Excitation spectra of chlorophyll fluorescence in spinach and barley chloroplasts at 4 K

Excitation spectra of chlorophyll a fluorescence in chloroplasts from spinach and barley were measured at 4.2 K. The spectra showed about the same resolution as the corresponding absorption spectra. Excitation spectra for long-wave chlorophyll a emission (738 or 733 nm) indicate that the main absorption maximum of the photosystem (PS) I complex is at 680 nm, with minor bands at longer wavelengths. From the corresponding excitation spectra it was concluded that the emission bands at 686 and 695 nm both originate from the PS II complex. The main absorption bands of this complex were at 676 and 684 nm. The PS I and PS II excitation spectra both showed a contribution by the light-harvesting chlorophyll $\text{a}\text{b}$ protein(s), but direct energy transfer from PS II to PS I was not observed at 4 K. Omission of Mg²⁺ from the suspension favored energy transfer from the light-harvesting protein to PS I. Excitation spectra of a chlorophyll b-less mutant of barley showed an average efficiency of 50–60% for energy transfer from β-carotene to chlorophyll a in the PS I and in the PS II complexes.     

Fluorescence emission spectra of Photosystem I,Photosystem II and the light-harvesting chlorophyll a/b complex of higher plants

Fluorescence emission spectra excited at 514 and 633 nm were measured at ?196 °C on dark-grown bean leaves which had been partially greened by a repetitive series of brief xenon flashes. Excitation at 514 nm resulted in a greater relative enrichment of the 730 nm emission band of Photosystem I than was obtained with 633 nm excitation. The difference spectrum between the 514 nm excited fluorescence and the 633 nm excited fluorescence was taken to be representative of a pure Photosystem I emission spectrum at ?196 °C. It was estimated from an extrapolation of low temperature emission spectra taken from a series of flashed leaves of different chlorophyll content that the emission from Photosystem II at 730 nm was 12% of the peak emission at 694 nm. Using this estimate, the pure Photosystem I emission spectrum was subtracted from the measured emission spectrum of a flashed leaf to give an emission spectrum representative of pure Photosystem II fluorescence at ?196 °C. Emission spectra were also measured on flashed leaves which had been illuminated for several hours in continuous light. Appreciable amounts of the light-harvesting chlorophyll a/b protein, which has a low temperature fluorescence emission maximum at 682 nm, accumulate during greening in continuous light. The emission spectra of Photosystem I and Photosystem II were subtracted from the measured emission spectrum of such a leaf to obtain the emission spectrum of the light-harvesting chlorophyll a/b protein at ?196 °C.     

Analyses of absorption and fluorescence spectra of water-soluble chlorophyll proteins,pigment System II particles and chlorophyll a in diethylether solution by the curve-fitting method

Absorption and fluorescence spectra in the red region of water-soluble chlorophyll proteins, Lepidium CP661, CP663 and Brassica CP673, pigment System II particles of spinach chloroplasts and chlorophyll a in diethylether solution at 25°C were analyzed by the curve-fitting method (French, C.S., Brown, J.S. and Lawrence, M.C. (1972) Plant Physiol. 49, 421–429). It was found that each of the chlorophyll forms of the chlorophyll proteins and the pigment System II particles had a corresponding fluorescence band with the Stokes shift ranging from 0.6 to 4.0 nm.The absorption spectrum of chlorophyll a in diethylether solution was analyzed to one major band with a peak at 660.5 nm and some minor bands, while the fluorescence spectrum was analyzed to one major band with a peak at 664.9 nm and some minor bands. A mirror image was clearly demonstrated between the resolved spectra of absorption and fluorescence. The absorption spectrum of Lepidium CP661 was composed of a chlorophyll b form with a peak at 652.8 nm and two chlorophyll a forms with peaks at 662.6 and 671.9 nm. The fluorescence spectrum was analyzed to five component bands. Three of them with peaks at 654.8, 664.6 and 674.6 nm were attributed to emissions of the three chlorophyll forms with the Stokes shift of 2.0–2.7 nm. The absorption spectrum of Brassica CP673 had a chlorophyll b form with a peak at 653.7 nm and four chlorophyll a forms with peaks at 662.7, 671.3, 676.9 and 684.2 nm. The fluorescence spectrum was resolved into seven component bands. Four of them with peaks at 666.7, 673.1, 677.5 and 686.2 nm corresponded to the four chlorophyll a forms with the Stokes shift of 0.6–4.0 nm. The absorption spectrum of the pigment System II particles had a chlorophyll b form with a peak at 652.4 nm and three chlorophyll a forms with peaks at 662.9, 672.1 and 681.6 nm. The fluorescence spectrum was analyzed to four major component bands with peaks at 674.1, 682.8, 692.0 and 706.7 nm and some minor bands. The former two bands corresponded to the chlorophyll a forms with peaks at 672.1 and 681.6 nm with the Stokes shift of 2.0 and 1.2 nm, respectively.Absorption spectra at 25°C and at ?196°C of the water-soluble chlorophyll proteins were compared by the curve-fitting method. The component bands at ?196°C were blue-shifted by 0.8–4.1 nm and narrower in half widths as compared to those at 25°C.     

