Effect of Supplementary UVB Radiation on Chlorophyll Synthesis and Accumulation of Photosystems during Chloroplast Development in Spirodela oligorrhiza

Abstract— Although the effects of ultraviolet B (UVB, 290–320 nm) radiation have been studied in plants extensively, little is known about the potential impacts on maturation of chloroplasts. To address this problem, the effects of supplementary UVB on chloroplast development were examined in the aquatic higher plant Spirodela oligorrhiza. Dark-grown Spirodela-containing proplastids were exposed to photosynthetically active radiation (PAR) and ultraviolet A (UVA, 320–400 nm), plus supplementary UVB equivalent to 1% of PAR on a photon basis. The biosynthesis and assembly of chlorophyll (Chi) into reaction centers was followed for 4 days in situ by low temperature (77 K) Chi fluorescence. Impacts on chloroplast development were detected after only 1 h incubation in light with supplementary UVB. Fluorescence emission signals from Chi associated with the photosystem (PS) II antenna, PSII reaction centers and PSI reaction centers were detected at the same time with or without UVB, but the magnitude of PS fluorescence was diminished up to 60% in plants incubated in UVB. The Chi content was also lower in UVB-treated plants, but to a lesser degree than anticipated by low temperature fluorescence, suggesting lack of organization and/or association of Chi with PS. Electron transport, measured with room temperature fluorescence induction, was not consistently different in plants exposed to UVB. These results suggest that with UVB, fewer and/or smaller PS form during chloroplast development, but there is not a large inhibition of Chi synthesis or PSII activity.     

EXCITATION ENERGY TRANSFER IN THE CRYPTOPHYTES. FLUORESCENCE EXCITATION SPECTRA AND PICOSECOND TIME-RESOLVED EMISSION SPECTRA OF INTACT ALGAE AT 77 K

Excitation spectra of chlorophyll- a (Chl- a ) fluorescence in intact cells of Cryptomonas ovata, Chroomonas pauciplastida and Chroomonas salina were determined at 77 K. For all species the excitation spectra for emission from Chl- a associated with photosystem II (PSII) showed increased contributions by a carotenoid (493 nm) and phycobiliproteins, and decreased contributions by carotenoid (417 nm, 505 nm) and Chl- a (445 nm) as compared to excitation spectra for emission from Chl- a associated with photosystem I (PSI). Excitation spectra of C. salina and C. ovata showed an increased contribution by Chl- c ² to PSII Chl- a fluorescence emission. In all three species the absorbance band positions of Chl- a , as determined from the excitation spectra, were similar to those previously described in green plants. green algae and phycobilisome-containing organisms. Time-resolved 77 K fluorescence emission spectra of C. ovata and C. salina showed successive emission from both phycoerythrin and Chl- c ², PSII Chl- a , and PSI Chl- a. C. pauciplastida showed successive emission from phycocyanin, PSII Chl- a , and PSI Chl- a. Spectral red-shifts with time were observed for the phycobiliprotein peaks in all three species. The fluorescence decay of phycoerythrin in C. ovata and C. salina was faster than that of phycocyanin in C. pauciplastida. The results are discussed in relation to the organization of the antenna pigments of PSII and PSI in the cryptophyte algae.     

Early reactions of light-induced protochlorophyllide and chlorophyllide transformations analyzed in vivo at room temperature with a diode array spectrofluorometer

The steps of protochlorophyllide (Pchlide) photoreduction and subsequent chlorophyllide (Chlide) transformations which occur in the seconds to minutes time-scale were studied using a diode array spectrofluorometer in dark-grown barley leaves. The intensity of the excitation light was varied between 3 and 2,500 micromol m(-2) s(-1) and a series of fluorescence spectra were recorded at room temperature in the seconds and minutes time scales. In certain experiments, 77-K emission spectra were measured with the same equipment. The high quality of the spectra allowed us to run spectral resolution studies which proved the occurrence, at room temperature, of multiple Pchlide and Chlide forms found previously in 77-K spectra. The comparison of the 77-K and room-temperature spectra showed that the fluorescence yields of the nonphotoactive 633-nm Pchlide form and of the Chlide product emitting at 678 nm were temperature independent. The fluorescence intensity of aggregated NADPH-pigment-POR complexes (photoactive 656-nm Pchlide and 693-nm Chlide forms) were strongly increased at 77 K, while that of the NADP(+)-Chlide-POR (684-686-nm Chlide form) was much less affected by temperature. Information was obtained also about the dynamics of the transformation of pigment forms in the light at different photon densities. At low light intensities, the phototransformation of the 642-644-nm Pchlide form was faster than that of the 654-656-nm form. The relative amplitudes of Gaussian components related to different Chlide forms found after exposure to a constant amount of photons strongly depended on the light intensity used. Strong quenching of all Chlide components occurred upon prolonged exposure to high intensity light. These effects are discussed by considering the interconversion processes between different forms of the pigment-protein complexes, their relative fluorescence yields and energy migration processes.     

