Reflective Light Properties of Tissue Layers in Beech (Fagus sylvatica L.) Leaves

Abstract— Reflective light properties of various tissue layers (adaxial epidermis, palisade and spongy parenchyma and abaxial epidermis) of beech (Fagus sylvatica L.) leaves were measured using paradermal (parallel to epidermis) and transverse sections in the visible and infrared spectral bands by a high-resolution reflectance cytophotometer. Results showed an increment of reflectances of rest leaves following successive removal of paradermal tissue layers (adaxial epidermis, palisade parenchyma, abaxial epidermis). Reflectances of palisade parenchyma with spongy parenchyma and abaxial epidermis increased linearly in the infrared and green spectral bands with increasing chloroplast count. The enhancement of paradermal reflectances with successive removal of paradermal tissue layers was due to various optical effects of tissue/cell layers, whereby removal of paradermal tissue layers containing light absorbers, e.g. chloroplasts/chlorophyll-protein complexes, is of importance. The parabolic relationships found between lateral reflectances and distances of various tissue/cell layers from adaxial epidermis indicated lateral reflectances of the mesophyll to be governed mainly by selective chlorophyll absorb-ances of light according to Lambert-Beer's law.     

ON THE INTERPRETATION OF ABSORPTION SPECTRA OF LEAVES–II. THE NON-ABSORBED RAY OF THE SIEVE EFFECT and THE MEAN OPTICAL PATHLENGTH IN THE REMAINDER OF THE LEAF

Abstract— The sieve effect and scattering within leaves are analysed by the use of a simple model. By plotting the leaf transmittance (corrected for light not entering the leaf) vs the transmittance of an equivalent amount of homogeneous plastid pigments, an intercept is found where the latter is zero. This minimum transmittance represents the fraction of the leaf area devoted to the ray of the sieve effect which strikes no chloroplasts. It varied between 7% and 0.2% in non-senescent leaves. When this was subtracted from the leaf spectrum, the peak absorbance was greater than that of the homogeneous leaf pigments in all cases. The ratio of the leaf absorbance to that of the homogeneous pigments, at the same wavelength, is the apparent optical pathlength, which increases with decreasing absorbance. By plotting this ratio vs the absorbance of the equivalent homogeneous pigment, an intercept is found where the latter is zero. This intercept is interpreted as an estimate of the true mean scattering pathlength. Leaves with high chlorophyll contents had low pathlengths (mean and SD = 2.30 ± 0.25); with moderate and low contents, the values were higher (2.75 ± 0.28, 3.95 ± 0.77). Another application of the model gave values between 3 and 4 for the true scattering pathlength.     

Quantitative study of fluorescence excitation and emission spectra of bean leaves

A quantitative and comprehensive knowledge of leaf fluorescence is required for the interpretation of fluorescence signals at the canopy level and also for the modelling of leaf and canopy fluorescence. In this work we present full range fluorescence excitation and emission spectra of intact leaves, expressed in units of apparent spectral fluorescence yield, from both the adaxial and the abaxial sides of the leaves, and for both front-side and back-side geometries. Emission spectra were measured for incident radiations in the blue and the green spectral range. The red/far-red fluorescence ratio depended on the measurement geometry and on the excitation wavelength. Excitation spectra were measured for emissions at 687 and 760 nm. When the abaxial side was illuminated, the measured spectra always had a larger intensity compared to adaxial side that is explained by the higher scattering of the spongy tissues. At 760 nm, the spectra had the same shape for front-side and back-side geometry, indicating that scattering predominated. At 687 nm, the shape of the spectra was very different for front-side and back-side geometry due to re-absorption of red fluorescence within the leaf. The comparison of excitation spectra measured from the adaxial or the abaxial side revealed differences in carotenoid absorption.     

PHYTOCHROME MACRO-DISTRIBUTION, LOCAL PHOTOCONVERSION AND INTERNAL PHOTON FLUENCE RATE FOR Cucurbita pepo L. COTYLEDONS

Abstract— Using 7-day-oId cotyledons of Cucurbita pepo L., local phytochrome photoconversions could be measured for blue, red and far-red light. For this purpose, after nonsaturating irradiation, cotyledons were sliced into discs 0.3 to 0.5 mm thick and signals measured. This method also yielded the internal phytochrome distribution of the cotyledons with maximal concentration near the adaxial surface, dropping to about 50% in the center and reaching again about 90% at the abaxial surface. Local phytochrome conversion rates were used to calculate internal fluence rates across the cotyledons. Relative internal fluence rates were also derived from measured reflectances and transmittance according to the Kubelka-Munk theory. The general shape of the internal fluence distribution calculated on the basis of these two methods coincided well. It was observed that the internal local photoconversion is proportional to the penetration depth over a wide range of incident fluences and for all wavelengths tested, showing in addition that reciprocity holds. A method to calculate internal fluence rates by a simplified procedure assuming either linear or exponential functions is described.     

