Skip to main content
SpringerLink
Log in

Redox and hormone profiling of a Nicotiana tabacum dedifferentiated protoplast culture suggests a role for a cytokinin and gibberellin in plant totipotency

  • Original Article
  • Published:
Plant Cell, Tissue and Organ Culture (PCTOC) Aims and scope Submit manuscript

Abstract

The ability of plant tissues to retain totipotency despite being fully differentiated has been documented for decades. The transition from mature plant tissue to rejuvenated tissue first requires dedifferentiation of mature tissue, followed by rejuvenation (re-entry into the cell cycle) and somatic embryogenesis. We used a Nicotiana tabacum protoplast-based culture system to elucidate the role played by redox and phytohormone networks during the process of dedifferentiation and rejuvenation. Classical markers of redox homeostasis were measured during the rejuvenation process and lipid peroxidation is proposed as the best marker for indicating recovery of cells from oxidative stress sustained during the process of protoplast preparation and culture, prior to rejuvenation. A transient increase at 24 h after culture (HAC) in levels of a cytokinin riboside, iPA, suggests a putative novel function in initiating a stem-cell niche in an auxin dependent manner. A sharp rise at 72 HAC of gibberellin GA4, furthermore suggests a function for this hormone during the process of rejuvenation. These two key findings are consistent with previously described plant models for lateral root developmental. Therein, iPA could be involved in ‘stem-cell-niche’ initiation. Subsequently, GA4 could be involved in rapidly suppressing this initiation step following the earliest cell divisions, thereby enabling the establishment of this ‘niche’ into a callus-like tissue.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

Abbreviations

ABA:

Abscisic acid

ACC:

1-Amino-cyclopropane-1-carboxylic acid

BAP:

6-Benzylaminopurine

BR:

Brassinosteroid

2,4D:

2,4-Dichlorophenoxyacetic acid

DHZ:

Dihydrozeatin

DHZR:

Dihydrozeatin riboside

GA:

Gibberellin

HAC:

Hours after culture

IAA:

Indole-3-acetic acid

2iP:

Isopentenyladenine

iPA:

Isopentenyladenosine

JA:

Jasmonic acid

MDA:

Malondialdehyde

NAA:

Naphthaleneacetic acid

SA:

Salicylic acid

UPLC:

Ultrahigh performance liquid chromatography

Z:

Zeatin

ZR:

Zeatin riboside

References

  • Aloni R (2013) Role of hormones in controlling vascular differentiation and the mechanism of lateral root initiation. Planta 238:819–830

    Article  CAS  PubMed  Google Scholar 

  • Alvarez Palomo ABA, McLenachan S, Osete JR et al (2014) Plant hormones increase efficiency of reprogramming mouse somatic cells to induced pluripotent stem cells and reduce tumorigenicity. Stem Cells Dev 23:586–593

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Amaral JS, Casal S, Torres D et al (2005) Simultaneous determination of tocopherols and tocotrienols in hazelnuts by a normal phase liquid chromatographic method. Anal Sci 21:1545–1548

    Article  CAS  PubMed  Google Scholar 

  • Atta R, Laurens L, Boucheron-Dubuisson E et al (2009) Pluripotency of Arabidopsis xylem pericycle underlies shoot regeneration from root and hypocotyl explants grown in vitro. Plant J 57:626–644

    Article  CAS  PubMed  Google Scholar 

  • Björklund S, Antti H, Uddestrand I et al (2007) Cross-talk between gibberellin and auxin in development of Populus wood: gibberellin stimulates polar auxin transport and has a common transcriptome with auxin. Plant J 52:499–511

    Article  PubMed  Google Scholar 

  • Bourgin JP, Chupeau Y, Missonier C (1979) Plant regeneration from mesophyll protoplasts of several Nicotiana species. Physiol Plant 45:288–292

    Article  CAS  Google Scholar 

  • Brady SM, Sarkar SF, Bonetta D et al (2003) The ABSCISIC ACID INSENSITIVE 3 (ABI3) gene is modulated by farnesylation and is involved in auxin signaling and lateral root development in Arabidopsis. Plant J 34:67–75

