The mosaic oat genome gives insights into a uniquely healthy cereal crop.

Cultivated oat (Avena sativa L.) is an allohexaploid (AACCDD, 2n = 6x = 42) thought to have been domesticated more than 3,000 years ago while growing as a weed in wheat, emmer and barley fields in Anatolia1,2. Oat has a low carbon footprint, substantial health benefits and the potential to replace animal-based food products. However, the lack of a fully annotated reference genome has hampered efforts to deconvolute its complex evolutionary history and functional gene dynamics. Here we present a high-quality reference genome of A. sativa and close relatives of its diploid (Avena longiglumis, AA, 2n = 14) and tetraploid (Avena insularis, CCDD, 2n = 4x = 28) progenitors. We reveal the mosaic structure of the oat genome, trace large-scale genomic reorganizations in the polyploidization history of oat and illustrate a breeding barrier associated with the genome architecture of oat. We showcase detailed analyses of gene families implicated in human health and nutrition, which adds to the evidence supporting oat safety in gluten-free diets, and we perform mapping-by-sequencing of an agronomic trait related to water-use efficiency. This resource for the Avena genus will help to leverage knowledge from other cereal genomes, improve understanding of basic oat biology and accelerate genomics-assisted breeding and reanalysis of quantitative trait studies.

Improved Salinity Tolerance-Associated Variables Observed in EMS Mutagenized Wheat Lines.

Salinity tolerance-associated phenotypes of 35 EMS mutagenized wheat lines originating from BARI Gom-25 were compared. Vegetative growth was measured using non-destructive image-based phenotyping. Five different NaCl concentrations (0 to 160 mM) were applied to plants 19 days after planting (DAP 19), and plants were imaged daily until DAP 38. Plant growth, water use, leaf Na+, K+ and Cl- content, and thousand kernel weight (TKW) were measured, and six lines were selected for further analysis. In saline conditions, leaf Na+, K+, and Cl- content variation on a dry weight basis within these six lines were ~9.3, 1.4, and 2.4-fold, respectively. Relative to BARI Gom-25, two (OA6, OA62) lines had greater K+ accumulation, three (OA6, OA10, OA62) had 50-75% lower Na+:K+ ratios, and OA62 had ~30% greater water-use index (WUI). OA23 had ~2.2-fold greater leaf Na+ and maintained TKW relative to BARI Gom-25. Two lines (OA25, OA52) had greater TKW than BARI Gom-25 when grown in 120 mM NaCl but similar Na+:K+, WUI, and biomass accumulation. OA6 had relatively high TKW, high leaf K+, and WUI, and low leaf Na+ and Cl-. Phenotypic variation revealed differing associations between the parameters measured in the lines. Future identification of the genetic basis of these differences, and crossing of lines with phenotypes of interest, is expected to enable the assessment of which combinations of parameters deliver the greatest improvement in salinity tolerance.

COVID-19 lockdown reveals tourists as seabird guardians.

The widespread lockdowns put in place to limit the spread of the new coronavirus disease (COVID-19) offers a rare opportunity in understanding how human presence influence ecosystems. Using data from long-term seabird monitoring, we reveal a previously concealed guarding effect by tourist groups on an iconic seabird colony in the Baltic Sea. The absence of tourists in 2020 lead to a sevenfold increase in presence of white-tailed eagles Haliaeetus albicilla, a sevenfold increase in their disturbance of breeding common murres Uria aalge and causing 26% lower murre productivity than the long-term average. Eagles did not prey on murres, but their frequent disturbances delayed egg laying and facilitated egg predation from herring gulls Larus argentatus and hooded crows Corvus cornix. Based on our findings, we suggest that human presence could be used as a strategic measure in guarding seabird colonies, and that a social-ecological systems perspective is vital for long-term success in protected area management.

Development and characterization of an EMS-mutagenized wheat population and identification of salt-tolerant wheat lines.

