Analysis of the Accase Mutation Profile of Italian Ryegrass (Lolium Perenne SSP. Multiflorum) Accessions Resistant to Accase Inhibitors

Lolium perenne ssp. multiflorum (Italian ryegrass) resistant to ACCase inhibiting herbicides has been reported in many wheat producing counties across Arkansas. Resistance is believed to be the result of point mutations creating amino acid substitutions in the CT domain of the plastidic ACCase gene. This study explores the occurrence of mutations in the ACCase gene of ryegrass populations. Plant material was collected and DNA was extracted from 10 Arkansas ryegrass populations. Six of the populations were known to be resistant to the ACCase inhibitor diclofop-methyl, while the remaining four populations were known to be susceptible to diclofop-methyl. Two highly conserved regions of the plastidic ACCase gene known to contain mutations that confer resistance to ACCase inhibiting herbicides were then amplified and sequenced. Analysis of the sequences revealed that only 41% of the resistant populations expressed a mutation known to confer resistance. Several resistant populations of ryegrass did not contain any of the known mutations in their plastidic ACCase gene. This result means that either a mutation in a different region of the CT domain affects the affinity to ACCase inhibiting herbicides or the plants harbor a different mechanism of resistance. Further, in some resistant populations, not all plants within that population possessed a mutation known to cause resistance to ACCase inhibitors. This suggests that within a population, multiple mechanisms of resistance may exist. Further research is needed to determine the mechanism of resistance in diclofop-resistant plants that do not harbor mutations in the tested ACCase herbicide-binding domains

Fitness costs associated with evolved herbicide resistance alleles in plants

Predictions based on evolutionary theory suggest that the adaptive value of evolved herbicide resistance alleles may be compromised by the existence of fitness costs. There have been many studies quantifying the fitness costs associated with novel herbicide resistance alleles, reflecting the importance of fitness costs in determining the evolutionary dynamics of resistance. However, many of these studies have incorrectly defined resistance or used inappropriate plant material and methods to measure fitness. This review has two major objectives. First, to propose a methodological framework that establishes experimental criteria to unequivocally evaluate fitness costs. Second, to present a comprehensive analysis of the literature on fitness costs associated with herbicide resistance alleles. This analysis reveals unquestionable evidence that some herbicide resistance alleles are associated with pleiotropic effects that result in plant fitness costs. Observed costs are evident from herbicide resistance-endowing amino acid substitutions in proteins involved in amino acid, fatty acid, auxin and cellulose biosynthesis, as well as enzymes involved in herbicide metabolism. However, these resistance fitness costs are not universal and their expression depends on particular plant alleles and mutations. The findings of this review are discussed within the context of the plant defence trade-off theory and herbicide resistance evolution

ACCase 6 is the essential acetyl-CoA carboxylase involved in fatty acid and mycolic acid biosynthesis in mycobacteria

Mycolic acids are essential for the survival, virulence and antibiotic resistance of the human pathogen Mycobacterium tuberculosis. Inhibitors of mycolic acid biosynthesis, such as isoniazid and ethionamide, have been used as efficient drugs for the treatment of tuberculosis. However, the increase in cases of multidrug-resistant tuberculosis has prompted a search for new targets and agents that could also affect synthesis of mycolic acids. In mycobacteria, the acyl-CoA carboxylases (ACCases) provide the building blocks for de novo fatty acid biosynthesis by fatty acid synthase (FAS) I and for the elongation of FAS I products by the FAS II complex to produce meromycolic acids. By generating a conditional mutant in the accD6 gene of Mycobacterium smegmatis, we demonstrated that AccD6 is the essential carboxyltransferase component of the ACCase 6 enzyme complex implicated in the biosynthesis of malonyl-CoA, the substrate of the two FAS enzymes of Mycobacterium species. Based on the conserved structure of the AccD5 and AccD6 active sites we screened several inhibitors of AccD5 as potential inhibitors of AccD6 and found that the ligand NCI-172033 was capable of inhibiting AccD6 with an IC50 of 8 ìM. The compound showed bactericidal activity against several pathogenic Mycobacterium species by producing a strong inhibition of both fatty acid and mycolic acid biosynthesis at minimal inhibitory concentrations. Overexpression of accD6 in M. smegmatis conferred resistance to NCI-172033, confirming AccD6 as the main target of the inhibitor. These results define the biological role of a key ACCase in the biosynthesis of membrane and cell envelope fatty acids, and provide a new target, AccD6, for rational development of novel anti-mycobacterial drugsFil: Kurth, Daniel German. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Gago, Gabriela Marisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: de la Iglesia, Agustina Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Bazet Lyonnet, Bernardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Lin, Ting Wang. University of California; Estados UnidosFil: Morbidoni, Héctor Ricardo. Universidad Nacional de Rosario. Facultad de Ciencias Médicas; ArgentinaFil: Tsai, Shiou Chuan. University of California; Estados UnidosFil: Gramajo, Hugo Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentin

