CHLOROPLAST PIGMENTS and THEIR BIOSYNTHESIS IN RELATION TO LIGHT INTENSITY*

Abstract— Depending on the light intensity that they received during growth, radish seedlings altered not only the pigment and quinone composition of the thylakoid membrane but also the chloroplast ultrastructure. In strong light, sun chloroplasts of radish were very similar to those from sun leaves of beech trees, while those developed under under dim light possessed a typical shade chloroplast. Radish shade chloroplasts contained a higher chlorophyll content and a higher concentration of xanthophylls resulting in a lower xanthophyll to carotene ratio as compared to sun chloroplasts. Chloroplasts from radish grown in strong light showed a much higher activity in their terpenoid metabolism than plastids from shade plants. Chlorophylls and carotenoids which are involved in the absorption of light and the transfer of energy during photosynthesis were labeled by [³H]-mevalonate to a much higher degree in plastids from sun leaves as compared to plastids from shade leaves. This shows that in strong light where pigments are continuously broken down and resynthesized in order to maintain photosynthesis, chlorophylls and carotenoids exhibit a much higher turnover rate than the pigments of shade plants.     

Reinvestigation of the triplet-minus-singlet spectrum of chloroplasts

A comparison of the triplet-minus-singlet (TmS) absorption spectrum of spinach chloroplasts, recorded some thirty years ago, with the more recently published TmS spectrum of isolated Chla/b LHCII (light-harvesting complexes associated with photosystem II of higher plants) shows that the two spectra are very similar, which is to be expected, since only the carotenoid pigments contribute to each spectrum. Be that as it may, the comparison also reveals a dissimilarity: photoexcitation of the sample does, or does not, affect the absorbance in the Qy region (650-700 nm), depending on whether the sample is a suspension of chloroplasts or of isolated LHCII. The Qy-signal in the TmS spectrum of LHCII decays, it should be noted, at the same rate as the rest of the difference spectrum, and its most prominent feature is a negative peak. As the carotenoids do not absorb in the Qy region, the presence of a signal in this region calls for an explanation: van der Vos, Carbonera and Hoff, the first to find as well as fathom the phenomenon, attributed the Qy-signal to a change, in the absorption spectrum of a chlorophyll a (Chla) molecule, brought about by the presence of triplet excitation on a neighbouring carotenoid (Car). The difference in the behaviours of chloroplasts and LHCII, if reproducible, would imply that the Car triplets which give rise to the TmS spectrum of chloroplasts do not influence the absorption spectra of their Chla neighbours. With a view to reaching a firm conclusion about this vexed issue, spinach chloroplasts and thylakoids have been examined with the aid of the same kinetic spectrometer as that used for investigating LHCII; the TmS spectra of both chloroplasts and thylakoids contain prominent bleaching signals centred at 680 nm, and the triplet decay time in each case is comparable to that of the Chla/b LHCII triplets. Results pertaining to other closely related systems are recalled, and it is concluded that, so far as the overall appearance of the TmS spectrum is concerned, spinach chloroplasts are by no means abnormal.     

Light absorption by the chlorophyll a-b complexes of photosystem II in a leaf with special reference to LHCII

To investigate the light-harvesting properties of the Photosystem II chlorophyll (chl) a-b complexes (major light-harvesting complex of Photosystem II [LHCII], CP24, CP26, CP29) in a mature leaf under natural "daylight" illumination, the absorption spectra of the isolated complexes were converted into the photon absorption spectrum (1-T) within a leaf, using the approach of Rivadossi et al. ([1999] Photosynth. Res. 60, 209-215). In the Qy region, significant enhancement of light harvesting by the chl b electronic transitions, with respect to the absorption spectra (optical density [OD]), as well as a large and generalized increase (between two- and four-fold) associated with the vibrational bands of both chl a and b, was observed, which acquires an important light-harvesting role (approximately 30-40% of total). In the Soret region, a small increase in light harvesting by chl b was indicated. To gain more detailed information on these aspects the light harvesting of LHCII in a leaf was investigated. This required describing the pigment absorption (chl a and b, carotenoids) in the LHCII OD spectrum in terms of spectral subbands, which were subsequently used to estimate the relative light harvesting of each pigment type in LHCII of a leaf. When the entire visible spectral interval between 400 and 730 nm is considered, the chl a light harvesting is essentially unchanged with respect to the absorption spectrum (OD) of isolated LHCII, whereas the chl b contribution is 20% higher and the carotenoids are 33% lower. The relative enhancement of the chl b absorption is principally associated with the Qy electronic transition region, the light-harvesting contribution of which becomes prominent in the leaf.     

