Myrosinase-binding Proteins Research Articles

Proteome-wide identification of methylglyoxalated proteins in rapeseed (Brassica napus L.)

Methylglyoxal (MG), a highly reactive cellular metabolite, is crucial for plant growth and environmental responses. MG may function by modifying its target proteins, but little is known about MG-modified proteins in plants. Here, MG-modified proteins were pulled down by an antibody against methylglyoxalated proteins and detected using liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis. We identified 543 candidate proteins which are involved in multiple enzymatic activities and metabolic processes. A great number of candidate proteins were predicted to localize to cytoplasm, chloroplast, and nucleus, consistent with the known subcellular compartmentalization of MG. By further analyzing the raw LC–MS/MS data, we obtained 42 methylglyoxalated peptides in 35 proteins and identified 10 methylglyoxalated lysine residues in a myrosinase-binding protein (BnaC06G0061400ZS). In addition, we demonstrated that MG modifies the glycolate oxidase and β-glucosidase to enhance and inhibit the enzymatic activity, respectively. Together, our study contributes to the investigation of the MG-modified proteins and their potential roles in rapeseed.

Cold modulated nuclear S-nitrosoproteome analysis indicates redox modulation of novel Brassicaceae specific, myrosinase and napin in Brassica juncea

Stress leads to production of reactive oxygen species (ROS) and reactive nitrogen species (RNS). Among the RNS, nitric oxide (NO) is an important signaling molecule that mediates its effect majorly via a reversible post-translational modification (PTM) S-nitrosylation. However, information on the NO mediated cold stress signaling is still missing for nucleus. Therefore, in the present study an attempt was made to analyze NO signaling, particularly, S-nitrosylation in the nuclei of Brassica juncea seedlings. Cold stress (4 °C) induced nitrate reductase (NR) mediated NO production along with the accumulation of available protein thiols (5 fold, at 96 h, 4 °C) and low molecular weight thiols (1.2 fold at 12 h, 4 °C). S-nitrosoglutathione (GSNO, a NO donor) and cold (endogenous, S-nitrosylation without the addition of NO donor) treated, neutravidin-agarose affinity chromatography purified S-nitrosylated proteins were resolved as 55- and 32-spots respectively on the 2-D gels. Functional categorization after MALDI-TOF/TOF identification showed 67% targets to be stress/defense/redox related and 33% to be metabolic, suggesting the potential role(s) of S-nitrosylation in these responses. Cold stress increased S-nitrosylation of Cu/Zn SOD, l-ascorbate oxidase, fructose bisphosphate aldolase (FBA2) and malate dehydrogenase 1 (MDH1), therefore, indicating NO mediated regulation of redox and metabolism related pathways. Identification of targets of glucosinolate hydrolysis pathway (myrosinase and myrosinase-binding protein 2-like) indicated the role of S-nitrosylation in regulating mustard oil bomb. Presence of myrosinase was confirmed by activity measurements and western blotting. Increased S-nitrosylation of myrosinase in cold stress in the nucleus is reported for the first time. Here, we proposed S-nitrosylation mediated nuclear trafficking of non-nuclear targets such as beta-d-xylosidase, daikon cysteine protease, and beta-glucosidase to the nucleus. Similarly, identification of FBA2 and MDH1 in the nucleus, indicated the role of NO in modulating their moonlighting function via S-nitrosylation in stress. Present study provided the first repertoire of cold modulated S-nitrosylated proteins in the nucleus.

Identification of lipopolysaccharide-interacting plasma membrane-type proteins in Arabidopsis thaliana

