Coordinated transcriptional regulation of the carotenoid biosynthesis contributes to fruit lycopene content in high-lycopene tomato genotypes

Abstract

Lycopene content in tomato fruit is largely under genetic control and varies greatly among genotypes. Continued improvement of lycopene content in elite varieties with conventional breeding has become challenging, in part because little is known about the underlying molecular mechanisms in high-lycopene tomatoes (HLYs). We collected 42 HLYs with different genetic backgrounds worldwide. High-performance liquid chromatography (HPLC) analysis revealed lycopene contents differed among the positive control wild tomato Solanum pimpinellifolium, HLYs, the normal lycopene cultivar “Moneymaker”, and the non-lycopene cultivar NC 1Y at the pink and red ripe stages. Real-time RT-PCR analysis of expression of the 25 carotenoid biosynthesis pathway genes of each genotype showed a significantly higher expression in nine upstream genes (GGPPS1, GGPPS2, GGPPS3, TPT1, SSU II, PSY2, ZDS, CrtISO and CrtISO-L1 but not the well-studied PSY1, PDS and Z-ISO) at the breaker and/or red ripe stages in HLYs compared to Moneymaker, indicating a higher metabolic flux flow into carotenoid biosynthesis pathway in HLYs. Further conversion of lycopene to carotenes may be prevented via the two downstream genes ( β -LCY2 and ε -LCY), which had low-abundance transcripts at either or both stages. Additionally, the significantly higher expression of four downstream genes (BCH1, ZEP, VDE, and CYP97C11) at either or both ripeness stages leads to significantly lower fruit lycopene content in HLYs than in the wild tomato. This is the first systematic investigation of the role of the complete pathway genes in regulating fruit lycopene biosynthesis across many HLYs, and enables tomato breeding and gene editing for increased fruit lycopene content.

References

[1] FAOSTAT. Retrieved June 16, 2020, from http://www.fao.org/faostat/en/#home

[2] Foolad MR, Merk HL, Ashrafi H . Genetics, genomics and breeding of late blight and early blight resistance in tomato. Crit Rev Plant Sci. 2008; 27: 75-107.

[3] Helyes L, Lugasi A, Pék Z . Effect of irrigation on processing tomato yield and antioxidant components. Turk J Agric For. 2012; 36: 702-9.

[4] Gu M, Fang H, Gao Y et al. Characterization of enzymatic modified soluble dietary fiber from tomato peels with high release of lycopene. Food Hydrocoll. 2020; 99: 105321.

[5] Rao AV, Agarwal S . Role of lycopene as antioxidant carotenoid in the prevention of chronic diseases: a review. Nutr Res. 1999; 19: 305-23.

[6] Yoo HJ, Park WJ, Lee GM et al. Inferring the genetic determinants of fruit colors in tomato by carotenoid profiling. Molecules. 2017; 22: 764.

[7] Oltman AE, Jervis SM, Drake MA . Consumer attitudes and preferences for fresh market tomatoes. J Food Sci. 2014; 79: S2091-7.

[8] di Mascio P, Kaiser S, Sies H . Lycopene as the most efficient biological carotenoid singlet oxygen quencher. Arch Biochem Biophys. 1989; 274: 532-8.

[9] Islamian JP, Mehrali H . Lycopene as a carotenoid provides radioprotectant and antioxidant effects by quenching radiation-induced free radical singlet oxygen: an overview. Cell J. 2015; 16: 386-91.

[10] Ilahy R, Siddiqui MW, Tlili I, Montefusco A . When color really matters: horticultural performance and functional quality of high-lycopene tomatoes. Crit Rev Plant Sci. 2018; 37: 15-53.

[11] Cazzonelli CI, Pogson BJ . Source to sink: regulation of carotenoid biosynthesis in plants. Trends Plant Sci. 2010; 15: 266-74.

[12] Namitha KK, Archana SN, Negi PS . Expression of carotenoid biosynthetic pathway genes and changes in carotenoids during ripening in tomato (Lycopersicon esculentum) . Food Funct. 2011; 2: 168.

