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Phytosulfokine promotes cell division in protoplast culture and adventitious shoot formation in protoplast-derived calluses of Nicotiana benthamiana

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Abstract

Phytosulfokines (PSK) belong to a new class of peptide growth factors involved in intercellular signaling in plants. In this study, we examined the effect of PSK on cell division and subsequent adventitious shoot formation in protoplast-derived callus culture of Nicotiana benthamiana. Protoplast cultures and protoplast-derived calluses of N. benthamiana were treated with different concentrations of PSK (0, 0.01, 0.03, 0.1, 0.3, and 1 µM). Cell division efficiency was the highest at 32% in the 0.1 µM PSK treatment, which was about two times higher than that of control treatment. PSK concentrations < 0.1 µM were more effective in inducing cell division in the protoplasts than those > 0.3 µM. In contrast, higher concentrations of PSK were more effective in inducing adventitious shoot formation in protoplast-derived calluses. The frequency of adventitious shoot formation was the highest at 8.5% when the calluses were treated with 1 µM PSK. qRT-PCR analysis showed an increase in the expression of genes involved in cell division and differentiation: G1/S specific cyclin (CYCD3-1), cytokinin dependent kinase (CDK), and WUSCHEL (WUS) at 0.1 µM PSK treatment. These results indicate that PSK plays a significant role in promoting cell division and adventitious shoot formation in N. benthamiana. The results obtained in this study could be applied to promote cell division and plant regeneration in protoplast cultures of diverse recalcitrant crop plant species.

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References

  • Adamus A (2012) An alginate-layer technique for culture of Brassica oleracea L. protoplasts. In Vitro Cell Dev Biol Plant 48(2):265–273

    Article  Google Scholar 

  • Aghdam MS, Sayyari M, Luo Z (2020) Exogenous application of phytosulfokine α (PSKα) delays yellowing and preserves nutritional quality of broccoli florets during cold storage. Food Chem 333:127481

    Article  CAS  Google Scholar 

  • Aghdam MS, Ebrahimi A, Sheikh-Assadi M (2021) Phytosulfokine α (PSKα) delays senescence and reinforces SUMO1/SUMO E3 ligase SIZ1 signaling pathway in cut rose flowers (Rosa hybrida cv. Angelina). Sci Rep 11(1):23227

    Article  CAS  Google Scholar 

  • Amano Y, Tsubouchi H, Shinohara H, Ogawa M, Matsubayashi Y (2007) Tyrosine-sulfated glycopeptide involved in cellular proliferation and expansion in Arabidopsis. Proc Natl Acad Sci 104:18333–18338

    Article  CAS  Google Scholar 

  • Asif M, Eudes F, Randhawa H, Amundsen E, Spaner D (2014) Phytosulfokine alpha enhances microspore embryogenesis in both triticale and wheat. Plant Cell Tissue Organ Cult 116(1):125–130

    Article  CAS  Google Scholar 

  • Benson EE (2000) In vitro plant recalcitrance: an introduction. In Vitro Cell Dev Biol Plant 36:141–148

    Article  Google Scholar 

  • Davey MR, Anthony P, Power JB, Lowe KC (2005) Plant protoplasts: status and biotechnological perspectives. Biotechnol Adv 23(2):131–171

    Article  CAS  Google Scholar 

  • Eun C-H, Ko S-M, Matsubayashi Y, Sakagami Y, Kamada H (2003) Phytosulfokine-α requires auxin to stimulate carrot non-embryogenic cell proliferation. Plant Physiol Biochem 41(5):447–452

    Article  CAS  Google Scholar 

  • Frearson EM, Power JB, Cocking EC (1973) The isolation, culture and regeneration of Petunia leaf protoplasts. Dev Biol 33:130–137

    Article  CAS  Google Scholar 

  • Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirement of suspension culture of soybean root cells. Exp Cell Res 50:151–158

    Article  CAS  Google Scholar 

  • Grzebelus E, Szklarczyk M, Gren J, Sniegowska K, Jopek M, Kacinska I, Mrozek K (2012) Phytosulfokine stimulates cell divisions in sugar beet (Beta vulgaris L.) mesophyll protoplast cultures. Plant Growth Regul 67(1):93–100

    Article  CAS  Google Scholar 

  • Grzebelus E, Maćkowska K, Macko-Podgórni A, Kiełkowska A, Szklarczyk M, Baranski R, Grzebelus D (2019) Application of protoplast technology to Apiaceae species. Acta Hortic 1264:931–944

