Table 1.
In vitro activity of recombinant grapevine GA oxidases Escherichia coli lysates expressing recombinant GA oxidases (column 1) were incubated with a GA substrate (column 2) for 4h at 30 °C. Each recombinant protein was tested at least three times with each of the following precursors: GA1, GA4, GA20, GA9, GA12, and GA53. The following GAs were detected in the reaction by UPLC-MS/MS: GA1, GA4, GA8, GA9, GA20, GA34, GA29, GA51, GA12, and GA53. The observed product is reported in column 3, and the corresponding activity in column 4. Recombinant GA2ox proteins were also tested for GA20ox activity with GA12 and GA53, and no product among those indicated was detected in multiple experiments.
| Protein | Substrate | Products | Activity |
|---|---|---|---|
| GST-VvGA20ox1 | GA12 | GA9 | GA20ox |
| GA53 | GA20 | GA20ox | |
| GST-VvGA20ox2 | GA12 | GA9 | GA20ox |
| GA53 | None | ||
| GST-VvGA20ox3 | GA12 | GA9 | GA20ox |
| GA53 | GA20 | GA20ox | |
| GST-VvGA3ox1 | GA9 | GA4 | GA3ox |
| GA20 | GA1 | GA3ox | |
| GA4 | — | GA3ox | |
| GA1 | — | ||
| GST-VvGA3ox2 | GA9 | GA4 | GA3ox |
| GA20 | GA1 | GA3ox | |
| GA4 | — | ||
| GA1 | — | ||
| GST-VvGA3ox3 | GA9 | GA4 | GA3ox |
| GA20 | GA1 | GA3ox | |
| GA4 | — | ||
| GA1 | — | ||
| VvGA2ox2 | GA9 | GA51 | GA2ox |
| GA20 | GA29 | GA2ox | |
| GA4 | GA34 | GA2ox | |
| GA1 | GA8 | GA2ox | |
| VvGA2ox3 | GA9 | — | |
| GA20 | — | ||
| GA4 | GA34 | GA2ox | |
| GA1 | GA8 | GA2ox | |
| VvGA2ox4 | GA9 | GA51 | GA2ox |
| GA20 | GA29 | GA2ox | |
| GA4 | GA34 | GA2ox | |
| GA1 | GA8 | GA2ox | |
| GST-VvGA2ox5 | GA9 | GA51 | GA2ox |
| GA20 | GA29 | GA2ox | |
| GA4 | GA34 | GA2ox | |
| GA1 | GA8 | GA2ox | |
| GST-VvGA2ox7 | GA9 | GA51 | GA 2ox |
| GA20 | — | ||
| GA4 | GA34 | GA2ox | |
| GA1 | GA8 | GA2ox |