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

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 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.Abbreviations ADH alcohol dehydrogenase - AMO 1618 2isopropyl-4-(trimethylammoniumchloride)-5-methylphenyl piperi-dine-1-carboxylate - BSA bovine serum albumin - DTT dithioth-reitol - GA_n gibberellin A_n - GAPDH NADP⁺-glyceraldehyde 3-phosphate dehydrogenase - GC-MS combined gas chromatography-mass spectrometry - GGPP all trans-isomer of geranyl-geranyl diphosphate - KS ent-kaurene synthetase - MDH malate dehydrogenase - MAA mevalonate activating activity - SOR shikimate oxidoreductase We thank Mrs. Gudrun Bodtke and Mrs. Dorothee Dasbach for able technical assistance, Prof. L.N. Mander (Australian National University, Canberra, Australia) for ent-[²H₂]kaurene and Dr. Yuji Kamiya (RIKEN, Saitama, Japan) for geranylgeraniol and copalol. The work was supported by the Deutsche Forschungsgemeinschaft.     

Gibberellins and pea seed development

The gibberellin (GA)-biosynthesis mutations, lh ⁱ , ls and Ie ⁵⁸³⁹ have been used to investigate the role(s) of the GAs in seed development of the garden pea (Pisum sativum L.). Seeds homozygous for lh ⁱ possess reduced GA levels, are more likely to abort during development, and weigh less at harvest, compared with wild-type seeds due to expression of the lh ⁱ mutation in the embryo and/ or endosperm. Compared with wild-type seeds, the lh ⁱ mutation reduces endogenous GA₁ and gibberellic acid (GA₃) levels in the embryo/endosperm a few days after anthesis and fertilizing lh ⁱ plants with wild-type pollen dramatically increases GA₁ and GA₃ levels in the embryo/ endosperm and restores normal seed development. By contrast, the ls and le ⁵⁸³⁹ mutations do not appear to reduce GA levels in the embryo/endosperm of seeds a few days after anthesis, and do not affect embryo or endosperm development. However, both the ls and lh ⁱ mutations substantially reduce endogenous GA levels in embryos at contact point (the first day the liquid endosperm disappears). Levels of GAs in seeds from crosses involving the ls and lh ⁱ mutations suggest that GAs are synthesised in both the embryo/endosperm and testa and that the expression of ls depends on the tissue and developmental stage examined. These results suggest that GAs (possibly GA₁ and/or GA₃) play an important role early in pea seed development by regulating the development of the embryo and/or endosperm. By contrast, the high GA levels found in wild-type seeds at contact point (and beyond) do not appear to have a physiological role in seed development.Abbreviations GA_n gibberellin A_n - DAA days after anthesis - WT wild-type We thank Noel Davies, Katherine McPherson and Peter Bobbi for technical assistance, Professor L. Mander (ANU, Canberra) for dideuterated GA standards, and the Australian Research Council and Frontier Research Program, The Institute of Physical and Chemical Research (RIKEN, Japan), for financial support.     

Developmental regulation of aldoxime formation in seedlings and mature plants of Chinese cabbage (Brassica campestris ssp. pekinensis) and oilseed rape (Brassica napus): Glucosinolate and IAA biosynthetic enzymes

The first steps in the biosynthesis of glucosinolates and indole-3-acetic acid (IAA) in oilseed rape (Brassica napus L.) and Chinese cabbage (Brassica campestris ssp. pekinensis) involve the formation of aldoximes. In rape the formation of aldoximes from chain-extended amino acids, for aromatic and aliphatic glucosinolate biosynthesis, is catalysed by microsomal flavin-containing monooxygenases. The formation of indole-3-aldoxime from l-tryptophan, the potential precursor of both indole-3-acetic acid and indolyl-glucosinolates, is catalysed by several microsomal peroxidases. The biosynthesis of glucosinolates and indole-3-acetic acid was shown to be under developmental control in oilseed rape and Chinese cabbage. No monooxygenase activities were detected in cotyledons or old leaves of either species. The highest monooxygenase activities were found in young expanding leaves; as the leaves reached full expansion and matured the activities decreased rapidly. The indole-aldoxime-forming activity was found in all of the tissues analysed, but there was also a clear decrease in foliar activity with maturity in leaves of rape and Chinese cabbage. Partial characterisation of the Chinese cabbage monooxygenases showed that they have essentially identical properties to the previously characterised rape enzymes; they are not cytochrome P450-type enzymes, but resemble flavin-containing monooxygenases. No monooxygenase inhibitors were detected in microsomes prepared from either cotyledons or old leaves.Abbreviations DHMet dihomomethionine - FMO flavin-containing monooxygenase - HPhe homophenylalanine - IAA indole-3-acetic acid - l-Phe l-phenylalanine - l-Trp l-tryptophan - MO monooxygenase - IAALD indole-3-acetaldehyde - IAOX indole-3-aldoxime - THMet trihomomethionine     

Expression of ferredoxin-dependent glutamate synthase in dark-grown pine seedlings

Pine seedlings are able to accumulate chlorophylls and develop green plastids in a light-independent manner. In this work, we have characterized ferredoxin-dependent glutamate synthase (EC 1.4.7.1; Fd-GOGAT), a key enzyme in nitrogen interconversion during this process. Fd-GOGAT has been purified about 170-fold from cotyledons of maritime pine (Pinus pinaster). As occurs in angiosperms, the native enzyme is a single polypeptide with an apparent molecular mass of 163–168 kDa that is confined to the chloroplast stroma. Polyclonal antibodies generated against the purified enzyme were used to immunoscreen a gt11 expression library from Scots pine (Pinus sylvestris) seedlings and partial cDNA clones were isolated and characterized. The clone with the longest cDNA insert (pGOP44) contained the codification for the C-terminal (550 amino acids) of the pine Fd-GOGAT polypeptide. Immunological cross-reactivity and comparative amino sequence analysis revealed that Fd-GOGAT is a well conserved protein in higher plants. Western blot analyses showed that protein was expressed in chloroplast-containing pine tissues and this expression pattern was not affected by exogenously supplied nitrogen. Fd-GOGAT mRNA, polypeptide and enzyme activity accumulated in substantial amounts in dark-grown pine seedlings. The presence of a functional Fd-GOGAT may be important to provide the required glutamate for the biosynthesis of nitrogen compounds during chloroplast biogenesis in the dark.     

