Showing papers in "Photosynthesis Research in 1987"

Primary photochemistry of reaction centers from the photosynthetic purple bacteria.

Abstract: Photosynthetic organisms transform the energy of sunlight into chemical potential in a specialized membrane-bound pigment-protein complex called the reaction center. Following light activation, the reaction center produces a charge-separated state consisting of an oxidized electron donor molecule and a reduced electron acceptor molecule. This primary photochemical process, which occurs via a series of rapid electron transfer steps, is complete within a nanosecond of photon absorption. Recent structural data on reaction centers of photosynthetic bacteria, combined with results from a large variety of photochemical measurements have expanded our understanding of how efficient charge separation occurs in the reaction center, and have changed many of the outstanding questions.

State 1/state 2 changes in higher plants and algae

Abstract: Current ideas regarding the molecular basis of State 1/State 2 transitions in higher plants and green algae are mainly centered around the view that excitation energy distribution is controlled by phosphorylation of the light-harvesting complex of photosystem II (LHC-II). The evidence supporting this view is examined and the relationship of the transitions occurring in these systems to the corresponding transitions seen in red and blue-green algae is explored.

Photosynthesis and stomatal conductance of potato leaves-effects of leaf age, irradiance, and leaf water potential.

Abstract: Potatoes (Solanum tuberosum L., cv. Bintje) were grown in a naturally lit glasshouse. Laboratory measurements on leaves at three insertion levels showed a decline with leaf age in photosynthetic capacity and in stomatal conductance at near saturating irradiance. Conductance declined somewhat more with age than photosynthesis, resulting in a smaller internal CO2 concentration in older relative to younger leaves. Leaves with different insertion number behaved similarly. The changes in photosynthesis rate and in nitrogen content with leaf age were closely correlated. When PAR exceeded circa 100 W m−2 the rate of photosynthesis and stomatal conductance changed proportionally as indicated by a constant internal CO2 concentration. The photosynthesis-irradiance data were fitted to an asymptotic exponential model. The parameters of the model are AMAX, the rate of photosynthesis at infinite irradiance, and EFF, the slope at low light levels. AMAX declined strongly with leaf age, as did EFF, but to a smaller extent. During drought stress photosynthetic capacity declined directly with decreasing water potential (range −0.6 to −1.1 MPa). Initially, stomatal conductance declined faster than photosynthetic capacity.

Photosynthesis of cotton plants exposed to elevated levels of carbon dioxide in the field

Abstract: The cotton (Gossypium hirsutum L) plant responds to a doubling of atmospheric CO2 with almost doubled yield Gas exchange of leaves was monitored to discover the photosynthetic basis of this large response Plants were grown in the field in open-top chambers with ambient (nominally 350 μl/l) or enriched (nominally either 500 or 650 μl/l) concentrations of atmospheric CO2 During most of the season, in fully-irrigated plants the relationship between assimilation (A) and intercellular CO2 concentration (ci) was almost linear over an extremely wide range of ci CO2 enrichment did not alter this relationship or diminish photosynthetic capacity (despite accumulation of starch to very high levels) until very late in the season, when temperature was somewhat lower than at midseason Stomatal conductance at midseason was very high and insensitive to CO2, leading to estimates of ci above 85% of atmospheric CO2 concentration in both ambient and enriched chambers Water stress caused A to show a saturation response with respect to ci, and it increased stomatal closure in response to CO2 enrichment In fully-irrigated plants CO2 enrichment to 650 μl/l increased A more than 70%, but in water-stressed plants enrichment increased A only about 52% The non-saturating response of A to ci, the failure of CO2 enrichment to decrease photosynthetic capacity for most of the season, and the ability of the leaves to maintain very high ci, form in part the basis for the very large response to CO2 enrichment

A model for the mechanism of chloride activation of oxygen evolution in photosystem II.

