Relationship between chloroplast structure and O2 evolution rate of leaf discs in plants from different biotopes in South Finland

Abstract The chloroplast ultrastructure, especially the thylakoid organization, the polypeptide composition of the thylakoid membranes and photosynthetic O₂ evolution rate, chlorophyll (Chl) content and Chi a/b ratio were studied in leaves of nine plants growing in contrasting biotopes in the wild in South Finland. All the measurements were made at the beginning of the period of main growth on leaves approaching full expansion, when the CO₂-saturated O₂ evolution rate (measured at 20°C and 1500 μmol photons m^?2s^?1) was at a maximum, ranging from 19.2 to 6.9 μmol O₂ cm^?2 h^?1. Among the species, the Chi a/b ratio varied between 3.75 and 2.71. In the mesophyll chloroplasts, the ratio of the total length of appressed to non-appressed thylakoid membranes varied between 1.07 and 1.79, the number of partitions per granum varied between 2.8 and 12.0 and the grana area between 21 and 42% of the chloroplast area. There was a significant relationship between the rate of O₂ evolution of the leaf discs and the thylakoid organization in the mesophyll chloroplasts. The higher the O₂ evolution rate, the lower was the ratio of the total length of appressed to non-appressed thylakoid membranes and also the lower the grana area. Although the relationship of the photosynthetic rate with the Chi content and the Chi a/b ratio of the leaves was not as clear, a significant negative correlation existed between the Chi a/b ratio and the ratio of appressed to non-appressed thylakoid membranes, indicating lateral heterogeneity in the distribution of different Chl- protein complexes.     

Dual action of ionophore A23187 on intact chloroplasts

1. Ionophore A23187 induces uncoupling of potassium ferricyanide-dependent O₂ evolution by envelope-free chloroplasts and oxaloacetate-dependent O₂ evolution by intact chloroplasts. The half maximal concentration ($\text{C}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) for stimulation of oxygen evolution in both cases is approximately 4 μM · 100 μg chlorophyll · ml^?1.2. Ionophore A23187 also induces inhibition of CO₂ and 3-phosphoglycerate-dependent O₂ evolution by intact chloroplasts in the presence of 3 mM MgCl₂. The half maximal concentrations ($\text{C}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) for inhibition of O₂ evolution are 3 μM and 5 μM respectively · 100 μg^?1 chlorophyll · ml^?1.3. A very high concentration of ionophore A23187 (10 μM · 20 μg^?1 chlorophyll · ml^?1) plus 0.1 mM EDTA lowers the fluorescence yield of intact chloroplasts suspended in a cation-free medium in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, indicating loss of divalent cation from the diffuse double layers of the thylakoid membranes.4. These results are discussed in relation to ionophore A23187-induced divalent cation/proton exchange at both the thylakoid and the envelope membranes of intact chloroplasts.     

Quantitative determination of coupling factor CF1 of chloroplasts

Repeated washes of isolated chloroplasts with dilute sodium pyrophosphate solution results in the removal of carboxydismutase and some minor proteins from the thylakoid membranes. A subsequent treatment of the membranes with hypertonic sucrose solution yields pure coupling factor CF₁ in the supernatant. Purity of the protein was demonstrated by disc electrophoresis.The amount of CF₁ protein liberated was quantitatively determined. The percentage of CF₁ removed by this treatment was calculated from the Ca²⁺-dependent ATPase activity retained at the thylakoid membranes. From these data the total CF₁ content of chloroplasts was calculated. An average value of 0.42 mg CF₁ protein/mg chlorophyll was obtained. Based on a molecular weight for CF₁ of 326000 (see Farron, F. (1970) Biochemistry 9, 3823–3828), a ratio of 1 mole CF₁ per 860 moles chlorophyll was computed.     

