Ryo Matsuda

Analysis of the relationship between blue-light photon flux density and the photosynthetic properties of spinach (Spinacia oleracea L.) leaves with regard to the acclimation of photosynthesis to growth irradiance

Abstract Blue light has been suggested to participate in the acclimation of photosynthesis to growth irradiance. We analyzed the effects of blue light intensity on the photosynthetic properties of leaves with regard to acclimation to irradiance. Spinach (Spinacia oleracea L.) plants were grown under mixtures of blue and red light with blue-light photon flux densities (PFDs) of 0, 30, 100 and 150 µmol m−2 s−1 at a total photosynthetic PFD of 300 µmol m−2 s−1. The light-saturated rate of photosynthesis under white light, leaf N content per unit leaf area, leaf dry weight per unit leaf area and the ratio of cytochrome (Cyt) f content to light-harvesting chlorophyll-binding protein of photosystem II (LHCII) content were evaluated. The photosynthetic rate tended to increase with increasing blue-light PFD up to 100 µmol m−2 s−1, and this was associated with an increase in leaf N content per unit leaf area. However, the increase in leaf N content per unit leaf area did not necessarily result from an increase in leaf dry weight per unit leaf area. The Cyt f to LHCII content ratio increased linearly with increasing blue-light PFD up to 100 µmol m−2 s−1, indicating that plants grown under higher blue-light PFD up to this value resembled plants grown under higher irradiance in terms of N partitioning between electron-transport components and light-harvesting components. This result suggests that the level of blue light is involved only in the acclimation to relatively low growth irradiances at the chloroplast level.

Growth of rice plants under red light with or without supplemental blue light

Abstract The effects of blue light supplementation to red light on growth, morphology and N utilization in rice plants (Oryza sativa L. cv. Sasanishiki and Nipponbare) were investigated. Plants were grown under two light quality treatments, red light alone (R) or red light supplemented with blue light (RB; red/blue-light photosynthetic photon flux density [PPFD] ratio was 4/1), at 380 mol m−2 s−1 PPFD. The biomass production of both cultivars grown under RB conditions was higher than that of plants grown under R conditions. This enhancement of biomass production was caused by an increase in the net assimilation rate (NAR). The higher NAR was associated with a higher leaf N content per leaf area at the whole-plant level, which was accompanied by higher contents of the key components of photosynthesis, including Rubisco and chlorophyll. In Sasanishiki, preferential biomass investment in leaf blades and expansion of wider and thinner leaves also contributed to the enhancement of biomass production. These morphological changes in the leaves were not observed in Nipponbare. Both the changes in physiological characteristics, including leaf photosynthesis, and the changes in morphological characteristics, including leaf development, contributed to the enhancement of biomass production under RB conditions, although the extent of these changes differed between the two cultivars.

Environment Control to Improve Recombinant Protein Yields in Plants Based on Agrobacterium-Mediated Transient Gene Expression.

Agrobacterium-mediated transient expression systems enable plants to produce a wide range of recombinant proteins on a rapid timescale. To achieve economically feasible upstream production and downstream processing, two yield parameters should be considered: (1) recombinant protein content per unit biomass and (2) recombinant protein productivity per unit area–time at the end of the upstream production. Because environmental factors in the upstream production have impacts on these parameters, environment control is important to maximize the recombinant protein yield. In this review, we summarize the effects of pre- and postinoculation environmental factors in the upstream production on the yield parameters and discuss the basic concept of environment control for plant-based transient expression systems. Preinoculation environmental factors associated with planting density, light quality, and nutrient supply affect plant characteristics, such as biomass and morphology, which in turn affect recombinant protein content and productivity. Accordingly, environment control for such plant characteristics has significant implications to achieve a high yield. On the other hand, postinoculation environmental factors, such as temperature, light intensity, and humidity, have been shown to affect recombinant protein content. Considering that recombinant protein production in Agrobacterium-mediated transient expression systems is a result of a series of complex biological events starting from T-DNA transfer from Agrobacterium tumefaciens to protein biosynthesis and accumulation in leaf tissue, we propose that dynamic environment control during the postinoculation process, i.e., changing environmental conditions at an appropriate timing for each event, may be a promising approach to obtain a high yield. Detailed descriptions of plant growth conditions and careful examination of environmental effects will significantly contribute to our knowledge to stably obtain high recombinant protein content and productivity, thus enhancing the utility of plant-based transient expression systems as recombinant protein factories.

