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Experimental systems to simulate future elevated CO 2 conditions in the field often have large, rapid fluctuations in CO 2 . To examine possible impacts of such fluctuations on photosynthesis, the intact leaves of the field-grown plants of five species were exposed to two-minute cycles of CO 2 between 400 and 800 μmol mol -1 , lasting a total of 10 min, with photosynthesis, stomatal conductance and PSII fluorescence measured at the end of each half-cycle and also 10 min after the end of the cycling. Prior to the cyclic CO 2 treatments, the steady-state responses of leaf gas exchange and fluorescence to CO 2 were determined. In four of the five species, in which stomatal conductance decreased with increasing CO 2 , the cyclic CO 2 treatments reduced stomatal conductance. In those species, both photosynthesis and the photochemical efficiency of PSII were reduced at limiting internal CO 2 levels, but not at saturating CO 2 . In the fifth species, there was no change in stomatal conductance with CO 2 and no change in either photosynthesis or PSII efficiency at any CO 2 level with CO 2 cycling. It is concluded that in many, but not all, species, fluctuations in CO 2 may reduce photosynthesis at low CO 2 , partly by decreasing the photochemical efficiency of photosystem II as well as by decreasing stomatal conductance.

CO 2 is currently a growth-limiting resource for plants with C 3 metabolism, and elevated CO 2 also often reduces stomatal conductance, reducing plant water stress. Increased photosynthesis and improved water status might be expected to result in increased leaf size. It is therefore unexpected that leaf size is in some cases reduced in plants grown at elevated CO 2 , and also unexpected that elevated CO 2 applied only during darkness can increase leaf size. These experiments compared leaf size responses to day and/or night elevated CO 2 in six cultivars of Phaseolus vulgaris grown with either constant or varying temperature in controlled environment chambers. Diverse responses of leaf size to elevated CO 2 were found among the cultivars, including increased leaf size with elevated CO 2 applied only during darkness in some cultivars and temperature regimes. However, leaf size responses to elevated CO 2 and cultivar differences in response were unrelated to differences in leaf water potential or turgor pressure.

The response of carbon fixation in C 3 plants to elevated CO 2 is relatively larger when photosynthesis is limited by carboxylation capacity (V C ) than when limited by electron transport (J). Recent experiments under controlled, steady-state conditions have shown that photosynthesis at elevated CO 2 may be limited by V C even at limiting PPFD. These experiments were designed to test whether this also occurs in dynamic field environments. Leaf gas exchange was recorded every 5 min using two identical instruments both attached to the same leaf. The CO 2 concentration in one instrument was controlled at 400 μmol mol -1 and one at 600 μmol mol -1 . Leaves were exposed to ambient sunlight outdoors, and cuvette air temperatures tracked ambient outside air temperature. The water content of air in the leaf cuvettes was kept close to that of the ambient air. These measurements were conducted on multiple, mostly clear days for each of three species, Glycine max , Lablab purpureus , and Hemerocallis fulva . The results indicated that in all species, photosynthesis was limited by V C rather than J at both ambient and elevated CO 2 both at high midday PPFDs and also at limiting PPFDs in the early morning and late afternoon. During brief reductions in PPFD due to midday clouds, photosynthesis became limited by J. The net result of the apparent deactivation of Rubisco at low PPFD was that the relative stimulation of diurnal carbon fixation at elevated CO 2 was larger than would be predicted when assuming limitation of photosynthesis by J at low PPFD.

Attempts to identify crop genetic material with larger growth stimulation at projected elevated atmospheric CO 2 concentrations are becoming more common. The probability of reductions in photosynthesis and yield caused by short-term variation in CO 2 concentration within elevated CO 2 treatments in the free-air CO 2 enrichment plots raises the question of whether similar effects occur in glasshouse or indoor chamber experiments. These experiments were designed to test whether even the normal, modest, cyclic variation in CO 2 concentration typical of indoor exposure systems have persistent impacts on photosynthesis and growth, and to explore mechanisms underlying the responses observed. Wheat, cotton, soybeans, and rice were grown from seed in indoor chambers at a mean CO 2 concentration of 560 μmol mol -1 , with "triangular" cyclic variation with standard deviations of either 4.5 or 18.0 μmol mol -1 measured with 0.1 s sampling periods with an open path analyzer. Photosynthesis, stomatal conductance, and above ground biomass at 20 to 23 days were reduced in all four species by the larger variation in CO 2 concentration. Tests of rates of stomatal opening and closing with step changes in light and CO 2 , and tests of responses to square-wave cycling of CO 2 were also conducted on individual leaves of these and three other species, using a leaf gas exchange system. Reduced stomatal conductance due to larger amplitude cycling of CO 2 during growth occurred even in soybeans and rice, which had equal rates of opening and closing in response to step changes in CO 2 . The gas exchange results further indicated that reduced mean stomatal conductance was not the only cause of reduced photosynthesis in variable CO 2 conditions.