Mass spectrometric characterization of photooxidative protein modifications in Arabidopsis thaliana thylakoid membranes

Oxidative and nitrosative stress leaves footprints in the plant chloroplast in the form of oxidatively modified proteins. Using a mass spectrometric approach, we identified 126 tyrosine and 12 tryptophan nitration sites in 164 nitrated proteolytic peptides, mainly from photosystem I (PSI), photosystem II (PSII), cytochrome b(6) /f and ATP-synthase complexes and 140 oxidation products of tyrosine, tryptophan, proline, phenylalanine and histidine residues. While a high number of nitration sites were found in proteins from four photosynthetic complexes indicating that the nitration belongs to one of the prominent posttranslational protein modifications in photosynthetic apparatus, amino acid oxidation products were determined mostly in PSII and to a lower extent in PSI. Exposure of plants to light stress resulted in an increased level of tyrosine and tryptophan nitration and tryptophan oxidation in proteins of PSII reaction center and the oxygen-evolving complex, as compared to low light conditions. In contrast, the level of nitration and oxidation of these amino acid residues strongly decreased for all light-harvesting proteins of PSII under the same conditions. Based on these data, we propose that oxidative modifications of proteins by reactive oxygen and nitrogen species might represent an important regulatory mechanism of protein turnover under light stress conditions, especially for PSII and its antenna proteins.     

Efficient energy transfer from the long-wavelength antenna chlorophylls to P700 in photosystem I complexes from Spirulina platensis

To study the role of the long-wavelength chlorophylls (Chl) in photosystem I (PSI), the action spectra of P700 photooxidation at 293 and 77 K have been measured for PSI trimeric and monomeric complexes isolated from Spirulina platensis. The long-wavelength Chls which absorb in the region 710dash740 nm transfer excitation energy to the reduced P700 with the same efficiency as bulk antenna Chls, causing the oxidation of P700. The relative quantum yield of P700 photooxidation is about unity (293-77 K) even under the direct excitation of Chl absorbing at 735 nm (Chl735). At 77 K Chl735 exhibits a fluorescence band at 760 nm (F760) whose intensity is quenched under illumination of the PSI trimeric complexes from Spirulina. The relative quantum yield of F760 quenching is not dependent on the wavelength of excitation in the region 620–750 nm. Since the value of the overlap integral between the band of F760 and the absorption band of the cation radical of P700 (P700⁺) is higher than that of the P700 band, it is suggested that Chl735 transfers energy to P700⁺ more efficiently than to reduced P700; energy transfer to P700⁺ causes the quenching of F760. A linear relationship between the photooxidation rate of P700 and the fraction of P700⁺ at 293 K indicates that the energy exchange between PSI subunits of the trimer is negligible. Thus, the antenna of PSI trimers of Spirulina is organized in separate photosynthetic units.     

High light-induced changes of 77 K fluorescence emission of pea thylakoid membranes with altered membrane fluidity

The effect of lipid phase order of isolated thylakoid membranes on fluorescent characteristics of both photosystems during illumination with high light intensity at 22 degrees C and 4 degrees C was investigated. For artificial modification of membrane fluidity two membrane perturbing agents were applied-cholesterol and benzyl alcohol. 77 K fluorescence emission and excitation spectra of control, cholesterol- and benzyl alcohol-treated thylakoid membranes were analysed in order to determine the high light-induced changes of emission bands attributed to different chlorophyll-protein complexes-F 735, emitted by photosystem I-light-harvesting complex I; and F 685 and F 695, emitted by photosystem II-light-harvesting complex II. Analysis of emission bands showed that high light treatment leads to a decrease of the area of band at 695 nm and a concomitant increase of intensity of the band at 735 nm. The involvement of different pigment pools (chlorophyll a and chlorophyll b) in the energy supply of both photosystems before and after photoinhibitory treatment was estimated on the basis of excitation fluorescence spectra. The dependence of the ratios F 735/F 685 and the band areas at 685 and 695 nm on the illumination time was studied at both temperatures. Data presented indicate that cholesterol incorporation stabilized the intersystem structure in respect to light-induced changes of fluorescence emission of PSI and PSII. It was shown that the effect of fluid properties of thylakoid membranes on the 77 K fluorescence characteristics of main pigment protein complexes of pea thyalkoid membranes depends on the temperature during high light treatment.     

