Design, Synthesis, and Herbicidal Evaluation of Pyrrolidinone-Containing 2-Phenylpyridine Derivatives as Novel Protoporphyrinogen Oxidase Inhibitors.

In this work, a series of pyrrolidinone-containing 2-phenylpyridine derivatives were synthesized and evaluated as novel protoporphyrinogen IX oxidase (PPO, EC 1.3.3.4) inhibitors for herbicide development. At 150 g ai/ha, compounds 4d, 4f, and 4l can inhibit the grassy weeds of Echinochloa crus-galli (EC), Digitaria sanguinalis (DS), and Lolium perenne (LP) with a range of 60 to 90%. Remarkably, at 9.375 g ai/ha, these compounds showed 100% inhibition effects against broadleaf weeds of Amaranthus retroflexus (AR) and Abutilon theophrasti (AT), which were comparable to the performance of the commercial herbicides flumioxazin (FLU) and saflufenacil (SAF) and better than that of acifluorfen (ACI). Molecular docking analyses revealed significant hydrogen bonding and π-π stacking interactions between compounds 4d and 4l with Arg98, Asn67, and Phe392, respectively. Additionally, representative compounds were chosen for in vivo assessment of PPO inhibitory activity, with compounds 4d, 4f, and 4l demonstrating excellent inhibitory effects. Notably, compounds 4d and 4l induced the accumulation of reactive oxygen species (ROS) and a reduction in the chlorophyll (Chl) content. Consequently, compounds 4d, 4f, and 4l are promising lead candidates for the development of novel PPO herbicides.

NOL-mediated functional stay-green traits in perennial ryegrass (Lolium perenne L.) involving multifaceted molecular factors and metabolic pathways regulating leaf senescence.

Loss of chlorophyll (Chl) is a hallmark of leaf senescence, which may be regulated by Chl catabolic genes, including NON-YELLOW COLORING 1 (NYC1)-like (NOL). The objective of this study was to determine molecular factors and metabolic pathways underlying NOL regulation of leaf senescence in perennial grass species. LpNOL was cloned from perennial ryegrass (Lolium perenne L.) and found to be highly expressed in senescent leaves. Transient overexpression of LpNOL accelerated leaf senescence and Chl b degradation in Nicotiana benthamiana. LpNOL RNA interference (NOLi) in perennial ryegrass not only significantly blocked Chl degradation in senescent leaves, but also delayed initiation and progression of leaf senescence. This study found that NOL, in addition to functioning as a Chl b reductase, could enact the functional stay-green phenotype in perennial grass species, as manifested by increased photosynthetic activities in NOLi plants. Comparative transcriptomic analysis revealed that NOL-mediated functional stay-green in perennial ryegrass was mainly achieved through the modulation of Chl catabolism, light harvesting for photosynthesis, photorespiration, cytochrome respiration, carbohydrate catabolism, oxidative detoxification, and abscisic acid biosynthesis and signaling pathways.

Host apoplastic cysteine protease activity is suppressed during the mutualistic association of Lolium perenne and Epichloë festucae.

Plants secrete various defence-related proteins into the apoplast, including proteases. Papain-like cysteine proteases (PLCPs) are central components of the plant immune system. To overcome plant immunity and successfully colonize their hosts, several plant pathogens secrete effector proteins inhibiting plant PLCPs. We hypothesized that not only pathogens, but also mutualistic microorganisms interfere with PLCP-meditated plant defences to maintain endophytic colonization with their hosts. Epichloë festucae forms mutualistic associations with cool season grasses and produces a range of secondary metabolites that protect the host against herbivores. In this study, we performed a genome-wide identification of Lolium perenne PLCPs, analysed their evolutionary relationship, and classified them into nine PLCP subfamilies. Using activity-based protein profiling, we identified four active PLCPs in the apoplast of L. perenne leaves that are inhibited during endophyte interactions. We characterized the L. perenne cystatin LpCys1 for its inhibitory capacity against ryegrass PLCPs. LpCys1 abundance is not altered during the mutualistic interaction and it mainly inhibits LpCP2. However, since the activity of other L. perenne PLCPs is not sensitive to LpCys1, we propose that additional inhibitors, likely of fungal origin, are involved in the suppression of apoplastic PLCPs during E. festucae infection.

