Mohamed Badawi

The CBF gene family in hexaploid wheat and its relationship to the phylogenetic complexity of cereal CBFs.

Most temperate plants tolerate both chilling and freezing temperatures whereas many species from tropical regions suffer chilling injury when exposed to temperatures slightly above freezing. Cold acclimation induces the expression of cold-regulated genes needed to protect plants against freezing stress. This induction is mediated, in part, by the CBF transcription factor family. To understand the evolution and function of this family in cereals, we identified and characterized 15 different CBF genes from hexaploid wheat. Our analyses reveal that wheat species, T. aestivum and T. monococcum, may contain up to 25 different CBF genes, and that Poaceae CBFs can be classified into 10 groups that share a common phylogenetic origin and similar structural characteristics. Six of these groups (IIIc, IIId, IVa, IVb, IVc and IVd) are found only in the Pooideae suggesting they represent the CBF response machinery that evolved recently during colonization of temperate habitats. Expression studies reveal that five of the Pooideae-specific groups display higher constitutive and low temperature inducible expression in the winter cultivar, and a diurnal regulation pattern during growth at warm temperature. The higher constitutive and inducible expression within these CBF groups is an inherited trait that may play a predominant role in the superior low temperature tolerance capacity of winter cultivars and possibly be a basis of genetic variability in freezing tolerance within the Pooideae subfamily.

Structure and Functional Analysis of Wheat ICE (Inducer of CBF Expression) Genes

Two different inducers of CBF expression (ICE1-like genes), TaICE41 and TaICE87, were isolated from a cDNA library prepared from cold-treated wheat aerial tissues. TaICE41 encodes a protein of 381 aa with a predicted MW of 39.5 kDa while TaICE87 encodes a protein of 443 aa with a predicted MW of 46.5 kDa. TaICE41 and TaICE87 share 46% identity while they share 50 and 47% identity with Arabidopsis AtICE1 respectively. Expression analysis revealed that mRNA accumulation was not altered by cold treatment suggesting that both genes are expressed constitutively. Gel mobility shift analysis showed that TaICE41 and TaICE87 bind to different MYC elements in the wheat TaCBFIVd-B9 promoter. Transient expression assays in Nicotiana benthamiana, showed that both TaICE proteins can activate TaCBFIVd-B9 transcription. The different affinities of TaICE41 and TaICE87 for MYC variants suggest that ICE binding specificity may be involved in the differential expression of wheat CBF genes. Furthermore, analysis of MYC elements demonstrates that a specific variant is present in the wheat CBF group IV that is associated with freezing tolerance. Overexpression of either TaICE41 or TaICE87 genes in Arabidopsis enhanced freezing tolerance only upon cold acclimation suggesting that other factors induced by low temperature are required for their activity. The increased freezing tolerance in transgenic Arabidopsis is associated with a higher expression of the cold responsive activators AtCBF2, AtCBF3, and of several cold-regulated genes.

Molecular and structural analyses of a novel temperature stress-induced lipocalin from wheat and Arabidopsis

Two cDNAs corresponding to a novel lipocalin were identified from wheat and Arabidopsis. The two cDNAs designated Tatil for Triticum aestivum L. temperature‐induced lipocalin and Attil for Arabidopsis thaliana temperature‐induced lipocalin encode polypeptides of 190 and 186 amino acids respectively. Structure analyses indicated the presence of the three structurally conserved regions that characterize lipocalins. Sequence analyses revealed that this novel class of plant lipocalin shares homology with three evolutionarily related lipocalins: the mammalian apolipoprotein D (ApoD), the bacterial lipocalin and the insect Lazarillo. The comparison of the putative tertiary structures of both the human ApoD and the wheat TaTIL suggest that the two proteins differ in membrane attachment and ligand interaction. Northern analyses demonstrated that Tatil and Attil transcripts are upregulated during cold acclimation and heat‐shock treatment. The putative functions of this novel class of plant lipocalins during temperature stresses are discussed.

