Butterfly wing colours: scale beads make white pierid wings brighter.

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.

Spatiotemporal variation of metabolism in a plant circadian rhythm: the biological clock as an assembly of coupled individual oscillators.

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 (phi(PSII)) over a single leaf. Noninvasive highly sensitive chlorophyll fluorescence imaging reveals randomly initiated patches of varying phi(PSII) that are propagated within minutes to hours in wave fronts, forming dynamically expanding and contracting clusters and clearly dephased regions of phi(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.

Expressing an RbcS Antisense Gene in Transgenic Flaveria bidentis Leads to an Increased Quantum Requirement for CO2 Fixed in Photosystems I and II.

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.

Imaging of chlorophyll-a-fluorescence in leaves: Topography of photosynthetic oscillations in leaves of Glechoma hederacea.

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.

Vacuolar pH oscillations in mesophyll cells accompany oscillations of photosynthesis in leaves: Interdependence of cellular compartments, and regulation of electron flow in photosynthesis.

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.

Chloroplast energization and oxidation of P700/plastocyanin in illuminated leaves at reduced levels of CO2 or oxygen.

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.

Regulation of chloroplast metabolism in leaves: Evidence that NADP-dependent glyceraldehydephosphate dehydrogenase, but not ferredoxin-NADP reductase, controls electron flow to phosphoglycerate in the dark-light transition.

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.

Oscillations in photosynthesis are initiated and supported by imbalances in the supply of ATP and NADPH to the Calvin cycle.

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.

Light-dependent pH changes in leaves of C3 plants : IV. Action spectra indicate indirect energization of proton transport into mesophyll vacuoles by cyclic photophosphorylation.

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.

Control of photosynthesis in leaves as revealed by rapid gas exchange and measurements of the assimilatory force FA.

The rapid transients of CO2 gas exchange have been measured in leaves ofHelianthus annuus L. In parallel experiments the assimilatory force FA, which is the product of the phosphorylation potential and the redox ratio NADPH/NADP, has been calculated from measured ratios of dihydroxyacetone phosphate to phosphoglycerate in the chloroplast stroma and in leaves. The following results were obtained: (i) When the light-dependent stroma alkalization was measured under steady-state conditions for photosynthesis in air containing 2000 µl · l(-1) CO2, alkalization increased with photosynthesis as the quantum flux density (irradiance) was increased. This contrasts to the light-dependent stroma alkalisation measured in dark-adapted leaves during the dark-light transient (Laisk et al. 1989, Planta177, 350-358) which reached a maximum at a quantum flux density far below that necessary to saturate photosynthesis. This maximum was about three times higher than the maximum stroma alkalization at light- and CO2-saturated photosynthesis. (ii) Accurate calculations of the assimilatory force FA require a consideration of the stromal pH. However, under many conditions, changes in the stromal pH resulting from changes in photosynthetic flux can be neglected because they are small. (iii) Stromal ratios of dihydroxyacetone phosphate to phosphoglycerate are generally lower than ratios measured in leaf extracts. The value of FA calculated from stromal metabolites was about 30% lower than FA calculated from cellular metabolites. Still, it appears sufficient for many purposes to calculate FA from metabolite measurements in leaf extracts. (iv) In the light, the catalytic capacity of the photosynthetic apparatus is adjusted to the level of irradiance. The response of carbon assimilation to large increases in irradiance is slow because it requires enzyme activation. Deactivation of the Calvin cycle induced by decreases in irradiance is slower than activation. (v) Changes in catalytic capacity and in the availability or level of substrates such as CO2 alter the flux resistance of the Calvin cycle. A decrease in flux resistance explains why FA often does not increase by much and may actually decrease when carbon flux is increased. Adjustments of flux resistances in the Calvin cycle and of photosystem-II activity in the electron-transport chain permit varying rates of photosynthesis at low levels of ATP and NADPH. As NADP remains available, the danger of over-reduction which leads to photoinactivation of electron transport is minimized.