The broad adaptability of wheat and barley is in part attributable to their flexible growth habit, in that spring forms have recurrently evolved from the ancestral winter growth habit. In diploid wheat and barley growth habit is determined by allelic variation at the VRN-1 and/or VRN-2 loci, whereas in the polyploid wheat species it is determined primarily by allelic variation at VRN-1. Dominant Vrn-A1 alleles for spring growth habit are frequently associated with mutations in the promoter region in diploid wheat and in the A genome of common wheat. However, several dominant Vrn-A1, Vrn-B1, Vrn-D1 (common wheat) and Vrn-H1 (barley) alleles show no polymorphisms in the promoter region relative to their respective recessive alleles. In this study, we sequenced the complete VRN-1 gene from these accessions and found that all of them have large deletions within the first intron, which overlap in a 4-kb region. Furthermore, a 2.8-kb segment within the 4-kb region showed high sequence conservation among the different recessive alleles. PCR markers for these deletions showed that similar deletions were present in all the accessions with known Vrn-B1 and Vrn-D1 alleles, and in 51 hexaploid spring wheat accessions previously shown to have no polymorphisms in the VRN-A1 promoter region. Twenty-four tetraploid wheat accessions had a similar deletion in VRN-A1 intron 1. We hypothesize that the 2.8-kb conserved region includes regulatory elements important for the vernalization requirement. Epistatic interactions between VRN-H2 and the VRN-H1 allele with the intron 1 deletion suggest that the deleted region may include a recognition site for the flowering repression mediated by the product of the VRN-H2 gene of barley.

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The meristematic tissues of temperate woody perennials must acclimate to freezing temperatures to survive the winter and resume growth the following year. To determine whether the C-repeat binding factor (CBF) family of transcription factors contributing to this process in annual herbaceous species also functions in woody perennials, we investigated the changes in phenotype and transcript profile of transgenic Populus constitutively expressing CBF1 from Arabidopsis ( AtCBF1 ). Ectopic expression of AtCBF1 was sufficient to significantly increase the freezing tolerance of non-acclimated leaves and stems relative to wild-type plants. cDNA microarray experiments identified genes upregulated by ectopic AtCBF1 expression in Populus , demonstrated a strong conservation of the CBF regulon between Populus and Arabidopsis and identified differences between leaf and stem regulons. We studied the induction kinetics and tissue specificity of four CBF paralogues identified from the Populus balsamifera subsp . trichocarpa genome sequence ( PtCBF s). All four PtCBF s are cold-inducible in leaves, but only PtCBF1 and PtCBF3 show significant induction in stems. Our results suggest that the central role played by the CBF family of transcriptional activators in cold acclimation of Arabidopsis has been maintained in Populus . However, the differential expression of the PtCBF s and differing clusters of CBFresponsive genes in annual (leaf) and perennial (stem) tissues suggest that the perennial-driven evolution of winter dormancy may have given rise to specific roles for these 'master-switches' in the different annual and perennial tissues of woody species.

With the aim of dissecting the genetic determinants of flowering time, vernalization response, and photoperiod sensitivity, we mapped the candidate genes for Vrn-H2 and Vrn-H1 in a facultative x winter barley mapping population and determined their relationships with flowering time and vernalization via QTL analysis. The Vrn-H2 candidate ZCCT-H genes were completely missing from the facultative parent and present in the winter barley parent. This gene was the major determinant of flowering time under long photoperiods in controlled environment experiments, irrespective of vernalization, and under spring-sown field experiments. It was the sole determinant of vernalization response, but the effect of the deletion was modulated by photoperiods when the vernalization requirement was fulfilled. There was no effect under short photoperiods. The Vrn-H1 candidate gene (HvBM5A) was mapped based on a microsatellite polymorphism we identified in the promoter of this gene. Otherwise, the HvBM5A alleles for the two parents were identical. Therefore, the significant flowering time QTL effect associated with this locus suggests tight linkage rather than pleiotropy. This QTL effect was smaller in magnitude than those associated with the Vrn-H2 locus and was significant in two-way interactions with Vrn-H2. The Vrn-H1 locus had no effect on vernalization response. Our results support the Vrn-H2/Vrn-H1 repressor/structural gene model for vernalization response in barley and suggest that photoperiod may also affect the Vrn genes or tightly linked loci.

SummaryPTLF, the Populus trichocarpa homolog of LEAFY (LFY) and FLORICAULA, was cloned to assess its function in a dioecious tree species. In situ hybridization studies showed that the gene was expressed most strongly in developing in¯orescences. Expression was also seen in leaf primordia and very young leaves, most notably in apical vegetative buds near in¯orescences, but also in seedlings. Although ectopic expression of the PTLF cDNA in Arabidopsis accelerated¯owering, only one of the many tested transgenic lines of Populus¯owered precociously. The majority of trees within a population of 3-year-old transgenic hybrid Populus lines with PTLF constitutively expressed showed few differences when compared to controls. However, phenotypic effects on growth rate and crown development, but not owering, were seen in some trees with strong PTLF expression and became manifest only as the trees aged. Competence to respond to overexpression of LFY varied widely among Populus genotypes, giving consistent early¯owering in only a single male P. tremula 3 P. tremuloides hybrid and causing gender change in another hybrid genotype. PTLF activity appears to be subject to regulation that does not affect heterologously expressed LFY, and is dependent upon tree maturation. Both genes provide tools for probing the mechanisms of delayed competence to¯ower in woody plants.