Lily WRKY factor LlWRKY22 promotes thermotolerance through autoactivation and activation of LlDREB2B

Abstract

Most of WRKY transcription factors play important roles in plant development, protection against disease, and response to abiotic stress; however, their roles in lily are largely unknown. Transcriptome analysis in lily (Lilium longiflorum) led to the identification and isolation of a WRKY-IIe gene, LlWRKY22, which was found to be activated at high temperature and play a positive role in thermotolerance regulation. LlWRKY22 expression was continuously activated by heat stress. We further found that LlWRKY22 protein localized to the nucleus and exhibited transactivation activity in both yeast and plant cells, and that its C terminus contributed to its transactivation activity. Meanwhile, overexpression of LlWRKY22 in lily improved thermotolerance and activated the expression of heat-related LlDREB2B gene; however, silencing of LlWRKY22 exerted the opposite effects. Further analysis revealed that LlWRKY22 directly activated the expression of LlDREB2B by binding to two tandem W-box elements on its promoter. Simultaneously, we also found that LlWRKY22 can directly bind its own promoter, thereby activating its own expression and forming a positive regulatory loop. Combined, our findings demonstrated that LlWRKY22 may be a new regulator of heat stress response and positively participates in the establishment of thermotolerance by activating itself and LlDREB2B.

References

[1] Hayes S, Schachtschabel J, Mishkind M et al. Hot topic: Thermosensing in plants. Plant Cell Environ. 2021; 44: 2018-33.

[2] Jagadish SVK, Way DA, Sharkey TD . Plant heat stress: concepts directing future research. Plant Cell Environ. 2021; 44: 1992-2005.

[3] Ohama N, Sato H, Shinozaki K et al. Transcriptional regulatory network of plant heat stress response. Trends Plant Sci. 2017; 22: 53-65.

[4] Eulgem T, Rushton PJ, Robatzek S et al. The WRKY superfamily of plant transcription factors. Trends Plant Sci. 2000; 5: 199-206.

[5] Ulker B, Somssich IE . WRKY transcription factors: from DNA binding towards biological function. Curr Opin Plant Biol. 2004; 7: 491-8.

[6] Rushton PJ, Somssich IE, Ringler P et al. WRKY transcription factors. Trends Plant Sci. 2010; 15: 247-58.

[7] Zhang Y, Wang L . The WRKY transcription factor superfamily: its origin in eukaryotes and expansion in plants. BMC Evol Biol. 2005; 5: 1.

[8] Jiang J, Ma S, Ye N et al. WRKY transcription factors in plant responses to stresses. J Integr Plant Biol. 2017; 59: 86-101.

[9] Wani SH, Anand S, Singh B et al. WRKY transcription factors and plant defense responses: latest discoveries and future prospects. Plant Cell Rep. 2021; 40: 1071-85.

[10] Ciolkowski I, Wanke D, Birkenbihl RP et al. Studies on DNA-binding selectivity of WRKY transcription factors lend structural clues into WRKY-domain function. Plant Mol Biol. 2008; 68: 81-92.

[11] Johnson CS, Kolevski B, Smyth DR . TRANSPARENT TESTA GLABRA2, a trichome and seed coat development gene of Arabidopsis, encodes a WRKY transcription factor. Plant Cell. 2002; 14: 1359-75.

[12] Devaiah BN, Karthikeyan AS, Raghothama KG . WRKY75 transcription factor is a modulator of phosphate acquisition and root development in Arabidopsis. Plant Physiol. 2007; 143: 1789-801.

[13] Kim KC, Lai Z, Fan B et al. Arabidopsis WRKY38 and WRKY62 transcription factors interact with histone deacetylase 19 in basal defense. Plant Cell. 2008; 20: 2357-71.

[14] Jiang W, Yu D . Arabidopsis WRKY2 transcription factor mediates seed germination and postgermination arrest of development by abscisic acid. BMC Plant Biol. 2009; 9: 96.

[15] Jing S, Zhou X, Song Y et al. Heterologous expression of OsWRKY23 gene enhances pathogen defense and dark-induced leaf senescence in Arabidopsis. Plant Growth Regul. 2009; 58: 181-90.

[16] Zhang CQ, Xu Y, Lu Y et al. The WRKY transcription factor OsWRKY78 regulates stem elongation and seed development in rice. Planta. 2011; 234: 541-54.

[17] Rishmawi L, Pesch M, Juengst C et al. Non-cell-autonomous regulation of root hair patterning genes by WRKY75 in Arabidopsis. Plant Physiol. 2014; 165: 186-95.

[18] Guo D, Zhang J, Wang X et al. The WRKY transcription factor WRKY71/EXB1 controls shoot branching by transcriptionally regulating RAX genes in Arabidopsis. Plant Cell. 2015; 27: 3112-27.

[19] Zhang L, Chen L, Yu D . Transcription factor WRKY75 interacts with DELLA proteins to affect flowering. Plant Physiol. 2018; 176: 790-803.

[20] Ma Z, Li W, Wang H et al. WRKY transcription factors WRKY12 and WRKY13 interact with SPL10 to modulate age-mediated flowering. J Integr Plant Biol. 2020; 62: 1659-73.