Evolved polygenic herbicide resistance in Lolium rigidum by low-dose herbicide selection within standing genetic variation

The interaction between environment and genetic traits under selection is the basis of evolution. In this study, we have investigated the genetic basis of herbicide resistance in a highly characterized initially herbicide-susceptible Lolium rigidum population recurrently selected with low (below recommended label) doses of the herbicide diclofop-methyl. We report the variability in herbicide resistance levels observed in F1 families and the segregation of resistance observed in F2 and back-cross (BC) families. The selected herbicide resistance phenotypic trait(s) appear to be under complex polygenic control. The estimation of the effective minimum number of genes (NE), depending on the herbicide dose used, reveals at least three resistance genes had been enriched. A joint scaling test indicates that an additive-dominance model best explains gene interactions in parental, F1, F2 and BC families. The Mendelian study of six F2 and two BC segregating families confirmed involvement of more than one resistance gene. Cross-pollinated L. rigidum under selection at low herbicide dose can rapidly evolve polygenic broad-spectrum herbicide resistance by quantitative accumulation of additive genes of small effect. This can be minimized by using herbicides at the recommended dose which causes high mortality acting outside the normal range of phenotypic variation for herbicide susceptibility

Transcriptome profiling of a spirodiclofen susceptible and resistant strain of the European red mite Panonychus ulmi using strand-specific RNA-seq

Background: The European red mite, Panonychus ulmi, is among the most important mite pests in fruit orchards, where it is controlled primarily by acaricide application. However, the species rapidly develops pesticide resistance, and the elucidation of resistance mechanisms for P. ulmi has not kept pace with insects or with the closely related spider mite Tetranychus urticae. The main reason for this lack of knowledge has been the absence of genomic resources needed to investigate the molecular biology of resistance mechanisms. Results: Here, we provide a comprehensive strand-specific RNA-seq based transcriptome resource for P. ulmi derived from strains susceptible and resistant to the widely used acaricide spirodiclofen. From a de novo assembly of the P. ulmi transcriptome, we manually annotated detoxification enzyme families, target-sites of commonly used acaricides, and horizontally transferred genes implicated in plant-mite interactions and pesticide resistance. In a comparative analysis that incorporated sequences available for Panonychus citri, T. urticae, and insects, we identified radiations for detoxification gene families following the divergence of Panonychus and Tetranychus genera. Finally, we used the replicated RNA-seq data from the spirodiclofen susceptible and resistant strains to describe gene expression changes associated with resistance. A cytochrome P450 monooxygenase, as well as multiple carboxylcholinesterases, were differentially expressed between the susceptible and resistant strains, and provide a molecular entry point for understanding resistance to spirodiclofen, widely used to control P. ulmi populations. Conclusions: The new genomic resources and data that we present in this study for P. ulmi will substantially facilitate molecular studies of underlying mechanisms involved in acaricide resistance

Glyphosate resistance in annual ryegrass (Lolium rigidum Gaud.) with multiple resistance mechanisms.