    Article  CAS  PubMed  Google Scholar 

  • Caboche M (1980) Nutritional-requirements of protoplast-derived, haploid tobacco cells grown at low cell densities in liquid-medium. Planta 149:7–18

    Article  CAS  PubMed  Google Scholar 

  • Chen GH, Liu CP, Chen SCG et al (2012) Role of ARABIDOPSIS A-FIFTEEN in regulating leaf senescence involves response to reactive oxygen species and is dependent on ETHYLENE INSENSITIVE2. J Exp Bot 63:275–292

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Chitteti BR, Tan F, Mujahid H et al (2008) Comparative analysis of proteome differential regulation during cell dedifferentiation in Arabidopsis. Proteomics 8:4303–4316

    Article  CAS  PubMed  Google Scholar 

  • Chupeau M-C, Granier F, Pichon O et al (2013) Characterization of the early events leading to totipotency in an Arabidopsis protoplast liquid culture by temporal transcript profiling. Plant Cell 25:2444–2463. doi:10.1105/tpc.113.109538

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Clark DG, Gubrium EK, Barrett JE et al (1999) Root formation in ethylene-insensitive plants. Plant Physiol 121:53–59

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Cocking EC (1960) Method for the isolation of plant protoplasts and vacuoles. Nature 187:962–963

    Article  Google Scholar 

  • Cowling RJ, Harberd NP (1999) Gibberellins control Arabidopsis hypocotyl growth via regulation of cellular elongation. J Exp Bot 50:1351–1357

    Article  CAS  Google Scholar 

  • Dubrovsky JG, Sauer M, Napsucialy-Mendivil S et al (2008) Auxin acts as a local morphogenetic trigger to specify lateral root founder cells. Proc Natl Acad Sci USA 105:8790–8794

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Eeckhaut T, Lakshmanan P, Deryckere D et al (2013) Progress in plant protoplast research. Planta 6:991–1003

    Article  Google Scholar 

  • Ernst D, Oesterhelt D (1984) Effect of exogenous cytokinins on growth and somatic embryogenesis in anise cells (Pimpinella anisum L.). Planta 161:246–248

    Article  CAS  PubMed  Google Scholar 

  • Fosket DE, Miksche JP (1966) Role of wound in callus initiation from carrot taproot phloem explants cultivated in vitro. Am J Bot 53:611–613

    Article  Google Scholar 

  • Foyer CH, Noctor G (2005) Redox homeostasis and antioxidant signaling: a metabolic interface between stress perception and physiological responses. Plant Cell 17:1866–1875

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Fukaki H, Tasaka M (2009) Hormone interactions during lateral root formation. Plant Mol Biol 69:437–449

    Article  CAS  PubMed  Google Scholar 

  • Galbraith DW (1994) Flow-cytometry and sorting of plant-protoplasts and cells. Methods Cell Biol 42:539–561

    Article  PubMed  Google Scholar 

  • Gechev T, Mehterov N, Denev I et al (2013) A simple and powerful approach for isolation of Arabidopsis mutants with increased tolerance to H2O2-induced cell death. Methods Enzymol 527:203–220

    Article  CAS  PubMed  Google Scholar 

  • Gliwicka M, Nowak K, Balazadeh S et al (2013) Extensive modulation of the transcription factor transcriptome during somatic embryogenesis in Arabidopsis thaliana. PLoS ONE 8:e69261. doi:10.1371/journal.pone.0069261

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Gou JQ, Strauss SH, Tsai CJ et al (2010) Gibberellins regulate lateral root formation in Populus through interactions with auxin and other hormones. Plant Cell 22:623–639

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Grafi G (2004) How cells dedifferentiate: a lesson from plants. Dev Biol 268:1–6

    Article  CAS  PubMed  Google Scholar 

  • Grafi G, Chalifa-Caspi V, Nagar T et al (2011) Plant response to stress meets dedifferentiation. Planta 233:433–438

    Article  CAS  PubMed  Google Scholar 

  • Griffith OW (1980) Determination of glutathione and glutathione disulfide using glutathione-reductase and 2-vinylpyridine. Anal Biochem 106:207–212

