RcbHLH59-RcPRs module enhances salinity stress tolerance by balancing Na+/K+ through callose deposition in rose (Rosa chinensis)

Abstract

Basic helix–loop–helix (bHLH) proteins play pivotal roles in plant growth, development, and stress responses. However, the molecular and functional properties of bHLHs have not been fully characterized. In this study, a novel XI subgroup of the bHLH protein gene RcbHLH59 was isolated and identified in rose (Rosa sp .). This gene was induced by salinity stress in both rose leaves and roots, and functioned as a transactivator. Accordingly, silencing RcbHLH59 affected the antioxidant system, Na⁺/K⁺ balance, and photosynthetic system, thereby reducing salt tolerance, while the transient overexpression of RcbHLH59 improved salinity stress tolerance. Additionally, RcbLHLH59 was found to regulate the expression of sets of pathogenesis-related (PR) genes in RcbHLH59-silenced (TRV- RcbHLH59) and RcbHLH59-OE) rose plants. The RcPR4/1 and RcPR5/1 transcript levels showed opposite changes in the TRV- RcbHLH59 and RcbHLH59-OE lines, suggesting that these two genes are regulated by RcbHLH59. Further analysis revealed that RcbHLH59 binds to the promoters of RcPR4/1 and RcPR5/1, and that the silencing of RcPR4/1 or RcPR5/1 led to decreased tolerance to salinity stress. Moreover, callose degradation- and deposition-related genes were impaired in RcPR4/1- or RcPR5/1-silenced plants, which displayed a salt tolerance phenotype by balancing the Na⁺/K⁺ ratio through callose deposition. Collectively, our data highlight a new RcbLHLH59-RcPRs module that positively regulates salinity stress tolerance by balancing Na⁺/K⁺ and through callose deposition in rose plants.

Acknowledgements

This research was supported by the National Key Research and Development Program (2018YFD1000400) and National Natural Science Foundation of China (Grant No. 32002084). We would like to thank Editage (www.editage.cn) for English language editing.

Author contributions

X.J. and L.S. designed and performed the experiments. Y.Z., S.Y., L.G., and S.L. conducted the experiments and analyzed the data. X.J., L.O., and L.S. wrote and revised the manuscript.

Data availability

Sequence data in this study can be found in the Genome Database for Rosaceae (https://www.rosaceae.org/) and the accession numbers are listed 804 in Supplementary Data Table S3.

Conflict of interest

The authors declare they have no competing interests.

Accession numbers

Sequence data in this study can be found in the Genome Database for Rosaceae (https://www.rosaceae.org/) and the accession numbers are listed in Supplementary Data Table S3.

Supplementary data

Supplementary data is available at Horticulture Research online.

References

[1] Zhang H, Zhu J, Gong Z et al. Abiotic stress responses in plants. Nat Rev Genet 2022; 23: 104-19.

[2] Zhao S, Zhang Q, Liu M et al. Regulation of plant responses to salt stress. Int J Mol Sci 2021; 22: 4609-25.

[3] Zhao C, Zhang H, Song C et al. Mechanisms of plant responses and adaptation to soil salinity. Innovation 2020; 1: 100017.

[4] Li M, Chen R, Jiang Q et al. GmNAC06, a NAC domain transcription factor enhances salt stress tolerance in soybean. Plant Mol Biol 2021; 105: 333-45.

[5] Zhang X, Long Y, Chen X et al. A NAC transcription factor OsNAC3 positively regulates ABA response and salt tolerance in rice. BMC Plant Biol 2021; 21: 546-59.

[6] Wu X, Xu J, Meng X et al. Linker histone variant HIS1-3 and WRKY1 oppositely regulate salt stress tolerance in Arabidopsis. Plant Physiol 2022; 189: 1833-47.

[7] Gao Y-F, Liu J-K, Yang F-M et al. The WRKY transcription factor WRKY8 promotes resistance to pathogen infection and mediates drought and salt stress tolerance in Solanum lycopersicum. Physiol Plant 2020; 168: 98-117.

[8] Liu Z-Y, Li X-P, Zhang T-Q et al. Overexpression of ThMYB8 mediates salt stress tolerance by directly activating stress-responsive gene expression. Plant Sci 2021; 302: 110668.

[9] Zhang X, Chen L, Shi Q et al. SlMYB102, an R2R3-type MYB gene, confers salt tolerance in transgenic tomato. Plant Sci 2020; 291: 110356.

[10] Zhao S, Wang H, Jia X et al. The HD-zip I transcription factor MdHB7-like confers tolerance to salinity in transgenic apple (Malus domestica). Physiol Plant 2021; 172: 1452-64.