A kinetic model for estimating net photosynthetic rates of cos lettuce leaves under pulsed light

Time-averaged net photosynthetic rate (Pn) under pulsed light (PL) is known to be affected by the PL frequency and duty ratio, even though the time-averaged photosynthetic photon flux density (PPFD) is unchanged. This phenomenon can be explained by considering that photosynthetic intermediates (PIs) are pooled during light periods and then consumed by partial photosynthetic reactions during dark periods. In this study, we developed a kinetic model to estimate Pn of cos lettuce (Lactuca sativa L. var. longifolia) leaves under PL based on the dynamics of the amount of pooled PIs. The model inputs are average PPFD, duty ratio, and frequency; the output is Pn. The rates of both PI accumulation and consumption at a given moment are assumed to be dependent on the amount of pooled PIs at that point. Required model parameters and three explanatory variables (average PPFD, frequency, and duty ratio) were determined for the simulation using Pn values under PL based on several combinations of the three variables. The model simulation for various PL levels with a wide range of time-averaged PPFDs, frequencies, and duty ratios further demonstrated that Pn under PL with high frequencies and duty ratios was comparable to, but did not exceed, Pn under continuous light, and also showed that Pn under PL decreased as either frequency or duty ratio was decreased. The developed model can be used to estimate Pn under various light environments where PPFD changes cyclically.

Light-induced systemic regulation of photosynthesis in primary and trifoliate leaves of Phaseolus vulgaris: effects of photosynthetic photon flux density (PPFD) versus spectrum.

The objectives of this work using Phaseolus vulgaris were to examine whether the light spectrum incident on mature primary leaves (PLs) is related to leaf-to-leaf systemic regulation of developing trifoliate leaves (TLs) in photosynthetic characteristics, and to investigate the relative importance of spectrum and photosynthetic photon flux density (PPFD) in light-induced systemic regulation. Systemic regulation was induced by altering PPFD and the spectrum of light incident on PLs using a shading treatment and lighting treatments including either white, blue, green or red light-emitting diodes (LEDs). Photosynthetic characteristics were evaluated by measuring the light-limited and light-saturated net photosynthetic rates and the amounts of nitrogen (N), chlorophyll (Chl) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39). Shading treatment on PLs decreased the amounts of N, Chl and Rubisco of TLs and tended to decrease the photosynthetic rates. However, we observed no systemic effects induced by the light spectrum on PLs in this study, except that a higher amount of Rubisco of TLs was observed when the PLs were irradiated with blue LEDs. Our results imply that photoreceptors in mature leaves have little influence on photosynthetic rates and amounts of N and Chl of developing leaves through systemic regulation, although the possibility of the action of blue light irradiation on the amount of Rubisco cannot be ruled out. Based on these results, we concluded that the light spectrum incident on mature leaves has little systemic effect on developing leaves in terms of photosynthetic characteristics and that the light-induced systemic regulation was largely accounted for by PPFD.

Interaction between the spectral photon flux density distributions of light during growth and for measurements in net photosynthetic rates of cucumber leaves.

The net photosynthetic rate of a leaf becomes acclimated to the plants environment during growth. These rates are often measured, evaluated and compared among leaves of plants grown under different light conditions. In this study, we compared net photosynthetic rates of cucumber leaves grown under white light-emitting diode (LED) light without and with supplemental far-red (FR) LED light (W- and WFR-leaves, respectively) under three different measuring light (ML) conditions: their respective growth light (GL), artificial sunlight (AS) and blue and red (BR) light. The difference in the measured photosynthetic rates between W- and WFR-leaves was greater under BR than under GL and AS. In other words, an interaction between supplemental FR light during growth and the spectral photon flux density distribution (SPD) of ML affected the measured net photosynthetic rates. We showed that the comparison and evaluation of leaf photosynthetic rates and characteristics can be biased depending on the SPD of ML, especially for plants grown under different photon flux densities in the FR waveband. We also investigated the mechanism of the interaction. We confirmed that the distribution of excitation energy between the two photosystems (PSs) changed in response to the SPD of GL, and that this change resulted in the interaction, as suggested in previous reports. However, changes in PS stoichiometry could not completely explain the adjustment in excitation energy distribution observed in this study, suggesting that other mechanisms may be involved in the interaction.

Effect of temperature post viral vector inoculation on the amount of hemagglutinin transiently expressed in Nicotiana benthamiana leaves

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.

Agroinfiltration of leaves for deconstructed viral vector-based transient gene expression: infiltrated leaf area affects recombinant hemagglutinin yield

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.