Glyphosate (N-(phosphonomethyl)glycine) is a post-emergent, systemic and non-selective herbicide for the control of annual and perennial weeds. This herbicide has very low toxicity to the mammals. The target enzyme for glyphosate in plants is 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Glyphosate inhibits the biosynthesis of the aromatic amino acids phenylalanine, tyrosine and tryptophan in the plant. The first case of glyphosate resistance was reported in Lolium rigidum in Australia after 15 years of persistence use of this herbicide and the number weeds reported resistant to glyphosate has increased around the world. So far, two mechanisms known to be involved in resistance to glyphosate are target-site mutation and reduced herbicide translocation. Recently, two populations of L. rigidum from Australia have been discovered with very high levels of resistance to glyphosate. This project aims to determine the levels of glyphosate resistance in these populations, investigate glyphosate resistance mechanisms in the populations and finally assess the mode of inheritance of resistance. In this project, four resistant (NLR70, SLR77, SLR80 and SLR88) and one susceptible (VLR1) L. rigidum populations were evaluated for their response to glyphosate. From the dose response experiments, the susceptible population of VLR1 was completely controlled with the recommended rate of glyphosate (450 g a.e ha⁻ ¹). In contrast, the resistant populations were not fully controlled by this herbicide rate. There was considerable variation between the populations in their resistance to glyphosate. In comparison to the susceptible population VLR1, SLR77 was 2.2 to 3.5 fold resistant to glyphosate, NLR70 was 3.7 to 8.4 fold resistant to glyphosate, SLR88 was 5.6 to 11.4 fold resistant to glyphosate and SLR80 was 8.2 to 76.7 fold resistant to glyphosate. The mechanism of glyphosate resistance in the populations was investigated. ¹⁴ C-glyphosate was used to determine the absorption and translocation of glyphosate among the populations. There was no significant difference on the absorption of ¹⁴ C-glyphosate 48 hours after treatment in the population. However, the accumulation of ¹⁴ C-glyphosate in the stem region was higher in the susceptible VLR1 population (25.9%) and in resistant SLR77 (25%) than the other three populations. The resistant populations NLR70, SLR88 and SLR80 had about half the amount of glyphosate accumulating in the stem region. These three resistant populations appear to be resistant to glyphosate as a result of reduced translocation of glyphosate to the shoot meristem. Part of the EPSP synthase gene of the susceptible and four resistant populations was amplified and sequenced to identify any changes in the nucleotide sequence. The predicted amino acid sequence from the susceptible population VLR1 was the same as the consensus sequence from other plant species in the conserved region sequenced. However, the resistant populations of NLR70, SLR77, SLR80 and SLR88 showed polymorphisms within the nucleotide sequence in this region. Single nucleotide substitutions of A for C at codon 106 were observed in the resistant populations SLR77 and SLR80. This nucleotide change is predicted to substitute threonine for proline at position 106. In the resistant population SLR88, a nucleotide substitution of T for C was observed at the same codon. This nucleotide substitution is predicted to change the amino acid from proline 106 to serine. Therefore, these three populations appear to be resistant to glyphosate as a result of a target-site mutation. An inheritance study was conducted by cross pollinating the susceptible VLR1 and resistant SLR88 population. From the dose response, the parent susceptible was completely killed with the recommended rate of glyphosate and higher rates of glyphosate were required to control parental resistant and both F₁ progenies (maternal susceptible and resistant). Both F₁ progenies showed an intermediate response to glyphosate compared with the parental populations. This indicated that the resistance to glyphosate in population SLR88 is inherited by nuclear gene(s) through the transfer of pollen during the cross pollination. It is suggested that SLR88 and SLR80 population contain both glyphosate resistant mechanisms due to the cross pollination between individuals with different resistant mechanisms. Having two resistant mechanisms results in populations being highly resistant to glyphosate compared to those with one resistance mechanism. The higher level of glyphosate resistance in these multiple glyphosate resistance populations will likely make them harder to manage.Thesis (M.Ag.Sc.) -- University of Adelaide, School of Agriculture, Food and Wine, 201

Underlying Resistance Mechanisms in the Cynosurus echinatus Biotype to Acetyl CoA Carboxylase-Inhibiting Herbicides

Hedgehog dogtail (Cynosurus echinatus) is an annual grass, native to Europe, but also widely distributed in North and South America, South Africa and Australia. Two hedgehog dogtail biotypes, one diclofop-methyl (DM)-resistant and one DM-susceptible were studied in detail for experimental dose-response resistance mechanisms. Herbicide rates that inhibited shoot growth by 50% (GR50) were determined for DM, being the resistance factor (GR50R/GR50S) of 43.81. When amitrole (Cyt. P450 inhibitor) was applied before treatment with DM, the R biotype growth was significantly inhibited (GR50 of 1019.9 g ai ha-1) compared with the GR50 (1484.6 g ai ha-1) found for the R biotype without pretreatment with amitrole. However, GR50 values for S biotype do not vary with or without amitrole pretreatment. Dose-response experiments carried out to evaluate cross-resistance, showed resistance to aryloxyphenoxypropionate (APP), cyclohexanodione (CHD) and phenylpyrazoline (PPZ) inhibiting herbicides. Both R and S biotypes had a similar 14C-DM uptake and translocation. The herbicide was poorly distributed among leaves, the rest of the shoot and roots with unappreciable acropetal and/or basipetal DM translocation at 96 HAT. The metabolism of 14C-DM, D-acid and D-conjugate metabolites were identified by thin-layer chromatography. The results showed that DM resistance in C. echinatus is likely due to enhanced herbicide metabolism, involving Cyt. P450 as was demonstrated by indirect assays (amitrole pretreatment). The ACCase in vitro assays showed that the target site was very sensitive to APP, CHD and PPZ herbicides in the C. echinatus S biotype, while the R biotype was insensitive to the previously mentioned herbicides. DNA sequencing studies confirmed that C. echinatus cross-resistance to ACCase inhibitors has been conferred by specific ACCase double point mutations Ile-2041-Asn and Cys-2088-Arg

