Connection between the rate of cooling and fluorescence properties at 77 K of isolated chloroplasts

Cooling of chloroplasts to ?196°C can under certain circumstances lead to an erroneous analysis of energy distribution. After minimizing influences of sample geometry and effects of plastid concentration it is shown that externally induced membrane change leads to an increase in the ratio $\text{F}{}_{740}\text{F}{}_{687}$ of the fluorescence emission spectrum. Similar alterations can be observed by variation of the rate of cooling the plastids to 77 K, especially if whole chloroplasts are used. The differences in emission ratios are indicative also of changes in initial energy distribution between the photosystems, given here by the value α_N. This is inferred from experiments with either osmotically induced thylakoid disturbances or those effected through a slow cooling process. The circumstances and the significance of these observations are discussed.     

Regulation of excitation energy in a wheat mutant deficient in light-harvesting pigment protein complex

Chloroplasts of the CD3 wheat mutant were deficient primarily in chlorophyll of light harvesting pigment proteins (LHPP) 1 and 2 and CP1a. The reduced level of protein associated with chlorophyll of LHPP1 and LHPP2 and the reduced level of low molecular weight polypeptides between 23 and 29 kilodaltons confirmed that the CD3 mutant was deficient in the LHPP complex. The high fluorescence emission ratio at 740 (F740) to 686 nanometers (F686) observed from chloroplasts of normal wheat following light induced phosphorylation of the LHPP complex was not noted from mutant chloroplasts. The long wavelength peak fluorescence emission (F740) was shifted to a shorter wavelength peak (F725) and was reduced in intensity compared to that of normal wheat thylakoids. The ratio of variable fluorescence to maximum fluorescence, a measure of PSII photochemical efficiency, was the same for the normal wheat and mutant leaves. The ratios of uncoupled photosystem I/photosystem II electron transport rates for mutant and normal wheat chloroplasts were similar at saturating light suggesting that absorbed excitation energy was distributed to the two photosystem reaction centers of the mutant in a similar manner as in the normal wheat. Proteins of the LHPP complex were differentially phosphorylated by action of a membrane protein kinase when both normal wheat and CD3 mutant thylakoids were irradiated without an electron transport chain acceptor. Even though the F740/F686 ratio was low in mutant thylakoids, the phosphorylation of the 27-kilodalton LHPP polypeptide was consistent with the mutant being in a state II condition. The data gave rise to the suggestion that the F740/F686 ratio might not indicate excitation energy distribution to the two photosystems in the mutant.     

The effect of detergent Triton X=100 on the light induced changes in the fluorescence yield of chloroplasts]

It is shown that light induced changes of the fluorescence yield (delta F) of isolated chloroplasts are affected by Triton X-100. delta F value descreases with the increase of the detergent concentration from 0 to 0.03%, increases in the range of 0.03--0.05% and is irreversibly blocked at concentrations more than 0.08--0.1%. The same dependence of delta F on the detergent concentration is obtained for "digitonin" fragments of chloroplasts enriched in the photosystem 2, but not for fragments enriched in the photosystem 1. Light induced delta F of chloroplasts treated by detergent were activated by hydroxylamine and saturated at lower light intensities than delta F of untreated chloroplasts. Addition of 0.01% Triton resulted in an activation of light induced delta F of chloroplasts with damaged donor part of photosystem 2. It is suggested that the complex dependence of delta F of chloroplasts on the Triton concentration is due to superposition of several effects: the uncoupling of photophosphorylation, inactivation of the electron transport chain in the donor and acceptor parts of photosystem 2, and changes of acting concentration of Triton X-100 within the range of critical micelle concentration.     

Changes in the chlorophyll fluorescence characteristics of chloroplasts from intact pumpkin cotyledons,caused by organ excision and kinetin treatment

The chlorophyll (Chl) fluorescence emission as well as excitation and polarization characteristics of chloroplasts from intact cotyledons were determined in pumpkin seedlings after removal of one cotyledon (co-cotyledon) or apical bud or primary root, or after kinetin treatment of derooted seedlings. Qualitatively, the fluorescence emission and excitation spectra of chloroplasts were similar. The fluorescence emission spectra showed a maximum at 685 (F685) and a hump at 735 nm (F735), whereas the excitation spectra showed peaks at 439, 471, 485, and 676 nm. The fluorescence intensities at F685 and F735 differed in various groups of seedlings, as indicated by changes in their ratios. Similarly, the ratios of 471/439, 485/439, and 676/439 nm were also different. Variability in the Chl fluorescence intensity values and the fluorescence polarization of chloroplasts prepared from various seedling types may suggest a different degree of binding between the pigment complexes and light-harvesting Chl-protein (LHCP), resulting in different rates of photoexcitation energy loss in the form of fluorescence emission. Kinetin treatment improved the coupling of pigment complexes with reaction centre, as indicated by low polarization values in derooted and kinetin-treated seedlings, which suggests the development of a suntype chloroplast.     