Cytochrome P450 CYP81A10v7 in Lolium rigidum confers metabolic resistance to herbicides across at least five modes of action.

Rapid and widespread evolution of multiple herbicide resistance in global weed species endowed by increased capacity to metabolize (degrade) herbicides (metabolic resistance) is a great threat to herbicide sustainability and global food production. Metabolic resistance in the economically damaging crop weed species Lolium rigidum is well known but a molecular understanding has been lacking. We purified a metabolic resistant (R) subset from a field evolved R L. rigidum population. The R, the herbicide susceptible (S) and derived F2 populations were used for candidate herbicide resistance gene discovery by RNA sequencing. A P450 gene CYP81A10v7 was identified with higher expression in R vs. S plants. Transgenic rice overexpressing this Lolium CYP81A10v7 gene became highly resistant to acetyl-coenzyme A carboxylase- and acetolactate synthase-inhibiting herbicides (diclofop-methyl, tralkoxydim, chlorsulfuron) and moderately resistant to hydroxyphenylpyruvate dioxygenase-inhibiting herbicide (mesotrione), photosystem II-inhibiting herbicides (atrazine and chlorotoluron) and the tubulin-inhibiting herbicide trifluralin. This wide cross-resistance profile to many dissimilar herbicides in CYP81A10v7 transgenic rice generally reflects what is evident in the R L. rigidum. This report clearly showed that a single P450 gene in a cross-pollinated weed species L. rigidum confers resistance to herbicides of at least five modes of action across seven herbicide chemistries.

Exogenous phosphorus treatment facilitates chelation-mediated cadmium detoxification in perennial ryegrass (Lolium perenne L.).

Cadmium (Cd) is an on-going environmental pollutant associated with hindered plant growth. In response, plants possess various strategies to alleviate Cd stress, including reactive oxygen species (ROS) scavenging and chelation-mediated Cd detoxification. The present study examined the Cd defense mechanism of perennial ryegrass (Lolium perenne L.), taking into account the effect of exogenous phosphorus (P) input. It was found that despite triggering antioxidant enzyme activity, Cd stress heightened lipid peroxidation levels. Exogenous P input partially mitigated the lipid peroxidation impact and decreased the levels of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) antioxidant enzymes, revealing reduced ROS-scavenging activity. Importantly, notable relationships were determined between the amount of Cd uptake in the root and the amount of non-protein thiols (R2 = 0.914), glutathione (R2 = 0.805) and phytochelatins (R2 = 0.904) in proportion to the amount of exogenous P applied. The levels of amino acids proline and cysteine were also enhanced by exogenous P input showing their influence in alleviating Cd stress. Overall, it is reported that Cd detoxification in ryegrass plants can be stimulated by exogenous P input, which facilitates chelation-mediated Cd detoxification processes.

Overexpression of the Lolium perenne L. delta1-pyrroline 5-carboxylate synthase (LpP5CS) gene results in morphological alterations and salinity tolerance in switchgrass (Panicum virgatum L.).

In plants, Δ1-pyrroline- 5-carboxylate synthase (P5CS) is the rate-limiting enzyme in proline biosynthesis. In this study, we introduced the LpP5CS (Lolium perenne L.) gene into switchgrass by Agrobacterium-mediated transformation. The transgenic lines (TG) were classified into two groups based on their phenotypes and proline levels. The group I lines (TG4 and TG6) had relatively high proline levels and improved biomass yield. The group II lines (TG1 and TG2) showed low proline levels, severely delayed flowering, stunted growth and reduced biomass yield. Additionally, we used RNA-seq analysis to detect the most significant molecular changes, and we analyzed differentially expressed genes, such as flowering-related and CYP450 family genes. Moreover, the biomass yield, physiological parameters, and expression levels of reactive oxygen species scavenger-related genes under salt stress all indicated that the group I plants exhibited significantly increased salt tolerance compared with that of the control plants, in contrast to the group II plants. Thus, genetic improvement of switchgrass by overexpressing LpP5CS to increase proline levels is feasible for increasing plant stress tolerance.

