title: 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.
authors.affiliation: Department of Plant Science, University of Manitoba, Winnipeg, MB R3T 2N2, Canada. stasolla@ms.umanitoba.ca
authors.forename: Claudio
authors.initials: C
authors.lastname: Stasolla
authors.forename: Mark F
authors.initials: MF
authors.lastname: Belmonte
authors.forename: Muhammad
authors.initials: M
authors.lastname: Tahir
authors.forename: Mohamed
authors.initials: M
authors.lastname: Elhiti
authors.forename: Khalil
authors.initials: K
authors.lastname: Khamiss
authors.forename: Ronny
authors.initials: R
authors.lastname: Joosen
authors.forename: Chris
authors.initials: C
authors.lastname: Maliepaard
authors.forename: Andrew
authors.initials: A
authors.lastname: Sharpe
authors.forename: Branimir
authors.initials: B
authors.lastname: Gjetvaj
authors.forename: Kim
authors.initials: K
authors.lastname: Boutilier
mesh_terms: D000963:Antimetabolites / Q000494:pharmacology*; D000975:Antioxidants / Q000378:metabolism; D001205:Ascorbic Acid / Q000378:metabolism*; D029688:Brassica napus / Q000187:drug effects / Q000196:embryology* / Q000254:growth & development / Q000378:metabolism; D019328:Buthionine Sulfoximine / Q000494:pharmacology*; D046508:Culture Techniques; D020869:Gene Expression Profiling; D005978:Glutathione / Q000378:metabolism; D018519:Meristem / Q000254:growth & development*; D020411:Oligonucleotide Array Sequence Analysis; D013997:Time Factors
chemical_list: D000963:Antimetabolites; D000975:Antioxidants; D019328:Buthionine Sulfoximine; D005978:Glutathione; D001205:Ascorbic Acid