Skip to main content
SpringerLink
Log in

The effects of reactive nitrogen and oxygen species on the regeneration and growth of cucumber cells from isolated protoplasts

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

Abstract

The present study investigated changes in the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in isolated mesophyll protoplasts and cell cultures of the cucumber Cucumis sativus cv. Marketer. Although only a minor increase in the level of nitrogen oxide (NO) was observed during the first 7 days of culture following protoplast isolation, a substantial accumulation of ROS was detected. Compounds known to modulate endogenous ROS and RNS levels were employed to study their role in cucumber protoplast regeneration and growth. Supplementing the culture medium with the NO donors S-nitrosoglutathione and sodium nitroprusside and the ROS scavenger ascorbate significantly increased protoplast viability and cell density. In contrast, cell density was significantly decreased following the addition of catalase to the medium. Scavenging of ROS and RNS induced the formation of cucumber microcalli, thus suggesting a differential role of NO in the maintenance of cell viability and in the control of cell division. Our findings confirm the crucial role of controlled ROS and RNS production in both protoplast regeneration and cellular growth and differentiation.

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
Fig. 9

Similar content being viewed by others

Abbreviations

BAP:

6-Benzylaminopurine

cPTIO:

2-(4-Carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide

CW:

Calcofluor White

DAF-FM DA:

4-Amino-5-methylamino-2′,7′-difluorofluorescein diacetate

FDA:

Fluorescein diacetate

GSNO:

S-nitrosoglutathione

H2DCF DA:

2′,7′-Dichlorofluorescein diacetate

IBA:

Indole-3-butyric acid

L-NAME:

NG-nitro-l-arginine methyl ester

L-NMMA:

NG-monomethyl-l-arginine

MES:

2-(N-Morpholino)ethanesulphonic Acid

MTT:

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NAA:

1-Naphthaleneacetic acid

NO:

Nitric oxide

NOS:

Nitric oxide synthase

RNS:

Reactive nitrogen species

ROS:

Reactive oxygen species

SNP:

Sodium nitroprusside

References

  • Abbasi BH, Khan M, Guo B, Bokhari SA, Khan MA (2011) Efficient regeneration and antioxidative enzyme activities in Brassica rapa var. turnip. Plant Cell Tiss Org Cult 105:337–344

    Article  CAS  Google Scholar 

  • Allan AC, Fluhr R (1997) Two distinct sources of elicited reactive oxygen species in tobacco epidermal cells. Plant Cell 9:1559–1572

    Article  PubMed  CAS  Google Scholar 

  • Beligni MV, Fath A, Bethke PC, Lamattina L, Jones RL (2002) Nitric oxide acts as an antioxidant and delays programmed cell death in barley aleurone layers. Plant Physiol 129:1642–1650

    Article  PubMed  CAS  Google Scholar 

  • Benet H, Ar Gall E, Asensi A, Kloareg B (1997) Protoplast regeneration from gametophytes and sporophytes of some species in the order Laminariales (Phaeophyceae). Protoplasma 199:39–48

    Article  Google Scholar 

  • Bonga JM, Klimaszewska KK, Aderkas P (2010) Recalcitrance in clonal propagation, in particular of conifers. Plant Cell Tiss Organ Cult 100:241–254

    Article  Google Scholar 

  • Burza W, Malepszy S (1995) In vitro culture of Cucumis sativus L. XVIII. Plants from protoplasts through direct somatic embryogenesis. Plant Cell Tiss Organ Cult 41:259–266

    Article  Google Scholar 

  • Burza W, Wozniak B, Tarkowska JA, Malepszy S (1994) Cytohistological analysis of somatic embryogenesis in cucumber (Cucumis sativus L.). II. Natural fluorescence and direct somatic embryogenesis from protoplasts. Acta Soc Bot Poloniae 63:265–268

    Google Scholar 

  • Cassells AC, Curry RF (2011) Oxidative stress and physiological, epigenetic and genetic variability in plant tissue culture: implications for micropropagators and genetic engineers. Plant Cell Tiss Organ Cult 64:145–157

