RtNAC055 promotes drought tolerance via a stomatal closure pathway linked to methyl jasmonate/hydrogen peroxide signaling in Reaumuria trigyna

Abstract

The stomata regulate CO₂ uptake and efficient water usage, thereby promoting drought stress tolerance. NAC proteins (NAM, ATAF1/2, and CUC2) participate in plant reactions following drought stress, but the molecular mechanisms underlying NAC-mediated regulation of stomatal movement are unclear. In this study, a novel NAC gene from Reaumuria trigyna, RtNAC055, was found to enhance drought tolerance via a stomatal closure pathway. It was regulated by RtMYC2 and integrated with jasmonic acid signaling and was predominantly expressed in stomata and root. The suppression of RtNAC055 could improve jasmonic acid and H₂O₂ production and increase the drought tolerance of transgenic R. trigyna callus. Ectopic expression of RtNAC055 in the Arabidopsis atnac055 mutant rescued its drought-sensitive phenotype by decreasing stomatal aperture. Under drought stress, overexpression of RtNAC055 in poplar promoted ROS (H₂O₂) accumulation in stomata, which accelerated stomatal closure and maintained a high photosynthetic rate. Drought upregulated the expression of PtRbohD/F, PtP5CS2, and PtDREB1.1, as well as antioxidant enzyme activities in heterologous expression poplars. RtNAC055 promoted H₂O₂ production in guard cells by directly binding to the promoter of RtRbohE, thus regulating stomatal closure. The stress-related genes RtDREB1.1/P5CS1 were directly regulated by RtNAC055. These results indicate that RtNAC055 regulates stomatal closure by maintaining the balance between the antioxidant system and H₂O₂ level, reducing the transpiration rate and water loss, and improving photosynthetic efficiency and drought resistance.

Acknowledgements

We would like to thank Prof. Xia Lanqin and Dr He Yubing for great support. We thank Han Ying of Inner Mongolia University for kindly providing the technical assistance with the Li-Cor 6400. We thank Prof. Zhang Lingang for providing the Nikon Eclipse Ci-S. We also thank Prof. Ha Da for kindly providing help for the dual-luciferase assay. This research was supported by the National Natural Science Foundation of China (grants 31760700 and 32060404), the Nanfan special project of CAAS (YBXM2307), and the Hainan Yazhou Bay Seed Laboratory (B23CJ0208).

Author contributions

B.J.M., L.L.Z., and Y.C.W. planned and designed the research; B.J.M., J.Z, S.Y.G., H.Y.Z., L.Y., H.J.C., X.Q.B., and X.L.X. performed the experiments; B.J.M. analyzed the data; B.J.M. and Y.C.W. wrote the manuscript. All authors read and approved the final manuscript.

Data availability

The authors confirm that all data from this study are avail-able and can be found in this article and in the supplementary information.

Conflict of interests

The authors declare that they have no competing interests.

Supplementary data

Supplementary data is available at Horticulture Research online.

References

[1] Basu S, Rabara R. Abscisic acid - an enigma in the abiotic stress tolerance of crop plants. Plant Gene. 2017; 11:90-8

[2] Wang W, Vinocur B, Altman A. Plant responses to drought, salinity and extreme temperatures: towards genetic engineering for stress tolerance. Planta. 2003; 218:1-14

[3] Krasensky J, Jonak C. Drought, salt, and temperature stress-induced metabolic rearrangements and regulatory networks. J Exp Bot. 2012; 63:1593-608

[4] Gupta A, Rico-Medina A, Cano-Delgado AI. The physiology of plant responses to drought. Science. 2020; 368:266-9

[5] Hedrich R, Shabala S. Stomata in a saline world. Curr Opin Plant Bio. 2018; 46:87-95

[6] Miller G, Suzuki N, Ciftci-Yilmaz S. et al. Reactive oxygen species homeostasis and signalling during drought and salinity stresses. Plant Cell Environ. 2010; 33:453-67

[7] de Ollas C, Arbona V, Gomez-Cadenas A. Jasmonoyl isoleucine accumulation is needed for abscisic acid build-up in roots of Arabidopsis under water stress conditions. Plant Cell Environ. 2015; 38:2157-70

