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°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 ≤ 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

A guide to barley mutants

BACKGROUND: Mutants have had a fundamental impact upon scientific and applied genetics. They have paved the way for the molecular and genomic era, and most of today's crop plants are derived from breeding programs involving mutagenic treatments.RESULTS: Barley (Hordeum vulgare L.) is one of the most widely grown cereals in the world and has a long history as a crop plant. Barley breeding started more than 100 years ago and large breeding programs have collected and generated a wide range of natural and induced mutants, which often were deposited in genebanks around the world. In recent years, an increased interest in genetic diversity has brought many historic mutants into focus because the collections are regarded as valuable resources for understanding the genetic control of barley biology and barley breeding. The increased interest has been fueled also by recent advances in genomic research, which provided new tools and possibilities to analyze and reveal the genetic diversity of mutant collections.CONCLUSION: Since detailed knowledge about phenotypic characters of the mutants is the key to success of genetic and genomic studies, we here provide a comprehensive description of mostly morphological barley mutants. The review is closely linked to the International Database for Barley Genes and Barley Genetic Stocks ( bgs.nordgen.org ) where further details and additional images of each mutant described in this review can be found.</p

A guide to barley mutants

BACKGROUND: Mutants have had a fundamental impact upon scientific and applied genetics. They have paved the way for the molecular and genomic era, and most of today's crop plants are derived from breeding programs involving mutagenic treatments.RESULTS: Barley (Hordeum vulgare L.) is one of the most widely grown cereals in the world and has a long history as a crop plant. Barley breeding started more than 100 years ago and large breeding programs have collected and generated a wide range of natural and induced mutants, which often were deposited in genebanks around the world. In recent years, an increased interest in genetic diversity has brought many historic mutants into focus because the collections are regarded as valuable resources for understanding the genetic control of barley biology and barley breeding. The increased interest has been fueled also by recent advances in genomic research, which provided new tools and possibilities to analyze and reveal the genetic diversity of mutant collections.CONCLUSION: Since detailed knowledge about phenotypic characters of the mutants is the key to success of genetic and genomic studies, we here provide a comprehensive description of mostly morphological barley mutants. The review is closely linked to the International Database for Barley Genes and Barley Genetic Stocks ( bgs.nordgen.org ) where further details and additional images of each mutant described in this review can be found.</p

A two-component response regulator is involved in cold sensing in oat

Plants have developed a number of different physiological and developmental responses to abiotic stress. One important process is acclimation, where mild stress conditions greatly enhance tolerance to later and more severe conditions. During the acclimation changes in gene expression patterns occur, which leads to a plant response involving the necessary modifications of growth, development and cellular homeostasis. During the last years, six different cellular signal transduction pathways between the initial cold-stress perception and the gene expression response have been documented. Here we suggest an additional pathway. Two component systems, first described in prokaryotes, have also been identified in yeast and plant systems, but not in animals. They are characterised by a phosphotransfer reaction between two types of signal transducers and involves a sensory histidine kinase receptor and a response regulator. In plants, two-component systems play important roles in ethylene and cytokinin signalling and osmosensing but have not been directly coupled to cold signalling so far. From a subtractive oat cDNA library enriched in cold induced sequences we isolated a full-length clone, denoted AsDP5. Northern and RT-PCR analysis showed that the AsDP5 was induced at +4oC in less than 1 h. Analysis of the deduced 621 amino acids long protein revealed that it had a N-terminal two component response regulator domain, putative nuclear localisation signals and a zink finger DNA binding domain at the C-terminus. Thus, the AsDP5 protein strongly resembles the response regulator protein of two-component systems. This cold signalling pathway is now carefully analysed in oat and in the model plant Arabidopsis, using various knock-out mutants and transgenic systems.vokMyynti MTT tietopalvelu

Ant13 Encodes Regulatory Factor WD40 Controlling Anthocyanin and Proanthocyanidin Synthesis in Barley (Hordeum vulgare L.)

Flavonoid compounds like anthocyanins and proanthocyanidins are important plant secondary metabolites having wide biological activities for humans. In this study, the molecular function of the Ant13 locus, which is one of the key loci governing flavonoid synthesis in barley, was determined. It was found that Ant13 encodes a WD40-type regulatory protein, which is required for transcriptional activation of a set of structural genes encoding enzymes of flavonoid biosynthesis at the leaf sheath base (colored by anthocyanins) and in grains (which accumulate proanthocyanidins). Besides its role in flavonoid biosynthesis, pleiotropic effects of this gene in plant growth were revealed. The mutants deficient in the Ant13 locus showed similar germination rates but a decreased rate of root and shoot growth and yield-related parameters in comparison to the parental cultivars. This is the seventh Ant locus (among 30) for which molecular functions in flavonoid biosynthesis regulation have been determined

Comparing two microarray platforms for identifying mutated genes in barley (Hordeum vulgare L.)

