Towards improved dynamic photosynthesis in C3 crops by utilizing natural genetic variation.

Under field environments, fluctuating light conditions induce dynamic photosynthesis, which affects carbon gain by crop plants. Elucidating the natural genetic variations among untapped germplasm resources and their underlying mechanisms can provide an effective strategy to improve dynamic photosynthesis and, ultimately, improve crop yields through molecular breeding approaches. In this review, we first overview two processes affecting dynamic photosynthesis, namely (i) biochemical processes associated with CO2 fixation and photoprotection and (ii) gas diffusion processes from the atmosphere to the chloroplast stroma. Next, we review the intra- and interspecific variations in dynamic photosynthesis in relation to each of these two processes. It is suggested that plant adaptations to different hydrological environments underlie natural genetic variation explained by gas diffusion through stomata. This emphasizes the importance of the coordination of photosynthetic and stomatal dynamics to optimize the balance between carbon gain and water use efficiency under field environments. Finally, we discuss future challenges in improving dynamic photosynthesis by utilizing natural genetic variation. The forward genetic approach supported by high-throughput phenotyping should be introduced to evaluate the effects of genetic and environmental factors and their interactions on the natural variation in dynamic photosynthesis.

Flavodiiron proteins enhance the rate of CO2 assimilation in Arabidopsis under fluctuating light intensity.

The proton concentration gradient (ΔpH) and membrane potential (Δψ) formed across the thylakoid membrane contribute to ATP synthesis in chloroplasts. Additionally, ΔpH downregulates photosynthetic electron transport via the acidification of the thylakoid lumen. K+ exchange antiporter 3 (KEA3) relaxes this downregulation by substituting ΔpH with Δψ in response to fluctuation of light intensity. In the Arabidopsis (Arabidopsis thaliana) line overexpressing KEA3 (KEA3ox), the rate of electron transport is elevated by accelerating the relaxation of ΔpH after a shift from high light (HL) to low light. However, the plant cannot control electron transport toward photosystem I (PSI), resulting in PSI photodamage. In this study, we crossed the KEA3ox line with the line (Flavodiiron [Flv]) expressing the Flv proteins of Physcomitrium patens. In the double transgenic line (Flv-KEA3ox), electrons overloading toward PSI were pumped out by Flv proteins. Consequently, photodamage of PSI was alleviated to the wild-type level. The rate of CO2 fixation was enhanced in Flv and Flv-KEA3ox lines during HL periods of fluctuating light, although CO2 fixation was unaffected in any transgenic lines in constant HL. Upregulation of CO2 fixation was accompanied by elevated stomatal conductance in fluctuating light. Consistent with the results of gas exchange experiments, the growth of Flv and Flv-KEA3ox plants was better than that of WT and KEA3ox plants under fluctuating light.

MIC-100, a new system for high-throughput phenotyping of instantaneous leaf photosynthetic rate in the field.

Photosynthesis occurs mainly in plant leaves and is a fundamental process in the global carbon cycle and in crop production. The exploitation of natural genetic variation in leaf photosynthetic capacity is a promising strategy to meet the increasing demand for crops. The present study reports the newly developed photosynthesis measurement system 'MIC-100,' with a higher throughput for measuring instantaneous photosynthetic rate in the field. MIC-100 is established based on the closed system and directly detects the CO2 absorption in the leaf chamber. The reproducibility, accuracy, and measurement throughput of MIC-100 were tested using soybean (Glycine max L. (Merr.)) and rice (Oryza sativa L.) grown under field conditions. In most cases, the coefficient of variance (CV) for repeated-measurements of the same leaf was less than 0.1. The photosynthetic rates measured with the MIC-100 model showed a significant correlation (R2 = 0.93-0.95) with rates measured by a widely used gas-exchange system. The measurement throughput of the MIC-100 is significantly greater than that of conventional open gas-exchange systems under field conditions. Although MIC-100 solely detects the instantaneous photosynthetic rate under a given environment, this study demonstrated that the MIC-100 enables the rough evaluation of leaf photosynthesis within the large-scale plant populations grown in the field.

Drought stress reduces crop carbon gain due to delayed photosynthetic induction under fluctuating light conditions.