BACKGROUND: Triticum aestivum (wheat) is one of the world's oldest crops and has been used for >8000 years as a food crop in North Africa, West Asia and Europe. Today, wheat is one of the most important sources of grain for humans, and is cultivated on greater areas of land than any other crop. As the human population increases and soil salinity becomes more prevalent, there is increased pressure on wheat breeders to develop salt-tolerant varieties in order to meet growing demands for yield and grain quality. Here we developed a mutant wheat population using the moderately salt-tolerant Bangladeshi variety BARI Gom-25, with the primary goal of further increasing salt tolerance. RESULTS: After titrating the optimal ethyl methanesulfonate (EMS) concentration, ca 30,000 seeds were treated with 1% EMS, and 1676 lines, all originating from single seeds, survived through the first four generations. Most mutagenized lines showed a similar phenotype to BARI Gom-25, although visual differences such as dwarfing, giant plants, early and late flowering and altered leaf morphology were seen in some lines. By developing an assay for salt tolerance, and by screening the mutagenized population, we identified 70 lines exhibiting increased salt tolerance. The selected lines typically showed a 70% germination rate on filter paper soaked in 200 mM NaCl, compared to 0-30% for BARI Gom-25. From two of the salt-tolerant OlsAro lines (OA42 and OA70), genomic DNA was sequenced to 15x times coverage. A comparative analysis against the BARI Gom-25 genomic sequence identified a total of 683,201 (OA42), and 768,954 (OA70) SNPs distributed throughout the three sub-genomes (A, B and D). The mutation frequency was determined to be approximately one per 20,000 bp. All the 70 selected salt-tolerant lines were tested for root growth in the laboratory, and under saline field conditions in Bangladesh. The results showed that all the lines selected for tolerance showed a better salt tolerance phenotype than both BARI Gom-25 and other local wheat varieties tested. CONCLUSION: The mutant wheat population developed here will be a valuable resource in the development of novel salt-tolerant varieties for the benefit of saline farming.

Acylated monogalactosyl diacylglycerol: prevalence in the plant kingdom and identification of an enzyme catalyzing galactolipid head group acylation in Arabidopsis thaliana.

The lipid phase of the thylakoid membrane is mainly composed of the galactolipids mono- and digalactosyl diacylglycerol (MGDG and DGDG, respectively). It has been known since the late 1960s that MGDG can be acylated with a third fatty acid to the galactose head group (acyl-MGDG) in plant leaf homogenates. In certain brassicaceous plants like Arabidopsis thaliana, the acyl-MGDG frequently incorporates oxidized fatty acids in the form of the jasmonic acid precursor 12-oxo-phytodienoic acid (OPDA). In the present study we further investigated the distribution of acylated and OPDA-containing galactolipids in the plant kingdom. While acyl-MGDG was found to be ubiquitous in green tissue of plants ranging from non-vascular plants to angiosperms, OPDA-containing galactolipids were only present in plants from a few genera. A candidate protein responsible for the acyl transfer was identified in Avena sativa (oat) leaf tissue using biochemical fractionation and proteomics. Knockout of the orthologous gene in A. thaliana resulted in an almost total elimination of the ability to form both non-oxidized and OPDA-containing acyl-MGDG. In addition, heterologous expression of the A. thaliana gene in E. coli demonstrated that the protein catalyzed acylation of MGDG. We thus demonstrate that a phylogenetically conserved enzyme is responsible for the accumulation of acyl-MGDG in A. thaliana. The activity of this enzyme in vivo is strongly enhanced by freezing damage and the hypersensitive response.

Genomic identification of expressed globulin storage proteins in oat.

Introduction: Oats, a highly nutritious cereal known for their health benefits, contain various macromolecules of significant biological value, including abundant and highly digestible proteins. Despite their importance, oat proteins have not been extensively studied. Here, we present a complete set of the expressed globulins genes, which code for the main storage protein in oats as well as their chromosomal positions. Methods: Published expressed sequence tags for globulins were used as queries in the Sang oat genome. In addition, globulin proteins were fractionated from oat flour by solvent extraction based on differential solubility with other classes of cereal proteins. The protein fractions were separated by gel electrophoresis and analyzed by tandem mass spectrometry to confirm their identity and expression in seed. Results and discussion: In total 32 globulin gene sequences were identified on the oat genome. Out of these, the expression on RNA level could be confirmed and 27 were also detected as expressed proteins by MS. Our results provide the most extensive set of salt-soluble oat globulin sequences to date, paving the way for further understanding their implications for human nutrition. In addition, a simple methodology to fractionate oat proteins is presented.

