Zahra Agharbaoui

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.

Transgenic tomato plants expressing a Lycopersicon chilense chitinase gene demonstrate improved resistance to Verticillium dahliae race 2

Abstract An acidic endochitinase gene (pcht28) isolated from Lycopersicon chilense was introduced into tomato (L. esculentum) through Agrobacterium-mediated transformation, using the CAMV 35S promoter. Transgenic plants demonstrated a high level of constitutive expression of pcht28 and chitinase enzyme activity. Kanamycin-resistant R1 plants (resulting from self-pollination of transgenic plants) as well as R2 plants were evaluated for their tolerance to Verticillium dahliae (race 1 and 2 for R1 plants and race 2 for R2 plants) in the greenhouse. They demonstrated a significantly (P<0.05) higher level of tolerance to the fungi compared to the nontransgenic plants, as measured by foliar disease symptoms, vascular discoloration, and vascular discoloration index. The transgenic plants produced in this study represent a source of genetic resistance to Verticillium dahliae.

Transformation of the wild tomatoLycopersicon chilense Dun. byAgrobacterium tumefaciens.

SummaryLeaf disc transformation-regeneration technique was applied to the drought tolerant wild relative of cultivated tomato,Lycopersicon chilense, using a plasmid construct which contained the coding sequences of neomycin phosphotransferase (NPTII) and chloramphenicol acetyltransferase (CAT) genes. The two genotypes used, LA2747 and LA1930, showed a distinct difference in their aptitude to transformation; a higher success rate was obtained for the first genotype in every stage of the process. Shoots were formed on the regeneration medium containing 100 μg/ml kanamycin through direct or indirect organogenesis. Root formation became only possible when the concentration of kanamycin was reduced to 50 μg/ml. Expression of chloramphenicol acetyltransferase gene was observed in all of the kanamycin-screened plants after they matured; the activity of the gene was absent or low in some of the young plants. The presence of the CAT gene in transgenic plants was further confirmed by Southern blot analysis. Although transgenic plants grew to maturity, they did not produce fruit, owing to the self incompatibility ofL. chilense.

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.

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.