Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Buthionine sulfoximine (BSO)-mediated improvement in cultured embryo quality in vitro entails changes in ascorbate metabolism, meristem development and embryo maturation


Applications of buthionine sulfoximine (BSO), an inhibitor of GSH (reduced glutathione), which switches the cellular glutathione pool towards the oxidized form GSSG, positively influences embryo quality by improving the structure of the shoot apical meristem and promoting embryo maturation, both of which improve the post-embryonic performance of the embryos. To investigate the mechanisms underlying BSO-mediated improvement in embryo quality the transcript profiles of developing Brassica napus microspore-derived embryos cultured in the absence (control) or presence of BSO were analyzed using a 15,000-element B. napus oligo microarray. BSO applications induced major changes in transcript accumulation patterns, especially during the late phases of embryogenesis. BSO affected the transcription and activities of key enzymes involved in ascorbate metabolism, which resulted in major fluctuations in cellular ascorbate levels. These changes were related to morphological characteristics of the embryos and their post-embryonic performance. BSO applications also activated many genes controlling meristem formation and function, including ZWILLE, SHOOTMERISTEMLESS, and ARGONAUTE 1. Increased expression of these genes may contribute to the improved structural quality of the shoot poles observed in the presence of BSO. Compared to their control counterparts, middle- and late-stage BSO-treated embryos also showed increased accumulation of transcripts associated with the maturation phase of zygotic embryo development, including genes encoding ABA-responsive proteins and storage- and late-embryogenic abundant (LEA) proteins. Overall these transcriptional changes support the observation that the BSO-induced oxidized glutathione redox state allows cultured embryos to reach both morphological and physiological maturity, which in turn guarantees successful regeneration and enhanced post-embryonic growth.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10
Fig. 11



Reduced ascorbate


Ascorbate peroxidase


Buthionine sulfoximine




Dehydroascorbate reductase


Glutathione reductase


Reduced glutathione


Oxidized glutathione


Microspore-derived embryos




Monodehydroascorbate reductase


Late embryogenic abundant


  1. 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

  2. Belmonte MF, Stasolla C (2007) Applications of dl-buthionine-[S, R]-sulfoximine deplete cellular glutathione and improve white spruce (Picea glauca) somatic embryo development. Plant Cell Rep 26:517–523

  3. Belmonte MF, Donald G, Reid DM, Yeung EC, Stasolla C (2005) 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

  4. Belmonte M, Ambrose SJ, Ross ARS, Abrams SR, Stasolla C (2006) Improved development of microspore derived embryo cultures of Brassica napus cv Topaz following changes in glutathione metabolism. Physiol Plant 127:690–700

  5. Belmonte MF, Tahir M, Schroeder D, Stasolla C (2007) Over-expression of HBK3, a class I KNOX homeobox gene, improves the development of Norway spruce (Picea abies) somatic embryos. J Exp Bot 58:2851–2861

  6. Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc Ser 57:289–300

  7. Bohmert K, Camus I, Bellini C, Bouchez D, Caboche M, Benning C (1998) AGO1 defines a novel locus of Arabidopsis controlling leaf development. EMBO J 17:170–180

  8. Boutilier K, Fiers M, Liu CM, van Der Geest AHM (2005) Biochemical and molecular aspects of haploid embryogenesis. In: Palmer CE, Keller WA, Kasha KJ (eds) Haploids in crop improvements II, vol 56. Springer, Heidelberg, pp 73–95

  9. Carmell MA, Xuan Z, Zhang MQ, Hannon G (2002) The Argonaute family: tentacles that reach into RNAi, developmental control, stem cell maintenance and tumorigenesis. Genes Dev 16:2733–2742

  10. 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

  11. Cleveland WS (1979) Robust logically weighted regression and smoothing scatterplots. J Am Stat Assoc 74:829–833

  12. Cox DN, Chao A, Baker J, Chang L, Qiao D, Lin H (1998) A novel class of evolutionarily conserved genes defined by piwi are essential for stem cell renewal. Genes Dev 12:3715–3727

  13. De Gara L, Tommasi F (1999) Ascorbate redox enzymes: a network of reactions involved in plant development. Recent Res Dev Phytochem 3:1–15

