PpBBX32 and PpZAT5 modulate temperature-dependent and tissue-specific anthocyanin accumulation in peach fruit

Abstract

Anthocyanins are important compounds for fruit quality and nutrition. The R2R3 MYB transcription factor PpMYB10.1 is known to be critical for regulating anthocyanin accumulation in peach. However, regulatory factors upstream of PpMYB10.1 which control temperature-dependent, cultivar-contrasted and tissue-specific anthocyanin accumulation remain to be determined. In this study, differential anthocyanin accumulation in the outer flesh near the peel (OF) of peach [Prunus persica (L.) Batsch] was observed between cultivars ‘Zhonghuashoutao’ and ‘Dongxuemi’, as well as among different storage temperatures and different fruit tissues of ‘Zhonghuashoutao’. By cross-comparisons of RNA-Seq data of samples with differential anthocyanin accumulation, transcription factor genes PpBBX32 and PpZAT5 were identified. These were functionally characterized as two positive regulators for anthocyanin accumulation via transient expression and genetic transformation. Various interaction assays revealed that both PpBBX32 and PpZAT5 can directly activate the PpMYB10.1 promoter and meanwhile interact at protein level as a PpZAT5-PpBBX32-PpMYB10.1 complex. Furthermore, the results of in silico analysis and exogenous application of methyl jasmonate (MeJA) indicated that MeJA favored anthocyanin accumulation,while it was also found that anthocyanin accumulation as well as PpBBX32 and PpZAT5 expression correlated significantly with endogenous JA and JA-Ile in different fruit tissues. In summary, PpBBX32 and PpZAT5 are upstream activators of PpMYB10.1, allowing JAs to take part in temperature-dependent and tissue-specific anthocyanin accumulation by modulating their expression. This work enriches the knowledge of the transcriptional regulatory mechanisms for differential anthocyanin accumulation under internal and external factors.

Acknowledgements

We would like to thank colleagues Mr Changqing Zhu for his assistance on anthocyanin measurement as well as Ms Rong Jin for her help on growing tobacco plants. We also gratefully acknowledge the effective guidance and helpful discussion on experiments from Ms Yun Zhao of Wuhan Botanical Garden, Chinese Academy of Sciences and Mr Yongchao Zhu of Guizhou University. This work was financially supported by the National Natural Science Foundation of China (32072542), Pao Yu-Kong International Fund, Zhejiang University, and the 111 project (B17039).

Author contributions

C.X. designed the study with the help from B.Z., A.C.A., and K.C.; C.X. obtained funding; D.H. performed the experiments and analyses with the help from L.X., Y.L., and M.L.; D.H. and C.X. wrote the manuscript; B.Z., A.C.A., K.L.-W., and K.C. contributed to revision. All authors read and approved the final manuscript.

Data availability

The transcriptome data newly generated in this study have been submitted to the Sequence Read Archive (SRA) database at the National Center for Biotechnology Information (NCBI) with accession numbers PRJNA1092796 and PRJNA1093033.

Conflict of interest statement

The authors declare no competing interests.

Supplementary data

Supplementary data is available at Horticulture Research online.

References

[1] Mol J, Grotewold E, Koes R. How genes paint flowers and seeds. Trends Plant Sci. 1998; 3:212-7

[2] Allan AC, Hellens RP, Laing WA. MYB transcription factors that colour our fruit. Trends Plant Sci. 2008; 13:99-102

[3] He J, Giusti MM. Anthocyanins: natural colorants with health-promoting properties. Annu Rev Food Sci Technol. 2010; 1:163-87

[4] Holton TA, Cornish EC. Genetics and biochemistry of anthocyanin biosynthesis. Plant Cell. 1995; 7:1071-83

[5] Boss PK, Davies C, Robinson SP. Analysis of the expression of anthocyanin pathway genes in developing Vitis vinifera L. cv Shiraz grape berries and the implications for pathway regulation. Plant Physiol. 1996; 111:1059-66