    Article  CAS  PubMed  Google Scholar 

  • He C, Chen X, Huang H et al (2012) Reprogramming of H3K27me3 is critical for acquisition of pluripotency from cultured Arabidopsis tissues. PLoS Genet 8:e1002911. doi:10.1371/journal.pgen.1002911

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Hodges DM, Delong JM, Forney CF et al (1999) Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. Planta 207:604–611

    Article  CAS  Google Scholar 

  • Ivanchenko MG, Muday GK, Dubrovsky JG (2008) Ethylene-auxin interactions regulate lateral root initiation and emergence in Arabidopsis thaliana. Plant J 55:335–347

    Article  CAS  PubMed  Google Scholar 

  • Iwase A, Mitsuda N, Koyama T et al (2011) The AP2/ERF transcription factor WIND1 controls cell dedifferentiation in Arabidopsis. Curr Biol 21:508–514

    Article  CAS  PubMed  Google Scholar 

  • Jiménez VM, Bangerth F (2001) Endogenous hormone levels in explants and in embryogenic and non-embryogenic cultures of carrot. Physiol Plant 111:389–395

    Article  PubMed  Google Scholar 

  • Jiménez VM, Guevara E, Herrera J et al (2005) Evolution of endogenous hormone concentration in embryogenic cultures of carrot during early expression of somatic embryogenesis. Plant Cell Rep 23:567–572

    Article  PubMed  Google Scholar 

  • Juvany M, Müller M, Munné-Bosch S (2013) Photo-oxidative stress in emerging and senescing leaves: a mirror image? J Exp Bot 64:3087–3098

    Article  CAS  PubMed  Google Scholar 

  • Kao KN, Michayluk MR (1975) Nutritional-requirements for growth of Vicia hajastana cells and protoplasts at a very low population-density in liquid-media. Planta 126:105–110

    Article  CAS  PubMed  Google Scholar 

  • Kim JI, Murphy AS, Baek D et al (2011) YUCCA6 over-expression demonstrates auxin function in delaying leaf senescence in Arabidopsis thaliana. J Exp Bot 62:3981–3992

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Kleczkowski K, Schell J (1995) Phytohormone conjugates - nature and function. Crit Rev Plant Sci 14:283–298

    Article  CAS  Google Scholar 

  • Kurakawa T, Ueda N, Maekawa M et al (2007) Direct control of shoot meristem activity by a cytokinin-activating enzyme. Nature 445:652–655

    Article  CAS  PubMed  Google Scholar 

  • Laplaze L, Benkova E, Casimiro I et al (2007) Cytokinins act directly on lateral root founder cells to inhibit root initiation. Plant Cell 19:3889–3900

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Lichtenthaler HK (1987) Chlorophylls and carotenoids - pigments of photosynthetic biomembranes. Methods Enzymol 148:350–382

    Article  CAS  Google Scholar 

  • Meyer Y, Cooke R (1979) Time course of hormonal-control of the 1st mitosis in tobacco mesophyll protoplasts cultivated in vitro. Planta 147:181–185

    Article  CAS  PubMed  Google Scholar 

  • Michalczuk L, Cooke TJ, Cohen JD (1992) Auxin levels at different stages of carrot somatic embryogenesis. Phytochemistry 31:1097–1103

    Article  CAS  Google Scholar 

  • Mittler R, Vanderauwera S, Suzuki N et al (2011) ROS signaling: the new wave? Trends Plant Sci 16:300–309

    Article  CAS  PubMed  Google Scholar 

  • Mok MC, Mok DWS, Dixon SC et al (1982) Cytokinin structure-activity-relationships and the metabolism of N6-(Δ2-isopentenyl)adenosine-8-14C in Phaseolus callus tissues. Plant Physiol 70:173–178

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Muday GK, Rahman A, Binder BM (2012) Auxin and ethylene: collaborators or competitors? Trends Plant Sci 17:181–195

    Article  CAS  PubMed  Google Scholar 

  • Müller M, Munné-Bosch S (2011) Rapid and sensitive hormonal profiling of complex plant samples by liquid chromatography coupled to electrospray ionization tandem mass spectrometry. Plant Methods 7:37. doi:10.1186/1746-4811-7-37