Synthesis and Functional Reconstitution of Light‐Harvesting Complex II into Polymeric Membrane Architectures

One of most important processes in nature is the harvesting and dissipation of solar energy with the help of light‐harvesting complex II (LHCII). This protein, along with its associated pigments, is the main solar‐energy collector in higher plants. We aimed to generate stable, highly controllable, and sustainable polymer‐based membrane systems containing LHCII–pigment complexes ready for light harvesting. LHCII was produced by cell‐free protein synthesis based on wheat‐germ extract, and the successful integration of LHCII and its pigments into different membrane architectures was monitored. The unidirectionality of LHCII insertion was investigated by protease digestion assays. Fluorescence measurements indicated chlorophyll integration in the presence of LHCII in spherical as well as planar bilayer architectures. Surface plasmon enhanced fluorescence spectroscopy (SPFS) was used to reveal energy transfer from chlorophyll b to chlorophyll a, which indicates native folding of the LHCII proteins.     

Kinetic nature of the thermal destabilization of LHCII macroaggregates

The main light-harvesting chl a/b pigment-protein complex of photosystem II (LHCII) in isolated state forms macroaggregates with different ultrastructure and lipid content [I. Simidjiev, V. Barzda, L. Mustardy, G. Garab, Anal. Biochem. 250 (1997) 169-175]. The thermodynamic stability of highly delipidated tightly bound LHCII macroaggregates is studied by differential scanning calorimetry and fluorescence spectroscopy. The calorimetric profile of LHCII is asymmetric, the denaturation transition is taking place at around 72 degrees C. A shoulder, which overlaps with the main denaturation transition, appears around 58 degrees C. The denaturation temperature strongly depends on the scanning rate indicating the kinetic nature of the thermal destabilization of LHCII macroaggregates. The fluorescence data prove that the thermal denaturation of LHCII is an irreversible and kinetically controlled process.     

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.     

REGULATION OF CHLOROPLAST DEVELOPMENT BY RED AND BLUE LIGHT

There are specific differences between red and blue light greening of etiolated seedlings of Hordevm vulgare L. Blue light results in a different prenyl lipid composition of chloroplast as compared to red light of equal quanta density. This is documented by a much higher prenylquinone content, higher chlorophyll a/b ratios, and lower values for the ratio xanthophylls to carotenes (x/c). The photosynthetic activity of “blue light” chloroplasts (Hill reaction) is higher than that of “red light” chloroplasts. These differences in prenylquinone composition and Hill-activity are associated with a different ultrastructure of chloroplasts. “Red light” chloroplasts exhibit a much higher grana content than “blue light” chloroplasts. The difference in thylakoid composition, photosynthetic activity and chloroplast structure found between blue and red light greening are similar to those found between sun and shade leaves and those between plants grown under high and low light intensities.     

ORGANIZATION OF PIGMENT-PROTEIN COMPLEXES INTO MACRODOMAINS IN THE THYLAKOID MEMBRANES OF WILD-TYPE and CHLOROPHYLL fo-LESS MUTANT OF BARLEY AS REVEALED BY CIRCULAR DICHROISM

