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.

Abscisic Acid, Stress, and Ripening (TtASR1) Gene as a Functional Marker for Salt Tolerance in Durum Wheat.

In semiarid Mediterranean agroecosystems, drought and salinity are the main abiotic stresses hampering wheat productivity and yield instability. Abscisic acid, stress, and ripening (ASR) are small plant proteins and play important roles in different biological processes. In the present study, the TtASR1 gene was isolated and characterized for the first time from durum wheat (Tritucum turgidum L. subsp. durum). TtASR1 is a small gene, about 684 bp long, located on chromosome 4AL, encoding a protein of 136 amino acid residues consisting of a histidine-rich N terminus and C-terminal conserved ABA-WDS domain (Pfam PF02496). Our results showed that TtASR1 protein could function as a chaperone-like protein and improve the viability of E. coli under heat and cold stress and increase the Saccharomyces cerevisiae tolerance under salt and osmotic stress. Transcript expression patterns of TtASR1 revealed that ASRs play important roles in abiotic stress responses in diverse organs. Indeed, TtASR1 was upregulated in leaves by different developmental (ABA) and environmental signals (PEG, salt). In cv. Mahmoudi (salt-tolerant Tunisian durum landraces) roots, TtASR1 was upregulated by salt stress, while it was downregulated in cv. Azizi (salt-sensitive Tunisian durum landraces), supporting the implication of this gene in the salt tolerance mechanism. Taken together and after validation in the plant system, the TtASR1 gene may provide a potential functional marker for marker-assisted selection in a durum wheat breeding program for salt tolerance.

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).