Abstract: A hypothesis is proposed to explain the function of Cl- in activating the oxygenevolving complex (OEC) of photosystem II (PS II), based on the results of recent 35Cl-NMR studies. The putative mechanism involves Cl- binding to two types of sites. An intrinsic site is suggested to be composed of three histidyl residues (His 332 and His 337 from D1 and His 337 D2). It is proposed that Cl- binding to this site accelerates the abstraction of H+ from water by raising the pKa's of the histidine imidazole groups. Cl- binding also stimulates the transfer of H+ from this intrinsic site to a set of extrinsic sites on the 33 kD extrinsic polypeptide. The extrinsic Cl- binding sites are suggested to involve four protein domains that are linked together by salt-bridge contacts. Chloride and H+ donated from the intrinsic site attack these intramolecular salt-bridges in a defined sequence, thereby exposing previously inaccessible Cl- and H+ binding sites and stimulating the oxidation of water. This hypothesis also proposes a possible structure for the Mn active site within the D1/D2 complex. Specific amino-acid residues that are likely to participate as Mn lignads are identified on the lumenal portions of the D1 and D2 proteins that are different from those in the L and M subunits of photosynthetic bacteria; the choice of these residues is based on the metal coordination chemistry of these residues, their location within the polypeptide chain, the regularity of their spacing, and their conservation through evolution. The catalytic Mn-binding residues are suggested to be D-61, E-65, E-92, E-98, D-103; D-308, E-329, E-342 and E-333 in D1, and H-62, E-70, H-88, E-97, D-101; E-313, D-334, E-338 and E-345 in D2. Finally, this hypothesis identifies sites on both D2 and the 33 kD extrinsic polypeptide that might be involved in high- and low-affinity Ca2+ binding.

The rate of formation of P700(+)-A 0 (-) in photosystem I particles from spinach as measured by picosecond transient absorption spectroscopy.

Abstract: Photosystem I particles containing 30-40 chlorophyll a molecules per primary electron donor P700 were subjected to 1.5 ps low density laser flashes at 610 nm resulting in excitation of the antenna chlorophyll a molecules followed by energy transfer to P700 and subsequent oxidation of P700. Absorbance changes were monitored as a function of time with 1.5 ps time resolution. P700 bleaching (decrease in absorbance) occurred within the time resolution of the experiment. This is attributed to the formation of (1)P700.(*) This observation was confirmed by monitoring the rise of a broad absorption band near 810 nm due to chlorophyll a excited singlet state formation. The appearance of the initial bleach at 700 nm was followed by a strong bleaching at 690 nm. The time constant for the appearance of the 690 nm bleach is 13.7±0.8 ps. In the near-infrared region of the spectrum, the 810 nm band (which formed upon the excitation of the photosystem I particles) diminished to about 60% of its original intensity with the same 13.7 ps time constant as the formation of the 690 nm band. The spectral changes are interpreted as due to the formation of the charge separated state P700(+)-A0 (-), where A0 is the primary electron acceptor chlorophyll a molecule.

Kinetics of oxidation of the bound cytochromes in reaction centers from Rhodopseudomonas viridis

Abstract: The initial oxidized species in the photochemical charge separation in reaction centers from Rps. viridis is the primary donor, P+, a bacteriochlorophyll dimer. Bound c-type cytochromes, two high potential (Cyt c 558) and two low potential (Cyt c 553), act as secondary electron donors to P+. Flash induced absorption changes were measured at moderate redox potential, when the high potential cytochromes were chemically reduced. A fast absorption change was due to the initial oxidation of one of the Cyt c 558 by P+ with a rate of 3.7×106s-1 (τ=270nsec). A slower absorption change was attributable to a transfer, or sharing, of the remaining electron from one high potential heme to the other, with a rate of 2.8×105s-1 (τ=3.5 μsec). The slow change was measured at a number of wavelengths throughout the visible and near infrared and revealed that the two high potential cytochromes have slightly different differential absorption spectra, with α-band maxima at 559 nm (Cyt c 559) and 556.5 nm (Cyt c 556), and dissimilar electrochromic effects on nearby pigments. The sequence of electron transfers, following a flash, is: Cyt c 556→Cyt c 559→P+. At lower redox potentials, a low midpoint potential cytochrome, Cyt c 553, is preferentially oxidized by P+ with a rate of 7×106s-1 (τ=140 nsec). The assignment of the low and high potential cytochromes to the four, linearly arranged hemes of the reaction center is discussed. It is concluded that the closest heme to P must be the high potential Cyt c 559, and it is suggested that a likely arrangement for the four hemes is: c 553 c 556 c 553 c 559P.

