Regulation of photosystem stoichiometry,chlorophyll a and chlorophyll b content and relation to chloroplast ultrastructure

The structural-functional organization of higher plant chloroplasts has been investigated in relation to the particular light conditions during plant growth. (1) Light intensity variations during growth caused changes in the $\text{Chl}\text{a}\text{Chl}\text{b}$ ratio, in the light-saturated uncoupled rates of electron transport to a Hill oxidant and in the distribution of the chloroplast volume between the membrane and stroma phases. (2) Light quality differences during growth had an effect on the PS II/PS I reaction center ratio and on the chloroplast membrane phase differentiation into grana and stroma thylakoids. Plants grown under far-red-enriched (680–710 nm) illumination contained higher (20–25%) amounts of PS II and simultaneously lower (20–25%) amounts of PS I reaction centers. They also showed a higher grana density along with thicker grana stacks in their chloroplasts. (3) The size of the light-harvesting antenna pool of PS II centers was estimated from the fluorescence time course of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea-poisoned chloroplasts and was found to be fairly constant (±10%) in spite of the variable PS II/PS I reaction center ratio. The results are compatible with the hypothesis that the structural entities of grana facilitated the centralization and relative concentration increase of a certain group of PS II reaction centers.     

Chloroplast culture IX chlorophyll(ide) a biosynthesis invitro at rates higher than inMvivo

Chlorophyll $\text{a}$ is the plant pigment which in nature catalyzes the conversion of solar energy into chemical energy. By pretreating etiolated cucumber cotyledons with kinetin and gibberellic acid in the dark, it was observed that the plastids which were isolated from such tissues, and incubated in a cofactor-fortified medium, under a repetitive light-dark regime, were capable of synthesizing chlorophyll(ide) $\text{a}$ from exogenous δ-aminolevulinic acid at a rate about twice as high as the highest rates observable in greening tissues $\text{in}\text{vivo}$.     

The yield of chlorophyll a fluorescence as a means to test the various deexcitation mechanisms in the antenna system of green plants

With the aid of measurements of the fluorescence yield, the efficiency of the various deexcitation mechanisms of an exciton in the light-harvesting system has been determined. For this purpose, the fluorescence of dark-adapted as well as of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-treated and preilluminated leaves of Zea mays L. was excited by single ultrashort laser pulses of different energies. The experimental results have served for the fitting of solutions of rate equations, which describe the deexcitation by linear relaxation processes like fluorescence and radiationless transitions, by annihiation of excitons, and by traps both in the ground state and in an excited state. We have obtained the following results: a ratio of antenna chlorophyll molecules to Photosystem II traps of 600:1, an annihilation constant γ = 2·10^?8 cm³·s^?1, a mean trapping time of $\text{t}\text{?}\text{=0.5}\text{ns}$, a trapping probability for traps in the ground state of 2·10^?8 cm³·s^?1, and 6·10^?9 cm³·s^?1 for traps in an excited state.     

The fluorescence lifetime and quantum yield of chlorophyll a in Triton X-100 solutions

The fluorescence of chlorophyll (Chl) a in 0.007–0.1% Triton X-100 was investigated by a phase-shift technique. The Chl a concentrations varied from 0.7 to 25 μM. Parallel measurements of fluorescence lifetime (τ) and quantum yield (ψ) were made. It was concluded that homogeneous energy transfer takes place at detergent concentrations above 0.025%: (i) the transfer between uniform molecules of the pigment solubilized in Triton X-100 micelles, when τ and ψ are constant; (ii) the transfer towards the quenching centers, resulting in a proportional decrease in τ and ψ. At a Triton X-100 concentration of about 0.025% the Chl a emission becomes heterogeneous. It is evident from the disproportional decrease in τ and ψ (greater in ψ than in τ) and also from the rise of the fluorescence at 730–750 nm. As the Triton X-100 concentration becomes lower than the critical one (0.021%), the number of micelles drops abruptly and Chl a forms colloid particles in the aqueous medium. This manifests itself as a decrease in τ and as a certain stabilization of ψ. Having analyzed the complex pattern of the τ/ψ ratio, we concluded that under these conditions more than 90% of Chl a is in a weakly fluorescent form (τ < 30 ps) and about 1% is in an aggregated state fluorescing at 732 nm with τ about 0.7 ns.     