Macromolecular characterization of high ß-glucan oat lines.

Oat (Avena sativa) is a cereal grain rich in fibers, proteins, vitamins and minerals. Oats have been linked to several health benefits, such as lowering blood cholesterol levels, counteracting cardiovascular disease and regulating blood sugar levels. This study aimed to characterize two new oat lines with high ß-glucan content emanating from ethyl methyl sulphonate mutagenesis on the Lantmännen elite variety Belinda. Two of the mutated lines, and the mother variety Belinda, were profiled for ß-glucan, arabinoxylan, total dietary fiber and starch composition. In addition, total lipid and protein content, amino acid composition and ß-glucan molecular weights were analyzed. The high levels of ß-glucan resulted in a significant increase in total dietary fiber, but no correlation could be established between higher or lower levels of the assayed macromolecules, i.e., between arabinoxylan-, starch-, lipid- or protein levels in the mutated lines compared to the reference. The results indicate separate biosynthetic pathways for ß-glucans and other macromolecules and an independent regulation of the different polysaccharides studied. Therefore, ethyl methyl sulphonate mutagenesis can be used to increase levels of multiple macromolecules in the same line.

Characterization of high Arabinoxylan oat lines identified from a mutagenized oat population.

Oat (Avena sativa) is a nutritionally important cereal crop that is rich in health-promoting dietary fibers, favorable proteins and polar lipids. In this work, ca. 500 random lines of a mutagenized oat population of high genetic variation were screened for arabinoxylan (AX) content. This identified lines with up to 60% higher AX levels in flour from whole seed and up to 100% higher in flour from dehulled seeds, as compared to the original Belinda variety. In addition, the cellular localization of AX was determined in cross-sections of dehulled seeds from three high and one low AX line using a xylan-specific antibody. This revealed variations in the amount and localization of AX between high and low AX lines. The high AX lines will now serve as a starting point in the development of oat varieties with superior health-promoting and rheological properties.

ERK/p90(RSK)/14-3-3 signalling has an impact on expression of PEA3 Ets transcription factors via the transcriptional repressor capicúa.

Compounds that inhibit signalling upstream of ERK (extracellular-signal-regulated kinase) are promising anticancer therapies, motivating research to define how this pathway promotes cancers. In the present study, we show that human capicúa represses mRNA expression for PEA3 (polyoma enhancer activator 3) Ets transcription factors ETV1, ETV4 and ETV5 (ETV is Ets translocation variant), and this repression is relieved by multisite controls of capicúa by ERK, p90(RSK) (p90 ribosomal S6 kinase) and 14-3-3 proteins. Specifically, 14-3-3 binds to p90(RSK)-phosphorylated Ser¹7³ of capicúa thereby modulating DNA binding to its HMG (high-mobility group) box, whereas ERK phosphorylations prevent binding of a C-terminal NLS (nuclear localization sequence) to importin α4 (KPNA3). ETV1, ETV4 and ETV5 mRNA levels in melanoma cells are elevated by siRNA (small interfering RNA) knockdown of capicúa, and decreased by inhibiting ERK and/or expressing a form of capicúa that cannot bind to 14-3-3 proteins. Capicúa knockdown also enhances cell migration. The findings of the present study give further mechanistic insights into why ETV1 is highly expressed in certain cancers, indicate that loss of capicúa can desensitize cells to the effects of ERK pathway inhibitors, and highlight interconnections among growth factor signalling, spinocerebellar ataxias and cancers.

Foraging range scales with colony size in high-latitude seabirds.

