Advances in field-based high-throughput photosynthetic phenotyping.

Gas exchange techniques revolutionized plant research and advanced understanding, including associated fluxes and efficiencies, of photosynthesis, photorespiration, and respiration of plants from cellular to ecosystem scales. These techniques remain the gold standard for inferring photosynthetic rates and underlying physiology/biochemistry, although their utility for high-throughput phenotyping (HTP) of photosynthesis is limited both by the number of gas exchange systems available and the number of personnel available to operate the equipment. Remote sensing techniques have long been used to assess ecosystem productivity at coarse spatial and temporal resolutions, and advances in sensor technology coupled with advanced statistical techniques are expanding remote sensing tools to finer spatial scales and increasing the number and complexity of phenotypes that can be extracted. In this review, we outline the photosynthetic phenotypes of interest to the plant science community and describe the advances in high-throughput techniques to characterize photosynthesis at spatial scales useful to infer treatment or genotypic variation in field-based experiments or breeding trials. We will accomplish this objective by presenting six lessons learned thus far through the development and application of proximal/remote sensing-based measurements and the accompanying statistical analyses. We will conclude by outlining what we perceive as the current limitations, bottlenecks, and opportunities facing HTP of photosynthesis.

An analysis of ozone damage to historical maize and soybean yields in the United States.

Numerous controlled experiments find that elevated ground-level ozone concentrations ([O3]) damage crops and reduce yield. There have been no estimates of the actual yield losses in the field in the United States from [O3], even though such estimates would be valuable for projections of future food production and for cost-benefit analyses of reducing ground-level [O3]. Regression analysis of historical yield, climate, and [O3] data for the United States were used to determine the loss of production due to O3 for maize (Zea mays) and soybean (Glycine max) from 1980 to 2011, showing that over that period production of rain-fed fields of soybean and maize were reduced by roughly 5% and 10%, respectively, costing approximately $9 billion annually. Maize, thought to be inherently resistant to O3, was at least as sensitive as soybean to O3 damage. Overcoming this yield loss with improved emission controls or more tolerant germplasm could substantially increase world food and feed supply at a time when a global yield jump is urgently needed.

Loss of photosynthetic efficiency in the shade. An Achilles heel for the dense modern stands of our most productive C4 crops?

The wild progenitors of major C4 crops grew as individuals subjected to little shading. Today they are grown in dense stands where most leaves are shaded. Do they maintain photosynthetic efficiency in these low light conditions produced by modern cultivation? The apparent maximum quantum yield of CO2 assimilation (ΦCO2max,app), a key determinant of light-limited photosynthesis, has not been systematically studied in field stands of C4 crops. ΦCO2max,app was derived from the initial slope of the response of leaf CO2 uptake (A) to photon flux (Q). Leaf fractional light absorptance (α) was measured to determine the absolute maximum quantum yield of CO2 assimilation on an absorbed light basis (ΦCO2max,abs). Light response curves were determined on sun and shade leaves of 49 field plants of Miscanthus × giganteus and Zea mays following canopy closure. ΦCO2max,app and ΦCO2max,abs declined significantly by 15-27% (P<0.05) with canopy depth. Experimentally, leaf age was shown unlikely to cause this loss. Modeling canopy CO2 assimilation over diurnal courses suggested that the observed decline in ΦCO2max,app with canopy depth costs 10% of potential carbon gain. Overcoming this limitation could substantially increase the productivity of major C4 crops.

Can the cyanobacterial carbon-concentrating mechanism increase photosynthesis in crop species? A theoretical analysis.

