Transcription factors MdEIL1 and MdHY5 integrate ethylene and light signaling to promote chlorophyll degradation in mature apple peels

Abstract

Although it is well established that ethylene and light stimulate the process of chlorophyll degradation in mature apple peels, there is still a need for further exploration of the molecular mechanisms that regulate this process. This study identified MdEIL1 and MdHY5 as promoters of the chlorophyll degradation pathway in apple peels, activated by ethylene and light. Physiological and molecular tests demonstrated that MdEIL1 and MdHY5 are responsible for activating the expression of genes associated with chlorophyll degradation, including MdERF17, MdNYC1, MdPPH, and MdPAO. Furthermore, the interaction between MdEIL1 and MdHY5 proteins enhances their regulatory activity on the target gene MdERF17. Moreover, MdEIL1 binds to the promoter of MdHY5, resulting in the upregulation of its expression, which is further enhanced in the presence of the MdEIL1-MdHY5 protein complex. These findings indicate that MdEIL1-MdHY5 module acts as positive regulator mediating ethylene and light signals that promote chlorophyll degradation in apple peels.

Acknowledgements

This work was supported by the National Key Research and Development Program of China (2023YFD230100, 2018YFD1000105), the National Natural Science Foundation of China (31872940).

Author contributions

S.-Q.F. was responsible for research design. L.-X.L., S.-K.Y., Y.F., S.W., D.L., and Z.-M.W. were in charge of experimental implementation and data analysis. S.-Q.F., H.-Y.M., and L.-X.L. were responsible for manuscript writing.

Data availability

Results supporting our results can be obtained in supplementary materials of this study.

Conflict of interests

A declaration of no competing interests has been made by the authors.

Supplementary Data

Supplementary data is available at Horticulture Research online.

References

[1] Giovannoni J, Nguyen C, Ampofo B. et al. The epigenome and transcriptional dynamics of fruit ripening. Annu Rev Plant Biol. 2017;68: 61-84

[2] Liu H, Jiang W, Cao J. et al. Changes in extractable and non-extractable polyphenols and their antioxidant properties during fruit on-tree ripening in five peach cultivars. Hortic Plant J. 2019;5: 137-44

[3] Goldschmidt EE. Chlorophyll decomposition in senescing leaves and ripening fruits: functional and evolutionary perspectives. Acta Hortic. 2001;553: 331-6

[4] Krautler B. Chlorophyll breakdown and chlorophyll catabolites in leaves and fruit. Photochem Photobiol Sci. 2008;7: 1114-20

[5] Giovannoni JJ. Genetic regulation of fruit development and ripening. Plant Cell. 2004;16: S170-80

[6] Zhu G, Wang S, Huang Z. et al. Rewiring of the fruit metabolome in tomato breeding. Cell. 2018;172: 249-261.e12

[7] Kusaba M, Ito H, Morita R. et al. Rice NON-YELLOW COLORING1 is involved in light-harvesting complex II and grana degradation during leaf senescence. Plant Cell. 2007;19: 1362-75

[8] Horie Y, Ito H, Kusaba M. et al. Participation of chlorophyll bre-ductase in the initial step of the degradation of light-harvesting chlorophyll a/bprotein complexes in Arabidopsis. JBiolChem. 2009;284: 17449-56

[9] Sato Y, Morita R, Katsuma S. et al. Two short-chain dehydrogenase/reductases, NON-YELLOW COLORING 1 and NYC1-LIKE, are required for chlorophyll b and light-harvesting complex II degradation during senescence in rice. Plant J. 2009; 57:120-31

[10] Meguro M, Ito H, Takabayashi A. et al. Identification of the 7-hydroxymethyl chlorophyll a reductase of the chlorophyll cycle in Arabidopsis. Plant Cell. 2011;23: 3442-53

[11] Schelbert S, Aubry S, Burla B. et al. Pheophytin pheophorbide hydrolase (pheophytinase) is involved in chlorophyll breakdown during leaf senescence in Arabidopsis. Plant Cell. 2009;21: 767-85

[12] Ren G, Zhou Q, Wu S. et al. Reverse genetic identification of CRN1 and its distinctive role in chlorophyll degradation in Arabidopsis. J Integr Plant Biol. 2010;52: 496-504

[13] Pruzinska A, Tanner G, Anders I. et al. Chlorophyll breakdown: pheophorbide a oxygenase is a Rieske-type iron-sulfur protein, encoded by the accelerated cell death 1 gene. Proc Natl Acad Sci USA. 2003;100: 15259-64

[14] Sakuraba Y, Jeong J, Kang M. et al. Phytochrome-interacting transcription factors PIF4 and PIF5 induce leaf senescence in Arabidopsis. Nat Commun. 2014;5:4636

[15] Charoenchongsuk N, Ikeda K, Itai A. et al. Comparison of the expression of chlorophyll-degradation-related genes during ripening between stay-green and yellow-pear cultivars. Sci Hor-tic. 2015;181: 89-94