Distribution, detection and genetic background of herbicide-resistant Alopecurus myosuroides (Huds.) in Germany

Weed control is an important part in agricultural practice. Since selective herbicides were introduced, the labour-intensive mechanical weed control was replaced by chemicals. The use of chemicals for weed control has become increasingly problematic due to the evolution of herbicide-resistant weeds. In Germany, Alopecurus myosuroides (Huds.) is one of the most problematic weeds concerning herbicide resistance. The first resistant black-grass biotype in Germany was found in 1982. More than 30 years after the first resistant black-grass was found in Germany (1982), there are still numerous unsolved questions and challenges concerning the problem of herbicide resistance. Further knowledge about the distribution, the detection, and the genetic background of different resistance mechanisms is needed to find comprehensive solutions for the future. Knowledge about the occurrence and distribution of herbicide-resistant black-grass in Germany, and the herbicides primarily affected may provide more detailed information for farmers to quickly react on upcoming resistances. Moreover, if the genetic background of resistance is better understood, practical conclusions regarding the choice of herbicides and management tools can be drawn. Furthermore, a reliable and easy-to-handle test system for the detection of resistances would enable farmers to react faster and in a more targeted manner. The aim of the present study was to investigate these aspects addressing herbicide-resistant black-grass in Germany. How widespread is TSR in Germany? Did the amount of TSR change over the years? Are there TSR-hot spots in Germany? The first paper addressed ACCase and ALS-resistant black-grass samples in Germany. It dealt with the distribution and development of TSR in Germany over a period of nine years. It could be demonstrated that TSR was more widespread than expected. The frequency of ACCase-TSR increased from 4.0% in 2004 to 38.5% in 2012. ALS-TSR rose from 0.8% in 2007 to 13.9% in 2012. Both TSRs significantly increased over time within a standing number of seed samples over the years. How many genes are involved in NTSR? Are there differences between the plants and can we detect cross-resistances? The second paper dealt with the inheritance of NTSR conferring resistance to chlorotoluron, fenoxaprop-P-ethyl, pinoxaden, mesosulfuron + iodosulfuron, and flufenacet in six different black-grass plants. Segregation analyses of the quantitative trait showed a minimum of five loci conferring specific resistances. The resistances against chlorotoluron and fenoxaprop-P-ethyl were mostly conferred by one locus, whereas resistances against pinoxaden and mesosulfuron + iodosulfuron were mostly conferred by a minimum of two loci. A minimum of one to three loci explained resistance to flufenacet. The accumulation of resistance loci in individual plants could be achieved by the study. Furthermore, the number of loci was shown to be herbicide- and plant-specific which further confirms the complexity of NTSR. How is it possible to test pre-emergence herbicides in black-grass? Which test system is the most reliable? The aim of the third paper was to find a reliable test system to monitor pre-emergence herbicide resistance in black-grass. It is widely known that diverse sites of action can be affected by NTSR. Moreover, resistance against pre-emergence herbicides belonging to the HRAC groups N, K3, and C2 occur. The outcomes of the study indicate that a soil-based greenhouse test with pre-germinated seeds is most suitable for resistance detection. Discriminating herbicide rates which were able to distinguish between the resistant and susceptible black-grass biotypes were found for all of the herbicides tested. This enables a reliable, quick, and easy way to identify pre-emergence resistance. In conclusion, herbicide-resistant black-grass has become an increasing problem in Germany. The high frequency of nearly 40% ACCase TSR on resistance suspected sites highlights the importance of changes in agricultural practices. The aim should be to avoid the repeated use of single site of action herbicides in short term crop-rotations with large quantities of winter cereals. The accumulation of NTSR loci in single plants increases the risk of biotypes with broad resistances against many different modes of action. Resistance linkages were found to be plant-specific which may result in unpredictable resistance situations in the field. Even pre-emergence herbicides can be affected by NTSR. An option to detect these resistances is provided by a soil-based greenhouse bioassay with pre-germinated seeds.Unkrautregulierung ist einer der wichtigsten Aspekte im Ackerbau. Seit der