Herbicidas inibidores da ACCase em plantas de Cenchrus echinatus em estresse hídrico

This project aimed to relate the efficiency of control of ACCase inhibiting herbicides applied post-emergence in Cenchrus echinatus under different soil water contents. The experiments were conducted in a greenhouse, with the application of three different herbicides (fluazifop-p-butyl, haloxyfop-methyl and sethoxydim + oil Assist) and the experimental design for each herbicide was completely randomized design with four replications, consisting a 3 x 4 factorial, with the combination of water management strategies (-0.03, -0.07 and -1.5 MPa) and four doses of these products (100, 50, 25 and 0% of the recommended dose). Herbicide application was made at vegetative stage of 2-3 tillers. The water management strategies were initiated in the development stage of two leaves, replacing the water until the soil reaches the potential of -0.01 MPa, when it came to severe pre-determined for each water management. The physiological parameters evaluated were: photosynthetic rate, stomatal conductance, transpiration, leaf temperature and plant dry matter. The visual assessments of phytotoxicity were performed at 7, 14, 21 and 28 days after application. The efficiency of these herbicides was influenced by soil management and water lowest in plants grown in the minimal potential of water in the soil of -1.5 MPa. All the herbicides were unsatisfactory controls in applications late (2-3 tiller plants).

Herbicide-binding Sites Revealed in the Structure of Plant Acetohydroxyacid Synthase

The sulfonylureas and imidazolinones are potent commercial herbicide families. They are among the most popular choices for farmers worldwide, because they are nontoxic to animals and highly selective. These herbicides inhibit branched-chain amino acid biosynthesis in plants by targeting acetohydroxyacid synthase (AHAS, EC 2.2.1.6). This report describes the 3D structure of Arabidopsis thaliana AHAS in complex with five sulfonylureas (to 2.5 angstrom resolution) and with the imidazolinone, imazaquin (IQ; 2.8 angstrom). Neither class of molecule has a structure that mimics the substrates for the enzyme, but both inhibit by blocking a channel through which access to the active site is gained. The sulfonylureas approach within 5 angstrom of the catalytic center, which is the C2 atom of the cofactor thiamin diphosphate, whereas IQ is at least 7 angstrom from this atom. Ten of the amino acid residues that bind the sulfonylureas also bind IQ. Six additional residues interact only with the sulfonylureas, whereas there are two residues that bind IQ but not the sulfonylureas. Thus, the two classes of inhibitor occupy partially overlapping sites but adopt different modes of binding. The increasing emergence of resistant weeds due to the appearance of mutations that interfere with the inhibition of AHAS is now a worldwide problem. The structures described here provide a rational molecular basis for understanding these mutations, thus allowing more sophisticated AHAS inhibitors to be developed. There is no previously described structure for any plant protein in complex with a commercial herbicide.

Dissecting cross-talk between microglia and motoneurons in ALS: signaling events and soluble factors

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

Chlorophyll a fluorescence in rice plants exposed of herbicides of group imidazolinone

The objective of this work was to investigate the injuries caused to the photosynthetic apparatus of three types of rice exposed to application of imidazolinone group herbicides. Two experiments were conducted using herbicides Imazethapyr+imazapic and Imazapyr+imazapic, in a split-plot experimental design, and a 3 x 3 factorial, with six replications. The first factor (A) consisted of the herbicide rates 0, 100 e 200 g ha-1 of Imazethapyr+imazapic and 0, 140 e 280 g ha-1 of Imazapyr+imazapic; factor B consisted of type of rice (cv. Puitá Inta CL, sensitive red rice ecotype and red rice ecotype with suspected herbicide tolerance to Imidazolinone). Chlorophyll a fluorescence parameters were evaluated in plants at 30 days after herbicide application, using a portable fluorometer (HandyPEA, Hanstech). The photosynthetic metabolism of cv. Puitá Inta CL was found to tolerate commercial dosages of both herbicides. High sensitivity to the herbicides was observed for the sensitive red rice ecotype, while the photosynthetic apparatus of red rice ecotype with suspected herbicide tolerance showed high tolerance to both herbicides applied at rates higher than the commercial rate. The application of chemical herbicides of the imidazolinone group on rice plants causes changes in the photosynthetic metabolism of plants, detected by evaluating the emission of transient chlorophyll a fluorescence. This method can be useful in helping detect resistance and/or tolerance of red rice plants to herbicides of the imidazolinone group.

