Acetolactate Synthase Activity Research Articles

Distinctive physiological and molecular responses of foxtail millet and maize to nicosulfuron.

Nicosulfuron is the leading acetolactate synthase inhibitor herbicide product, and widely used to control gramineous weeds. Here, we investigated the metabolic process of nicosulfuron into foxtail millet and maize, in order to clarify the mechanism of the difference in sensitivity of foxtail millet and maize to nicosulfuron from the perspective of physiological metabolism and provide a theoretical basis for the breeding of nicosulfuron-resistant foxtail millet varieties. We treated foxtail millet (Zhangzagu 10, Jingu 21) and maize (Nongda 108, Ditian 8) with various doses of nicosulfuron in both pot and field experiments. The malonaldehyde (MDA) content, target enzymes, detoxification enzymes, and antioxidant enzymes, as well as related gene expression levels in the leaf tissues of foxtail millet and maize were measured, and the yield was determined after maturity. The results showed that the recommended dose of nicosulfuron caused Zhangzagu 10 and Jingu 21 to fail to harvest; the yield of the sensitive maize variety (Ditian 8) decreased by 37.09%, whereas that of the resistant maize variety (Nongda 108) did not decrease. Nicosulfuron stress increased the CYP450 enzyme activity, MDA content, and antioxidant enzyme activity of foxtail millet and maize, reduced the acetolactate synthase (ALS) activity and ALS gene expression of foxtail millet and Ditian 8, and reduced the glutathione S-transferase (GST) activity and GST gene expression of foxtail millet. In conclusion, target enzymes, detoxification enzymes, and antioxidant enzymes were involved in the detoxification metabolism of nicosulfuron in plants. ALS and GST are the main factors responsible for the metabolic differences among foxtail millet, sensitive maize varieties, and resistant maize varieties. These findings offer valuable insights for exploring the target resistance (TSR) and non-target resistance (NTSR) mechanisms in foxtail millet under herbicide stress and provides theoretical basis for future research of develop foxtail millet germplasm with diverse herbicide resistance traits.

Effect of Plant Growth-Promoting Bacteria on Antioxidant Status, Acetolactate Synthase Activity, and Growth of Common Wheat and Canola Exposed to Metsulfuron-Methyl.

Metsulfuron-methyl, a widely used herbicide, could cause damage to the sensitive plants in crop-rotation systems at extremely low levels in the soil. The potential of plant growth-promoting bacteria (PGPB) for enhancing the resistance of plants against herbicide stress has been discovered recently. Therefore, it is poorly understood how physiological processes occur in plants, while PGPB reduce the phytotoxicity of herbicides for agricultural crops. In greenhouse studies, the effect of strains Pseudomonas protegens DA1.2 and Pseudomonas chlororaphis 4CH on oxidative damage, acetolactate synthase (ALS), enzymatic and non-enzymatic antioxidants in canola (Brassica napus L.), and wheat (Triticum aestivum L.) were investigated under two levels (0.05 and 0.25 mg∙kg-1) of metsulfuron-methyl using spectrophotometric assays. The inoculation of herbicide-exposed wheat with bacteria significantly increased the shoots fresh weight (24-28%), amount of glutathione GSH (60-73%), and flavonoids (5-14%), as well as activity of ascorbate peroxidase (129-140%), superoxide dismutase SOD (35-49%), and ALS (50-57%). Bacterial treatment stimulated the activity of SOD (37-94%), ALS (65-73%), glutathione reductase (19-20%), and the accumulation of GSH (61-261%), flavonoids (17-22%), and shoots weight (27-33%) in herbicide-exposed canola. Simultaneous inoculation prevented lipid peroxidation induced by metsulfuron-methyl in sensitive plants. Based on the findings, it is possible that the protective role of bacterial strains against metsulfuron-metil is linked to antioxidant system activation.