[13] Walter MH, Strack D . Carotenoids and their cleavage products: biosynthesis and functions. Nat Prod Rep. 2011; 28: 663.

[14] Gallagher CE, Matthews PD, Li F, Wurtzel ET . Gene duplication in the carotenoid biosynthetic pathway preceded evolution of the grasses. Plant Physiol. 2004; 135: 1776-83.

[15] Ronen G, Carmel-Goren L, Zamir D, Hirschberg J . An alternative pathway to beta-carotene formation in plant chromoplasts discovered by map-based cloning of beta and old-gold color mutations in tomato. Proc Natl Acad Sci U S A. 2000; 97: 11102-7.

[16] Fantini E, Falcone G, Frusciante S et al. Dissection of tomato lycopene biosynthesis through virus-induced gene silencing. Plant Physiol. 2013; 163: 986-98.

[17] Isaacson T, Ronen G, Zamir D, Hirschberg J . 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.

[18] Pandurangaiah S, Ravishankar KV, Shivashankar KS et al. Differential expression of carotenoid biosynthetic pathway genes in two contrasting tomato genotypes for lycopene content. J Biosci. 2016; 41: 257-64.

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

[20] Stigliani AL, Giorio G, D’Ambrosio C . Characterization of P450 carotenoid beta- and epsilon-hydroxylases of tomato and transcriptional regulation of xanthophyll biosynthesis in root, leaf, petal and fruit. Plant Cell Physiol. 2011; 52: 851-65.

[21] Yuan H, Zhang J, Nageswaran D, Li L . Carotenoid metabolism and regulation in horticultural crops. Hortic Res. 2015; 2: 15036.

[22] Zhou F, Pichersky E . The complete functional characterisation of the terpene synthase family in tomato. New Phytol. 2020; 226: 1341-60.

[23] Bramley PM . Regulation of carotenoid formation during tomato fruit ripening and development. J Exp Bot. 2002; 53: 2107-13.

[24] Fraser PD, Kiano JW, Truesdale MR et al. Phytoene synthase-2 enzyme activity in tomato does not contribute to carotenoid synthesis in ripening fruit. Plant Mol Biol. 1999; 40: 687-98.

[25] Pecker I, Chamovitz D, Linden H et al. A single polypeptide catalyzing the conversion of phytoene to zeta-carotene is transcriptionally regulated during tomato fruit ripening. Proc Natl Acad Sci U S A. 1992; 89: 4962-6.

[26] D’Ambrosio C, Giorio G, Marino I et al. Virtually complete conversion of lycopene into β-carotene in fruits of tomato plants transformed with the tomato lycopene β-cyclase (tlcy-b) cDNA . Plant Sci. 2004; 166: 207-14.

[27] McQuinn RP, Wong B, Giovannoni JJ . AtPDS overexpression in tomato: exposing unique patterns of carotenoid self-regulation and an alternative strategy for the enhancement of fruit carotenoid content . Plant Biotechnol J. 2018; 16: 482-94.

[28] Li X, Wang Y, Chen S et al. Lycopene is enriched in tomato fruit by CRISPR/Cas9-mediated multiplex genome editing. Front Plant Sci. 2018; 9: 559.

[29] Rothan C, Diouf I, Causse M . Trait discovery and editing in tomato. Plant J. 2018; 97: 73-90.

[30] Zsogon A, Čermák T, Naves ER et al. De novo domestication of wild tomato using genome editing. Nat Biotechnol. 2018; 36: 1211-6.

[31] Bai Y, Lindhout P . Domestication and breeding of tomatoes: what have we gained and what can we gain in the future? Ann Bot. 2007; 100: 1085-94.

[32] Razifard H, Ramos A, Della Valle AL et al. Genomic evidence for complex domestication history of the cultivated tomato in Latin America. Mol Biol Evol. 2020; 37: 1118-32.