Density-dependent prey depletion around breeding colonies has long been considered an important factor controlling the population dynamics of colonial animals.1-4 Ashmole proposed that as seabird colony size increases, intraspecific competition leads to declines in reproductive success, as breeding adults must spend more time and energy to find prey farther from the colony.1 Seabird colony size often varies over several orders of magnitude within the same species and can include millions of individuals per colony.5,6 As such, colony size likely plays an important role in determining the individual behavior of its members and how the colony interacts with the surrounding environment.6 Using tracking data from murres (Uria spp.), the world's most densely breeding seabirds, we show that the distribution of foraging-trip distances scales to colony size0.33 during the chick-rearing stage, consistent with Ashmole's halo theory.1,2 This pattern occurred across colonies varying in size over three orders of magnitude and distributed throughout the North Atlantic region. The strong relationship between colony size and foraging range means that the foraging areas of some colonial species can be estimated from colony sizes, which is more practical to measure over a large geographic scale. Two-thirds of the North Atlantic murre population breed at the 16 largest colonies; by extrapolating the predicted foraging ranges to sites without tracking data, we show that only two of these large colonies have significant coverage as marine protected areas. Our results are an important example of how theoretical models, in this case, Ashmole's version of central-place-foraging theory, can be applied to inform conservation and management in colonial breeding species.

Identification of gliadin-binding peptides by phage display.

BACKGROUND: Coeliac disease (CD) is a common and complex disorder of the small intestine caused by intolerance to wheat gluten and related edible cereals like barley and rye. Peptides originating from incomplete gliadin digestion activate the lamina propria infiltrating T cells to release proinflammatory cytokines, which in turn cause profound tissue remodelling of the small intestinal wall. There is no cure for CD except refraining from consuming gluten-containing products. RESULTS: Phage from a random oligomer display library were enriched by repeated pannings against immobilised gliadin proteins. Phage from the final panning round were plated, individual plaques picked, incubated with host bacteria, amplified to a population size of 1011 to 1012 and purified. DNA was isolated from 1000 purified phage populations and the region covering the 36 bp oligonucleotide insert from which the displayed peptides were translated, was sequenced. Altogether more than 150 different peptide-encoding sequences were identified, many of which were repeatedly isolated under various experimental conditions. Amplified phage populations, each expressing a single peptide, were tested first in pools and then one by one for their ability to inhibit binding of human anti-gliadin antibodies in ELISA assays. These experiments showed that several of the different peptide-expressing phage tested inhibited the interaction between gliadin and anti-gliadin antibodies. Finally, four different peptide-encoding sequences were selected for further analysis, and the corresponding 12-mer peptides were synthesised in vitro. By ELISA assays it was demonstrated that several of the peptides inhibited the interaction between gliadin molecules and serum anti-gliadin antibodies. Moreover, ELISA competition experiments as well as dot-blot and western blot revealed that the different peptides interacted with different molecular sites of gliadin. CONCLUSIONS: We believe that several of the isolated and characterised gliadin-binding peptides described here could provide valuable tools for researchers in the field of CD by facilitating studies on localisation and uptake of various gliadin peptides in the small intestine. In future work, the potential of these peptides to detoxify gluten will be investigated.

In vitro digestive stability of complexes between gliadin and synthetic blocking peptides.

Celiac disease is caused by an inappropriate immune response to incompletely digested gluten proteins. We investigated whether synthetic peptides with high affinity to wheat gliadin could be selected with a phage display technique and whether complexes between such peptides and gliadin could sustain gastric and pancreatic digestion. Two synthetic peptides, P61 and P64, were selected because of their high affinity to immobilized gliadin. They were allowed to form complexes with gliadin, whereafter the complexes were subjected to in vitro digestion with gastric and pancreatic enzymes. The digestion products were analyzed with Western blot and RP HPLC. The results showed that both peptides formed stable complexes with intact gliadin and that complexes between gliadin and peptide P64 partly resisted gastrointestinal digestion. The two peptides reduced the binding of serum anti-gliadin IgA antibodies by 12%, and 11.5%, respectively, and the binding of anti-gliadin antibodies of the IgG isotype by 13% and 10%. Thus peptides produced by a phage display technique could interact stably with gliadin partly masking epitopes for antibody binding. A combination of peptides of this kind may be used to block gliadin-immune system interactions.

