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The dynamic and efficient coordination of primary photosynthetic reactions with leaf energization and metabolism under a wide range of environmental conditions is a fundamental property of plants involving processes at all functional levels. The present historical perspective covers 60 years of research aiming to understand the underlying mechanisms, linking major breakthroughs to current progress. It centers on the contributions of Ulrich Heber who had pioneered novel concepts, fundamental methods, and mechanistic understanding of photosynthesis. An important first step was the development of non-aqueous preparation of chloroplasts allowing the investigation of chloroplast metabolites ex vivo (meaning that the obtained results reflect the in vivo situation). Later on, intact chloroplasts, retaining their functional envelope membranes, were isolated in aqueous media to investigate compartmentation and exchange of metabolites between chloroplasts and external medium. These studies elucidated metabolic interaction between chloroplasts and cytoplasm during photosynthesis. Experiments with isolated intact chloroplasts clarified that oxygenation of ribulose-1.5-bisphosphate generates glycolate in photorespiration. The development of non-invasive optical methods enabled researchers identifying mechanisms that balance electron flow in the photosynthetic electron transport system avoiding its over-reduction. Recording chlorophyll a (Chl a) fluorescence allowed one to monitor, among other parameters, thermal energy dissipation by means of 'nonphotochemical quenching' of the excited state of Chl a. Furthermore, studies both in vivo and in vitro led to basic understanding of the biochemical mechanisms of freezing damage and frost tolerance of plant leaves, to SO2 tolerance of tree leaves and dehydrating lichens and mosses.

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.

There are very few investigations of cold hardiness in native Australian insects, and no such studies on insects from Tasmania. The Apteropanorpidae is a family of wingless Mecoptera endemic to Tasmania, comprising four described species that can be active in winter. In this study, we used infrared video thermography to investigate the physiological and behavioural responses of Apteropanorpa tasmanica to fast (0.3 degrees Cmin(-1)) and slow (0.03 degrees Cmin(-1)) rates of temperature reduction down to -10 degrees C. No adults survived cooling to -10 degrees C at either cooling rate. Mean supercooling points (SCPs) from fast cooling were -7.0 and -4.6 degrees C in 2002 and 2003, respectively. Ice nucleation always began in the abdomen, however, the position of nucleation within the abdomen varied between individuals. There was no relationship between SCP and body length, and no significant difference in SCPs between males and females. Stress-induced fast walking began when insects reached approximately -1.5 degrees C. Cooling rate did not affect the SCP or the temperature at which the behavioural stress response began. Adults survived for only short periods of time in the supercooled state; however they survived in the laboratory for up to 60 days at 4 degrees C, indicating their longevity at more favourable temperatures. Members of the Apteropanorpidae are adapted to the relatively warm, maritime climate currently influencing Tasmania.

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.

We used chlorophyll fluorescence imaging to examine the homogeneity of photosynthetic metabolism during CAM in the thick leaves of Kalanchoe daigremontiana Hamet et Perrier de la Bathie. Intense, persistent fluorescence from a DCMU treated thin leaf of Clematis sp placed beneath a K. daigremontiana leaf was readily detected through the thick leaf. Evidently reabsorption of fluorescence was qualitatively unimportant in the system used. Chlorophyll fluorescence images from 7 mm tissue discs excised from Kalanchoe leaves were collected at 60 s intervals during 20 min transients elicited by red excitation light. Information about patchiness and subsurface processes was gained by statistical factor analysis and Fourier transform. Although small, highly resolved rings of bright chlorophyll fluorescence surrounding discs of low fluorescence were observed from cells near the surface, no independent regional temporal variation in fluorescence was evident in the surface-biased images. Temporally independent chlorophyll fluorescence was present in images biased towards sub-epidermal sources, in most phases of CAM, and during endogenous rhythm. These asynchronous changes were several millimetres apart. This patchy fluorescence was confirmed when attached leaves were excited with blue light in a leaf chamber while CO2 and H2O exchange was monitored. Large spatio-temporal variations in the efficiency of photosystem II were always observed during phases II and IV of CAM, when both CO2 fixation cycles are active, and during the maximum rate of CO2 fixation during the endogenous rhythm in continuous light. These data are discussed in terms of metabolic isolation in the thick but uniform tissues in which gas diffusion may be largely confined to wet cell walls, thereby rendering the tissue functionally heterobaric. Prolonged, but in some instances, reversible alterations in PSII efficiency could be produced by injection of metabolic inhibitors, confirming that patchy fluorescence may reflect the differing energy costs of photosynthesis in different CAM phases.

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].

