BpSPL2 promotes drought tolerance in Betula platyphylla Suk by modulating lignin accumulation

Abstract

Drought stress severely impairs plant growth and development, and lignin plays a critical role in plant resistance. In this study, we investigated the physiological and molecular mechanisms underlying drought adaptation regulated by BpSPL2 in birch (Betula platyphylla Suk), using transgenic overexpressing lines (OE3 and OE5) and SRDX repressor lines suppressed BpSPL2 expression (S2 and S4). The results showed that, compared with the wild type, the drought tolerance of two-year-old OE3 and OE5 birch lines was significantly enhanced under natural drought conditions, with reduced accumulation of malondialdehyde (MDA) and H₂O₂ content, increased proline content, and elevated antioxidant enzyme activities. In contrast, S2 and S4 lines exhibited greater drought sensitivity. RNA-seq analysis revealed that, under both normal and drought conditions, differentially expressed genes (DEGs) between genotypes were consistently enriched in pathways related to phenylpropanoid biosynthesis, cell wall formation, and lignin synthesis. Overexpression of BpSPL2 significantly increased the expression of lignin synthesis-related genes in birch and promoted lignin accumulation, indicating that BpSPL2 enhances birch drought tolerance by promoting lignin synthesis. To uncover potential molecular mechanisms, we performed a multi-omics correlation analysis integrating transcriptome and DAP-seq data, identifying 12 DEGs in the phenylpropanoid biosynthesis pathway as potential target genes of BpSPL2. DAP-seq results revealed that both the BpHCT25 and BpCSE5 promoter regions contain conserved BpSPL2 binding motifs. RT-qPCR analysis demonstrated that BpHCT25 and BpCSE5 expression levels were significantly elevated in the OE5 line, whereas they were downregulated in the S4 line. Further validation using biochemical approaches such as yeast one hybrid (Y1H) and dual luciferase reporter (DLR) assays confirmed that BpSPL2 binds to the promoters of its downstream genes BpHCT25 and BpCSE5, thereby promoting their expression. In summary, we report a novel molecular mechanism in birch where BpSPL2 mediates lignin biosynthesis to regulate drought resistance. BpSPL2 serves as a potential target for drought-tolerant breeding in birch, and these findings may hold significant application value for developing molecular breeding strategies for drought-resistant forest trees.

Keywords

Betula platyphylla / Drought stress / BpSPL2 / Lignin

References

[1] Bachir IH, Ployet R, Teulières C, Cassan-Wang H, Mounet F, Grima-Pettenati J. Regulation of secondary cell wall lignification by abiotic and biotic constraints. Adv Bot Res. 2022, 104: 363-392.

[2] Bang SW, Lee DK, Jung H, Chung PJ, Kim YS, Do Choi Y, Suh JW, Kim JK. Overexpression of OsTF1L, a rice HD-Zip transcription factor, promotes lignin biosynthesis and stomatal closure that improves drought tolerance. Plant Biotechnol J. 2019, 17(1): 118-131.

[3] Bang SW, Choi S, Jin XJ, Jung SE, Choi JW, Seo JS, Kim JK. Transcriptional activation of rice cinnamoyl-CoA reductase 10 by OsNAC5, contributes to drought tolerance by modulating lignin accumulation in roots. Plant Biotechnol J. 2022, 20(4): 736-747.

[4] Bartlett A, O’Malley RC, Huang SC, Galli M, Nery JR, Gallavotti A, Ecker JR. Mapping genome-wide transcription-factor binding sites using DAP-seq. Nat Protoc. 2017, 12(8): 1659-1672.

[5] Chen T, Qin GZ, Tian SP. Regulatory network of fruit ripening: current understanding and future challenges. New Phytol. 2020, 228(4): 1219-1226.