  14. 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, Geneva, pp 157–162

  15. 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

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

  17. Dunstan DI, Tautorus TE, Thorpe TA (1995) Somatic embryogenesis in woody plants. In: Thorpe TA (ed) In vitro embryogenesis in plants. Kluwer, Dordrecht, pp 267–308

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

  19. 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

  20. Fletcher JC, Brand U, Running MP, Simon R, Meyerowitz EM (1999) Signaling of cell fate decisions by CLAVATA3 in Arabidopsis shoot meristems. Science 283:1911–1914

  21. Galland R, Randoux B, Vasseur J, Hilbert JL (2001) A glutathione-S-transferase cDNA identified by mRNA differential display is upregulated during somatic embryogenesis in Cichorium. Biochim Biophys Acta 1522:212–216

  22. Gardiner CS, Salmen JJ, Brandt CJ, Stover SK (1998) Glutathione is present in reproductive tract secretions and improves development of mouse embryos after chemically induced glutathione depletion. Biol Rep 59:431–436

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

  24. Girke T, Todd J, Ruuska S, White J, Benning C, Ohlrogge J (2000) Microarray analysis of developing Arabidopsis seeds. Plant Physiol 124:1570–1581

  25. Griffith OW, Meister A (1979) Potent and specific inhibition of glutathione synthesis by buthionine sulfoximine (s-n-Butyl Homocysteine Sulfoximine). J Biochem Chem 254:7558–7560

  26. Haberlandt G (1902) Culturvesuche mit isolierten Pflanzenzellen. Sitzungsberg Kais Aked Wiss Wien Mat-Natuirwiss Kl. Abt 111:69–92

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

  28. Joosen R, Cordewener J, Supena EDJ, Vorst O, Lammers M, Maliepaard C, Zeilmaker T, Miki B, America T, Custers J, Boutilier K (2007) Combined transcriptome and proteome analysis identifies pathways and markers associated with the establishment of rapeseed microspore-derived embryo development. Plant Physiol 144:1–18

  29. Kataoka Y, Takeichi M, Uemura T (2001) Developmental role and molecular characterization of Drosophila homologue of Arabidopsis Argonaute 1, the founder of a novel gene superfamily. Gene Cells 6:313–325

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

  31. Kidner CA, Martienssen RA (2005) The developmental role of microRNA in plants. Curr Opin Plant Biol 8:38–44

  32. Kong L, Yeung EC (1992) Development of white spruce somatic embryos: II. Continual shoot meristem development during germination. In Vitro Cell Dev Biol Plant 28:125–131

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

  34. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods 25:402–408

  35. Long JA, Moan EI, Medford JI, Barton MK (1996) A member of the KNOTTED class of homeodomain proteins encoded by the STM gene of Arabidopsis. Nature 379:66–69

  36. Lotan T, Ohto M-A, Matsudaira K, West MAL, Lo R, Kwong RW, Yamagishi K, Fischer RL, Goldberg RB, Harada JJ (1998) Arabidopsis LEAFY COTYLEDON1 is sufficient to induce embryo development in vegetative cells. Cell 93:1195–1205

  37. 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

  38. Malik MR, Wang F, Dirpaul JM, Zhou N, Polowick PI, Ferrie AMR, Krochko JE (2007) Transcript profiling and identification of molecular markers for early embryogenesis in Brassica napus. Plant Physiol 144:134–154

  39. 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

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

  41. Nagasaki H, Sato Y (2007) The small interfering RNA production pathway is required for shoot meristem initiation in rice. Proc Natl Acad Sci USA 104:14867–14871

  42. Ouellet T, Routledge RG, Miki B (1992) Members of the acetohydroxyacid synthase multigene family of Brassica napus have divergent expression patterns of expression. Plant J 2:321–330

  43. Potters G, De Gara L, Asard H, Horemans N (2005) Ascorbate and glutathione: guardians of the cell cycle, partners in crime? Plant Physiol Biochem 40:537–548

  44. Raijmakers MT, Steegers EA, Peters WH (2001) Glutathione S-transferases and thiol concentrations in embryonic and early fetal tissues. Hum Reprod 16:2445–2450