Molecular characterization of acetolactate synthase genes in lentil (Lens culinaris Medik.): A key target enzyme of imazethapyr herbicide resistance

Weed infestation severely affects the productivity of lentils. Imazethapyr, a Group 2 herbicide controls prominent broadleaf weeds and grasses in the field. Imazethapyr blocks the first step of branched-chain amino acids (BCAA) biosynthesis by inhibiting the activity of acetolactate synthase (ALS) activity. The present study aims to explore the effect of herbicide stress on the ALS gene expression and enzyme activity in lentil genotypes on different days after spray. The study revealed the presence of two homologous genes in the lentil genome, LcALS2 and LcALS3, present on chromosomes 2 and 3, respectively. qRT-PCR of the LcALS2 gene in the selected herbicide-tolerant (LL1397 and LL1612) and susceptible (FLIP 2004-7 L and PL07) genotypes indicated a significant decrease of the ALS gene expression in the susceptible genotypes. The results were also supported by the in vitro ALS enzyme activity assay, indicating significant inhibition of ALS activity in FLIP 2004-7 L (17.33% and 49.21%) followed by PL07 (33.11% and 19.7%) at 4 and 7 days after spray (DAS), respectively compared to the control. This is the first-ever report on the effect of herbicide stress on ALS gene expression and its correlation with ALS enzyme activity on different days after spraying in lentil crop. It is important from an agronomic perspective to understand herbicide tolerance mechanism at the genetic level for selecting an efficient variety and improving crop productivity.

Optimized prime editing efficiently generates heritable mutations in maize.

Low efficiency is the main obstacle to using prime editing in maize (Zea mays). Recently, prime-editing efficiency was greatly improved in mammalian cells and rice (Oryza sativa) plants by engineering prime-editing guide RNAs (pegRNAs), optimizing the prime editor (PE) protein, and manipulating cellular determinants of prime editing. In this study, we tested PEs optimized via these three strategies in maize. We demonstrated that the ePE5max system, composed of PEmax, epegRNAs (pegRNA-evopreQ. 1), nicking single guide RNAs (sgRNAs), and MLH1dn, efficiently generated heritable mutations that conferred resistance to herbicides that inhibit 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), acetolactate synthase (ALS), or acetyl CoA carboxylase (ACCase) activity. Collectively, we demonstrate that the ePE5max system has sufficient efficiency to generate heritable (homozygous or heterozygous) mutations in maize target genes and that the main obstacle to using PEs in maize has thus been removed.

Target-site and metabolic mechanisms of tolerance to penoxsulam in pond lovegrass (Eragrostis japonica)

AbstractThe identification of herbicide tolerance is essential for effective chemical weed control. According to whole-plant dose–response assays, none of 29 pond lovegrass [Eragrostis japonica (Thunb.) Trin.] populations were sensitive to penoxsulam. The effective dose values of penoxsulam causing 50% inhibition of fresh weight (GR50: 105.14 to 148.78 g ai ha−1) in E. japonica populations were much higher than the label rate of penoxsulam (15 to 30 g ai ha−1) in the field. This confirmed that E. japonica was tolerant to penoxsulam. Eragrostis japonica populations showed 52.83- to 74.76-fold higher tolerance to penoxsulam than susceptible barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.]. The mechanisms of tolerance to penoxsulam in E. japonica were also identified. In vitro activity assays revealed that the penoxsulam concentration required to inhibit 50% of the acetolactate synthase (ALS) activity (IC50) was 12.27-fold higher in E. japonica than in E. crus-galli. However, differences in the ALS gene, previously found to endow target-site resistance in weeds, were not detected in the sequences obtained. Additionally, the expression level of genes encoding ALS in E. japonica was approximately 2-fold higher than in E. crus-galli after penoxsulam treatment. Furthermore, penoxsulam tolerance can be significantly reversed by three cytochrome P450 monooxygenase (CytP450) inhibitors (1-aminobenzotriazole, piperonyl butoxide, and malathion), and the activity of NADPH-dependent cytochrome P450 reductase toward penoxsulam in E. japonica increased significantly (approximately 7-fold higher) compared with that of treated E. crus-galli. Taken together, these results indicate that lower ALS sensitivity, relatively higher ALS expression levels, and stronger metabolism of CytP450s combined to bring about penoxsulam tolerance in E. japonica.

