Please wait a minute...
Journal of Integrative Agriculture
Advanced Online Publication | Current Issue | Archive | Adv Search
A single nucleotide mutation in BrECB2 impaired RNA editing efficiency and early chloroplast biosynthesis

Zifan Zhao, Feng Pan, Tianxiang Zhao, Luyao Zhang, Qingli Hou, Tianer Tang, Nan Wang, Chong Tan, Yun Zhang#, Zhiyong Liu#

Liaoning Key Laboratory of Genetics and Breeding for Cruciferous Vegetable Crops, College of Horticulture, Shenyang Agricultural University, Shenyang, 110866, China

 Highlights 

A delayed chloroplast development mutant ‘M136’ during self-crossing of the pak choi inbred ‘136’.

BrECB2 is identified as the candidate gene by BSA-seq and InDel makers.

BrECB2 encodes a chloroplast-localized DYW-type pentatricopeptide repeat protein.

BrECB2 is involved in early chloroplast development and RNA editing.

Download:  PDF in ScienceDirect  
Export:  BibTeX | EndNote (RIS)      
摘要  

叶绿体是进行光合作用的重要器官,而光合作用对提高青梗菜产量至关重要。在本研究中,我们对白菜自交系'136'自交过程中出现的叶绿体发育延迟突变体'M136'进行了评估。M136'的新生真叶是黄色的,随着成熟逐渐变绿。M136'的叶绿体发育、色素含量和光合作用参数受到影响,并随着生长逐渐恢复,叶绿素荧光参数也受到影响。根据遗传分析和集群分离分析法BSA)-seq,突变体的表型由一个单隐性基因控制,该基因被鉴定为BraA06g011520.3.5CBrECB2),它编码一种DYW型五肽重复(PPR)蛋白。在'M136'中,BrECB2的第4个PPR基序中的T-to-C单核苷酸多态性(SNP)导致苏氨酸到异亮氨酸德氨基酸置换。BrECB2主要在幼叶中表达。‘M136'的叶绿体RNA编辑效率受到影响,在叶片变绿后完全恢复,在互补品系中编辑效率部分恢复。‘M136'的质体编码RNA聚合酶活性未受影响。功能互补分析表明,BrECB2的瞬时过表达部分挽救了'M136'的突变体表型和RNA编辑效率。综上所述,本研究表明BrECB2参与叶绿体早期发育和RNA编辑,为了解青梗菜叶绿体发育调控网络提供了理论依据。



Abstract  

Chloroplasts are important organs for photosynthesis, which is essential for increasing the yields of pak choi. In this study, we evaluated a delayed chloroplast development mutant ‘M136’ during self-crossing of the pak choi inbred ‘136’. The newborn true leaves of ‘M136’ were yellow and gradually green with maturation. Chloroplast development, pigment contents and photosynthesis parameters were impaired and gradually recovered with growth in ‘M136’, and chlorophyll fluorescence parameters were also impaired in ‘M136’. Based on genetic analysis and bulk segregant analysis (BSA)-seq, the mutant phenotype was controlled by a single recessive gene, identified as BraA06g011520.3.5C (BrECB2), which encoded a DYW-type pentatricopeptide repeat (PPR) protein. In ‘M136’, a T-to-C single nucleotide polymorphism (SNP) in the 4th PPR motif of BrECB2 caused a Threonine-to-Isoleucine amino acid substitution. BrECB2 was mainly expressed in young leaves. The chloroplast RNA editing efficiency of ‘M136’ was affected and fully recovered after the leaf turned green, and the editing efficiency was partially restored in complementary lines. The plastid-encoded RNA polymerase activity was not affected in ‘M136’. Functional complementation analyses revealed that the transient overexpression of BrECB2 partially rescued the mutant phenotype and the RNA editing efficiency of ‘M136’. In summary, this study indicate that BrECB2 is involved in early chloroplast development and RNA editing, providing a theoretical basis for understanding the regulatory network involved in chloroplast development in pak choi.

Keywords:  pak choi       BSA-seq        BrECB2        pentatricopeptide repeat protein        RNA editing        chloroplast development  
Online: 03 November 2025  
Fund: 

This work was supported by the National key research and development program (2022YFF1003004 and 2023YFD1200101), the Earmarked Fund for CARS-23, and the National Natural Science Foundation of China (32272736 and 32472762).