  45. Shi Z-Z, Osei-Frimpong J, Kala G, Kala SV, Barrios RJ, Habib GM, Lukin DJ, Danney CM, Lieberman MW (2000) Glutathione synthesis is essential for mouse development but not cell growth in culture. Proc Natl Acad Sci USA 97:5101–5106

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

  47. Stasolla C, Yeung EC (2006) 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

  48. Stasolla C, Craig D, Li Z, Wenbin L, van Zyl L, Sederoff RR (2003) The effect of polyethylene glycol (PEG) on gene expression of developing white spruce somatic embryos. Plant Physiol 131:49–60

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

  50. Stasolla C, Bozhkov PV, Chu TM, Van Zyl L, Egertsdotter U, Suarez MF, Craig D, Wolfinger RD, Von Arnold S, Sederoff RR (2004b) Variation in transcript abundance during somatic embryogenesis in gymnosperms. Tree Physiol 24:1073–1085

  51. Tahir M, Law DL, Stasolla C (2006) Molecular characterization of PgAGO, a novel conifer genes expressed in the apical cells and required during white spruce (Picea glauca) somatic embryogenesis. Tree Physiol 26:1257–1270

  52. Teo WL-L, Kumar P, Goch J, Swarup S (2004) The expression of BroSTM, a KNOTTED1-like gene, marks the cell type and timing of in vitro shoot induction in Brassica oleracea. Plant Mol Biol 46:567–580

  53. Thibaud-Nissen F, Shealy RT, Khanna A, Vodkin LO (2003) Clustering of microarray data reveals transcript patterns associated with somatic embryogenesis in soybean. Plant Physiol 132:118–136

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

  55. Tommasi F, Paciolla C, de Pinto MC, De Gara L (2001) A comparative study of glutathione and ascorbate metabolism during germination of Pinus pinea L. seeds. J Exp Bot 52:1647–1654

  56. Vrinten PL, Nakamura T, Kasha KJ (1999) Characterization of cDNAs expressed in early stages of microspore embryogenesis in barley (Hoedeum vulgare) L. Plant Mol Biol 41:455–465

  57. West M, Yee KM, Danao J, Zimmerman JL, Fischer RL, Goldberg RB, Harada JJ (1994) LEAFY COTYLEDON1 is an essential regulator of late embryogenesis and cotyledon identity in Arabidopsis. Plant Cell 6:1731–1745

  58. 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

  59. Yeung EC (1995) Structural developmental patterns in somatic embryogenesis. In: Thorpe TA (ed) In vitro embryogenesis in plants. Kluwer, Dordrecht, pp 205–249

  60. Yeung EC (1999) The use of histology in the study of plant tissue culture systems—some practical comments. In Vitro Cell Dev Biol Plant 35:137–143

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

  62. Yeung EC, Stasolla C (2001) Somatic embryogenesis—apical meristems and embryo conversion. Korean J Plant Tissue Cult 27:299–307

  63. Yeung E, Rahman MH, Thorpe TA (1996) Comparative development of zygotic and microspore-derived embryos in Brassica napus L. cv Topas. I. Histodifferentiation. Int J Plant Sci 157:27–39

  64. Yeung EC, Belmonte MF, Tu L, Stasolla C (2005) Glutathione modulation of in vitro development. In Vitro Cell Dev Biol Plant 41:584–590

Download references


This research was supported by a fellowship from the Nederlandse Organisatie voor Wetenschappelijke Onderzoek (NWO) to CS and KB, and Natural Sciences and Engineering Research Council of Canada Grants to CS. The technical assistance of Mr. B. Luit is also appreciated.

Author information

Correspondence to Claudio Stasolla.

Electronic supplementary material

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Stasolla, C., Belmonte, M.F., Tahir, M. et al. Buthionine sulfoximine (BSO)-mediated improvement in cultured embryo quality in vitro entails changes in ascorbate metabolism, meristem development and embryo maturation. Planta 228, 255 (2008). https://doi.org/10.1007/s00425-008-0735-z

Download citation


  • Brassica napus
  • Buthionine sulfoximine
  • Canola
  • Meristem
  • Transcript levels