The organization of pigment-protein complexes into large chiral macrodomains was investigated in wild-type and chlorophyll b-less mutant thylakoid membranes of barley. The variations in the anomalous circular dichroism bands and in the angular-dependence of circular intensity differential scattering showed that in wild-type chloroplasts, the formation of macrodomains was governed by interactions of the light-harvesting chlorophyll alb complexes (LHCII). Two external factors could be identified which regulate the parameters of the anomalous circular dichroism signal: (i) electrostatic screening by divalent cations under conditions that favor membrane stacking and (ii) the osmotic pressure of the medium, which is suggested to affect the lateral interactions between complexes and influence the packing-density of particles. These two factors governed preferentially the negative and the positive anomalous circular dichroism signals, respectively. In the chlorina f-2 mutant thylakoid membranes, deficient in most chlorophyll b binding proteins, the formation of macrodomains which gave rise to the anomalous circular dichroism signals was still regulated by these same external factors. However, in the absence of major LHCII polypeptides the formation of macrodomains was apparently mediated by other complexes having weaker interaction capabilities. As a consequence, the size of the macrodomains under comparable conditions appeared smaller in the mutant than in the wild-type thylakoid membranes. Circular dichroism is a valuable probe for examining the long-range interactions between pigment-protein complexes which participate in the formation and stabilization of membrane ultrastruc-ture. A functional role of macrodomains in long-range energy migration processes is proposed.     

Equilibrium between quenched and nonquenched conformations of the major plant light-harvesting complex studied with high-pressure time-resolved fluorescence

Nonphotochemical quenching (NPQ) of chlorophyll fluorescence plays an important role in the protection of plants against excessive light. Fluorescence quenching of the major light-harvesting complex (LHCII) provides a model system to study the mechanism of NPQ. The existence of both quenched and nonquenched states of LHCII has been postulated. We used time-resolved fluorescence and hydrostatic pressure to study differences between these states. Pressure shifts the thermodynamic equilibrium between the two states. The estimated volume difference was 5 mL/mol, indicating a local conformational switch. The estimated free energy difference was 7.0 kJ/mol: high enough to keep the quenched state population low under normal conditions, but low enough to switch in a controlled way. These properties are physiologically relevant properties, because they guarantee efficient light harvesting, while at the same time maintaining the capacity to switch to a quenched state. These results indicate that conformational changes of LHCII can play an important role in NPQ.     

Assembly of Photosystem I and II during the Early Phases of Light-Induced Development of Chloroplasts from Proplastids in Spirodela oligorrhiza

The aquatic higher plant Spirodela oligorrhiza , which contains proplastids when grown in the dark, was used to study light-dependent chloroplast development. Low-temperature (77 K) and room temperature fluorescence were utilized in situ on whole plants to examine plastid development. The dark-grown plants contain two 77 K fluorescence peaks, at 633 nm (F633) and at 657 nm (F657), with F633 dominating. The F657 species represents protochlorophyllide that is bound to protochloro-phyllide oxidoreductase. It was rapidly phototrans-formed to chlorophyllide (within 5 s) via a monomolec-ular reaction. Free protochlorophyllide (F633) was converted to chlorophyllide during a 3 h exposure to light. Photosystem (PS) assembly in Spirodela could be detected 2 h after the plants were first exposed to light, with the PSII reaction center (77 K fluorescence at 684 nm) appearing slightly before the PSI reaction center (77 K fluorescence at 725 nm). After the first reaction centers were formed the antenna complexes were added; the light-harvesting complex (LHC) I of PSI appeared after 8 h, and 47 kDa chlorophyll protein of PSII appeared between 12 h and 24 h. After 30 h of exposure to light, the plants acquired the ability to perform a light state transition, marking the appearance of functional LHCII complexes in the developing chloroplast. Finally, it was found that photosynthetic activity, as measured by room temperature chlorophyll fluorescence, accelerated con-comitantly with detection of the antenna complexes. Therefore, although reaction centers are detected very early during the proplastid to chloroplast conversion, they may have little activity or be unstable until the antennae are present.     