Role of Glutamine Synthetase Isogenes and Herbicide Metabolism in the Mechanism of Resistance to Glufosinate in Lolium perenne L. spp. multiflorum Biotypes from Oregon.

Glufosinate-resistant Lolium perenne L. spp. multiflorum biotypes from Oregon exhibited resistance levels up to 2.8-fold the field rate. One resistant biotype (MG) had an amino acid substitution in glutamine synthetase 2 (GS2), whereas the other (OR) exhibited the wild-type genotype. We hypothesized that the amino acid substitution in GS2 is involved in the resistance mechanism in MG and that non-target site resistance mechanisms are present in OR. OR metabolized glufosinate faster than the other two biotypes, with >75% of the herbicide metabolized in comparison to 50% in MG and the susceptible biotype. A mutation in GS2 co-segregating with resistance in MG did not reduce the enzyme activity, with results further supported by our enzyme homology models. This research supports the conclusion that a metabolism mechanism of glufosinate resistance is present in OR and that glufosinate resistance in MG is not due to an altered target site.

Low pyrrolizidine alkaloid levels in perennial ryegrass is associated with the absence of a homospermidine synthase gene.

BACKGROUND: Pyrrolizidine alkaloids (PAs) are a class of secondary metabolites that function as feeding deterrents in a range of different plant species. In perennial ryegrass (Lolium perenne L.) the only PAs that have been identified are the thesinine-rhamnoside group, which displays significant genetic variation. Homospermidine synthase (HSS) has evolved from deoxyhypusine synthase (DHS) and catalyses the first step in the PA pathway, making it a key candidate for the investigation of genes influencing observed PA trait variation. RESULTS: During PCR amplification and sequence analysis of DHS we identified two putative HSS genes in perennial ryegrass. One of the genes (LpHSS1) was absent in some perennial ryegrass plants. Thesinine-rhamnoside levels were measured using liquid chromatography coupled with mass spectrometry in a diverse association mapping population, consisting of 693 plants free of fungal endophytic symbionts. Association tests that accounted for population structure identified a significant association of absence of the LpHSS1 gene with lower levels of thesinine-rhamnoside PAs. HSS-like gene sequences were identified for other grass species of the Poaceae, including tall fescue, wheat, maize and sorghum. CONCLUSION: HSS is situated at the crucial first step in the PA pathway making it an important candidate gene for investigation of involvement in PA phenotypic variation. In this study, PA level in perennial ryegrass was strongly associated with the presence or absence of the LpHSS1 gene. A genetic marker, developed for the presence/absence of LpHSS1, may be used for marker-assisted breeding to either lower or increase PAs in breeding populations of perennial or Italian ryegrass to investigate a potential role in the deterrence of herbivore pests. The presence of HSS-like genes in several other Poaceae species suggests that PA biosynthesis may occur in plant family members beyond perennial ryegrass and tall fescue and identifies a potential route for manipulating PA levels.

Nanoscale zero-valent iron assisted phytoremediation of Pb in sediment: Impacts on metal accumulation and antioxidative system of Lolium perenne.

Lead (Pb) is a highly toxic environmental pollutant, and could result in toxic effects on living organisms. The effects of 0, 100, 200, 500, 1000 and 2000 mg/kg of nZVI on plant growth, Pb accumulation and antioxidative responses of Lolium perenne were investigated. Results showed that the total Pb contents in L. perenne with the treatment of low concentrations of nZVI (100, 200 and 500 mg/kg) were higher than those in the non-nZVI treatments, and the highest Pb accumulation capacity of 1175.40 µg per pot was observed in L. perenne with the treatment of 100 mg/kg nZVI. However, the total Pb contents in L. perenne decreased at high concentrations of nZVI (1000 and 2000 mg/kg). This might be resulted from the decrease of photosynthetic chlorophyll content and the aggravated oxidative stress induced by the high concentration of nZVI, which caused the decrease of plant biomass and metal accumulation capacity in plant. Moreover, the sequential extraction experiments results showed that the lowest acid soluble fraction of Pb in the sediments was found in the treatment with 100 mg/kg of nZVI, indicating that 100 mg/kg was the optimum concentration for nZVI to assist the phytoremediation of Pb-polluted sediment. To conclude, these findings provide a promising method to remediate Pb-polluted sediment by nZVI assisted phytoremediation.

