Effect of additional water supply during grain filling on protein composition and epitope characteristics of winter oats.

Pure oats in gluten-free diets (GFD) represent important nutritional benefits for people suffering from celiac disease (CD). However, oat cultivars do not contain the typical CD-related wheat gliadin analog polypeptides. Emerging evidence suggests that oat cultivars containing gluten-like epitopes in avenin sequences may pose potential health risks for celiac patients in rare cases, depending on the individual's susceptibility. Consequently, it is necessary to screen oats in terms of protein and epitope composition, to be able to select safe varieties for gluten-free applications. The overall aim of our study is to investigate the variation of oat protein composition directly related to health-related and techno-functional properties and to examine how the protein compositional parameters change due to irrigation during the grain-filling period as compared to the natural rain-fed grown, in a large winter oat population of different geographic origin. Elements of an oat sample population representing 164 winter oat varieties from 8 countries and the protein composition of resulting samples have been characterized. Size distribution of the total protein extracts has been analyzed by SE-HPLC, while the 70% ethanol extracted proteins were analyzed by RP-HPLC. Protein extracts are separated into 3 main groups of fractions on the SE-HPLC column; polymeric, avenin, and non-avenin monomeric protein groups, representing 59.17-80.87%, 12.89-31.03%, and 3.40-9.41% of total protein content, respectively. The ratio of polymeric to monomeric proteins varied between 1.71 and 6.07. 91 RP-HPLC-separated peaks have been differentiated from the ethanol extractable proteins of the entire population. The various parameters identified a lot of variation, confirming the significance of genotypic variation. In addition, it was also established that the additional water supply during grain filling significantly affected the various quantitative parameters of protein content, but not its qualitative structure. This environmental effect, however, was strongly genotype-dependent. Winter oat genotypes with low levels of epitope content were identified and it was proven that these characteristics were independent of the environmental factor of water availability. These genotypes are appropriate for initiating a specific breeding program to yield oat cultivars suitable for CD patients.

Effects of genetic components of plant development on yield-related traits in wheat (Triticum aestivum L.) under stress-free conditions.

The dynamics of plant development not only has an impact on ecological adaptation but also contributes to the realization of genetically determined yield potentials in various environments. Dissecting the genetic determinants of plant development becomes urgent due to the global climate change, which can seriously affect and even disrupt the locally adapted developmental patterns. In order to determine the role plant developmental loci played in local adaptation and yield formation, a panel of 188 winter and facultative wheat cultivars from diverse geographic locations were characterized with the 15K Illumina Single Nucleotide Polymorphism (SNP) chip and functional markers of several plant developmental genes and included into a multiseason field experiment. Genome-wide association analyses were conducted on five consecutive developmental phases spanning from the first node appearance to full heading together with various grain yield-related parameters. The panel was balanced for the PPD-D1 photoperiod response gene, which facilitated the analyses in the two subsets of photoperiod-insensitive and -sensitive genotypes in addition to the complete panel. PPD-D1 was the single highest source, explaining 12.1%-19.0% of the phenotypic variation in the successive developmental phases. In addition, 21 minor developmental loci were identified, each one explaining only small portions of the variance, but, together, their effects amounted to 16.6%-50.6% of phenotypic variance. Eight loci (2A_27, 2A_727, 4A_570, 5B_315, 5B_520, 6A_26, 7A_1-(VRN-A3), and 7B_732) were independent of PPD-D1. Seven loci were only detectable in the PPD-D1-insensitive genetic background (1A_539, 1B_487, 2D_649, 4A_9, 5A_584-(VRN-A1), 5B_571-(VRN-B1), and 7B_3-(VRN-B3)), and six loci were only detectable in the sensitive background, specifically 2A_740, 2D_25, 3A_579, 3B_414, 7A_218, 7A_689, and 7B_538. The combination of PPD-D1 insensitivity and sensitivity with the extremities of early or late alleles in the corresponding minor developmental loci resulted in significantly altered and distinct plant developmental patterns with detectable outcomes on some yield-related traits. This study examines the possible significance of the above results in ecological adaptation.

Investigation of Protein and Epitope Characteristics of Oats and Its Implications for Celiac Disease.