Changes in the room-temperature emission spectrum of chlorophyll during fast and slow phases of the Kautsky effect in intact leaves

Changes in the room-temperature emission spectrum of chlorophyll (Chl) were analyzed using fast diode-array recordings during the Kautsky effect in mature and in greening barley leaves. In mature leaves, the comparison of F(O) (basal level of fluorescence yield at transient O) and F(M) (maximum level of fluorescence yield at transient M) spectra showed that the relative amplitude of total variable fluorescence was maximal for the 684 nm Photosystem II (PSII) band and minimal for the 725 nm Photosystem I band. During the increase from F(O) to F(M), a progressive redshift of the spectrum of variable fluorescence occurred. This shift reflected the different fluorescence rise kinetics of different layers of chloroplasts inside the leaf. This was verified by simulating the effect of screening on the emission spectrum of isolated chloroplasts and by experiments on greening leaves with low Chl content. In addition, experiments performed at different greening stages showed that the presence of uncoupled Chl at early-greening stages and light-harvesting complex II (LHCII) at later stages have detectable but minor effects on the shape of room-temperature emission spectra. When strong actinic light was applied to mature green leaves, the slow fluorescence yield, which declined from F(M) to F(T) (steady-state level of fluorescence yield at transient T), was accompanied by a slight redshift of the 684 nm PSII band because of nonphotochemical quenching of short-wavelength-emitting Chl ascribed to LHCII.     

THE LIGHT REQUIREMENT OF ACID-BASE TRANSITION INDUCED LUMINESCENCE OF CHLOROPLASTS

Abstract— The luminescence that occurs when chloroplasts are taken from an acid environment to a basic one is shown to be dependent on prior illumination of the chloroplasts. The relation between the light absorbed and luminescence is given by the following equation L = L _max(1-e ^al where L and L _max are the light emitted and maximum light emission at high flash energy, respectively, J quanta absorbed per chlorlphyll molecule, and α a constant with a value of approximately 200 chlorophyll molecules per quanta absorbed. The action spectrum of the luminescence is consistent with that of photosystem II. The metastable state formed during illumination decays in the dark via a temperature dependent second order process.     

Determination of rare earth elements in chloroplasts of Brassia napus by INAA and biochemical separation techniques

Intact chloroplasts were isolated from mesophyll protoplasts of Brassia napus. Concentrations of 8 rare earth elements (REEs) in the chloroplasts were determined by instrumental neutron activation analysis (INAA). The results showed that there were trace amounts of REEs in the chloroplasts, which corresponded to 1 atom of REEs per 2000 chlorophyll molecules. About 30% of the total REEs in the leaves are localized in the chloroplasts and the light REEs were enriched with respect to the heavy elements of the series.     

Energy transfer between surface-immobilized light-harvesting chlorophyll a/b complex (LHCII) studied by surface plasmon field-enhanced fluorescence spectroscopy (SPFS)

The major light-harvesting chlorophyll a/b complex (LHCII) of the photosynthetic apparatus in green plants can be viewed as a protein scaffold binding and positioning a large number of pigment molecules that combines rapid and efficient excitation energy transfer with effective protection of its pigments from photobleaching. These properties make LHCII potentially interesting as a light harvester (or a model thereof) in photoelectronic applications. Most of such applications would require the LHCII to be immobilized on a solid surface. In a previous study we showed the immobilization of recombinant LHCII on functionalized gold surfaces via a 6-histidine tag (His tag) in the protein moiety. In this work the occurrence and efficiency of Fo?rster energy transfer between immobilized LHCII on a functionalized surface have been analyzed by surface plasmon field-enhanced fluorescence spectroscopy (SPFS). A near-infrared dye was attached to some but not all of the LHC complexes, serving as an energy acceptor to chlorophylls. Analysis of the energy transfer from chlorophylls to this acceptor dye yielded information about the extent of intercomplex energy transfer between immobilized LHCII.     

Isolation and characteristics of the PSI-LHCI-LHCII supercomplex under high light

We developed a novel method for the isolation of the PSI-LHCI-LHCII complex from spinach leaves. The supercomplex was resolved into a core complex (CPI), LHCII trimers, LHCI dimers and LHCII monomers using green gel electrophoresis. We then investigate changes in the fluorescence and absorption spectra of PSI-LHCI-LHCII under high light. In addition, we compared light-induced denaturation of the core protein subunits in both PSI-LHCI and PSI-LHCI-LHCII. Differences in denaturation and photochemical activity indicated that binding of LHCII increased the photosensitivity of the PSI core. Increased energy delivered to the PSI core during illumination accelerated damage to the core complex.     

