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1.
Characterization and evolution of a myrosinase from the cabbage aphid Brevicoryne brassicae. 总被引:2,自引:0,他引:2
A M E Jones P Winge A M Bones R Cole J T Rossiter 《Insect biochemistry and molecular biology》2002,32(3):275-284
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. 相似文献
2.
Arabidopsis myrosinases TGG1 and TGG2 have redundant function in glucosinolate breakdown and insect defense 总被引:3,自引:0,他引:3
In Arabidopsis and other Brassicaceae, the enzyme myrosinase (beta-thioglucoside glucohydrolase, TGG) degrades glucosinolates to produce toxins that deter herbivory. A broadly applicable selection for meiotic recombination between tightly linked T-DNA insertions was developed to generate Arabidopsis tgg1tgg2 double mutants and study myrosinase function. Glucosinolate breakdown in crushed leaves of tgg1 or tgg2 single mutants was comparable to that of wild-type, indicating redundant enzyme function. In contrast, leaf extracts of tgg1tgg2 double mutants had undetectable myrosinase activity in vitro, and damage-induced breakdown of endogenous glucosinolates was apparently absent for aliphatic and greatly slowed for indole glucosinolates. Maturing leaves of myrosinase mutants had significantly increased glucosinolate levels. However, developmental decreases in glucosinolate content during senescence and germination were unaffected, showing that these processes occur independently of TGG1 and TGG2. Insect herbivores with different host plant preferences and feeding styles varied in their responses to myrosinase mutations. Weight gain of two Lepidoptera, the generalist Trichoplusia ni and the facultative Solanaceae-specialist Manduca sexta, was significantly increased on tgg1tgg2 double mutants. Two crucifer-specialist Lepidoptera had differing responses. Whereas Plutella xylostella was unaffected by myrosinase mutations, Pieris rapae performed better on wild-type, perhaps due to reduced feeding stimulants in tgg1tgg2 mutants. Reproduction of two Homoptera, Myzus persicae and Brevicoryne brassicae, was unaffected by myrosinase mutations. 相似文献
3.
The chemical nature of the hydrolysis products from the glucosinolate-myrosinase system depends on the presence or absence of supplementary proteins, such as epithiospecifier proteins (ESPs). ESPs (non-catalytic cofactors of myrosinase) promote the formation of epithionitriles from terminal alkenyl glucosinolates and as recent evidence suggests, simple nitriles at the expense of isothiocyanates. The ratio of ESP activity to myrosinase activity is crucial in determining the proportion of these nitriles produced on hydrolysis. Sulphoraphane, a major isothiocyanate produced in broccoli seedlings, has been found to be a potent inducer of phase 2 detoxification enzymes. However, ESP may also support the formation of the non-inductive sulphoraphane nitrile. Our objective was to monitor changes in ESP activity during the development of broccoli seedlings and link these activity changes with myrosinase activity, the level of terminal alkenyl glucosinolates and sulphoraphane nitrile formed. Here, for the first time, we show ESP activity increases up to day 2 after germination before decreasing again to seed activity levels at day 5. These activity changes paralleled changes in myrosinase activity and terminal alkenyl glucosinolate content. There is a significant relationship between ESP activity and the formation of sulforaphane nitrile in broccoli seedlings. The significance of these findings for the health benefits conferred by eating broccoli seedlings is briefly discussed. 相似文献
4.
