Light Sheet Microscopy Imaging of Light Absorption and Photosynthesis Distribution in Plant Tissue.

In vivo variable chlorophyll fluorescence measurements of photosystem II (PSII) quantum yields in optically dense systems are complicated by steep tissue light gradients due to scattering and absorption. Consequently, externally measured effective PSII quantum yields may be composed of signals derived from cells differentially exposed to actinic light, where cells located deeper inside tissues receive lower irradiance than cells closer to the surface and can display distinct photophysiological status. We demonstrate how measured distributions of PSII quantum yields in plant tissue change under natural tissue light gradients as compared with conventionally measured quantum yields with even exposure to actinic light. This was achieved by applying actinic irradiance perpendicular to one side of thallus cross sections of the aquatic macrophyte Fucus vesiculosus with laser light sheets of defined spectral composition, while imaging variable chlorophyll fluorescence from cross sections with a microscope-mounted pulse amplitude-modulated imaging system. We show that quantum yields are highly affected by light gradients and that traditional surface-based variable chlorophyll fluorescence measurements result in substantial underestimations and/or overestimations, depending on incident actinic irradiance. We present a method for using chlorophyll fluorescence profiles in combination with integrating sphere measurements of reflectance and transmittance to calculate depth-resolved photon absorption profiles, which can be used to correct apparent PSII electron transport rates to photons absorbed by PSII. Absorption profiles of the investigated aquatic macrophyte were different in shape from what is typically observed in terrestrial leaves, and based on this finding, we discuss strategies for optimizing photon absorption via modulation of the structural organization of phytoelements according to in situ light environments.

Measurement of the optical properties of leaves under diffuse light.

Measuring leaf light absorptance is central to many areas of plant biology including photosynthesis and energy balance. Absorptance is calculated from measured values of transmittance and reflectance, and most such measurements have used direct beam light. However, photosynthesis and other processes can differ under direct and diffuse light. Optical properties under diffuse light may be different, but there have been technical difficulties involved in measuring total reflectance of diffuse light. We developed instrumentation to measure this reflectance using a chopped measuring beam delivered alternately to sample and reference integrating spheres, and lock-in detection. We also built instrumentation for measuring transmittance of diffuse light. We developed standards to calibrate our instruments and correct for substitution error, a known systematic error with integrating sphere-based measurements. Helianthus annuus leaves measured under diffuse light reflected 5-10% more and transmitted a few percent less 400-700 nm light than under direct light. Overall absorptance was only a few percent higher under direct light, but leaves may utilize absorbed direct and diffuse light differently. For example, of the light entering the leaf, significantly more direct light than diffuse light is transmitted through the leaf, suggesting differences in localization of absorption within the leaf.

Anthocyanin influence on light absorption within juvenile and senescing sugar maple leaves - do anthocyanins function as photoprotective visible light screens?

Foliar anthocyanins are hypothesised to function as photoprotective visible light screens, preventing over-excitation of the photosynthetic system, and decreasing the likelihood of photo-oxidative stress by absorbing green light and reducing the amount of light available to be absorbed by chloroplasts in deeper tissue layers. Chlorophyll fluorescence imaging was used to test the hypothesis that anthocyanins in the palisade mesophyll of juvenile and senescing sugar maple (Acer saccharum Marsh.) leaves function as visible light screens by assessing their influence on light absorption profiles within leaves. We hypothesised that an effective anthocyanic light screen should reduce light absorption, particularly of green wavelengths, by chloroplasts in the spongy mesophyll. Both anthocyanic juvenile and senescing leaves absorbed greater amounts of green light than corresponding nonanthocyanic leaves. However, profiles of green light absorption by chlorophyll within anthocyanic leaves were not shifted to reflect reduced absorption of green light by spongy mesophyll chloroplasts. Further, the spongy mesophyll of both anthocyanic juvenile and senescing leaves absorbed proportions of green light equal to or greater than the spongy mesophyll of corresponding nonanthocyanic leaves. These results indicate that though they may provide a general source of photoprotection by reducing the total quantity of light available to be absorbed by chlorophyll, the anthocyanins in juvenile and senescing sugar maple leaves do not attenuate light in a manner consistent with that expected for an anthocyanic screen in the palisade mesophyll.

Optical effects of abaxial anthocyanin on absorption of red wavelengths by understorey species: revisiting the back-scatter hypothesis.

A red/purple coloration of lower (abaxial) leaf surfaces is commonly observed in deeply-shaded understorey plants, especially in the tropics. However, the functional significance of red abaxial coloration, including its role in photosynthetic adaptation, remains unclear. The objective of this study was to test the back-scatter hypothesis for abaxial leaf coloration, which posits that red pigments internally reflect/scatter red light transmitted by the upper leaf surface back into the mesophyll, thereby enhancing photon capture in light-limited environments. Abaxially red/non-red variegated leaves of Begonia heracleifolia (Cham. & Schltdl.) were used to compare reflectance spectra and chlorophyll fluorescence profiles of abaxially anthocyanic (red) and acyanic (non-red) tissues under red light. Photosynthetic gas exchange in response to red light was also compared for abaxially red/non-red leaf sections. The results did not support a back-scattering function, as anthocyanic leaf surfaces were not more reflective of red light than acyanic surfaces. Anthocyanic tissues also did not exhibit any increases in the mesophyll absorbance of red light, or increased photosynthetic gas exchange under red light at any intensity, relative to acyanic tissues. These results suggest that abaxial anthocyanins do not significantly enhance the absorption of red light in the species tested, and alternative functions are discussed.

