Abstract
Gibberellin (GA) biosynthesis in cell-free systems from Cucurbita maxima L. endosperm was reinvestigated using incubation conditions different from those employed in previous work. The metabolism of GA12 yielded GA13, GA43 and 12α-hydroxyGA43 as major products, GA4, GA37, GA39, GA46 and four unidentified compounds as minor products. The intermediates GA15, GA24 and GA25 accumulated at low protein concentrations. The structure of the previously uncharacterised 12α-hydroxyGA43 was inferred from its mass spectrum and by its formation from both GA39 and GA43. Gibberellin A39 and 12α-hydroxyGA43 were formed by a soluble 12α-hydroxylase that had not been detected before. Gibberellin A12-aldehyde was metabolised to essentially the same products as GA12 but with less efficiency. A new 13-hydroxylation pathway was found. Gibberellin A53, formed from GA12 by a microsomal oxidase, was converted by soluble 2-oxoglutarate-dependent oxidases to GA1 GA23, GA28, GA44, and putative 2β-hydroxyGA28. Minor products were GA19, GA20, GA38 and three unidentified GAs. Microsomal 13-hydroxylation (the formation of GA53) was suppressed by the cofactors for 2-oxoglutarate-dependent enzymes. Reinvestigation of the endogenous GAs confirmed the significance of the new metabolic products. In addition to the endogenous GAs reported by Blechschmidt et al. (1984, Phytochemistry 23, 553–558), GA1, GA8, GA25, GA28, GA36, GA48 and 12α-hydroxyGA43 were identified by full-scan capillary gas chromatography-mass spectrometry and Kovats retention indices. Thus both the 12α-hydroxylation and the 13-hydroxylation pathways found in the cell-free system operate also in vivo, giving rise to 12α-hydroxyGA43 and GA1 (or GA8), respectively, as their end products. Evidence for endogenous GA20 and GA24 was also obtained but it was less conclusive due to interference.
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Abbreviations
- BSA:
-
bovine serum albumin
- GAn :
-
gibberellin An
- DTT:
-
dithiothreitol
- GC-MS:
-
combined gas chromatography-mass spectrometry
- HPLC:
-
high-performance liquid chromatography
- MeTMSi:
-
methyl ester trimethylsilyl ether
- m/z :
-
mass to charge ratio
References
Blechschmidt, S., Castel, U., Gaskin, P., Hedden, P., Graebe, J.E., MacMillan, J. (1984) GC/MS analysis of the plant hormones in seeds of Cucurbita maxima. Phytochemistry 23, 553–558
Castel, U. (1980) Veränderungen im Hormonhaushalt von Kürbiskeimlingen während der Entwicklung und die biologische Aktivität von 12α-Hydroxygibberellin A4. Staatsexamensarbeit für das Lehramt an Gymnasien, Universität Göttingen
Gilmour, S.J., Bleecker, A.B., Zeevaart, J.A.D. (1987) Partial purification of gibberellin oxidases from spinach leaves. Plant Physiol. 85, 87–90
Graebe, J.E. (1972) The biosynthesis of gibberellin precursors in a cell-free system from Cucurbita pepo L. In: Plant growth substances 1970, pp. 151–157, Carr, D.J., ed. Springer, Berlin Heidelberg New York
Graebe, J.E. (1987) Gibberellin biosynthesis and control. Annu. Rev. Plant Physiol. 38, 419–465
Graebe, I.E., Lange, T. (1990) The dioxygenases in gibberellin biosynthesis after Gibberellin A12-aldehyde. In: Plant growth substances 1988, pp. 314–321, Pharis, R.P., Rood, S.B., eds. Springer, Berlin Heidelberg
Graebe, J.E., Bowen, D.H., MacMillan, J. (1972) The conversion of mevalonic acid into gibberellin A12-aldehyde in a cell-free system from Cucurbita pepo. Planta 102, 261–271
Graebe, J.E., Hedden, P., Gaskin, P., MacMillan, J. (1974a) Biosynthesis of gibberellins A12, A15, A24, A36 and A37 by a cellfree system from Cucurbita maxima. Phytochemistry 13, 1433–1440
Graebe, J.E., Hedden, P., Gaskin, P., MacMillan, J. (1974b) The biosynthesis of a C19-gibberellin from mevalonic acid in a cellfree system from a higher plant. Planta 120, 307–309