Kayla J. Vastenhout Ruthellen H. TornbergAmanda L. Johnson Michael W. AmolinsJared R. Mays 《Analytical biochemistry》2014
Isothiocyanates (ITCs) are one of several hydrolysis products of glucosinolates, plant secondary metabolites that are substrates for the thioglucohydrolase myrosinase. Recent pursuits toward the development of synthetic non-natural ITCs have consequently led to an exploration of generating these compounds from non-natural glucosinolate precursors. Evaluation of the myrosinase-dependent conversion of select non-natural glucosinolates to non-natural ITCs cannot be accomplished using established ultraviolet–visible (UV–Vis) spectroscopic methods. To overcome this limitation, an alternative high-performance liquid chromatography (HPLC)-based analytical approach was developed where initial reaction velocities were generated from nonlinear reaction progress curves. Validation of this HPLC method was accomplished through parallel evaluation of three glucosinolates with UV–Vis methodology. The results of this study demonstrate that kinetic data are consistent between both analytical methods and that the tested glucosinolates respond similarly to both Michaelis–Menten and specific activity analyses. Consequently, this work resulted in the complete kinetic characterization of three glucosinolates with Sinapis alba myrosinase, with results that were consistent with previous reports. 相似文献
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6.
Guard cell- and phloem idioblast-specific expression of thioglucoside glucohydrolase 1 (myrosinase) in Arabidopsis 总被引:9,自引:0,他引:9
Thioglucoside glucohydrolase 1 (TGG1) is one of two known functional myrosinase enzymes in Arabidopsis. The enzyme catalyzes the hydrolysis of glucosinolates into compounds that are toxic to various microbes and herbivores. Transgenic Arabidopsis plants carrying beta-glucuronidase and green fluorescent protein reporter genes fused to 0.5 or 2.5 kb of the TGG1 promoter region were used to study spatial promoter activity. Promoter activity was found to be highly specific and restricted to guard cells and distinct cells of the phloem. No promoter activity was detected in the root or seed. All guard cells show promoter activity. Positive phloem cells are distributed in a discontinuous pattern and occur more frequent in young tissues. Immunocytochemical localization of myrosinase in transverse and longitudinal sections of embedded material show that the TGG1 promoter activity reflects the position of the myrosinase enzyme. In the flower stalk, the myrosinase-containing phloem cells are located between phloem sieve elements and glucosinolate-rich S cells. Our results suggest a cellular separation of myrosinase enzyme and glucosinolate substrate, and that myrosinase is contained in distinct cells. We discuss the potential advantages of locating defense and communication systems to only a few specific cell types. 相似文献
7.
Revised determination of free and complexed myrosinase activities in plant extracts. 总被引:2,自引:0,他引:2
Nora Travers-Martin Franziska Kuhlmann Caroline Müller 《Plant Physiology and Biochemistry》2008,46(4):506-516
The enzyme myrosinase (thioglucoside glucohydrolase, EC 3.2.1.147, formerly EC 3.2.3.1) catalyzes the hydrolysis of glucosinolates after tissue damage in plants of the order Brassicales. The various myrosinase isoforms occur either as free soluble dimers or as insoluble complexes. We propose a reliable method for determination of both soluble and insoluble myrosinase activity concentrations in partially purified plant extracts. The procedure requires the removal of endogenous glucosinolates through ion-exchange columns previous to enzyme measurements. Myrosinase activity was assayed in continuous mode by photometric quantification of the released glucose using glucose-oxidase with peroxidase and colorimetric indicators. The measurement of the colored product at 492nm has a favorable signal to noise ratio both in clear extract solutions (free dimers) and in turbid pellet suspensions (insoluble complexes). No interferences by ascorbic acid were found in continuous analyses. With the recommended sample preparation methods and assay conditions potential activities in damaged plant tissues can be characterized which are involved in plant defense mechanisms. 相似文献
