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Environmental control of plant incubation conditions before and after agroinfiltration is crucial during recombinant protein production using an Agrobacterium-mediated transient gene expression system to ensure high and consistent productivity. In an effort to design a low-cost and energy-saving upstream production process, we assessed the lighting effects during the post-agroinfiltration process on the leaf content of recombinant influenza hemagglutinin (HA) expressed by the magnICON® deconstructed viral vector system. Detached Nicotiana benthamiana leaves were incubated for 6 days post-agroinfiltration under five different lighting conditions: (1) 6-day dark (D–D), (2) 3-day dark followed by 3-day lighting at a photosynthetic photon flux density (PPFD) of 100 µmol m−2 s−1 (D–L100) and (3) its reversed lighting condition (L100–D), and (4) 6-day lighting at a PPFD of 100 µmol m−2 s−1 (L100–L100) and (5) 500 µmol m−2 s−1 (L500–L500). The HA content at 6 days post-agroinfiltration was 25 µg gFM−1 (D–D), 408 µg gFM−1 (D–L100), 566 µg gFM−1 (L100–L100), 693 µg gFM−1 (L500–L500) and 756 µg gFM−1 (L100–D), respectively. The HA content in L100–D was not less than that in the other lighting conditions, indicating that lighting during the latter half of the post-agroinfiltration process is not crucial for recombinant protein production. Based on these findings, we propose to implement a multiphase environment control in the upstream production process of transient expression system, wherein reduced number of light sources may provide a low-cost and energy-saving design for the post-agroinfiltration phase. Post-agroinfiltration 3-day lighting and subsequent 3-day dark enabled detached Nicotiana benthamiana leaves to produce and accumulate a high amount of recombinant influenza hemagglutinin using the magnICON® transient expression system.

The morphology of plants growing under combined blue- and red-light irradiation is affected by the presence or absence of time slots of blue- and red-light mono-irradiation. The purposes of this study were to investigate the morphology and growth of cos lettuce grown under light irradiation combining several durations of blue and red light simultaneously and independent mono-irradiations of blue and red light during the day, and to clarify the effects of the durations of blue-light mono-irradiation and blue-light irradiation. Young cos lettuce seedlings were grown under 24-h blue-light irradiation with a photosynthetic photon flux density (PPFD) of 110μmol m−2 s−1 (B+0R) or under 24-h blue-light irradiation with a PPFD of 100μmol m−2 s−1 supplemented with 8 (B+8R), 16 (B+16R), and 24-h (B+24R) red-light irradiation with PPFDs of 30, 15, and 10μmol m−2 s−1, respectively (Experiment 1). The daily light integral was 9.50mol m−2 in all treatments. In Experiment 1, leaf elongation was promoted as the duration of red-light irradiation decreased and the duration of blue-light mono-irradiation increased. The maximum shoot dry weight was observed under the B+8R treatment. Growth was likely promoted by the expansion of the light-receptive area caused by moderate leaf elongation without tilting. In Experiment 2, young cos lettuce seedlings were grown as for Experiment 1, but blue- and red-light irradiation intensities were reversed (R+0B, R+8B, R+16B, and R+24B). Leaf elongation was promoted by the absence of blue-light irradiation (R+0B). The leaf surface was increasingly flattened, and the shoot dry weight was enhanced, as the duration of blue-light irradiation increased. Thus, cos lettuce leaf morphology may be manipulated by adjusting each duration of blue-light mono-irradiation, red-light mono-irradiation, and blue- and red-light simultaneous irradiation, which can, in turn, promote cos lettuce growth.