About author:  Zifan Zhao, E-mail: zhaozifan818@163.com; #Correspondence Yun Zhang, Tel: +86-13889241423, E-mail: zhangyun511@syau.edu.cn; Zhiyong Liu, Tel: +86-13224252099, E-mail: liuzhiyong99@syau.edu.cn

Cite this article: 

Zifan Zhao, Feng Pan, Tianxiang Zhao, Luyao Zhang, Qingli Hou, Tianer Tang, Nan Wang, Chong Tan, Yun Zhang, Zhiyong Liu. 2025. A single nucleotide mutation in BrECB2 impaired RNA editing efficiency and early chloroplast biosynthesis. Journal of Integrative Agriculture, Doi:10.1016/j.jia.2025.11.001

Abe A, Kosugi S, Yoshida K, Natsume S, Takagi H, Kanzaki H, Matsumura H, Yoshida K, Mitsuoka C, Tamiru M, Innan H, Cano L, Kamoun S, Terauchi R. 2012. Genome sequencing reveals agronomically important loci in rice using MutMap. Nature Biotechnology, 30, 174-178.

Barkan A, Small I. 2014. Pentatricopeptide repeat proteins in plants. Annual Review of Plant Biology, 65, 415-442.

Bentolila S, Oh J, Hanson M R, Bukowski R. 2013. Comprehensive High-Resolution Analysis of the Role of an Arabidopsis Gene Family in RNA Editing. PLoS Genetics, 9, e1003584.

Börner T, Aleynikova A Y, Zubo Y O, Kusnetsov V V. 2015. Chloroplast RNA polymerases: Role in chloroplast biogenesis. Biochimica et Biophysica Acta, 1847, 761-769.

Cao Z L, Yu Q B, Sun Y, Lu Y, Cui Y L, Yang Z N. 2011. A point mutation in the pentatricopeptide repeat motif of the AtECB2 protein causes delayed chloroplast development. Journal of Integrative Plant Biology, 53, 258-269.

Feng X, Yang S, Zhang Y, Zhiyuan C, Tang K, Li G, Yu H, Leng J, Wang Q. 2021. GmPGL2, Encoding a Pentatricopeptide Repeat Protein, Is Essential for Chloroplast RNA Editing and Biogenesis in Soybean. Front Plant Sci, 12, 690973.

Gao L L, Hong Z H, Wang Y, Wu G Z. 2023. Chloroplast proteostasis: A story of birth, life, and death. Plant Communication, 4, 100424.

Gao Y, Huang S, Qu G, Fu W, Zhang M, Liu Z, Feng H. 2020. The mutation of ent-kaurene synthase, a key enzyme involved in gibberellin biosynthesis, confers a non-heading phenotype to Chinese cabbage (Brassica rapa L. ssp. pekinensis). Horticulture Research, 7, 178.

Ge W, Zhang J, Feng H, Wang Y, Ji R. 2023. The root meristem growth factor BrRGF6 positively regulates Chinese cabbage to infection of clubroot disease caused by Plasmodiophora Brassicae. Horticulture Research, 10, uhac292.

Hedtke B, Börner T, Weihe A. 1997. Mitochondrial and chloroplast phage-type RNA polymerases in Arabidopsis. Science, 277, 809-811.

Hill J T, Demarest B L, Bisgrove B W, Gorsi B, Su Y C, Yost H J. 2013. MMAPPR: mutation mapping analysis pipeline for pooled RNA-seq. Genome Research, 23, 687-697.

Hoch B, Maier R M, Appel K, Igloi G L, Kössel H. 1991. Editing of a chloroplast mRNA by creation of an initiation codon. Nature, 353, 178-180.

Huang C, Yu Q B, Li Z R, Ye L S, Xu L, Yang Z N. 2017. Porphobilinogen deaminase HEMC interacts with the PPR-protein AtECB2 for chloroplast RNA editing. Plant Journal, 92, 546-556.

Huang X, Zhang X, Yang S. 2009. A novel chloroplast-localized protein EMB1303 is required for chloroplast development in Arabidopsis. Cell Research, 19, 1205-1216.

Knoop V. 2011. When you can't trust the DNA: RNA editing changes transcript sequences. Cellular and Molecular Life Sciences, 68, 567-586.

Kotera E, Tasaka M, Shikanai T. 2005. A pentatricopeptide repeat protein is essential for RNA editing in chloroplasts. Nature, 433, 326-330.

Lan J, Lin Q, Zhou C, Liu X, Miao R, Ma T, Chen Y, Mou C, Jing R, Feng M, Nguyen T, Ren Y, Cheng Z, Zhang X, Liu S, Jiang L, Wan J. 2023. Young Leaf White Stripe encodes a P-type PPR protein required for chloroplast development. Journal of Integrative Plant Biology, 65, 1687-1702.

Li H, Li L, Wu W, Wang F, Zhou F, Lin Y. 2022. SvSTL1 in the large subunit family of ribonucleotide reductases plays a major role in chloroplast development of Setaria viridis. Plant Journal, 111, 625-641.

