Mitogen-activated protein kinase 14-mediated phosphorylation of MaMYB4 negatively regulates banana fruit ripening

Abstract

Mitogen-activated protein kinase (MAPK/MPK) cascades play crucial parts in plant growth, development processes, immune ability, and stress responses; however, the regulatory mechanism by which MAPK affects fruit ripening remains largely unexplored. Here, we reported that MaMPK14 cooperated with MaMYB4 to mediate postharvest banana fruit ripening. Transient overexpression of individual MaMPK14 and MaMYB4 in banana fruit delayed fruit ripening, confirming the negative roles in the ripening. The ripening negative regulator MaMYB4 could repress the transcription of genes associated with ethylene biosynthesis and fruit softening, such as MaACS1, MaXTH5, MaPG3, and MaEXPA15. Furthermore, MaMPK14 phosphorylated MaMYB4 at Ser160 via a direct interaction. Mutation at Ser160 of MaMYB4 reduced its interaction with MaMPK14 but did not affect its subcellular localization. Importantly, phosphorylation of MaMYB4 by MaMPK14 enhanced the MaMYB4-mediated transcriptional inhibition, binding strength, protein stability, and the repression of fruit ripening. Taken together, our results delineated the regulation pathway of MAPK module during banana fruit ripening, which involved the phosphorylation modification of MaMYB4 mediated by MaMPK14.

Acknowledgements

This work was funded by the National Natural Science Foundation of China (Grant No. 31830071) and China Agriculture Research System of Ministry of Finance (MOF) and Ministry of Agriculture and Rural affairs (MARA) (Grant No. CARS-31).

Author contributions

W.L. conceived and designed the experiments. Y.Y. performed most of the experiments and analysed the data. C.W., W.W., and Y. Z. performed some of the experiments. Y.Y. wrote the manuscript. J.K. revised the manuscript. W.S., J.C., and Y. J. gave advice for the manuscript. All the authors have read and approved the final version of the manuscript.

Data availability

All relevant data can be found within the manuscript and its supporting materials.

Conflict of interest

The authors declare no conflicts of interest.

References

[1] Brumos J. Gene regulation in climacteric fruit ripening. Curr Opin Plant Biol. 2021; 63: 102042.

[2] Chen T, Qin G, Tian S . Regulatory network of fruit ripening: current understanding and future challenges. New Phytol. 2020; 228: 1219-26.

[3] Xiang Y, Bian X, Wei T et al. ZmMPK5 phosphorylates ZmNAC49 to enhance oxidative stress tolerance in maize. New Phytol. 2021; 232: 2400-17.

[4] Zhang M, Zhang S . Mitogen-activated protein kinase cascades in plant signaling. J Integr Plant Biol. 2022; 64: 301-41.

[5] Sun T, Zhang Y . MAP kinase cascades in plant development and immune signaling. EMBO Rep. 2022; 23: e53817.

[6] Mao G, Meng X, Liu Y et al. Phosphorylation of a WRKY transcription factor by two pathogen-responsive MAPKs drives phytoalexin biosynthesis in Arabidopsis. Plant Cell. 2011; 23: 1639-53.

[7] Gui J, Luo L, Zhong Y et al. Phosphorylation of LTF1, an MYB transcription factor in Populus, acts as a sensory switch regulating lignin biosynthesis in wood cells. Mol Plant. 2019; 12: 1325-37.

[8] Tian X, Li X, Zhou W et al. Transcription factor OsWRKY53 positively regulates brassinosteroid signaling and plant architecture. Plant Physiol. 2017; 175: 1337-49.

[9] Shan W, Guo YF, Wei W et al. Banana MaBZR1/2 associate with MaMPK14 to modulate cell wall modifying genes during fruit ripening. Plant Cell Rep. 2020; 39: 35-46.

[10] Wu CJ, Shan W, Liu XC et al. Phosphorylation of transcription factor bZIP21 by MAP kinase MPK6-3 enhances banana fruit ripening. Plant Physiol. 2022; 188: 1665-85.

[11] Liu Z, Mei E, Tian X et al. OsMKKK70 regulates grain size and leaf angle in rice through the OsMKK4-OsMAPK6-OsWRKY53 signaling pathway. J Integr Plant Biol. 2021; 63: 2043-57.

