Orientation of the pigments in Photosystem II: A low-temperature linear dichroism and polarized fluorescence emission study of chlorophyll-protein complexes isolated from Chlamydomonas reinhardtii

The orientation of the various absorbing and fluorescing dipoles in Photosystem II have been investigated by linearly polarized light spectroscopy at 5 K, performed on macroscopically oriented PS II complexes derived from Chlamydomonas reinhardtii. Linear dichroism and absorption spectra show that the Q_Y transitions of the chlorophyll molecules are mostly tilted at less than 35° from the plane of largest cross-section of the particle (which in vivo coincides with the plane of the thylakoid membrane). The chlorophyll forms absorbing at 676 and 683 nm are oriented closer to the membrane than the forms absorbing at 665 and 670 nm which are tilted at approximately 35° from the plane. A dip observed around 680 nm in the LD/absorption spectra indicates a component tilted at a larger angle away from the membrane plane than the 676 nm- and 683 nm-absorbing species. A component weakly absorbing around 693 nm and exhibiting a negative LD (tilt larger than 35°) is clearly resolved. The amplitude of the LD at 693 nm relative to that observed at the maximum (676 nm) varies from sample to sample. In the blue spectral region, two populations of carotenoids are observed; one absorbs around 460 and 490 nm, while the other absorbs around 510 nm. They are oriented out of and near to the thylakoid plane, respectively. Comparison of polarized absorption and fluorescence spectra from the same oriented samples allows the assignment of the 695 nm fluorescence emission to the dipoles responsible for the LD signal at 693 nm.     

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.     

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.     

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.     

Origin of the F685 and F695 fluorescence in Photosystem II

The emission spectra of CP47-RC and core complexes of Photosystem II (PS II) were measured at different temperatures and excitation wavelengths in order to establish the origin of the emission and the role of the core antenna in the energy transfer and charge separation processes in PS II. Both types of particles reveal strong dependences of spectral shape and yield on temperature. The results indicate that the well-known F-695 emission at 77 K arises from excitations that are trapped on a red-absorbing CP47 chlorophyll, whereas the F-685 nm emission at 77 K arises from excitations that are transferred slowly from 683 nm states in CP47 and CP43 to the RC, where they are trapped by charge separation. We conclude that F-695 at 77 K originates from the low-energy part of the inhomogeneous distribution of the 690 nm absorbing chlorophyll of CP47, while at 4 K the fluorescence originates from the complete distribution of the 690 nm chlorophyll of CP47 and from the low-energy part of the inhomogeneous distribution of one or more CP43 chlorophylls.     

Excitation dynamics and heterogeneity of energy equilibration in the core antenna of photosystem I from the cyanobacterium Synechocystis sp. PCC 6803

Energy equilibration in the photosystem I core antenna from the cyanobacterium Synechocystis sp. PCC 6803 was studied using femtosecond transient absorption spectroscopy at 298 K. The photosystem I core particles were excited at 660, 693, and 710 nm with 150 fs spectrally narrow laser pulses (fwhm = 5 nm). Global analysis revealed three kinetic processes in the core antenna with lifetimes of 250-500 fs, 1.5-2.5 ps, and 20-30 ps. The first two components represent strongly excitation wavelength-dependent energy equilibration processes while the 20-30 ps phase reflects the trapping of energy by the reaction center. Excitation into the blue and red edge of the absorption band induces downhill and uphill energy flows, respectively, between different chlorophyll a spectral forms of the core. Excitation at 660 nm induces a 500 fs downhill equilibration process within the bulk of antenna while the selective excitation of long-wavelength-absorbing chlorophylls at 710 nm results in a 380 fs uphill energy transfer to the chlorophylls absorbing around 695-700 nm, presumably reaction center pigments. The 1.5-2.5 ps phases of downhill and uphill energy transfer are largely equivalent but opposite in direction, indicating energy equilibration between bulk antenna chlorophylls at 685 nm and spectral forms absorbing below 700 nm. Transient absorption spectra with excitation at 693 nm exhibit spectral evolution within approximately 2 ps of uphill energy transfer to major spectral forms at 680 nm and downhill energy transfer to red pigments at 705 nm. The 20-30 ps trapping component and P(700) photooxidation spectra derived from data on the 100 ps scale are largely excitation wavelength independent. An additional decay component of red pigments at 710 nm can be induced either by selective excitation of red pigments or by decreasing the temperature to 264 K. This component may represent one of the phases of energy transfer from inhomogeneously broadened red pigments to P(700). The data are discussed based on the available structural model of the photosystem I reaction center and its core antenna.     