Characterisation of the effects of Antimycin A upon high energy state quenching of chlorophyll fluorescence (qE) in spinach and pea chloroplasts.

Abstract: High energy state quenching of chlorophyll fluorescence (qE) is inhibited by low concentrations of the inhibitor antimycin A in intact and osmotically shocked chloroplasts isolated from spinach and pea plants. This inhibition is independent of any effect upon ΔpH (as measured by 9-aminoacridine fluorescence quenching). A dual control of qE formation, by ΔpH and the redox state of an unidentified chloroplast component, is implied. Results are discussed in terms of a role for qE in the dissipation of excess excitation energy within photosystem II.

Large scale preparation of pure phycobiliproteins

Abstract: This paper describes simple procedures for the purification of large amounts of phycocyanin and allophycocyanin from the cyanobacterium Microcystis aeruginosa. A homogeneous natural bloom of this organism provided hundreds of kilograms of cells. Large samples of cells were broken by freezing and thawing. Repeated extraction of the broken cells with distilled water released phycocyanin first, then allophycocyanin, and provides supporting evidence for the current models of phycobilisome structure. The very low ionic strength of the aqueous extracts allowed allophycocyanin release in a particulate form so that this protein could be easily concentrated by centrifugation. Other proteins in the extract were enriched and concentrated by large scale membrane filtration. The biliproteins were purified to homogeneity by chromatography on DEAE cellulose. Purity was established by HPLC and by N-terminal amino acid sequence analysis. The proteins were examined for stability at various pHs and exposures to visible light.

The transmembrane distribution of galactolipids in chloroplast thylakoids is universal in a wide variety of temperate climate plants.

Abstract: The transmembrane distribution of monogalactosyldiacylglycerol and digalactosyldiacylglycerol was determined in chloroplast thylakoids from a range of temperate climate plants. These plants included dicotyledons, monocotyledons, C16:3 and C18:3 plants and herbicide-resistant species. In all the thylakoids examined monogalactosyldiacylglycerol was enriched in the outer leaflet (53–65%) while digalactosyldiacylglycerol was highly enriched in the inner leaflet (78–90%). The non-bilayer forming monogalactosyldiacylglycerol represented 55–81% of the total acyl lipids of the outer monolayer. The relative acyl lipid composition of both leaflets of the thylakoid membrane indicates that the lamellar structure is strongly favored in the inner monolayer, whereas the outer one presents a metastable character which allows the probable coexistence of both lamellar and non-lamellar phases. The consequence of this asymmetry for the stability and function of the thylakoid membrane is discussed.

C3-C4 intermediate photosynthetic characteristics of cassava (Manihot esculenta Crantz)

Abstract: Cassava, bean and maize leaves were fed with14CO2 in light and the primary products of photosynthesis identified 5 and 10 seconds after assimilation. In maize, approximately three quarters of the labelled carbon was incorporated in C4 acids, in beans about two thirds in PGA, and in cassava approximately 40–60% in C4 acids with 30–50% in PGA. These data indicate that cassava possesses the C4 photosynthetic cycle, however due to the lack of typical Kranz anatomy appreciable carbon assimilation takes place directly through the Calvin-Benson-Bassham cycle.

Induction kinetics of heat emission before and after photoinhibition in cotyledons of Raphanus sativus

Abstract: Heat emitted during non-radiative de-excitation was determined in vivo by the photoacoustic method. The dependence of the photoacoustic signal on the length of the pulses (modulation frequency) of the excitation light and the effect of continuous light, which saturates photosynthesis but does not directly contribute to the signal, are described. The induction kinetic of heat emission measured with intact leaves differed only slightly from the induction kinetic of fluorescence (Kautsky effect) detected in parallel. The photoacoustic signal at high modulation frequencies (279 Hz), which represents the signal of heat emission, and the photoacoustic signal at low modulation frequencies (17 Hz), interpreted as a signal of pulsed oxygen evolution superimposed on the heat emission, were measured with leaves before and after photoinhibition. It was demonstrated that after photoinhibition the decrease in fluorescence yield and in photosynthetic activity (here detected as photoacoustic signal at 17 Hz) are paralleled by an increase in the yield of non-radiative deexcitation (photoacoustic signal at 279 Hz). The increase of heat emission, which has been hypothized for photoinhibited leaves, could now be proved by measuring the induction kinetics of the photoacoustic signal.