Analysis of absorption spectra changes induced by temperature lowering on phycobilisomes,thylakoids and chlorophyll-protein complexes

Using fourth derivative analysis, differences between room and low temperature absorption spectra were studied. The positions of most absorption bands of the water-soluble, accessory pigment complex, the phycobilisome, remained unchanged after cooling. The stability of the wavelength positions of chlorophyll a forms in vivo as a function of temperature (Gulyaev, B.A. and Litvin, F.F. (1967) Biofizika 12, 845–854) was generally confirmed. The wavelength positions of all chlorophyll a forms in the P-700 chlorophyll a protein complex were unchanged when the preparations were cooled to ?196°C. Likewise, with other chlorophyll-containing materials: the light-harvesting chlorophyll $\text{a}\text{b}$ protein complex and the thylakoids of higher plants, algae, and cyanobacteria, the wavelength positions of most chlorophyll a forms were stable upon cooling. An exception was a 680 nm chlorophyll a band which was generally split at low temperature into two bands with the materials investigated. An interpretation of the multiplicity of chlorophyll spectral forms and the spectral changes induced by cooling for these forms is given using exciton theory and the energy-coupling variation of chlorophyll a molecules.     

Macromolecular organization of chlorophyll a in aggregated chlorophyll ab protein complex as shown by circular dichroism at room and cryogenic temperatures

This report concerns the large circular dichroic (CD) signal of intact chloroplasts of higher plants. The CD spectra of chloroplasts are compared with the aggregated form of the light-harvesting chlorophyll $\text{a}\text{b}$ complex at 25°C and ?250°C. The light-harvesting chlorophyll aggregate has a CD of magnitude equal to or greater than chloroplasts, but of opposite sign, and it is not related to the CD of the unaggregated form, and hence its arrangement is an artefact compared to the arrangement in the chloroplast. We suggest that this preparation, which has pseudo-lamellar structure, is a clear example of a large CD signal being generated by macromolecular association. The asymmetry of organization in the chloroplast has an opposite sense to that of the aggregate, but affects only chlorophyll a, not chlorophyll b.     

Survival of tissue culture cells frozen by a two-step procedure to -196 degrees C. I. Holding temperature and time.

A two-step freezing procedure has been examined in order to separate some of the causes of damage following freezing and thawing. Different holding temperatures and times have been studied during the freezing of Chinese hamster tissue culture cells in dimethyl sulphoxide (5%, $\text{v}\text{v}$). Damage following rapid cooling to, time at, and thawing from different holding temperatures was found to increase at lower holding temperatures and at longer times. Damage on subsequent cooling from the holding temperature to ?196 °C and thawing was found to diminish at lower holding temperatures and longer times. The net result was that optimal survival from ?196 °C was obtained after 10 min at ?25 °C. Protection against the second step of cooling to ?196 °C was acquired at the holding temperature itself and was absent at ?15 °C without freezing.It seems that this technique will allow the different phases of freezing injury to be separated. These phases may include thermal shock to the holding temperature, hypertonic damage at the holding temperature and dilution shock on thawing from ?196 °C.     

