Pag4CL3 is a key regulator of lignin and melatonin biosynthesis for enhancing cold tolerance in 84 K poplar (Populus alba ×  P. glandulosa)

Abstract

Precise phenological regulation is critical for temperate trees to survive the winter. However, the underlying mechanism is still unclear. Here, we found that Pag4CL3 coordinately modulates lignin biosynthesis and melatonin accumulation in 84 K poplar (Populus alba ×  P. glandulosa). Overexpression of Pag4CL3 or Pag4CL5 increased the lignin content in stem but reduced plant growth. In contrast, knockout of either gene reduced stem lignin monomers, promoted growth, and improved cold tolerance, with Pag4CL3 mutants (4cl3) exhibiting more pronounced resistance. PagSNAT2, which encodes a key enzyme in melatonin (MT) biosynthesis, is markedly upregulated in the 4cl3 mutant. Consistent with this, overexpression of PagSNAT2 promoted MT accumulation in 84 K poplar, and the 4cl3 mutant exhibited significantly higher MT levels in both autumn dormant and spring sprouting buds compared to the wild-type. Yeast two-hybrid (Y2H) and luciferase complementation assays further confirmed that Pag4CL3 directly interacts with PagSNAT2. Additionally, low temperature inhibited the binding of transcription factor PagDRS1 to the Pag4CL3 promoter and attenuating its suppression of melatonin synthesis. This study thus unveils a cold-responsive PagDRS1–Pag4CL3–PagSNAT2 regulatory module that balances structural formation and stress adaptation in trees, providing a theoretical basis and breeding strategy for developing poplar varieties with enhanced biomass and winter resilience.

Keywords

Pag4CL3 / Pag4CL5 / 84 K poplar / Melatonin / Cold

References

[1] Aphalo PJ, Sadras VO. Explaining pre-emptive acclimation by linking information to plant phenotype. J Exp Bot, 2022, 73(15): 5213-5234.

[2] Bhuiyan NH, Selvaraj G, Wei YD, King J. Role of lignification in plant defense. Plant Signal Behav, 2009, 4(2): 158-159.

[3] Campbell MM, Sederoff RR. Variation in lignin content and composition (mechanisms of control and implications for the genetic improvement of plants). Plant Physiol, 1996, 110(1): 3-13.

[4] Chang CY, Bräutigam K, Hüner NPA, Ensminger I. Champions of winter survival: cold acclimation and molecular regulation of cold hardiness in evergreen conifers. New Phytol, 2021, 229(2): 675-691.

[5] Chen F, Dixon RA. Lignin modification improves fermentable sugar yields for biofuel production. Nat Biotechnol, 2007, 25(7): 759-761.

[6] Chen HC, Song JN, Williams CM, Shuford CM, Liu J, Wang JP, Li QZ, Shi R, Gokce E, Ducoste J, Muddiman DC, Sederoff RR, Chiang VL. Monolignol pathway 4-coumaric acid: coenzyme A ligases in Populus trichocarpa: novel specificity, metabolic regulation, and simulation of coenzyme A ligation fluxes. Plant Physiol, 2013, 161(3): 1501-1516.

[7] Chen HC, Song JN, Wang JP, Lin YC, Ducoste J, Shuford CM, Liu J, Li QZ, Shi R, Nepomuceno A, Isik F, Muddiman DC, Williams C, Sederoff RR, Chiang VL. Systems biology of lignin biosynthesis in Populus trichocarpa: heteromeric 4-coumaric acid: coenzyme A ligase protein complex formation, regulation, and numerical modeling. Plant Cell, 2014, 26(3): 876-893.

[8] Chen KQ, Song MR, Guo YN, Liu LF, Xue H, Dai HY, Zhang ZH. MdMYB46 could enhance salt and osmotic stress tolerance in apple by directly activating stress-responsive signals. Plant Biotechnol J, 2019, 17(12): 2341-2355.