    Article  Google Scholar 

  • Crow JP (1997) Dichlorodihydrofluorescein and dihydrorhodamine 123 are sensitive indicators of peroxynitrite in vitro: Implications for intracellular measurement of reactive ritrogen and oxygen species. Nitric Oxide 1:145–157

    Article  PubMed  CAS  Google Scholar 

  • Dabauza M, Roig LA, Moreno V (1991) Selective methods for the recovery of somatic hybrids of Cucumis melo × metuliferus and C. sativus × C. metuliferus. Cucurbit Gen Coop Rep 14:81–84

    Google Scholar 

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

    Article  CAS  Google Scholar 

  • De Marco A, Roubelakis-Angelakis KA (1996) The complexity of enzymic control of hydrogen peroxide concentration may affect the regeneration potential of plant protoplasts. Plant Physiol 110:137–145

    PubMed  Google Scholar 

  • Debeaujon I, Branchard M (1992) Induction of static embryogenesis and callogenesis from cotyledons and leaf protoplasts-derived colonies of melon (Cucumis melo L.). Plant Cell Rep 12:37–40

    Article  Google Scholar 

  • Del Rio LA, Corpas FJ, Barroso JB (2004) Nitric oxide and nitric oxide synthase activity in plants. Phytochemistry 65:783–792

    Article  PubMed  CAS  Google Scholar 

  • Delledonne M, Zeier J, Marocco A, Lamb C (2001) Signal interactions between nitric oxide and reactive oxygen intermediates in the plant hypersensitive disease resistance response. Proc Natl Acad Sci USA 98:13454–13459

    Article  PubMed  CAS  Google Scholar 

  • Fellner M, Lebeda A (1998) Cyclus induction and protoplast isolation from tissues of Cucumis sativus L. and C. melo L. seedlings. Biol Plantarum 41:11–24

    Article  Google Scholar 

  • Ferrer MA, Ros Barceló A (1999) Differential effects of nitric oxide on peroxidase and H2O2 production by the xylem of Zinnia elegans. Plant Cell Environ 22:891–897

    Article  CAS  Google Scholar 

  • Gajdová J, Lebeda A, Navrátilová B (2004) Protoplast cultures of Cucumis and Cucurbita spp. In: Lebeda A, Paris HS (eds) Progress in cucurbit genetics and breeding research. Proceedings of Cucurbitaceae 2004, the 8th EUCARPIA meeting on cucurbit genetics and breeding. Palacký University in Olomouc, Olomouc, pp 441–454

  • Gajdová J, Navrátilová B, Smolná I, Lebeda A (2007a) Effects of genotype, source tissue and media composition on Cucumis and Cucurbita protoplast isolation and regeneration. Acta Hortic 731:89–94

    Google Scholar 

  • Gajdová J, Navrátilová B, Smolná I, Lebeda A (2007b) Factors affecting protoplast isolation and cultivation of Cucumis spp. J Appl Bot Food Qual 81:1–6

    Google Scholar 

  • Han X, Yang H, Duan K, Zhang X, Zhao H, You S, Jiang Q (2009) Sodium nitroprusside promotes multiplication and regeneration of Malus hupehensis in vitro plantlets. Plant Cell Tiss Organ Cult 96:29–34

    Article  CAS  Google Scholar 

  • Hao G, Du X, Zhao F, Shi R, Wang J (2009) Role of nitric oxide in UV-B-induced activation of PAL and stimulation of flavonoid biosynthesis in Ginkgo biloba callus. Plant Cell Tiss Organ Cult 97:175–185

    Article  CAS  Google Scholar 

  • Harrington BJ, Raper KB (1968) Use of a fluorescent brightener to demonstrate cellulose in the cellular slime molds. Appl Microbiol 16:106–113

    PubMed  CAS  Google Scholar 

  • Ishii S (1988) Factors influencing protoplast viability of suspension-cultured rice cells during isolation process. Plant Physiol 88:26–29

    Article  PubMed  CAS  Google Scholar 

  • Kalra C, Babbar SB (2010) Nitric oxide promotes in vitro organogenesis in Linum usitatissimum L. Plant Cell Tiss Organ Cult 103:353–359

