Marta Goodrich-Tanrikulu

The plant growth regulator methyl jasmonate inhibits aflatoxin production by Aspergillus flavus.

Aflatoxins are highly toxic and carcinogenic compounds produced by certain Aspergillus species on agricultural commodities. The presence of fatty acid hydroperoxides, which can form in plant material either preharvest under stress or postharvest under improper storage conditions, correlates with high levels of aflatoxin production. Effects on fungal growth and aflatoxin production are known for only a few of the numerous plant metabolites of fatty acid hydroperoxides. Jasmonic acid (JA), a plant growth regulator, is a metabolite of 13-hydroperoxylinolenic acid, derived from alpha-linolenic acid. The volatile methyl ester of JA, methyl jasmonate (MeJA), is also a plant growth regulator. In this study we report the effect of MeJA on aflatoxin production and growth of Aspergillus flavus. MeJA at concentrations of 10(-3)-10(-8) M in the growth medium inhibited aflatoxin production, by as much as 96%. Exposure of cultures to MeJA vapour similarly inhibited aflatoxin production. The amount of aflatoxin produced depended on the timing of the exposure. MeJA treatment also delayed spore germination and inhibited the production of a mycelial pigment. These fungal responses resemble plant jasmonate responses.

Changes in fatty acid composition of Neurospora crassa accompany sexual development and ascospore germination.

Fatty acid composition was determined during several stages of sexual development in Neurospora crassa. Triacylglycerol was the predominant acyl lipid in cultures undergoing sexual development. The absolute amounts of triacylglycerol in fertilized cultures varied over time, in contrast to control (unfertilized or mock-fertilized) cultures, in which the amount of triacylglycerol decreased linearly with age. In cultures competent to undergo sexual development, alpha-linoleate was the predominant fatty acid, ranging from 53 to 65% of the total fatty acid mass. alpha-Linolenate was 3% or less of the total fatty acid, in marked contrast to the much higher levels (10-35%) typically reported for vegetative cultures. In fertilized cultures, a slightly higher mass ratio of oleate was also observed. This difference was due to the developing asci: in developing asci and mature ascospores, oleate replaced alpha-linoleate as the predominant fatty acid (45 to 50% of the total). In germinating ascospores, the fatty acid composition approached that of vegetative cultures 6 h after inducing germination by heat activation. These results show that the fatty acid composition of sexual tissues of Neurospora differs substantially from the composition of asexual tissues, and that extensive changes in fatty acid composition correlate with several events in the sexual stage of development.

Characterization of Oleoyl-12-Hydroxylase in Castor Microsomes Using the Putative Substrate, 1-Acyl-2-Oleoyl-sn-Glycero-3-Phosphocholine

We have characterized the oleoyl-12-hydroxylase in the microsomal fraction of immature castor bean using the putative substrate, 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine (2-oleoyl-PC). Previous characterizations of this enzyme used oleoyl-CoA as substrate and relied on the enzyme transferring oleate from oleoyl-CoA to lysophosphatidylcholine to form 2-oleoyl-PC (acyl-CoA:lysophosphatidylcholine acyltransferase) in addition to oleoyl-12-hydroxylase. The present assay system and characterization use 2-oleoyl-PC as substrate (oleoyl-12-hydroxylase alone). Use of the actual substrate for assay purposes is important for the eventual purification of the oleoyl-12-hydroxylase. Ricinoleate (product of oleoyl-12-hydroxylase) and linoleate (product of oleoyl-12-desaturase) were identified as metabolites of oleate of 2-oleoyl-PC by high-performance liquid chromatography and gas chromatography/mass spectrometry. The activity of oleoyl-12-hydroxylase in the microsomal fraction reached a peak about 44 d after anthesis of castor, while the activity of oleoyl-12-desaturase reached a peak about 23 d after anthesis. The optimal temperature for the oleoyl-12-hydroxylase was about 22.5°C, and the optimal pH was 6.3. Catalase stimulated oleoyl-12-hydroxylase while bovine serum albumin and CoA did not activate oleoyl-12-hydroxylase. The phosphatidylcholine analogue, oleoyloxyethyl phosphocholine, inhibited the activity of oleoyl-12-hydroxylase. These results further support the hypothesis that the actual subtrate of oleoyl-12-hydroxylase is 2-oleoyl-PC.

Fatty acid biosynthesis in novel ufa mutants of Neurospora crassa

New mutants of Neurospora crassa having the ufa phenotype have been isolated. Two of these mutants, like previously identified ufa mutants, require an unsaturated fatty acid for growth and are almost completely blocked in the de novo synthesis of unsaturated fatty acids. The new mutations map to a different chromosomal location than previously characterized ufa mutations. This implies that at least one additional genetic locus controls the synthesis of unsaturated fatty acids in Neurospora.