The multiple roles of light-harvesting chlorophyll a/b-protein complexes define structure and optimize function of Arabidopsis chloroplasts: A study using two chlorophyll b-less mutants

The multiple roles of light-harvesting chlorophyll a/b-protein complexes in the structure and function of Arabidopsis chloroplasts were investigated using two chlorophyll b-less mutants grown under metal halide lamps with a significant far-red component. In ch1-3, all six light-harvesting proteins of photosystem (PS) II were greatly decreased; in ch1-3lhcb5, Lhcb5 was completely absent while the other five proteins were further decreased. The thylakoids of ch1-3 were less negatively-charged than the wild type, and those of ch1-3lhcb5 were even less so. Despite the expected weaker electrostatic repulsion, however, thylakoids in leaves of the mutants were not well stacked, an effect we attribute to lower van der Waals attraction, lower electrostatic attraction between opposite charges, and the absence or instability of PSII supercomplexes and peripheral light-harvesting trimers. The quantum yield of oxygen evolution in leaves decreased from 0.109 (wild type) to 0.087 (ch1-3) and 0.081 (ch1-3lhcb5) O₂ (photon absorbed)^− 1; we attribute this decrease to an excessive spillover from PSII to PSI, a limited PSII antenna, and increased light-independent thermal dissipation in PSII in the mutants. Destabilization of the donor side of PSII, indicated by slower electron donation to the redox-active tyrosine Y_Z in ch1-3, probably enhanced PSII susceptibility to photoinactivation, increased the non-functional PSII complexes in vivo, and further inactivated PSII complexes in vitro. The evolution of chlorophyll b-containing chloroplasts seems to fine-tune oxygenic photosynthesis.     

Bicarbonate effects on chlorophyll a fluorescence transients in the presence and the absence of diuron

We investigated the effect of HCO^?₃ addition to CO₂-depleted thylakoids by means of fluorescence techniques. (1) In the presence of diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea), the net reduction of the primary quinone-type electron acceptor (Q) of Photosystem (PS) II is about 2-times faster in the absence of HCO^?₃ than in its presence, whether normal, heat-treated or NH₂OH-treated samples are used. This effect of HCO^?₃ is, therefore, not on the O₂-evolving apparatus. It is, however, interpreted to be due to an influence of HCO^?₃ on the kinetics of the reduction of Q, perhaps combined with an effect on the back reaction of Q^? with P-680⁺, the oxidized form of the PS II reaction center chlorophyll a. (2) Fluorescence experiments in the absence of diuron indicate that the absence of HCO^?₃ results in a complete block at the quinone level; the area over the fluorescence induction curve in the absence of HCO^?₃ was found to be 2.2-times higher in the absence than in the presence of diuron, pointing to a complete block of BH₂ oxidation in the absence of HCO^?₃. (3) No change in the midpoint potential of Q is observed when HCO^?₃ is added to CO₂-depleted membranes. HCO^?₃ not only has a large (on/off) effect on the reoxidation of BH₂, but also a smaller effect between P-680 and Q. We propose that HCO^?₃ binding to its specific site in the thylakoid membrane results in a conformational change, allowing normal electron transport between the two photosystems.     

Molecular mechanism of uncoupling in brown adipose tissue mitochondria. The non-identity of proton and chloride conducting pathways

Specific permeability properties of the inner membrane of brown adipose tissue mitochondria were analysed with the aid of simultaneous pH measurements outside mitochondria and of mitochondria swelling. It was shown that valinomycin-induced potassium diffusion potential drives a parallel passive uptake of chlorides and extrusion of proton. Electrogenic H+ -extrusion was independent on anion transport, no competition was found between the two processes and the former process exerted a lower sensitivity to the inhibitory effect of GDP. The existence of two distinct, independent pathways for translocation of protons and halide anions across the membrane is suggested.     

