Daylength and spatial expression of a gibberellin 20-oxidase isolated from hybrid aspen (Populus tremula L. x P-tremuloides Michx.)

Physiologically active gibberellins (GAs) are key regulators of shoot growth in trees. To investigate this mechanism of GA-controlled growth in hybrid aspen, we cloned cDNAs encoding gibberellin 20-oxidase (GA 20-oxidise), a key, highly regulated enzyme in the biosynthesis of GAs. Clones were isolated from leaf and cambium cDNA libraries using probes generated by polymerise chain reaction, based on conserved domains of GA 20-oxidises. Upon expression in Escherichia coli, the GST-fusion protein was shown to oxidise GA(12) as well as oxidising the 13-hydroxylated substrate GA(53), successively to GA(9) and GA(20), respectively. The gene PttGA20ox1 was expressed in meristematic cells and growing tissues such as expanding internodes, leaves and roots. The expression was negatively regulated by both GA(4) and overexpression of phytochrome A. RNA analysis also showed that the expression was down-regulated in late-expanding leaf tissue in response to short days (SDs). Actively growing tissues such as early elongating internodes, petioles and leaf blades had the highest levels of C-19-GAs. Upon transfer to SDs an accumulation of GA(19) was observed in early elongating internodes and leaf blades. The levels of C-19-GAs were also to some extent changed upon transfer to SDs. The levels of GA(20) were down-regulated in internodes, and those of GA(1) were significantly reduced in early expanding leaf blades. In roots the metabolites GA(19) and GAs decreased upon shifts to SDs, while GA(20) accumulated slightly. The down-regulation of GA 20-oxidise activity in response to SDs was further indicated by studies of [C-14]GA(12) metabolism in shoots, demonstrating that the substrate for GA 20-oxidise, [C-14]GA(53), accumulates in SDs.