Efforts to optimize photosynthesis can be informed by measurement of it's underlying and coordinated sub-processes. For decades, non-invasive techniques have been integrated to enable assessment of the coordination that exists within and between the gaseous diffusional, energy productive, and energy consumptive reactions of leaf-level photosynthesis. One of the more prevalent techniques measures chlorophyll a fluorescence using a fluorometer that employes pulsed amplitude modulation (PAM). The maximum fluorescence yield observed under steady-state actinic illumination, or Fm', has been shown to be prone to underestimation. Errors can be propagated to various derivative parameters representative of photosynthetic phenomena that have been identified as targets for photosynthetic optimization. A technique involving a multiphase flash of variable irradiance has been shown to be capable of correcting for underestimation of Fm'. The technique reveals that whereas Fm' is prone to underestimation, the maximum fluorescence yield following brief cessation of actinic illumination, or Fm", is not. The differential susceptibility of Fm' and Fm" to misestimation renders essential features of photosynthetic regulation, which are governed by coordinated sub-processes of leaf level photosynthesis, subject to misinterpretation.

Early plant selection for desirable traits is important in tree improvement programs and sustainable forest management. In this study, we demonstrate the use of image based high-throughput plant phenotyping (HTPP, LemnaTec 3D Scanalyzer, Germany), with Red, Green, Blue (RGB), and hyperspectral cameras, to quantify Quercus bicolor and Quercus prinoides seedlings growth and development [plant height, projected leaf area (LA), plant/canopy width, ConvexHull, and plant aspect ratio], and assess their response to a dry-down period, under controlled environment. HTPP images were validated against low throughput measurements, including gas exchange, leaf spectral properties, and morphological traits. Using HTPP, we recorded significant differences in growth dynamic in examined species, with faster initial growth rate early in the growing season, higher photosynthetic rates, larger LA, and seedling dimension, in Q. bicolor, compared to Q. prinoides. This has ecological implications on species responses to shading and timing of drought stress, as well as their competitive relationship with each other and with other species under changing climate. HTTP showed that both growth and leaf expansion ceased under dry-down treatment. Image derived and measured morphological traits were highly and significantly correlated under both well-watered and dry-down conditions for both species. To obtain meaningful physiological information using spectrometry, we calculated 12 vegetation indices (VIs) from both HTPP and handheld spectrometers. Vogelmann and Maccioni indices had the highest correlations across methods, suggesting their potential use for assessing oak seedlings performance and health. Our results emphasized the importance of VIs ground truthing, since VIs performance can vary significantly between species and treatments. HTPP tools can successfully be used to effectively assess forest seedlings of the two Quercus species, important for early plant selection for forest management purposes and tree improvement programs.

