Induction of myrosinase gene expression and myrosinase activity in radish hypocotyls by phototropic stimulation

The role of myrosinase (beta-thioglucoside glucohydrolase, EC 3.2.3.1) in the phototropic response in radish hypocotyls was investigated. Unilateral illumination with blue light abruptly up-regulated the activity of myrosinase, which releases bioactive 4-methylthio-3-butenyl isothiocyanate (MTBI) from inactive 4-methylthio-3-butenyl glucosinolate (MTBG), in the illuminated halves of radish hypocotyls 10 min after onset of phototropic stimulation, peaking after 30 min and decreasing thereafter. The myrosinase activity in the shaded halves also increased, but was significantly lower than that in the illuminated halves. Furthermore, whether blue light illumination induces myrosinase gene expression was studied. Northern blotting analysis indicated that myrosinase mRNA levels were increased markedly in unilaterally illuminated hypocotyls, reaching maximum signal intensity within 10 min after onset of blue illumination, declining nearly to the control level thereafter. These results suggested that phototropic stimulation promotes myrosinase gene expression and myrosinase activity in the illuminated side, resulting in the conversion of inactive MTBG to active MTBI and simultaneously producing more active raphanusanins, causing a phototropic response.     

Identification of the major glucosinolate (4-mercaptobutyl glucosinolate) in leaves of Eruca sativa L. (salad rocket)

The major and structurally unique glucosinolate (GLS) in leaves of Eruca sativa L. (salad rocket) was identified as 4-mercaptobutyl GLS. Both 4-methylthiobutyl GLS and 4-methylsulfinylbutyl GLS were also present, but at lower concentrations. The 4-mercaptobutyl GLS was observed to oxidise under common GLS extraction conditions, generating a disulfide GLS that may be reduced efficiently by tris(2-carboxyethyl) phosphine hydrochloride (TCEP) to reform the parent molecule. The identities of 4-mercaptobutyl GLS and of the corresponding dimeric GLS were confirmed by LC/MS, MS/MS and NMR. Myrosinase treatment of an enriched GLS fraction or of the purified dimer GLS generated a mixture of unique bi-functional disulfides, including bis-(4-isothiocyanatobutyl) disulfide (previously identified elsewhere). TCEP reduction of the purified dimer, followed by myrosinase treatment, yielded only 4-mercaptobutyl ITC. GLS-derived volatiles generated by autolysis of fresh seedlings and true leaves were 4-mercaptobutyl ITC (from the newly identified GLS), 4-methylthiobutyl ITC (from 4-methylthiobutyl GLS) and 4-methylsulfinylbutyl ITC (from 4-methylsulfinyl-butyl GLS); no unusual bi-functional disulfides were found in fresh leaf autolysate. These results led to the conclusion that, in planta, the new GLS must be present as 4-mercaptobutyl GLS and not as the disulfide found after extraction and sample concentration. This new GLS and its isothiocyanate are likely to contribute to the unique odour and flavour of E. sativa.     

Aversion and attraction to harmful plant secondary compounds jointly shape the foraging ecology of a specialist herbivore

Most herbivorous insect species are restricted to a narrow taxonomic range of host plant species. Herbivore species that feed on mustard plants and their relatives in the Brassicales have evolved highly efficient detoxification mechanisms that actually prevent toxic mustard oils from forming in the bodies of the animals. However, these mechanisms likely were not present during the initial stages of specialization on mustard plants ~100 million years ago. The herbivorous fly Scaptomyza nigrita (Drosophilidae) is a specialist on a single mustard species, bittercress (Cardamine cordifolia; Brassicaceae) and is in a fly lineage that evolved to feed on mustards only in the past 10–20 million years. In contrast to many mustard specialists, S. nigrita does not prevent formation of toxic breakdown products (mustard oils) arising from glucosinolates (GLS), the primary defensive compounds in mustard plants. Therefore, it is an appealing model for dissecting the early stages of host specialization. Because mustard oils actually form in the bodies of S. nigrita, we hypothesized that in lieu of a specialized detoxification mechanism, S. nigrita may mitigate exposure to high GLS levels within plant tissues using behavioral avoidance. Here, we report that jasmonic acid (JA) treatment increased GLS biosynthesis in bittercress, repelled adult female flies, and reduced larval growth. S. nigrita larval damage also induced foliar GLS, especially in apical leaves, which correspondingly displayed the least S. nigrita damage in controlled feeding trials and field surveys. Paradoxically, flies preferred to feed and oviposit on GLS‐producing Arabidopsis thaliana despite larvae performing worse in these plants versus non‐GLS‐producing mutants. GLS may be feeding cues for S. nigrita despite their deterrent and defensive properties, which underscores the diverse relationship a mustard specialist has with its host when lacking a specialized means of mustard oil detoxification.     

