Anders Falk

The glucosinolate-degrading enzyme myrosinase in Brassicaceae is encoded by a gene family.

A full-length cDNA clone (MB3) and three partial clones (MA1, MB1 and MB2) which encode myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1) were isolated from a Sinapis alba (white mustard) cDNA library. Nucleotide sequence analysis of these clones revealed that they are encoded by a gene family. Southern blot analysis with gene-specific probes showed that the gene family consists of a least two subfamilies (MA and MB) each with several members both in S. alba and in Brassica napus (oilseed rape). In Arabidopsis thaliana (wall cress) only three myrosinase genes seem to be present. Northern blot analysis indicated that all the myrosinase mRNA species have the same size, approximately 1.95 kb.

Expression of a zeatin-O-glucoside-degrading beta-glucosidase in Brassica napus.

A [beta]-glucosidase was purified from seeds of Brassica napus L. (oilseed rape). The 130-kD native enzyme consisted of a disulfide-linked dimer of 64-kD monomers. Internal amino acid sequences were used to construct degenerate primers for polymerase chain reaction-mediated cloning of cDNA for the enzyme. One nearly full-length and one partial [beta]-glucosidase-encoding cDNA clone were isolated and sequenced. Southern hybridization showed that [beta]-glucosidase is encoded by a small gene family in B. napus. Northern hybridization showed that the genes are expressed in the seed, with a low degree of expression in other tissues. In the seed, the expression started at 30 days after pollination (DAP), with the highest expression at 40 DAP. The size of the transcript was approximately 1900 nucleotides. In situ hybridization to developing seeds of B. napus showed that the [beta]-glucosidase expression started at 30 DAP around the provascular tissue in the embryo axis. In the cotyledons, mRNA initially accumulated around the provascular tissues but was detected first at 35 DAP. At 40 DAP, expression occurred in most parts of the seed. In situ hybridization also detected [beta]-glucosidase mRNA in shoots, young roots, and the basal part of the hypocotyls. Zeatin-O-glucoside was identified as a natural substrate for B. napus [beta]-glucosidase.

A Wound- and Methyl Jasmonate-Inducible Transcript Coding for a Myrosinase-Associated Protein with Similarities to an Early Nodulin

Myrosinase is regarded as a defense-related enzyme in the Brassicaceae and is capable of hydrolyzing glucosinolates into various compounds, some of which are toxic. Several myrosinase isoenzymes exist, and some of them have been found in association with nonmyrosinase proteins. One of these associated proteins, myrosinase-associated protein (MyAP), was purified from seeds of Brassica napus both in complexes with myrosinase and in a free form. MyAP is a glycosylated, 40-kD protein with at least one intramolecular disulfide bridge. A monoclonal anti-MyAP antibody precipitated myrosinase activity from B. napus seed extracts and in these complexes both a 65- and a 70-kD myrosinase were present. The subsequent cloning and analysis revealed the existence of a gene family encoding MyAP or MyAP-related protein and that transcripts corresponding to MyAP in nonwounded plants are found predominantly in seeds. At least some members of the gene family exhibited responsiveness toward wounding and methyl jasmonate vapor. MyAP displayed considerable similarity to an early nodulin (ENOD8) from Medicago sativa and to a proline-rich protein (APG), described as anther specific, from Arabidopsis thaliana and B. napus. Similarity to expressed sequence tags from both A. thaliana and Oryza sativa has also been found.

Differential expression of myrosinase gene families

In mature seeds of Brassica napus three major and three minor myrosinase isoenzymes were identified earlier. These myrosinases are known to be encoded by at least two different families of myrosinase genes, denoted MA and MB. In the work described in this paper the presence of different myrosinase isoenzymes in embryos, seedlings, and vegetative mature tissues of B. napus was studied and related to the expression of myrosinase MA and MB genes in the same tissues to facilitate future functional studies of these enzymes. In developing seeds, myrosinases of 75, 73, 70, 68, 66, and 65 kD were present. During seedling development there was a turnover of the myrosinase pool such that in 5-d-old seedlings the 75-, 70-, 66-, and 65-kD myrosinases were present, with the 70- and 75-kD myrosinases predominating. In 21-d-old seedlings the same myrosinases were present, but the 66- and 65-kD myrosinase species were most abundant. At flowering the mature organs of the plant contained only a 72-kD myrosinase. MA genes were expressed only in developing seeds, whereas MB genes were most highly expressed in seeds, seedling cotyledons, young leaves, and to a lesser extent other organs of the mature plant. During embryogenesis of B. napus, myrosinase MA and MB gene transcripts started to accumulate approximately 20 d after pollination and reached their highest level approximately 15 d later. MB transcripts accumulated to about 3 times the amount of MA transcripts. In situ hybridization analysis of B. napus embryos showed that MA transcripts were present predominantly in myrosin cells in the axis, whereas MB genes were expressed in myrosin cells of the entire embryo. The embryo axis contained 75-, 70-, and 65-kD myrosinases, whereas the cotyledons contained mainly 70- and 65-kD myrosinases. Amino acid sequencing revealed the 75-kD myrosinase to be encoded by the MA gene family. The high degree of cell and tissue specificity of the expression of myrosinase genes suggests that studies of their transcription should provide interesting information concerning a complex type of gene regulation.

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.