Graebe, J.E., Hedden, P., MacMillan, J. (1974c) Gibberellin biosynthesis: new intermediates in the Cucurbita system. In: Plant growth substances 1973, pp. 260–266, Sumiki, Y., ed. Hirokawa, Tokyo
Graebe, J.E., Hedden, P., Rademacher, W. (1980) Gibberellin biosynthesis. In: Gibberellins-Chemistry, physiology and use (Monograph 5), pp. 31–47, Lenton, J.R., ed. British Plant Growth Regulator Group, Wantage
Graebe, J.E., Lange, T., Pertsch, S., Stöckl, D. (1991) The relationship of different gibberellin biosynthetic pathways in Cucurbita maxima endosperm and embryos and the purification of a C-20 oxidase from the endosperm. In: Gibberellins, pp. 51–61, Takahashi, N., Phinney, B.O., MacMillan, J., eds. Springer, New York
Hedden, P. (1983) In vitro metabolism of gibberellins. In: The biochemistry and physiology of gibberellins, vol. 1, pp. 99–149, Crozier, A., ed. Praeger Press, New York
Hedden, P., Graebe, J.E. (1982) Cofactor requirements for the soluble oxidases in the metabolism of the C20-gibberellins. J. Plant Growth Regul. 1, 105–116
Hedden, P., Croker, S.J., Rademacher, W., Jung, J. (1989) Effects of the triazole plant growth retardant BAS 111. W on gibberellin levels in oilseed rape, Brassica napus. Physiol. Plant. 75, 445–451
Hedden, P., Graebe, J.E., Beale, M.H., Gaskin, P., MacMillan, J. (1984) The biosynthesis of 12α-hydroxylated gibberellins in a cell-free system from Cucurbita maxima endosperm. Phytochemistry 23, 569–574
Kamiya, Y., Graebe, J.E. (1983) The biosynthesis of all major pea gibberellins in a cell-free system from Pisum sativum. Phytochemistry 22, 681–689
Lange, T., Graebe, J.E. (1989) The partial purification and characterization of a gibberellin C-20 hydroxylase from immature Pisum sativum L. seeds. Planta 179, 211–221
Lange, T., Graebe, J.E. (1993) Enzymes of gibberellin synthesis. In: Methods in plant biochemistry, vol. 9, pp. 403–430, Lea, P.J., ed. Academic Press, London
Lange, T., Hedden, P., Graebe, J.E. (1993) Gibberellin biosynthesis in cell-free extracts from developing Cucurbita maxima embryos and the identification of new endogenous gibberellins. Planta 189, 350–358
Pharis, R.P., King, R.W. (1985) Gibberellins and reproductive development in seed plants. Annu. Rev. Plant Physiol. 36, 517–568
Reeve, D.R., Crozier, A. (1974) An assessment of gibberellin structure-activity relationships. J. Exp. Bot. 25, 431–445
Scopes, R.K. (1982) Protein purification. Springer, New York Berlin Heidelberg
Sedmak, J.J., Grossberg, S.E. (1977) A rapid, sensitive and versatile assay for protein using Coomassie Brilliant Blue G250. Anal. Biochem. 79, 544–552
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We thank Dr. Dietmar Stöckl (Pflanzenphysiologisches Institut und Botanischer Garten, Göttingen, FRG) and Mr. Mervyn J. Lewis (Long Ashton Research Station, Bristol, UK) for the GC-MS measurements and Mr. Paul Gaskin (Long Ashton Research Station, Bristol, UK) for assistance with mass-spectra interpretation. We also thank the Deutsche Forschungsgemeinschaft, FRG, for financial support, the Agricultural and Food Research Council, UK, for a Scientific Collaboration award as well as the Deutsche Akademische Austauschdienst and the British Council for a joint Academic Research Collaboration award.
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Lange, T., Hedden, P. & Graebe, J.E. Biosynthesis of 12α-and 13-hydroxylated gibberellins in a cell-free system from Cucurbita maxima endosperm and the identification of new endogenous gibberellins. Planta 189, 340–349 (1993). https://doi.org/10.1007/BF00194430
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DOI: https://doi.org/10.1007/BF00194430