Slow stomatal oscillations (70–95 min), associated with feedback within the plant hydraulic systems, were studied in cotton (Gossypium hirsutum L.). Oscillations were only evident when the whole plant was exposed to light, and were not influenced by reductions in intercellular CO2 concentrations (Ci) in intact, attached leaves. Oscillations were synchronised among different leaves of the same plant, even when the leaf-to-air vapour pressure difference (VPD) was reduced in a cuvette enclosing one of the leaves. In the trough phase of stomatal oscillations the apparent Ci was higher than expected from the combination of the observed assimilation rate and the A(Ci) relationship measured in the absence of oscillations. Using chlorophyll fluorescence imaging we found evidence of stomatal heterogeneity in this phase. Finally, we found that stomatal oscillations appeared to be correlated with xylem embolism, with more vessels filled with gas at the peak than at the troughs of stomatal oscillations.

The control of stomatal aperture is multi-factorial. The signal perception and transduction chains within the guard cells are still unknown. In transgenic tobacco plants (Nicotiana tobaccum) with a reduced amount of b6f complexes the capacity of the linear electron transport rate is decreased. Because of it’s role in the control of stomatal opening, it should be expected, that the transgenic plants have a lower stomatal opening. Different signals were given to induce short term opening and responses were examined. Although the aim of the experiments was primarily to show a different response of wildtype and B6F plants to opening signals, which involve the linear electron transport chain, we also want to draw your attention to the differences in the kinetic response of stomata depending on the quality of the triggering signal regardless of the plants.

Images of chlorophyll-a-fluorescence oscillations were recorded using a camera-based fluorescence imaging system. Oscillations with frequencies around 1 per min were initiated by a transient decrease in light intensity during assimilation at an elevated CO2-concentration. The oscillation was inhomogenously distributed over the leaf. In cells adjacent to minor veins, frequency and damping rate was high, if there was any oscillation. In contrast, the amplitude was highest in cells most distant from phloem elements (maximal distance about 300 μm). The appearance of minor veins in oscillation images is explained by a gradient in the metabolic control in the mesophyll between minor veins and by transport of sugar from distant cells to phloem elements. The potential of fluorescence imaging to visualize ‘microscopic’ source-sink interactions and metabolic domains in the mesophyll is discussed.

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.

Chlorophyll fluorescence, light scattering, the electrochromic shift P515 and levels of some photosynthetic intermediates were measured in illuminated leaves. Oxygen and CO2 concentrations in the gas phase were varied in order to obtain information on control of Photosystem II activity under conditions such as produced by water stress, when stomatal closure restricts access of CO2 to the photosynthetic apparatus. Light scattering and energy-dependent fluorescence quenching indicated a high level of chloroplast energization under high intensity illumination even when linear electron transport was curtailed in CO2-free air or in 1% oxygen with 35 μll(-1) CO2. Calculations of the phosphorylation potential based on measurements of phosphoglycerate, dihydroxyacetone phosphate and NADP revealed ratios of intrathylakoid to extrathylakoid proton concentrations, which were only somewhat higher in air containing 35 μl l(-1) CO2 than in CO2-free air or 1% oxygen/35 μl l(-1) CO2. Anaerobic conditions prevented appreciable chloroplast energization. Acceptor-limitation of electron flow resulted in a high reduction level of the electron transport chain, which is characterized by decreased oxidation of P700, not only under anaerobic conditions, but also in air, when CO2 was absent, and in 1% oxygen, when the CO2 concentration was reduced to 35 μll(-1). Efficient control of electron transport was indicated by the photoaccumulation of P700 (+) at or close to the CO2 compensation point in air. It is proposed to require the interplay between photorespiratory and photosynthetic electron flows, electron flow to oxygen and cyclic electron flow. The field-indicating electrochromic shift (P515) measured as a rapid absorption decrease on switching the light off followed closely the extent of photoaccumulation of P700 (+) in the light.

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.

Despite the obvious benefits of using thermography under field conditions, most infrared studies at the leaf level are generally conducted in the laboratory. One reason for this bias is that accuracy can potentially be compromised in sunlight because reflected radiation from the leaf might affect the calculation of the temperature measurement. We have developed a method for measuring leaf temperature in sunlight by using thermal imagery to generate cooling curves from which the time constant for cooling, τ, can be calculated. The original temperature of the sunlit leaf may be determined by extrapolating backwards in time. In the absence of specular reflection, there is close agreement between the extrapolated sunlit temperature and the sunlit temperature recorded by the camera. However, when reflected radiation is high, the difference between the initial (incorrect) temperature determined from the sunlit image and the temperature extrapolated from the cooling curve can be > 2°C. Notably, our results demonstrate a close agreement between the extrapolated sunlit temperature and the temperature of the leaf approximately 1 s after being shaded, suggesting that this shaded image provides a good estimate of the original sunlit temperature. Thus, our technique provides two means for measuring leaf surface temperature in sunlight.

