The Breeding of Winter-Hardy Malting Barley.

In breeding winter malting barley, one recurring strategy is to cross a current preferred spring malting barley to a winter barley. This is because spring malting barleys have the greatest amalgamation of trait qualities desirable for malting and brewing. Spring barley breeding programs can also cycle their material through numerous generations each year-some managing even six-which greatly accelerates combining desirable alleles to generate new lines. In a winter barley breeding program, a single generation per year is the limit when the field environment is used and about two generations per year if vernalization and greenhouse facilities are used. However, crossing the current favored spring malting barley to a winter barley may have its downsides, as winter-hardiness too may be an amalgamation of desirable alleles assembled together that confers the capacity for prolonged cold temperature conditions. In this review I touch on some general criteria that give a variety the distinction of being a malting barley and some of the general trends made in the breeding of spring malting barleys. But the main objective of this review is to pull together different aspects of what we know about winter-hardiness from the seemingly most essential aspect, which is survival in the field, to molecular genetics and gene regulation, and then finish with ideas that might help further our insight for predictability purposes.

Cold Conditioned: Discovery of Novel Alleles for Low-Temperature Tolerance in the Vavilov Barley Collection.

Climate changes leading to higher summer temperatures can adversely affect cool season crops like spring barley. In the Upper Midwest region of the United States, one option for escaping this stress factor is to plant winter or facultative type cultivars in the autumn and then harvest in early summer before the onset of high-temperature stress. However, the major challenge in breeding such cultivars is incorporating sufficient winter hardiness to survive the extremely low temperatures that commonly occur in this production region. To broaden the genetic base for winter hardiness in the University of Minnesota breeding program, 2,214 accessions from the N. I. Vavilov Institute of Plant Industry (VIR) were evaluated for winter survival (WS) in St. Paul, Minnesota. From this field trial, 267 (>12%) accessions survived [designated as the VIR-low-temperature tolerant (LTT) panel] and were subsequently evaluated for WS across six northern and central Great Plains states. The VIR-LTT panel was genotyped with the Illumina 9K SNP chip, and then a genome-wide association study was performed on seven WS datasets. Twelve significant associations for WS were identified, including the previously reported frost resistance gene FR-H2 as well as several novel ones. Multi-allelic haplotype analysis revealed the most favorable alleles for WS in the VIR-LTT panel as well as another recently studied panel (CAP-LTT). Seventy-eight accessions from the VIR-LTT panel exhibited a high and consistent level of WS and select ones are being used in winter barley breeding programs in the United States and in a multiparent population.

Transcriptional differentiation of UV-B protectant genes in maize landraces spanning an elevational gradient in Chiapas, Mexico.

Globally, farmers cultivate and maintain crop landraces (i.e., traditional varieties). Landraces contain unique diversity shaped in part by natural and human-mediated selection and are an indispensable resource for farmers. Since environmental conditions change with elevation, crop landraces grown along elevational gradients have provided ideal locations to explore patterns of local adaptation. To further probe traits underlying this differentiation, transcriptome signatures can help provide a foundation for understanding the ways in which functional genetic diversity may be shaped by environment. In this study, we returned to an elevational gradient in Chiapas, Mexico, to assess transcriptional differentiation of genes underlying UV-B protection in locally adapted maize landraces from multiple elevations. We collected and planted landraces from three elevational zones (lowland, approximately 600 m; midland, approximately 1,550 m; highland approximately 2,100 m) in a common garden at 1,531 m. Using RNA-seq data derived from leaf tissue, we performed differential expression analysis between maize from these distinct elevations. Highland and lowland landraces displayed differential expression in phenylpropanoid and flavonoid biosynthesis genes involved in the production of UV-B protectants and did so at a rate greater than expected based on observed background transcriptional differentiation across the genome. These findings provide evidence for the differentiation of suites of genes involved in complex ecologically relevant pathways. Thus, while neutral evolutionary processes may have played a role in the observed patterns of differentiation, UV-B may have also acted as a selective pressure to differentiate maize landraces in the region. Studies of the distribution of functional crop genetic diversity across variable landscapes can aid us in understanding the response of diversity to abiotic/biotic change and, ultimately, may facilitate its conservation and utilization.