Experimental elevation of [CO2] around C3 crops in the field has been shown to increase yields by suppressing the Rubisco oxygenase reaction and, in turn, photorespiration. Bioengineering a cyanobacterial carbon-concentrating mechanism (CCM) into C3 crop species provides a potential means of elevating [CO2] at Rubisco, thereby decreasing photorespiration and increasing photosynthetic efficiency and yield. The cyanobacterial CCM is an attractive alternative relative to other CCMs, because its features do not require anatomical changes to leaf tissue. However, the potential benefits of engineering the entire CCM into a C3 leaf are unexamined. Here, a CO2 and HCO3⁻ diffusion-reaction model is developed to examine how components of the cyanobacterial CCM affect leaf light-saturated CO2 uptake (A(sat)) and to determine whether a different Rubisco isoform would perform better in a leaf with a cyanobacterial CCM. The results show that the addition of carboxysomes without other CCM components substantially decreases A(sat) and that the best first step is the addition of HCO3⁻ transporters, as a single HCO3⁻ transporter increased modeled A(sat) by 9%. Addition of all major CCM components increased A(sat) from 24 to 38 µmol m⁻² s⁻¹. Several Rubisco isoforms were compared in the model, and increasing ribulose bisphosphate regeneration rate will allow for further improvements by using a Rubisco isoform adapted to high [CO2]. Results from field studies that artificially raise [CO2] suggest that this 60% increase in A(sat) could result in a 36% to 60% increase in yield.

Reduction of transpiration and altered nutrient allocation contribute to nutrient decline of crops grown in elevated CO(2) concentrations.

Plants grown in elevated [CO(2) ] have lower protein and mineral concentrations compared with plants grown in ambient [CO(2) ]. Dilution by enhanced production of carbohydrates is a likely cause, but it cannot explain all of the reductions. Two proposed, but untested, hypotheses are that (1) reduced canopy transpiration reduces mass flow of nutrients to the roots thus reducing nutrient uptake and (2) changes in metabolite or enzyme concentrations caused by physiological changes alter requirements for minerals as protein cofactors or in other organic complexes, shifting allocation between tissues and possibly altering uptake. Here, we use the meta-analysis of previous studies in crops to test these hypotheses. Nutrients acquired mostly by mass flow were decreased significantly more by elevated [CO(2) ] than nutrients acquired by diffusion to the roots through the soil, supporting the first hypothesis. Similarly, Mg showed large concentration declines in leaves and wheat stems, but smaller decreases in other tissues. Because chlorophyll requires a large fraction of total plant Mg, and chlorophyll concentration is reduced by growth in elevated [CO(2) ], this supports the second hypothesis. Understanding these mechanisms may guide efforts to improve nutrient content, and allow modeling of nutrient changes and health impacts under future climate change scenarios.

To have value, comparisons of high-throughput phenotyping methods need statistical tests of bias and variance.

The gap between genomics and phenomics is narrowing. The rate at which it is narrowing, however, is being slowed by improper statistical comparison of methods. Quantification using Pearson's correlation coefficient (r) is commonly used to assess method quality, but it is an often misleading statistic for this purpose as it is unable to provide information about the relative quality of two methods. Using r can both erroneously discount methods that are inherently more precise and validate methods that are less accurate. These errors occur because of logical flaws inherent in the use of r when comparing methods, not as a problem of limited sample size or the unavoidable possibility of a type I error. A popular alternative to using r is to measure the limits of agreement (LOA). However both r and LOA fail to identify which instrument is more or less variable than the other and can lead to incorrect conclusions about method quality. An alternative approach, comparing variances of methods, requires repeated measurements of the same subject, but avoids incorrect conclusions. Variance comparison is arguably the most important component of method validation and, thus, when repeated measurements are possible, variance comparison provides considerable value to these studies. Statistical tests to compare variances presented here are well established, easy to interpret and ubiquitously available. The widespread use of r has potentially led to numerous incorrect conclusions about method quality, hampering development, and the approach described here would be useful to advance high throughput phenotyping methods but can also extend into any branch of science. The adoption of the statistical techniques outlined in this paper will help speed the adoption of new high throughput phenotyping techniques by indicating when one should reject a new method, outright replace an old method or conditionally use a new method.

Two decades of fumigation data from the Soybean Free Air Concentration Enrichment facility.