[33] Liu L, Shao Z, Zhang M, Wang Q . Regulation of carotenoid metabolism in tomato. Mol Plant. 2015; 8: 28-39.

[34] Kachanovsky DE, Filler S, Isaacson T, Hirschberg J . Epistasis in tomato color mutations involves regulation of phytoene synthase 1 expression by cis-carotenoids. Proc Natl Acad Sci U S A. 2012; 109: 19021-6.

[35] Galpaz N, Wang Q, Menda N et al. Abscisic acid deficiency in the tomato mutant high-pigment 3 leading to increased plastid number and higher fruit lycopene content. Plant J. 2008; 53: 717-30.

[36] Kolotilin I, Koltai H, Tadmor Y et al. Transcriptional profiling of high pigment-2^dg tomato mutant links early fruit plastid biogenesis with its overproduction of phytonutrients. Plant Physiol. 2007; 145: 389-401.

[37] Lieberman M, Segev O, Gilboa N et al. The tomato homolog of the gene encoding UV-damaged DNA binding protein 1 (DDB1) underlined as the gene that causes the high pigment-1 mutant phenotype. Theor Appl Genet. 2004; 108: 1574-81.

[38] Djidonou D, Simonne AH, Koch KE et al. Nutritional quality of field-grown tomato fruit as affected by grafting with interspecific hybrid rootstocks. HortScience. 2016; 51: 1618-24.

[39] Armendariz R, Macua JI, Lahoz I et al. Lycopene content in commercial tomato cultivars for paste in Navarra. Acta Hortic. 2006; 724: 259-62.

[40] Carli P, Caruso G, Fogliano V et al. Development of a methodology to forecast the nutritional value of new tomato hybrids. Euphytica. 2011; 180: 291-300.

[41] Foolad MR . Genome mapping and molecular breeding of tomato. Int J Plant Genomics. 2007; 2007: 52.

[42] Gardner RG . ‘Carolina gold’, a hybrid tomato, and its parents, NC 1Y and NC 2Y. HortScience. 2000; 35: 966-7.

[43] Sharma A, Zhang L, Niño-Liu D et al. A Solanum lycopersicum × Solanum pimpinellifolium linkage map of tomato displaying genomic locations of R-genes, RGAs, and candidate resistance/defense-response ESTs . Int J Plant Genomics. 2008; 2008: 1-18.

[44] Panthee DR, Perkins-Veazie P, Randall D, Brown AF . Lycopene estimation in tomato lines using infrared absorbance and tomato analyzer. Int J Veg Sci. 2013; 19: 240-55.

[45] Exposito-Rodríguez M, Borges AA, Borges-Pérez A, Pérez JA . Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process. BMC Plant Biol. 2008; 8: 131.

[46] Gonzalez-Aguilera KL, Saad CF, Chávez Montes RA et al. Selection of reference genes for quantitative real-time RT-PCR studies in tomato fruit of the genotype MT-Rg1. Front Plant Sci. 2016; 7: 1386.

[47] Zhao F et al. An optimized protocol for stepwise optimization of real-time RT-PCR analysis. Hortic Res. 2021; 8: 179.

[48] Dorak M, ed. Real-Time PCR . New York: Taylor & Francis Group; 2007.

[49] 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.

[50] Vandesompele J, de Preter K, Pattyn F et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002; 3: RESEARCH0034.

[51] Davis AR, Fish WW, Perkins-Veazie P . A rapid spectrophotometric method for analyzing lycopene content in tomato and tomato products. Postharvest Biol Technol. 2003; 28: 425-30.

[52] Fish WW, Perkins-Veazie P, Collins JK . A quantitative assay for lycopene that utilizes reduced volumes of organic solvents. J Food Compos Anal. 2002; 15: 309-17.

[53] Miller JC, Tanksley SD . RFLP analysis of phylogenetic relationships and genetic variation in the genus Lycopersicon . Theor Appl Genet. 1990; 80: 437-48.