The Rapid A/Ci Response (RACiR) is a dynamic method of leaf-level gas exchange that allows the calculation of fundamental parameters for photosynthetic capacity in much shorter times than standard steady-state methods. This is accomplished by using CO2 ramps instead of discrete values under steady-state conditions. Here, we present data describing potential pitfalls and provide a list of best practices that are important to follow in order to ensure the data acquired are of high quality. Stinziano et al. (2017) demonstrated that the RACiR generates robust estimates of Vcmax (maximal carboxylation rate) and J (potential rate of electron transport) similar to the standard A/Ci, which can be very useful in phenotyping applications. They also suggested that the RACiR technique may generate new biological insights that are unattainable from slower measurements (Stinziano et al., 2017). Here, we respond to the Letter by Taylor & Long (2019; in this issue of New Phytologist, pp. 621–624), reiterating the points from Stinziano et al. (2017) and emphasizing that applying RACiR methodology outside of the specific conditions and recommendations of Stinziano et al. (2017) requires further testing. Fig. 1(a,b) of Taylor & Long (2019) shows data using a ramp of 200 μmol mol−1 min−1, which Stinziano et al. (2017) stated as being a rate that may compromise the estimate of J. Therefore, it is not surprising to see examples at that speed that generate larger deviations from a standard A/Ci than was in our data, provided in both figures and supplementary files of Stinziano et al. (2017) as well as in Fig. 1(c) of Taylor & Long (2019). Here, we present results demonstrating that higher ramp rates generate larger differences in the apparent compensation point (Fig. 1). These results also demonstrate that CO2 ramping rate can result in apparent assimilation offsets. Such offsets may be due to instrumentation artifacts such as an imperfect empty chamber correction or other effects that are not yet demonstrated. At sufficiently high ramp rates, differences between standard steady-state and dynamic RACiR gas exchange methods would be expected and could reflect real differences in the underlying biology. When CO2 can be changed faster than enzyme activation states, stomatal conductance, mesophyll conductance, or faster than some biochemical pools can respond, then gas exchange data may need reinterpretation, and this new approach may offer opportunities to test model assumptions. However, we note that higher CO2 ramp rates are complicated and we recommend limiting ramp rates to 100 ppm min−1, unless one is explicitly examining the mechanisms behind high ramp rate offsets. Taylor & Long (2019) extended data analyses to parameters that were beyond the scope of Stinziano et al. (2017). Based on potential procedural or biological concerns mentioned earlier, at high ramp rates RACiR may not generate compensation points similar to a standard A/Ci. However, we also point out that standard A/Ci methods used to generate estimates of other parameters presented in Taylor & Long (2019), including gm (mesophyll conductance), Rd (dark respiration), and Γ* (photorespiratory compensation point) are potentially problematic (Pons et al., 2009; Walker & Cousins, 2013; Walker & Ort, 2015; Farquhar & Busch, 2017) and we would not recommend using them. Taylor & Long (2019) also combine data from the 500 to 0 and 300 to 800 μmol mol−1 RACiRs of Stinziano et al. (2017) when performing model fits. While compiling a larger data set for curve fitting is understandable, we do not think it is appropriate here. Notably, the RACiRs in Stinziano et al. (2017) were not conducted in a way to maximize alignment between ramp ranges due to the way the CO2 ramping loops were set up. The order of measurements is known to affect the results as evidenced by the common practice, in a standard A/Ci, of carefully returning to a common mid-point value between low and high CO2 ranges. This is required because of the biological effects occurring during slow measurements, such as enzyme deactivation, in each CO2 range. Therefore, we recommend against combining RACiRs from multiple ranges. When we performed a re-analysis of the Stinziano et al. (2017) data using the Taylor & Long (2019) script, but restricted the fit to the 300–800 μmol mol−1 RACiRs, most of the significant differences presented in Taylor & Long (2019) were no longer significant. However, we do not present the table here because we believe a larger data set is needed for proper statistical analyses. Every parameter estimate has some sort of error associated with it. When relatively small numbers of parameter estimates are compared using a standard statistical test (such as a t-test) this error is essentially ignored, and so we urge caution in declaring true differences between parameter estimates based on relatively limited data. Essentially, a larger data set that includes analyses of all errors would be helpful in order to more fully investigate these issues. Taylor & Long (2019) raise an excellent point: curve fitting approach matters. This issue was thoroughly examined by Gu et al. (2010) and has been repeatedly addressed by multiple groups for well over a decade (e.g. Ethier & Livingston, 2004; Dubois et al., 2007; Sharkey et al., 2007). It is important to recognize that different methods for fitting A/Ci responses can yield different parameter estimates. This makes assigning ‘truth’ more difficult since the method used can affect the outcome. This is especially true for values like gm, Rd, and Γ* such that it is not widely accepted that these are optimally derived from a single standard A/Ci (see methods Pons et al., 2009; Walker & Cousins, 2013; Walker & Ort, 2015; Farquhar & Busch, 2017). The most widely accepted use for the A/Ci is to estimate Vcmax and J (with much less certainty about J) and those values are in ‘reasonably close agreement’ between the standard and RACiR approaches in Taylor & Long (2019) and in Stinziano et al. (2017). While it is logical and valuable to compare standard A/Ci and RACiR approaches for fitting other parameters as Taylor & Long (2019) have done, assigning truth becomes more difficult given the uncertainty of model fitting as well as the methodological points we have raised earlier. Given the importance of careful consideration of ramping conditions and preliminary testing with a species of interest, we are providing a more detailed and explicit procedural guide for new users. RACiR requires characterization of the species in question to determine the best way to setup the CO2 ramps, especially the ramping rate. To date, a ramping rate of 100 μmol mol−1 min−1 has provided the best comparisons with steady-state Vcmax and J. RACiR empty-chamber calibration curves are specific to flow rate, temperature, and ramp rate, and may be sensitive to large changes in water mole fraction during the ramp. As such, we are clarifying key recommendations whenever RACiR is used: Taken together, we think the main conclusions of Stinziano et al. (2017) still hold in that RACiR can generate estimates of Vcmax and J that are substantially similar to estimates derived from the standard approach, and that RACiR is a useful screening tool for rapid phenotyping. The work of Taylor & Long (2019) is helpful in determining where differences in parameter estimates between the two methods may exist, and points to the need for additional research as RACiR is further developed.