Changes in the chlorophyll fluorescence characteristics of chloroplasts from intact pumpkin cotyledons, caused by organ excision and kinetin treatment

The chlorophyll (Chl) fluorescence emission as well as excitation and polarization characteristics of chloroplasts from intact cotyledons were determined in pumpkin seedlings after removal of one cotyledon (co-cotyledon) or apical bud or primary root, or after kinetin treatment of derooted seedlings. Qualitatively, the fluorescence emission and excitation spectra of chloroplasts were similar. The fluorescence emission spectra showed a maximum at 685 (F685) and a hump at 735 nm (F735), whereas the excitation spectra showed peaks at 439, 471, 485, and 676 nm. The fluorescence intensities at F685 and F735 differed in various groups of seedlings, as indicated by changes in their ratios. Similarly, the ratios of 471/439, 485/439, and 676/439 nm were also different. Variability in the Chl fluorescence intensity values and the fluorescence polarization of chloroplasts prepared from various seedling types may suggest a different degree of binding between the pigment complexes and light-harvesting Chl-protein (LHCP), resulting in different rates of photoexcitation energy loss in the form of fluorescence emission. Kinetin treatment improved the coupling of pigment complexes with reaction centre, as indicated by low polarization values in derooted and kinetin-treated seedlings, which suggests the development of a suntype chloroplast. This revised version was published online in June 2006 with corrections to the Cover Date.     

Competition between the 735 nm fluorescence and the photochemistry of Photosystem I in chloroplasts at low temperature

Fluorescence emission spectra of chloroplasts, initially frozen to--196 degrees C, were measured at various temperatures as the sample was allowed to warm. The 735 nm emission band attributed to fluorescence from Photosystem I was approx. 10-fold greater at--196 degrees C than at--78 degrees C. The initial rate of photooxidation of P-700 was also measured at--196 degrees C and--78 degrees C and was found to be approximately twice as large at the higher temperature. It is proposed that the 735 nm emission band is fluorescence from a long wavelength form of chlorophyll, C-705, which acts as a trap for excitation energy in the antenna chlorophyl system of Photosystem I. Furthermore, it is proposed that C-705 only forms on cooling to low temperatures and that the temperature dependence of the 735 nm emission is the temperature dependence for the formation of C-705. C-705 and P-700 compete to trap the excitation energy in Photosystem I. It is estimated from the data that at--78 degrees C P-700 traps approx. 20 times more energy than C-705 while, at--196 degrees C, the two traps are approximately equally effective. By analogy, the 695 nm fluorescence which also appears on cooling to--196 degrees C is attributed to traps in Photosystem II which form only on cooling to temperatures near--196 degrees C.     

Effects of sodium and magnesium cations on the “dark-” and light-induced chlorophyll a fluorescence yields in sucrose-washed spinach chloroplasts

The effects of Na⁺ and Mg²⁺ on the “dark” level (O level) and light-induced (P level) fluorescence in sucrose-washed spinach chloroplasts were studied. Low concentrations of NaCl (2–10 mM) cause a significant decrease in both the O and P levels in the chlorophyll fluorescence transient. The effect on the O level may reflect changes in the bulk chlorophyll a. At 77 °K NaCl increases the $\text{F735}\text{F685}$ emission peak ratio in dark-adapted and preilluminated chloroplasts, but has no significant effect on this ratio in sucrose-washed Photosystem II particles. This evidence is consistent with a sodium-induced excitation-energy distribution in favor of Photosystem I.In the presence of MgCl₂, with or without NaCl, there is a slight decrease in the O and P level fluorescence as compared with the salt-free control, but an increase as compared with the NaCl-treated sample. Magnesium appears to override the sodium-induced changes. At low temperatures in chloroplasts and Photosystem II particles, MgCl₂ has different effects on the $\text{F735}\text{F685}$ ratio apparently depending on the state of the membrane. Magnesium, however, always induces an increase in the $\text{F695}\text{F685}$ ratio. These results suggest that magnesium may influence Photosystem II reaction centers as well as energy distribution between the two photosystems.     

