Fine mapping of a HvCBF gene cluster at the frost resistance locus Fr-H2 in barley.

Barley is an economically important model for the Triticeae tribe. We recently developed a new resource: the 'Nure' x 'Tremois' mapping population. Two low temperature QTLs were found to segregate on the long arm of chromosome 5H (Fr-H1, distal; Fr-H2, proximal). With the final aim of positional cloning of the genetic determinants of Fr-H1 and Fr-H2, a large segregating population of 1,849 F(2) plants between parents 'Nure' and 'Tremois' was prepared. These two QT loci were first validated by using a set of F(3) families, marker-selected to harbor pairs of reciprocal haplotypes, with one QTL fixed at homozygosity and the alternate one in heterozygous phase. The study was then focused towards the isolation of the determinant of Fr-H2. Subsequent recombinant screens and phenotypic evaluation of F(4) segregants allowed us to estimate (P < or = 0.01) a refined genomic interval of Fr-H2 (4.6 cM). Several barley genes with the CBF transcription factor signature had been already roughly mapped in cluster at Fr-H2, and they represent likely candidate genes underlying this QTL. Using the large segregating population (3,698 gametes) a high-resolution genetic map of the HvCBF gene cluster was then constructed, and after fine mapping, six recombinations between the HvCBFs were observed. It was therefore possible to genetically divide seven HvCBF subclusters in barley, in a region spanning 0.81 cM, with distances among them varying from 0.03 to 0.32 cM. The few recombinants between the different HvCBF subclusters are being marker-selected and taken to homozygosity, to phenotypically separate the effects of the single HvCBF genes.

Mapping regulatory genes as candidates for cold and drought stress tolerance in barley.

Cereal crop yield is greatly affected in many growing areas by abiotic stresses, mainly low temperature and drought. In order to find candidates for the tolerance genes for these stresses, 13 genes encoding for transcription factors and upstream regulators were screened by amplification and SSCP on six parental genotypes of three barley mapping populations ('Nure' x 'Tremois', 'Proctor' x 'Nudinka', and 'Steptoe' x 'Morex'), and mapped as newly developed STS, SNP, and SSCP markers. A new consensus function map was then drawn using the three maps above, including 16 regulatory candidate genes (CGs). The positions of barley cold and drought tolerance quantitative trait loci (QTLs) presently described in the literature were added to the consensus map to find positional candidates from among the mapped genes. A cluster of six HvCBF genes co-mapped with the Fr-H2 cold tolerance QTL, while no QTLs for the same trait were positioned on chromosome 7H, where two putative barley regulators of CBF expression, ICE1 and FRY1, found by homology search, were mapped in this work. These observations suggest that CBF gene(s) themselves, rather than their two regulators, are at present the best candidates for cold tolerance. Four out of 12 drought tolerance QTLs of the consensus map are associated with regulatory CGs, on chromosomes 2H, 5H, and 7H, and two QTLs with effector genes, on chromosomes 5H and 6H. The results obtained could be used to guide MAS applications, allowing introduction into an ideal genotype of favourable alleles of tolerance QTLs.

Transcriptional adaptor and histone acetyltransferase proteins in Arabidopsis and their interactions with CBF1, a transcriptional activator involved in cold-regulated gene expression.

The ARABIDOPSIS CBF transcriptional activators bind to the CRT/DRE regulatory element present in the promoters of many cold-regulated genes and stimulate their transcription. Expression of the CBF1 proteins in yeast activates reporter genes carrying a minimal promoter with the CRT/DRE as an upstream regulatory element. Here we report that this ability of CBF1 is dependent upon the activities of three key components of the yeast Ada and SAGA complexes, namely the histone acetyltransferase (HAT) Gcn5 and the transcriptional adaptor proteins Ada2 and Ada3. This result suggested that CBF1 might function through the action of similar complexes in ARABIDOPSIS In support of this hypothesis we found that ARABIDOPSIS has a homolog of the GCN5 gene and two homologs of ADA2, the first report of multiple ADA2 genes in an organism. The ARABIDOPSIS GCN5 protein has intrinsic HAT activity and can physically interact in vitro with both the ARABIDOPSIS ADA2a and ADA2b proteins. In addition, the CBF1 transcriptional activator can interact with the ARABIDOPSIS GCN5 and ADA2 proteins. We conclude that ARABIDOPSIS encodes HAT-containing adaptor complexes that are related to the Ada and SAGA complexes of yeast and propose that the CBF1 transcriptional activator functions through the action of one or more of these complexes.

Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activators as an early step in cold-induced COR gene expression.

