Peptide phytosulfokine-α improves the efficiency of somatic embryogenesis in hybrid sweetgum leaves

Abstract

Somatic embryogenesis (SE) from vegetative organs remains a major bottleneck for the clonal propagation of forest trees, and conventional plant-hormone treatments are insufficient for establishing efficient regeneration systems. Here, we evaluated the effect of phytosulfokine-α (PSK-α) on leaf-derived SE in hybrid sweetgum (Liquidambar styraciflua × L. formosana). During the embryogenic callus (EC) induction stage, application of 0.2 mg L⁻¹ PSK-α increased EC induction efficiency by 25%. During the EC maturation stage, 0.4 mg L⁻¹ PSK-α increased the yield of cotyledonary embryos from 38 to 82 per 0.2 g of EC. Histological analysis suggested that EC originates from parenchyma cells of the spongy mesophyll and indicated that PSK-α facilitates EC induction by promoting the formation of dense meristematic cell clusters. Transcriptomic profiling revealed a stage-specific gene regulatory cascade modulated by PSK-α: The PSK gene was associated with leaf cell dedifferentiation at 1 d post-induction; GDSL-family genes mediated cell wall remodeling and also served as candidate regulatory genes controlling cell proliferation at 15–30 d post-induction; and receptor-like kinase genes may function in late-stage EC maturation. Auxin signaling genes (including PLT2, IAA4, GH3 and SAUR) mediate the early phase of EC induction, whereas core developmental regulators such as SERK1, GRF5, YUC6, LEA14 and multiple WRKY family transcription factors drive the subsequent differentiation of somatic embryos. All these genes were significantly upregulated following PSK-α treatment. This study established an efficient leaf-derived SE system for a hybrid sweetgum genotype and demonstrated the positive regulatory role of PSK-α. Our findings provide a promising basis for the large-scale propagation of elite hybrid sweetgum cultivars and lay a foundation for further investigation of the regulatory mechanisms of PSK-α.

Keywords

Leaf / Liquidambar styraciflua × L. formosana / Peptide signaling / Phytosulfokine-α / Somatic embryogenesis

References

[1] Ahmadi B, Shariatpanahi ME, Teixeira da Silva JA. Efficient induction of microspore embryogenesis using abscisic acid, jasmonic acid and salicylic acid in Brassica napus L. Plant Cell Tissue Organ Cult, 2014, 1163343351.

[2] Asif M, Eudes F, Randhawa H, Amundsen E, Spaner D. Phytosulfokine alpha enhances microspore embryogenesis in both Triticale and wheat. Plant Cell Tissue Organ Cult, 2014, 1161125130.

[3] Chen JY, Yang YH, Xu HX, Chen XY, Chen XJ. Stimulatory effect of phytosulfokine-alpha on the proliferation of tobacco “Bright Yellow 2” suspension cells. J Plant Physiol Mol Biol, 2007, 3313945.

[4] Chen BJ, Fiers M, Dekkers BJW, Maas L, van Esse GW, Angenent GC, Zhao Y, Boutilier K. ABA signalling promotes cell totipotency in the shoot apex of germinating embryos. J Exp Bot, 2021, 721864186436.

[5] Chen XY, Zhang LY, Liu CB, Wang R, Dai JF, Kong LS, Zhang JF, Zhao J. The transcription factor LpWRKY65 enhances embryogenic capacity through reactive oxygen species scavenging during somatic embryogenesis of larch. Plant Physiol, 2025, 1983kiaf286

[6] Du L, Kang XL, Guo HR, Zhu ZF, Wu R, Yuan MJ, Ding CY. Cotyledonary somatic embryo is one kind of intermediate material similar to callus in the process of in vitro tissue culture from Rosa hybrida ‘John F. Kennedy’. BMC Genomics, 2024, 251362

[7] Ferrari IF, Marques GA, Junior WLS, Biazotti BB, Pena Passos M, de Almeida JAS, Mondego JMC, Mayer JLS. Comparative ontogenesis of Coffea Arabica L. somatic embryos reveals the efficiency of regeneration modulated by the explant source and the embryogenesis pathway. In Vitro Cell Dev Biol Plant, 2021, 575796810.