CBF2A-CBF4B genomic region copy numbers alongside the circadian clock play key regulatory mechanisms driving expression of FR-H2 CBFs.

The C-Repeat Binding Factors (CBFs) are DNA-binding transcriptional activators that were identified using Arabidopsis thaliana. In barley, Hordeum vulgare, a cluster of CBF genes reside at FROST RESISTANCE-H2, one of two loci having major effects on winter-hardiness. FR-H2 was revealed in a population derived from the winter barley 'Nure' and the spring barley 'Trèmois'. 'Nure' harbors two to three copies of CBF2A and CBF4B as a consequence of tandem iteration of the genomic region encompassing these genes whereas 'Trèmois' harbors single copies, and these copy number differences are associated with their transcript level differences. Here we explore further the relationship between FR-H2 CBF gene copy number and transcript levels using 'Admire', a winter barley accumulating FR-H2 CBF gene transcripts to very high levels, and a group of lines related to 'Admire' through descent. DNA blot hybridization indicated the CBF2A-CBF4B genomic region is present in 7-8 copies in 'Admire' and is highly variable in copy number across the lines related to 'Admire'. At normal growth temperatures transcript levels of CBF12, CBF14, and CBF16 were higher in lines having greater CBF2A-CBF4B genomic region copy numbers than in lines having fewer copy numbers at peak expression level time points controlled by the circadian clock. Chromatin immunoprecipitation indicated CBF2 was at the CBF12 and CBF16 promoters at normal growth temperatures. These data support a scenario in which CBF2A-CBF4B genomic region copy numbers affect expression of other FR-H2 CBFs through a mechansim in which these other FR-H2 CBFs are activated by those in the copy number variable unit.

Hv-CBF2A overexpression in barley accelerates COR gene transcript accumulation and acquisition of freezing tolerance during cold acclimation.

C-Repeat Binding Factors (CBFs) are DNA-binding transcriptional activators of gene pathways imparting freezing tolerance. Poaceae contain three CBF subfamilies, two of which, HvCBF3/CBFIII and HvCBF4/CBFIV, are unique to this taxon. To gain mechanistic insight into HvCBF4/CBFIV CBFs we overexpressed Hv-CBF2A in spring barley (Hordeum vulgare) cultivar 'Golden Promise'. The Hv-CBF2A overexpressing lines exhibited stunted growth, poor yield, and greater freezing tolerance compared to non-transformed 'Golden Promise'. Differences in freezing tolerance were apparent only upon cold acclimation. During cold acclimation freezing tolerance of the Hv-CBF2A overexpressing lines increased more rapidly than that of 'Golden Promise' and paralleled the freezing tolerance of the winter hardy barley 'Dicktoo'. Transcript levels of candidate CBF target genes, COR14B and DHN5 were increased in the overexpressor lines at warm temperatures, and at cold temperatures they accumulated to much higher levels in the Hv-CBF2A overexpressors than in 'Golden Promise'. Hv-CBF2A overexpression also increased transcript levels of other CBF genes at FROST RESISTANCE-H2-H2 (FR-H2) possessing CRT/DRE sites in their upstream regions, the most notable of which was CBF12. CBF12 transcript levels exhibited a relatively constant incremental increase above levels in 'Golden Promise' both at warm and cold. These data indicate that Hv-CBF2A activates target genes at warm temperatures and that transcript accumulation for some of these targets is greatly enhanced by cold temperatures.

Cbf14 copy number variation in the A, B, and D genomes of diploid and polyploid wheat.

