The Evolution of C4 Photosynthesis in Flaveria (Asteraceae): Insights from the Flaveria linearis Complex.

Flaveria is a leading model for C4 plant evolution due to the presence of a dozen C3-C4 intermediate species, many of which are associated with a phylogenetic complex centered around Flaveria linearis. To investigate C4 evolution in Flaveria, we updated the Flaveria phylogeny and evaluated gas exchange, starch δ13C, and activity of C4 cycle enzymes in 19 Flaveria species and 28 populations within the F. linearis complex. A principal component analysis identified six functional clusters: (1) C3, (2) sub-C2, (3) full C2, (4) enriched C2, (5) sub-C4, and (6) fully C4 species. The sub-C2 species lacked a functional C4 cycle, while a gradient was present in the C2 clusters from little to modest C4 cycle activity as indicated by δ13C and enzyme activities. Three Yucatan populations of F. linearis had photosynthetic CO2 compensation points equivalent to C4 plants but showed little evidence for an enhanced C4 cycle, indicating they have an optimized C2 pathway that recaptures all photorespired CO2 in the bundle sheath (BS) tissue. All C2 species had enhanced aspartate aminotransferase activity relative to C3 species and most had enhanced alanine aminotransferase activity. These aminotransferases form aspartate and alanine from glutamate and in doing so could help return photorespiratory nitrogen (N) from BS to mesophyll cells, preventing glutamate feedback onto photorespiratory N assimilation. Their use requires upregulation of parts of the C4 metabolic cycle to generate carbon skeletons to sustain N return to the mesophyll, and thus could facilitate the evolution of the full C4 photosynthetic pathway.

Stomatal response to VPD in C4 plants with different biochemical sub-pathways.

C4 NAD-malic enzyme (NAD-ME) species occurs in drier regions and exhibit different drought responses compared to C4 NADP-malic enzyme (NADP-ME) species. However, a physiological mechanism explaining the geographical discrepancies remains uncertain. This study examined gas exchange patterns that might explain different distributions observed between two subtypes of C4 photosynthesis. We measured the response of leaf gas exchange to vapour pressure deficit (VPD) and CO2 in plants from six distinct C4 clades having closely related NAD-ME and NADP-ME species using a Li-Cor 6400 gas exchange system. We found that NAD-ME species exhibited greater relative reductions in stomatal conductance with increases in VPD than NADP-ME species but observed no consistent subtype differences in C4 cycle activity as indicated by the initial slope of the A response to intercellular CO2 concentration. Based on these results, we hypothesise the greater response of gs to increasing VPD may enable NAD-ME plants to outperform NADP-ME plants in hot, dry environments where VPD is normally high.

CAM photosynthesis in Bulnesia retama (Zygophyllaceae), a non-succulent desert shrub from South America.

BACKGROUND AND AIMS: Bulnesia retama is a drought-deciduous, xerophytic shrub from arid landscapes of South America. In a survey of carbon isotope ratios (δ13C) in specimens from the field, B. retama exhibited less negative values, indicative of CAM or C4 photosynthesis. Here, we investigate whether B. retama is a C4 or CAM plant. METHODS: Gas-exchange responses to intercellular CO2, diurnal gas-exchange profiles, δ13C and dawn vs. afternoon titratable acidity were measured on leaves and stems of watered and droughted B. retama plants. Leaf and stem cross-sections were imaged to determine whether the tissues exhibited succulent CAM or C4 Kranz anatomy. KEY RESULTS: Field-collected stems and fruits of B. retama exhibited δ13C between -16 and -19 ‰. Plants grown in a glasshouse from field-collected seeds had leaf δ13C values near -31 ‰ and stem δ13C values near -28 ‰. The CO2 response of photosynthesis showed that leaves and stems used C3 photosynthesis during the day, while curvature in the nocturnal response of net CO2 assimilation rate (A) in all stems, coupled with slightly positive rates of A at night, indicated modest CAM function. C4 photosynthesis was absent. Succulence was absent in all tissues, although stems exhibited tight packing of the cortical chlorenchyma in a CAM-like manner. Tissue titratable acidity increased at night in droughted stems. CONCLUSIONS: Bulnesia retama is a weak to modest C3 + CAM plant. This is the first report of CAM in the Zygophyllaceae and the first showing that non-succulent, xerophytic shrubs use CAM. CAM alone in B. retama was too limited to explain less negative δ13C in field-collected plants, but combined with effects of low stomatal and mesophyll conductance it could raise δ13C to observed values between -16 and -19 ‰. Modest CAM activity, particularly during severe drought, could enable B. retama to persist in arid habitats of South America.