Viral vector-based transient gene expression in Nicotiana benthamiana: effects of light source on leaf temperature and hemagglutinin content

Plants may serve as a promising platform for the production of recombinant proteins, including biopharmaceuticals. Two main biotechnologies for transgene expression in plants exist: these are stable transformation and transient gene expression. Transient gene expression with viral vectors enables rapid and mass production of recombinant proteins in plant tissues (Marillonnet et al. 2005). In such a system, plants are often grown in a fully contained, environmentally controlled facility to prevent transgene flow to the outside as well as to ensure uniform batch-to-batch productivity. As artificial light sources for plant growth, several kinds of light sources, including fluorescent lamps (FLs) (e.g., Norikane 2015) and light-emitting diodes (LEDs) (e.g., Holtz et al. 2015; Norikane 2015), can be used. These light sources are different in radiative properties, e.g., the spectral distribution of physiologically active radiation (300–800 nm) and radiative emittance of thermal radiation that increases with increasing temperature of the light source. Such radiative properties of light sources can affect the level of recombinant protein accumulation through changes in light and/or thermal environments of plants. Here, we compared FLs and LEDs as light sources for the growth of Nicotiana benthamiana plants post viralvector inoculation to examine the effects on the accumulation of hemagglutinin (HA), an influenza vaccine antigen. Seeds were sown into rockwool cubes (AO36/40; ROCKWOOL B.V., Roermond, The Netherlands) and seedlings were grown in a temperature-controlled growth room equipped with natural white FLs at 210 lmol m s photosynthetic photon flux density (PPFD) for 16 h day. Daily mean air temperatures (AT) were 28 ± 3/23 ± 3 C (day/night). At 14 days after sowing, seedlings were transplanted onto rockwool blocks (Delta 6.5G, ROCKWOOL B.V.). The rockwool blocks were subirrigated with a nutrient solution (prescription A; OAT Agrio Co., Ltd., Tokyo, Japan) adjusted at an electrical conductivity of 0.18 S m and a pH of 6.0. A ‘‘deconstructed’’ tobamoviral replicon system (magnICON; Marillonnet et al. 2005) was used to express a gene encoding the ectodomain of HA derived from the influenza A virus and to localize the protein in the endoplasmic reticulum (Matsuda et al. 2012). A suspension of transformed Agrobacterium tumefaciens GV3101::pMP90 carrying the plasmid vector was vacuum infiltrated into leaves of plants of 35 days (experiments 1 and 3) or 40 days (experiment 2) in age for inoculation as described previously (Matsuda et al. 2012). Plants were thereafter grown in growth chambers equipped with natural white FLs (FPL55EX-N, Iwasaki Electric Co., Ltd., Tokyo, Japan) or phosphor-based white LEDs (NSPW310DS-b2w, Nichia Corp., Tokushima, Japan) (Fig. 1a) at 200 lmol m s PPFD for 16 h day. AT was maintained at 14 ± 1, 17 ± 1, 20 ± 1, 23 ± 1, 26 ± 1, or 29 ± 1 C throughout the day. Experiments were repeated three times, and different combinations of AT treatment were subjected to the respective experiments: in experiment 1, 17, 20, 23, and 26 C for the FL treatment, and 20, 23, 26, and 29 C for Communicated by Neal Stewart.

Effects of plant density on recombinant hemagglutinin yields in an Agrobacterium-mediated transient gene expression system using Nicotiana benthamiana plants

Agrobacterium‐mediated transient expression systems enable plants to rapidly produce a wide range of recombinant proteins. To achieve economically feasible upstream production and downstream processing, it is beneficial to obtain high levels of two yield‐related quantities of upstream production: recombinant protein content per fresh mass of harvested biomass (g gFM−1) and recombinant protein productivity per unit area‐time (g m−2/month). Here, we report that the density of Nicotiana benthamiana plants during upstream production had significant impacts on the yield‐related quantities of recombinant hemagglutinin (HA). The two quantities were smaller at a high plant density of 400 plants m−2 than at a low plant density of 100 plants m−2. The smaller quantities at the high plant density were attributed to: (i) a lower HA content in young leaves, which usually have high HA accumulation potentials; (ii) a lower biomass allocation to the young leaves; and (iii) a high area‐time requirement for plants. Thus, plant density is a key factor for improving upstream production in Agrobacterium‐mediated transient expression systems. Biotechnol. Bioeng. 2017;114: 1762–1770.