[6] Chen S, Wang YC, Yu LL, Zheng T, Wang S, Yue Z, Jiang J, Kumari S, Zheng CF, Tang HB, Li J, Li YQ, Chen JJ, Zhang WB, Kuang HH, Robertson JS, Zhao PX, Li HY, Shu SQ, Yordanov YS, Huang HJ, Goodstein DM, Gai Y, Qi Q, Min JM, Xu CY, Wang SB, Qu GZ, Paterson AH, Sankoff D, Wei HR, Liu GF, Yang CP. Genome sequence and evolution ofBetula platyphylla. Hortic Res. 2021, 8. 37

[7] Choi SJ, Lee Z, Kim S, Jeong E, Shim JS. Modulation of lignin biosynthesis for drought tolerance in plants. Front Plant Sci. 2023, 14. 1116426

[8] de Oliveira Gomes LE, Bernardino AF. Drought effects on tropical estuarine benthic assemblages in Eastern Brazil. Sci Total Environ. 2020, 703. 135490

[9] Diekmann S, Dürr C, Herrmann A, Lindner I, Jozic D. Single use bioreactors for the clinical production of monoclonal antibodies–a study to analyze the performance of a CHO cell line and the quality of the produced monoclonal antibody. BMC Proc. 2011, 5(8. P103

[10] Dong NQ, Lin HX. Contribution of phenylpropanoid metabolism to plant development and plant–environment interactions. J Integr Plant Biol. 2021, 631): 180-209.

[11] Eziz A, Yan ZB, Tian D, Han WX, Tang ZY, Fang JY. Drought effect on plant biomass allocation: a meta-analysis. Ecol Evol. 2017, 7(24): 11002-11010.

[12] Fang QL, Wu D, Sun H, Wang LY, Liu YP, Mei WF, Guo HY. A bHLH transcription factor confers salinity stress tolerance in Betula platyphylla. Plant Direct. 2024, 8(12. e70029

[13] Fei XT, Li JM, Kong LJ, Hu HC, Tian JY, Liu YL, Wei AZ. MiRNAs and their target genes regulate the antioxidant system of Zanthoxylum bungeanum under drought stress. Plant Physiol Biochem. 2020, 150: 196-203.

[14] Feng C, Zhang X, Du BY, Xiao YQ, Wang YY, Sun YT, Zhou X, Wang C, Liu Y, Li TH. MicroRNA156ab regulates apple plant growth and drought tolerance by targeting transcription factor MsSPL13. Plant Physiol. 2023, 1923): 1836-1857.

[15] Gambino G, Perrone I, Gribaudo I. A rapid and effective method for RNA extraction from different tissues of grapevine and other woody plants. Phytochem Anal. 2008, 19(6): 520-525.

[16] Gao ZH, Gao Y, Jia HH, Wang WH, Xue BW, Su YH, Guo XQ, Wang C. The GhMPK7-GhSDIRIP1 module enhances drought tolerance in cotton by regulating ABA signaling. New Phytol. 2025, 248(3): 1351-1367.

[17] Guo HY, Wang YC, Wang LQ, Hu P, Wang YM, Jia YY, Zhang CR, Zhang Y, Zhang YM, Wang C, Yang CP. Expression of the MYB transcription factor gene BplMYB46 affects abiotic stress tolerance and secondary cell wall deposition in Betula platyphylla. Plant Biotechnol J. 2017, 15(1): 107-121.

[18] Heinz S, Benner C, Spann N, Bertolino E, Lin YC, Laslo P, Cheng JX, Murre C, Singh H, Glass CK. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol Cell. 2010, 38(4): 576-589.

[19] Hu X, Zheng T, Chen W, Duan H, Yuan Z, An J, Zhang H, Liu X. Genome-wide identification and expression analysis of the GST gene family of Betula platyphylla. J Forestry Res. 2024, 35: 123.

