Acute stress reduces population-level metabolic and proteomic variation.

BACKGROUND: Variation in omics data due to intrinsic biological stochasticity is often viewed as a challenging and undesirable feature of complex systems analyses. In fact, numerous statistical methods are utilized to minimize the variation among biological replicates. RESULTS: We demonstrate that the common statistics relative standard deviation (RSD) and coefficient of variation (CV), which are often used for quality control or part of a larger pipeline in omics analyses, can also be used as a metric of a physiological stress response. Using an approach we term Replicate Variation Analysis (RVA), we demonstrate that acute physiological stress leads to feature-wide canalization of CV profiles of metabolomes and proteomes across biological replicates. Canalization is the repression of variation between replicates, which increases phenotypic similarity. Multiple in-house mass spectrometry omics datasets in addition to publicly available data were analyzed to assess changes in CV profiles in plants, animals, and microorganisms. In addition, proteomics data sets were evaluated utilizing RVA to identify functionality of reduced CV proteins. CONCLUSIONS: RVA provides a foundation for understanding omics level shifts that occur in response to cellular stress. This approach to data analysis helps characterize stress response and recovery, and could be deployed to detect populations under stress, monitor health status, and conduct environmental monitoring.

Constitutive redox and phosphoproteome changes in multiple herbicide resistant Avena fatua L. are similar to those of systemic acquired resistance and systemic acquired acclimation.

Plants are routinely confronted with numerous biotic and abiotic stressors, and in response have evolved highly effective strategies of systemic acquired resistance (SAR) and systemic acquired acclimation (SAA), respectively. A much more evolutionarily recent abiotic stress is the application of herbicides to control weedy plants, and their intensive use has selected for resistant weed populations that cause substantial crop yield losses and increase production costs. Non-target site resistance (NTSR) to herbicides is rapidly increasing worldwide and is associated with alterations in generalized stress defense networks. This work investigated protein post-translational modifications associated with NTSR in multiple herbicide resistant (MHR) Avena fatua, and their commonalities with those of SAR and SAA. We used proteomic, biochemical, and immunological approaches to compare constitutive protein profiles in MHR and herbicide susceptible (HS) A. fatua populations. Phosphoproteome and redox proteome surveys showed that post-translational modifications of proteins with functions in core cellular processes were reduced in MHR plants, while those involved in xenobiotic and stress response, reactive oxygen species detoxification and redox maintenance, heat shock response, and intracellular signaling were elevated in MHR as compared to HS plants. More specifically, MHR plants contained constitutively elevated levels of three protein kinases including the lectin S-receptor-like serine/threonine-protein kinase LecRK2, a well-characterized component of SAR. Analyses of superoxide dismutase enzyme activity and protein levels did not reveal constitutive differences between MHR and HS plants. The overall results support the idea that herbicide stress is perceived similarly to other abiotic stresses, and that A. fatua NTSR shares analogous features with SAR and SAA. We speculate that MHR A. fatua's previous exposure to sublethal herbicide doses, as well as earlier evolution under a diversity of abiotic and biotic stressors, has led to a heightened state of stress preparedness that includes NTSR to a number of unrelated herbicides.

Proteomic and biochemical assays of glutathione-related proteins in susceptible and multiple herbicide resistant Avena fatua L.

Extensive herbicide usage has led to the evolution of resistant weed populations that cause substantial crop yield losses and increase production costs. The multiple herbicide resistant (MHR) Avena fatua L. populations utilized in this study are resistant to members of all selective herbicide families, across five modes of action, available for A. fatua control in U.S. small grain production, and thus pose significant agronomic and economic threats. Resistance to ALS and ACCase inhibitors is not conferred by target site mutations, indicating that non-target site resistance mechanisms are involved. To investigate the potential involvement of glutathione-related enzymes in the MHR phenotype, we used a combination of proteomic, biochemical, and immunological approaches to compare their constitutive activities in herbicide susceptible (HS1 and HS2) and MHR (MHR3 and MHR4) A. fatua plants. Proteomic analysis identified three tau and one phi glutathione S-transferases (GSTs) present at higher levels in MHR compared to HS plants, while immunoassays revealed elevated levels of lambda, phi, and tau GSTs. GST specific activity towards 1-chloro-2,4-dinitrobenzene was 1.2-fold higher in MHR4 than in HS1 plants and 1.3- and 1.2-fold higher in MHR3 than in HS1 and HS2 plants, respectively. However, GST specific activities towards fenoxaprop-P-ethyl and imazamethabenz-methyl were not different between untreated MHR and HS plants. Dehydroascorbate reductase specific activity was 1.4-fold higher in MHR than HS plants. Pretreatment with the GST inhibitor NBD-Cl did not affect MHR sensitivity to fenoxaprop-P-ethyl application, while the herbicide safener and GST inducer mefenpyr reduced the efficacy of low doses of fenoxaprop-P-ethyl on MHR4 but not MHR3 plants. Mefenpyr treatment also partially reduced the efficacy of thiencarbazone-methyl or mesosulfuron-methyl on MHR3 or MHR4 plants, respectively. Overall, the GSTs described here are not directly involved in enhanced rates of fenoxaprop-P-ethyl or imazamethabenz-methyl metabolism in MHR A. fatua. Instead, we propose that the constitutively elevated GST proteins and related enzymes in MHR plants are representative of a larger, more global suite of abiotic stress-related changes.