Application of an Enzymatic Hydrolysed L-α-Amino Acid Based Biostimulant to Improve Sunflower Tolerance to Imazamox.

Herbicides, commonly used in agriculture to control weeds, often cause negative effects on crops. Safeners are applied to reduce the damage to crops without affecting the effectiveness of herbicides against weeds. Plant biostimulants have the potential to increase tolerance to a series of abiotic stresses, but very limited information exists about their effects on herbicide-stressed plants. This study aims to verify whether the application of a potential safener such as Terra-Sorb®, an L-α-amino acid-based biostimulant, reduces the phytotoxicity of an Imazamox-based herbicide and to elucidate which tolerance mechanisms are induced. Sunflower plants were treated with Pulsar® 40 (4% Imazamox) both alone and in combination with Terra-Sorb®. Plants treated with the herbicide in combination with Terra-Sorb® showed higher growth, increased acetolactate synthase (ALS) activity, and amino acid concentration with respect to the plants treated with Imazamox alone. Moreover, the biostimulant protected photosynthetic activity and reduced oxidative stress. This protective effect could be due to the glutathione S-transferase (GST) induction and antioxidant systems dependent on glutathione (GSH). However, no effect of the biostimulant application was observed regarding phenolic compound phenylalanine ammonium-lyase (PAL) activity. Therefore, this study opens the perspective of using Terra-Sorb® in protecting sunflower plants against an imazamox-based herbicide effect.

Behaviour of bispyribac sodium in soil and its impact on biochemical constituents of rice

ABSTRACT The present study evaluates the dissipation behaviour of bispyribac sodium in acidic soils and its effect on biochemical constituents of rice. Dissipation kinetics followed first-order kinetics and >75% of applied bispyribac sodium dissipated within 7 days after application. Half life of bispyribac sodium ranged from 4.06 to 13.93 and 3.19 to 11.95 days in 2016 and 2017, respectively at studied application rates. The amount of bispyribac-sodium adsorbed increased with increase in concentration of herbicide and log KF of bispyribac-sodium was 1.528 µg1−1/n g−1 ml1/n in studied silty clay loam soil. Terminal residues in rice grain were below the maximum residue limit (<0.01). Acetolactate synthase activity and total carotenoids decreases significantly as compared to control up to 30 days after bispyribac sodium application and thereafter it recovered with the passage of time. Total chlorophyll and total sugar content in rice leaves increased significantly and maximum increase was observed when treated with bispyribac sodium at 20 g ha−1. Quality parameters, i.e. total protein and total sugars were not significantly influenced by application of bispyribac sodium. The use of bispyribac sodium at studied application rate in rice crop is safe both in terms of crop quality and food safety.

Microemulsions of tribenuron-methyl using Pluronic F127: Physico-chemical characterization and efficiency on wheat weed

Microemulsions have received particular interest as novel vehicles for drug delivery. Herein, a new tribenuron-methyl (TM) loaded oil-in-water Pluronic F127-stabilized microemulsion is developed as a promising delivery nanocarrier for herbicides to control the growth of wheat weed (Convolvulus arvensis). The diameter for the TM-loading microemulsions was determined to be 7.5 nm. Experimental results revealed a high entrapment (83% ± 6%) and loading efficiency (0.44% ± 0.07%) of TM in F127-stabilized microemulsions. The in-vitro release profile from the microemulsions ensured a controlled and sustained release of the loaded TM, with ca. 50% release of TM was achieved from the microemulsions within the first 35 h, in comparison to a complete release of free TM within the first 5 h. After 5 weeks, TM-microemulsions were able to reduce the fresh weight (76%), dry weight (55%), acetolactate synthase activity (83%), and plant height (79%) of C. arvensis in comparison with controls. Using a herbicide concentration as low as ca. 50% [15 g of active ingredient per hectare,g a.i. ha−1)], of the recommended dose of commercial herbicides (Granstar) great inhibitions of weed growth were obtained. Therefore, the present microemulsion system opens the door to new nanocarriers for effective weed control to reduce economic costs and potential pollution issues.

