Optical properties and nondestructive estimation of anthocyanin content in plant leaves.

Absorption and reflectance spectra of maple (Acer platanoides), cotoneaster (Cotoneaster alaunica), dogwood (Cornus alba) and pelargonium (Pelargonium zonale) leaves with a wide range of pigment content and composition were studied in visible and near-infrared spectra in order to reveal specific anthocyanin (Anth) spectral features in leaves. Comparing absorption spectra of Anth-containing and Anth-free leaves with the same chlorophyll (Chl) content, absorption spectra of Anth in leaves were derived. The main spectral feature of Anth absorption in vivo was a peak around 550 nm; the peak magnitude was closely related to Anth content. A quantitative nondestructive technique was developed to subtract Chl contribution to reflectance in this spectral region and retrieve Anth content from reflectance over a wide range of pigment content and composition. Anth reflectance index in the form ARI = (R550)-1 - (R700)-1, where (R550)-1 and (R700)-1 are inverse reflectances at 550 and 700 nm, respectively, allowed an accurate estimation of Anth accumulation, even in minute amounts, in intact senescing and stressed leaves.     

New vegetation indices for remote measurement of chlorophylls based on leaf directional reflectance spectra

Directional reflectance (R) spectra from 380 to 780 nm for nadir illuminated leaves of four different plants (croton, Codiaeum variegatum; spotted eleagnus, Eleagnus pungens Maculata; Japanese pittosporum, Pittosporum tobira and Benjamin fig, Ficus benjamina Starlight) were acquired at a viewing angle of 30 degrees from the nadir direction. Chlorophyll-a and -b content of leaves covered a range of 1-60 and 0.5-21 microg/cm(2), respectively. In contrast with previous results from hemispherical reflectance measurements, directional reflectance data does not correlate well with chlorophyll concentration. This is mainly due to the external reflectance (R(E)) at the leaf epidermis, caused by the mismatch of the refractive index at the air-epidermis and epidermis-inner layer boundary. The external reflectance can be identified with the blue flat reflectance between 380 and 480 nm. The inner reflectance (R(I)), obtained by subtracting the external reflectance from the measured spectra, was found to be linearly related to the logarithm of the chlorophyll content. Good fitting of the log (Chl) versus R(I)(lambda) curves were obtained for R(I) in the green band (around 550 nm) and close to the inflection point in the red edge (around 700 nm). The coefficient of determination, r(2), of curve fitting improved (up to 0.97) when the normalised inner reflectance NR(I)(lambda)=R(I)(lambda)/R(I)(lambda(0)), with lambda(0)>or=750 nm, was used instead of the absolute reflectance. The best indices for Chl, Chl-a and Chl-b determination were R(I)(542)/R(I)(750), R(I)(706)/R(I)(750) and R(I)(556)/R(I)(750), respectively. However, since the content of Chl-a relative to Chl-b was almost constant for the plants investigated, the two last indices must be further validated on leaves with a high variability in the Chl-a:Chl-b ratio. The error in the determination of chlorophyll content was found to be of the order of 10%. This value was lower than those obtained by applying the vegetation indices previously suggested. Therefore, the normalised inner reflectance in the green and in the red edge represents a more suitable index for the chlorophyll determination than those up to now used.     

In vivo noninvasive detection of chlorophyll distribution in cucumber (Cucumis sativus) leaves by indices based on hyperspectral imaging