Atmospheric CO2 decline and the timing of CAM plant evolution.

BACKGROUND AND AIMS: CAM photosynthesis is hypothesized to have evolved in atmospheres of low CO2 concentration in recent geological time because of its ability to concentrate CO2 around Rubisco and boost water use efficiency relative to C3 photosynthesis. We assess this hypothesis by compiling estimates of when CAM clades arose using phylogenetic chronograms for 73 CAM clades. We further consider evidence of how atmospheric CO2 affects CAM relative to C3 photosynthesis. RESULTS: Where CAM origins can be inferred, strong CAM is estimated to have appeared in the past 30 million years in 46 of 48 examined clades, after atmospheric CO2 had declined from high (near 800 ppm) to lower (<450 ppm) values. In turn, 21 of 25 clades containing CAM species (but where CAM origins are less certain) also arose in the past 30 million years. In these clades, CAM is probably younger than the clade origin. We found evidence for repeated weak CAM evolution during the higher CO2 conditions before 30 million years ago, and possible strong CAM origins in the Crassulaceae during the Cretaceous period prior to atmospheric CO2 decline. Most CAM-specific clades arose in the past 15 million years, in a similar pattern observed for origins of C4 clades. CONCLUSIONS: The evidence indicates strong CAM repeatedly evolved in reduced CO2 conditions of the past 30 million years. Weaker CAM can pre-date low CO2 and, in the Crassulaceae, strong CAM may also have arisen in water-limited microsites under relatively high CO2. Experimental evidence from extant CAM species demonstrates that elevated CO2 reduces the importance of nocturnal CO2 fixation by increasing the contribution of C3 photosynthesis to daily carbon gain. Thus, the advantage of strong CAM would be reduced in high CO2, such that its evolution appears less likely and restricted to more extreme environments than possible in low CO2.

The CAM lineages of planet Earth.

BACKGROUND AND SCOPE: The growth of experimental studies of crassulacean acid metabolism (CAM) in diverse plant clades, coupled with recent advances in molecular systematics, presents an opportunity to re-assess the phylogenetic distribution and diversity of species capable of CAM. It has been more than two decades since the last comprehensive lists of CAM taxa were published, and an updated survey of the occurrence and distribution of CAM taxa is needed to facilitate and guide future CAM research. We aimed to survey the phylogenetic distribution of these taxa, their diverse morphology, physiology and ecology, and the likely number of evolutionary origins of CAM based on currently known lineages. RESULTS AND CONCLUSIONS: We found direct evidence (in the form of experimental or field observations of gas exchange, day-night fluctuations in organic acids, carbon isotope ratios and enzymatic activity) for CAM in 370 genera of vascular plants, representing 38 families. Further assumptions about the frequency of CAM species in CAM clades and the distribution of CAM in the Cactaceae and Crassulaceae bring the currently estimated number of CAM-capable species to nearly 7 % of all vascular plants. The phylogenetic distribution of these taxa suggests a minimum of 66 independent origins of CAM in vascular plants, possibly with dozens more. To achieve further insight into CAM origins, there is a need for more extensive and systematic surveys of previously unstudied lineages, particularly in living material to identify low-level CAM activity, and for denser sampling to increase phylogenetic resolution in CAM-evolving clades. This should allow further progress in understanding the functional significance of this pathway by integration with studies on the evolution and genomics of CAM in its many forms.

The crucial roles of mitochondria in supporting C4 photosynthesis.

C4 photosynthesis involves a series of biochemical and anatomical traits that significantly improve plant productivity under conditions that reduce the efficiency of C3 photosynthesis. We explore how evolution of the three classical biochemical types of C4 photosynthesis (NADP-ME, NAD-ME and PCK types) has affected the functions and properties of mitochondria. Mitochondria in C4 NAD-ME and PCK types play a direct role in decarboxylation of metabolites for C4 photosynthesis. Mitochondria in C4 PCK type also provide ATP for C4 metabolism, although this role for ATP provision is not seen in NAD-ME type. Such involvement has increased mitochondrial abundance/size and associated enzymatic capacity, led to changes in mitochondrial location and ultrastructure, and altered the role of mitochondria in cellular carbon metabolism in the NAD-ME and PCK types. By contrast, these changes in mitochondrial properties are absent in the C4 NADP-ME type and C3 leaves, where mitochondria play no direct role in photosynthesis. From an eco-physiological perspective, rates of leaf respiration in darkness vary considerably among C4 species but does not differ systematically among the three C4 types. This review outlines further mitochondrial research in key areas central to the engineering of the C4 pathway into C3 plants and to the understanding of variation in rates of C4 dark respiration.