Target-site mutation and enhanced metabolism confer resistance to thifensulfuron-methyl in a multiple-resistant redroot pigweed (Amaranthus retroflexus) population

AbstractRedroot pigweed (Amaranthus retroflexus L.) is a troublesome dicot weed species widely distributed across China. A population of A. retroflexus that survived the recommended label rate of thifensulfuron-methyl was collected from the main soybean [Glycine max (L.) Merr.] production area in China. Whole-plant dose–response assays indicated that the resistant (R) population was highly resistant (61.80-fold) to thifensulfuron-methyl compared with the susceptible (S1 and S2) populations. In vitro acetolactate synthase (ALS) activity experiments showed that the thifensulfuron-methyl I50 value for the R population was 40.17 times higher than that for the S1 population. A preliminary malathion treatment study indicated that the R population might have cytochrome P450–mediated metabolic resistance. The R population exhibited a high level of cross-resistance to representative ALS herbicides (imazethapyr, flumetsulam, and bispyribac-sodium) and multiple resistance to the commonly used protoporphyrinogen oxidase (PPO)-inhibiting herbicides lactofen and fomesafen. Two common mutations, Trp-574-Leu in ALS and Arg-128-Gly in PPO2, were identified within the R population. This study identified possible enhanced metabolism of thifensulfuron-methyl coexisting with target-site mutations in both ALS and PPO2 in a multiple-resistant A. retroflexus population.

Evolving Multiple Resistance to EPSPS, GS, ALS, PSI, PPO, and Synthetic Auxin Herbicides in Dominican Republic Parthenium hysterophorus Populations. A Physiological and Biochemical Study

Two Parthenium hysterophorus populations resistant (R) and susceptible (S) harvested in banana crop from the Dominican Republic were studied. All S plants died when the herbicides were applied at field dose, except with paraquat. For the R population, the order of plant survival was as follows: glyphosate and paraquat &gt; flazasulfuron &gt; glufosinate &gt; fomesafen &gt; 2,4-D. The resistance factors obtained in the dose–response assays showed a high resistance to glyphosate, flazasulfuron, and fomesafen, medium resistance to glufosinate and 2,4-D, and a natural tolerance to paraquat (resistance factor (RF) = 1.0). The I50 values obtained in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), acetolactate synthase (ALS), and glutamine synthetase (GS) activity studies with glyphosate, flazasulfuron, and glufosinate, respectively, were greater in R than in S. The effect of fomesafen was measured by the Proto IX levels, obtaining five times more Proto IX in the S than in the R population. The resistance to 2,4-D in the R was determined by the lower accumulation of ethylene compared to the S population. The studies with 14C-paraquat conclude that the lower absorption and translocation in both the R and S populations would explain the natural tolerance of P. hysterophorus. This is the first case of multiple resistance to herbicides with different mechanisms of action confirmed in P. hysterophorus.

Dipteryx alata, a tree native to the Brazilian Cerrado, is sensitive to the herbicide nicosulfuron.

The expansion of land use for agricultural interests and the excessive use of herbicides are among the causes of biodiversity losses in the Brazilian Cerrado biome. Therefore, we aimed to test the hypothesis that Dipteryx alata Vogel, a common species in this biome, is sensitive to nicosulfuron because of its high phytotoxicity. We evaluated physiological, biochemical and morphological responses in D. alata plants exposed to increasing doses of the herbicide. Young plants were transplanted to 10 L pots containing substrate composed of soil and sand (2:1) after fertilization. After an acclimation period, the following doses of nicosulfuron were applied: 0 (control), 6, 12, 24, 48, and 60 g a.e. ha-1. The experiment was conducted in a randomized block design factorial scheme with six doses of nicosulfuron, three evaluation times, and five replicates per treatment. The effects of the herbicide were assessed by measuring gas exchange, chlorophyll a fluorescence, photosynthetic pigments, membrane permeability, antioxidant enzymes and acetolactate synthase. Nicosulfuron altered the photosynthetic machinery and enzymatic metabolism of D. alata. Reductions in physiological traits, increased catalase and ascorbate peroxidase activities, enhanced malondialdehyde concentrations rate of electrolyte leakage and decreased acetolactate synthase activity in response to nicosulfuron all suggest that D. alata is sensitive to this herbicide.