Chl fluorescence has been used widely to calculate photosynthetic electron transport rates. Portable photosynthesis instruments allow for combined measurements of gas exchange and Chl fluorescence. We analyzed the influence of spectral quality of actinic light on Chl fluorescence and the calculated electron transport rate, and compared this with photosynthetic rates measured by gas exchange in the absence of photorespiration. In blue actinic light, the electron transport rate calculated from Chl fluorescence overestimated the true rate by nearly a factor of two, whereas there was closer agreement under red light. This was consistent with the prediction made with a multilayer leaf model using profiles of light absorption and photosynthetic capacity. Caution is needed when interpreting combined measurements of Chl fluorescence and gas exchange, such as the calculation of CO2 partial pressure in leaf chloroplasts.

Phenotyping for photosynthetic gas exchange parameters is limiting our ability to select plants for enhanced photosynthetic carbon gain and to assess plant function in current and future natural environments. This is due, in part, to the time required to generate estimates of the maximum rate of ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) carboxylation (Vc,max ) and the maximal rate of electron transport (Jmax ) from the response of photosynthesis (A) to the CO2 concentration inside leaf air spaces (Ci ). To relieve this bottleneck, we developed a method for rapid photosynthetic carbon assimilation CO2 responses [rapid A-Ci response (RACiR)] utilizing non-steady-state measurements of gas exchange. Using high temporal resolution measurements under rapidly changing CO2 concentrations, we show that RACiR techniques can obtain measures of Vc,max and Jmax in ~5 min, and possibly even faster. This is a small fraction of the time required for even the most advanced gas exchange instrumentation. The RACiR technique, owing to its increased throughput, will allow for more rapid screening of crops, mutants and populations of plants in natural environments, bringing gas exchange into the phenomic era.

Ozone (O(3)) uptake by plants leads to an increase in reactive oxygen species (ROS) in the intercellular space of leaves and induces signalling processes reported to involve the membrane-bound heterotrimeric G-protein complex. Therefore, potential G-protein-mediated response mechanisms to O(3) were compared between Arabidopsis thaliana L. lines with null mutations in the α- and β-subunits (gpa1-4, agb1-2 and gpa1-4/agb1-2) and Col-0 wild-type plants. Plants were treated with a range of O(3) concentrations (5, 125, 175 and 300 nL L(-1)) for 1 and 2 d in controlled environment chambers. Transcript levels of GPA1, AGB1 and RGS1 transiently increased in Col-0 exposed to 125 nL L(-1) O(3) compared with the 5 nL L(-1) control treatment. However, silencing of α and β G-protein genes resulted in little alteration of many processes associated with O(3) injury, including the induction of ROS-signalling genes, increased leaf tissue ion leakage, decreased net photosynthesis and stomatal conductance, and increased peroxidase activity, especially in the leaf apoplast. These results indicated that many responses to O(3) stress at physiological levels were not detectably influenced by α and β G-proteins.