[21] Chen L, Zhang L, Xiang S et al. The transcription factor WRKY75 positively regulates jasmonate-mediated plant defense to necrotrophic fungal pathogens. J Exp Bot. 2021; 72: 1473-89.

[22] Chen L, Song Y, Li S et al. The role of WRKY transcription factors in plant abiotic stresses. Biochim Biophys Acta. 2012;1819:120-8.

[23] Kasajima I, Ide Y, Yokota Hirai M et al. WRKY6 is involved in the response to boron deficiency in Arabidopsis thaliana . Physiol Plant. 2010; 139: 80-92.

[24] Zou C, Jiang W, Yu D . Male gametophyte-specific WRKY34 transcription factor mediates cold sensitivity of mature pollen in Arabidopsis. J Exp Bot. 2010; 61: 3901-14.

[25] Yan JY, Li CX, Sun L et al. A WRKY transcription factor regulates Fe translocation under Fe deficiency. Plant Physiol. 2016; 171: 2017-27.

[26] Luo X, Li C, He X et al. ABA signaling is negatively regulated by GbWRKY1 through JAZ1 and ABI1 to affect salt and drought tolerance. Plant Cell Rep. 2020; 39: 181-94.

[27] Zhao K, Zhang D, Lv K et al. Functional characterization of poplar WRKY75 in salt and osmotic tolerance. Plant Sci. 2019; 289: 110259.

[28] Zhu D, Hou L, Xiao P et al. VvWRKY30, a grape WRKY transcription factor, plays a positive regulatory role under salinity stress. Plant Sci. 2019; 280: 132-42.

[29] Wen W, Wang R, Su L et al. MsWRKY11, activated by MsWRKY22, functions in drought tolerance and modulates lignin biosynthesis in alfalfa (Medicago sativa L.) . Environ Exp Bot. 2021; 184: 104373.

[30] Li S, Fu Q, Huang W et al. Functional analysis of an Arabidopsis transcription factor WRKY25 in heat stress. Plant Cell Rep. 2009; 28: 683-93.

[31] Li S, Fu Q, Chen L et al. Arabidopsis thaliana WRKY25, WRKY26, and WRKY33 coordinate induction of plant thermotolerance. Planta. 2011; 233: 1237-52.

[32] Li S, Zhou X, Chen L et al. Functional characterization of Arabidopsis thaliana WRKY39 in heat stress. Mol Cells. 2010; 29: 475-83.

[33] Ding L, Wu Z, Teng R et al. LlWRKY39 is involved in thermotolerance by activating LlMBF1c and interacting with LlCaM3 in lily (Lilium longiflorum) . Hortic Res. 2021; 8: 36.

[34] Niu CF, Wei W, Zhou QY et al. Wheat WRKY genes TaWRKY2 and TaWRKY19 regulate abiotic stress tolerance in transgenic Arabidopsis plants. Plant Cell Environ. 2012; 35: 1156-70.

[35] Li F, Li M, Wang P et al. Regulation of cotton (Gossypium hirsutum) drought responses by mitogen-activated protein (MAP) kinase cascade-mediated phosphorylation of GhWRKY59 . New Phytol. 2017; 215: 1462-75.

[36] Wu Z, Liang J, Zhang S et al. A canonical DREB2-type transcription factor in lily is post-translationally regulated and mediates heat stress response. Front Plant Sci. 2018; 9: 243.

[37] Davletova S, Rizhsky L, Liang H et al. Cytosolic ascorbate peroxidase 1 is a central component of the reactive oxygen gene network of Arabidopsis. Plant Cell. 2005; 17: 268-81.

[38] Zhou X, Jiang YYD . WRKY22 transcription factor mediates dark-induced leaf senescence in Arabidopsis. Mol Cells. 2011; 31: 303-13.

[39] Hsu FC, Chou MY, Chou SJ et al. Submergence confers immunity mediated by the WRKY22 transcription factor in Arabidopsis. Plant Cell. 2013; 25: 2699-713.

[40] Kloth KJ, Wiegers GL, Busscher-Lange J et al. AtWRKY22 promotes susceptibility to aphids and modulates salicylic acid and jasmonic acid signalling. J Exp Bot. 2016; 67: 3383-96.

[41] Asai T, Tena G, Plotnikova J et al. MAP kinase signalling cascade in Arabidopsis innate immunity. Nature. 2002; 415: 977-83.

[42] Abbruscato P, Nepusz T, Mizzi L et al. OsWRKY22, a monocot WRKY gene, plays a role in the resistance response to blast . Mol Plant Pathol. 2012; 13: 828-41.

[43] Wang L, Chen S, Peng A et al. CRISPR/Cas9-mediated editing of CsWRKY22 reduces susceptibility to Xanthomonas citri subsp. citri in Wanjincheng orange (Citrus sinensis (L.) Osbeck) . Plant Biotechnol Rep. 2019; 13: 501-10.

[44] Li C, Wang J, Ji N et al. PpHOS1, a RING E3 ubiquitin ligase, interacts with PpWRKY22 in the BABA-induced priming defense of peach fruit against Rhizopus stolonifer . Postharvest Biol Tec. 2020; 159: 111029.