Different crystal morphologies lead to slightly different conformations of light-harvesting complex II as monitored by variations of the intrinsic fluorescence lifetime

In 2005, it was found that the fluorescence of crystals of the major light-harvesting complex LHCII of green plants is significantly quenched when compared to the fluorescence of isolated LHCII (A. A. Pascal et al., Nature, 2005, 436, 134-137). The Raman spectrum of crystallized LHCII was also found to be different from that of isolated LHCII but very similar to that of aggregated LHCII, which has often been considered a good model system for studying nonphotochemical quenching (NPQ), the major protection mechanism of plants against photodamage in high light. It was proposed that in the crystal LHCII adopts a similar (quenching) conformation as during NPQ and indeed similar changes in the Raman spectrum were observed during NPQ in vivo (A. V. Ruban et al., Nature, 2007, 450, 575-579). We now compared the fluorescence of various types of crystals, differing in morphology and age. Each type gave rise to its own characteristic mono-exponential fluorescence lifetime, which was 5 to 10 times shorter than that of isolated LHCII. This indicates that fluorescence is not quenched by random impurities and packing defects (as proposed recently by T. Barros et al., EMBO Journal, 2009, 28, 298-306), but that LHCII adopts a particular structure in each crystal type, that leads to fluorescence quenching. Most interestingly, the extent of quenching appears to depend on the crystal morphology, indicating that also the crystal structure depends on this crystal morphology but at the moment no data are available to correlate the crystals' structural changes to changes in fluorescence lifetime.     

BLUE AND WHITE LIGHT EFFECTS ON CHLOROPLAST DEVELOPMENT IN A SOYBEAN MUTANT

Phenotypic difference for chloroplast development between the normal green (CL1) and the Cy₉y₉ soybean mutant was observed when the plants were grown under 18W m^?2 white or blue light. Under these conditions the mutant soybean accumulated less Chi b, neoxanthin, carotene and less total pigment than the CL1 genotype. Chloroplasts of the Cy₉y₉ line were deficient in the LHP complex relative to that of chloroplasts from the normal soybean. Specific differences were noted between chloroplasts from plants grown under blue and white light. Accumulations of a 34 kD (PSII) and a 16–17 kD (PSI) membrane polypeptide were decreased by blue light in both soybean genotypes. Blue light induced a greater accumulation of a 32 kD (PSII) polypeptide than white light. Blue light reduced granal thylakoid stacking and increased the proportion of stroma thylakoids compared to those that developed under white light. PSI electron transport activity was stimulated by the blue light treatment more than that of PSII.     

Circular Polarized Light Activated Chiral Satellite Nanoprobes for the Imaging and Analysis of Multiple Metal Ions in Living Cells

Here, we construct a handedness‐dependent circular polarized light (CPL)‐activated chiral satellite assemblies formed from DNAzymes and spiny platinum modified with gold nanorods and upconversion nanoparticles (UCNPs), enabling the simultaneous quantitative analysis of multiple divalent metal ions in living cells. The chiral nanoprobes, in coordination with their corresponding divalent metal ions under 980 nm left circular polarized (LCP) light illumination, served as an in situ confocal bioimaging platform for the quantitation of the given intracellular metal ions. The limit of detection (LOD) of the chiral probes in living cells is 1.1 nmol/10⁶ cells, 1.02 nmol/10⁶ cells and 0.45 nmol/10⁶ cells for Zn²⁺, Mg²⁺, and Cu²⁺, respectively.     

A METHOD FOR MEASURING PHYTOCHROME IN PLANTS GROWN IN WHITE LIGHT

Abstract— Quantitative spectrophotometric measurement of phytochrome in plants grown for relatively long periods of time in white light is not possible due to the presence of chlorophyll. A method is described that prevents the accumulation of chlorophyll permitting such direct spectrophotometric measurement in light-grown tissue. Oat seedlings grown in the presence of the herbicide San 9789† (Norflurazon) for 6 days in constant light have considerably less chlorophyll than etiolated seedlings exposed to 1 min of light. Phytochrome concentrations measured in vivo and in vitro in these herbicide-treated plants were found to be about 2% of the level in etiolated tissue.