Production of Biopharmaceuticals in Nicotiana benthamiana-Axillary Stem Growth as a Key Determinant of Total Protein Yield.

Data are scarce about the influence of basic cultural conditions on growth patterns and overall performance of plants used as heterologous production hosts for protein pharmaceuticals. Higher plants are complex organisms with young, mature, and senescing organs that show distinct metabolic backgrounds and differ in their ability to sustain foreign protein expression and accumulation. Here, we used the transient protein expression host Nicotiana benthamiana as a model to map the accumulation profile of influenza virus hemagglutinin H1, a clinically promising vaccine antigen, at the whole plant scale. Greenhouse-grown plants submitted to different light regimes, submitted to apical bud pruning, or treated with the axillary growth-promoting cytokinin 6-benzylaminopurine were vacuum-infiltrated with agrobacteria harboring a DNA sequence for H1 and allowed to express the viral antigen for 7 days in growth chamber under similar environmental conditions. Our data highlight the importance of young leaves on H1 yield per plant, unlike older leaves which account for a significant part of the plant biomass but contribute little to total antigen titer. Our data also highlight the key contribution of axillary stem leaves, which contribute more than 50% of total yield under certain conditions despite representing only one-third of the total biomass. These findings underline the relevance of both considering main stem leaves and axillary stem leaves while modeling heterologous protein production in N. benthamiana. They also demonstrate the potential of exogenously applied growth-promoting hormones to modulate host plant architecture for improvement of protein yields.

Plant extracts and essential oil product as feed additives to control rabbit meat microbial quality.

The present work evaluated the effects of feed supplementation with plant extracts (onion, cranberry) and a commercial essential oil product (Xtract™) on rabbit meat quality. Five groups of 48 weaned Grimaud female rabbits each received a control ration (C) or a diet supplemented with onion extract (500 or 1000 ppm), cranberry extracts (500 ppm) and essential oil product (100 ppm) alone or in combination. Microbiological quality was evaluated on whole hind legs stored under aerobic and anaerobic conditions at 4 °C. Growth performances, feed intake and both meat composition and quality were similar amongst the experimental groups. Anyhow, meat total phenolic content was significantly higher in all supplemented ones (P < 0.001). Diet supplementation effect was observed (P < 0.05) and microbial control was improved more importantly under anaerobic conditions, notably for Total Aerobic Mesophilic counts, presumptive Pseudomonas and Enterobacteriaceae (P < 0.03). Overall, supplementation with onion extract (500 ppm) suppressed microbial growth more effectively.

Characterization of industrial broccoli discards (Brassica oleracea var. italica) for their glucosinolate, polyphenol and flavonoid contents using UPLC MS/MS and spectrophotometric methods.

The agrifood industry produces tons of waste and substandard products that are discarded at great expense. Valorization of industrial residues curbs issues related to food security and environmental problems. Broccoli (Brassica oleracea var. italica) is associated with varied beneficial health effects, but its production yields greater than 25% rejects. We aimed to characterize and quantify industrial broccoli by-products for their glucosinolate and polyphenol contents as a first step towards industrial bio-refining. Broccoli segments and rejected lots of 10 seed cultivars were analyzed using UPLC MS/MS. Variability in the contents of bioactive molecules was observed within and between the cultivars. Broccoli by-products were rich in glucosinolates (0.2-2% dry weight sample), predominantly glucoraphanin (32-64% of the total glucosinolates), whereas the polyphenolic content was less than 0.02% dry weight sample. Valorization of industrial residues facilitates the production of high value functional food ingredients along with socio-economic sustainability.

Persistent negative temperature response of mesophyll conductance in red raspberry (Rubus idaeus L.) leaves under both high and low vapour pressure deficits: a role for abscisic acid?

The temperature dependence of mesophyll conductance (gm ) was measured in well-watered red raspberry (Rubus idaeus L.) plants acclimated to leaf-to-air vapour pressure deficit (VPDL) daytime differentials of contrasting amplitude, keeping a fixed diurnal leaf temperature (Tleaf ) rise from 20 to 35 °C. Contrary to the great majority of gm temperature responses published to date, we found a pronounced reduction of gm with increasing Tleaf irrespective of leaf chamber O2 level and diurnal VPDL regime. Leaf hydraulic conductance was greatly enhanced during the warmer afternoon periods under both low (0.75 to 1.5 kPa) and high (0.75 to 3.5 kPa) diurnal VPDL regimes, unlike stomatal conductance (gs ), which decreased in the afternoon. Consequently, the leaf water status remained largely isohydric throughout the day, and therefore cannot be evoked to explain the diurnal decrease of gm . However, the concerted diurnal reductions of gm and gs were well correlated with increases in leaf abscisic acid (ABA) content, thus suggesting that ABA can induce a significant depression of gm under favourable leaf water status. Our results challenge the view that the temperature dependence of gm can be explained solely from dynamic leaf anatomical adjustments and/or from the known thermodynamic properties of aqueous solutions and lipid membranes.​.