Drought stress is a major limiting factor for crop growth and yield. Water availability in the field can cyclically change between drought and rewatering conditions, depending on precipitation patterns. Concurrently, light intensity under field conditions can fluctuate, inducing dynamic photosynthesis and transpiration during the crop growth period. The present study aimed to characterize carbon gain and water use in fluctuating light under drought and rewatering conditions in two major crops, namely rice and soybean. We conducted gas exchange measurements under fluctuating light conditions with rice and soybean plants exposed to drought treatment (9-13 days) imposed by withholding water and subsequent rewatering treatment (8-9 days). Drought stress significantly reduced the maximum CO2 assimilation rate (A) in soybean but not in rice. Under drought conditions, A increased after a step increase in light and then gradually decreased in both crops, resulting in the significant reduction of steady-state A in rice and soybean. Moreover, drought stress delayed photosynthetic induction in both crops even when it had relatively small impact on maximum A. These results suggest that the drought effects on photosynthesis should be evaluated based on induction, maximum, and steady states. The delayed photosynthetic induction under drought owing to the reduced gas diffusional conductance via stomata resulted in a substantial loss of leaf carbon gain under fluctuating light conditions. Meanwhile, rewatering, after drought, completely recovered photosynthesis under fluctuating light in both crops. Therefore, the stability of photosynthetic induction can be a promising target to improve drought tolerance during crop breeding in the future.

Minimizing VPD Fluctuations Maintains Higher Stomatal Conductance and Photosynthesis, Resulting in Improvement of Plant Growth in Lettuce.

Vapor pressure deficit (VPD) is considered to be one of the major environmental factors influencing stomatal functions and photosynthesis, as well as plant growth in crop and horticultural plants. In the greenhouse cultivation, air temperature and relative air humidity are regulated by switching on/off the evaporative systems and opening/closing the roof windows, which causes VPD fluctuation. However, it remains unclear how VPD fluctuation affects photosynthetic and growth performance in plants. Here, we examined the effects of the VPD fluctuation on the photosynthetic and growth characteristics in lettuce (Lactuca sativa L.). The parameters for gas exchange and chlorophyll fluorescence and biomass production were evaluated under the conditions of drastic (1.63 kPa for 6 min and 0.63 for 3 min) or moderate (1.32 kPa for 7 min and 0.86 kPa for 3 min) VPD fluctuation. The drastic VPD fluctuation induced gradual decrease in stomatal conductance and thus CO2 assimilation rate during the measurements, while moderate VPD fluctuation caused no reduction of these parameters. Furthermore, data showed moderate VPD fluctuation maintained leaf expansion and the efficiency of CO2 diffusion across leaf surface, resulting in enhanced plant growth compared with drastic VPD fluctuation. Taken together, fine regulation of VPD can be crucial for better plant growth by maintaining the photosynthetic performance in lettuce. The present work demonstrates the importance of VPD control during plant cultivation in plant factories and greenhouses.

Overexpression of both Rubisco and Rubisco activase rescues rice photosynthesis and biomass under heat stress.

Global warming threatens food security by decreasing crop yields through damage to photosynthetic systems, especially Rubisco activation. We examined whether co-overexpression of Rubisco and Rubisco activase improves the photosynthetic and growth performance of rice under high temperatures. We grew three rice lines-the wild-type (WT), a Rubisco activase-overexpressing line (oxRCA) and a Rubisco- and Rubisco activase-co-overexpressing line (oxRCA-RBCS)-and analysed photosynthesis and biomass at 25 and 40°C. Compared with the WT, the Rubisco activase content was 153% higher in oxRCA and 138% higher in oxRCA-RBCS, and the Rubisco content was 27% lower in oxRCA and similar in oxRCA-RBCS. The CO2 assimilation rate (A) of WT was lower at 40°C than at 25°C, attributable to Rubisco deactivation by heat. On the other hand, that of oxRCA and oxRCA-RBCS was maintained at 40°C, resulting in higher A than WT. Notably, the dry weight of oxRCA-RBCS was 26% higher than that of WT at 40°C. These results show that increasing the Rubisco activase content without the reduction of Rubisco content could improve yield and sustainability in rice at high temperature.

Stomatal, mesophyll conductance, and biochemical limitations to photosynthesis during induction.

The dynamics of leaf photosynthesis in fluctuating light affects carbon gain by plants. Mesophyll conductance (gm) limits CO2 assimilation rate (A) under the steady state, but the extent of this limitation under non-steady-state conditions is unknown. In the present study, we aimed to characterize the dynamics of gm and the limitations to A imposed by gas diffusional and biochemical processes under fluctuating light. The induction responses of A, stomatal conductance (gs), gm, and the maximum rate of RuBP carboxylation (Vcmax) or electron transport (J) were investigated in Arabidopsis (Arabidopsis thaliana (L.)) and tobacco (Nicotiana tabacum L.). We first characterized gm induction after a change from darkness to light. Each limitation to A imposed by gm, gs and Vcmax or J was significant during induction, indicating that gas diffusional and biochemical processes limit photosynthesis. Initially, gs imposed the greatest limitation to A, showing the slowest response under high light after long and short periods of darkness, assuming RuBP-carboxylation limitation. However, if RuBP-regeneration limitation was assumed, then J imposed the greatest limitation. gm did not vary much following short interruptions to light. The limitation to A imposed by gm was the smallest of all the limitations for most of the induction phase. This suggests that altering induction kinetics of mesophyll conductance would have little impact on A following a change in light. To enhance the carbon gain by plants under naturally dynamic light environments, attention should therefore be focused on faster stomatal opening or activation of electron transport.

