Mutation of YFT3, an isomerase in the isoprenoid biosynthetic pathway, impairs its catalytic activity and carotenoid accumulation in tomato fruit

Abstract

Tomato fruit colors are directly associated with their appearance quality and nutritional value. However, tomato fruit color formation is an intricate biological process that remains elusive. In this work we characterized a tomato yellow fruited tomato 3 (yft3, e9292, Solanum lycopersicum) mutant with yellow fruits. By the map-based cloning approach, we identified a transversion mutation (A2117C) in the YFT3 gene encoding a putative isopentenyl diphosphate isomerase (SlIDI1) enzyme, which may function in the isoprenoid biosynthetic pathway by catalyzing conversion between isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). The mutated YFT3 (A2117C) (designated YFT3 allele) and the YFT3 genes did not show expression difference at protein level, and their encoded YFT3 allelic (S126R) and YFT3 proteins were both localized in plastids. However, the transcript levels of eight genes (DXR, DXS, HDR, PSY1, CRTISO, CYCB, CYP97A, and NCED) associated with carotenoid synthesis were upregulated in fruits of both yft3 and YFT3 knockout (YFT3-KO) lines at 35 and 47 days post-anthesis compared with the red-fruit tomato cultivar (M82). In vitro and in vivo biochemical analyses indicated that YFT3 (S126R) possessed much lower enzymatic activities than the YFT3 protein, indicating that the S126R mutation can impair YFT3 activity. Molecular docking analysis showed that the YFT3 allele has higher ability to recruit isopentenyl pyrophosphate (IPP), but abolishes attachment of the Mg²⁺ cofactor to IPP, suggesting that Ser126 is a critical residue for YTF3 biochemical and physiological functions. As a result, the yft3 mutant tomato line has low carotenoid accumulation and abnormal chromoplast development, which results in yellow ripe fruits. This study provides new insights into molecular mechanisms of tomato fruit color formation and development.

Acknowledgements

The authors are grateful to Dr Dani Zamir and the TGRC (Tomato Genetic Resource Center, UC Davis, CA, USA) for providing tomato seeds. Many thanks to Dr Xin Li and Ge Wang (Instrumental Analysis Center, Shanghai Jiao Tong University, Shanghai, China) for their assistance with carotenoid and TEM analyses. We thank PlantScribe (www.plantscribe.com) and Dr Muhammad Naeem for carefully editing this manuscript. This work was supported by the National Natural Science Foundation of China (32072583, 32372694), Shanghai Collaborative Innovation Center of Agri-Seeds Foundation (ZXWH2150201/010), Shanghai and Kunshan Creation Center of Tomato Novel Germplasm Foundation (SJYY2022-T001), and Collection and Conservation of the Characteristic Crop Germplasm Resource in Kunshan City (Kunshan-AGR-001).

Author contributions

L.Zhao conceived and designed the study and wrote the manuscript. W.L. performed the main experimental work and wrote the manuscript. W.Z. L.C.,and Y.L. conducted the carotenoid analysis and ultrastructural observations. L.Z. performed molecular docking analysis. C.H. assayed YFT3 protein activity in vivo and vitro. T.W. and Y.C. carried out tomato cultivation and greenhouse management. L.Zhang performed data analysis.

Data availability

The data underlying this article are available in the article and online supplementary material.

Conflict of interest

The authors declare no conflicts of interest.

Supplementary data

Supplementary data are available at Horticulture Research online.

References

[1] Liu LH, Shao ZY, Zhang M. et al. Regulation of carotenoid metabolism in tomato. Mol Plant. 2015; 8:28-39

[2] Zhou M, Deng L, Guo SG. et al. Alternative transcription and feedback regulation suggest that SlIDI1 is involved in tomato carotenoid synthesis in a complex way. Hortic Res. 2022;9:uhab045

[3] Botella-Pavía P, Besumbes O, Phillips MA. et al. Regulation of carotenoid biosynthesis in plants: evidence for a key role of hydroxymethylbutenyl diphosphate reductase in controlling the supply of plastidial isoprenoid precursors. Plant J. 2004; 40:188-99

