Functional characterization of gibberellin oxidases from cucumber, Cucumis sativus L.

Cucurbits have been used widely to elucidate gibberellin (GA) biosynthesis. With the recent availability of the genome sequence for the economically important cucurbit Cucumis sativus, sequence data became available for all genes potentially involved in GA biosynthesis for this species. Sixteen cDNAs were cloned from root and shoot of 3-d to 7-d old seedlings and from mature seeds of C. sativus. Two cDNAs code for GA 7-oxidases (CsGA7ox1, and -2), five for GA 20-oxidases (CsGA20ox1, -2, -3, -4, and -5), four for GA 3-oxidases (CsGA3ox1, -2, -3, and -4), and another five for GA 2-oxidases (CsGA2ox1, -2, -3, -4, and 5). Their enzymatic activities were investigated by heterologous expression of the cDNAs in Escherichia coli and incubation of the cell lysates with C-14-labelled, D2-labelled, or unlabelled GA-substrates. The two GA 7-oxidases converted GA(12)-aldehyde to GA(12) efficiently. CsGA7ox1 converted GA(12) to GA(14), to 15 alpha-hydroxyGA(12), and further to 15 alpha-hydroxyGA(14). CsGA7ox2 converted GA(12) to its 12 alpha-hydroxylated analogue GA(111). All five GA 20-oxidases converted GA(12) to GA(9) as a major product, and to GA(25) as a minor product. The four GA 3-oxidases oxidized the C-19-GA GA(9) to GA(4) as the only product. In addition, three of them (CsGA3ox2, -3, and -4) converted the C-20-GA GA(12) to GA(14). The GA 2-oxidases CsGA2ox1, -2, -3, and -4 oxidized the C-19-GAs GA(9) and GA(4) to GA(34) and GA(51), respectively. CsGA2ox2, -3, and -4 converted GA(51) and GA(34) further to respective GA-catabolites. In addition to C-19-GAs, CsGA2ox4 also converted the C-20-GA GA(12) to GA(110). In contrast, CsGA2ox5 oxidized only the C-20 GA(12) to GA(110) as the sole product. As shown for CsGA20ox1 and CsGA3ox1, similar reactions were catalysed with 13-hydroxlyated GAs as substrates. It is likely that these enzymes are also responsible for the biosynthesis of 13-hydroxylated GAs in vivo that occur at low levels in cucumber. (C) 2013 Elsevier Ltd. All rights reserved.