Fluorescence emission spectra of chloroplasts and subchloroplast preparations at low temperature

A study was made of the chlorophyll fluorescence spectra between 100 and 4.2 K of chloroplasts of various species of higher plants (wild strains and chlorophyll b mutants) and of subchloroplast particles enriched in Photosystem I or II. The chloroplast spectra showed the well known emission bands at about 685, 695 and 715–740 nm; the System I and II particles showed bands at about 675, 695 and 720 nm and near 685 nm, respectively. The effect of temperature lowering was similar for chloroplasts and subchloroplast particles; for the long wave bands an increase in intensity occurred mainly between 100 and 50 K, whereas the bands near 685 nm showed a considerable increase in the region of 50-4.2 K. In addition to this we observed an emission band near 680 nm in chloroplasts, the amplitude of which was less dependent on temperature. The band was missing in barley mutant no. 2, which lacks the lightharvesting chlorophyll a/b-protein complex. At 4.7 K the spectra of the variable fluorescence (F_v) consisted mainly of the emission bands near 685 and 695 nm, and showed only little far-red emission and no contribution of the band at 680 nm.From these and other data it is concluded that the emission at 680 nm is due to the light-harvesting complex, and that the bands at 685 and 695 nm are emitted by the System II pigment-protein complex. At 4.2 K, energy transfer from System II to the light-harvesting complex is blocked, but not from the light-harvesting to the System I and System II complexes. The fluorescence yield of the chlorophyll species emittting at 685 nm appears to be directly modulated by the trapping state of the reaction center.     

Observation of two quenchers of chlorophyll fluorescence in chloroplasts at −196°C

Fluorescence induction at ?196°C has been monitored in chloroplasts rapidly frozen after poising at different redox potentials at room temperature. It was found that, as at room temperature, the initial level of fluorescence observed upon shutter opening (F_o), relative to the final level observed after 10 seconds of illumination (F_m) increased as the redox potential of the chloroplasts was lowered. Redox titration revealed the presence of two quenching components with E_m,7.8 at ?70 mV and ?275 mV accounting for approx. 75% and 25% of the variable fluorescence (F_v). Parallel observation of fluorescence yield at room temperature similarly gave two components, with E_m,7.8 at ?95 mV and ?290 mV, also accounting for approx. 75% and 25%. Simultaneous measurement of fluorescence emission at ?196°C at 695 nm and 735 nm indicated that both emissions are quenched by the same redox components.     

Quenching of chlorophyll fluorescence and primary photochemistry in chloroplasts by dibromothymoquinone

The quenching action of dibromothymoquinone on fluorescence and on primary photochemistry was examined in chloroplasts at ?196 °C. Both the initial (F₀) and final (F_M) levels of fluorescence as well as the fluorescence of variable yield (F_v = F_M ? F₀) were quenched at ?196 °C to a degree which depended on the concentration of dibromothymoquinone added prior to freezing. The initial rate of photoreduction of C-550 at — 196 °C, which was assumed to be proportional to maximum yield for primary photochemistry, ?_Po, was also decreased in the presence of dibromothymoquinone. Simple theory predicts that the ratio $\text{F}{}_{\mathrm{<mtext>V</mtext>}}\text{F}{}_{\mathrm{<mtext>M</mtext>}}$ should equal ?_Po. Excellent agreement was found in a comparison of relative values of ?_Po with relative values of $\text{F}{}_{\mathrm{<mtext>V</mtext>}}\text{F}{}_{\mathrm{<mtext>M</mtext>}}$ at various degrees of quenching by dibromothymoquinone. These results are taken to indicate that F₀ and F_V are the same type of fluorescence, both emanating from the bulk chlorophyll of Photosystem II.Dibromothymoquinone appears to create quenching centers in the bulk chlorophyll of Photosystem II which compete with the reaction centers for excitation energy. The rate constant for the quenching of excitation energy by dibromothymoquinone is directly proportional to the concentration of the quencher. Rate constants for the de-excitation of excited chlorophyll molecules by fluorescence, k_F, by nonradiative decay processes, k_D, by photochemistry, k_P, and by the specific quenching of dibromothymoquinone, k_Q, were calculated assuming the absolute yield of fluorescence at F₀ to be either 0.02 or 0.05.     

