Allan K. Stobart

ISOLATION OF A DELTA 5-FATTY ACID DESATURASE GENE FROM MORTIERELLA ALPINA

Arachidonic acid (C20:4 Δ5,8,11,14) is a polyunsaturated fatty acid synthesized by the Δ5-fatty acid desaturation of di-homo-γ-linolenic acid (C20:3 Δ8,11,14). In mammals, it is known to be a precursor of the prostaglandins and the leukotrienes but it is also accumulated by the filamentous fungusMortierella alpina. We have isolated a cDNA encoding the Δ5-fatty acid desaturase from M. alpinavia a polymerase chain reaction-based strategy using primers designed to the conserved histidine box regions of microsomal desaturases, and confirmed its function by expression in the yeast Saccharomyces cerevisiae. Analysis of the lipids from the transformed yeast demonstrated the accumulation of arachidonic acid. The M. alpina Δ5-desaturase is the first example of a cloned Δ5-desaturase, and differs from other fungal desaturases previously characterized by the presence of an N-terminal domain related to cytochrome b 5.

The effect of kinetin on chlorophyll synthesis in ageing etiolated barley leaves exposed to light

Abstract Etiolated barley seedlings lose the ability to produce chlorophyll and soluble protein on exposure to light with increasing age. Similarly, the production of δ-aminolaevulinic acid-dehydratase and succinyl-CoA synthetase is decreased in older etiolated leaves exposed to light. The rate of protochlorophyllide 652 regeneration decreased well before the rates of exogenous δ-aminolaevulinic acid conversion to protochlorophyllide 632 was affected by ageing. Application of kinetin retarded these ageing symptoms in the etiolated leaves.

TOBACCO CYTOCHROME B5 : CDNA ISOLATION, EXPRESSION ANALYSIS AND IN VITRO PROTEIN TARGETING

A full-length clone encoding cytochromeb5 has been isolated from a tobacco leaf cDNA library in αgt11 by PCR using degenerate primers. This cDNA encodes a protein of 139 residues which exhibits a high degree of homology to other cytochromeb5s, the message for which is expressed predominantly in developing seeds and in pigmented flower tissue. In the developing tobacco seed the mRNA is abundant at very early stages (<10 days after flowering). Southern analysis indicated that more than one gene encodes cytochromeb5 in the tobacco genome.In vitro transcription and translation studies of the cDNA indicated that the protein inserts into the ER membrane by a non-SRP-mediated pathway and that theC-terminus of the protein is required for targeting and insertion.

The turnover of chlorophyll in greening wheat leaves

Abstract A method for the estimation of chlorophyll turnover in wheat leaves is presented. This is based on the inhibition of chlorophyll synthesis by treatment of the cut leaves with laevulinic acid (LA), a competitive inhibitor of δ-aminolaevulinic acid dehydratase. The turnover of chlorophyll in young, greening leaves, given short periods of light was a relatively rapid process. However, in seedlings exposed to light for longer periods the turnover became progressively slower, and was measured in days rather than hours.

The hormonal control of betacyanin synthesis in Amaranthus caudatus

Abstract Gibberellic acid (GA 3 ) inhibits amaranthin synthesis whereas the growth retardant, phosphon D, enhances pigment levels in A. caudatus seedlings exposed to light. No effect was observed on chlorophyll and carotenoid synthesis. Radioactive tyrosine and DOPA were incorporated into amaranthin. The specific activity of amaranthin synthesised in the presence of 14 C-tyrosine or 14 C-DOPA in seedlings treated with GA 3 is higher than water controls. The specific activity of pigment from phosphon D treated tissue is relatively low. GA 3 treated tissue has lower active tyrosine and DOPA pools compared to phosphon treated seedlings. Tyrosine and DOPA-oxidase activity increases in GA 3 treated and H 2 O control seedlings exposed to light. Kinetin stimulates the synthesis of amaranthin in dark-grown seedlings and this is not overcome by simultaneous GA 3 application. Dark-grown seedlings treated with different kinetin concentrations and incubated in 14 C-tyrosine synthesise radioactive amaranthin of similar specific activity. Kinetin treatment of dark-grown seedlings brings about an increased tyrosine and DOPA-oxidase activity. The results indicate that GA 3 controls the production and/or availability of tyrosine whereas kinetin can mimic light treatment and controls the utilisation of tyrosine probably by bringing about the synthesis or activation of tyrosine and DOPA-oxidase protein.

The effects of hormones and inhibitors on amaranthin synthesis in seedlings of Amaranthus tricolor.