[20] Huang X, Chu GM, Wang J, Luo HH, Yang ZN, Sun LP, Rong WW, Wang M. Integrated metabolomic and transcriptomic analysis of specialized metabolites and isoflavonoid biosynthesis in Sophora alopecuroides L. under different degrees of drought stress. Ind Crops Prod. 2023, 197. 116595

[21] Huang EX, Tang J, Song SM, Yan H, Yu XY, Luo CL, Chen YN, Ji HY, Chen AQ, Zhou JY, Liao H. Caffeic acid O-methyltransferase from Ligusticum chuanxiong alleviates drought stress, and improves lignin and melatonin biosynthesis. Front Plant Sci. 2024, 15. 1458296

[22] Huang HD, Zheng ML, Jenks MA, Yang PF, Zhao HY, Lü SY. Squamosa promoter binding protein-like 13 slp1 together with spl9 redundantly regulates wax biosynthesis under drought stress. J Exp Bot. 2024, 75(16): 4978-4992.

[23] Isasa E, Link RM, Jansen S, Tezeh FR, Kaack L, Sarmento Cabral J, Schuldt B. Addressing controversies in the xylem embolism resistance–vessel diameter relationship. New Phytol. 2023, 2381283-296.

[24] Jia YQ, Niu YN, Zhao HM, Wang ZB, Gao CQ, Wang C, Chen S, Wang YC. Hierarchical transcription factor and regulatory network for drought response in Betula platyphylla. Hortic Res. 2022, 9. uhac040

[25] Jia YQ, Zhao HM, Niu YN, Wang YC. Long noncoding RNA from Betula platyphylla, BplncSIR1, confers salt tolerance by regulating BpNAC2 to mediate reactive oxygen species scavenging and stomatal movement. Plant Biotechnol J. 2024, 22(1): 48-65.

[26] Jiang T, Liu FC, Rodriguez K, Rabanal N, Lian ZY, Lin MF, Gong HJ, Wang SH, Huo HQ. Genome-wide analysis of the SPL gene family in lettuce and its role in miR156/SPL module-mediated plant development and regeneration. Hortic Plant J. 2025.

[27] Jiao P, Jiang ZZ, Miao M, Wei XT, Wang CL, Liu SY, Guan SY, Ma YY. Zmhdz9, an HD-Zip transcription factor, promotes drought stress resistance in maize by modulating ABA and lignin accumulation. Int J Biol Macromol. 2024, 258. 128849

[28] Jing Y, Yu Y, Wang H, Wu Y, Li C. Genome-wide identification and expression analysis of the bZIP gene family in silver birch Betula pendula Roth. J Forestry Res. 2022, 33: 1616-1631.

[29] Jung SE, Kim TH, Shim JS, Bang SW, Bin Yoon H, Oh SH, Kim YS, Oh SJ, Seo JS, Kim JK. Rice NAC17 transcription factor enhances drought tolerance by modulating lignin accumulation. Plant Sci. 2022, 323. 111404

[30] Kriegshauser L, Knosp S, Grienenberger E, Tatsumi K, Gütle DD, Sørensen I, Herrgott L, Zumsteg J, Rose JKC, Reski R, Werck-Reichhart D, Renault H. Function of the hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyl transferase is evolutionarily conserved in embryophytes. Plant Cell. 2021, 33(5): 1472-1491.

[31] Kurjak D, Petrík P, Konôpková AS, Link RM, Gömöry D, Hajek P, Liesebach M, Leuschner C, Schuldt B. Inter-provenance variability and phenotypic plasticity of wood and leaf traits related to hydraulic safety and efficiency in seven European beech Fagus sylvatica L. provenances differing in yield. Ann for Sci. 2024, 81(1. 11

[32] Li JJ, Tang B, Li YX, Li CG, Guo MJ, Chen HY, Han SC, Li J, Lou QJ, Sun WQ, Wang P, Guo HF, Ye W, Zhang ZY, Zhang HL, Yu SB, Zhang L, Li ZC. Rice SPL10 positively regulates trichome development through expression of HL6 and auxin-related genes. J Integr Plant Biol. 2021, 638): 1521-1537.