Lipopolysaccharide (LPS) is an amphiphatic bacterial glycoconjugate found on the external membrane of Gram-negative bacteria. This endotoxin is considered as a microbe-associated molecular pattern (MAMP) molecule and has been shown to elicit defense responses in plants. Here, LPS-interacting proteins from Arabidopsis thaliana plasma membrane (PM)-type fractions were captured and identified in order to investigate those involved in LPS perception and linked to triggering of innate immune responses. A novel proteomics-based affinity-capture strategy coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed for the enrichment and identification of LPS-interacting proteins. As such, LPS isolated from Burkholderia cepacia (LPSB.cep.) was immobilized on three independent and distinct affinity-based matrices to serve as bait for interacting proteins from A. thaliana leaf and callus tissue. These were resolved by 1D electrophoresis and identified by mass spectrometry. Proteins specifically bound to LPSB.cep. have been implicated in membrane structure (e.g. COBRA-like and tubulin proteins), membrane trafficking and/or transport (e.g. soluble NSF attachment protein receptor (SNARE) proteins, patellin, aquaporin, PM instrinsic proteins (PIP) and H+-ATPase), signal transduction (receptor-like kinases and calcium-dependent protein kinases) as well as defense/stress responses (e.g. hypersensitive-induced response (HIR) proteins, jacalin-like lectin domain-containing protein and myrosinase-binding proteins). The novel affinity-capture strategy for the enrichment of LPS-interacting proteins proved to be effective, especially in the binding of proteins involved in plant defense responses, and can thus be used to elucidate LPS-mediated molecular recognition and disease mechanism(s).

Proteomic analysis of stress-related proteins in transgenic broccoli harboring a gene for cytokinin production during postharvest senescence

Our previous study revealed a cytokinin-related retardation of post-harvest floret yellowing in transgenic broccoli ( Brassica oleracea var. italica) that harbored the bacterial isopentenyltransferase ( ipt) gene. We aimed to investigate the underlining mechanism of this delayed post-harvest senescence. We used 2D electrophoresis and liquid chromatography–electrospray ionization–mass spectrometry/mass spectrometry for a proteomics analysis of heads of ipt-transgenic and non-transgenic inbred lines of broccoli at harvest and after four days post-harvest storage. At harvest, we found an accumulation of stress-responsive proteins involved in maintenance of protein folding (putative protein disulfide isomerase, peptidyl-prolyl cis–trans isomerase and chaperonins), scavenging of reactive oxygen species (Mn superoxide dismutase), and stress protection [myrosinase-binding protein, jasmonate inducible protein, dynamin-like protein, NADH dehydrogenase (ubiquinone) Fe-S protein 1 and stress-inducible tetratricopeptide repeat-containing protein]. After four days’ post-harvest storage of non-transgenic broccoli florets, the levels of proteins involved in protein folding and carbon fixation were decreased, which indicates cellular degradation and a change in metabolism toward senescence. In addition, staining for antioxidant enzyme activity of non-transgenic plants after post-harvest storage revealed a marked decrease in activity of Fe-superoxide dismutase and ascorbate peroxidase. Thus, the accumulation of stress-responsive proteins and antioxidant enzyme activity in ipt-transgenic broccoli are most likely associated with retardation of post-harvest senescence.

Putative 32 kDa myrosinase binding protein At3g16450.1 from Arabidopsis

Putative 32 kDa myrosinase binding protein At3g16450.1 from Arabidopsis

Structure of the putative 32 kDa myrosinase‐binding protein from Arabidopsis (At3g16450.1) determined by SAIL‐NMR

The product of gene At3g16450.1 from Arabidopsis thaliana is a 32 kDa, 299-residue protein classified as resembling a myrosinase-binding protein (MyroBP). MyroBPs are found in plants as part of a complex with the glucosinolate-degrading enzyme myrosinase, and are suspected to play a role in myrosinase-dependent defense against pathogens. Many MyroBPs and MyroBP-related proteins are composed of repeated homologous sequences with unknown structure. We report here the three-dimensional structure of the At3g16450.1 protein from Arabidopsis, which consists of two tandem repeats. Because the size of the protein is larger than that amenable to high-throughput analysis by uniform (13)C/(15)N labeling methods, we used stereo-array isotope labeling (SAIL) technology to prepare an optimally (2)H/(13)C/(15)N-labeled sample. NMR data sets collected using the SAIL protein enabled us to assign (1)H, (13)C and (15)N chemical shifts to 95.5% of all atoms, even at a low concentration (0.2 mm) of protein product. We collected additional NOESY data and determined the three-dimensional structure using the cyana software package. The structure, the first for a MyroBP family member, revealed that the At3g16450.1 protein consists of two independent but similar lectin-fold domains, each composed of three beta-sheets.