Recent in vivo assays of the responses of Rubisco to temperature in C 3 plants have revealed substantial diversity. Three cultivars of soybean ( Glycine max L. Merr.), Holt, Fiskeby V, and Spencer, were grown in indoor chambers at 15, 20, and 25 °C. Leaf photosynthesis was measured over the range of 15 to 30 °C, deliberately avoiding higher temperatures which may cause deactivation of Rubisco, in order to test for differences in temperature responses of photosynthesis, and to investigate in vivo Rubisco kinetic characteristics responsible for any differences observed. The three cultivars differed in the optimum temperature for photosynthesis (from 15 to 30 °C) at 400 mmol mol -1 external CO 2 concentration when grown at 15 °C, and in the shapes of the response curves when grown at 25 °C. The apparent activation energy of the maximum carboxylation rate of Rubisco differed substantially between cultivars at all growth temperatures, as well as changing with growth temperature in two of the cultivars. The activation energy ranged from 58 to 84 kJ mol -1 , compared with the value of 64 kJ mol -1 used in many photosynthesis models. Much less variation in temperature responses occurred in photosynthesis measured at nearly saturating CO 2 levels, suggesting more diversity in Rubisco than in electron transport thermal properties among these soybean cultivars.

High intrinsic water-use efficiency (WUE i ), the ratio of leaf photosynthesis to stomatal conductance, may be a useful trait in adapting crops to water-limited environments. In soybean, cultivar differences in stomatal response to vapor pressure deficit have not consistently translated into differences in WUE i in the field. In this study, six cultivars of soybeans previously shown to differ in WUE i in indoor experiments were grown in the field in Beltsville, Maryland, and tested for mid-day WUE i on nine clear days during the mid-seasons of two years. Measurement dates were chosen for diverse temperatures, and air temperatures ranged from 21 to 34 °C on the different dates. Air saturation deficits for water vapor ranged from 0.9 to 2.2 kPa. Corrected carbon isotope delta values for 13 C (CID) were determined on mature, upper canopy leaves harvested during early pod filling each year. WUE i differed among cultivars in both years and the differences were consistent across measurement dates. Correlations between mean WUE i and CID were not significant in either year. It is concluded that consistent cultivar differences in WUE i exist in these soybean cultivars under field conditions, but that carbon isotope ratios may not be useful in identifying them because of cultivar differences in mesophyll conductance.

Whether the mesophyll conductance to CO₂ movement (g m ) within leaves of C₃ plants changes with CO₂ concentration remains a matter of debate, particularly at low CO₂ concentrations. We tested for changes in g m over the range of sub-stomatal CO₂ concentrations (C i ) for which Rubisco activity limited photosynthesis (A) in three plant species grown under the same conditions. Mesophyll conductance was estimated by three independent methods: the oxygen sensitivity of photosynthesis, variable J fluorescence combined with gas exchange, and the curvature of the Rubisco-limited A vs. C i curve. The latter assay used a new method of rapidly obtaining data points at approximately every 3 μmol mol -1 for Rubisco-limited A vs. C i curves, allowing separate estimates of curvature over limited C i ranges. In two species, soybean and sunflower, no change in g m with C i was detected using any of the three methods of estimating g m . In common bean measured under the same conditions as the other species, all three methods indicated large decreases in g m with increasing C i . Therefore, change in g m with C i in the Rubsico-limited region of A vs. C i curves depended on the species, but not on the method of estimating g m .

Seeds of three C₃ and one C₄ annual weedy species were collected from agricultural fields in Beltsville, Maryland in 1966 and 2006, when atmospheric CO₂ concentrations averaged about 320 and 380 mol mol -1 , respectively. Plants from each collection year were grown over a range of CO₂ concentrations to test for adaptation of these weedy species to recent changes in atmospheric CO₂. In all three of the C₃ species, the increase in CO₂ concentration from 320 mol mol -1 to 380 mol mol -1 increased total dry mass at 24 days in plants from seeds collected in 2006, but not in plants from seeds collected in 1966. Shoot and seed dry mass at maturity was greater at the higher growth CO₂ in plants collected in 2006 than in 1966 in two of the species. Down-regulation of photosynthetic carboxylation capacity during growth at high CO₂ was less in the newer seed lots than in the older in two of the species. Overall, the results indicate that adaptation to recent changes in atmospheric CO₂ has occurred in some of these weedy species., Competing Interests: The author declares no conflict of interest.

Improving water use efficiency (WUE) may prove a useful way to adapt crop species to drought. Since the recognition of the importance of mesophyll conductance to CO₂ movement from inside stomatal pores to the sites of photosynthetic carboxylation, there has been interest in how much intraspecific variation in mesophyll conductance (g m ) exists, and how such variation may impact leaf WUE within C₃ species. In this study, the g m and leaf WUE of fifteen cultivars of soybeans grown under controlled conditions were measured under standardized environmental conditions. Leaf WUE varied by a factor of 2.6 among the cultivars, and g m varied by a factor of 8.6. However, there was no significant correlation (r = -0.047) between g m and leaf WUE. Leaf WUE was linearly related to the sub-stomatal CO₂ concentration. The value of g m affected the ratio of maximum Rubisco carboxylation capacity calculated from the sub-stomatal CO₂ concentration to that calculated from the CO₂ concentration at the site of carboxylation. That is, variation in g m affected the efficiency of Rubisco carboxylation, but not leaf WUE. Nevertheless, there is considerable scope for genetically improving soybean leaf water use efficiency., Competing Interests: The author declares no conflict of interest.