[33] Li DD, Yang JL, Pak S, Zeng MZ, Sun JL, Yu S, He YT, Li CH. PuC3H35 confers drought tolerance by enhancing lignin and proanthocyanidin biosynthesis in the roots of Populus ussuriensis. New Phytol. 2022, 233(1): 390-408.

[34] Li YX, Han SC, Sun XM, Khan NU, Zhong Q, Zhang ZY, Zhang HL, Ming F, Li ZC, Li JJ. Variations in OsSPL10 confer drought tolerance by directly regulating OsNAC2 expression and ROS production in rice. J Integr Plant Biol. 2023, 65(4): 918-933.

[35] Li JY, Ren JJ, Lei XY, Fan WM, Tang L, Zhang QQ, Bao Z, Zhou WF, Bai J, Zhang YZ, Gong CM. CsREV-CsTCP4-CsVND7 module shapes xylem patterns differentially between stem and leaf to enhance tea plant tolerance to drought. Cell Rep. 2024, 43(4. 113987

[36] Li W, Lin YJ, Chen YL, Zhou CG, Li S, De Ridder N, Oliveira DM, Zhang LJ, Zhang BC, Wang JP, Xu CZ, Fu XK, Luo KM, Wu AM, Demura T, Lu MZ, Zhou YH, Li LG, Umezawa T, Boerjan W, Chiang VL. Woody plant cell walls: fundamentals and utilization. Mol Plant. 2024, 171): 112-140.

[37] Li PC, Zhu TZ, Wang YY, Zhang XM, Yang XY, Fang S, Li W, Rui WY, Yang AQ, Duan YM, Yan YX, Pan QC, Jia ZT, Wang HM, Yang ZF, Yu P, Xu CW. Natural variation in a cortex/epidermis-specific transcription factor bZIP89 determines lateral root development and drought resilience in maize. Sci Adv. 2025, 11(15. eadt1113

[38] Liao ZG, Zhang YC, Yu Q, Fang WC, Chen MY, Li TF, Liu Y, Liu ZC, Chen L, Yu SW, Xia H, Xue HW, Yu H, Luo LJ. Coordination of growth and drought responses by GA-ABA signaling in rice. New Phytol. 2023, 2403): 1149-1161.

[39] Liu QQ, Luo L, Zheng LQ. Lignins: biosynthesis and biological functions in plants. Int J Mol Sci. 2018, 19(2): 335.

[40] Liu W, Jiang Y, Wang CH, Zhao LL, Jin YZ, Xing QJ, Li M, Lv TH, Qi HY. Lignin synthesized by CmCAD2 and CmCAD3 in oriental melon Cucumis melo L. seedlings contributes to drought tolerance. Plant Mol Biol. 2020, 1036689-704.

[41] Liu D, An Y, Li Z, Wang Z, Zhao Y, Wang X. Differences and similarities in radial growth of Betula species to climate change. J for Res. 2024, 35: 40.

[42] Liu WH, Yang Y, Hu YY, Peng X, He LS, Ma TF, Zhu SQ, Xiang LE, Chen N. Overexpression of squamosa promoter binding protein-like 4a (NtSPL4a) alleviates Cd toxicity in Nicotiana tabacum. Plant Physiol Biochem. 2024, 210. 108656

[43] Luan YT, Chen ZJ, Meng JS, Tao J, Zhao DQ. PoWRKY17 promotes drought tolerance in Paeonia ostii by modulating lignin accumulation. Ind Crops Prod. 2023, 204. 117228

[44] Luan YT, Yu H, An HL, Chen ZJ, Zhao DQ, Tao J. Phosphorylation of the transcription factor PoWRKY31 by PoKIN10 activates lignin biosynthesis to improve tree peony drought tolerance. New Phytol. 2025, 248(4): 1784-1801.

[45] Ma HY, Su LW, Zhang W, Sun Y, Li DN, Li S, Lin YJ, Zhou CG, Li W. Epigenetic regulation of lignin biosynthesis in wood formation. New Phytol. 2025, 24541589-1607.

