Kinetic imaging of chlorophyll fluorescence using modulated light

Fluorometers that measure the kinetics of chlorophyll fluorescence have become invaluable tools for determining the photosynthetic performance of plants. Many of these instruments use high frequency modulated light to measure the rate, efficiency and regulation of photosynthesis. The technique is non-invasive and is effective under diverse environmental conditions. Recently, imaging fluorometers have been introduced that reveal variability in photosynthesis over the surface of a leaf or between individual plants. Most imaging instruments depend on continuous light or low frequency modulated light for fluorescence excitation, which imposes serious limitations on measurements of the fluorescence parameters, especially the minimum fluorescence (F₀) and variable fluorescence (F_V). Here, we describe a new instrument that combines the advantage of high frequency modulated light with two-dimensional imaging of chlorophyll fluorescence. The fluorometer produces dynamic images of chlorophyll fluorescence from leaves or plants, providing accurate mapping of F₀ and F_V, and non-photochemical quenching. A significant feature of the instrument is that it can record fluorescence images of leaves in daylight under field conditions. This revised version was published online in June 2006 with corrections to the Cover Date.     

A new paradigm in leaf-level photosynthesis: direct and diffuse lights are not equal

Global-change scenarios suggest a trend of increasing diffuse light due to expected increases in cloud cover. Canopy-level measurements of plant-community photosynthesis under diffuse light show increased productivity attributed to more uniform distribution of light within the forest canopy, yet the effect of the directional quality of light at the leaf level is unknown. Here we show that leaf-level photosynthesis in sun leaves of both C(3) and C(4) plants can be 10-15% higher under direct light compared to equivalent absorbed irradiances of diffuse light. High-light-grown leaves showed significant photosynthetic enhancement in direct light, while shade-adapted leaves showed no preference for direct or diffuse light at any irradiance. High-light-grown leaves with multiple palisade layers may be adapted to better utilize direct than diffuse light, while shade leaf structure does not appear to discriminate light based on its directionality. Based upon our measurements, it appears that leaf-level and canopy-level photosynthetic processes react differently to the directionality of light, and previously observed increases in canopy-level photosynthesis occur even though leaf-level photosynthesis decreases under diffuse light.     

Modeling the influence of differential sectoriality on the photosynthetic responses of understory saplings to patchy light and water availability

Exploitation of patchy light is a key determinant of plant performance in the forest understory. While many adaptive traits are known, the role of stem vasculature in understory photosynthesis is not established. Sectoriality—the degree of vascular constraint to long distance transport—has been hypothesized to limit growth in heterogeneous light. We simulated the photosynthetic potential of sectored and integrated plants in patchy light, as a function of soil water potential (patchy or uniform). We used hydraulic parameters typical of temperate woody species in an Ohm’s law model including a tangential resistance parameter, and simulated cavitation by varying axial resistance of leaves, leaves and roots, or the whole plant. Our results suggest that differential sectoriality will not affect photosynthesis when water is plentiful, but can constrain stomatal conductance at more negative soil water potentials, especially when only a small portion of the crown receives light. This effect is strongest just below the turgor loss point, and depends on axial resistance and soil water heterogeneity. Increased resistance in high light leaves decreases photosynthesis regardless of sectoriality. However, when resistance is increased for leaves and roots or the whole plant, photosynthesis decreases more for sectored than for integrated plants. Moreover, the simulations suggest that sectoriality can further depress photosynthesis when water availability is asymmetrical. These results might explain why integrated species, such as Betula lenta, B. alleghaniensis, and Acer saccharum thrive in the forest understory and grow rapidly into canopy gaps, while sectored species, such as Quercus rubra, do not.     

