Transcriptome meta-analysis of abiotic stresses-responsive genes and identification of candidate transcription factors for broad stress tolerance in wheat.

Under field conditions, wheat is subjected to single or multiple stress conditions. The elucidation of the molecular mechanism of stress response is a key step to identify candidate genes for stress resistance in plants. In this study, RNA-seq data analysis identified 17.324, 10.562, 5.510, and 8.653 differentially expressed genes (DEGs) under salt, drought, heat, and cold stress, respectively. Moreover, the comparison of DEGs from each stress revealed 2374 shared genes from which 40% showed highly conserved expression patterns. Moreover, co-expression network analysis and GO enrichment revealed co-expression modules enriched with genes involved in transcription regulation, protein kinase pathway, and genes responding to phytohormones or modulating hormone levels. The expression of 15 selected transcription factor encoding genes was analyzed under abiotic stresses and ABA treatment in durum wheat. The identified transcription factor genes are excellent candidates for genetic engineering of stress tolerance in wheat.

Overexpression of durum wheat NAC transcription factor TtNTL3A promotes early flowering and increases multiple stress tolerance in transgenic Arabidopsis.

Plant-specific NAC transcription factors play important roles in plant development processes, hormone signaling and response to biotic and abiotic stresses. Here, a newly identified membrane-bound NAC gene, designated as TtNTL3A, was isolated from durum wheat. TtNTL3A was homologous to bread wheat TaNAC8 and rice OsNAC8. Moreover, yeast trans-activation assays revealed that TtNTL3A is a transcriptional activator. TtNTL3A was highly expressed in developing seeds and was induced by abiotic stresses, abscisic acid treatment and the infection with Fusarium graminearum. Besides, Transgenic Arabidopsis overexpressing TtNTL3A showed early flowering phenotype and higher tolerance to salt and drought stresses. RT-qPCR analysis revealed that drought and salt-responsive genes were highly expressed in transgenic lines compared to WT plants. Besides, these lines showed resistance to Fusarium graminearum, which was accompanied by a higher expression of pathogenesis-related genes (AtPR-1, Atpdf1.2, and AtNPR1) in TtNTL3A-OE lines. These findings suggest that TtNTL3A is an interesting target of genetic engineering to improve wheat tolerance to biotic and abiotic stresses.

Abscisic Acid-Stress-Ripening Genes Involved in Plant Response to High Salinity and Water Deficit in Durum and Common Wheat.

In the dry and hot Mediterranean regions wheat is greatly susceptible to several abiotic stresses such as extreme temperatures, drought, and salinity, causing plant growth to decrease together with severe yield and quality losses. Thus, the identification of gene sequences involved in plant adaptation to such stresses is crucial for the optimization of molecular tools aimed at genetic selection and development of stress-tolerant varieties. Abscisic acid, stress, ripening-induced (ASR) genes act in the protection mechanism against high salinity and water deficit in several plant species. In a previous study, we isolated for the first time the TtASR1 gene from the 4A chromosome of durum wheat in a salt-tolerant Tunisian landrace and assessed its involvement in plant response to some developmental and environmental signals in several organs. In this work, we focused attention on ASR genes located on the homoeologous chromosome group 4 and used for the first time a Real-Time approach to "in planta" to evaluate the role of such genes in modulating wheat adaptation to salinity and drought. Gene expression modulation was evaluated under the influence of different variables - kind of stress, ploidy level, susceptibility, plant tissue, time post-stress application, gene chromosome location. ASR response to abiotic stresses was found only slightly affected by ploidy level or chromosomal location, as durum and common wheat exhibited a similar gene expression profile in response to salt increase and water deficiency. On the contrary, gene activity was more influenced by other variables such as plant tissue (expression levels were higher in roots than in leaves), kind of stress [NaCl was more affecting than polyethylene glycol (PEG)], and genotype (transcripts accumulated differentially in susceptible or tolerant genotypes). Based on such experimental evidence, we confirmed Abscisic acid, stress, ripening-induced genes involvement in plant response to high salinity and drought and suggested the quantification of gene expression variation after long salt exposure (72 h) as a reliable parameter to discriminate between salt-tolerant and salt-susceptible genotypes in both Triticum aestivum and Triticum durum.

Structural and Functional Characterization of the ABA-Water Deficit Stress Domain from Wheat and Barley: An Intrinsically Disordered Domain behind the Versatile Functions of the Plant Abscissic Acid, Stress and Ripening Protein Family.