Chloroplast culture VIII a new effect of kinetin in enhancing the synthesis and accumulation of protochlorophyllide invitro

By pretreating etiolated cucumber cotyledons with kinetin in the dark, it was observed that the plastids isolated from such tissues were 400% more active in the conversion of δ-aminolevulinic acid into protochlorophyllide, than plastids prepared from water-treated controls. The experimental evidence is consistent with the hypothesis that (a) the kinetin dark-pretreatment of the etiolated tissue, uncouples the joint biosynthesis of prothylakoids and protochlorophyll and results in the accumulation of excess prothylakoid membranes poorly supplied with protochlorophyllide (b) upon isolation of the plastids and incubation with δ-aminolevulinic acid, the latter is very rapidly converted into membrane-bound protochlorophyllide.     

Comparison of the effect of excessive light on chlorophyll fluorescence (77K) and photon yield of O2 evolution in leaves of higher plants

High-light treatments (1750–2000 mol photons m^–2 · s^–1) of leaves from a number of higher-plant species invariably resulted in quenching of the maximum 77K chlorophyll fluorescence at both 692 and 734 nm (F _M, 692 and F _M, 734). The response of instantaneous fluorescence at 692 nm (F _O, 692) was complex. In leaves of some species F _O, 692 increased dramatically in others it was quenched, and in others yet it showed no marked, consistent change. Regardless of the response of F _O, 692 an apparently linear relationship was obtained between the ratio of variable to maximum fluorescence (F _V/F _M, 692) and the photon yield of O₂ evolution, indicating that photoinhibition affects these two variables to approximately the same extent. Treatment of leaves in a CO₂–free gas stream containing 2% O₂ and 98% N₂ under weak light (100 mol · m^–2 · s^–1) resulted in a general and fully reversible quenching of 77K fluorescence at 692 and 734 nm. In this case both F _O, 692 and F _M, 692 were invariably quenched, indicating that the quenching was caused by an increased non-radiative energy dissipation in the pigment bed. We propose that high-light treatments can have at least two different, concurrent effects on 77K fluorescence in leaves. One results from damage to the photosystem II (PSII) reaction-center complex and leads to a rise in F _O, 692; the other results from an increased non-radiative energy dissipation and leads to quenching of both F _O, 692 and F _M, 692 This general quenching had a much longer relaxation time than reported for pH-dependent quenching in algae and chloroplasts. Sun leaves, whose F _V/F _M, 692 ratios were little affected by high-light exposure in normal air, suffered pronounced photoinhibition when the exposure was made under conditions that prevent photosynthetic gas exchange (2% O₂, 0% CO₂). However, they were still less susceptible than shade leaves, indicating that the higher capacity for energy dissipation via photosynthesis is not the only cause of their lower susceptibility. The rate constant for recovery from photoinhibition was much higher in mature sun leaves than in mature shade leaves, indicating that differences in the capacity for continuous repair may in part account for the difference in their susceptibility to photoinhibition.Abbreviations and symbols kDa kilodalton - LHC-II light-harvesting chlorophyll-protein complex - PFD photon flux density (photon fluence rate) - PSI, PSII photosystem I, II - F _O, F _M, F _V instantaneous, maximum, variable fluorescence emission - absorptance - _a photon yield of O₂ evolution (absorbed light) C.I.W.-D.P.B. Publication No. 925     