[8] Wei H, Jing Y, Zhang L. et al. Phytohormones and their crosstalk in regulating stomatal development and patterning. J Exp Bot. 2021; 72:2356-70

[9] Sierla M, Waszczak C, Vahisalu T. et al. Reactive oxygen species in the regulation of stomatal movements. Plant Physiol. 2016; 171:1569-80

[10] Zhu J. Abiotic stress signaling and responses in plants. Cell. 2016; 167:313-24

[11] Neill S, Barros R, Bright J. et al. Nitric oxide, stomatal closure, and abiotic stress. J Exp Bot. 2008; 59:165-76

[12] Shi YC, Fu YP, Liu WQ. NADPH oxidase in plasma membrane is involved in stomatal closure induced by dehydroascorbate. Plant Physiol Biochem. 2012; 51:26-30

[13] Gemes K, Kim YJ, Park KY. et al. An NADPH-oxidase/polyamine oxidase feedback loop controls oxidative burst under salinity. Plant Physiol. 2016; 172:1418-31

[14] Islam MM, Ye W, Akter F. et al. Reactive carbonyl species mediate methyl jasmonate-induced stomatal closure. Plant Cell Physiol. 2020; 61:1788-97

[15] Pei Z, Murata Y, Benning G. et al. Calcium channels activated by hydrogen peroxide mediate abscisic acid signalling in guard cells. Nature. 2000; 406:731-4

[16] Zhou Y, Feng J, Li Q. et al. SmMYC2b enhances tanshinone accumulation in Salvia miltiorrhiza by activating pathway genes and promoting lateral root development. Front. Plant Sci. 2020; 11:559438

[17] Verma D, Jalmi SK, Bhagat PK. et al. A bHLH transcription factor, MYC2, imparts salt intolerance by regulating proline biosynthesis in Arabidopsis. FEBS J. 2020; 287:2560-76

[18] Min D, Li F, Zhang X. et al. SlMYC2 involved in methyl jasmonate-induced tomato fruit chilling tolerance. J Agric Food Chem. 2018; 66:3110-7

[19] Du M, Zhao J, Tzeng DTW. et al. MYC2 orchestrates a hierarchical transcriptional cascade that regulates jasmonate-mediated plant immunity in tomato. Plant Cell. 2017; 29:1883-906

[20] Zhang C, Lei Y, Lu C. et al. MYC2, MYC3, and MYC4 function additively in wounding-induced jasmonic acid biosynthesis and catabolism. J Integr Plant Biol. 2020; 62:1159-75

[21] 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

[22] Jiang G, Yan H, Wu F. et al. Litchi fruit LcNAC1 is a target of LcMYC2 and regulator of fruit senescence through its interaction with LcWRKY1. Plant Cell Physiol. 2017; 58:1075-89

[23] Li T, Xu Y, Zhang L. et al. The jasmonate-activated transcription factor MdMYC2 regulates ETHYLENE RESPONSE FACTOR and ethylene biosynthetic genes to promote ethylene biosynthesis during apple fruit ripening. Plant Cell. 2017; 29:1316-34

[24] Zhang Y, Ji TT, Li TT. et al. Jasmonic acid promotes leaf senescence through MYC2-mediated repression of CATALASE2 expression in Arabidopsis. Plant Sci. 2020; 299:110604

[25] Zhang Q, Xie Z, Zhang R. et al. Blue light regulates secondary cell wall thickening via MYC2/MYC4 activation of the NST1-directed transcriptional network in Arabidopsis. Plant Cell. 2018; 30:2512-28

[26] Maruta T, Inoue T, Tamoi M. et al. Arabidopsis NADPH oxidases, AtrbohD and AtrbohF, are essential for jasmonic acid-induced expression of genes regulated by MYC 2 transcription factor. Plant Sci. 2011; 180:655-60

[27] Hiroshi TYIS, et al.Nobuhiro Akiyoshi TMIM, Shimada H. Allantoin, a stress-related purine metabolite, can activate jasmonate signaling in a MYC2-regulated and abscisic acid-dependent manner. J Exp Bot. 2016; 67:2519-32