Plants and water: the search for a comprehensive understanding.

We learn early in life sciences classes that water is the solution of life, working in tandem with carbon to make life as we know it possible. Globally, the abundance of water can be misleading, as most of this water is unavailable, being overly salinized in the oceans or locked in deep underground reserves. On land, the critical supply is of freshwater, which is unevenly distributed in space and time. Even the wettest environments can experience episodic water deficit, and flash flooding periodically occurs in arid landscapes. While humanity can capture, store and transport freshwater over large distances to ensure sustained supply, such options are not apparent for plants except in an immediate local context. Plants must make do with the water in their immediate surroundings, whether it be abundant or scarce. How they do this has led to a myriad of adaptive solutions, involving capturing, storing and transporting water. The traits that enable them to optimize water use in a range of hydraulic environments, subject to multivariate selective constraints, are the essence of the discipline of plant-water relations.

Seed size effects on plant establishment under low atmospheric CO2, with implications for seed size evolution.

BACKGROUND AND AIMS: Low atmospheric CO2 concentration depresses photosynthesis and resource use efficiency, and therefore can inhibit phases of the life cycle such as seedling establishment. Seed reserves can compensate for photosynthetic inhibition by accelerating seedling growth. We therefore hypothesize that seedlings arising from large seeds show less inhibition from low atmospheric CO2 than young plants from small seeds. Seed size effects on seedling responses to low CO2 may also be enhanced in warm environments, due to greater photorespiration at high temperature. METHODS: Phaseolus and Vigna seeds differing in mass by over two orders of magnitude were planted and grown for 14 d in growth chambers with CO2 concentrations of 370, 180 or 100 ppm, in thermal regimes of 25 °C/19 °C, 30 °C/24 °C or 35 °C/29 °C (day/night). We measured leaf area expansion, shoot growth and mortality of the seedlings arising from the variously sized seeds at 14 days after planting (14 DAP). KEY RESULTS: Relative to small-seeded plants, large-seeded genotypes produced greater leaf area and shoot mass at 14 DAP across the range of CO2 treatments in the 25 °C/19 °C and 30 °C/24 °C regimes, and at 100 ppm in the 35 °C/29 °C treatment. The proportional decline in leaf area and seed mass with CO2 reduction was generally greater for seedlings arising from small than from large seeds. Reductions in leaf area due to CO2 reduction increased in the warmer temperature treatments. In the 35 °C/19 °C treatment at 100 ppm CO2, seedling mortality was greater in small- than in large-seeded genotypes, and the small-seeded genotypes were unable to exit the seedling stage by the end of the experiment. CONCLUSIONS: The results support a hypothesis that seedlings from large seeds grow and establish better than seedlings from small seeds in warm, low CO2 environments. During low CO2 episodes in Earth's history, such as the past 30 million years, large seeds may have been favoured by natural selection in warm environments. With the recent rise in atmospheric CO2 due to human activities, trade-offs between seed size and number may already be affected, such that seed size today may be non-optimal in their natural habitats.

The Evolutionary Origin of C4 Photosynthesis in the Grass Subtribe Neurachninae.

The Australian grass subtribe Neurachninae contains closely related species that use C3, C4, and C2 photosynthesis. To gain insight into the evolution of C4 photosynthesis in grasses, we examined leaf gas exchange, anatomy and ultrastructure, and tissue localization of Gly decarboxylase subunit P (GLDP) in nine Neurachninae species. We identified previously unrecognized variation in leaf structure and physiology within Neurachne that represents varying degrees of C3-C4 intermediacy in the Neurachninae. These include inverse correlations between the apparent photosynthetic carbon dioxide (CO2) compensation point in the absence of day respiration (C * ) and chloroplast and mitochondrial investment in the mestome sheath (MS), where CO2 is concentrated in C2 and C4 Neurachne species; width of the MS cells; frequency of plasmodesmata in the MS cell walls adjoining the parenchymatous bundle sheath; and the proportion of leaf GLDP invested in the MS tissue. Less than 12% of the leaf GLDP was allocated to the MS of completely C3 Neurachninae species with C * values of 56-61 µmol mol-1, whereas two-thirds of leaf GLDP was in the MS of Neurachne lanigera, which exhibits a newly-identified, partial C2 phenotype with C * of 44 µmol mol-1 Increased investment of GLDP in MS tissue of the C2 species was attributed to more MS mitochondria and less GLDP in mesophyll mitochondria. These results are consistent with a model where C4 evolution in Neurachninae initially occurred via an increase in organelle and GLDP content in MS cells, which generated a sink for photorespired CO2 in MS tissues.