Vanadium in photosynthesis of Chlorella fusca and higher plants

The influence of vanadium compounds (vanadate, vanadyl citrate) on photosynthesis in Chlorella fusca and in algal and spinach chloroplasts has been investigated. It was found that: 1. At moderately high concentrations (at least 0.1 mM) both vanadate and vanadyl citrate enhance photosynthetic O₂ production in intact C. fusca cells. At lower V concentration (about 2 μM) only vanadate stimulates photosynthesis. The increase is dependent on culture conditions and on light intensity. 2. Up to 1 mM V, neither vanadium compound influences PS II activity, either in intact cells or in algal or spinach chloroplasts. 3. The PS I reaction in algal and spinach chloroplasts is maximally enhanced (3-fold) in presence of vanadium (20 μM). The increase is independent of light intensity. 4. Cr(VI), Mo(VI), and W(VI) (1 mM) stimulate photosynthesis in intact C. fusca cells, but do not influence the photosystems of isolated chloroplasts. Vanadium is suggested to act as a redox catalyst in the electron transport from PS II to PS I.     

The chlorophyll-protein complexes of Acetabularia. A novel chlorophyll ab complex which forms oligomers

(1) Five minor chlorophyll-protein complexes were isolated from thylakoid membranes of the green alga Acetabularia by SDS-polyacrylamide gel electrophoresis, after SDS or octylglucoside solubilization. None of them were related to CP I (Photosystem I reaction center core) or CP II (chlorophyll $\text{a}\text{b}$ light-harvesting complex). (2) Two complexes (CPa-1 and CPa-2) contained only chlorophyll (Chl) a, with absorption maxima of 673 and 671 nm, and fluorescence emission maxima of 683 nm compared to 676 nm for CP II. The complexes had apparent molecular masses of 43–47 and 38–40 kDa, and contained a single polypeptide of 41 and 37 kDa, respectively. They each account for about 3% of the total chlorophyll. (3) Three complexes had identical spectra, with Chl $\text{a}\text{b}$ ratios of 3–4 compared to 2 for thylakoid membranes, and a pronounced shoulder around 485 nm indicating enrichment in carotenoids. One of them was the complex ‘CP 29’ (Camm, E.L. and Green, B.R. (1980) Plant Physiol. 66, 428–432) and the other two were slightly different oligomeric forms of CP 29. They could be formed from CP 29 during reelectrophoresis; but about half the complex was isolated originally in an oligomeric form. Together they account for at least 7% of the total chlorophyll. Their function is unknown.     

Analysis of chlorophyll fluorescence induction curves in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea as a function of magnesium concentration and NADPH-activated light-harvesting chlorophyll ab-protein phosphorylation

The effect of Mg²⁺ concentration and phosphorylation of light-harvesting chlorophyll $\text{a}\text{b-}\text{protein}$ on various chlorophyll fluorescence induction parameters of isolated pea thylakoids has been studied. (1) Lowering the Mg²⁺ concentration from 3 to 0.4 mM decreases only the variable fluorescence (F_v) and the area above the induction curve while at the same time increasing the slow exponential component of the rise (β_max). (2) A further decrease in Mg²⁺ concentration from 0.4 to 0 mM decreases the initial (F₀) fluorescence level such that the ratio $\text{F}{}_{\mathrm{<mtext>v</mtext>}}\text{F}{}_{\mathrm{<mtext>m</mtext>}}$ increases slightly as does the area above the induction curve and β_max. (3) Thylakoid membranes, phosphorylated at 5 mM Mg²⁺, show an equal decrease in F_v and F₀, no change in the area above the induction curve and an increase in β_max. At 2 mM Mg²⁺, however, phosphorylation induced a more extensive quenching of F_v so that the $\text{F}{}_{\mathrm{<mtext>v</mtext>}}\text{F}{}_{\mathrm{<mtext>m</mtext>}}$ ratio was lowered and the area above the induction curve decreased while β_max increased. (4) When phosphorylated membranes were subsequently suspended in an Mg²⁺-free medium the effect on F₀ due to phosphorylation was found to be additive to that due to the absence of Mg²⁺. The effect of membrane phosphorylation on fluorescence is discussed in relation to the control of excitation energy distribution and shows that different mechanisms operate depending on the background Mg²⁺ levels. At high Mg²⁺ the phosphorylation seems to affect the absorption cross-section of Photosystem II while at lower Mg²⁺ levels there is an additional effect of increased spillover from Photosystem II to I.     