Intensive herbicide use has selected for constitutively elevated levels of stress-responsive mRNAs and proteins in multiple herbicide-resistant Avena fatua L.

BACKGROUND: Intensive use of herbicides has led to the evolution of two multiple herbicide-resistant (MHR) Avena fatua (wild oat) populations in Montana that are resistant to members of all selective herbicide families available for A. fatua control in US small grain crops. We used transcriptome and proteome surveys to compare constitutive changes in MHR and herbicide-susceptible (HS) plants associated with non-target site resistance. RESULTS: Compared to HS plants, MHR plants contained constitutively elevated levels of differentially expressed genes (DEGs) with functions in xenobiotic catabolism, stress response, redox maintenance and transcriptional regulation that are similar to abiotic stress-tolerant phenotypes. Proteome comparisons identified similarly elevated proteins including biosynthetic and multifunctional enzymes in MHR plants. Of 25 DEGs validated by RT-qPCR assay, differential regulation of 21 co-segregated with flucarbazone-sodium herbicide resistance in F3 families, and a subset of 10 of these were induced or repressed in herbicide-treated HS plants. CONCLUSION: Although the individual and collective contributions of these DEGs and proteins to MHR remain to be determined, our results support the idea that intensive herbicide use has selected for MHR populations with altered, constitutively regulated patterns of gene expression that are similar to those in abiotic stress-tolerant plants. © 2017 Society of Chemical Industry.

Impact of biotic and abiotic stresses on the competitive ability of multiple herbicide resistant wild oat (Avena fatua).

Ecological theory predicts that fitness costs of herbicide resistance should lead to the reduced relative abundance of resistant populations upon the cessation of herbicide use. This greenhouse research investigated the potential fitness costs of two multiple herbicide resistant (MHR) wild oat (Avena fatua) populations, an economically important weed that affects cereal and pulse crop production in the Northern Great Plains of North America. We compared the competitive ability of two MHR and two herbicide susceptible (HS) A. fatua populations along a gradient of biotic and abiotic stresses The biotic stress was imposed by three levels of wheat (Triticum aestivum) competition (0, 4, and 8 individuals pot(-1)) and an abiotic stress by three nitrogen (N) fertilization rates (0, 50 and 100 kg N ha(-1)). Data were analyzed with linear mixed-effects models and results showed that the biomass of all A. fatua populations decreased with increasing T. aestivum competition at all N rates. Similarly, A. fatua relative growth rate (RGR) decreased with increasing T. aestivum competition at the medium and high N rates but there was no response with 0 N. There were no differences between the levels of biomass or RGR of HS and MHR populations in response to T. aestivum competition. Overall, the results indicate that MHR does not confer growth-related fitness costs in these A. fatua populations, and that their relative abundance will not be diminished with respect to HS populations in the absence of herbicide treatment.

Functional analysis of TaABF1 during abscisic acid and gibberellin signalling in aleurone cells of cereal grains.

The wheat transcription factor TaABF1 physically interacts with the protein kinase PKABA1 and mediates both abscisic acid (ABA)-induced and ABA-suppressed gene expression. In bombarded aleurone cells of imbibing grains, the effect of TaABF1 in down-regulating the gibberellin (GA)-induced Amy32b promoter was stronger in the presence of exogenous ABA. As these grains contained low levels of endogenous ABA, the effect of TaABF1 may also be mediated by ABA-induced activation even in the absence of exogenous ABA. Levels of TaABF1 protein decreased slightly during imbibition of afterripened grains. However, TaABF1 levels (especially in aleurone layers) were not substantially affected by exogenous ABA or GA, indicating that changes in TaABF1 protein level are not an important part of regulating its role in hormone signalling. We found that TaABF1 was phosphorylated in vivo in aleurone cells, suggesting a role for post-translational modification in regulating TaABF1 activity. Induction of Amy32b by overexpression of the transcription factor GAMyb could not be prevented by TaABF1, indicating that TaABF1 acts upstream of GAMyb transcription in the signalling pathway. Supporting this view, knockdown of TaABF1 by RNA interference resulted in increased expression from the GAMyb promoter. These results are consistent with a model in which TaABF1 is constitutively present in aleurone cells, while its ability to down-regulate GAMyb is regulated in response to ABA.

Resistance to wheat streak mosaic virus in transgenic wheat engineered with the viral coat protein gene.

Wheat (Triticum aestivum) plants were stably transformed with the coat protein (CP) gene of wheat streak mosaic virus (WSMV) by the biolistic method. Eleven independently transformed plant lines were obtained and five were analyzed for gene expression and resistance to WSMV. One line showed high resistance to inoculations of two WSMV strains. This line had milder symptoms and lower virus titer than control plants after inoculation. After infection, new growth did not show symptoms. The observed resistance was similar to the 'recovery' type resistance described previously using WSMV NIb transgene and in other systems. This line looked morphologically normal but had an unusually high transgene copy number (approximately 90 copies per 2C homozygous genome). Northern hybridization analysis indicated a high level of degraded CP mRNA expression. However, no coat protein expression was detected.