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completed projects

Asparagine metabolism in barley

Nematode resistance

Molecular farming in clover

Asparagine metabolism in barley

In protein crops like the legumes nearly all nitrogen transported from the vegetative parts to the developing seeds is in the form of the amino acid asparagine. In stach crops like barley glutamin is the primary vehicle for nitrogen transport. The aim of the project has been to isolate the genes encoding the enzymes asparagine synthetase and asparaginase; to study their expression and via transformation of barley to change the amino acid metabolism to evaluate possible effects on the protein content and amino acvid composition of the grain. Two genes (HvAS1 and HvAS2) encoding asparagine synthetase were cloned from barley and the genes mapped to chromosomes 5 and 3. Expression analysis of HvAS1 showed that gene expression is induced only after prolonged darkness. Likewise, there is an increase in the amount of HvAS1 protein as measured in Western blots of leaf proteins. The HvAS2 gene is expressed at a lower level, the expression being higher in the light than in darkness in leaves while light/darkness had no effect on the expression level in roots. Two genes with similar expression patternes have formerly been identified in Arabidopsis. A transgenic barley line constitutively expressing the HvAS1 gene was generated by microprojectile bombardment of immature embryos. The gene is expressed but apparently at a low level. The T1 progeny show a distinct phenotype. They grow slower than wild type and have a delayed senescence and possess larger leaves and grains as well as thicker roots and tillers.

A cDNA and genomic clone encoding asparaginase has been cloned from barley. The gene has been mapped to chromosome 2. The asparaginase gene has also been isolated from Lolium perenne. It can be predicted from the derived amino acid sequence that there are no signal or transit sequences but a minimum of one and possibly three trans membrane sequences. Expression studies have shown that the gene is transcribed in nearly all tissues and immuno detection analyses indicate that the asparaginase protein is present in young developing leaves. About 20 transgene barley lines have been generated containing the asparginase gene under the control of the constitutive ubiquitin promoter from maize as well as a barley line where the expression of the GUS reporter gene is driven by the asparaginase promoter. These lines are at present being analysed. Likewise, an HPLC-based method for measurements of free amino acids has been implemented.

Scientists: Marianne Geller Møller (asparagine synthetase) and Knud G. Madsen (asparaginase)

Møller, M.G., Taylor, C., Rasmussen, S.K. and Holm, P.B.: Molecular cloning and characterisation of two genes encoding asparagine synthetase in barley (Hordeum vulgare). Biochim. Biophys. Acta 1628, 123-132

Nematode resistance

Nematode parasitism and plant root nematode resistance is an outstanding example of a biological interaction between a parasite and a host. The juvenile roundworms of genera such as Heterodera and Meloidogyne penetrate the root epidermis cells and invade a parenchyma cell in the developing vascular cylinder. The cell is stimulated to increase metabolic activity and growth, the walls between the adjacent cells are gradually broken down and the protoplasted cells fuse to a larger syncytium that act as a feeding structure for the nematodes. It is apparent that the nematode in addition to excreting cell wall degrading enzymes, as well as other enzymes for syncytium formation and nutrient uptake, also takes control over the development of the surrounding cells which results in an up-regulation of gene activities beneficial for the formation of the surrounding syncytium and synthesis of compounds necessary for the nematodes. It is also apparent that the invading nematode can suppress the normal defence response of the plant, initiated during the initial stages of the infection. Plant nematode resistance thus constitutes a fascinating research area of great relevance to the understanding of plant - parasite interactions which also may be of relevance for other interactions such as mycorrizha and fungal infections.

On a worldwide basis nematode attacks on cultivated plants cause losses of the order of 80-100 billion US$ per year. At sexual maturity the females swell and burst the surrounding tissue thus damaging the root. After mating the females transform into a capsule filled with eggs and embryos that fall off the root. The eggs and the embryos in the capsule can remain viable in the soil for several years. Nematode attacks can be treated chemically, but current nematicides are very toxic and have been forbidden in a number of countries.

Genes conferring resistance to nematode attacks have been identified in a number of crop plants and in particular in their wild relatives. In a few cases the incorporation of such genes by conventional breeding has been successful but in many cases, e.g. in sugar beet, a transfer of resistance by traditional breeding methods with wild species have proven inefficient due to gain losses and chromosomal instability in the hybrids and the back-cross material. Therefore, at present the transfer of resistance genes by transformation is the most realistic and efficient way for achieving good resistance in plant systems.

Shortly before the initiation of the current project the first nematode resistance gene termed Hs pro-1 was isolated via positional cloning from a wild beet, Beta procumbens, in a concerted effort of 16 laboratories in 8 European countries. The data published (Cai et al. 1997) indicated that it was a resistance gene of the leucine rich repeat type and that sugar beet hairy root cultures constitutively expressing the resistance gene had an increased tolerance to the nematodes. The objective of the present project has been to clone homologues to this resistance gene from barley, to study the expression of these genes, to generate transgenic plants with up regulated resistance genes, to identify the localization of the protein at the histological and sub cellular level and eventually to establish the function of the resistance gene.

