Leaf microscopy applications in photosynthesis research: identifying the gaps.

Leaf imaging via microscopy has provided critical insights into research on photosynthesis at multiple junctures, from the early understanding of the role of stomata, through elucidating C4 photosynthesis via Kranz anatomy and chloroplast arrangement in single cells, to detailed explorations of diffusion pathways and light utilization gradients within leaves. In recent decades, the original two-dimensional (2D) explorations have begun to be visualized in three-dimensional (3D) space, revising our understanding of structure-function relationships between internal leaf anatomy and photosynthesis. In particular, advancing new technologies and analyses are providing fresh insight into the relationship between leaf cellular components and improving the ability to model net carbon fixation, water use efficiency, and metabolite turnover rate in leaves. While ground-breaking developments in imaging tools and techniques have expanded our knowledge of leaf 3D structure via high-resolution 3D and time-series images, there is a growing need for more in vivo imaging as well as metabolite imaging. However, these advances necessitate further improvement in microscopy sciences to overcome the unique challenges a green leaf poses. In this review, we discuss the available tools, techniques, challenges, and gaps for efficient in vivo leaf 3D imaging, as well as innovations to overcome these difficulties.

On the shape of cicada's wing leading-edge cross section.

An important role that the leading-edge cross-section shape plays in the wing flight performance is well known in aeronautics. However, little is known about the shape of the leading-edge cross section of an insect's wing and its contribution to remarkable qualities of insect flight. In this paper, we reveal, in the first time, the shape of the leading-edge cross section of a cicada's wing and analyze its variability along the wing. We also identify and quantify similarities in characteristic dimensions of this shape in the wings of three different cicada species.

Aerosol generation with various approaches to oxygenation in healthy volunteers in the emergency department.

OBJECTIVES: Health care workers experience an uncertain risk of aerosol exposure during patient oxygenation. To improve our understanding of these risks, we sought to measure aerosol production during various approaches to oxygenation in healthy volunteers in an emergency department. METHODS: This was a prospective study conducted in an empty patient room in an academic ED. The room was 10 ft. long x 10 ft. wide x 9 ft. tall (total volume 900 ft3) with positive pressure airflow (1 complete turnover of air every 10 minutes). Five oxygenation conditions were used: humidified high-flow nasal cannula (HFNC) at 3 flow rates [15, 30, and 60 liters per minute (LPM)], non-rebreather mask (NRB) at 1 flow rate (15 LPM), and closed-circuit continuous positive airway pressure (CPAP) using the ED ventilator; in all cases a simple procedural mask was used. The NRB and HFNC at 30 LPM maneuvers were also repeated without the procedural mask, and CPAP was applied both with and without a filter. Each subject then sequentially underwent 8 total oxygenation conditions, always in the same order. Each oxygenation condition was performed with the participant on a standard ED bed. Particles were measured by laser aerosol spectrometer, with the detector sampling port positioned directly over the center of the bed, 0.35 meters away and at a 45-degree angle from the subject's mouth. Each approach to oxygenation was performed for 10 minutes, followed by a 20-minute room washout (≈ 2 complete room air turnovers). Particle counts were summated for 2 size ranges (150-300 nm and 0.5-2.0 µm) and compared before, during, and after each of the 8 oxygenation conditions. RESULTS: Eight adult subjects were enrolled (mean age 42 years, body mass index 25). All subjects completed 8 oxygenation procedures (64 total). Mean particle counts per minute across all oxygenation procedures was 379 ± 112 (mean ± SD) for smaller aerosols (150-300 nm) and 9.3 ± 4.6 for larger aerosols (0.5-2.0 µm). HFNC exhibited a flow-dependent increase in particulate matter (PM) generation-at 60 LPM, HFNC had a substantial generation of small (55% increase) and large particles (70% increase) compared to 15 LPM. CPAP was associated with lowered small and large particle generation (≈ 10-15% below baseline for both sizes of PM). A patient mask limited particle generation with the NRB, where it was associated with a reduction in small and large particulates (average 40% and 20% lower, respectively). CONCLUSION: Among 3 standard oxygenation procedures, higher flow rates generally were associated with greater production of both small and large aerosols. A patient mask lowered aerosol counts in the NRB only. Protocol development for oxygenation application should consider these factors to increase health care worker safety.

What is the fate of xylem-transported CO2 in Kranz-type C4 plants?

