Measurement of gradients of absorbed light in spinach leaves from chlorophyll fluorescence profiles

Profiles of chlorophyll fluorescence were measured in spinach leaves irradiated with monochromatic light. The characteristics of the profiles within the mesophyll were determined by the optical properties of the leaf tissue and the spectral quality of the actinic light. When leaves were infiltrated with 10^?4M DCMU [3‐(3,4‐dichlorophenyl)‐1, 1‐dimethyl‐urea] or water, treatments that minimized light scattering, irradiation with 2000 μmol m^?2 s^?1 green light produced broad Gaussian‐shaped fluorescence profiles that spanned most of the mesophyll. Profiles for chlorophyll fluorescence in the red (680 ± 16 nm) and far red (λ > 710 nm) were similar except that there was elevated red fluorescence near the adaxial leaf surface relative to far red fluorescence. Fluorescence profiles were narrower in non‐infiltrated leaf samples where light scattering increased the light gradient. The fluorescence profile was broader when the leaf was irradiated on its adaxial versus abaxial surface due to the contrasting optical properties of the palisade and spongy mesophyll. Irradiation with blue, red and green monochromatic light produced profiles that peaked 50, 100 and 150 μm, respectively, beneath the irradiated surface. These results are consistent with previous measurements of the light gradient in spinach and they agree qualitatively with measurements of carbon fixation under monochromatic blue, red and green light. These results suggest that chlorophyll fluorescence profiles may be used to estimate the distribution of quanta that are absorbed within the leaf for photosynthesis.     

Measurement of chlorophyll fluorescence within leaves using a fibreoptic microprobe

Abstract. The distribution of chlorophyll fluorescence was measured within leaves of Medicago saliva with a fibre optic microprobe. Leaves were irradiated with broad band blue light (1000 μmol m⁻²s⁻¹) and chlorophyll fluorescence was measured at 688 nm. The amount of fluorescence measured within the leaf depended upon the direction in which the probe was inserted. When the probe was advanced directly through the leaf from the shaded towards the irradiated surface, the maximum amount of detected fluorescence occurred near the boundary between the palisade and spongy mesophyll. When the probe was advanced through the leaf from the opposite direction maximum detected fluorescence was at the boundary between the epidermis and palisade. These results appear to be a consequence of the blue light gradient, which declined exponentially within the palisade but was counterbalanced by increasing chlorophyll content within the leaf. Modelling indicates that the measured distribution of chlorophyll fluorescence can be explained by relatively uniform emission of fluorescence throughout the palisade layer, indicating that the chloroplasts may be photosynthetically specialized to their light environment within the leaf.     

Profiles of photosynthetic oxygen-evolution within leaves of Spinacia oleracea

Oxygen evolution was measured from mesophyll tissues in spinach leaves using a photoacoustic technique. The photosynthetic capacity of individual cell layers was measured by directing microscopic beams of light, 40 μm wide, to cells exposed within a leaf cross section. The resulting profile for oxygen-evolution potential was relatively flat, indicating a uniform capacity for photosynthesis in leaf mesophyll tissues. Two experimental approaches were used to estimate the photosynthetic performance of individual mesophyll cell layers when white light was applied to the adaxial leaf surface. These experiments indicated that oxygen was produced relatively uniformly across the mesophyll and that oxygen evolution increased with irradiance of the white light applied to the leaf surface. The measured profiles for oxygen evolution and capacity are flatter than previous measurements of profiles of fixed carbon and estimates of profiles for absorbed light within spinach leaves.     