The ‘mustard oil bomb’: not so easy to assemble?! Localization, expression and distribution of the components of the myrosinase enzyme system

Glucosinolates are plant secondary metabolites that are hydrolysed by the action of myrosinases into various products (isothiocyanates, thiocyanates, epithionitriles, nitriles, oxazolidines). Massive hydrolysis of glucosinolates occurs only upon tissue damage but there is also evidence indicating metabolism of glucosinolates in intact plant tissues. It was originally believed that the glucosinolate–myrosinase system in intact plants was stable due to a spatial separation of the components. This has been coined as the ‘mustard oil bomb’ theory. Proteins that form complexes with myrosinases have been described: myrosinase-binding proteins (MBPs) and myrosinase-associated proteins (MyAPs/ESM). The roles of these proteins and their biological relevance are not yet completely known. Other proteins of the myrosinase enzyme system are the epithiospecifier protein (ESP) and the thiocyanate-forming protein (TFP) that divert the glucosinolate hydrolysis from isothiocyanate production to nitrile/epithionitrile or thiocyanate production. Some glucosinolate hydrolysis products act as plant defence compounds against insects and pathogens or have beneficial health effects on humans. In this review, we survey and critically assess the available information concerning the localization, both at the tissular/cellular and subcellular level, of the different components of the myrosinase enzyme system. Data from the model plant Arabidopsis thaliana is compared to that from other glucosinolate-producing Brassicaceae in order to show common as well as divergent features of the ‘mustard oil bomb’ among these species.

The Genetic Basis of Constitutive and Herbivore-Induced ESP-Independent Nitrile Formation in Arabidopsis

Glucosinolates are a group of thioglucosides that are components of an activated chemical defense found in the Brassicales. Plant tissue damage results in hydrolysis of glucosinolates by endogenous thioglucosidases known as myrosinases. Spontaneous rearrangement of the aglucone yields reactive isothiocyanates that are toxic to many organisms. In the presence of specifier proteins, alternative products, namely epithionitriles, simple nitriles, and thiocyanates with different biological activities, are formed at the expense of isothiocyanates. Recently, simple nitriles were recognized to serve distinct functions in plant-insect interactions. Here, we show that simple nitrile formation in Arabidopsis (Arabidopsis thaliana) ecotype Columbia-0 rosette leaves increases in response to herbivory and that this increase is independent of the known epithiospecifier protein (ESP). We combined phylogenetic analysis, a screen of Arabidopsis mutants, recombinant protein characterization, and expression quantitative trait locus mapping to identify a gene encoding a nitrile-specifier protein (NSP) responsible for constitutive and herbivore-induced simple nitrile formation in Columbia-0 rosette leaves. AtNSP1 is one of five Arabidopsis ESP homologues that promote simple nitrile, but not epithionitrile or thiocyanate, formation. Four of these homologues possess one or two lectin-like jacalin domains, which share a common ancestry with the jacalin domains of the putative Arabidopsis myrosinase-binding proteins MBP1 and MBP2. A sixth ESP homologue lacked specifier activity and likely represents the ancestor of the gene family with a different biochemical function. By illuminating the genetic and biochemical bases of simple nitrile formation, our study provides new insights into the evolution of metabolic diversity in a complex plant defense system.

Myrosinase in horseradish ( Armoracia rusticana) root: Isolation of a full-length cDNA and its heterologous expression in Spodoptera frugiperda insect cells