Quercetin interaction with the chloroplast ATPase complex

1. Quercetin, a flavonoid which acts as an energy transfer inhibitor in photophosphorylation is shown to inhibit the P-ATP exchange activity of membrane-bound CF1 and the ATPase activity of isolated CF1. Quercetin, affects also the proton uptake in chloroplasts in a manner similar to that of dicyclohexylcarbodiimide. 2. The light-dependent proton uptake in EDTA-treated chloroplasts is stimulated by quercetin. In untreated chloroplasts quercetin has a dual effect: it enhances at pH above 7.5 while at lower pH values it decreases the extent of H+ uptake. Similar effects were obtained with dicyclohexylcarbodiimide. 3. Like quercetin, dicyclohexylcarbodiimide was also found to inhibit the ATPase activity of isolated CF1. 4. Quercetin inhibits uncoupled electron transport induced by either EDTA-treatment of chloroplasts or by addition of uncouplers. Quercetin restores H+ uptake in both types of uncoupled chloroplasts. 5. The mode of action of quercetin and dicyclohexylcarbodiimide in photophosphorylation is discussed, and interaction with both CF1 and F0 is suggested.     

A Comparative Study on Function and Membrane Proteins of Chloroplasts of Three Species of Cephalotaxus in Late Winter

Three species of evergreen Cephalotaxus (C. fortunei Hook. f., C. sinensis Li and C. harringtonia cv. Fastigiata) were used as materials to study some functional properties of chloroplasts. It is found that the oxygenevolving capacity of the chloroplasts from these plants is inhibited but partial reaction of PS-II and effect of Mg²⁺ on energy distribution between two photosystems are detectable during the winter. Seasonal effects on the functional properties of chloroplasts from evergreen Cephalotaxus are similar to that of conifer chloroplasts. The ratio between F₆₈₅, F₆₉₅ and F₇₃₅ of fluorescence emission spectra at 77°K of chloroplasts is different among these three species. It is found by using SDS-PAGE that the number of polypeptide resolved from thylakoid membrane of C. harringtonia cv. Fastigiata substantially differs from that of C. fortunei Hook.f. and C. sinensis Li. The result shows that the fluorescence emission spectrum feature and polypeptide composition of thylakoid membrane may be used as a tool for systematics of the genus Cephalotaxus.     

Fluorescence and energy transfer in phycobiliprotein-containing algae at low temperature

Fluorescence emission spectra of Anacystis nidulans, Porphyridium cruentum and Cyanidium caldarium, three phycobiliprotein-containing algae, were measured at temperatures between 4 and 120 K in the absence and in the presence of quinones as quenchers of chlorophyll fluorescence. In all species three major emission bands were observed in the chlorophyll a region, near 685 nm (F-685), 695 nm (F-695) and between 710 and 730 nm. Additional bands were observed at shorter wavelengths; these were preferentially excited by light absorbed by the phycobiliproteins and are presumably due to phycocyanins and allophycocyanins.

The amplitudes of F-685, F-695 and the long-wave emission showed a distinct increase upon cooling. For F-685 and F-695 the temperature dependence was similar to that earlier observed with spinach chloroplasts, for the long-wave emission it appeared to depend on the location of the emission bands, which was different for different species. All three bands were strongly quenched by quinones. These and other data suggest that the origin of these bands is the same as in higher plants, and that the fluorescence increase upon cooling can be explained by a lowering of the efficiency of energy transfer between chlorophyll molecules. It is concluded that at most a small percentage of the emission at 685 nm can be ascribed to allophycocyanin B, and that the efficiency of energy transfer between allophycocyanin B and chlorophyll a probably exceeds 99% both at 77 and 4 K. Experiments with isolated phycobilisomes suggest that energy transfer from allophycocyanin to allophycocyanin B occurs with an efficiency of about 90% at low temperature.

The effect of quenchers can be understood by the assumption that the quenching is caused by the formation of non-fluorescent traps in the bulk chlorophyll. Of three quinones tested, the strongest quenching was observed with dibromothymoquinone, which quenched F-685, F-695 and the long-wave emission approximately equally. Menadione and 1,4-naphthoquinone, however, preferentially quenched the long-wave bands, indicating a stronger interaction with Photosystem I than with Photosystem II chlorophylls.     