High concentrations of dissolved inorganic carbon in stems of herbaceous and woody C3 plants exit leaves in the dark. In the light, C3 species use a small portion of xylem-transported CO2 for leaf photosynthesis. However, it is not known if xylem-transported CO2 will exit leaves in the dark or be used for photosynthesis in the light in Kranz-type C4 plants. Cut leaves of Amaranthus hypochondriacus were placed in one of three solutions of [NaH13 CO3 ] dissolved in KCl water to measure the efflux of xylem-transported CO2 exiting the leaf in the dark or rates of assimilation of xylem-transported CO2 * in the light, in real-time, using a tunable diode laser absorption spectroscope. In the dark, the efflux of xylem-transported CO2 increased with increasing rates of transpiration and [13 CO2 *]; however, rates of 13 Cefflux in A. hypochondriacus were lower compared to C3 species. In the light, A. hypochondriacus fixed nearly 75% of the xylem-transported CO2 supplied to the leaf. Kranz anatomy and biochemistry likely influence the efflux of xylem-transported CO2 out of cut leaves of A. hypochondriacus in the dark, as well as the use of xylem-transported CO2 * for photosynthesis in the light. Thus increasing the carbon use efficiency of Kranz-type C4 species over C3 species.

Contribution and consequences of xylem-transported CO2 assimilation for C3 plants.

Traditionally, leaves were thought to be supplied with CO2 for photosynthesis by the atmosphere and respiration. Recent studies, however, have shown that the xylem also transports a significant amount of inorganic carbon into leaves through the bulk flow of water. However, little is known about the dynamics and proportion of xylem-transported CO2 that is assimilated, vs simply lost to transpiration. Cut leaves of Populus deltoides and Brassica napus were placed in either KCl or one of three [NaH13 CO3 ] solutions dissolved in water to simultaneously measure the assimilation and the efflux of xylem-transported CO2 exiting the leaf across light and CO2 response curves in real-time using a tunable diode laser absorption spectroscope. The rates of assimilation and efflux of xylem-transported CO2 increased with increasing xylem [13 CO2 *] and transpiration. Under saturating irradiance, rates of assimilation using xylem-transported CO2 accounted for c. 2.5% of the total assimilation in both species in the highest [13 CO2 *]. The majority of xylem-transported CO2 is assimilated, and efflux is small compared to respiration. Assimilation of xylem-transported CO2 comprises a small portion of total photosynthesis, but may be more important when CO2 is limiting.

Using the rapid A-Ci response (RACiR) in the Li-Cor 6400 to measure developmental gradients of photosynthetic capacity in poplar.

The rapid A-Ci response (RACiR) technique alleviates limitations of measuring photosynthetic capacity by reducing the time needed to determine the maximum carboxylation rate (Vcmax ) and electron transport rate (Jmax ) in leaves. Photosynthetic capacity and its relationships with leaf development are important for understanding ecological and agricultural productivity; however, our current understanding is incomplete. Here, we show that RACiR can be used in previous generation gas exchange systems (i.e., the LI-6400) and apply this method to rapidly investigate developmental gradients of photosynthetic capacity in poplar. We compared RACiR-determined Vcmax and Jmax as well as respiration and stomatal conductance (gs ) across four stages of leaf expansion in Populus deltoides and the poplar hybrid 717-1B4 (Populus tremula × Populus alba). These physiological data were paired with leaf traits including nitrogen concentration, chlorophyll concentrations, and specific leaf area. Several traits displayed developmental trends that differed between the poplar species, demonstrating the utility of RACiR approaches to rapidly generate accurate measures of photosynthetic capacity. By using both new and old machines, we have shown how more investigators will be able to incorporate measurements of important photosynthetic traits in future studies and further our understanding of relationships between development and leaf-level physiology.

Using multirate rapid A/Ci curves as a tool to explore new questions in the photosynthetic physiology of plants.

Steady-state photosynthetic CO2 responses (A/Ci curves) are used to assess environmental responses of photosynthetic traits and to predict future vegetative carbon uptake through modeling. The recent development of rapid A/Ci curves (RACiRs) permits faster assessment of these traits by continuously changing [CO2 ] around the leaf, and may reveal additional photosynthetic properties beyond what is practical or possible with steady-state methods. Gas exchange necessarily incorporates photosynthesis and (photo)respiration. Each process was expected to respond on different timescales due to differences in metabolite compartmentation, biochemistry and diffusive pathways. We hypothesized that metabolic lags in photorespiration relative to photosynthesis/respiration and CO2 diffusional limitations can be detected by varying the rate of change in [CO2 ] during RACiR assays. We tested these hypotheses through modeling and experiments at ambient and 2% oxygen. Our data show that photorespiratory delays cause offsets in predicted CO2 compensation points that are dependent on the rate of change in [CO2 ]. Diffusional limitations may reduce the rate of change in chloroplastic [CO2 ], causing a reduction in apparent RACiR slopes under high CO2 ramp rates. Multirate RACiRs may prove useful in assessing diffusional limitations to gas exchange and photorespiratory rates.