Chlorophyll fluorescence transients from the diatom Phaeodactylum tricornutum: relative rates of cyclic phosphorylation and chlororespiration.

Abstract: In Phaeodactylum tricornutum cells kept 30 min in the dark, induction of fluorescence showed the well-known levels OIDPSMT. The decrease of MT was the most important when the intensity of excitation light was high. It was mainly due to the photochemical quenching. After addition of DCMU (2 to 20 μM), a quenching qE was still observed: this quenching, cancelled by NH4Cl (2 to 20 mM) is attributed to ΔpH. This qE was also inhibited by antimycin, an inhibitor of cyclic phosphorylation and may be of chlororespiration above plastoquinones. Anaerobiosis also decreased it. We can infer that chlororespiration also plays a part in the formation of the ΔpH in the presence of DCMU. After 30 mn of preillumination in red light, the levels P and M were lower and the quenching in presence of DCMU was no more observed: thus, neither the chlororespiration nor the cyclic phosphorylation were active, unless the activity of ATPase was much more important. So, in diatoms, one at least of the above cited phenomena can be modulated by light.

Studies on the protolytic reactions coupled with water cleavage in photosystem II membrane fragments from spinach

Abstract: The protolytic reactions of PSII membrane fragments were analyzed by measurements of absorption changes of the water soluble indicator dye bromocresol purple induced by a train of 10 μs flashes in dark-adapted samples. It was found that: a) in the first flash a rapid H+-release takes place followed by a slower H+-uptake. The deprotonation is insensitive to DCMU but is completely eliminated by linolenic acid treatment of the samples; b) the extent of the H+-uptake in the first flash depends on the redox potential of the suspension. In this time domain no H+-uptake is observed in the subsequent flashes; c) the extent of the H+-release as a function of the flash number in the sequence exhibits a characteristic oscillation pattern. Multiphasic release kinetics are observed. The oscillation pattern can be satisfactorily described by a 1, 0, 1, 2 stoichiometry for the redox transitions Si → Si+1 (i=0, 1, 2, 3) in the water oxidizing enzyme system Y. The H+-uptake after the first flash is assumed to be a consequence of the very fast reduction of oxidized Q400(Fe3+) formed due to dark incubation with K3[Fe(CN)6]. The possible participation of component Z in the deprotonation reactions at the PSII donor side is discussed.

The effect of the dark interval in intermittent light on thylakoid development: photosynthetic unit formation and light harvesting protein accumulation

Abstract: Etiolated bean plants were grown in intermittent light with dark intervals of shorter or longer duration, to modulate the rate of chlorophyll accumulation, relative to that of the other thylakoid components formed. We thus produced conditions under which chlorophyll becomes more or less a limiting factor. We then tested whether LHC complexes can be incorporated in the thylakoid. It was found that an equal amount of chlorophyll, formed under the same total irradiation received, may be used for the stabilization of few and large-in-size PS units containing LHC components (short dark-interval intermittent light), or for the stabilization of many and small-in-size PS units with no LHC components (long dark-interval intermittent light). The size of the PS units diminishes as the dark-interval duration is increased, with no further change after 98 minutes. The PSII/cytf ratio remains constant throughout development in intermittent light and equal to that of mature chloroplasts (PSII/cytf = 1) except in the case of very long dark-interval regimes, where about half PSII units per cytf are present. The PSII/PSI ratio was found to be correlated with the PSII unit size (the larger the size, the lower the ratio). The number of PSI units operating on the same electron transfer chain varied depending on the size of the PSII unit (the larger the PSII unit size, the more the PSI units per chain). The results suggest that it is not the chlorophyll content per se which regulates the stabilization of LHC in developing thylakoids and consequently the size of the PS units, but rather the rate by which it is accumulated, relative to that of the other thylakoid components.

Regulation of photosynthetic carbon metabolism during phosphate limitation of photosynthesis in isolated spinach chloroplasts.

