Four novel Cit7GlcTs functional in flavonoid 7-O-glucoside biosynthesis are vital to flavonoid biosynthesis shunting in citrus

Abstract

Citrus fruits have abundant flavonoid glycosides (FGs), an important class of natural functional and flavor components. However, there have been few reports about the modification of UDP-glycosyltransferases (UGTs) on flavonoids in citrus. Notably, in flavonoid biosynthesis, 7-O-glucosylation is the initial and essential step of glycosylation prior to the synthesis of flavanone disaccharides, the most abundant and iconic FGs in citrus fruits. Here, based on the accumulation of FGs observed at the very early fruit development stage of two pummelo varieties, we screened six novel flavonoid 7-O-glucosyltransferase genes (7GlcTs) via transcriptomic analysis and then characterized them in vitro. The results revealed that four Cg7GlcTs possess wide catalytic activities towards various flavonoid substrates, with CgUGT89AK1 exhibiting the highest catalytic efficiency. Transient overexpression of CgUGT90A31 and CgUGT89AK1 led to increases in FG synthesis in pummelo leaves. Interestingly, these two genes had conserved sequences and consistent functions across different germplasms. Moreover, CitUGT89AK1 was found to play a role in the response of citrus to Huanglongbing infection by promoting FG production. The findings improve our understanding of flavonoid 7-O-glucosylation by identifying the key genes, and may help improve the benefits of flavonoid biosynthesis for plants and humans in the future.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (NSFC, No. 32002010) and the National Key Research and Development Program of China (Grant No. 2023YFD2300600).

Author contributions

J.X. and J.J.C. designed the experiment; Z.Y.Y. performed the experiments and wrote the manuscript; G.L., H.X.Z. and H.W. provided technical support for the experiments. W.Y.D. and H.X.Z. analyzed flavonoids and gene expression levels; J.W.Z. and Z.X.P. revised the manuscript.

Conflict of interest statement

The authors declare no competing interests.

Data availability

The data used to support this study’s findings are included within the article. The RNA-seq data have been deposited in the NCBI Sequence Read Archive, accession number: PRJNA1072851 (http://www.ncbi.nlm.nih.gov/bioproject/1072851).

References

[1] Lu XM, Zhao C, Shi H. et al. Nutrients and bioactives in citrus fruits: different citrus varieties, fruit parts, and growth stages. Crit Rev Food Sci Nutr. 2023; 63:2018-41

[2] Zema DA, Calabrò PS, Folino A. et al. Valorisation of citrus processing waste: a review. Waste Manag. 2018; 80:252-73

[3] Zheng WK, Zhang W, Liu D. et al. Evolution-guided multiomics provide insights into the strengthening of bioactive flavone biosynthesis in medicinal pummelo. Plant Biotechnol J. 2023; 21:1577-89

[4] Zhao CY, Wang F, Lian YH. et al. Biosynthesis of citrus flavonoids and their health effects. Crit Rev Food Sci Nutr. 2020; 60:566-83

[5] Tripoli E, La Guardia M, Giammanco S. et al. Citrus flavonoids: molecular structure, biological activity and nutritional properties: a review. Food Chem. 2007; 104:466-79

[6] Winkel-Shirley B. Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiol. 2001; 126:485-93

[7] Frydman A, Liberman R, Huhman DV. et al. The molecular and enzymatic basis of bitter/non-bitter flavor of citrus fruit: evolution of branch-forming rhamnosyl-transferases under domestication. Plant J. 2013; 73:166-78

[8] Peters DJ, Constabel CP. Molecular analysis of herbivore-induced condensed tannin synthesis: cloning and expression of dihydroflavonol reductase from trembling aspen (Populus tremuloides). Plant J. 2003; 33:605-5

[9] Wang LJ, Guo D, Zhao G. et al. Group IIc WRKY transcription factors regulate cotton resistance to Fusarium oxysporum by promoting GhMKK2-mediated flavonoid biosynthesis. New Phytol. 2022; 236:249-65

[10] Schulz E, Tohge T, Zuther E. et al. Natural variation in flavonol and anthocyanin metabolism during cold acclimation in Arabidopsis thaliana accessions. Plant Cell Environ. 2015; 38:1658-72