The long-wavelength chlorophyll states of plant LHCI at room temperature: a comparison with PSI-LHCI

The red antenna states of the external antenna complexes of higher plant photosystem I, known as LHCI, have been analyzed by measurement of their preequilibrium fluorescence upon direct excitation at 280 K. In addition to the previously detected F735 state, a hitherto undetected low-energy state with emission maximum around 713 nm was observed. The 280 K bandwidths (FWHM) are 55 nm for the F735 state and approximately 27 nm for the F713-nm state, much greater than for non-red-shifted antenna chlorophylls. The origin absorption band for the F735-nm state was directly detected by determination of its excitation (action) spectrum and lies at 709-710 nm. The absorption spectrum for F735, calculated using the Stepanov expression, closely overlaps the excitation spectrum, indicating that the very large Stokes shift (25 nm) is due to vibrational relaxation within the excited-state manifold and solvent effects can be excluded. Fluorescence anisotropy measurements, with direct excitation of F735, indicate that the transition dipoles of the two red states are parallel. Similar experiments performed in the long-wavelength absorbing tail of PSI-LHCI indicate the presence of emission state(s) that are red-shifted with respect to F735 of isolated LHCI. It is suggested that these are brought about by interactions between the complexes in PSI-LHCI, which occur in some yet undefined way, and which are broken upon solubilization of the component parts.     

Nuclear pea mutants deficient in chlorophyll b and the major polypeptide of the light-harvesting chlorophyll a/b protein of photosystem II

Eight chlorophyll b deficient nuclear mutants of pea (Pisum sativum L.) have been characterized by low temperature fluorescence emission spectra of their leaves and by the ultrastructure, photochemical activities and polypeptide compositions of the thylakoid membranes. The room temperature fluorescence induction kinetics of leaves and isolated thylakoids have also been recorded. In addition, the effects of Mg²⁺ on the fluorescence kinetics of the membranes have been investigated. The mutants are all deficient in the major polypeptide of the light-harvesting chlorophyll a/b protein of photosystem II. The low temperature fluorescence emission spectra of aurea-5106, xantha-5371 and –5820 show little or no fluorescence around 730 nm (photosystem I fluorescence), but possess maxima at 685 and 695 nm (photosystem II fluorescence). These three mutants have low photosystem II activities, but significant photosystem I activities. The long-wavelength fluorescence maximum is reduced for three other mutants. The Mg²⁺ effect on the variable component of the room temperature fluorescence (685 nm) induction kinetics is reduced in all mutants, and completely absent in aurea-5106 and xantha-5820. The thylakoid membranes of these 2 mutants are appressed pairwise in 2-disc grana of large diameter. Chlorotica-1-206A and–130A have significant long-wavelength maxima in the fluorescence spectra and show the largest Mg²⁺ enhancement of the variable part of the fluorescence kinetics. These two mutants have rather normally structured chloroplast membranes, though the stroma regions are reduced. The four remaining mutants are in several respects of an intermediate type.Abbreviations Chl chlorophyll - CPI Chi-protein complex I, F_o, F_v - F_m parameters of room temperature chlorophyll fluorescence induction kinetics - F685, F695 and F-1 components of low temperature chlorophyll emission with maximum at 685, 695 and ca 735 nm, respectively - PSI photosystem I - PSII photosystem II - LHCI and LHCII light-harvesting chlorophyll a/b complexes associated with PSI and PSII, respectively - SDS sodium dodecyl sulfate     

Effects of urea and o-phenanthroline on F-695 emission in chloroplasts

To clarify the nature of the chlorophyll species which fluorescesat about 695 nm in vivo (F-695), effects of the addition ofurea and related compounds and of urea plus o-phenanthrolineon the emission spectra of spinach chloroplast fragments, at77°K, were examined. F-695 emission was partially decreased by the presence of alow concentration (0.1 M) of urea, thiourea, guanidine hydrochloride,methylguanidine hydrochloride, acetamide, N-methylurea, or dimethylurea. The concurrent addition of o-phenanthroline with the reagents(0.1–1 M) caused a marked increase in F-695 emission anda decrease in F-685 emission. Methyl-substituted ureas and acetamide,however, were less effective. The effect was largely dependenton the pH of the sample. These two effects, the decrease and increase in F-695 emission,were reversible and were inhibited by the presence of ethanol. The phenomena are probably due to specific interactions betweenadded reagents and the component(s) in chloroplasts which havean intimate connection with a postulated energy trap; an invivo species of chlorophyll responsible for the fluorescenceemission near 695 nm. (Received August 17, 1971; )     

Orientation of emitting dipoles of chlorophyll A in thylakoids: considerations on the orientation factor in vivo.