[8] Ganguly A, Humnabadkar A, Gautam K, Willemsen V, Xu L, Dagdas Y, Prasad K. PLETHORA–autophagy axis activates organ regeneration through ROS modulation. Proc Natl Acad Sci U S A, 2026, 1236e2513954123

[9] Guan Y, Li SG, Fan XF, Su ZH. Application of somatic embryogenesis in woody plants. Front Plant Sci, 2016, 7: 938.

[10] Hanai H, Matsuno T, Yamamoto M, Matsubayashi Y, Kobayashi T, Kamada H, Sakagami Y. A secreted peptide growth factor, phytosulfokine, acting as a stimulatory factor of carrot somatic embryo formation. Plant Cell Physiol, 2000, 4112732.

[11] Hao L, Zhou LN, Xu X, Cao J, Xi T. The role of salicylic acid and carrot embryogenic callus extracts in somatic embryogenesis of naked oat (Avena nuda). Plant Cell Tissue Organ Cult, 2006, 851109113.

[12] Hao ZD, Wu H, Zheng RH, Li R, Zhu ZL, Chen Y, Lu Y, Cheng TL, Shi JS, Chen JH. The plant peptide hormone phytosulfokine promotes somatic embryogenesis by maintaining redox homeostasis in Cunninghamia lanceolata. Plant J, 2023, 1134716733.

[13] Hecht V, Vielle-Calzada JP, Hartog MV, Schmidt ED, Boutilier K, Grossniklaus U, de Vries SC. The Arabidopsis somatic embryogenesis receptor kinase 1 gene is expressed in developing ovules and embryos and enhances embryogenic competence in culture. Plant Physiol, 2001, 1273803816.

[14] Hong PI, Chen JT, Chang WC. Effects of salicylic and acetylsalicylic acid on direct somatic embryogenesis in Oncidium. J Plant Biochem Biotechnol, 2008, 172149153.

[15] Hutchinson MJ, Saxena PK. Acetylsalicylic acid enhances and synchronizes thidiazuron-induced somatic embryogenesis in Geranium (Pelargonium x hortorum Bailey) tissue cultures. Plant Cell Rep, 1996, 157512515.

[16] Igasaki T, Akashi N, Shinohara K. Somatic embryogenesis in Cryptomeria japonica D. don: gene for phytosulfokine (PSK) precursor. Somatic embryogenesis, 2006Springer-Verlag201213.

[17] Kępczyńska E, Ruduś I, Kępczyński J. Endogenous ethylene in indirect somatic embryogenesis of Medicago sativa L.. Plant Growth Regul, 2009, 5916373.

[18] Kiełkowska A, Adamus A. Peptide growth factor Phytosulfokine-α stimulates cell divisions and enhances regeneration from B. oleracea var. capitata L. protoplast culture. J Plant Growth Regul, 2019, 383931944.

[19] Kim D, Langmead B, Salzberg SL. HISAT: a fast spliced aligner with low memory requirements. Nat Methods, 2015, 124357360.

[20] Langfelder P, Horvath S. WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics, 2008, 9: 559.

[21] Lee HG, Jang SY, Jie EY, Choi SH, Park OS, Bae SH, Kim HS, Kim SW, Hwang GS, Seo PJ. Adenosine monophosphate enhances callus regeneration competence for de novo plant organogenesis. Mol Plant, 2023, 161218671870.

[22] Li MF, Wrobel-Marek J, Heidmann I, Horstman A, Chen BJ, Reis R, Angenent GC, Boutilier K. Auxin biosynthesis maintains embryo identity and growth during BABY BOOM-induced somatic embryogenesis. Plant Physiol, 2022, 188210951110.

[23] Li Y, Qi SZ, Chen SY, Li HX, Zhang T, Bao F, Zhan DJ, Pang ZW, Zhang JF, Zhao J. Genome-wide identification and expression analysis of late embryogenesis abundant (LEA) genes reveal their potential roles in somatic embryogenesis in hybrid sweetgum (Liquidambar styraciflua × Liquidambar formosana). For Res, 2023.

