Identification and characterization of CsYP in regulating chloroplast development and cucumber peel color

Abstract

Skin color is a crucial quality trait in cucumber fruit, yet the regulatory mechanisms underlying cucumber skin color remain poorly understood. In this study, we characterized a cucumber natural mutant displaying yellow peel, and identified a key gene yellow peel ( CsYP) through map-based cloning. CsYP encodes a rhodanese-like protein with a Rhod domain. A single-base insertion results in premature termination of protein translation, leading to the yellowing pericarp phenotype in the natural mutant. To further investigate the function of CsYP, two knockout lines, yp-1 and yp-2, were generated using CRISPR-Cas9 technology. Phenotypic investigation of yp-1 and yp-2 revealed a significant yellowing of the pericarp starting from 6 days after pollination, consistent with the natural mutant phenotype. Additionally, our study revealed an interaction between CsYP and Cscytb6f, a cytochrome b6-f complex iron–sulfur subunit, suggesting a collaborative role of CsYP and iron–sulfur proteins in regulating cucumber peel color. These findings provide novel insights into the regulatory mechanisms underlying cucumber peel color and broaden our understanding of this important trait.

Acknowledgements

This study was supported by the National Key Research and Development Program ‘Strategic Science and Technology Innovation Cooperation’ Key Special Project (2023YFE0206900), the 2115 Talent Development Program of China Agricultural University. Heartfelt thanks to all the people who have ever helped me in this paper. Special thanks to professor Yiqun Weng for his great efforts in proofreading this article.

Author contributions

X.L. configured and supervised research, and revised the manuscript. Y.C., H.W., Xi Zhao performed the research. Y.C., H.W., S.L., Xiao Zhang drafted of manuscript. Y.D., Yaru Wang, M.A. contributed to experimental design, data interpretation, and manuscript editing. L.Y., Y.D., X.C. contributed to phenotypic data collection in field trials. Yiqun Weng, H.R. participated in data analysis, and manuscript writing.

Data availability

The data supporting this article are accessible within the article itself and the online supplementary material.

Conflicts of interest statement

None declared.

Supplementary material

Supplementary material is available at Horticulture Research online.

References

[1] Xie J . Gene list 2001 for cucumber. Cucurbit Genet Coop Rpt. 2001; 24: 110-36

[2] Grumet R, Garcia-Mas J, Katzir N. Cucurbit Genetics and Genomics: A Look to the Future. In: Grumet R, Katzir N, Garcia-Mas J , eds. Genetics and Genomics of Cucurbitaceae. Springer International Publishing: Cham, 2017, 409-15

[3] Gebretsadik K, Qiu X, Dong S. et al. Molecular research progress and improvement approach of fruit quality traits in cucumber. Theor Appl Genet. 2021; 134: 3535-52

[4] Barrett DM, Beaulieu JC, Shewfelt R . Color, flavor, texture, and nutritional quality of fresh-cut fruits and vegetables: desirable levels, instrumental and sensory measurement, and the effects of processing. Crit Rev Food Sci Nutr. 2010; 50: 369-89

[5] Zhou Q, Wang S, Hu B. et al. An accumulation and replication of chloroplasts 5 gene mutation confers light green peel in cucumber. J Integr Plant Biol. 2015; 57: 936-42

[6] Liu H, Jiao J, Liang X. et al. Map-based cloning, identification and characterization of the w gene controlling white immature fruit color in cucumber (Cucumis sativus L.) . Theor Appl Genet. 2016; 129: 1247-56

[7] Lun Y, Wang X, Zhang C. et al. A CsYcf54 variant conferring light green coloration in cucumber. Euphytica. 2016; 208: 509-17

[8] Hao N, Du Y, Li H. et al. CsMYB36 is involved in the formation of yellow green peel in cucumber (Cucumis sativus L.) . Theor Appl Genet. 2018; 131: 1659-69

[9] Zhai X, Wu H, Wang Y. et al. The fruit glossiness locus, dull fruit (D), encodes a C2H2-type zinc finger transcription factor, CsDULL, in cucumber (Cucumis sativus L.) . Hortic Res. 2022; 9: uhac146

[10] Li S, Yang WC . Rumei, research progress on yellow green leaf mutants in rice. Biotechnol Bull. 2018; 34: 15-21

[11] Chen C, Zhou G, Liu X. et al. Genetic law and molecular mechanism of cucumber peel color. Mol Plant Breed. 2020; 18: 4029-35

[12] Hollingshead S, Kopečná J, Jackson PJ. et al. Conserved chloroplast open-reading frame ycf54 is required for activity of the magnesium protoporphyrin monomethylester oxidative cyclase in Synechocystis PCC 6803 . J Biol Chem. 2012; 287: 27823-33

[13] Arya GC, Dong Y, Heinig U. et al. The metabolic and proteomic repertoires of periderm tissue in skin of the reticulated Sikkim cucumber fruit. Hortic Res. 2022; 9: uhac092

[14] He S, Ye Y, Yuan Y. et al. Insights into flavonoid biosynthesis during cucumber fruit peel coloration based on metabolite profiling and transcriptome analyses. Hortic Plant J. 2023; 9: 763-76

[15] Fu D-Q, Meng L-H, Zhu B-Z. et al. Silencing of the SlNAP7 gene influences plastid development and lycopene accumulation in tomato. Sci Rep . 2016; 6: 38664