Local weak light induces the improvement of photosynthesis in adjacent illuminated leaves in maize seedlings

To copy with highly heterogeneous light environment, plants can regulate photosynthesis locally and systemically, thus, maximizing the photosynthesis of individual plants. Therefore, we speculated that local weak light may induce the improvement of photosynthesis in adjacent illuminated leaves in plants. In order to test this hypothesis, maize seedlings were partially shaded, and gas exchange, chlorophyll a fluorescence and biochemical analysis were carefully assessed. It was shown that local shading exacerbated the declines in the photosynthetic rates, chlorophyll contents, electron transport and carbon assimilation‐related enzyme activities in shaded leaves as plants growth progressed. While, the decreases of these parameters in adjacent illuminated leaves of shaded plants were considerably alleviated compared to the corresponding leaves of control plants. Obviously, the photosynthesis in adjacent illuminated leaves in shaded plants was improved by local shading, and the improvement in adjacent lower leaves was larger than that in adjacent upper ones. As growth progressed, local shading induced higher abscisic acid contents in shaded leaves, but it alleviated the increase in the abscisic acid contents in adjacent leaves in shaded plants. Moreover, the difference in sugar content between shaded leaves and adjacent illuminated ones was gradually increased. Consequently, local weak light suppressed the photosynthesis in shaded leaves, while it markedly improved the photosynthesis of adjacent illuminated ones. Sugar gradient between shaded leaves and adjacent illuminated ones might play a key role in photosynthetic regulation of adjacent illuminated leaves.     

Effect of Aminoacetonitrile on the CO2 Exchange Rate in Rice Leaves

Aminoacetonitrile (AAN), a specific inhibitor of glycine oxidation in the photorespiratory glycolate pathway, did not inhibit photosynthetic CO₂ fixation, but inhibited the apparent photosynthesis of rice leaves under high photosynthetic conditions. However, under such low photosynthetic conditions as low light intensity or senescent leaves, the apparent photosynthesis was not inhibited by AAN. The application of AAN to the leaves led to a greater accumulation of glycine under a high photosynthetic condition like strong light intensity.

From these results, it can be postulated that the inhibition of apparent photosynthesis by AAN was due to the accumulation of intermediate metabolites in the photorespiratory glycolate pathway which was induced by AAN treatment.     

Instantaneous canopy photosynthesis: analytical expressions for sun and shade leaves based on exponential light decay down the canopy and an acclimated non-rectangular hyperbola for leaf photosynthesis

Analytical expressions for the contributions of sun and shade leaves to instantaneous canopy photosynthesis are derived. The analysis is based on four assumptions. First, that the canopy is closed in the sense that it is horizontally uniform. Secondly, that there is an exponential profile of light down the canopy with the same decay constant for light from different parts of the sky. Thirdly, that the leaf photosynthetic response to incident irradiance can be described by a three-parameter non-rectangular hyperbola (NRH). And lastly, that light acclimation at the leaf level occurs in only one parameter of the NRH, that describing the light-saturated photosynthetic rate, which is assumed to be proportional to the local averaged leaf irradiance. These assumptions have been extensively researched empirically and theoretically and their limitations are quite well understood. They have been widely used when appropriate. Combining these four assumptions permits the derivation of algebraic expressions for instantaneous canopy photosynthesis which are computationally efficient because they avoid the necessity for numerical integration down the canopy. These are valuable for modelling plant and crop ecosystems, for which canopy photosynthesis is the primary driver. Ignoring the sun/shade dichotomy can result in overestimates of canopy photosynthesis of up to 20 %, but using a rectangular hyperbola instead of a non-rectangular hyperbola to estimate canopy photosynthesis taking account of sun and shade leaves can lead to a similarly sized underestimate.     

In vivo studies of gross photosynthesis in attached leaves by means of photothermal radiometry

In photothermal radiometry, heat radiation from an illuminated object, in synchronism with incident chopped light, is observed using an infrared detector with suitable electronics. By thus measuring the heat released during pulse-wise irradiation of leaves, conclusions can be drawn as to the gross efficiency of photosynthesis: More heat means less photochemically stored energy. Saturation of photosynthesis, by employing additional strong continuous-wave background light, affords an internal photothermal radiometry signal reference corresponding to the photochemical zero efficiency level, against which the signal in the absence of saturation can be compared. Through such means, gross energy storage efficiencies approaching 30% have been observed in Caragana arborescens Lam. at low light intensities. Several other examples are given, including measurements on dark-adapted leaves and leaves infiltrated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea, to support our conclusion that photothermal radiometry provides a powerful new method for in vivo studies of photosynthesis in whole, attached leaves.     