[9] Chen L, Cui YM, Yao YH, An LK, Bai YX, Li X, Yao XH, Wu KL. Genome-wide identification of WD40 transcription factors and their regulation of the MYB-bHLH-WD40 (MBW) complex related to anthocyanin synthesis in Qingke (Hordeum vulgare L. var. nudum Hook. f.). BMC Genomics, 2023, 24(1): 166

[10] Chen XX, Liu K, Luo TT, Zhang BL, Yu JY, Ma D, Sun XQ, Zheng HW, Xin BN, Xia JX. Four MYB transcription factors regulate suberization and nonlocalized lignification at the root endodermis in rice. Plant Cell, 2024, 37(1): koae278

[11] Chen Q, Chen YL, Li X, Zhang LP, Rengel Z. Phytomelatonin: biosynthesis, signaling, and functions. Annu Rev Plant Biol, 2025, 76(1): 171-195.

[12] Colombage R, Singh MB, Bhalla PL. Melatonin and abiotic stress tolerance in crop plants. Int J Mol Sci, 2023, 24(8): 7447

[13] Cooke JEK, Eriksson ME, Junttila O. The dynamic nature of bud dormancy in trees: environmental control and molecular mechanisms. Plant Cell Environ, 2012, 35(10): 1707-1728.

[14] Ding YL, Yang SH. Surviving and thriving: how plants perceive and respond to temperature stress. Dev Cell, 2022, 57(8): 947-958.

[15] Ding F, Wang G, Zhang SX. Exogenous melatonin mitigates methyl viologen-triggered oxidative stress in poplar leaf. Molecules, 2018, 23(11): 2852

[16] Du MC, Xu C, Wang A, Lv PC, Xu ZQ, Zhang XL. Different drought recovery strategy between Larix spp. and Quercus mongolica in temperate forests. Sci Total Environ, 2024, 938: 173521

[17] Feikema PM, Baker TG. Effect of soil salinity on growth of irrigated plantation Eucalyptus in south-eastern Australia. Agric Water Manag, 2011, 98(7): 1180-1188.

[18] Figueroa-Macías JP, García YC, Núñez M, Díaz K, Olea AF, Espinoza L. Plant growth-defense trade-offs: molecular processes leading to physiological changes. Int J Mol Sci, 2021, 22(2): 693

[19] Forster EJ, Styles D, Healey JR. Temperate forests can deliver future wood demand and climate-change mitigation dependent on afforestation and circularity. Nat Commun, 2025, 16(1): 3872

[20] Galleguillos C, Acuña-Rodríguez IS, Torres-Díaz C, Gundel PE, Molina-Montenegro MA. Genetic control underlying the flowering-drought tolerance trade-off in the Antarctic plant Colobanthus quitensis. Plant Cell Environ, 2023, 46(10): 3158-3169.

[21] Gao S, Liu RS, Zhou T, Fang W, Yi CX, Lu RJ, Zhao X, Luo H. Dynamic responses of tree-ring growth to multiple dimensions of drought. Glob Chang Biol, 2018, 24(11): 5380-5390.

[22] Hu WJ, Harding SA, Lung J, Popko JL, Ralph J, Stokke DD, Tsai CJ, Chiang VL. Repression of lignin biosynthesis promotes cellulose accumulation and growth in transgenic trees. Nat Biotechnol, 1999, 17(8): 808-812.

[23] Huot B, Yao J, Montgomery BL, He SY. Growth-defense tradeoffs in plants: a balancing act to optimize fitness. Mol Plant, 2014, 7(8): 1267-1287.

