Edward W. Underhill

Storage-protein regulation and lipid accumulation in microspore embryos of Brassica napus L.

Embryos derived in vitro from isolated microspores of Brassica napus L. were compared with their zygotic counterparts. Parameters investigated included storage-protein accumulation and gene expression, fattyacid composition, storage-lipid biosynthesis, and the appearance of oil-body proteins. The microspore embryos accumulate storage-protein and show increases in levels of their transcripts during the torpedo stage. These embryos were sensitive to abscisic acid (ABA) with respect to accumulation of storage-protein mRNA and oil-body proteins. Post-transcriptional regulation of cruciferin accumulation is indicated by a disparity between ABA-enhanced transcript accumulation and a less marked effect at the level of protein accumulation. To investigate storage-lipid profiles, two cultivars of Brassica napus, Reston and Topas, were used. The former accumulates major quantities of C20 (11.2%) and C22 (39.9%) fatty acids in its seeds, the latter predominantly C18 fatty acids. The higher-molecular-weight fatty acids (>C18) normally occur only in seeds and were used as biochemical markers for seed-specific metabolism in microspore embryos. Microspore embryos from Reston were found to accumulate C20 (10.6%) and C22 (31.2%) fatty acids after 35 d in culture at levels and proportions comparable to those found in seeds. Similarly, microspore embryos of Topas had a fatty-acid profile similar to that of mature Topas seed. Activities of enzymes involved in the accumulation of storage lipids (erucoyl-CoA synthetase [EC 6.2.1.3], erucoyl-CoA thioesterase [EC 3.1.2.2] and erucoyl-CoA acyltransferase [EC 2.3.1.15 or EC 2.3.1.20]) were detected in torpedostage microspore embryos. Their specific activities were higher than have been reported to date for analogous preparations from zygotic embryos of B. napus. The similarities in storage-lipid and protein composition of these embryos to their zygotic counterparts, along with their sensitivity to ABA, indicate that microspore embryos might be exploited to facilitate studies of biochemistry and gene regulation in oilseeds.

A simple enzymatic method for the preparation of radiolabeled erucoyl-CoA and other long-chain fatty acyl-CoAs and their characterization by mass spectrometry

A simple two-step method for the biosynthesis of radiolabeled erucoyl-coenzyme A of high specific activity and other long-chain fatty acyl-coenzyme A (acyl-CoA) thioesters is reported. 1-14C-labeled erucic and oleic acids, as well as unlabeled ricinoleic and nervonic acids, were incubated at 35 degrees C with coenzyme A in the presence of ATP, MgCl2, and acyl-CoA synthetase (EC 6.2.1.3) from Pseudomonas spp. to yield the corresponding CoA thioesters. Following incubation, each thioester was purified by rapid passage through a disposable reverse-phase C18 extraction column. The overall yields were greater than 90% and the purities greater than 95%, based on the distribution of radioactivity, and chromatographic and spectral properties. Fast ion bombardment-mass spectrometry was employed to confirm the structures of the various acyl-CoAs.

Identification of chemical imposition stimulants for the diamondback moth, Plutella xylostella, present in three species of Brassicaceae

Plant chemicals in three cruciferous crop species, Brassica napus L., B. juncea (L.) Czerniak, and Sinapis alba L., that stimulate oviposition in the diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae) were investigated in laboratory bioassays. Aerial portions of 4‐ to 6‐week‐old plants were extracted and fractionated using ion‐exchange liquid chromatography. The oviposition stimulants were identified as glucosinolates, which are found in all Brassicaceae species. Activity of extracts was largely eliminated by treatment with myrosinase or sulphatase, enzymes which degrade glucosinolates. Reference standards of the same glucosinolates and in the same concentrations as in the extracts were equally stimulatory. A test with eight different glucosinolates demonstrated that the moths do not discriminate between glucosinolates with different side‐chain structures. However, in tests using allylglucosinolate the oviposition response was dose‐dependent. One of the species tested, S. alba, contained a possible oviposition deterrent.

