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

International audienceThe 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..This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC B

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

Abstract 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

Allelic Variants of Glutamine Synthetase and Glutamate Synthase Genes in a Collection of Durum Wheat and Association with Grain Protein Content

Wheat is one of the most important crops grown worldwide. Despite the fact that it accounts for only 5% of the global wheat production, durum wheat (Triticum turgidum L. subsp. durum) is a commercially important tetraploid wheat species, which originated and diversified in the Mediterranean basin. In this work, the candidate gene approach has been applied in a collection of durum wheat genotypes; allelic variants of genes glutamine synthetase (GS2) and glutamate synthase (GOGAT) were screened and correlated with grain protein content (GPC). Natural populations and collections of germplasms are quite suitable for this approach, as molecular polymorphisms close to a locus with evident phenotypic effects may be closely associated with their character, providing a better physical resolution than genetic mapping using ad hoc constituted populations. A number of allelic variants were detected both for GS2 and GOGAT genes, and regression analysis demonstrated that some variations are positively and significantly related to the GPC effect. Additionally, these genes map into homoeologous chromosome groups 2 and 3, where several authors have localized important quantitative trait loci (QTLs) for GPC. The information outlined in this work could be useful in breeding and marker-assisted selection programs

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