Abstract: Intact chloroplasts isolated from spinach were illuminated in the absence of inorganic phosphate (Pi) or with optimum concentrations of Pi added to the reaction medium. In the absence of Pi photosynthesis declined after the first 1–2 min and was less than 10% of the maximum rate after 5 min. Export from the chloroplast was inhibited, with up to 60% of the 14C fixed being retained in the chloroplast, compared to less than 20% in the presence of Pi. Despite the decreased export, chloroplasts depleted of Pi had lower levels of triose phosphate while the percentage of total phosphate in 3-phosphoglycerate was increased. Chloroplast ATP declined during Pi depletion and reached dark levels after 3–4 min in the light without added Pi. At this point, stromal Pi concentration was 0.2 mM, which would be limiting to ATP synthesis. Addition of Pi resulted in a rapid burst of oxygen evolution which was not initially accompanied by net CO2 fixation. There was a large decrease in 3-phosphoglycerate and hexose plus pentose monophosphates in the chloroplast stroma and a lesser decrease in fructose-1,6-bisphosphate. Stromal levels of triose phosphate, ribulose-1,5-bisphosphate and ATP increased after resupply of Pi. There was an increased export of 14-labelled compounds into the medium, mostly as triose phosphate. Light activation of both fructose-1,6-bisphosphatase and ribulose-1,5-bisphosphate carboxylase was decreased in the absence of Pi but increased following Pi addition.

Comparison of the electron spin polarized spectrum found in plant photosystem I and in iron-depleted bacterial reaction centers with time-resolved K-band EPR; evidence that the photosystem I acceptor A1 is a quinone.

Abstract: The suggestion that the electron acceptor A1 in plant photosystem I (PSI) is a quinone molecule is tested by comparisons with the bacterial photosystem The electron spin polarized (ESP) EPR signal due to the oxidized donor and reduced quinone acceptor (P 870 + Q-) in iron-depleted bacterial reaction centers has similar spectral characteristics as the ESP EPR signal in PSI which is believed to be due to P 700 + A 1 - , the oxidized PSI donor and reduced A1 This is also true for better resolved spectra obtained at K-band (∼24 GHz) These same spectral characteristics can be simulated using a powder spectrum based on the known g-anisotropy of reduced quinones and with the same parameter set for Q- and A1 - The best resolution of the ESP EPR signal has been obtained for deuterated PSI particles at K-band Simulation of the A1 - contribution based on g-anisotropy yields the same parameters as for bacterial Q- (except for an overall shift in the anisotropic g-factors, which have previously been determined for Q-) These results provide evidence that A1 is a quinone molecule The electron spin polarized signal of P700 + is part of the better resolved spectrum from the deuterated PSI particles The nature of the P700 + ESP is not clear; however, it appears that it does not exhibit the polarization pattern required by mechanisms which have been used so far to explain the ESP in PSI

Is there significant cyclic electron flow around photoreaction 1 in cyanobacteria

Abstract: Evidence for a cyclic electron flow has been sought by study of the steady-state poise of P700 and rate of photoreaction 1 in three cyanobacteria. Under an actinic light 1 (440 or 680 nm) the rate of photoreaction 1 is limited by the rate of electron supply provided by photoreaction 2 and by all return electron flow from low potential donors such as ferredoxin and NAD(P)H. Plots of p, the steady-state fraction of P700 reduced, versus the reciprocal intensity, 1/I, yield linear segments of slope Ip. From considerations of a simple model the slopes and extrapolated intercepts of the linear segments provide estimates of the rate of return electron flow. Analysis shows that the total return electron flow cannot be large, by one estimate not more than three times the rate of dark respiration. This result leads to a conclusion that cyclic electron flow (and any dependent phosphorylation) is not a significant process in these cyanobacteria at ordinary light intensities.

Differences between Indica and Japonica rice varieties in CO2 exchange rates in response to leaf nitrogen and temperature.

Abstract: Four Indica and five Japonica varieties of rice (Oryza sativa L.) were examined to elucidate their differences in photosynthetic activity and dark respiratory rate as influenced by leaf nitrogen levels and temperatures. The photosynthetic rates of single leaf showed correlations with total nitrogen and soluble protein contents in the leaves. Respiratory rate was also positively correlated with the leaf nitrogen content. When compared at the same level of leaf nitrogen or soluble protein content, the four Indica varieties and one of Japonica varieties, Tainung 67, which have some Indica genes derived from one of its parents, showed higher photosynthetic rates than the remaining four Japonica varieties. At the same photosynthetic rate, the Indica varieties showed lower respiratory rate than Japonica varieties. When the leaf temperature rose from 20°C to 30°C, the photosynthetic rate increased by 18 to 41%, whereas the respiratory rate increased by 100 to 150%. These increasing rates in response to temperature were higher in the Japonica than in the Indica varieties. In this respect, Tainung 67 showed the same behavior as of the other four Japonica varieties.