Characterization and antioxidant activity of avenanthramides from selected oat lines developed by mutagenesis technique.

From a mutagenized oat population, produced by ethyl methanesulfonate mutagenesis, hulled grains from 17 lines with elevated avenanthramide (AVN) content were selected and their AVN structures, concentrations and antioxidant potentials were determined by HPLC-MS2 and HPLC equipped with an on-line ABTS+ antioxidant detection system. The data obtained showed qualitative and quantitative differences in the synthesis of AVNs in the different lines, with a total AVN concentration up to 227.5 µg/g oat seed flour in the highest line, compared with 78.2 µg/g seed in the commercial line, SW Belinda. In total, 25 different AVNs were identified with avenanthramide B structures being among the most abundant, and AVN C structures having the highest antioxidant activity. The findings indicate the potential of oat mutagenesis in combination with a high precision biochemical selection method for the generation of stable mutagenized lines with a high concentration of total and/or individual AVNs in the oat seed grain.

Development and characterization of an oat TILLING-population and identification of mutations in lignin and beta-glucan biosynthesis genes.

BACKGROUND: Oat, Avena sativa is the sixth most important cereal in the world. Presently oat is mostly used as feed for animals. However, oat also has special properties that make it beneficial for human consumption and has seen a growing importance as a food crop in recent decades. Increased demand for novel oat products has also put pressure on oat breeders to produce new oat varieties with specific properties such as increased or improved beta-glucan-, antioxidant- and omega-3 fatty acid levels, as well as modified starch and protein content. To facilitate this development we have produced a TILLING (Targeting Induced Local Lesions IN Genomes) population of the spring oat cultivar SW Belinda. RESULTS: Here a population of 2600 mutagenised M2 lines, producing 2550 M3 seed lots were obtained. The M2 population was initially evaluated by visual inspection and a number of different phenotypes were seen ranging from dwarfs to giants, early flowering to late flowering, leaf morphology and chlorosis. Phloroglucinol/HCl staining of M3 seeds, obtained from 1824 different M2 lines, revealed a number of potential lignin mutants. These were later confirmed by quantitative analysis. Genomic DNA was prepared from the M2 population and the mutation frequency was determined. The estimated mutation frequency was one mutation per 20 kb by RAPD-PCR fingerprinting, one mutation per 38 kb by MALDI-TOF analysis and one mutation per 22.4 kb by DNA sequencing. Thus, the overall mutation frequency in the population is estimated to be one mutation per 20-40 kb, depending on if the method used addressed the whole genome or specific genes. During the investigation, 6 different mutations in the phenylalanine ammonia-lyase (AsPAL1) gene and 10 different mutations in the cellulose synthase-like (AsCslF6) beta-glucan biosynthesis gene were identified. CONCLUSION: The oat TILLING population produced in this work carries, on average, hundreds of mutations in every individual gene in the genome. It will therefore be an important resource in the development of oat with specific characters. The population (M5) will be available for academic research via Nordgen http://www.nordgen.org as soon as enough seeds are obtained.[Genbank accession number for the cloned AsPAL1 is GQ373155 and GQ379900 for AsCslF6].

In silico analysis of promoter regions from cold-induced genes in rice (Oryza sativa L.) and Arabidopsis thaliana reveals the importance of combinatorial control.

MOTIVATION: Cold acclimation involves a number of different cellular processes that together increase the freezing tolerance of an organism. The DREB1/CBFs are transcription factors (TFs) that are prominent in the regulation of cold responses in Arabidopsis thaliana, rice and many other crops. We investigated if the expression of DREB1/CBFs and co-expressed genes relies on combinatorial control by several TFs. Our results support this notion and indicate that methods for studying the regulation of complex cellular processes should include identification of combinations of motifs, in addition to searching for individual overrepresented binding sites.

