Transcription factor CsTT8 promotes fruit coloration by positively regulating the methylerythritol 4-phosphate pathway and carotenoid biosynthesis pathway in citrus (Citrus spp.)

Abstract

Carotenoids directly influence citrus fruit color and nutritional value, which is critical to consumer acceptance. Elucidating the potential molecular mechanism underlying carotenoid metabolism is of great importance for improving fruit quality. Despite the well-established carotenoid biosynthetic pathways, the molecular regulatory mechanism underlying carotenoid metabolism remains poorly understood. Our previous studies have reported that the Myc-type basic helix–loop–helix (bHLH) transcription factor (TF) regulates citrus proanthocyanidin biosynthesis. Transgenic analyses further showed that overexpression of CsTT8 could significantly promote carotenoid accumulation in transgenic citrus calli, but its regulatory mechanism is still unclear. In the present study, we found that overexpression of CsTT8 enhances carotenoid content in citrus fruit and calli by increasing the expression of CsDXR, CsHDS, CsHDR, CsPDS, CsLCYE, CsZEP, and CsNCED2, which was accompanied by changes in the contents of abscisic acid and gibberellin. The in vitro and in vivo assays indicated that CsTT8 directly bound to the promoters of CsDXR, CsHDS, and CsHDR, the key metabolic enzymes of the methylerythritol 4-phosphate (MEP) pathway, thus providing precursors for carotenoid biosynthesis and transcriptionally activating the expression of these three genes. In addition, CsTT8 activated the promoters of four key carotenoid biosynthesis pathway genes, CsPDS, CsLCYE, CsZEP, and CsNCED2, directly promoting carotenoid biosynthesis. This study reveals a novel network of carotenoid metabolism regulated by CsTT8. Our findings will contribute to manipulating carotenoid metabolic engineering to improve the quality of citrus fruit and other crops.

Acknowledgements

This research was supported by the National Natural Science Foundation of China (No. 31930095, 32172527) and the Modern Agro-industry Technology Research System (CARS-26).

Author contributions

X.X.D. supervised the research; Q.S. and X.X.D. designed the experiments; Q.S. and Z.C.H. performed the experiments with contributions from R.R.W. and Y.Z.Y.; Z.Z.X. provided the plant materials. Q.S. and Z.C.H. wrote the manuscript; Q.S. and X.X.D. revised the manuscript; J.L.Y., L.J.C., J.X., W.W.G., Y.J.C., and Q.X. provided critical comments on manuscript editing.

Data availability

All relevant data are included in the paper and its supplementary files. Sequence data can be found in CPBD (http://citrus.hzau.edu.cn/). All accession numbers are listed in Supplementary Data Table S1.

Conflict of interest

There are no competing interests.

References

[1] Idamokoro EM, Hosu YS . Out-look on worldwide trends of related studies on citrus waste as feed for livestock production: a scientometric analysis. Front Res Metr Anal. 2022; 7: 869974

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

[3] Zhu K, Zheng X, Ye J, et al. Regulation of carotenoid and chlorophyll pools in hesperidia, anatomically unique fruits found only in citrus. Plant Physiol. 2021; 187: 829-45

[4] Cazzonelli CI . Carotenoids in nature: insights from plants and beyond. Funct Plant Biol. 2011; 38: 833-47

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

[6] Obulesu M, Dowlathabad MR, Bramhachari PV . Carotenoids and Alzheimer’s disease: an insight into therapeutic role of retinoids in animal models. Neurochem Int. 2011; 59: 535-41

[7] Carretero-Paulet L, Ahumada Í, Cunillera N, et al. Expression and molecular analysis of the Arabidopsis DXR gene encoding 1-deoxy-d-xylulose 5-phosphate reductoisomerase, the first committed enzyme of the 2-C-methyl-d-erythritol 4-phosphate pathway. Plant Physiol. 2002; 129: 1581-91

[8] Rodríguez-Concepción M, Ahumada I, Diez-Juez E, et al. 1-Deoxy-d-xylulose 5-phosphate reductoisomerase and plastid isoprenoid biosynthesis during tomato fruit ripening. Plant J. 2001; 27: 213-22

[9] Cordoba E, Salmi M, León P . Unravelling the regulatory mechanisms that modulate the MEP pathway in higher plants. J Exp Bot. 2009; 60: 2933-43