Identification of chemical oviposition 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.

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Résumé Les produits chimiques trouvés dans trois espèces de crucifères cultivées, Brassica napus L., B. juncea (L.) Czerniak, et Sinapis alba L., qui stimulent l'oviposition chez la teigne des crucifères, Plutella xylostella (L.) (Lepidoptera: Plutellidae) ont été examinés. La partie aérienne des plants agés de 4 à 6 semaines a été extraite avec le methanol bouillant à 80%, le methanol pur, et l'éther. La concentration par filtration sur célite a donné comme resultat un extrait aqueux. Le materiel restant sur la célite a été dissout avec hexane pour donner un extrait lipophillique. L'extrait aqueux a été fractionné à l'aide de la chromatographie liquide sur échangeur d'ion pour donner trois fractions: neutre, cationique, et anionique. Les extraits et les fractions ont été ajustés à 1 g poids frais de tissu de plant par ml, appliqués sur du papier filtre, et exposés aux papillons femelles dans les essais de choix d'oviposition. L'oviposition a été stimulée fortement en présence de l'extrait aqueux, la fraction anionique, et quelque sous-fractions anioniques. Plus tard, il a été déterminé que ces derniers contenaient des glucosinolates.Chez les trois espèces de crucifères, les stimulants d'oviposition ont été identifiés comme étant des glucosinolates, que l'on retrouve dans toutes les espèces de crucifères. L'activité des extraits a été éliminée en grande partie par traitement avec myrosinase ou sulphatase, des enzymes qui dégradent spécifiquement les glucosinolates. Des standards de references des mêmes glucosinolates et aux même concentrations que dans les extraits ont eu également un effet stimulant. Un essai avec huit glucosinolates differentes à une concentration de 50 g/ml appliquées sur du papier filter à 3.2 g/cm² a démontré que les papillons ne discriminent pas entre les glucosinolates possédant des chaines secondaires differentes. Par contre, dans les essais utilisant l'allylglucosinolate, la réponse d'oviposition a été dépendente de la dose. S. alba a semblé contenir un inhibiteur de l'oviposition, qui est retrouvé dans l'extrait aqueux mais non pas dans la fraction anionique.

     

Effects of different secondary metabolite profiles in plant defense syndromes on specialist and generalist herbivores

Plants defend themselves against herbivores not only by a single trait but also by diversified multiple defense strategies. It remains unclear how these multiple defense mechanisms are effectively organized against herbivores. In this study, we focused on Brassicaceae plants, which have one of the most diversified secondary metabolites, glucosinolates (GSLs), as a defense against herbivores. By analyzing various defense traits including GSL profiles among 12 species (11 genera) of Brassicaceae plants, it is revealed that their defense strategies can be divided into three categories as multiple defenses. The GSL profiles differed between these three categories: (i) high nutritional level with long‐chain aliphatic GSLs; (ii) low nutritional level and high physical defenses with short‐chain aliphatic GSLs; and (iii) high nutritional level and low defense. The feeding experiment was conducted using two types of herbivores, Pieris rapae (Lepidoptera: Pieridae) as a specialist herbivore and the Eri silkmoth Samia cynthia ricini (Lepidoptera: Saturniidae) as a generalist, to assess the ability of each plant in multiple defense strategy. It was observed that the Eri silkmoth's performance differed according to which defense strategy it was exposed to. However, the growth rate of P. rapae did not vary among the three categories of defense strategy. These results suggest that the diversified defense strategies of Brassicaceae species have evolved to cope with diversified herbivores.     