Mean surface ozone concentration is predicted to increase 23% by 2050. Previous chamber studies of crops report large yield losses caused by elevation of tropospheric ozone, and have been the basis for projecting economic loss. This is the first study with a food crop (soybean, Glycine max) using free-air gas concentration enrichment (FACE) technology for ozone fumigation. A 23% increase in ozone concentration from an average daytime ambient 56 p.p.b. to a treatment 69 p.p.b. over two growing seasons decreased seed yield by 20%. Total above-ground net primary production decreased by 17% without altering dry mass allocation among shoot organs, except seed. Fewer live leaves and decreased photosynthesis in late grain filling appear to drive the ozone-induced losses in production and yield. These results validate previous chamber studies suggesting that soybean yields will decrease under increasing ozone exposure. In fact, these results suggest that when treated under open-air conditions yield losses may be even greater than the large losses already reported in earlier chamber studies. Yield losses with elevated ozone were greater in the second year following a severe hailstorm, suggesting that losses caused by ozone might be exacerbated by extreme climatic events.

It is anticipated that enrichment of the atmosphere with CO(2) will increase photosynthetic carbon assimilation in C3 plants. Analysis of controlled environment studies conducted to date indicates that plant growth at concentrations of carbon dioxide ([CO(2)]) anticipated for 2050 ( approximately 550 micromol mol(-1)) will stimulate leaf photosynthetic carbon assimilation (A) by 20 to 40%. Simultaneously, concentrations of tropospheric ozone ([O(3)]) are expected to increase by 2050, and growth in controlled environments at elevated [O(3)] significantly reduces A. However, the simultaneous effects of both increases on a major crop under open-air conditions have never been tested. Over three consecutive growing seasons > 4700 individual measurements of A, photosynthetic electron transport (J(PSII)) and stomatal conductance (g(s)) were measured on Glycine max (L.) Merr. (soybean). Experimental treatments used free-air gas concentration enrichment (FACE) technology in a fully replicated, factorial complete block design. The mean A in the control plots was 14.5 micromol m(-2) s(-1). At elevated [CO(2)], mean A was 24% higher and the treatment effect was statistically significant on 80% of days. There was a strong positive correlation between daytime maximum temperatures and mean daily integrated A at elevated [CO(2)], which accounted for much of the variation in CO(2) effect among days. The effect of elevated [CO(2)] on photosynthesis also tended to be greater under water stress conditions. The elevated [O(3)] treatment had no statistically significant effect on mean A, g(s) or J(PSII) on newly expanded leaves. Combined elevation of [CO(2)] and [O(3)] resulted in a slightly smaller increase in average A than when [CO(2)] alone was elevated, and was significantly greater than the control on 67% of days. Thus, the change in atmospheric composition predicted for the middle of this century will, based on the results of a 3 year open-air field experiment, have smaller effects on photosynthesis, g(s) and whole chain electron transport through photosystem II than predicted by the substantial literature on relevant controlled environment studies on soybean and likely most other C3 plants.

Plant growth is typically stimulated at elevated carbon dioxide concentration ([CO2]), but a sustained and maximal stimulation of growth requires acquisition of additional N in proportion to the additional C fixed at elevated [CO2]. We hypothesized that legumes would be able to avoid N limitation at elevated [CO2]. Soybean was grown without N fertilizer from germination to final senescence at elevated [CO2] over two growing seasons under fully open-air conditions, providing a model legume system. Measurements of photosynthesis and foliar carbohydrate content showed that plants growing at elevated [CO2] had a c. 25% increase in the daily integral of photosynthesis and c. 58% increase in foliar carbohydrate content, suggesting that plants at elevated [CO2] had a surplus of photosynthate. Soybeans had a low leaf N content at the beginning of the season, which was a further c. 17% lower at elevated [CO2]. In the middle of the season, ureide, total amino acid and N content increased markedly, and the effect of elevated [CO2] on leaf N content disappeared. Analysis of individual amino acid levels supported the conclusion that plants at elevated [CO2] overcame an early-season N limitation. These soybean plants showed a c. 16% increase in dry mass at final harvest and showed no significant effect of elevated [CO2] on leaf N, protein or total amino acid content in the latter part of the season. One possible explanation for these findings is that N fixation had increased, and that these plants had acclimated to the increased N demand at elevated [CO2].

