ENT-KAURENE BIOSYNTHESIS IN A CELL-FREE SYSTEM FROM WHEAT (TRITICUM-AESTIVUM L) SEEDLINGS AND THE LOCALIZATION OF ENT-KAURENE SYNTHETASE INPLASTIDS OF 3 SPECIES

A cell-free system capable of converting [C-14]geranylgeranyl diphosph ate to ent-[C-14]kaurene and to an unidentified acid-hydrolysable comp ound was obtained from the basal portions of 5-d-old shoots of wheat s eedlings (Triticum aestivum L.). By means of marker enzyme activities, the synthesis of ent-kaurene and the unknown compound could be quanti tatively assigned to a plastid fraction obtained by Percoll-gradient c entrifugation of the homogenate. The enzyme activities were located wi thin the plastids, probably in the stroma, because they withstood tryp sin treatment of the intact plastids, and the plastids had to be broke n to release the activity, which was then obtained in soluble form. Pl astid membranes had no activity. Plastid stroma preparations obtained from pea (Pisum sativum L.) shoot tips and pumpkin (Cucurbita maxima L .) endosperm also yielded ent-kaurene synthetase activity, but did not form the unknown compound. The exact nature of the active plastids wa s not ascertained, but the use of methods for proplastid isolation was essential for full activity, and the active tissues are all known to contain high proportions of proplastids, developing chloroplasts or le ucoplasts. We therefore believe that ent-kaurene synthesis may be limi ted to these categories. Mature chloroplasts from the wheat leaves did not contain ent-kaurene synthetase activity and did not yield the unk nown component. Incorporation of [C-14]geranylgeranyl diphosphate into ent-[C-14]kaurene and the unknown component was assayed by high-perfo rmance liquid chromatography with on-line radiocounting. ent-[C-14]Kau rene was identified by Kovats retention index and full mass spectra ob tained by combined gas chromatography-mass spectrometry. The unknown c omponent was first believed to be copalyl diphosphate, because it yiel ded a compound on acid hydrolysis, which migrated like copalol on high -performance liquid chromatography and gave a mass spectrum very simil ar to that of authentic copalol. However, differences in the mass spec trum and in retention time on capillary gas chromatography excluded id entity with copalol. Furthermore, the unhydrolysed compound was not co nverted to ent-kaurene by a cell-free system from C. maxima endosperm as copalyl diphosphate would have been.