    Google Scholar 

  • Hall RD, Pedersen C, Krens FA (1993) Improvement of protoplast culture protocols for Beta vulgaris L (sugar beet). Plant Cell Rep 12(6):339–342

    Article  CAS  Google Scholar 

  • Hanai H, Matsuno T, Yamamoto M, Matsubayashi Y, Kobayashi T, Kamada H, Sakagami Y (2000) A secreted peptide growth factor, phytosulfokine, acting as a stimulatory factor of carrot somatic embryo formation. Plant Cell Physiol 41:27–32

    Article  CAS  Google Scholar 

  • Huttner WB (1982) Sulphation of tyrosine residues: a widespread modification of proteins. Nature 299:273–276

    Article  CAS  Google Scholar 

  • Igarashi D, Tsuda K, Katagiri F (2012) The peptide growth factor, phytosulfokine, attenuates pattern-triggered immunity. Plant J 71:194–204

    Article  CAS  Google Scholar 

  • Igasaki T, Akashi N, Ujino-Ihara T, Matsubayashi Y, Sakagami Y, Shinohara K (2003) Phytosulfokine stimulates somatic embryogenesis in Cryptomeria japonica. Plant Cell Physiol 44:1412–1416

    Article  CAS  Google Scholar 

  • Jeong YY, Lee HY, Kim SW, Noh YS, Seo PJ (2021) Optimization of protoplast regeneration in the model plant Arabidopsis thaliana. Plant Methods 17(1):1–16

    Article  Google Scholar 

  • Kiełkowska A, Adamus A (2017) Early studies on the effect of peptide growth factor phytosulfokine-α on Brassica oleracea var. capitata L protoplasts. Acta Soc Bot Pol Pol Tow Bot 86(3):3558

    Google Scholar 

  • Kiełkowska A, Adamus A (2019) Peptide growth factor phytosulfokine-α stimulates cell divisions and enhances regeneration from B. oleracea var. capitata L. protoplast culture. J Plant Growth Regul 38(3):931–944

    Article  Google Scholar 

  • Kim BJ, Gibson DM, Shuler ML (2006) Effect of the plant peptide regulator, phytosulfokine-α, on the growth and taxol production from Taxus sp. suspension cultures. Biotechnol Bioeng 95(1):8–14

    Article  CAS  Google Scholar 

  • Kou X, Liu Q, Sun Y, Wang P, Zhang S, Wu J (2020) The peptide PbrPSK2 from phytosulfokine family induces reactive oxygen species (ROS) production to regulate pear pollen tube growth. Front Plant Sci 11:601993

    Article  Google Scholar 

  • Kutschmar A, Rzewuski G, Stührwohldt N, Beemster GTS, Inze D, Sauter M (2009) PSK-α promotes root growth in Arabidopsis. New Phytol 181(4):820–831

    Article  CAS  Google Scholar 

  • Mackowska K, Jarosz A, Grzebelus E (2014) Plant regeneration from leaf-derived protoplasts within the Daucus genus: effect of different conditions in alginate embedding and phytosulfokine application. Plant Cell Tissue Organ Cult 117(2):241–252

    Article  CAS  Google Scholar 

  • Matsubayashi Y (2011) Post-translational modifications in secreted peptide hormones in plants. Plant Cell Physiol 52(1):5–13

    Article  CAS  Google Scholar 

  • Matsubayashi Y, Sakagami Y (1996) Phytosulfokine, sulfated peptides that induce the proliferation of single mesophyll cells of Asparagus officinalis L. Proc Natl Acad Sci U S A 93:7623–7627

    Article  CAS  Google Scholar 

  • Matsubayashi Y, Sakagami Y (1998) Effects of the medium ammonium-nitrate ratio on competence for Asparagus cell division induced by phytosulfokine-α. Plant Cell Rep 11(5):368–372

  • Matsubayashi Y, Takagi L, Sakagami Y (1997) Pytosulfokine-α, a sulfated pentapeptide, stimulates the proliferation of rice cells by means of specific high- and low-affinity binding sites. Proc Natl Acad Sci USA 94:13357–13362

    Article  CAS  Google Scholar 

  • Matsuzaki Y, Ogawa-Ohnishi M, Mori A, Matsubayashi Y (2010) Secreted peptide signals required for maintenance of root stem cell niche in Arabidopsis. Science 329:1065–1067

    Article  CAS  Google Scholar 

  • Moore KL (2009) Protein tyrosine sulfation: a critical posttranslation modification in plants and animals. Proc Natl Acad Sci U S A 106(35):14741–14742