[16] Ahn CS, Lee JH, Pai H-S . Silencing of NbNAP1 encoding a Plastidic SufB-like protein affects chloroplast development in Nicotiana benthamiana . Mol Cells. 2005; 20: 112-8

[17] Pierik AJ, Netz DJA, Lill R . Analysis of iron-sulfur protein maturation in eukaryotes. Nat Protoc. 2009; 4: 753-66

[18] Nagane T, Tanaka A, Tanaka R . Involvement of AtNAP1 in the regulation of chlorophyll degradation in Arabidopsis thaliana . Planta. 2010; 231: 939-49

[19] Balk J, Pilon M . Ancient and essential: the assembly of iron-sulfur clusters in plants. Trends Plant Sci. 2011; 16: 218-26

[20] Beinert H, Holm RH, Münck E . Iron-sulfur clusters: nature’s modular, multipurpose structures. Science. 1997; 277: 653-9

[21] Møller SG, Kunkel T, Chua N-H . A plastidic ABC protein involved in intercompartmental communication of light signaling. Genes Dev. 2001; 15: 90-103

[22] Léon S, Touraine B, Briat J-F. et al. The AtNFS2 gene from Arabidopsis thaliana encodes a NifS-like plastidial cysteine desulphurase . Biochem J. 2002; 366: 557-64

[23] Pilon-Smits EAH, Garifullina GF, Abdel-Ghany S. et al. Characterization of a NifS-like chloroplast protein from Arabidopsis. Implications for its role in sulfur and selenium metabolism. Plant Physiol. 2002; 130: 1309-18

[24] Palmer G, Sands RH . On the magnetic resonance of spinach ferredoxin. J Biol Chem. 1966; 241: 253

[25] Hall DO, Gibson JF, Whatley FR . Electron spin resonance spectra of spinach ferredoxin. Biochem Biophys Res Commun. 1966; 23: 81-4

[26] Bonomi F, Pagani S, Kurtz DM Jr . Enzymic synthesis of the 4Fe-4S clusters of Clostridium pasteurianum ferredoxin . Eur J Biochem. 1985; 148: 67-73

[27] Bordo D, Bork P . The rhodanese/Cdc25 phosphatase superfamily. EMBO Rep . 2002; 3: 741-6

[28] Couturier J, Touraine B, Briat J-F. et al. The iron-sulfur cluster assembly machineries in plants: current knowledge and open questions. Front Plant Sci. 2013; 4: 259

[29] Bartels A, Mock H-P, Papenbrock J . Differential expression of Arabidopsis sulfurtransferases under various growth conditions. Plant Physiol Biochem. 2007; 45: 178-87

[30] Cui Y, Li S, Dong Y. et al. Genetic regulation and molecular mechanism of immature cucumber peel color: a review. Veg Res . 2023; 3: 1-6

[31] Moseler A, Selles B, Rouhier N. et al. Novel insights into the diversity of the sulfurtransferase family in photosynthetic organisms with emphasis on oak. New Phytol. 2020; 226: 967-77

[32] Aminlari M, Gilanpour H . Comparative studies on the distribution of rhodanese in different tissues of domestic animals. Comp Biochem Physiol B: Comp Biochem. 1991; 99: 673-7

[33] Ramírez P, Toledo H, Guiliani N. et al. An exported Rhodanese-like protein is induced during growth of Acidithiobacillus ferrooxidans in metal sulfides and different sulfur compounds . Appl Environ Microbiol. 2002; 68: 1837-45

[34] Bonomi F, Pagani S, Cerletti P. et al. Rhodanese-mediated sulfur transfer to succinate dehydrogenase. Eur J Biochem. 1977; 72: 17-24

[35] Pagani S, Galante YM . Interaction of rhodanese with mitochondrial NADH dehydrogenase. Biochim Biophys Acta. 1983; 742: 278-84

[36] Okegawa Y, Basso L, Shikanai T. et al. Cyclic electron transport around PSI contributes to photosynthetic induction with thioredoxin f. Plant Physiol . 2020; 184: 1291-302

[37] Lan Y, Chen Q, Kong M. et al. PetM is essential for the stabilization and function of the cytochrome b6f complex in Arabidopsis. Plant Cell Physiol. 2021; 62: 1603-14

[38] Talib EA, Outten CE . Iron-sulfur cluster biogenesis, trafficking, and signaling: roles for CGFS glutaredoxins and BolA proteins. Biochim Biophys Acta Mol Cell Res . 2021; 1868: 118847

[39] Ibrahim IM, Wu H, Ezhov R. et al. An evolutionarily conserved iron-sulfur cluster underlies redox sensory function of the chloroplast sensor kinase. Commun Biol . 2020; 3: 13

[40] Nie J, Shan N, Liu H. et al. Transcriptional control of local auxin distribution by the CsDFB1-CsPHB module regulates floral organogenesis in cucumber. Proc Natl Acad Sci . 2021; 118: e2023942118

[41] Livak KJ, Schmittgen TD . Analysis of relative gene expression data using real-time quantitative PCR and the 2 ^−ΔΔCT method . Methods. 2001; 25: 402-8

[42] Bolger AM, Lohse M, Usadel B . Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014; 30: 2114-20

[43] Calzadilla PI, Carvalho FEL, Gomez R. et al. Assessing photosynthesis in plant systems: a cornerstone to aid in the selection of resistant and productive crops. Environ Exp Bot. 2022; 201: 104950