[10] Welsch R, Beyer P, Hugueney P, et al. Regulation and activation of phytoene synthase, a key enzyme in carotenoid biosynthesis, during photomorphogenesis. Planta. 2000; 211: 846-54

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

[12] Feng K, Hou X-L, Xing G-M, et al. Advances in AP2/ERF superfamily transcription factors in plant. Crit Rev Biotechnol. 2020; 40: 750-76

[13] Dubos C, Stracke R, Grotewold E, et al. MYB transcription factors in Arabidopsis. Trends Plant Sci. 2010; 15: 573-81

[14] Feller A, Machemer K, Braun EL, et al. Evolutionary and comparative analysis of MYB and bHLH plant transcription. Plant J. 2011; 66: 94-116

[15] Abdullah-Zawawi M-R, Ahmad-Nizammuddin N-F, Govender N, et al. Comparative genome-wide analysis of WRKY, MADS-box and MYB transcription factor families in Arabidopsis and rice. Sci Rep. 2021; 11: 19678

[16] Yuan Y, Ren S, Liu X, et al. SlWRKY35 positively regulates carotenoid biosynthesis by activating the MEP pathway in tomato fruit. New Phytol. 2022; 234: 164-78

[17] Wu M, Xu X, Hu X, et al. SlMYB72 regulates the metabolism of chlorophylls, carotenoids, and flavonoids in tomato fruit. Plant Physiol. 2020; 183: 854-68

[18] Fu CC, Han YC, Fan ZQ, et al. The papaya transcription factor CpNAC1 modulates carotenoid biosynthesis through activating phytoene desaturase genes CpPDS2/4 during fruit ripening. J Agric Food Chem. 2016; 64: 5454-63

[19] Fu CC, Han YC, Kuang JF, et al. Papaya CpEIN3a and CpNAC2 co-operatively regulate carotenoid biosynthesis-related genes CpPDS2/4, CpLCY-e and CpCHY-b during fruit ripening. Plant Cell Physiol. 2017; 58: 2155-65

[20] Lu S, Zhang Y, Zhu K, et al. The citrus transcription factor CsMADS6 modulates carotenoid metabolism by directly regulating carotenogenic genes. Plant Physiol. 2018; 176: 2657-76

[21] Lu S, Ye J, Zhu K, et al. A fruit ripening-associated transcription factor CsMADS5 positively regulates carotenoid biosynthesis in citrus. J Exp Bot. 2021; 72: 3028-43

[22] Zhu K, Chen H, Mei X, et al. Transcription factor CsMADS3 coordinately regulates chlorophyll and carotenoid pools in citrus hesperidium. Plant Physiol. 2023; 193: 519-36

[23] Zhang Y, Zhang Y, Sun Q, et al. Citrus transcription factor CsHB5 regulates abscisic acid biosynthetic genes and promotes senescence. Plant J. 2021; 108: 151-68

[24] Zhu K, Sun Q, Chen H, et al. Ethylene activation of carotenoid biosynthesis by a novel transcription factor CsERF061. J Exp Bot. 2021; 72: 3137-54

[25] Hao Y, Zong X, Ren P, et al. Basic helix-loop-helix (bHLH) transcription factors regulate a wide range of functions in Arabidopsis. Int J Mol Sci. 2021; 22: 7152

[26] Guo H, Yang H, Mockler TC, et al. Regulation of flowering time by Arabidopsis photoreceptors. Science. 1998; 279: 1360-3

[27] Nambara E, Marion-Poll A . Abscisic acid biosynthesis and catabolism. Annu Rev Plant Biol. 2005; 56: 165-85

[28] Tanabe N, Noshi M, Mori D, et al. The basic helix-loop-helix transcription factor, bHLH11 functions in the iron-uptake system in Arabidopsis thaliana. J Plant Res. 2019; 132: 93-105

[29] Murre C, McCaw PS, Baltimore D . A new DNA binding and dimerization motif in immunoglobulin enhancer binding, daughterless, MyoD, and myc proteins. Cell. 1989; 56: 777-83

[30] Toledo-Ortiz G, Johansson H, Lee KP, et al. The HY5-PIF regulatory module coordinates light and temperature control of photosynthetic gene transcription. PLoS Genet. 2014; 10: e1004416

