Determining nitrogen isotopes discrimination under drought stress on enzymatic activities, nitrogen isotope abundance and water contents of Kentucky bluegrass.

Drought stress is the most pervasive threat to plant growth, which predominantly encumbers turf grass growth by causing alterations in plant functions. This study appraised the role of nitrogen isotopes in providing a theoretical basis for developing and improving Kentucky bluegrass cultivar performance under drought stress. Nitrogen isotopes labelled 15NH4Cl and K15NO3 were prepared to replace KNO3 in Hoagland's solution at concentrations of 15NH4+ and 15NO3 at 1.5, 15, and 30 mM; the solutions were imposed on stressed plants under glasshouse conditions. Nitrogenous nutrition reduced oxidative stress by elevating the enzymatic activities and proline contents of all three clonal ramet leaves, particularly under stress conditions. Apart from nitrogen content, nitrogen isotope abundance, relative water content and water potential within controls were enhanced in treated with 15NH4+ than in with 15NO3 in both the roots and leaves of Kentucky bluegrass. Nevertheless, an application of 15NH4Cl and K15NO3 at 30 mM had a positive influence to some extent on these attributes under drought stress. Overall, our results suggested that nitrogen isotopes contributed to drought tolerance in all three clonal ramets of Kentucky bluegrass by maintaining a better osmoprotectant and antioxidant defence system, which helped the plants eliminate reactive oxygen species.

Molecular characteristics of the first case of haloxyfop-resistant Poa annua.

Haloxyfop is one of two acetyl-coenzyme A carboxylase (ACCase) inhibitors that is recommended for controlling Poa annua. We have characterised a population of P. annua that had developed resistance to haloxyfop. This resistant population was found to be almost 20 times less sensitive to haloxyfop than a susceptible population based on percentage survival of individuals in two dose-response experiments. However, the haloxyfop-resistant population was still susceptible to clethodim. Pre-treatment of resistant individuals with a cytochrome P450 inhibitor, malathion, did not change the sensitivity level of the resistant plants to haloxyfop, suggesting that a non-target site mechanism of resistance involving enhanced metabolism, was not responsible for this resistance in P. annua. Gene sequencing showed that a target site mutation at position 2041, which replaced isoleucine with threonine in the carboxyltransferase (CT) domain of the ACCase enzyme, was associated with resistance to haloxyfop in the resistant population. An evaluation of the 3-D structure of the CT domain suggested that, unlike Asn-2041, which is the most common mutation at this position reported to date, Thr-2041 does not change the conformational structure of the CT domain. This is the first study investigating the molecular mechanism involved with haloxyfop resistance in P. annua.

EPSPS duplication and mutation involved in glyphosate resistance in the allotetraploid weed species Poa annua L.

BACKGROUND: Poa annua is a widespread winter annual weed species in California. Recently, poor control of this species with glyphosate was reported by growers in an almond orchard in California with a history of repetitive glyphosate use. The objectives of this research were to evaluate the level of glyphosate resistance in a developed S4 P. annua line (R) and identify the mechanisms of resistance involved. RESULTS: Whole-plant dose-response experiments confirmed glyphosate resistance in R, which required 18-fold more glyphosate to achieve a 50% growth reduction compared with a susceptible line (S), results that were supported by the lower shikimate accumulation observed in R compared with S. No differences in glyphosate absorption, translocation, or metabolism were observed, suggesting that non-target-site mechanisms of resistance are not involved in the resistance phenotype. A missense single nucleotide polymorphism was observed in EPSPS coding position 106 in R, resulting in a leucine to proline substitution. This polymorphism was observed exclusively in P. supina EPSPS homeologs. A seven-fold increase in the number of copies of EPSPS alleles was observed in R compared with S. CONCLUSIONS: We report the first case of glyphosate resistance associated with both EPSPS duplication and target-site mutation at position 106, leading to high levels of glyphosate resistance in the allotetraploid weed species Poa annua L. Data obtained in this research will be useful for the development of diagnostic tools for rapid glyphosate resistance identification, monitoring and containment. © 2018 Society of Chemical Industry.

A new amino acid substitution (Ala-205-Phe) in acetolactate synthase (ALS) confers broad spectrum resistance to ALS-inhibiting herbicides.

