In this study, gas exchange characteristics and temperature of Nicotiana benthamiana leaves transiently overexpressing hemagglutinin (HA), an influenza vaccine antigen, with an Agrobacterium tumefaciens-assisted viral vector were investigated. Inoculation of leaves with an empty viral vector not containing the HA gene decreased the net photosynthetic rate (Pn) and transpiration rate (T) from 2 to 3 days post-infiltration (DPI) in the A. tumefaciens suspension. Expression of HA with the vector decreased Pn and T to much lower levels until 4 DPI. Such significant decreases were not observed in leaves infiltrated with suspension of A. tumefaciens not carrying the viral vector or in uninfiltrated leaves. Thus, viral vector inoculation itself decreased Pn and T to a certain extent and the HA expression further decreased them. The decreases in Pn and T in empty vector-inoculated and HA expression vector-inoculated leaves were associated with decreases in stomatal conductance, suggesting that the reduction of gas exchange rates was caused at least in part by stomatal closure. More detailed gas exchange and chlorophyll fluorescence analyses revealed that in HA vector-inoculated leaves, the capacity of ribulose-1,5-bisphosphate carboxylase/oxygenase to assimilate CO2 and the capacity of photosynthetic electron transport in planta were downregulated, which contributed also to the decrease in Pn. Leaf temperature (LT) increased in viral vector-inoculated leaves, which was associated with the decrease in T. When HA vector-inoculated leaves were grown at air temperatures (ATs) of 21, 23, and 26°C post-infiltration, HA accumulated earlier in leaves and the days required for HA content to attain its peak became shorter, as AT was higher. The highest LT was found 1–2 days earlier than the highest leaf HA content under all post-infiltration AT conditions. This phenomenon could be applicable in a non-destructive technique to detect the optimum harvesting date for individual plants to determine the day when leaf HA content reaches its maximum level, irrespective of spatiotemporal variation of AT, in a plant growth facility.

We investigated the effects of photosynthetic photon flux density PPFD , air temperature, and CO 2 concentration before gene transfer on the accumulation level of hemagglutinin HA , an influenza vaccine antigen, in Nicotiana benthamiana leaves in a transient gene expression system. Plants were treated for two weeks before gene transfer with different levels of PPFD, air temperature or CO 2 concentration in separate experiments. Leaf fresh mass FM , leaf HA content per unit FM, and leaf HA content per plant at 6 d post infiltration 6 DPI for gene transfer all tended to increase with increasing average PPFD during the treatment period. Leaf FM and leaf HA content per plant at 6 DPI were significantly greater at an average day air temperature of 25 C-30 C during the treatment period than at 20 C-25 C. No clear effects of CO 2 concentration were found on either growth or leaf HA content under the conditions tested in the present study. There was a positive correlation between leaf HA content per unit FM at 6 DPI and leaf FM at 0 or 6 DPI in plants grown under different PPFDs and air temperatures. Preparing plants with increased leaf FM by applying high PPFD and / or high air temperature before gene transfer should increase not only total HA yield per plant but also HA content per unit leaf biomass at harvest.