We have previously described the evaluation of a cDNA microarray platform to identify and clone mutated barley (Hordeum vulgare L.) genes, using their transcriptionally defective mutant alleles (S. Zakhrabekova, C.G. Karmangara, D. von Wettstein, M. Hansson, A microarray approach for identification of mutated genes, Plant Physiol. Biochem. 40 (2002) 189-197). It was concluded that competitive hybridization between phenotypically similar mutants could specifically highlight an arrayed clone, corresponding to the mutated gene. In this study we evaluate whether the Affymetrix microarray platform can be used for the same purpose. The Affymetrix barley microarray contains a large number of clones (22,792 probe sets). In this and the previous study we used two barley mutant strains, xantha-h.57 and xantha-f.27, with known mutations in different subunit genes of the chlorophyll biosynthetic enzyme magnesium chelatase (EC 6.6. 1. 1). Mutant xantha-h.57 produces no Xantha-h mRNA whereas in xantha-f27 the nonsense mutation in the last exon of the gene, results in nonsense-mediated decay of Xantha-f mRNA. We conclude that the Affyinetrix platform meets our requirements and that our approach successfully highlighted the arrayed Xantha-h clone and that Xantha-f was among the top fourteen candidates

A microarray approach for identifying mutated genes

This study was performed to evaluate if microarray technology can identify genes using their transcriptionally defective mutant alleles. Three barley (Hordeum vulgare L.) mutant strains, xantha-h57, xantha-f27 and xantha-g28, with mutations in the genes encoding the three subunits of the chlorophyll biosynthetic enzyme magnesium chelatase, were used in a reconstruction experiment. The mutation xantha-h57 prevents transcription of Xantha-h mRNA. Microarrays were prepared by robotic spotting of PCR products from 968 clones at 1760 positions. Most of the materials were from cloned expressed sequence tags (ESTs). The barley Xantha-h gene was printed at six positions in duplicate. Crude bacterial lysates as well as purified plasmids were used as template in the PCR reactions. The bacterial lysates did not affect the printing quality of the DNA. This simplification is important, as it increases the throughput capacity and decreases costs. cDNA from the three mutants and the wild-type strain were differentially labeled with fluorescing nucleotides. Labeled cDNA from one mutant was mixed in equal amounts with labeled cDNA of another mutant or wild type and competitively hybridized to the microarrayed clones. The combination of labeled cDNA from xantha-h57 with that of xantha-f27 or xantha-g28 specifically highlighted the positions representing the Xantha-h gene on the microarrays. Therefore, we regard this experiment as a demonstration that microarrays provide a very promising method for screening large DNA libraries in order to clone genes known only through their mutant phenotype. This opens up a new way of using the microarray technology for cloning genes from eukaryotes with complex genomes for which genome sequencing is unlikely to proceed. Our results also put the many available plant mutant collections in focus as treasuries for gene hunters

Analysis of early-flowering genes at barley chromosome 2H expands the repertoire of mutant alleles at the Mat-c locus

Key message: Analyses of barley mat-c loss of function mutants reveal deletions, splice-site mutations and nonsynonymous substitutions in a key gene regulating early flowering. Abstract: Optimal timing of flowering is critical for reproductive success and crop yield improvement. Several major quantitative trait loci for flowering time variation have been identified in barley. In the present study, we analyzed two near-isogenic lines, BW507 and BW508, which were reported to carry two independent early-flowering mutant loci, mat-b.7 and mat-c.19, respectively. Both introgression segments are co-localized in the pericentromeric region of chromosome 2H. We mapped the mutation in BW507 to a 31 Mbp interval on chromosome 2HL and concluded that BW507 has a deletion of Mat-c, which is an ortholog of Antirrhinum majus CENTRORADIALIS (AmCEN) and Arabidopsis thaliana TERMINAL FLOWER1 (AtTFL1). Contrary to previous reports, our data showed that both BW507 and BW508 are Mat-c deficient and none of them are mat-b.7 derived. This work complements previous studies by identifying the uncharacterized mat-c.19 mutant and seven additional mat-c mutants. Moreover, we explored the X-ray structure of AtTFL1 for prediction of the functional effects of nonsynonymous substitutions caused by mutations in Mat-c

Molecular characterization of CBF transcription factor genes in oat

By EST sequencing we identified 2866 different genes from a cold induced cDNA library from the British winter oat variety Gerald (see other abstract by Bräutigam et al). Among these there are several genes similar to cold and/or drought-induced genes previously identified in rice, wheat, rye, barley and Arabidopsis. More than 100 genes encoded putative transcription factors. Of particular importance for the regulation of cold acclimation is the CBF transcription factor family genes, which have important regulatory roles in the cold signalling pathway. In all CBF proteins analysed so far, a characteristic AP2-binding domain has been found. This interacts with a DRE/CRT regulatory element and activates transcription of downstream cold and dehydration responsive genes. Four CBF factor sequences were found in the oat EST collection, belonging to at least 2 different gene families. A full-length cDNA clone from one such gene, denoted AsCBF1, was isolated and sequenced. This revealed a 909 bp long open reading frame (ORF) encoding a putative protein of 303 aa that contain an AP2-binding domain. By semiquantitative RT-PCR analysis we showed that expression of the AsCBF1 gene was induced already 30 minutes after cold induction at +4oC. By chromosomal walking an additional 457 bp upstream of the AsCBF1 translational start codon was isolated. A TATAA-box was then found as well as several other potential regulatory elements including a Myc-recognition site. These elements will now be further characterised both in vitro and in vivo. CBF genes will also be used as genetic engineering tools to develop more cold tolerant oat.vokMyynti MTT tietopalvelu