[11] Bian XH, Li W, Niu CF. et al. A class B heat shock factor selected for during soybean domestication contributes to salt tolerance by promoting flavonoid biosynthesis. New Phytol. 2020; 225:268-83

[12] Peng M, Shahzad R, Gul A. et al. Differentially evolved glucosyltransferases determine natural variation of rice flavone accumulation and UV-tolerance. Nat Commun. 2017; 8:1975

[13] Alseekh S, de Souza LP, Benina M. et al. The style and substance of plant flavonoid decoration; towards defining both structure and function. Phytochemistry. 2020; 174:112347

[14] Duan L, Guo L, Dou L-L. et al. Comparison of chemical profiling and antioxidant activities of fruits, leaves, branches, and flowers of Citrus grandis ’Tomentosa’. J Agric Food Chem. 2014; 62:11122-9

[15] Chen R, Qi QL, Wang MT. et al. Therapeutic potential of naringin: an overview. Pharm Biol. 2016; 54:3203-10

[16] Roeschlin RA, Favaro MA, Chiesa MA. et al. Resistance to citrus canker induced by a variant of Xanthomonas citri ssp citri is associated with a hypersensitive cell death response involving autophagy-associated vacuolar processes. Mol Plant Pathol. 2017; 18:1267-81

[17] Hijaz F, Al-Rimawi F, Manthey JA. et al. Phenolics, flavonoids and antioxidant capacities in citrus species with different degree of tolerance to Huanglongbing. Plant Signal Behav. 2020; 15:1752447

[18] Wang ZX, Niu Y, Vashisth T. et al. Nontargeted metabolomics-based multiple machine learning modeling boosts early accurate detection for citrus Huanglongbing. Horticulture Research. 2022;9:uhac145

[19] Massenti R, Lo Bianco R, Sandhu AK. et al. Huanglongbing modifies quality components and flavonoid content of ’Valencia’ oranges. J Sci Food Agr. 2016; 96:73-8

[20] Gordon RM, Washington TL, Sims CA. et al. Performance of macroporous resins for debittering HLB-affected grapefruit juice and its impacts on furanocoumarin and consumer sensory acceptability. Food Chem. 2021; 352:129367

[21] Moriguchi T, Kita M, Tomono Y. et al. Gene expression in flavonoid biosynthesis: correlation with flavonoid accumulation in developing citrus fruit. Physiol Plant. 2001; 111:66-74

[22] Chen JJ, Li G, Zhang H. et al. Primary bitter taste of citrus is linked to a functional allele of the 1,2-Rhamnosyltransferase gene originating from Citrus grandis. J Agric Food Chem. 2021; 69:9869-82

[23] Frydman A, Weisshaus O, Bar-Peled M. et al. Citrus fruit bitter flavors: isolation and functional characterization of the gene Cm1,2RhaT encoding a 1,2 rhamnosyltransferase, a key enzyme in the biosynthesis of the bitter flavonoids of citrus. Plant J. 2004; 40:88-100

[24] Jones P, Messner B, Nakajima JI. et al. UGT73C6 and UGT78D1, glycosyltransferases involved in flavonol glycoside biosynthesis in Arabidopsis thaliana. J Biol Chem. 2003; 278:43910-8

[25] Dai XL, Shi X, Yang C. et al. Two UDP-glycosyltransferases catalyze the biosynthesis of bitter flavonoid 7-O-Neohesperidoside through sequential glycosylation in tea plants. J Agric Food Chem. 2022; 70:2354-65

[26] Hirotani M, Kuroda R, Suzuki H. et al. Cloning and expression of UDP-glucose: flavonoid 7-O-glucosyltransferase from hairy root cultures of Scutellaria baicalensis. Planta. 2000; 210:1006-13

[27] Liu X, Lin C, Ma X. et al. Functional characterization of a flavonoid glycosyltransferase in sweet Orange (Citrus sinensis). Front Plant Sci. 2018; 9:166

[28] Shen SQ, Wang S, Yang C. et al. Elucidation of the melitidin biosynthesis pathway in pummelo. J Integr Plant Biol. 2023; 65:2505-18

[29] Chen JJ, Yuan Z, Zhang H. et al. Cit1,2RhaT and two novel CitdGlcTs participate in flavor-related flavonoid metabolism during citrus fruit development. J Exp Bot. 2019; 70:2759-71

