Myrosinase-binding Proteins Research Articles

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.

Functional expression and characterization of the myrosinase MYR1 from Brassica napus in Saccharomyces cerevisiae.

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.

Characterization of a Flower-Specific Gene Encoding a Putative Myrosinase Binding Protein in Arabidopsis thaliana

A cDNA clone, 4B-1, previously isolated by differential screening is preferentially expressed in floral organs of Arabidopsis thaliana. Characterization of the full length cDNA and the genetic locus corresponding to 4B-1 cDNA revealed that it potentially encodes a myrosinase binding protein (MBP) which is presumably present in a large myrosinase complex. The deduced amino acid sequence of the polypeptide encoded by cDNA clone (designated f-AtMBP) appeared to consist of two parts: one region at the C-terminal half representing overall homology with AtMBP, an MBP homologue in A. thaliana, and the other at an extended N-terminal region of about 150 amino acids showing significant identity with the N-terminal region of the MBP-related protein reported in Brassica. Expression analysis by RNA blot and in situ hybridization showed that f-AtMBP was specifically expressed in floral meristems, pistils, stamens, petals, and ovules of immature flowers, but no expression was observed in the specialized cells called the myrosin cells in the hypocotyl and cotyledons of developing seeds where myrosinase enzymes are normally found. Although MBPs and MBP-related proteins are considered to be inducible by exogenous application of signal molecules and physical wounding, we found that f-AtMBP expression was not activated by such treatment, suggesting that f-AtMBP is a novel type of MBP specific to floral organs.

Co-localization of myrosinase- and myrosinase-binding proteins in grains of myrosin cells in cotyledon of Brassica napus seedlings

The localization of myrosinase and its binding proteins, MBP97 and MBP70, was analysed during early seedling growth of Brassica napus L. Myrosinase and MBP97 are both present in the mature seeds while MBP70 is not detectable. All three proteins accumulate during germination. The MBPs, although lacking cleavable signal peptides, are synthesized on the ER membrane. Among the three major myrosinase isozymes in the young seedlings, the 65 and the 70 kDa isoforms are present exclusively as complexes with MBP97 and MBP70 in a 20 000 × g pellet, while the 75 kDa myrosinase is not associated with MBPs and is predominantly present in the 20 000 × g supernatant. Light microscopic immunocytochemical analyses show myrosinase and MBP97 to be co-localized to the myrosin cells found scattered in cotyledons. Immunoelectron microscopic analyses prove that the myrosinase, the MBP97 and the MBP70 are co-localized in the myrosin grains of the myrosin cells suggesting that complexed myrosinases exist in planta.

Two jasmonate-inducible myrosinase-binding proteins from Brassica napus L. seedlings with homology to jacalin.

Two homologous but different cDNAs encoding a 97-kDa and a 70-kDa protein from Brassica napus L. seedlings have been characterized. Both proteins contain sequence motifs with high homology to the IgA binding lectin, jacalin, and the deduced 97-kDa protein contains the peptide sequences of myrosinase-binding protein. The 70-kDa and the 97-kDa protein can both be isolated as a complex containing myrosinase, indicating they indeed are myrosinase-binding proteins. We provide evidence that the 70-kDa protein binds IgA in vitro, and therefore classify the protein as a jacalin-type lectin. Both the 97-kDa and the 70-kDa proteins are encoded by a small number of genes in the Brassica genome. The mRNA for the 97-kDa protein is detected in both light- and dark-grown seedlings, whereas the mRNA for the 70-kDa protein is mainly detected in etiolated seedlings. The transcript levels for both proteins are transient and are rapidly increased by methyl jasmonate. The 70-kDa protein is synthesized de novo during germination and accumulates mainly in the hypocotyl and in the root. By immunogold labeling we show that a few cells scattered in the cotyledons of young seedlings (approx. 5% of total cells), contain protein-body-like structures with the 70-kDa protein. These bodies are present in a 10,000 g pellet from which the 70-kDa protein can be extracted by sodium carbonate. In addition, the 70-kDa protein is detected in the amorphous structures of the vacuole in a few cells of the cotyledon, the hypocotyl and the root.

The myrosinase-binding protein from Brassica napus seeds possesses lectin activity and has a highly similar vegetatively expressed wound-inducible counterpart.

