The development of low-alkaloid (LA) tobacco varieties is an important target in the tobacco breeding industry. However, LA Burley 21 plants, in which the Nic1 and Nic2 loci controlling nicotine biosynthesis are deleted, are characterized by impaired leaf maturation that leads to poor leaf quality before and after curing. K E Y W O R D Sethylene, inhibition of biosynthesis, maturation, nic1/nic2 mutation, nicotine, ornithine

Arabidopsis thaliana plants were treated simultaneously with the fungicide tebuconazole and the phytohormone abscisic acid (ABA). We carried out comparative proteomic and transcriptomic analysis against untreated controls under different stress regimes. The chemicals were applied 24 h before the onset of drought stress (removal of the nutrient medium) or salinity stress (hydroponic culture using 150 mM NaCl), and samples were taken during the stress treatments and after a 24 h recovery period. The combined chemical treatment protected plants against both forms of stress. Difference in-gel electrophoresis revealed 18 and 34 unique protein markers representing induced tolerance to drought and salinity stress, respectively. Most of the markers represented plastid functions (such as CO(2) fixation and photosystem II activity), and their abundance was reduced under stress conditions but maintained at near normal levels in the treated plants. The corresponding transcripts were reduced in abundance primarily under drought stress but not salinity stress, indicating that the signal transduction pathways activated by tebuconazole/ABA treatment depend on the nature of the stress stimulus.

Plants adapt to abiotic stress by undergoing diverse biochemical and physiological changes that involve hormone-dependent signaling pathways. The effects of plant hormones can be mimicked by exogenous chemical regulators such as herbicide safeners, which not only enhance stress tolerance but also confer hormetic benefits such as increased vigor and yield. In this study, rice plants growing in normal and saline soils were exposed to abscisic acid (ABA), the safener cyprosulfamide or both compounds together. We found that cyprosulfamide, either alone or in combination with ABA, protected the plants from salinity stress and induced vigorous growth, including the formation of new tillers and early flowering. Proteomic analysis identified several proteins that were induced by stress and/or the chemical treatments, including the late embryogenesis abundant protein OsLEA3, a putative mitochondrial translocase and a putative fumarylacetoacetate hydrolase. The corresponding gene s were induced by stress and/or the individual chemical treatments, but expression dropped back when the stress was removed. However, the combination of ABA and cyprosulfamide prolonged the expression of all three genes beyond the stress period, and allowed the plants to maintain their enhanced growth characteristics. These data support a model involving cooperation between the cyprosulfamide and ABA signaling pathways. Accordingly, it was found that cyprosulfamide induces ABA synthesis more robustly than salinity stress, allowing the two regulators to converge on certain downstream target genes. We discuss the impact of our results on current models for the hormonal regulation of stress response pathways in rice and other plants

BackgroundThe high incidence and mortality rate of malaria remains a serious burden for many developing countries, and a vaccine that induces durable and highly effective immune responses is, therefore, desirable. An earlier analysis of the stage-specific in vitro efficacy of a malaria vaccine candidate cocktail (VAMAX) considered the general properties of complex multi-component, multi-stage combination vaccines in rabbit immunization experiments using a hyper-immunization protocol featuring six consecutive boosts and a strong, lipopolysaccharide-based adjuvant. This follow-up study investigates the effect of antigen dose on the in vitro efficacy of the malaria vaccine cocktail using a conventional vaccination scheme (one prime and two boosts) and a human-compatible adjuvant (Alhydrogel®).ResultsIgG purified from rabbits immunized with 0.1, 1, 10 or 50 µg doses of the VAMAX vaccine candidate cocktail was analysed for total IgG and antigen-cocktail-specific titers. An increase in cocktail-specific titers was observed between 0.1 and 1 µg and between 10 and 50 µg, whereas no significant difference in titers was observed between 1 and 10 µg. Antigen component-specific antibody titers and stage-specific in vitro efficacy assays were performed with pooled IgG from animals immunized with 1 and 50 µg of the VAMAX cocktail. Here, the component-specific antibody levels showed clear dose dependency whereas the determined stage-specific in vitro IC50 values (as a correlate of efficacy) were only dependent on the titer amounts of stage-specific antibodies.ConclusionsThe stage-specific in vitro efficacy of the VAMAX cocktail strongly correlates with the corresponding antigen-specific titers, which for their part depend on the antigen dose, but there is no indication that the dose has an effect on the in vitro efficacy of the induced antibodies. A comparison of these results with those obtained in the previous hyper-immunization study (where higher levels of antigen-specific IgG were observed) suggests that there is a significant need to induce an immune response matching efficacy requirements, especially for a PfAMA1-based blood stage vaccine, by using higher doses, better adjuvants and/or better formulations.

Pichia pastoris is a simple and powerful expression platform that has the ability to produce a wide variety of recombinant proteins, ranging from simple peptides to complex membrane proteins. A well-established fermentation strategy is available comprising three main phases: a batch phase, followed by a glycerol fed-batch phase that increases cell density, and finally an induction phase for product expression using methanol as the inducer. We previously used this three-phase strategy at the 15-L scale to express three different AMA1-DiCo-based malaria vaccine candidates to develop a vaccine cocktail. For two candidates, we switched to a two-phase strategy lacking the intermediate glycerol fed-batch phase. The new strategy not only provided a more convenient process flow but also achieved 1.5-fold and 2.5-fold higher space-time yields for the two candidates, respectively, and simultaneously reduced the final cell mass by a factor of 1.3, thus simplifying solid–liquid separation. This strategy also reduced the quantity of host cell proteins that remained to be separated from the two vaccine candidates (by 34% and 13%, respectively), thus reducing the effort required in the subsequent purification steps. Taken together, our new fermentation strategy increased the overall fermentation performance for the production of two different AMA1-DiCo-based vaccine candidates.