Silicon Transporters and Effects of Silicon Amendments in Strawberry under High Tunnel and Field Conditions.

Together with longer production periods, the commercial transition to day-neutral strawberry (Fragaria × ananassa) varieties has favored the development of diseases such as powdery mildew (Podosphaera aphanis) that thrives in late summer-early fall. In an attempt to find alternative solutions to fungicides currently employed to curb the disease, we wanted to investigate the potential of silicon (Si) amendments that have been associated with prophylactic properties against powdery mildews. To this end, our first objective was to determine if strawberry was a Si-competent species following the recent characterization of the properties of Si transporters that plants must carry to uptake silicic acid. Based on genomic data, we were able to conclude that strawberry contained both functional influx (Lsi1) and efflux (Lsi2) transporters for Si uptake. Subsequently commercial experiments under high tunnel and field conditions were conducted with different Si fertilization regimes: constant soluble Si feeding in high tunnel, and bi-weekly soluble Si feeding or three concentrations of calcium silicate fertilization in the field. Results from high tunnel experiments showed that strawberry could accumulate as much as 3% Si on a dry-weight basis, the highest concentration ever reported for this species. All six tested cultivars contained roughly the same concentration, thereby confirming the limited genetic variability, also observed in other species, associated with the trait. Silicon fertilization under high tunnel led to a significant reduction of powdery mildew severity in both years and on all cultivars, and a significant increase in yield of marketable fruits reaching as much as 300% with cv. Monterey. By contrast, Si fertilization under field conditions in soils deficient in plant available Si, either in soluble or solid form, did not result in significant accumulation of Si in plants, regardless of the cultivars, year or concentrations. Our results have thus provided both genotypic and phenotypic proof that strawberry can greatly benefit from Si fertilization, but have also highlighted the importance of validating the fertilization regime to ensure that Si is properly absorbed and/or available to the plant.

Fatty acid profiles of polar and neutral lipids of ten species of higher basidiomycetes indigenous to eastern Canada.

Neutral and polar lipid contents of ten species of edible mushrooms indigenous to Eastern Canada belonging to the families Agaricaceae, Amanitaceae, Boletaceae, Coprinaceae, Ganodermataceae, and Lycoperdaceae were analysed. The total lipid content of the species analysed ranged from 3.1% (Ganoderma applanatum) to 16% (w/w) d.w. (Amanita vaginata) and averaged 8.6% (w/w) d.w. Polar lipids accounted for more than 50% of the total lipids in most species and differences were observed between neutral and polar lipid contents according to the species analysed. In both lipid fractions, high proportions of unsaturated fatty acids (FAs) ranging from 62.7 to 82.3% (polar lipids) and 59.8 to 82.5% (neutral lipids) of the total FAs were observed. Analysis of FA profiles showed that both neutral and polar lipids were mainly composed of linoleic (18:2 Delta9c,12c), oleic (18:1 Delta9c), and palmitic (16:0) acids. Significant differences (P<0.05) in the contents of specific FAs were observed between mushroom species. Among the 44 FAs detected in the species analysed, the occurrence of cis-11-heptadecenoic (17:1 Delta11c) acid is reported for the first time in basidiomycetes, while elaidic acid (18:1 Delta9t) is reported for the first time in fungi.

Fatty acid profiles of polar and non-polar lipids of Pleurotus ostreatus and P. cornucopiae var. 'citrino-pileatus' grown at different temperatures.

The application of fatty acid (FA) composition data has now extended to studies of physiology, chemotaxonomy, and intrageneric differentiation, as well as to studies of human nutrition. Environmental factors such as nutritional components, oxygen, and temperature are known to affect lipid content and composition in living organisms, including fungi. In the present study, the polar and non-polar lipid content of Pleurotus ostreatus and P. cornucopiae var. citrino-pileatus fruiting bodies produced at temperatures ranging from 12-27 degrees C and from 17-27 degrees C, respectively, were analysed to evaluate the effect of temperature on lipid composition in these mushrooms. Results showed that lowering the growth temperature below 17 degrees C generally provided an expected increase in FA unsaturation in polar and non-polar lipids of P. ostreatus. Although raising the temperature above 17 degrees C did not show any clear-cut tendency in FA unsaturation, it did reveal that growth temperature had a differential effect on the FA profiles in fruiting bodies of P. ostreatus and P. cornucopiae. This study suggests that care should be taken when using FA content and unsaturation data for physiological, chemotaxonomic, and intrageneric differentiation studies, and that it may be possible to manipulate lipid unsaturation in Pleurotus spp. through modified growth temperatures.