Myrosinase (β-thioglucoside glucohydrolase, EC. 3.2.3.1), is the only known S-glucosidase in plants, catalyzing the hydrolysis of glucosinolates into compounds that have diverse biological activities. In the present study, a full-length cDNA encoding myrosinase ( ArMY1) was cloned from horseradish ( Armoracia rusticana) root. ArMY1 has an open reading frame of 1614 nucleotides with a deduced protein of 538 amino acids and molecular mass of 61.6 kD. ArMY1 shows highest overall amino acid identity (72%) with Arabidopsis thaliana myrosinase TGG2. Heterologous expression of ArMY1 in baculovirus-infected Sf9 cells resulted in an immunologically active recombinant ArMY1 protein, when probed with myrosinase-specific monoclonal antibody 3D7, with apparent mass 65 kD. ArMY1 mRNA signal of about 1.95 kb was detected in the leaves and roots of horseradish, but not in the florets of broccoli. Phylogenetic analysis showed that ArMY1 does not cluster with any of the currently known myrosinase subfamilies, MA, MB, MC, and may represent a novel myrosinase subfamily. This is the first report of cloning of myrosinase cDNA from horseradish root. It provides important sequence information that will enable further studies of myrosinase expression patterns and their interaction with myrosinase-binding proteins and other myrosinase-associated proteins.

Glucosinolate hydrolysis in Lepidium sativum––identification of the thiocyanate-forming protein

Glucosinolates are a class of thioglycosides found predominantly in plants of the order Brassicales whose function in anti-herbivore defense has been attributed to the products formed by myrosinase-catalyzed hydrolysis upon plant tissue damage. The most common type of hydrolysis products, the isothiocyanates, are toxic to a wide range of organisms. Depending on the glucosinolate side-chain structure and the presence of certain protein factors, other types of hydrolysis products, such as simple nitriles, epithionitriles and organic thiocyanates, can be formed whose biological functions are not well understood. Of the proteins controlling glucosinolate hydrolysis, only epithiospecifier proteins (ESPs) that promote the formation of simple nitriles and epithionitriles have been identified on a molecular level. We investigated glucosinolate hydrolysis in Lepidium sativum and identified a thiocyanate-forming protein (TFP) that shares 63-68% amino acid sequence identity with known ESPs and up to 55% identity with myrosinase-binding proteins from Arabidopsis thaliana, but differs from ESPs in its biochemistry. TFP does not only catalyze thiocyanate and simple nitrile formation from benzylglucosinolate but also the formation of simple nitriles and epithionitriles from aliphatic glucosinolates. Analyses of glucosinolate hydrolysis products in L. sativum autolysates and TFP transcript accumulation revealed an organ-specific regulation of thiocyanate formation. The identification of TFP defines a new family of proteins that control glucosinolate hydrolysis and challenges the previously proposed reaction mechanism of epithionitrile formation. As a protein that promotes the formation of a wide variety of hydrolysis products, its identification provides an important tool for further elucidating the mechanisms of glucosinolate hydrolysis as well as the ecological role and the evolutionary origin of the glucosinolate-myrosinase system.

(275) Cloning of a Myrosinase cDNA from Horseradish (Armoracia rusticana) Root and Its Heterologous Expression in Spodoptera frugiperda Insect Cells

A full-length cDNA (ArMY1) encoding myrosinase (β-thioglucoside glucohydrolase, EC 3.2.3.1) was cloned from horseradish (Armoracia rusticana) root. ArMY1 has an open reading frame of 1614 nucleotides with a deduced protein of 538 amino acids and molecular mass of 61.6 kD. ArMY1 shows highest overall amino acid identity (72%) with Arabidopsis thaliana myrosinase TGG2. ArMY1 mRNA signal of about 1.95 kb was detected in the leaves and roots of horseradish, but not in the leaves of broccoli. Heterologous expression of ArMY1 in baculovirus-infected Sf9 cells resulted in an immunologically active recombinant ArMY1 protein when probed with myrosinase-specific monoclonal antibody 3D7 with apparent mass 65 kD. Phylogenetic analysis showed that ArMY1 does not cluster with any of the current myrosinase subfamilies, i.e., the MA, MB, and MC subfamilies, and may represent a novel myrosinase subfamily in root tissue. This is the first report of cloning of myrosinase cDNA from horseradish root. It provides important sequence information that will enable further studies of myrosinase expression patterns and their interaction with myrosinase–binding proteins and other myrosinase-associated proteins.

