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

Abstract

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.

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Abbreviations

ASC:

Reduced ascorbate

APX:

Ascorbate peroxidase

BSO:

Buthionine sulfoximine

DHA:

Dehydroascorbate

DHAR:

Dehydroascorbate reductase

GR:

Glutathione reductase

GSH:

Reduced glutathione

GSSG:

Oxidized glutathione

MDEs:

Microspore-derived embryos

MDHA:

Monodehydroascorbate

MDHAR:

Monodehydroascorbate reductase

LEA:

Late embryogenic abundant

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Acknowledgments

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.

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Correspondence to Claudio Stasolla.

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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

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Keywords

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