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Changes in the Glutathione and Ascorbate Redox State Trigger Growth During Embryo Development and Meristem Reactivation at Germination

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Ascorbate-Glutathione Pathway and Stress Tolerance in Plants

Abstract

In vitro embryogenesis via androgenesis and somatic embryogenesis represents an efficient propagation tool as well as a suitable model system for investigating structural, physiological, and molecular events governing embryo development. One of the major problems encountered by tissue culturists is the poor efficiency of embryos produced in vitro and their inability to regenerate viable plants, which denote sub-optimal culture conditions. Judicious alterations of the glutathione (GSH) and ascorbate (AsA) redox state have a profound effect on morphogenesis and improve the quality of the embryos by promoting a zygotic-like histodifferentiation pattern and producing well organized meristems. This approach has been investigated successfully during the development and germination of both Brassica napus (canola, an angiosperm) microspore-derived embryos (MDEs) and Picea glauca (white spruce, a conifer) somatic embryos. The imposition of a reduced glutathione environment during the early embryonic phases induces cellular proliferation and increases the number of immature embryos, possibly by promoting the synthesis of nucleotides required for energetic processes and mitotic activity. Continuation of embryo development is best conducted if the glutathione pool is experimentally switched towards an oxidized state; a condition favoring histodifferentiation and post-embryonic growth in both canola and spruce. Structural analyses showed that the oxidized glutathione environment favors the proper formation of the shoot apical meristem (SAM), which acquires a “zygotic-like” appearance. The apical poles of glutathione-treated embryos are well organized and display a proper expression and localization of meristem marker genes. These conditions are not found in control embryos which develop abnormal SAMs characterized by the presence of intercellular spaces and differentiation of meristematic cells. Such meristems fail to reactivate at germination resulting in embryo abortion. Physiological and molecular studies have further demonstrated that the oxidized glutathione environment induces several responses, including changes in ascorbate metabolism, abscisic acid and ethylene synthesis, as well as alterations in storage product deposition patterns. If an oxidized glutathione environment favors embryo formation, a reduced ascorbate redox state is required to promote germination and conversion, i.e. the emergence of functional shoots and roots. Specifically, a highly reduced ascorbate environment induces cell proliferation and de-novo meristem formation within those SAMs with an abnormal architecture. Overall it appears that precise changes in glutathione and ascorbate metabolism are required to ensure proper embryo formation and regeneration.

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References

  • Arrigoni O, De Gara L, Tommasi F, Liso R (1992) Changes in the ascorbate system during seed development in Vicia faba L. Plant Physiol 99:235–238

    Article  PubMed  CAS  Google Scholar 

  • Arrigoni O, and De Tullio MC (2002) Ascorbic acid: much more than just an antioxidant. Biochim Biophys Acta 1569:1–9

    Article  PubMed  CAS  Google Scholar 

  • Belmonte MF, Stasolla C, Loukanina N, Yeung EC, Thorpe TA (2003) Glutathione modulation of purine metabolism in cultured white spruce embryogenic tissue. Plant Sci 165:1377–1385

    Article  CAS  Google Scholar 

  • Belmonte MF, Donald G, Reid DM, Yeung EC, Stasolla C (2005a) Alterations of the glutathione redox state improve apical meristem structure and somatic embryo quality in white spruce (Picea glauca). J Exp Bot 56:2355–2364

    Article  PubMed  CAS  Google Scholar 

  • Belmonte MF, Stasolla C, Katahira R, Loukanina N, Yeung EC, Thorpe TA (2005b) Glutathione-induced growth of embryogenic tissue of white spruce correlates with changes in pyrimidine nucleotide metabolism. Plant Sci 168:803–812

    Article  CAS  Google Scholar 

  • Belmonte MF, Ambrose SJ, Ross ARS, Abrams S, Stasolla C (2006) Improved development of microspore-derived embryo cultures of B. napus cv Topas following changes in glutathione metabolism. Physiol Plant 127:690–700

    Article  CAS  Google Scholar 

  • Bielawski W, Joy KW (1986) Reduced and oxidized glutathione and glutathione reductase activity in tissues of Pisum sativum. Planta 169:267–272

    Article  CAS  Google Scholar 

  • Bozhkov PV, Filonova LH, von Arnold S (2002) A key developmental switch during Norway spruce somatic embryogenesis is induced by withdrawal of growth regulators and is associated with cell death and extracellular acidification. Biotech Bioeng 77:658–667

