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Recent advances in the retrieval of Chl fluorescence from space using passive methods (solar-induced Chl fluorescence, SIF) promise improved mapping of plant photosynthesis globally. However, unresolved issues related to the spatial, spectral, and temporal dynamics of vegetation fluorescence complicate our ability to interpret SIF measurements. We developed an instrument to measure leaf-level gas exchange simultaneously with pulse-amplitude modulation (PAM) and spectrally resolved fluorescence over the same field of view – allowing us to investigate the relationships between active and passive fluorescence with photosynthesis. Strongly correlated, slope-dependent relationships were observed between measured spectra across all wavelengths (Fλ, 670–850 nm) and PAM fluorescence parameters under a range of actinic light intensities (steady-state fluorescence yields, Ft) and saturation pulses (maximal fluorescence yields, Fm). Our results suggest that this method can accurately reproduce the full Chl emission spectra – capturing the spectral dynamics associated with changes in the yields of fluorescence, photochemical (ΦPSII), and nonphotochemical quenching (NPQ). We discuss how this method may establish a link between photosynthetic capacity and the mechanistic drivers of wavelength-specific fluorescence emission during changes in environmental conditions (light, temperature, humidity). Our emphasis is on future research directions linking spectral fluorescence to photosynthesis, ΦPSII, and NPQ.

Arundo donax has attracted interest as a potential bioenergy crop due to a high apparent productivity. It uses C3 photosynthesis yet appears competitive with C4 grass biomass feedstock's and grows in warm conditions where C4 species might be expected to be that productive. Despite this there has been no systematic study of leaf photosynthetic properties. This study determines photosynthetic and photorespiratory parameters for leaves in a natural stand of A. donax growing in southern Portugal. We hypothesise that A. donax has a high photosynthetic potential in high and low light, stomatal limitation to be small and intrinsic water use efficiency unusually low. High photosynthetic rates in A. donax resulted from a high capacity for both maximum Rubisco (Vc,max 117 μmol CO2 m(-2) s(-1)) and ribulose-1:5-bisphosphate limited carboxylation rate (Jmax 213 μmol CO2 m(-2) s(-1)) under light-saturated conditions. Maximum quantum yield for light-limited CO2 assimilation was also high relative to other C3 species. Photorespiratory losses were similar to other C3 species under the conditions of measurement (25%), while stomatal limitation was high (0.25) resulting in a high intrinsic water use efficiency. Overall the photosynthetic capacity of A. donax is high compared to other C3 species, and comparable to C4 bioenergy grasses.

The wing-scale morphologies of the pierid butterflies Pieris rapae (small white) and Delias nigrina (common jezabel), and the heliconine Heliconius melpomene are compared and related to the wing-reflectance spectra. Light scattering at the wing scales determines the wing reflectance, but when the scales contain an absorbing pigment, reflectance is suppressed in the absorption wavelength range of the pigment. The reflectance of the white wing areas of P. rapae, where the scales are studded with beads, is considerably higher than that of the white wing areas of H. melpomene, which has scales lacking beads. The beads presumably cause the distinct matt-white colour of the wings of pierids and function to increase the reflectance amplitude. This will improve the visual discrimination between conspecific males and females.

C 4 grasses of the NAD-ME type ( Astrebla lappacea , Eleusine coracana , Eragrostis superba , Leptochloa dubia , Panicum coloratum , Panicum decompositum ) and the NADP-ME type ( Bothriochloa bladhii, Cenchrus ciliaris, Dichanthium sericeum , Panicum antidotale , Paspalum notatum , Pennisetum alopecuroides , Sorghum bicolor ) were used to investigate the role of O 2 as an electron acceptor during C 4 photosynthesis. Mass spectrometric measurements of gross O 2 evolution and uptake were made concurrently with measurements of net CO 2 uptake and chlorophyll fluorescence at different irradiances and leaf temperatures of 30 and 40 ∞ ∞ ∞ C. In all C 4 grasses gross O 2 uptake increased with increasing irradiance at very high CO 2 partial pressures ( p CO 2 ) and was on average 18% of gross O 2 evolution. Gross O 2 uptake at high irradiance and high p CO 2 was on average 3.8 times greater than gross O 2 uptake in the dark. Furthermore, gross O 2 uptake in the light increased with O 2 concentration at both high CO 2 and the compensation point, whereas gross O 2 uptake in the dark was insensitive to O 2 concentration. This suggests that a significant amount of O 2 uptake may be associated with the Mehler reaction, and that the Mehler reaction varies with irradiance and O 2 concentration. O 2 exchange characteristics at high p CO 2 were similar for NAD-ME and NADP-ME species. NAD-ME species had significantly greater O 2 uptake and evolution at the compensation point particularly at low irradiance compared to NADP-ME species, which could be related to different rates of photorespiratory O 2 uptake. There was a good correlation between electron transport rates estimated from chlorophyll fluorescence and gross O 2 evolution at high light and high p CO 2 .