The use of pure oats (oats cultivated with special care to avoid gluten contamination from wheat, rye, and barley) in the gluten-free diet (GFD) represents important nutritional benefits for the celiac consumer. However, emerging evidence suggests that some oat cultivars may contain wheat gliadin analog polypeptides. Consequently, it is necessary to screen oats in terms of protein and epitope composition to be able to select safe varieties for gluten-free applications. The overall aim of our study is to investigate the variability of oat protein composition directly related to health-related and techno-functional properties. Elements of an oat sample population representing 162 cultivated varieties from 20 countries and the protein composition of resulting samples have been characterized. Size distribution of the total protein extracts has been analyzed by size exclusion-high performance liquid chromatography (SE-HPLC) while the 70% ethanol-extracted proteins were analyzed by RP-HPLC. Protein extracts separated into three main groups of fractions on the SE-HPLC column: polymeric proteins, avenins (both containing three subgroups based on their size), and soluble proteins, representing respectively 68.79-86.60, 8.86-27.72, and 2.89-11.85% of the total protein content. The ratio of polymeric to monomeric proteins varied between 1.37 and 3.73. Seventy-six reversed phase-HPLC-separated peaks have been differentiated from the ethanol extractable proteins of the entire population. Their distribution among the cultivars varied significantly, 6-23 peaks per cultivar. The number of appearances of peaks also showed large variation: one peak has been found in 107 samples, while 15 peaks have been identified, which appeared in less than five cultivars. An estimation method for ranking the avenin-epitope content of the samples has been developed by using MS spectrometric data of collected RP-HPLC peaks and bioinformatics methods. Using ELISA methodology with the R5 antibody, a high number of the investigated samples were found to be contaminated with wheat, barley, or rye.

Single versus repeated heat stress in wheat: What are the consequences in different developmental phases?

With a possible reference to heat priming and to characterize the extent and variation in the heat stress responses in wheat, the effects of single vs. repeated heat stresses were examined by measuring the changes in morphological and grain yield-related traits and photosynthetic parameters. To achieve these objectives, 51 winter wheat cultivars of various geographic origins were included in two independent experiments covering different phenological stages. In Experiment I, a single heat stress event was applied at stem elongation (SE) and booting (B), and the repeated heat stress was applied at both of these stages (SE+B). In Experiment II, the single heat stress was applied at stem elongation (SE) and full heading (CH), while the repeated heat stress was applied at both stages (SE+CH). While genotype was a more important factor for determining the morphological and yield-related traits, it was the treatment effect that mostly influenced the photosynthetic parameters, with the exception of the chlorophyll content. The heading stage was more sensitive to heat stress than the booting stage, which was primarily due to the larger decrease in the average seed number. The importance of biomass in contributing to grain yield intensified with the heat stress treatments. There was a large variation between the wheat cultivars not only in yielding abilities under control conditions but also in sensitivities to the various heat stresses, based on which 7 distinct groups with specific response profiles could be identified at a highly significant level. The 7 wheat groups were also characterized by their reaction patterns of different magnitudes and directions in their responses to single vs. repeated heat stresses, which depended on the phenological phases during the second cycle of heat stress. The possible association between these findings and heat priming is discussed.

Heat stress responses in a large set of winter wheat cultivars (Triticum aestivum L.) depend on the timing and duration of stress.

The adverse effects of heat on plant yield strongly depend on its duration and the phenological stage of the crops when the heat occurs. To clarify the effects of these two aspects of heat stress, systematic research was conducted under controlled conditions on 101 wheat cultivars of various geographic origin. Different durations of heat stress (5, 10 and 15 days) were applied starting from three developmental stages (ZD49: booting stage, ZD59: heading, ZD72: 6th day after heading). Various morphological, yield-related traits and physiological parameters were measured to determine the stress response patterns of the wheat genotypes under combinations of the duration and the timing of heat stress. Phenological timing significantly influenced the thousand-kernel weight and reproductive tiller number. The duration of heat stress was the most significant component in determining both seed number and seed weight, as well as the grain yield consequently, explaining 51.6% of its phenotypic variance. Irrespective of the developmental phase, the yield-related traits gradually deteriorated over time, and even a 5-day heat stress was sufficient to cause significant reductions. ZD59 was significantly more sensitive to heat than either ZD49 or ZD72. The photosynthetic activity of the flag leaf was mostly determined by heat stress duration. No significant associations were noted between physiological parameters and heat stress response as measured by grain yield. Significant differences were observed between the wheat genotypes in heat stress responses, which varied greatly with developmental phase. Based on the grain yield across developmental phases and heat stress treatments, eight major response groups of wheat genotypes could be identified, and among them, three clusters were the most heat-tolerant. These cultivars are currently included in crossing schemes, partially for the identification of the genetic determinants of heat stress response and partially for the development of new wheat varieties with better heat tolerance.

Rising atmospheric CO2 concentration may imply higher risk of Fusarium mycotoxin contamination of wheat grains.

