John T. Rossiter

Spatial organization of the glucosinolate–myrosinase system in brassica specialist aphids is similar to that of the host plant

Secondary metabolites are important in plant defence against pests and diseases. Similarly, insects can use plant secondary metabolites in defence and, in some cases, synthesize their own products. The paper describes how two specialist brassica feeders, Brevicoryne brassicae (cabbage aphid) and Lipaphis erysimi (turnip aphid) can sequester glucosinolates (thioglucosides) from their host plants, yet avoid the generation of toxic degradation products by compartmentalizing myrosinase (thioglucosidase) into crystalline microbodies. We propose that death, or damage, to the insect by predators or disease causes disruption of compartmentalized myrosinase, which results in the release of isothiocyanate that acts as a synergist for the alarm pheromone E–β–farnesene.

Purification and characterisation of epithiospecifier protein from Brassica napus: enzymic intramolecular sulphur addition within alkenyl thiohydroximates derived from alkenyl glucosinolate hydrolysis.

Epithiospecifier protein (ESP), a ferrous ion dependent protein, has a potential role in regulating the release of elemental sulphur, nitriles, isothiocyanates and cyanoepithioalkanes from glucosinolates. Two classes of ESP polypeptides were purified with molecular masses of 39 and 35 kDa, and we show that the previously reported instability was conditionally dependent. The 39 kDa polypeptide was made up of two distinct isozymes (5.00, 5.14) whilst several were present for the 35 kDa form of ESP (5.40–5.66). An anti‐ESP antibody reacted with both the 39 and 35 kDa ESP forms in Brassica napus and strongly with a polypeptide corresponding to the 35 kDa ESP form in Crambe abyssinica, but did not detect any ESP in Sinapis alba or Raphanus sativus. A cytochrome P‐450 mediated iron dependent epoxidation type mechanism is suggested for ESP.

An investigation on structural aspects influencing product formation in enzymic and chemical oxidation of quercetin and related flavonols

Abstract Quercetin oxidation, with mushroom polyphenol oxidase (PPO), horseradish peroxidase (POD) and potassium ferricyanide, was studied by means of reversed-phase high-performance liquid chromatography (RP-HPLC). In order to establish structural requirements for product formation, two other flavonols, morin and rutin (quercetin 3-O-rutinoside), which possess key structural differences, were also treated. Oxidations were performed, either in aqueous acetonitrile (MeCN) or aqueous N,N-dimethylformamide (DMF), due to poor solubility of flavonols in aqueous media. The chromatographic profiles obtained from quercetin solutions, treated with PPO, POD, or potassium ferricyanide, were very similar, yielding the same set of products. In contrast, morin and rutin oxidations proceeded via different pathways. From the examinations carried out, it became clear that some products which possess significantly lower polarity compared with quercetin have, as a strict requirement, both the o-catechol function and the 3-hydroxyl group.

Characterization and evolution of a myrosinase from the cabbage aphid Brevicoryne brassicae

The aphid myrosinase gene has been elucidated using Rapid Amplification of cDNA Ends-PCR. Sequencing has shown that aphid myrosinase has significant sequence similarity (35%) to plant myrosinases and other members of glycosyl hydrolase family 1 (GHF1). The residues acting as proton donor and nucleophile, in the hydrolysis of glucosinolates by aphid myrosinase, are identified as Glu 167 and Glu 374 respectively. The equivalent residues in plant myrosinase are Gln 187 and Glu 409 and for the cyanogenic beta-glucosidase Glu 183 and Glu 397. Thus it would appear that the absence of a proton donor is not necessary for the hydrolysis of glucosinolates as was thought to be the case for the plant myrosinases. Aphid myrosinase appears to be more similar to animal beta-O-glucosidases than to plant myrosinases, as assessed by sequence similarity and phylogenetic techniques. These results strongly suggest that myrosinase activity has twice arisen from beta-O-glucosidases in plants and animals. Comparison of aphid myrosinase with plant myrosinase has highlighted Lys 173 and Arg 312 as possibly playing a crucial role in the hydrolysis of glucosinolates by aphid myrosinase.

Biosynthesis of 2-hydroxy-3-butenylglucosinolate and 3-butenylglucosinolate in Brassica napus

Abstract Desulpho-3-butenylglucosinolate is considered to be a potential late stage precursor in the biosynthesis of ( R )-2-hydroxy-3-butenylglucosinolate (progoitrin) and 3-butenylglucosinolate (gluconapin) in Brassica . Chemically synthesized [4,5- 3 H](β- d -glucopyranosyl)- 4-pentenethiohydroxamic acid when administered to developing seedlings of Brassica napus cv Bienvenu was found to be well incorporated into both 2-hydroxy-3-butenylglucosinolate and 3-butenylglucosinolate, while feeding experiments with [ 14 C]methionine have shown that de novo biosynthesis is slow. Analysis of seeds and developing seedlings has shown that levels of 2-hydroxy-3-butenylglucosinolate are maintained while 3-butenylglucosinolate levels are considerably reduced.

Inhibition of thioglucosidase-catalysed glucosinolate hydrolysis by castanospermine and related alkaloids

Abstract The ability of some naturally occurring polyhydroxyalkaloids to inhibit the hydrolysis of the glucosinolates, sinigrin (allylglucosinolate) and progoitrin [(2R)-2-hydroxybut-3-enylglucosinolate], by thioglucosidases from mustard and the cabbage aphid, Brevicoryne brassicae, was assessed. Several compounds, in particular castanospermine, DMDP and alexine were effective. The inhibitory activity varied according to substrate, buffer composition and pH, and enzyme source.