Activation of MAP kinases by green leaf volatiles in grasses.

OBJECTIVE: Previously we have shown that mechanical wounding and volatiles released from cut grass, activated a 46 and 44 kDa mitogen-activated protein kinase (MAPK) in the model grass species Lolium temulentum (Lt). MAPKs play an important role as signal relays that connect incoming stress signals and stress responses. Since green leaf volatiles (GLV) are released during wounding, we wanted determine if specific compounds contained in the GLV mixture or if GLV generated from other plant species could activate these Lt MAPKs. RESULTS: Our analysis found that just a 1-min exposure to GLV was enough to activate the Lt 46 kDa MAPK within 3 min and the 44 kDa MAPK within 15 min. This activation pattern showed similar kinetics to those observed after wounding, and the GLV and wound activated bands associated with these MAPKs displayed identical migration on sodium dodecyl sulfate polyacrylamide gels. Thirteen different commercially available plant volatiles (alcohols, aldehydes and ketones) were tested and all thirteen volatile compounds were able to activate these same Lt MAPKs. Furthermore, GLV derived from three other grass species as well as tomato, a dicot, were also shown to activate these MAPKs in Lt.

Horizontal transfer of a ß-1,6-glucanase gene from an ancestral species of fungal endophyte to a cool-season grass host.

Molecular characterisation has convincingly demonstrated some types of horizontal gene transfer in eukaryotes, but nuclear gene transfer between distantly related eukaryotic groups appears to have been rare. For angiosperms (flowering plants), nuclear gene transfer events identified to date have been confined to genes originating from prokaryotes or other plant species. In this report, evidence for ancient horizontal transfer of a fungal nuclear gene, encoding a ß-1,6-glucanase enzyme for fungal cell wall degradation, into an angiosperm lineage is presented for the first time. The gene was identified from de novo sequencing and assembly of the genome and transcriptome of perennial ryegrass, a cool-season grass species. Molecular analysis confirmed the presence of the complete gene in the genome of perennial ryegrass. No corresponding sequence was found in other plant species, apart from members of the Poeae sub-tribes Loliinae and Dactylidinae. Evidence suggests that a common ancestor of the two sub-tribes acquired the gene from a species ancestral to contemporary grass-associated fungal endophytes around 9-13 million years ago. This first report of horizontal transfer of a nuclear gene from a taxonomically distant eukaryote to modern flowering plants provides evidence for a novel adaptation mechanism in angiosperms.

Molecular and physiological responses of Iranian Perennial ryegrass as affected by Trinexapac ethyl, Paclobutrazol and Abscisic acid under drought stress.

Drought stress is the major limiting factor which affects turfgrass management in area with restricted rainfall or irrigation water supply. Trinexapac ethyl (TE), Paclobutrazol (PAC) and Abscisic acid (ABA) are three plant growth regulators (PGRs) that are commonly used on turf species for increasing their tolerance to different environmental stresses such as drought. However, little is known about the impact of PGRs on stress tolerance of Iranian Perennial ryegrass (Lolium perenne). The present study was conducted to examine the visual and physiological changes of Iranian Perennial ryegrass in response to foliar application of TE, PAC, and ABA under drought stress conditions. According to the obtained results, application of all three PGRs considerably restored visual quality of drought exposed plants. TE treatment increased chlorophyll content, proline content and resulted in less malondialdehyde (MDA) in drought stressed Perennial ryegrass. Application of all PGRs enhanced the relative water content (RWC) and decreased the electrolyte leakage (EL) and Hydrogen peroxide contents (H2O2 content) of plants under drought stress, though the impact of TE was more pronounced. Throughout the experiment, TE- and ABA-treated plant showed greater soluble sugar (SSC) content as compared to the control. Antioxidant enzymes activities of drought exposed plants were considerably increased by PGRs application. Catalase (CAT) and Superoxide dismutase (SOD) activities were greater in TE-treated grasses followed by PAC-treated plants. Ascorbate peroxidase (APX) and peroxidase (POD) activities were significantly enhanced by TE and ABA application. The results of the present investigation suggest that application of TE, ABA and PAC enhances drought tolerance in Perennial ryegrass. TE, PAC and ABA were all effective in mitigating physiological damages resulting from drought stress, however the beneficial effects of TE were more pronounced. The result obtained of real time-PCR suggested that regulation of CAT, APX, POD and SOD genes expression at translational levels highly depended on the application of TE, PAC and ABA. Also, the results showed that deletion mutation in SOD and POD genes were not leading to enzyme inactivation.