Orientation angles of five emitting dipoles of chlorophyll a in thylakoids were estimated from low temperature fluorescence polarization ratio spectra of magnetically oriented chloroplasts. A simple expression is given also for the evaluation of data from linear dichroism measurements. It is shown that the Qy dipoles of chlorophylls lie more in the plane of the membranes and span a larger angular interval than was previously thought. Values for the orientation factor are calculated using various models corresponding to different degrees of local order of the Qy dipoles of chlorophylls in the thylakoid. We show that the characteristic orientation pattern of the Qy dipoles of chlorophylls in the membrane, i.e., increasing dichroism toward longer wavelengths, may favour energy transfer between the antenna chlorophylls as well as funnel the excitation energy into the reaction centers.     

Variability of light-induced circular dichroism spectra of photosystem I complexes of cyanobacteria

Circular dichroism (CD) spectra of photosystem I (PSI) complexes of the cyanobacteria Thermosynechococcus elongatus, Arthrospira platensis and Synechocystis sp. PCC 6803 were studied. CD spectra of dark-adapted PSI trimers and monomers, measured at 77 K, show common bands at 669–670(+), 673(+), 680(−), 683–685(−), 696–697(−), 702(−) and 711(−) nm. The intensities of these bands are species specific. In addition, bands at 683–685(−) and 673(+) nm differ in intensity for trimeric and monomeric PSI complexes. CD difference spectra (P700⁺–P700) of PSI complexes at 283 K exhibit conservative bands at 701(−) and 691(+) nm due to changes in resonance interaction of chlorophylls in the reaction center upon oxidation of P700. Additional bands are observed at 671(−), 678(+), 685(−), 693(−) nm and in the region 720–725 nm those intensities correlate with intensities of analogous bands of antenna chlorophylls in dark-adapted CD spectra. It is suggested that the variability of CD difference spectra of PSI complexes is determined by changes in resonance interaction of reaction center chlorophylls with closely located antenna chlorophylls.     

Differently oriented chlorophylls in Mesotaenium caldariorum detected by microphotometrical dichroism measurements in vivo

The chloroplasts of individual cells of Mesotaenium caldariorum were examined microphotometrically under non-polarized and polarized measuring light. The measurement with non-polarized light showed different absorption bands of the thylakoids depending on the position of their surface with respect to the incident light beam: in the edge position, the absorption bands lie at 672 nm, in the face position at 678 nm. From this difference in absorption maxima, we conclude that the molecules related to the sub-bands at the two wavelengths are oriented differently. The Q_y transition of the molecules which absorb light at 678 nm must be oriented parallel to the face of the thylakoids (fraction I), while that of the molecules absorbing at 672 nm is oriented perpendicular to the face (fraction II). Measurement with polarized light leads to the same conclusion that two fractions of differently oriented chlorophylls exist: In the edge position, a very large difference between E_∥ and E_⊥ (dichroism) was found in red light, with a maximum of E_∥ lying at 675 nm and a maximum of E_⊥ at 670 nm, with a shoulder at 650 nm. In the blue region, especially in the Soret band zone, the chloroplast showed a negative dichroism in the edge position, which changes over to positive values when the wavelength exceeds 450 nm. In the face position no dichroism in red or blue light could be detected. Comparison of the ‘edge position dichroism’ in red light with that in blue light justifies the supposition that the chlorin planes of the chlorophyll molecules may be oriented perpendicular or parallel to the thylakoid face, in the case of perpendicular orientation with the Q_y transitions of fraction II and the x-transitions (B_x, Q_x) of fraction I projecting out of the plane, and for parallel orientation with all transition moments lying parallel to the plane (fraction I). The relative dichroism, $\text{(E}{}_{\mathrm{\parallel }}\text{? E}{}_{\mathrm{\perp }}\text{)}\text{(E}{}_{\mathrm{\parallel }}\text{+ E}{}_{\mathrm{\perp }}\text{)}$, measured at the edge position amounts to 0.34 (i.e., 34% of the total absorption) at 680 nm. These data probably do not reflect the total quantity of oriented chlorophyll because from the opposite orientations of the Q_y transition moments of fraction I and II pigment a partial quenching of the measurable dichroism results. The red light absorption bands of the two chlorophylls oriented in an opposite manner (fractions I and II) correspond to the known bands of Photosystem I and II.     