Properties of diacylglycerol acyltransferase from microspore-derived embryos ofBrassica napus

Abstract Particulate diacylglycerol acyltransferase (EC 2.3.1.20) from microspore-derived embryos of Brassica napus cv Reston was characterized. Diacylglycerol acyltransferase activity was mainly associated with the 10 000 and 100 000 g pellets prepared by differential centrifugation of embryo homogenate. The 100 000 g microsomal pellet was characterized in greater detail. Microsomal diacylglycerol acyltransferase activity was optimal near neutral pH and was affected by buffer composition. The microsomal enzyme was assayed optimally in a temperature range of 30–40° but appeared unstable at temperatures exceeding 40°. The enzyme activity reached a plateau at a concentration of ca 5 μM oleoyl-CoA but beyond this concentration, erucoyl-CoA was a more effective acyl donor. Considerably higher utilization rates for erucoyl-CoA were found using microsomes prepared from both high and low-erucic acid lines of Brassica napus . 2-Bromooctanoate inhibited microsomal diacylglycerol acyltransferase by 50% at a concentration of 1.5 mM. Detergents which might solubilize diacylglycerol acyltransferase were tested for enzyme inhibition. n -Octyl β- d -glucopyranoside was found to be the least inhibitory of the detergents, when tested at equivalent final concentrations.

Synthesis and field testing of enantiomers of 6Z,9Z-cis-3,4-epoxydienes as sex attractants for geometrid moths : Interactions of enantiomers and regioisomers.

Stereoselective syntheses of chiral C17 to C21 6Z,9Z-cis-3,4-epoxydienes were developed. Field tests of the enantiomerically enriched epoxides as components of synthetic sex attractant lures were carried out, and those with C17 and C19 chain lengths, particularly, were attractive to male moths of several species. Moths were usually specifically attracted by one of a pair of enantiomers, and the opposite enantiomer could actually be a behavioral antagonist. Males belonging to nine species of Geometridae were captured.Probole amicaria (Herrich-Schäffer) males were taken in traps baited with the mixture (6Z,9Z,3S,4R)-epoxy-nonadecadiene (6Z,9Z,3S,4R-epoxy-19∶H) + 3Z,9Z,6R,7S-epoxy-19∶H + 3Z,6Z,9Z-19∶H(9∶1∶8). Other species responding to the C19 compounds included (attractant components follow in parentheses);Sicya macularia (Harris) (6Z,9Z,3S,4R-epoxy-19∶H + 3Z,6Z,9Z-19∶H),Anavitrinella pampinaria (Guenée) (6Z,9Z-cis-3,4-epoxy-19∶H + 3Z,9Z,6S,7R-epoxy-19∶H), andLycia ursaria (Walker) (6Z,9Z-3S, 4R-epoxy-19∶H + 3Z,6Z,9Z-19∶H). Males of the following species were captured byC17 epoxides:Itame occiduaria (Packard) (6Z,9Z,3R,4S-epoxy-17∶H + 3Z,6Z,9Z-17∶H),Itame brunneata (Thunberg) (6Z,9Z,3S,4R-epoxy-17∶H),Epelis truncataria (Walker) (both enantiomers of 6Z,9Z-cis-3,4-epoxy-17∶H),Semiothisa ulsterata (Pearsall) (3Z,9Z-6S,7R-epoxy-17∶H), andS. signaria dispuncta (Walker) (3Z,9Z-cis-6,7-epoxy-17∶H + 3Z,6Z,9Z-17∶H). The interactions among enantiomers and regioisomers are discussed as a mechanism by which cross attraction between sympatric species is limited.

Formation of trierucoylglycerol (trierucin) from 1,2-Dierucoylglycerol by a homogenate of microspore-derived embryos ofBrassica napus L

Homogenates of microspore-derived embryos of rape (Brassica napus L.) incubated with [1-14C]erucoyl-CoA and 1,2-dierucoylglycerol are able to assemble trierucoyl-glycerol (trierucin). In addition, radioactive triacylglycerols are formed by transferring [1-14C]-erucoyl moieties to endogenous lipid precursors. Stereospecific analysis of radioactive triacylglycerols revealed that labeled erucoyl moieties had been incorporated exclusively into thesn-1,3 positions with more than 95% of radioactivity in thesn-3 position. No incorporation of labeled erucic acid into thesn-2 position has been observed. All data agree with the involvement of 1,2-diacylglycerol acyltransferase (E.C. 2.3.1.20), which utilized 1,2-dierucoylglycerol as well as endogenous 1,2-diacylglycerols as acceptors of erucoyl moieties. This result is of particular interest for the genetic modification of rape and other Cruciferae for producing trierucin in their seed oils.