The ASR protein family has been discovered thirty years ago in many plant species and is involved in the tolerance of various abiotic stresses such as dehydration, salinity and heat. Despite its importance, nothing is known about the conserved ABA-Water Deficit Stress Domain (ABA-WDS) of the ASR gene family. In this study, we characterized two ABA-WDS domains, isolated from durum wheat (TtABA-WDS) and barley (HvABA-WDS). Bioinformatics analysis shows that they are both consistently predicted to be intrinsically disordered. Hydrodynamic and circular dichroism analysis indicate that both domains are largely disordered but belong to different structural classes, with HvABA-WDS and TtABA-WDS adopting a PreMolten Globule-like (PMG-like) and a Random Coil-like (RC-like) conformation, respectively. In the presence of the secondary structure stabilizer trifluoroethanol (TFE) or of increasing glycerol concentrations, which mimics dehydration, the two domains acquire an α-helical structure. Interestingly, both domains are able to prevent heat- and dehydration-induced inactivation of the enzyme lactate dehydrogenase (LDH). Furthermore, heterologous expression of TtABA-WDS and HvABA-WDS in the yeast Saccharomyces cerevisiae improves its tolerance to salt, heat and cold stresses. Taken together our results converge to show that the ABA-WDS domain is an intrinsically disordered functional domain whose conformational plasticity could be instrumental to support the versatile functions attributed to the ASR family, including its role in abiotic stress tolerance. Finally, and after validation in the plant system, this domain could be used to improve crop tolerance to abiotic stresses.

Structural disorder and induced folding within two cereal, ABA stress and ripening (ASR) proteins.

Abscisic acid (ABA), stress and ripening (ASR) proteins are plant-specific proteins involved in plant response to multiple abiotic stresses. We previously isolated the ASR genes and cDNAs from durum wheat (TtASR1) and barley (HvASR1). Here, we show that HvASR1 and TtASR1 are consistently predicted to be disordered and further confirm this experimentally. Addition of glycerol, which mimics dehydration, triggers a gain of structure in both proteins. Limited proteolysis showed that they are highly sensitive to protease degradation. Addition of 2,2,2-trifluoroethanol (TFE) however, results in a decreased susceptibility to proteolysis that is paralleled by a gain of structure. Mass spectrometry analyses (MS) led to the identification of a protein fragment resistant to proteolysis. Addition of zinc also induces a gain of structure and Hydrogen/Deuterium eXchange-Mass Spectrometry (HDX-MS) allowed identification of the region involved in the disorder-to-order transition. This study is the first reported experimental characterization of HvASR1 and TtASR1 proteins, and paves the way for future studies aimed at unveiling the functional impact of the structural transitions that these proteins undergo in the presence of zinc and at achieving atomic-resolution conformational ensemble description of these two plant intrinsically disordered proteins (IDPs).

Understanding the Effects of Genotype, Growing Year, and Breeding on Tunisian Durum Wheat Allergenicity. 1. The Baker's Asthma Case.

Baker's asthma is a serious airway disease triggered by wheat protein CM3 α-amylase/trypsin inhibitor. The purpose of the present study was to investigate the impact of genotype and crop year on allergen CM3 α-amylase/trypsin inhibitor associated with baker's asthma. A historical series of Tunisian durum wheat (100 accessions), derived from three crop years, was used to compare the amount of CM3 from landraces to advanced cultivars. CM3 protein quantification was assessed after an enzymatic cleavage of the soluble protein extracts on a UPLC/ESI-MS system, using a marker peptide for its quantification. Combined data analysis of variance revealed an important effect of genotype, crop year, and their interaction. The CM3 allergenic proteins were found to significantly vary among studied genotypes, as confirmed by genetic variability, coefficient of variance, heritability, and genetic advance.

Understanding the Effects of Genotype, Growing Year, and Breeding on Tunisian Durum Wheat Allergenicity. 2. The Celiac Disease Case.

The aim of this study was to compare immunogenic and toxic gluten peptides related to celiac disease (CD). 100 accessions of genotypes selected during the 20th century in Tunisia were in vitro digested and then analyzed by UPLC/ESI-MS technique using an isotopically labeled internal standard. The first MANOVA confirmed a high variability in the content of immunogenic and toxic peptides reflecting high genetic diversity in the germplasm released during the past century in Tunisia, consistently with PCA and clustering analysis results. Our finding showed also important variability in CD epitopes due to growing season's climate scenarios. Moreover, the second MANOVA revealed significant differences between abandoned and modern cultivars' CD-related peptide amounts. Although we could not conclude that there was an augment of allergens in newly selected durum wheat lines compared to abandoned ones, we demonstrated that modern genotype peptides were less sensitive to climate variation, which is a useful indicator for wheat breeders.

Bridging Between Proline Structure, Functions, Metabolism, and Involvement in Organism Physiology.

Much is now known about proline multifunctionality and metabolism; some aspects of its biological functions are still unclear. Here, we discuss some cases in the proline, structure, definition, metabolism, compartmentalization, accumulation, plausible functions and also its implication in homeostasis and organism physiology. Indeed, we report the role of proline in cellular homeostasis, including redox balance and energy status and their implication as biocatalyst for aldolase activity. Proline can act as a signaling molecule to modulate mitochondrial functions, influence cell proliferation or cell death, and trigger specific gene expression, which can be essential for plant recovery from stresses. Although, the regulation and the function of proline accumulation, during abiotic stresses, are not yet completely understood. The engineering of proline metabolism could lead to new opportunities to improve plant tolerance against environmental stresses. This atypical amino acid has a potential role in the toxicity during growth of some microorganism, vegetal, and mammalian species. Furthermore, we note that the purpose through the work is to provide a rich, concise, and mostly cohesive source on proline, considered as a platform and an anchor between several disciplines and biological functions.