PpARF6 acts as an integrator of auxin and ethylene signaling to promote fruit ripening in peach

Abstract

Although auxin is known to induce ethylene biosynthesis in some Rosaceae fruit crops, the mechanisms underlying the auxin–ethylene interaction during fruit ripening remain largely unknown. Here, the regulatory role of an auxin response factor, PpARF6, in fruit ripening was investigated in peach. Peach fruits showed accelerated ripening after treatment with auxin and PpARF6 was found to be significantly induced. PpARF6 not only could induce ethylene synthesis by directly activating the transcription of ethylene biosynthetic genes, but also competed with EIN3-binding F-box proteins PpEBF1/2 for binding to ethylene-insensitive3-like proteins PpEIL2/3, thereby keeping PpEIL2/3 active. Moreover, PpARF6 showed an interaction with PpEIL2/3 to enhance the PpEIL2/3-activated transcription of ethylene biosynthetic genes . Additionally, ectopic overexpression of PpARF6 in tomato accelerated fruit ripening by promoting the expression of genes involved in ethylene synthesis and fruit texture. In summary, our results revealed a positive regulatory role of PpARF6 in peach fruit ripening via integrating auxin and ethylene signaling.

Acknowledgements

This project was supported by grants from the Strategic Priority Research Program of the Chinese Academy of Sciences (Precision Seed Design and Breeding, XDA24030404), the National Natural Science Foundation of China (32102363 and 32272687), the China Agriculture Research System (CARS-30), and Hubei Hongshan Laboratory (2021hszd017).

Author contributions

R.-X.Z. and Y.H. planned and designed the experiments. X.C., Y.L., and X.Z. performed the experiments. X.C. and B.Z. performed data analysis. X.C. and R.-X.Z. wrote the manuscript. Y.H. revised the manuscript.

Data availability

All the data supporting the findings of this study are available in the paper and supplementary data.

Conflict of interest

None declared.

References

[1] Haji T, Yaegaki H, Yamaguchi M . Inheritance and expression of fruit texture melting, non-melting and stony hard in peach. Sci Hortic. 2005; 105: 241-8

[2] Gu C, Wang L, Wang W et al. Copy number variation of a gene cluster encoding endopolygalacturonase mediates flesh texture and stone adhesion in peach. J Exp Bot. 2016; 67: 1993-2005

[3] Pan L, Zeng WF, Niu L et al. PpYUC11, a strong candidate gene for the stony hard phenotype in peach (Prunus persica L. Batsch), participates in IAA biosynthesis during fruit ripening. J Exp Bot. 2015; 66: 7031-44

[4] Cirilli M, Giovannini D, Ciacciulli A et al. Integrative genomics approaches validate PpYUC11-like as candidate gene for the stony hard trait in peach (P. persica L. Batsch). BMC Plant Biol. 2018; 18: 1293-6

[5] Mashiguchi K, Tanaka K, Sakai T et al. The main auxin biosynthesis pathway in Arabidopsis. Proc Natl Acad Sci USA. 2011; 108: 18512-7

[6] Tatsuki M, Soeno K, Shimada Y et al. Insertion of a transposon-like sequence in the 5 ′-flanking region of the YUCCA gene causes the stony hard phenotype . Plant J. 2018; 96: 815-27

[7] Haji T, Yaegaki H, Yamaguchi M . Varietal differences in the relationship between maturation characteristics, storage life and ethylene production in peach fruit. J Jpn Soc Hortic Sci. 2004; 73: 97-104

[8] Tatsuki M, Haji T, Yamaguchi M . The involvement of 1-aminocyclopropane-1-carboxylic acid synthase isogene, ppACS1, in peach fruit softening . J Exp Bot. 2006; 57: 1281-9

[9] Hayama H, Tatsuki M, Ito A et al. Ethylene and fruit softening in the stony hard mutation in peach. Postharvest Biol Technol. 2006; 41: 16-21

[10] 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

[11] Lü PT, Yu S, Zhu N et al. Genome encode analyses reveal the basis of convergent evolution of fleshy fruit ripening. Nat Plants. 2018; 4: 784-91

[12] Martin-Pizarro C, Vallarino JG, Osorio S et al. The NAC transcription factor FaRIF controls fruit ripening in strawberry. Plant Cell. 2021; 33: 1574-93

[13] Li T, Jiang ZY, Zhang LC et al. Apple (Malus domestica) MdERF2 negatively affects ethylene biosynthesis during fruit ripening by suppressing MdACS1 transcription . Plant J. 2016; 88: 735-48

[14] An JP, Wang XF, Li YY et al. EIN3-LIKE1, MYB1, and ETHYLENE RESPONSE FACTOR3 act in a regulatory loop that synergistically modulates ethylene biosynthesis and anthocyanin accumulation. Plant Physiol. 2018; 178: 808-23

[15] Xiao YY, Chen JY, Kuang JF et al. Banana ethylene response factors are involved in fruit ripening through their interactions with ethylene biosynthesis genes. J Exp Bot. 2013; 64: 2499-510