Photoacclimation in spathiphyllum

We studied photoacclimation in Spathiphyllum grown at an irradiance of 40 or 420 micromol/m2 s (LL or HL, respectively). All parameters studied responded to acclimation. Leaves at LL, in contrast to HL, were thinner and oriented perpendicular to the incident light, had more chlorophyll per g f w, fewer stomata on the upper leaf surface and a reduced layer of mesophyll cells. Their chloroplasts at HL had wider grana with less thylakoids per granum, and better organized photosystems than at LL. PSI and PSII activities per mg chlorophyll ( Vmax ), and PSI and PSII content (total activity per g f w), were lower at LL than at HL and so was the light requirement for saturation of the PSI or PSII partial photoreactions, suggesting that fewer photosystems with larger antenna size prevail at LL, but many more with smaller antenna size at HL. Analysis of chlorophyll distribution among the thylakoid pigment-protein complexes showed less antenna chlorophyll serving PSII (CPa+LHCP1+LHCP3) than that serving PSI (CPIa+CPI+LHCP2) at LL as compared to HL, and thus a lower PSII/PSI ratio at LL, in agreement with the general finding that LL plants, with larger PSII antenna size, have lower PSII/PSI ratio. The increase in PSI antenna size at LL was correlated with the increase in the distribution of chlorophyll in pigment-protein complexes serving PSI, and a very large chlorophyll/protein molar ratio in the isolated CPI complex. On the other hand, the PSII antenna chlorophyll (CPa+LHCP1+LHCP3) on a g f w basis, and the chlorophyll a/b ratio remained more or less constant at LL or HL. This may reflect our finding that Spathiphyllum contains mainly the 27 kDa inner LHCII antenna protein, the size of which remains unaffected by photoacclimation. The increase in the distribution of chlorophyll in pigment-protein complexes serving PSII at HL, therefore, reflects the higher population of PSII at HL. Very high PSI activity was found at HL, which we attribute to the highly organized small in size PSI.     

Spectroscopic properties of protochlorophyllide analyzed in situ in the course of etiolation and in illuminated leaves

The spectroscopic properties of photoactive (i.e. flash-transformable) and nonphotoactive protochlorophyll(ide)s (Pchl(ide)) were reinvestigated during the development of bean leaves in darkness. Two phases in the process of Pchl(ide) accumulation were apparent from quantitative measurements of pigment content: a lag phase (first week) during which photoactive Pchl(ide) accumulated faster than nonphotoactive Pchl(ide); and a fast phase (second week), showing parallel accumulation of both types of Pchl(ide). 'Flashed-minus-dark' absorbance difference spectra recorded in situ at 77 K showed that P650-655 was the predominant form of photoactive protochlorophyllide regardless of developmental stage. Quantitative analysis of energy migration processes between the Pchl(ide) forms showed the existence of energy transfer units containing a 1:8 ratio of nonphotoactive and photoactive Pchl(ide)s during development. Gaussian deconvolution of in situ 77 K fluorescence spectra indicated that the 633 nm band of nonphotoactive Pchl(ide) was made of four bands, at 625, 631, 637 and 643 nm, whose relative amplitudes only slightly changed during development. The emission band of photoactive Pchlide was also analyzed using the same method. Three components were found at 644, 652 and 657 nm. The emission band of P650-655 included the last two components, which become predominant only in fully etiolated plants. Photoactive Pchlide with an emission maximum at 653 nm was detected in the light during development of leaves of photoperiodically grown plants.     