Sequence of a cDNA clone encoding the enzyme myrosinase and expression of myrosinase in different tissues of Brassica napus

We have isolated and sequenced a cDNA clone for the glucosinolate-degrading enzyme myrosinase from Brassica napus (oilseed rape). Using a probe derived from the cDNA clone we have studied the expression of myrosinase genes in different tissues of B. napus. The myrosinase genes are most highly expressed in the young leaves, the cotyledons and the developing seed. Other parts of the plant only show a relatively low expression of myrosinase. In the developing seed the expression of myrosinase is first detected at 15 days after pollination and at the highest levels around 30 days after pollination. The myrosinase genes are turned on earlier, but also silenced earlier than the genes for the storage protein cruciferin.

Conversion of an amplified fragment length polymorphism marker into a co-dominant marker in the mapping of the Rph15 gene conferring resistance to barley leaf rust, Puccinia hordei Otth.

Leaf rust, caused by Puccinia hordei, is an important disease afflicting barley (Hordeum vulgare) in many production regions of the world. The leaf rust resistance gene Rph15 was identified in an accession of wild barley (Hordeum vulgare subsp. spontaneum) and is one of the most broadly effective resistance genes known. Using amplified fragment length polymorphism (AFLP) and simple sequence repeat markers, Rph15 was mapped to chromosome 2HS in an F2 population derived from a cross between Bowman (Rph15), a Bowman backcross-derived line carrying Rph15, and the susceptible cultivar Bowman. AFLP marker P13M40 co-segregated with Rph15 in this mapping population and two others involving Bowman (Rph15) and cultivars Proctor and Nudinka. The dominant AFLP marker P13M40 was converted to a co-dominant PCR-based marker that may be useful in breeding programs employing marker-assisted selection. The allelic relationship between Rph15 and the gene Rph16, also mapping to chromosome 2HS, was studied. The lack of segregation in F2 progeny derived from the two resistance sources indicates that Rph15 and Rph16 are alleles of the same locus.

Characterization of a new myrosinase in Brassica napus

A full-length cDNA clone defining the new myrosinase gene family MC in Brassica napus was isolated and sequenced. Southern hybridization showed that the MC family probably consists of 3 or 4 genes in B. napus. MC genes are expressed in the developing seed, but not in the vegetative tissues investigated. In situ hybridizations to developing seeds showed that the MC genes are expressed in the myrosin cells of the embryo axis and the cotyledons. Complexes with myrosinase and myrosinase-binding protein (MBP) were purified and characterized. Sequencing of peptides from myrosinases occurring in the complexes showed that the 70 kDa myrosinase is encoded by the MC genes, whereas the 65 kDa myrosinase is encoded by the MB genes. This is in contrast to the 75 kDa myrosinase which occurs in free form and is encoded by the MA genes. Deglycosylations of the myrosinase complexes and the free myrosinase showed that the molecular sizes of the myrosinases could be reduced significantly by this treatment, and that the size differences between the different myrosinases are mainly due to differences in glycosylation.

Characterization of a Brassica napus myrosinase pseudogene: myrosinases are members of the BGA family of beta-glycosidases

Myrosinase isoenzymes are known to be encoded by two different families of genes denoted MA and MB. Nucleotide sequence analysis of a Brassica napus genomic clone containing a gene for myrosinase revealed it to be a pseudogene of the MA family. The gene spans more than 5 kb and contains at least 12 exons. The exon sequence of the gene is highly similar to myrosinase cDNA sequences. However, the gene displays three potential or actual pseudogene characters. Southern blot analysis using probes from the 3′ portions of the genomic and B. napus MA and MB cDNA clones showed that MA type myrosinases are encoded by approximately 4 genes, while MB type myrosinases are encoded by more than 10 genes in B. napus. Northern blots with mRNA from seeds and young leaves probed with the MA-and MB-specific probes showed that the MA and MB myrosinase gene families are differentially expressed. Myrosinases are highly similar to proteins of a β-glycosidase enzyme family comprising both β-glycosidases and phospho-β-glycosidases of as diverged species as archaebacteria, bacteria, mammals and plants. By homology to these β-glycosidases, putative active site residues in myrosinase are discussed on the basis of the similarity between β-glycosidases and cellulases.

Sequencing of 15 622 gene-bearing BACs clarifies the gene-dense regions of the barley genome

Summary Barley (Hordeum vulgare L.) possesses a large and highly repetitive genome of 5.1 Gb that has hindered the development of a complete sequence. In 2012, the International Barley Sequencing Consortium released a resource integrating whole‐genome shotgun sequences with a physical and genetic framework. However, because only 6278 bacterial artificial chromosome (BACs) in the physical map were sequenced, fine structure was limited. To gain access to the gene‐containing portion of the barley genome at high resolution, we identified and sequenced 15 622 BACs representing the minimal tiling path of 72 052 physical‐mapped gene‐bearing BACs. This generated ~1.7 Gb of genomic sequence containing an estimated 2/3 of all Morex barley genes. Exploration of these sequenced BACs revealed that although distal ends of chromosomes contain most of the gene‐enriched BACs and are characterized by high recombination rates, there are also gene‐dense regions with suppressed recombination. We made use of published map‐anchored sequence data from Aegilops tauschii to develop a synteny viewer between barley and the ancestor of the wheat D‐genome. Except for some notable inversions, there is a high level of collinearity between the two species. The software HarvEST:Barley provides facile access to BAC sequences and their annotations, along with the barley–Ae. tauschii synteny viewer. These BAC sequences constitute a resource to improve the efficiency of marker development, map‐based cloning, and comparative genomics in barley and related crops. Additional knowledge about regions of the barley genome that are gene‐dense but low recombination is particularly relevant.