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Stress responsive miRNAs and isomiRs in cereals

Abiotic and biotic stress conditions are vital determinants in the production of cereals, the major caloric source in human nutrition. Small RNAs, miRNAs and isomiRs are central to post-transcriptional regulation of gene expression in a variety of cellular processes including development and stress responses. Several miRNAs have been identified using new technologies and have roles in stress responses in plants, including cereals. The overall knowledge about the cereal miRNA repertoire, as well as an understanding of complex miRNA mediated mechanisms of target regulation in response to stress conditions, is far from complete. Ongoing efforts that add to our understanding of complex miRNA machinery have implications in plant response to stress conditions. Additionally, sequence variants of miRNAs (isomiRNAs or isomiRs), regulation of their expression through dissection of upstream regulatory elements, the role of Processing-bodies (P-bodies) in miRNA exerted gene regulation and yet unveiled organellar plant miRNAs are newly emerging topics, which will contribute to the elucidation of the miRNA machinery and its role in cereal tolerance against abiotic and biotic stresses.

Proteome changes in wild and modern wheat leaves upon drought stress by two-dimensional electrophoresis and nanoLC-ESI–MS/MS

To elucidate differentially expressed proteins and to further understand post-translational modifications of transcripts, full leaf proteome profiles of two wild emmer (Triticum turgidum ssp. dicoccoides TR39477 and TTD22) and one modern durum wheat (Triticum turgidum ssp. durum cv. Kızıltan) genotypes were compared upon 9-day drought stress using two-dimensional gel electrophoresis and nano-scale liquid chromatographic electrospray ionization tandem mass spectrometry methods. The three genotypes compared exhibit distinctive physiological responses to drought as previously shown by our group. Results demonstrated that many of the proteins were common in both wild emmer and modern wheat proteomes; of which, 75 were detected as differentially expressed proteins. Several proteins identified in all proteomes exhibited drought regulated patterns of expression. A number of proteins were observed with higher expression levels in response to drought in wild genotypes compared to their modern relative. Eleven protein spots with low peptide matches were identified as candidate unique drought responsive proteins. Of the differentially expressed proteins, four were selected and further analyzed by quantitative real-time PCR at the transcriptome level to compare with the proteomic data. The present study provides protein level differences in response to drought in modern and wild genotypes of wheat that may account for the differences of the overall responses of these genotypes to drought. Such comparative proteomics analyses may aid in the better understanding of complex drought response and may suggest candidate genes for molecular breeding studies to improve tolerance against drought stress and, thus, to enhance yields.

The drought response displayed by a DRE-binding protein from Triticum dicoccoides.

Drought is one of the major causes of dramatic yield loss in crop plants. Knowledge of how to alleviate this loss is still limited due to the complexity of both the stress condition and plant responses. Wild emmer wheat (Triticum turgidum ssp. dicoccoides) is a potential source of important drought-resistance genes for its cultivated relatives. The gene for an emmer DRE-binding protein, TdicDRF1, was cloned and shown to be drought-responsive with orthologs in other plants. This is the first report of the cloning of TdicDRF1, and its expression was further characterized by RT-PCR in both drought-sensitive and drought-resistant accessions of Triticum dicoccoides. Analysis of the AP2/ERF DNA-binding domain of TdicDRF1 as a GST-fusion protein and its binding to DRE by electrophoretic mobility shift assay (EMSA) indicate functional differences between wheat DREBs and those characterized in Arabidopsis thaliana. DREB expression increased in drought-stressed roots, correlating with the RT-PCR results, but not in leaf, showing that tissue-specific regulation occurs at the protein level. Hence, the DREB-DRE interaction undergoes subtle multi-level regulation.

Root precursors of microRNAs in wild emmer and modern wheats show major differences in response to drought stress.