    Article  CAS  Google Scholar 

  • Motose H, Iwamoto K, Endo S, Demura T, Sakagami Y, Matsubayashi Y, Moore KL, Fukuda H (2009) Involvement of phytosulfokine in the attenuation of stress response during the transdifferentiation of Zinnia mesophyll cells into tracheary elements. Plant Physiol 150:437–447

    Article  CAS  Google Scholar 

  • Murashige T, Skoog FA (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Mustafa NR, De Winter W, Van Iren F, Verpoorte R (2011) Initiation, growth and cryopreservation of plant cell suspension cultures. Nat Protoc 6(6):715–742

    Article  CAS  Google Scholar 

  • Nakayama T, Shinohara H, Tanaka M, Baba K, Ogawa-Ohnishi M, Matsubayashi Y (2017) A peptide hormone required for Casparian strip diffusion barrier formation in Arabidopsis roots. Science 355(6322):284–286

    Article  CAS  Google Scholar 

  • Ochatt S, Conreux C, Moussa Mcolo R, Despierre G, Magnin-Robert J-B, Raffiot B (2018) Phytosulfokine-alpha, an enhancer of in vitro regeneration competence in recalcitrant legumes. Plant Cell, Tissue Organ Cult 135(2):189–201

    Article  CAS  Google Scholar 

  • Patel N, Mohd-Radzman NA, Corcilius L, Crossett B, Connolly A, Cordwell SJ, Ivanovici A, Taylor K, Williams J, Binos S, Mariani M, Payne RJ, Djordjevic MA (2018) Diverse peptide hormones affecting root growth identified in the Medicago truncatula secreted peptidome. Mol Cell Proteomics 17(1):160–174

    Article  CAS  Google Scholar 

  • Reichardt S, Piepho H-P, Stintzi A, Schaller A (2020) Peptide signaling for drought-induced tomato flower drop. Science 377(6485):1482–1485

    Article  Google Scholar 

  • Ryan CA, Pearce G (2001) Polypeptide hormones. Plant Physiol 125(1):65–68

    Article  CAS  Google Scholar 

  • Sasaki K, Ishise T, Shimomura K, Kobayashi T, Matsubayashi Y, Sakagami Y, Umetsu H, Kamada H (2002) Effects of phytosulfokine-α on growth and tropane alkaloid production in transformed roots of Atropa belladonna. Plant Growth Regul 36(1):87–90

    Article  CAS  Google Scholar 

  • Shinohara H (2021) Root meristem growth factor RGF, a sulfated peptide hormone in plants. Peptides 142:170556

    Article  CAS  Google Scholar 

  • Sizolwenkosi, Mlotshwa Zhiyong, Yang YunJu, Kim Xuemei, Chen (2006) Floral patterning defects induced by Arabidopsis. APETALA2 and microRNA172 expression in Nicotiana benthamiana. Plant Molecular Biology 61(4-5) 781–793. https://doi.org/10.1007/s11103-006-0049-0

    Article  CAS  Google Scholar 

  • Stuhrwohldt N, Dahlke RI, Steffens B, Johnson A, Sauter M (2011) Phytosulfokine-α controls hypocotyl length and cell expansion in Arabidopsis thaliana through phytosulfokine receptor 1. PLoS ONE 6(6):e21054

    Article  Google Scholar 

  • Stuhrwohldt N, Dahlke R, Kutschmar A, Peng X, Sun M-X, Sauter M (2015) Phytosulfokine peptide signaling controls pollen tube growth and funicular pollen tube guidance in Arabidopsis thaliana. Physiologia Plant 153(4):643–653

    Article  Google Scholar 

  • Umehara M, Ogita S, Sasamoto H, Eun C-H, Matsubayashi Y, Sakagami Y, Kamada H (2005) Two stimulatory effects of the peptidyl growth factor phytosulfokine during somatic embryogenesis in Japanese larch (Larix leptolepis Gordon). Plant Sci 169(5):901–907

    Article  CAS  Google Scholar 

  • Wang C, Yu H, Zhang Z, Yu L, Xu X, Hong Z, Luo L (2015) Phytosulfokine is involved in positive regulation of Lotus japonicus nodulation. Mol Plant Microbe Interact 28(8):847–855

    Article  CAS  Google Scholar 

  • Wu H, Zheng R, Hao Z, Meng Y, Weng Y, Zhou X, Zhu L, Hu X, Wang G, Shi J, Chen J (2019) Cunninghamia lanceolata PSK peptide hormone genes promote primary root growth and adventitious root formation. Plants (basel) 8(11):520