First report of molecular basis of resistance to imazethapyr in common lambsquarters (Chenopodium album)

Abstract Common lambsquarters (Chenopodium album L.) is one of the most troublesome weeds in soybean [Glycine max (L.) Merr.] and corn (Zea mays L.) fields in northeast China. In 2017, a C. album population that survived imazethapyr at the recommended field rate was collected from a soybean field in Heilongjiang Province in China. Experiments were conducted to determine the basis of resistance to imazethapyr and investigate the herbicide-resistance pattern in C. album. Dose–response tests showed that the resistant population (R) displayed high resistance to imazethapyr (20-fold) compared with the susceptible population (S). An in vitro acetolactate synthase (ALS) activity assay indicated that the ALS of the R population was resistant to imazethapyr compared with the ALS of the S population. Sequence analysis of the ALS gene revealed that the GCA was replaced by ACA at amino acid position 122, which resulted in an alanine to threonine substitution (Ala-122-Thr) in the R population. The R population displayed cross-resistance to thifensulfuron-methyl and flumetsulam but susceptibility to bispyribac-sodium, flucarbazone, glyphosate, mesotrione, and fomesafen. These results confirmed that the basis of imazethapyr resistance in C. album was conferred by the Ala-122-Thr substitution in the ALS enzyme. This is the first report of the target-site basis of ALS-inhibiting herbicide resistance in C. album.

Alleviation of chlorimuron-ethyl toxicity to soybean by branched-chain amino acids or naphthalic anhydride

Soybean seeds, soaked in branched-chain amino acids [Valine, Isoleucine and Leucine (VIL)] mixture or dressed in naphthalic anhydride (NA), were germinated for 10 days and treated with chlorimuron-ethyl for 120 h. Chlorimuron-ethyl significantly inhibited acetolactate synthase (ALS) activity in shoots of all samples during the first 72 h. Moreover, it increased Km but decreased Vmax, Kcat, Kcat/Km and Vmax/Km as well as the endogenous levels of valine, isoleucine and leucine particularly during the first 72 h. In concomitance, there were significant decreases in protein content and activities of nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), glutamate synthase (GOGAT) as well as growth parameters during the first 72 h. However, VIL or NA mitigated the herbicide effect. So, the drop in Vmax, Kcat and Kcat/Km by chlorimuron-ethyl declares decreases in enzyme concentration, catalytic rate and catalytic efficiency, respectively. Whereas the increase in Km could indicate that chlorimuron-ethyl induced an inhibition for ALS of the mixed-type which would affect synthesis and structural integrity of the enzyme. This inhibition would diminish the formation of branched-chain amino acids and protein, particularly with the inhibition of N incorporation enzyme (NR, NiR, GS and GOGAT). Consequently, retardation and disturbances in formation of structural and functional protein would arise. Nonetheless, VIL mixture overcame chlorimuron-ethyl toxicity via compensating the drop in the endogenous branched-chain amino acids while NA might cause mitigation by detoxifying the herbicide.

Nicosulfuron-resistant Amaranthus retroflexus L. in Northeast China

Amaranthus retroflexus L. is a troublesome broadleaf weed in autumn crop fields in China. Farmers complain that A. retroflexus cannot be controlled by nicosulfuron at the recommended field rate in maize fields in Heilongjiang Province. The aim of this study was to determine the molecular basis of nicosulfuron resistance in A. retroflexus. Whole-plant response assays revealed that two resistant populations (R1 and R2) exhibited resistance (14.50- and 44.24-fold) to nicosulfuron. In vitro acetolactate synthase (ALS) activity assays indicated that the nicosulfuron I50 values for R1 and R2 populations were 8.12 and 22.32 times higher than that for the susceptible population (S), respectively. Sequence analysis of ALS showed amino acid mutations Ser-653-Asn in R1 and Trp-574-Leu in R2. These two target-site mutations seem to play a primary role in A. retroflexus resistance to nicosulfuron, and this is the first time that the Ser-653-Asn mutation has been reported in this species.