Flavonoid distribution in tissues of Phillyrea latifolia L. leaves as estimated by microspectrofluorometry and multispectral fluorescence microimaging

A new method for detecting the tissue-specific distribution of flavonoids has been developed by coupling microspectrofluorometry and multispectral fluorescence microimaging techniques. Fluorescence responses of cross sections taken from 1 year old Phillyrea latifolia leaves exposed to full (sun leaves) or 15% (shade leaves) solar radiation in a coastal area of Southern Tuscany were analyzed. Fluorescence spectra of different tissue layers, each normalized at its fluorescence maximum, that were stained or not stained with Naturstoff reagent A (in ethanol), under excitation with UV light (lambdaexc = 365 nm) or blue light (lambdaexc = 436 nm) were recorded. The shape of the fluorescence spectra of tissue layers from shade and sun leaves differed only under UV excitation. The fluorescence of stained cross sections from sun and shade leaves as well as from different layers of sun leaves received a markedly different contribution from the blue (470 nm) and the yellow-red (580 nm) wavebands. Such changes in tissue fluorescence signatures were related to light-induced changes of extractable caffeic acid derivatives and flavonoid glycosides, namely quercetin 3-O-rutinoside and luteolin 7-O-glucoside. Wall-bound phenolics, i.e. hydroxycinnamic acids (p-coumaric, ferulic and caffeic acid) and flavonoids (apigenin and luteolin derivatives), did not substantially differ between sun and shade leaves. A Gaussian deconvolution analysis of fluorescence spectra was subsequently performed to estimate the contribution of flavonoids (emitting at 600 nm, F600 [red fluorescence contribution = signal integrated over a Gaussian band centered at about 600 nm]) relative to the tissue fluorescence (Ftot [total fluorescence = signal integrated over the whole fluorescence spectrum]). The F600/ Ftot ratios sharply differed between analogous tissues of sun and shade leaves, as well as among tissue layers within each leaf type. A highly resolved picture of the tissue flavonoid distribution was finally provided through a fluorescence microimaging technique by acquiring fluorescence images at the blue (fluorescence at about 470 nm [F470]) and yellow-red (fluorescence at about 580 nm [F580]) wavelengths and correcting the F580 image for the contribution of nonflavonoids to the fluorescence at 580 nm. Monochrome images were elaborated by adequate computing functions to visualize the exclusive accumulation of flavonoids in different layers of P. latifolia leaves. Our data show that in shade leaves flavonoids almost exclusively occurred in the adaxial epidermal layer. In sun leaves flavonoids largely accumulated in the adaxial epidermal and subepidermal cells and followed a steep gradient passing from the adaxial epidermis to the inner spongy layers. Flavonoids also largely occurred in the abaxial epidermal cells and constituted the exclusive class of phenylpropanoids synthesized by the cells of glandular trichomes. The proposed method also allowed for the discrimination of the relative abundance of hydroxycinnamic derivatives and flavonoids in different layers of the P. latifolia leaves.     

Responses of epidermal phenolic compounds to light acclimation: in vivo qualitative and quantitative assessment using chlorophyll fluorescence excitation spectra in leaves of three woody species

Chlorophyll fluorescence (ChlF) excitation spectra were measured to assess the UV-sunscreen compounds accumulated in fully expanded leaves of three woody species belonging to different chemotaxons, (i.e. Morus nigra L., Prunus mahaleb L. and Lagerstroemia indica L.), grown in different light microclimates. The logarithm of the ratio of ChlF excitation spectra (logFER) between two leaves acclimated to different light microclimates was used to assess the difference in epidermal absorbance (EAbs). EAbs increased with increasing solar irradiance intercepted for the three species. This epidermal localisation of UV-absorbers was confirmed by the removal of the epidermis. It was possible to simulate EAbs as a linear combination of major phenolic compounds (Phen) identified in leaf methanol extracts by HPLC-DAD. Under UV-free radiation conditions, shaded leaves of M. nigra accumulated chlorogenic acid. Hydroxybenzoic acid (HBA) derivatives and hydroxycinnamic acid (HCA) derivatives greatly increased with increasing PAR irradiance under the low UV-B conditions found in the greenhouse. These traits were also observed for the HCA of the two other species. Flavonoid (FLAV) accumulation started under low UV-A irradiance, and became maximal in the adaxial epidermis of sun-exposed leaves outdoors. A decrease in the amount of HCA was observed concomitantly to the intense accumulation of FLAV for both leaf sides of the three species. Judging from the logFER, under low UV-B conditions, larger amounts of HCA are present in the epidermis in comparison to FLAV for the three species. Upon transition from the greenhouse to full sunlight outdoors, there was a decrease in leaf-soluble HCA that paralleled FLAV accumulation in reaction to increasing solar UV-B radiation in the three species. In M. nigra, that contains large amounts of HCA, the logFER analysis showed that this decrease occurred in the adaxial epidermis, whereas the abaxial epidermis, which is protected from direct UV-B radiation, continued to accumulate large amounts of HCA.     