Photosynthesis within isobilateral Eucalyptus pauciflora leaves

Adult Eucalyptus pauciflora leaves are vertically displayed. They have multiple palisade cell layers beneath both surfaces, interrupted by numerous oil glands. Here, we characterized light absorption, chlorophyll, photosynthetic capacity and CO2 fixation profiles through these leaves. Multiple chlorophyll fluorescence images of leaves viewed in cross-section were made by applying light from different directions. 14CO2 labelling, followed by paradermal cryosectioning, was used to measure profiles of photosynthesis. Photosynthetic capacity peaked 75 microm into the mesophyll beneath each surface and was lowest in the centre of the 600-microm-thick leaf. Predictions by a multilayer model using Beer's law matched the observed profiles of 14C fixation. When constrained to the horizontal, a vertically acclimated leaf gains only 79% of the daily photosynthesis achieved by a horizontally acclimated leaf. However, it outperforms the horizontally acclimated leaf when both are oriented vertically. Each half of the observed profile of photosynthetic capacity closely matches the profile of light absorption through the leaf with unilateral illumination to that surface. Derivation of biochemical parameters from gas exchange measured under unilateral illumination would underestimate the real photosynthetic capacity of these leaves by 21%.     

May photoinhibition be a consequence, rather than a cause, of limited plant productivity?

Photoinhibition in leaves in response to high and/or excess light, consisting of a decrease in photosynthesis and/or photosynthetic efficiency, is frequently equated to photodamage and often invoked as being responsible for decreased plant growth and productivity. However, a review of the literature reveals that photoinhibited leaves characterized for foliar carbohydrate levels were invariably found to possess high levels of sugars and starch. We propose that photoinhibition should be placed in the context of whole-plant source–sink regulation of photosynthesis. Photoinhibition may represent downregulation of the photosynthetic apparatus in response to excess light when (1) more sugar is produced in leaves than can be utilized by the rest of the plant and/or (2) more light energy is harvested than can be utilized by the chloroplast for the fixation of carbon dioxide into sugars.     

Effect of light interception by non-photosynthetic organs on canopy photosynthetic production

A canopy photosynthesis model was derived on the assumption that the light diminution within a canopy is caused by both leaves and non-photosynthetic organs. The light diminution by leaves and that by non-photosynthetic organs were taken into account separately in the Lambert-Beer equation of light extinction. The light flux density on the leaf surface at each depth was evaluated from the leaf's share of light. The light flux density on the leaf surface thus obtained was incorporated into the Monsi-Saeki model of canopy photosynthesis. The proposed model was applied for estimating gross canopy photosynthesis in a 19-year-oldLarix leptolepis plantation where 38% of the light diminution was due to non-photosynthetic organs. The daily canopy photosynthesis on one summer day calculated using the present model was about 22% less than that calculated by the conventional Monsi-Saeki model, in which light interception by non-photosynthetic organs is neglected. The degree of such reduction in canopy photosynthesis through shading by non-photosynthetic organs was assessed in relation to parameters affecting light extinction, leaf photosynthetic characteristics, and light regime above the canopy.     

What is the most prominent factor limiting photosynthesis in different layers of a greenhouse cucumber canopy?

Background and Aims

Maximizing photosynthesis at the canopy level is important for enhancing crop yield, and this requires insights into the limiting factors of photosynthesis. Using greenhouse cucumber (Cucumis sativus) as an example, this study provides a novel approach to quantify different components of photosynthetic limitations at the leaf level and to upscale these limitations to different canopy layers and the whole plant.

Methods

A static virtual three-dimensional canopy structure was constructed using digitized plant data in GroIMP. Light interception of the leaves was simulated by a ray-tracer and used to compute leaf photosynthesis. Different components of photosynthetic limitations, namely stomatal (S_L), mesophyll (M_L), biochemical (B_L) and light (L_L) limitations, were calculated by a quantitative limitation analysis of photosynthesis under different light regimes.