Unsaturation of vapour pressure inside leaves of two conifer species.

Stomatal conductance (gs) impacts both photosynthesis and transpiration, and is therefore fundamental to the global carbon and water cycles, food production, and ecosystem services. Mathematical models provide the primary means of analysing this important leaf gas exchange parameter. A nearly universal assumption in such models is that the vapour pressure inside leaves (ei) remains saturated under all conditions. The validity of this assumption has not been well tested, because so far ei cannot be measured directly. Here, we test this assumption using a novel technique, based on coupled measurements of leaf gas exchange and the stable isotope compositions of CO2 and water vapour passing over the leaf. We applied this technique to mature individuals of two semiarid conifer species. In both species, ei routinely dropped below saturation when leaves were exposed to moderate to high air vapour pressure deficits. Typical values of relative humidity in the intercellular air spaces were as low 0.9 in Juniperus monosperma and 0.8 in Pinus edulis. These departures of ei from saturation caused significant biases in calculations of gs and the intercellular CO2 concentration. Our results refute the longstanding assumption of saturated vapour pressure in plant leaves under all conditions.

The Sphagnome Project: enabling ecological and evolutionary insights through a genus-level sequencing project.

Considerable progress has been made in ecological and evolutionary genetics with studies demonstrating how genes underlying plant and microbial traits can influence adaptation and even 'extend' to influence community structure and ecosystem level processes. Progress in this area is limited to model systems with deep genetic and genomic resources that often have negligible ecological impact or interest. Thus, important linkages between genetic adaptations and their consequences at organismal and ecological scales are often lacking. Here we introduce the Sphagnome Project, which incorporates genomics into a long-running history of Sphagnum research that has documented unparalleled contributions to peatland ecology, carbon sequestration, biogeochemistry, microbiome research, niche construction, and ecosystem engineering. The Sphagnome Project encompasses a genus-level sequencing effort that represents a new type of model system driven not only by genetic tractability, but by ecologically relevant questions and hypotheses.

Inside out: efflux of carbon dioxide from leaves represents more than leaf metabolism.

High concentrations of inorganic carbon in the xylem, produced from root, stem, and branch respiration, travel via the transpiration stream and eventually exit the plant through distant tissues as CO2. Unlike previous studies that focused on the efflux of CO2 from roots and woody tissues, we focus on efflux from leaves and the potential effect on leaf respiration measurements. We labeled transported inorganic carbon, spanning reported xylem concentrations, with 13C and then manipulated transpiration rates in the dark in order to vary the rates of inorganic carbon supply to cut leaves from Brassica napus and Populus deltoides. We used tunable diode laser absorbance spectroscopy to directly measure the rate of gross 13CO2 efflux, derived from inorganic carbon supplied from outside of the leaf, relative to gross 12CO2 efflux generated from leaf cells. These experiemnts showed that 13CO2 efflux was dependent upon the rate of inorganic carbon supply to the leaf and the rate of transpiration. Our data show that the gross leaf efflux of xylem-transported CO2 is likely small in the dark when rates of transpiration are low. However, gross leaf efflux of xylem-transported CO2 could approach half the rate of leaf respiration in the light when transpiration rates and branch inorganic carbon concentrations are high, irrespective of the grossly different petiole morphologies in our experiment.

The rapid A-Ci response: photosynthesis in the phenomic era.

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.

The carbon fertilization effect over a century of anthropogenic CO2 emissions: higher intracellular CO2 and more drought resistance among invasive and native grass species contrasts with increased water use efficiency for woody plants in the US Southwest.