Raman spectroscopic investigations of sarcoplasmic reticulum membranes

Raman spectra are presented for sarcoplasmic reticulum membranes. Interpretation of the 1000–1130 cm^?1 region of the spectrum indicates that the sarcoplasmic reticulum membrane may be more fluid than erythrocyte membranes that have been examined by the same technique. The fluidity of the membrane also manifests itself in the amide I portion of the membrane spectrum with a strong 1658 cm^?1 band characteristic of CC stretching in hydrocarbon side chains exhibiting cis conformation. This band is unaltered in intensity and position in H₂O and in ²H₂O thus obscuring amide I protein conformation. Of particular interest is the appearance of strong, resonantly enhanced bands at 1160 and 1527 cm^?1 attributable to membrane-associated carotenoids.     

Temperature-sensitivity of D1 protein metabolism in isolated Zea mays chloroplasts

The effects of low temperature on the synthesis and stability of the 32 kDa D1 protein of photosystem II were investigated in chloroplasts isolated from maize (Zea mays cv. LG11) leaves. The synthesis of D1 by intact chloroplasts in vitro was strongly dependent on temperature; the Q₁₀ for the initial rate of incorporation of [³⁵S]-methionine into D1 was ca. 2.6 over the range 13–25°C. The synthesis of other thylakoid polypeptides exhibited a similar temperature dependence, whilst synthesis of stromal proteins was considerably less temperature-dependent, with the exception of two polypeptides of ca. 56 and 59.5 kDa. The stability of newly-synthesized D1 in the thylakoid membranes was dependent both on the temperature at which the plants were grown and on the temperature during the pulse-labelling period when the protein was synthesized. In chloroplasts isolated from maize leaves grown at 25°C, D1 that was synthesized and assembled at 25 °C in vitro was rapidly degraded during the chase period. At lower chase temperatures the protein was more stable. When chloroplasts from 25°C-grown leaves were pulse-labelled at 13°C, the stability of D1 was markedly enhanced at all temperatures during the chase period. This effect was even more pronounced in chloroplasts isolated from plants grown at 14°C. The implications of these results are discussed with regard to the ability of maize to recover from photoinhibitory damage at low temperatures.     

Redox dependence of the blue-light-induced phosphorylation of a 100-kDa protein on isolated plasma membranes from tips of coleoptiles

A blue-light-induced rapid phosphorylation of a 100-kDa protein localized in plasma membranes of phototropically sensitive tips of maize (Zea mays L.) coleoptiles was studied. Since, under in-vivo conditions or in a crude homogenate of tips, cytosolic ATP is the phosphate donor for the light-induced phosphorylation of this protein, a subsequent in-vitro phosphorylation by [³²P]ATP is prevented. However, in-vitro irradiation of microsomal membranes isolated from non-irradiated tips followed by a 1-min incubation with [³²P]ATP resulted in a strong phosphorylation (labelling) of the 100-kDa plasma-membrane protein. This process was saturated by a 7-s light pulse (200 μmol photons·m^?2·s^?1). In the absence of [³²P]ATP the capacity for in-vitro phosphorylation of the 100-kDa protein after a 30-s light pulse declined slowly within 60 min but could be reconstituted by a new light pulse in the presence of reducing compounds. Moreover, when plasma membranes which had been stored frozen were used, reducing compounds such as NADH, NADPH, ascorbate, glutathione or dithiotreithol enhanced the light-triggered in-vitro phosphorylation. These compounds were unable to elicit or enhance the phosphorylation in the dark. It is suggested that the transfer of (blue-light) excited electrons from the chromophore moiety of the receptor to the target (either the 100-kDa protein or the protein kinase itself) is facilitated when reducing compounds instantly eliminate the positive charge generated at the chromophore. The transferred electrons could finally alter the redox state and-or the conformation of either the 100-kDa protein, rendering it susceptible to the action of a protein kinase, or the protein kinase which would then be capable of phospho-rylating the 100-kDa protein.     

Site of methylation of 2-phytyl-1,4-naphthoquinol in phylloquinone (vitamin K1) synthesis in spinach chloroplasts

Phylloquinol (the quinol form of vitamin K₁) is synthesized from 2-phytyl-1,4-naphthoquinol and S-adenosylmethionine at the thylakoid membranes of spinach chloroplasts. The addition of soluble stroma protein (chloroplast extract) is necessary S-Adenosylhomocysteine acts as strong competitive inhibitor.     

