An intronless tau class glutathione transferase detoxifies several herbicides in flufenacet-resistant ryegrass.

Resistance to preemergence herbicides, e.g. inhibitors of the biosynthesis of very-long-chain fatty acids (VLCFAs), is evolving in response to increased use of these compounds. Grass weeds such as ryegrasses (Lolium spp.) have accumulated resistance to various herbicide modes of action. Here, an RNA-seq analysis was conducted using 3 ryegrass populations resistant to the VLCFA biosynthesis inhibitor flufenacet to investigate this phenomenon. Besides various transcripts, including putative long noncoding RNAs (lncRNAs), a single putatively functional tau class glutathione transferase (GST) was constitutively differentially expressed. It was further induced by herbicide application. This GST was expressed as a recombinant protein in Escherichia coli along with other GSTs and detoxified flufenacet rapidly in vitro. Detoxification rates of other herbicides tested in vitro were in accordance with cross-resistance patterns previously determined in vivo. A genome-wide GST analysis revealed that the candidate GST was located in a cluster of 3 intronless GSTs. Their intronless nature possibly results from the retroposition of cellular mRNAs followed by tandem duplication and may affect gene expression. The large number of GSTs (≥195) in the genome of rigid ryegrass (Lolium rigidum) compared with other plant organisms is likely a key factor in the ability of this weed to evolve resistance to different herbicide chemistries. However, in the case of flufenacet resistance, a single upregulated GST with high affinity for the substrate flufenacet possibly contributes overproportionally to rapid herbicide detoxification in planta. The regulation of this gene and the role of differentially expressed transcripts, including various putative lncRNAs, require further investigation.

Sucrose-Phosphate Synthase and Sucrose Synthase contribute to refoliation in ryegrass, a grassland fructan-accumulating species.

The perennity of grassland species such as Lolium perenne greatly depends on their ability to regrow after cutting or grazing. Refoliation largely relies on the mobilization of fructans in the remaining tissues and on the associated sucrose synthesis and transport towards the basal leaf meristems. However, nothing is known yet about the sucrose synthesis pathway. Sucrose Phosphate Synthase (SPS) and Sucrose Synthase (SuS) activities, together with their transcripts, were monitored during the first hours after defoliation along the leaf axis of mature leaf sheaths and elongating leaf bases (ELB) where the leaf meristems are located. In leaf sheaths, which undergo a sink-source transition, fructan and sucrose contents declined while SPS and SuS activities increased, along with the expression of LpSPSA, LpSPSD.2, LpSuS1, LpSuS2, and LpSuS4. In ELB, which continue to act as a strong carbon sink, SPS and SuS activities increased to varying degrees while the expression of all the LpSPS and LpSuS genes decreased after defoliation. SPS and SuS both contribute to refoliation but are regulated differently depending on the source or sink status of the tissues. Together with fructan metabolism, they represent key determinants of ryegrass perennity and, more generally, of grassland sustainability.

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.

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.

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.

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.

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.

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.

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.

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.

Cloning and characterization of a novel fructan 6-exohydrolase strongly inhibited by sucrose in Lolium perenne.

MAIN CONCLUSION: The first 6-fructan exohydrolase (6-FEH) cDNA from Lolium perenne was cloned and characterized. Following defoliation, Lp6 - FEHa transcript level unexpectedly decreased together with an increase in total FEH activity. Lolium perenne is a major forage grass species that accumulates fructans, mainly composed of ß(2,6)-linked fructose units. Fructans are mobilized through strongly increased activities of fructan exohydrolases (FEHs), sustaining regrowth following defoliation. To understand the complex regulation of fructan breakdown in defoliated grassland species, the objective was to clone and characterize new FEH genes in L. perenne. To find FEH genes related to refoliation, a defoliated tiller base cDNA library was screened. Characterization of the recombinant protein was performed in Pichia pastoris. In this report, the cloning and enzymatic characterization of the first 6-FEH from L. perenne is described. Following defoliation, during fructan breakdown, Lp6-FEHa transcript level unexpectedly decreased in elongating leaf bases (ELB) and in mature leaf sheaths (tiller base) in parallel to increased total FEH activities. In comparison, transcript levels of genes coding for fructosyltransferases (FTs) involved in fructan biosynthesis also decreased after defoliation but much faster than FEH transcript levels. Since Lp6-FEHa was strongly inhibited by sucrose, mechanisms modulating FEH activities are discussed. It is proposed that differences in the regulation of FEH activity among forage grasses influence their tolerance to defoliation.