The influence of epidermal windows on the light environment within the leaves of six succulents.

An omni-directional fibre optic microprobe was used to measure the quantity and quality of light within the leaves of six succulents having epidermal windows, three species having a subterranean growth habit (Haworthia truncata, Lithops olivacea, and Opthalmophyllum longum) and three growing above ground (Peperomia dolabriformis, P. graveolens, and the sprawling vine Senecio rowleyanus). Although light levels at most locations inside the leaves of all species were high, near those incident on the window surfaces, light levels inside the leaves of the two species of Peperomia often greatly exceeded incident light levels, indicating considerable light scattering and focusing by the leaf tissue. The spectral quality of light inside the leaves of all taxa reflected the absorption properties of chlorophyll, with most of the photons in the green wavelengths. Light quality and quantity inside the leaves did not correlate with the growth habit of the plants, the size of the window (as a proportion of the total leaf area), or location inside the leaf, although light levels generally declined and wavelengths increased deeper in the leaves. Application of reflective tape to the windows reduced internal light levels in L. olivacea and S. rowleyanus, although reductions were not always statistically significant. Although light levels throughout the leaves of P. graveolens were substantially and significantly reduced as a result of the application of reflective tape to its windows, the light levels even at the basal chlorenchyma on the abaxial side of the leaf remained high. In all species investigated, the levels of near-infrared radiation inside the leaves were surprisingly high, yet also declined deeper inside the succulent leaves. This near-infrared radiation may add to the heat load of these plants. Furthermore, application of reflective tape to the windows also reduced the amount of near-infrared radiation inside the leaves of the three succulents examined. These results led to a novel, testable hypothesis that may help to explain previous findings that application of reflective tape to the windows of the leaves of these succulents did not effect a reduction in photosynthetic activity.

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.

Do epidermal lens cells facilitate the absorptance of diffuse light?

Many understory plants rely on diffuse light for photosynthesis because direct light is usually scattered by upper canopy layers before it strikes the forest floor. There is a considerable gap in the literature concerning the interaction of direct and diffuse light with leaves. Some understory plants have well-developed lens-shaped epidermal cells, which have long been thought to increase the absorption of diffuse light. To assess the role of epidermal cell shape in capturing direct vs. diffuse light, we measured leaf reflectance and transmittance with an integrating sphere system using leaves with flat (Begonia erythrophylla, Citrus reticulata, and Ficus benjamina) and lens-shaped epidermal cells (B. bowerae, Colocasia esculenta, and Impatiens velvetea). In all species examined, more light was absorbed when leaves were irradiated with direct as opposed to diffuse light. When leaves were irradiated with diffuse light, more light was transmitted and more was reflected in both leaf types, resulting in absorptance values 2-3% lower than in leaves irradiated with direct light. These data suggest that lens-shaped epidermal cells do not aid the capture of diffuse light. Palisade and mesophyll cell anatomy and leaf thickness appear to have more influence in the capture and absorption of light than does epidermal cell shape.

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%.

A photoacoustic method for rapid assessment of temperature effects on photosynthesis.

The photosynthetic and photoacoustic properties of leaf samples were studied using a photoacoustic system modified for precise temperature control. Data were collected over a temperature range of -10 degrees C to +60 degrees C. A distinct acoustic noise transient marked the freezing temperature of the samples. A positive absorption transient and a brief period of oxygen uptake marked the thermal denaturing temperature of the samples. Between these extremes, the effects of temperature on light absorption, oxygen evolution, and photochemical energy storage were quantified quickly and easily. Oxygen evolution could be measured as low as -5 degrees C and showed a broad temperature peak that was 10 degrees C lower under limiting light intensity than under saturating light intensity. Photochemical energy storage showed a narrower temperature peak that was only slightly lower for limiting light intensities than for saturating light intensities. In a survey of diverse plants, temperature response curves for oxygen evolution were determined readily for a variety of leaf types, including ferns and conifer needles. These results demonstrate that temperature-controlled photoacoustics can be useful for rapid assessment of temperature effects on photosynthesis and other leaf properties.

Leaf architecture and direction of incident light influence mesophyll fluorescence profiles.