Comparative structural and catalytic properties of oxygen-evolving photosystem II preparations

Abstract: Biochemical techniques now exist to produce the oxygen-evolving complex of photosystem II (PSII) and its associated photochemical redox reactions in various states of purity. These preparations permit one to assess the structural roles of polypeptides in promoting activity by using selective extraction techniques which remove certain polypeptides, to carry out reconstitution studies which re-establish activity, and, in the case of more recently developed, highly purified preparations discussed in this overview, to identify the minimal polypeptide complement necessary for photosynthetic oxygen evolution activity. These comparative investigations also suggest a tentative structure for an oxygen-evolving PSII core complex whose primary constituents are a hydrophobic complex of polypeptide, manganese, calcium and chloride, and the 33 kDa extrinsic polypeptide.

Characteristics of a photorespiratory mutant of barley (Hordeum vulgare L.) deficient in phosphogly collate phosphatase.

Abstract: A barley mutant RPr84/90 has been isolated by selecting for plants which grow poorly in natural air, but normally in air enriched to 0.8% CO2. After 5 minutes of photosynthesis in air containing14CO2 this mutant incorporated 26% of the14C carbon into phosphoglycollate, a compound not normally labelled in wild type (cv. Maris Mink) leaves.

C3-C 4 intermediate photosynthetic characteristics of cassava (Manihot esculenta Crantz) : I. Gas exchange.

Abstract: The gas exchange characteristics of cassava were compared with one C3 species, common bean, and two C4 species, maize and amaranthus Cassava leaf photorespiration, about 12% of maximum net photosynthesis in normal air, and the CO2 release in CO2-free air under intense light were lower than the values typically reported for C3 species The CO2 compensation point of whole leaves (25 cm(3) CO2m(-3)) was intermediate between C3 and C4 species valuesGas exchange was restricted to either the upper or lower surface of amphistomatous leaves by covering one side of the leaf with silicone grease The CO2 compensation point of the upper leaf surface was less than 6 cm(3) CO2m(-3) and the CO2 release into CO2-free air in the light was essentially zero On the lower leaf surface considerable CO2 release occurred in both the light and the darkThe hypothesis presented to explain these results is the existence of an efficient CO2 recycling mechanism in the palisade layer in the upper half of the leaf In the light of recent data (presented in the second paper of this series) indicating that cassava produces C4 acids as primary products of photosynthesis, it is proposed that this hypothesis is consistent with the possible existence of the C4 photosynthetic pathway in the palisade layer of cassava leavesThe results and hypothesis are discussed in relation to the crop's adaptation to the environmental conditions where it is normally grown The implications of variation in anatomical features such as stomatal distribution on both surfaces of the leaf are analyzed with a view to enhancing the potential productivity of cassava under stress conditions

Investigation of the spatial relationships between photosystem 2 polypeptides by reversible crosslinking and diagonal electrophoresis.

Abstract: Nearest neighbour relationships within the LHC2-PS2 complex were investigated by using the reversible crosslinking agent dithiobis(succinimidyl propionate) (DSP). This was accomplished by treating PS2-enriched membranes, prepared from chloroplasts of Pisum sativum, with the crosslinker followed by diagonal electrophoresis of the solubilised polypeptides. Analysis of the off-diagonal spot patterns produced by crosslinker cleavage and second dimension electrophoresis was made on the basis of: staining with Coomassie blue or silver, labelling with [35S]-methionine, and sensitivity to 1 M NaCl washing. It was concluded that LHC2 polypeptides crosslinked with several components of the PS2 complex and that the extrinsic polypeptides associated with water oxidation, having approximate molecular weights of 16 and 23 kDa, crosslink to form homodimers. The latter finding suggests that there may be more than one copy of each of these polypeptides per PS2 complex.

Reversal of heat-induced alterations in photochemical activities in wheat primary leaves.