Physiological analysis of transgenic Arabidopsis thaliana plants expressing one nitrilase isoform in sense or antisense direction

Summary The biosynthesis of IAA in Arabidopsis thaliana can proceed via different pathways, which involve indole-3-acetonitrile (IAN) as an intermediate. The enzyme nitrilase, which converts IAN to the auxin indole-3-acetic acid (IAA), might be a key enzyme in this biosynthetic pathway. To elucidate the role of nitrilase during the development of the plant, we have investigated transgenic Arabidopsis plants, carrying one of the four nitrilase isoforms so far identified in Arabidopsis in either sense or antisense direction. The plants have been analyzed for their phenotype, auxin content, nitrilase transcript and protein amounts, and nitrilase activity. Analysis of free IAA content in seedlings did not reveal any prominent differences between the different lines, whereas the total IAA content was slightly increased in the line overexpressing NIT2 (sNIT2). In two of the antisense lines (aNIT1 and aNIT2) the total auxin content was about 75% lower than in the wild type, while IAN was increased in these two antisense lines by about 2-fold. In sNIT2 plants the IAN content was slightly decreased compared with the wild type. When IAN was added to the medium, a strong increase in IAA content was found in sNIT2 seedlings (ca. 22-fold), which was much lower in wild type and antisense plants. In mature plants levels of free IAA were decreased by about 25% and 50% in the antisense lines aNIT2 and aNIT1, respectively, whereas the IAA content in the other lines did not differ much. sNIT2/aNIT2 plants were further characterized because it was shown by Northern and Western analysis as well as enzyme activity measurements that sNIT2 plants overexpress nitrilase constitutively. The results with these plants have shown that i) both nitrilase 1 and nitrilase 2 accept the same substrates, and ii) overexpression of nitrilase does not lead to a visible phenotype because in vivo IAN might be the limiting factor in these plants. Application of IAN leads to an increase in total nitrilase protein as shown by Western blotting. Using transgenic lines with promoter-GUS fusions for each NIT gene, it was shown that i) NIT1 is highly expressed in seedlings and ii) NIT2 is strongly induced (21-fold) after IAN application.

Accumulation of the phytoalexin lettucenin a and changes in 3-hydroxy-3-methylglutaryl coenzyme a reductase activity in lettuce seedlings with the red spot disorder

The sesquiterpenoid lactone phytoalexin lettucenin A was isolated from seedlings of lettuce with the red spot physiological disorder. Accumulation of lettucenin A was preceded by increased activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase. The red spot syndrome may provide a tractable mimic of the hypersensitive reaction of lettuce to microbial challenge.

Microautoradiographic localisation of a glucosinolate precursor to specific cells in Brassica napus L. embryos indicates a separate transport pathway into myrosin cells

Abstract. The in-situ localisation of a desulpho-glucosinolate precursor has been studied by microautoradiography of cryo-sections from immature seeds and pods of the high-glucosinolate Brassica napus L. cv. Argentine collected 23xa0days after pollination. After feeding with the tritium-labelled glucosinolate precursor [4,5-3H](β-D-glucopyranosyl)-4-pentenethiohydroxamic acid, embryo radicles, cotyledons and pod-wall were frozen in liquid nitrogen. Cryotome sections were freeze-dried and coated with nuclear emulsion autoradiographic film. A distinct pattern of radioactivity derived from the glucosinolate precursor was found in specific cells in both radicle and cotyledons. In contrast, the labelling in pod walls was not cell specific, but general at the inner side of the pod wall. The results show that the glucosinolate/desulphoglucosinolate was localised in specific cells, in a pattern resembling that of myrosin cells known to contain myrosinase (EC 3.2.3.1). In addition [4,5-3H](β-D-glucopyranosyl)-4-pentenethiohydroxamic acid was fed to immature seeds and pods of B. napus and a quantitative incorporation into 2-hydroxy-3-butenylglucosinolate and 3-butenylglucosinolate was observed. When [4,5-3H](β-D-glucopyranosyl)-4-pentenethiohydroxamic acid was fed to 4-day-old seedlings the label was taken up by all tissues. We propose a model in which glucosinolate/desulphoglucosinolates are transported to myrosin cells to participate in the myrosinase-glucosinolate multifunctional defence system.

Discovery of a Bacterial Glycoside Hydrolase Family 3 (GH3) β-Glucosidase with Myrosinase Activity from a Citrobacter Strain Isolated from Soil.

A Citrobacter strain (WYE1) was isolated from a UK soil by enrichment using the glucosinolate sinigrin as sole carbon source. The enzyme myrosinase was purified using a combination of ion exchange and gel filtration to give a pure protein of approximately 66 kDa. The N-terminal amino acid and internal peptide sequence of the purified protein were determined and used to identify the gene, which, based on InterPro sequence analysis, belongs to the family GH3, contains a signal peptide, and is a periplasmic protein with a predicted molecular mass of 71.8 kDa. A preliminary characterization was carried out using protein extracts from cell-free preparations. The apparent KM and Vmax were 0.46 mM and 4.91 mmol dm(-3) min(-1) mg(-1), respectively, with sinigrin as substrate. The optimum temperature and pH for enzyme activity were 25 °C and 6.0, respectively. The enzyme was marginally activated with ascorbate by a factor of 1.67.