This paper originates from a presentation at the IIIrd International Congress on Crassulacean Acid Metabolism, Cape Tribulation, Queensland, Australia, August 2001. We investigated photosynthetic electron transport in leaves of the facultative crassulacean acid metabolism (CAM) plant Mesembryanthemum crystallinum L. After CAM induction, electron transport exhibited variable redox kinetics during the diurnal CAM cycle. In CAM Phase IV, most of PSI (P700) and chlorophyll a fluorescence relaxed with a halftime of 20 ms after a saturating light pulse. This time-constant may reflect the overall linear electron flux from PSII to PSI in saturating light. Comparable relaxation kinetics were also determined for C3 plants. At the end of CAM Phase I and during Phase II, slow components (> 50% of signal amplitude) appeared in both P700 reduction and fluorescence relaxation. They displayed halftimes > 250 ms and > 1 s, suggesting a strong restriction of the linear electron flux from H2O to NADP. The appearance of the slow redox components was accompanied by a decrease in the Fv/Fm ratio of chlorophyll a fluorescence, suggesting a reversible detachment of light-harvesting complex (LHC) II from PSII. The slow redox fractions and the depression of Fv/Fm disappeared again in parallel to malate decarboxylation during CAM Phase III. We discuss a reversible downregulation of linear electron flux during CAM Phases II and III, due to a reversible deprivation of cytochrome-b6f complexes (cyt-bfs) and PSI from the linear system. In parallel, a redistribution of some LHCIIs could also occur. This could be an adaptive response to a reduced metabolic demand for NADPH due to a limited carbon flux through the Calvin cycle, resulting from low Rubisco activation. Furthermore, the cyt-bfs and PSIs deprived of linear electron transport could support cyclic electron flux to cover an increased ATP demand during gluconeogenesis in CAM Phase III.

We have used transgenic tobacco (Nicotiana tabacum L. cv. W38) plants expressing an antisense gene directed against the transcript of the Rieske FeS protein of the chloroplast bf complex to examine the effect a reduction in chloroplast Rieske FeS content on leaf gas exchange and photosynthetic metabolite pools. Plants with chloroplast Rieske FeS protein content ranging from 5 to 80% of wild-type were analysed. CO2 assimilation rate declined linearly with the reduction in Rieske FeS content when expressed on a leaf area basis. In contrast to photosynthesis, there was no change in stomatal conductance except for plants with less than 10% of wild-type Rieske FeS content. There was a close correlation between Rieske FeS content and cytochrome f content, showing that the reduction in Rieske FeS content lead to a similar reduction in other components of the cytochrome bf complex. While lower Rieske FeS content was associated with declines in chlorophyll content, ATPδ subunit and ribulosebisphosphate carboxylase–oxygenase (Rubisco) contents, these declines were considerably smaller than the reduction in cytochrome bf content. As Rieske FeS content was reduced, there was a slight lowering of the chlorophyll a/b ratio. Lower CO2 assimilation rates at ambient CO2 and high light were associated with dramatic reductions in ribulose bisphosphate (RuBP) and modest declines in 3- phosphoglycerate (PGA). Rubisco carbamylation declined to 40–50% in plants with Rieske FeS content lower than 20% of wild-type. We conclude that, at high light, a reduction in chloroplast Rieske FeS protein content leads to inhibition of CO2 assimilation rate via reductions in the rate of RuBP regeneration caused by a restriction in electron transport and via effects on in vivo Rubisco activity.

Respiration (R, non-photorespiratory mitochondrial CO2 release) in leaves is inhibited by light. However, exposure to darkness after a period of illumination can also result in R being temporarily stimulated (termed ‘light enhanced dark respiration’, LEDR). We used a fast-response CO2 exchange system to investigate these observations in tobacco leaves. After switching off the light, there were two peaks of CO2 release, the first at 15–20 s (the photorespiratory post-illumination burst) and the second at 180–250 s (LEDR). LEDR occurred in all post-illumination experiments, independent of O2 or CO2 concentration. However, LEDR increased with increasing irradiance during the pre-dark period, suggesting some dependency on prior photosynthesis. We investigated the inhibition of R by light at low CO2 concentrations (?*): ?* is the intercellular CO2 concentration at which net CO2 release represents R in the light. The inhibition of R in the light took about 50 s and was even evident at 3 mmol photons m-2 s-1, regardless of the light quality (red, blue or white). The inhibition of R by light showed similar dependency on irradiance as LEDR, such that the degree of inhibition was positively correlated with the level of LEDR. In the light, switching from 350 ppm to a low CO2 concentration that resulted in the intercellular CO2 concentration being at ?*, resulted in R initially increasing and then stabilising. Maintaining the leaf at ?* did not, therefore, lead to an underestimation of R. Our data suggest that a common mechanism may be responsible for both the inhibition of R by light and LEDR.