Cold-induced expression of the Arabidopsis COR (cold-regulated) genes is mediated by a DNA regulatory element termed the CRT (C-repeat)/DRE (dehydration-responsive element). Recently, we identified a transcriptional activator, CBF1, that binds to the CRT/DRE and demonstrated that its overexpression in transgenic Arabidopsis plants at non-acclimating temperatures induces COR gene expression and increases plant freezing tolerance. Here we report that CBF1 belongs to a small family of closely related proteins which includes CBF2 and CBF3. DNA sequencing of an 8.7 kb region of the Arabidopsis genome along with genetic mapping experiments indicated that the three CBF genes are organized in direct repeat on chromosome 4 at 72.8 cM, closely linked to molecular markers PG11 and m600. Like CBF1, both CBF2 and CBF3 activated expression of reporter genes in yeast that contained the CRT/DRE as an upstream activator sequence. The transcript levels for all three CBF genes increased within 15 min of transferring plants to low temperature, followed by accumulation of COR gene transcripts at about 2 h. CBF transcripts also accumulated rapidly in response to mechanical agitation. The promoter regions of the CBF genes do not contain the CRT sequence, CCGAC, and overexpression of CBF1 did not have a detectable effect on CBF3 transcript levels, suggesting that the CBF gene family is not subject to autoregulation. We propose that cold-induced expression of CRT/DRE-containing COR genes involves a low temperature-stimulated signalling cascade in which CBF gene induction is an early event.

Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit.

Recent efforts have defined a cis-acting DNA regulatory element in plants, the C-repeat/dehydration responsive element (DRE), that stimulates transcription in response to low temperature and water deficit. Here we report the isolation of an Arabidopsis thaliana cDNA that encodes a C-repeat/DRE binding factor, CBF1 (C-repeat/DRE Binding Factor 1). Analysis of the deduced CBF1 amino acid sequence indicates that the protein has a molecular mass of 24 kDa, a potential nuclear localization sequence, and a possible acidic activation domain. CBF1 also has an AP2 domain, which is a DNA-binding motif of about 60 aa present in the Arabidopsis proteins APETALA2, AINTEGUMENTA, and TINY; the tobacco ethylene response element binding proteins; and numerous other plant proteins of unknown function. The transcript levels for CBF1, which appears to be a single or low copy number gene, did not change appreciably in plants exposed to low temperature or in detached leaves subjected to water deficit. Binding of CBF1 to the C-repeat/DRE was demonstrated in gel shift assays using recombinant CBF1 protein expressed in Escherichia coli. Moreover, expression of CBF1 in yeast was found to activate transcription of reporter genes containing the C-repeat/DRE as an upstream activator sequence but not mutant versions of the DNA element. We conclude that CBF1 can function as a transcriptional activator that binds to the C-repeat/DRE DNA regulatory element and, thus, is likely to have a role in cold- and dehydration-regulated gene expression in Arabidopsis.

A linkage map of sweet cherry based on RAPD analysis of a microspore-derived callus culture population.

A partial linkage map was constructed for the sweet cherry (Prunus avium L.) cultivar Emperor Francis from a population of 56 microspore-derived callus culture individuals. The callus cultures were genotyped for two allozymes and 90 random amplified polymorphic DNA (RAPD) markers using 79 random decanucleotide DNA primers and the polymerase chain reaction (PCR). Eighty-nine markers mapped to 10 linkage groups totaling 503.3 cM. DNA blot and hybridization analysis using five cloned RAPDs as probes demonstrated that one of the decanucleotide primers amplified a region of the Emperor Francis genome containing a unique sequence, whereas the other four decanucleotide primers amplified regions of the Emperor Francis genome containing repeated sequences. The five cloned RAPD probes also recognized putative homologous regions in ground cherry, P.fruticosa Pall., and sour cherry, P. cerasus L., a naturally occurring allopolyploid between P.fruticosa and P.avium.

Pto3 and Pto4: novel genes from Lycopersicon hirsutum var. glabratum that confer resistance to Pseudomonas syringae pv tomato.

Accessions of wild Lycopersicon germplasm were screened for resistance to Pseudomonas syringae pv tomato (P.s. tomato). Resistance to both race-0 and race-1 strains of P.s. tomato was identified in L. pimpinellifolium, L. peruvianum and L. hirsutum var. glabratum. Resistance to race-0 derived from L. hirsutum var. glabratum (Pto3) appeared to be inherited independently of Pto1 and Pto2. Filial and backcross generations derived from interspecific crosses between L. esculentum and L. hirsutum var. glabratum revealed that Pto3 resistance was inherited in a complex fashion and was incompletely dominant under conditions of high bacteria inocula. Resistance to P.s. tomato race-1 (Pto4) was also identified in L. hirsutum var. glabratum. Pto3 and Pto4 segregated independently of each other.

Phylogenetic relationships between the chlorophyll a/b binding protein (CAB) multigene family: an intra- and interspecies study.

The genome of Glycine max (L.) Merr. cv. "Dare" contains a chlorophyll a/b binding (Cab) protein gene family consisting of 10 genes. The primary structures of two linked Cab genes (Cab 4 and Cab 5) were determined. A comparison of the nucleic acid and predicted amino acid sequences of Cab 4 and Cab 5 revealed a high degree of similarity (96% and 98%, respectively). Phylogenetic inferences drawn from sequence comparisons between previously characterized soybean Cab 1, 2, and 3 and Cab 4 and 5 suggested that soybean Cab 3 was an evolutionarily distant member within this family. We further investigated the molecular evolution of the Cab gene family by comparing nucleotide sequences from 25 different Cab genes representing diverse phylogenetic taxa including monocot and dicot species. Phylogenetic inferences from these data support existing morphological phylogenies in that all species within one family clustered together. These data suggested that the Solanaceae were more evolutionarily distant from the monocots than the Fabaceae and Brassicacea. In addition, these data supported the theory that Cab Type I and II genes originated prior to divergence of the monocots and dicots.