Investigations of the Blue-green Fluorescence Emission of Plant Leaves

The UV light (337 nm) induced blue-green fluorescence emission of green leaves is characterized at room temperature (298 K) by a maximum near 450 nm (blue region) and a shoulder near 525 nm (green region) and was here also studied at 77 K. At liquid nitrogen temperature (77 K) the blue (F450) and green fluorescence (F525) are much enhanced as is the red chlorophyll fluorescence near 735 nm. During development of green tobacco leaves the blue fluorescence F450 (77 K) is shifted towards longer wavelengths from about 410 nm to 450 nm. The isolated leaf epidermis of tobacco showed only slight fluorescence emission with a maximum near 410 nm. The green fluorescence F525 was found to mainly originate from the mesophyll of the leaf, its intensity increased when the epidermis was removed. The red chlorophyll fluorescence emission was also enhanced when the epidermis was stripped off; this considerably changed the blue/red fluorescence ratios F450/F690 and F450/F735. The epidermis, with its cell wall and UV-light-absorbing substances in its vacuole, plays the role of a barrier for the exciting UV-light. In contrast to intact and homogenized leaves, isolated intact chloroplasts and thylakoid membranes did not exhibit a blue-green fluorescence emission.     

Effects of Cucumber Mosaic Virus Infection on Photosynthetic Activities of Tobacco Leaves and Chloroplasts

Changes in photosynthetic activities were studied with tobacco (Nicotiana tabacum L.) leaves and chloroplasts infected by cucumber mosaic virus (CMV) at the top, middle and bottom located leaves. Net photosynthetic rate was reduced at all three positioned leaves, with the maximum reduction occurring at the top leaves (31.9% of control). The infected chloroplasts showed a reduction in electron transport rates of the whole chain electron transport, photosystem Ⅱ (PSⅡ) and photosystem Ⅰ (PSⅠ). Since the decline in the whole chain electron transport (15.6% of control, H2O→MV) closely paralleled the decline in PSⅡ activity (20.9% of control, H2O→PBQ), the inhibition of the latter was probably responsible for the overall decrease. Chlorophyll a fluorescence measurements showed a variable reduced fluorescence yield (Fv/Fo) which indicated that PSⅡ was impaired and the CO2 assimilation was disturbed by CMV infection. Fluorescence emission spectra at 77 K indicated that energy distribution between PSⅡ and PSⅠ was affected. F686/F734 of infected leaves and chloroplasts increased and the greatest increase (331.1% of control ) was found in the top leaves. These data may conclude that the infection inhibited mainly the PSⅡ activity.     

Possible influence of the rate of specimen cooling on the determination of energy distribution in photosynthesis by fluorescence emission at 77 K

The rate of cooling to 77 K appears to be a determining factor in obtaining valid low temperature emission spectra of photosynthetic organisms. Evidence is shown that the usual method of cooling the algae or chloroplasts in suspension leads to artefacts in the spectra and considerable discrepancies in quantitative determinations.     

Does the rate of cooling affect fluorescence properties of chloroplasts at -196 degrees C?

The question addressed in the title was examined by measuring fluorescence emission spectra and light-induced fluorescence-yield changes of chloroplasts which had been frozen to -196 degrees C rapidly, as very thin samples adsorbed into substrates whick were plunged directly into liquid nitrogen, or slowly by the cooling action of liquid nitrogen through the wall of the cuvette. Contrary to previous reports, we found that the rate of cooling had no influence on the shape of the emission spectrum, the extent of the variable fluorescence or the fraction of the absorbed quanta which are delivered initially to Photosystem I.     

Studies on the ferrochelatase activity of mitochondria and submitochondrial particles with special reference to the regulatory function of the mitochondrial inner membrane

The question addressed in the title was examined by measuring fluorescence emission spectra and light-induced fluorescence-yield changes of chloroplasts which had been frozen to ?196 °C rapidly, as very thin samples adsorbed into substrates which were plunged directly into liquid nitrogen, or slowly by the cooling action of liquid nitrogen through the wall of the cuvette. Contrary to previous reports, we found that the rate of cooling had no influence on the shape of the emission spectrum, the extent of the variable fluorescence or the fraction of the absorbed quanta which are delivered initially to Photosystem I.     