Freezing tolerance and winter hardiness are complex traits. In the Triticeae, two loci on the group 5 chromosome homoeologs are repeatedly identified as having major effects on these traits. Recently, we found that segments of the genomic region at one of these loci, Frost resistance-2 (Fr-2) is copy number variable in barley. Freezing-tolerant winter-hardy genotypes have greater tandem copy numbers of the genomic region encompassing the C-repeat binding factor genes Cbf2A and Cbf4B at Fr-H2 than the less freezing-tolerant nonwinter-hardy genotypes. Here we report that in wheat the Cbf14 gene at Fr-2 is copy number variable. Using DNA blot hybridizations, we estimated copy numbers of Cbf14 across the different genomes of diploid and polyploid wheat. Copy numbers of Cbf14 are lower in the B genome than in the A and D genomes across all ploidy levels. Among hexaploid red wheats, winter genotypes harbor greater Cbf14 copy numbers than spring genotypes. Cbf14 copy numbers also vary across the red winter wheats such that hard wheats harbor greater copy numbers than soft wheats. Analysis of hexaploid wheat chromosome 5 substitution lines indicates that Cbf14 copy numbers in the introgressions are stable in the different backgrounds. Taken together our data suggest that higher copy number states existed in the diploid wild ancestors prior to the polyploidization events and that the loss of Cbf14 copies occurred in the cultivated germplasm.

Comparative analyses reveal potential uses of Brachypodium distachyon as a model for cold stress responses in temperate grasses.

BACKGROUND: Little is known about the potential of Brachypodium distachyon as a model for low temperature stress responses in Pooideae. The ice recrystallization inhibition protein (IRIP) genes, fructosyltransferase (FST) genes, and many C-repeat binding factor (CBF) genes are Pooideae specific and important in low temperature responses. Here we used comparative analyses to study conservation and evolution of these gene families in B. distachyon to better understand its potential as a model species for agriculturally important temperate grasses. RESULTS: Brachypodium distachyon contains cold responsive IRIP genes which have evolved through Brachypodium specific gene family expansions. A large cold responsive CBF3 subfamily was identified in B. distachyon, while CBF4 homologs are absent from the genome. No B. distachyon FST gene homologs encode typical core Pooideae FST-motifs and low temperature induced fructan accumulation was dramatically different in B. distachyon compared to core Pooideae species. CONCLUSIONS: We conclude that B. distachyon can serve as an interesting model for specific molecular mechanisms involved in low temperature responses in core Pooideae species. However, the evolutionary history of key genes involved in low temperature responses has been different in Brachypodium and core Pooideae species. These differences limit the use of B. distachyon as a model for holistic studies relevant for agricultural core Pooideae species.

Regulation of freezing tolerance and flowering in temperate cereals: the VRN-1 connection.

In winter wheat (Triticum spp.) and barley (Hordeum vulgare) varieties, long exposures to nonfreezing cold temperatures accelerate flowering time (vernalization) and improve freezing tolerance (cold acclimation). However, when plants initiate their reproductive development, freezing tolerance decreases, suggesting a connection between the two processes. To better understand this connection, we used two diploid wheat (Triticum monococcum) mutants, maintained vegetative phase (mvp), that carry deletions encompassing VRN-1, the major vernalization gene in temperate cereals. Homozygous mvp/mvp plants never flower, whereas plants carrying at least one functional VRN-1 copy (Mvp/-) exhibit normal flowering and high transcript levels of VRN-1 under long days. The Mvp/- plants showed reduced freezing tolerance and reduced transcript levels of several cold-induced C-REPEAT BINDING FACTOR transcription factors and COLD REGULATED genes (COR) relative to the mvp/mvp plants. Diploid wheat accessions with mutations in the VRN-1 promoter, resulting in high transcript levels under both long and short days, showed a significant down-regulation of COR14b under long days but not under short days. Taken together, these studies suggest that VRN-1 is required for the initiation of the regulatory cascade that down-regulates the cold acclimation pathway but that additional genes regulated by long days are required for the down-regulation of the COR genes. In addition, our results show that allelic variation in VRN-1 is sufficient to determine differences in freezing tolerance, suggesting that quantitative trait loci for freezing tolerance previously mapped on this chromosome region are likely a pleiotropic effect of VRN-1 rather than the effect of a separate closely linked locus (FROST RESISTANCE-1), as proposed in early freezing tolerance studies.

Increasing omega-3 desaturase expression in tomato results in altered aroma profile and enhanced resistance to cold stress.