Energetics of photosynthesis in Zea mays. II. Studies of the flash-induced electrochromic shift and fluorescence induction in mesophyll chloroplasts

Patterns of electron transfer in isolated mesophyll chloroplasts of maize (Zea mays L.) were studied in the presence of the physiological substrates, oxaloacetate, 3-phosphoglycerate and pyruvate. Flash-induced absorbance changes due to the electrochromic pigment band-shift (P-518) were used to estimate relative electron flow rates through the cyclic and non-cyclic pathways of electron transport. Further information on the redox state of electron carriers and the activity of coupled electron flow was obtained from measurements of fluorescence induction and of actinic-light-induced fluorescence changes. The results demonstrate the importance of correct redox poising for optimal rates of photosynthesis and are discussed in relation to the operation and regulation of photosynthesis in the C₄ system.     

Photosynthetic nature of nitrate uptake and reduction in the cyanobacterium Anacystis nidulans

The photosynthetic nature of the initial stages of nitrate assimilation, namely, uptake and reduction of nitrate, has been investigated in cells of the cyanobacterium Anacystis nidulans treated with l-methionine dl-sulfoximine to prevent further assimilation of the ammonium resulting from nitrate reduction. The light-driven utilization of nitrate or nitrite by these cells results in ammonium release and is associated with concomitant oxygen evolution. Stoichiometry values of about 2 mol oxygen evolved per mol nitrate reduced to ammonium and 1.5 mol oxygen per mol nitrite have been determined in the presence of CO₂, as well as in its absence, with nitrate or nitrite as the only Hill reagent. This indicates that in A. nidulans water photolysis directly provides, without the need for carbon metabolites, the reducing power required for the in vivo reduction of nitrate and nitrite to ammonium, processes which are besides strongly inhibited when the operation of the photosynthetic noncyclic electron flow is blocked. Evidence indicating the participation of concentrative transport system(s) in the uptake of nitrate and nitrite by A. nidulans is also presented. The operation of these energy-requiring systems seems to account for the sensitivity to ATP-synthesis inhibitors exhibited by nitrate and nitrite utilization in l-methionine dl-sulfoximine-treated cells. The utilization of nitrate by A. nidulans cells, concomitant with oxygen evolution, can therefore be considered as a genuinely CO₂-independent photosynthetic process that makes direct use of photosynthetically generated assimilatory power.     

Photoinduced quenching of chlorophyll fluorescence in intact chloroplasts and algae. Resolution into two components

The light-induced decline of chlorophyll a fluorescence from a peak (P) to a low stationary level (S) in intact, physiologically active isolated chloroplasts and in intact Chlorella cells is shown to be predominantly composed of two components: (1) fluorescence quenching by partial reoxidation of the quencher Q, the primary acceptor of Photosystem II and (2) energy-dependent fluorescence quenching related to the photoinduced acidification of the intrathylakoid space. These two mechanisms of fluorescence quenching can be distinguished by the different kinetics of the relaxation of quenching observed upon addition of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU). The relaxation of quenching by addition of DCMU is biphasic. The fast phase with a half-time of about 1 s is attributed to the reversal of Q-dependent quenching. The slow phase with a half-time of about 15 s in chloroplasts and 5 s in Chlorella cells is ascribed to relaxation of energy-dependent quenching. As shown by fluorescence spectroscopy at 77 K, the energy-dependent fluorescence quenching essentially is not caused by increased transfer of excitation energy to Photosystem I. By analyzing the energy- and Q-dependent components of quenching, information on the energy state of the thylakoid membranes and on the redox state of Q under various physiological conditions is obtained.     