The objective of this study was to investigate the spectral behavior of the relationship between reflectance and chlorophyll content and to develop a technique for non-destructive chlorophyll estimation and distribution in leaves using hyperspectral imaging. The hyperspectral imaging data cube of cucumber (Cucumis sativus) leaves in the range of 450-850 nm was investigated and preprocessed. Sixty optical signatures or indices as a function of the associated reflectance (R(λ)) at the special wavelength (λ) nm which proposed in the literatures were used to predict the total chlorophyll content in cucumber leaves. Finally, R(710)/R(760), (R(780)-R(710))/(R(780)-R(680)), (R(750)-R(705))/(R(750)+R(705)), (R(680)-R(430))/(R(680)+R(430)), R(860)/(R(550)×R(708)), (R(695-705))(-1)-(R(750-800))(-1), and REP-LEM (a index based on red edge position and estimated with a linear extrapolation method) were identified as optimum indices. Red-edge waveband (680-780 nm) appeared in all these optimum indices, indicating the importance of REP (red edge position) in chlorophyll estimation. When (R(695-705))(-1)-(R(750-800))(-1), the best index was applied to an independent validation set, chlorophyll content (r=0.8286) were reasonably well predicted, indicating model robustness. Depending on the sample, this technique enables to identify and characterize the relative content of various chlorophyll that distribution in the cucumber leaves. The map shows a relatively low level of chlorophyll at margins. Higher level can be noticed in the regions along the main veins and in some areas exhibiting dark green tissue. Our results indicate that hyperspectral imaging has considerable promise for predicting pigments in leaves and, the pigments can be detected in situ in living plant samples non-destructively.     

Time-resolved two-photon spectroscopy of photosystem I determines hidden carotenoid dark-state dynamics

We present time-resolved fs two-photon pump-probe data measured with photosystem I (PS I) of Thermosynechococcus elongatus. Two-photon excitation (lambda(exc)/2 = 575 nm) in the spectral region of the optically forbidden first excited singlet state of the carotenoids, Car S1, gives rise to a 800 fs and a 9 ps decay component of the Car S1 --> S(n) excited-state absorption with an amplitude of about 47 +/- 16% and 53 +/- 10%, respectively. By measuring a solution of pure beta-carotene under exactly the same conditions, only a 9 ps decay component can be observed. Exciting PS I at exactly the same spectral region via one-photon excitation (lambda(exc) = 575 nm) also does not show any sub-ps component. We ascribe the observed constant of 800 fs to a portion of about 47 +/- 16% beta-carotene states that can potentially transfer their energy efficiently to chlorophyll pigments via the optically dark Car S1 state. We compared these data with conventional one-photon pump-probe data, exciting the optically allowed second excited state, Car S2. This comparison demonstrates that the fast dynamics of the optically forbidden state can hardly be unravelled via conventional one-photon excitation only because the corresponding Car S1 populations are too small after Car S2 --> Car S1 internal conversion. A direct comparison of the amplitudes of the Car S1 --> S(n) excited-state absorption of PS I and beta-carotene observed after Car S2 excitation allows determination of a quantum yield for the Car S1 formation in PS I of 44 +/- 5%. In conclusion, an overall Car S2 --> Chl energy-transfer efficiency of approximately 69 +/- 5% is observed at room temperature with 56 +/- 5% being transferred via Car S2 and probably very hot Car S1 states and 13 +/- 5% being transferred via hot and "cold" Car S1 states.     

Spectral characterization of chlorophyll fluorescence in barley leaves during linear heating. Analysis of high-temperature fluorescence rise around 60 degrees C

The spectral characteristics of chlorophyll fluorescence and absorption during linear heating of barley leaves within the range 25-75 degreesC (fluorescence temperature curve, FTC) were studied. Leaves with various content of light harvesting complexes (green, Chl b-less chlorina f2 and intermittent light grown) revealing different types of FTC were used. Differential absorption, emission and excitation spectra documented four characteristic phases of the FTC. The initial two FTC phases (a rise in the 46-49 degreesC region and a subsequent decrease to about 55 degreesC) mostly reflected changes in the fluorescence quantum yield peaking at about 685 nm. A steep second fluorescence rise at 55-61 degreesC was found to originate from a short-wavelength Chl a spectral form (emission maximum at 675 nm) causing a gradual blue shift of the emission spectra. In this temperature range, a clear correspondence of the blue shift in the emission and absorption spectra was found. We suggest that the second fluorescence rise in FTC reflects a weakening of the Chl a-protein interaction in the thylakoid membrane.     