Differential wound induction of the myrosinase system in oilseed rape ( Brassica napus): contrasting insect damage with mechanical damage

Diamond-back moth ( Plutella xylostella) is a serious insect pest specialised for herbivory on Crucifers although such plants contains the glucosinolate–myrosinase system implied as an important resource to withstand insect pests. We have compared the effects of mechanical wounding using forceps or scissors with herbivory by diamond-back moth larvae on oilseed rape ( Brassica napus). The transcript levels of myrosinase binding protein (MBP), myrosinase associated protein (MyAP) and myrosinase were studied of leaf samples at different time points together with myrosinase enzyme activity measurements. In all cases, wounding gave a transient local increase in transcript levels of MBP and MyAP that were similar to control levels again within 24–48 h. Systemic induction of MBP and MyAP transcripts was observed after diamond-back moth herbivory and mechanical wounding by scissors while wounding by forceps caused down-regulation. In contrast, the myrosinase transcript levels were induced less and in wounded leaves after diamond-back moth feeding only. The myrosinase activity decreased especially in the soluble fraction prepared from wounded leaves after herbivory. The changes in MBP and myrosinase transcript levels were reflected in protein levels according to Western blot analysis. Mechanical wounding with forceps is often used for convenience as a substitute for insect attack but does not mimic well the effects of diamond-back moth herbivory to the plants.

Infestation by cabbage aphid (Brevicoryne brassicae) on oilseed rape (Brassica napus) causes a long lasting induction of the myrosinase system

AbstractCabbage aphid, Brevicoryne brassicae (L.) (Hemiptera: Aphididae), is specialised on cruciferous plants and constitutes a world‐wide problem with a substantial negative impact on agriculture and horticulture. The myrosinase‐glucosinolate system present in crucifers is believed to serve as an important barrier to general herbivores, whereas specialist insects rely on this system for host recognition. Two proteins with unknown function, myrosinase binding protein (MBP) and myrosinase associated protein (MyAP), are also present in such plants and bind to specific myrosinases. In order to study regulation and overall effects on the myrosinase system, one leaf of oilseed rape (Brassica napus L.) (Brassicaceae) plants at the two‐leaf stage was exposed to cabbage aphids for 1 h. After removal of the insects, infested and non‐infested leaves were analysed by Northern blot analysis for the presence of MBP, MyAP, and myrosinase transcripts at different time points up to 48 h. MBP transcript levels continued to rise during the whole experiment, and were seven‐ to eight‐fold higher compared with control plants 48 h after removal of the aphids. At 24 h the MyAP transcripts reached maximal levels and were four‐fold higher than the control levels. Myrosinase transcripts were down regulated by aphid infestation and the level was less than half that of the control plants at 48 h. Apparently cabbage aphid infestation causes both transient and more long lasting (up to 48 h) effects on the myrosinase system of the host.

Identification by suppression subtractive hybridization and expression analysis of Arabidopsis thaliana putative defence genes during Orobanche ramosa infection

A suppression subtractive hybridization strategy was used to identify genes that were induced 2 h after infection of Arabidopsis thaliana by broomrape ( Orobanche ramosa) seedlings. Among these genes, the expression of twelve was analysed from the first hour to the seventh day of this compatible interaction by cDNA blot analyses. The twelve genes showed a transient expression, which occurred in seven cases as early as the first or second hour of interaction and ended 2 or 3 h later. Most of the proteins encoded by these genes are already known to be involved in different A. thaliana response pathways to pathogen attack. The first two genes to be induced (Rc kinase and ACaM5) encoded a receptor and a calmodulin, and could be involved in signal transduction. The two following genes encoding a sucrose carrier ( SUC1) and a protein with a pectin methylesterase inhibitor domain were found to be overexpressed; their roles are consistent with plant defence emergence. The gst1, gst11 and peptide methionine sulfoxide reductase genes, which were turned on early, are known to play a role in the detoxification of reactive oxygen species. The accumulation of mRNAs for lox1, a latex allergen and a myrosinase binding protein could be related to a jasmonate dependent pathway, while genes for a class III peroxidase and a caffeoyl-CoA 3- O-methyltransferase, both likely to be involved in cell wall reinforcement, were also upregulated.