[4] Pankratov I, McQuinn R, Schwartz J. et al. Fruit carotenoid-deficient mutants in tomato reveal a function of the plastidial isopentenyl diphosphate isomerase (IDI1) in carotenoid biosynthesis. Plant J. 2016; 88:82-94

[5] Zhong SL, Fei ZJ, Chen YR. et al. Single-base resolution methylomes of tomato fruit development reveal epigenome modifications associated with ripening. Nat Biotechnol. 2013; 31:154-9

[6] Sun TH, Rao S, Zhou XS. et al. Plant carotenoids: recent advances and future perspectives. Mol Hortic. 2022; 2:3

[7] Schwartz SH, Qin XQ, Zeevaart JAD. Characterization of a novel carotenoid cleavage dioxygenase from plants. J Biol Chem. 2001; 276:25208-11

[8] Vogel JT, Tan BC, McCarty DR. et al. The carotenoid cleavage dioxygenase 1 enzyme has broad substrate specificity, cleaving multiple carotenoids at two different bond positions. J Biol Chem. 2008; 283:11364-73

[9] Vogel JT, Tieman DM, Sims CA. et al. Carotenoid content impacts flavor acceptability in tomato (Solanum lycopersicum). J Sci Food Agric. 2010; 90:2233-40

[10] Tieman D, Zhu GT, Resende MFR Jr. et al. A chemical genetic roadmap to improved tomato flavor. Science. 2017; 355:391-4

[11] Chen LL, Li WZ, Li YP. et al. Identified trans-splicing of YELLOW-FRUITED TOMATO 2 encoding the PHYTOENE SYNTHASE 1 protein alters fruit color by map-based cloning, functional complementation and RACE. Plant Mol Biol. 2019; 100:647-58

[12] Fray RG, Grierson D. Identification and genetic analysis of normal and mutant phytoene synthase genes of tomato by sequencing, complementation and co-suppression. Plant Mol Biol. 1993; 22:589-602

[13] Ronen G, Cohen M, Zamir D. et al. Regulation of carotenoid biosynthesis during tomato fruit development: expression of the gene for lycopene epsilon-cyclase is down-regulated during ripening and is elevated in the mutant Delta. Plant J. 1999; 17:341-51

[14] Ronen G, Carmel-Goren L, Zamir D. et al. An alternative pathway to β-carotene formation in plant chromoplasts discovered by map-based cloning of Beta and old-gold color mutations in tomato. Proc Natl Acad Sci USA. 2000; 97:11102-7

[15] Isaacson T, Ronen G, Zamir D. et al. Cloning of tangerine from tomato reveals a carotenoid isomerase essential for the production of beta-carotene and xanthophylls in plants. Plant Cell. 2002; 14:333-42

[16] Barry CS, Giovannoni JJ. Ethylene and fruit ripening. J Plant Growth Regul. 2007; 26:143-59

[17] Gao L, Zhao WH, Qu HO. et al. The yellow-fruited tomato 1 (yft1) mutant has altered fruit carotenoid accumulation and reduced ethylene production as a result of a genetic lesion in ETHYLENE INSENSITIVE2. Theor Appl Genet. 2016; 129:717-28

[18] Wang WH, Wang YY, Chen T. et al. Current insights into post-transcriptional regulation of fleshy fruit ripening. Plant Physiol. 2023; 192:1785-98

[19] Martel C, Vrebalov J, Tafelmeyer P. et al. The tomato MADS-box transcription factor RIPENING INHIBITOR interacts with promoters involved in numerous ripening processes in a COLORLESS NONRIPENING-dependent manner. Plant Physiol. 2011; 157:1568-79

[20] Vrebalov J, Ruezinsky D, Padmanabhan V. et al. A MADS-box gene necessary for fruit ripening at the TOMATO RIPENING-INHIBITOR (Rin) locus. Science. 2002; 296:343-6

[21] Manning K, Tör M, Poole M. et al. A naturally occurring epigenetic mutation in a gene encoding an SBP-box transcription factor inhibits tomato fruit ripening. Nat Genet. 2006; 38:948-52

[22] Gao Y, Wei W, Fan ZQ. et al. Re-evaluation of the nor mutation and the role of the NAC-NOR transcription factor in tomato fruit ripening. J Exp Bot. 2020; 71:3560-74