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The defensive function of the glucosinolate-myrosinase system in plants of the order Capparales results from the formation of isothiocyanates when glucosinolates are hydrolysed by myrosinases upon tissue damage. In some glucosinolate-containing plant species, as well as in the insect herbivore Pieris rapae, protein factors alter the outcome of myrosinase-catalysed glucosinolate hydrolysis, leading to the formation of products other than isothiocyanates. To date, two such proteins have been identified at the molecular level, the epithiospecifier protein (ESP) from Arabidopsis thaliana and the nitrile-specifier protein (NSP) from P. rapae. These proteins share no sequence similarity although they both promote the formation of nitriles. To understand the biochemical bases of nitrile formation, we compared some of the properties of these proteins using purified preparations. We show that both proteins appear to be true enzymes rather than allosteric cofactors of myrosinases, based on their substrate and product specificities and the fact that the proportion of glucosinolates hydrolysed to nitriles does not remain constant when myrosinase activity varies. No stable association between ESP and myrosinase could be demonstrated during affinity chromatography, nevertheless some proximity of ESP to myrosinase is required for epithionitrile formation to occur, as evidenced by the lack of ESP activity when it was spatially separated from myrosinase in a dialysis chamber. The significant difference in substrate- and product specificities between A. thaliana ESP and P. rapae NSP is consonant with their different ecological functions. Furthermore, ESP and NSP differ remarkably in their requirements for metal ion cofactors. We found no indications of the involvement of a free radical mechanism in epithionitrile formation by ESP as suggested in earlier reports. 相似文献
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Induction of plant responses by a sequestering insect: Relationship of glucosinolate concentration and myrosinase activity 总被引:4,自引:0,他引:4
Induction of plant allelochemicals is of particular ecological importance for interactions with herbivores that can make use of induced metabolites by incorporating them for their own defence. Induction patterns in white mustard, Sinapis alba, were investigated following herbivory of the turnip sawfly, Athalia rosae, which sequesters plant glucosinolates. Larvae of different age were allowed to feed for 24 h on young leaves of premature, non-flowering plants. Changes in primary and secondary metabolites were recorded in the damaged leaves (local) and in the adjacent leaves and stems (systemic) for several days. Organ- and time-specific patterns were evident. Local responses included increases in glucosinolate concentrations, soluble and insoluble myrosinase activity and glucose levels, while systemic responses in leaves were restricted to increases in myrosinase activities and glucose. All effects were strongest immediately after feeding and declined mostly within a day. Stems had overall lower constitutive levels of glucosinolates and myrosinase activities than leaves. Feeding by one large larva had a greater impact on the plant's physiology than feeding by three small ones, even though both treatments resulted in quantitatively similar leaf destruction. Local increase in glucosinolate concentration could be beneficial for larvae, while conspecifics feeding on induced adjacent leaves might be negatively affected due to higher myrosinase activity levels. The results are discussed in the context of the ‘optimal defence theory’ and the ‘lethal plant defence paradox’. 相似文献
12.
Determination of myrosinase (thioglucoside glucohydrolase) activity by a spectrophotometric coupled enzyme assay 总被引:1,自引:0,他引:1
The hexokinase/glucose-6-phosphate dehydrogenase coupled enzyme system was used to assay for plant thioglucoside glucohydrolase (myrosinase, EC 3.2.3.1) by measuring the rate of glucose released during hydrolysis of glucosinolates. This coupled assay was compared with two other assays for myrosinase: a pH-stat assay that measures the rate of acid released during glucosinolate hydrolysis, and a spectrophotometric assay in which the decrease in the absorbance at 227.5 nm is used to measure the disappearance of the substrate, 2-propenylglucosinolate (DSA assay). The coupled and pH-stat assays were found to give comparable activities and were linear with enzyme concentration over the range 0 to 30 micrograms. The DSA assay gave lower myrosinase activity in comparison to the coupled and pH-stat assays. This is due to the lower concentrations of substrate and activator (ascorbate) which must be used in the assay. The DSA assay was found to give a nonlinear relationship with enzyme concentration over the range 2 to 30 micrograms. For these reasons this assay was found to be unsatisfactory. The coupled assay was found to be more sensitive and more widely applicable than the pH-stat assay as a routine continuous assay for myrosinase activity. 相似文献
13.