[30] Kubo A, Arai Y, Nagashima S. et al. Alteration of sugar donor specificities of plant glycosyltransferases by a single point mutation. Arch Biochem Biophys. 2004; 429:198-203

[31] Yin QG, Han X, Chen J. et al. Identification of specific glycosyltransferases involved in Flavonol glucoside biosynthesis in ginseng using integrative metabolite profiles, DIA proteomics, and phylogenetic analysis. J Agric Food Chem. 2021; 69:1714-26

[32] Dong NQ, Lin HX. Contribution of phenylpropanoid metabolism to plant development and plant-environment interactions. J Integr Plant Biol. 2021; 63:180-209

[33] Yin QG, Shen G, di S. et al. Genome-wide identification and functional characterization of UDP-glucosyltransferase genes involved in flavonoid biosynthesis in Glycine max. Plant Cell Physiol. 2017; 58:1558-72

[34] Yin QG. et al. Involvement of three putative glucosyltransferases from the UGT72 family in flavonol glucoside/rhamnoside biosynthesis in Lotus japonicus seeds. J Exp Bot. 2017; 68:594-609

[35] Wang X, Li CF, Zhou ZL. et al. Identification of three (Iso)flavonoid glucosyltransferases from Pueraria lobata. Front Plant Sci. 2019; 10:28

[36] Mcintosh CA, Latchinian L, Mansell RL. Flavanone-specific 7-O-glucosyltransferase activity in citrus Paradisi seedlings: purification and characterization. Arch Biochem Biophys. 1990; 282:50-7

[37] Berhow MA, Smolensky D. Developmental and substrate specificity of hesperetin-7-O-glucosyltransferase activity in Citrus Limon tissues using high-performance liquid chromatographic analysis. Plant Sci. 1995; 112:139-47

[38] Li PL, Liu MH, Hu JH. et al. Systematic chemical profiling of Citrus grandis ’Tomentosa’ by ultra-fast liquid chromatography/diode-array detector/quadrupole time-of-flight tandem mass spectrometry. J Pharm Biomed Anal. 2014; 90:167-79

[39] Hu B, Rao MJ, Deng X. et al. Molecular signatures between citrus and Candidatus Liberibacter asiaticus. PLoS Pathog. 2021; 17:e1010071

[40] Hijaz F, Manthey JA, Van der Merwe D. et al. Nucleotides, micro- and macro-nutrients, limonoids, flavonoids, and hydroxycinnamates composition in the phloem sap of sweet orange. Plant Signal Behav. 2016; 11:e1183084

[41] Ma WX, Pang Z, Huang X. et al. Citrus Huanglongbing is a pathogen-triggered immune disease that can be mitigated with antioxidants and gibberellin. Nat Commun. 2022; 13:529

[42] Wang SC, Yang C, Tu H. et al. Characterization and metabolic diversity of flavonoids in citrus species. Sci Rep-Uk. 2017; 7:10549

[43] Peng ZX, Zhang H, Li W. et al. Comparative profiling and natural variation of polymethoxylated flavones in various citrus germplasms. Food Chem. 2021; 354:129499

[44] Wu BP, Gao L, Gao J. et al. Genome-wide identification, expression patterns, and functional analysis of UDP glycosyltransferase family in peach (Prunus persica L. Batsch). Front Plant Sci. 2017; 8:389

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

[46] Lu SW, 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

[47] Wang X, Xu Y, Zhang S. et al. Genomic analyses of primitive, wild and cultivated citrus provide insights into asexual reproduction. Nat Genet. 2017; 49:765-72

[48] Zhu F, Luo T, Liu C. et al. An R2R3-MYB transcription factor represses the transformation of α- and β-branch carotenoids by negatively regulating expression of CrBCH2 and CrNCED5 in flavedo of citrus reticulate. New Phytol. 2017; 216:178-92

[49] Li WY, Li G, Yuan Z. et al. Illustration of the variation in the content of flavanone rutinosides in various citrus germplasms from genetic and enzymatic perspectives. Horticulture Research. 2022;9:uhab017

[50] Hussain SB, Shi CY, Guo LX. et al. Type I H+-pyrophosphatase regulates the vacuolar storage of sucrose in citrus fruit. J Exp Bot. 2020; 71:5935-47