Plant-made pharmaceutical (PMP) protein production or molecular farming is attracting considerable interest as a novel system for production of therapeutic proteins. Using plant-based expression systems, various PMP proteins, including vaccines, antibodies, and other proteins such as hormones, growth factors, blood proteins, cytokines, and enzymes can be synthesized (De Muynck et al., 2010; Matoba et al., 2011; Rybicki, 2010). Several PMP products have been or are tested in early phase clinical trials and show safety and efficacy (Yusibov et al., 2011). The biotechnology-based pharmaceutical market is more than $80 billion in 2008 (Strohl and Knight, 2009), and PMP may be expected to account for a part of it in the near future. Greenhouse tomato production is considered as a suitable system for PMPs in terms of availability of a relatively efficient transformation system (Mason et al., 2002), high biomass yield (Twyman et al., 2003), containment to prevent transgene flow to the outside (Twyman et al., 2003), and capability of environmental control for steering the plant growth to maximize the protein productivity with minimum input of available resources (Matsuda et al., 2009).
Alvarez et al. (2006) developed transgenic tomato lines transformed with the Y. pestis f1-v fusion gene encoding the F1-V fusion protein, a subunit vaccine candidate against bubonic and pneumonic plague, and driven by the constitutive cauliflower mosaic virus (CaMV) 35S promoter. They demonstrated with mice that orally delivered freeze-dried fruit of the transgenic plants was immunogenic (Alvarez et al., 2006) and protective against a challenge of Y. pestis (Alvarez and Cardineau, 2010), suggesting that it can be used as an edible vaccine. Our previous studies showed that the transgenic plants grown in a growth chamber (Alvarez et al., 2006) had a 6-fold higher fruit F1-V protein concentration than those in a greenhouse (Matsuda et al., 2009), suggesting that the fruit F1-V protein concentrations were possibly affected by growing conditions, cultural practice, or both. However, partly because protein is not a typical quality attribute in tomato fruit, there is limited information available for environmental and cultural factors affecting fruit protein concentrations. We believe that target protein productivity can be maximized by carefully optimizing environmental conditions around the plants and that inappropriate control of abiotic environments could decrease the production of transgenic proteins in plants. In the commercial context, the optimal environments for PMP production would be indispensable information for enhancing protein productivity per unit area and time or for preventing the potential loss of a protein product.
Stevens et al. (2007) showed that moderate water stress due to less irrigation enhanced the concentration of subunit vaccine candidate against anthrax in leaves of transgenic tobacco (Nicotiana tabacum). Providing water stress to plants by lowering water potential in the root zone is simply, predictably, and economically achievable in greenhouse tomato hydroponics, by increasing the EC of the nutrient solution by adding sodium chloride (NaCl) (Wu and Kubota, 2008a) and has been commercially practiced to improve the flavor in tomato (Wu and Kubota, 2008a). Applying low water potential in root zone generally “concentrates” soluble solids in tomato fruit (Adams, 1991; Krauss et al., 2006; Lin and Glass, 1999; Mitchell et al., 1991; Wu et al., 2004; Wu and Kubota, 2008b). Similarly, fruit TSP and F1-V in fruit may be increased in the transgenic tomato grown under a high EC (more negative water potential in the root zone). However, to our knowledge, there is no information available on the dynamics of TSP or a target protein concentration in tomato fruit under high EC.
The aim of this study was thus to investigate the effects of an increased EC of hydroponic nutrient solution on TSP and F1-V protein concentrations in fruit of the transgenic tomato grown in a greenhouse. Whole-plant growth and development, and leaf gas exchange characteristics were also evaluated to examine whether there were adverse effects of the high EC (Adams, 1991; Romero-Arande et al., 2001). Results would provide information on how important strict management of nutrient solution EC is for optimizing practical F1-V production with the transgenic tomato in greenhouse.
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