[12] Zhang Y, Long Y, Liu Y et al. MAPK5 and MAPK10 overexpression influences strawberry fruit ripening, antioxidant capacity and resistance to Botrytis cinerea . Planta. 2022; 255: 1-11.

[13] Fan ZQ, Ba LJ, Shan W et al. A banana R2R3-MYB transcription factor MaMYB3 is involved in fruit ripening through modulation of starch degradation by repressing starch degradation-related genes and MabHLH6. Plant J. 2018; 96: 1191-205.

[14] Liu M, Gomes BL, Mila I et al. Comprehensive profiling of ethylene response factors expression identifies ripening-associated ERF genes and their link to key regulators of fruit ripening in tomato (Solanum lycopersicum). Plant Physiol. 2016; 170: 1732-44.

[15] Xiao YY, Kuang JF, Qi XN et al. A comprehensive investigation of starch degradation process and identification of a transcriptional activator MabHLH6 during banana fruit ripening. Plant Biotechnol J. 2018; 16: 151-64.

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

[17] Xie Y, Chen P, Yan Y et al. An atypical R2R3 MYB transcription factor increases cold hardiness by CBF-dependent and CBF-independent pathways in apple. New Phytol. 2018; 218: 201-18.

[18] Kirik V, Lee MM, Wester K et al. Functional diversification of MYB23 and GL1 genes in trichome morphogenesis and initiation. Development. 2005; 132: 1477-85.

[19] Lee MM, Schiefelbein J . Cell pattern in the Arabidopsis root epidermis determined by lateral inhibition with feedback. Plant Cell. 2002; 14: 611-8.

[20] Zhong R, Lee C, McCarthy RL et al. Transcriptional activation of secondary wall biosynthesis by rice and maize NAC and MYB transcription factors. Plant Cell Physiol. 2011; 52: 1856-71.

[21] Lin S, Singh RK, Navarre DA . R2R3-MYB transcription factors, StmiR858 and sucrose mediate potato flavonol biosynthesis. Hortic Res. 2021; 8: 1-13.

[22] Fornalé S, Shi X, Chai C et al. ZmMYB31 directly represses maize lignin genes and redirects the phenylpropanoid metabolic flux. Plant J. 2010; 64: 633-44.

[23] Ampomah-Dwamena C, Thrimawithana AH, Dejnoprat S et al. A kiwifruit (Actinidia deliciosa) R2R3-MYB transcription factor modulates chlorophyll and carotenoid accumulation . New Phytol. 2019; 221: 309-25.

[24] Jian W, Cao H, Yuan S et al. SlMYB75, an MYB-type transcription factor, promotes anthocyanin accumulation and enhances volatile aroma production in tomato fruits. Hortic Res. 2019; 6: 1-15.

[25] An JP, Wang XF, Zhang XW et al. An apple MYB transcription factor regulates cold tolerance and anthocyanin accumulation and undergoes MIEL1-mediated degradation. Plant Biotechnol J. 2020; 18: 337-53.

[26] Morse AM, Whetten RW, Dubos C et al. Post-translational modification of an R2R3-MYB transcription factor by a MAP kinase during xylem development. New Phytol. 2009; 183: 1001-13.

[27] Kim SH, Kim HS, Bahk S et al. Phosphorylation of the transcriptional repressor MYB15 by mitogen-activated protein kinase 6 is required for freezing tolerance in Arabidopsis. Nucleic Acids Res. 2017; 45: 6613-27.

[28] Dóczi R, Brader G, Pettkó-Szandtner A et al. The Arabidopsis mitogen-activated protein kinase kinase MKK3 is upstream of group C mitogen-activated protein kinases and participates in pathogen signaling. Plant Cell. 2007; 19: 3266-79.

[29] Yang YY, Shan W, Yang TW et al. MaMYB4 is a negative regulator and a substrate of RING-type E3 ligases MaBRG2/3 in controlling banana fruit ripening. Plant J. 2022; 110: 1651-69.

[30] Asif MH, Lakhwani D, Pathak S et al. Genome-wide identification and expression analysis of the mitogen-activated protein kinase gene family from banana suggest involvement of specific members in different stages of fruit ripening. Funct Integr Genomic. 2014; 14: 161-75.

[31] Kuang JF, Chen JY, Liu XC et al. The transcriptional regulatory network mediated by banana (Musa acuminata) dehydration-responsive element binding (MaDREB) transcription factors in fruit ripening . New Phytol. 2017; 214: 762-81.