Ricinoleate biosynthesis in castor microsomes

Abstract Oleoyl-12-hydroxylase is the enzyme responsible for ricinoleate biosynthesis in castor (Ricinus communis). The hydroxylase introduces the mid-chain hydroxyl group, resulting in a fatty acid with numerous chemical uses. To determine conditions for high levels of ricinoleate production, it is important to characterize the hydroxylase activity in isolation from interfering activities. However, progress in purifying this enzyme has been limited by the inability to solubilize and reconstitute the enzyme activity from microsomes. By using low levels of microsomal protein (12–35 μg) and following conversion of 14C-oleoyl-CoA to 14C-ricinoleate, the reaction is linear as a function of time and the rate is directly proportional to amount of protein. Moreover, at the lower levels of microsomal protein, microsomes treated under conditions that solubilize the hydroxylase recover full activity. This represents an important step in developing the ability to reconstitute the hydroxylase activity. Other factors involved in the high level of ricinoleate production by castor are the steady increase in hydroxylase activity throughout development and the decline in oleoyl desaturase. The glycol lipid oleoyloxyethyl phosphocholine is an effective inhibitor of hydroxylase activity, and should prove to be a useful tool in following the movement of labelled fatty acids through lipid pools.

Characterization of Neurospora crassa mutants isolated following repeat-induced point mutation of the beta subunit of fatty acid synthase

Neurospora crassacel-2 mutants were isolated following repeat-induced point mutation using part of the gene encoding β-fatty acid synthase. These mutants are phenotypically less leaky than cel-1, which has a defective α-fatty acid synthase. The cel-2 mutant had a strict fatty acid (16:0) requirement for growth, and syn-thesized less fatty acid de novo than cel-1. Unlike cel-1, cel-2 has impaired fertility, and homozygous crosses are infertile, suggesting a low but strict requirement for fatty acid synthesis during sexual development. Like cel-1, cel-2 synthesized unusually high levels of the polyunsaturate 18:3Δ9,12,15, and elongated 18:2Δ9,12 and 18:3Δ9,12,15 to 20:2Δ11,14 and 20:3Δ11,14,17, respectively. These fatty acids are not synthesized by wild-type, except following treatment with cerulenin (a fatty acid synthase inhibitor), demonstrating that inhibition of fatty acid biosynthesis results in a relative increase in both fatty acid desaturation and elongation activity.

Novel Neurospora crassa mutants with altered synthesis of polyunsaturated fatty acids

Five new mutants of Neurospora crassa that require supplementation with unsaturated fatty acids have been isolated. The mutants, designated pfa, are impaired in the synthesis of the polyunsaturated fatty acids α-linoleic or α-linolenic acid, but are able to synthesize oleic acid. The pfa mutants are thus distinct from previously described ufa mutants, which are unable to synthesize oleic acid. The five pfa mutants map to distinct loci, and have characteristic patterns of incorporation of [14C]acetate and [14C]oleate into their fatty acids.

A systematic survey of the fatty acid composition of Neurospora strains

Numerous studies have examined aspects of lipid metabolism in Neurospora crassa, N. tetrasperma and N. sitophila, but little systematic comparison has been done on lipids of the different species. Most obviously missing, however, is comparison of the fatty acid composition of lipids among Neurospora species. Fatty acid composition (especially the production of particular polyunsaturated fatty acids) is often a key factor in genus or species identification, particularly in bacteria, but also in other fungi such as Aspergillus, Penicillium and Mortierella (Kock and Botha 1998 In Frisvad et al. (eds), Chemical Fungal Taxonomy, Marcel Dekker, NY, p. 219-246). Until this study, reports of the fatty acid composition of Neurospora lipids have been limited to a few laboratory strains of N. crassa. Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This regular paper is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol46/iss1/3

Metabolism of ricinoleate by Neurospora crassa.

Neurospora crassa is a potential expression system for evaluating fatty-acid-modifying genes from plants producing uncommon fatty acids. One such gene encodes the hydroxylase that converts oleate to ricinoleate, a fatty acid with important industrial uses. To develop this expression system, it is critical to evaluate the metabolism and physiological effects of the expected novel fatty acid(s). We therefore examined effects of ricinoleate on lipid biosynthesis and growth of N. crassa. Ricinoleate inhibited growth and reduced levels of phospholipids and of 2-hydroxy fatty acids in glycolipids, but led to increased lipid accumulation on a mass basis. To evaluate incorporation and metabolism of ricinoleate, we followed the fate of 14 μM–3 mM [1-14C]ricinoleate. The fate of the [14C]ricinoleate was concentration-dependent. At higher concentrations, ricinoleate was principally incorporated into triacylglycerols. At lower concentrations, ricinoleate was principally metabolized to other compounds. Thus, N. crassa transformants expressing the hydroxylase gene can be detected if the level of hydroxylase expression allows both growth and ricinoleate accumulation.

Isotope discrimination by the stearoyl desaturase of Neurospora crassa

In wild type Neurospora crassa, we previously observed an unexpectedly low proportion of label in unsaturated fatty acids that should have been derived from deuterated palmitate (16:0). Interpretation of whether the position of deuteration could account for these results was inconclusive, because wild type had a low proportion of total fatty acids derived from the deuterated supplement. The N. crassa cel-1 strain is ideally suited for studies of fatty acid enrichment, because cel-1 must obtain its fatty acids exogenously. We supplemented cel-1 with 16:0s deuterated at different positions. With each of the labelled 16:0s, cel-1 desaturated the stearate (18:0) derived from the 16:0 more efficiently than wild type. However, when the 16:0 was deuterated at the site of future desaturation, we indeed observed effects on fatty acid composition and label distribution consistent with inhibition of the 18:0 desaturase. Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This regular paper is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol46/iss1/4