Sulphur uptake and distribution in double and single low varieties of oilseed rape (Brassica napus L.)

Sulphur (S) uptake and distribution in double low (Cobra) and single low (Bienvenu) winter oilseed rape were studied in field experiments at Cockle Park, Northumberland, at a site where the S supply was adequate. Total S uptake at maturity of between 80–100 kg ha^-1 was similar in both varieties. Applications of S at a rate of 100 kg ha^-1 increased S uptake by 10–15 kg ha^-1. while applications of nitrogen (N) at a rate of 300 kg ha^-1 increased S uptake by 29–34 kg ha^-1. Sulphur distribution in the vegetative tissues varied little between the two varieties but the distribution within the pods differed significantly between the two varieties. In Bienvenu 65.8% of pod S was located in the seeds, while in Cobra 57.4 and 68.8% in the 1988–89 and 1989–90 seasons, respectively, was retained in the pod walls. The high S content of the seeds of Bienvenu was due to their high glucosinolate content, whereas the high content of S in the pod walls of Cobra was associated with the presence of free SO₄ ^2-, which accounted for 70.6 to 89.4% of total S in the pod walls. The percentages of total plant S present in the pods were significantly increased by N applications and slightly decreased by S applications.     

Isothiocyanates and thioureas in enzyme hydrolysates of Tropaeolum tuberosum

Analysis of the isothiocyanates arising from enzymatic hydrolysis of glucosinolate extracts of Tropaeolum tuberosum supports the assessment of two subspecies. Seeds, tubers, leaves and flowers of T tuberosum subsp. tuberosum produced p-methoxybenzyl isothiocyanate. Subspecies silvestre produced benzyl-, 2-propyl- and 2-butylisothiocyanates. N,N-Di(4-methoxybenzyl)thiourea was detected in tuber extracts of subsp. tuberosum by HPLC.     

Myzus persicae (green peach aphid) feeding on Arabidopsis induces the formation of a deterrent indole glucosinolate

Cruciferous plants produce a wide variety of glucosinolates as a protection against herbivores and pathogens. However, very little is known about the importance of individual glucosinolates in plant defense and the regulation of their production in response to herbivory. When Myzus persicae (green peach aphid) feeds on Arabidopsis aliphatic glucosinolates pass through the aphid gut intact, but indole glucosinolates are mostly degraded. Although aphid feeding causes an overall decrease in Arabidopsis glucosinolate content, the production of 4-methoxyindol-3-ylmethylglucosinolate is induced. This altered glucosinolate profile is not a systemic plant response, but is limited to the area in which aphids are feeding. Aphid feeding on detached leaves causes a similar change in the glucosinolate profile, demonstrating that glucosinolate transport is not required for the observed changes. Salicylate-mediated signaling has been implicated in other plant responses to aphid feeding. However, analysis of eds5, pad4, npr1 and NahG transgenic Arabidopsis, which are compromised in this pathway, demonstrated that aphid-induced changes in the indole glucosinolate profile were unaffected. The addition of purified indol-3-ylmethylglucosinolate to the petioles of cyp79B2 cyp79B3 mutant leaves, which do not produce indole glucosinolates, showed that this glucosinolate serves as a precursor for the aphid-induced synthesis of 4-methoxyindol-3-ylmethylglucosinolate. In artificial diets, 4-methoxyindol-3-ylmethylglucosinolate is a significantly greater aphid deterrent in the absence of myrosinase than its metabolic precursor indol-3-ylmethylglucosinolate. Together, these results demonstrate that, in response to aphid feeding, Arabidopsis plants convert one indole glucosinolate to another that provides a greater defensive benefit.