Effect of Metsulfuron Methyl on Acetolactate Synthase Activity and Protein Content of Wheat

Results of field studies laid out during Rabi 2013–14 and 2014–15 in randomized block design to study the effect of metsulfuron methyl applied at 2, 4, 8 g ha−1 on biochemical attributes viz. acetolactate synthase activity and total soluble protein content of wheat crop are reported. Wheat plant samples were collected at 0 (2 h), 1, 3, 5, 7, 10, 15, 30, 45, 60, 75 and 90 d after herbicide application and at harvest for biochemical analysis. A significant decrease was observed in acetolactate synthase activity and total soluble protein upto 10 d after herbicide application. Quality indices i.e. protein and gluten content of wheat grain increased significantly with the metsulfuron methyl treatments. It has been concluded that the herbicide has a non-significant effect on the biochemical attributes of wheat.

The response of weedy sunflower (Helianthus annuus L.) to nicosulfuron: An examination of vegetative parameters and acetolactate synthase activity

Genetic and morphological interpopulation variability of weed species is often responsible for variable responses to herbicides. As weedy sunflower, an invasive form of Helianthus annuus L., possesses high morphological and genetic variability, very different responses of its populations to herbicides can be expected. This species is one of the dominant weed species in row crops, including maize, in many European countries in which nicosulfuron is intensively used for weed control. There are little available data about the response of this sunflower form to nicosulfuron or of the interpopulation variability of its response to other herbicides. The responses of three weedy sunflower populations to nicosulfuron were studied in field dose-response experiments, and acetolactate synthase (ALS) enzyme activity at different herbicide concentrations was determined in vitro. Interpopulation variability in the response to nicosulfuron was confirmed. Populations WS2 and WS3 were more that 20-fold and 30-fold less susceptible to nicosulfuron, respectively, than population WS1, based on fresh weight, whereas the differences were not so prominent based on other parameters, including plant height, leaf area and ALS activity, and ranged from 2 to 12-fold.

Effects of ZJ0273 on barley and growth recovery of herbicide-stressed seedlings through application of branched-chain amino acids.

In this study, we evaluated the effect of the herbicide propyl 4-(2-(4,6-dimethoxypyrimidin-2-yloxy)benzylamino) benzoate (ZJ0273) on barley growth and explored the potential to trigger growth recovery through the application of branched-chain amino acids (BCAAs). Barley plants were foliar-sprayed with various concentrations of ZJ0273 (100, 500, or 1000 mg/L) at the four-leaf stage. Increasing either the herbicide concentration or measurement time after herbicide treatment significantly impaired plant morphological parameters such as plant height and biomass, and affected physiological indexes, i.e. maximal photochemical efficiency (Fv/Fm), quantum yield of photosystem II (ФPSII), net photosynthetic rate (Pn), and chlorophyll meter value (soil and plant analyzer development (SPAD)). Cellular injury of herbicide-treated plants was also evidenced by increased levels of reactive oxygen species (ROS) and antioxidative enzyme activity. Elevated levels of herbicide significantly reduced the activity of acetolactate synthase (ALS)-a key enzyme in the biosynthesis of BCAAs. In a separate experiment, growth recovery in herbicide-stressed barley plants was studied using various concentrations of BCAAs (10, 50, 100, and 200 mg/L). Increasing BCAA concentration in growth media significantly increased the biomass of herbicide-stressed barley seedlings, but had no significant effect on non-stressed plants. Further, BCAAs (100 mg/L) significantly down-regulated ROS and consequently antioxidant enzyme levels in herbicide-stressed plants. Our results showed that exogenous application of BCAAs could reverse the inhibitory effects of ZJ0273 by restoring protein biosynthesis in barley seedlings.

Multiple Resistance Evolution in Bipyridylium-Resistant Epilobium ciliatum After Recurrent Selection.