[28] Zhang M, Jin X, Chen Y. et al. TcMYC2a, a basic helix-loop-helix transcription factor, transduces JA-signals and regulates taxol biosynthesis in Taxus chinensis. Front. Plant Sci. 2018; 9:863

[29] Wei X, Mao L, Wei X. et al. MYB41, MYB107, and MYC2 promote ABA-mediated primary fatty alcohol accumulation via activation of AchnFAR in wound suberization in kiwifruit. Hortic Res. 2020; 7:86

[30] Nguyen KH, Mostofa MG, Watanabe Y. et al. Overexpression of GmNAC085 enhances drought tolerance in Arabidopsis by regulating glutathione biosynthesis, redox balance and glutathione-dependent detoxification of reactive oxygen species and methylglyoxal. Environ Exp Bot. 2019; 161:242-54

[31] Li Y, Yang X, Li X. Role of jasmonate signaling pathway in resistance to dehydration stress in Arabidopsis. Acta Physiol Plant. 2019; 41:1-12

[32] Olsen AN, Ernst HA, Leggio LL. et al. NAC transcription factors: structurally distinct, functionally diverse. Trends Plant Sci. 2005; 10:79-87

[33] Wang T, Tohge T, Ivakov A. et al. Salt-related MYB1 coordinates abscisic acid biosynthesis and signaling during salt stress in Arabidopsis. Plant Physiol. 2015; 169:1027-41

[34] Erpen L, Devi HS, Grosser JW. et al. Potential use of the DREB/ERF, MYB, NAC and WRKY transcription factors to improve abiotic and biotic stress in transgenic plants. Plant Cell Tissue Organ Cult. 2018; 132:1-25

[35] Kim MH, Cho JS, Lee JH. et al. Poplar MYB transcription factor PtrMYB012 and its Arabidopsis AtGAMYB orthologs are differentially repressed by the Arabidopsis miR159 family. Tree Physiol. 2018; 38:801-12

[36] Chen K, Song M, Guo Y. et al. MdMYB46 could enhance salt and osmotic stress tolerance in apple by directly activating stress-responsive signals. Plant Biotechnol J. 2019; 17:2341-55

[37] Devkar V, Thirumalaikumar VP, Xue GP. et al. Multifaceted regulatory function of tomato SlTAF1 in the response to salinity stress. New Phytol. 2020; 225:1681-98

[38] Liu XH, Lyu YS, Yang W. et al. A membrane-associated NAC transcription factor OsNTL3 is involved in thermotolerance in rice. Plant Biotechnol J. 2020; 18:1317-29

[39] Yang J, Wang H, Zhao S. et al. Overexpression levels of LbDREB6 differentially affect growth, drought, and disease tolerance in poplar. Front. Plant Sci. 2020; 11:528550

[40] Huang X, Amee M, Chen L. Bermudagrass CdWRKY50 gene negatively regulates plants’ response to salt stress. Environ Exp Bot. 2021; 188:104513

[41] Jiang Y, Tong S, Chen N. et al. The PalWRKY77 transcription factor negatively regulates salt tolerance and abscisic acid signaling in Populus. Plant J. 2021; 105:1258-73

[42] Ma B, Liu X, Guo S. et al. RtNAC100 involved in the regulation of ROS, Na⁺ accumulation and induced salt-related PCD through MeJA signal pathways in recretohalophyte Reaumuria trigyna. Plant Sci. 2021; 310:110976

[43] Han F, Wang P, Chen X. et al. An ethylene-induced NAC transcription factor acts as a multiple abiotic stress responsor in conifer. Hortic Res. 2023; 10:uhad130

[44] Gray J. Guard cells: transcription factors regulate stomatal movements. Curr Biol. 2005; 15:R593-5

[45] Li Y, Han S, Sun X. et al. Variations in OsSPL10 confer drought tolerance by directly regulating OsNAC2 expression and ROS production in rice. J Integr Plant Biol. 2023; 65:918-33

[46] Bang SW, Lee DK, Jung H. et al. Overexpression of OsTF1L, a rice HD-zip transcription factor, promotes lignin biosynthesis and stomatal closure that improves drought tolerance. Plant Biotechnol J. 2019; 17:118-31