Evergreen leaves of temperate climate plants are often subject to frosts. Changes in carbon gain patterns arise from freezing-related tissue damage, and from interactions between light and temperature stress. We examined relationships between spatial patterns in freezing and concentrations of chlorophyll. Spatial patterns in pigmentation in leaves that had or had not been exposed to naturally occurring frosts were determined by conventional extraction techniques combined with high-resolution hyperspectral imaging of reflectance from intact leaves. Predictive indices were developed to relate reflectance to chlorophyll content and chlorophyll a/b ratios within intact leaves. Leaves exposed to frosts had lower chlorophyll contents and more variable a/b ratios than protected leaves. In frost-affected leaves, chlorophyll content was highest near leaf centres and decreased toward leaf tips and margins. Decline in chlorophyll content was associated with shifts in chlorophyll a/b ratios and increases in red pigmentation due to anthocyanin, with effects being greater on leaf sides exposed directly to the sun. These altered pigmentation patterns were consistent with patterns in freezing. The present results illustrate the fine scale of spatial variation in leaf response to freezing, and raise important questions about impacts of freezing on photosynthetic function in over-wintering evergreens.

The complex dynamic properties of biological timing in organisms remain a central enigma in biology despite the increasingly precise genetic characterization of oscillating units and their components. Although attempts to obtain the time constants from oscillations of gene activity and biochemical units have led to substantial progress, we are still far from a full molecular understanding of endogenous rhythmicity and the physiological manifestations of biological clocks. Applications of nonlinear dynamics have revolutionized thinking in physics and in biomedical and life sciences research, and spatiotemporal considerations are now advancing our understanding of development and rhythmicity. Here we show that the well known circadian rhythm of a metabolic cycle in a higher plant, namely the crassulacean acid metabolism mode of photosynthesis, is expressed as dynamic patterns of independently initiated variations in photosynthetic efficiency (φ PSII ) over a single leaf. Noninvasive highly sensitive chlorophyll fluorescence imaging reveals randomly initiated patches of varying φ PSII that are propagated within minutes to hours in wave fronts, forming dynamically expanding and contracting clusters and clearly dephased regions of φ PSII . Thus, this biological clock is a spatiotemporal product of many weakly coupled individual oscillators, defined by the metabolic constraints of crassulacean acid metabolism. The oscillators operate independently in space and time as a consequence of the dynamics of metabolic pools and limitations of CO 2 diffusion between tightly packed cells.

Summary • The sink‐source transition of developing Nicotiana tabacum (tobacco) leaves was studied here using chlorophyll fluorescence imaging. • In accordance with leaf development, the quantum efficiency of PSII, showed a steep gradient across the leaf with increasing values towards the tip. • The linear electron transport rate (ETR) saturated at higher CO 2 concentrations in the younger, than in the mature, part of the leaf, probably due to a lower Rubisco activity or a higher CO 2 diffusion resistance. • The induction of ETR at CO 2 concentrations near the compensation point after long-term dark adaptation of the young leaf, showed distinct responses; ETR rose rapidly in the basal but more slowly in the apical regions. There was a correlation between fast induction and carbohydrate import, as measured by 14 C-translocation. In the basal regions, larger pools of metabolic intermediates are expected due to imported carbohydrates. These might be used in the Calvin cycle directly after dark‐light transition providing the electron acceptors for the faster induction of ETR. Additionally, a higher mitochondrial respiration can provide CO 2 for the Calvin cycle in these regions.

