Louis-Philippe Vezina

New cold- and drought-regulated gene from Medicago sativa

Plants are known to differ in their ability to withstand freezing temperatures, but the molecular/genetic basis of this differential freezing tolerance is unclear. Exposure of plants to low, nonfreezing temperatures (cold acclimation) increases their tolerance to subsequent freezing (see recent review by Guy, 1990). Significant biochemical modifications occur during cold acclimation of plants, including changes in gene expression (Thomashow, 1990). Three cold acclimation-specific cDNAs (Mohapatra et al., 1989) and one cDNA responsive to environmental stress (cold and drought) and ABA (Mohapatra et al., 1988) have previously been isolated from alfalfa (Medicago sativa L. cv Apica). DNA sequence determination of four alfalfa cDNAs that were shown to be responsive to environmental stresses (low temperature and drought) and ABA indicated that they are part of a family of genes encoding Gly-rich proteins containing many repeated peptide motifs (Lu0 et al., 1991, 1992). We report here the isolation of a new full-length cDNA clone (MsaciA) that is cold and drought regulated in alfalfa. Plants of the cold-tolerant alfalfa were grown at 21OC for 5 weeks and cold acclimated for 2 weeks at 2OC. A XgtlO library was constructed with mRNA isolated from cold-acclimated crowns. The cDNA of a cold-inducible transcript was isolated by differential hybridization using single-strand cDNA synthesized from cold-acclimated and nonacclimated crowns. The nucleotide sequence of the full-length cDNA and the deduced amino acid sequence of MsaciA have been determined (Table I). MsaciA encodes a putative Gly-rich protein (38%), which contains many repeated motifs. This putative protein shares homology in the range of 68 to 88% (amino acid identity) with the previously isolated environmental stressand ABA-regulated putative proteins from alfalfa (Lu0 et al., 1991, 1992) and, thus, represents a new member of this gene family.

Modification of Phospholipid Catabolism in Microsomal Membranes of [gamma]-Irradiated Cauliflower (Brassica oleracea L.)

Acceleration of membrane deterioration has been observed recently during storage of [gamma]-irradiated cauliflower (Brassica oleracea L., Botrytis group). In the present study, the activity of microsome-associated lipolytic enzymes was investigated in cauliflower florets exposed to 0 or 4 kilograys of [gamma] radiation and stored for 8 d at 13[deg]C. Radiolabeled breakdown products obtained from the metabolism of (16:0/18:2*)-phosphatidylcholine and (16:0/16:0)-phosphatidyl-[N-methyl-3H]choline by microsomal membranes indicated that phospholipase D (EC 3.1.4.4), phosphatidic acid phosphatase (EC 3.1.3.4), and lipolytic acyl hydrolase were associated with the membranes. The rate of phosphatidylcholine catabolism by the membranes increased slowly in control cauliflower during storage. [gamma] irradiation caused an immediate rise in phosphatidylcholine catabolism that remained higher than that of the controls during subsequent storage. Collectively, the data suggest that enhancement of membrane lipolytic activity results from free-radical-induced stress. Rapid increase of the membrane-associated phospholipase D activity may be a key event leading to accelerated membrane deterioration following [gamma] irradiation.

Plant N-glycan profiling of minute amounts of material.

Development of convenient strategies for identification of plant N-glycan profiles has been driven by the emergence of plants as an expression system for therapeutic proteins. In this article, we reinvestigated qualitative and quantitative aspects of plant N-glycan profiling. The extraction of plant proteins through a phenol/ammonium acetate procedure followed by deglycosylation with peptide N-glycosidase A (PNGase A) and coupling to 2-aminobenzamide provides an oligosaccharide preparation containing reduced amounts of contaminants from plant cell wall polysaccharides. Such a preparation was also suitable for accurate qualitative and quantitative evaluation of the N-glycan content by mass spectrometry. Combining these approaches allows the profiling to be carried out from as low as 500 mg of fresh leaf material. We also demonstrated that collision-induced dissociation (CID) mass spectrometry in negative mode of N-glycans harboring alpha(1,3)- or alpha(1,6)-fucose residue on the proximal GlcNAc leads to specific fragmentation patterns, thereby allowing the discrimination of plant N-glycans from those arising from mammalian contamination.

Biochemical composition of haemagglutinin‐based influenza virus‐like particle vaccine produced by transient expression in tobacco plants

Influenza virus-like particles (VLPs) are noninfectious particles resembling the influenza virus representing a promising vaccine alternative to inactivated influenza virions as antigens. Medicago inc. has developed a plant-based VLP manufacturing platform allowing the large-scale production of GMP-grade influenza VLPs. In this article, we report on the biochemical compositions of these plant-based influenza candidate vaccines, more particularly the characterization of the N-glycan profiles of the viral haemagglutinins H1 and H5 proteins as well as the tobacco-derived lipid content and residual impurities. Mass spectrometry analyses showed that all N-glycosylation sites of the extracellular domain of the recombinant haemagglutinins carry plant-specific complex-type N-glycans having core α(1,3)-fucose, core β(1,2)-xylose epitopes and Lewis(a) extensions. Previous phases I and II clinical studies have demonstrated that no hypersensibility nor induction of IgG or IgE directed against these glycans was observed. In addition, this article showed that the plant-made influenza vaccines are highly pure VLPs preparations while detecting no protein contaminants coming either from Agrobacterium or from the enzymes used for the enzyme-assisted extraction process. In contrast, VLPs contain few host cell proteins and glucosylceramides associated with plant lipid rafts. Identification of such raft markers, together with the type of host cell impurity identified, confirmed that the mechanism of VLP formation in planta is similar to the natural process of influenza virus assembly in mammals.