[46] Ma XT, Umar AW, Zhou Y, Yu LL, Liu XY, Ahmad N, Zhang J, Liu XM. Hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase CtHCT061 enhances lignin biosynthesis and drought tolerance in safflower (Carthamus tinctorius L.). Ind Crops Prod. 2025, 236. 121929

[47] Ning K, Chen S, Huang HJ, Jiang J, Yuan HM, Li HY. Molecular characterization and expression analysis of the SPL gene family with BpSPL9 transgenic lines found to confer tolerance to abiotic stress in Betula platyphylla Suk. Plant Cell Tissue Organ Cult. 2017, 130(3): 469-481.

[48] Niu MX, Feng CH, He F, Zhang H, Bao Y, Liu SJ, Liu X, Su YY, Liu C, Wang HL, Yin WL, Xia XL. The miR6445-NAC029 module regulates drought tolerance by regulating the expression of glutathione S-transferase U23 and reactive oxygen species scavenging in *Populus*. New Phytol. 2024, 242(5): 2043-2058.

[49] Pereira L, Domingues-Junior AP, Jansen S, Choat B, Mazzafera P. Is embolism resistance in plant xylem associated with quantity and characteristics of lignin?. Trees. 2018, 32(2): 349-358.

[50] Pérez-Antón M, Schneider I, Kroll P, Hofhuis H, Metzger S, Pauly M, Hay A. Explosive seed dispersal depends on SPL7 to ensure sufficient copper for localized lignin deposition via laccases. Proc Natl Acad Sci U S A. 2022, 119(24. e2202287119

[51] Petek-Petrik A, Petrík P, Lamarque LJ, Cochard H, Burlett R, Delzon S. Drought survival in conifer species is related to the time required to cross the stomatal safety margin. J Exp Bot. 2023, 7421): 6847-6859.

[52] Qi SY, Yao TT, Ren ZQ, Wang JC, Wang ZY, Liu HZ, Song JQ, Li HJ, Liu TT, Ding CJ, Hu YB, Zhang HH. Integrative miRNA-mRNA analysis reveals key regulatory mechanisms of the miR156g-PagSPL1B module in poplar drought tolerance. Ind Crops Prod. 2025, 237. 122235

[53] Ragas REG, Scanlon MJ. Plant development: the role of SPL9 in age-dependent sculpting of leaf shape. Curr Biol. 2024, 34(19): R891-R893.

[54] Shen MX, Liu YD, Xu YM, Sun Y, Sun LQ, Feng YP, Chen MY, Zhang C, Li H, Lu H. PtrLAC17 act as a key laccase in catalyzing lignin polymerization in both the stem xylem cell walls and the guard cell walls, coordinately regulating drought tolerance in *Populus*. Int J Biol Macromol. 2025, 321. 146547

[55] Sun XH, Zhang LL, Xu WH, Zheng JP, Yan ML, Zhao M, Wang XY, Yin Y. A comprehensive analysis of the peanut squamosa promoter binding protein-like gene family and how AhSPL5 enhances salt tolerance in transgenic *Arabidopsis*. Plants Basel. 2024, 13(8. 1057

[56] Sun N, Yang SW, Zhang TH, Xu JJ, Wang KX, Liang YL, Sun CQ, Zhang XL, Zhang HH. MYB37 enhances drought tolerance by maintaining ROS homeostasis and alleviating photosynthetic inhibition in *Arabidopsis thaliana*. Plant Physiol Biochem. 2025, 228. 110289

[57] Taylor LP, Grotewold E. Flavonoids as developmental regulators. Curr Opin Plant Biol. 2005, 83): 317-323.

[58] Vanholme R, Cesarino I, Rataj K, Xiao YG, Sundin L, Goeminne G, Kim H, Cross J, Morreel K, Araujo P, Welsh L, Haustraete J, McClellan C, Vanholme B, Ralph J, Simpson GG, Halpin C, Boerjan W. Caffeoyl shikimate esterase (CSE) is an enzyme in the lignin biosynthetic pathway in Arabidopsis. Science. 2013, 341(6150): 1103-1106.