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.

Enhanced rates of herbicide metabolism in low herbicide-dose selected resistant Lolium rigidum.

Lolium rigidum is an obligately cross-pollinated, genetically diverse species and an economically important herbicide resistance-prone weed. Our previous work has demonstrated that recurrent selection of initially susceptible L. rigidum populations with low herbicide rates results in rapid herbicide resistance evolution. Here we report on the mechanisms endowing low-dose-selected diclofop-methyl resistance in L. rigidum. Results showed that resistance was not due to target-site ACCase mutations or overproduction, or differential herbicide leaf uptake and translocation. The in vivo de-esterification of diclofop-methyl into phytotoxic diclofop acid was rapid and similar in resistant versus susceptible populations. However, further metabolism of diclofop acid into non-toxic metabolites was always faster in resistant plants than susceptible plants, resulting in up to 2.6-fold lower level of diclofop acid in resistant plants. This corresponded well with up to twofold higher level of diclofop acid metabolites in resistant plants. The major polar metabolites of diclofop acid chromatographically resembled those of wheat, a naturally tolerant species. Clearly, recurrent selection at reduced herbicide rates selected for non-target-site-based enhanced rates of herbicide metabolism, likely involving cytochrome P450 monooxygenases.

Leaf Rubisco turnover in a perennial ryegrass (Lolium perenne L.) mapping population: genetic variation, identification of associated QTL, and correlation with plant morphology and yield.

This study tested the hypotheses that: (i) genetic variation in Rubisco turnover may exist in perennial ryegrass (Lolium perenne L.); (ii) such variation might affect nitrogen use efficiency and plant yield; and (iii) genetic control of Rubisco turnover might be amenable to identification by quantitative trait loci (QTL) mapping. A set of 135 full-sib F1 perennial ryegrass plants derived from a pair cross between genotypes from the cultivars 'Grasslands Impact' and 'Grasslands Samson' was studied to test these hypotheses. Leaf Rubisco concentration at different leaf ages was measured and modelled as a log-normal curve described by three mathematical parameters: D (peak Rubisco concentration), G (time of D), and F (curve standard deviation). Herbage dry matter (DM) yield and morphological traits (tiller weight (TW), tiller number (TN), leaf lamina length (LL), and an index of competitive ability (PI)) were also measured. The progeny exhibited continuous variation for all traits. Simple correlation and principal component analyses indicated that plant productivity was associated with peak Rubisco concentration and not Rubisco turnover. Lower DM was associated with higher leaf Rubisco concentration indicating that Rubisco turnover effects on plant productivity may relate to energy cost of Rubisco synthesis rather than photosynthetic capacity. QTL detection by a multiple QTL model identified seven significant QTL for Rubisco turnover and nine QTL for DM and morphological traits. An indication of the genetic interdependence of DM and the measures of Rubisco turnover was the support interval overlap involving QTL for D and QTL for TN on linkage group 5 in a cluster involving QTL for DM and PI. In this region, alleles associated with increased TN, DM, and PI were associated with decreased D, indicating that this region may regulate Rubisco concentration and plant productivity via increased tillering. A second cluster involving QTL for LL, TN, PI and DM was found on linkage group 2. The two clusters represent marker-trait associations that might be useful for marker-assisted plant breeding applications. In silico comparative analysis indicated conservation of the genetic loci controlling Rubisco concentration in perennial ryegrass and rice.

