Gibberellin metabolism in cell-free extracts from spinach leaves in relation to photoperiod

Cell-free extracts capable of converting [14C]-labeled gibberellins (GAs) were prepared from spinach (Spinacia oleracea L.) leaves. [14C]-labeled GAs, prepared enzymically from [14C]mevalonic acid, were incubated with these extracts, and products were identified by gas chromatography-mass spectrometry. The following pathway was found to operate in extracts from spinach leaves grown under long day (LD) conditions: $\text{GA}_{12}$ → $\text{GA}_{53}$ → $\text{GA}_{44}$ → $\text{GA}_{19}$ → $\text{GA}_{20}$. The pH optima for the enzymic conversions of $[{}^{14}\text{C}]\text{GA}_{53}$, $[{}^{14}\text{C}]\text{GA}_{44}$ and $[{}^{14}\text{C}]\text{GA}_{19}$ were approximately 7.0, 8.0, and 6.5, respectively. These three enzyme activities required Fe2+, α-ketoglutarate and O2 for activity, and ascorbate stimulated the conversion of $[{}^{14}\text{C}]\text{GA}_{53}$ and $[{}^{14}\text{C}]\text{GA}_{19}$. Extracts from plants given LD or short days (SD) were examined, and enzymic activities were measured as a function of exposure to LD, as well as to darkness following 8 LD. The results indicate that the activities of the enzymes oxidizing $\text{GA}_{53}$ and $\text{GA}_{19}$ are increased in LD and decreased in SD or darkness, but that the enzyme activity oxidizing $\text{GA}_{44}$ remains high irrespective of light or dark treatment. This photoperiodic control of enzyme activity is not due to the presence of an inhibitor in plants grown in SD. These observations offer an explanation for the higher $\text{GA}_{20}$ content of spinach plants in LD than in SD.