[6] Tanaka Y, Sasaki N, Ohmiya A. Biosynthesis of plant pigments: anthocyanins, betalains and carotenoids. Plant J. 2008; 54:733-49

[7] Zhao Y, Dong WQ, Zhu YC. et al. PpGST1, an anthocyanin-related glutathione S-transferase gene, is essential for fruit coloration in peach. Plant Biotechnol J. 2019; 18:1284-95

[8] Albert NW, Davies KM, Lewis DH. et al. A conserved network of transcriptional activators and repressors regulates anthocyanin pigmentation in eudicots. Plant Cell. 2014; 26:962-80

[9] Xue L, Liu XF, Wang WL. et al. MYB transcription factors encoded by diversified tandem gene clusters cause varied Morella rubra fruit color. Plant Physiol. 2024; 195:598-616

[10] Gu KD, Wang CK, Hu DG. et al. How do anthocyanins paint our horticultural products? Sci Hortic. 2019; 249:257-62

[11] Zhao Y, Sun JL, Cherono S. et al. Colorful hues: insight into the mechanisms of anthocyanin pigmentation in fruit. Plant Physiol. 2023; 192:1718-32

[12] Tao RY, Bai SL, Ni JB. et al. The blue light signal transduction pathway is involved in anthocyanin accumulation in ‘red Zaosu’ pear. Planta. 2018; 248:37-48

[13] Zhao Y, Min T, Chen MJ. et al. The photomorphogenic transcription factor PpHY5 regulates anthocyanin accumulation in response to UVA and UVB irradiation. Front Plant Sci. 2020; 11:603178

[14] Lin-Wang K, Micheletti D, Palmer J. et al. High temperature reduces apple fruit colour via modulation of the anthocyanin regulatory complex. Plant Cell Environ. 2011; 34:1176-90

[15] Butelli E, Licciardello C, Zhang Y. et al. Retrotransposons control fruit-specific, cold-dependent accumulation of anthocyanins in blood oranges. Plant Cell. 2012; 24:1242-55

[16] Zhu YC, Zhang B, Allan AC. et al. DNA demethylation is involved in the regulation of temperature-dependent anthocyanin accumulation in peach. Plant J. 2020; 102:965-76

[17] Qi TC, Song SS, Ren QC. et al. The jasmonate-ZIM-domain proteins interact with the WD-repeat/bHLH/MYB complexes to regulate jasmonate-mediated anthocyanin accumulation and trichome initiation in Arabidopsis thaliana. Plant Cell.. 2011; 23:1795-814

[18] Ni JB, Zhao Y, Tao RY. et al. Ethylene mediates the branching of the jasmonate-induced flavonoid biosynthesis pathway by suppressing anthocyanin biosynthesis in red Chinese pear fruits. Plant Biotechnol J. 2020; 18:1223-40

[19] Bai SL, Tao RY, Tang YX. et al. BBX16, a B-box protein, positively regulates light-induced anthocyanin accumulation by activating MYB10 in red pear. Plant Biotechnol J. 2019; 17:1985-97

[20] Ni JB, Wang SM, Yu WJ. et al. The ethylene-responsive transcription factor PpERF9 represses PpRAP2.4 and PpMYB114 via histone deacetylation to inhibit anthocyanin biosynthesis in pear. Plant Cell. 2023; 35:2271-92

[21] Liu WJ, Mei ZX, Yu L. et al. The ABA-induced NAC transcription factor MdNAC1 interacts with a bZIP-type transcription factor to promote anthocyanin synthesis in red-fleshed apples. Hort Res. 2023; 10:uhad049

[22] Cheng J, Wei GC, Zhou H. et al. Unraveling the mechanism underlying the glycosylation and methylation of anthocyanins in peach. Plant Physiol. 2014; 166:1044-58

[23] Ravaglia D, Espley RV, Henry-Kirk RA. et al. Transcriptional regulation of flavonoid biosynthesis in nectarine (Prunus persica) by a set of R2R3 MYB transcription factors. BMC Plant Biol. 2013; 13:68