Enhanced expression of Rubisco activase splicing variants differentially affects Rubisco activity during low temperature treatment in Lolium perenne.

Alternative splicing of the Rubisco activase gene was shown to be a point for optimization of photosynthetic carbon assimilation. It can be expected to be a stress-regulated event that depends on plant freezing tolerance. The aim of the study was to examine the relationships among Rubisco activity, the expression of two Rubisco activase splicing variants and photoacclimation to low temperature. The experiment was performed on two Lolium perenne genotypes with contrasting levels of freezing tolerance. The study investigated the effect of pre-hardening (15°C) and cold acclimation (4°C) on net photosynthesis, photosystem II photochemical activity, Rubisco activity and the expression of two splicing variants of the Rubisco activase gene. The results showed an induction of Rubisco activity at both 15°C and 4°C only in a highly freezing-tolerant genotype. The enhanced Rubisco activity after pre-hardening corresponded to increased expression of the splicing variant representing the large isoform, while the increase in Rubisco activity during cold acclimation was due to the activation of both transcript variants. These boosts in Rubisco activity also corresponded to an activation of non-photochemical mechanism of photoacclimation induced at low temperature exclusively in the highly freezing-tolerant genotype. In conclusion, enhanced expression of Rubisco activase splicing variants caused an increase in Rubisco activity during pre-hardening and cold acclimation in the more freezing-tolerant Lolium perenne genotype. The induction of the transcript variant representing the large isoform may be an important element of increasing the carbon assimilation rate supporting the photochemical mechanism of photosynthetic acclimation to cold.

Functional characterization and hormonal regulation of the PHEOPHYTINASE gene LpPPH controlling leaf senescence in perennial ryegrass.

Chlorophyll (Chl) degradation occurs naturally during leaf maturation and senescence, and can be induced by stresses, both processes involving the regulation of plant hormones. The objective of this study was to determine the functional roles and hormonal regulation of a gene encoding pheophytin pheophorbide hydrolyase (PPH) that catabolizes Chl degradation during leaf senescence in perennial grass species. A PPH gene, LpPPH, was cloned from perennial ryegrass (Lolium perenne L.). LpPPH was localized in the chloroplast. Overexpressing LpPPH accelerated Chl degradation in wild tobacco, and rescued the stay-green phenotype of the Arabidopsis pph null mutant. The expression level of LpPPH was positively related to the extent of leaf senescence. Exogenous application of abscisic acid (ABA) and ethephon (an ethylene-releasing agent) accelerated the decline in Chl content in leaves of perennial ryegrass, whereas cytokinin (CK) and aminoethoxyvinylglycine (AVG; an ethylene biosynthesis inhibitor) treatments suppressed leaf senescence, corresponding to the up- or down-regulation of LpPPH expression. The promoters of five orthologous PPH genes were predicted to share conserved cis-elements potentially recognized by transcription factors in the ABA and CK pathways. Taken together, the results suggested that LpPPH-mediated Chl breakdown could be regulated positively by ABA and ethylene, and negatively by CK, and LpPPH could be a direct downstream target gene of transcription factors in the ABA and CK signaling pathways.

Widespread occurrence of both metabolic and target-site herbicide resistance mechanisms in Lolium rigidum populations.