The Soybean Free Air Concentration Enrichment (SoyFACE) facility is the longest running open-air carbon dioxide and ozone enrichment facility in the world. For over two decades, soybean, maize, and other crops have been exposed to the elevated carbon dioxide and ozone concentrations anticipated for late this century. The facility, located in East Central Illinois, USA, exposes crops to different atmospheric concentrations in replicated octagonal ~280 m2 Free Air Concentration Enrichment (FACE) treatment plots. Each FACE plot is paired with an untreated control (ambient) plot. The experiment provides important ground truth data for predicting future crop productivity. Fumigation data from SoyFACE were collected every four seconds throughout each growing season for over two decades. Here, we organize, quality control, and collate 20 years of data to facilitate trend analysis and crop modeling efforts. This paper provides the rationale for and a description of the SoyFACE experiments, along with a summary of the fumigation data and collation process, weather and ambient data collection procedures, and explanations of air pollution metrics and calculations.

Greater antioxidant and respiratory metabolism in field-grown soybean exposed to elevated O3 under both ambient and elevated CO2.

Antioxidant metabolism is responsive to environmental conditions, and is proposed to be a key component of ozone (O(3)) tolerance in plants. Tropospheric O(3) concentration ([O(3)]) has doubled since the Industrial Revolution and will increase further if precursor emissions rise as expected over this century. Additionally, atmospheric CO(2) concentration ([CO(2)]) is increasing at an unprecedented rate and will surpass 550 ppm by 2050. This study investigated the molecular, biochemical and physiological changes in soybean exposed to elevated [O(3) ] in a background of ambient [CO(2)] and elevated [CO(2)] in the field. Previously, it has been difficult to demonstrate any link between antioxidant defences and O(3) stress under field conditions. However, this study used principle components analysis to separate variability in [O(3)] from variability in other environmental conditions (temperature, light and relative humidity). Subsequent analysis of covariance determined that soybean antioxidant metabolism increased with increasing [O(3)], in both ambient and elevated [CO(2)]. The transcriptional response was dampened at elevated [CO(2)], consistent with lower stomatal conductance and lower O(3) flux into leaves. Energetically expensive increases in antioxidant metabolism and tetrapyrrole synthesis at elevated [O(3)] were associated with greater transcript levels of enzymes involved in respiratory metabolism.

Genomic basis for stimulated respiration by plants growing under elevated carbon dioxide.

Photosynthetic and respiratory exchanges of CO(2) by plants with the atmosphere are significantly larger than anthropogenic CO(2) emissions, and these fluxes will change as growing conditions are altered by climate change. Understanding feedbacks in CO(2) exchange is important to predicting future atmospheric [CO(2)] and climate change. At the tissue and plant scale, respiration is a key determinant of growth and yield. Although the stimulation of C(3) photosynthesis by growth at elevated [CO(2)] can be predicted with confidence, the nature of changes in respiration is less certain. This is largely because the mechanism of the respiratory response is insufficiently understood. Molecular, biochemical and physiological changes in the carbon metabolism of soybean in a free-air CO(2) enrichment experiment were investigated over 2 growing seasons. Growth of soybean at elevated [CO(2)] (550 micromol x mol(-1)) under field conditions stimulated the rate of nighttime respiration by 37%. Greater respiratory capacity was driven by greater abundance of transcripts encoding enzymes throughout the respiratory pathway, which would be needed for the greater number of mitochondria that have been observed in the leaves of plants grown at elevated [CO(2)]. Greater respiratory quotient and leaf carbohydrate content at elevated [CO(2)] indicate that stimulated respiration was supported by the additional carbohydrate available from enhanced photosynthesis at elevated [CO(2)]. If this response is consistent across many species, the future stimulation of net primary productivity could be reduced significantly. Greater foliar respiration at elevated [CO(2)] will reduce plant carbon balance, but could facilitate greater yields through enhanced photoassimilate export to sink tissues.