[11] Bi C, Yu Y, Dong C et al. The bZIP transcription factor TabZIP15 improves salt stress tolerance in wheat. Plant Biotechnol J 2021; 19: 209-11.

[12] Song Y, Li S, Sui Y et al. SbbHLH85, a bHLH member, modulates resilience to salt stress by regulating root hair growth in sorghum. Theor Appl Genet 2021; 135: 201-16.

[13] Li J, Wang T, Han J et al. Genome-wide identification and characterization of cucumber bHLH family genes and the functional characterization of CsbHLH041 in NaCl and ABA tolerance in Arabidopsis and cucumber. BMC Plant Biol 2020; 20: 272.

[14] Jones S . An overview of the basic helix-loop-helix proteins. Genome Biol 2004; 5: 226-32.

[15] Nesi N, Debeaujon I, Jond C et al. The TT8 gene encodes a basic helix-loop-helix domain protein required for expression of DFR and BAN genes in Arabidopsis siliques. Plant Cell 2000; 12: 1863-78.

[16] Atchley WR, Fitch WM . A natural classification of the basic helix-loop-helix class of transcription factors. Proc Natl Acad Sci USA 1997; 94: 5172-6.

[17] Liu W, Tai H, Li S et al. bHLH122 is important for drought and osmotic stress resistance in Arabidopsis and in the repression of ABA catabolism. New Phytol 2014; 201: 1192-204.

[18] Jiang Y, Yang B, Deyholos MK . Functional characterization of the Arabidopsis bHLH92 transcription factor in abiotic stress. Mol Genet Genomics 2009; 282: 503-16.

[19] Chen H-C, Cheng W-H, Hong C-Y et al. The transcription factor OsbHLH035 mediates seed germination and enables seedling recovery from salt stress through ABA-dependent and ABA-independent pathways, respectively. Rice 2018; 11: 50.

[20] Toda Y, Tanaka M, Ogaw D et al. RICE SALT SENSITIVE3 forms a ternary complex with JAZ and class-C bHLH factors and regulates jasmonate-induced gene expression and root cell elongation. Plant Cell 2013; 25: 1709-25.

[21] 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.

[22] Teng Y, Lv M, Zhang X et al. BEAR1, a bHLH transcription factor, controls salt response genes to regulate rice salt response. J Plant Biol 2022; 65: 217-30.

[23] Zhang H, Guo J, Chen X et al. Pepper bHLH transcription factor CabHLH035 contributes to salt tolerance by modulating ion homeostasis and proline biosynthesis. Hortic Res 2022; 9: uhac203.

[24] Zhao R, Song X, Yang N et al. Expression of the subgroup IIIf bHLH transcription factor CpbHLH1 from Chimonanthus praecox (L.) in transgenic model plants inhibits anthocyanin accumulation. Plant Cell Rep 2020; 39: 891-907.

[25] Jiang L, Tian X, Li S et al. The AabHLH35 transcription factor identified from Anthurium andraeanum is involved in cold and drought tolerance. Plants (Basel) 2019; 8: 216.

[26] Zuo Z, Kang G, Hong C et al. A novel basic helix-loop-helix transcription factor, ZjICE2 from Zoysia japonica confers abiotic stress tolerance to transgenic plants via activating the DREB/CBF regulon and enhancing ROS scavenging. Plant Mol Biol 2020; 102: 447-62.

[27] Zarattini M, Farjad M, Launay A et al. Every cloud has a silver lining: how abiotic stresses affect gene expression in plant-pathogen interactions. J Exp Bot 2021; 72: 1020-33.

[28] Jain D, Khurana JP . Role of pathogenesis-related (PR) proteins in plant defense mechanism. Mol Aspects Plant-Pathog Interact 2018; 12: 265-81.

[29] Zribi I, Ghorbel M, Brini F . Pathogenesis related proteins (PRs): from cellular mechanisms to plant defense. Curr Protein Pept Sci 2021; 22: 396-412.

[30] van Loon LC, Rep M, Pieterse CM . Significance of inducible defense-related proteins in infected plants. Annu Rev Phytopathol 2006; 44: 135-62.

[31] Muoki RC, Paul A, Kaachra A et al. Membrane localized thaumatin-like protein from tea (CsTLP) enhanced seed yield and the plant survival under drought stress in Arabidopsis thaliana. Plant Physiol Biochem 2021; 163: 36-44.

[32] Myagmarjav D, Sukweenadhi J, Kim YJ et al. Molecular characterization and expression analysis of pathogenesis related protein 6 from Panax ginseng. Plant Genet 2017; 53: 1211-20.