One of the drawbacks in improving the aroma properties of tomato (Solanum lycopersicum) fruit is the complexity of this organoleptic trait, with a great variety of volatiles contributing to determine specific quality features. It is well established that the oxylipins hexanal and (Z)-hex-3-enal, synthesized through the lipoxygenase pathway, are among the most important aroma compounds and impart in a correct proportion some of the unique fresh notes in tomato. Here, we confirm that all enzymes responsible for the synthesis of these C6 compounds are present and active in tomato fruit. Moreover, due to the low odor threshold of (Z)-hex-3-enal, small changes in the concentration of this compound could modify the properties of the tomato fruit aroma. To address this possibility, we have overexpressed the omega-3 fatty acid desaturases FAD3 and FAD7 that catalyze the conversion of linoleic acid (18:2) to linolenic acid (18:3), the precursor of hexenals and its derived alcohols. Transgenic OE-FAD tomato plants exhibit altered fatty acid composition, with an increase in the 18:3/18:2 ratio in leaves and fruits. These changes provoke a clear variation in the C6 content that results in a significant alteration of the (Z)-hex-3-enal/hexanal ratio that is particularly important in ripe OE-FAD3FAD7 fruits. In addition to this effect on tomato volatile profile, OE-FAD tomato plants are more tolerant to chilling. However, the different behaviors of OE-FAD plants underscore the existence of separate fatty acid fluxes to ensure plant survival under adverse conditions.

CBF gene copy number variation at Frost Resistance-2 is associated with levels of freezing tolerance in temperate-climate cereals.

Frost Resistance-1 (FR-1) and FR-2 are two loci affecting freezing tolerance and winter hardiness of the temperate-climate cereals. FR-1 is hypothesized to be due to the pleiotropic effects of VRN-1. FR-2 spans a cluster of C-Repeat Binding Factor (CBF) genes. These loci are genetically and functionally linked. Recent studies indicate CBF transcripts are downregulated by the VRN-1 encoded MADS-box protein or a factor in the VRN-1 pathway. Here, we report that barley genotypes 'Dicktoo' and 'Nure' carrying a vrn-H1 winter allele at VRN-H1 harbor increased copy numbers of CBF coding sequences relative to Vrn-H1 spring allele genotypes 'Morex' and 'Tremois'. Sequencing bacteriophage lambda genomic clones from these four genotypes alongside DNA blot hybridizations indicate approximately half of the eleven CBF orthologs at FR-H2 are duplicated in individual genomes. One of these duplications discriminates vrn-H1 genotypes from Vrn-H1 genotypes. The vrn-H1 winter allele genotypes harbor tandem segmental duplications through the CBF2A-CBF4B genomic region and maintain two distinct CBF2 paralogs, while the Vrn-H1 spring allele genotypes harbor single copies of CBF2 and CBF4. An additional CBF gene, CBF13, is a pseudogene interrupted by multiple non-sense codons in 'Tremois' whereas CBF13 is a complete uninterrupted coding sequence in 'Dicktoo' and 'Nure'. DNA blot hybridization with wheat DNAs reveals greater copy numbers of CBF14 also occurs in winter wheats than in spring wheats. These data indicate that variation in CBF gene copy numbers is widespread in the Triticeae and suggest selection for winter hardiness co-selects winter alleles at both VRN-1 and FR-2.

The low temperature-responsive, Solanum CBF1 genes maintain high identity in their upstream regions in a genomic environment undergoing gene duplications, deletions, and rearrangements.