Einführung selektiver Herbizide wurde die intensive Handarbeit durch chemische Unkrautbekämpfung ersetzt. Mittlerweile ist der verstärkte Herbizideinsatz zunehmend problematisch, da sich herbizid-resistente Unkräuter entwickeln. In Deutschland gilt der Ackerfuchsschwanz (Alopecurus myosuroides Huds.) als eines der problematischsten Unkräuter hinsichtlich der Herbizidresistenz. Obwohl seitdem dem ersten Resistenzfund im Jahr 1982 mehr als 30 Jahre vergangen sind, bestehen in der Wissenschaft noch viele ungelöste Fragen bezüglich Herbizidresistenz. Zusätzliches Wissen über die Verbreitung, die Erkennung und den genetischen Hintergrund verschiedener Resistenzmechanismen ist nötig, um umfassende Lösungsansätze für die Zukunft zu entwickeln. Die Kenntnis über das Auftreten und die Verbreitung von herbizid-resistentem Ackerfuchsschwanz in Deutschland und darüber, welche Herbizide am häufigsten betroffen sind, kann Landwirten zielgerichtet Informationen geben, um schnell auf aufkommende Resistenzen zu reagieren. Weiterhin kann ein besseres Verständnis des genetischen Hintergrunds von Resistenz zu praktischen Schlussfolgerungen für die Herbizidwahl benutzt werden. Außerdem kann ein zuverlässiges und einfach zu handhabendes Testsystem zur Resistenzerkennung den Landwirten eine schnelle und zielgerichtete Reaktion ermöglichen. Das Ziel der vorliegenden Arbeit war es, diese verschiedenen Aspekte bezüglich herbizid-resistenten Ackerfuchsschwanz in Deutschland näher zu beleuchten. Wie verbreitet ist Zielortresistenz in Deutschland? Veränderte sich der Anteil von Zielortresistenz in den letzten Jahren? Existieren Orte mit besonders hohem Zielortresistenzanteil? Die erste Publikation untersuchte ACCase und ALS-resistenten Ackerfuchsschwanzproben in Deutschland und betrachtete die Verbreitung und Entwicklung von TSR über neun Jahre in Deutschland. Zielortresistenz zeigte sich als weiter verbreitet als vermutet. Die Häufigkeiten von ACCase-TSR stieg von 4,0% im Jahr 2004 auf 38,5% im Jahr 2012. ALS-TSR stieg von 0,8% im Jahr 2007 auf 13,9% im Jahr 2012. Beide TSR Anteile stiegen über die Jahre signifikant an. Wie viele Genorte sind an Nicht-Zielortresistenz (NTSR) beteiligt? Bestehen Unterschiede zwischen Pflanzen und gibt es Kreuz-Resistenzen? Die weite Publikation handelte von der Vererbung von NTSR gegen Chlortoluron, Fenoxaprop-P-Ethyl, Pinoxaden, Mesosulfuron + Iodosulfuron und Flufenacet in sechs verschiedenen Ackerfuchsschwanzpflanzen. Die Aufspaltungsverhältnisse des quantitativen Merkmals zeigten ein Minimum von fünf Loci, die herbizid-spezifische Resistenzen bewirken. Resistenzen gegenüber Chlortoluron und Fenoxaprop-P-ethyl wurden meistens durch einen Genort bedingt, während an den Resistenzen gegenüber Pinoxaden und Mesosulfuron + Iodosulfuron meistens mindestens zwei Genorte beteiligt waren. Mindestens drei Genorte zeigten sich an der Resistenz gegenüber Flufenacet bei den meisten der untersuchten Pflanzen beteiligt. Die Anreicherung von Resistenz-Genorten in einzelnen Pflanzen konnte in der Studie gezeigt werden. Weiter zeigte sich die Anzahl Genorte als pflanzen-spezifisch, was die Komplexität von NTSR bekräftigt. Wie kann Resistenz gegen Vorauflaufherbizide bei Ackerfuchsschwanz getestet werden? Welches ist das geeignetste Testsystem? Das Ziel der dritten Veröffentlichung war es, ein verlässliches Testsystem für Resistenz-Monitorings von Vorauflaufherbiziden bei Ackerfuchsschwanz zu finden. Es ist bekannt, dass NTSR verschiedene Wirkmechanismen betreffen kann. Auch Resistenzen gegenüber Vorauflaufherbiziden, die zu den HRAC Gruppen C2, N und K3 gehören, treten auf. Die Ergebnisse der dritten Untersuchung zeigen, dass ein Gewächshaustest mit Boden und vorgekeimten Samen eine geeignete Methode ist, um diese Resistenzen nachzuweisen. Kritische Dosierungen für die Unterscheidung zwischen resistenten und sensitiven Ackerfuchsschwanzbiotypen konnten für alle getesteten Herbizide gefunden werden. Dies ermöglicht eine verlässliche, schnelle und praktikable Durchführung von Resistenztests. Zusammenfassend ergibt sich, dass herbizid-resistenter Ackerfuchsschwanz in Deutschland ein zunehmendes Problem darstellt. Der hohe Anteil von fast 40% ACCase-TSR bei Resistenzverdachtsfällen hebt die Wichtigkeit von Veränderungen in der ackerbaulichen Praxis deutlich hervor. Die Anreicherung von NTSR Loci in Einzelpflanzen erhöht das Risiko für Biotypen mit breiten Resistenzen gegen viele verschiedene Wirkstoffe. Pflanzenspezifische Resistenzverknüpfungen wurden gefunden, wodurch nicht vorhersagbare Resistenzsituationen auf den Feldern möglich sind. Sogar Vorauflaufherbizide können durch NTSR unwirksam werden. Eine Möglichkeit, diese Resistenzen zu überprüfen ist durch einen Gewächshaustest mit Boden und vorgekeimten Samen für die geprüften Wirkstoffe gegeben