The kinetics of in vivo state transitions in mesophyll and guard cell chloroplasts monitored by 77 k fluorescence emission spectra

Fluorescence emission spectral peaks at 685, 695 and 730 nanometers (F685, F695, and F730) were recorded 77 K from diluted leaf tissue and epidermal powders prepared from Saxifraga cernua. The time course for state 1 to state 2 transitions was monitored as changes in the ratios of the three emission peaks. During illumination with light 2 (580 nm) the F730/F695 and F730/F685 ratios increased within minutes to establish a condition characteristic of state 2. A major difference between the two chloroplast types was the more rapid establishment of state 2 by mesophyll chloroplasts. An increase in light 2 intensity caused an increase in the magnitude of the F730/F695 ratio for both chloroplast types and, for guard cell chloroplasts, a decrease in the time required to establish the new ratio. The role of reversible phosphorylation of the light-harvesting chlorophyll a/b protein complex in regulating state transitions for both mesophyll and guard cell chloroplasts was assessed using DCMU and sodium fluoride, a specific phosphatase inhibitor. DCMU-treated mesophyll and epidermal tissues failed to show a state 1-state 2 transition. NaF-treated tissues attained state 2 but lacked the ability to revert back to state 1.     

Thylakoid protein phosphorylation during State 1—State 2 transitions in osmotically shocked pea chloroplasts

In osmotically shocked pea chloroplasts illuminated with modulated blue-green light (light 2), phosphorylation of the light-harvesting chlorophyll $\text{a}\text{b-}\text{protein}$ complex (LHCP) accompanies the slow decrease in modulated fluorescence that indicates adaptation to light absorbed predominantly by Photosystem II (State 2). On subsequent additional illumination with continuous far-red light (absorbed predominantly by Photosystem I; light 1) both effects are reversed: modulated chlorophyll fluorescence emission increases (indicating adaptation towards State 1) and LHCP is dephosphorylated. Net phosphorylation and dephosphorylation of LHCP induced by light 2 and excess light 1, respectively, occur on the same time scale as the ATP-dependent chlorophyll fluorescence changes indicative of State 2 and State 1 transitions. The phosphatase inhibitor NaF (10 mM), stimulates the effect of blue-green light on fluorescence and prevents the effect of far-red light. These results provide a demonstration that light of different wavelengths can control excitation energy distribution between the two photosystems via the plastoquinol-activated LHCP phosphorylation mechanism suggested previously (Allen, J.F., Bennett, J., Steinback, K.E. and Arntzen, C.J. (1981) Nature 291, 25–29; and Horton, P. and Black, M.T. (1980) FEBS Lett. 119, 141–144).     

Factors Affecting Ethanol Induced Luminescence in Dark-Treated Chloroplasts

Dark-treated chloroplasts emit light when treated with a high ethanol concentration. The ethanol treatment causes chlorophyll solvation. The light is red and emanates from a singlet excited molecule, probably a chlorophyll peroxide. It is quenched by acetone, sodium dodecyl sulfate treatment of the chloroplasts before the addition of ethanol, boiling, reducing substances and low pH. It is enhanced by ferricyanide and high pH. This is interpreted as a requirement for an organized structure and for an energy transfer system for light emission to occur.     

Light-induced changes of NADPH fluorescence in isolated chloroplasts: a spectral and fluorescence lifetime study

Isolated chloroplasts show a light-induced reversible increase in blue-green fluorescence (BGF), which is only dependent on NADPH changes. In the present communication, we report a time-resolved and spectral analysis of this BGF in reconstituted chloroplasts and intact isolated chloroplasts, in the dark and under actinic illumination. From these measurements we deduced the contribution of the different forms of NADPH (free and bound to proteins) to the light-induced variation of BGF and conclude that this variation is due only to the redox change of the NADP pool. A simple model estimating the distribution of NADPH between the free and bound form was designed, that explains the differences measured for the BGF of reconstituted chloroplasts and intact chloroplasts. From the decay-associated spectra of the chloroplast BGF, we also deduced the participation of flavins to the green peak of chloroplast fluorescence emission spectrum, and the existence of excitation energy transfer from proteins to bound NADPH in chloroplasts. In addition, we re-examined the use of chloroplast BGF as a quantitative measure of NADPH concentration, and confirmed that chloroplast BGF can be used for non-destructive, continuous and probably quantitative monitoring of light-induced changes in NADP redox state.