Some plants like Arabidopsis thaliana increase in freezing tolerance when exposed to low nonfreezing temperatures, a process known as cold acclimation. Other plants including tomato, Solanum lycopersicum, are chilling sensitive and incur injury during prolonged low temperature exposure. A key initial event that occurs upon low temperature exposure is the induction of genes encoding the CBF transcription factors. In Arabidopsis three CBF genes, present in a tandemly-linked cluster, are induced by low temperatures. Tomato also harbors three tandemly-linked CBF genes, Sl-CBF3-CBF1-CBF2, but only one of these, Sl-CBF1, is low-temperature responsive. Here we report that Solanum species that are closely-allied to cultivated tomato essentially share this structural organization, but the locus is in a dynamic state of flux. Additional paralogs and in-frame deletions between adjacent genes occur, and the genomic regions flanking the CBF genes are dissimilar across Solanum species. Nevertheless, the CBF1 upstream region remains intact and highly conserved. This feature differed for CBF2 and CBF3, whose upstream regions were far less conserved. CBF1 was also the only low-temperature responsive gene in the cluster and its expression was greatly affected by a circadian clock. The tuber-bearing S. tuberosum and S. commersonii also harbored a fourth gene, CBF4, which was also low temperature responsive. CBF4 was physically linked to CBF5 in S. tuberosum, but CBF5 was absent from S. commersonii. Phylogenic analyses suggest that CBF5-CBF4 resulted from the duplication of the CBF3-CBF1-CBF2 cluster. DNA sequence motifs shared between the Solanum CBF1 and CBF4 upstream regions were identified, portions of which were also present in the Arabidopsis CBF1-3 upstream regions. These results suggest that much greater functional constraints are placed upon the Solanum CBF1 upstream regions over the other CBF upstream regions and that CBF4 has retained the capacity for low temperature responsiveness following the duplication event that gave rise to CBF4.

A retrotransposon-mediated gene duplication underlies morphological variation of tomato fruit.

Edible fruits, such as that of the tomato plant and other vegetable crops, are markedly diverse in shape and size. SUN, one of the major genes controlling the elongated fruit shape of tomato, was positionally cloned and found to encode a member of the IQ67 domain-containing family. We show that the locus arose as a result of an unusual 24.7-kilobase gene duplication event mediated by the long terminal repeat retrotransposon Rider. This event resulted in a new genomic context that increased SUN expression relative to that of the ancestral copy, culminating in an elongated fruit shape. Our discovery demonstrates that retrotransposons may be a major driving force in genome evolution and gene duplication, resulting in phenotypic change in plants.

Identification of candidate CBF genes for the frost tolerance locus Fr-Am2 in Triticum monococcum.

A cluster of eleven CBF genes was recently mapped to the Frost resistance-2 (Fr-Am2) locus on chromosome 5 of diploid wheat (Triticum monococcum) using a cross between frost tolerant accession G3116 and frost sensitive DV92. The Fr-Am2 locus was mapped at the peak of two overlapping quantitative trait loci (QTL), one for frost survival and the other for differential expression of the cold regulated gene COR14b. Seven lines with recombination events within the CBF cluster were used to identify CBF candidate genes for these QTL. The lines carrying the critical recombination events were tested for whole plant frost survival and for differential transcript levels of cold induced COR14b and DHN5 genes. The strongest effect for these traits was associated to the linked TmCBF12, TmCBF14 and TmCBF15 genes, with the G3116 allele conferring improved frost tolerance and higher levels of COR14b and DHN5 transcript at mild cold temperatures (12-15 degrees C) than the DV92 allele. Comparison of CBF protein sequences revealed that the DV92 TmCBF12 protein contains a deletion of five amino acids in the AP2 DNA binding domain. Electrophoretic Mobility Shift Assays (EMSA) confirmed that the protein encoded by this allele cannot bind to the CRT/DRE (C-repeat/ dehydration-responsive element) motif present in the promoters of several cold induced genes. A smaller effect on frost tolerance was mapped to the distal group of CBF genes including TmCBF16. Transcript levels of TmCBF16, as well as those of TmCBF12 and TmCBF15 were up-regulated at mild cold temperatures in G3116 but not in DV92. Higher threshold induction temperatures can result in earlier initiation of the cold acclimation process and better resistance to subsequent freezing temperatures. The non-functional TmCBF12 allele in DV92 can also contribute to its lower frost tolerance.

Comparative genomic sequence and expression analyses of Medicago truncatula and alfalfa subspecies falcata COLD-ACCLIMATION-SPECIFIC genes.