BACKGROUND: Lolium rigidum populations in Australia and globally have demonstrated rapid and widespread evolution of resistance to acetyl coenzyme A carboxylase (ACCase)-inhibiting and acetolactate synthase (ALS)-inhibiting herbicides. Thirty-three resistant L. rigidum populations, randomly collected from crop fields in a most recent resistance survey, were analysed for non-target-site diclofop metabolism and all known target-site ACCase gene resistance-endowing mutations. RESULTS: The HPLC profile of [(14) C]-diclofop-methyl in vivo metabolism revealed that 79% of these resistant L. rigidum populations showed enhanced capacity for diclofop acid metabolism (metabolic resistance). ACCase gene sequencing identified that 91% of the populations contain plants with ACCase resistance mutation(s). Importantly, 70% of the populations exhibit both non-target-site metabolic resistance and target-site ACCase mutations. CONCLUSIONS: This work demonstrates that metabolic herbicide resistance is commonly occurring in L. rigidum, and coevolution of both metabolic resistance and target-site resistance is an evolutionary reality. Metabolic herbicide resistance can potentially endow resistance to many herbicides and poses a threat to herbicide sustainability and thus crop production, calling for major research and management efforts.

Effect of decabromodiphenyl ether (BDE-209) on a soil-biota system: Role of earthworms and ryegrass.

In the present study, the toxic effect of decabromodiphenyl ether (BDE-209), an important brominated fire retardant, on soil was evaluated by amending with different concentrations (0 mg/kg, 1 mg/kg, 10 mg/kg, and 500 mg/kg dry wt) for 40 d. The activities of 3 soil enzymes (urease, catalase, and alkaline phosphatase) were measured as the principal assessment endpoints. Meanwhile, the effects of natural environmental factors, such as light conditions and soil biota, on BDE-209 intoxication were studied. For the latter, 30 earthworms (Metaphire guillelmi) with fully matured clitella or ryegrass (Lolium perenne) with fully matured leaves were exposed in soil amended with BDE-209. The activities of the soil enzymes were adversely affected by BDE-209, especially for the high-concentration treatments, with greater adverse effects in the dark than in the light. The presence of earthworms reduced toxicity to BDE-209, whereas ryegrass did not. The calculated integrated biomarker response index, which provides a general indicator of the health status of test species by combining different biomarker signals, further validated these findings. Moreover, the antioxidant status (oxidant-antioxidant balance) of these 2 biota was assessed. Results indicated that BDE-209 significantly affected the activities of antioxidant enzymes (superoxide dismutase and catalase) and enhanced the levels of malondialdehyde in both species. The present study may facilitate a better understanding of the toxicity of BDE-209 toward the soil environment. Environ Toxicol Chem 2016;35:1349-1357. © 2015 SETAC.

Effects of GA3 on Plant Physiological Properties, Extraction, Subcellular Distribution and Chemical Forms of Pb in Lolium perenne.

The effects of growth-promoting hormone gibberellic acid 3 (GA3) on physiology, Pb phytoextraction, and metal detoxification mechanisms in Lolium perenne were studied. Results showed that addition of GA3 alone at lower doses (1 or 10 µM) facilitated antioxidant defense of L. perenne under Pb stress, decreased the toxicity of Pb in plant shoot by increasing the proportion of Pb in cell wall, hence significantly enhanced photosynthesis and plant growth, as well as Pb uptake and accumulation in L. perenne (P < 0.05). However, these indicators showed the opposite changes when treated with GA3 at a higher dose (100 µM). Of the total Pb in plant shoot, 36-51% was associated with cell wall, and 31-40% was soluble fraction, while 41.4-49.7% was NaCl extractable, 24.6-35.4% HAc extractable followed by other fractions. These findings suggest that Pb fixation by pectates and proteins in cell wall and sequestration in vacuole are responsible for Pb detoxification in plant, and the GA3 at 1 µM appears to be optimal for enhancing Pb phytoextraction by L. perenne from Pb polluted soils.

