A trial of production of the plant-derived high-value protein in a plant factory: photosynthetic photon fluxes affect the accumulation of recombinant miraculin in transgenic tomato fruits

Abstract

One of the ultimate goals of plant science is to test a hypothesis obtained by basic science and to apply it to agriculture and industry. A plant factory is one of the ideal systems for this trial. Environmental factors affect both plant yield and the accumulation of recombinant proteins for industrial applications within transgenic plants. However, there have been few reports studying plant productivity for recombinant protein in closed cultivation systems called plant factories. To investigate the effects of photosynthetic photon flux (PPF) on tomato fruit yield and the accumulation of recombinant miraculin, a taste-modifying glycoprotein, in transgenic tomato fruits, plants were cultivated at various PPFs from 100 to 400 (µmol m(-2) s(-)1) in a plant factory. Miraculin production per unit of energy used was highest at PPF100, although miraculin production per unit area was highest at PPF300. The commercial productivity of recombinant miraculin in transgenic tomato fruits largely depended on light conditions in the plant factory. Our trial will be useful to consider the trade-offs between the profits from production of high-value materials in plants and the costs of electricity.

Figure 1

Cross no. 2 plants grown at various photosynthetic photon fluxes (PPFs) two weeks after starting experimental light conditions in a closed cultivation system. Seedlings were grown at PPF400 for approximately one month after germination and were then transferred to PPF100, 200, 300 and 400. Scale bar indicates 5 cm.

Figure 2

Phenotypes of cross no. 2 plants grown at various photosynthetic photon fluxes (PPFs) 2, 3 and 4 weeks after starting experimental light conditions in a closed cultivation system. Plant height (A), plant diameter (B), SPAD value (C) and photosynthetic rates of old and new leaves 2 weeks after starting experimental light conditions (D) were investigated. Photosynthetic rates of old leaves under a first truss and new leaves grown after starting experimental light conditions were measured. The means were statistically separated by Tukey's test (A–C, n = 10; D, n = 5); means that are significantly different at the 5% level are identified by different letters. Vertical bars indicate the standard error (A–C, n = 10; D, n = 5).

Figure 3

Number of fruits on cross no. 2 plants grown at various photosynthetic photon fluxes (PPFs) in a closed cultivation system. Red ripening fruits harvested were also counted 14 weeks after starting experimental light conditions. The means were statistically separated by Tukey's test (n = 8); means that are significantly different at the 5% level are identified by different letters. Vertical bars indicate the standard error (n = 8).

Figure 4

Fruit yield per plant (A) and per area per year in a layer (B) in cross no. 2 plants grown at various photosynthetic photon fluxes (PPFs) in the closed cultivation system. The data presented in (A) are the means ± standard errors of eight plants. The data presented in (B) were calculated from the data presented in (A) and include the planting density (plants m⁻²) and the growing period per year (days year⁻¹).

Figure 5

Accumulation levels and concentrations of recombinant miraculin and soluble protein concentration in the pericarp of cross no. 2 plants. Red-ripening fruits were harvested. Miraculin accumulation was determined by immunoblot analysis. Protein extracts (the equivalent of 50 µgFW per lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Miraculin concentration was measured by ELISA. Miraculin accumulation and concentration data were obtained from three different bulk pericarp tissues from five fruits. The miraculin and soluble protein concentration data were the means ± standard errors of three different bulk pericarp tissues. The means were statistically separated by Tukey's test (n = 3); means that are significantly different at the 5% level are identified by different letters.

Figure 6

Characteristics of the first fruits of cross no. 2 plants grown at various photosynthetic photon fluxes (PPFs) in a closed cultivation system. The first fruits were harvested at red-ripening stage. Harvesting time (A) and miraculin concentration in the exocarp and mesocarp (B). The data presented are the means ± standard errors of eight fruits. The means were statistically separated by Tukey's test (n = 8); means that are significantly different at the 5% level are identified by different letters. Vertical bars indicate standard error (n = 8).

Figure 7

Effect of prolonging harvesting time on miraculin concentration in the exocarp and mesocarp of cross no. 2 fruits grown at a photosynthetic photon flux of 400 µmol m⁻² s⁻¹ in a closed cultivation system. Fruits were harvested from 0 to 6 weeks after orange stage. The miraculin concentration in the pericarp was calculated using the miraculin concentration and the percentage of weight ratio of the exocarp and mesocarp (Sup. Table S2). The means were statistically separated by Tukey's test (n = 8); means that are significantly different at the 5% level are identified by different letters. Vertical bars indicate the standard error (n = 8).

Figure 8

Relative ratio of miraculin production per unit of area and energy from the pericarp of cross no. 2 plants grown at various photosynthetic photon fluxes (PPFs) in a closed cultivation system. Relative ratio of miraculin production per unit of area was calculated based on the miraculin production per layer (Table S3). The values represent means of relative to the value for PPF400 (assigned a value of 1.0; 4.2 g m⁻² year⁻¹). Relative ratio of miraculin production per unit of energy was calculated based on the miraculin production per layer and relative ratios of energy that are 1, 2, 3 and 4 at PPF100, 200, 300 and 400, respectively. The values per unit of energy also represent means of relative to the value for PPF400.