A number of COR genes (COld-Regulated genes) have been implicated in the acquisition of low temperature (LT) tolerance in wheat (Triticum aestivum L.). This study compared the relative expression patterns of selected COR genes in leaf and crown tissues of wheat near-isogenic lines to increase understanding of the molecular mechanisms underlying LT acclimation. Reciprocal near-isogenic lines were generated such that the dominant Vrn-A1 and recessive vrn-A1 loci were interchanged in a spring cv. Manitou and a winter cv. Norstar. Phenological development, acquisition of LT tolerance, and WCS120 polypeptide accumulation in these genotypes proceeded at rates similar to those previously reported for 6 °C acclimation from 0 to 98 d. However, a differential accumulation of WCS120 polypeptide and expression of the COR genes Wcs120, Wcor410, and Wcor14 was observed in the leaf and crown tissues. COR gene transcript levels peaked at 2 d of the acclimation period in both tissues and differences among genotypes were most evident at this time. COR gene expression was highest for the LT-tolerant and lowest for the tender genotypes. However, expression rates were divergent enough in genotypes with intermediate hardiness that comparisons among tissues and/or times during acclimation often resulted in variable interpretations of the relative expression of the COR genes in the determination of LT tolerance. These observations emphasize the need to pay close attention to experimental conditions, sampling times, and genotype and tissue selection in experiments designed to identify the critical genetic components that interact to determine LT acclimation.

BackgroundTo identify the genes involved in the development of low temperature (LT) tolerance in hexaploid wheat, we examined the global changes in expression in response to cold of the 55,052 potentially unique genes represented in the Affymetrix Wheat Genome microarray. We compared the expression of genes in winter-habit (winter Norstar and winter Manitou) and spring-habit (spring Manitou and spring Norstar)) cultivars, wherein the locus for the vernalization gene Vrn-A1 was swapped between the parental winter Norstar and spring Manitou in the derived near-isogenic lines winter Manitou and spring Norstar. Global expression of genes in the crowns of 3-leaf stage plants cold-acclimated at 6°C for 0, 2, 14, 21, 38, 42, 56 and 70 days was examined.ResultsAnalysis of variance of gene expression separated the samples by genetic background and by the developmental stage before or after vernalization saturation was reached. Using gene-specific ANOVA we identified 12,901 genes (at p < 0.001) that change in expression with respect to both genotype and the duration of cold-treatment. We examined in more detail a subset of these genes (2,771) where expression was highly influenced by the interaction between these two main factors. Functional assignments using GO annotations showed that genes involved in transport, oxidation-reduction, and stress response were highly represented. Clustering based on the pattern of transcript accumulation identified genes that were up or down-regulated by cold-treatment. Our data indicate that the cold-sensitive lines can up-regulate known cold-responsive genes comparable to that of cold-hardy lines. The levels of expression of these genes were highly influenced by the initial rate and the duration of the gene's response to cold. We show that the Vrn-A1 locus controls the duration of gene expression but not its initial rate of response to cold treatment. Furthermore, we provide evidence that Ta.Vrn-A1 and Ta.Vrt1 originally hypothesized to encode for the same gene showed different patterns of expression and therefore are distinct.ConclusionThis study provides novel insight into the underlying mechanisms that regulate the expression of cold-responsive genes in wheat. The results support the developmental model of LT tolerance gene regulation and demonstrate the complex genotype by environment interactions that determine LT adaptation in winter annual cereals.

The in vitro competency of mature cereal embryos (winter, spring and durum wheats, oat, barley and triticale) was assessed for direct multiple shoot production on culture media containing the plant growth regulators, thidiazuron (TDZ) and/or 6-benzylaminopurine (BAP). Mature embryos of CDC Dancer oat showed the best response, with 69 shoots per explant on culture medium containing a combination of 4.5 lM TDZ and 4.4 lM BAP. TDZ alone induced about 16 shoots per explant from the oat. Among the wheat genotypes, durum wheat showed the most number of shoots (35) per explant on culture medium containing 4.5 lM of TDZ and 4.4 lM of BAP. With TDZ alone, shoot regeneration for durum wheat ranged from 27-32 shoots per explant. The regeneration frequency from the three winter wheat genotypes ranged from 11-25 shoots per explant and was highest on culture medium containing 9.1 lM TDZ and 4.4 lM BAP. The latter culture medium was also effective for a triticale genotype, inducing 34 shoots per explant. The regeneration from mature embryos of barley genotypes ranged from 5-9 shoots per explant. The mature embryos of all the cereals tested could be used for in vitro regeneration with TDZ and TDZ+BAP combinations.

An in vitro spike culture method was optimized to evaluate Fusarium head blight (FHB) resistance in wheat (Triticum aestivum) and used to screen a population of ethyl methane sulfonate treated spike culture-derived variants (SCDV). Of the 134 SCDV evaluated, the disease severity score of 47 of the variants was ≤30%. Single nucleotide polymorphisms (SNP) in the UDP-glucosyltransferase (UGT) genes, TaUGT-2B, TaUGT-3B, and TaUGT-EST, differed between AC Nanda (an FHB-susceptible wheat variety) and Sumai-3 (an FHB-resistant wheat cultivar). SNP at 450 and 1,558 bp from the translation initiation site in TaUGT-2B and TaUGT-3B, respectively were negatively correlated with FHB severity in the SCDV population, whereas the SNP in TaUGT-EST was not associated with FHB severity. Fusarium graminearum strain M7-07-1 induced early expression of TaUGT-2B and TaUGT-3B in FHB-resistant SCDV lines, which were associated with deoxynivalenol accumulation and reduced FHB disease progression. At 8 days after inoculation, deoxynivalenol concentration varied from 767 ppm in FHB-resistant variants to 2,576 ppm in FHB-susceptible variants. The FHB-resistant SCDV identified can be used as new sources of FHB resistance in wheat improvement programs.

An in vitro culture system is useful to study grain development under defined conditions to minimize confounding effects associated with whole plant studies and metabolite movement into the developing grains. The objective of this study was to monitor the expression patterns of carbohydrate metabolism genes during grain development in an in vitro wheat spike culture system. Immature spikes were cultured prior to anthesis, and grains were collected at various days postanthesis (DPA). Grains from cultured spikes showed maximum expression of starch metabolic genes by 10 DPA, with a rapid decline thereafter. The rapid increase and decrease in expression rate in the in vitro system was thought to be due to fructan exohydrolase (1-FEH and 6-FEH) or sucrose transporter 1 (SUT1) and sucrose synthase (SuSy) genes being highly expressed. SUT1 reached peak expression at 8 DPA, two days earlier than the other genes, and may account for the rapid early stage trigger in expression of the other genes. However, expression of 1-FEH and 6-FEH genes in in vitro-cultured spikes peaked at 12 DPA, two days later than the other genes, and could indicate that fructan catabolism was not a factor in the rapid accumulation of starch in the in vitro-cultured spikes. Accumulation of GBSSI polypeptides generally showed similar patterns in both systems, with the maximum amount in the in vitro system observed four days later than in the in planta spikes, reflecting different turnover controls of GBSSI transcripts. The in vitro system offers opportunities for further refinement and detailed grain development studies.