Effect of herbicide resistance endowing Ile-1781-Leu and Asp-2078-Gly ACCase gene mutations on ACCase kinetics and growth traits in Lolium rigidum.

The rate of herbicide resistance evolution in plants depends on fitness traits endowed by alleles in both the presence and absence (resistance cost) of herbicide selection. The effect of two Lolium rigidum spontaneous homozygous target-site resistance-endowing mutations (Ile-1781-Leu, Asp-2078-Gly) on both ACCase activity and various plant growth traits have been investigated here. Relative growth rate (RGR) and components (net assimilation rate, leaf area ratio), resource allocation to different organs, and growth responses in competition with a wheat crop were assessed. Unlike plants carrying the Ile-1781-Leu resistance mutation, plants homozygous for the Asp-2078-Gly mutation exhibited a significantly lower RGR (30%), which translated into lower allocation of biomass to roots, shoots, and leaves, and poor responses to plant competition. Both the negligible and significant growth reductions associated, respectively, with the Ile-1781-Leu and Asp-2078-Gly resistance mutations correlated with their impact on ACCase activity. Whereas the Ile-1781-Leu mutation showed no pleiotropic effects on ACCase kinetics, the Asp-2078-Gly mutation led to a significant reduction in ACCase activity. The impaired growth traits are discussed in the context of resistance costs and the effects of each resistance allele on ACCase activity. Similar effects of these two particular ACCase mutations on the ACCase activity of Alopecurus myosuroides were also confirmed.

RNA-Seq analysis of rye-grass transcriptomic response to an herbicide inhibiting acetolactate-synthase identifies transcripts linked to non-target-site-based resistance.

Non-target-site resistance (NTSR) to herbicides that disrupts agricultural weed control is a worldwide concern for food security. NTSR is considered a polygenic adaptive trait driven by differential gene regulation in resistant plants. Little is known about its genetic determinism, which precludes NTSR diagnosis and evolutionary studies. We used Illumina RNA-sequencing to investigate transcriptomic differences between plants from the global major weed rye-grass sensitive or resistant to the acetolactate-synthase (ALS) inhibiting herbicide pyroxsulam. Plants were collected before and along a time-course after herbicide application. De novo transcriptome assembly yielded a resource (LOLbase) including 92,381 contigs representing potentially active transcripts that were assigned putative annotations. Early effects of ALS inhibition consistent with the literature were observed in resistant and sensitive plants, proving LOLbase data were relevant to study herbicide response. Comparison of resistant and sensitive plants identified 30 candidate NTSR contigs. Further validation using 212 plants resistant or sensitive to pyroxsulam and/or to the ALS inhibitors iodosulfuron + mesosulfuron confirmed four contigs (two cytochromes P450, one glycosyl-transferase and one glutathione-S-transferase) were NTSR markers which combined expression levels could reliably identify resistant plants. This work confirmed that NTSR is driven by differential gene expression and involves different mechanisms. It provided tools and foundation for subsequent NTSR investigations.

Evidence for alternative splicing mechanisms in meadow fescue (Festuca pratensis) and perennial ryegrass (Lolium perenne) Rubisco activase gene.

Rubisco activase is required to regulate the catalytic activity of Rubisco in plants, in an ATP-dependent manner. One or two Rubisco activase proteins have been identified in different plant species. In some species, the two isoforms are the products of alternative splicing of the Rubisco activase gene. The aim of this study was to confirm that Lolium perenne and Festuca pratensis plants have two isoforms of Rubisco activase and that they are the products of alternative splicing of common pre-mRNA. Protein gel blot analyses indicated that L. perenne and F. pratensis leaves contained two Rubisco activase proteins. Sequence analysis of cDNA and genomic DNA showed that differential splicing generated two mRNAs that differed in sequence only in the inclusion of 48 bp. The insertion contains a stop codon leading to the synthesis of a shorter polypeptide. Under the conditions of our experiment, the shorter splicing variant of L. perenne and F. pratensis Rubisco activase gene was preferentially produced. Any further studies concerning Rubisco activase genes in L. perenne and/or F. pratensis plants should take into consideration the mechanism of its expression.