    Article  PubMed Central  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Nagata T, Takebe I (1970) Cell wall regeneration and cell division in isolated tobacco mesophyll protoplasts. Planta 92:301–308

    Article  CAS  PubMed  Google Scholar 

  • Nagata T, Takebe I (1971) Plating of isolated tobacco mesophyll protoplasts on agar medium. Planta 99:12–20

    Article  CAS  PubMed  Google Scholar 

  • Negi S, Ivanchenko MG, Muday GK (2008) Ethylene regulates lateral root formation and auxin transport in Arabidopsis thaliana. Plant J 55:175–187

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Niu SH, Li ZX, Yuan HW et al (2013) Proper gibberellin localization in vascular tissue is required to regulate adventitious root development in tobacco. J Exp Bot 64:3411–3424

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Papadakis AK, Roubelakis-Angelakis KA (2002) Oxidative stress could be responsible for the recalcitrance of plant protoplasts. Plant Physiol Biochem 40:549–559

    Article  CAS  Google Scholar 

  • Papadakis AK, Siminis CI, Roubelakis-Angelakis KA (2001) Reduced activity of antioxidant machinery is correlated with suppression of totipotency in plant protoplasts. Plant Physiol 126:434–444

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Pasternak TP, Prinsen E, Ayaydin F et al (2002) The role of auxin, pH, and stress in the activation of embryogenic cell division in leaf protoplast-derived cells of alfalfa. Plant Physiol 129:1807–1819

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Pasternak T, Potters G, Caubergs R et al (2005) Complementary interactions between oxidative stress and auxins control plant growth responses at plant, organ, and cellular level. J Exp Bot 56:1991–2001

    Article  CAS  PubMed  Google Scholar 

  • Queval G, Noctor G (2007) A plate reader method for the measurement of NAD, NADP, glutathione, and ascorbate in tissue extracts: application to redox profiling during Arabidopsis rosette development. Anal Biochem 363:58–69

    Article  CAS  PubMed  Google Scholar 

  • Sattler SE, Gilliland LU, Magallanes-Lundback M et al (2004) Vitamin E is essential for seed longevity and for preventing lipid peroxidation during germination. Plant Cell 16:1419–1432. doi:10.1105/tpc.021360

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Sembdner G, Atzorn R, Schneider G (1994) Plant hormone conjugation. Plant Mol Biol 26:1459–1481

    Article  CAS  PubMed  Google Scholar 

  • Shivakumar AG, Padayatty JD (1979) Correlation between the levels of N6-(Δ2-isopentenyl)-adenosine and synthesis of DNA in germinating rice seeds. Indian J Exp Biol 17:187–190

    CAS  Google Scholar 

  • Skoog F (1944) Growth and organ formation in tobacco tissue cultures. Am J Bot 31:19–24

    Article  Google Scholar 

  • Skoog F (1955) Initiation of cell division and organ formation in plant tissues. J Cell Comp Physiol 46:365

    Google Scholar 

  • Su Y-H, Liu Y-B, Zhang X-S (2011) Auxin–cytokinin interaction regulates meristem development. Mol Plant 4:616–625

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Sugimoto K, Jiao YL, Meyerowitz EM (2010) Arabidopsis regeneration from multiple tissues occurs via a root development pathway. Dev Cell 18:463–471

    Article  CAS  PubMed  Google Scholar 

  • Sugimoto K, Gordon SP, Meyerowitz EM (2011) Regeneration in plants and animals: dedifferentiation, transdifferentiation, or just differentiation? Trends Cell Biol 21:212–218

    Article  CAS  PubMed  Google Scholar 

  • Szymanska R, Kruk J (2008) Gamma-tocopherol dominates in young leaves of runner bean (Phaseolus coccineus) under a variety of growing conditions: the possible functions of gamma-tocopherol. Phytochemistry 69:2142–2148

    Article  CAS  PubMed  Google Scholar 

  • Takebe I, Otsuki Y, Aoki S (1968) Isolation of tobacco mesophyll cells in intact and active state. Plant Cell Physiol 9:115–124

    Google Scholar 

  • Tessadori F, Chupeau MC, Chupeau Y et al (2007) Large-scale dissociation and sequential reassembly of pericentric heterochromatin in dedifferentiated Arabidopsis cells. J Cell Sci 120:1200–1208