From 1890 to 2015, anthropogenic carbon dioxide emissions have increased atmospheric CO2 concentrations from 270 to 400 mol mol-1 . The effect of increased carbon emissions on plant growth and reproduction has been the subject of study of free-air CO2 enrichment (FACE) experiments. These experiments have found (i) an increase in internal CO2 partial pressure (ci ) alongside acclimation of photosynthetic capacity, (ii) variable decreases in stomatal conductance, and (iii) that increases in yield do not increase commensurate with CO2 concentrations. Our data set, which includes a 115-year-long selection of grasses collected in New Mexico since 1892, is consistent with an increased ci as a response to historical CO2 increase in the atmosphere, with invasive species showing the largest increase. Comparison with Palmer Drought Sensitivity Index (PDSI) for New Mexico indicates a moderate correlation with Δ13 C (r2  = 0.32, P < 0.01) before 1950, with no correlation (r2  = 0.00, P = 0.91) after 1950. These results indicate that increased ci may have conferred some drought resistance to these grasses through increased availability of CO2 in the event of reduced stomatal conductance in response to short-term water shortage. Comparison with C3 trees from arid environments (Pinus longaeva and Pinus edulis in the US Southwest) as well as from wetter environments (Bromus and Poa grasses in New Mexico) suggests differing responses based on environment; arid environments in New Mexico see increased intrinsic water use efficiency (WUE) in response to historic elevated CO2 while wetter environments see increased ci . This study suggests that (i) the observed increases in ci in FACE experiments are consistent with historical CO2 increases and (ii) the CO2 increase influences plant sensitivity to water shortage, through either increased WUE or ci in arid and wet environments, respectively.

Why small fluxes matter: the case and approaches for improving measurements of photosynthesis and (photo)respiration.

Since its inception, the Farquhar et al. (1980) model of photosynthesis has been a mainstay for relating biochemistry to environmental conditions from chloroplast to global levels in terrestrial plants. Many variables could be assigned from basic enzyme kinetics, but the model also required measurements of maximum rates of photosynthetic electron transport (J max ), carbon assimilation (Vcmax ), conductance of CO2 into (g s ) and through (g m ) the leaf, and the rate of respiration during the day (R d ). This review focuses on improving the accuracy of these measurements, especially fluxes from photorespiratory CO2, CO2 in the transpiration stream, and through the leaf epidermis and cuticle. These fluxes, though small, affect the accuracy of all methods of estimating mesophyll conductance and several other photosynthetic parameters because they all require knowledge of CO2 concentrations in the intercellular spaces. This review highlights modified methods that may help to reduce some of the uncertainties. The approaches are increasingly important when leaves are stressed or when fluxes are inferred at scales larger than the leaf.

Evolution of photorespiration from cyanobacteria to land plants, considering protein phylogenies and acquisition of carbon concentrating mechanisms.

Photorespiration and oxygenic photosynthesis are intimately linked processes. It has been shown that under the present day atmospheric conditions cyanobacteria and all eukaryotic phototrophs need functional photorespiration to grow autotrophically. The question arises as to when this essential partnership evolved, i.e. can we assume a coevolution of both processes from the beginning or did photorespiration evolve later to compensate for the generation of 2-phosphoglycolate (2PG) due to Rubisco's oxygenase reaction? This question is mainly discussed here using phylogenetic analysis of proteins involved in the 2PG metabolism and the acquisition of different carbon concentrating mechanisms (CCMs). The phylogenies revealed that the enzymes involved in the photorespiration of vascular plants have diverse origins, with some proteins acquired from cyanobacteria as ancestors of the chloroplasts and others from heterotrophic bacteria as ancestors of mitochondria in the plant cell. Only phosphoglycolate phosphatase was found to originate from Archaea. Notably glaucophyte algae, the earliest branching lineage of Archaeplastida, contain more photorespiratory enzymes of cyanobacterial origin than other algal lineages or land plants indicating a larger initial contribution of cyanobacterial-derived proteins to eukaryotic photorespiration. The acquisition of CCMs is discussed as a proxy for assessing the timing of periods when photorespiratory activity may have been enhanced. The existence of CCMs also had marked influence on the structure and function of photorespiration. Here, we discuss evidence for an early and continuous coevolution of photorespiration, CCMs and photosynthesis starting from cyanobacteria via algae, to land plants.

The arc mutants of Arabidopsis with fewer large chloroplasts have a lower mesophyll conductance.

Photosynthetic cells of most land plant lineages have numerous small chloroplasts even though most algae, and even the early diverging land plant group the hornworts, tend to have one or a few large chloroplasts. One constraint that small chloroplasts could improve is the resistance to CO2 diffusion from the atmosphere to the chloroplast stroma. We examined the mesophyll conductance (inverse of the diffusion resistance) of mutant Arabidopsis thaliana plants with one or only a few large chloroplasts per cell. The accumulation and replication of chloroplasts (arc) mutants of A. thaliana were studied by model fitting to gas exchange data and (13)CO2 discrimination during carbon fixation. The two methods generally agreed, but the value of the CO2 compensation point of Rubisco (Γ *) used in the model had a large impact on the estimated photosynthetic parameters, including mesophyll conductance. We found that having only a few large chloroplasts per cell resulted in a 25-50 % reduction in the mesophyll conductance at ambient CO2.