Structure-function analyses of a caffeic acid O-methyltransferase from perennial ryegrass reveal the molecular basis for substrate preference.

Lignin forms from the polymerization of phenylpropanoid-derived building blocks (the monolignols), whose modification through hydroxylation and O-methylation modulates the chemical and physical properties of the lignin polymer. The enzyme caffeic acid O-methyltransferase (COMT) is central to lignin biosynthesis. It is often targeted in attempts to engineer the lignin composition of transgenic plants for improved forage digestibility, pulping efficiency, or utility in biofuel production. Despite intensive investigation, the structural determinants of the regiospecificity and substrate selectivity of COMT remain poorly defined. Reported here are x-ray crystallographic structures of perennial ryegrass (Lolium perenne) COMT (Lp OMT1) in open conformational state, apo- and holoenzyme forms and, most significantly, in a closed conformational state complexed with the products S-adenosyl-L-homocysteine and sinapaldehyde. The product-bound complex reveals the post-methyl-transfer organization of COMT's catalytic groups with reactant molecules and the fully formed phenolic-ligand binding site. The core scaffold of the phenolic ligand forges a hydrogen-bonding network involving the 4-hydroxy group that anchors the aromatic ring and thereby permits only metahydroxyl groups to be positioned for transmethylation. While distal from the site of transmethylation, the propanoid tail substituent governs the kinetic preference of ryegrass COMT for aldehydes over alcohols and acids due to a single hydrogen bond donor for the C9 oxygenated moiety dictating the preference for an aldehyde.

Functional analyses of caffeic acid O-Methyltransferase and Cinnamoyl-CoA-reductase genes from perennial ryegrass (Lolium perenne).

Cinnamoyl CoA-reductase (CCR) and caffeic acid O-methyltransferase (COMT) catalyze key steps in the biosynthesis of monolignols, which serve as building blocks in the formation of plant lignin. We identified candidate genes encoding these two enzymes in perennial ryegrass (Lolium perenne) and show that the spatio-temporal expression patterns of these genes in planta correlate well with the developmental profile of lignin deposition. Downregulation of CCR1 and caffeic acid O-methyltransferase 1 (OMT1) using an RNA interference-mediated silencing strategy caused dramatic changes in lignin level and composition in transgenic perennial ryegrass plants grown under both glasshouse and field conditions. In CCR1-deficient perennial ryegrass plants, metabolic profiling indicates the redirection of intermediates both within and beyond the core phenylpropanoid pathway. The combined results strongly support a key role for the OMT1 gene product in the biosynthesis of both syringyl- and guaiacyl-lignin subunits in perennial ryegrass. Both field-grown OMT1-deficient and CCR1-deficient perennial ryegrass plants showed enhanced digestibility without obvious detrimental effects on either plant fitness or biomass production. This highlights the potential of metabolic engineering not only to enhance the forage quality of grasses but also to produce optimal feedstock plants for biofuel production.

Ice recrystallization inhibition mediated by a nuclear-expressed and -secreted recombinant ice-binding protein in the microalga Chlamydomonas reinhardtii.

A Lolium perenne ice-binding protein (LpIBP) demonstrates superior ice recrystallization inhibition (IRI) activity and has proposed applications in cryopreservation, food texturing, as well as in being a "green" gas hydrate inhibitor. Recombinant production of LpIBP has been previously conducted in bacterial and yeast systems for studies of protein characterization, but large-scale applications have been hitherto limited due to high production costs. In this work, a codon-optimized LpIBP was recombinantly expressed and secreted in a novel one-step vector system from the nuclear genome of the green microalga Chlamydomonas reinhardtii. Both mixotrophic and photoautotrophic growth regimes supported LpIBP expression, indicating the feasibility of low-cost production using minimal medium, carbon dioxide, and light energy as input. In addition, multiple growth and bioproduct extraction cycles were performed by repetitive batch cultivation trials, demonstrating the potential for semi-continuous production and biomass harvesting. Concentrations of recombinant protein reached in this proof of concept approach were sufficient to demonstrate IRI activity in culture media without additional purification or concentration, with activity further verified by thermal hysteresis and morphology assays. The incorporation of the recombinant LpIBP into a model gas hydrate offers the promise that algal production may eventually find application as a "green" hydrate inhibitor.