Lichtenthaler H K, Wellburn A R. 1983. Determinations of total carotenoids and chlorophylls a and b of leaf extracts in different solvents. Biochemical Society Transactions, 11, 591-592.

Liu D, Li Z A, Li Y, Molloy D P, Huang C. 2023. The DYW domain of RARE1 plays an indispensable role in regulating accD-C794 RNA editing in Arabidopsis thaliana. Plant Science (Amsterdam, Netherlands), 334, 111751.

Liu G, Yu H, Yuan L, Li C, Ye J, Chen W, Wang Y, Ge P, Zhang J, Ye Z, Zhang Y. 2021a. SlRCM1, which encodes tomato Lutescent1, is required for chlorophyll synthesis and chloroplast development in fruits. Horticulture Research, 8, 128.

Liu X Y, Jiang R C, Wang Y, Tang J J, Sun F, Yang Y Z, Tan B C. 2021b. ZmPPR26, a DYW-type pentatricopeptide repeat protein, is required for C-to-U RNA editing at atpA-1148 in maize chloroplasts. Journal of Experimental Botany, 72, 4809-4821.

Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods, 25, 402-408.

Lurin C, Andrés C, Aubourg S, Bellaoui M, Bitton F, Bruyère C, Caboche M, Debast C, Gualberto J, Hoffmann B, Lecharny A, Le Ret M, Martin-Magniette M L, Mireau H, Peeters N, Renou J P, Szurek B, Taconnat L, Small I. 2004. Genome-wide analysis of Arabidopsis pentatricopeptide repeat proteins reveals their essential role in organelle biogenesis. Plant Cell, 16, 2089-2103.

Okuda K, Myouga F, Motohashi R, Shinozaki K, Shikanai T. 2007. Conserved domain structure of pentatricopeptide repeat proteins involved in chloroplast RNA editing. Proceedings of the National Academy of Sciences of the United States of America, 104, 8178-8183.

Pan M, Xia C, Gu S, He H, Wang G, Weng Y, Pan J, Pan J. 2024. Loss-function-of a UMP kinase leads to impaired chloroplast development and photosynthesis efficiency in cucumber. Vegetable Research, 4.

Small I D, Peeters N. 2000. The PPR motif - a TPR-related motif prevalent in plant organellar proteins. Trends in Biochemical Sciences, 25, 46-47.

Song G, Dong S, Liu C, Zou J, Ren J, Feng H. 2023. BrKCS6 mutation conferred a bright glossy phenotype to Chinese cabbage. Theoretical and Applied Genetics, 136, 216.

Tang J, Zhang W, Wen K, Chen G, Sun J, Tian Y, Tang W, Yu J, An H, Wu T, Kong F, Terzaghi W, Wang C, Wan J. 2017. OsPPR6, a pentatricopeptide repeat protein involved in editing and splicing chloroplast RNA, is required for chloroplast biogenesis in rice. Plant Molecular Biology, 95, 345-357.

Thomson S M, Pulido P, Jarvis R P. 2020. Protein import into chloroplasts and its regulation by the ubiquitin-proteasome system. Biochemical Society Transactions, 48, 71-82.

Tseng C C, Sung T Y, Li Y C, Hsu S J, Lin C L, Hsieh M H. 2010. Editing of accD and ndhF chloroplast transcripts is partially affected in the Arabidopsis vanilla cream1 mutant. Plant Molecular Biology, 73, 309-323.

Wang H, Liu J, Zhao W, Terzaghi W, Deng L, Liu H, Zheng Q, Fan S, Hua W, Zheng M. 2023a. DELAYED GREENING 409 encodes a dual-localized pentatricopeptide repeat protein required for chloroplast and mitochondrial development. Plant Physiology, 192, 2768-2784.

Wang N, Liu Z, Zhang Y, Li C, Feng H. 2018. Identification and fine mapping of a stay-green gene (Brnye1) in pakchoi (Brassica campestris L. ssp. chinensis). Theoretical and Applied Genetics, 131, 673-684.

Wang Y, Duan Y, Ai P. 2023b. OsTHA8 encodes a pentatricopeptide repeat protein required for RNA editing and splicing during rice chloroplast development. The Crop Journal, 11, 1353-1367.

Yamazaki H, Tasaka M, Shikanai T. 2004. PPR motifs of the nucleus-encoded factor, PGR3, function in the selective and distinct steps of chloroplast gene expression in Arabidopsis. Plant Journal, 38, 152-163.

Yu Q B, Jiang Y, Chong K, Yang Z N. 2009. AtECB2, a pentatricopeptide repeat protein, is required for chloroplast transcript accD RNA editing and early chloroplast biogenesis in Arabidopsis thaliana. Plant Journal, 59, 1011-1023.