The biosynthesis of the oxygenated monoterpenes in mint

Abstract Radioactive acetate and mevalonate were found to be incorporated into the volatile oil of Mentha piperita L. var. Mitcham and M. arvensis L. var. glabrata Ray. 14 C-Labelled acetate, mevalonate and CO 2 were fed under light and dark conditions and the results indicate that acetate and mevalonate are incorporated per se and not via prior degradation to CO 2 . Acetate-1- and 2- 14 C and mevalonate-2- 14 C were incorporated into the oil more slowly than 14 CO 2 in the light; all terpenes studied including menthol and menthofuran showed radioactivity within 5 min of feeding 14 CO 2 . Isolation of individual monoterpenes by GLC and determination of their specific activities upon feeding 14 CO 2 for various periods of metabolism confirmed Reitsemas biosynthetic sequences (piperitenone→ piperitone→menthone→menthol; piperitenone→pulegone→menthone and menthofuran) in its major aspects, but it could not be confirmed that piperitenone is the first isolable precursor of these two sequences. Also, isomenthone (which was not considered previously) was found to play a major role, and evidence suggesting the sequence: an unknown precursor→isomenthone→menthone→menthol was obtained.

Biosynthesis of mustard oil glucosides: N-Hydroxyphenylalanine, a precursor of glucotropaeolin and a substrate for the enzymatic and nonenzymatic formation of phenylacetaldehyde oxime☆☆☆

Abstract The incorporation of DL - N -hydroxyphenylalanine-2- 14 C into the mustard oil glucoside glucotropaeolin was demonstrated by plant feeding experiments. The efficiency of conversion of 14 C from this acid into the aglycone moiety of glucotropaeolin was higher than that from DL -phenylalanine-2- 14 C or -3- 14 C, and was comparable with that observed previously from phenylacetaldehyde oxime-1- 14 C. These results support the reaction sequence: phenylalanine → N -hydroxyphcnylalanine → phenylacetaldehyde oxime → glucotropaeolin. Enzyme preparations were obtained from Sinapis alba L., Tropoeolum majus L. and Nasturtium officinale R.Br. which catalyze the transformation of N -hydroxyphenylalanine to phenylacetaldehyde oxime. Although no absolute requirement for cofactors could be found, an increase in the rate of formation of the aldehyde oxime and consumption of oxygen was observed using FMN and enzyme preparations from T . majus and S . alba . Using the enzyme from T . majus it was demonstrated that the μmoles of phenylacetaldehyde oxime formed and of oxygen consumed were equivalent. In this case FMN appeared to be the prosthetic group and oxygen the physiological hydrogen acceptor. In addition to this enzymatic conversion, an unspecific and nonenzymatic oxidation of the N -hydroxyamino acid to phenylacetaldehyde oxime and phenylacetonitrile was observed.

Gas-liquid chromatography of terpenes—XII : Seasonal variation in the volatile oil from Tanacetum vulgare L.

Abstract The seasonal variation in the composition of the volatile oil from locally grown wild tansy plants was determined. Some plants were found to produce 80–90 per cent l-thujone, whereas others gave the normal content (70–85 per cent) of d-isothujone throughout the growing season. Significant variations in the content of the minor components were found to occur only in very young plants, when appreciable amounts of ϵ-, γ- and δ-cadinene and an unidentified labile (possibly C5) alcohol were detected.

Purification and properties of a UDP glucose: Thiohydroximate glucosyltransferase from higher plants☆

Abstract An enzyme which catalyses the formation of desulfobenzylglucosinolate by glucosyl transfer from UDP glucose to phenylacetothiohydroximate has been isolated from leaves of Tropaeolum majus L. and purified 20-fold. The enzyme possessed a high degree of specificity for the sugar acceptor molecule (thiohydroximates), the donor nucleotide (UPD) and its sugar (glucose). The activity of the enzyme was increased by sulfhydryl, chelating and reducing compounds: the activity disappeared in the absence of β-mercaptoethanol and was strongly inhibited by mercuric chloride and p -chloromercuribenzoate. Cell-free extracts of other glucosinolate-containing plants, including Sinapis alba L., Nasturtium officinale R. Br. and Armoracia lapathifolia Gilib. contained a similar glucosyl-transferase activity. It is concluded that the glycosylation of phenylacetothiohydroximate is an integral step in the biosynthesis of benzylglucosinolate.