Linear ion trap MS(n) of enzymatically synthesized 13C-labeled fructans revealing differentiating fragmentation patterns of ß (1-2) and ß (1-6) fructans and providing a tool for oligosaccharide identification in complex mixtures.

Fructans are polymeric carbohydrates, which play important roles as plant reserve carbohydrates and stress protectants, and are beneficial for human health and animal production. Fructans are formed by the addition of ß-d-fructofuranosyl units to sucrose, leading to very complex mixtures of 1-kestose based inulins, 6-kestose linked levans, and 6G-kestose derived neoseries inulins and levans in cool season grasses such as Lolium perenne. The identification of isomeric fructan oligomers in chromatographic analysis of crude plant extracts is often hampered by the lack of authentic standards, and unambiguous peak assignment usually requires time-consuming analyses of purified fructan oligomers. We have developed a LC-MS(n) method for the separation and detection of fructan isomers and present here evidence for specific MS(n) fragmentation patterns associated with ß 1-2 (inulins) and ß 2-6 (levans) fructans. LC-MS(n) analysis of (13)C labeled fructan oligomers produced by L. perenne fructosyltransferases expressed in yeast has enabled us to account for the observed fragmentation patterns in terms of preferential cleavage of the glycosidic bond between O- and fructose C2 in both inulins and levans and to differentiate reducing-end from nonreducing end cross ring cleavages in levans. We propose that higher order MS fragmentation patterns can be used to distinguish between the two major classes of fructan, i.e., inulins and levans, without the need for authentic standards.

Selection for low dormancy in annual ryegrass (Lolium rigidum) seeds results in high constitutive expression of a glucose-responsive α-amylase isoform.

BACKGROUND AND AIMS: α-Amylase in grass caryopses (seeds) is usually expressed upon commencement of germination and is rarely seen in dry, mature seeds. A heat-stable α-amylase activity was unexpectedly selected for expression in dry annual ryegrass (Lolium rigidum) seeds during targeted selection for low primary dormancy. The aim of this study was to characterize this constitutive activity biochemically and determine if its presence conferred insensitivity to the germination inhibitors abscisic acid and benzoxazolinone. METHODS: α-Amylase activity in developing, mature and germinating seeds from the selected (low-dormancy) and a field-collected (dormant) population was characterized by native activity PAGE. The response of seed germination and α-amylase activity to abscisic acid and benzoxazolinone was assessed. Using an alginate affinity matrix, α-amylase was purified from dry and germinating seeds for analysis of its enzymatic properties. KEY RESULTS: The constitutive α-amylase activity appeared late during seed development and was mainly localized in the aleurone; in germinating seeds, this activity was responsive to both glucose and gibberellin. It migrated differently on native PAGE compared with the major activities in germinating seeds of the dormant population, but the enzymatic properties of α-amylase purified from the low-dormancy and dormant seeds were largely indistinguishable. Seed imbibition on benzoxazolinone had little effect on the low-dormancy seeds but greatly inhibited germination and α-amylase activity in the dormant population. CONCLUSIONS: The constitutive α-amylase activity in annual ryegrass seeds selected for low dormancy is electrophoretically different from that in germinating seeds and its presence confers insensitivity to benzoxazolinone. The concurrent selection of low dormancy and constitutive α-amylase activity may help to enhance seedling establishment under competitive conditions.