    Article  CAS  Google Scholar 

  • Buchanan BB (1980) Role of light in the regulation of chloroplast enzymes. Annu Rev Plant Physiol 31:341–374

    Article  CAS  Google Scholar 

  • Cerrutti L, Mian N, Bateman A (2000) Domains in gene silencing and cell differentiation proteins: the novel PAZ domain and redefinition of the PIWI domain. Trends Biochem Sci 25:481–482

    Article  Google Scholar 

  • Chen J, Goldsborough PB (1994) Increased activity of glutamylcysteine synthetase in tomato cells selected for cadmium tolerance. Plant Physiol 10:233–239

    Google Scholar 

  • De Gara L, de Pinto M, Paciolla A, Cappetti V, Arrigoni O (1996) Is ascorbate peroxidase only a scavenger of hydrogen peroxide? In: Obinger C, Burner U, Pennel C, Greppin H (eds) Plant peroxidases: biochemistry and physiology. University of Geneva, Switzerland, pp 157–162

    Google Scholar 

  • De Gara L, de Pinto MC, Arrigoni O (1997) Ascorbate synthesis and ascorbate peroxidase activity during the early stage of wheat germination. Physiol Plant 100:894–900

    Article  Google Scholar 

  • De Gara L, Tommasi F (1999) Ascorbate redox enzymes: a network of reactions involved in plant development. Recent Res Dev Phytochem 3:1–5

    Google Scholar 

  • De Gara L, de Pinto M, Moliterni VM, D’Egidio MG (2003) Redox regulation and storage processes during maturation in kernels of Triticum durum. J Exp Bot 54:249–258

    Article  PubMed  Google Scholar 

  • de Pinto M, Francis D, De Gara L (1999) The redox state of the ascorbate-dehydroascorbate pair as a specific sensor of cell division in tobacco BY2 cells. Protoplasma 209:90–97

    Article  PubMed  Google Scholar 

  • De Tullio MC, Paciolla C, Dalla Vecchia F, Rascio N, D’Emerico S, De Gara L, Liso R, Arrigoni O (1999) Changes in onion root development induced by the inhibition of peptidyl-prolyl hydroxylase and influence of the ascorbate system on cell division and elongation. Planta 21:424–434

    Article  Google Scholar 

  • De Tullio MC, Arrigoni O (2006) The ascorbic acid system in seeds: to protect and serve. Seed Sci Res 13:249–260

    Google Scholar 

  • Earnshaw BA, Johnson MA (1985) The effect of glutathione on development of white carrot suspension cultures. Biochem Biophys Res Comm 133:988–993

    Article  PubMed  CAS  Google Scholar 

  • Earnshaw BA, Johnson MA (1987) Control of wild carrot somatic embryo development by antioxidants. Plant Physiol 85:273–276

    Article  PubMed  CAS  Google Scholar 

  • Fiers M, Golemiec E, Xu J, van der Geest L, Heidstra R, Stiekema W, Liu CM (2005) The 14-amino acid CLV3, CLE19, and CLE40 peptides trigger consumption of the root meristem in Arabidopsis through a CLAVATA2-dependent pathway. Plant Cell 17:2542–2553

    Article  PubMed  CAS  Google Scholar 

  • Filonova LH, Bozhkov PV, Brukhin VB, Daniel G, Zhivotovsky B, von Arnold S (2000) Two waves of programmed cell death occur during formation and development of somatic embryos in the gymnosperm Norway spruce. J Cell Sci 113:4399–4411

    PubMed  CAS  Google Scholar 

  • Fiorani M, De Sanctis R, Scarlatti F, Vallorani L, De Bellis R, Serafini G, Bianchi M, Stocchi V (2000) Dehydroascorbic acid irreversibly inhibits hexokinase activity. Mol Cell Biochem 209:145–153

    Article  PubMed  CAS  Google Scholar 

  • Fukushima S, Imaida K, Shibat MA, Tamano T, Kurata Y, Shirai T (1988) L-ascorbic acid amplification of second-stage bladder carcinogenesis promotion by NaHCO3. Cancer Res 48:6317–6320