[45] Li C, Lei C, Huang Y et al. PpWRKY22 physically interacts with PpHOS1/PpTGA1 and positively regulates several SA-responsive PR genes to modulate disease resistance in BABA-primed peach fruit. Sci Hortic. 2021; 290: 110479.

[46] Long Q, Du M, Long J et al. Transcription factor WRKY22 regulates canker susceptibility in sweet orange (Citrus sinensis Osbeck) by enhancing cell enlargement and CsLOB1 expression. Hortic Res. 2021; 8: 50.

[47] Li GZ, Wang ZQ, Yokosho K et al. Transcription factor WRKY22 promotes aluminum tolerance via activation of OsFRDL4 expression and enhancement of citrate secretion in rice (Oryza sativa) . New Phytol. 2018; 219: 149-62.

[48] Robatzek S, Somssich IE . Targets of AtWRKY6 regulation during plant senescence and pathogen defense. Genes Dev. 2002; 16: 1139-49.

[49] Miao Y, Smykowski A, Zentgraf U . A novel upstream regulator of WRKY53 transcription during leaf senescence in Arabidopsis thaliana . Plant Biol. 2008; 10: 110-20.

[50] Liu S, Kracher B, Ziegler J et al. Negative regulation of ABA signaling by WRKY33 is critical for Arabidopsis immunity towards Botrytis cinerea 2100. elife. 2015; 4: e07295.

[51] Sakuma Y, Maruyama K, Qin F et al. Dual function of an Arabidopsis transcription factor DREB2A in water-stress-responsive and heat-stress-responsive gene expression. P Natl Acad Sci USA. 2006; 103: 18822-7.

[52] Yoshida T, Ohama N, Nakajima J et al. Arabidopsis HsfA1 transcription factors function as the main positive regulators in heat shock-responsive gene expression. Mol Gen Genomics. 2011; 286: 321-32.

[53] Schramm F, Larkindale J, Kiehlmann E et al. A cascade of transcription factor DREB2A and heat stress transcription factor HsfA3 regulates the heat stress response of Arabidopsis. Plant J. 2008; 53: 264-74.

[54] Chen H, Hwang JE, Lim CJ et al. Arabidopsis DREB2C functions as a transcriptional activator of HsfA3 during the heat stress response. Biochem Bioph Res Co. 2010; 401: 238-44.

[55] Yoshida T, Sakuma Y, Todaka D et al. Functional analysis of an Arabidopsis heat-shock transcription factor HsfA3 in the transcriptional cascade downstream of the DREB2A stress-regulatory system. Biochem Bioph Res Co. 2008; 368: 515-21.

[56] Sato H, Mizoi J, Tanaka H et al. Arabidopsis DPB3-1, a DREB2A interactor, specifically enhances heat stress-induced gene expression by forming a heat stress-specific transcriptional complex with NF-Y subunits. Plant Cell. 2014; 26: 4954-73.

[57] Suzuki N, Sejima H, Tam R et al. Identification of the MBF1 heat-response regulon of Arabidopsis thaliana . Plant J. 2011; 66: 844-51.

[58] 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.

[59] Liu ZQ, Shi LP, Yang S et al. A conserved double-W box in the promoter of CaWRKY40 mediates autoregulation during response to pathogen attack and heat stress in pepper. Mol Plant Pathol. 2021; 22: 3-18.

[60] Liu YG, Chen Y . High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences. BioTechniques. 2007; 43: 649-56.

[61] Wu Z, Liang J, Wang C et al. Overexpression of lily HsfA3s in Arabidopsis confers increased thermotolerance and salt sensitivity via alterations in proline catabolism. J Exp Bot. 2018; 69: 2005-21.

[62] Jiang X, Chi X, Zhou R et al. Transcriptome profiling to identify tepal cell enlargement and pigmentation genes and the function of LtEXLB1 in Lilium tsingtauense . Funct Plant Biol. 2021; 48: 241-56.

[63] Luo J, Li R, Xu X et al. SMRT and Illumina RNA sequencing and characterization of a key NAC gene LoNAC29 during the flower senescence in Lilium oriental ‘Siberia’ . Genes. 2021; 12: 869.

[64] Oren-Shamir M, Gal D . Color fading of rose petals due to a transient high temperature stress. HortScience. 2000; 35: 415D-415.

[65] Khunmuang S, Kanlayanarat S, Wongs-Aree C et al. Ethylene induces a rapid degradation of petal anthocyanins in cut vanda ‘Sansai blue’ orchid flowers. Front Plant Sci. 2019; 10: 1004.

[66] Movahed N, Pastore C, Cellini A et al. The grapevine VviPrx31 peroxidase as a candidate gene involved in anthocyanin degradation in ripening berries under high temperature. J Plant Res. 2016; 129: 513-26.

[67] Niu J, Zhang G, Zhang W et al. Anthocyanin concentration depends on the counterbalance between its synthesis and degradation in plum fruit at high temperature. Sci Rep. 2017; 7: 7684.