Global Expression Patterns of Three Festuca Species Exposed to Different Doses of Glyphosate Using the Affymetrix GeneChip Wheat Genome Array

Glyphosate has been shown to act as an inhibitor of an aromatic amino acid biosynthetic pathway, while other pathways that may be affected by glyphosate are not known. Cross species hybridizations can provide a tool for elucidating biological pathways conserved among organisms. Comparative genome analyses have indicated a high level of colinearity among grass species and Festuca, on which we focus here, and showed rearrangements common to the Pooideae family. Based on sequence conservation among grass species, we selected the Affymetrix GeneChip Wheat Genome Array as a tool for the analysis of expression profiles of three Festuca (fescue) species with distinctly different tolerances to varying levels of glyphosate. Differences in transcript expression were recorded upon foliar glyphosate application at 1.58 mM and 6.32 mM, representing 5% and 20%, respectively, of the recommended rate. Differences highlighted categories of general metabolic processes, such as photosynthesis, protein synthesis, stress responses, and a larger number of transcripts responded to 20% glyphosate application. Differential expression of genes encoding proteins involved in the shikimic acid pathway could not be identified by cross hybridization. Microarray data were confirmed by RT-PCR and qRT-PCR analyses. This is the first report to analyze the potential of cross species hybridization in Fescue species and the data and analyses will help extend our knowledge on the cellular processes affected by glyphosate

Molecular analysis of glyphosate and osmotic stress responsive genes

Cross species hybridization can provide a tool for elucidating biological pathways conserved among organisms. Based on sequence conservation among grass species, we selected Affymetrix GeneChip® wheat genome array as a tool to analyze changes in gene expression profiles of three Festuca species in response to varying levels of glyphosate. Differences in transcript expression upon glyphosate application at 5% and 20% of the recommended rate were recorded. Differences highlighted metabolic categories, including photosynthesis, protein synthesis, and stress responses. Expression levels of a larger number of transcripts altered with 20% glyphosate. RT-PCR analysis was conducted for experimental validation. This is the first report to analyze the potential of cross species hybridization in Festuca species and the data help extend our knowledge on the cellular processes affected by glyphosate. Autophagy related gene, Atg8 has been used for monitoring autophagy in various organisms. In this study, Atg8 gene was identified in Brachypodium distachyon (named as BdAtg8) under osmotic stress. Expression profile of BdAtg8 was examined in a variety of tissues of different ages and osmotic stress conditions. Expression level of BdAtg8 elevated with osmotic stress, especially in the roots. BdAtg8 complemented atg8Δ[delta]::kan MX yeast mutants grown under starvation conditions. Monodansylcadaverine was used to observe autophagosomes, and autophagy was shown to be constitutively active in Brachypodium. Autophagy was more active in plants exposed to osmotic stress. BdATG8 protein was expressed in yeast and analyzed with western blotting. In conclusion, under osmotic stress conditions, BdAtg8 gene is required for induction of autophagy in Brachypodium

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