Dean E. Riechers

Joint action of the effects of atrazine and mesotrione can lead to synergistic herbicidal activity in broadleaf weed species. The objective of these experiments was to determine if specific rates are required to provide synergistic joint activity between mesotrione and atrazine in both triazine-sensitive (TS) and triazine-resistant (TR) redroot pigweed. Herbicide rates were evaluated in TS and TR redroot pigweed in two experiments: a dose response of mesotrione alone and in mixture with a constant rate of atrazine and a dose response of atrazine alone and in mixture with a constant rate of mesotrione. Results from these experiments revealed that synergism was detected in the TS pigweed when 56 g ai ha−1 mesotrione was mixed with 126 g ai ha−1 atrazine. In the TR pigweed, synergism was detected when mesotrione rate at 10 to 56 g ha−1 was mixed with a constant rate of atrazine at 126 g ha−1. Additionally, when mesotrione was held constant at 10 g ha−1, synergism was detected in mixture with atrazine from 31 to 3556 g ha−1 in TR pigweed. Furthermore, in TR pigweed, analysis of slope deviation across the dose-response curves of mesotrione with and without atrazine revealed a divergence that increased in magnitude as the rate of mesotrione increased. In other words, increased synergism was observed with increased mesotrione rate in the TR pigweed, which was also supported by biomass reduction and atrazine-like injury to the leaves. An additional experiment investigated synergism between mesotrione and bromoxynil in both TS and TR pigweed. Mesotrione at 10 g ha−1 was synergistic when paired with bromoxynil from 70 to 210 g ha−1 in both the TS and the TR pigweed.

Genome sequencing of many model systems, including the human, yeast, Arabidopsis, and rice genomes, has been completed. As a complement to genomic techniques, proteomics has emerged to study the composition, structure, function, and interaction of the expressed proteins or proteome in a given cell, tissue, or organism. During the past several years, tremendous progress has been made with proteomic techniques in human and yeast, but progress has been slower in plants due to several factors. Proteomic techniques have been used in plants, especially in agriculturally important crops and weeds, to understand mechanisms of herbicide tolerance and weed resistance. Proteomic techniques, starting from protein extraction to protein identification, have been developed and advanced for large-scale proteomics studies, but limitations and problems for these techniques still exist for plant proteomics. Further technological improvements are needed to enhance quantitative and comparative large-scale proteomics studies. Applications of these techniques may help weed scientists to understand stress tolerance in crops and investigate weediness traits further. Proteomic techniques have unique strengths but also offer several challenges. Together with transcriptomics and metabolomics, these techniques for analyzing global patterns of gene expression offer new and novel techniques for better understanding biological questions of interest to weed scientists in the future.

Chemicals called safeners protect cereal crops from herbicide toxicity. Proteomic methods (2‐D PAGE and LC‐MS/MS) were utilized to identify safener‐ and/or herbicide‐regulated proteins in three tissues (root, leaf, and coleoptile) of Triticum tauschii seedlings to better understand a safener's mechanism of action. Growth experiments showed that the safener cloquintocet‐mexyl protected seedlings from injury by the herbicide dimethenamid. In total, 29 safener‐induced and 10 herbicide‐regulated proteins were identified by LC‐MS/MS. These proteins were classified into two major categories based on their expression patterns, and were further classified into several functional groups. Surprisingly, mutually exclusive sets of proteins were identified following herbicide or safener treatment, suggesting that different signaling pathways may be recruited. Safener‐responsive proteins, mostly involved in xenobiotic detoxification, also included several new proteins that had not been previously identified as safener‐responsive, whereas herbicide‐regulated proteins belonged to several classes involved in general stress responses. Quantitative RT‐PCR revealed that multidrug resistance‐associated protein (MRP) transcripts were highly induced by safeners and two MRP genes were differentially expressed. Our results indicate that safeners protect T. tauschii seedlings from herbicide toxicity by coordinately inducing proteins involved in an entire herbicide detoxification pathway mainly in the coleoptile and root, thereby protecting new leaves from herbicide injury.

The expression of a candidate auxin-responsive gene was evaluated for use in a diagnostic assay for plant growth regulator (PGR) herbicide injury in soybean leaves. Expression ofGH3, a primary auxin-responsive gene, was evaluated in response to dicamba and clopyralid at the RNA and protein levels, and proteomic analysis evaluated global expression of proteins in response to dicamba. Expression ofGH3was also analyzed in response to heat, drought, salt stress, and infection by soybean mosaic virus (SMV) and bean pod mottle virus (BPMV) to determine the specificity ofGH3expression as a diagnostic marker for PGR herbicide injury. At the RNA level,GH3was strongly induced by dicamba and clopyralid within 8 h after application. Expression peaked 1 to 3 d after treatment (DAT) in response to 10% and 1% of a labeled dose of dicamba and clopyralid, with higher expression levels detected at higher herbicide rates. At the protein level, GH3 expression was also strongly induced at 1, 2, and 3 DAT by 10% vs. 1% of a labeled dose of dicamba and clopyralid. Heat, drought, and salt stress and infection with SMV or BPMV had no effect onGH3expression at either the RNA or protein level. Proteomic analysis identified three proteins that were up-regulated in response to dicamba. Two were induced for less than 7 DAT, and a third was identified as a stress-response enzyme (superoxide dismutase) that is likely not specific to PGR herbicide injury. Expression ofGH3was highly induced by PGR herbicides at the RNA and protein level and was not affected by environmental stresses or viral infection, indicating thatGH3expression has excellent potential for use in a diagnostic assay for PGR herbicide injury.