Resistance of Amaranthus retroflexus to acetolactate synthase inhibitor herbicides in Brazil

When in competition with cotton, Amaranthus retroflexus can cause high yield losses. Due to the limited availability of selective herbicides registered for post emergence control of this weed, the same herbicides have been used repeated times over the last few years, which may have selected resistant biotypes. Biotypes of A. retroflexus collected from the main areas of cotton cultivation in Brazil were submitted to dose-response trials, by applying the herbicides trifloxysulfuron-sodium and pyrithiobac-sodium in doses equivalent to 0, ¼, ½, 1, 2 and 4 times the recommended rates. Resistance to ALS inhibitors was confirmed in biotypes of A. retroflexus. Biotype MS 2 from Mato Grosso do Sul, was cross-resistant to both trifloxysulfuron-sodium and pyrithiobac-sodium, while biotype MS 1 was resistant to trifloxysulfuron-sodium only. Likewise, singular and cross resistance was also confirmed in biotypes from Goiás (GO 3, GO 4 and GO 6), in relation to trifloxysulfuron­sodium and pyrithiobac-sodium. One biotype from Mato Grosso (MT 13) was not resistant to any of the ALS inhibitors evaluated in this work.

Potential of plant species for bioremediation of soils applied with imidazolinone herbicides

Imidazolinone herbicides present physicochemical characteristics that allow them to persist longer in environment, with increased chances of soil and water contamination, as well as carryover effects on subsequent crops. Phytoremediation is shown as a promising technique to decontaminate soils polluted by herbicides. The aim of this study was to assess the potential of some winter grown species in removing residuals from soils contaminated with imazethapyr + imazapic and imazapic + imazapyr, using pre-emergence to control weeds in summer grown rice fields. The experiment was conducted in a completely randomized design, with four replications. All species were subjected to herbicide application at different doses. Imazethapyr + imazapic and imazapyr + imazapic were applied at doses of 0.0, 1.0 and 2.0 L ha-1, and 0.0, 140 and 280 g ha-1, respectively, in pre-emergence of the species. Brassica napus and Festuca arundinaceae are not tolerant to herbicides, with 100% of phytotoxicity (plant death) for all doses assessed. The herbicide imazapyr + imazapic proved to be less selective, causing the highest phytotoxicity in the species tested. The most tolerant species to the herbicides was Vicia sativa, which may be the most suitable one for phytoremediation programs in areas contaminated with imazethapyr + imazapic and imazapyr + imazapic.

Desenvolupament de metodologia analítica per al seguiment d'herbicides fenoxiacètics i cafeïna en el medi ambient

El control d'herbicides i altres anàlits orgànics presents en el medi ambient constitueix una pràctica habitual en els laboratoris des de l'establiment de legislacions que limiten la seva concentració. Per aquesta raó, cal el desenvolupament de noves metodologies analítiques per al seguiment de compostos orgànics en el medi. Molt sovint aquests anàlits es troben a nivells traça en aigües i sòls, conjuntament amb un alt contingut de substàncies húmiques i fúlviques. Així, un dels reptes existents és el tractament de la mostra (extracció, concentració i "clean-up" d'aquests anàlits per a una bona quantificació). Aquests processos han de venir complementats per tècniques cromatogràfiques que permetin la mesura final dels anàlits. La investigació que es presenta en aquesta tesi es centra en el desenvolupament d'un mètode per a la determinació de 2,4-D i MCPA i els seus metabòlits fenòlics i d'un altre per a la determinació de cafeïna. El primer dels procediments desenvolupats s'ha aplicat al seguiment dels herbicides i els metabòlits fenòlics en sòls d'un camp de golf, mentre que el segon s'ha emprat per a la determinació de cafeïna en aigües naturals i, posteriorment, en aigües residuals.