[59] Wang XY, Zhang D, Cui N, Yu Y, Yu GC, Fan HY. Transcriptome and miRNA analyses of the response to Corynespora cassiicola in cucumber. Sci Rep. 2018, 8(1): 7798.

[60] Xiang Y, Liu WJ, Niu YX, Li Q, Zhao CY, Pan YT, Li GD, Bian XL, Miao YD, Zhang AY. The maize GSK3-like kinase ZmSK1 negatively regulates drought tolerance by phosphorylating the transcription factor ZmCPP2. Plant Cell. 2025, 372. koaf032

[61] Yamasaki K, Kigawa T, Inoue M, Tateno M, Yamasaki T, Yabuki T, Aoki M, Seki E, Matsuda T, Nunokawa E, Ishizuka Y, Terada T, Shirouzu M, Osanai T, Tanaka A, Seki M, Shinozaki K, Yokoyama S. A novel zinc-binding motif revealed by solution structures of DNA-binding domains of Arabidopsis SBP-family transcription factors. J Mol Biol. 2004, 337149-63.

[62] Yao YD, Wu XT, Yang Y, Hou XM, Xia XJ, Liu ZS, Wang CL, Liao WB. SPL transcription factors in plants: a potential stress regulator. Hortic Plant J. 2025.

[63] Yu YB, Yu Y, Cui N, Ma LF, Tao R, Ma ZT, Meng XN, Fan HY. Lignin biosynthesis regulated by CsCSE1 is required for Cucumis sativus defence to Podosphaera xanthii. Plant Physiol Biochem. 2022, 186: 88-98.

[64] Zhai XZ, Yan XC, Zenda T, Wang N, Dong AY, Yang Q, Zhong Y, Xing Y, Duan HJ. Overexpression of the peroxidase gene ZmPRX1 increases maize seedling drought tolerance by promoting root development and lignification. Crop J. 2024, 12(3): 753-765.

[65] Zhang Y, Liu T, Meyer CA, Eeckhoute J, Johnson DS, Bernstein BE, Nusbaum C, Myers RM, Brown M, Li W, Liu XS. Model-based analysis of ChIP-seq MACS. Genome Biol. 2008, 9(9. R137

[66] Zhang HM, Zhu JH, Gong ZZ, Zhu JK. Abiotic stress responses in plants. Nat Rev Genet. 2022, 232): 104-119.

[67] Zhang WZ, Wang L, Zhang LP, Kong XQ, Zhang J, Wang X, Pei YX, Jin ZP. H2S-mediated balance regulation of stomatal and non-stomatal factors responding to drought stress in Chinese cabbage. Hortic Res. 2023, 10(3. uhac284

[68] Zhao HM, Jia YQ, Niu YN, Wang YC. The BpPP2C-BpMADS11-BpERF61 signaling confers drought tolerance in Betula platyphylla. New Phytol. 2024, 2446): 2364-2381.

[69] Zhao Y, He JQ, Liu MM, Miao JN, Ma C, Feng YJ, Qian JJ, Li HH, Bi HH, Liu WX. The SPL transcription factor TaSPL6 negatively regulates drought stress response in wheat. Plant Physiol Biochem. 2024, 206. 108264

[70] Zheng K, Cai YS, Qu YY, Teng L, Wang CY, Gao J, Chen QJ. Effect of the HCT gene on lignin synthesis and fiber development in Gossypium barbadense. Plant Sci. 2024, 338. 111914

[71] Zhou XM, Ren RY, Sun H, Wang LY, He WJ, Guo HY. BpMYB06 acts as a positive regulatory factor in saline–alkaline stress resistance by binding to two novel elements. Plant Cell Physiol. 2025, 66(3): 318-332.

RIGHTS & PERMISSIONS

The Author(s)