BackgroundPhosphor-converted white LEDs (PCW-LEDs) of numerous types with different relative spectral photon-flux-density distributions (SPDs) are commercially available today. Some are regarded as promising light sources for plant factories with artificial lighting. Therefore, some methods must be developed for rapidly selecting appropriate ones from numerous types of PCW-LEDs with relevant evaluation criteria. For rapid determination of leaf net photosynthetic rates (Pn) under dozens of PCW-LED lights with different SPDs, we have developed a rapid and mostly automated Pn-quantification method.ResultsThe method uses a modified LED-artificial sunlight source system (LASS system) and a Pn-measurement system. The modified LASS system includes LEDs of 30 types with different relative SPDs: monochromatic LEDs of 27 types (426–826 nm peak wavelengths) and PCW-LEDs of three types. The system combines the LED lights to produce light with an SPD, which can accurately approximate that of any PCW-LED light at a photosynthetic photon flux density (PPFD) of 150 μmol m−2 s−1. The Pn-quantification method includes two steps: Step 1 – producing the selected PCW-LED lights; Step 2 – using the modified LASS system to supply all the produced lights automatically and successively to an identical leaf and simultaneously measuring Pn using a Pn-measurement system. We produced PCW-LED lights of 30 types at a PPFD of 150 μmol m−2 s−1 within 2.5 h. Then we individually measured the Pn of cos lettuce, red-leaf lettuce, and green-leaf lettuce (Lactuca sativa L.) plants under all produced lights in 16 h per cultivar per repetition. Results show that the mean Pn values of the cos lettuce, red-leaf lettuce, and green-leaf lettuce under the 30 produced lights were, respectively, 7.11–8.02, 5.76–7.11, and 4.83–6.17 μmol m−2 s−1 at 20 days after sowing.ConclusionsA rapid and greatly automated method was developed for successive measurement of Pn under dozens of combined lights, of which each SPD approximated that of the selected PCW-LED lights, within days, which indicates that the method can determine the Pn quickly under numerous PCW-LED lights. Consequently, it contributes to the rapid selection of PCW-LED lights performing high Pn.

Deconstructed viral vector systems for large-scale production of recombinant proteins in Nicotiana benthamiana plants require Agrobacterium tumefaciens-assisted delivery into mesophyll cells by vacuum infiltration of leaves. To clarify the importance of uniform infiltration over the leaf surface and to propose a possible method for uniform infiltration, we quantified the extent of leaf infiltration and evaluated the potential effect of uniform infiltration on recombinant protein yield. We also investigated the effects of plant characteristics (e.g., plant age, leaf dry mass per area) and leaf detachment treatment on the extent of infiltration. First, a simple method was developed to measure the extent of leaf infiltration using a red dye solution. The quantitative results showed that the extent of infiltration in young and old leaves was substantially lower than in mature leaves. However, recombinant hemagglutinin (HA), an influenza vaccine antigen, accumulated in the infiltrated area of young and old leaves, indicating that they can synthesize and accumulate HA at detectable levels. The extent of infiltration was affected by the plant age but not by leaf dry mass per area. Improving the extent of infiltration by supplemental syringe infiltration significantly increased total HA content in leaves. Thus, increasing the infiltrated leaf area represents a potential strategy for increasing the recombinant protein yield in deconstructed viral vector-based transient gene expression systems with A. tumefaciens. The extent of infiltration was also improved without the need for time-consuming syringe infiltration when detached leaves were subjected to vacuum infiltration, suggesting that this may be a potential method to increase the extent of infiltration.

To enable us to analyze more systematically the effects of the spectral distribution of light (i.e. light quality) on photosynthetic electron transport, we propose a simple mathematical model which describes electron transport reactions under light-limited conditions based on the excitation energy distributed to the photosystems. The model assumes that the rate-limiting photosystem performs the photochemical reaction at its maximum yield, while the yield in the other photosystem is passively down-regulated to equalize the rates of linear electron transport through the photosystems. Using intact cucumber leaves, we tested the model by comparing actual and estimated photosynthetic parameters under several combinations of photon flux densities of red and far-red lights (R and FR, respectively). Simultaneously provided R and FR yielded greater gross photosynthetic rates than the sums of the rates under only R and only FR, which is known as the 'enhancement effect'. The present model reproduced these non-additive increases in the gross photosynthetic rates in response to supplemental FR to R and provided more accurate estimates than an existing method that did not take the enhancement effect into account (root mean square errors: 0.11 and 0.21 μmol m-2 s-1, respectively). Using the present model, the photon flux density of the supplemental FR which gives the changing point of rate-limiting photosystem and the photochemical yields of the non-rate-limiting photosystems were estimated reasonably well. The present study has therefore formulated a simplified quantitative electron transport model in response to the light spectrum based on generally accepted concepts and demonstrated its validity experimentally.

Absorbed light energy is converted into excitation energy. The excitation energy is distributed to photosystems depending on the wavelength and drives photochemical reactions. A non-destructive, mechanistic, and quantitative method for estimating the fraction of the excitation energy distributed to photosystem II (f) was developed. To estimate the f values for two simultaneously provided actinic lights (ALs) with different spectral distributions, photochemical yields of the photosystems were measured under the ALs and were then fitted to an electron transport model assuming the balance between the electron transport rates through the photosystems. To test the method using leaves with different properties in terms of the long- and short-term acclimation (adjustment of photosystem stoichiometry and state transition, respectively), the f values for red and far-red light (R and FR) were estimated in leaves grown (~1 week) under white light without and with supplemental FR and adapted (~10 min) to R without and with supplemental FR. The f values for R were clearly greater than those for FR and those of leaves grown with and adapted to supplemental FR tended to be higher than the controls. These results are consistent with previous studies and therefore support the validity of the proposed method.