Abstract: Chloroplasts isolated from elevated temperature treated 8-day-old continuous-white-light-grown wheat primary leaves lost the ability to photo-oxidize water. Also, the ability of ascorbate to donate electrons to photosystem II declined. However, a significant increase in reduced dichlorophenolindophenol-supported photosystem-I-mediated methylviologen photo-reduction activity was observed. The plants stressed at 45°C and 47°C were subsequently grown at 25°C and the partial photochemical activities were measured in chloroplasts isolated from the plants at 24-h intervals. The post stress alterations observed are (1) a significant restoration of water oxidation capacity in 45°C- and partial restoration in 47°C-treated leaves. Ascorbate-supported photochemical activities recovered more or less in similar fashion; (2) reversal of enhanced photosystem I activity in both 45°C- and 47°C-treated leaves. These results suggest that the restoration in water oxidation capacity is possible in 45°C-treated leaves and is limited by the severity of heat stress in 47°C-treated leaves. Restoration of water oxidation capacity vis-a-vis to the reversal of heat-enhanced photosystem I activity also indicates the existence of possible endogenous control for repair of alterations during the post stress.

A solid-state, portable instrument for measurement of chlorophyll luminescence induction in plants.

Abstract: A newly developed compact instrument is described for the measurement of chlorophyll luminescence induction in plants. The instrument operates with a pulsed light emitting diode (LED) as light source and a photodiode as luminescence detector. A special emitter-detector geometry provides for high irradiance of the sample and efficient collection of luminescence by the detector. With insertion of appropriate filters the same probe is also suited for measuring prompt chlorophyll fluorescence. The instrument shows considerable flexibility with respect to pulse frequency, relative lengths of light/dark intervals and luminescence sampling periods. Due to a selective amplifier system only that part of luminescence is processed which is induced by the individual excitation pulses. By this approach, the problem of "slow phase accumulation", encountered with conventional phosphoroscopes, is eliminated. Some examples are given for system operation, demonstrating satisfactory performance in measurements with intact leaves and isolated chloroplasts.

Crassulacean acid metabolism, CO2-recycling, and tissue desiccation in the Mexican epiphyte Tillandsia schiedeana Steud (Bromeliaceae).

Abstract: After 23 days without water in a greenhouse, rates of nocturnal CO2 uptake in Tillandsia schiedeana decreased substantially and maximum rates occurred later in the dark period eventually coinciding with the onset of illumination. Nocturnal CO2 uptake accounted for less than half the total nighttime increase in acidity measured in well-watered plants. With increased tissue desiccation, only 11–12% of measured acid accumulation was attributable to atmospheric CO2 uptake. Plants desiccated for 30 days regained initial levels of nocturnal acid accumulation and CO2 uptake after rehydration for 10h. These results stress the importance of CO2 recycling via CAM in this epiphytic bromeliad, especially during droughts.

Energy transfer and pigment composition in three chlorophyll b-containing light-harvesting complexes isolated from Mantoniella squamata (Prasinophyceae), Chlorella fusca (Chlorophyceae) and Sinapis alba.

Abstract: Light-harvesting Chl a/b protein complexes were isolated from the higher plant Sinapis alba, the green alga Chlorella fusca, and the prasinophycean alga Mantoniella squamata by mild gel electrophoresis. The energy transfer from chlorophyll b and the accessory xanthophyll was measured by means of fluoresence spectroscopy at 77 K. The pigment composition of the isolated antenna complexes was determined by high performance liquid chromatography in order to calculate the number of light absorbing molecules per chlorophyll a in the different light-harvesting complexes. These results were complemented by the quantitation of the pigments in total thylakoids as well as in the different electrophoretic fractions. On the basis of these data the in vivo ratios of xanthophylls per chlorophyll a could be estimated. The results show that the light-harvesting complexes from Chlorella and from Sinapis exhibit identical ratios of total xanthophylls per chlorophyll a. By contrast, in the prasinophycean alga Mantoniella, the light-harvesting complex markedly differs from the other chlorophyll b containing proteins. It contains, in addition to neoxanthin and violaxanthin, high amounts of prasinoxanthin and its epoxide, which contribute significantly to light absorption. The concentration of chlorophyll b in the complex is very much higher in the antenna of Mantoniella than in those of Chlorella and Sinapis. Furthermore, it must be emphasized that in addition to chlorophyll b, a third chlorophyll species acts in the energy transfer to chlorophyll a. This chlorophyll c-like pigment is found to be present in a concentration which improves very efficiently the absorption in blue light. In light of these results it can be concluded that the absorption cross section in Mantoniella is higher not only because of an enhanced number of light-harvesting particles in the membrane, but also because of a higher ratio of accessory pigments to chlorophyll a.