MFP1 is a thylakoid-associated,nucleoid-binding protein with a coiled-coil structure

Plastid DNA, like bacterial and mitochondrial DNA, is organized into protein–DNA complexes called nucleoids. Plastid nucleoids are believed to be associated with the inner envelope in developing plastids and the thylakoid membranes in mature chloroplasts, but the mechanism for this re-localization is unknown. Here, we present the further characterization of the coiled-coil DNA-binding protein MFP1 as a protein associated with nucleoids and with the thylakoid membranes in mature chloroplasts. MFP1 is located in plastids in both suspension culture cells and leaves and is attached to the thylakoid membranes with its C-terminal DNA-binding domain oriented towards the stroma. It has a major DNA-binding activity in mature Arabidopsis chloroplasts and binds to all tested chloroplast DNA fragments without detectable sequence specificity. Its expression is tightly correlated with the accumulation of thylakoid membranes. Importantly, it is associated in vivo with nucleoids, suggesting a function for MFP1 at the interface between chloroplast nucleoids and the developing thylakoid membrane system.     

Biosynthetic cause of in vivo acquired thermotolerance of photosynthetic light reactions and metabolic responses of chloroplasts to heat stress

Thermotolerance of photosynthetic light reactions in vivo is correlated with a decrease in the ratio of monogalactosyl diacylglycerol to digalactosyl diacylglycerol and an increased incorporation into thylakoid membranes of saturated digalactosyl diacylglycerol species. Although electron transport remains virtually intact in thermotolerant chloroplasts, thylakoid protein phosphorylation is strongly inhibited. The opposite is shown for thermosensitive chloroplasts in vivo. Heat stress causes reversible and irreversible inactivation of chloroplast protein synthesis in heat-adapted and nonadapted plants, respectively, but doe not greatly affect formation of rapidly turned-over 32 kilodalton proteins of photosystem II. The formation on cytoplasmic ribosomes and import by chloroplasts of thylakoid and stroma proteins remain preserved, although decreased in rate, at supraoptimal temperatures. Thermotolerant chloroplasts accumulate heat shock proteins in the stroma among which 22 kilodalton polypeptides predominate. We suggest that interactions of heat shock proteins with the outer chloroplast envelope membrane might enhance formation of digalactosyl diacylglycerol species. Furthermore, a heat-induced recompartmentalization of the chloroplast matrix that ensures effective transport of ATP from thylakoid membranes towards those sites inside the chloroplast and the cytoplasm where photosynthetically indispensable components and heat shock proteins are being formed is proposed as a metabolic strategy of plant cells to survive and recover from heat stress.     

The effect of chloroplast coupling factor removal on thylakoid membrane ion permeability

Removal of coupling factor protein (CF₁) from spinach thylakoid membranes results in an enhancement of proton permeability but has no effect on chloride or potassium permeability. Anion permeability was measured by the rate of thylakoid packed volume changes. Potassium permeability was monitored by turbidity changes, packed thylakoid volume changes and ion flux studies using ⁸⁶Rb⁺ as a tracer. ⁴⁵Ca²⁺ was used to measure divalent cation fluxes. CF₁-depleted chloroplasts had an unaltered rate of Ca²⁺ uptake, but the rate of Ca²⁺ efflux appeared to be increased. Calcium efflux rates could also be increased by the addition of a proton specific uncoupler, FCCP.     

Light-induced slow changes in chlorophyll a fluorescence in isolated chloroplasts: Effects of magnesium and phenazine methosulfate