[23] Vrebalov J, Pan IL, Arroyo AJM. et al. Fleshy fruit expansion and ripening are regulated by the tomato SHATTERPROOF gene TAGL1. Plant Cell. 2009; 21:3041-62

[24] Fujisawa M, Nakano T, Shima Y. et al. A large-scale identification of direct targets of the tomato MADS box transcription factor RIPENING INHIBITOR reveals the regulation of fruit ripening. Plant Cell. 2013; 25:371-86

[25] Fujisawa M, Shima Y, Higuchi N. et al. Direct targets of the tomato-ripening regulator RIN identified by transcriptome and chromatin immunoprecipitation analyses. Planta. 2012; 235:1107-22

[26] Fujisawa M, Shima Y, Nakagawa H. et al. Transcriptional regulation of fruit ripening by tomato FRUITFULL homologs and associated MADS box proteins. Plant Cell. 2014; 26:89-101

[27] Lin ZF, Hong YG, Yin MG. et al. A tomato HD-zip homeobox protein, LeHB-1, plays an important role in floral organogenesis and ripening. Plant J. 2008; 55:301-10

[28] Chung MY, Vrebalov J, Alba R. et al. A tomato (Solanum lycopersicum) APETALA2/ERF gene, SlAP2a, is a negative regulator of fruit ripening. Plant J. 2010; 64:936-47

[29] Zhao WH, Li YH, Fan SZ. et al. The tomato WRKY32 transcription factor affects ripe fruit color by regulating YFT1, a core component of ethylene signal transduction. J Exp Bot. 2021; 72:4269-82

[30] Lanahan MB, Yen Hsiao-Ching C, Giovannoni JJ. et al. The never ripe mutation blocks ethylene perception in tomato. Plant Cell. 1994; 6:521-30

[31] Jin X, Baysal C, Gao LH. et al. The subcellular localization of two isopentenyl diphosphate isomerases in rice suggests a role for the endoplasmic reticulum in isoprenoid biosynthesis. Plant Cell Rep. 2020; 39:119-33

[32] Vranová E, Coman D, Gruissem W. Network analysis of the MVA and MEP pathways for isoprenoid synthesis. Annu Rev Plant Biol. 2013; 64:665-700

[33] Athanasakoglou A, Grypioti E, Michailidou S. et al. Isoprenoid biosynthesis in the diatom Haslea ostrearia. New Phytol. 2019; 222:230-43

[34] Ganjewala D, Kumar S, Luthra R. An account of cloned genes of methyl-erythritol-4- phosphate pathway of isoprenoid biosynthesis in plants. Curr Issues Mol Biol. 2008;11:i35-45

[35] Rodriguez-Concepcion M, Boronat A. Elucidation of the methylerythritol phosphate pathway for isoprenoid biosynthesis in bacteria and plastids. A metabolic milestone achieved through genomics. Plant Physiol. 2002; 130:1079-89

[36] Rohdich F, Hecht S, Gärtner K. et al. Studies on the non-mevalonate terpene biosynthetic pathway: metabolic role of IspH (LytB) protein. Proc Natl Acad Sci USA. 2002; 99:1158-63

[37] Tritsch D, Hemmerlin A, Bach TJ. et al. Plant isoprenoid biosynthesis via the MEP pathway: in vivo IPP/DMAPP ratio produced by (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase in tobacco BY-2 cell cultures. FEBS Lett. 2010; 584:129-34

[38] Okada K, Kasahara H, Yamaguchi S. et al. Genetic evidence for the role of isopentenyl diphosphate isomerases in the mevalonate pathway and plant development in Arabidopsis. Plant Cell Physiol. 2008; 49:604-16

[39] Chen HL, Li MJ, Liu CQ. et al. Enhancement of the catalytic activity of isopentenyl diphosphate isomerase (IDI) from Saccharomyces cerevisiae through random and site-directed mutagenesis. Microb Cell Fact. 2018; 17:65