SummaryBoth gibberellic acid and abscisic acid inhibit the light induced synthesis of amaranthin in Amaranthus tricolor seedlings. The auxin, indolyl-3-acetic acid has no effect. The protein/RNA inhibitors, cycloheximide and 8-azaguanine, also reduced the levels of amaranthin produced.

A gibberellin bioassay based on betacyanin production in Amaranthus caudatus seedlings

SummaryAmaranthus caudatus L. seedlings produce less amaranthin in the presence of gibberellic acid A3 (GA3). This phenomenon has been tested as a bioassay for gibberellic acid and has been compared to the bioassay based on lettuce hypocotyl extension. The inhibition of pigment synthesis is much more sensitive to GA3 at concentrations from 1 to 0.01 μg/ml. Of the gibberellins tested GA1, GA3, GA4, and GA7 are the most active in the described bioassay.

Biosynthesis of triacylglycerol in the filamentous fungus Mucor circinelloides

Lipid metabolism was studied in 2-d-old liquid cultures of Mucor circinelloides grown at 25 degrees C. Under these conditions, oil accumulated to 0.5 g l-1 with a gamma-linolenic acid content (gamma 18:3) of 60 mg l-1. The major labelled lipids in cultures incubated with [14C]acetate were triacylglycerol (TAG), phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The proportion of label declined in the phospholipids and increased in TAG with time. [14C]18:1 and [14C]18:2 rapidly appeared in PC and PE and later accumulated in [14C]gamma 18:3. TAG-synthesizing capacity was greatest in the microsomal membrane fraction, which accumulated high levels of phosphatidic acid in the presence of glycerol 3-phosphate and acyl-CoA substrates at pH 7.0. Further metabolism of phosphatidic acid to diacylglycerol and TAG was achieved by increasing the pH to 8.0. Lysophosphatidic acid: acyl-CoA acyltransferase (LPAAT) activity was particularly high and may have accounted for the rapid accumulation of phosphatidic acid in the membranes. The glycerol-3-phosphate: acyl-CoA acyltransferase (GPAAT) and LPAAT were non-specific for a range of saturated and unsaturated species of acyl-CoA although the GPAAT showed a marked selectivity for palmitoyl-CoA and the LPAAT for oleoyl- and linoleoyl-CoA. gamma-Linolenic acid was detected at all three positions of sn-TAG and was particularly enriched at the sn-3 position. The preparation of active in vitro systems (microsomal membranes) capable of the complete biosynthetic pathway for TAG assembly may be valuable in understanding the assembly of oils in future transgenic applications.

Mevalonate-activating enzymes in greening tissue cultures

Abstract Isopentenyl pyrophosphate (IPP) was identified as one of the products in assays of mevalonate-activating enzymes in acetone powders prepared from callus culture of Kalanchoe crenata . Mevalonate-5-phosphate (MVAP) and mevalonate-5-pyrophosphate (MVAPP) were the main products isolated from assay mixture. There was an increase in mevalonate-activating enzyme activity in illuminated tissue cultures in various stages of greening and this was associated with the development of chloroplasts.

Effect of 2,2′-bipyridyl on porphyrin synthesis in etiolated and light-treated barley leaves

Abstract The effects of 2,2′-bipyridyl on porphyrin formation differed in illuminated and dark-treated barley leaves. In the dark, bipyridyl treatment increased photoconvertible protochlorophyllide (Pchlide, P650) and decreased the protohaem content. The increase in Pchlide could not be wholly accounted for by a diversion of ‘substrate’ from protohaem synthesis. The rate of Pchlide regeneration was slightly higher in chelator treated leaves which suggests increased δ-aminolaevulinic acid (ALA) synthesis. Only small quantities of Mg-protoporphyrinmonomethylester (Mg-protoME) were detected in etiolated leaves treated with bipyridyl in the dark. Protochlorophyll (P630) synthesis from exogenously supplied ALA was lower in the chelator treatments. The results suggest that only when substantial quantities of ALA are being utilized in dark-grown leaves does a ‘metal’ become limiting in the bipyridyl treated leaves. In the light, bipyridyl inhibited chlorophyll synthesis, again suggesting that when substantial amounts of ALA were being utilized a ‘metal’ becomes rate limiting. Bipyridyl treatment also inhibited ALA production in light-treated leaves. The incorporation of glycine-[ 14 C] into ALA in the presence of bipyridyl was severely restricted compared to the incorporation of glutamate-[ 14 C]. The data suggest two pathways for ALA synthesis; the classical ALA-synthetase which utilizes glycine and is operative in dark-grown leaves and a second enzyme system, which uses glutamate, and is of quantitative importance in the light.