In Arabidopsis (Arabidopsis thaliana) the low-temperature induction of genes encoding the C-REPEAT BINDING FACTOR (CBF) transcriptional activators is a key step in cold acclimation. CBFs in turn activate a battery of downstream genes known as the CBF regulon, which collectively act to increase tolerance to low temperatures. Fundamental questions are: What determines the size and scope of the CBF regulon, and is this is a major determinant of the low-temperature tolerance capacity of individual plant species? Here we have begun to address these questions through comparative analyses of Medicago truncatula and Medicago sativa subsp. falcata. M. truncatula survived to -4 degrees C but did not cold acclimate, whereas Medicago falcata cold acclimated and survived -14 degrees C. Both species possessed low-temperature-induced CBFs but differed in the expression of the COLD-ACCLIMATION-SPECIFIC (CAS) genes, which are candidate CBF targets. M. falcata CAS30 was robustly cold-responsive whereas the MtCAS31 homolog was not. M. falcata also possessed additional CAS30 homologs in comparison to the single CAS31 gene in M. truncatula. MfCAS30 possessed multiple pairs of closely spaced C-REPEAT/DEHYDRATION RESPONSIVE ELEMENT (CRT/DRE) motifs, the cognate CBF binding site in its upstream region whereas MtCAS31 lacked one CRT/DRE partner of the two proximal partner pairs. CAS genes also shared a promoter structure comprising modules proximal and distal to the coding sequence. CAS15, highly cold-responsive in both species, harbored numerous CRT/DRE motifs, but only in the distal module. However, fusion of the MtCAS15 promoter, including the distal module, to a reporter gene did not result in low-temperature responsiveness in stably transformed Arabidopsis. In contrast, both MtCAS31 and MfCAS30 promoter fusions were low-temperature responsive, although the MfCAS31 fusion was less robust than the MfCAS30 fusion. From these studies we conclude that CAS genes harbor CRT/DRE motifs, their proximity to one another is likely key to regulatory output in Medicago, and they may be located kilobases distal to the transcriptional start site. We hypothesize that these differences in CRT/DRE copy numbers in CAS30/CAS31 upstream regions combined with differences in gene copy numbers may be a factor in determining differences in low-temperature tolerance between M. truncatula and M. falcata.

Expression levels of barley Cbf genes at the Frost resistance-H2 locus are dependent upon alleles at Fr-H1 and Fr-H2.

Genetic analyses have identified two loci in wheat and barley that mediate the capacity to overwinter in temperate climates. One locus co-segregates with VRN-1, which affects the vernalization requirement. This locus is known as Frost resistance-1 (Fr-1). The second locus, Fr-2, is coincident with a cluster of more than 12 Cbf genes. Cbf homologs in Arabidopsis thaliana play a key regulatory role in cold acclimatization and the acquisition of freezing tolerance. Here we report that the Hordeum vulgare (barley) locus VRN-H1/Fr-H1 affects expression of multiple barley Cbf genes at Fr-H2. RNA blot analyses, conducted on a 'Nure'x'Tremois' barley mapping population segregating for VRN-H1/Fr-H1 and Fr-H2, revealed that transcript levels of all cold-induced Cbf genes at Fr-H2 were significantly higher in recombinants harboring the vrn-H1 winter allele than in recombinants harboring the Vrn-H1 spring allele. Steady-state Cbf2 and Cbf4 levels were also significantly higher in recombinants harboring the Nure allele at Fr-H2. Additional experiments indicated that, in vrn-H1 genotypes requiring vernalization, Cbf expression levels were dampened after plants were vernalized, and dampened Cbf expression was accompanied by robust expression of Vrn-1. Cbf levels were also significantly higher in plants grown under short days than under long days. Experiments in wheat and rye indicated that similar regulatory mechanisms occurred in these plants. These results suggest that VRN-H1/Fr-H1 acts in part to repress or attenuate expression of the Cbf at Fr-H2; and that the greater level of low temperature tolerance attributable to the Nure Fr-H2 allele may be due to the greater accumulation of Cbf2 and Cbf4 transcripts during normal growth and development.

Mapping of barley homologs to genes that regulate low temperature tolerance in Arabidopsis.