    Article  CAS  Google Scholar 

  • Kim HJ, Tsoy I, Park MK, Lee YS, Lee JH, Seo HG, Chang KC (2006) Iron released by sodium nitroprusside contributes to heme oxygenase-1 induction via the cAMP-protein kinase A-mitogen-activated protein kinase pathway in RAW 264.7 cells. Mol Pharmacol 69:1633–1640

    Article  PubMed  CAS  Google Scholar 

  • Kopyra M, Gwozdz EA (2004) The role of nitric oxide in plant growth regulation and responses to abiotic stresses. Acta Physiol Plantarm 26:459–472

    Article  CAS  Google Scholar 

  • Lebeda A (1999) Pseudoperonospora cubensis on Cucumis spp. and Cucurbita spp.—resistance breeding aspects. Acta Hortic 492:363–370

    Google Scholar 

  • Love AJ, Miller JJ, Sadanandom A (2008) Timing is everything: regulatory overlap in plant cell death. Trends Plant Sci 13:585–595

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Navrátilová B, Luhová L, Petřivalský M (2008) Effect of UV-C irradiation on mesophyll protoplasts of Cucumis sativus. Plant Cell Tiss Organ Cult 94:313–318

    Article  Google Scholar 

  • Neill SJ, Desikan R, Clarke A, Hancock JT (2002) Nitric oxide is a novel component of abscisic acid signaling in stomatal guard cells. Plant Physiol 128:13–16

    Article  PubMed  CAS  Google Scholar 

  • Neill S, Bright J, Desikan R, Hancock J, Harrison J, Wilson I (2008) Nitric oxide evolution and perception. J Exp Bot 59:25–35

    Article  PubMed  CAS  Google Scholar 

  • Ondřej V, Navrátilová B, Lebeda A (2009) The heterochromatin as a marker for protoplast differentiation of Cucumis sativus. Plant Cell Tiss Organ Cult 96:229–234

    Article  Google Scholar 

  • Ondřej V, Navrátilová B, Protivánková I, Piterková J, Sedlářová M, Luhová L, Lebeda L (2010) Recondensation level of repetitive sequences in the plant protoplast nucleus is limited by oxidative stress. J Exp Bot 61:2395–2401

    Article  PubMed  Google Scholar 

  • Orczyk W, Malepszy S (1985) In vitro culture of Cucumis sativus L. V. Stabilizing effect of glycine on leaf protoplasts. Plant Cell Rep 4:269–273

    Article  CAS  Google Scholar 

  • Ötvös K, Pasternak TP, Miskolczi P, Domoki M, Dorjgotov D, Szücs A, Bottka S, Dudits D, Fehér A (2005) Nitric oxide is required for, and promotes auxin-mediated activation of, cell division and embryogenic cell formation but does not influence cell cycle progression in alfalfa cell cultures. Plant J 43:849–860

    Article  PubMed  Google Scholar 

  • Papadakis AK, Roubelakis-Angelakis KA (1999) The generation of active oxygen species differs in tobacco and grapevine mesophyll protoplasts. Plant Physiol 121:197–206

    Article  PubMed  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  CAS  Google Scholar 

  • Pasternak TP, Potters G, Caubergs R, Jansen MAK (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  PubMed  CAS  Google Scholar 

  • Pasternak TP, Ötvös K, Domoki M, Fehér A (2007) Linked activation of cell division and oxidative stress defense in alfalfa leaf protoplast-derived cells is dependent on exogenous auxin Plant Growth Regul 51:109–117

    CAS  Google Scholar 

  • Plader W, Malpszy S, Burza W, Rusinowski Z (1998) The relationship between the regeneration system and genetic variability in the cucumber (Cucumis sativus L). Euphytica 103:9–15

    Article  Google Scholar 

  • Sagi M, Fluhr R (2006) Production of reactive oxygen species by plant NADPH oxidases. Plant Physiol 141:336–340

    Article  PubMed  CAS  Google Scholar 

  • Sheen J (2001) Signal transduction in maize and Arabidopsis mesophyll protoplasts. Plant Physiol 127:1466–1475

    Article  PubMed  CAS  Google Scholar 

  • Siminis CI, Kanellis AK, Roubelakis-Angelakis KA (1994) Catalase is differentially expressed in dividing and nondividing protoplasts. Plant Physiol 105:1375–1383