[24] Li Y, Di Q, Luo L, Yu LL. Phytosulfokine peptides, their receptors, and functions. Front Plant Sci, 2024, 14. 1326964

[25] Li HX, Fan YM, Kang JD, Qi SZ, Bao F, Li Y, Cheng L, Zhan DJ, Pang ZW, Zhao J, Zhang JF. Standardized protocol for somatic embryogenesis from vegetative organs in hybrid sweetgum (L. styraciflua × L. Formosana). Forests (Basel), 2025, 164670

[26] Li Y, Ma WH, Qi SZ, Ma HY, Li HX, Bao F, Fan YM, Zhan DJ, Pang ZW, Zhao J, Zhang JF. Genome-wide characterization of the WRKY gene family and the role of LsfWRKY29 in regulating somatic embryogenesis in hybrid sweetgum (Liquidambar styraciflua × L. Formosana). Int J Biol Macromol, 2025, 293. 139287

[27] Li Y, Ma WH, Wang PT, Ma HY, Li HX, Bao F, Fan YM, Qi SZ, Zhan DJ, Pang ZW, Zhao J, Zhang JF, Chen SY. BZR2-GRF5 acts as a hub module in regulating in vitro regeneration through brassinosteroid–auxin crosstalk in hybrid sweetgum. New Phytol, 2025, 247416941711.

[28] Matsubayashi Y, Sakagami Y. Phytosulfokine, sulfated peptides that induce the proliferation of single mesophyll cells of Asparagus officinalis L. Proc Natl Acad Sci U S A, 1996, 931576237627.

[29] Merkle SA, Neu KA, Battle PJ, Bailey RL. Somatic embryogenesis and plantlet regeneration from immature and mature tissues of sweetgum (Liquidambar styraciflua). Plant Sci, 1998, 1322169178.

[30] Motose H, Iwamoto K, Endo S, Demura T, Sakagami Y, Matsubayashi Y, Moore KL, Fukuda H. Involvement of phytosulfokine in the attenuation of stress response during the transdifferentiation of Zinnia mesophyll cells into tracheary elements. Plant Physiol, 2009, 1501437447.

[31] Ochatt S, Conreux C, Moussa Mcolo R, Despierre G, Magnin-Robert JB, Raffiot B. Phytosulfokine-alpha, an enhancer of in vitro regeneration competence in recalcitrant legumes. Plant Cell Tissue Organ Cult, 2018, 1352189201.

[32] Ogawa-Ohnishi M, Yamashita T, Kakita M, Nakayama T, Ohkubo Y, Hayashi Y, Yamashita Y, Nomura T, Noda S, Shinohara H, Matsubayashi Y. Peptide ligand-mediated trade-off between plant growth and stress response. Science, 2022, 3786616175180.

[33] Park JE, Park JY, Kim YS, Staswick PE, Jeon J, Yun J, Kim SY, Kim J, Lee YH, Park CM. GH3-mediated auxin homeostasis links growth regulation with stress adaptation response in Arabidopsis. J Biol Chem, 2007, 282131003610046.

[34] Pertea M, Pertea GM, Antonescu CM, Chang TC, Mendell JT, Salzberg SL. StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat Biotechnol, 2015, 333290295.

[35] Qi SZ, Zhao RR, Yan JC, Fan YM, Huang C, Li HX, Chen SY, Zhang T, Kong LS, Zhao J, Zhang JF. Global transcriptome and coexpression network analyses reveal new insights into somatic embryogenesis in hybrid sweetgum (Liquidambar styraciflua × Liquidambar formosana). Front Plant Sci, 2021, 12. 751866

[36] Roustan JP, Latche A, Fallot J. Stimulation of Daucus carota somatic embryogenesis by inhibitors of ethylene synthesis: cobalt and nickel. Plant Cell Rep, 1989, 83182185.

[37] Ruduś I, Weiler EW, Kępczyńska E. Do stress-related phytohormones, abscisic acid and jasmonic acid play a role in the regulation of Medicago sativa L. somatic embryogenesis?. Plant Growth Regul, 2009, 592159169.

[38] Ruduś I, pczy ska EK, pczy ski JK, Wasternack C, Miersch O (2005) Changes in jasmonates and 12-oxophytodienoic acid contents of Medicago sativa L. during somatic embryogenesis. Acta Physiol Plant 27(4): 497–504. https://doi.org/10.1007/s11738-005-0055-x

[39] Salaün C, Lepiniec L, Dubreucq B. Genetic and molecular control of somatic embryogenesis. Plants (Basel), 2021, 1071467

[40] Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, Amin N, Schwikowski B, Ideker T. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res, 2003, 131124982504.