Photoactive protochlorophyllide regeneration in cotyledons and leaves from higher plants

Chlorophyll accumulation during greening implies the continuous transformation of photoactive protochlorophyllide (Pchlide) to chlorophyllide. Since this reaction is a light-dependent step, the study of regeneration of photoactive Pchlide under a continuous illumination is difficult. Therefore this process is best studied on etiolated plants during a period of darkness following the initial photoreduction of photoactive Pchlide. In this study, the regeneration process has been studied using spinach cotyledons, as well as barley and bean leaves, illuminated by a single saturating flash. The regeneration was characterized using 77 K fluorescence emission and excitation spectra and high-performance liquid chromatography. The fluorescence data indicated that the same spectral forms of photoactive Pchlide are regenerated by different pathways: (1) photoactive Pchlide regeneration starts immediately after the photoreduction through the formation of a nonphotoactive Pchlide form, emitting fluorescence at approximately 651 nm. This form is similar to the large aggregate of photoactive Pchlide present before the illumination, but it contains oxidized form of nicotinamide adenine dinucleotide phosphate, instead of the reduced form (NADPH), in the ternary complexes; and (2) after the dislocation of the large aggregates of chlorophyllide-light-dependent NADPH:Pchlide a photooxidoreductase-NADPH ternary complexes, the regeneration occurs at the expense of the several nonphotoactive Pchlide spectral forms present before the illumination.     

Effects of Pb2+ on energy distribution and photochemical activity of spinach chloroplast

Lead (Pb(2+)) is a well-known highly toxic element. The mechanisms of the Pb(2+) toxicity are not well understood for photosynthesis. In this paper, we reported the effect of Pb(2+) on light absorption, distribution and conversion of spinach chloroplast by spectroscopy, and photochemical reaction activities. Several effects of Pb(2+) were observed: (1) the absorption peak intensity of chloroplast obviously decreased in red and blue region and produced optical flattering; (2) fluorescence quantum yield nearby 680 nm of chloroplast greatly declined; (3) the excitation band nearby 440 nm of chloroplast significantly descended; (4) Pb(2+) treatments reduced of the rate of whole chain electron transport, photochemical activities of PSII DCPIP photoreduction and oxygen evolution, but the photoreduction activities of PSI were little changed. Together, the studies of the experiments showed that Pb(2+) decreased absorption of light on spinach chloroplast and inhibited excitation energy to be absorbed by LHCII and transferred to PSII, then reduced the conversion from light energy to electron energy, and decelerated electron transport, water photolysis and oxygen evolution.     

LIGHT-ABSORPTION AND ELECTRON-TRANSPORT BALANCE BETWEEN PHOTOSYSTEM II AND PHOTOSYSTEM I IN SPINACH CHLOROPLASTS

Abstract— The distribution of absorbed light and the turnover of electrons by the two photosystems in spinach chloroplasts was investigated. This was implemented upon quantitation of photochemical reaction centers, chlorophyll antenna size and composition of each photosystem (PS), and rate of light absorption in situ. In spinach chloroplasts, the photosystem stoichiometry was PSIIJPSII_α/PSII_β/PSI= 1.3/0.4/1.0. The number (N) of chlorophyll (a+b) molecules associated with each PS was N(PSII_α)/N(PSII_β)/N(PSI)=230/100/200, i.e. about 65% of all Chl is associated with PSII and about 35% with PSI. Light absorption by PSII in vivo is selectively attenuated at the molecular, membrane and leaf levels, (a) The rate of light absorption by PSII was only 0.85 that of PSI because of the lower rate of light absorption by Chl b as compared to Chl a (approximately 80% of all Chl b in the chloroplast is associated with PSII). (b) The exclusive localization of PSII_α in the membrane of the grana partition regions and of PSI in intergrana lamellae resulted in a differential “sieve effect” or “flattening of absorbance” by the photosystems in the two membrane regions. Due to this phenomenon, the rate of light absorption by PSII was lower than that of PSI by 15-20%. (c) Selective filtering of sunlight through the spinach leaf results in a substantial distortion of the effective absorbance spectra and concomitant attenuation of light absorption by the two photosystems. Such attenuation was greater for PSII than for PSI because the latter benefits from light absorption in the 700-730 nm region. It is concluded that, in spite of its stoichiometric excess in spinach chloroplasts, light absorption by PSII is not greater than that by PSI due to the different molecular composition of the two light-harvesting antenna systems, due to the localization of PSII in the grana, and also because of the light transmission properties through the leaf. The elevated PSII/PSI reaction center ratio of 1.7 and the association of 65% of all Chl with PSII help to counter the multilevel attenuation of light absorption by PSII and ensure a balanced PSII/PSI electron turnover ratio of about 1:1.     