MAIN CONCLUSION: This is a first report of an Ala-205-Phe substitution in acetolactate synthase conferring resistance to imidazolinone, sulfonylurea, triazolopyrimidines, sulfonylamino-carbonyl-triazolinones, and pyrimidinyl (thio) benzoate herbicides. Resistance to acetolactate synthase (ALS) and photosystem II inhibiting herbicides was confirmed in a population of allotetraploid annual bluegrass (Poa annua L.; POAAN-R3) selected from golf course turf in Tennessee. Genetic sequencing revealed that seven of eight POAAN-R3 plants had a point mutation in the psbA gene resulting in a known Ser-264-Gly substitution on the D1 protein. Whole plant testing confirmed that this substitution conferred resistance to simazine in POAAN-R3. Two homeologous forms of the ALS gene (ALSa and ALSb) were detected and expressed in all POAAN-R3 plants sequenced. The seven plants possessing the Ser-264-Gly mutation conferring resistance to simazine also had a homozygous Ala-205-Phe substitution on ALSb, caused by two nucleic acid substitutions in one codon. In vitro ALS activity assays with recombinant protein and whole plant testing confirmed that this Ala-205-Phe substitution conferred resistance to imidazolinone, sulfonylurea, triazolopyrimidines, sulfonylamino-carbonyl- triazolinones, and pyrimidinyl (thio) benzoate herbicides. This is the first report of Ala-205-Phe mutation conferring wide spectrum resistance to ALS inhibiting herbicides.

Protective effect of spermidine on salt stress induced oxidative damage in two Kentucky bluegrass (Poa pratensis L.) cultivars.

To improve the salinity tolerance of turfgrass and investigate the effect of spermidine (Spd) on antioxidant metabolism and gene expression under salinity stress condition, exogenous Spd was applied before two kentucky bluegrass (Poa pratensis L.) cultivars ('Kenblue' and 'Midnight') were exposed to 200 mM sodium chloride (NaCl) stress for 28 d. Salinity stress decreased the turfgrass quality, increased the content of malonyldialdehyde (MDA), superoxide anion (O2(·-)) and hydrogen peroxide (H2O2), and enhanced activities of superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (POD) and ascorbate peroxidase (APX) and isozymes intensity in both cultivars. In addition, the expression level of Cu/ZnSOD was down-regulated in 'Kenblue' but up-regulated in 'Midnight' after salt treatment. Salinity stress also enhanced the expression of APX but inhibited the expression of CAT and POD in both cultivars. Exogenous Spd treatment alleviated the salinity-induced oxidative stress through decreasing MDA, H2O2 and O2(·-) contents in both cultivars. Besides, exogenous Spd further enhanced the activities of SOD, CAT, POD and APX accompanied with the increased intensity of specific isozymes of SOD, CAT and APX in both cultivars and POD in 'Kenblue'. Moreover, Spd further up-regulated expression levels of Cu/ZnSOD and APX, but down-regulated those of CAT and POD in both cultivars. These results indicated that exogenous Spd might improve turfgrass quality and promote the salinity tolerance in the two cultivars of kentucky bluegrass through reducing oxidative damages and increasing enzyme activity both at protein and transcriptional levels.

Indaziflam herbicidal action: a potent cellulose biosynthesis inhibitor.

Cellulose biosynthesis is a common feature of land plants. Therefore, cellulose biosynthesis inhibitors (CBIs) have a potentially broad-acting herbicidal mode of action and are also useful tools in decoding fundamental aspects of cellulose biosynthesis. Here, we characterize the herbicide indaziflam as a CBI and provide insight into its inhibitory mechanism. Indaziflam-treated seedlings exhibited the CBI-like symptomologies of radial swelling and ectopic lignification. Furthermore, indaziflam inhibited the production of cellulose within <1 h of treatment and in a dose-dependent manner. Unlike the CBI isoxaben, indaziflam had strong CBI activity in both a monocotylonous plant (Poa annua) and a dicotyledonous plant (Arabidopsis [Arabidopsis thaliana]). Arabidopsis mutants resistant to known CBIs isoxaben or quinoxyphen were not cross resistant to indaziflam, suggesting a different molecular target for indaziflam. To explore this further, we monitored the distribution and mobility of fluorescently labeled CELLULOSE SYNTHASE A (CESA) proteins in living cells of Arabidopsis during indaziflam exposure. Indaziflam caused a reduction in the velocity of YELLOW FLUORESCENT PROTEIN:CESA6 particles at the plasma membrane focal plane compared with controls. Microtubule morphology and motility were not altered after indaziflam treatment. In the hypocotyl expansion zone, indaziflam caused an atypical increase in the density of plasma membrane-localized CESA particles. Interestingly, this was accompanied by a cellulose synthase interacting1-independent reduction in the normal coincidence rate between microtubules and CESA particles. As a CBI, for which there is little evidence of evolved weed resistance, indaziflam represents an important addition to the action mechanisms available for weed management.

Resistance to ACCase-inhibiting herbicides in an Asia minor bluegrass (Polypogon fugax) population in China.