We investigated the allelic nature and map locations of Hordeum vulgare (barley) homologs to three classes of Arabidopsis low temperature (LT) regulatory genes-CBFs, ICE1, and ZAT12-to determine if there were any candidates for winterhardiness-related quantitative trait loci (QTL). We phenotyped the Dicktoo x Morex (DxM) mapping population under controlled freezing conditions and in addition to the previously reported 5H-L Fr-H1 QTL, observed three additional LT tolerance QTLs on 1H-L, 4H-S, and 4H-L. We identified and assigned either linkage map or chromosome locations to 1 ICE1 homolog, 2 ZAT12 homologs, and 17 of 20 CBF homologs. Twelve of the CBF genes were located on 5H-L and the 11 with assigned linkage map positions formed 2 tandem clusters on 5H-L. A subset of these CBF genes was confirmed to be physically linked, validating the map position clustering. The tandem CBF clusters are not candidates for the DxM LT tolerance Fr-H1 QTL, as they are approximately 30 cM distal to the QTL peak. No LT tolerance QTL was detected in conjunction with the CBF gene clusters in Dicktoo x Morex. However, comparative mapping using common markers and BIN positions established the CBF clusters are coincident with reported Triticeae LT tolerance and COR gene accumulation QTLs and suggest one or more of the CBF genes may be candidates for Fr-H2 in some germplasm combinations. These results suggest members of the CBF gene family may function as components of winter-hardiness in the Triticeae and underscore both the importance of extending results from model systems to economically important crop species and in viewing QTL mapping results in the context of multiple germplasm combinations.

Structural, functional, and phylogenetic characterization of a large CBF gene family in barley.

CBFs are key regulators in the Arabidopsis cold signaling pathway. We used Hordeum vulgare (barley), an important crop and a diploid Triticeae model, to characterize the CBF family from a low temperature tolerant cereal. We report that barley contains a large CBF family consisting of at least 20 genes (HvCBFs) comprising three multigene phylogenetic groupings designated the HvCBF1-, HvCBF3-, and HvCBF4-subgroups. For the HvCBF1- and HvCBF3-subgroups, there are comparable levels of phylogenetic diversity among rice, a cold-sensitive cereal, and the cold-hardy Triticeae. For the HvCBF4-subgroup, while similar diversity levels are observed in the Triticeae, only a single ancestral rice member was identified. The barley CBFs share many functional characteristics with dicot CBFs, including a general primary domain structure, transcript accumulation in response to cold, specific binding to the CRT motif, and the capacity to induce cor gene expression when ectopically expressed in Arabidopsis. Individual HvCBF genes differed in response to abiotic stress types and in the response time frame, suggesting different sets of HvCBF genes are employed relative to particular stresses. HvCBFs specifically bound monocot and dicot cor gene CRT elements in vitro under both warm and cold conditions; however, binding of HvCBF4-subgroup members was cold dependent. The temperature-independent HvCBFs activated cor gene expression at warm temperatures in transgenic Arabidopsis, while the cold-dependent HvCBF4-subgroup members of three Triticeae species did not. These results suggest that in the Triticeae - as in Arabidopsis - members of the CBF gene family function as fundamental components of the winter hardiness regulon.

Multiple hydrophobic motifs in Arabidopsis CBF1 COOH-terminus provide functional redundancy in trans-activation.

The Arabidopsis CBF proteins activate expression of a set of genes whose upstream regulatory sequences typically harbor one or more copies of the CRT/DRE low temperature cis-acting DNA regulatory element. Using domain swap experiments in both yeast and Arabidopsis we show that the NH3-terminal 115 amino acids direct CBF1 to target genes and the COOH-terminal 98 amino acids function in trans-activation. Mutational analysis through the COOH-terminus using truncation and alanine-substitution mutants in yeast revealed four motifs that contribute positively towards activation. Overexpression of mutants in plants support this conclusion and also indicated that disruption of a single motif did not seriously compromise activity unless combined with the disruption of a second. These motifs consist of clusters of hydrophobic residues which are delimited from one another by short stretches of Asp, Glu, Pro and other residues favoring the formation of loops. This structural pattern is conserved across plant taxa as revealed through alignment of Arabidopsis CBF1 with homologous sequences from a diverse array of plant species. Overexpression in plants of the CBF1 COOH-terminus as a fusion with the yeast GAL4 DNA binding domain also resulted in severe stunting of growth, a phenotype which was alleviated if the activation domain was rendered ineffective. Taken together these results suggest that high level overexpression of an active, CBF activation domain compromises plant growth.