[24] Jalil A, Faisal H, Ummara K, Nazir A, Juan L, Sezai E, Shahid I, Umer JH, Tahira A, Panfeng T, Jiezhong C (2024) Melatonin: A promising approach to enhance abiotic stress tolerance in horticultural plants. S Afr J Bot 164:66–76. https://doi.org/10.1016/j.sajb.2023.10.045

[25] Jiang LP, Pei XN, Hu YB, Chiang VL, Zhao XY. Effects of environment and genotype on growth traits in poplar clones in Northeast China. Euphytica, 2021, 217(8): 169

[26] Jiang LY, Yue ML, Liu YQ, Zhang NT, Lin YX, Zhang YT, Wang Y, Li MY, Luo Y, Zhang Y, Wang XR, Chen Q, Tang HR. A novel R2R3-MYB transcription factor FaMYB5 positively regulates anthocyanin and proanthocyanidin biosynthesis in cultivated strawberries (Fragaria × ananassa). Plant Biotechnol J, 2023, 21(6): 1140-1158.

[27] Jung JH, Domijan M, Klose C, Biswas S, Ezer D, Gao M, Khattak AK, Box MS, Charoensawan V, Cortijo S, Kumar M, Grant A, Locke JC, Schäfer E, Jaeger KE, Wigge PA. Phytochromes function as thermosensors in Arabidopsis. Science, 2016, 354(6314): 886-889.

[28] Kaixin W, Qufan X, Jalal AG, Jie Z (2022) Functions and prospects of melatonin in plant growth, yield and quality. J Exp Bot 73(17):5928–5946. https://doi.org/10.1093/jxb/erac233

[29] Kim JH, Hilleary R, Seroka A, He SY. Crops of the future: building a climate-resilient plant immune system. Curr Opin Plant Biol, 2021, 60: 101997

[30] Koch GW, Sillett SC, Jennings GM, Davis SD. The limits to tree height. Nature, 2004, 428(6985): 851-854.

[31] Li CF, Wang XQ, Ran LY, Tian QY, Fan D, Luo KM. PtoMYB92 is a transcriptional activator of the lignin biosynthetic pathway during secondary cell wall formation in Populus tomentosa. Plant Cell Physiol, 2015, 56(12): 2436-2446.

[32] Li RQ, Jiang M, Song Y, Zhang HL. Melatonin alleviates low-temperature stress via ABI5-mediated signals during seed germination in rice (Oryza sativa L.). Front Plant Sci, 2021, 12: 727596.

[33] Li W, Jimmy Lin YC, 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, 18(5): 909-910.

[34] Liu YK, Zhou J. MAPping kinase regulation of ICE1 in freezing tolerance. Trends Plant Sci, 2018, 23(2): 91-93.

[35] Liu DM, He J, Li Q, Zhang X, Wang YS, Sun QG, Wang WH, Zhang ML, Wang YL, Xu HS, Fang L, Jiang L, Liu SJ, Chen LM, Tian YL, Liu X, Wang RY, Zhang ZG, Chern M, Dong XO, Wang HY, Liu YQ, Ronald PC, Wan JM. A WRKY transcription factor confers broad-spectrum resistance to biotic stresses and yield stability in rice. Proc Natl Acad Sci U S A, 2025, 122(10): e2411164122

[36] Ma JY, Zuo DJ, Zhang XD, Li HC, Ye H, Zhang NJ, Li MD, Dang M, Geng FD, Zhou HJ, Zhao P. Genome-wide identification analysis of the 4-Coumarate: CoA ligase (4CL) gene family expression profiles in Juglans regia and its wild relatives J. Mandshurica resistance and salt stress. BMC Plant Biol, 2024, 24(1): 211

[37] Manfre A, Glenn M, Nuñez A, Moreau RA, Dardick C. Light quantity and photosystem function mediate host susceptibility to Turnip mosaic virus via a salicylic acid-independent mechanism. Mol Plant Microbe Interact, 2011, 24(3): 315-327.