Zhang B, Wu Y, Li S, Ren W, Yang L, Zhuang M, Lv H, Wang Y, Ji J, Hou X, Zhang Y. 2024. Chloroplast C-to-U editing, regulated by a PPR protein BoYgl-2, is important for chlorophyll biosynthesis in cabbage. Horticulture Research, 11, uhae006.

Zhang H, Li X, Yang Y, Hu K, Zhou X, Wen J, Yi B, Shen J, Ma C, Fu T, Tu J. 2022. BnaA02.YTG1, encoding a tetratricopeptide repeat protein, is required for early chloroplast biogenesis in Brassica napus. The Crop Journal, 10, 597-610.

Zhao X, Chen L, Yuan K, Liu Y, Yang L, Zhuang M, Zhang Y, Wang Y, Ji J, Fujimoto R, Lv H. 2024. Genetic mapping and gene editing reveal BoAP1 as a crucial factor regulating chloroplast development in Brassica oleracea. Vegetable Research, 4.

Zhao Y, Huang S, Wang N, Zhang Y, Ren J, Zhao Y, Feng H. 2022. Identification of a biomass unaffected pale green mutant gene in Chinese cabbage (Brassica rapa L. ssp. pekinensis). Bioscience Reports, 12, 7731.

Zhao Z, Tan C, Zhang J, Zhang L, Hou Q, Tang T, Wang B, Zhang Y, Ye X, Zhang Y, Liu Z. 2025. BrSWN mutation reduces the H3K27me3 level at the BrFLC2 and BrFLC3 loci and confers a late-bolting phenotype in Chinese cabbage. Plant Journal, 122, e70151.

Zhou K, Zhang C, Xia J, Yun P, Wang Y, Ma T, Li Z. 2021. Albino seedling lethality 4; Chloroplast 30S Ribosomal Protein S1 is Required for Chloroplast Ribosome Biogenesis and Early Chloroplast Development in Rice. Rice, 14, 47.

Zhou W, Cheng Y, Yap A, Chateigner-Boutin A L, Delannoy E, Hammani K, Small I, Huang J. 2009. The Arabidopsis gene YS1 encoding a DYW protein is required for editing of rpoB transcripts and the rapid development of chloroplasts during early growth. Plant Journal, 58, 82-96.

[1] Jian Ma, Guoliang Yuan, Xinyang Xu, Haijun Zhang, Yanhong Qiu, Congcong Li, Huijun Zhang. Identification and molecular marker development for peel color gene in melon (Cucumis melo L.)[J]. >Journal of Integrative Agriculture, 2025, 24(7): 2589-2600.
[2] Guilong Lu, Chang Zhang, Qibin Wu, Tingting Sun, Shaolin Yang, Erya Wei, Junhui Li, Youxiong Que. Multiple chromosomal configurations and phylogenetic implications in Saccharum mitochondrial genomes[J]. >Journal of Integrative Agriculture, 2025, 24(10): 3909-3925.
[3] Jiawei Pan, Jia Song, Rahat Sharif, Xuewen Xu, Shutong Li, Xuehao Chen.

A mutation in the promoter of the yellow stripe-like transporter gene in cucumber results in a yellow cotyledon phenotype [J]. >Journal of Integrative Agriculture, 2024, 23(3): 849-862.

[4] Liang Ma, Tingli Hu, Meng Kang, Xiaokang Fu, Pengyun Chen, Fei Wei, Hongliang Jian, Xiaoyan Lü, Meng Zhang, Yonglin Yang. Identification of candidate genes for early-maturity traits by combining BSA-seq and QTL mapping in upland cotton (Gossypium hirsutum L.)[J]. >Journal of Integrative Agriculture, 2024, 23(10): 3472-3486.
[5] CAO Peng-hui, WANG Di, GAO Su, LIU Xi, QIAO Zhong-ying, XIE Yu-lin, DONG Ming-hui, DU Tan-xiao, ZHANG Xian, ZHANG Rui, JI Jian-hui. OsDXR interacts with OsMORF1 to regulate chloroplast development and the RNA editing of chloroplast genes in rice[J]. >Journal of Integrative Agriculture, 2023, 22(3): 669-678.
[6] ZHENG Yin-jian, ZHANG Yi-ting, LIU Hou-cheng, LI Ya-min, LIU Ying-liang, HAO Yan-wei, LEI Bing-fu. Supplemental blue light increases growth and quality of greenhouse pak choi depending on cultivar and supplemental light intensity[J]. >Journal of Integrative Agriculture, 2018, 17(10): 2245-2256.
No Suggested Reading articles found!