Complex formation of myrosinase isoenzymes in oilseed rape seeds are dependent on the presence of myrosinase-binding proteins.

The enzyme myrosinase (EC 3.2.3.1) degrades the secondary compounds glucosinolates upon wounding and serves as a defense to generalist pests in Capparales. Certain myrosinases are present in complexes together with other proteins such as myrosinase-binding proteins (MBP) in extracts of oilseed rape (Brassica napus) seeds. Immunhistochemical analysis of wild-type seeds showed that MBPs were present in most cells but not in the myrosin cells, indicating that the complex formation observed in extracts is initiated upon tissue disruption. To study the role of MBP in complex formation and defense, oilseed rape antisense plants lacking the seed MBPs were produced. Western blotting and immunohistochemical staining confirmed depletion of MBP in the transgenic seeds. The exclusive expression of myrosinase in idioblasts (myrosin cells) of the seed was not affected by the down-regulation of MBP. Using size-exclusion chromatography, we have shown that myrosinases with subunit molecular masses of 62 to 70 kD were present as free dimers from the antisense seed extract, whereas in the wild type, they formed complexes. In accordance with this, MBPs are necessary for myrosinase complex formation of the 62- to 70-kD myrosinases. The product formed from sinalbin hydrolysis by myrosinase was the same whether MBP was present or not. The performance of a common beetle generalist (Tenebrio molitor) fed with seeds, herbivory by flea beetles (Phyllotreta undulata) on cotyledons, or growth rate of the Brassica fungal pathogens Alternaria brassicae or Lepthosphaeria maculans in the presence of seed extracts were not affected by the down-regulation of MBP, leaving the physiological function of this protein family open.

Characterization of a Brassica napus myrosinase expressed and secreted by Pichia pastoris.

In Brassica napus three different gene families with different temporal and tissue-specific expression and distribution patterns encode myrosinases (thioglucoside glucohydrolases, EC 3.2.3.1). Myrosinases encoded by the MA gene family are found as free and soluble dimers, while myrosinases encoded by the MB and MC gene families are mainly found in large insoluble complexes associated with myrosinase-binding proteins and myrosinase-associated proteins. These large complexes impede purification and characterization of MB and MC myrosinases from the plant. We used Pichia pastoris to express and secrete functional recombinant MYR1 myrosinase from B. napus to allow further characterization of myrosinase belonging to the MB gene family. The purified recombinant myrosinase hydrolyzes sinigrin with a K(m) of 1.0 mM; the specific activity and calculated k(cat)/K(m) were 175 U/mg and 1.9 x 10(5) s(-1) M(-1), respectively. A novel in-gel staining method for myrosinase activity is presented.

Different myrosinase and idioblast distribution in Arabidopsis and Brassica napus.

Myrosinase (EC 3.2.3.1) is a glucosinolate-degrading enzyme mainly found in special idioblasts, myrosin cells, in Brassicaceae. This two-component system of secondary products and degradative enzymes is important in plant-insect interactions. Immunocytochemical analysis of Arabidopsis localized myrosinase exclusively to myrosin cells in the phloem parenchyma, whereas no myrosin cells were detected in the ground tissue. In Brassica napus, myrosinase could be detected in myrosin cells both in the phloem parenchyma and in the ground tissue. The myrosin cells were similar in Arabidopsis and B. napus and were found to be different from the companion cells and the glucosinolate-containing S-cells present in Arabidopsis. Confocal laser scanning immunomicroscopy analysis of myrosin cells in B. napus embryos showed that the myrosin grains constitute a continuous reticular system in the cell. These findings indicate that in the two species studied, initial cells creating the ground tissue have different potential for making idioblasts and suggest that the myrosinase-glucosinolate system has at least partly different functions. Several myrosinases in B. napus extracts are recovered in complex together with myrosinase-binding protein (MBP), and the localization of MBP was therefore studied in situ. The expression of MBP was highest in germinating seedlings of B. napus and was found in every cell except the myrosin cells of the ground tissue. Rapid disappearance of the MBP from the non-myrosin cells and emergence of MBP in the myrosin cells resulted in an apparent colocalization of MBP and myrosinase in 7-d-old seedlings.

