	9.85
OsClpP6	LOC_Os03g29810.1	780	259	7.94
OsClp7	LOC_Os04g44400.1	930	309	7.30
OsClp8	LOC_Os05g51450.1	1161	386	9.30
OsClp9	LOC_Os06g04530.1	894	297	9.50
OsClp10	LOC_Os06g39712.1	651	216	4.38
/	LOC_Os08g15270.1	651	216	4.38
/	LOC_Os10g21300.1	651	216	4.38
OsClp11	LOC_Os10g43050.1	882	293	4.69
OsClp13	LOC_Os12g10590.1	651	216	4.49

Fig. 2. Analysis of expression pattern of OsClpP6 A represents the tissue expression pattern of OsClpP6 in rice, and B represents the subcellular localization of the OsClpP6 protein. P3 and P8 represent young panicles at the 3rd and 8th stages of panicle differentiation, respectively.

Fig. 3. OsClpP6 gene structure and mutation types

Fig. 4. Phenotypic observation of OsClpP6 mutants A shows the phenotypic images of WT, clpp6-6s-ko-1, and clpp6-6s-ko-2 at the mature stage; B shows the flag leaves of WT, clpp6-6s-ko-1, and clpp6-6s-ko-2; C-H are the transmission electron microscopy images of the leaves of WT, clpp6-6s-ko-1, and clpp6-6s-ko-2; I is the statistical chart of plant height; J is the statistical chart of chlorophyll b content in the flag leaf; K is the statistical chart of chlorophyll a content in the flag leaf; Cw stands for cell wall; S stands for starch granules; Og stands for osmiophilic globules; Thy stands for thylakoids.

Fig. 5. Observation of panicle phenotypes of OsClpP6 mutants A shows the panicle phenotypes of WT, clpp6-6s-ko-1, and clpp6-6s-ko-2; B and C show comparison of grain shape among WT, clpp6-6s-ko-1, and clpp6-6s-ko-2; D-I are statistical charts of agronomic traits.

Fig. 6. RNA-seq quality assessment A represents the number of reads aligned to the reference genome; B indicates the number of reads aligned to unique locations in the reference genome; C refers to the selection of 20 up- and 20 down-regulated genes for qRT-PCR validation of transcriptome results; D represents the qRT-PCR analysis results of genes related to chloroplast development and photosynthesis in WT and the mutants. Data are presented as mean ± standard deviation (n = 3). ** P < 0.01.

Fig. 7. Analysis of RNA-seq result The enrichment significance q-value less than 0.05, and the bubble size representing the number of genes in the pathway.

Table 3. Differentially expressed genes in photosynthesis-related pathways

KEGG通路 KEGG pathway	基因编号 Gene ID	基因 Gene	log₂(FC_clpp6-6s-ko/WT)
光合作用Photosynthesis	Os01g0773700		−1.8675
	Os02g0103800	OsLFNR1	−2.5355
	Os02g0103850		−2.5179
	Os02g0578400		−3.1080
	Os06g0107700	OsLFNR2	−1.2132
	Os07g0148900		−2.7024
	Os08g0119800		−2.0123
	Os09g0481200		−2.2606
	Os10g0355800		2.1703
	Os12g0189400		−2.0962
	Os12g0420400		−1.8026
	Os04g0414700		−1.8744
光合作用-天线蛋白Photosynthesis-antenna proteins	Os01g0600900		−2.0603
	Os02g0197600		−1.2470
	Os06g0320500		−1.8205
	Os08g0435900	Lhca4	−1.9374
	Os09g0346500	OsCAB1R	−1.2537
光合组织中的碳固定作用Carbon fixation in photosynthetic organisms	Os02g0601300		1.1576
	Os04g0682100		−4.7302
	Os06g0133800		−1.9227
	Os06g0608700	OsAld-Y	−2.7748
	Os10g0390500	FLO12	1.6028
	Os11g0171300	AldP	−1.9355
	Os12g0169600		−3.9710
	Os12g0291066		−3.1725
	Os12g0291100	OsRBCS3	−3.6935
	Os12g0291400	OsRBCS5	−3.5781
	Os12g0292301		−5.1414
	Os12g0292400	OsRBCS4	−4.3509

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