Key Results

In the virtual cucumber canopy, B_L and L_L were the most prominent factors limiting whole-plant photosynthesis. Diffusional limitations (S_L + M_L) contributed <15 % to total limitation. Photosynthesis in the lower canopy was more limited by the biochemical capacity, and the upper canopy was more sensitive to light than other canopy parts. Although leaves in the upper canopy received more light, their photosynthesis was more light restricted than in the leaves of the lower canopy, especially when the light condition above the canopy was poor. An increase in whole-plant photosynthesis under diffuse light did not result from an improvement of light use efficiency but from an increase in light interception. Diffuse light increased the photosynthesis of leaves that were directly shaded by other leaves in the canopy by up to 55 %.

Conclusions

Based on the results, maintaining biochemical capacity of the middle–lower canopy and increasing the leaf area of the upper canopy would be promising strategies to improve canopy photosynthesis in a high-wire cucumber cropping system. Further analyses using the approach described in this study can be expected to provide insights into the influences of horticultural practices on canopy photosynthesis and the design of optimal crop canopies.     

Leaf photosynthetic light response: a mechanistic model for scaling photosynthesis to leaves and canopies

1. The response of photosynthesis to radiation is an often-studied but poorly understood process, represented empirically in most photosynthesis models. However, in scaling photosynthesis from leaf to canopy, predictions of canopy photosynthesis are very sensitive to parameters describing the response of leaves to Photosynthetic Photon Flux Density (PPFD).
2. In this study, a mechanistic, yet still simple, approach is presented that models the degree of light saturation in leaves explicitly, assuming a heterogeneous environment of PPFD and chlorophyll.
3. Possible mechanisms determining the ratio of chlorophyll to nitrogen are considered, including a direct dependence on PPFD, a mechanism involving the red/far-red ratio of light in the canopy, and an approach based upon maximizing photosynthesis.
4. Comparison of model predictions with two data sets of light, nitrogen and chlorophyll from canopies of Populus and Corylus suggests that the red/far-red mechanism is the most realistic. The data also show that the trees studied do not always optimize their nitrogen partitioning to maximize photosynthetic yield.
5. We then apply the model to the data sets, to predict the shape of light response curves of leaves within canopies and assess the applicability of simple scaling schemes, in which full acclimation of photosynthesis to PPFD justifies the use of big-leaf models. We conclude that, at least for the data used, basic assumptions of such schemes do not hold.     

Acclimation of photosynthesis to lightflecks in tomato leaves: interaction with progressive shading in a growing canopy

Plants in natural environments are often exposed to fluctuations in light intensity, and leaf‐level acclimation to light may be affected by those fluctuations. Concurrently, leaves acclimated to a given light climate can become progressively shaded as new leaves emerge and grow above them. Acclimation to shade alters characteristics such as photosynthetic capacity. To investigate the interaction of fluctuating light and progressive shading, we exposed three‐week old tomato (Solanum lycopersicum ) plants to either lightflecks or constant light intensities. Lightflecks of 20 s length and 1000 μmol m^?2 s^?1 peak intensity were applied every 5 min for 16 h per day, for 3 weeks. Lightfleck and constant light treatments received identical daily light sums (15.2 mol m^?2 day^?1). Photosynthesis was monitored in leaves 2 and 4 (counting from the bottom) during canopy development throughout the experiment. Several dynamic and steady‐state characteristics of photosynthesis became enhanced by fluctuating light when leaves were partially shaded by the upper canopy, but much less so when they were fully exposed to lightflecks. This was the case for CO₂‐saturated photosynthesis rates in leaves 2 and 4 growing under lightflecks 14 days into the treatment period. Also, leaf 2 of plants in the lightfleck treatment showed significantly faster rates of photosynthetic induction when exposed to a stepwise change in light intensity on day 15. As the plants grew larger and these leaves became increasingly shaded, acclimation of leaf‐level photosynthesis to lightflecks disappeared. These results highlight continuous acclimation of leaf photosynthesis to changing light conditions inside developing canopies.     