Light and chlorophyll gradients within Cucurbita cotyledons

Abstract. Measurement of light within 10–14-d-old green and etiolated Cucurbita pepo cotyledons were made with fibre-optic microprobes to assess the influence of chlorophyll distribution and anatomical variations in mesophyll cell type (spongy versus palisade) on internal light pattern. More than 50% of the pigment in green cotyledons occurred in the upper (adaxial) 300 μm and this gradient strongly influenced the internal propagation of 680 nm light. When the upper (adaxial) surface was irradiated with 680 nm light, almost complete absorption occurred within the first 400 μm (palisade) of approximately 1200-μm-thick cotyledons. In contrast, when lower (abaxial) surfaces were irradiated with 680 nm light, penetration extended throughout the spongy mesophyll to about the 700 μm depth. Measurements of collimaled and scattered light gradients at 550, 680 and 750 nm indicated that collimaled light was rapidly scattered by mesophyll cells. In cotyledons irradiated on the upper surface, spongy mesophyll cells received only scattered light. Furthermore, comparisons of scattered light gradients obtained from cotyledons irradiated on upper and lower surfaces suggested that spongy mesophyll cells scatter light more effectively than palisade cells, probably due to the greater proportion of intercellular air spaces in spongy mesophyll tissue. These data also indicate that both the spectral quality and quantity of light incident on palisade versus spongy mesophyll cells differs, perhaps contributing to developmental and physiological differences between these two mesophyll cell types.     

Chlorophyll and light gradients in sun and shade leaves of Spinacia oleracea

Abstract. Light gradients were measured and correlated with chlorophyll concentration and anatomy of leaves in spinach (Spinacia oleracea L.). Light gradients were measured at 450, 550 and 680 nm within thin (455 μm) and thick (630 μm) leaves of spinach grown under sun and shade conditions. The light gradients were relatively steep in both types of leaves and 90% of the light at 450 and 680 nm was absorbed by the initial 140 μm of the palisade. In general, blue light was depleted faster than red light which, in turn was depleted faster than green light. Light penetrated further into the thicker palisade of sun leaves in comparison to the shade leaves. The distance that blue light at 450 nm travelled before it became 90% depleted was 120 μm in sun leaves versus 76 μm in shade leaves. Red light at 680 nm and green light at 550 nm travelled further but the trends were similar to that measured at 450nm. The steeper light gradients within the palisade-of shade leaves were caused by increased scattering of light within the intercellular air spaces and/or cells which were less compact than those in sun leaves. The decline in the amount of light within the leaf appeared to be balanced by a gradient in chlorophyll concentration measured in paradermal sections. Progressing from the adaxial epidermis, chlorophyll content increased through the palisade and then declined through the spongy mesophyll. Chlorophyll content was similar in the palisade of both sun and shade leaves. Chloroplast distribution within both sun and shade leaves was relatively uniform so that the chlorophyll gradient appeared to be caused by greater amounts of chlorophyll within chloroplasts located deeper within the leaf. These results indicate that the anatomy of the palisade may be of special importance for controlling the penetration of photo-synthetically active radiation into the leaf. Changing the structural characteristics of individual palisade cells or their arrangement may be an adaptation that maximizes the absorption of light in leaves with varying mesophyll thickness due to different ambient light regimes.     

pH dependent chlorophyll fluorescence quenching in spinach thylakoids from light treated or dark adapted leaves

The pH dependence of maximum chlorophyll fluorescence yield (F_m) was examined in spinach thylakoids in the presence of nigericin to dissipate the transthylakoid pH gradient. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was present to eliminate photochemical quenching. Thylakoids were prepared from dark adapted leaves (dark thylakoids) or preilluminated leaves (light thylakoids). In the latter there had been approximately 50% conversion of the xanthophyll violaxanthin to zeaxanthin, while no conversion had occurred in the former. In the presence of a reductant such as ascorbate, antimycin A sensitive quenching was observed (half maximal quenching at 5 M), whose pH dependence differed between the two types of thylakoid. Preillumination of leaves resulted in more quenching at pH values where very little quenching was observed in dark thylakoids (pH 5–7.6). This was similar to activation of high-energy-state quenching (qE) observed previously (Rees D, Young A, Noctor G, Britton G and Horton P (1989) FEBS Lett 256: 85–90). Thylakoids isolated from preilluminated DTT treated leaves, that contained no zeaxanthin, behaved like dark thylakoids. A second form of quenching was observed in the presence of ferricyanide, that could be reversed by the addition of ascorbate. This was not antimycin A sensitive and showed the same pH dependence in both types of thylakoid. The former type of quenching, but not the latter, showed similar low temperature fluorescence emission spectra to qE, and was considered to occur by the same mechanism.Abbreviations DCMU 3(3,4-dichlorophenyl)-1,1-dimethylurea - DTT dithiothreitol - EDTA Ethylenediaminetetra-acetic acid - F₀ dark level fluorescence yield - F_m maximum fluorescence yield - F_v/F_m ratio of variable to total fluorescence yield - Hepes 4-(2-hydroxyethyl)1-piperazineethanesul-phonic acid - Mes 2-(N-morpholino) ethanesulfonate - pH transthylakoid pH gradient - PS I Photosystem I - PS II Photosystem II - Q_A primary stable electron acceptor of Photosystem II - qE high-energy-state fluorescence quenching     