Evidence is presented contrary to the suggestion that C4 plants grow larger at elevated CO2 because the C4 pathway of young C4 leaves has C3-like characteristics, making their photosynthesis O2 sensitive and responsive to high CO2. We combined PAM fluorescence with gas exchange measurements to examine the O2 dependence of photosynthesis in young and mature leaves of Panicum antidotale (C4, NADP-ME) and P. coloratum (C4, NAD-ME), at an intercellular CO2 concentration of 5 Pa. P. laxum (C3) was used for comparison. The young C4 leaves had CO2 and light response curves typical of C4 photosynthesis. When the O2 concentration was gradually increased between 2 and 40%, CO2 assimilation rates (A) of both mature and young C4 leaves were little affected, while the ratio of the quantum yield of photosystem II to that of CO2 assimilation (ΦPSII/ΦCO2) increased more in young (up to 31%) than mature (up to 10%) C4 leaves. A of C3 leaves decreased by 1·3 and ΦPSII/ΦCO2 increased by 9-fold, over the same range of O2 concentrations. Larger increases in electron transport requirements in young, relative to mature, C4 leaves at low CO2 are indicative of greater O2 sensitivity of photorespiration. Photosynthesis modelling showed that young C4 leaves have lower bundle sheath CO2 concentration, brought about by higher bundle sheath conductance relative to the activity of the C4 and C3 cycles and/or lower ratio of activities of the C4 to C3 cycles.

It was previously shown with concurrent measurements of gas exchange and carbon isotope discrimination that the reduction of ribulose-1,5-bisphosphate carboxylase/oxygenase by an antisense gene construct in transgenic Flaveria bidentis (a C4 species) leads to reduced CO2 assimilation rates, increased bundle-sheath CO2 concentration, and leakiness (defined as the ratio of CO2 leakage to the rate of C4 acid decarboxylation; S. von Caemmerer, A. Millegate, G.D. Farquhar, R.T. Furbank [1997] Plant Physiol 113: 469–477). Increased leakiness in the transformants should result in an increased ATP requirement per mole of CO2 fixed and a change in the ATP-to-NADPH demand. To investigate this, we compared measurements of the quantum yield of photosystem I and II ([phi]PSI and [phi]PSII) with the quantum yield of CO2 fixation ([phi]CO2) in control and transgenic F. bidentis plants in various conditions. Both [phi]PSI/[phi]CO2 and [phi]PSII/[phi]CO2 increased with a decrease in ribulose-1,5-bisphosphate carboxylase/oxygenase content, confirming an increase in leakiness. In the wild type the ratio of [phi]PSI to [phi]PSII was constant at different irradiances but increased with irradiance in the transformants, suggesting that cyclic electron transport may be higher in the transformants. To evaluate the relative contribution of cyclic or linear electron transport to extra ATP generation, we developed a model that links leakiness, ATP/NADP requirements, and quantum yields. Despite some uncertainties in the light distribution between photosystem I and II, we conclude from the increase of [phi]PSII/[phi]CO2 in the transformants that cyclic electron transport is not solely responsible for ATP generation without NADPH production.

Technical features and examples of application of a special emitter–detector module for highly sensitive measurements of the electrochromic pigment absorbance shift (ECS) via dual-wavelength (550–520 nm) transmittance changes (P515) are described. This device, which has been introduced as an accessory of the standard, commercially available Dual-PAM-100 measuring system, not only allows steady-state assessment of the proton motive force (pmf) and its partitioning into ΔpH and ΔΨ components, but also continuous recording of the overall charge flux driven by photosynthetic light reactions. The new approach employs a double-modulation technique to derive a continuous signal from the light/dark modulation amplitude of the P515 signal. This new, continuously measured signal primarily reflects the rate of proton efflux via the ATP synthase, which under quasi-stationary conditions corresponds to the overall rate of proton influx driven by coupled electron transport. Simultaneous measurements of charge flux and \(CO_2\) uptake as a function of light intensity indicated a close to linear relationship in the light-limited range. A linear relationship between these two signals was also found for different internal \(CO_2\) concentrations, except for very low \(CO_2\), where the rate of charge flux distinctly exceeded the rate of CO2 uptake. Parallel oscillations in \(CO_2\) uptake and charge flux were induced by high \(CO_2\) and \(O_2\). The new device may contribute to the elucidation of complex regulatory mechanisms in intact leaves.