The Routes of ships are mainly determined based on their speed and roll angles. The routes are also determined based on the estimated weather and wave situation predicted using numerical simulation, economic and political situations, and the captain’s experience. Many researchers have used data assimilation techniques for improving the accuracy of wave prediction. However, there are few studies that focus on data assimilation using ensemble Kalman filter (hereinafter, EnKF) for wave prediction. In this study, we focused on one factor, namely wave prediction for improving the accuracy of the wave prediction. We constructed a data assimilation system that applies EnKF to a wave prediction model SWAN, in order to make wave predictions using real-time observational data obtained from ships. We discuss its applicability by targeting typhoon No. 23 which occurred on October 13, 2004. We also performed sensitivity analysis for examining the accuracy of estimation by changing the number of ensemble members and the parameters of random number generation. In the cases using EnKF, the errors in the significant wave height and peak wave periods between the observational data and simulation results were smaller than those in the original case. It was shown that the accuracy of wave prediction both offshore and near the coast can be improved by using NOWPHAS wave data. Thus, the proposed wave prediction model has enough accuracy to predict the wave situation. Moreover, in cases where a small observation error was assumed, the error in the estimated significant wave height could be reduced by about 93.8 % (at the maximum) compared to the case without using data assimilation.

MR angiography using the silent MR angiography algorithm (silent MRA), which combines arterial spin labeling and an ultrashort time echo, has not been used for the evaluation of cerebral arteriovenous malformations (CAVMs). We aimed to determine the usefulness of silent MRA for the evaluation of CAVMs. Twenty-nine CAVMs of 28 consecutive patients diagnosed by 4D CT angiography or digital subtraction angiography, who underwent both time-of-flight (TOF) MRA and silent MRA, were enrolled. Two observers independently assessed the TOF-MRA and silent MRA images of CAVMs. Micro AVM was defined as AVM with a nidus diameter less than 10 mm. The detection rate, visualization of the components, and accuracy of Spetzler–Martin grade were evaluated with statistical software R. For all 29 CAVMs, 23 (79%) lesions were detected for TOF-MRA and all for silent MRA. Of 10 micro AVMs, only 4 (40%) lesions were detectable on TOF-MRA and all (100%) on silent MRA. The visibility of the nidus and drainer was significantly better for silent MRA than TOF-MRA (p < 0.001), while there was no significant difference in the feeder between the two sequences. The accuracy rates of the Spetzler–Martin grade for the TOF and silent MRA were 38% (11/29) and 79.3% (23/29), respectively (p < 0.001). Silent MRA is useful for evaluating CAVM components and detecting micro AVM.

This study examined the growth and morphology of cos lettuce ( Lactuca sativa L.) to ascertain the effects of irradiation patterns with combinations of blue and red LED light, with spectral photon flux density changing with a period of 24 h. In experiment 1, plants were grown under irradiation patterns in which plants were irradiated with blue and red LED lights at a PPFD of 90 μmol m −2 s −1 for 14 h per day respectively. The red LED light irradiation starting time was simultaneous to or delayed 1, 4, or 7 h from the blue LED light irradiation starting time. Results showed that the shoot fresh weight of plants grown under irradiation patterns with red LED light irradiation being delayed 4 or 7 h from blue LED light irradiation was significantly greater than that under irradiation patterns with blue and red LED light irradiation starting simultaneously. This result showed that the growth of plants can be promoted merely by temporally shifting the irradiation hours of blue and red LED lights. We then conducted experiments to assess the mechanism of the observed growth-promoting effect from viewpoints of the diurnal PPFD change and duration of blue and red light monochromatic irradiation. The results of these experiments revealed that the diurnal PPFD change contributed to the growth-promoting effect and that blue and/or red light monochromatic irradiation increased the total leaf area of plants.