Pre-visual detection of iron and phosphorus deficiency by transformed reflectance spectra

Reflectance spectroscopy and strategies for spectral analysis over the visible range from 380 to 780 nm were used to provide diagnostic information on iron (Fe) and phosphorus (P) status of Brassica chinensis L. var parachinensis (Bailey) grown under hydroponics conditions. Leaf reflectance (R) spectra were collected and normalized inner reflectance (NR(I)) spectra were calculated. The regression coefficients (B-matrix) and variable importance for projection (VIP) in partial least squares regression were used to determine important wavelengths that correlate with total chlorophyll (Chl) content. No single wavelength that showed good correlation with Chl content was found. Therefore, NR(I) was transformed into CIELAB color values, which simplified the whole visible spectrum into three values. Our results showed that upon Fe deprivation, plants entered into a deficiency state very rapidly, highlighting the importance of early diagnosis. The direct effect of Fe on leaf Chl content allowed CIELAB color values to be used for pre-visual detection of Fe deficiency 2 days before the appearance of visually distinguishable morphological changes. On the other hand, P-deprived plants showed a marked decline in cellular P levels but remained above critical threshold concentrations after 7 days. The Chl content was not affected by the leaf P content and CIELAB color values showed no difference with control plants.     

Multichannel flash spectroscopy of the reaction centers of wild-type and mutant Rhodobacter sphaeroides: bacteriochlorophyllB-mediated interaction between the carotenoid triplet and the special pair

Multichannel flash spectroscopy (with microsecond time resolution) has been applied to carotenoid (Car)-containing and Car-less reaction centers (RC) of Rhodobacter sphaeroides with a view to investigate the interaction between the Car and its neighboring pigments at room temperature. Under neutral redox potential conditions, where the primary quinone acceptor (QA) is oxidized, the light-induced spectral changes in the 350-1000 nm region are attributed to the photochemical oxidation of the special pair (denoted here as P870), the generation of P870(+)QA(-), and the attendant electrochromism of adjacent chromophores. A bathochromic shift of <1 nm in the visible absorption region of Car reveals the sensitivity of Car to the P870 photooxidation. Under low redox potential conditions, where QA is reduced, P870 triplets (P870(+)) are formed. The time-resolved triplet-minus-singlet (TmS) spectrum of Car-less RC shows a deep bleaching at 870 nm, which belongs to P870(+), and additional (but smaller) bleaching at 800 nm; the entire spectrum decays at the same rate (with a lifetime of about 50 micros). The bleaching at 800 nm arises from the pigment interaction between P870(+) and the accessory bacteriochlorophylls on A and B branches (BA,B). In Car-containing RC, the TmS spectra of Car are accompanied by two smaller, negative signals--a sharp peak at 809 +/- 2 nm and a broad band at 870 nm--which decay at the same rate as the TmS spectrum of Car (ca 10 micros). The former is ascribed to the perturbation, by Car(+), of the absorption spectrum of BB; the latter, to the TmS spectrum of P870(+), a species that appears to be in approximate thermal equilibrium with Car(+). These assignments are consistent with the absorption-detected magnetic resonance spectra obtained by other workers at low temperatures.     

CHANGES IN SPECTRAL PROPERTIES OF CHLOROPHYLL DURING GREENING OF BARLEY LEAVES

Abstract— In order to study the formation of photochemical systems in the early phase of greening of barley leaves, we examined the changes in chlorophyll (Chl) spectral components of the absorption spectrum of greening tissues using a curvc analysis method. Peak positions of the fourth-derivative spectrum were changing during the first 30 min of illumination hut became stable thereafter. Results of curve analysis showed that about half of the spectral components of mature leaves were already found at 30 min after onset of illumination, and the rest appeared by 4 h. After 30 min the half widths of some spectral componcnts changed slightly. The 683 nm form of Chl a of the photosystem 11 (PSII) core was present after 30 min of illumination and increased up to 2 h, followed by a decrease. The 641 nm form of Chl u of photosystem I (PSI) appeared at 30 min and increased until 2.5 h. The 699 nm form of Chl a also appeared at 1.5 h. The 640 nm form of Chl b appeared at 2 h, while the 649 nm Chl b form was found earlier (1.5 h). Both components were increasing with prolonged time of illumination. Finally, the 704 nm form of Chl a characteristic of the PSI peripheral antenna appeared at 4 h of greening. Ruorescence of PSI and PSII began to appear at 1 and 2 h, respectively. These spectral changes were discussed in relation to the formation of chlorophyll-protein complexes and the development of photochemical activities.     

