ent-Kaurene biosynthesis in a cell-free system from wheat (Triticum aestivum L.) seedlings and the localisation of ent-kaurene synthetase in plastids of three species

Abstract

Abstract.A cell-free system capable of converting [-14C]geranylgeranyl diphosphate to ent- [14C]kaurene and to an unidentified acid-hydrolysable compound was obtained from the basal portions of 5-d-old shoots of wheat seedlings (Triticum aestivum L.). By means of marker enzyme activities, the synthesis of ent-kaurene and the unknown compound could be quantitatively assigned to a plastid fraction obtained by Percoll-gradient centrifugation of the homogenate. The enzyme activities were located within the plastids, probably in the stroma, because they withstood trypsin treatment of the intact plastids, and the plastids had to be broken to release the activity, which was then obtained in soluble form. Plastid 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 was 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 leucoplasts. We therefore believe that ent-kaurene synthesis may be limited to these categories. Mature chloroplasts from the wheat leaves did not contain ent-kaurene synthetase activity and did not yield the unknown component. Incorporation of [14C]geranylgeranyl diphosphate into ent-[14C]kaurene and the unknown component was assayed by high-performance liquid chromatography with on-line radiocounting. ent-[14C]Kaurene was identified by Kovats retention index and full mass spectra obtained by combined gas Abbreviations: ADH = alcohol dehydrogenase; AMO 1618 = 2'-isopropyl-4'-(trimethylammonium chloride)-5'-methylphenyl piperidine-1-carboxylate; BSA = bovine serum albumin; DTT = dithiothreitol; GA. = gibberellin A.; GAPDH = NADP+-glyceraldehyde 3-phosphate dehydrogenase; GC-MS = combined gas chromatography-mass spectrometry; GGPP = all trans-isomer of geranylgeranyl diphosphate; KS = ent-kaurene synthetase; MDH = malate dehydrogenase; MAA = mevalonate activating activity; SOR= shikimate oxidoreductase Correspondence to: J.E. Graebe; FAX: 49 (551)39 78 23 chromatography-mass spectrometry. The unknown component was first believed to be copalyl diphosphate, because it yielded a compound on acid hydrolysis, which migrated like copalol on high-performance liquid chromatography and gave a mass spectrum very similar to that of authentic copalol. However, differences in the mass spectrum and in retention time on capillary gas chromatography excluded identity with copalol. Furthermore, the unhydrolysed compound was not converted to ent-kaurene by a cell-free system from C. maxima endosperm as copalyl diphosphate would have been.

Synergistic Substrate Inhibition of ent-Copalyl Diphosphate Synthase: A Potential Feed-Forward Inhibition Mechanism Limiting Gibberellin Metabolism

“…secondary metabolism) is significantly less sensitive to Mg 21 substrate inhibition effects than the GA biosynthesis-specific AtCPS. However, any such biochemical regulation of CPS enzymatic activity must operate in concert with previously observed transcriptional (Silverstone et al, 1997) and posttranslational (Aach et al, 1995) regulatory mechanisms.…”

“…However, despite the enormous increase in flux toward photosynthetic pigment production through their common GGPP precursor (RodriguezConcepcion et al, 2004), relatively little increase in levels of GA metabolites is observed (Reid et al, 2002). Whereas CPS is preferentially expressed in nonphotosynthetic cell types (Silverstone et al, 1997) and its activity is associated with proplastids and seems to be essentially absent from chloroplasts (Aach et al, 1995), indicating transcriptional and posttranscriptional regulation during chloroplast development, we hypothesize that the kinetic properties of CPS reported here represent an additional regulatory mechanism limiting GA metabolism, at least in deetiolation, although potentially also more broadly in chloroplast development. Specifically, it has been demonstrated that light induces an increase in Mg 21 levels in plastids (Ishijima et al, 2003), along with up-regulation of the 2-Cmethyl-D-erythritol-4-P pathway that provides isoprenoid precursors for the production of GGPP in plastids (Carretero-Paulet et al, 2002;Rodriguez-Concepcion et al, 2004).…”

“…Notably, futile flux into GA metabolism seems to be suppressed because only relatively small increases are seen in the level of GA intermediates and catabolites (Reid et al, 2002), although CPS gene transcription is not affected by light (Chang et al, 2005). Whereas it has been reported that CPS is preferentially expressed in nonphotosynthetic cell types (Silverstone et al, 1997) and its enzymatic activity is associated with proplastids and is essentially absent from mature chloroplasts (Aach et al, 1995), presumably due to some level of posttranscriptional regulation during plastid development, no such regulatory mechanism has been reported during deetiolation. It also has been reported that CPS exhibits significant GGPP-dependent substrate inhibition (Frost and West, 1977;Kawaide et al, 2000), which has been suggested to play a role in regulating GA biosynthesis (Kawaide et al, 2000).…”

Terpenoids and Gibberellic Acids Interaction in Plants

“…In higher plants these reactions are catalyzed by two enzymes, CPP synthase formerly Kaurene synthase A and ent-Kaurene (K) synthase formerly Kaurene synthase B, renaming was suggested by Mcmillan 1979. Both enzymes were localized in isolated proplastids of meristematic shoot tissues, but not in mature chloroplasts of pea and wheat (Aachet al, 1995(Aachet al, , 1997 or of pumpkins endosperm (Simcox et al, 1975;Aach et al, 1995).…”

ent-Kaurene Synthase from the FungusPhaeosphaeria sp. L487

“…On the other hand, the two activities were successfully separated from a plant enzyme preparation from Marah macrocarpus by chromatographic methods (8). The two enzymes are possibly localized in plastids (9). Quite recently, genes encoding both enzymes have been cloned from plants: CPS from Arabidopsis (10), maize (11) and pea (12), and KS from pumpkin (13).…”