[47] Cominelli E, Galbiati M, Tonelli C. Transcription factors controlling stomatal movements and drought tolerance. Transcription. 2010; 1:41-5

[48] Cominelli E, Galbiati M, Vavasseur A. et al. A guard-cell-specific MYB transcription factor regulates stomatal movements and plant drought tolerance. Curr Biol. 2005; 15:1196-200

[49] Gimenez Ibanez S, Boter M, Ortigosa A. JAZ2 controls stomata dynamics during bacterial invasion. New Phytol. 2017; 213:1378-92

[50] Du M, Zhai Q, Deng L. et al. Closely related NAC transcription factors of tomato differentially regulate stomatal closure and reopening during pathogen attack. Plant Cell. 2014; 26:3167-84

[51] Liang YK, Dubos C, Dodd IC. et al. AtMYB61, an R2R3-MYB transcription factor controlling stomatal aperture in Arabidopsis thaliana. Curr Biol. 2005; 15:1201-6

[52] Huang X, Chao DY, Gao JP. et al. A previously unknown zinc finger protein, DST, regulates drought and salt tolerance in rice via stomatal aperture control. Genes Dev. 2009; 23:1805-17

[53] You J, Zong W, Li X. et al. The SNAC1-targeted gene OsSRO1c modulates stomatal closure and oxidative stress tolerance by regulating hydrogen peroxide in rice. J Exp Bot. 2013; 64:569-83

[54] Negi S, Tak H, Ganapathi TR. A banana NAC transcription factor (MusaSNAC1) impart drought tolerance by modulating stomatal closure and H2O2 content. Plant Mol Biol. 2018; 96:457-71

[55] Du C, Ma B, Wu Z. et al. Reaumuria trigyna transcription factor RtWRKY23 enhances salt stress tolerance and delays flowering in plants. J Plant Physiol. 2019; 239:38-51

[56] Li N, Du C, Ma B. et al. Functional analysis of ion transport properties and salt tolerance mechanisms of RtHKT 1 from the recretohalophyte Reaumuria trigyna. Plant Cell Physiol. 2019; 60:85-106

[57] Li N, Wang X, Ma B. et al. Expression of a Na⁺/H⁺ antiporter RtNHX1 from a recretohalophyte Reaumuria trigyna improved salt tolerance of transgenic Arabidopsis thaliana. J Plant Physiol. 2017; 218:109-20

[58] Dang ZH, Zheng LL, Wang J. et al. Transcriptomic profiling of the salt-stress response in the wild recretohalophyte Reaumuria trigyna. BMC Genomics. 2013; 14:29

[59] Zhang J, Cheng K, Ma B. et al. CaCl2 promotes the cross adaptation of Reaumuria trigyna to salt and drought by regulating Na⁺, ROS accumulation and programmed cell death. Plant Physiol Biochem. 2023; 195:214-27

[60] Zhang J, Zhang M, Zhang J. et al. Overexpression of RtSYP121 confers cadmium colerance by promoting vesicle trafficking, maintaining ion homeostasis, and alleviating photosynthetic inhibition in Arabidopsis. Ecotoxicol Environ Saf. 2023; 252:114620

[61] Wang J, Li Q, Zhang M. et al. The high pH value of alkaline salt destroys the root membrane permeability of Reaumuria trigyna and leads to its serious physiological decline. J Plant Res. 2022; 135:785-98

[62] Wang H, Wang C, Liu H. et al. An efficient Agrobacterium-mediated transformation and regeneration system for leaf explants of two elite aspen hybrid clones Populus alba × P. berolinensis and Populus davidiana × P. bolleana. Plant Cell Rep. 2011; 30:2037-44

[63] Higo K, Ugawa Y, Iwamoto M. et al. Plant cis-acting regulatory DNA elements (PLACE) database:1999. Nucleic Acids Res. 1999; 27:297-300

[64] Lescot M, Déhais P, Thijs G. et al. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 2002; 30:325-7

[65] Clough SJ, Bent AF. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 1998; 16:735-43

[66] Yang Y, Li HG, Wang J. et al. ABF3 enhances drought tolerance via promoting ABA-induced stomatal closure by directly regulating ADF5 in Populus euphratica. J Exp Bot. 2020; 71:7270-85