IMMEDIATE PIGMENT DARKENING: VISUAL AND REFLECTANCE SPECTROPHOTOMETRIC ANALYSIS OF ACTION SPECTRUM

Immediate pigment darkening (IPD) occurs in human skin upon exposure to ultraviolet-A and visible radiation. The spectral changes that occur during IPD were measured with a rapid scanning reflectance spectrophotometer (RS) which employs optical fiber bundles for delivery and detection of light between 400 and 750 nm. The radiation dose dependence and wavelength dependence (334-549 nm irradiation) of IPD were studied by both the classical visual grading method and by spectrophotometric scoring using the RS system. The spectral changes that occur at long wavelengths with IPD mimic the natural absorption spectrum of melanin. Therefore, the IPD was scored in terms of the apparent change in melanin optical density, using the method Kollias and Baqer [Photochem. Photobiol. 43, 49-54 (1986)], based on reflectance in the 620-720 nm range. The nonlinearity of the visual grading method is demonstrated. The degree of IPD is first-order with respect to delivered dose and saturates after high doses. The maximum amount of IPD attained at saturation is greater for shorter wavelengths. Extrapolation of the reflectance data suggests the longest wavelength capable of eliciting IPD is about 470 nm.     

Assignment of the lowest Qy-state and spectral dynamics of the CP29 chlorophyll a/b antenna complex of green plants: a hole-burning study

Low-temperature absorption, fluorescence and persistent non-photochemical hole-burned spectra are reported for the CP29 chlorophyll (Chl) a/b antenna complex of photosystem II of green plants. The absorption-origin band of the lowest Qy-state lies at 678.2 nm and carries a width of approximately 130 cm-1 that is dominated by inhomogeneous broadening at low temperatures. Its absorption intensity is equivalent to that of one of the six Chl a molecules of CP29. The absence of a significant satellite hole structure produced by hole burning, within the absorption band of the lowest state, indicates that the associated Chl a molecule is weakly coupled to the other Chl and, therefore, that the lowest-energy state is highly localized on a single Chl a molecule. The electron-phonon coupling of the 678.2 nm state is weak with a Huang-Rhys factor S of 0.5 and a peak phonon frequency (omega m) of approximately 20 cm-1. These values give a Stokes shift (2S omega m) in good agreement with the measured positions of the absorption band at 678.2 nm and a fluorescence-origin band at 679.1 nm. Zero-phonon holes associated with the lowest state have a width of approximately 0.05 cm-1 at 4.2 K, corresponding to a total effective dephasing time of approximately 400 ps. The temperature dependence of the zero-phonon holewidth indicates that this time constant is dominated at temperatures below 8 K by pure dephasing/spectral diffusion due to coupling of the optical transition to the glass-like two-level systems of the protein. Zero-phonon hole-widths obtained for the Chl b bands at 638.5 and 650.0 nm, at 4.2 K, lead to lower limits of 900 +/- 150 fs and 4.2 +/- 0.3 ps, respectively, for the Chl b-->Chl a energy-transfer times. Downward energy transfer from the Chl a state(s) at 665.0 nm occurs in 5.3 +/- 0.6 ps at 4.2 K.     