[33] Liu W-X, Zhang F-C, Zhang W-Z et al. Arabidopsis Di19 functions as a transcription factor and modulates PR1, PR2, and PR5 expression in response to drought stress. Mol Plant 2013; 6: 1487-502.

[34] Ellinger D, Voigt CA . Callose biosynthesis in Arabidopsis with a focus on pathogen response: what we have learned within the last decade. Ann Bot 2014; 114: 1349-58.

[35] Kierszniowska S, Seiwert B, Schulze WX . Definition of Arabidopsis sterol-rich membrane microdomains by differential treatment with methyl-beta-cyclodextrin and quantitative proteomics. Mol Cell Proteomics 2009; 8: 612-23.

[36] Luna E, Pasto V, Robert J et al. Callose deposition: a multifaceted plant defense response. Mol Plant Microbe Interact 2011; 24: 183-93.

[37] Bilska A, Sowinski P . Closure of plasmodesmata in maize (Zea mays) at low temperature: a new mechanism for inhibition of photosynthesis. Ann Bot 2010; 106: 675-86.

[38] Kierszniowska M . The cell wall in plant cell response to trace metals: polysaccharide remodeling and its role in defense strategy. Acta Physiol Plant 2011; 33: 35-51.

[39] Cui W, Lee JY . Arabidopsis callose synthases CalS1/8 regulate plasmodesmal permeability during stress. Nat Plants 2016; 2: 16034.

[40] Oide S, Bejai S, Staal J et al. A novel role of PR2 in abscisic acid (ABA) mediated, pathogen-induced callose deposition in Arabidopsis thaliana. New Phytol 2013; 200: 1187-99.

[41] Hunter K, Kimura S, Rokka A et al. CRK2 enhances salt tolerance by regulating callose deposition in connection with PLDα1. Plant Physiol 2019; 180: 2004-21.

[42] Geng L, Su L, Fu L et al. Genome-wide analysis of the rose (Rosa chinensis) NAC family and characterization of RcNAC091. Plant Mol Biol 2022; 108: 605-19.

[43] Li X, Wan S, Kang Y et al. Chinese rose (Rosa chinensis) growth and ion accumulation under irrigation with waters of different salt contents. Agric Water Manage 2016; 163: 180-9.

[44] Tian X, Wang Z, Zhang Q et al. Genome-wide transcriptome analysis of the salt stress tolerance mechanism in Rosa chinensis. PLoS One 2018; 13: e0200938.

[45] Su L, Zhao X, Geng L et al. Analysis of the thaumatin-like genes of Rosa chinensis and functional analysis of the role of RcTLP6 in salt stress tolerance. Planta 2021; 254: 118.

[46] Bailey PC, Martin C, Toledo-Ortiz G et al. Update on the basic helix-loop-helix transcription factor gene family in Arabidopsis thaliana. Plant Cell 2003; 15: 2497-502.

[47] He Z, Wang Z, Nie X et al. UNFERTILIZED EMBRYO SAC 12 phosphorylation plays a crucial role in conferring salt tolerance. Plant Physiol 2022; 188: 1385-401.

[48] Yang J, Gao M, Huang L et al. Identification and expression analysis of the apple (Malus × domestica) basic helix-loop-helix transcription factor family. Sci Rep 2017; 7: 28.

[49] Waseem M, Rong X, Li Z . Dissecting the role of a basic helix-loop-helix transcription factor, SlbHLH22, under salt and drought stresses in transgenic Solanum lycopersicum L. Front Plant Sci 2019; 10: 734.

[50] Qiu J-R, Huang Z, Xiang X-Y et al. MfbHLH38, a Myrothamnus flabellifolia bHLH transcription factor, confers tolerance to drought and salinity stresses in Arabidopsis. BMC Plant Biol 2020; 20: 542.

[51] Mittler R, Zandalinas SI, Fichman Y et al. Reactive oxygen species signalling in plant stress responses. Nat Rev Mol Cell Biol 2022; 23: 663-79.

[52] Liu J, Fu C, Li G et al. ROS homeostasis and plant salt tolerance: plant nanobiotechnology updates. Sustainability 2021; 13: 3552.

[53] Li Z, Chen W, Zhang C et al. RcMYBPA2 of Rosa chinensis functions in proanthocyanidin biosynthesis and enhances abiotic stress tolerance in transgenic tobacco. Plant Cell Tiss Org 2019; 137: 441-54.

[54] Gilroy S, Białasek M, Suzuki N et al. ROS, calcium, and electric signals: key mediators of rapid systemic signaling in plants. Plant Physiol 2016; 171: 1606-15.

[55] Liu D, Li Y, Zhou Z et al. Tobacco transcription factor bHLH123 improves salt tolerance by activating NADPH oxidase NtRbohE expression. Plant Physiol 2021; 186: 1706-20.

