Cloning and Overproduction of Gibberellin 3-Oxidase in Hybrid Aspen Trees. Effects on Gibberellin Homeostasis and Development

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Abstract
To broaden our understanding of gibberellin (GA) biosynthesis and the mechanism whereby GA homeostasis is maintained in plants, we have investigated the degree to which the enzyme GA 3-oxidase (GA3ox) limits the formation of bioactive GAs in elongating shoots of hybrid aspen (Populus tremula × Populus tremuloides). We describe the cloning of a hybrid aspen GA3ox and its functional characterization, which confirmed that it has 3β-hydroxylation activity and more efficiently converts GA9 to GA4 than GA20 to GA1. To complement previous studies, in which transgenic GA 20-oxidase (GA20ox) overexpressers were found to produce 20-fold higher bioactive GA levels and subsequently grew faster than wild-type plants, we overexpressed an Arabidopsis GA3ox in hybrid aspen. The generated GA3ox overexpresser lines had increased 3β-hydroxylation activity but exhibited no major changes in morphology. The nearly unaltered growth pattern was associated with relatively small changes in GA1 and GA4 levels, although tissue-dependent differences were observed. The absence of increases in bioactive GA levels did not appear to be due to feedback or feed-forward regulation of dioxygenase transcripts, according to semiquantitative reverse transcription polymerase chain reaction analysis of PttGA20ox1, PttGA3ox1, and two putative PttGA2ox genes. We conclude that 20-oxidation is the limiting step, rather than 3β-hydroxylation, in the formation of GA1 and GA4 in elongating shoots of hybrid aspen, and that ectopic GA3ox expression alone cannot increase the flux toward bioactive GAs. Finally, several lines of evidence now suggest that GA4 has a more pivotal role in the tree hybrid aspen than previously believed.

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