Equations for non-destructive determination of chlorophyll b : a ratios in grasses were developed from reflectance spectra of intact leaves of barley (Hordeum vulgare L.) and two barley mutants: clorina f2, which lacks chlorophyll b and clorina f104, which has a low chlorophyll b content. These plants enabled separation of effects of chlorophyll composition on reflectance spectra due to differential light absorption by chlorophylls a and b and to measure the effects of chlorophyll b on the contribution of fluorescence emitted by chlorophyll a to the reflectance spectra. Indices developed from these data were then tested on growth chamber-grown leaves from six C3 and 17 C4 grass species (7 NAD-ME and 10 NADP-ME subtypes). We used the chlorophyll b : a ratio because the data were less skewed than the chlorophyll a : b ratio. The best index for determination of the chlorophyll b : a ratio utilised wavelengths affected by chlorophyll absorbance: [R626 – 0.5 (R603 + R647)]/[R552– R626]. The chlorophyll b : a ratio was significantly lower in the C4 than C3 grasses, but was not sufficient in itself to separate these two functional groups. However, because of differences in fluorescence characteristics, C3 and C4 species could be distinguished by an index based on wavelengths affected by chlorophyll fluorescence: [R696 to 709/R545 to 567].

An experimental system is described to analyze the spacial distribution of leaf assimilation from chlorophyll-a fluorescence images. Fluorescence parameters were determined with a digitized camera system pixel-by-pixel and calibrated against the assimilation rate from integrated gas exchange. The approach is an extension of the work by P.F. Daley, et al. (1989, Plant Physiol. 90, 1233–1238) and allows the visualisation of assimilation images of leaves with high resolution. With a leaf of Glechoma hederacea L., assimilation images were taken at short time intervals during stomata-related oscillations of the assimilation rate (period about 13 min) over a time period of more than 3 h. Mathematical pixel-by-pixel analysis of images shows nearly Gaussian distribution of assimilation values around a minimum (5 μmol CO2 · m−2 · s−1) and maximum (13 μmol CO2 · m−2 · s−1) peak value during oscillations. Synchronous oscillations, initiated by rapid changes in gas composition, changed into non-synchronous oscillations, due to slight local variations in the period. As a consequence, the oscillation of gas exchange (H2O and CO2) died out whilst leaf assimilation continued to oscillate non-synchronously and patchily. Patchy distribution of frequencies largely followed gas-exchange compartmentation (‘alveoli’), whilst non-homogeneous distribution of the amplitude was on a larger scale, with a decrease in amplitudes from the leaf base towards distal areas. The data are discussed considering the framework of physiological processes involved in stomatal control.

Oscillations of photosynthesis induced in leaves of Vicia faba L. were accompanied by oscillations not only in the pH of the chloroplast stroma, but also by pH oscillations in the cytosol and in the vacuole of leaf mesophyll cells. Cytosolic pH oscillations were in phase with stromal oscillations, but antiparallel to vacuolar pH oscillations. During maxima of photosynthesis, the cytosolic pH exhibited maxima and the vacuolar pH minima. Vacuolar acidification is interpreted to be the result of energized proton transport from the cytosol into the vacuole. Since the ratio of dihydroxyacetone phosphate to phosphoglycerate is maximal during the peaks of photosynthesis (Stitt et al., 1988, J. Plant Physiol. 133, 133-143; Laisk et al., 1991, Planta 185, 554-562), while the activity of NADP-malic dehydrogenase is highest during minima of photosynthesis (Scheibe and Stitt, 1988, Plant Physiol. Biochem. 26, 473-481), the present data indicate in agreement with earlier observations (Yin et al., 1991, Planta 184, 30-34) that light-dependent cytosolic energization is brought about by the oxidation of dihydroxyacetone phosphate rather than of malate. They also indicate that the over-reduction of the electrontransport chain observed during minima of photosynthesis is relieved not predominantly by oxaloacetate reduction and export of the resulting malate from the chloroplasts but by another reaction, presumably oxygen reduction.