[41] Spartz AK, Ren H, Park MY, Grandt KN, Lee SH, Murphy AS, Sussman MR, Overvoorde PJ, Gray WM. SAUR inhibition of PP2C-D phosphatases activates plasma membrane H⁺-ATPases to promote cell expansion in Arabidopsis. Plant Cell, 2014, 26521292142.

[42] Thi LT, Pleschka E. Somatic embryogenesis of some Daucus species influenced by ABA. J Appl Bot Food Qual, 2005, 79114

[43] Umehara M, Ogita S, Sasamoto H, Eun CH, Matsubayashi Y, Sakagami Y, Kamada H. Two stimulatory effects of the peptidyl growth factor phytosulfokine during somatic embryogenesis in Japanese larch (Larix leptolepis Gordon). Plant Sci, 2005, 1695901907.

[44] Vendrame W, Holliday C, Merkle S. Clonal propagation of hybrid sweetgum (Liquidambar styraciflua × L. Formosana) by somatic embryogenesis. Plant Cell Rep, 2001, 208691695.

[45] Wang C, Yu HX, Zhang ZM, Yu LL, Xu XS, Hong ZL, Luo L. Phytosulfokine is involved in positive regulation of Lotus japonicus nodulation. Mol Plant Microbe Interact, 2015, 288847855.

[46] Yamaguchi YL, Ishida T, Sawa S. CLE peptides and their signaling pathways in plant development. J Exp Bot, 2016, 671648134826.

[47] Yang WT, Zhai HW, Wu FM, Deng L, Chao Y, Meng XW, Chen Q, Liu CH, Bie XM, Sun CL, Yu Y, Zhang XF, Zhang XY, Chang ZQ, Xue M, Zhao YJ, Meng XB, Li BS, Zhang XS, Zhang DJ, Zhao XY, Gao CX, Li JY, Li CY. Peptide REF1 is a local wound signal promoting plant regeneration. Cell, 2024, 1871230243038.e14.

[48] Yuan JC, Liu XX, Zhao H, Wang Y, Wei X, Wang P, Zhan JJ, Liu LS, Li FG, Ge XY. GhRCD1 regulates cotton somatic embryogenesis by modulating the GhMYC3–GhMYB44–GhLBD18 transcriptional cascade. New Phytol, 2023, 2401207223.

[49] Zeng JJ, Deng Y, Iqbal S, Zhang JR, Wu KL, Ma GH, Li L, Dai GY, Deng RF, Fang L, Zeng SJ. Direct somatic embryogenesis and related gene expression networks in leaf explants of Hippeastrum ‘Bangkok Rose’. Hortic Plant J, 2024, 102556572.

[50] Zhang H, Hu ZJ, Lei C, Zheng CF, Wang J, Shao SJ, Li X, Xia XJ, Cai XZ, Zhou J, Zhou YH, Yu JQ, Foyer CH, Shi K. A plant phytosulfokine peptide initiates auxin-dependent immunity through cytosolic Ca²⁺ signaling in tomato. Plant Cell, 2018, 303652667.

[51] Zhang JJ, Hao ZD, Ruan XX, Weng YH, Chen XY, Zhu JJ, Lu L, Lu Y, Ma YX, Chen JH, Shi JS. Role of BABY BOOM transcription factor in promoting somatic embryogenesis and genetic transformation in a woody magnoliid Liriodendron. Plant Cell Environ, 2025, 48748594872.

[52] Zheng QL, Zheng YM, Perry SE. AGAMOUS-Like15 promotes somatic embryogenesis in Arabidopsis and soybean in part by the control of ethylene biosynthesis and response. Plant Physiol, 2013, 161421132127.

[53] Zheng LW, Ma JJ, Song CH, An N, Zhang D, Zhao CP, Qi SY, Han MY. Genome-wide identification and expression profiling analysis of brassinolide signal transduction genes regulating apple tree architecture. Acta Physiol Plant, 2017, 398177.

Funding

Natural Science Foundation of Henan Province((252300420713)

Fundamental Research Funds of State Key Laboratory of Tree Genetics and Breeding（Beijing Forestry University(TGBFRF202506)

National Key R&D Program of China(2023YFD2200602)

National Natural Science Foundation of China(32271836)

Fundamental Research Funds for the Central Universities(2019ZY39)

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Northeast Forestry University