Russ Monson and the evolution of C4 photosynthesis.

Early in his career, Russ Monson produced a series of influential eco-physiological papers that helped lay the foundation for the study of C4 plant evolution. Among the most important was a 1984 paper with Maurice Ku and Gerry Edwards that outlined the pathway for the evolutionary bridge from C3 to C4 photosynthesis. This model proposed C4 photosynthesis arose out of a shuttle that imported photorespiratory metabolites into bundle sheath (BS) cells, where glycine decarboxylase cleaved off CO2, allowing it to accumulate and be efficiently refixed by BS Rubisco. By the mid-1990's, Monson's research focus had shifted away from C4 plants, save for one 2003 paper on C3 versus C4 stomatal control with Travis Huxman, and a series of critical reviews on C4 evolution. These reviews heavily influenced the modern synthesis of C4 evolutionary studies, which incorporates phylogenomic understanding with physiological, molecular, and structural characterizations of trait shifts in multiple evolutionary lineages. Subsequent research supported the Monson et al. model from 1984, by showing a glycine shuttle occurs in nearly all C3-C4 intermediate species identified. Monson also examined the physiological controls over the ecological distribution of C3, C3-C4 intermediate, and C4 photosynthesis, building our understanding of the fitness value of the intermediate and C4 pathway in relevant microenvironments. By establishing the foundation for discoveries that followed, Russ Monson can rightly be considered a leading pioneer contributing to the evolutionary biology of C4 photosynthesis.

Global change biology: A primer.

Because of human action, the Earth has entered an era where profound changes in the global environment are creating novel conditions that will be discernable far into the future. One consequence may be a large reduction of the Earth's biodiversity, potentially representing a sixth mass extinction. With effective stewardship, the global change drivers that threaten the Earth's biota could be alleviated, but this requires clear understanding of the drivers, their interactions, and how they impact ecological communities. This review identifies 10 anthropogenic global change drivers and discusses how six of the drivers (atmospheric CO2 enrichment, climate change, land transformation, species exploitation, exotic species invasions, eutrophication) impact Earth's biodiversity. Driver impacts on a particular species could be positive or negative. In either case, they initiate secondary responses that cascade along ecological lines of connection and in doing so magnify the initial impact. The unique nature of the threat to the Earth's biodiversity is not simply due to the magnitude of each driver, but due to the speed of change, the novelty of the drivers, and their interactions. Emphasizing one driver, notably climate change, is problematic because the other global change drivers also degrade biodiversity and together threaten the stability of the biosphere. As the main academic journal addressing global change effects on living systems, GCB is well positioned to provide leadership in solving the global change challenge. If humanity cannot meet the challenge, then GCB is positioned to serve as a leading chronicle of the sixth mass extinction to occur on planet Earth.

Facultative crassulacean acid metabolism in a C3-C4 intermediate.

The Portulacaceae enable the study of the evolutionary relationship between C4 and crassulacean acid metabolism (CAM) photosynthesis. Shoots of well-watered plants of the C3-C4 intermediate species Portulaca cryptopetala Speg. exhibit net uptake of CO2 solely during the light. CO2 fixation is primarily via the C3 pathway as indicated by a strong stimulation of CO2 uptake when shoots were provided with air containing 2% O2. When plants were subjected to water stress, daytime CO2 uptake was reduced and CAM-type net CO2 uptake in the dark occurred. This was accompanied by nocturnal accumulation of acid in both leaves and stems, also a defining characteristic of CAM. Following rewatering, net CO2 uptake in the dark ceased in shoots, as did nocturnal acidification of the leaves and stems. With this unequivocal demonstration of stress-related reversible, i.e. facultative, induction of CAM, P. cryptopetala becomes the first C3-C4 intermediate species reported to exhibit CAM. Portulaca molokiniensis Hobdy, a C4 species, also exhibited CAM only when subjected to water stress. Facultative CAM has now been demonstrated in all investigated species of Portulaca, which are well sampled from across the phylogeny. This strongly suggests that in Portulaca, a lineage in which species engage predominately in C4 photosynthesis, facultative CAM is ancestral to C4. In a broader context, it has now been demonstrated that CAM can co-exist in leaves that exhibit any of the other types of photosynthesis known in terrestrial plants: C3, C4 and C3-C4 intermediate.