    Article  CAS  PubMed  Google Scholar 

  • Ubeda-Tomás S, Federici F, Casimiro I et al (2009) Gibberellin signaling in the endodermis controls Arabidopsis root meristem size. Curr Biol 19:1194–1199

    Article  PubMed  Google Scholar 

  • Wang SP, Xu ZH, Wei ZM (1990) Genetic transformation of leaf explants of Populus tomentosa. Acta Bot Sin 32:172–177

    Google Scholar 

  • Wang XD, Nolan KE, Irwanto RR et al (2011) Ontogeny of embryogenic callus in Medicago truncatula: the fate of the pluripotent and totipotent stem cells. Ann Bot 107:599–609

    Article  PubMed Central  PubMed  Google Scholar 

  • Widholm JM (1972) Use of fluorescein diacetate and phenosafranine for determining viability of cultured plant-cells. Stain Technol 47:189–194

    CAS  PubMed  Google Scholar 

  • Xiao LH, Zhang LC, Yang G et al (2012) Transcriptome of protoplasts reprogrammed into stem cells in Physcomitrella patens. PLoS ONE 7:e0035961. doi:10.1371/journal.pone.0035961

    Google Scholar 

  • Xu K, Liu J, Fan MZ et al (2012) A genome-wide transcriptome profiling reveals the early molecular events during callus initiation in Arabidopsis multiple organs. Genomics 100:116–124

    Article  CAS  PubMed  Google Scholar 

  • Yang XY, Zhang XL (2010) Regulation of somatic embryogenesis in higher plants. Crit Rev Plant Sci 29:36–57

    Article  CAS  Google Scholar 

  • Yang XY, Zhang XL, Yuan DJ et al (2012) Transcript profiling reveals complex auxin signalling pathway and transcription regulation involved in dedifferentiation and redifferentiation during somatic embryogenesis in cotton. BMC Plant Biol 12:110. doi:10.1186/1471-2229-12-110

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Zelcer A, Galun E (1976) Culture of newly isolated tobacco protoplasts - precursor incorporation into protein, RNA and DNA. Plant Sci Lett 7:331–336

    Article  CAS  Google Scholar 

  • Zhao J, Morozova N, Williams L et al (2001) Two phases of chromatin decondensation during dedifferentiation of plant cells—distinction between competence for cell fate switch and a commitment for S phase. J Biol Chem 276:22772–22778

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We are very grateful to Dr. Maren Müller for her assistance with phytohormone analysis. Support for the research in the S.M.-B. laboratory was provided by the Spanish Ministry of Science and Innovation (Grant Nos. BFU2012-32057 and PRI-AIBNZ-2011-0833). Research in the P.P.D laboratory was supported by a Grant from the Royal Society of New Zealand. J.A.M. holds a FPU fellowship from the Spanish Government.

Author contributions

JJ and JAM performed the majority of the experiments. PPD and SMB conceived of the study and participated in the design and coordination of the work. All authors contributed to the draft and read and approved the final manuscript.

Author information

Authors and Affiliations

  1. Institute of Fundamental Sciences, Massey University, Palmerston North, 4442, New Zealand

    J. Jayaraman & P. P. Dijkwel

  2. Departament de Biologia Vegetal, Facultat de Biologia, Universitat de Barcelona, Avinguda Diagonal 643, 08028, Barcelona, Spain

    J. A. Miret & S. Munné-Bosch

Authors
  1. J. Jayaraman
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. J. A. Miret
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. S. Munné-Bosch
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. P. P. Dijkwel
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to P. P. Dijkwel.

Additional information

J. Jayaraman and J. A. Miret: Shared first authors.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOC 249 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Jayaraman, J., Miret, J.A., Munné-Bosch, S. et al. Redox and hormone profiling of a Nicotiana tabacum dedifferentiated protoplast culture suggests a role for a cytokinin and gibberellin in plant totipotency. Plant Cell Tiss Organ Cult 124, 295–306 (2016). https://doi.org/10.1007/s11240-015-0893-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11240-015-0893-1

Keywords

Search

Navigation