We have investigated the possible relationships between the cation-induced and phenazine methosulfate (PMS)-induced fluorescence changes and their relation to light induced conformational changes of the thylakoid membrane.1. In isolated chloroplasts, PMS markedly lowers the quantum yield of chlorophyll a fluorescence (φ_f) when added either in the presence or the absence of dichloro-phenyldimethylurea (DCMU). In contrast, Mg²⁺ causes an increase in φ_f. However, these effects are absent in isolated chloroplasts fixed with glutaraldehyde that retain (to a large extent) the ability to pump protons, suggesting that structural alteration of the membrane—not the pH changes—is required for the observed changes in φ_f. The PMS triggered decrease in φ_f is not accompanied by any changes in the emission (spectral) characteristics of the two pigment systems, whereas room temperature emission spectra with Mg²⁺ and Ca²⁺ show that there is a relative increase of System II to System I fluorescence.2. Washing isolated chloroplasts with 0.75 mM EDTA eliminates (to a large extent) the PMS-induced quenching and Mg²⁺-induced increase of φ_f, and these effects are not recovered by the further addition of dicyclohexyl carbodiimide. It is known that washing with EDTA removes the coupling factor, and thus, it seems that the coupling factor is (indirectly) involved in conformational change of thylakoid membranes leading to fluorescence yield changes.3. In purified pigment System II particles, neither PMS nor Mg²⁺ causes any change in φ_f. Our data, taken together with those of the others, suggest that a structural modification of the thylakoid membranes (not macroscopic volume changes of the chloroplasts) containing both Photosystems I and II is necessary for the PMS-induced quenching and Mg²⁺-induced increase of φ_f. These two effects can be explained with the assumption that the PMS effect is due to an increase in the rate of internal conversion (k_h), whereas the Mg²⁺ effect is due to a decrease in the rate of energy transfer (k_t), between the two photosystems.4. From the relative ratio of φ_f with DCMU and DCMU plus Mg²⁺, we have calculated k_t (the rate constant of energy transfer between Photosystems II and I to be 4.2·10⁸ s^?1, and φ_t (quantum yield of this transfer) to be 0.12.     

A Protein Phosphorylation Threshold for Functional Stacking of Plant Photosynthetic Membranes

Phosphorylation of photosystem II (PSII) proteins affects macroscopic structure of thylakoid photosynthetic membranes in chloroplasts of the model plant Arabidopsis. In this study, light-scattering spectroscopy revealed that stacking of thylakoids isolated from wild type Arabidopsis and the mutant lacking STN7 protein kinase was highly influenced by cation (Mg⁺⁺) concentrations. The stacking of thylakoids from the stn8 and stn7stn8 mutants, deficient in STN8 kinase and consequently in light-dependent phosphorylation of PSII, was increased even in the absence of Mg⁺⁺. Additional PSII protein phosphorylation in wild type plants exposed to high light enhanced Mg⁺⁺-dependence of thylakoid stacking. Protein phosphorylation in the plant leaves was analyzed during day, night and prolonged darkness using three independent techniques: immunoblotting with anti-phosphothreonine antibodies; Diamond ProQ phosphoprotein staining; and quantitative mass spectrometry of peptides released from the thylakoid membranes by trypsin. All assays revealed dark/night-induced increase in phosphorylation of the 43 kDa chlorophyll-binding protein CP43, which compensated for decrease in phosphorylation of the other PSII proteins in wild type and stn7, but not in the stn8 and stn7stn8 mutants. Quantitative mass spectrometry determined that every PSII in wild type and stn7 contained on average 2.5±0.1 or 1.4±0.1 phosphoryl groups during day or night, correspondingly, while less than every second PSII had a phosphoryl group in stn8 and stn7stn8. It is postulated that functional cation-dependent stacking of plant thylakoid membranes requires at least one phosphoryl group per PSII, and increased phosphorylation of PSII in plants exposed to high light enhances stacking dynamics of the photosynthetic membranes.     

Events surrounding the early development of Euglena chloroplasts. 15. Origin of plastid thylakoid polypeptides in wild-type and mutant cells