Charge separation is virtually irreversible in photosystem II core complexes with oxidized primary quinone acceptor

X-ray structures of the Photosystem II (PSII) core revealed relatively large interpigment distances between the CP43 and CP47 antenna complexes and the reaction center (RC) with respect to the interpigment distances in a single unit. This finding questions the possibility of fast energy equilibration among the antenna and the RC, which has been the basic explanation for the measured PSII fluorescence kinetics for more than two decades. In this study, we present time-resolved fluorescence measurements obtained with a streak-camera setup on PSII core complexes from Thermosynechococcus elongatus at room temperature (RT) and at 77 K. Kinetic modeling of the RT data obtained with oxidized quinone acceptor Q(A), reveals that the kinetics are best described by fast primary charge separation at a time scale of 1.5 ps and slow energy transfer from the antenna into the RC, which results in an energy equilibration time between the antenna and the RC of about 44 ps. This model is consistent with structure-based computations. Primary radical pair formation was found to be a virtually irreversible process. Energy equilibration within the CP43 and CP47 complexes is shown to occur at a time scale of 8 ps. Kinetic modeling of the 77 K data reveals similar energy transfer time scales in the antenna units and among the antenna and the RC as at RT, respectively, 7 and 37 ps. We conclude that the energy transfer from the CP43/CP47 antenna to the RC is the dominant factor in the total charge separation kinetics in intact PSII cores.     

The effect of decreasing temperature up to chilling values on the in vivo F685/F735 chlorophyll fluorescence ratio in Phaseolus vulgaris and Pisum sativum: the role of the photosystem I contribution to the 735 nm fluorescence band

The effect of leaf temperature (T), between 23 and 4 degrees C, on the chlorophyll (Chl) fluorescence spectral shape was investigated under moderate (200 microE m-2 s-1) and low (30-35 microE m-2 s-1) light intensities in Phaseolus vulgaris and Pisum sativum. With decreasing temperature, an increase in the fluorescence yield at both 685 and 735 nm was observed. A marked change occurred at the longer emission band resulting in a decrease in the Chl fluorescence ratio, F685/F735, with reducing T. Our fluorescence analysis suggests that this effect is due to a temperature-induced state 1-state 2 transition that decreases and increases photosystem II (PSII) and photosystem I (PSI) fluorescence, respectively. Time-resolved fluorescence life-time measurements support this interpretation. At a critical temperature (about 6 degrees C) and low light intensity a sudden decrease in fluorescence intensity was observed, with a larger effect at 685 than at 735 nm. This is probably linked to a modification of the thylakoid membranes, induced by chilling temperatures, which can alter the spill-over from PSII to PSI. The contribution of photosystem I to the long-wavelength Chl fluorescence band (735 nm) at room temperature was estimated by both time-resolved fluorescence lifetime and fluorescence yield measurements at 685 and 735 nm. We found that PSI contributes to the 735 nm fluorescence for about 40, 10 and 35% at the minimal (F0), maximal (Fm) and steady-state (Fs) levels, respectively. Therefore, PSI must be taken into account in the analysis of Chl fluorescence parameters that include the 735 nm band and to interpret the changes in the Chl fluorescence ratio that can be induced by different agents.     

Quantum effects in photosynthesis

In photosynthesis light is absorbed by the light-harvesting antenna and within several tens of picoseconds transferred to the photosynthetic reaction center (RC) where an ultrafast charge separation is initiated. Photosynthetic purple bacteria employ a single reaction center. In contrast, in photosynthesis of plants, algae and cyanobacteria, two reaction centers, Photosystem II (PSII) and Photosystem I (PSI), operate in series. PSII uses light to extract electrons from water (to produce oxygen); PSI uses light to reduce NADP + to NADPH. The electron transfer from PSII to PSI is coupled to the build-up of a proton motive force (pmf) that is used to form ATP. NADPH and ATP are required in the Calvin-Benson cycle to produce a reduced sugar. In the following we will discuss photosynthetic charge separation and photosynthetic light-harvesting with an emphasis on the role of quantum mechanics.     