Drought imprints on crops can reduce yield loss: Nature's insights for food security.

The Midwestern "Corn-Belt" in the United States is the most productive agricultural region on the planet despite being predominantly rainfed. In this region, global climate change is driving precipitation patterns toward wetter springs and drier mid- to late-summers, a trend that is likely to intensify in the future. The lack of precipitation can lead to crop water limitations that ultimately impact growth and yields. Young plants exposed to water stress will often invest more resources into their root systems, possibly priming the crop for any subsequent mid- or late-season drought. The trend toward wetter springs, however, suggests that opportunities for crop priming may lessen in the future. Here, we test the hypothesis that early season dry conditions lead to drought priming in field-grown crops and this response will protect crops against growth and yield losses from late-season droughts. This hypothesis was tested for the two major Midwestern crop, maize and soybean, using high-resolution daily weather data, satellite-derived phenological metrics, field yield data, and ecosystem-scale model (Agricultural Production System Simulator) simulations. The results from this study showed that priming mitigated yield losses from a late season drought of up to 4.0% and 7.0% for maize and soybean compared with unprimed crops experiencing a late season drought. These results suggest that if the trend toward wet springs with drier summers continues, the relative impact of droughts on crop productivity is likely to worsen. Alternatively, identifying opportunities to breed or genetically modify pre-primed crop species may provide improved resilience to future climate change.

Altered physiological function, not structure, drives increased radiation-use efficiency of soybean grown at elevated CO2.

Previous studies of elevated carbon dioxide concentration ([CO(2)]) on crop canopies have found that radiation-use efficiency is increased more than radiation-interception efficiency. It is assumed that increased radiation-use efficiency is due to changes in leaf-level physiology; however, canopy structure can affect radiation-use efficiency if leaves are displayed in a manner that optimizes their physiological capacity, even though the canopy intercepts the same amount of light. In order to determine the contributions of physiology and canopy structure to radiation-use and radiation-interception efficiency, this study relates leaf-level physiology and leaf display to photosynthetic rate of the outer canopy. We used a new imaging approach that delivers three-dimensional maps of the outer canopy during the growing season. The 3D data were used to model leaf orientation and mean photosynthetic electron transport of the outer canopy to show that leaf orientation changes did not contribute to increased radiation-use; i.e. leaves of the outer canopy showed similar diurnal leaf movements and leaf orientation in both treatments. Elevated [CO(2)] resulted in an increased maximum electron transport rate (ETR(max)) of light reactions of photosynthesis. Modeling of canopy light interception showed that stimulated leaf-level electron transport at elevated [CO(2)], and not alterations in leaf orientation, was associated with stimulated radiation-use efficiency and biomass production in elevated [CO(2)]. This study provides proof of concept of methodology to quantify structure-function relationships in combination, allowing a quantitative estimate of the contribution of both effects to canopy energy conversion under elevated [CO(2)].

Effects of chronic elevated ozone concentration on antioxidant capacity, photosynthesis and seed yield of 10 soybean cultivars.

Crops losses to tropospheric ozone (O(3)) in the United States are estimated to cost $1-3 billion annually. This challenge is expected to increase as O(3) concentrations ([O(3)]) rise over the next half century. This study tested the hypothesis that there is cultivar variation in the antioxidant, photosynthetic and yield response of soybean to growth at elevated [O(3)]. Ten cultivars of soybean were grown at elevated [O(3)] from germination through maturity at the Soybean Free Air Concentration Enrichment facility in 2007 and six were grown in 2008. Photosynthetic gas exchange, leaf area index, chlorophyll content, fluorescence and antioxidant capacity were monitored during the growing seasons in order to determine if changes in these parameters could be used to predict the sensitivity of seed yield to elevated [O(3)]. Doubling background [O(3)] decreased soybean yields by 17%, but the variation in response among cultivars and years ranged from 8 to 37%. Chlorophyll content and photosynthetic parameters were positively correlated with seed yield, while antioxidant capacity was negatively correlated with photosynthesis and seed yield, suggesting a trade-off between antioxidant metabolism and carbon gain. Exposure response curves indicate that there has not been a significant improvement in soybean tolerance to [O(3)] in the past 30 years.