Cytochromes of plant plasma membranes. Characterization by absorbance difference spectrophotometry and redox titration

The cytochrome composition of plasma membranes (PM) obtained by phase partitioning of microsomal fractions from spinach leaves (Spinacea oleracea L. cv. Medania), cauliflower inflorescences (Brassica oleracea L.), sugar beer leaves (Beta vulgaris L.) and barley (Hordeum vulgare L. cv. Kristina) roots and leaves was characterized by absorbance difference spectrophotometry at different reducing conditions at 20 and – 196°C, by redox titration, and by heme staining of polypeptide bands after lithium dodecyl sulfate polyacrylamide gel electrophoresis (LDS-PAGE). The location of the α-bands in the difference spectra and the loss of heme after treatment with LDS indicated that predominantly cytochromes of the b-type were present in all species tested. The total concentration of cytochrome was ca 0.35 nmol (mg protein)^?1. The main component (ca 70% of total) was completely reduced by ascorbate and partly by NADH and had a midpoint potential of ca 150 mV. At – 196°C, ascorbate reduction revealed a symmetrical α-band at ca 557 nm with PM from spinach leaves, cauliflower and sugar beet leaves, but with barley root and leaf PM ascorbate reduction resulted in an asymmetrical α-band (shoulder at 552, maximum at 559 nm). In the dithionite-reduced minus ascorbate-reduced spectrum at –196°C a split α-band (552 + 558 nm) was seen with PM from all species. This minor component had a midpoint potential of ca – 50 mV and is probably identical to cytochrome b₅, the presence of which would explain the relatively high NADH-cytochrome c reductase activities observed with plant PM. With PM from cauliflower, CO-difference spectra indicated that cytochromes P-420 and P-450 were present at concentrations up to 0.06 and 0.03 nmol (mg protein)^?1, respectively. Visualization of cytochromes by heme staining after LDS-PAGE was complicated by endogenous peroxidase activity and by loss of heme during solubilisation. A presumptive b-cytochrome (heme-stained band at 94 kDa) was only detected with barley leaf PM.     

Effect of aVirido-Xantha mutation in barley on the content of individual plastid pigments

Summary A virido-xantha (vx) barley mutant with reduced chlorophyll content when grown at 15°C was investigated to determine quantitative and qualitative changes in the plastid pigments. Both chlorophyll and the capacity to produce protochlorophyllide from exogeneously supplied δ-aminolevulinic acid are reduced in the mutant. The total carotenoid content is also reduced in the mutant, but individual carotenoids are not reduced coordinately. Antheraxanthin actually accumulates in the mutant, and zeaxanthin was found in significant amounts in mutant seedlings but could not be detected in normal barley seedlings. Included in the methods is a procedure for the preliminary identification of zeaxanthin employing spectrophotometric analysis of fractions eluted from a sucrose column. The presence of recessive suppressors of thevx mutant results in increased contents of the chlorophyll pigments accompanied by a partial reversal of the changes in carotenoid indicated above. The changes in chlorophyll and carotenoid contents are also partially reversed when mutant seedlings are grown at 21°C. Research supported under contract AT(11-1)-332 with the United States Atomic Energy Commission.     