Functional expression and characterization of the myrosinase MYR1 from Brassica napus in Saccharomyces cerevisiae 总被引:5,自引:0,他引:5
Myrosinases are thioglucosidases that hydrolyze the natural plant products glucosinolates. We have expressed the myrosinase MYR1 from Brassica napus in Saccharomyces cerevisiae. The recombinant myrosinase was enzymatically active which shows that the MYR1, which in the plant is complex bound with myrosinase-binding proteins and myrosinase-associated proteins, is functional in its free form. Characterization of the recombinant MYR1 with respect to pH optimum, substrate specificity, activation by ascorbic acid, and inhibitors showed similar characteristics as previously observed for other plant myrosinases. The indolizidine alkaloid castanospermine, an inhibitor of O-glycosidases, inhibited the hydrolysis of p-hydroxybenzylglucosinolate with a K(i) value of 0.3 microM and 2-deoxy-2-fluoroglucotropaeolin, a specific inhibitor of thioglucosidases, inhibited the enzyme with a K(i) value of 1 mM. The expression of the myrosinase in yeast was transient and the growth of the yeast cells was significantly reduced during the period of expression of the myrosinase. Immunoblot analysis showed that the highest level of expression of MYR1 was obtained 24 h after induction with galactose. The amount of myrosinase protein correlated with the level of enzyme activity. The transient expression of myrosinase indicates that myrosinase is toxic to the cells. This is the first report on successful heterologous expression of a myrosinase and provides an important tool for, e.g., further characterization of myrosinase by site-directed mutagenesis and for studying the interaction between myrosinase and myrosinase-binding proteins, myrosinase-associated proteins, and epithiospecifier proteins. 相似文献
14.
Bioactive compounds were produced from natural glucosinolates, secondary plant metabolites, using myrosinase (thioglucoside glucohydrolase EC 3.2.3.1) isolated from ripe seeds of Sinapis alba. The enzyme was immobilized on granular nylon 6.6 with the crosslinking technique. Immobilized myrosinase displayed extraordinary operational and storage stability. Using a small thermostatted continuous packed-bed bioreactor, the enzyme activity was unchanged after 15 days of continuous use at 37 degrees C and after >1 year of storage at room temperature. The bioreactor was particularly efficient in producing pure isothiocyanates, but it was less efficient for pure nitrile production. 相似文献
15.
Husebye H Arzt S Burmeister WP Härtel FV Brandt A Rossiter JT Bones AM 《Insect biochemistry and molecular biology》2005,35(12):1311-1320
The aphid Brevicoryne brassicae is a specialist feeding on Brassicaceae plants. The insect has an intricate defence system involving a beta-D-thioglucosidase (myrosinase) that hydrolyses glucosinolates sequestered from the host plant into volatile isothiocyanates. These isothiocyanates act synergistically with the pheromone E-beta-farnesene to form an alarm system when the aphid is predated. In order to investigate the enzymatic characteristics of the aphid myrosinase and its three-dimensional structure, milligram amounts of pure recombinant aphid myrosinase were obtained from Echerichia coli. The recombinant enzyme had similar physiochemical properties to the native enzyme. The global structure is very similar to Sinapis alba myrosinase and plant beta-O-glucosidases. Aphid myrosinase has two catalytic glutamic acid residues positioned as in plant beta-O-glucosidases, and it is not obvious why this unusual enzyme hydrolyses glucosinolates, the common substrates of plant myrosinases which are normally not hydrolyzed by plant beta-O-glucosidases. The only residue specific for aphid myrosinase in proximity of the glycosidic linkage is Tyr180 which may have a catalytic role. The aglycon binding site differs strongly from plant myrosinase, whereas due to the presence of Trp424 in the glucose binding site, this part of the active site is more similar to plant beta-O-glucosidases, as plant myrosinases carry a phenylalanine residue at this position. 相似文献
16.
Jones AM Bridges M Bones AM Cole R Rossiter JT 《Insect biochemistry and molecular biology》2001,31(1):1-5
Plant myrosinases and glucosinolates constitute a defence system in cruciferous plants towards pests and diseases. We have purified for the first time a non-plant myrosinase from the cabbage aphid Brevicoryne brassicae (L.) to homogeneity. The protein was N-terminally blocked and protease (trypsin and lys c) degradation gave peptides of which five were sequenced. The protein is a dimer with subunits of mass 54 kDa+/-500 Da. Western blot analysis with an anti-aphid myrosinase antibody showed a strong cross reaction with a protein extract from the Brassica specialist, B. brassicae. The anti-aphid myrosinase antibody does not cross react with plant myrosinase neither does an anti-plant myrosinase antibody cross react with aphid myrosinase. 相似文献
17.