[24] Zhao Y, Dong WQ, Wang K. et al. Differential sensitivity of fruit pigmentation to ultraviolet light between two peach cultivars. Front Plant Sci. 2017; 8:1552

[25] Zhou H, Lin-Wang K, Wang HL. et al. Molecular genetics of blood-fleshed peach reveals activation of anthocyanin biosynthesis by NAC transcription factors. Plant J. 2015; 82:105-21

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

[27] An JP, Qu FJ, Yao JF. et al. The bZIP transcription factor MdHY5 regulates anthocyanin accumulation and nitrate assimilation in apple. Hortic Res. 2017; 4:17023

[28] Bai SL, Tao RY, Yin L. et al. Two B-box proteins, PpBBX18 and PpBBX21, antagonistically regulate anthocyanin biosynthesis via competitive association with Pyrus pyrifolia ELONGATED HYPOCOTYL 5 in the peel of pear fruit. Plant J. 2019; 100:1208-23

[29] Mao ZL, Jiang HY, Wang S. et al. The MdHY5-MdWRKY41-MdMYB transcription factor cascade regulates the anthocyanin and proanthocyanidin biosynthesis in red-fleshed apple. Plant Sci. 2021; 306:110848

[30] Wang WQ, Moss SMA, Zeng LH. et al. The red flesh of kiwifruit is differentially controlled by specific activation-repression systems. New Phytol. 2022; 235:630-45

[31] Zhao L, Zhang YQ. et al. PpHY5 is involved in anthocyanin coloration in the peach flesh surrounding the stone. Plant J. 2023; 114:951-64

[32] Gangappa SN, Botto JF. The BBX family of plant transcription factors. Trends Plant Sci. 2014; 19:460-70

[33] Datta S, Hettiarachchi GH, Deng XW. et al. Arabidopsis CONSTANS-LIKE 3 is a positive regulator of red light signaling and root growth. Plant Cell. 2006; 18:70-84

[34] Bai SL, Saito T, Honda C. et al. An apple B-box protein, MdCOL11, is involved in UV-B-and temperature-induced anthocyanin biosynthesis. Planta. 2014; 240:1051-62

[35] An JP, Wang XF, Zhang XW. et al. MdBBX22 regulates UV-B-induced anthocyanin biosynthesis through regulating the function of MdHY5 and is targeted by MdBT 2 for 26S proteasome-mediated degradation. Plant Biotechnol J. 2019; 17:2231-3

[36] Fang HC, Dong YH, Yue XX. et al. MdCOL4 interaction mediates crosstalk between UV-B and high temperature to control fruit coloration in apple. Plant Cell Physiol. 2019; 60:1055-66

[37] Fang HC, Dong YH, Yue XX. et al. The B-box zinc finger protein MdBBX20 integrates anthocyanin accumulation in response to ultraviolet radiation and low temperature. Plant Cell Environ. 2019; 42:2090-104

[38] Zhao L, Sun JL, Cai YM. et al. PpHYH is responsible for light-induced anthocyanin accumulation in fruit peel of Prunus persica. Tree Physiol.. 2022; 42:1662-77

[39] Holtan HE, Bandong S, Marion CM. et al. BBX32, an Arabidopsis B-box protein, functions in light signaling by suppressing HY5-regulated gene expression and interacting with STH2/BBX21. Plant Physiol. 2011; 156:2109-23

[40] An JP, Wang XF, Espley RV. et al. An apple B-box protein MdBBX37 modulates anthocyanin biosynthesis and hypocotyl elongation synergistically with MdMYBs and MdHY5. Plant Cell Physiol. 2020; 61:130-43

[41] Englbrecht CC, Schoof H, Bohm S. Conservation, diversification and expansion of C2H2 zinc finger proteins in the Arabidopsis thaliana genome. BMC Genomics. 2004; 5:39

[42] Kubo KI, Sakamoto A, Kobayashi A. et al. Cys2/His2 zinc-finger protein family of petunia: evolution and general mechanism of target-sequence recognition. Nucleic Acids Res. 1998; 26:608-15