MicroRNAs, small regulatory molecules with significant impacts on the transcriptional network of all living organisms, have been the focus of several studies conducted mostly on modern wheat cultivars. In this study, we investigated miRNA repertoires of modern durum wheat and its wild relatives, with differing degrees of drought tolerance, to identify miRNA candidates and their targets involved in drought stress response. Root transcriptomes of Triticum turgidum ssp. durum variety Kızıltan and two Triticum turgidum ssp. dicoccoides genotypes TR39477 and TTD-22 under control and drought conditions were assembled from individual RNA-Seq reads and used for in silico identification of miRNAs. A total of 66 miRNAs were identified from all species, across all conditions, of which 46 and 38 of the miRNAs identified from modern durum wheat and wild genotypes, respectively, had not been previously reported. Genotype- and/or stress-specific miRNAs provide insights into our understanding of the complex drought response. Particularly, miR1435, miR5024, and miR7714, identified only from drought-stress roots of drought-tolerant genotype TR39477, can be candidates for future studies to explore and exploit the drought response to develop tolerant varieties.

Identification of the VERNALIZATION 4 gene reveals the origin of spring growth habit in ancient wheats from South Asia

Significance A precise regulation of flowering time is critical for plant reproductive success and for cereal crops to maximize grain production. In wheat, barley, and other temperate cereals, vernalization genes play an important role in the acceleration of reproductive development after long periods of low temperatures during the winter (vernalization). In this study, we identified VERNALIZATION 4 (VRN-D4), a vernalization gene that was critical for the development of spring growth habit in the ancient wheats from South Asia. We show that mutations in regulatory regions of VRN-D4 are shared with other VRN-A1 alleles and can be used to modulate the vernalization response. These previously unknown alleles provide breeders new tools to engineer wheat varieties better adapted to different or changing environments. Wheat varieties with a winter growth habit require long exposures to low temperatures (vernalization) to accelerate flowering. Natural variation in four vernalization genes regulating this requirement has favored wheat adaptation to different environments. The first three genes (VRN1–VRN3) have been cloned and characterized before. Here we show that the fourth gene, VRN-D4, originated by the insertion of a ∼290-kb region from chromosome arm 5AL into the proximal region of chromosome arm 5DS. The inserted 5AL region includes a copy of VRN-A1 that carries distinctive mutations in its coding and regulatory regions. Three lines of evidence confirmed that this gene is VRN-D4: it cosegregated with VRN-D4 in a high-density mapping population; it was expressed earlier than other VRN1 genes in the absence of vernalization; and induced mutations in this gene resulted in delayed flowering. VRN-D4 was found in most accessions of the ancient subspecies Triticum aestivum ssp. sphaerococcum from South Asia. This subspecies showed a significant reduction of genetic diversity and increased genetic differentiation in the centromeric region of chromosome 5D, suggesting that VRN-D4 likely contributed to local adaptation and was favored by positive selection. Three adjacent SNPs in a regulatory region of the VRN-D4 first intron disrupt the binding of GLYCINE-RICH RNA-BINDING PROTEIN 2 (TaGRP2), a known repressor of VRN1 expression. The same SNPs were identified in VRN-A1 alleles previously associated with reduced vernalization requirement. These alleles can be used to modulate vernalization requirements and to develop wheat varieties better adapted to different or changing environments.

Autophagy-related gene, TdAtg8, in wild emmer wheat plays a role in drought and osmotic stress response

An autophagy-related gene Atg8 was cloned for the first time from wild emmer wheat, named as TdAtg8, and its role on autophagy under abiotic stress conditions was investigated. Examination of TdAtg8 expression patterns indicated that Atg8 expression was strongly upregulated under drought stress, especially in the roots when compared to leaves. LysoTracker® red marker, utilized to observe autophagosomes, revealed that autophagy is constitutively active in Triticum dicoccoides. Moreover, autophagy was determined to be induced in plants exposed to osmotic stress when compared to plants grown under normal conditions. Functional studies were executed in yeast to confirm that the TdATG8 protein is functional, and showed that the TdAtg8 gene complements the atg8∆::kan MX yeast mutant strain grown under nitrogen deficiency. For further functional analysis, TdATG8 protein was expressed in yeast and analyzed using Western immunoblotting. Atg8-silenced plants were exposed to drought stress and chlorophyll and malondialdehyde (MDA) content measurements demonstrated that Atg8 plays a key role on drought stress tolerance. In addition, Atg8-silenced plants exposed to osmotic stress were found to have decreased Atg8 expression level in comparison to controls. Hence, Atg8 is a positive regulator in osmotic and drought stress response.