The reconstitution of a Photosystem II protein complex, P-700-chlorophyll a-protein complex and light-harvesting chlorophyll ab-protein

A Photosystem II reaction centre protein complex was extracted from spinach chloroplasts using digitonin. This complex showed (i) high rates of dichloroindophenol and ferricyanide reduction in the presence of suitable donors, (ii) low-temperature fluorescence at 685 nm with a variable shoulder at 695 nm which increased as the complex aggregated due to depletion of digitonin and (iii) four major polypeptides of 47, 39, 31 and 6 kDa on dissociating polyacrylamide gels. The Photosystem II protein complex, together woth the P-700-chlorophylla protein complex and light-harvesting chlorophyll $\text{a}\text{b-}\text{protein}$ complex (LHCP) also isolated using digitonin, were reconstituted with lipids from spinach chloroplasts to form proteoliposomes. The low-temperature (77 K) fluorescence properties of the various proteoliposomes were analysed. The $\text{F}{}_{685}\text{F}{}_{695}$ ratios of the Photosystem II reaction centre protein complex-liposomes decreased as the lipid to protein ratios were increased. The $\text{F}{}_{681}\text{F}{}_{697}$ ratios of LHCP-liposomes were found to behave similarly. Light excitation of chlorophyll b at 475 nm stimulated emission from both the Photosystem II protein complex (F₆₈₅ and F₆₉₅) and the P-700-chlorophyll a-protein complex (F₇₃₅) when LHCP was reconstituted with either of these complexes, demonstrating energy transfer between LHCP and PS I or II complexes in liposomes. No evidence was found for energy transfer from the PS II complex to the P-700-chlorophyll a-protein complex reconstituted in the same proteoliposome preparation. Proteoliposome preparations containing all three chlorophyll-protein complexes showed fluorescence emission at 685, 700 and 735 nm.     

Comparison of chlorophyll a spectra in wild-type and mutant barley chloroplasts grown under day or intermittent light

The absorption (640–710 nm) and fluorescence emission (670–710 nm) spectra (77 K) of wild-type and Chl b-less, mutant, barley chloroplasts grown under either day or intermittent light were analysed by a RESOL curve-fitting program. The usual four major forms of Chl a at 662, 670, 678 and 684 nm were evident in all of the absorption spectra and three major components at 686, 693 and 704 nm in the emission spectra. A broad Chl a component band at 651 nm most likely exists in all chlorophyll spectra in vivo. The results show that the mutant lacks not only Chl b, but also the Chl a molecules which are bound to the light-harvesting, Chl a/b, protein complex of normal plants. It also appears that the absorption spectrum of this antenna complex is not modified appreciably by its isolation from thylakoid membranes.Abbreviations Chl chlorophyll - DL daylight - ImL intermittent light - WT wildtype - LHC light-harvesting Chl a/b protein complex - S.E. standard error of the mean DBP-CIW No. 763.     

Inhibition of photosystem I and photosystem II in chloroplasts by UV radiation

The effects of UV radiation on the low temperature fluorescenceand primary photochemistry of PSII and PSI of spinach chloroplastswere studied. Fluorescence induction curves at –196°Cwere measured at 695 nm for PSII fluorescence and at 730 nmfor PSI fluorescence to determine both the initial F_o and finalF_M levels. The primary photochemistry of PSII was measured asthe rate of photoreduction of C-550 at – 196°C, thatof PSI as the rate of photooxidation of P₇₀₀ at –196°C.The results were analyzed in terms of a model for the photosyntheticapparatus which accounts for the yields of fluorescence andprimary photochemistry. According to this analysis UV radiationincreases nonradiative decay processes at the reaction centerchlorophyll of PSII. However, the effect of UV radiation isnot uniform throughout the sample during irradiation so thataccount must be taken of the fraction of PSII reaction centerswhich have been irradiated at any given time. UV radiation alsoinactivates P700 and causes a slight increase in nonradiativedecay in the antenna chlorophyll of PSI. All fluorescence ofvariable yield, F_V = F_M–F_o, at 730 nm is due to energytransfer from PSII to PSI so that the sensitivity of Fv to UVradiation is the same at 730 and 695 nm. ¹Present address: Department of Biology, Faculty of Science,Toho University, Narashino, Chiba 275, Japan. ²Present address: Central Research Laboratories, Fuji PhotoFilm Co., Ltd., 105 Mizonuma, Asaka-Shi, Saitama 351, Japan. (Received September 10, 1975; )     