[43] Ciftci-Yilmaz S, Mittler R. The zinc finger network of plants. Cell Mol Life Sci. 2008; 65:1150-60

[44] Hichri I, Muhovski Y, Zizkova E. et al. The Solanum lycopersicum zinc Finger2 cysteine-2/histidine-2 repressor-like transcription factor regulates development and tolerance to salinity in tomato and arabidopsis. Plant Physiol. 2014; 164:1967-90

[45] Shi H, Liu G, Wei Y. et al. The zinc-finger transcription factor ZAT6 is essential for hydrogen peroxide induction of anthocyanin synthesis in Arabidopsis. Plant Mol Biol.. 2018; 97:165-76

[46] Wang DR, Yang K, Wang X. et al. Overexpression of MdZAT5, an C2H2-type zinc finger protein, regulates anthocyanin accumulation and salt stress response in apple calli and Arabidopsis. Int J Mol Sci.. 2022; 23:1897-913

[47] Zhang L, Tao RY, Wang SM. et al. PpZAT5 suppresses the expression of a B-box gene PpBBX18 to inhibit anthocyanin biosynthesis in the fruit peel of red pear. Front Plant Sci. 2022; 13:1022034

[48] Sakamoto H, Maruyama K, Sakuma Y. et al. Arabidopsis Cys2/His2-type zinc-finger proteins function as transcription repressors under drought, cold, and high-salinity stress conditions. Plant Physiol. 2004; 136:2734-46

[49] Yu L, Liu XF, Guo ZW. et al. Interaction between MdZAT10a-like and MdbHLH100 negatively regulates salt tolerance in apple (Malus domestica Borkh.). Environ Exp Bot. 2022; 200:104938

[50] Saniewski M, Miyamoto K, Ueda J. Methyl jasmonate induces gums and stimulates anthocyanin accumulation in peach shoots. J Plant Growth Regul. 1998; 17:121-4

[51] Tang TT, Zhou HS, Wang LB. et al. Post-harvest application of methyl jasmonate or prohydrojasmon affects color development and anthocyanins biosynthesis in peach by regulation of sucrose metabolism. Frontiers Nutrition. 2022; 9:871467

[52] An XH, Tian Y, Chen KQ. et al. MdMYB9 and MdMYB11 are involved in the regulation of the JA-induced biosynthesis of anthocyanin and proanthocyanidin in apples. Plant Cell Physiol. 2015; 56:650-62

[53] Xie Y, Tan HJ, Ma ZX. et al. DELLA proteins promote anthocyanin biosynthesis via sequestering MYBL2 and JAZ suppressors of the MYB/bHLH/WD 40 complex in Arabidopsis thaliana. Mol Plant.. 2016; 9:711-21

[54] Wasternack C, Hause B. Jasmonates: biosynthesis, perception, signal transduction and action in plant stress response, growth and development. Ann Bot. 2013; 111:1021-58

[55] Ruan JJ, Zhou YX, Zhou ML. et al. Jasmonic acid signaling pathway in plants. Int J Mol Sci. 2019; 20:2479

[56] Chen CJ, Chen H, Zhang Y. et al. TBtools: an integrative toolkit developed for interactive analyses of gig biological data. Mol Plant. 2020; 13:1194-202

[57] Hellens RP, Allan AC, Friel EN. et al. Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants. Plant Methods. 2005; 1:13

[58] Morris GM, Huey R, Olson AJ. Using AutoDock for ligand-receptor docking. Current Protocols Bioinformatics. 2008; 8:8.14

[59] Katchalski-Katzir E, Shariv I, Eisenstein M. et al. Molecular surface recognition: determination of geometric fit between proteins and their ligands by correlation techniques. Proc Natl Acad Sci USA. 1992; 89:2195-9

[60] Pan XQ, Welti R, Wang XM. Simultaneous quantification of major phytohormones and related compounds in crude plant extracts by liquid chromatography-electrospray tandem mass spectrometry. Phytochemistry. 2008; 69:1773-81