To identify cereal homologues to the Hs1pro-1 gene a careful database search for monocot homologues was performed with different regions of the protein. This resulted in the identification of four EST clones from rice (UAa). These clones were purchased, sequenced and shown by sequence comparisons to originate from the same gene. Together they comprised a full-length rice cDNA of 1422bp encoding for a protein that is about 50% identical with the Hs1pro-1.

Alignment of the B. procumbens, rice and an A. thaliana derived protein sequences revealed several conserved regions, which was used in the design of degenerate primers. The degenerated primers were utilized in RT-PCR based amplification to identify homologous genes in barley and wheat. mRNA from leaves and root of the barley cultivar Alexis and the wheat cultivar Bob White was used in the first round of cDNA synthesis. PCR amplification products were obtained from both the barley and wheat cultivar and shown to comprise the 3´region of the gene.

Subsequently five genomic barley clones were isolated by screening a genomic library with the Alexis fragment as a probe. Sub cloning and sequencing showed that all four clones contained the same gene. A 7.5 kb long fragment was sequenced and shown to contain a large 5´upstream region, the structural gene and a 3´downstream sequence. Structural analysis showed that the gene has a coding region of 1401 bp. As in B. procumbens, the barley coding sequence has no introns and comparisons of the derived protein sequence revealed a 83% identity to the rice coding region and a 50% amino acid sequence identity with the Hs1pro-1 protein. Furthermore, the barley and rice homologues possess the same structural features as the B. procumbens protein i.e. a putative membrane-spanning region and a leucine rich region (Cai et al. 1997). The genes show no obvious homology to other known genes in either plants or animals.

Genomic Southern analysis of the rice and barley genes under high stringency indicated that they are single copy genes. This is supported by the finding of only one Hs1pro-1 homologue in the complete rice sequence (Goff et al. 2002). The coding regions from the barley cultivar Alexis and rice were isolated by PCR to be used for expression and transgene analyses.

Mapping of the Hs1pro-1barley homologue (DIAS)
The barley gene was mapped at the level of the chromosome arm using the Betzes wheat/barley addition lines. Both Southern analysis and gene specific PCR showed that the gene is located on the long arm of chromosome 3 from barley. This implies that the barley Hs1pro-1 homologue is different from the well-known Ha2 nematode resistance gene in barley that is known to reside on the long arm of chromosome 2 (Andersen and Andersen 1973; Kretchmer et al. 1997; Barr et al. 1998).

A large number of transgenic A. thaliana lines, hosting the rice and barley genes under the control of the constitutive CaMV 35S-promoter, were generated by the floral dip method and were in collaboration with Danisco Seeds in Holeby infected with nematodes. The test was performed on the lines showing the highest transgene expression level as evidenced by RT-PCR. The experiments were repeated three times. The assays did not provide conclusive evidence for increased nematode resistance in the transgenic plants expressing the barley and rice Hs1 genes.

Induction of the barley and rice Hs1 gene in A. thaliana by abiotic and biotic stress
Promoter sequence analysis identified two cis-elements identical to the DRE (dehydration-responsive element), involved in drought, salt and low-temperature stress induced gene expression (Yamaguchi-Shinozaki and Shinizaki 1994). The elements are positioned 2.4 kb upstream of the start codon. These regions led us to believe that the gene was involved in response to abiotic stress.

The transgenic A. thaliana lines used for the nematode infection studies were tested for enhanced tolerance to salt stress in root bending assays. The experiment was also repeated three times and showed in some cases a weak but not at all conclusive evidence of tolerance to salt stress.

In B. procumbens the Hs1pro-1 gene is mainly expressed in the root and is induced upon nematode attack. On Northern blots of poly-A RNA from wild type barley we found conclusive evidence for a right size 1.5 kb transcript that is more abundant in roots than in leaves.

The regulation of the gene with respect to salt stress was studied in both wild type barley and wheat plants. The salt stress experiments were performed on seedlings exposed to 200 mM NaCl solution for 30 min, 1 hour and 2 hours. RNA was extracted from leaves and roots and showed that the gene is up regulated 3-4 times during the first hours of salt stress. Together with presence of the DRE cis elements in the promoter this indicates that the gene is involved in response to abiotic stress.

Generation of transgenic barley lines over expressing the barley and the rice Hs1 gene (DIAS)
For the barley transgene experiments we use the malting spring barley cultivar Golden Promise, which is known to be susceptible to the cereal nematode Heterodera avenae. Constructs were generated consisting of the rice and barley Hs1pro-1 homologues under control of the strong constitutive rice actin promoter while the maize ubiquitin promoter was used for driving expression of the B. procumbens Hs1pro-1 gene. Transformation was achieved using particle bombardment of immature embryos and the bar-Bialaphos selection technique. Three lines of plants were obtained hosting the coding sequence of barley Hs1 and one line contained the B. procumbens Hs1pro-1 gene. The transgenes were shown to be transmitted to the progeny. In one line with the barley Hs1 gene the bar gene had segregated out. This incident is very rare since the Bar gene and the gene of interest normally insert very close and linked on the chromosomes.
We are currently trying to find means to further characterize these lines with respect to copy number of the transgenes and the expression levels of the Hs1 genes.

Scientist: Søren Borg
Publication: In preparation