Inhibitory effect of CDP, a polysaccharide extracted from the mushroom Collybia dryophila, on nitric oxide synthase expression and nitric oxide production in macrophages.

The effect of Collybia dryophila polysaccharide (CDP), a (1-->3), (1-->4)-beta-D-glucan extracted from the mushroom C. dryophila, was evaluated on nitric oxide (NO) production induced by lipopolysaccharide (LPS) and gamma interferon (IFNgamma) or by LPS alone in RAW 264.7 cells. CDP significantly inhibited NO production in a dose-dependent manner without affecting cell viability. The inhibition of NO by CDP was consistent with decreases in both inducible nitric oxide synthase (iNOS) protein and mRNA expression suggesting that CDP exerts its effect by inhibiting iNOS gene expression. In addition, CDP at concentrations of 400 and 800 microg/ml was shown to significantly increase prostaglandin E2 (PGE2) production in LPS- and IFNgamma-induced macrophages when compared to the control.

A bioactive (1-->3)-, (1--4)-beta-D-glucan from Collybia dryophila and other mushrooms.

Polysaccharides from higher Basidiomycete mushrooms, mainly beta-D-glucans, are considered to be potent bioactive fungal compounds. In this study a beta-glucan (1.237 x 10(6) Da) consisting of (1-->3) and (1-->4) glucosidic linkages, named Collybia dryophila polysaccharide (CDP), was extracted from the wild mushroom C. dryophila. CDP was shown to strongly inhibit nitric oxide production in activated macrophages suggesting that this polysaccharide displays a potential anti-inflammatory activity. In addition it was shown that polysaccharides similar to CDP (CDP-like) are present in Lentinus edodes and different wild mushrooms collected in northeastern North America.

Understory light and root ginsenosides in forest-grown Panax quinquefolius.

The objective of this study was to determine the relationship between light levels in the understory of a broadleaf forest and the content of six ginsenosides (Rg(1), Re, Rb(1), Rc, Rb(2,) and Rd) in 1- and 2-year-old American ginseng (Panax quinquefolius L.) roots. Our results revealed that ginsenoside contents in 1- and 2 year-old roots collected in September were significantly related to direct and total light levels, and duration of sunflecks. At this time, the effect of light levels accounted for up to 48 and 62% of the variation in ginsenoside contents of 1- and 2-year-old American ginseng roots. Also, red (R) and far red (FR) light, and the R:FR ratio significantly affected Rd, Rc, and Rg(1) contents in 2-year-old roots, accounting for up to 40% of the variation in ginsenoside contents.

Examination of environmental quality of raw and composting de-inking paper sludge.

Paper sludges were traditionally landfilled or burned. Over the years, the use of paper sludges on soils has increased, as well as the concerns about their environmental effects. Therefore, the chemical characterization of paper sludges and their young (immature) compost needed to be investigated, and over 150 inorganic and organic chemicals were analyzed in de-inking paper sludge (DPS). In general, nitrogen, phosphorus and potassium contents were low but variable in raw DPS and its young compost. The contents of arsenic, boron, cadmium, cobalt, chromium, manganese, mercury, molybdenum, nickel, lead, selenium, and zinc were also low and showed low variability. However, the copper contents were above the Canadian compost regulation for unrestricted use and required a follow-up. The fatty- and resin acids, and polycyclic aromatic hydrocarbons were the organic chemicals measured at the highest concentrations. For resinic acids, care should be taken to avoid that leachates reach aquatic life. For polycyclic aromatic hydrocarbons, naphthalene should be followed until soil content reaches 0.1 microg g(-1), the maximum allowed for soil use for agricultural purposes according to Canadian Environmental Quality Guidelines. In young compost, the concentration of these chemical families decreased over time and most compounds were below the detection limits after 24 weeks of composting. In raw DPS, among the phenol, halogenated and monoaromatic hydrocarbons, dioxin and furan, and polychlorinated biphenyl families, most compounds were below the detection limits. The raw DPS and its young compost do not represent a major threat for the environment but can require an environmental follow-up.