Asia minor bluegrass (Polypogon fugax) is a common annual grass weed of winter crops distributed across China. We conducted a study on the resistance level and the mechanism of resistance to ACCase-inhibiting herbicides in a P. fugax population from China. Whole-plant dose-response experiments in greenhouse showed that the resistant P. fugax population was 1991, 364, 269, 157, and 8-fold resistant to clodinafop-propargyl, fluazifop-p-butyl, haloxyfop-R-methyl, quizalofop-p-ethyl and fenoxaprop-p-ethyl relative to the reference susceptible population, which was susceptible to all the five AOPP herbicides. Much lower R/S values of 3.5, 2.4 and 3.5, respectively, were detected for clethodim, sethoxydim and pinoxaden. Molecular analysis of resistance confirmed that the Ile2041 to Asn mutation in the resistant population conferred resistance to AOPP herbicides, but not to CHD and DEN herbicides. This is the first report of a target site mutation that corresponded to resistance to AOPP herbicides in P. fugax. Proper resistance management practices are necessary to prevent ACCase-inhibiting herbicides from becoming ineffective over wide areas.

The role of cytochrome P450 monooxygenase in the different responses to fenoxaprop-P-ethyl in annual bluegrass (Poa annua L.) and short awned foxtail (Alopecurus aequalis Sobol.).

Herbicide resistance or tolerance in weeds mediated by cytochrome P450 monooxygenase is a considerable problem. However, cytochrome P450 mediated resistance or tolerance in weeds was less studied. Thus, in this work, the role of the cytochrome P450 monooxygenase in the different responses of Poa annua and Alopecurus aequalis to fenoxaprop-P-ethyl was studied. We found that the effect of fenoxaprop-P-ethyl could be synergized by piperonyl butoxide (PBO) in P. annua, but not by malathion. After being treated with fenoxaprop-P-ethyl (containing mefenpyr-diethyl), the contents of cytochrome P450 and cytochrome b5 in P. annua increased significantly compared to plants treated with mefenpyr-diethyl only or untreated plants. However, the increase was less in A. aequalis, which was susceptible to fenoxaprop-P-ethyl. The activities of ρ-nitroanisole O-demethylase (PNOD), ethoxyresorufin O-deethylase (EROD), ethoxycoumarin oxidase (ECOD) and NADPH-dependent cytochrome P450 reductase mediated by cytochrome P450 monooxygenase increased in P. annua after treatment with fenoxaprop-P-ethyl, especially the activities of ECOD and cytochrome P450 reductase. Besides this, cytochrome P450 monooxygenase activity toward fenoxaprop-P-ethyl in P. annua increased significantly compared to untreated or treated with mefenpyr-diethyl plants and treated or untreated A. aequalis. Cytochrome P450 monooxygenase may play an important role in the different responses to fenoxaprop-P-ethyl in P. annua and A. aequalis.

Origin and timing of the horizontal transfer of a PgiC gene from Poa to Festuca ovina.

A segregating second locus, PgiC2, for the enzyme phosphoglucose isomerase (PGIC) is found in the grass sheep's fescue, Festuca ovina. We have earlier reported that a phylogenetic analysis indicates that PgiC2 has been horizontally transferred from the reproductively separated grass genus Poa. Here we extend our analysis to include intron and exon information on 27 PgiC sequences from 18 species representing five genera, and confirm our earlier finding. The origin of PgiC2 can be traced to a group of closely interrelated, polyploid and partially asexual Poa species. The sequence most similar to PgiC2 is found in Poa palustris with a divergence, based on synonymous substitutions, of only 0.67%. This value suggests that the transfer took place less than 600,000 years ago (late Pleistocene), at a time when most extant Poa and Festuca species already existed.

Use of plant cell cultures to study graminicide effects on lipid metabolism.

Graminicides belonging to the cyclohexanedione and aryloxyphenoxypropionate classes are well established to act by disrupting acyl lipid biosynthesis via specific inhibition of acetyl-CoA carboxylase. Species of grass inherently resistant to such herbicides, or biotypes of grassy weed species which display acquired resistance to recommended rates of graminicide application, are known to possess an altered plastidic multifunctional acetyl-CoA carboxylase showing reduced sensitivity to these herbicides in vitro. Studies reported here demonstrate that cell suspension cultures of maize, a graminicide-sensitive species and Poa annua, a graminicide-insensitive species, display a similar differential sensitivity of acyl lipid biosynthesis as tissue from corresponding intact plants. Acyl lipid biosynthesis in P. annua can be inhibited if sufficiently high concentrations of graminicide are used. The major plastidic form and the minor cytosolic forms of acetyl-CoA carboxylase were successfully purified from maize cell suspensions, were compared to those from leaf tissue and were shown to be differentially inhibited by graminicides in a similar manner to their counterparts from leaf tissue. These studies demonstrate that cell suspensions are useful for studying the mode of action of graminicides, especially in view of the limited amount of material obtainable from many grassy species which are very fine-growing.