Measurement of light within thin plant tissues with fiber optic microprobes

Vogelmann, T. C., Knapp, A. K., McClean, T. M. and Smith, W. K. 1988. Measurement of light within thin plant tissues with fiber optic microprobes. - Physiol. Plant. 72: 623–630.
The measurement of light with fiber optic microprobes has been extended to thin (200–300 μm) plant tissue samples. To test the method, light measurements were made in thin aqueous films and paradermal sections from 10-day-old etiolated Cucurbita pepo L. cv. Fordhook cotyledons. The measurements obtained were highly reproducible. Paradermal sections of spongy mesophyll that were irradiated with collimated light scattered light more effectively than the palisade layer of intact cotyledons. These results demonstrate that different plant tissues have different light scattering characteristics. The successful extension of the fiber optic microprobe technique to thin systems makes it possible to examine the optical properties of different cell layers within leaves and other plant organs.     

The functional significance of palisade tissue: penetration of directional versus diffuse light

Light gradients were measured in leaves that had different types of anatomical development of the mesophyll but similar pigment content. Leaves of the legume, Thermopsis montana, had columnar palisade and spongy mesophyll whereas leaves of the monocot, Smilacina stellata, had spongy mesophyll only. Light gradients were measured at 550 nm in both types of leaves when they were irradiated with collimated or diffuse light. When irradiated with collimated light, light gradients were steeper in leaves with spongy mesophyll in comparison to those that had palisade tissue. On the other hand, light gradients were similar between both leaf types when they were irradiated with diffuse light. Thus, columnar palisade cells facilitated the penetration of collimated light over diffuse light. These results suggest that palisade tissue may help distribute light more uniformly to chloroplasts within the leaf. Moreover, the functional significance of palisade tissue may be related to the amount of collimated light within the natural environment.     

Control of Photosystem II in spinach leaves by continuous light and by light pulses given in the dark

The light-induced induction of components of non-photochemical quenching of chlorophyll fluorescence which are distinguished by different rates of dark relaxation (qNf, rapidly relaxing and qNs, slowly relaxing or not relaxing at all in the presence brief saturating light pulses which interrupt darkness at low frequencies) was studied in leaves of spinach.After dark adaptation of the leaves, a fast relaxing component developed in low light only after a lag phase. Quenching increased towards a maximum with increasing photon flux density. This fast component of quenching was identified as energy-dependent quenching qE. It required formation of an appreciable transthylakoid pH and was insignificant when darkened spinach leaves received 1 s pulses of light every 30 s even though zeaxanthin was formed from violaxanthin under these conditions.Another quenching component termed qNs developed in low light without a lag phase. It was not dependent on a transthylakoid pH gradient, decayed exponentially with a long half time of relaxation and was about 20% of total quenching irrespective of light intensity. When darkened leaves were flashed at frequencies higher than 0.004 Hz with 1 s light pulses, this quenching also appeared. Its extent was very considerable, and it did not require formation of zeaxanthin. Relaxation was accelerated by far-red light, and this acceleration was abolished by NaF.We suggest that qNs is the result of a so-called state transition, in which LHC II moves after its phosphorylation from fluorescent PS II to nonfluorescent PS I. This state transition was capable of decreasing in darkened leaves the potential maximum quantum efficiency of electron flow through Photosystem II by about 20%.Abbreviations PFD photon flux density - PS photosystem     