[67] Zhao W, Huang H, Wang J. et al. Jasmonic acid enhances osmotic stress responses by MYC2-mediated inhibition of protein phosphatase 2C1 and response regulators 26 transcription factor in tomato. Plant J. 2023; 113:546-61

[68] Shen C, Zhang Y, Li Q. et al. PdGNC confers drought tolerance by mediating stomatal closure resulting from NO and H₂O₂ production via the direct regulation of PdHXK1 expression in Populus. New Phytol. 2021; 230:1868-82

[69] Lu K, Shen D, Dong S. et al. Uptake of graphene enhanced the photophosphorylation performed by chloroplasts in rice plants. Nano Res. 2020; 13:3198-205

[70] Tran LS, Nakashima K, Sakuma Y. et al. Isolation and functional analysis of Arabidopsis stress-inducible NAC transcription factors that bind to a drought-responsive cis-element in the early responsive to dehydration stress 1 promoter. Plant Cell. 2004; 16:2481-98

[71] Fu Y, Ma H, Chen S. et al. Control of proline accumulation under drought via a novel pathway comprising the histone methylase CAU1 and the transcription factor ANAC055. J Exp Bot. 2018; 69:579-88

[72] Xu Z, Kim SY, Hyeon DY. et al. The Arabidopsis NAC transcription factor ANAC096 cooperates with bZIP-type transcription factors in dehydration and osmotic stress responses. Plant Cell. 2013; 25:4708-24

[73] Ye H, Liu S, Tang B. et al. RD26 mediates crosstalk between drought and brassinosteroid signalling pathways. Nat Commun. 2017; 8:14573-3

[74] Hu P, Zhang K, Yang C. BpNAC012 positively regulates abiotic stress responses and secondary wall biosynthesis. Plant Physiol. 2019; 179:700-17

[75] Goossens J, Mertens J, Goossens A. Role and functioning of bHLH transcription factors in jasmonate signalling. J Exp Bot. 2017; 68:1333-47

[76] Huysmans M, Buono RA, Skorzinski N. et al. NAC transcription factors ANAC087 and ANAC046 control distinct aspects of programmed cell death in the Arabidopsis columella and lateral root cap. Plant Cell. 2018; 30:2197-213

[77] Mao C, Ding J, Zhang B. et al. OsNAC2 positively affects salt-induced cell death and binds to the OsAP37 and OsCOX11 promoters. Plant J. 2018; 94:454-68

[78] Zhang L, Yao L, Zhang N. et al. Lateral root development in potato is mediated by Stu-mi164 regulation of NAC transcription factor. Front. Plant Sci. 2018; 9:383

[79] Lee DK, Chung PJ, Jeong JS. et al. The rice OsNAC6 transcription factor orchestrates multiple molecular mechanisms involving root structural adaptions and nicotianamine biosynthesis for drought tolerance. Plant Biotechnol J. 2017; 15:754-64

[80] Zhu F, Luo T, Liu C. et al. A NAC transcription factor and its interaction protein hinder abscisic acid biosynthesis by synergistically repressing NCED5 in Citrus reticulata. J Exp Bot. 2020; 71:3613-25

[81] 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

[82] Gao Y, Zhang YP, Fan ZQ. et al. Mutagenesis of SlNAC4 by CRISPR/Cas9 alters gene expression and softening of ripening tomato fruit. Vegetable Res. 2021; 1:1-12

[83] Nakano Y, Yamaguchi M, Endo H. et al. NAC-MYB-based transcriptional regulation of secondary cell wall biosynthesis in land plants. Front. Plant Sci. 2015; 6:288

[84] Zhang Q, Luo F, Zhong Y. et al. Modulation of NAC transcription factor NST1 activity by XYLEM NAC DOMAIN1 regulates secondary cell wall formation in Arabidopsis. J Exp Bot. 2020; 71:1449-58

[85] Chen K, Guo Y, Song M. et al. Dual role of MdSND1 in the biosynthesis of lignin and in signal transduction in response to salt and osmotic stress in apple. Hortic Res. 2020; 7:204