[16] Liang C, Wang Y, Zhu Y. et al. OsNAP connects abscisic acid and leaf senescence by fine-tuning abscisic acid biosynthesis and directly targeting senescence-associated genes in rice. Proc Natl Acad Sci USA. 2014;111: 10013-8

[17] Zhu X, Chen J, Xie Z. et al. Jasmonic acid promotes degreening via MYC2/3/4- and ANAC019/055/072-mediated regulation of major chlorophyll catabolic genes. Plant J. 2015;84: 597-610

[18] Qiu K, Li Z, Yang Z. et al. EIN3 and ORE1 accelerate degreening during ethylene-mediated leaf senescence by directly activating chlorophyll catabolic genes in Arabidopsis. PLoS Genet. 2015;11: e1005399

[19] Schommer C, Palatnik JF, Aggarwal P. et al. Controlofjas-monate biosynthesis and senescence by miR319 targets. PLoS Biol. 2008;6:e230

[20] Yin XR, Xie XL, Xia XJ. et al. Involvement of an ethylene response factor in chlorophyll degradation during citrus fruit degreening. Plant J. 2016;86: 403-12

[21] John I, Drake R, Farrell A. et al. Delayed leaf senescence in ethylene-deficient ACC-oxidase antisense tomato plants: molecular and physiological analysis. Plant J. 1995;7: 483-90

[22] Tsuchisaka A, Yu G, Jin H. et al. A combinatorial interplay among the 1-aminocyclopropane-1-carboxylate isoforms regulates ethylene biosynthesis in Arabidopsis thaliana. Genetics. 2009;183: 979-1003

[23] Oh SA, Park JH, Lee GI. et al. Identification of three genetic loci controlling leaf senescence in Arabidopsis thaliana. Plant J. 1997;12: 527-35

[24] Chao Q, Rothenberg M, Solano R. et al. Activation of the ethylene gas response pathway in Arabidopsis by the nuclear protein ETHYLENE-INSENSITIVE3 and related proteins. Cell. 1997;89: 1133-44

[25] Yang TF, Gonzalez-Carranza ZH, Maunders MJ. et al. Ethylene and the regulation of senescence processes in transgenic Nico-tiana sylvestris plants. Ann Bot. 2008;101: 301-10

[26] Kim JH, Woo HR, Kim J. et al. Trifurcate feed-forward regulation of age-dependent cell death involving miR164 in Arabidopsis. Science. 2009;323: 1053-7

[27] Kim HJ, Hong SH, Kim YW. et al. Gene regulatory cascade of senescence associated NAC transcription factors activated by ETHYLENE-INSENSITIVE2-mediated leaf senescence signalling in Arabidopsis. JExp Bot. 2014;65: 4023-36

[28] Li ZH, Peng JY, Wen X. et al. Ethylene-insensitive3 is a senescence-associated gene that accelerates age-dependent leaf senescence by directly repressing miR164 transcription in Arabidopsis. Plant Cell. 2013;25: 3311-28

[29] Han ZH, Hu YN, Lv YD. et al. Natural variation underlies differ-ences in ETHYLENE RESPONSE FACTOR17 activity in fruit peel degreening. Plant Physiol. 2018;176: 2292-304

[30] Wang S, Wang T, Li QQ. et al. Phosphorylation of MdERF17 by MdMPK4 promotes apple fruit peel degreening during light/dark transitions. Plant Cell. 2022;34: 1980-2000

[31] Wei Y, Jin JT, Xu YX. et al. Ethylene-activated MdPUB 24 mediates ubiquitination of MdBEL7 to promote chlorophyll degradation in apple fruit. Plant J. 2021;108: 169-82

[32] Gangappa SN, Botto JF. The multifaceted roles of HY5 in plant growth and development. Mol Plant. 2016;9: 1353-65

[33] Zhang ZJ, Zhang HW, Quan RD. et al. Transcriptional regulation of ethylene response factor LeERF2 in the expression of ethylene biosynthesis genes controls ethylene production in tomato and tobacco. Plant Physiol. 2009;150: 365-77

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

[35] Song Y, Yang C, Gao S. et al. Age-triggeredand dark -induced leaf senescence require the bHLH transcription factors PIF3,4, and 5. Mol Plant. 2014;7: 1776-87

[36] Zhang Y, Liu Z, Chen Y. et al. PHYTOCHROMEINTERACTING FACTOR 5 (PIF5) positively regulates dark-induced senescence and chlorophyll degradation in Arabidopsis. Plant Sci. 2015;237: 57-68

[37] Koornneef M, Rolff E, Spruit CJP. Genetic control of light-inhibited hypocotyl elongation in Arabidopsis thaliana (L.) Heynh. ZPflanzenphysiol. 1980;100: 147-60

[38] Deikman J, Hammer PE. Induction of anthocyanin accumulation by cytokinins in Arabidopsis thaliana. Plant Physiol. 1995;108: 47-57