    PubMed  CAS  Google Scholar 

  • Skálová D, Navrátilová B, Ondřej V, Lebeda A (2010) Optimizing culture for in vitro pollination and fertilization in Cucumis sativus and C. melo. Acta Biol Cracov 52:111–115

    Google Scholar 

  • Song L, Ding W, Zhao MG, Sun B, Zhang L (2006) Nitric oxide protects against oxidative stress under heat stress in the calluses from two ecotypes of reed. Plant Sci 171:449–458

    Article  CAS  Google Scholar 

  • Song L, Ding W, Shen J, Zhang Z, Bi Y, Zhang L (2008) Nitric oxide mediates abscisic acid induced thermotolerance in the calluses from two ecotypes of reed under heat stress. Plant Sci 175:826–832

    Article  CAS  Google Scholar 

  • Sun J, Li L, Liu M, Wang M, Ding M, Deng S, Lu C, Zhou X, Shen X, Zheng X, Chen S (2010) Hydrogen peroxide and nitric oxide mediate K+/Na+ homeostasis and antioxidant defense in NaCl-stressed callus cells of two contrasting poplars. Plant Cell Tiss Org Cult 103:205–215

    Article  CAS  Google Scholar 

  • Szechynska-Hebda M, Skrzypek E, Dabrowska G, Biesaga-Koscielniak J, Filek M, Wedzony M (2007) The role of oxidative stress induced by growth regulators in the regeneration process of wheat. Acta Physiol Plantarum 29:327–337

    Article  CAS  Google Scholar 

  • Wang Y, Lin JS, Wang GX (2006) Calcium-mediated mitochondrial permeability transition involved in hydrogen peroxide-induced apoptosis in tobacco protoplasts. J Integ Plant Biol 48:433–439

    Article  CAS  Google Scholar 

  • Xu J, Yin H, Wang W, Mi Q, Liu X (2009) Effects of sodium nitroprusside on callus induction and shoot regeneration in micropropagated Dioscorea opposita. Plant Growth Regul 59:279–285

    Article  CAS  Google Scholar 

  • Xu M, Sheng J, Wang H, Dong J (2011) Involvement of NADPH oxidase-mediated H2O2 signaling in PB90-induced hypericin accumulation in Hypericum perforatum cells. Plant Cell Tiss Organ Cult 105:47–53

    Article  CAS  Google Scholar 

  • Yao N, Greenberg JT (2006) Arabidopsis ACCELERATED CELL DEATH2 modulates programmed cell death. Plant Cell 18:397–411

    Article  PubMed  CAS  Google Scholar 

  • Yasuda K, Watanabe M (2007) Generation of intracellular reactive oxygen species during the generation of intracellular reactive oxygen species during the isolation of Brassica napus leaf protoplasts. Plant Biotech 24:361–366

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The cooperation of the Olympus Czech Group (Prague, Czech Republic) is greatly appreciated. This research was supported by the Czech Ministry of Education, Youth and Sports through research grants Kontakt ME 08048 and MSM 6198959215.

Author information

Authors and Affiliations

  1. Department of Biochemistry, Faculty of Science, Palacký University, Šlechtitelů 11, 783 71, Olomouc, Czech Republic

    Marek Petřivalský, Pavla Vaníčková, Markéta Ryzí, Jana Piterková & Lenka Luhová

  2. Department of Botany, Faculty of Science, Palacký University, Šlechtitelů 11, 783 71, Olomouc, Czech Republic

    Božena Navrátilová & Michaela Sedlářová

Authors
  1. Marek Petřivalský
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Pavla Vaníčková
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Markéta Ryzí
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Božena Navrátilová
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jana Piterková
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Michaela Sedlářová
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Lenka Luhová
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Marek Petřivalský.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Petřivalský, M., Vaníčková, P., Ryzí, M. et al. The effects of reactive nitrogen and oxygen species on the regeneration and growth of cucumber cells from isolated protoplasts. Plant Cell Tiss Organ Cult 108, 237–249 (2012). https://doi.org/10.1007/s11240-011-0035-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11240-011-0035-3

Keywords

Search

Navigation