    PubMed  CAS  Google Scholar 

  • Gaspar T, Kevers C, Penel C, Greppin H, Reid DM, Thorpe TA (1996) Plant hormones and plant growth regulators in plant tissue culture. In Vitro Cell Dev Biol Plant 32:272–289

    Article  CAS  Google Scholar 

  • Grant JJ, Loake GJ (2001) Role of reactive oxygen intermediates and cognate redox signaling in disease resistance. Plant Physiol 124:21–29

    Article  Google Scholar 

  • Hays DB (1996) The role of hormones in Brassica napus embryo development. PhD thesis. University of Calgary, Calgary, Alberta, Canada

    Google Scholar 

  • Henmi K, Tsuboi S, Demura T, Fukuda H, Iwabuchi M, Ogawa KA (2001) A possible role of glutathione and glutathione disulfide in tracheary element differentiation in the cultured mesophyll cells of Zinnia elegans. Plant Cell Physiol 42:673–676

    Article  PubMed  CAS  Google Scholar 

  • Ito Y, Nakanomyo I, Motose H, Iwamoto K, Sawa S, Dohmae N, Fukuda H, Dodeca H (2006) CLE peptides as suppressors of plant stem cell differentiation. Science 313:842–845

    Article  PubMed  CAS  Google Scholar 

  • Kermode AR (1995) Regulatory mechanisms involved in the transition from seed development to germination. Crit Rev Plant Sci 9:155–195

    Article  Google Scholar 

  • Jain SM, Newton RJ, Soltes EJ (1988) Enhancement of somatic embryogenesis in norway spruce (Picea abies). Theor Appl Genet 76:501–506

    Article  Google Scholar 

  • Jiang K, Meng YL, Feldman L (2004) Quiescent center formation in maize is associated with an auxin-related oxidizing environment. Development 130:1429–1438

    Article  Google Scholar 

  • Jiang K, Feldman LJ (2005) Regulation of root apical meristem development. Annu Rev Cell Dev Biol 21:485–509

    Article  PubMed  CAS  Google Scholar 

  • Levine M (1947) Differentiation of carrot root tissue grown in culture. Bull Torrey Bot Club 74:321–323

    Article  Google Scholar 

  • Kerk NM, Feldman LJ (1995) A biochemical model for the initiation and maintenance of quiescent center: implications for organization of root meristems. Development 121:2825–2833

    CAS  Google Scholar 

  • Luwe M (1996) Antioxidants in the apoplast and symplast of beech (Fagus sylvatica L.) leaves: seasonal variations and responses to changing ozone concentration in air. Plant Cell Environ 19:321–328

    Article  CAS  Google Scholar 

  • Lynn K, Fernandez A, Aida M, Sedbrook J, Tasaka M, Masson P, Barton MK (1999) The PINHEAD/ZWILLE gene acts pleiotropically in Arabidopsis development and has overlapping functions with the ARGONAUTE1 gene. Development 126:469–481

    PubMed  CAS  Google Scholar 

  • Mahalingam R, Fedoroff N (2003) Stress response, cell death and signaling: the many faces of reactive oxygen species. Physiol Plant 119:56–68

    Article  CAS  Google Scholar 

  • Maraschin SF, Caspers M, Potokinad E, Wulfert F, Granerd A, Spaink HP, Wang M (2006) cDNA array analysis of stress-induced gene expression in barely androgenesis. Physiol Plant 127:535–550

    Article  CAS  Google Scholar 

  • Marre E, Arrigoni O (1957) Metabolic reactions to auxin. I. The effects of auxin on glutathione and the effects of glutathione on growth of isolated plant parts. Physiol Plant 10:289–301

    Article  CAS  Google Scholar 

  • May MJ, Leaver CJ (1994) Arabidopsis thaliana γ-glutamylcysteine synthetase is structurally unrelated to mammalian, yeast, and E.coli homologs. Proc Natl Acad Sci USA 91:10059–10063

    Article  PubMed  CAS  Google Scholar 

  • May MJ, Leaver CJ (1993) Oxidative stimulation of glutathione synthesis in Arabidopsis thaliana suspension cultures. Plant Physiol 103:621–627

    PubMed  CAS  Google Scholar 

  • May MJ, Vernoux T, Leaver CJ, Montagu V, Inez D (1998) Glutathione homeostasis in plants: implications for environmental sensing and plant development. J Exp Bot 49:649–667