A system for imaging transverse distribution of scattered light and chlorophyll fluorescence in intact rice leaves

In order to examine the transverse distribution of scattered light and chlorophyll fluorescence in intact rice leaves, a micro-fluorescence imaging system was devised using a microscope, a CCD camera with an image intensifier, an Ar and a He-Ne laser light source, an image processor, and a microcomputer. A laser light was projected vertically on to the surface of a rice leaf segment at a cut-edge, and scattered light and induced fluorescence were observed at the cut-section from a 90° angle to the axis of the laser beam. The intensity of scattered light showed a maximum at several micrometres depth from the leaf surface and a steep gradient afterwards. Fluorescence reached a maximum crossing with the decline curve of the scattered light. The maximum of fluorescence measured at 741 nm was observed at a greater depth from the leaf surface than that at 687 nm, suggesting that part of the fluorescence of the longer wavelength was emitted due to absorption of fluorescence of the shorter wavelength. Profiles of the scattered light and the chlorophyll fluorescence depended on leaf anatomy.     

Studies on light absorption and photochemical activity changes in chloroplast suspensions and leaves due to light scattering and light filtration across chloroplast and vegetation layers

An experimental analysis is presented concerning the effect on relative light absorption by the two photosystems caused by (a) a highly light scattering environment (the detour effect) and (b) light filtration across successive chloroplast layers (the light attenuation effect). Both suspensions of isolated chloroplasts and leaves were employed.It is concluded that within a single spinach leaf these phenomena are likely to lead to only rather small increases in relative photosystem I absorption and activity with respect to photosystem II and will thus not exert a significant effect on non cyclic electron transport. On the contrary when light is filtrated across successive vegetation layers (shade light) significant increases in the relative PSI absorption and activity may be encountered.It is determined that the detour effect in mature leaves from a variety of plants increases overall photosynthetically useful light absorption by 35–40%.Abbreviations F_M maximal fluorescence - LHCP₂ light-harvesting chlorophyl a/b protein complex II - Q_A-primary quinone acceptor of photosystem II     

Light scattering,chlorophyll fluorescence and state of the adenylate system in illuminated spinach leaves

Scattering of green light and chlorophyll fluorescence by spinach leaves kept in a stream of air or nitrogen were compared with leaf adenylate levels during illumination with blue, red or far-red light. Energy charge and ATP-ADP ratios exhibited considerable variability in different leaves both in the dark and in the light. Variability is explained by different possible states of the reaction oxidizing triose phosphate or reducing 3-phosphoglycerate. Except when oxygen levels were low, there was an inverse relationship between light scattering and chlorophyll fluorescence during illumination with blue or red light. When CO₂ was added to a stream of CO₂-free air, chlorophyll fluorescence increased, sometimes after a transient decrease, and both light scattering and leaf $\text{ATP}\text{ADP}$ ratios decreased. Similar observations were made when air was replaced by nitrogen under blue or high-intensity red light. Under these conditions, over-reduction caused inhibition of electron transport and phosphorylation in chloroplasts. However, when air was replaced by nitrogen during illumination with low-intensity red light or far-red light, light scattering increased instead of decreasing. Under these light conditions, $\text{ATP}\text{ADP}$ ratios were maintained in the light. They decreased drastically only after darkening. Although $\text{ATP}\text{ADP}$ ratios responded faster than light scattering or the slow secondary decline of chlorophyll fluorescence due to illumination, it appeared that in the steady state, light scattering and chlorophyll fluorescence are useful indicators of the phosphorylation state of the leaf adenylate system at least under aerobic conditions, when chloroplast and extrachloroplast adenylate systems can effectively communicate.     