Expression of vernalization responsive genes in wheat is associated with histone H3 trimethylation

The transition to flowering in winter wheat requires prolonged exposure to low temperature, a process called vernalization. This process is regulated by a genetic pathway that involves at least three genes, Triticum aestivumVERNALIZATION 1 (TaVRN1), Triticum aestivumVERNALIZATION 2 (TaVRN2) and Triticum aestivumFLOWERING LOCUS T-like 1 (TaFT1). These genes regulate flowering by integrating environmental and developmental cues. To determine whether the expression of these genes is associated with the chromatin methylation state during vernalization in wheat, the level of two markers of histone modifications, the activator histone H3 trimethylation of lysine 4 (H3K4me3) and the repressor histone H3 trimethylation of lysine 27 (H3K27me3) were measured at the promoter regions of these three genes. Bioinformatics analysis of these promoters demonstrates the presence of conserved cis-acting elements in the promoters of the three vernalization genes, TaVRN1, TaVRN2 and TaFT1. These elements are targeted by common transcription factors in the vernalization responsive cereals. These promoters also contain the functional “units” PRE/TRE targeted by Polycomb and Trithorax proteins that maintain repressed or active transcription states of developmentally regulated genes. These proteins are known to be associated with the regulation of H3K4me3 and H3K27me3. Expression studies indicate that TaVRN1 and TaFT1 are up-regulated by vernalization in winter wheat. This up-regulation is associated with increased level of the activator H3K4me3 with no change in the level of the repressor H3K27me3 at the promoter region. This study shows that the flowering transition induced by vernalization in winter wheat is associated with histone methylation at the promoter level of TaVRN1 and TaFT1 while the role of these markers is less evident in TaVRN2 repression. This may represent part of the cellular memory of vernalization in wheat.

Transcriptome analysis of an mvp mutant reveals important changes in global gene expression and a role for methyl jasmonate in vernalization and flowering in wheat.

Summary Molecular and physiological analyses of a wheat mvp mutant, and winter and spring wheats suggest that methyl jasmonate is involved in modulating vernalization and floral transition in wheat.

Long-Term Growth Under Elevated CO2 Suppresses Biotic Stress Genes in Non-Acclimated, But Not Cold-Acclimated Winter Wheat

This study compared the photosynthetic performance and the global gene expression of the winter hardy wheat Triticum aestivum cv Norstar grown under non-acclimated (NA) or cold-acclimated (CA) conditions at either ambient CO2 or elevated CO2. CA Norstar maintained comparable light-saturated and CO2-saturated rates of photosynthesis but lower quantum requirements for PSII and non-photochemical quenching relative to NA plants even at elevated CO2. Neither NA nor CA plants were sensitive to feedback inhibition of photosynthesis at elevated CO2. Global gene expression using microarray combined with bioinformatics analysis revealed that genes affected by elevated CO2 were three times higher in NA (1,022 genes) compared with CA (372 genes) Norstar. The most striking effect was the down-regulation of genes involved in the plant defense responses in NA Norstar. In contrast, cold acclimation reversed this down-regulation due to the cold induction of genes involved in plant pathogenesis resistance; and cellular and chloroplast protection. These results suggest that elevated CO2 has less impact on plant performance and productivity in cold-adapted winter hardy plants in the northern climates compared with warmer environments. Selection for cereal cultivars with constitutively higher expression of biotic stress defense genes may be necessary under elevated CO2 during the warm growth period and in warmer climates.

Heterologous Expression of Wheat VERNALIZATION 2 (TaVRN2) Gene in Arabidopsis Delays Flowering and Enhances Freezing Tolerance