Changes in the structure of chlorophyll-protein complexes and excitation energy transfer during photoinhibitory treatment of isolated photosystem I submembrane particles

The activity of light-induced oxygen consumption, absorption spectra, low temperature (77 K) chlorophyll fluorescence emission and excitation spectra were studied in suspensions of photosystem (PS) I submembrane particles illuminated by 2000 microE m(-2) s(-1) strong white light (WL) at 4 degrees C. A significant stimulation of oxygen uptake was observed during the first 1-4 h of photoinhibitory treatment, which rapidly decreased during further light exposure. Chlorophyll (Chl) content gradually declined during the exposure of isolated PSI particles to strong light. In addition to the Chl photobleaching, pronounced changes were found in Chl absorption and fluorescence spectra. The position of the major peak in the red part of the absorption spectrum shifted from 680 nm towards shorter wavelengths in the course of strong light exposure. A 6-nm blue shift of that peak was observed after 5-h illumination. Even more pronounced changes were found in the characteristics of Chl fluorescence. The magnitude of the dominating long-wavelength emission band at 736 nm located in untreated particles was five times reduced after 2-h exposure, whereas the loss in absolute Chl contents did not exceed 10% of its initial value. The major peak in low-temperature Chl fluorescence emission spectra shifted from 736 to 721 nm after 6-h WL treatment. Individual Chl-protein complexes differed in the response of their absorption spectra to strong WL. Unlike light-harvesting complexes (LHC), LHCI-680 and LHC-730, which did not exhibit changes in the major peak position, its maximum was shifted from 678 to 671 nm in CPIa complex after PSI submembrane particles were irradiated with strong light for 6 h. The results demonstrated that excitation energy transfer represents the stage of photosynthetic utilization of absorbed quanta which is most sensitive to strong light in isolated PSI particles.     

Protochlorophyllide phototransformation in the bundle sheath cells of Zea mays

The protochlorophyllide transformation process was investigated by using comparative analysis of 77 K fluorescence spectral changes occurring in isolated bundle sheath (BS) cells of etiolated Zea mays leaves after being exposed to a 200 ms saturating flash. Deconvolution analysis of the fluorescence spectra showed essential differences in the ratio of protochlorophyll(ides) and chlorophyll(ides) spectral forms indicating for BS cells to have a characteristic pathway of protochlorophyllide transformation. Bundle sheath cells showed a high ratio between non-photoactive protochlorophyll(ide)-F632 and photoactive protochlorophyllide-F655. In those cells, the 200 ms flash triggered a preferential formation of chlorophyll(ide)-F675 which remained stable in the dark for at least 90 min. Isolated BS cells showed an accumulation of chlorophyll(ide)-F675 resulting in the formation of inactive photosystem II. However for mesophyll cells of intact leaves, it was found to have a high ratio between photoactive and non-photoactive protochlorophyll(ide), showing the succession of chlorophyll(ide) forms usually known in C(3) plants. Protochlorophyllide phototransformation pathway in BS cells related to early stages of plastid differentiation triggered by light may indicate specific conditions for PSII assembly process leading to inactive PSII forms.     

THE PRIMARY REACTION IN THE PHOTOREDUCTION OF PROTOCHLOROPHYLLIDE MONITORED BY NANOSECOND FLUORESCENCE MEASUREMENTS

Abstract— Time resolved fluorescence measurements, carried out on protochlorophyllide reductase enriched membranes from oat ( Avena sativa ), are described. A fast (1 ns at 293 K) decaying fluorescence component is assigned to the photoactive NADPH-protochlorophyllide-enzyme complex, while a slower (5 ns) component is ascribed to non-photoactive protochlorophyllide. The results are interpreted in terms of a new fast primary step in the light requiring step of chlorophyll synthesis. The temperature dependence of the rate of this reaction has been studied by measuring the decay time of the fast fluorescence component at various temperatures from 77 to 293 K. Complete spectra of the kinetic fluorescence components have been measured at 293, 160 and 77 K.     