Fluorescence emission spectra and thylakoid protein kinase activities of three higher plant mutants deficient in chlorophyll b

The properties of three higher plant mutants having less than normal amounts of chlorophyll b were compared with their respective wild-types. These mutants included the chlorophyll-b-lacking U374 sweet clover (Melilotus alba) and chlorina-f2 barley (Hordeum vulgare) as well as the chlorophyll b-deficient CD3 wheat (Triticum aestivum). Fluorescence emission spectra from leaves of the sweet-clover mutant at 77 K show great similarity to the previously published spectrum of the barley mutant; rather than the predominant long-wavelength emission at approx. 740 nm in the wild-type plants, an emission maximum at approx. 720 nm is observed. The wheat mutant, containing reduced but measurable amounts of chlorophyl b, had 77 K long-wavelength fluorescence emissions at both 720 and 740 nm. These data indicate that these PS-I-derived fluorescence emissions are strongly influenced by the presence of antennae components. When examined for the ability to perform a light-induced State 1-State 2 transition in vivo, none was detected in the U374 sweet clover, whereas the CD3 wheat was capable of this process. The phosphorylation of endogenous polypeptides in isolated thylakoid membranes was examined using [γ-³²P]ATP as substrate for the thylakoid protein kinase activities. All three mutants had higher thylakoid protein kinase activity than the respective normal plants on a chlorophyll basis. The response of the mutant and normal sweet clover thylakoid protein kinase activities to ATP concentration was essentially identical. In contrast, the thylakoid protein kinase activities in the barley and wheat mutants appeared to saturate at markedly lower ATP concentrations than in the respective normal plants. These data suggest that the chlorina-f2 and CD3 mutants may be lacking one of the thylakoid protein kinases normally present in wild-type plants and that mutants lacking chlorophyll b may be of at least two different types.     

Anisotropy of the emission and absorption bands of spinach chloroplasts measured by fluorescence polarization and polarized excitation spectra at low temperature

Spectra of fluorescence polarization were measured between 4 and 120 K of spinach chloroplasts, oriented in a magnetic field. At least seven emission bands were observed. The well known bands near 685 nm (‘F-685’) and 735–740 nm (‘F-735’) and the band near 680 nm (‘F-680’) were strongly polarized parallel to the plane of the thylakoid membrane, whereas emission bands near 695 nm (‘F-695’), 710, 730–735 and 760 nm showed perpendicular polarization. Assuming perfect orientation of the thylakoid membranes, we calculated orientation angles of 64, 47 and 66.5° for the emission dipoles of F-685, F-695 and F-735, respectively, with respect to the normal of the membrane. Excitation spectra of F-695 and F-735 in polarized light at 4 K provided information about the orientation of the absorption dipoles of chlorophylls a and b. The spectra thus obtained were in very good agreement with the linear dichroism spectrum. Moreover, they allowed us to distinguish between the pigments associated with Photosystems I and Ii, which is not possible from measurement of linear dichroism alone. The results indicate that a high degree of orientation is not confined to the long-wave absorbing bands, but also bands at shorter wavelength show a clear anisotropy. The calculated orientations were in quantitative agreement with the hypothesis that F-685 and F-735 are associated with chlorophylls absorbing at 676 and 710–715 nm, respectively.     

Organization of the photosynthetic apparatus of the chlorina-f2 mutant of barley using chlorophyll fluorescence decay kinetics