Techniques are described for the isolation of plastid thylakoid membranes from light-grown and dark-grown cells of Euglena gracilis var. bacillaris, and from mutants affecting plastid development. These membranes, which have minimal contamination with other cell fractions, are localized in sucrose gradients by using the thylakoid membrane sulfolipid as a specific marker. The plastid thylakoid membrane polypeptides isolated from these membranes were separated on SDS polyacrylamide gels and yielded patterns containing 30–40 polypeptides. Light-grown strain Z gave patterns identical with bacillaris. Since the plastid thylakoid polypeptide patterns obtained from dark-grown wild-type cells and from a bleached mutant W₃BUL in which plastid DNA is undetectable are identical, it appears that the proplastid thylakoid polypeptides of wild-type cannot be coded in plastid DNA and are probably coded in nuclear DNA. The plastid thylakoid polypeptide patterns obtained from various dark-grown mutants are identical to those obtained from dark-grown wild-type cells. Light-grown mutants, making large but abnormal chloroplasts, show a correlation between the amount of chlorophyll formed and the amount of a plastid thylakoid polypeptide thought to be associated with one of the pigment-protein light-harvesting complexes. Treatment with SAN 9789 (4-chloro-5-(methyl-amino)-2-(α,α,α,-trifluoro-m-tolyl)-3-(2H(pyridazinone) known to block carotenoid synthesis at the level of phytoene, causes a progressive loss of all plastid thylakoid polypeptides during growth in darkness and results in the establishment of a new, lower steady-state level of sulfolipid. At least ten of the plastid thylakoid polypeptides become labeled when isolated chloroplasts are supplied with radioactive amino acids; of these six are undectable in W₃BUL and are, therefore, candidates for coding by plastid DNA.     

Involvement of Singlet Oxygen in 5-Aminolevulinic Acid-Induced Photodynamic Damage of Cucumber (Cucumis sativus L.) Chloroplasts

Cucumber (Cucumis sativus L., cv Poinsette) plants were sprayed with 20 millimolar 5-aminolevulinic acid and then incubated in the dark for 14 hours. The intact chloroplasts were isolated from the above plants in the dark and were exposed to weak light (250 micromoles per square meter per second). Within 30 minutes, photosystem II activity was reduced by 50%. The singlet oxygen (¹O₂) scavengers, histidine and sodium azide (NaN₃) significantly protected against the damage caused to photosystem II. The hydroxyl radical scavenger formate failed to protect the thylakoid membranes. The production of ¹O₂ monitored as N,N-dimethyl p-nitrosoaniline bleaching increased as a function of light exposure time of treated chloroplasts and was abolished by the ¹O₂ quencher, NaN₃. Membrane lipid peroxidation monitored as malondialdehyde production was also significantly reduced when chloroplasts were illuminated in the presence of NaN₃ and histidine. Protochlorophyllide was the most abundant pigment accumulated in intact chloroplasts isolated from 5-aminolevulinic acid-treated plants and was probably acting as type II photosensitizer.     

Protein rotational mobility in thylakoid membranes of different polypeptide composition in the wild type and mutant strains of Chlamydomonas reinhardtii

The rotational mobility of thylakoid membrane proteins labeled with a paramagnetic analog of N-ethylmaleimide was investigated by saturation transfer electron spin resonance. In the wild type strain of Chlamydomonas reinhardtii two polypeptides are prominently labeled. They correspond to the 19-kDa subunit of the reaction center I protein and to the 30-kDa subunit of the light harvesting complex. Several polypeptides, most of which are either trypsin or alkaline sensitive, are also labeled. In order to circumvent the lack of specificity during the labeling, we have compared the rotational mobilities of labeled proteins in thylakoid membranes from several mutant strains which lack in photosystem I., ATPase or light harvesting complexes. Comparison of the saturation transfer electron spin resonance spectra obtained with these mutant membranes as well as with trypsin- and alkaline-treated membranes allowed us to characterize the rotational contribution of some of the labeled proteins to the overall protein dynamics observed in the wild type strain. The reaction center I protein undergoes slow rotation as compared to the other labeled proteins. The rotational characteristics of the labeled light harvesting complexes are those of a peptide fragment in the complex which is in rapid motion in unstacked membranes. Stacking of the thylakoid membranes upon Mg²⁺ addition is accompanied by a marked change in shape of the saturation transfer spectra, and corresponds to the appearance of highly immobilized nitroxides. We interpret these changes as arising mainly from the hindrance upon membrane appression, of the labeled fragment of the light harvesting complexes which protrude at the thylakoid outer surface.     