[56] Qiu J-R, Huang Z, Xiang X-Y et al. MfbHLH38, a Myrothamnus flabellifolia bHLH transcription factor, confers tolerance to drought and salinity stresses in Arabidopsis. BMC Plant Biol 2020; 20: 542.

[57] Dong Y, Wang C, Han X et al. A novel bHLH transcription factor PebHLH35 from Populus euphratica confers drought tolerance through regulating stomatal development, photosynthesis and growth in Arabidopsis. Biochem Biophys Res Commun 2014; 450: 453-8.

[58] Brindha C, Vasantha S, Raja AK et al. Characterization of the salt overly sensitive pathway genes in sugarcane under salinity stress. Physiol Plant 2021; 171: 677-87.

[59] Guan Q, Wu J, Yue X et al. A nuclear calcium-sensing pathway is critical for gene regulation and salt stress tolerance in Arabidopsis. PLoS Genet 2013; 9: e1003755.

[60] Liu T, Chen T, Kan J et al. The GhMYB36 transcription factor confers resistance to biotic and abiotic stress by enhancing PR1 gene expression in plants. Plant Biotechnol J 2022; 20: 722-35.

[61] Anisimova OK, Shchennikova AV, Kochieva EZ et al. Pathogenesis-related genes of PR1, PR2, PR4, and PR5 families are involved in the response to Fusarium infection in garlic (Allium sativum L.). Int J Mol Sci 2021; 22: 6688.

[62] Zhou Z, Zhu Y, Tian Y et al. MdPR4, a pathogenesis-related protein in apple, is involved in chitin recognition and resistance response to apple replant disease pathogens. J Plant Physiol 2021; 260: 153390.

[63] Sun W, Zhou Y, Movahedi A et al. Thaumatin-like protein (Pe-TLP) acts as a positive factor in transgenic poplars enhanced resistance to spots disease. Physiol Mol Plant Pathol 2020; 112: 101512.

[64] Pillai SE, Kumar C, Patel HK et al. Overexpression of a cell wall damage induced transcription factor, OsWRKY42, leads to enhanced callose deposition and tolerance to salt stress but does not enhance tolerance to bacterial infection. BMC Plant Biol 2018; 18: 177.

[65] Jach G, Görnhardt B, Mundy J et al. Enhanced quantitative resistance against fungal disease by combinatorial expression of different barley antifungal proteins in transgenic tobacco. Plant J 1995; 8: 97-109.

[66] Jongedijk E, Tigelaar H, van Roekel JSC et al. Synergistic activity of chitinases and β-1,3-glucanases enhances fungal resistance in transgenic tomato plants. Euphytica 1995; 85: 173-80.

[67] Dorokhov YL, Ershova NM, Sheshukova EV et al. Plasmodesmata conductivity regulation: a mechanistic model. Plants (Basel) 2019; 8: 595.

[68] Saheed SA, Cierlik I, Larsson KAE et al. Stronger induction of callose deposition in barley by Russian wheat aphid than bird cherry-oat aphid is not associated with differences in callose synthase β-1,3-glucanase transcript abundance. Physiol Plant 2009; 135: 150-61.

[69] O’Lexy R, Kasai K, Clark N et al. Exposure to heavy metal stress triggers changes in plasmodesmatal permeability via deposition and breakdown of callose. J Exp Bot 2018; 69: 3715-28.

[70] Bao Y, Chen C, Fu L et al. Comparative transcriptome analysis of Rosa chinensis ‘Old Blush’ provides insights into the crucial factors and signaling pathways in salt stress response. Agron J 2021; 113: 3031-50.

[71] Bashandy H, Jalkanen S, Teeri T-H . Within leaf variation is the largest source of variation in agroinfiltration of Nicotiana benthamiana. Plant Methods 2015; 11: 47.

[72] Nakashima J, Laosinchai W, Cui X et al. New insight into the mechanism of cellulose and callose biosynthesis: proteases may regulate callose biosynthesis upon wounding. Cellulose 2003; 10: 369-89.

[73] Kumar S, Stecher G, Li M et al. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 2018; 35: 1547-9.

[74] Li W, Fu L, Geng Z et al. Physiological characteristic changes and full-length transcriptome of rose (Rosa chinensis) roots and leaves in response to drought stress. Plant Cell Physiol 2021; 61: 2153-66.

[75] Luo J, Chen S, Cao S et al. Rose (Rosa hybrida) ethylene responsive factor 3 promotes rose flower senescence via direct activation of the abscisic acid synthesis-related 9-CIS-EPOXYCAROTENOID DIOXYGENASE gene. Plant Cell Physiol 2021; 62: 1030-43.