Extended depth-of-focus imaging of chlorophyll fluorescence from intact leaves

Imaging dynamic changes in chlorophyll a fluorescence provides a valuable means with which to examine localised changes in photosynthetic function. Microscope-based systems provide excellent spatial resolution which allows the response of individual cells to be measured. However, such systems have a restricted depth of focus and, as leaves are inherently uneven, only a small proportion of each image at any given focal plane is in focus. In this report we describe the development of algorithms, specifically adapted for imaging chlorophyll fluorescence and photosynthetic function in living plant cells, which allow extended-focus images to be reconstructed from images taken in different focal planes. We describe how these procedures can be used to reconstruct images of chlorophyll fluorescence and calculated photosynthetic parameters, as well as producing a map of leaf topology. The robustness of this procedure is demonstrated using leaves from a number of different plant species. This revised version was published online in June 2006 with corrections to the Cover Date.     

Evaluation of a technique for the measurement of chlorophyll fluorescence from leaves exposed to continuous white light

Abstract An instrument for the generation and measurement of modulated chlorophyll fluorescence signals from leaves exposed to continuous, highintensity white light is described. Modulated fluorescence is generated in the leaf by pulsed diodes emitting low-intensity yellow radiation and is detected with a photodiode whose output is fed to an amplifier locked in to the frequency of the lightemitting diodes. Comparisons are made between the modulated fluorescence signals measured with this instrument and the continuous fluorescence signals emitted from dark-adapted leaf tissue and isolated thylakoids when photosynthetic activity is induced by exposure to a range of intensities of continuous broad-band, blue-green light. The modulated fluorescence signals were similar to the continuous fluorescence signals, but they were not always identical. The small differences between the two signals are mainly attributable to differences in the populations of chloroplasts being monitored in the two measurements as a result of differential penetration of the modulated and actinic light sources into the sample.     

The importance of PS I chlorophyll red forms in light-harvesting by leaves

We have investigated the importance of the long wavelength absorbing spectral forms (red forms) of Photosystem I in photosynthetic light harvesting by leaves. To this end leaf spectra were simulated by using a linear combination of absorption (OD) spectra of purified Photosystem I, Photosystem II and LHC II, multiplied by an empirical multiple scattering chloroplast/leaf conversion function. In this way it is demonstrated that while the PS I red forms account for only about 4–5% of light absorption in a normal daylight environment, in different shadelight environments these long wavelength pigments may be responsible for up to 40% of total photon capture. In the context of maximising the photosynthetic quantum efficiency under the low light conditions of shadelight, this relative increase in the absorption cross section of PS I can be understood by considering the increased synthesis of the major PS II antenna complex, LHC II, known to occur in plants growing under these light conditions. It is demonstrated that for plants in a moderate to deep shadelight regime the PS II cross section needs to increase by 50% to 100% via LHC II synthesis to balance the increased PS I absorption by the red forms. The possibility that under shade light conditions the increased PS I cross section may serve in cyclic phosphorylation is also discussed.     