Surface charges on chloroplast membranes as studied by particle electrophoresis

1. Particle microelectrophoresis mobility studies have been conducted with chloroplast thylakoid membranes and with isolated intact chloroplasts.2. The pH dependence of the electrophoretic mobility indicated that at pH values above 4.3 both membrane systems carry a net negative charge.3. Chemical treatment of thylakoids has shown that neither the sugar residues of the galactolipids in the membrane nor the basic groups of the membrane proteins having pK values between 6 and 10 are exposed at the surface.4. However, treatment with 1-ethyl-3(3-dimethylaminopropyl)carbodiimide, together with glycine methyl ester, neutralized the negative charges on the thylakoid membrane surface indicating the involvement of carboxyl groups which, because of their pH sensitivity, are likely to be the carboxyl groups of aspartic and glutamic acid residues.5. The nature of the protein giving rise to the negative surface charges on the thylakoids is not known but is shown not to involve the coupling factor or the light harvesting $\text{chlorophyl}\text{a}\text{chlorophyll}\text{b}\text{pigment · protein complex}$.6. No significant effect of light was observed on the electrophoretic mobility of either thylakoids or intact chloroplasts.7. The striking difference in the ability of divalent and monovalent cations to screen the surface charges was demonstrated and explained in terms of the Gouy-Chapman theory.8. Calculations of the ζ-potentials for thylakoid membranes gave values for the charge density at the plane of shear to be in the region of one electronic charge per 1500–2000 Ų.9. The significance of the results is discussed in terms of cation distribution in chloroplasts and the effect of cations on photosynthetic phenomena.     

Suppression of Oxygen Evolving System in Spinach Chloroplast Membranes due to Release of Manganese on Exposure to Osmotic Stress in vitro in Presence of Magnesium

When spinach chloroplast membranes were exposed to osmotic stress in vitro, by incubation in 1.0 M sorbitol + 10 mM MgCl₂ their oxygen evolving system was suppressed. The possible reasons for such inactivation of PS II mediated oxygen evolution were examined. There were conformational changes in the chloroplast membranes, as indicated by their absorption spectra. The pattern of sensitivity to DCMU was not altered. The sensitivity of PS II to water stress remained, even after a pre-wash treatment with NaCI (which removed 18 and 24 kD proteins) but not when the thylakoids were pretreated with NH₂0H or CaCl₂ (removed manganese and 33 kD). The manganese content of thylakoid membranes was markedly reduced under osmotic stress in presence of magnesium. We suggest that exposure of chloroplasts to 1.0 M sorbitol in presence of Mg₂₊ released manganese from thylakoid membranes, thereby leading to a suppression in oxygen evolution.     

Light-, pH- and uncoupler-dependent association of chloride with chloroplast thylakoids

Studies of the association of Cl^? with Photosystem (PS) II in CF₁-containing thylakoid membranes revealed that photosynthetically active Cl^? is retained in a Cl^?-free medium unless it is sufficiently alkaline, uncoupling conditions are established and light is excluded. After treatment under such conditions, electron transport from water became dependent on added Cl^? under all conditions. Quantitative measurements of ³⁶Cl^? retention in the light revealed that there were about five Cl^? anions present in Cl^?-sufficient chloroplasts per PS II reaction center, and one-fourth of that in Cl^?-deficient samples. Uncouplers representing three different types of uncoupling mechanism were found to be effective mediators of Cl^? release from thylakoids. Since the ability to collapse a proton gradient probably is the only property shared by all the tested uncouplers, a proton gradient may be involved in the retention of Cl^?. As uncoupler-mediated Cl^? release did not depend on preillumination of our samples, a long-lived proton gradient must exist in dark-adapted chloroplasts which may not span the whole thickness of the thylakoid membrane. It is postulated that the Cl^? active in PS II reactions resides in a special membrane domain from which protons slowly equilibrate with those in the bulk solutions. Cl^? is thought to be released to the bulk phases only when the pH of the membrane domain is raised above a certain threshold by the action of uncouplers. This domain may be identical to the intramembranous compartment which has been postulated to be associated with PS II (Prochaska, L.J. and Dilley, R.A., (1978) Front. Biol. Res. Energ. 1, 265–274).