There is strong evidence to suggest that global warming is leading to an extended growing season by altering the timing of autumnal events such as bud set and leaf abscission1,2,3, with important impacts on ecosystem productivity and global carbon cycling. However, while temperature is an important driver of spring phenological events, the relationship between temperature and autumn phenology is weak4. Here, we present results from three open-air field experiments in which elevated atmospheric CO2 concentration [CO2] at the concentration likely to exist in 2050, extended the growing season of: (1) three abundant North American forest trees; (2) the world’s most extensively grown broad-leaved crop (soybean); and (3) two European poplars. Across experiments and over multiple years, elevated [CO2] delayed autumnal declines in leaf area, chlorophyll concentration, photosynthesis and normalized vegetation difference index (NVDI) by 2-7 days for soybean and 5-15 days for trees. These findings indicate that [CO2] alters growing season length and the rise in atmospheric [CO2] over the past 30 years could explain 26-52% of the extended growing season now ascribed to warming3.

How forests will respond to rising [CO2] in the long term is uncertain, most studies having involved juvenile trees in chambers prior to canopy closure. Poplar free-air CO2 enrichment (Viterbo, Italy) is one of the first experiments to grow a forest from planting through canopy closure to coppice, entirely under open-air conditions using free-air CO2 enrichment technology. Three Populus species: P. alba, P. nigra and P. x euramericana, were grown in three blocks, each containing one control and one treatment plot in which CO2 was elevated to the expected 2050 concentration of 550 ppm. The objective of this study was to estimate gross primary production (GPP) from recorded leaf photosynthetic properties, leaf area index (LAI) and meteorological conditions over the complete 3-year rotation cycle. From the meteorological conditions recorded at 30 min intervals and biweekly measurements of LAI, the microclimate of leaves within the plots was estimated with a radiation transfer and energy balance model. This information was in turn used as input into a canopy microclimate model to determine light and temperature of different leaf classes at 30 min intervals which in turn was used with the steady-state biochemical model of leaf photosynthesis to compute CO2 uptake by the different leaf classes. The parameters of these models were derived from measurements made at regular intervals throughout the coppice cycle. The photosynthetic rates for different leaf classes were summed to obtain canopy photosynthesis, i.e. GPP. The model was run for each species in each plot, so that differences in GPP between species and treatments could be tested statistically. Significant stimulation of GPP driven by elevated [CO2] occurred in all 3 years, and was greatest in the first year (223-251%), but markedly lower in the second (19-24%) and third years (5-19%). Increase in GPP in elevated relative to control plots was highest for P. nigra in 1999 and for P. x euramericana in 2000 and 2001, although in 1999 P. alba had a higher GPP than P. x euramericana. Our analysis attributed the decline in stimulation to canopy closure and not photosynthetic acclimation. Over the 3-year rotation cycle from planting to harvest, the cumulative GPP was 4500, 4960 and 4010 g C m(-2) for P. alba, P. nigra and P. x euramericana, respectively, in current [CO2] and 5260, 5800 and 5000 g C m(-2) in the elevated [CO2] treatments. The relative changes were consistent with independent measurements of net primary production, determined independently from biomass increments and turnover.