New DArT markers for oat provide enhanced map coverage and global germplasm characterization.

BACKGROUND: Genomic discovery in oat and its application to oat improvement have been hindered by a lack of genetic markers common to different genetic maps, and by the difficulty of conducting whole-genome analysis using high-throughput markers. This study was intended to develop, characterize, and apply a large set of oat genetic markers based on Diversity Array Technology (DArT). RESULTS: Approximately 19,000 genomic clones were isolated from complexity-reduced genomic representations of pooled DNA samples from 60 oat varieties of global origin. These were screened on three discovery arrays, with more than 2000 polymorphic markers being identified for use in this study, and approximately 2700 potentially polymorphic markers being identified for use in future studies. DNA sequence was obtained for 2573 clones and assembled into a non-redundant set of 1770 contigs and singletons. Of these, 705 showed highly significant (Expectation < 10E-10) BLAST similarity to gene sequences in public databases. Based on marker scores in 80 recombinant inbred lines, 1010 new DArT markers were used to saturate and improve the 'Kanota' x 'Ogle' genetic map. DArT markers provided map coverage approximately equivalent to existing markers. After binning markers from similar clones, as well as those with 99% scoring similarity, a set of 1295 non-redundant markers was used to analyze genetic diversity in 182 accessions of cultivated oat of worldwide origin. Results of this analysis confirmed that major clusters of oat diversity are related to spring vs. winter type, and to the presence of major breeding programs within geographical regions. Secondary clusters revealed groups that were often related to known pedigree structure. CONCLUSION: These markers will provide a solid basis for future efforts in genomic discovery, comparative mapping, and the generation of an oat consensus map. They will also provide new opportunities for directed breeding of superior oat varieties, and guidance in the maintenance of oat genetic diversity.

Putative cold acclimation pathways in Arabidopsis thaliana identified by a combined analysis of mRNA co-expression patterns, promoter motifs and transcription factors.

BACKGROUND: With the advent of microarray technology, it has become feasible to identify virtually all genes in an organism that are induced by developmental or environmental changes. However, relying solely on gene expression data may be of limited value if the aim is to infer the underlying genetic networks. Development of computational methods to combine microarray data with other information sources is therefore necessary. Here we describe one such method. RESULTS: By means of our method, previously published Arabidopsis microarray data from cold acclimated plants at six different time points, promoter motif sequence data extracted from approximately 24,000 Arabidopsis promoters and known transcription factor binding sites were combined to construct a putative genetic regulatory interaction network. The inferred network includes both previously characterised and hitherto un-described regulatory interactions between transcription factor (TF) genes and genes that encode other TFs or other proteins. Part of the obtained transcription factor regulatory network is presented here. More detailed information is available in the additional files. CONCLUSION: The rule-based method described here can be used to infer genetic networks by combining data from microarrays, promoter sequences and known promoter binding sites. This method should in principle be applicable to any biological system. We tested the method on the cold acclimation process in Arabidopsis and could identify a more complex putative genetic regulatory network than previously described. However, it should be noted that information on specific binding sites for individual TFs were in most cases not available. Thus, gene targets for the entire TF gene families were predicted. In addition, the networks were built solely by a bioinformatics approach and experimental verifications will be necessary for their final validation. On the other hand, since our method highlights putative novel interactions, more directed experiments could now be performed.

Wholegrain oat diet changes the expression of genes associated with intestinal bile acid transport.