Proteomic methods such as two‐dimensional gel electrophoresis and liquid chromatography tandem mass spectrometry, as well as immunoblotting, were used to identify herbicide safener‐induced proteins in the coleoptile of Triticum tauschii , a diploid wheat containing the D genome also found in the cultivated, hexaploid wheat Triticum aestivum . The herbicide safener fluxofenim dramatically increased protein abundance in the molecular weight ( M _r ) range of 24 to 30 kDa, as well as a few higher M _r proteins, in the coleoptile of T. tauschii seedlings. In total, twenty proteins were identified in this study. Eleven proteins were highly safener induced and only weakly expressed in the control; seven proteins were new safener induced proteins that were not detected in the control. Two other proteins were constitutively expressed in both the control and safener‐treated coleoptiles. Among the eighteen inducible proteins, fifteen were glutathione S ‐transferase (GST) subunits that fall into three subclasses: eight proteins were from the tau subclass, six proteins were from the phi subclass, and one protein was from the lambda class. Another three safener inducible proteins showed homology to the aldo/keto reductase family and with proteins that have roles in glycolysis and the Krebs cycle. Two constitutively expressed proteins were identified, one having highest homology to the dehydroascorbate reductase subclass of GSTs and one with an ascorbate peroxidase. Immunoblot analyses, using two different antisera raised against the same GST protein but differing in their specificity, were used to further characterize the GST proteins expressed in response to safener treatment. Results from immunoblotting, combined with mass spectral analysis, showed that post‐translational modification of GST proteins in control and safener‐treated coleoptiles may occur.

Glutathione S-transferase (GST; EC 2.5.1.18) gene expression was examined in the coleoptile and new leaf tissue of etiolated shoots of the diploid wheat species Triticum tauschii (Coss.) Schmal., which is considered to be a progenitor and the D-genome donor to cultivated, hexaploid bread wheat Triticum aestivum L. GST expression (mRNA, protein, and enzyme activity with a herbicide substrate) in these shoot tissues was examined in response to herbicide safener treatment. Two different antibody probes, raised against the same safener-inducible GST protein (TtGSTU1) but differing in their specificity, were utilized to determine tissue distribution and subcellular localization of GST proteins in etiolated shoots. GST transcripts, immunoreactive GST proteins, and herbicide-metabolizing activity were all highest in the coleoptile of etiolated, safener-treated T. tauschii shoots. Anti-GST immunolabeling was strongest in the outer epidermal and adjoining sub-epidermal cells in both coleoptiles and new leaves following safener treatment. Our data indicate that safeners protect grass crops from herbicide injury by dramatically inducing the expression of GST proteins in the outer cell layers of the coleoptile, which prevents the herbicide from reaching the sensitive new leaves of etiolated shoots as they emerge from the soil.

Glutathione S-transferase (GST) gene expression was examined in several Triticum species, differing in genome constitution and ploidy level, to determine genome contribution to GST expression in cultivated, hexaploid bread wheat (Triticum aestivum). Two tandemly duplicated tau class GST genes (TtGSTU1 and TtGSTU2) were isolated from a single bacterial artificial chromosome clone in a library constructed from the diploid wheat and D genome progenitor to cultivated wheat, Triticum tauschii. The genes are very similar in genomic structure and their encoded proteins are 95% identical. Gene-specific reverse transcriptase-polymerase chain reaction analysis revealed differential transcript accumulation ofTtGSTU1 and TtGSTU2 in roots and shoots. Expression of both genes was induced by herbicide safeners, 2,4-dichlorophenoxyacetic acid and abscisic acid, in the shoots of T. tauschii; however, expression ofTtGSTU1 was always higher than TtGSTU2. In untreated seedlings, TtGSTU1 was expressed in both shoots and roots, whereas TtGSTU2 expression was only detected in roots. RNA gel-blot analysis of ditelosomic, aneuploid lines that are deficient for 6AS, 6BS, or 6DS chromosome arms of cultivated, hexaploid bread wheat showed differential genome contribution to safener-induced GST expression in shoots compared with roots. The GST genes from the D genome of hexaploid wheat contribute most to safener-induced expression in the shoots, whereas GSTs from the B and D genomes contribute to safener-induced expression in the roots.