Predictions of yield for the globe's major grain and legume arable crops suggest that, with a moderate temperature increase, production may increase in the temperate zone, but decline in the tropics. In total, global food supply may show little change. This security comes from inclusion of the direct effect of rising carbon dioxide (CO2) concentration, [CO2], which significantly stimulates yield by decreasing photorespiration in C3 crops and transpiration in all crops. Evidence for a large response to [CO2] is largely based on studies made within chambers at small scales, which would be considered unacceptable for standard agronomic trials of new cultivars or agrochemicals. Yet, predictions of the globe's future food security are based on such inadequate information. Free-Air Concentration Enrichment (FACE) technology now allows investigation of the effects of rising [CO2] and ozone on field crops under fully open-air conditions at an agronomic scale. Experiments with rice, wheat, maize and soybean show smaller increases in yield than anticipated from studies in chambers. Experiments with increased ozone show large yield losses (20%), which are not accounted for in projections of global food security. These findings suggest that current projections of global food security are overoptimistic. The fertilization effect of CO2 is less than that used in many models, while rising ozone will cause large yield losses in the Northern Hemisphere. Unfortunately, FACE studies have been limited in geographical extent and interactive effects of CO2, ozone and temperature have yet to be studied. Without more extensive study of the effects of these changes at an agronomic scale in the open air, our ever-more sophisticated models will continue to have feet of clay.

The Intergovernmental Panel on Climate Change projects that atmospheric [CO2] will reach 550 ppm by 2050. Numerous assessments of plant response to elevated [CO2] have been conducted in chambers and enclosures, with only a few studies reporting responses in fully open-air, field conditions. Reported yields for the world's two major grain crops, wheat and rice, are substantially lower in free-air CO2 enrichment (FACE) than predicted from similar elevated [CO2] experiments within chambers. This discrepancy has major implications for forecasting future global food supply. Globally, the leguminous-crop soybean (Glycine max (L.) Merr.) is planted on more land than any other dicotyledonous crop. Previous studies have shown that total dry mass production increased on average 37% in response to increasing [CO2] to approximately 700 ppm, but harvestable yield will increase only 24%. Is this representative of soybean responses under open-air field conditions? The effects of elevation of [CO2] to 550 ppm on total production, partitioning and yield of soybean over 3 years are reported. This is the first FACE study of soybean (http://www.soyface.uiuc.edu) and the first on crops in the Midwest of North America, one of the major food production regions of the globe. Although increases in both aboveground net primary production (17-18%) and yield (15%) were consistent across three growing seasons and two cultivars, the relative stimulation was less than projected from previous chamber experiments. As in previous studies, partitioning to seed dry mass decreased; however, net production during vegetative growth did not increase and crop maturation was delayed, not accelerated as previously reported. These results suggest that chamber studies may have over-estimated the stimulatory effect of rising [CO2], with important implications on global food supply forecasts.

Rising atmospheric carbon dioxide concentration ([CO2]) is widely recognized, but less appreciated is a concomitant rise in tropospheric ozone concentration ([O-3]). In industrialized countries, [O-3] has risen by 0.5% to 2.5% per year. Tropospheric [O-3] is predicted to reach a global mean of >60 nL L-1 by 2050 with greater averages locally. Previous studies in enclosures suggest that this level of [O-3] will decrease leaf photosynthesis, thereby limiting growth and yield of Glycine max L. Merr. SoyFACE (Soybean Free Air gas Concentration Enrichment) is the first facility to elevate atmospheric [O-3] (approximately 1.2X current) in replicated plots under completely open-air conditions within an agricultural field. Measurements of gas exchange (assimilation versus light and assimilation versus intercellular [CO2]) were made on excised leaves from control and treatment plots (n = 4). In contrast to expectations from previous chamber studies, elevated [O-3] did not alter light-saturated photosynthesis (A(sat), P = 0.09), carboxylation capacity (V-c,V-max,V- P = 0.82), or maximum electron transport (J(max), P = 0.66) for the topmost most recently fully expanded leaf at any stage crop development. Leaves formed during the vegetative growth stage did not show a significant ozone-induced loss of photosynthetic capacity as they aged. Leaves formed during flowering did show a more rapid loss of photosynthetic capacity as they aged in elevated [O-3]. A(sat), V-c,V-max, and J(max) (P = 0.04, 0.004, and 0.002, respectively) were decreased 20% to 30% by treatment with ozone. This is noteworthy since these leaves provide photosynthate to the developing grain. In conclusion, a small (approximately 20%) increase in tropospheric [O-3] did not significantly alter photosynthetic capacity of newly expanded leaves, but as these leaves aged, losses in photosynthetic carbon assimilation occurred.