霸王鞭和麒麟掌叶片的表面微结构及超疏水性

霸王鞭(Euphorbia antiquorum)和麒麟掌(Euphorbia neriifolia var. cristata)是2种特殊的叶片, 正面不疏水而叶片背面超疏水的沙漠植物. 本文通过接触角测试仪、 电子显微镜和表面张力测试仪分别对叶片的超疏水性、 表面微观形貌和表面黏附力进行了测试和表征. 采用模板法, 以聚乙烯醇为模板、 以聚苯乙烯为基底制备仿叶片背面结构的聚苯乙烯薄膜, 并对薄膜表面的超疏水性、 表面微观形貌和表面黏附力进行了测试和表征, 发现这2种叶片背面的平均间距为1~3 μm的层片状微观结构可以构建出具有超疏水高黏附力特性的表面.     

Influence of Surface Structure,Pigmentation and Particulate Matter on Plant Reflectance and Fluorescence

Optical properties of plant leaves are relevant to evaluate their physiological state and stress effect. The main objective of this work was to study how variegation, pigment composition or reflective features modifies leaves' photophysical behavior. For this purpose, green leaves (Ficus benjamina), purple leaves (Tradescantia pallida), green leaves covered by white trichomes (Cineraria maritima) and variegated leaves (Codiaeum aucubifolium) were analyzed. Firstly, foliar surface morphology was evaluated by scanning electron microscopy. UV‐vis and near‐IR reflectance and transmittance spectra were obtained to calculate absorption (k) and scattering (s) coefficients. The theoretical approaches of Pile of Plates and Kubelka–Munk's theory resulted still valid for nonstandard leaves with differing surface conditions. However, frequently used spectral indices were not reliable for predicting water content, when leaves differed from conventional ones. The proportionality between the absorption factor and chromophore/pigment concentration was also lost for hairy leaves. A simplified model to describe these facts was presented here. Fluorescence spectra were recorded and corrected, due to light re‐absorption. Water‐optical parameter connection and pigment‐optical parameter connection were thoroughly discussed. Leaf surface morphology and pigmentation have not only influenced the optical features of leaves but also played a role in the effect that particulate matter could cause on leaf photosynthesis.     

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.     

Measurement of the Optical Properties of Leaves Under Diffuse Light

Measuring leaf light absorptance is central to many areas of plant biology including photosynthesis and energy balance. Absorptance is calculated from measured values of transmittance and reflectance, and most such measurements have used direct beam light. However, photosynthesis and other processes can differ under direct and diffuse light. Optical properties under diffuse light may be different, but there have been technical difficulties involved in measuring total reflectance of diffuse light. We developed instrumentation to measure this reflectance using a chopped measuring beam delivered alternately to sample and reference integrating spheres, and lock-in detection. We also built instrumentation for measuring transmittance of diffuse light. We developed standards to calibrate our instruments and correct for substitution error, a known systematic error with integrating sphere-based measurements. Helianthus annuus leaves measured under diffuse light reflected 5–10% more and transmitted a few percent less 400–700 nm light than under direct light. Overall absorptance was only a few percent higher under direct light, but leaves may utilize absorbed direct and diffuse light differently. For example, of the light entering the leaf, significantly more direct light than diffuse light is transmitted through the leaf, suggesting differences in localization of absorption within the leaf.     

ANALYTICAL MODELING FOR THE OPTICAL PROPERTIES OF THE SKIN WITH IN VITRO AND IN VIVO APPLICATIONS

Abstract— Analytical modeling that interrelates the optical properties of multilayered structures is applied to the skin. The mathematical approach is based on relations of diffuse reflectance and transmittance of a multilayered system and the diffuse reflectance and transmittance of each component layer. The formula can also be derived from the Kubelka–Munk theory of radiation transfer. Using both collimated and diffuse incident irradiance, the applicability of the model to human epidermis over the UV and visible region has been verified. The model has been applied to calculate the absorption and scattering coefficients of human epidermis in vitro , and to estimate the epidermal transmittance under simulated in vivo condition.     