Eucalyptus ESTs associated with resistance to herbicide inhibitors of aromatic and branched-chain amino acid synthesis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Eficiência de herbicidas inibidores da ALS e PROTOX sob condições de déficit hídrico no comportamento bioquímico de plantas daninhas

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Glukose-konjugierte Hemmstoffe als Strategie der gezielten Hemmung des DNA-Reparaturproteins O 6 -Methylguanin-DNA-Methyltransferase in Tumorzellen und der Einfluss von ATP-binding cassette Transportern

Das DNA-Reparaturprotein O6-Methylguanin-DNA-Methyltransferase [MGMT] ist der Hauptresistenzfaktor gegenüber der zytotoxischen Wirkung von SN1-alkylierenden Zytostatika in der Tumortherapie. Die Verwendung der MGMT-Hemmstoffe O6-Benzylguanin [O6BG] und O6-(4-Bromothenyl)guanin [O6BTG] führte zu einer Sensibilisierung des Normalgewebes, was eine Dosis-Reduktion der Zytostatika erforderlich machte und die erhoffte Therapieverbesserung verhinderte. Aus diesem Grund ist eine Strategie der selektiven Hemmung des MGMT-Proteins (Targeting-Strategie) erforderlich, um die systemische Toxizität in der Kombinationsbehandlung zu reduzieren. In dieser Arbeit wurde die Anwendbarkeit der Glukose-Konjugation als Targeting-Strategie untersucht, da Tumorzellen einen erhöhten Glukoseverbrauch aufweisen und demzufolge Glukosetransporter überexprimieren. Die Glukose-Konjugate O6BG-Glu und O6BTG-Glu inhibierten MGMT in Tumorzellen und sensibilisierten die Zellen gegenüber den alkylierenden Agenzien Temozolomid [TMZ] und Lomustin [CCNU]. Des Weiteren inaktivierten die Glukose-Konjugate die MGMT-Aktivität im Tumor eines Xenograft-Mausmodells und reduzierten das Tumorwachstum nach einer TMZ-Behandlung im gleichen Ausmass wie die Inhibitoren O6BG und O6BTG. Trotzdem war auch mit den Glukose-Konjugaten keine Steigerung der Zytostatika-Dosis im Mausmodell möglich. Die Untersuchungen der Aufnahme von O6BG-Glu und O6BTG-Glu wiederlegten eine Involvierung der Glukosetransporter. Der Einsatz von spezifischen Glukosetransporter-Inhibitoren und Kompetitions-Experimenten führte zu keiner Verminderung der MGMT-Hemmung oder Aufnahme vom radioaktiven H3-O6BTG-Glu in die Zelle. Dies legt nahe, dass die Glukose-Konjugate über einen unspezifischen Mechanismus (aktiv) in die Zellen gelangen. Der Grund für eine mögliche unselektive Aufnahme könnte im hydrophoben Alkyllinker, der für die Konjugation des Glukosemoleküls verwendet wurde, begründet sein. Dies führt zur Generierung von amphipathischen Konjugaten, die eine initiale Bindung an die Plasmamembran aufweisen und eine Aufnahme über den Flip-Flop-Mechanismus (transbilayer transport) wahrscheinlich machen. Die amphipathische Molekülstruktur der Glukose-Konjugate führte zu einer Partikelbildung in wässrigen Lösungen, die eine Reduktion der Menge an aktiven Monomeren von O6BG-Glu und O6BTG-Glu bewirken, die zur Hemmung von MGMT zur Verfügung stehen. Der zweite Teil der Arbeit befasste sich mit der Rolle von ABC-Transportern hinsichtlich einer Targeting-Strategie von MGMT-Hemmstoffen. Obwohl eine hohe Expression dieser ABC-Transporter in Tumoren zur Resistenzentwicklung gegenüber Zytostatika führt, wurde ihr Einfluss auf MGMT-Hemmstoffe oder einer MGMT-Targeting-Strategie niemals untersucht. In dieser Arbeit wurde zum ersten Mal ein aktiver Efflux von MGMT-Hemmstoffen durch ABC-Transporter nachgewiesen. Die Inhibition von ABC-Transportern bewirkte eine schnellere Inaktivierung von MGMT durch die Glukose-Konjugate. Des Weiteren zeigten Kompetitions-Experimente mit den MGMT-Hemmstoffen eine verminderte Efflux-Rate von Fluoreszenzfarbstoffen, die spezifisch von ABC-Transportern exportiert werden. ABC-Transporter reduzieren die wirksame Konzentration des Hemmstoffes in der Zelle und beeinträchtigen somit die Effektivität der MGMT-Inhibition. Eine simultane Hemmung der ABC-Transporter P-glycoprotein (P-gp), multi resistance protein 1 (MRP1) and breast cancer resistance protein (BCRP) erhöhte die Effektivität der MGMT-Hemmstoffe (O6BG, O6BTG, O6BG-Glu, O6BTG-Glu) und verstärkte auf diese Weise die TMZ-induzierte Toxizität in Tumorzelllinien. Die Involvierung von ABC-Transportern in der intrazellulären Speicherung von MGMT-Hemmstoffen ist wahrscheinlich die Ursache für die beobachteten Unterschiede in der Sensibilisierung verschiedener Tumorzelllinien gegenüber Zytostatika durch das Glukose-Konjugat O6BG-Glu. Eine Strategie, den Einfluss von ABC-Transportern zu reduzieren und zukünftliche MGMT-Targeting-Strategien effizienter umzusetzen, ist die Verwendung von O6BTG als Ausgangssubstanz. Die höhere Inhibitionsfähigkeit der Bromthiophenmoleküle vermindert die erforderliche intrazelluläre Konzentration für eine vollständige MGMT-Hemmung und reduziert auf diese Weise den Einfluss von ABC-Transportern.