[40] Ma DM, Li G, Alejos-Gonzalez F. et al. Overexpression of a type-I isopentenyl pyrophosphate isomerase of Artemisia annua in the cytosol leads to high arteannuin B production and artemisinin increase. Plant J. 2017; 91:466-79

[41] Zhao L, Zhang J, Liu T. et al. Design, synthesis, and antiviral activities of coumarin derivatives containing dithioacetal structures. J Agric Food Chem. 2020; 68:975-81

[42] Durbecq V, Sainz G, Oudjama Y. et al. Crystal structure of isopentenyl diphosphate: dimethylallyl diphosphate isomerase. EMBO J. 2001; 20:1530-7

[43] Zhu F, Wen WW, Cheng YJ. et al. The metabolic changes that effect fruit quality during tomato fruit ripening. Mol Hortic. 2022; 2:2

[44] Nakamura A, Shimada H, Masuda T. et al. Two distinct isopentenyl diphosphate isomerases in cytosol and plastid are differentially induced by environmental stresses in tobacco. FEBS Lett. 2001; 506:61-4

[45] Zheng W, Sun F, Bartlam M. et al. The crystal structure of human isopentenyl diphosphate isomerase at 1.7 Å resolution reveals its catalytic mechanism in isoprenoid biosynthesis. J Mol Biol. 2007; 366:1447-58

[46] Wouters J, Oudjama Y, Barkley SJ. et al. Catalytic mechanism of Escherichia coli isopentenyl diphosphate isomerase involves Cys-67, Glu-116, and Tyr-104 as suggested by crystal structures of complexes with transition state analogues and irreversible inhibitors. J Biol Chem. 2003; 278:11903-8

[47] Yan N, Liu Y, Zhang H. et al. Solanesol biosynthesis in plants. Molecules. 2017; 22:510

[48] Broman KW, Wu H, Sen S. et al. R/QTL: QTL mapping in experimental crosses. Bioinformatics. 2003; 19:889-90

[49] Deng L, Wang H, Sun CL. et al. Efficient generation of pink-fruited tomatoes using CRISPR/Cas9 system efficient generation of pink-fruited tomatoes using CRISPR/Cas9 system. J Genet Genomics. 2018; 45:51-4

[50] Weigel D, Glazebrook J. Transformation of Agrobacterium using the freeze-thaw method. Cold Spring Harb Protoc. 2006; 2006:pdb.prot4666-1036

[51] Zhao WH, Gao L, Li YH. et al. Yellow-fruited phenotype is caused by 573 bp insertion at 5′ UTR of YFT1 allele in yft1 mutant tomato. Plant Sci. 2020; 300:110637

[52] Mao J, Zhang YC, Sang Y. et al. A role for Arabidopsis cryptochromes and COP1 in the regulation of stomatal opening. Proc Natl Acad Sci USA. 2005; 102:12270-5

[53] Nelson BK, Cai X, Nebenführ A. A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. Plant J. 2007; 51:1126-36

[54] Voinnet O, Rivas S, Mestre P. et al. Retracted: an enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J. 2003; 33:949-56

[55] Kilambi HV, Kumar R, Sharma R. et al. Chromoplast-specific carotenoid associated protein appears to be important for enhanced accumulation of carotenoids in hp1 tomato fruits. Plant Physiol. 2013; 161:2085-101

[56] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976; 72:248-54

[57] Li ZD, Ji J, Wang G. et al. Cloning and heterologous expression of isopentenyl diphosphate isomerase gene from Lycium chinense. J Plant Biochem Biotechnol. 2016; 25:40-8

[58] Rampogu S, Gajula RG, Lee G. et al. Unravelling the therapeutic potential of marine drugs as SARS-CoV-2 inhibitors: an insight from essential dynamics and free energy landscape. Comput Biol Med. 2021; 135:104525

[59] Wu GS, Robertson DH, Brooks CL. et al. Detailed analysis of grid-based molecular docking: a case study of CDOCKER - a CHARMm-based MD docking algorithm. J Comput Chem. 2003; 24:1549-62

[60] Rampogu S, Zeb A, Baek A. et al. Discovery of potential plant-derived peptide deformylase (PDF) inhibitors for multidrug-resistant bacteria using computational studies. J Clin Med. 2018; 7:563