    Article  CAS  Google Scholar 

  • Xu M, Du Q, Tian C, Wang Y, Jiao Y (2021) Stochastic gene expression drives mesophyll protoplast regeneration. Sci Adv 7(33):8466

    Article  Google Scholar 

  • Yamakawa S, Sakuta C, Matsubayashi Y, Sakagami Y, Kamada H, Satoh S (1998) The promotive effects of a peptidyl plant growth factor, phytosulfokine-α, on the formation of adventitious roots and expression of a gene for a root-specific cystatin in cucumber hypocotyls. J Plant Res 111(3):453–458

    Article  CAS  Google Scholar 

  • Yang G, Shen S, Kobayashi T, Matsubayashi Y, Sakagami Y, Kamada H (1999) Stimulatory effects of a novel peptidyl plant growth factor, phytosulfokine-α, on the adventitious bud formation from callus of Antirrhinum majus. Plant Biotechnol 16(3):231–234

    Article  CAS  Google Scholar 

  • Yang H, Matsubayashi Y, Hanai H, Nakamura K, Sakagami Y (2000) Molecular cloning and characterization of OsPSK, a gene encoding a precursor for phytosulfokine-α, required for rice cell proliferation. Plant Mol Biol 635:635–647

    Article  Google Scholar 

  • Yang H, Matsubayashi Y, Nakamura K, Sakagami Y (2001) Diversity of Arabidopsis genes encoding precursors for phytosulfokine, a peptide growth factor. Plant Physiol 127:842–851

    Article  CAS  Google Scholar 

  • Yang F, Xia X-R, Ke X, Ye J, Zhu L-H (2020) Somatic embryogenesis in slash pine (Pinus elliottii Engelm): improving initiation of embryogenic tissues and maturation of somatic embryos. Plant Cell Tissue Organ Cult 143(1):159–171

    Article  CAS  Google Scholar 

  • Zhang H, Hu Z, Lei C, Zheng C, Wang J, Shao S, Li X, Xia X, Cai X, Zhou J, Zhou Y, Yu J, Foyer CH, Shi K (2018) A plant phytosulfokine peptide initiates auxin-dependent immunity through cytosolic Ca2+ signaling in tomato. Plant Cell 30:652–667

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program (KGM 5282223) and New Breeding Technologies Development Program (Project No. PJ01653001), Rural Development Administration, Republic of Korea.

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Authors and Affiliations

  1. Biological Resource Center, Korea Research Institute of Bioscience and Biotechnology, 181 Ipsingil, Jeongeup-Si, 56212, Jeollabuk-Do, Korea

    Su Ji Joo, Seung Hee Choi, Eun Yee Jie & Suk Weon Kim

  2. Department of Applied Plant Science, Chonnam National University, Gwangju, 61186, Korea

    Su Ji Joo & Ok Ran Lee

  3. Interdisciplinary Program in IT-Bio Convergence System, Chonnam National University, 61186, Gwangju, Republic of Korea

    Ok Ran Lee

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  1. Su Ji Joo
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Correspondence to Suk Weon Kim.

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11816_2022_805_MOESM1_ESM.jpg

Supplementary Figure1. Effect of PSK and KCLO3 on cell division from mesophyll protoplasts in protoplast culture medium. In comparison to PSK and KCLO3 combinatory treatment, the frequency of cell division from KCLO3 alone treatment was decreased after 1 week of culture. After 2 weeks of incubation, sustained cell division was observed in KCLO3 alone treatment. Scale bars represent 100 µm (JPG 799 KB)

11816_2022_805_MOESM2_ESM.jpg

Supplementary Figure 2 Effect of PSK on cell division from mesophyll protoplasts in protoplast culture medium in the absence of plant growth regulators. After 1 week of culture, mesophyll protoplasts could not divide in the protoplast culture medium without plant growth regulators. Cell elongation and budding was observed in the 0.1 µM PSK treatment, but cell division did not occur even when the incubation period was increased. Scale bars represent 100 µm (JPG 835 KB)

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Joo, S.J., Choi, S.H., Jie, E.Y. et al. Phytosulfokine promotes cell division in protoplast culture and adventitious shoot formation in protoplast-derived calluses of Nicotiana benthamiana. Plant Biotechnol Rep 16, 633–643 (2022). https://doi.org/10.1007/s11816-022-00805-6

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