Spectroscopic properties of the reaction center and of the 47 kDa chlorophyll protein of Photosystem II

The D1-D2-cytochrome b-559 reaction center complex and the 47 kDa antenna chlorophyll protein isolated from spinach Photosystem II were characterized by means of low temperature absorption and fluorescence spectroscopy. The low temperature absorption spectrum of the D1-D2-cytochrome b-559 complex showed two bands in the Q_y region located at 670 and 680 nm. On the basis of its absorption maximum and orientation the latter component may be attributed at least in part to P-680, the primary electron donor of Photosystem II. The 47 kDa antenna complex showed absorption bands at 660, 668 and 677 nm and a minor component at 690 nm. The latter transition appeared to be associated with the characteristic low temperature 695 nm fluorescence band of Photosystem II. The 695 nm emission band was absent in the D1-D2 complex, which indicates that it does not originate from the reaction center pheophytin, as earlier proposed. The transition dipole responsible for the main fluorescence at 684 nm from this complex had a parallel orientation with respect to the membrane plane in the native structure. The reaction center preparation contains two spectrally distinct carotenoids with different orientations.     

The effects of Triton X-100 and n-octyl beta-D-glucopyranoside on energy transfer in photosynthetic membranes.

The effects of the non-ionic detergents Triton X-100 and n-octyl beta-D-glucopyranoside on energy transfer between pigment-protein complexes of Pisum sativum thylakoids were investigated. This was done by monitoring the 77K fluorescence-emission characteristics of stacked and unstacked thylakoids exposed to a range of detergent concentrations. At sub-critical micellar concentrations, the detergents had little effect, whereas above these concentrations they caused increases of up to 20-fold in short-wavelength fluorescence intensity and a shift in its maximum wavelength from 685 to 680 nm. Fluorescence-emission intensities at 695 and 735 nm were relatively unaffected by detergent treatments, although Triton X-100 caused a wavelength shift in the emission peak from 735 to 728 nm. The results are discussed in terms of reversible dissociation of pigment-protein complexes induced by mild detergent solubilization and the consequent cessation of inter-complex energy transfer.     

Phosphorylation of the light-harvesting chlorophyll-protein regulates excitation energy distribution between photosystem II and photosystem I

The kinetics of thylakoid membrane protein phosphorylation in the presence of light and adenosine triphosphate is correlated to an incease in the 77 °K fluorescence emission at 735 nm (F735) relative to that at 685 nm (F685). Analysis of detergent-derived submembrane fractions indicate phosphorylation only of the polypeptides of Photosystem II, and the light-harvesting chlorophyll-protein complex serving Photosystem II (LHC-II). Although several polypeptides are phosphorylated, only the dephosphorylation kinetics of LHC-II follow the kinetics of the decrease of the $\text{F735}\text{F685}$ fluorescence emission ratios. The relative quantum yield of Photosystem II was significantly lower in phosphorylated membranes compared to dephosphorylated membranes. Reversible LHC-II phosphorylation thus provides the physiological mechanism for the control of the distribution of absorbed excitation energy between the two photosystems.     

Spectral inhomogeneity of photosystem I and its influence on excitation equilibration and trapping in the cyanobacterium Synechocystis sp. PCC6803 at 77 K.

Ultrafast transient absorption spectroscopy was used to probe excitation energy transfer and trapping at 77 K in the photosystem I (PSI) core antenna from the cyanobacterium Synechocystis sp. PCC 6803. Excitation of the bulk antenna at 670 and 680 nm induces a subpicosecond energy transfer process that populates the Chl a spectral form at 685--687 nm within few transfer steps (300--400 fs). On a picosecond time scale equilibration with the longest-wavelength absorbing pigments occurs within 4-6 ps, slightly slower than at room temperature. At low temperatures in the absence of uphill energy transfer the energy equilibration processes involve low-energy shifted chlorophyll spectral forms of the bulk antenna participating in a 30--50-ps process of photochemical trapping of the excitation by P(700). These spectral forms might originate from clustered pigments in the core antenna and coupled chlorophylls of the reaction center. Part of the excitation is trapped on a pool of the longest-wavelength absorbing pigments serving as deep traps at 77 K. Transient hole burning of the ground-state absorption of the PSI with excitation at 710 and 720 nm indicates heterogeneity of the red pigment absorption band with two broad homogeneous transitions at 708 nm and 714 nm (full-width at half-maximum (fwhm) approximately 200--300 cm(-1)). The origin of these two bands is attributed to the presence of two chlorophyll dimers, while the appearance of the early time bleaching bands at 683 nm and 678 nm under excitation into the red side of the absorption spectrum (>690 nm) can be explained by borrowing of the dipole strength by the ground-state absorption of the chlorophyll a monomers from the excited-state absorption of the dimeric red pigments.     