[16] Shan W, Kuang JF, Chen L et al. Molecular characterization of banana NAC transcription factors and their interactions with ethylene signalling component EIL during fruit ripening. J Exp Bot. 2012; 63: 5171-87

[17] Han YC, Kuang JF, Chen JY et al. Banana transcription factor MaERF11 recruits histone deacetylase MaHDA1 and represses the expression of MaACO1 and expansins during fruit ripening. Plant Physiol. 2016; 171: 1070-84

[18] Wang XB, Pan L, Wang Y et al. PpIAA1 and PpERF4 form a positive feedback loop to regulate peach fruit ripening by integrating auxin and ethylene signals. Plant Sci. 2021; 313: 111084

[19] Zhou H, Zhao L, Yang QR et al. Identification of EIL and ERF genes related to fruit ripening in peach. Int J Mol Sci. 2020; 21: 2846

[20] Yang C, Lu X, Ma B et al. Ethylene signaling in rice and Arabidopsis: conserved and diverged aspects . Mol Plant. 2015; 8: 495-505

[21] Trainotti L, Tadiello A, Casadoro G . The involvement of auxin in the ripening of climacteric fruits comes of age: the hormone plays a role of its own and has an intense interplay with ethylene in ripening peaches. J Exp Bot. 2007; 58: 3299-308

[22] Böttcher C, Burbidge CA, Boss PK et al. Interactions between ethylene and auxin are crucial to the control of grape (Vitis vinifera L.) berry ripening. BMC Plant Biol. 2013; 13: 222

[23] Godoy F, Kühn N, Muñoz M et al. The role of auxin during early berry development in grapevine as revealed by transcript profiling from pollination to fruit set. Hortic Res. 2021; 8: 140

[24] Korasick DA, Jez JM, Strader LC . Refining the nuclear auxin response pathway through structural biology. Curr Opin Plant Biol. 2015; 27: 22-8

[25] Powers SK, Strader LC . Up in the air: untethered factors of auxin response. F1000Res. 2016; 5: 133

[26] Leyser O. Auxin signaling. Plant Physiol. 2018; 176: 465-79

[27] Hao YW, Hu GJ, Breitel D et al. Auxin response factor SlARF2 is an essential component of the regulatory mechanism controlling fruit ripening in tomato. PLoS Genet. 2015; 11: e1005649

[28] Liu MC, Chen Y, Chen Y et al. The tomato ethylene response factor Sl-ERF.B3 integrates ethylene and auxin signaling via direct regulation of Sl-aux/IAA27. New Phytol. 2018; 219: 631-40

[29] Yue PT, Lu Q, Liu Z et al. Auxin-activated MdARF5 induces the expression of ethylene biosynthetic genes to initiate apple fruit ripening. New Phytol. 2020; 226: 1781-95

[30] Zhang T, Li WJ, Xie RX et al. CpARF2 and CpEIL1 interact to mediate auxin-ethylene interaction and regulate fruit ripening in papaya. Plant J. 2020; 103: 1318-37

[31] Diao DH, Hu X, Guan D et al. Genome-wide identification of the ARF (auxin response factor) gene family in peach and their expression analysis. Mol Biol Rep. 2020; 47: 4331-44

[32] Zheng BB, Zhao L, Jiang XH et al. Assessment of organic acid accumulation and its related genes in peach. Food Chem. 2021; 334: 127567

[33] Li Y, Cao K, Li N et al. Genomic analyses provide insights into peach local adaptation and responses to climate change. Genome Res. 2021; 31: 592-606

[34] Li Y, Wang LR . Genetic resources, breeding programs in China, and gene mining of peach: a review. Hortic Plant J. 2020; 6: 205-15

[35] Lieberman-Lazarovich M, Yahav C, Israeli A et al. Deep conservation of cis-element variants regulating plant hormonal responses. Plant Cell. 2019; 31: 2559-72

[36] Given NK, Venis MA, Gierson D . Hormonal regulation of ripening in the strawberry, a non-climacteric fruit. Planta. 1988; 174: 402-6

[37] Chirinos X, Ying S, Rodrigues MA et al. Transition to ripening in tomato requires hormone-controlled genetic reprogramming initiated in gel tissue. Plant Physiol. 2022; 191: 610-25

[38] El-Sharkawy I, Mila I, Bouzayen M et al. Regulation of two germin-like protein genes during plum fruit development. J Exp Bot. 2010; 61: 1761-70

[39] Busatto N, Tadiello A, Moretto M et al. Ethylene-auxin crosstalk regulates postharvest fruit ripening process in apple. Fruit Res. 2021; 1: 1-13

[40] Tatsuki M, Nakajima N, Fujii H et al. Increased levels of IAA are required for system 2 ethylene synthesis causing fruit softening in peach (Prunus persica L. Batsch). J Exp Bot. 2013; 64: 1049-59

[41] Barry CS, Llop-Tous MI, Grierson D . The regulation of 1-aminocyclopropane-1-carboxylic acid synthase gene expression during the transition from system-1 to system-2 ethylene synthesis in tomato. Plant Physiol. 2000; 123: 979-86

