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.

The wheat TdRL1 is the functional homolog of the rice RSS1 and promotes plant salt stress tolerance.

KEY MESSAGE: Rice rss1 complementation assays show that wheat TdRL1 and RSS1 are true functional homologs. TdRL1 over-expression in Arabidopsis conferred salt stress tolerance and alleviated ROS accumulation. Plants have developed highly flexible adaptive responses to their ever-changing environment, which are often mediated by intrinsically disordered proteins (IDP). RICE SALT SENSITIVE 1 and Triticum durum RSS1-Like 1 protein (TdRL1) are both IDPs involved in abiotic stress responses, and possess conserved D and DEN-Boxes known to be required for post-translational degradation by the APC/Ccdc20 cyclosome. To further understand their function, we performed a computational analysis to compare RSS1 and TdRL1 co-expression networks revealing common gene ontologies, among which those related to cell cycle progression and regulation of microtubule (MT) networks were over-represented. When over-expressed in Arabidopsis, TdRL1::GFP was present in dividing cells and more visible in cortical and endodermal cells of the Root Apical Meristem (RAM). Incubation with the proteasome inhibitor MG132 stabilized TdRL1::GFP expression in RAM cells showing a post-translational regulation. Moreover, immuno-cytochemical analyses of transgenic roots showed that TdRL1 was present in the cytoplasm and within the microtubular spindle of mitotic cells, while, in interphasic cells, it was rather restricted to the cytoplasm with a spotty pattern at the nuclear periphery. Interestingly in cells subjected to stress, TdRL1 was partly relocated into the nucleus. Moreover, TdRL1 transgenic lines showed increased germination rates under salt stress conditions as compared to wild type. This enhanced salt stress tolerance was associated to an alleviation of oxidative damage. Finally, when expressed in the rice rss1 mutant, TdRL1 suppressed its dwarf phenotype upon salt stress, confirming that both proteins are true functional homologs required for salt stress tolerance in cereals.

Genome wide identification of wheat and Brachypodium type one protein phosphatases and functional characterization of durum wheat TdPP1a.

Reversible phosphorylation is an essential mechanism regulating signal transduction during development and environmental stress responses. An important number of dephosphorylation events in the cell are catalyzed by type one protein phosphatases (PP1), which catalytic activity is driven by the binding of regulatory proteins that control their substrate specificity or subcellular localization. Plants harbor several PP1 isoforms accounting for large functional redundancies. While animal PP1s were reported to play relevant roles in controlling multiple cellular processes, plant orthologs remain poorly studied. To decipher the role of plant PP1s, we compared PP1 genes from three monocot species, Brachypodium, common wheat and rice at the genomic and transcriptomic levels. To gain more insight into the wheat PP1 proteins, we identified and characterized TdPP1a, the first wheat type one protein phosphatase from a Tunisian durum wheat variety Oum Rabiaa3. TdPP1a is highly conserved in sequence and structure when compared to mammalian, yeast and other plant PP1s. We demonstrate that TdPP1a is an active, metallo-dependent phosphatase in vitro and is able to interact with AtI2, a typical regulator of PP1 functions. Also, TdPP1a is capable to complement the heat stress sensitivity of the yeast mutant indicating that TdPP1a is functional also in vivo. Moreover, transient expression of TdPP1a::GFP in tobacco leaves revealed that it is ubiquitously distributed within the cell, with a strong accumulation in the nucleus. Finally, transcriptional analyses showed similar expression levels in roots and leaves of durum wheat seedlings. Interestingly, the expression in leaves is significantly induced following salinity stress, suggesting a potential role of TdPP1a in wheat salt stress response.

Molecular and functional characterization of the durum wheat TdRL1, a member of the conserved Poaceae RSS1-like family that exhibits features of intrinsically disordered proteins and confers stress tolerance in yeast.

Because of their fixed lifestyle, plants must acclimate to environmental changes by orchestrating several responses ranging from protective measures to growth control. Growth arrest is observed upon abiotic stress and can cause penalties to plant production. But, the molecular interface between stress perception and cell cycle control is poorly understood. The rice protein RSS1 is required at G1/S transition ensuring normal dividing activity of proliferative cells during salt stress. The role of RSS1 in meristem maintenance together with its flexible protein structure implies its key function as molecular integrator of stress signaling for cell cycle control. To study further the relevance of RSS1 and its related proteins in cereals, we isolated the durum wheat homolog, TdRL1, from Tunisian durum wheat varieties and extended our analyses to RSS1-like proteins from Poaceae. Our results show that the primary sequences of TdRL1 and the Graminae RSS1-like family members are highly conserved. In silico analyses predict that TdRL1 and other RSS1-like proteins share flexible 3-D structures and have features of intrinsically disordered/unstructured proteins (IDP). The disordered structure of TdRL1 is well illustrated by an electrophoretical mobility shift of the purified protein. Moreover, heterologous expression of TdRL1 in yeast improves its tolerance to salt and heat stresses strongly suggesting its involvement in abiotic stress tolerance mechanisms. Such finding adds new knowledge to our understanding of how IDPs may contribute as central molecular integrators of stress signaling into improving plant tolerance to abiotic stresses.