P700 is rapidly, but only transiently photooxidized upon illuminating dark-adapted leaves. Initial oxidation is followed by a reductive phase even under far-red illumination which excites predominantly photosystem (PS) I. In this phase, oxidized P700 is reduced by electrons coming from PSII. Charge separation in the reaction center of PSI is prevented by the unavailability of electron acceptors on the reducing side of PSI. It is subsequently made possible by the opening of an electron gate which is situated between PSI and the electron acceptor phosphoglycerate. Electron acceptors immediately available for reduction while the gate is closed corresponded to 10 nmol · (mg chlorophyll)(-1) electrons in geranium leaves, 16 nmol · (mg chlorophyll)(-1) in sunflower and 22 nmol · (mg chlorophyll)(-1) in oleander. Reduction of NADP during the initial phase of P700 oxidation showed that the electron gate was not represented by ferredoxin-NADP reductase. Availability of ATP indicated that electron flow was not hindered by deactivation of the thylakoid ATP synthetase. It is concluded that NADP-dependent glyceraldehydephosphate dehydrogenase is completely deactivated in the dark and activated in the light. The rate of activation depends on the length of the preceding dark period. As chloroplasts contain both NAD- and NADP-dependent glyceraldehydephosphate dehydrogenases, deactivation of the NADP-dependent enzyme disconnects chloroplast NAD and NADP systems and prevents phosphoglycerate reduction in the dark at the expense of NADPH and ATP which are generated by glucose-6-phosphate oxidation and glycolytic starch breakdown, respectively.

Action spectra in the red region of the spectrum for light-dependent cytosolic alkalization in leaves of C3 plants which also received a low background of blue light differed from the action spectra for light-dependent vacuolar acidification. Light above 680 nm was less effective in supporting the cytosolic alkalization reaction than light below 680 nm. In contrast, in leaves illuminated in CO2-free air the light-dependent vacuolar acidification exhibited a maximum at or even above 700 nm. When photorespiratory carbohydrate oxidation was suppressed in low oxygen, a substantially changed action spectrum of the acidification reaction resembled in shape that of the cytosolic alkalization with the exception that it was extended towards the far-red. From the presented data and from previously published data (Yin et al., 1990b, Planta 182, 253-261; Yin et al., 1990c, Planta 182, 262-269) it is concluded that in the presence of a weak background of blue light, and in the absence of CO2 which drains electrons from the electron transport chain, cyclic photophosphorylation induced by far-red light permits increased export of dihydroxyacetone phosphate from the chloroplasts into the cytosol where its oxidation increases the cytosolic energy state giving rise to increased proton transport across the tonoplast. The data do not lend support to the view that export of malate from the chloroplasts and its oxidation in the mitochondria contribute significantly to cytosolic energization in the light.

Oscillations in the rate of photosynthesis of sunflower (Helianthus annuus L.) leaves were induced by subjecting leaves, whose photosynthetic apparatus had been activated, to a sudden transition from darkness or low light to high-intensity illumination, or by transfering them in the light from air to an atmosphere containing saturating CO2. It was found that at the first maximum, light-and CO2-saturated photosynthesis can be much faster than steady-state photosynthesis. Both QA in the reaction center of PS II and P700 in the reaction center of PS I of the chloroplast electron-transport chain were more oxidized during the maxima of photosynthesis than during the minima. Maxima of P700 oxidation slightly preceded maxima in photosynthesis. During a transition from low to high irradiance, the assimilatory force FA, which was calculated from ratios of dihydroxyacetone phosphate to phosphoglycerate under the assumption that the reactions catalyzed by NADP-dependent glyceraldehydephosphate dehydrogenase, phosphoglycerate kinase and triosephosphate isomerase are close to equilibrium, oscillated in parallel with photosynthesis. However, only one of its components, the calculated phosphorylation potential (ATP)/(ADP)(Pi), paralleled photosynthesis, whereas calculated NADPH/NADP ratios exhibited antiparallel behaviour. When photosynthetic oscillations were initiated by a transition from low to high CO2, the assimilatory force FA declined, was very low at the first minimum of photosynthesis and increased as photosynthesis rose to its second maximum. The observations indicate that the minima in photosynthesis are caused by lack of ATP. This leads to overreduction of the electron-transport chain which is indicated by the reduction of P700. During photosynthetic oscillations the chloroplast thylakoid system is unable to adjust the supply of ATP and NADPH rapidly to demand at the stoichiometric relationship required by the carbonreduction cycle.