Neuroprotection through excitability and mTOR required in ALS motoneurons to delay disease and extend survival.

Delaying clinical disease onset would greatly reduce neurodegenerative disease burden, but the mechanisms influencing early preclinical progression are poorly understood. Here, we show that in mouse models of familial motoneuron (MN) disease, SOD1 mutants specifically render vulnerable MNs dependent on endogenous neuroprotection signaling involving excitability and mammalian target of rapamycin (mTOR). The most vulnerable low-excitability FF MNs already exhibited evidence of pathology and endogenous neuroprotection recruitment early postnatally. Enhancing MN excitability promoted MN neuroprotection and reversed misfolded SOD1 (misfSOD1) accumulation and MN pathology, whereas reducing MN excitability augmented misfSOD1 accumulation and accelerated disease. Inhibiting metabotropic cholinergic signaling onto MNs reduced ER stress, but enhanced misfSOD1 accumulation and prevented mTOR activation in alpha-MNs. Modulating excitability and/or alpha-MN mTOR activity had comparable effects on the progression rates of motor dysfunction, denervation, and death. Therefore, excitability and mTOR are key endogenous neuroprotection mechanisms in motoneurons to counteract clinically important disease progression in ALS.

Molecular Characterization of Photomixotrophic Tobacco Cells Resistant to Protoporphyrinogen Oxidase-Inhibiting Herbicides1

Peroxidizing herbicides inhibit protoporphyrinogen oxidase (Protox), the last enzyme of the common branch of the chlorophyll- and heme-synthesis pathways. There are two isoenzymes of Protox, one of which is located in the plastid and the other in the mitochondria. Sequence analysis of the cloned Protox cDNAs showed that the deduced amino acid sequences of plastidial and mitochondrial Protox in wild-type cells and in herbicide-resistant YZI-1S cells are the same. The level of plastidial Protox mRNA was the same in both wild-type and YZI-1S cells, whereas the level of mitochondrial Protox mRNA YZI-1S cells was up to 10 times the level of wild-type cells. Wild-type cells were observed by fluorescence microscopy to emit strong autofluorescence from chlorophyll. Only a weak fluorescence signal was observed from chlorophyll in YZI-1S cells grown in the Protox inhibitor N-(4-chloro-2-fluoro-5-propagyloxy)-phenyl-3,4,5,6-tetrahydrophthalimide. Staining with DiOC6 showed no visible difference in the number or strength of fluorescence between wild-type and YZI-1S mitochondria. Electron micrography of YZI-1S cells showed that, in contrast to wild-type cells, the chloroplasts of YZI-1S cells grown in the presence of N-(4-chloro-2-fluoro-5-propagyloxy)-phenyl-3,4,5,6-tetrahydrophthalimide exhibited no grana stacking. These results suggest that the herbicide resistance of YZI-1S cells is due to the overproduction of mitochondrial Protox.