[42] Zeng WF, Pan L, Liu H et al. Characterization of 1-aminocyclopropane-1-carboxylic acid synthase (ACS) genes during nectarine fruit development and ripening . Tree Genet Genomes. 2015; 11: 18

[43] Aizat WM, Able JA, Stangoulis JCR et al. Characterisation of ethylene pathway components in non-climacteric capsicum. BMC Plant Biol. 2013; 13: 191

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

[45] Cancé C, Martin-Arevalillo R, Boubekeur K et al. Auxin response factors are keys to the many auxin doors. New Phytol. 2022; 235: 402-19

[46] Chandler JW . Auxin response factors. Plant Cell Environ. 2016; 39: 1014-28

[47] Guo HW, Ecker JR . Plant responses to ethylene gas are mediated by SCF EBF1/EBF2 dependent proteolysis of EIN3 transcription factor. Cell. 2003; 115: 667-77

[48] Potuschak T, Lechner E, Parmentier Y et al. EIN3-dependent regulation of plant ethylene hormone signaling by two Arabidopsis F box proteins: EBF1 and EBF2 . Cell. 2003; 115: 679-89

[49] Binder BM . Ethylene signaling in plants. J Biol Chem. 2020; 295: 7710-25

[50] Shi H, Liu RL, Xue C et al. Seedlings transduce the depth and mechanical pressure of covering soil using COP1 and ethylene to regulate EBF1/EBF2 for soil emergence. Curr Biol. 2016; 26: 139-49

[51] Tao S, Estelle M . Mutational studies of the aux/IAA proteins in Physcomitrella reveal novel insights into their function . New Phytol. 2018; 218: 1534-42

[52] Dougherty L, Zhu YD, Xu KN . Assessing the allelotypic effect of two aminocyclopropane carboxylic acid synthase-encoding genes MdACS1 and MdACS3a on fruit ethylene production and softening in Malus. Hortic Res. 2016; 3: 16024

[53] Hu SH, Liu LH, Li S et al. Regulation of fruit ripening by the brassinosteroid biosynthetic gene SlCYP90B3 via an ethylene-dependent pathway in tomato. Hortic Res. 2020; 7: 163

[54] Li YL, He H, Hou YY et al. Salicylic acid treatment delays apricot (Prunus armeniaca L.) fruit softening by inhibiting ethylene biosynthesis and cell wall degradation . Sci Hortic. 2022; 300: 111061

[55] Ma JJ, Li J, Zhao JB et al. Inactivation of a gene encoding carotenoid cleavage dioxygenase (CCD4) leads to carotenoid-based yellow coloration of fruit flesh and leaf midvein in peach. Plant Mol Biol Rep. 2014; 32: 246-57

[56] Nagpal P, Ellis CM, Weber H et al. Auxin response factors ARF6 and ARF8 promote jasmonic acid production and flower maturation. Development. 2005; 132: 4107-18

[57] Liu N, Wu S, Van Houten J et al. Down-regulation of AUXIN RESPONSE FACTORS 6 and 8 by microRNA 167 leads to floral development defects and female sterility in tomato. J Exp Bot. 2014; 65: 2507-20

[58] 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

[59] Yang QR, Yang XP, Wang L et al. Two R2R3-MYB genes cooperatively control trichome development and cuticular wax biosynthesis in Prunus persica. New Phytol. 2022; 234: 179-96

[60] Tong ZG, Gao ZH, Wang F et al. Selection of reliable reference genes for gene expression studies in peach using real-time PCR. BMC Plant Biol. 2009; 10: 71

[61] Sun CL, Deng L, Du M et al. A transcriptional network promotes anthocyanin biosynthesis in tomato flesh. Mol Plant. 2020; 13: 42-58

[62] Liu ZY, Jia YX, Ding YL et al. Plasma membrane CRPK1-mediated phosphorylation of 14-3-3 proteins induces their nuclear import to fine-tune CBF signaling during cold response. Mol Cell. 2017; 66: 117-128.e5

[63] Zhou H, Lin-Wang K, Wang FR et al. Activator-type R2R3-MYB genes induce a repressor-type R2R3-MYB gene to balance anthocyanin and proanthocyanidin accumulation. New Phytol. 2019; 221: 1919-34

[64] Zhou ZY, Bi GZ, Zhou JM . Luciferase complementation assay for protein-protein interactions in plants. Curr Protoc Plant Biol. 2018; 3: 42-50

[65] Zhang R, Min Y, Holappa LD et al. A role for the auxin response factors ARF6 and ARF8 homologs in petal spur elongation and nectary maturation in Aquilegia. New Phytol. 2020; 227: 1392-405

[66] Li CY, Liu GH, Xu CC et al. The tomato suppressor of prosystemin-mediated responses2 gene encodes a fatty acid desaturase required for the biosynthesis of jasmonic acid and the production of a systemic wound signal for defense gene expression. Plant Cell. 2003; 15: 1646-61

[67] Arnon DI . Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 1949; 24: 1-15

[68] Wellburn AR . The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. J Plant Physiol. 1994; 144: 307-13