COI1 affects myrosinase activity and controls the expression of two flower-specific myrosinase-binding protein homologues in Arabidopsis

Two cDNA clones homologous to myrosinase-binding proteins (MBPs) were identified by differential display in Arabidopsis thaliana (L.) Heynh. The cDNAs (MBP1 and MBP2) correspond to two open-reading frames found in a gene cluster of seven putative MBP genes located on chromosome 1. The predicted proteins MBP1 and MBP2 are similar to lectins and plant aggregating factors. In addition. MBP2 contains a region of high content of proline and alanine residues, commonly found in arabinogalactan proteins and hydroxyproline-rich glycoproteins. Transcripts corresponding to MBP1 and MBP2 genes are exclusively and abundantly expressed in flowers but are not detected in male-sterile flowers of coi1 plants, insensitive to jasmonic acid. Northern analysis and in situ hybridization revealed that MBP mRNAs are present in higher levels in immature flowers and are localized in several floral organs, including the ovary, ovules, style, anthers and filament. Transcripts of the Arabidopsis myrosinase gene TGG1 show a pattern of expression similar to that observed for the MBP genes during flower development; however, they are also abundant in green tissues and are only partially affected by COI1. Crude preparations of soluble proteins from leaf and flower extracts of wild-type Arabidopsis showed myrosinase activity when sinigrin was used as substrate. In contrast, coi1 plants showed significantly reduced myrosinase activities in both leaves and flowers. The results show that COI1 controls MBP expression in flowers and significantly affects the expression and activity of myrosinase in Arabidopsis.

The myrosinase‐glucosinolate system in the interaction between Leptosphaeria maculans and Brassica napus

summary Leptosphaeria maculans causes blackleg disease, and resistance to this fungal pathogen is an important trait in the breeding of oilseed rape. A better comprehension of the role of the myrosinase-glucosinolate system in this context is of great value. The present study is the first to address effects on multiple components of this complex system, including concentrations of individual glucosinolates, product formation, myrosinase isoform distribution and activity, and levels of myrosinase binding proteins during the infection process. One resistant B. napus cultivar (Maluka) and one susceptible cultivar (Westar) were compared in the investigation. Our results show that the two cultivars had the same histological distribution, isoform expression, and activity of the myrosinase enzymes. The glucosinolate levels were also similar, with the exception of glucobrassicin and neoglucobrassicin, which were significantly lower in the resistant cultivar at 11 days post-infection. Growth of the fungus on the plant tissues did not alter glucosinolate levels, suggesting that L. maculans does not degrade these compounds. When the plants were starved of sulphur, and thereby depleted of glucosinolates, no increased susceptibility was observed. Hence, we suggest that the myrosinase-glucosinolate system does not determine the outcome of the interaction between B. napus and L. maculans.

B-genome derived resistance to Leptosphaeria maculans in near isogenic Brassica napus lines is independent of glucosinolate profile

The role of the glucosinolate-myrosinase system in resistance to Leptosphaeria maculans was studied by monitoring changes in glucosinolate profiles in leaf tissue surrounding the site of inoculation. Susceptible Brassica napus cv. Hanna, resistant B. nigra and near isogenic lines derived from interspecific hybrids between the two species were compared. Expression of myrosinase binding protein and presence of genetic markers were also assayed. No correlation between degree of resistance and amount of sinigrin or other aliphatic glucosinolates was found. However, in time course experiments the glucosinolate profile of the L. maculans inoculated plants differed significantly from the water-inoculated control plants in the amount of 4-methoxy-glucobrassicin observed. Five to eight days post-inoculation an increased level of 4-methoxy glucobrassicin, ranging from 30% to 47% on average, was found in the inoculated plants, whilst controls varied between 7.6% and 9.2%. This increase was seen both in susceptible and resistant material. Other changes observed could mainly be assigned as effects of wounding. Although inoculation with L. maculans elicited changes in the leaf indolyl glucosinolate profiles, in the material studied, these changes could not be correlated to resistance against the fungus.