[38] McGregor IR, Helcoski R, Kunert N, Tepley AJ, Gonzalez-Akre EB, Herrmann V, Zailaa J, Stovall AEL, Bourg NA, McShea WJ, Pederson N, Sack L, Anderson-Teixeira KJ. Tree height and leaf drought tolerance traits shape growth responses across droughts in a temperate broadleaf forest. New Phytol, 2021, 231(2): 601-616.

[39] Nakanishi K, Li H, Ichino T, Tatsumi K, Osakabe K, Watanabe B, Shimomura K, Yazaki K. Peroxisomal 4-coumaroyl-CoA ligases participate in shikonin production in Lithospermum erythrorhizon. Plant Physiol, 2024, 195(4): 2843-2859.

[40] Novaes E, Kirst M, Chiang V, Winter-Sederoff H, Sederoff R. Lignin and biomass: a negative correlation for wood formation and lignin content in trees. Plant Physiol, 2010, 154(2): 555-561.

[41] Petruccelli R, Bartolini G, Ganino T, Zelasco S, Lombardo L, Perri E, Durante M, Bernardi R. Cold stress, freezing adaptation, varietal susceptibility of Olea europaea L.: a review. Plants, 2022, 11(10): 1367

[42] Plomion C. Wood formation in trees. Plant Physiol, 2001, 127(4): 1513-1523.

[43] Polle A, Chen SL. On the salty side of life: molecular, physiological and anatomical adaptation and acclimation of trees to extreme habitats. Plant Cell Environ, 2015, 38(9): 1794-1816.

[44] Pu YQ, Kosa M, Kalluri UC, Tuskan GA, Ragauskas AJ. Challenges of the utilization of wood polymers: how can they be overcome?. Appl Microbiol Biotechnol, 2011, 91(6): 1525-1536.

[45] Qiu DY, Bai SL, Ma JC, Zhang LS, Shao FJ, Zhang KK, Yang YF, Sun T, Huang JL, Zhou Y, Galbraith DW, Wang ZS, Sun GL. The genome of Populus alba x Populus tremula var. glandulosa clone 84K. DNA Res, 2019, 26(5): 423-431.

[46] Ramsay NA, Glover BJ. MYB-bHLH-WD40 protein complex and the evolution of cellular diversity. Trends Plant Sci, 2005, 10(2): 63-70.

[47] Rosso L, Cantamessa S, Bergante S, Biselli C, Fricano A, Chiarabaglio PM, Gennaro M, Nervo G, Secchi F, Carra A. Responses to drought stress in poplar: what do we know and what can we learn?. Life, 2023, 13(2): 533

[48] Shao RX, Zhang JJ, Shi WY, Wang YC, Tang YL, Liu ZK, Sun W, Wang H, Guo JM, Meng YJ, Kang GZ, Jagadish KS, Yang QH. Mercury stress tolerance in wheat and maize is achieved by lignin accumulation controlled by nitric oxide. Environ Pollut, 2022, 307: 119488

[49] Shi YX, Zhang J, Li JX, He JQ, Wu S, Yu M, Yang D, Ju LC. USP15, activated by TFAP4 transcriptionally, stabilizes SHC1 via deubiquitination and deteriorates renal cell carcinoma. Cancer Sci, 2024, 115(8): 2617-2629.

[50] Sun M, Yang XL, Zhu ZP, Xu QY, Wu KX, Kang YJ, Wang H, Xiong AS. Comparative transcriptome analysis provides insight into nitric oxide suppressing lignin accumulation of postharvest okra (Abelmoschus esculentus L.) during cold storage. Plant Physiol Biochem, 2021, 167: 49-67.

[51] Theocharis A, Clément C, Barka EA. Physiological and molecular changes in plants grown at low temperatures. Planta, 2012, 235(6): 1091-1105.

[52] Wang JP, Liu BG, Sun Y, Chiang VL, Sederoff RR. Enzyme-enzyme interactions in monolignol biosynthesis. Front Plant Sci, 2018, 9: 1942.