Field-grown tobacco plants maintain robust growth while accumulating large quantities of a bacterial cellulase in chloroplasts.

High accumulation of heterologous proteins expressed from the plastid genome has sometimes been reported to result in compromised plant phenotypes. Comparisons of transplastomic plants to wild-type (WT) are typically made in environmentally controlled chambers with relatively low light; little is known about the performance of such plants under field conditions. Here, we report on two plastid-engineered tobacco lines expressing the bacterial cellulase Cel6A. Field-grown plants producing Cel6A at ~20% of total soluble protein exhibit no loss in biomass or Rubisco content and only minor reductions in photosynthesis compared to WT. These experiments demonstrate that, when grown in the field, tobacco possesses sufficient metabolic flexibility to accommodate high levels of recombinant protein by increasing total protein synthesis and accumulation and/or by reallocating unneeded endogenous proteins. Based on current tobacco cultivation practices and readily achievable recombinant protein yields, we estimate that specific proteins could be obtained from field-grown transgenic tobacco plants at costs three orders of magnitude less than current cell culture methods.

Intensifying drought eliminates the expected benefits of elevated carbon dioxide for soybean.

Stimulation of C3 crop yield by rising concentrations of atmospheric carbon dioxide ([CO2]) is widely expected to counteract crop losses that are due to greater drought this century. But these expectations come from sparse field trials that have been biased towards mesic growth conditions. This eight-year study used precipitation manipulation and year-to-year variation in weather conditions at a unique open-air field facility to show that the stimulation of soybean yield by elevated [CO2] diminished to zero as drought intensified. Contrary to the prevalent expectation in the literature, rising [CO2] did not counteract the effect of strong drought on photosynthesis and yield because elevated [CO2] interacted with drought to modify stomatal function and canopy energy balance. This new insight from field experimentation under hot and dry conditions, which will become increasingly prevalent in the coming decades, highlights the likelihood of negative impacts from interacting global change factors on a key global commodity crop in its primary region of production.

Spring leaf flush in aspen (Populus tremuloides) clones is altered by long-term growth at elevated carbon dioxide and elevated ozone concentration.

Early spring leaf out is important to the success of deciduous trees competing for light and space in dense forest plantation canopies. In this study, we investigated spring leaf flush and how long-term growth at elevated carbon dioxide concentration ([CO(2)]) and elevated ozone concentration ([O(3)]) altered leaf area index development in a closed Populus tremuloides (aspen) canopy. This work was done at the Aspen FACE experiment where aspen clones have been grown since 1997 in conditions simulating the [CO(2)] and [O(3)] predicted for approximately 2050. The responses of two clones were compared during the first month of spring leaf out when CO(2) fumigation had begun, but O(3) fumigation had not. Trees in elevated [CO(2)] plots showed a stimulation of leaf area index (36%), while trees in elevated [O(3)] plots had lower leaf area index (-20%). While individual leaf area was not significantly affected by elevated [CO(2)], the photosynthetic operating efficiency of aspen leaves was significantly improved (51%). There were no significant differences in the way that the two aspen clones responded to elevated [CO(2)]; however, the two clones responded differently to long-term growth at elevated [O(3)]. The O(3)-sensitive clone, 42E, had reduced individual leaf area when grown at elevated [O(3)] (-32%), while the tolerant clone, 216, had larger mature leaf area at elevated [O(3)] (46%). These results indicate a clear difference between the two clones in their long-term response to elevated [O(3)], which could affect competition between the clones, and result in altered genotypic composition in future atmospheric conditions.

Hourly and seasonal variation in photosynthesis and stomatal conductance of soybean grown at future CO(2) and ozone concentrations for 3 years under fully open-air field conditions.

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.