Contribution of intercellular reflectance to photosynthesis in shade leaves

The potential contribution of intercellular light reflectance to photosynthesis was investigated by infiltrating shade leaves with mineral oil. Infiltration of leaves of Hydrophyllum canadense and Asarum canadense with mineral oil decreased adaxial leaf reflectance but increased transmittance. As a result of the large increase in transmittance, infiltration caused a decrease in absorptance of 25% and 30% at 550 and 750 nm, respectively. Thus, intercellular reflectance increased absorptance in these species by this amount. In a comparison of sun and shade leaves of Acer saccharum and Parthenocissus quinquefolia, oil infiltration decreased absorptance more in shade than in sun leaves. This difference suggests that the higher proportion of spongy mesophyll in shade leaves may increase internal light scattering and thus absorptance. The importance of the spongy mesophyll in increasing internal reflectance was also evident in comparisons of the optics of Populus leaves and in the fluorescence yield of oil-infiltrated leaves of several sun and shade species. Oil infiltration decreased the quantum yield of fluorescence (F_o) by 39–52% for shade leaves but only 21–25% for sun leaves. We conclude that the greater proportion of spongy parenchyma in shade leaves increased intercellular light scattering and thus absorptance. Direct measurements with fibre-optic light probes of the distribution of light inside leaves of Hydrophyllum canadense confirmed that oil infiltration decreased the amount of back-scattered light and that most of the light scattering for this species occurred from the middle of the palisade layer to the middle of the spongy mesophyll. We were not, however, able to assess the potential contribution of reflectance from the internal abaxial epidermis to total internal light scattering in these experiments. Using a mathematical model to compare the response of net photosynthesis (O₂, flux) to incident irradiance for control leaves of H. canadense and theoretical leaves with no intercellular reflectance, we calculated that intercellular reflectance caused a 1.97-fold increase in photosynthesis at 20 μmol m^?2s^?1 (incident photon flux density). This enhancement of absorption and photosynthesis by inter-cellular reflectance, without additional production and maintenance of photosynthetic pigments, may maintain shade leaves above the photosynthetic light compensation point between sunflecks and maintain the light induction state during protracted periods of low diffuse light.     

An integrated portable apparatus for the simultaneous field measurement of photosynthetic CO2 and water vapour exchange, light absorption and chlorophyll fluorescence emission of attached leaves

Abstract. A portable apparatus has been constructed to measure simultaneously the quantum yield of CO₂ assimilation, light absorption, chlorophyll fluorescence emission and water vapour exchange of attached intact leaves in the field. The core of the instrument is a light-integrating spherical leaf chamber which includes ports for a light source, photosynthetically active radiation sensor, fluorescence probes and gas inlet and outlet manifolds. Measurement of the quantum flux inside the empty chamber and with a leaf present allows determination of leaf absorptance. An open gas exchange system is employed using an infra-red analyser to measure leaf CO₂ exchange. Using a DC white light source the quantum yield of CO₂ assimilation based on absorbed light (φ_abs) may be determined rapidly in either ambient air or artificial gas mixtures. Inclusion of capacitance humidity probes into the gas inlet and outlet ports allows simultaneous determination of water vapour exchange and subsequent estimation of stomatal conductance to CO₂ and intercellular CO₂ concentration. Measurement of fluorescence emission by the sample leaf exposed to white light is achieved by a modulated fluorescence detection system. In addition to determination of the minimal, maximal and variable fluorescence levels, a further analysis allows the photochemical and non-photochemical components of fluorescence quenching, to be estimated. The theory and design of this apparatus is described in detail. The use of the apparatus in the field is demonstrated through a study of the photosynthetic performance of a maize and bean crop during the growing season and by analysis of the photosynthetic performance of crops subjected to nitrogen-stress and a herbicide treatment.     

Measurement of chlorophyll fluorescence within leaves using a modified PAM Fluorometer with a fiber-optic microprobe

By using a fiber-optic microprobe in combination with a modified PAM Fluorometer, chlorophyll fluorescence yield was measured within leaves with spatial resolution of approximately 20 m. The new system employs a miniature photomultiplier for detection of the pulse-modulated fluorescence signal received by the 20 m fiber tip. The obtained signal/noise ratio qualifies for recordings of fluorescence induction kinetics (Kautsky effect), fluorescence quenching by the saturation pulse method and determination of quantum yield of energy conversion at Photosystem II at different sites within a leaf. Examples of the system performance and of practical applications are given. It is demonstrated that the fluorescence rise kinetics are distinctly faster when chloroplasts within the spongy mesophyll are illuminated as compared to palisade chloroplasts. Photoinhibition is shown to affect primarily the quantum yield of the palisade chloroplasts when excessive illumination is applied from the adaxial leaf side. The new system is envisaged to be used in combination with light measurements within leaves for an assessment of the specific contributions of different leaf regions to overall photosynthetic activity and for an integrative modelling of leaf photosynthesis.This paper is dedicated to Ulrich Heber on the occasion of his 65th birthday, with great respect for his outstanding achievements in photosynthesis research.