Red-shifted chlorophyll a bands allow uphill energy transfer to photosystem II reaction centers in an aerial green alga,Prasiola crispa,harvested in Antarctica

An aerial green alga, Prasiola crispa (Lightf.) Menegh, which is known to form large colonies in Antarctic habitats, is subject to severe environmental stresses due to low temperature, draught and strong sunlight in summer. A considerable light-absorption by long-wavelength chlorophylls (LWC) at around 710 nm, which seem to consist of chlorophyll a, was detected in thallus of P. crispa harvested at a terrestrial environment in Antarctica. Absorption level at 710 nm against that at 680 nm was correlated with fluorescence emission intensity at 713 nm at room temperature and the 77 K fluorescence emission band from LWC was found to be emitted at 735 nm. We demonstrated that the LWC efficiently transfer excitation energy to photosystem II (PSII) reaction center from measurements of action spectra of photosynthetic oxygen evolution and P700 photo-oxidation. The global quantum yield of PSII excitation in thallus by far-red light was shown to be as high as by orange light, and the excitation balance between PSII and PSI was almost same in the two light sources. It is thus proposed that the LWC increase the photosynthetic productivity in the lower parts of overlapping thalli and contribute to the predominance of alga in the severe environment.     

Excitonic interactions in wild-type and mutant PSI reaction centers

Femtosecond excitation of the red edge of the chlorophyll a Q(Y) transition band in photosystem I (PSI), with light of wavelength > or = 700 nm, leads to wide transient (subpicosecond) absorbance changes: positive DeltaA between 635 and 665 nm, and four negative DeltaA bands at 667, 675, 683, and 695 nm. Here we compare the transient absorbance changes after excitation at 700, 705, and 710 nm at 20 K in several PSI preparations of Chlamydomonas reinhardtii where amino acid ligands of the primary donor, primary acceptor, or connecting chlorophylls have been mutated. Most of these mutations influence the spectrum of the absorbance changes. This supports the view that the chlorophylls of the electron transfer chain as well as the connecting chlorophylls are engaged in the observed absorbance changes. The wide absorption spectrum of the electron transfer chain revealed by the transient measurements may contribute to the high efficiency of energy trapping in photosystem 1. Exciton calculations, based on the recent PSI structure, allow an assignment of the DeltaA bands to particular chlorophylls: the bands at 675 and 695 nm to the dimers of primary acceptor and accessory chlorophyll and the band at 683 nm to the connecting chlorophylls. The subpicosecond transient absorption bands decay may reflect rapid charge separation in the PSI reaction center.     

Chlorophyll Synthesis in Dark-Grown Pine Primary Needles

The pigment content of dark-grown primary needles of Pinus jeffreyi L. and Pinus sylvestris L. was determined by high-performance liquid chromatography. The state of protochlorophyllide a and of chlorophylls during dark growth were analyzed by in situ 77 K fluorescence spectroscopy. Both measurements unambiguously demonstrated that pine primary needles are able to synthesize chlorophyll in the dark. Norflurazon strongly inhibited both carotenoid and chlorophyll synthesis. Needles of plants treated with this inhibitor had low chlorophyll content, contained only traces of xanthophylls, and accumulated carotenoid precursors. The first form of chlorophyll detected in young pine needles grown in darkness had an emission maximum at 678 nm. Chlorophyll-protein complexes with in situ spectroscopic properties similar to those of fully green needles (685, 695, and 735 nm) later accumulated in untreated plants, whereas in norflurazon-treated plants the photosystem I emission at 735 nm was completely lacking. To better characterize the light-dependent chlorophyll biosynthetic pathway in pine needles, the 77 K fluorescence properties of in situ protochlorophyllide a spectral forms were studied. Photoactive and nonphotoactive protochlorophyllide a forms with emission properties similar to those reported for dark-grown angiosperms were found, but excitation spectra were substantially red shifted. Because of their lower chlorophyll content, norflurazon-treated plants were used to study the protochlorophyllide a photoreduction process triggered by one light flash. The first stable chlorophyllide photoproduct was a chlorophyllide a form emitting at 688 nm as in angiosperms. Further chlorophyllide a shifts usually observed in angiosperms were not detected. The rapid regeneration of photoactive protochlorophyllide a from nonphotoactive protochlorophyllide after one flash was demonstrated.