Carotenoid radical cations as a probe for the molecular mechanism of nonphotochemical quenching in oxygenic photosynthesis

Nonphotochemical quenching (NPQ) is a fundamental mechanism in photosynthesis which protects plants against excess excitation energy and is of crucial importance for their survival and fitness. Recently, carotenoid radical cation (Car*+) formation has been discovered to be a key step for the feedback deexcitation quenching mechanism (qE), a component of NPQ, of which the molecular mechanism and location is still unknown. We have generated and characterized carotenoid radical cations by means of resonant two color, two photon ionization (R2C2PI) spectroscopy. The Car*+ bands have maxima located at 830 nm (violaxanthin), 880 nm (lutein), 900 nm (zeaxanthin), and 920 nm (beta-carotene). The positions of these maxima depend strongly on solution conditions, the number of conjugated C=C bonds, and molecular structure. Furthermore, R2C2PI measurements on the light-harvesting complex of photosystem II (LHC II) samples with or without zeaxanthin (Zea) reveal the violaxanthin (Vio) radical cation (Vio*+) band at 909 nm and the Zea*+ band at 983 nm. The replacement of Vio by Zea in the light-harvesting complex II (LHC II) has no influence on the Chl excitation lifetime, and by exciting the Chls lowest excited state, no additional rise and decay corresponding to the Car*+ signal observed previously during qE was detected in the spectral range investigated (800-1050 nm). On the basis of our findings, the mechanism of qE involving the simple replacement of Vio with Zea in LHC II needs to be reconsidered.     

Origin of chlorophyll fluorescence in plants at 55-75 degrees C

The origin of heat-induced chlorophyll fluorescence rise that appears at about 55-60 degrees C during linear heating of leaves, chloroplasts or thylakoids (especially with a reduced content of grana thylakoids) was studied. This fluorescence rise was earlier attributed to photosystem I (PSI) emission. Our data show that the fluorescence rise originates from chlorophyll a (Chl a) molecules released from chlorophyll-containing protein complexes denaturing at 55-60 degrees C. This conclusion results mainly from Chl a fluorescence lifetime measurements with barley leaves of different Chl a content and absorption and emission spectra measurements with barley leaves preheated to selected temperatures. These data, supported by measurements of liposomes with different Chl a/lipid ratios, suggest that the released Chl a is dissolved in lipids of thylakoid membranes and that with increasing Chl a content in the lipid phase, the released Chl a tends to form low-fluorescing aggregates. This is probably the reason for the suppressed fluorescence rise at 55-60 degrees C and the decreasing fluorescence course at 60-75 degrees C, which are observable during linear heating of plant material with a high Chl a/lipid ratio (e.g. green leaves, grana thylakoids, isolated PSII particles).     

Normal-phase HPLC separation of possible biosynthetic intermediates of pheophytin a and chlorophyll a'.

Normal-phase HPLC conditions have been developed for separating the C17(3) isoprenoid isomers, which are expected to be formed as biosynthetic intermediates of chlorophyll (Chl) a, Chl a' (C13(2)-epimer of Chl a), pheophytin (Pheo) a and protochlorophyll (PChl). The application of these conditions to pigment composition analysis of greening etiolated barley leaves allowed us to detect, for the first time, the C17(3) isomers of Chl a', a possible constituent of the primary electron donor of photosystem (PS) I, P700, and those of Pheo a, the primary electron acceptor of PS II, in the very early stage of greening. The C17(3) isomer distribution patterns were approximately the same between Chl a and Chl a', but significantly different between Pheo a and Chl a', probably reflecting the similarity and difference, respectively, in the biosynthetic pathways of these pigment pairs.     

Characterization of crude oils using fluorescence lifetime data

The average fluorescence lifetimes of nine North Sea crude oils with API gravities of between 20 and 51 were measured using a modular, filter based, instrument developed in-house. Two pulsed light emitting diode (LED) excitation sources (460 and 510 nm) were used to excite fluorescence, the lifetime of which was measured at a range of emission wavelengths. Fluorescence lifetimes were found to vary from 1.8 to 8.2 ns with confidence intervals of +/- 0.11 ns. The average lifetimes at all emission wavelengths were linearly correlated with API gravity and with aromatic concentration with the best results being obtained with the 460 nm excitation source. Predictive models with an accuracy of +/- 7.6 API degrees were generated using partial least-squares methods from average fluorescence lifetimes measured at an emission wavelength of 500 nm using 460 nm excitation. A better correlation was found between the aromatic concentration of the oils and the ratio of the average fluorescence lifetimes at measured at 550 and 650 nm using 460 nm excitation. This led to a quantitative model with an accuracy of +/- 5.4% for aromatic concentration.     