Influence of gibberellin biosynthesis inhibitors on stem elongation and floral initiation on in vitro chicory root explants under dark and light conditions

Root explants of chicory (Cichorium intybus L.) were cultured in vitro under continuous light or darkness. On a standard medium (no plant growth regulators added), flowering-stems were initiated under continuous light while under continuous dark, vegetative-stems were formed. Different types of GA (gibberellin) biosynthesis inhibitors were added to the culture medium. Paclobutrazol and compounds belonging to the group of cyclohexanetriones clearly reduced flowering-stem growth under light conditions and vegetative-stem growth under dark conditions. Under light conditions, flower bud initiation was not affected. These and other results suggest that GA₁ may be synthesized during the in vitro culture period and that it controls flowering-stem growth but not floral initiation.Abbreviations CCC chlormequat chloride - GA gibberellin - LAB 198 999 3,5-dioxo-4-butyryl-cyclohexane carboxylic acid ethyl ester - BAS 111..W 1-phenoxy-3-(1H-1,2,4-triazol-1-yl)-4-hydroxy-5,5-dimethylhexane     

Alterations in Phosphatidylcholine Metabolism of Stretch-Injured Cultured Rat Astrocytes

Abstract: The primary objective of this study was to determine the influence of stretch-induced cell injury on the metabolism of cellular phosphatidylcholine (PC). Neonatal rat astrocytes were grown to confluency in Silastic-bottomed tissue culture wells in medium that was usually supplemented with 10 µM unlabeled arachidonate. Cell injury was produced by stretching (5–10 mm) the Silastic membrane with a 50-ms pulse of compressed air. Stretch-induced cell injury increased the incorporation of [³H]choline into PC in an incubation time- and stretch magnitude-dependent manner. PC biosynthesis was increased three- to fourfold between 1.5 and 4.5 h after injury and returned to control levels by 24 h postinjury. Stretch-induced cell injury also increased the activity of several enzymes involved in the hydrolysis [phospholipase A₂ (EC 3.1.1.4) and C (PLC; EC 3.1.4.3)] and biosynthesis [phosphocholine cytidylyltransferase (PCT; EC 2.7.7.15)] of PC. Stretch-induced increases in PC biosynthesis and PCT activity correlated well (r = 0.983) and were significantly reduced by pretrating (1 h) the cells with an iron chelator (deferoxamine) or scavengers of reactive oxygen species such as superoxide dismutase and catalase. The stretch-dependent increase in PC biosynthesis was also reduced by antioxidants (vitamin E, vitamin E succinate, vitamin E phosphate, melatonin, and n-acetylcysteine). Arachidonate-enriched cells were more susceptible to stretch-induced injury because lactate dehydrogenase release and PC biosynthesis were significantly less in non-arachidonate-enriched cells. In summary, the data suggest that stretch-induced cell injury is (a) a result of an increase in the cellular level of hydroxyl radicals produced by an iron-catalyzed Haber-Weiss reaction, (b) due in part to the interaction of oxyradicals with the polyunsaturated fatty acids of cellular phospholipids such as PC, and (c) reversible as long as the cell's membrane repair functions (PC hydrolysis and biosynthesis) are sufficient to repair injured membranes. These results suggest that stretch-induced cell injury in vitro may mimic in part experimental traumatic brain injury in vivo because alterations in cellular PC biosynthesis and PLC activity are similar in both models. Therefore, this in vitro model of stretch-induced injury may supplement or be a reasonable alternative to some in vivo models of brain injury for determining the mechanisms by which traumatic cell injury results in cell dysfunction.     

Posttranslational Maturation of the Prohormone Convertase SPC3 In Vitro

Abstract: The subtilisin-like prohormone convertase SPC3 is likely to play a role in the biosynthesis of a variety of biologically active peptides. SPC3 undergoes a series of posttranslational processing events during its biosynthesis. Multiple forms have been identified that show varying degrees of truncation at the carboxyl terminus. In this study we show that the 86-kDa form of recombinant SPC3 with an intact carboxyl terminus can undergo rapid carboxyl-terminus truncation to produce a 64-kDa form. We have defined the optimal conditions for carboxyl-terminus truncation in vitro. The carboxyl-terminus truncation reaction was less calcium sensitive, active over a broader pH range, and showed differences in inhibitor sensitivity compared with the enzymatic activities of full-length and truncated forms of SPC3 toward a fluorescent peptide substrate. Increases in enzymatic activity of 86-kDa SPC3 were also measured over a time frame consistent with conversion to the 64-kDa form. However, similar specific activities for both forms of the enzyme suggest such activity increases may not be due to carboxyl-terminus truncation. The different enzymatic properties of the major molecular forms of SPC3 highlight the importance of understanding the molecular events regulating carboxyl-terminal processing of this endoprotease.