Measurement of degree of chlorophyll fluorescence polarization in relation to the regulation of excitation energy transfer between Photosystems I and II in pea chloroplasts

The degree of chlorophyll fluorescence polarization (p) at 740 nm was measured at room temperature for pea chloroplasts subjected to various conditions. (1) In agreement with previous published observations, p decreased when the Photosystem (PS) II traps were closed by illumination in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. (2) Under these conditions, the magnitude of p was also sensitive to the presence of salts. Under conditions when ‘spillover’ of the excitation energy from PS II to PS I was low, p was also low, being consistent with increased migration of energy between the PS II light-harvesting chlorophylls. In contrast, when spillover was at a maximum p increased. (3) The change in p in the presence of salts was dependent on the concentration and valency of the cations in such a way as to suggest the changes were mediated through electrostatic forces. The dependency of p on ionic composition of the experimental medium was closely related to the associated changes in fluorescence yield. (4) Membrane stacking, caused by lowering pH of the chloroplast suspension, did not induce a significant change in p, suggesting that this pH-induced process is different from the membrane stacking brought about by manipulating the salt levels. (5) Incubation of thylakoids with ATP induces light-dependent phosphorylation of the light-harvesting chlorophyll-protein complexes, and regulates excitation energy transfer between PS I and PS II (Bennett, J., Steinback, K.R. and Arntzen, C.J. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 5253–5257). Under conditions which bring about this phosphorylation it was found that p increased to a value indicative of spillover.     

Energetics of photosynthesis in Zea mays. I. Studies of the flash-induced electrochromic shift and fluorescence induction in bundle sheath cells

Bundle sheath strands free of mesophyll contamination were isolated from 3–4-week-old leaves of maize (Zea mays L.). Patterns of electron flow in the preparations were studied in the presence of physiological substrates. Relative electron flow rates were estimated from the flash-induced electrochromic band shift changes (P-518) and cytochrome f turnover. Induction of chlorophyll fluorescence was also measured. Little Photosystem II activity was found to be present, the principal pathway of electron flow being Photosystem I-driven cyclic electron transfer. The latter was activated through reductive poising by NADPH, generated via malate decarboxylation or (less efficiently) from dihydroxyacetone phosphate. The actions of these electron donors and of oxygen, nitrite and methyl viologen as electron acceptors in redox poising the Photosystem I-driven cycle were investigated and are discussed in relation to the regulation of photosynthesis in the bundle sheath.     

The ratio of variable to maximum chlorophyll fluorescence from photosystem II,measured in leaves at ambient temperature and at 77K,as an indicator of the photon yield of photosynthesis

The response of a number of species to high light levels was examined to determine whether chlorophyll fluorescence from photosystem (PS) II measured at ambient temperature could be used quantitatively to estimate the photon yield of O₂ evolution. In many species, the ratio of the yield of the variable (F_V) and the maximum chlorophyll fluorescence (F_M) determined from leaves at ambient temperature matched that from leaves frozen to 77K when reductions in F_V/F_M and the photon yield resulted from exposure of leaves to high light levels under favorable temperatures and water status. Under conditions which were less favorable for photosynthesis, F_V/F_M at ambient temperature often matched the photon yield more closely than F_V/F_M measured at 77K. Exposure of leaves to high light levels in combination with water stress or chilling stress resulted in much greater reductions in the photon yield than in F_V/F_M (at both ambient temperature and 77K) measured in darkness, which would be expected if the site of inhibition was beyond PSII. Following chilling stress, F_V/F_M determined during measurement of the photon yield in the light was depressed to a degree more similar to that of the depression of photon yield, presumably as a result of regulation of PSII in response to greatly reduced electron flow.Abbreviations and Symbols F_o yield of instantaneous fluorescence - F_M yield of maximum fluorescence - F_V yield of variable fluorescence - PFD photon flux density (400–700 nm) - PSI (II) photosystem I (II) This work was supported by the Deutsche Forschungsgemeinchaft. W.W.A. gratefully acknowledges the support of Fellowships from the North Atlantic Treaty Organization and the Alexander von Humboldt-Stiftung. We also thank Maria Lesch for plant maintenance.