A comparative study between an endoglucanase IV and its fused protein complex Cel5-CBM6

The recombinant endoglucanase IV (Cel5; encoded by egIV) of Ruminococcus albus was compared with protein Cel5-CBM6 comprised of Cel5 fused at the C-terminus with the single-cellulose binding domain II (CBM6) of Clostridium stercorarium xylanase A, in order to improve its binding ability. Previous analyses using ball-milled cellulose had suggested that a cellulose binding domain of xylanase A could enhance cellulase activity, especially with insoluble substrates. Comparison of the catalytic activities of Cel5 and Cel5-CBM6 were determined using carboxymethylcellulose, Avicel, and filter paper as substrates. This study confirmed previous findings, and provided further evidence suggesting that Cel5-CBM6 exhibits enhanced activity with insoluble cellulose compared to native Cel5. However, its hydrolytic activity with soluble substrates such as carboxymethylcellulose was comparable to Cel5. For both cellulases, central linkages of cellulooligosaccharides (up to six glucose residues) were found to be the preferred points of cleavage. The rates of hydrolysis with both cellulases increased with cellulooligosaccharide chain length, and at least three consecutive glycosyl residues seemed to be necessary for hydrolysis to occur. Cel5-CBM6 showed a higher affinity for cellulose substrates than did Cel5, as demonstrated by transmission electron microscopy. Taken together, these results suggest that CBM6 increases the affinity of Cel5 for insoluble substrates, and this increased binding capacity seems to result in increased catalytic activity.

PLANT EXPRESSION SYSTEM

A plant expression system and methods for expressing a protein of interest in a plant are provided. The plant expression system comprises a first nucleic acid sequence regulatory region sequence, operatively linked with a one or more than one comovirus enhancer, a nucleotide sequence of interest, one or more than one geminivirus amplification elements, and a second nucleic acid encoding a geminivirus replicase. The method of producing a protein of interest in a plant, involves introducing the plant expression system into a plant, or portion of the plant, and incubating the plant or the portion of the plant under conditions that permit the expression of the nucleotide sequence and producing the protein of interest.

Modulating secretory pathway pH by proton channel co-expression can increase recombinant protein stability in plants

Eukaryotic expression systems are used for the production of complex secreted proteins. However, recombinant proteins face considerable biochemical challenges along the secretory pathway, including proteolysis and pH variation between organelles. As the use of synthetic biology matures into solutions for protein production, various host-cell engineering approaches are being developed to ameliorate host-cell factors that can limit recombinant protein quality and yield. We report the potential of the influenza M2 ion channel as a novel tool to neutralize the pH in acidic subcellular compartments. Using transient expression in the plant host, Nicotiana benthamiana, we show that ion channel expression can significantly raise pH in the Golgi apparatus and that this can have a strong stabilizing effect on a fusion protein separated by an acid-susceptible linker peptide. We exemplify the utility of this effect in recombinant protein production using influenza hemagglutinin subtypes differentially stable at low pH; the expression of hemagglutinins prone to conformational change in mildly acidic conditions is considerably enhanced by M2 co-expression. The co-expression of a heterologous ion channel to stabilize acid-labile proteins and peptides represents a novel approach to increasing the yield and quality of secreted recombinant proteins in plants and, possibly, in other eukaryotic expression hosts.

Alfalfa, A Perennial Source of Recombinant Proteins

Alfalfa (Medicago sativa L.), the most important forage crop in the world, is now finding a new mission as a bioreactor for the production of useful proteins. Its advantageous agronomic characteristics, as well as its simple composition with regards to secondary metabolites, has motivated its choice as cellular factory for protein production. Alfalfa is a perennial crop that will yield 10 to 12 tons of dry matter for up to 6 years in the field, and more than 10 years when grown in a greenhouse. It fixes atmospheric nitrogen through a symbiotic association with root-invading bacteria, and thus has no fertilization requirements, if not for microelement deficiencies in some soil types. The plant is also propagated easily by stem cuttings, and has a strong regenerative capacity, which gives the possibility to produce large clonal populations within a short time frame. Finally, purification of recombinant proteins from its sub-fractions is fast and easy due to past industrial developments on wet fractionation processes for animal feed production.

Cell wall biochemical alterations during Agrobacterium-mediated expression of hemagglutinin-based influenza virus-like vaccine particles in tobacco

Summary Influenza virus‐like particles (VLPs) have been shown to induce a safe and potent immune response through both humoral and cellular responses. They represent promising novel influenza vaccines. Plant‐based biotechnology allows for the large‐scale production of VLPs of biopharmaceutical interest using different model organisms, including Nicotiana benthamiana plants. Through this platform, influenza VLPs bud from the plasma membrane and accumulate between the membrane and the plant cell wall. To design and optimize efficient production processes, a better understanding of the plant cell wall composition of infiltrated tobacco leaves is a major interest for the plant biotechnology industry. In this study, we have investigated the alteration of the biochemical composition of the cell walls of N. benthamiana leaves subjected to abiotic and biotic stresses induced by the Agrobacterium‐mediated transient transformation and the resulting high expression levels of influenza VLPs. Results show that abiotic stress due to vacuum infiltration without Agrobacterium did not induce any detectable modification of the leaf cell wall when compared to non infiltrated leaves. In contrast, various chemical changes of the leaf cell wall were observed post‐Agrobacterium infiltration. Indeed, Agrobacterium infection induced deposition of callose and lignin, modified the pectin methylesterification and increased both arabinosylation of RG‐I side chains and the expression of arabinogalactan proteins. Moreover, these modifications were slightly greater in plants expressing haemagglutinin‐based VLP than in plants infiltrated with the Agrobacterium strain containing only the p19 suppressor of silencing.