High-yielding rice Takanari has superior photosynthetic response to a commercial rice Koshihikari under fluctuating light.

Leaves within crop canopies experience variable light over the course of a day, which greatly affects photosynthesis and crop productivity. Little is known about the mechanisms of the photosynthetic response to fluctuating light and their genetic control. Here, we examined gas exchange, metabolite levels, and chlorophyll fluorescence during the photosynthetic induction response in an Oryza sativa indica cultivar with high yield (Takanari) and a japonica cultivar with lower yield (Koshihikari). Takanari had a faster induction response to sudden increases in light intensity than Koshihikari, as demonstrated by faster increases in net CO2 assimilation rate, stomatal conductance, and electron transport rate. In a simulated light regime that mimicked a typical summer day, the faster induction response in Takanari increased daily CO2 assimilation by 10%. The faster response of Takanari was explained in part by its maintenance of a larger pool of Calvin-Benson cycle metabolites. Together, the rapid responses of electron transport rate, metabolic flux, and stomatal conductance in Takanari contributed to the greater daily carbon gain under fluctuating light typical of natural environments.

Reproductive heat tolerance in a Mojave Desert annual plant, Trianthema portulacastrum.

PREMISE OF THE STUDY: Reproduction in many crop species is impaired above 30° to 35°C; however, the sensitivity of reproduction in the natural flora remains uncertain. Studies focusing on the effect of high temperature on plant reproduction in wild species are necessary to improve our understanding of how rising global temperatures will impact global plant reproductive success and may ultimately inform models of plant distribution in the future. Additionally, these studies may highlight candidates for thermotolerance that could be further explored for crop improvement. METHODS: We studied reproductive heat tolerance in Trianthema portulacastrum, a weedy species found in hot microsites throughout the tropics and subtropics. Plants were grown at seven day/night temperature combinations: 30°/24°C, 33°/24°C, 36°/24°C, 40°/24°C, 44°/24°C, 24°/40°C, and 40°/40°C to study the effect of both high-day and high-night temperatures. The reproductive parameters measured include anther dehiscence, pollen viability, germination, ovule number, and seed set. KEY RESULTS: Pollen viability and germination declined with increasing daytime temperature up to 44°C, but this did not affect fruit production or seed set. Seed set was reduced under high night temperature. Continuous high temperature over the day and night (40°C day/40°C night) decreased pollen viability by half and reduced seed set by two-thirds. CONCLUSIONS: Our results demonstrate Trianthema portulacastrum has much higher reproductive thermotolerance than commonly identified in crop species, and though inhibited, can retain fecundity at 40°C. Through a combination of night escape and increased thermal tolerance, it maintains fertility in the hot microsites of its natural environment.

Some like it hot: the physiological ecology of C4 plant evolution.

The evolution of C4 photosynthesis requires an intermediate phase where photorespiratory glycine produced in the mesophyll cells must flow to the vascular sheath cells for metabolism by glycine decarboxylase. This glycine flux concentrates photorespired CO2 within the sheath cells, allowing it to be efficiently refixed by sheath Rubisco. A modest C4 biochemical cycle is then upregulated, possibly to support the refixation of photorespired ammonia in sheath cells, with subsequent increases in C4 metabolism providing incremental benefits until an optimized C4 pathway is established. 'Why' C4 photosynthesis evolved is largely explained by ancestral C3 species exploiting photorespiratory CO2 to improve carbon gain and thus enhance fitness. While photorespiration depresses C3 performance, it produces a resource (photorespired CO2) that can be exploited to build an evolutionary bridge to C4 photosynthesis. 'Where' C4 evolved is indicated by the habitat of species branching near C3-to-C4 transitions on phylogenetic trees. Consistent with the photorespiratory bridge hypothesis, transitional species show that the large majority of > 60 C4 lineages arose in hot, dry, and/or saline regions where photorespiratory potential is high. 'When' C4 evolved has been clarified by molecular clock analyses using phylogenetic data, coupled with isotopic signatures from fossils. Nearly all C4 lineages arose after 25 Ma when atmospheric CO2 levels had fallen to near current values. This reduction in CO2, coupled with persistent high temperature at low-to-mid-latitudes, met a precondition where photorespiration was elevated, thus facilitating the evolutionary selection pressure that led to C4 photosynthesis.