    CAS  Google Scholar 

  • Moussian B, Schoof H, Haecker A, Jurgens G, Laux G (1998) Role of ZWILLE gene in the regulation of central shoot meristem cell fate during Arabidopsis embryogenesis. EMBO J 17:1799–1809

    Article  PubMed  CAS  Google Scholar 

  • Noctor G, Foyer CH (1998) Ascorbate and glutathione: keeping active oxygen under control. Annu Rev Plant Physiol Plant Mol Biol 49:249–279

    Article  PubMed  CAS  Google Scholar 

  • Noctor G, Arisi A-CM, Jouanin L, Kunert KJ, Rennenberg H, Foyer CH (1998) Glutathione: biosynthesis, metabolism and relationship to stress tolerance explored in transformed plants. J Exp Bot 49:623–647

    CAS  Google Scholar 

  • Ogawa K, Tasaka Y, Mino M, Tanaka T, Iwabuchi M (2001) Association of glutathione with flowering in Arabidopsis thaliana. Plant Cell Physiol 42:524–530

    Article  PubMed  CAS  Google Scholar 

  • Piyathilake CJ, Bell WC, Johanning GL, Cornwell PE, Heimburger DC, Grizzle WE (2000) The accumulation of ascorbic aicd by squamous cell carcinomas of the lung and larynx is associated with global methylation of DNA. Cancer 89:171–176

    Article  PubMed  CAS  Google Scholar 

  • Potters G, De Gara L, Asard H, Horemans N (2002) Ascorbate and glutathione: guardians of the cell cycle, pattern in crime? Plant Physiol Biochem 40:537–548

    Article  CAS  Google Scholar 

  • Raghavan V (1976) Experimental embryogenesis in vascular plants. Academic, London

    Google Scholar 

  • Ramesar-Fortner NS, Yeung EC (2001) Tri-iodobenzoic acid affects shoot apical meristem formation in zygotic embryos of Brassica napus. Can J Bot 79:265–273

    CAS  Google Scholar 

  • Rawlins MR, Leaver CJ, May MJ (1995) Characterization of Arabidopsis thaliana cDNA encoding glutathione synthetase. FEBS Lett 376:81–86

    Article  PubMed  CAS  Google Scholar 

  • Sanchez-Fernandez R, Fricker M, Corben L, White NS, Sheard N, Leaver CJ, Van Montagu M, Inze D, May MJ (1997) Cell proliferation and hair tip growth in the Arabidopsis root under mechanistically different forms of redox control. Proc Natl Acad Sci USA 94:2745–2750

    Article  PubMed  CAS  Google Scholar 

  • Shafer FQ, Buettner GR (2001) Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple. Free Radic Biol Med 30:1191–1198

    Article  Google Scholar 

  • Schutzendubel A, Polle A (2002) Plant responses to abiotic stresses: heavy metal-induced oxidative stress ad protection by micorrhization. J Exp Bot 53:1351–1365

    Article  PubMed  CAS  Google Scholar 

  • Shibata MA, Fukushima S, Asakawa E, Hirose M, Ito N (1992) The modifying effects of indomethacin or ascorbic acid on cell proliferation induced by different types of bladder tumor promoters in rat urinary bladder and forestomach mucosal epithelium. Jpn J Cancer Res 83:31–39

    Article  PubMed  CAS  Google Scholar 

  • Stasolla C, Yeung EC (2001) Ascorbic acid metabolism during white spruce somatic embryogenesis. Physiol Plant 111:196–205

    Article  CAS  Google Scholar 

  • Stasolla C, Yeung EC (2003) Recent advances in conifer somatic embryogenesis: improving somatic embryo quality. Plant Cell Tissue Org Cult 74:15–35

    Article  CAS  Google Scholar 

  • Stasolla C, Yeung EC, Thorpe TA (2003) Somatic embryogenesis in white spruce: physiology and biochemistry. In: Bhojawani SS (ed) Agrobiotechnology and plant tissue culture. Science Publishers Inc., Enfield, NH, pp 10–27

    Google Scholar 

  • Stasolla C, Thorpe TA (2004) Purine and pyrimidine nucleotide synthesis and degradation during in vitro morphogenesis of white spruce (Picea glauca). Front Biosci 9:1506–1519