Higher Stomatal Density Improves Photosynthetic Induction and Biomass Production in Arabidopsis Under Fluctuating Light.

Stomatal density (SD) is closely associated with photosynthetic and growth characteristics in plants. In the field, light intensity can fluctuate drastically within a day. The objective of the present study is to examine how higher SD affects stomatal conductance (g s ) and CO2 assimilation rate (A) dynamics, biomass production and water use under fluctuating light. Here, we compared the photosynthetic and growth characteristics under constant and fluctuating light among three lines of Arabidopsis thaliana (L.): the wild type (WT), STOMAGEN/EPFL9-overexpressing line (ST-OX), and EPIDERMAL PATTERNING FACTOR 1 knockout line (epf1). ST-OX and epf1 showed 268.1 and 46.5% higher SD than WT (p < 0.05). Guard cell length of ST-OX was 10.0% lower than that of WT (p < 0.01). There were no significant variations in gas exchange parameters at steady state between WT and ST-OX or epf1, although these parameters tended to be higher in ST-OX and epf1 than WT. On the other hand, ST-OX and epf1 showed faster A induction than WT after step increase in light owing to the higher g s under initial dark condition. In addition, ST-OX and epf1 showed initially faster g s induction and, at the later phase, slower g s induction. Cumulative CO2 assimilation in ST-OX and epf1 was 57.6 and 78.8% higher than WT attributable to faster A induction with reduction of water use efficiency (WUE). epf1 yielded 25.6% higher biomass than WT under fluctuating light (p < 0.01). In the present study, higher SD resulted in faster photosynthetic induction owing to the higher initial g s . epf1, with a moderate increase in SD, achieved greater biomass production than WT under fluctuating light. These results suggest that higher SD can be beneficial to improve biomass production in plants under fluctuating light conditions.

Two novel quantitative trait loci affecting the variation in leaf photosynthetic capacity among soybeans.

There is a large variation in CO2 assimilation rate per unit of leaf area (A) within or among crop species, which can be exploited to improve A by elucidating the mechanisms underlying such variation. The objective of the present study is to elucidate the genetic factors affecting the variation in leaf photosynthetic capacity among soybeans. Here, we conducted field experiments over three years, using Enrei, a leading variety in Japan, Peking, a landrace from China and the chromosome segment substitution lines derived from their progenies. The gas exchange measurements were conducted to evaluate A among soybean. Peking showed higher A than Enrei after the flowering in all the years. The genetic analysis identified two novel quantitative trait loci (QTLs) related to variation in A, which were located on chromosome 13 (qLPC13) and 20 (qLPC20). The Peking allele at qLPC13 increased A by 8.3 % in the Enrei genetic background, while the Peking allele at qLPC20 decreased A by 15.3 %. The present study is the first report on QTLs affecting a genotypic variation in leaf photosynthetic capacity among field-grown soybeans. The identification of the causal genes in these QTLs can provide a novel strategy to enhance leaf photosynthetic capacity with soybean breeding.

Genetic Diversity in Stomatal Density among Soybeans Elucidated Using High-throughput Technique Based on an Algorithm for Object Detection.

The stomatal density (SD) can be a promising target to improve the leaf photosynthesis in soybeans (Glycine max (L.) Merr). In a conventional SD evaluation, the counting process of the stomata during a manual operation can be time-consuming. We aimed to develop a high-throughput technique for evaluating the SD and elucidating the variation in the SD among various soybean accessions. The central leaflet of the first trifoliolate was sampled, and microscopic images of the leaflet replica were obtained among 90 soybean accessions. The Single Shot MultiBox Detector, an algorithm for an object detection based on deep learning, was introduced to develop an automatic detector of the stomata in the image. The developed detector successfully recognized the stomata in the microscopic image with high-throughput. Using this technique, the value of R2 reached 0.90 when the manually and automatically measured SDs were compared in the 150 images. This technique discovered a variation in SD from 93 ± 3 to 166 ± 4 mm-2 among the 90 accessions. Our detector can be a powerful tool for a SD evaluation with a large-scale population in crop species, accelerating the identification of useful alleles related to the SD in future breeding programs.