The vernalization gene 2 (VRN2), is a major flowering repressor in temperate cereals that is regulated by low temperature and photoperiod. Here we show that the gene from Triticum aestivum (TaVRN2) is also regulated by salt, heat shock, dehydration, wounding and abscissic acid. Promoter analysis indicates that TaVRN2 regulatory region possesses all the specific responsive elements to these stresses. This suggests pleiotropic effects of TaVRN2 in wheat development and adaptability to the environment. To test if TaVRN2 can act as a flowering repressor in species different from the temperate cereals, the gene was ectopically expressed in the model plant Arabidopsis. Transgenic plants showed no alteration in morphology, but their flowering time was significantly delayed compared to controls plants, indicating that TaVRN2, although having no ortholog in Brassicaceae, can act as a flowering repressor in these species. To identify the possible mechanism by which TaVRN2 gene delays flowering in Arabidopsis, the expression level of several genes involved in flowering time regulation was determined. The analysis indicates that the late flowering of the 35S::TaVRN2 plants was associated with a complex pattern of expression of the major flowering control genes, FCA, FLC, FT, FVE and SOC1. This suggests that heterologous expression of TaVRN2 in Arabidopsis can delay flowering by modulating several floral inductive pathways. Furthermore, transgenic plants showed higher freezing tolerance, likely due to the accumulation of CBF2, CBF3 and the COR genes. Overall, our data suggests that TaVRN2 gene could modulate a common regulator of the two interacting pathways that regulate flowering time and the induction of cold tolerance. The results also demonstrate that TaVRN2 could be used to manipulate flowering time and improve cold tolerance in other species.

An integrative approach to identify hexaploid wheat miRNAome associated with development and tolerance to abiotic stress

BackgroundWheat is a major staple crop with broad adaptability to a wide range of environmental conditions. This adaptability involves several stress and developmentally responsive genes, in which microRNAs (miRNAs) have emerged as important regulatory factors. However, the currently used approaches to identify miRNAs in this polyploid complex system focus on conserved and highly expressed miRNAs avoiding regularly those that are often lineage-specific, condition-specific, or appeared recently in evolution. In addition, many environmental and biological factors affecting miRNA expression were not yet considered, resulting still in an incomplete repertoire of wheat miRNAs.ResultsWe developed a conservation-independent technique based on an integrative approach that combines machine learning, bioinformatic tools, biological insights of known miRNA expression profiles and universal criteria of plant miRNAs to identify miRNAs with more confidence. The developed pipeline can potentially identify novel wheat miRNAs that share features common to several species or that are species specific or clade specific. It allowed the discovery of 199 miRNA candidates associated with different abiotic stresses and development stages. We also highlight from the raw data 267 miRNAs conserved with 43 miRBase families. The predicted miRNAs are highly associated with abiotic stress responses, tolerance and development. GO enrichment analysis showed that they may play biological and physiological roles associated with cold, salt and aluminum (Al) through auxin signaling pathways, regulation of gene expression, ubiquitination, transport, carbohydrates, gibberellins, lipid, glutathione and secondary metabolism, photosynthesis, as well as floral transition and flowering.ConclusionThis approach provides a broad repertoire of hexaploid wheat miRNAs associated with abiotic stress responses, tolerance and development. These valuable resources of expressed wheat miRNAs will help in elucidating the regulatory mechanisms involved in freezing and Al responses and tolerance mechanisms as well as for development and flowering. In the long term, it may help in breeding stress tolerant plants.

Identification of oxidative stress-responsive C2H2 zinc fingers associated with Al tolerance in near-isogenic wheat lines

Background and aimsAl tolerance is one of the most important trait for worldwide crop production. Using microarrays, we previously identified a transcription factor belonging to the C2H2 zinc finger protein (ZFP) family associated with Al tolerance in wheat (Houde and Diallo, BMC Genomics 9:400, 2008). The current work aimed to identify specific members of the C2H2 ZFP family that are associated with Al tolerance.MethodsWheat ESTs were used to assemble C2H2 ZFP family members that do not contain a classical EAR repressor domain. Specific primers were designed for qRT-PCR expression profiling of wheat root tips exposed to Al and H2O2. Two Al-tolerant and sensitive wheat cultivars including a pair of near-isogenic lines differing in Al tolerance were used.ResultsWe reconstituted 16 wheat Q-type C2H2 ZFP. Expression profiling identified two transcripts (TaZFP2 and TaZFP3) that accumulate rapidly upon exposure to Al or in response to H2O2 in two tolerant wheat cultivars, including the tolerant near-isogenic line.ConclusionThe responsiveness of these transcripts to H2O2 suggests that they may represent the wheat orthologs of ZFP transcription factors ZAT7 and ZAT12 that were shown to improve ROS tolerance in Arabidopsis. Thus, they may play a crucial role in the improvement of oxidative stress tolerance in wheat.