[31] Shi Y, Guo J, Zhang W, et al. Cloning of the lycopene β-cyclase gene in Nicotiana tabacum and its overexpression confers salt and drought tolerance. Int J Mol Sci. 2015; 16: 30438-57

[32] Zhou D, Shen Y, Zhou P, et al. Papaya CpbHLH1/2 regulate carotenoid biosynthesis-related genes during papaya fruit ripening. Hortic Res. 2019; 6: 80

[33] Rahim MA, Busatto N, Trainotti L . Regulation of anthocyanin biosynthesis in peach fruits. Planta. 2014; 240: 913-29

[34] Franco-Zorrilla JM, López-Vidriero I, Carrasco JL, et al. DNA-binding specificities of plant transcription factors and their potential to define target genes. Proc Natl Acad Sci USA. 2014; 111: 2367-72

[35] Eggersdorfer M, Wyss A . Carotenoids in human nutrition and health. Arch Biochem Biophys. 2018; 652: 18-26

[36] Mitra S, Rauf A, Tareq AM, et al. Potential health benefits of carotenoid lutein: an updated review. Food Chem Toxicol. 2021; 154: 112328

[37] Atchley WR, Fitch WM . A natural classification of the basic helix-loop-helix class of transcription factors. Proc Natl Acad Sci USA. 1997; 94: 5172-6

[38] Nisar N, Li L, Lu S, et al. Carotenoid metabolism in plants. Mol Plant. 2015; 8: 68-82

[39] Estévez JM, Cantero A, Reindl A, et al. 1-Deoxy-d-xylulose-5-phosphate synthase, a limiting enzyme for plastidic isoprenoid biosynthesis in plants. J Biol Chem. 2001; 276: 22901-9

[40] Bailey PC, Martin C, Toledo-Ortiz G, et al. Update on the basic helix-loop-helix transcription factor gene family in Arabidopsis thaliana. Plant Cell. 2003; 15: 2497-502

[41] Wille A, Zimmermann P, Vranová E, et al. Sparse graphical Gaussian modeling of the isoprenoid gene network in Arabidopsis thaliana. Genome Biol. 2004; 5: R92

[42] Sauret-Güeto S, et al. Plastid cues posttranscriptionally regulate the accumulation of key enzymes of the methylerythritol phosphate pathway in Arabidopsis. Plant Physiol. 2006; 141: 75-84

[43] Lee SY, Jang SJ, Jeong HB, et al. A mutation in zeaxanthin epoxidase contributes to orange coloration and alters carotenoid contents in pepper fruit (Capsicum annuum). Plant J. 2021; 106: 1692-707

[44] Feng G, Wu J, Xu Y, et al. High-spatiotemporal-resolution transcriptomes provide insights into fruit development and ripening in Citrus sinensis. Plant Biotechnol J. 2021; 19: 1337-53

[45] Wu J, Xu Z, Zhang Y, et al. An integrative analysis of the transcriptome and proteome of the pulp of a spontaneous late-ripening sweet orange mutant and its wild type improves our understanding of fruit ripening in citrus. J Exp Bot. 2014; 65: 1651-71

[46] Liu Q, Xu J, Liu Y, et al. A novel bud mutation that confers abnormal patterns of lycopene accumulation in sweet orange fruit (Citrus sinensis L. Osbeck). J Exp Bot. 2007; 58: 4161-71

[47] Zhang Y, Ye J, Liu C, et al. Citrus PH4-Noemi regulatory complex is involved in proanthocyanidin biosynthesis via a positive feedback loop. J Exp Bot. 2020; 71: 1306-21

[48] Zheng X, Zhu K, Sun Q, et al. Natural variation in CCD4 promoter underpins species-specific evolution of red coloration in citrus peel. Mol Plant. 2019; 12: 1294-307

[49] Cao H, Zhang J, Xu J, et al. Comprehending crystalline β-carotene accumulation by comparing engineered cell models and the natural carotenoid-rich system of citrus. J Exp Bot. 2012; 63: 4403-17

[50] Liu C, Long J, Zhu K, et al. Characterization of a citrus R2R3-MYB transcription factor that regulates the flavonol and hydroxycinnamic acid biosynthesis. Sci Rep. 2016; 6: 25352