Structure-activity relationships for a new family of sulfonylurea herbicides

Acetohydroxyacid synthase (AHAS; EC 2.2.1.6) catalyzes the first common step in branched-chain amino acid biosynthesis. The enzyme is inhibited by several chemical classes of compounds and this inhibition is the basis of action of the sulfonylurea and imidazolinone herbicides. The commercial sulfonylureas contain a pyrimidine or a triazine ring that is substituted at both meta positions, thus obeying the initial rules proposed by Levitt. Here we assess the activity of 69 monosubstituted sulfonylurea analogs and related compounds as inhibitors of pure recombinant Arabidopsis thaliana AHAS and show that disubstitution is not absolutely essential as exemplified by our novel herbicide, monosulfuron (2-nitro-N-(4'-methyl-pyrimidin-2'-yl) phenyl-sulfonylurea), which has a pyrimidine ring with a single meta substituent. A subset of these compounds was tested for herbicidal activity and it was shown that their effect in vivo correlates well with their potency in vitro as AHAS inhibitors. Three-dimensional quantitative structure-activity relationships were developed using comparative molecular field analysis and comparative molecular similarity indices analysis. For the latter, the best result was obtained when steric, electrostatic, hydrophobic and H-bond acceptor factors were taken into consideration. The resulting fields were mapped on to the published crystal structure of the yeast enzyme and it was shown that the steric and hydrophobic fields are in good agreement with sulfonylurea-AHAS interaction geometry.

Herbicides cross resistance of a multiple resistant short-awn foxtail ( Alopecurus aequalis Sobol.) population in wheat field

Alopecurus aequalis Sobol. is a common grass weed, which has become increasingly troublesome to control in China wheat fields. One A. aequalis population, collected from Anhui Province China, was suspected to be resistant to fenoxaprop-P-ethyl and mesosulfuron-methyl. This study aimed to establish the cross-resistance pattern using the purified subpopulation and explore the potential targetsite and non-target-site based resistance mechanisms. Sequencing results showed that a single nucleotide change of ATT to AAT was present in acetyl-CoA carboxylase (ACCase) gene of the resistant (R) plants, resulting in an Ile2041Asn amino acid substitution. Besides, another single nucleotide change of CCC to CGC was present in acetolactate synthase (ALS) gene of the R plants, resulting in a Pro197Arg amino acid substitution. The homozygous resistant plants were isolated and the seeds were used in whole-plant herbicide bioassays. Compared with the susceptible (S) population, R population displayed high level resistance to fenoxaprop-P-ethyl and mesosulfuronmethyl. Cross resistance patterns showed that the R population was highly resistant to clodinafop-propargyl, moderately resistant to pyroxsulam and flucarbazoncsodium, lowly resistant to pinoxaden, and susceptible to tralkoxydim, sethoxydim, and isoproturon. The pretreatment of piperonyl butoxide reduced the 50% growth reduction (GR50) value of fenoxaprop-P-ethyl, suggesting that target-site resistance and non-target-site resistance mechanisms were both present in fenoxaprop- P-ethyl-resistance of A. aequalis. This is the first report of ACCase Ile2041Asn and ALS Pro197Arg mutation in A. aequalis.

Mitotic crossing-over induced by two commercial herbicides in diploid strains of the fungus Aspergillus nidulans

Some herbicides are suspected of promoting teratogenic, carcinogenic and mutagenic events. Detection of induced mitotic crossing-over has proven to be an indirect way of testing the carcinogenic properties of suspicious substances, because mitotic crossing-over is involved in the multistep process of carcinogenesis. We examined mitotic crossing-over induced by two commercial herbicides (diuron and trifluralin) in diploid strains of Aspergillus nidulans based on the homozygotization index. Low doses (2.5 mu g/mL) of diuron were sufficient to increase the mean homozygotization index in 2.1 and 11.3 times for UT448//UT196 and Dp II-I//UT196, respectively, whereas the same dose of trifluralin increased this mean only 1.2 (UT448//UT196) and 3.5 (Dp II-I//UT196) times, respectively. The lower homozygotization index value found for trifluralin could be due to its interference with mitotic crossing-over in eukaryotic cells. We concluded that the diploid Dp II-I//UT196 of A. nidulans is more sensitive to organic compounds than UT448//UT196; these compounds cause recombinational events at a greater frequency in the latter diploid. This system holds promise as an initial test for carcino-genicity of organic compounds, including herbicides.