[54] Van der Beek JG, Verkerk R, Zabel P, Lindhout P . Mapping strategy for resistance genes in tomato based on RFLPs between cultivars: Cf 9 (resistance to Cladosporium fulvum) on chromosome 1. Theor Appl Genet. 1992; 84: 106-12.

[55] Villand J, Skroch PW, Lai T et al. Genetic variation among tomato accessions from primary and secondary centers of diversity. Crop Sci. 1998; 38: 1339-47.

[56] Park YH, West MAL, St Clair DA . Evolution of AFLPs for germplasm fingerprinting and assessment of genetic diversity in cultivars of tomato (Lycopersicon esculentum L.) . Genome. 2004; 47: 510-8.

[57] Garcia-Martinez S, Andreani L, Garcia-Gusano M et al. Evolution of amplified length polymorphism and simple sequence repeats for tomato germplasm fingerprinting: utility for grouping closely related traditional cultivars. Genome. 2005; 49: 648-56.

[58] Tam SM, Mhiri C, Vogelaar A et al. Comparative analysis of genetic diversities within tomato and pepper collections detected by retrotransposon-based SSAP, AFLP and SSR. Theor Appl Genet. 2005; 110: 819-31.

[59] Ament K, Van Schie CC, Bouwmeester HJ et al. Induction of a leaf specific geranylgeranyl pyrophosphate synthase and emission of (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene in tomato are dependent on both jasmonic acid and salicylic acid signaling pathways. Planta. 2006; 224: 1197-208.

[60] Giorio G, Stigliani AL, D’Ambrosio C . Phytoene synthase genes in tomato (Solanum lycopersicum L.) - new data on the structures, the deduced amino acid sequences and the expression patterns. FEBS J. 2008; 275: 527-35.

[61] Giuliano G, Bartley GE, Scolnik PA . Regulation of carotenoid biosynthesis during tomato development. Plant Cell. 1993; 5: 379-87.

[62] Lawrence SD, Cline K, Moore GA . Chromoplast development in ripening tomato fruit: identification of CDNAs for chromoplast-targeted proteins and characterization of a cDNA encoding a plastid-localized low-molecular-weight heat shock protein. Plant Mol Biol. 1997; 33: 483-92.

[63] Lois LM, Rodríguez-Concepción M, Gallego F et al. Carotenoid biosynthesis during tomato fruit development: regulatory role of 1-deoxy-D-xylulose 5-phosphate synthase. Plant J. 2000; 22: 503-13.

[64] Mohan V, Pandey A, Sreelakshmi Y, Sharma R . Neofunctionalization of chromoplast specific lycopene β cyclase gene (CYC-B) in tomato clade. PLoS One. 2016; 11: e0153333.

[65] Enfissi EMA, Nogueira M, Bramley PM, Fraser PD . The regulation of carotenoid formation in tomato fruit. Plant J. 2017; 89: 774-88.

[66] Azari R, Reuveni M, Evenor D et al. Overexpression of UV-DAMAGED DNA BINDING PROTEIN 1 links plant development and phytonutrient accumulation in high pigment-1 tomato . J Exp Bot. 2010; 61: 3627-37.

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

[68] Li C, Li W, Zhou Z et al. A new rice breeding method: CRISPR/Cas9 system editing of the Xa13 promoter to cultivate transgene-free bacterial blight-resistant rice. Plant Biotechnol J. 2020; 18: 313-5.

[69] Xu X, Xu Z, Li Z et al. Increasing resistance to bacterial leaf streak in rice by editing the promoter of susceptibility gene OsSULRT3;6 . Plant Biotechnol J. 2021; 19: 1101-3.

[70] Huang D, Kosentka PZ, Liu W . Synthetic biology approaches in regulation of targeted gene expression. Curr Opin Plant Biol. 2021; 63: 102036.

[71] Yang Y, Lee JH, Poindexter MR et al. Rational design and testing of abiotic stress-inducible synthetic promoters from poplar cis-regulatory elements. Plant Biotechnol J. 2021; 19: 1354-69.