ON THE INTERPRETATION OF ABSORPTION SPECTRA OF LEAVES–I. INTRODUCTION and THE CORRECTION OF LEAF SPECTRA FOR SURFACE REFLECTION

Abstract— Reflectance and transmittance spectra of leaves and their sum can be corrected to relate only to the light actually entering the leaf, if the reflectance of the epidermal surface is known. The latter is found if the leaf reflectances at several wavelengths near the transmittance minimum in the red are plotted vs the transmittances of a homogeneous suspension of the native pigment-proteins at the same chlorophyll content per unit area and at the same wavelengths. With non-senescent leaves, the relation is linear and the extrapolation of the pigment transmittance to zero gives the value for the surface reflection. Surface reflectance data (both adaxial and abaxial) are given for the leaves of a number of trees and a few herbs, plus examples of the raw and corrected spectra. With normal, glaucous leaves, the adaxial reflectance averaged 4.5% of the incident light ( n = 23, range = 3.7 −5.9, standard deviation = 0.4). The reflectances of the abaxial surfaces ranged between 7 and 13% since additional near-surface reflection occurred at the inside of the epidermis and in the spongy mesophyll. Reflectance and transmittance data demonstrated strong absorption in the epidermis below 480 nm.     

Non-invasive Monitoring of Biochemical Response of Wheat Seedlings Toward Titanium Dioxide Nanoparticles Treatment Using Attenuated Total Reflectance Fourier Transform Infrared and Laser Induced Fluorescence Spectroscopy

Widespread commercial application of titanium dioxide nanoparticles leads to their dispersion in the environment and inevitable interaction with living organisms. Their presence necessitates the monitoring of nanoparticle interactions with plants using advanced techniques that are capable of noninvasively and sensitively estimating the changes involved in the biochemical profile. The current study aims to investigate the effects of titanium dioxide nanoparticles on biochemicals of wheat leaves using label free, nondestructive, rapid, sensitive, and advanced spectroscopic probes: laser induced fluorescence and attenuated total reflectance Fourier transform infrared spectroscopy coupled with multivariate analysis. The fluorescence and infrared spectra of control and titanium dioxide nanoparticle treated wheat leaves were acquired in the region from 400 to 800?nm and 4000 to 485?cm^?1. The treatment of titanium dioxide nanoparticles decreases the chlorophyll content and the concentrations of cellulose, hemicellulose, xyloglucans, pectin, and lignin indicating interferences in the biosynthesis and structure of cell walls of the wheat leaves. The level of amide I, carbonyl, and methylene groups also increases following the treatment of titanium dioxide nanoparticles indicating lipid and protein peroxidation and the accumulation of carbonyl compounds. The changes in the integrated area ratios of the amide II/amide I, carbonyl/methyl, and methylene/amide II bands demonstrate disorder in the membrane integrity. This study establishes the efficiency of noninvasive, label-free, and rapid protocols based on attenuated total reflectance Fourier transform infrared and laser induced fluorescence to monitor the interactions of nanoparticles with plants at early stage of plant growth before visual signs of toxicity appear.     

表面吸附质对银亚胶体吸光特性的影响

纯银溶胶在390nm处有一吸收峰。当银溶胶吸附了1-苯基-5-巯基四氮唑(PMT)或2-巯基苯骈噻唑(MBT)时, 银溶胶由亮黄色转变为橙红色, 即在510～550nm处出现一个新的吸收峰。研究发现, 卤素离子在银溶胶颗粒上与PMT和MBT有竞争吸附作用。但是卤离子对银溶胶的光谱吸收的影响完全不同于PMT和MBT。在讨论这种差别时应首先考虑金属银溶胶颗粒表面性质因吸附不同物质所产生的变化。     

Non-destructive Phenotyping of Chili Pepper Ripening Using Spectroscopic Probes: A Potential Approach for Shelf-Life Measurement