[53] Wang DM, Liang XX, Bao YZ, Yang SX, Zhang X, Yu H, Zhang Q, Xu GY, Feng XZ, Dou DL. A malectin-like receptor kinase regulates cell death and pattern-triggered immunity in soybean. EMBO Rep, 2020, 21(11): e50442

[54] Wang QF, Qi CF, Wang LX, Li M, Niu YH, Muhammad N, Liu MJ, Liu ZG, Wang LX. ZjMAPKK4 interacted with ZjNAC78 regulates cold tolerance response in jujube. Plant Cell Environ, 2025, 48(5): 3691-3707.

[55] Xiao JC, Cao BY, Tang W, Sui XY, Tang Y, Lai YS, Sun B, Huang Z, Zheng YX, Li HX. The CaCAD1-CaPOA1 module positively regulates pepper resistance to cold stress by increasing lignin accumulation. Int J Biol Macromol, 2025, 290: 139979

[56] Xie JM, Chen YR, Cai GJ, Cai RL, Hu Z, Wang H. Tree visualization by one table (tvBOT): a web application for visualizing, modifying and annotating phylogenetic trees. Nucleic Acids Res, 2023

[57] Yan Y, Wang P, Lu Y, Bai YJ, Wei YX, Liu GY, Shi HT. MeRAV5 promotes drought stress resistance in cassava by modulating hydrogen peroxide and lignin accumulation. Plant J, 2021, 107(3): 847-860.

[58] Yao JW, Ma Z, Ma YQ, Zhu Y, Lei MQ, Hao CY, Chen LY, Xu ZQ, Huang X. Role of melatonin in UV-B signaling pathway and UV-B stress resistance in Arabidopsis thaliana. Plant Cell Environ, 2021, 44(1): 114-129.

[59] Ye TT, Yin XM, Yu L, Zheng SJ, Cai WJ, Wu Y, Feng YQ. Metabolic analysis of the melatonin biosynthesis pathway using chemical labeling coupled with liquid chromatography-mass spectrometry. J Pineal Res, 2019, 66(1): e12531

[60] Yuan LQ, Dang J, Zhang JY, Wang LY, Zheng H, Li GB, Li JX, Zhou F, Khan A, Zhang ZD, Hu XH. A glutathione S-transferase regulates lignin biosynthesis and enhances salt tolerance in tomato. Plant Physiol, 2024, 196(4): 2989-3006.

[61] Zeng YL, Wu HL, Ouyang S, Chen L, Fang X, Peng CH, Liu SR, Xiao WF, Xiang WH. Ecosystem service multifunctionality of Chinese fir plantations differing in stand age and implications for sustainable management. Sci Total Environ, 2021, 788: 147791

[62] Zeng D, Dai LJ, Li X, Li W, Qu GZ, Li S. Genome-wide identification of the ERF transcription factor family for structure analysis, expression pattern, and response to drought stress in Populus alba × Populus glandulosa. Int J Mol Sci, 2023, 24(4): 3697

[63] Zhang H, Zhao Y, Zhu JK. Thriving under stress: how plants balance growth and the stress response. Dev Cell, 2020, 55(5): 529-543.

[64] Zhang BF, Wang ZW, Dai XF, Gao JH, Zhao JF, Ma R, Chen YJ, Sun Y, Ma HY, Li S, Zhou CG, Wang JP, Li W. A COMPASS histone H3K4 trimethyltransferase pentamer transactivates drought tolerance and growth/biomass production in Populus trichocarpa. New Phytol, 2024, 241(5): 1950-1972.

[65] Zhou W, Wang Y, Li B, Petijová L, Hu SY, Zhang Q, Niu JF, Wang DH, Wang SQ, Dong Y, Čellárová E, Wang ZZ. Whole-genome sequence data of Hypericum perforatum and functional characterization of melatonin biosynthesis by N-acetylserotonin O-methyltransferase. J Pineal Res, 2021, 70(2): e12709

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