Tolerance of subzero winter cold in kudzu (Pueraria montana var. lobata).

The use of species distribution as a climate proxy for ecological forecasting is thought to be acceptable for invasive species. Kudzu (Pueraria montana var. lobata) is an important invasive whose northern distribution appears to be limited by winter survival; however, kudzu's cold tolerance thresholds are uncertain. Here, we used biogeographic evidence to hypothesize that exposure to - 20 °C is lethal for kudzu and thus determines its northern distribution limit. We evaluated this hypothesis using survival tests and electrolyte leakage to determine relative conductivity, a measure of cell damage, on 14 populations from eastern North America. Relative conductivity above 36% was lethal. Temperatures causing this damage averaged - 19.6 °C for northern and - 14.4 °C for southern populations, indicating kudzu acclimates to winter cold. To assess this, we measured relative conductivity of above- and belowground stems, and roots collected throughout the winter at a kudzu population in southern Ontario, Canada. Consistent with acclimation, the cold tolerance threshold of aboveground stems at the coldest time of year was - 26 °C, while stems insulated from cold extremes survived to - 17 °C-colder than the survival limits indicated by kudzu's biogeographic distribution. While these results do not rule out alternative cold limitations, they indicate kudzu can survive winters north of its current distribution. For kudzu, biogeography is not a proxy for climatic tolerance and continued northward migration is possible. Efforts to limit its spread are therefore prudent. These results demonstrate that physiological constraints inform predictions of climate-related changes in species distribution and should be considered where possible.

The sensitivity of photosynthesis to O2 and CO2 concentration identifies strong Rubisco control above the thermal optimum.

The biochemical model of C3 photosynthesis by Farquhar, von Caemmerer and Berry (FvCB) assumes that photosynthetic CO2 assimilation is limited by one of three biochemical processes that are not always easily discerned. This leads to improper assessments of biochemical limitations that limit the accuracy of the model predictions. We use the sensitivity of rates of CO2 assimilation and photosynthetic electron transport to changes in O2 and CO2 concentration in the chloroplast to evaluate photosynthetic limitations. Assessing the sensitivities to O2 and CO2 concentrations reduces the impact of uncertainties in the fixed parameters to a minimum and simultaneously entirely eliminates the need to determine the variable parameters of the model, such as Vcmax , J, or TP . Our analyses demonstrate that Rubisco limits carbon assimilation at high temperatures, while it is limited by triose phosphate utilization at lower temperatures and at higher CO2 concentrations. Measurements can be assigned a priori to one of the three functions of the FvCB model, allowing testing for the suitability of the selected fixed parameters of the model. This approach can improve the reliability of photosynthesis models on scales from the leaf level to estimating the global carbon budget.

Leaf photosynthetic rate and mesophyll cell anatomy changes during ontogenesis in backcrossed indica × japonica rice inbred lines.

The high-yielding indica rice variety, 'Takanari', has the high rate of leaf photosynthesis compared with the commercial japonica varieties. Among backcrossed inbred lines from a cross between 'Takanari' and a japonica variety, 'Koshihikari', two lines, BTK-a and BTK-b, showed approximately 20% higher photosynthetic rate than that of 'Takanari' for a flag leaf at full heading. This is a highest recorded rate of rice leaf photosynthesis. Here, the timing and cause of the increased leaf photosynthesis in the BTK lines were investigated by examining the photosynthesis and related parameters, as well as mesophyll cell anatomy during ontogenesis. Their photosynthetic rate was greater than that of 'Takanari' in the 13th leaf, as well as the flag leaf, but there were no differences in the 7th and 10th leaves. There were no consistent differences in the stomatal conductance, or the leaf nitrogen and Rubisco contents in the 13th and flag leaves. The total surface area of mesophyll cells per leaf area (TAmes) in the 13th and flag leaves increased significantly in the BTK lines due to the increased number and developed lobes of mesophyll cells compared with in 'Takanari'. The mesophyll conductance (g m) became greater in the BTK lines compared with 'Takanari' in the flag leaves but not in the 10th leaves. A close correlation was observed between TAmes and g m. We concluded that the increased mesophyll conductance through the development of mesophyll cells during the reproductive period is a probable cause of the greater photosynthetic rate in the BTK lines.