This study explores the application of spectroscopic techniques (laser induced fluorescence, Raman and attenuated total reflectance Fourier transform infrared) coupled with principal component analysis for the nondestructive, extraction free and rapid evaluation of biochemical changes associated with ripening of chili peppers at four stages (mature, pre-ripe, ripe and post-ripe). The analysis of the fluorescence spectra of the exocarp of chili pepper shows a decrease in the intensity of chlorophyll bands at 685 and 735?nm and an increase in the intensity of carotenoid fluorescence bands at 490–500?nm and 565–580?nm with progress in the ripening stages. These changes are regarded to be significant phenotypic markers for the ripening of chili peppers. The observed changes in the position of carotenoid bands in Raman spectra at 1004, 1156, 1188, and 1524?cm^?1 with increase in their intensity indicating the accumulation of carotenoids and change in the carotenoid composition from β-carotene in the mature chilis to capsanthin in the ripe chilis. In addition, the infrared spectra show changes in the carbohydrates, amide II, amide I and cutin at various stages of ripening. Also, the variation in the position of pectin bands indicates change in its molecular mass with decreasing content. The determined spectral signatures can be used as biomolecular index for effective monitoring of the ripening of chili peppers. The commercial application of noninvasive spectroscopic probes will be advantageous for the phenotyping of economically important plant parts, screening, grading, shelf life estimation and quality standardization.     

Light-stress-induced pigment changes and evidence for anthocyanin photoprotection in apples

Fruit of two apple (Malus domestica Borkh.) cultivars, differing in their ability to produce anthocyanin pigments when exposed to sunlight, have been studied using reflectance spectroscopy. Comparison of the spectra shows that apple anthocyanins in vivo possess a symmetric absorption band at 500-600 nm with a maximum near 550 nm. Anthocyanins considerably increase light absorption by apples. In on-tree-ripening Zhigulevskoe apples, accumulating high amounts of anthocyanin pigments, chlorophyll contents in sunlit and shaded sides of the fruits are found to be similar. In contrast, frequently considerably lower chlorophyll content is estimated in sunlit compared with shaded sides of Antonovka apples exhibiting low potential for anthocyanin formation. Sunlight also brings about an increase of carotenoid content over that of chlorophylls and accumulation of substances responsible for light absorption in the range 350-400 nm. The rates of high-light-induced chlorophyll bleaching in red zones of fruit containing anthocyanins are considerably lower than those in green zones and decrease with an increase in the pigment content. Anthocyanins show more stability to irradiation than chlorophylls. A protective function of anthocyanins against both light-induced stress in, and damage to, apples is suggested. It is proposed that anthocyanins function as an effective internal light trap filling the chlorophyll absorption gap in the green-orange part of the visible spectrum.     

Spectral heterogeneity in single light-harvesting chlorosomes from green sulfur photosynthetic bacterium chlorobium tepidum

The fluorescence emission properties of single chlorosomes from the green sulfur photosynthetic bacterium Chlorobium (Chl.) tepidum are studied for the first time, using a total internal reflection fluorescence microscope. The fluorescence peak positions of bacteriochlorophyll (BChl)-c self-aggregates in a single chlorosome of Chl. tepidum were widely distributed in the wavelength region between 750 and 768 nm, and the standard deviation (s.d. = 4.1 nm, n = 51) was larger than that of single chlorosomes of Chloroflexus (Cfl.) (s.d. = 1.9 nm, n = 50). The spectral heterogeneity among single chlorosomes from Chl. tepidum was in sharp contrast to those from Cfl. aurantiacus. The difference of chlorosomal spectral properties between Chl. tepidum and Cfl. aurantiacus at the single-unit level would be ascribed to the homolog composition of BChl-c--chlorosomes of Chl. tepidum have BChl substituted with various alkyl groups at both the 8- and 12-positions, whereas light-harvesting BChl-c molecules in Cfl. chlorosomes have the same substituents at the 8- (ethyl group) and 12- (methyl group) positions.