Increasing atmospheric CO2 concentration not only has a direct impact on plants but also affects plant-pathogen interactions. Due to economic and health-related problems, special concern was given thus in the present work to the effect of elevated CO2 (750 µmol mol-1) level on the Fusarium culmorum infection and mycotoxin contamination of wheat. Despite the fact that disease severity was found to be not or little affected by elevated CO2 in most varieties, as the spread of Fusarium increased only in one variety, spike grain number and/or grain weight decreased significantly at elevated CO2 in all the varieties, indicating that Fusarium infection generally had a more dramatic impact on the grain yield at elevated CO2 than at the ambient level. Likewise, grain deoxynivalenol (DON) content was usually considerably higher at elevated CO2 than at the ambient level in the single-floret inoculation treatment, suggesting that the toxin content is not in direct relation to the level of Fusarium infection. In the whole-spike inoculation, DON production did not change, decreased or increased depending on the variety × experiment interaction. Cooler (18 °C) conditions delayed rachis penetration while 20 °C maximum temperature caused striking increases in the mycotoxin contents, resulting in extremely high DON values and also in a dramatic triggering of the grain zearalenone contamination at elevated CO2. The results indicate that future environmental conditions, such as rising CO2 levels, may increase the threat of grain mycotoxin contamination.

Effects of ambient temperature in association with photoperiod on phenology and on the expressions of major plant developmental genes in wheat (Triticum aestivum L.).

In addition to its role in vernalization, temperature is an important environmental stimulus in determining plant growth and development. We used factorial combinations of two photoperiods (16H, 12H) and three temperature levels (11, 18 and 25 °C) to study the temperature responses of 19 wheat cultivars with established genetic relationships. Temperature produced more significant effects on plant development than photoperiod, with strong genotypic components. Wheat genotypes with PPD-D1 photoperiod sensitive allele were sensitive to temperature; their development was delayed by higher temperature, which intensified under non-inductive conditions. The effect of temperature on plant development was not proportional; it influenced the stem elongation to the largest extent, and warmer temperature lengthened the lag phase between the detection of first node and the beginning of intensive stem elongation. The gene expression patterns of VRN1, VRN2 and PPD1 were also significantly modified by temperature, while VRN3 was more chronologically regulated. The associations between VRN1 and VRN3 gene expression with early apex development were significant in all treatments but were only significant for later plant developmental phases under optimal conditions (16H and 18 °C). Under 16H, the magnitude of the transient peak expression of VRN2 observed at 18 and 25 °C associated with the later developmental phases.

Allele frequencies in the VRN-A1, VRN-B1 and VRN-D1 vernalization response and PPD-B1 and PPD-D1 photoperiod sensitivity genes, and their effects on heading in a diverse set of wheat cultivars (Triticum aestivum L.).

Heading of cereals is determined by complex genetic and environmental factors in which genes responsible for vernalization and photoperiod sensitivity play a decisive role. Our aim was to use diagnostic molecular markers to determine the main allele types in VRN-A1, VRN-B1, VRN-D1, PPD-B1 and PPD-D1 in a worldwide wheat collection of 683 genotypes and to investigate the effect of these alleles on heading in the field. The dominant VRN-A1, VRN-B1 and VRN-D1 alleles were present at a low frequency. The PPD-D1a photoperiod-insensitive allele was carried by 57 % of the cultivars and was most frequent in Asian and European cultivars. The PPD-B1 photoperiod-insensitive allele was carried by 22 % of the genotypes from Asia, America and Europe. Nine versions of the PPD-B1-insensitive allele were identified based on gene copy number and intercopy structure. The allele compositions in PPD-D1, PPD-B1 and VRN-D1 significantly influenced heading and together explained 37.5 % of the phenotypic variance. The role of gene model increased to 39.1 % when PPD-B1 intercopy structure was taken into account instead of overall PPD-B1 type (sensitive vs. insensitive). As a single component, PPD-D1 had the most important role (28.0 % of the phenotypic variance), followed by PPD-B1 (12.3 % for PPD-B1_overall, and 15.1 % for PPD-B1_intercopy) and VRN-D1 (2.2 %). Significant gene interactions were identified between the marker alleles within PPD-B1 and between VRN-D1 and the two PPD1 genes. The earliest heading genotypes were those with the photoperiod-insensitive allele in PPD-D1 and PPD-B1, and with the spring allele for VRN-D1 and the winter alleles for VRN-A1 and VRN-B1. This combination could only be detected in genotypes from Southern Europe and Asia. Late-heading genotypes had the sensitivity alleles for both PPD1 genes, regardless of the allelic composition of the VRN1 genes. There was a 10-day difference in heading between the earliest and latest groups under field conditions.