The time-resolved chlorophyll fluorescence emission of higher plant chloroplasts monitors the primary processes of photosynthesis and reflects photosynthetic membrane organization. In the present study we compare measurements of the chlorophyll fluorescence decay kinetics of the chlorophyll-b-less chlorina-f2 barley mutant and wild-type barley to investigate the effect of alterations in thylakoid membrane composition on chlorophyll fluorescence. Our analysis characterizes the fluorescence decay of chlorina-f2 barley chloroplasts by three exponential components with lifetimes of approx. 100 ps, 400 ps and 2 ns. The majority of the chlorophyll fluorescence originates in the two faster decay components. Although photo-induced and cation-induced effects on fluorescence yields are evident, the fluorescence lifetimes are independent of the state of the Photosystem-II reaction centers and the degree of grana stacking. Wild-type barley chloroplasts also exhibit three kinetic fluorescence components, but they are distinguished from those of the chlorina-f2 chloroplasts by a slow decay component which displays cation- and photo-induced yield and lifetime changes. A comparison is presented of the kinetic analysis of the chlorina-f2 barley fluorescence to the decay kinetics previously measured for intermittent-light-grown peas (Karukstis, K. and Sauer, K. (1983) Biochim. Biophys. Acta 725, 384–393). We propose that similarities in the fluorescence decay kinetics of both species are a consequence of analogous rearrangements of the thylakoid membrane organization due to the deficiencies present in the light-harvesting chlorophyll $\text{a}\text{b}$ complex.     

The study of the primary photoprocesses in Photosystem I of chloroplasts Recombination luminescence,chlorophyll triplet state and triplet-triplet annihilation

The dependence of the delayed luminescence of Photosystem I on the state of the reaction centers has been studied. Light flash induces a charge separation in the centers: $\text{P-700 · P-430}\textcolor{red}{}\text{P-700}{}^{\mathrm{+}}\text{· P-430}{}^{\mathrm{?}}$. Dark recombination of charges is accompanied by the recombination luminescence with $\text{τ}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 20}\text{ms}$.If the centers are in the P-700 · P-430^? state or if P-430 is inactivated by heat, then flashing of Photosystem I generates the triplet state chlorophyll with $\text{τ}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 0.5}\text{ms}$. The triplet state has been measured by the delayed fluorescence of chlorophyll at 20 °C and 77 °K and by the chlorophyll phosphorescence at 77 °K. The delayed fluorescence at 20 °C arises from the thermal activation of the triplet state up to the excited singlet level of chlorophyll and at 77 °K it is due to triplet-triplet annihilation. The quantum yield of the triplet formation, estimated by a comparison of the light saturation curves of delayed fluorescence at 20 °C and of P-700 photooxidation under the same experimental (optical) conditions, is ≈ 0.9 of the P-700⁺ yield. Only one triplet of chlorophyll can be generated per P-700. Under heat inactivation of P-430 the triplet formation is not observed when P-700 is oxidized.It is assumed that the triplet-triplet annihilation at 77 °K is related with the strong interaction between the chlorophyll molecules in the pigment complex of Photosystem I. The possibility of a triplet participation in the primary processes of photosynthesis is discussed.     

Fluorescence emission spectra of chloroplasts and subchloroplast preparations at low temperature.

A study was made of the chlorophyll fluorescence spectra between 100 and 4.2 K of chloroplasts of various species of higher plants (wild strains and chlorophyll b mutants) and of subchloroplast particles enriched in Photosystem I or II. The chloroplast spectra showed the well known emission bands at about 685, 695 and 715--740 nm; the System I and II particles showed bands at about 675, 695 and 720 nm and near 685 nm, respectively. The effect of temperature lowering was similar for chloroplasts and subchloroplast particles; for the long wave bands an increase in intensity occurred mainly between 100 and 50 K, whereas the bands near 685 nm showed a considerable increase in the region of 50--4.2 K. In addition to this we observed an emission band near 680 nm in chloroplasts, the amplitude of which was less dependent on temperature. The band was missing in barley mutant no. 2, which lacks the light-harvesting chlorophyll a/b-protein complex. At 4.7 K the spectra of the variable fluorescence (Fv) consisted mainly of the emission bands near 685 and 695 nm, and showed only little far-red emission and no contribution of the band at 680 nm. From these and other data it is concluded that the emission at 680 nm is due to the light-harvesting complex, and that the bands at 685 and 695 nm are emitted by the System II pigment-protein complex. At 4.2 K, energy transfer from System II to the light-harvesting complex is blocked, but not from the light-harvesting to the System I and System II complexes. The fluorescence yield of the chlorophyll species emitting at 685 nm appears to be directly modulated by the trapping state of the reaction center.