Fluorescence lifetimes of protochlorophyllide in plants with different proportions of short-wavelength and long-wavelength protochlorophyllide spectral forms

Dark-grown leaves of maize (Zea mays), wheat (Triticum aestivum), wild-type pea (Pisum sativum) and its light-independent photomorphogenesis mutant (lip1) have different proportions of protochlorophyllide (Pchlide) forms as revealed by low-temperature fluorescence emission spectra. Four discrete spectral forms of Pchlide, with emission peaks around 633, 640, 656 and 670 nm, could be distinguished after Gaussian deconvolution. In maize and wheat the 656 nm component was the most prominent, whereas for wild-type pea and its lip1 mutant, the 633 and 640 nm components contributed mostly to the fluorescence emission spectra. For the fluorescence lifetimes measured at 77 K a double exponential model was the most adequate to describe the Pchlide fluorescence decay not only for the Pchlide(650-656) form but also for the short-wavelength Pchlide forms. A fast component in the range 0.3-0.8 ns and a slow component in the range 5.1-7.1 ns were present in all samples, but the values varied, depending on species. The long-wavelength Pchlide(650-656) form had a slow component with a lifetime between 5.1 and 6.7 ns, probably reflecting the fluorescence from aggregated Pchlide. The short-wavelength Pchlide(628-633) form had values of the slow component varying between 6.2 and 7.1 ns. This represents a monomeric but probably protein-bound Pchlide form because the free Pchlide in solution has a much longer lifetime around 10 ns at 77 K. The contribution of different Pchlide forms to the measured lifetime values is discussed.     

Analysis of Fluorescence Lifetime of Protochlorophyllide and Chlorophyllide in Isolated Etioplast Membranes Measured from Multifrequency Cross-correlation Phase Fluorometry

The fluorescence decays of protochlorophyllide (Pchlide) and of chlorophyllide (Chlide) in wheat etioplast membranes were analyzed using a multiexponential fluorescence decay model. Using different excitation wavelengths from 430 to 470 nm, we found that a triple-exponential model at 14°C and a double-exponential model at — 170°C were adequate to describe the Pchlide fluorescence decay. We discuss the origin of the three fluorescence lifetime components at 14°C on the basis of the dependence of their fractional intensities on the excitation wavelength and by correlating the fractional intensities with integrated fluorescence intensities of different Pchlide forms in steady-state fluorescence spectra. The fluorescence decay of the main Pchlide form, photoactive Pchlide-F657, is shown to have a complex character with a fast component of 0.25 ns and a slower component of about 2 ns. Two lifetime components of 2 ns and 5.5–6.0 ns are ascribed to the second photoactive form, Pchlide-F645, and to nonphotoactive Pchlide forms, respectively. In etioplast membranes preilluminated by a short saturating light pulse, we found a single 5.0 ns component for Chlide-F688 (the Chlide-NADPH: protochlorophyllide oxidoreductase [PORJ-NADP⁺complex) and an additional 1.6 ns component when the formation of Chlide-F696 (the Chlide-POR-NADPH complex) was promoted by exogenous NADPH. From the fluorescence lifetime results we evaluated the quantum yield of the primary photoreaction by Chlide-F696 as being 70%.     

Carotenoid dependence of the protochlorophyllide to chlorophyllide phototransformation in dark-grown wheat seedlings.

The influence of carotenoids on partial protochlorophyllide (Pchlide) photoreduction and the successive formation of long-wavelength chlorophyllide (Chlide) forms was studied by low-temperature fluorescence spectroscopy (77 K). Wheat leaves with a decreased content of carotenoids obtained from norflurazon-treated seedlings (10 and 100 micromol l(-1)) were compared with leaves containing normal amounts of these pigments. Partial photoreduction of Pchlide was achieved by irradiation of the leaves with one light flash in combination with a number of neutral gray and/or red Perspex filters. There were significant differences between the fluorescence emission spectra (the position and height of the peaks) of dark-grown normal and carotenoid-deficient leaves irradiated with non-saturating white light of increasing intensity. The long-wavelength Chlide forms appeared first in the leaves nearly devoid of carotenoids (treated with 100 micromol l(-1) norflurazon), then in the leaves with carotenoid deficiency (treated with 10 micromol l(-1) norflurazon), and finally in normal leaves. After irradiation with non-saturating light of the same intensity, the ratio Chlide/Pchlide(657) was always the highest in the leaves nearly deficient of carotenoids, medium in the leaves with carotenoid deficiency and lowest in the normal leaves. Similarly to white light, red light of low intensity induced faster formation of long-wavelength Chlide species in the leaves with carotenoid deficiency in comparison to the normal leaves. We propose that, in leaves with reduced carotenoid content, a greater number of Pchlide molecules transform to Chlide per light flash than in normal leaves. The results are discussed in relation to the involvement of carotenoids in competitive absorption and light screening, as well as to their influence on Pchlide-Chlide interactions.