    Article  PubMed  CAS  Google Scholar 

  • Stasolla C, Belmonte MF, van Zyl L, Craig DL, Liu W, Yeung EC, Sederoff R (2004) The effect of reduced glutathione on morphology and gene expression of white spruce (Picea glauca) somatic embryos. J Exp Bot 55:695–709

    Article  PubMed  CAS  Google Scholar 

  • Stasolla C, Yeung EC (2006a) Exogenous applications of ascorbic acid induce shoot apical meristem growth in germinating white spruce (Picea glauca) somatic embryos. Int J Plant Sci 167:429–436

    Article  CAS  Google Scholar 

  • Stasolla C, Yeung EC (2006b) Endogenous ascorbic acid affects thymidine and uridine metabolism and modulates meristem reactivation in white spruce somatic embryos. Tree Physiol 26:1197–1206

    Article  PubMed  CAS  Google Scholar 

  • Stasolla C, Yeung EC (2007) Cellular ascorbic acid regulates the activity of major peroxidases in the apical poles of germinating white spruce (Picea glauca) somatic embryos. Plant Physiol Biochem 45:188–198

    Article  PubMed  CAS  Google Scholar 

  • Stasolla C, Loukanina N, Yeung EC, Thorpe TA (2007) Comparative studies on pyrimidine metabolism of Pinus radiata cotyledons cultured under shoot-forming and non shoot-forming conditions. J Plant Physiol 164:429–441

    Article  PubMed  CAS  Google Scholar 

  • Stasolla C, Belmonte M, Tahir M, Elhiti M, Joosen R, Maliepaard C, Sharpe A, Boutilier K (2008) Buthionine sulfoximine (BSO)–mediated improvement in in vitro cultured embryo quality is associated with major changes in transcripts involved with ascorbate metabolism, meristem development and embryo maturation. Planta 228:255–272

    Article  PubMed  CAS  Google Scholar 

  • Sundås-Larsson A, Svenson M, Liao H, Engström P (1998) A homeobox gene with potential developmental control function in the meristem of the conifer Picea abies. Proc Natl Acad Sci USA 95:15118–15122

    Article  PubMed  Google Scholar 

  • Thair M, Stasolla C (2005) Shoot apical development during in vitro embryogenesis. Can J Bot 84:1650–1659

    Article  Google Scholar 

  • Thorpe TA, Stasolla C (2001) Somatic embryogenesis. In: Bhojwani SS, Soh WY (eds) Current trends in the embryology of angiosperms. Kluwer, Boston, MA, pp 279–336

    Google Scholar 

  • Yao QA, Simion E, William M, Krochko J, Kasha KJ (1997) Biolistic transformation of haploid isolated microspores of barley (Hordeum vulgare L.). Genome 40:570–581

    Article  PubMed  CAS  Google Scholar 

  • Yeung EC, Aitken C, Biondi S, Thorpe TA (1981) Shoot histogenesis in cotyledon explants of radiata pine. Bot Gaz 142:494–501

    Article  Google Scholar 

  • Yeung EC (2002) The canola microspore-derived embryo as a model system to study developmental processes in plants. J Plant Biol 45:119–133

    Article  Google Scholar 

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Authors and Affiliations

  1. Department of Plant Science, University of Manitoba, Winnipeg, R3T 2N2, Manitoba, Canada

    Claudio Stasolla

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  1. Claudio Stasolla
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Correspondence to Claudio Stasolla .

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Editors and Affiliations

  1. Biotechnology Center in Southern Taiwan, Academia Sinica, Huandong Rd., Sinshih, Tainan, 74146, Taiwan R.O.C.

    Naser A. Anjum

  2. Biotechnology Center in Southern Taiwan, Academia Sinica, Huandong Rd., Sinshih, Tainan, 74146, Taiwan R.O.C.

    Ming-Tsair Chan

  3. Dept. Botany, Hamdard University, New Delhi, 110062, India

    Shahid Umar

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Stasolla, C. (2010). Changes in the Glutathione and Ascorbate Redox State Trigger Growth During Embryo Development and Meristem Reactivation at Germination. In: Anjum, N., Chan, MT., Umar, S. (eds) Ascorbate-Glutathione Pathway and Stress Tolerance in Plants. Springer, Dordrecht. https://doi.org/10.1007/978-90-481-9404-9_8

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