Light propagation and distribution inside leaves have been recognized as important processes influencing photosynthesis. Monochromatic light absorption across the mesophyll was measured using chlorophyll fluorescence generated from illumination of the cut edge (epi-illumination), as well as the adaxial or abaxial surfaces of the leaf. Species were selected that had basic leaf types: laminar leaf with adaxial palisade layer (Rhododendron catawbiense), needle with palisade (Abies fraseri), and needle without palisade (Picea rubens). Fluorescence was more evenly distributed across the mesophyll for adaxially illuminated leaves with a palisade cell layer, as well as for the needles (cylindrical) without palisade, when compared to fluorescence generated by abaxial illumination. Moreover, fluorescence from green light illumination remained high across the mesophyll of adaxially illuminated R. catawbiense, indicating a possible influence of mesophyll structure on internal light distribution beyond that of chlorophyll levels. These data support the idea that light propagation within the mesophyll is associated with asymmetric mesophyll structure, in particular the presence of palisade cell layers. In addition, we propose that the evolution of a more cylindrical leaf form, such as found in conifer species, may be a structural solution to excessive sunlight that replaces the highly differentiated mesophyll found in most laminar-leaved species.

Photoacoustic analysis indicates that chloroplast movement does not alter liquid-phase CO2 diffusion in leaves of Alocasia brisbanensis.

Light-mediated chloroplast movements are common in plants. When leaves of Alocasia brisbanensis (F.M. Bailey) Domin are exposed to dim light, mesophyll chloroplasts spread along the periclinal walls normal to the light, maximizing absorbance. Under high light, the chloroplasts move to anticlinal walls. It has been proposed that movement to the high-light position shortens the diffusion path for CO(2) from the intercellular air spaces to the chloroplasts, thus reducing CO(2) limitation of photosynthesis. To test this hypothesis, we used pulsed photoacoustics to measure oxygen diffusion times as a proxy for CO(2) diffusion in leaf cells. We found no evidence that chloroplast movement to the high-light position enhanced gas diffusion. Times for oxygen diffusion were not shorter in leaves pretreated with white light, which induced chloroplast movement to the high-light position, compared with leaves pretreated with 500 to 700 nm light, which did not induce movement. From the oxygen diffusion time and the diffusion distance from chloroplasts to the intercellular gas space, we calculated an oxygen permeability of 2.25 x 10(-)(6) cm(2) s(-)(1) for leaf cells at 20 degrees C. When leaf temperature was varied from 5 degrees C to 40 degrees C, the permeability for oxygen increased between 5 degrees C and 20 degrees C but changed little between 20 degrees C and 40 degrees C, indicating changes in viscosity or other physical parameters of leaf cells above 20 degrees C. Resistance for CO(2) estimated from oxygen permeability was in good agreement with published values, validating photoacoustics as another way of assessing internal resistances to CO(2) diffusion.

Ultraviolet radiation and the snow alga Chlamydomonas nivalis (Bauer) Wille.

Aplanospores of Chlamydomonas nivalis are frequently found in high-altitude, persistent snowfields where they are photosynthetically active despite cold temperatures and high levels of visible and ultraviolet (UV) radiation. The goals of this work were to characterize the UV environment of the cells in the snow and to investigate the existence and localization of screening compounds that might prevent UV damage. UV irradiance decreased precipitously in snow, with UV radiation of wavelengths 280-315 nm and UV radiation of wavelengths 315-400 nm dropping to 50% of incident levels in the top 1 and 2 cm, respectively. Isolated cell walls exhibited UV absorbance, possibly by sporopollenin, but this absorbance was weak in images of broken or plasmolyzed cells observed through a UV microscope. The cells also contained UV-absorbing cytoplasmic compounds, with the extrachloroplastic carotenoid astaxanthin providing most of the screening. Additional screening compound(s) soluble in aqueous methanol with an absorption maximum at 335 nm played a minor role. Thus, cells are protected against potentially high levels of UV radiation by the snow itself when they live several centimeters beneath the surface, and they rely on cellular screening compounds, chiefly astaxanthin, when located near the surface where UV fluxes are high.

Surface gas-exchange processes of snow algae.

The red-colored chlorophyte Chlamydomonas nivalis is commonly found in summer snowfields. We used a modified Li-Cor gas-exchange system to investigate surface gas-exchange characteristics of snow colonized by this alga, finding rates of CO(2) uptake up to 0.3 micromol.m(-2).s(-1) in dense algal blooms. Experiments varying the irradiance resulted in light curves that resembled those of the leaves of higher plants. Red light was more effective than white and much more effective than green or blue, because of the red astaxanthin that surrounds and masks the algal chloroplasts. Integrating daily course measurements of gas exchange showed CO(2) uptake around 2,300 micromol.m(-2).day(-1) in heavily colonized patches, indicating that summer snowfields can be surprisingly productive.

Profiles of photosynthesis within red and green leaves of Quintinia serrata.

We have measured photosynthesis at the cellular, tissue, and whole leaf levels to understand the role of anthocyanin pigments on patterns of light utilization. Profiles of chlorophyll fluorescence through sections of red and green leaves of Quintinia serrata showed that anthocyanins in the mesophyll restricted absorption of green light to the uppermost palisade mesophyll. The distribution was further restricted when anthocyanins were also present in the upper epidermis. Mesophyll cells located beneath a cyanic light-filter assumed the characteristic photosynthetic features of shade-adapted cells. As a result, red leaves showed a 23% reduction in CO2 assimilation under light-saturating conditions, and a lower threshold irradiance for light-saturation, relative to those of green leaves. The photosynthetic characteristics of red leaves are comparable to those of shade-acclimated plants.