Comparative ecophysiology of leaf and canopy photosynthesis

Leaves and herbaceous leaf canopies photosynthesize efficiently although the distribution of light, the ultimate resource of photosynthesis, is very biased in these systems. As has been suggested in theoretical studies, if a photosynthetic system is organized such that every photosynthetic apparatus photosynthesizes in concert, the system as a whole has the sharpest light response curve and is most adaptive. This condition can be approached by (i) homogenization of the light environment and (ii) acclimation of the photosynthetic properties of leaves or chloroplasts to their local light environments. This review examines these two factors in the herbaceous leaf canopy and in the leaf. Changes in the inclination of leaves in the canopy and differentiation of mesophyll into palisade and spongy tissue contribute to the moderation of the light gradient. Leaf and chloroplast movements in the upper parts of these systems under high irradiances also moderate light gradients. Moreover, acclimation of leaves and chloroplasts to the local light environment is substantial. These factors increase the efficiency of photosynthesis considerably. However, the systems appear to be less efficient than the theoretical optimum. When the systems are optically dense, the light gradients may be too great for leaves or chloroplasts to acclimate. The loss of photosynthetic production attributed to the imperfect adjustment of photosynthetic apparatus to the local light environment is most apparent when the photosynthesis of the system is in the transition between the light-limited and light-saturated phases. Although acclimation of the photosynthetic apparatus and moderation of light gradients are imperfect, these markedly raise the efficiency of photosynthesis. Thus more mechanistic studies on these adaptive attributes are needed. The causes and consequences of imperfect adjustment should also be investigated.     

Leaf display and photosynthesis of tree seedlings in a cool-temperate deciduous broadleaf forest understorey

To examine a possible convergence in leaf photosynthetic characteristics and leaf display responses to light environment in seedlings of three canopy and two shrub tree species in understorey of cool-temperate deciduous broadleaf forest, relationships between light environment, leaf orientation and leaf light-photosynthetic response were measured. Light capture of the seedlings (17-24 individuals with 2-12 leaves for each species) was assessed with a three dimensional geometric modeling program Y-plant. Leaf photosynthetic characteristics of the five species were found to have acclimated to the understorey light environment, i.e., low light compensation point and high apparent quantum yield. In addition, light-saturated photosynthetic rates were higher in seedlings inhabiting microsites with higher light availability. Efficiencies of light capture and carbon gain of the leaf display were evaluated by simulating the directionalities of light capture and daily photosynthesis for each seedling using hemispherical canopy photography. The results showed that most of the seedlings orientated their leaves in a way to increase the daily photosynthesis during the direct light periods (sunflecks) rather than maximize daily photosynthesis by diffuse light. Simulations also showed that daily photosynthesis would increase only 10% of that on actual leaf display when the leaves orientated to maximize the diffuse light interception. Simulations in which leaf orientations were varied showed that when the leaf display fully maximized direct light interception, the time that leaves were exposed to excessive photon flux density of >800 mumol photons m(-2) s(-1) were doubled. The understorey seedlings studied responded to the given light environments in a way to maximize the efficiency of acquisition and use of light during their short (approximately 3 month) seasonal growth period.     

Photosystem II efficiency and mechanisms of energy dissipation in iron-deficient,field-grown pear trees (Pyrus communis L.)

The dark-adapted Photosystem II efficiency of field-grown pear leaves, estimated by the variable to maximum chlorophyll fluorescence ratio, was little affected by moderate and severe iron deficiency. Only extremely iron-deficient leaves showed a decreased Photosystem II efficiency after dark adaptation. Midday depressions in Photosystem II efficiency were still found after short-term dark-adaptation in iron-deficient leaves, indicating that Photosystem II down-regulation occurred when the leaves were illuminated by excessive irradiance. The actual Photosystem II efficiency at steady-state photosynthesis was decreased by iron deficiency both early in the morning and at midday, due to closure of Photosystem II reaction centers and decreases of the intrinsic Photosystem II efficiency. Iron deficiency decreased the amount of light in excess of that which can be used in photosynthesis not only by decreasing absorptance, but also by increasing the relative amount of light dissipated thermally by the Photosystem II antenna. When compared to the controls, iron-deficient pear leaves dissipated thermally up to 20% more of the light absorbed by the Photosystem II, both early in the morning and at midday. At low light iron-deficient leaves with high violaxanthin cycle pigments to chlorophyll ratios had increases in pigment de-epoxidation, non-photochemical quenching and thermal dissipation. Our data suggest that pH could be the major factor controlling thermal energy dissipation, and that large (more than 10-fold) changes in the zeaxanthin plus antheraxanthin to chlorophyll molar ratio caused by iron deficiency were associated only to moderate increases in the extent of photoprotection.This revised version was published online in October 2005 with corrections to the Cover Date.     