ABSORPTION SPECTRA OF LEAVES CORRECTED FOR SCATTERING and DISTRIBUTIONAL ERROR: A RADIATIVE TRANSFER and ABSORPTION STATISTICS TREATMENT

A comprehensive treatment of light propagation through intact leaves based on the theories of radiative transfer and absorption statistics was used to calculate the theoretical absorption spectra of the chlorophyll-containing particles under conditions of multiple scattering and pigment spatial distribution equivalent to those in a leaf. These spectra were compared with the experimental in vivo spectra of leaves and in vitro spectra of chlorophyll-protein complexes extracted form these leaves. We conclude that the main discrepancies between the in vivo and in vitro spectra are apparently due to the optical artifacts specific for light propagation in leaves-multiple scattering and distributional error. Alterations of the pigment properties upon extraction significantly contribute to these discrepancies. The method has an estimated accuracy of about 10% and can be applied to derive the intrinsic optical properties of the photosynthetic mechanism in a leaf, as well as for the systematic study of their changes in the course of light adaptation.     

IN VIVO and POST MORTEM MEASUREMENTS OF THE ATTENUATION SPECTRA OF LIGHT IN MAMMALIAN TISSUES

Abstract— The attenuation of light in rabbit liver and striated muscle and in pig brain has been measured in the wavelength range400–800 nrn. A variable-wavelength monochromatic light source was used to illuminate the surface of the exposed tissues in situ. Optical fibers, coupled to a single photon counter, were positioned within the tissues at different depths to determine the distribution of light flux along the central axis of the light beam. The wavelength-dependence of attenuation was measured in vivo, and changes occurring in the attenuation spectra immediately post mortem were recorded. Strong absorption bands were observed around 425 nm and 550 nm in all three tissues, both in vivo and post mortem. At longer wavelengths, the attenuation decreased slowly, and no significant structure was observed. Substantial changes occurred immediately post mortem, particularly in the absorption bands. These effects are attributed mainly to de-oxygenation of blood, and changes in blood concentration in the tissues. The implications for light dosimetry in photodynamic therapy are discussed.     

Optical modeling of nanocrystalline TiO2 films

The light transmittance, T, in nanocrystalline TiO2 films was studied as a function of the light wavelength, lambda, the nanocrystal radius, a, and the film thickness, d. Two types of TiO2 nanoparticles were employed: a commercial powder (P25) and synthesized particles from titanium isopropoxide (SP). The X-ray diffraction measurements revealed that both P25 and SP are mainly anatase and the average crystal sizes, 2a, of P25 and SP are 50.3 and 23.7 nm, respectively. Despite the visual difference between micron-order thin films of P25 and SP, the light hemispherical transmittance corrected with the surface specular reflectance has a clear dependence of ln(T) = -0.5beta lambda(-4)a(3)d, with beta = 1.5 x 10(3) from visible to near-infrared wavelengths. The dependence and beta value were successfully explained by the simplest model on the basis of the Rayleigh scattering theory. This indicates that the nanocrystalline TiO2 thin films are a typical medium where the simplest scattering model is a good approximation. However, the model was inapplicable to light scattering in relatively thick P25 films of 1.5-3.0 microm because of nonnegligible internal multiple scattering. For the moderate thickness films, ln(T) proportional to lambda(gamma), where gamma increases from -4 in proportion to the film thickness is an alternative approximation. With these light scattering models, the light absorption rate of the TiO2 crystal was successfully evaluated from experimental extinction rates.     

ACTION SPECTRA FOR HYPHAL AGGREGATION,THE FIRST STAGE OF FRUITING,IN THE BASIDIOMYCETE Pleurotus ostreatus*

Abstract. Action spectra were determined for hyphal aggregation in Pleurotus ostreatus at wavelengths between 360 and 600 nm. The action spectrum for a 50% response had two maxima, one at 370 nm in the near-UV and the other a broad peak at 440–450 nm in the blue. Both were approximately of the same magnitude. A minimum was present at 400 nm and wavelengths greater than 530 nm invoked no response. Action spectra for a range of responses, 10–90%, were also determined which showed gradual changes in the peaks in the blue region. It was concluded that hyphal aggregation in P. ostreatus is under the control of a cryptochrome-like photoreceptor system.     