[39] Kim BC, Soh MC, Kang BJ. et al. Two dominant photomorphogenic mutations of Arabidopsis thaliana identi®ed as suppressor mutations of hy2. Plant J. 1996;9: 441-56

[40] Cluis CP, Mouchel CF, Hardtke CS. The Arabidopsis transcription factor HY5 integrates light and hormone signaling pathways. Plant J. 2004;38: 332-47

[41] Liu CC, Chi C, Jin LJ. et al. The bZIP transcription factor HY5 mediates CRY1a-induced anthocyanin biosynthesis in tomato. Plant Cell Environ. 2018;41: 1762-75

[42] 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

[43] 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 accumula-tion. Plant Physiol. 2018;178: 808-23

[44] WenG WuB, WangY. et al. Natural variations in MdNAC18 exert major genetic effect on apple fruit harvest date by regulating ethylene biosynthesis genes. J Integr Plant Biol. 2024;00: 1-20

[45] Zhu X, Chen J, Qiu K. et al. Phytohormone and light regulation of chlorophyll degradation. Front Plant Sci. 2017;8:1911

[46] Hörtensteiner S. Update on the biochemistry of chlorophyll breakdown. Plant Mol Biol. 2013;82: 505-17

[47] Trebitsh T, Goldschmidt EE, Riov J. Ethylene induces de novo synthesis of chlorophyllase, a chlorophyll degrading enzyme, in citrus fruit peel. Proc Natl Acad Sci USA. 1993;90: 9441-5

[48] Zhang X, Zhang Z, Li J. et al. Correlation of leaf senescence and gene expression/activities of chlorophyll degradation enzymes in harvested Chinese flowering cabbage (Brassica rapa var. parachinensis). J Plant Physiol. 2011;168: 2081-7

[49] Lv JY, Zhang MY, Bai L. et al. Effects of 1-methylcyclopropene (1-MCP) on the expression of genes involved in the chlorophyll degradation pathway of apple fruit during storage. Food Chem. 2020;308:125707

[50] Kami C, Lorrain S, Hornitschek P. et al. Light-regulated plant growth and development. Curr Top Dev Biol. 2010;91: 29-66

[51] Salin ML. Chloroplast and mitochondrial mechanisms for pro-tection against oxygen toxicity. Free Radic Res Commun. 1991;13: 851-8

[52] Shigenaga MK, Hagen TM, Ames BN. Oxidative damage and mitochondrial decay in aging. Proc Natl Acad Sci US A. 1994;91: 10771-8

[53] 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

[54] Wang S, Li LX, Zhang Z. et al. Ethylene precisely regulates anthocyanin synthesis in apple via a module comprising MdEIL1, MdMYB1,and MdMYB17. Hortic Res. 2022;9:uhac034

[55] Jung S, Krippner J, Schubert S. Is the photometric method using acetone extracts to determine chlorophyll concentrations appli-cable to Mg-deficient plants? J Plant Nutr Soil Sci. 2016;179: 439-42

[56] Wang YC, Liu WJ, Jiang HY. et al. The R2R3-MYB transcrip-tion factor MdMYB24-like is involved in methyl jasmonate induced anthocyanin biosynthesis in apple. Plant Physiol Biochem. 2019;139: 273-82

[57] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 2001;25: 402-8

[58] Wang S, Zhang Z, Li LX. et al. Apple MdMYB306-like inhibits anthocyanin synthesis by directly interacting with MdMYB17 and MdbHLH33. Plant J. 2022;110: 1021-34

[59] Lv YD, Zhang ML, Wu T. et al. The infiltration efficiency of Agrobacterium-mediated transient transformation in four apple cultivars. Sci Hortic. 2019;256: 108-597

[60] 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

[61] Romer P, Recht S, Strauß T. et al. Promoter elements of rice susceptibility genes are boundand activated by specific TAL effectors from the bacterialblight pathogen, Xanthomonas oryzae pv. Oryzae. New Phytol. 2010;187: 1048-57

[62] Bao C, Qin G, Cao F. et al. MdZAT5 regulates drought tolerance via mediating accumulation of drought-responsive miRNAs and mRNAs in apple. New Phytol. 2022;236: 2131-50

[63] Wang N, Qu CZ, Jiang SH. et al. The proanthocyanidin-specific transcription factor MdMYBPA1 initiates anthocyanin synthesis under low-temperature conditions in red-fleshed apples. Plant J. 2018;96: 39-55

[64] Jia DJ, Shen F, Wang Y. et al. Apple fruit acidity is genetically diversified by natural variations in three hierarchical epistatic genes MdSAUR37, MdPP2CH and MdALMTII. Plant J. 2018;95: 427-43

[65] Wang N, Liu WJ, Yu L. et al. HEAT SHOCK FACTOR A8a modu-lates flavonoid synthesis and drought tolerance. Plant Physiol. 2020;184: 1273-90