We investigated the effects of spectral distribution and photosynthetic photon flux density (PPFD) for overnight light irradiation supplied by light-emitting diodes (LEDs) on the growth and degree of continuous light (CL)-induced leaf injury of tomato seedlings in a growth-chamber experiment. Over a 14-day treatment period, all plants were irradiated daily for 12 h with white LED light at 300 μmol m−2 s−1 PPFD. During each 12-h nighttime interval, all plants except for the controls were irradiated with white LED light at 150 or 300 μmol m−2 s−1, or alternatively with blue, orange, or red LED light at 150 μmol m−2 s−1. Plants irradiated overnight exhibited injuries, with their visually rated severity of injury increased as the PPFD of nighttime white light was increased. In plants irradiated overnight at 150 μmol m−2 s−1, a higher degree of injury was experienced under blue light than under orange and red light. Overnight white-light irradiation at 150 μmol m−2 s−1 PPFD promoted dry matter production compared with the no-irradiation control, but no further increase was observed at a PPFD of 300 μmol m−2 s−1. Among different spectra, blue light was less effective than orange light at promoting dry matter production when white light was supplied during the day. Thus, both spectral distribution and PPFD of overnight LED light irradiation were demonstrated to affect tomato seedling growth and degree of injury.

The use of detached leaves instead of whole plants provides an alternative means for recombinant protein production based on Agrobacterium tumefaciens-mediated transient gene overexpression. However, the process for high-level protein production in detached leaves has not yet been established. In this study, we focused on leaf handling and maintenance conditions immediately after infiltration with Agrobacterium suspension (agroinfiltration) to improve recombinant protein expression in detached Nicotiana benthamiana leaves. We demonstrated that the residual water of bacterial suspension in detached leaves had significant impact on the yield of recombinant influenza hemagglutinin (HA). Immediately after agroinfiltration, detached leaves were stored in a dehumidified chamber to allow bacterial suspension water occupying intercellular space to be removed by transpiration. We varied the duration of this water removal treatment from 0.7 to 4.4 h, which resulted in leaf fresh weights ranging from 0.94 to 1.28 g g(-1) relative to weights measured just before agroinfiltration. We used these relative fresh weights (RFWs) as an indicator of the amount of residual water. The detached leaves were then incubated in humidified chambers for 6 days. We found that the presence of residual water significantly decreased HA yield, with a clear inverse correlation observed between HA yield and RFW. We next compared HA yields in detached leaves with those obtained from intact leaves by whole-plant expression performed at the same time. The maximum HA yield obtained from a detached leaf with a RFW of approximately 1.0, namely, 800 μg gFW(-1), was comparable to the mean HA yield of 846 μg gFW(-1) generated in intact leaves. Our results indicate the necessity of removing bacterial suspension water from agroinfiltrated detached leaves in transient overexpression systems and point to a critical factor enabling the detached-leaf system as a viable recombinant protein factory.

We investigated the effects of photosynthetic active radiation generated by blue (465 nm peak wavelength) light-emitting diodes (LEDs), red (625 nm peak wavelength) LEDs, or an electroluminescent sheet (EL) (two peak wavelengths: 454 and 565 nm), and dark conditions on the quality preservation of tomato (Solanum lycopersicum) seedlings during low-light irradiation (LLI) storage. The photosynthetic photon flux density (PPFD) at the plant canopy level was automatically controlled using a proportional-integral-derivative (PID) controller to maintain the CO2 exchange rate of the seedlings at zero. Nine seedlings were stored for 21 d in a storage case at 10 ± 0.5°C and at greater than 95% RH. The total dry weight of the tomato seedlings after storage under any LLI was significantly greater compared to that before storage and during dark storage. The PPFD required for maintaining the CO2 exchange rate of the seedlings at zero under all LLI treatments over a 21 d storage period gradually decreased due to the low-light adaptation of the plant. The mean PPFDs to maintain a light compensation point under the blue LED-, red LED-, and EL-LLI for the last week of storage were 5.15, 5.03, and 5.20 µmol·m−2·s−1, respectively. The spectral distribution of the EL light may help preserve seedling quality better than that of the blue or red LED lights and the dark condition, providing a vigorous seedlings with upright morphology and significantly lower specific leaf area (cm2·g−1 DW), which reflects a thick leaf. For green plant storage, an EL or other kind of mixed light is able to control PPFD automatically, which may help maintain seedling quality for more than 3 weeks longer than with blue or red monochromatic light.

Square-wave pulsed light is characterized by three parameters, namely average photosynthetic photon flux density (PPFD), pulsed-light frequency, and duty ratio (the ratio of light-period duration to that of the light–dark cycle). In addition, the light-period PPFD is determined by the averaged PPFD and duty ratio. We investigated the effects of these parameters and their interactions on net photosynthetic rate (P n) of cos lettuce leaves for every combination of parameters. Averaged PPFD values were 0–500 µmol m−2 s−1. Frequency values were 0.1–1000 Hz. White LED arrays were used as the light source. Every parameter affected P n and interactions between parameters were observed for all combinations. The P n under pulsed light was lower than that measured under continuous light of the same averaged PPFD, and this difference was enhanced with decreasing frequency and increasing light-period PPFD. A mechanistic model was constructed to estimate the amount of stored photosynthetic intermediates over time under pulsed light. The results indicated that all effects of parameters and their interactions on P n were explainable by consideration of the dynamics of accumulation and consumption of photosynthetic intermediates.