ULTRAVIOLET-RADIATION-ACCELERATED LEAF CHLOROSIS: PREVENTION OF CHLOROSIS BY REMOVAL OF EPIDERMIS OR BY FLOATING LEAF DISCS ON WATER*

Abstract— Low doses (1800–7200 ergs/mm²) of ultraviolet (U V) radiation accelerated chlorosis in the mesophyll of Nicotiana glutinosa leaves when the lower epidermis of the leaves was irradiated. This occurred in either a subsequent light or dark incubation. However, within 12 h after irradiation, peeling of the lower epidermis prevented this accelerated chlorosis. The accelerated chlorosis was also prevented by floating irradiated leaf discs on water during the incubation period without removal of the epidermis. These results suggest that accelerated chlorosis in mesophyll tissue caused by low doses of UV is due to an indirect effect of the UV-damaged epidermis, possibly mediated by some toxic substance released from the epidermal cells. High UV doses (36,000–108,000 ergs/mm²) prevented normal yellowing of the leaf. The irradiated portion of the leaf remained green, while the nonirradiated area turned yellow upon dark incubation. However, if the irradiated leaf was incubated in continuous light, bleaching of the irradiated area took place, and the irradiated area became yellow faster than the nonirradiated area. Peeling of the epidermis did not affect the outcome of these experiments. These results suggest that high UV doses directly damage the mesophyll tissue.     

FLUORESCENCE SPECTRA OF BLOWFLY METAXANTHOPSINS

Abstract— The main visual pigment of blowflies (xanthopsin, Vogt, 1983 Z. Naturforsch. 38c ,329–333) photoconverts into two thermostable metaxanthopsin states M and M'(e.g. Stavenga et al. , 1984 Photochem. Photobiol. 40 ,653–659). The fluorescence spectra of the two photoproducts were studied by microspectrofluorometry in vivo. The emission spectra of M and M'are very similar and peak at 660 nm. The excitation spectra of M and M'have peak wavelengths at Λ_max ' 584 nm and = 568 nm respectively.     

SIMILAR SPECTRA FOR THE INACTIVATION BY MONOCHROMATIC LIGHT OF TWO DISTINCT LEUCINE TRANSPORT SYSTEMS IN ESCHERICHIA COLI

Abstract—Kinetics of inactivation of two separate leucine transport systems (leucine specific and LIV) in E. coli by seven wavelengths of monochromatic light have been studied. Loss of leucine uptake is not due to generalized membrane damage causing non-specific leucine leakage. Inactivations are usually exponential but some wavelengths show shoulders at low doses. Two-component spectra between 254 and 435 nm occur for both transport systems. Inactivation is most efficient at 290 nm and a second peak occurs at 365 nm. Both leucine transport systems are inactivated similarly.     

Pigmentation of White,Brown, and Green Chicken Eggshells Analyzed by Reflectance,Transmittance, and Fluorescence Spectroscopy

We report on the reflectance, transmittance and fluorescence spectra (λ=200–1200 nm) of four types of chicken eggshells (white, brown, light green, dark green) measured in situ without pretreatment and after ablation of 20–100 μm of the outer shell regions. The color pigment protoporphyrin IX (PPIX) is embedded in the protein phase of all four shell types as highly fluorescent monomers, in the white and light green shells additionally as non-fluorescent dimers, and in the brown and dark green shells mainly as non-fluorescent poly-aggregates. The green shell colors are formed from an approximately equimolar mixture of PPIX and biliverdin. The axial distribution of protein and colorpigments were evaluated from the combined reflectances of both the outer and inner shell surfaces, as well as from the transmittances. For the data generation we used the radiative transfer model in the random walk and Kubelka-Munk approaches.     

ACTION SPECTRA FOR KILLING NON-DIVIDING NORMAL HUMAN AND XERODERMA PIGMENTOSUM CELLS

Abstract— Non-dividing human cells degenerate and eventually detach from a culture vessel surface when exposed to UV light. Action spectra for this kind of cell inactivation were determined using eight monochromatic wavelengths from 240 to 313 nm and both a normal DNA excision-repair-proficient strain and a repair-deficient Xeroderma pigmentosum (XP12BE) strain. The action spectra for both strains have similar shapes with a broad peak between 254 and 280 nm followed by a steep decline at wavelengths greater than 280 nm. The relative action spectra are similar to those for inactivation of reproductive capacity and pyrimidine dimer formation in rodent cells suggesting that the critical target and critical damage for inactivation of non-dividing human cells is DNA and damage to DNA, respectively. Normal repair-proficient cells are 5–7 times more resistant at all wavelengths, based on a comparison of D_o values, than repair-deficient XP12BE cells, supporting the conclusion that the inactivating damage at all wavelengths is to DNA.