Effect of heat and drought stress on the structure and composition of arabinoxylan and ß-glucan in wheat grain.

The effects of heat (H), drought (D) and H+D (from 12th day after heading for 15 days) on the dietary fiber content and composition (arabinoxylan (AX) and ß-glucan) of three winter wheat varieties (Plainsman V, Mv Magma and Fatima 2) were determined. Results showed that H and D stress decreased the TKW, the ß-glucan contents of the seeds and the quantity of the DP3+DP4 units, while the protein and AX contents increased. The highest amounts of AX and proteins were in the H+D stressed samples with heat stress also increasing the water extractability (WE) of the AX. However, while the content of AX content was generally increased by all stresses, drought stress had negative effect on the AX content of the drought tolerant Plainsman V. Fatima 2 behaved similarly to Plainsman V as regards to its drought tolerance, but was very sensitive to heat stress, while Mv Magma was the most resistant to heat stress.

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.

The quantitative response of wheat vernalization to environmental variables indicates that vernalization is not a response to cold temperature.

The initiation of flowering is a crucial trait that allows temperate plants to flower in the favourable conditions of spring. The timing of flowering initiation is governed by two main mechanisms: vernalization that defines a plant's requirement for a prolonged exposure to cold temperatures; and photoperiod sensitivity defining the need for long days to initiate floral transition. Genetic variability in both vernalization and photoperiod sensitivity largely explains the adaptability of cultivated crop plants such as bread wheat (Triticum aestivum L.) to a wide range of climatic conditions. The major genes controlling wheat vernalization (VRN1, VRN2, and VRN3) and photoperiod sensitivity (PPD1) have been identified, and knowledge of their interactions at the molecular level is growing. However, the quantitative effects of temperature and photoperiod on these genes remain poorly understood. Here it is shown that the distinction between the temperature effects on organ appearance rate and on vernalization sensu stricto is crucial for understanding the quantitative effects of the environmental signal on wheat flowering. By submitting near isogenic lines of wheat differing in their allelic composition at the VRN1 locus to various temperature and photoperiod treatments, it is shown that, at the whole-plant level, the vernalization process has a positive response to temperature with complex interactions with photoperiod. In addition, the phenotypic variation associated with the presence of different spring homoeoalleles of VRN1 is not induced by a residual vernalization requirement. The results demonstrate that a precise definition of vernalization is necessary to understand and model temperature and photoperiod effects on wheat flowering. It is suggested that this definition should be used as the basis for gene expression studies and assessment of functioning of the wheat flowering gene network, including an explicit account of the quantitative effect of environmental variables.

Adaptation of barley to mild winters: a role for PPDH2.

BACKGROUND: Understanding the adaptation of cereals to environmental conditions is one of the key areas in which plant science can contribute to tackling challenges presented by climate change. Temperature and day length are the main environmental regulators of flowering and drivers of adaptation in temperate cereals. The major genes that control flowering time in barley in response to environmental cues are VRNH1, VRNH2, VRNH3, PPDH1, and PPDH2 (candidate gene HvFT3). These genes from the vernalization and photoperiod pathways show complex interactions to promote flowering that are still not understood fully. In particular, PPDH2 function is assumed to be limited to the ability of a short photoperiod to promote flowering. Evidence from the fields of biodiversity, ecogeography, agronomy, and molecular genetics was combined to obtain a more complete overview of the potential role of PPDH2 in environmental adaptation in barley. RESULTS: The dominant PPDH2 allele is represented widely in spring barley cultivars but is found only occasionally in modern winter cultivars that have strong vernalization requirements. However, old landraces from the Iberian Peninsula, which also have a vernalization requirement, possess this allele at a much higher frequency than modern winter barley cultivars. Under field conditions in which the vernalization requirement of winter cultivars is not satisfied, the dominant PPDH2 allele promotes flowering, even under increasing photoperiods above 12 h. This hypothesis was supported by expression analysis of vernalization-responsive genotypes. When the dominant allele of PPDH2 was expressed, this was associated with enhanced levels of VRNH1 and VRNH3 expression. Expression of these two genes is needed for the induction of flowering. Therefore, both in the field and under controlled conditions, PPDH2 has an effect of promotion of flowering. CONCLUSIONS: The dominant, ancestral, allele of PPDH2 is prevalent in southern European barley germplasm. The presence of the dominant allele is associated with early expression of VRNH1 and early flowering. We propose that PPDH2 promotes flowering of winter cultivars under all non-inductive conditions, i.e. under short days or long days in plants that have not satisfied their vernalization requirement. This mechanism is indicated to be a component of an adaptation syndrome of barley to Mediterranean conditions.