SCOPE: The molecular mechanisms underlying the cholesterol-lowering properties of oats are only partly known. To study possible pathways involved, we investigated gene expressions in the liver and small intestine of mice fed oats. METHOD AND RESULTS: Cholesterol and bile acids were analyzed in plasma and feces from LDL-receptor deficient (LDLr-/- ) mice fed Western diet with wholegrain oats. A transcriptome analysis of mRNA from liver and jejunum was performed together with quantitative RT-PCR. Oat-fed mice had lower levels of plasma lipids and increased levels of bile acids and cholesterol in feces compared with controls. Two hundred thirty nine genes in jejunum and 25 genes in liver were differentially expressed (FDR corrected p < 0.05). The most affected biological process in jejunum was lipid biosynthesis and regulation. The apical sodium-dependent bile acid transporter (ASBT, Slc10a) and the intracellular bile acid binding protein (Fabp6) were both upregulated, whereas small heterodimer partner-1 (Shp-1) and apolipoprotein CII (Apoc2) were downregulated. CONCLUSIONS: Whole oats attenuated responses typically induced by high-fat diet. Increased expression of genes for intestinal bile acid uptake following oat consumption suggests retention in the gut lumen rather than decreased uptake capacity as cause for the increased bile acid excretion and the concomitant reduction of plasma cholesterol.

Generation and analysis of 9792 EST sequences from cold acclimated oat, Avena sativa.

BACKGROUND: Oat is an important crop in North America and northern Europe. In Scandinavia, yields are limited by the fact that oat cannot be used as a winter crop. In order to develop such a crop, more knowledge about mechanisms of cold tolerance in oat is required. RESULTS: From an oat cDNA library 9792 single-pass EST sequences were obtained. The library was prepared from pooled RNA samples isolated from leaves of four-week old Avena sativa (oat) plants incubated at +4 degrees C for 4, 8, 16 and 32 hours. Exclusion of sequences shorter than 100 bp resulted in 8508 high-quality ESTs with a mean length of 710.7 bp. Clustering and assembly identified a set of 2800 different transcripts denoted the Avena sativa cold induced UniGene set (AsCIUniGene set). Taking advantage of various tools and databases, putative functions were assigned to 1620 (58%) of these genes. Of the remaining 1180 unclassified sequences, 427 appeared to be oat-specific since they lacked any significant sequence similarity (Blast E values > 10(-10)) to any sequence available in the public databases. Of the 2800 UniGene sequences, 398 displayed significant homology (BlastX E values < or = 10(-10)) to genes previously reported to be involved in cold stress related processes. 107 novel oat transcription factors were also identified, out of which 51 were similar to genes previously shown to be cold induced. The CBF transcription factors have a major role in regulating cold acclimation. Four oat CBF sequences were found, belonging to the monocot cluster of DREB family ERF/AP2 domain proteins. Finally in the total EST sequence data (5.3 Mbp) approximately 400 potential SSRs were found, a frequency similar to what has previously been identified in Arabidopsis ESTs. CONCLUSION: The AsCIUniGene set will now be used to fabricate an oat biochip, to perform various expression studies with different oat cultivars incubated at varying temperatures, to generate molecular markers and provide tools for various genetic transformation experiments in oat. This will lead to a better understanding of the cellular biology of this important crop and will open up new ways to improve its agronomical properties.

Cloning and molecular characterisation of a gene encoding cytochrome P450 C-23 steroid hydroxylase (CYP90) from aspen.

Gene CPD (constitutive photomorphogenesis and dwarf) encodes a cytochrome P450 steroid side-chain hydroxylase (CYP90) involved in biosynthesis of brassinosteroids (BRs) in Arabidopsis thanalia. To dissect the molecular mechanism underlying the biosynthesis and action of the new phytohormone in perennial woody plants, a 1442 bp cDNA and a 1900 bp genomic DNA fragments whose 3'- half was well overlapped with the 5'-half of the former were obtained from a hybrid aspen (Populus tremula x tremudelois) by screening of the cDNA and genomic DNA libraries of this tree using a cDNA fragment from the Arabidopsis CPD gene in the present study. Sequence analysis revealed that the open reading frame comprising the cDNA and genomic DNA fragments encoded a protein of 476 amino acids with a molecular weight of 63 kD. This protein is four amino acid residues longer than Arabidopsis CYP90, and 78.32% identical to the latter. Also it shared high homologies with the Arabidopsis CYP90 in all known functional domains,including steroid-binding region closely related to BR biosynthesis, indicating a high conservatism of the putative CDP gene between annual herb and perennial woody plant species.