[86] Lou HQ, Fan W, Jin JF. et al. A NAC-type transcription factor confers aluminium resistance by regulating cell wall-associated receptor kinase 1 and cell wall pectin. Plant Cell Environ. 2020; 43:463-78

[87] Xiang Y, Sun X, Bian X. et al. The transcription factor ZmNAC49 reduces stomatal density and improves drought tolerance in maize. J Exp Bot. 2021; 72:1399-410

[88] Huang G, Dong B, Jiang J. et al. CmNAC 083 regulates resistance to Alternaria alternata via reactive oxygen species and jasmonic acid signaling pathways in Chrysanthemum morifolium. Ornamental Plant Res. 2023; 3:0.

[89] Zhang Y, Zhou Y, Zhang D. et al. PtrWRKY75 overexpression reduces stomatal aperture and improves drought tolerance by salicylic acid-induced reactive oxygen species accumulation in poplar. Environ Exp Bot. 2020; 176:104117

[90] Niu F, Wang C, Yan J. et al. Functional characterization of NAC55 transcription factor from oilseed rape (Brassica napus L.) as a novel transcriptional activator modulating reactive oxygen species accumulation and cell death. Plant Mol Biol. 2016; 92:89-104

[91] He F, Wang HL, Li HG. et al. PeCHYR1, a ubiquitin E3 ligase from Populus euphratica. enhances drought tolerance via ABA-induced stomatal closure by ROS production in Populus. Plant Biotechnol J. 2018; 16:1514-28

[92] Yan S, Luo S, Dong S. et al. Heterotrimeric G-proteins involved in the MeJA regulated ion flux and stomatal closure in Arabidopsis thaliana. Funct Plant Biol. 2015; 42:126-35

[93] Khokon MA, Salam MA, Jammes F. et al. Two guard cell mitogen-activated protein kinases, MPK9 and MPK12, function in methyl jasmonate-induced stomatal closure in Arabidopsis thaliana. Plant Biol (Stuttg). 2015; 17:946-52

[94] Li S, Wang N, Ji D. et al. A GmSIN1/GmNCED3s/GmRbohBs feed-forward loop acts as a signal amplifier that regulates root growth in soybean exposed to salt stress. Plant Cell. 2019; 31:2107-30

[95] Lata C, Prasad M. Role of DREBs in regulation of abiotic stress responses in plants. J Exp Bot. 2011; 62:4731-48

[96] Agarwal PK, Agarwal P, Reddy MK. et al. Role of DREB transcription factors in abiotic and biotic stress tolerance in plants. Plant Cell Rep. 2006; 25:1263-74

[97] Liu Q, Kasuga M, Sakuma Y. et al. Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperature-responsive gene expression, respectively, in Arabidopsis. Plant Cell. 1998; 10:1391-406

[98] Li X, Xie Y, Lu L. et al. Contribution of methylation regulation of MpDREB2A promoter to drought resistance of Mauls prunifolia. Plant Soil. 2019; 441:15-32

[99] Liao X, Guo X, Wang Q. et al. Overexpression of MsDREB6.2 results in cytokinin-deficient developmental phenotypes and enhances drought tolerance in transgenic apple plants. Plant J. 2017; 89:510-26

[100] Alshareef NO, Wang JY, Ali S. et al. Overexpression of the NAC transcription factor JUNGBRUNNEN1 (JUB1) increases salinity tolerance in tomato. Plant Physiol Biochem. 2019; 140:113-21

[101] Hao YJ, Wei W, Song QX. et al. Soybean NAC transcription factors promote abiotic stress tolerance and lateral root formation in transgenic plants. Plant J. 2011; 68:302-13

[102] Wang J, Zhang L, Cao Y. et al. CsATAF1 positively regulates drought stress tolerance by an ABA-dependent pathway and by promoting ROS scavenging in cucumber. Plant Cell Physiol. 2018; 59:930-45

[103] Ashraf M, Foolad MR. Roles of glycine betaine and proline in improving plant abiotic stress resistance. Environ Exp Bot. 2007; 59:206-16

[104] Yang C, Huang Y, Lv W. et al. GmNAC8 acts as a positive regulator in soybean drought stress. Plant Sci. 2020; 293:110442