This communication demonstrates that proteins in the family of myrosinase-binding proteins (MBP) present in seeds of Brassica napus possess lectin activity, binding most efficiently to p-aminophenyl alpha-D-mannopyranoside-agarose, and to some extent to N-acetylglucosamine-agarose. A cDNA encoding a vegetatively expressed, wound-inducible counterpart to these seed MBP was isolated and characterised. Upon wounding, this MBP transcript accumulated in old and young leaves, and was systemically expressed in the young plant. Additionally, the wound-induced MBP transcript increased in abundance after treating the young plants with methyl jasmonate (MeJA), jasmonic acid (JA) or abscisic acid (ABA), and to some extent in response to the ethylene precursor 1-aminocyclopropane-1-carboxylic acid. Expression induced by wounding, ABA or JA was antagonised by simultaneous feeding of the plants with salicylic acid. MBP polypeptides accumulated in MeJA-treated plants. The myrosinases redistributed from the soluble fraction into the insoluble fraction of a tissue extract after induction. The most abundant MBP (94 kDa) partitioned in the insoluble fraction, while two larger MBP (103 kDa and 108 kDa) were present only in the soluble fraction of extracts obtained from the control or MeJA-treated plant tissues.

Myrosinase-binding proteins are derived from a large wound-inducible and repetitive transcript.

Several non-myrosinase proteins have been found in association with some of the myrosinases extracted from rape (Brassica napus) seed. Most of these proteins seemed to belong to a large family of proteins ranging in size over approximately 30-110 kDa, namely the myrosinase-binding protein (MBP) family. Potentially all of these MBPs might be derived from a single large precursor, encoded by a 3.3-kb transcript. This transcript coded for a 99-kDa glycine-rich protein with a highly repetitive structure. The mature 50-kDa and 52-kDa MBP amino-terminal was located 255 amino acids from the putative initiation methionine. Also, a more divergently related transcript, the protein product of which was unknown, has been cloned. However, the largest open reading frame suggested a proline-rich protein. While this transcript seemed to be expressed predominantly in seeds, the MBP transcripts were expressed in several tissues and also exhibited a responsiveness to wounding and methyl jasmonate. Both proteins exhibited significant similarities to lectins from Artocarpus integer and from Maclura pomifera.

Characterization of rapeseed myrosinase-binding protein.

Myrosinase-binding proteins (MBPs) were purified from seeds of Brassica napus L. (oilseed rape). The proteins were characterized with respect to amino-acid composition, peptide sequence and isoelectric points. Gel electrophoresis and Western blotting of protein extracts from mature seeds showed the existence of at least ten proteins reacting with a monoclonal anti-MBP antibody and ranging in molecular size from 110 to 30 kDa. Proteins other than MBP reacting with the anti-MBP antibody were assigned as myrosinase-binding protein-related proteins (MBPRPs). Two MBPRPs were purified by immunoaffinity chromatography and characterized with respect to partial amino-acid sequence. Sequence identities were found between MBP and MBPRP. Western blot analysis of protein extracts from different tissues of B. napus showed that MBPRP is present in the whole plant, whereas MBP mostly occurs in the mature seed. A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was used to investigate the occurrence of MBP and MBPRP in developing seeds of some species in the Brassicaceae family.

Immunological characterization of rapeseed myrosinase

A purified 75-kDa myrosinase and a crude rapeseed myrosinase fraction were used as antigens to produce mouse anti-myrosinase monoclonal antibodies. The 75-kDa myrosinase was also used to produce a polyclonal rabbit antiserum. The antiserum and one monoclonal antibody reacted with three distinct rapeseed polypeptides of 75, 70 and 65 kDa (M75, M70 and M65, respectively). A second set of monoclonal antibodies reacted exclusively with the 75-kDa form of myrosinase, and a third set showed specificity towards two components of 52 and 50 kDa (myrosinase-binding proteins, MBP52 and MBP50, respectively). MBP52 and MBP50 lack inherent myrosinase activity, but are nevertheless capable of mediating immunoprecipitation of myrosinase due to their interaction with myrosinase. Gel chromatography and glycerol gradient centrifugation experiments resolved two myrosinase-containing fractions. One of these had an approximate molecular mass of 140 kDa and consisted of disulfide-linked dimers of the 75-kDa myrosinase. The other fraction was heterogeneous in size with molecular masses ranging from 250 kDa to approximately 1 MDa. The high-molecular-mass fractions contained complexes consisting of disulfide-linked 70-kDa and 65-kDa myrosinases and non-covalently bound 52-kDa and 50-kDa myrosinase-binding proteins.