[32] Song C, Yang Y, Yang T et al. MaMYB4 recruits histone deacetylase MaHDA2 and modulates the expression of ω-3 fatty acid desaturase genes during cold stress response in banana fruit . Plant Cell Physiol. 2019; 60: 2410-22.

[33] Ichimura K, Shinozaki K, Tena G et al. Mitogen-activated protein kinase cascades in plants: a new nomenclature. Trends Plant Sci. 2002; 7: 301-8.

[34] Reyna NS, Yang Y . Molecular analysis of the rice MAP kinase gene family in relation to Magnaporthe grisea infection. Mol Plant. 2006; 19: 530-40.

[35] Kovtun Y, Chiu WL, Tena G, Sheen J . Functional analysis of oxidative stress-activated mitogen-activated protein kinase cascade in plants. Proc Natl Acad Sci U S A. 2000; 97: 2940-5.

[36] Jammes F, Song C, Shin D et al. MAP kinases MPK9 and MPK12 are preferentially expressed in guard cells and positively regulate ROS-mediated ABA signaling. Proc Natl Acad Sci U S A. 2009; 106: 20520-5.

[37] Walia A, Lee JS, Wasteneys G et al. Arabidopsis mitogen-activated protein kinase MPK18 mediates cortical microtubule functions in plant cells. Plant J. 2009; 59: 565-75.

[38] Jeong MJ, Lee SK, Kim BG et al. A rice (Oryza sativa L.) MAP kinase gene, OsMAPK44, is involved in response to abiotic stresses. Plant Cell. 2006; 85: 151-60.

[39] Lee SK, Kim BG, Kwon TR et al. Overexpression of the mitogen-activated protein kinase gene OsMAPK33 enhances sensitivity to salt stress in rice (Oryza sativa L.). J Biosci. 2011; 36: 139-51.

[40] Wu CJ, Shan W, Liang S et al. MaMPK2 enhances MabZIP93-mediated transcriptional activation of cell wall modifying genes during banana fruit ripening. Plant Mol Biol. 2019; 101: 113-27.

[41] Liang SM, Chen SC, Liu ZL et al. MabZIP74 interacts with MaMAPK11-3 to regulate the transcription of MaACO1/4 during banana fruit ripening . Postharvest Biol Technol. 2020; 169: 111293.

[42] Li S, Wang W, Gao J et al. MYB75 phosphorylation by MPK4 is required for light-induced anthocyanin accumulation in Arabidopsis. Plant Cell. 2016; 28: 2866-83.

[43] Sun Y, Zhao J, Li X, Li Y . E2 conjugases UBC1 and UBC2 regulate MYB42-mediated SOS pathway in response to salt stress in Arabidopsis. New Phytol. 2020; 227: 455-72.

[44] Zhang F, Xiang L, Yu Q et al. ARTEMISININ BIOSYNTHESIS PROMOTING KINASE 1 positively regulates artemisinin biosynthesis through phosphorylating AabZIP1. J Exp Bot. 2018; 69: 1109-23.

[45] Han X, Zhang L, Zhao L et al. SnRK1 phosphorylates and destabilizes WRKY3 to enhance barley immunity to powdery mildew. Plant Communications 2020; 1: 100083.

[46] Hu C, Sheng O, Deng G et al. CRISPR/Cas9-mediated genome editing of MaACO1 (aminocyclopropane-1-carboxylate oxidase1) promotes the shelf life of banana fruit . Plant Biotechnol J. 2020; 19: 654-6.

[47] Chen S, Songkumarn P, Liu J et al. A versatile zero background T-vector system for gene cloning and functional genomics. Plant Physiol. 2009; 150: 1111-21.

[48] Walter M, Chaban C, Schütze K et al. Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J. 2004; 40: 428-38.

[49] Sainsbury F, Thuenemann EC, Lomonossoff GP . pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants. Plant Biotechnol J. 2009; 7: 682-93.

[50] Einarson MB, Pugacheva EN, Orlinick JR . Identification of protein-protein interactions with glutathione-S-transferase (GST) fusion proteins. CSH Protoc. 2007; 2007: pdb.top11.

[51] Zhou Z, Bi G, Zhou JM . Luciferase luciferase complementation assay for protein-protein interactions in plants. Curr Opin Plant Biol. 2018; 3: 42-50.