Proteomic analysis of resistance mediated by Rcm 2.0 and Rcm 5.1, two loci controlling resistance to bacterial canker of tomato.

Two quantitative trait loci from Lycopersicon hirsutum, Rcm 2.0 and Rcm 5.1, control resistance to Clavibacter michiganensis subsp. michiganensis, the causal agent of bacterial canker of tomato. Lines containing Rcm 2.0 and Rcm 5.1 and a susceptible control line were compared at 72 and 144 h postinoculation, using 2-dimensional gel electrophoresis to identify proteins regulated in response to C. michiganensis subsp. michiganensis infection. A total of 47 proteins were subjected to tandem mass spectrometry. Database queries with resulting spectra identified tomato genes for 26 proteins. The remaining 21 proteins were either identified in other species or possessed no homology to known proteins. Spectra were interpreted to deduce peptide amino acid sequences that were then used to query publicly available data. This approach identified tomato genes or expressed sequence tags for 44 of the proteins analyzed. Three superoxide dismutase (SOD) enzymes were differentially regulated among genotypes, and patterns of hydrogen peroxide accumulation were genotype- and tissue-specific, indicating a role for oxidative stress in response to C. michiganensis subsp. michiganensis. Steady-state mRNA and protein levels for SOD, thioredoxin M-type, S-adenosylhomocysteine hydrolase, and pathogenesis-related proteins demonstrated similar patterns of differential regulation. Lines containing Rcm 2.0 and Rcm 5.1 accumulate different proteins and steady-state mRNAs in response to inoculation, suggesting that the two loci may confer resistance through distinct mechanisms.

Freezing-sensitive tomato has a functional CBF cold response pathway, but a CBF regulon that differs from that of freezing-tolerant Arabidopsis.

Many plants increase in freezing tolerance in response to low temperature, a process known as cold acclimation. In Arabidopsis, cold acclimation involves action of the CBF cold response pathway. Key components of the pathway include rapid cold-induced expression of three homologous genes encoding transcriptional activators, CBF1, 2 and 3 (also known as DREB1b, c and a, respectively), followed by expression of CBF-targeted genes, the CBF regulon, that increase freezing tolerance. Unlike Arabidopsis, tomato cannot cold acclimate raising the question of whether it has a functional CBF cold response pathway. Here we show that tomato, like Arabidopsis, encodes three CBF homologs, LeCBF1-3 (Lycopersicon esculentum CBF1-3), that are present in tandem array in the genome. Only the tomato LeCBF1 gene, however, was found to be cold-inducible. As is the case for Arabidopsis CBF1-3, transcripts for LeCBF1-3 did accumulate in response to mechanical agitation, but not in response to drought, ABA or high salinity. Constitutive overexpression of LeCBF1 in transgenic Arabidopsis plants induced expression of CBF-targeted genes and increased freezing tolerance indicating that LeCBF1 encodes a functional homolog of the Arabidopsis CBF1-3 proteins. However, constitutive overexpression of either LeCBF1 or AtCBF3 in transgenic tomato plants did not increase freezing tolerance. Gene expression studies, including the use of a cDNA microarray representing approximately 8000 tomato genes, identified only four genes that were induced 2.5-fold or more in the LeCBF1 or AtCBF3 overexpressing plants, three of which were putative members of the tomato CBF regulon as they were also upregulated in response to low temperature. Additional experiments indicated that of eight tomato genes that were likely orthologs of Arabidopsis CBF regulon genes, none were responsive to CBF overexpression in tomato. From these results, we conclude that tomato has a complete CBF cold response pathway, but that the tomato CBF regulon differs from that of Arabidopsis and appears to be considerably smaller and less diverse in function.