The glucosinolate composition and content in various tissues of Arabidopsis thaliana (L.) Heynh. ecotype Columbia during development from seeds to bolting plants were determined in detail by high-performance liquid chromatography. Comparison of the glucosinolate profiles of leaves, roots and stems from mature plants with those of green siliques and mature seeds indicated that a majority of the seed glucosinolates were synthesized de novo in the silique. A comparison of the glucosinolate profile of mature seeds with that of cotyledons indicated that a major part of seed glucosinolates was retained in the cotyledons. Turnover of glucosinolates was studied by germination of seeds containing radiolabelled p-hydroxybenzylglucosinolate (p-OHBG). Approximately 70% of the content of [14C]p-OHBG in the seeds was detected in seedlings at the cotyledon stage and [14C]p-OHBG was barely detectable in young plants with rosettes of six to eight leaves. The turn-over of p-OHBG was found to coincide with the expression of the glucosinolate-degrading enzyme myrosinase, which was detectable at very low levels in seedlings at the cotyledon stage, but which dramatically increased in leaves from plants at later developmental stages. This indicates that there is a continuous turnover of glucosinolates during development and not only upon tissue disruption. 相似文献
18.
Ahuja I Borgen BH Hansen M Honne BI Müller C Rohloff J Rossiter JT Bones AM 《Journal of experimental botany》2011,62(14):4975-4993
Oilseed rape and other crop plants of the family Brassicaceae contain a unique defence system known as the glucosinolate-myrosinase system or the 'mustard oil bomb'. The 'mustard oil bomb' which includes myrosinase and glucosinolates is triggered by abiotic and biotic stress, resulting in the formation of toxic products such as nitriles and isothiocyanates. Myrosinase is present in specialist cells known as 'myrosin cells' and can also be known as toxic mines. The myrosin cell idioblasts of Brassica napus were genetically reprogrammed to undergo controlled cell death (ablation) during seed development. These myrosin cell-free plants have been named MINELESS as they lack toxic mines. This has led to the production of oilseed rape with a significant reduction both in myrosinase levels and in the hydrolysis of glucosinolates. Even though the myrosinase activity in MINELESS was very low compared with the wild type, variation was observed. This variability was overcome by producing homozygous seeds. A microspore culture technique involving non-fertile haploid MINELESS plants was developed and these plants were treated with colchicine to produce double haploid MINELESS plants with full fertility. Double haploid MINELESS plants had significantly reduced myrosinase levels and glucosinolate hydrolysis products. Wild-type and MINELESS plants exhibited significant differences in growth parameters such as plant height, leaf traits, matter accumulation, and yield parameters. The growth and developmental pattern of MINELESS plants was relatively slow compared with the wild type. The characteristics of the pure double haploid MINELESS plant are described and its importance for future biochemical, agricultural, dietary, functional genomics, and plant defence studies is discussed. 相似文献
19.
BONES A. M.; THANGSTAD O. P.; HAUGEN O. A.; ESPEVIK T. 《Journal of experimental botany》1991,42(12):1541-1550
20.
芥子油苷在植物-生物环境关系中的作用 总被引:21,自引:2,他引:19
芥子油苷是一类含氮、含硫的植物次生代谢物质,主要分布于白花菜目的十字花科植物。芥子油苷及其降解产物具有多种生化活性,近年来人们更多地关注芥子油苷代谢与其它物质代谢途径的相互联系以及与植物生存环境的相互作用。介绍了芥子油苷及其分布、芥子油苷-黑芥子酶系统以及由芥子油苷介导的植物对昆虫、病原体的防御作用和植物与植物之间的化感作用的研究概况。 相似文献