On-line stable isotope gas exchange reveals an inducible but leaky carbon concentrating mechanism in Nannochloropsis salina.

Carbon concentrating mechanisms (CCMs) are common among microalgae, but their regulation and even existence in some of the most promising biofuel production strains is poorly understood. This is partly because screening for new strains does not commonly include assessment of CCM function or regulation despite its fundamental role in primary carbon metabolism. In addition, the inducible nature of many microalgal CCMs means that environmental conditions should be considered when assessing CCM function and its potential impact on biofuels. In this study, we address the effect of environmental conditions by combining novel, high frequency, on-line (13)CO2 gas exchange screen with microscope-based lipid characterization to assess CCM function in Nannochloropsis salina and its interaction with lipid production. Regulation of CCM function was explored by changing the concentration of CO2 provided to continuous cultures in airlift bioreactors where cell density was kept constant across conditions by controlling the rate of media supply. Our isotopic gas exchange results were consistent with N. salina having an inducible "pump-leak" style CCM similar to that of Nannochloropsis gaditana. Though cells grew faster at high CO2 and had higher rates of net CO2 uptake, we did not observe significant differences in lipid content between conditions. Since the rate of CO2 supply was much higher for the high CO2 conditions, we calculated that growing cells bubbled with low CO2 is about 40 % more efficient for carbon capture than bubbling with high CO2. We attribute this higher efficiency to the activity of a CCM under low CO2 conditions.

The physiological basis for genetic variation in water use efficiency and carbon isotope composition in Arabidopsis thaliana.

Ecologists and physiologists have documented extensive variation in water use efficiency (WUE) in Arabidopsis thaliana, as well as association of WUE with climatic variation. Here, we demonstrate correlations of whole-plant transpiration efficiency and carbon isotope composition (δ(13)C) among life history classes of A. thaliana. We also use a whole-plant cuvette to examine patterns of co-variation in component traits of WUE and δ(13)C. We find that stomatal conductance (g s) explains more variation in WUE than does A. Overall, there was a strong genetic correlation between A and g s, consistent with selection acting on the ratio of these traits. At a more detailed level, genetic variation in A was due to underlying variation in both maximal rate of carboxylation (V cmax) and maximum electron transport rate (Jmax). We also found strong effects of leaf anatomy, where lines with lower WUE had higher leaf water content (LWC) and specific leaf area (SLA), suggesting a role for mesophyll conductance (g m) in variation of WUE. We hypothesize that this is due to an effect through g m, and test this hypothesis using the abi4 mutant. We show that mutants of ABI4 have higher SLA, LWC, and g m than wild-type, consistent with variation in leaf anatomy causing variation in g m and δ(13)C. These functional data also add further support to the central, integrative role of ABI4 in simultaneously altering ABA sensitivity, sugar signaling, and CO2 assimilation. Together our results highlight the need for a more holistic approach in functional studies, both for more accurate annotation of gene function and to understand co-limitations to plant growth and productivity.

Spatio-temporal decoupling of stomatal and mesophyll conductance induced by vein cutting in leaves of Helianthus annuus.

Reduction of hydraulic conductance to the canopy has been shown to result in stomatal responses to limit transpiration. To test for similar responses to perturbations of the hydraulic network in leaves, we simultaneously measured leaf gas exchange with spatially explicit chlorophyll-a fluorescence and leaf temperature to examine the effects of cutting a primary leaf vein in Helianthus annuus. We repeated the leaf treatment at each of three different vapor pressure deficits and monitored the short-term dynamics of gas exchange following the treatment. Immediately after treatment, photosynthesis and stomatal conductance (g s) showed a transient "wrong way" response in which photosynthesis declined despite increased g s. Comparisons of fluorescence and temperature across the leaf showed that both photosynthesis and g s were transiently patchy across the measured leaf area, but that the patchiness of the two processes did not correspond in space or time. This suggests that photosynthesis and g s respond to vein cutting-induced cavitation via different mechanisms. Because the stomatal response varied by vapor pressure difference condition but photosynthesis did not, it is likely that g s, but not photosynthesis, responded to a hydraulic signal. In contrast, we hypothesize that photosynthesis declined due to a wound-induced electrical signal that has recently been shown to transiently decrease mesophyll conductance to CO2. The interaction of epidermal hydraulics and the electrical signal across the leaf likely created a patchy pattern of chlorophyll fluorescence and leaf temperature that cannot be explained through the action of a single signal.