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Journal of Integrative Agriculture  2025, Vol. 24 Issue (6): 2190-2202    DOI: 10.1016/j.jia.2024.11.027
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ZF protein C2H2-71 regulates the soluble solids content in tomato by inhibiting LIN5

Fangman Li1, Junshen Lin1, John Kojo Ahiakpa1, Wenxian Gai1, Jinbao Tao1, Pingfei Ge1, Xingyu Zhang1, Yizhuo Mu5, Jie Ye1, Yuyang Zhang1, 2, 3, 4#

1 National Key Laboratory for Germplasm Innovation & Utilization of Horticultural Crops, Huazhong Agricultural University, Wuhan 430070, China

2 Hubei Hongshan Laboratory, Wuhan 430070, China

3 Shenzhen Institute of Nutrition and Health, Huazhong Agricultural University, Wuhan 430070, China

4 Shenzhen Branch, Guangdong Laboratory for Lingnan Modern Agriculture/Genome Analysis Laboratory of the Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518000, China

5 College of Agricultural and Environmental Science, University of California, Davis, California 95616, USA

 Highlights 
Zinc finger protein C2H2-71 regulates tomato fruit flavor and reproductive development.
C2H2-71 negatively regulates soluble solids content in tomato by suppressing SlLIN5 expression.
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摘要  

可溶性固形物含量(SSC)是番茄果实风味的重要影响因素。研究表明,果实可溶性固形物积累在糖代谢过程中受到转录调控。之前对于C2H2型锌指蛋白的研究主要集中在生长发育方向,但C2H2锌指蛋白对果实可溶性固形物积累的调控机制尚不清楚。本研究选取了8种不同可溶性固形物含量的番茄材料,通过对红熟果实转录组数据中SlC2H2家族基因表达分析,发现SlC2H2-71基因在高SSC材料中的表达量明显低于低SSC材料。利用CRISPR-Cas9系统构建SlC2H2-71敲除系,发现转基因材料红熟果实中可溶性固形物、果糖、葡萄糖、苹果酸和柠檬酸的含量上升,而蔗糖含量却下降。转录组分析表明,突变体果实中糖酸代谢通路相关基因表达被调控,其中细胞壁酸性转化酶SlLIN5表达上调,并通过实时荧光定量PCR证实了这一点。酵母单杂交(Y1H)、35S::UAS-GUS报告系统、双荧光素酶报告系统和凝胶迁移实验(EMSA)表明,SlC2H2-71锌指蛋白可直接结合SlLIN5启动子并抑制其表达。酶活实验表明SlC2H2-71敲除系果实中的细胞壁酸性转化酶活性显著高于对照系。综上所述,SlC2H2-71锌指蛋白可通过抑制细胞壁酸性转化酶SlLIN5基因的表达和其编码的细胞壁酸性转化酶活性来影响番茄红熟果实可溶性固形物积累。



Abstract  

Soluble solids content (SSC) plays an important role in determining the flavor of tomato fruits.  Tomato fruit SSC is transcriptionally regulated via sugar metabolism.  Previous studies have predominantly focused on the role of C2H2-type zinc finger proteins in tomato growth and development.  However, the specific regulatory mechanisms of C2H2 in the accumulation of soluble solids in tomato fruits are not fully understood.  This study used eight tomato accessions with varying levels of SSC to study the expression of SlC2H2 family genes in red ripe fruits.  The levels of SlC2H2-71 expression were found to be significantly reduced in high-SSC accessions compared to low-SSC accessions.  Several Slc2h2-71 mutant lines were developed using the CRISPR-Cas9 system, which led to elevated levels of soluble solids, fructose, glucose, malic acid, and citric acid in mature red ripe fruits.  However, the sucrose content in the edited Slc2h2-71 mutant lines was generally lower.  RNA-seq analysis revealed that fruits from the mutant lines had altered expression of genes related to the sugar and acid metabolic pathways, which was further confirmed by quantitative real-time PCR.  Specifically, the expression of SlLIN5 encoding the cell wall invertase (CWIN) was elevated.  The yeast one-hybrid (Y1H) assay, 35S::UAS-GUS, dual-luciferase reporter systems and electrophoretic mobility shift assay (EMSA) demonstrated that C2H2-71 regulates tomato sugar metabolism by directly binding to the promoter region of SlLIN5, culminating in the repression of its transcriptional activity.  The activity of acid invertase exhibited a significantly higher level in the SlC2H2-71 knock-out lines compared to the control lines.  In summary, the regulation of tomato fruit SSC by C2H2-71 involves the inhibition of SlLIN5 expression.

Keywords:  Solanum lycopersicum L.       C2H2 zinc finger protein        transcription factor        fruit flavor        cell wall invertase        LIN5  
Received: 22 January 2024   Online: 13 November 2024   Accepted: 11 June 2024
Fund: 
This work was supported by the grants from the National Key Research & Development Plan of China (2022YFF1003002; 2022YFD1200502), the National Natural Science Foundation of China (32372696), the Wuhan Biological Breeding Major Project, China (2022021302024852), the HZAU–AGIS Cooperation Fund, China (SZYJY2023022), the Funds for High Quality Development of Hubei Seed Industry, China (HBZY2023B004), the Hubei Agriculture Research System, China (2024HBSTX4-06), and the Funds of National Key Laboratory for Germplasm Innovation & Utilization of Horticultural Crops, China (Horti-3Y-2024-008).
About author:  Fangman Li, E-mail: lfm@mail.hzau.edu.cn; #Correspondence Yuyang Zhang, Tel: +86-27-87282010, E-mail: yyzhang@mail.hzau.edu.cn

Cite this article: 

Fangman Li, Junshen Lin, John Kojo Ahiakpa, Wenxian Gai, Jinbao Tao, Pingfei Ge, Xingyu Zhang, Yizhuo Mu, Jie Ye, Yuyang Zhang. 2025. ZF protein C2H2-71 regulates the soluble solids content in tomato by inhibiting LIN5. Journal of Integrative Agriculture, 24(6): 2190-2202.

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