Experimental methods for estimation of plant fitness costs associated with herbicide-resistance genes

Since the beginning of agriculture, crops have been exposed to recurrent invasion by weeds that can impose severe reductions in crop quality and yield. There have been continuing efforts to reduce the impacts of weeds on production. More than 40 yr ago, overreliance on herbicide technology to reduce weed infestations resulted in the selection of adaptive traits that enabled weed survival and reproduction under herbicide treatments. As a result, herbicide resistance in > 200 weed species has evolved worldwide.Fil: Vila Aiub, Martin Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Fisiológicas y Ecológicas Vinculadas a la Agricultura; ArgentinaFil: Gundel, Pedro Emilio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Fisiológicas y Ecológicas Vinculadas a la Agricultura; ArgentinaFil: Preston, Christopher. University of Adelaide. School of Agriculture, Food and Wine; Australi

Improving Production of Malonyl Coenzyme A-Derived Metabolites by Abolishing Snf1-Dependent Regulation of Acc1

Acetyl coenzyme A (acetyl-CoA) carboxylase (ACCase) plays a central role in carbon metabolism and has been the site of action for the development of therapeutics or herbicides, as its product, malonyl-CoA, is a precursor for production of fatty acids and other compounds. Control of Acc1 activity in the yeast Saccharomyces cerevisiae occurs mainly at two levels, i.e., regulation of transcription and repression by Snf1 protein kinase at the protein level. Here, we demonstrate a strategy for improving the activity of ACCase in S. cerevisiae by abolishing posttranslational regulation of Acc1 via site-directed mutagenesis. It was found that introduction of two site mutations in Acc1, Ser659 and Ser1157, resulted in an enhanced activity of Acc1 and increased total fatty acid content. As Snf1 regulation of Acc1 is particularly active under glucose-limited conditions, we evaluated the effect of the two site mutations in chemostat cultures. Finally, we showed that our modifications of Acc1 could enhance the supply of malonyl-CoA and therefore successfully increase the production of two industrially important products derived from malonyl-CoA, fatty acid ethyl esters and 3-hydroxypropionic acid. IMPORTANCE ACCase is responsible for carboxylation of acetyl-CoA to produce malonyl-CoA, which is a crucial step in the control of fatty acid metabolism. ACCase opened the door for pharmaceutical treatments of obesity and diabetes as well as the development of new herbicides. ACCase is also recognized as a promising target for developing cell factories, as its malonyl-CoA product serves as a universal precursor for a variety of high-value compounds in white biotechnology. Yeast ACCase is a good model in understanding the enzyme's catalysis, regulation, and inhibition. The present study describes the importance of protein phosphorylation in regulation of yeast ACCase and identifies potential regulation sites. This study led to the generation of a more efficient ACCase, which was applied in the production of two high-value compounds derived from malonyl-CoA, i.e., fatty acid ethyl esters that can be used as biodiesel and 3-hydroxypropionic acid that is considered an important platform chemical