The use of herbicides with different modes of action is the primary strategy used to control weeds possessing resistance to a single mechanism of action (MOA). However, this practice can lead to selection for generalist resistance mechanisms and may cause resistance to all MOAs. In this research, we characterized the resistance to diquat/paraquat (bipyridiliums) in an Epilobium ciliatum biotype (R1) collected in an olive orchard from Chile, where alternatives herbicides (2,4-D, glyphosate, glufosinate, flazasulfuron and pyraflufen-ethyl) with different MOAs were used, but they have also showed failure in controlling this species. Because the resistance/susceptibility patterns of the R1 biotype to glufosinate, 2,4-D and pyraflufen-ethyl were not clear, a recurrent resistance selection was carried out in field and greenhouse using these herbicides on R1 plants for three generations (R2 biotype). One biotype that was never treated with herbicides (S) was included as control. Results indicated that the S biotype was controlled at the field dose of all herbicides tested. The biotype R1 exhibited resistance to diquat, paraquat and flazasulfuron and natural tolerance to glyphosate. The R2 biotype displayed resistance to glufosinate, 2,4-D and pyraflufen-ethyl with LD50 (herbicide dose to kill 50% of plants) values higher than field doses in all assays. Physiological and biochemical studies determined the resistance to diquat of the R1 biotype, which was due to impaired translocation. The resistance to flazasulfuron in the R1 and R2 biotypes was confirmed by the low sensitivity of the acetolactate synthase (ALS) activity compared to the S biotype. The similar accumulation of shikimate in treated S, R1, and R2 plants with glyphosate supported the existence of innate tolerance to this herbicide in E. ciliatum. Resistance to glufosinate, 2,4-D and pyraflufen-ethyl in the R2 biotype, acquired after recurrent selection, was determined by low sensitivity of the glutamine synthetase, low accumulation of ethylene and protoporphyrinogen IX oxidase, respectively, in comparison to the S biotype. Epilobium ciliatum from Chilean olive orchards had resistance to only two MAOs (photosystem I and ALS inhibitors), but resistance to five MOAs could occur in the next cropping seasons, if alternatives to weed management, other than herbicides, are not included.

Expression and biochemical characterization of α-ketoglutarate decarboxylase from cyanobacterium Synechococcus sp. PCC7002

α-Ketoglutarate decarboxylase (α-KGD), one member of α-keto acid decarboxylases, catalyzing non-oxidative decarboxylation of α-ketoglutarate to form succinic semialdehyde, was proposed to play critical role in completing tricarboxylic acid (TCA) cycle of cyanobacteria. Although the catalytic function of α-KGD from Synechococcus sp. PCC7002 was demonstrated previously, there was no detailed biochemical characterization of α-KGD from Synechococcus sp. PCC7002 yet. In this study, the gene encoding α-KGD from Synechococcus sp. PCC7002 was amplified and soluble expression of recombinant α-KGD was achieved by coexpressing with pTf16 chaperone plasmid in E. coli BL21 (DE3). Kinetic analysis showed that the activity of α-KGD was dependent on cofactors of thiamine pyrophosphate and divalent cation. Meanwhile this α-KGD was specific for α-ketoglutarate with respect to the decarboxylation activity despite of the pretty low activity of acetolactate synthase. The catalytic efficiency of α-KGD (the values of kcat and kcat/Km for α-ketoglutarate were 1.2s−1 and 6.3×103M−1s−1, respectively) might provide evidence for its physiological role in TCA cycle of Synechococcus sp. PCC7002.

Effects of monosulfuron-ester on metabolic processes of nitrogen-fixing cyanobacteria Anabaena flos-aquae and Anabaena azotica.

Presence of the relatively new sulfonylurea herbicide monosulfuron-ester at 0.03–300 nmol/L affected the growth of two non-target nitrogen-fixing cyanobacteria (Anabaena flos-aquae and Anabaena azotica) and substantially inhibited in vitro Acetolactate synthase activity, with IC50 of 3.3 and 101.3 nmol/L for A. flos-aquae and A. azotica, respectively. Presenting in 30–300 nmol/L, it inhibited protein synthesis of the cyanobacteria with less amino acids produced as its concentration increased. Our findings support the view that monosulfuron-ester toxicity in both nitrogen-fixing cyanobacteria is due to its interference with protein metabolism via inhibition of branch-chain amino acid biosynthesis, and particularly Acetolactate synthase activity.