Subgenomic analysis of microRNAs in polyploid wheat

In this study, a survey of miRNAs using the next-generation sequencing data was performed at subgenomic level. After analyzing shotgun sequences from chromosome 4A of bread wheat (Triticum aestivum L.), a total of 68 different miRNAs were predicted in silico, of which 37 were identified in wheat for the first time. The long arm of the chromosome was found to harbor a higher variety (51) and representation (3,928) of miRNAs compared with the short arm (49; 2,226). Out of the 68 miRNAs, 32 were detected to be common to both arms, revealing the presence of separate miRNA clusters in the two chromosome arms. The differences in degree of representation of the different miRNAs were found to be highly variable, ranging 592-fold, which may have an effect on target regulation. Targets were retrieved for 62 (out of 68) of wheat-specific, newly identified miRNAs indicated that fundamental aspects of plant morphology such as height and flowering were predicted to be affected. In silico expression blast analysis indicated 24 (out of 68) were found to give hits to expressed sequences. This is the first report of species- and chromosome-specific miRNAs.

Plant abiotic stress signaling

Stress signaling is central to plants which—as immobile organisms—have to endure environmental fluctuations that constantly interfere with vigorous growth. As a result, plant-specific, elaborate mechanisms have evolved to perceive and respond to stress conditions. Currently, these stress responses are plausibly being revealed to involve crosstalks with energy signaling pathways as any growth-limiting factor alters plant’s energy status. Among these, autophagy, conventionally regarded as the mechanism whereby plants recycle and remobilize nutrients in bulk, has frequently been associated with stress responses. With the recent discoveries, however, autophagy has attained a novel role in stress signaling. In this review, major elements of abitoic stress signaling are summarized along with autophagy pathway, and in the light of recent discoveries, a putative, state-of-art role of autophagy is discussed.

Physical Mapping Integrated with Syntenic Analysis to Characterize the Gene Space of the Long Arm of Wheat Chromosome 1A

Background Bread wheat (Triticum aestivum L.) is one of the most important crops worldwide and its production faces pressing challenges, the solution of which demands genome information. However, the large, highly repetitive hexaploid wheat genome has been considered intractable to standard sequencing approaches. Therefore the International Wheat Genome Sequencing Consortium (IWGSC) proposes to map and sequence the genome on a chromosome-by-chromosome basis. Methodology/Principal Findings We have constructed a physical map of the long arm of bread wheat chromosome 1A using chromosome-specific BAC libraries by High Information Content Fingerprinting (HICF). Two alternative methods (FPC and LTC) were used to assemble the fingerprints into a high-resolution physical map of the chromosome arm. A total of 365 molecular markers were added to the map, in addition to 1122 putative unique transcripts that were identified by microarray hybridization. The final map consists of 1180 FPC-based or 583 LTC-based contigs. Conclusions/Significance The physical map presented here marks an important step forward in mapping of hexaploid bread wheat. The map is orders of magnitude more detailed than previously available maps of this chromosome, and the assignment of over a thousand putative expressed gene sequences to specific map locations will greatly assist future functional studies. This map will be an essential tool for future sequencing of and positional cloning within chromosome 1A.

Genomics approaches for crop improvement against abiotic stress.

As sessile organisms, plants are inevitably exposed to one or a combination of stress factors every now and then throughout their growth and development. Stress responses vary considerably even in the same plant species; stress-susceptible genotypes are at one extreme, and stress-tolerant ones are at the other. Elucidation of the stress responses of crop plants is of extreme relevance, considering the central role of crops in food and biofuel production. Crop improvement has been a traditional issue to increase yields and enhance stress tolerance; however, crop improvement against abiotic stresses has been particularly compelling, given the complex nature of these stresses. As traditional strategies for crop improvement approach their limits, the era of genomics research has arisen with new and promising perspectives in breeding improved varieties against abiotic stresses.