The Minami-Kanto gas field, where gases are dissolved in formation water, is a potential analogue for a marine gas hydrate area because both areas are characterized by the accumulation of microbial methane in marine turbidite sand layers interbedded with mud layers. This study examined the physicochemical impacts associated with natural gas production and well drilling on the methanogenic activity and composition in this gas field. Twenty-four gas-associated formation water samples were collected from confined sand aquifers through production wells. The stable isotopic compositions of methane in the gases indicated their origin to be biogenic via the carbonate reduction pathway. Consistent with this classification, methanogenic activity measurements using radiotracers, culturing experiments and molecular analysis of formation water samples indicated the predominance of hydrogenotrophic methanogenesis. The cultivation of water samples amended only with methanogenic substrates resulted in significant increases in microbial cells along with high-yield methane production, indicating the restricted availability of substrates in the aquifers. Hydrogenotrophic methanogenic activity increased with increasing natural gas production from the corresponding wells, suggesting that the flux of substrates from organic-rich mudstones to adjacent sand aquifers is enhanced by the decrease in fluid pressure in sand layers associated with natural gas/water production. The transient predominance of methylotrophic methanogens, observed for a few years after well drilling, also suggested the stimulation of the methanogens by the exposure of unutilized organic matter through well drilling. These results provide an insight into the physicochemical impacts on the methanogenic activity in biogenic gas deposits including marine gas hydrates.

Effects of continuous lighting (CL) with or without a diurnal temperature difference (DIF) on leaf photosynthesis, plant growth and development, and carbohydrate accumulation were investigated with tomato. Plants were grown for four weeks under a photoperiod of 12 h d −1 or CL of 24 h d −1 , each combined with a DIF of 10 or 0 °C. Measurements included: leaf net photosynthetic rates, the maximal quantum efficiency of PSII, growth analysis, and quantification of leaf carbohydrates. Severe and moderate CL-induced injury was found on leaves grown under CL with a DIF of 0 and 10 °C, respectively, while it was not observed for plants under 12 h d −1 photoperiod. On the other hand, the apparent severity of the CL-induced injury was not necessarily correlated with leaf net photosynthetic rates. At the initial treatment phase, CL reduced the light-limited photosynthesis while zero DIF reduced the light-saturated photosynthesis, suggesting that CL and zero DIF independently affected different processes in leaf photosynthetic metabolism. Thereafter, CL and zero DIF additively reduced both the light-limited and -saturated photosynthesis. Net photosynthetic rate of leaves under the respective growth conditions were also lowered under CL and/or zero DIF. Such reductions of leaf photosynthesis were reflected in dry matter production at the whole-plant level through decreases in net assimilation rate. Analysis of leaf carbohydrate contents revealed that apparent severity of the CL-induced injury was associated with the diurnal turnover of soluble sugars, suggesting it to be a factor possibly causing the injury.

Transient gene expression in whole plants by using viral vectors is promising as a rapid, mass production system for biopharmaceutical proteins. Recent studies have indicated that plant growth conditions such as air temperature markedly influence the accumulation levels of target proteins. Here, we investigated time course of the amount of recombinant hemagglutinin (HA), a vaccine antigen of influenza virus, in leaves of Nicotiana benthamiana plants grown at 20°C or 25°C post viral vector inoculation. The HA content per unit of leaf biomass increased and decreased from 4 to 6 days post inoculation at 20°C and 25°C, respectively, irrespective of the subcellular localization of HA. The overall HA contents were higher when HA was targeted to the endoplasmic reticulum (ER) rather than the apoplast. Necrosis of leaf tissues was specifically observed in plants inoculated with the ER-targeting vector and grown at 25°C. With the ER-targeting vector, the maximum HA contents at 20°C and 25°C were recorded at 6 and 4 days post inoculation, respectively, and were comparable to each other. HA contents thereafter decreased at both temperatures; the rate of reduction appeared faster at 25°C than at 20°C. From a practical point of view, our results indicate that the strategy of targeting HA to the ER, growing plants at a lower temperature of 20°C, and harvesting leaves at around a week after vector inoculation should be implemented to obtain a high HA yield stably and efficiently.