Limitation of Photosynthesis by Carbon Metabolism : I. Evidence for Excess Electron Transport Capacity in Leaves Carrying Out Photosynthesis in Saturating Light and CO(2)

It has been investigated how far electron transport or carbon metabolism limit the maximal rates of photosynthesis achieved by spinach leaves in saturating light and CO₂. Leaf discs were illuminated with high light until a steady state rate of O₂ evolution was attained, and then subjected to a 30 second interruption in low light, to generate an increased demand for the products of electron transport. Upon returning to high light there is a temporary enhancement of photosynthesis which lasts 15 to 30 seconds, and can be up to 50% above the steady state rate of O₂ evolution. This temporary enhancement is only found when saturating light intensities are used for the steady state illumination, is increased when low light rather than darkness is used during the interruption, and is maximal following a 30 to 60 seconds interruption in low light. Decreasing the temperature over the 10 to 30°C range led to the transient enhancement becoming larger. The temporary enhancement is associated with an increased ATP/ADP ratio, a decreased level of 3-phosphoglycerate, and increased levels of triose phosphate and ribulose 1,5-bisphosphate. Since electron transport can occur at higher rates than in steady state conditions, and generate a higher energy status, it is concluded that leaves have a surplus electron transport capacity in saturating light and CO₂. From the alterations of metabolites, it can be calculated that the enhanced O₂ evolution must be accompanied by an increased rate of ribulose 1,5-bisphosphate regeneration and carboxylation. It is suggested that the capacity for sucrose synthesis ultimately limits the maximal rates of photosynthesis, by restricting the rate at which inorganic phosphate can be recycled to support electron transport and carbon fixation in the chloroplast.     

The photosynthesis of ryegrass leaves grown in a simulated sward

Plants were taken from simulated swards of perennial ryegrass (Lolium perenne) grown in a controlled environment and the rates of photosynthesis of the youngest fully expanded leaves, and the second and third youngest leaves on the same tillers were measured. The youngest leaves had the highest rates and the third the lowest, with the second leaves intermediate. The rate of photosynthesis in bright light of successive youngest expanded leaves decreased as the swards increased in leaf area, but did not when plants were grown so that the main stem was not shaded. When plants were grown at different densities and the photosynthetic rates of leaves of a particular ontogenetic rank were measured, it was found that leaves on plants from higher densities had lower rates of photosynthesis. Also leaves on plants grown in bright light had higher photosynthetic rates than those on plants grown in dim light. It is concluded that the decline in the photosynthetic capacity of successive leaves in a rapidly growing simulated sward is due to the intense shading to which they are subjected during their expansion.     

The diurnal time course of net photosynthesis of soybean leaves: Analysis with a physiologically based steady-state photosynthesis model

Summary A physiologically based steady-state model of whole leaf photosynthesis (WHOLEPHOT) is used to analyze observed net photosynthesis daily time courses of soybean, Glycine max (L.) Merr., leaves. Observations during two time periods of the 1978 growing season are analyzed and compared. After adjustment of the model for soybean, net photosynthesis rates are calculated with the model in response to measured incident light intensity, leaf temperature, air carbon dioxide concentration, and leaf diffusion resistance. The steady-state calculations closely approximate observed net photosynthesis. Results of the comparison reveal a decrease in photosynthetic capacity in leaves sampled during the second time period, which is associated with decreasing ability of leaves to respond to light intensity and internal air space carbon dioxide concentration, increasing mesophyll resistance, and increasing stomatal resistance.