Formation of the S2 state and structure of the Mn complex in photosystem II lacking the extrinsic 33 kilodalton polypeptide.

Abstract: Electron paramagnetic resonance (EPR) spectroscopy and O2 evolution assays were performed on photosystem II (PSII) membranes which had been treated with 1 M CaCl2 to release the 17, 23 and 33 kilodalton (kDa) extrinsic polypeptides. Manganese was not released from PSII membranes by this treatment as long as a high concentration of chloride was maintained. We have quantitated the EPR signals of the several electron donors and acceptors of PSII that are photooxidized or reduced in a single stable charge separation over the temperature range of 77 to 240 K. The behavior of the samples was qualitatively similar to that observed in samples depleted of only the 17 and 23 kDa polypeptides (de Paula et al. (1986) Biochemistry25, 6487–6494). In both cases, the S2 state multiline EPR signal was observed in high yield and its formation required bound Ca2+. The lineshape of the S2 state multiline EPR signal and the magnetic properties of the manganese site were virtually identical to those of untreated PSII membranes. These results suggest that the structure of the manganese site is unaffected by removal of the 33 kDa polypeptide. Nevertheless, in samples lacking the 33 kDa polypeptide a stable charge separation could only be produced in about one half of the reaction centers below 160 K, in contrast to the result obtained in untreated or 17 and 23 kDa polypeptide-depleted PSII membranes. This suggests that one function of the 33 kDa polypeptide is to stabilize conformations of PSII that are active in secondary electron transfer events.

An immunological method for quantitative determination of photosynthetic fructose-1,6-bisphosphatase in leaf crude extracts.

Abstract: An immunological method for quantitative determination of photosynthetic fructose-1,6-bisphosphatase in crude extracts of leaves is proposed. It is based on the ELISA technique, and offers two modifications. A non-competitive technique has a higher sensitivity and is the right option for samples of low fructose-1,6-bisphosphatase content. However, this method is not sufficiently specific when the total protein is higher than 5 μg/cm3; so, despite its lower sensitivity, in these circumstances a competitive technique is more suitable. Thus photosynthetic fructose-1,6-bisphosphatase can be measured without interferences from the gluconeogenic cytosolic enzyme of the photosynthetic cell or from a non-specific phosphatase present in the chloroplast.

Assocation of the 33 kDa extrinsic polypeptide (water-splitting) with PS II particles: immunochemical quantification of residual polypeptide after membrane extraction.

Abstract: Various washing procedures were tested on Triton-prepared PS II particles for their ability to remove the 33 kDa extrinsic polypeptide (33 kDa EP) associated with the water-splitting complex. Residual 33 kDa EP was evaluated by Coomassie blue staining of SDS gels of washed particles and by Western blotting with an antibody specific for the 33 kDa EP. A wash with 16 mM Tris buffer, pH 8.3, inhibited water-splitting activity but did not remove all the 33 kDa EP. Sequential washes with 30 mM octyl glucoside (pH 8.0 and 6.8), and a single wash with 0.8 M Tris were also ineffective in removing all the 33 kDa EP. Washing with 1 M CaCl2 was more effective in removing 33 kDa EP; while only a faint trace of protein was detectable by Coomassie-staining, immunoblotting revealed a considerable remainder. The treated particles retained some water-splitting activity. The two step procedure of Miyao and Murata (1984) involving 1 M NaCl and 2.3 M urea was most effective, removing all but a trace of antibody positive protein. Our finding suggests that (1) the degree of depletion of the 33 kDa EP cannot be judged on the basis of Coomassie stain alone, and (2) this extrinsic protein is very tightly associated with the membrane, perhaps via a hydrophilic portion of this otherwise hydrophilic protein. The results also suggest that the presence or absence of the 33 kDa protein per se is not the primary determinant of residual water splitting activity.