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

Photochemistry and Photobiology, 1996, 63(4): 498-506

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

Enamul Hoque*' and Guido Remus2 'GSF-Forschungszentrum fur Umwelt und Gesundheit Neuherberg, OberschleiOheim, Germany and 'DLR-Deutsche Versuchs- und Forschunmanstalt fur Luft- und Raumfahrt, lnstitut fur Optoelektronik, Oberpfaffenhofen, Germany

Received 21 April 1995; accepted 2 January 1996

ABSTRACT

Reflective light properties of various tissue layers (adaxial epidermis, palisade and spongy parenchyma and abaxial epidermis) of beech (Fagus sylvuticu 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 tissuekell 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 absorbances of light according to Lambert-Beer's law.

INTRODUCTION

The interaction of sunlight with plant leaves and the absorbance of sunlight by photosynthetic pigments, chlorophyll fluorescence, light transmittance and the reflectances of light by the leaf's cellular structures determine the light's micro- environment within the leaves. The leaf's reflectance, absorbance and transmission of light are influenced by (1) internal leaf structure, controlling in part the diffusion or internal scattering and optical pathlength of incident solar irradiance, as well as the external structural features like the surface roughness of the leaves and the refractive index of the cu-

~~

*To whom correspondence should be addressed at: GSF-Forschung- szentrum fiir Umwelt und Gesundheit Neuherberg, Institut fiir Bio- chemische Pflanzenpathologie, IngolsWter Landstr. 1, D-85758 OberschleiRheim, Postfach 1129, Germany. Fax: +49 89 3187 3383; e-mail: [email protected].

0 I996 American Society for Photobiology 003 1-8655196 $5.00+0.00

ticular wax of the epidermis controlling the reflectance from the surface (l) , (2) pigment (composition, concentration and distribution) and chloroplast (count, distribution, discoloration and loss of compartmentalization) (24 ) status controlling the absorbance in the UV and visible spectral range, (3) leaf water status (concentration and distribution) controlling the refractive index gradient in the visible spectral range (1) and absorbance in the infrared spectral range and (4) angle of exposition of leaves controlling the diffusion or scattering and optical pathlength of incident light (5).

Plant reflectances involve specular reflection and mainly diffuse reflection (6). The phenomenon of light scattering, a basic component of the reflectance models, is complex and may be due to complex cytoplasmic contents, refractive index differences, irregular cellular shapes, microfibrils and various geometric organizations of tissues (l,6). One of the earliest theories to describe plant reflectances is known as the Willstatter-Stoll theory, which explains that leaf reflectances, termed refractive-reflective scattering (l,6), take place at cellular refractive interfaces within the leaf. Incident light is transmitted by total or critical reflectance through palisade parenchyma to the spongy parenchyma, characterized by spherical-irregular cells and large intercellular spaces, where the major portion of diffuse reflectance takes place (7). Ray tracing of light passed through the leaves in the spectral region 0.70-1.30 p,m showed that plant reflectance could be inadequately explained by Willstatter-Stoll theory; however, could be explained using a modified Kubelka- Munk model (8-10). Allen et al. (9) devised the modified model of Willstatter-Stoll theory considering leaf cellular interfaces as circular arcs in the medium with various refractive indices. It was further hypothesized by Sinclair et al. (6) that the high levels of leaf reflectances occur on the basis that cell walls act largely as diffusive reflective surfaces, with lesser amounts of specular reflectance. The mi- crofibril structure of the cell walls was suggested to induce the scattering necessary to be diffuse, as defined by Lam- bert's cosine law. The cell membranes and the middle la- mella were shown to reflect more infrared light than the surrounding protoplasm and cell organelles (1 1), obviously due to weak absorbance of infrared light by the intracellular structures. On the basis of an algorithm developed for absorbance and reflectance constants of paint layers of various thickness (8), the generation of a plant's reflectance was ex-

498

Photochemistry and Photobiology, 1996, 63(4) 499

plained by Allen and Richardson ( 1 2). Tucker and Garrat (1) developed a stochastic leaf radiation model based upon physical and physiological properties of dicot leaves to pre- dict the absorbed, reflected and transmitted radiations of nor- mal incidence. The optical system was described as a Mar- kov process considering probabilities of transitions of incident light (1,13).

Light absorption by a leaf's palisade and spongy parenchyma, and hence, the magnitude of reflectance and chlorophyll fluorescence, are affected by enhancement of absorption by multiple scattering due to lengthening of optical pathlength, e.g. by the detour or random or pathlength statistics effect (14-16), and by lowering of absorption efficiency due to the nonhomogeneous spatial distribution of absorbers, for example, by the sieve effect of pigments or absorption statistics effect or distributional error effect (15- 18). The absorbance of intact tissue, which is a turbid medium with particulate absorbers, could also be explained on the basis of wave-particle interaction of chloroplasts with incident light, e.g. using van de Hulsts' anomalous diffrac- tion (16). Recently, it has been shown that the light gradient of leaves could be described by solving the inverse problem of four-flux radiative transfer-a theory considering forward and backward diffuse as well as directed components of the overall radiation in a multiple scattering sample ( 1 6,19). The more simple treatment of this, the Kubelka-Munk theory (two-flux treatment), could also be used for the calculation of light gradients in plant leaves (16,19). On the other hand, theories based on single scattering, e.g. the Mie scattering theory, are inadequate for the calculation of light gradients in plant leaves (16). Mie scattering is caused by particles, whereby the intensity of scattered light in the forward direction is higher than that in the backward direction (20). Ray- leigh scattering, a special case of Mie scattering, may be ascribed to particles or molecules of sizes equal to or less than the wavelength of the incident light, and is approximately inversely proportional to the fourth power of the wavelength. The contribution of Mie and Rayleigh scattering to reflectance and photosynthetic light gradient in the leaf is insignificant (21) and was found to be true only in special cases of highly transparent tissues, where sometimes even the scattering proportional to

This article describes the reflective light gradient of beech leaves with special reference to the optical properties of various leaf tissues and generation of reflectances and discusses the ecophysiological significance of internal light gradients for plant leaves. This knowledge could also be useful for the assessment of photosynthetic light saturation and responses of plants to light stimuli as well as to understand the internal mechanisms of generation of the remote sensing signal reflectance.

can be observed (16).

MATERIALS AND METHODS Plant materials. In September and October 1990, sun and shade branches (30-50 cm long) were cut at a height of about 3 m from a healthy beech (Fagus sylvatica L.) tree (<60 years old) grown in the Ostfriedhof Park, Munich (about 457 m above sea level), trans- ported to the laboratory, recut under water and then watered in order to avoid water loss. Prior to cytophotometric measurement, turgid green leaves were detached from the sunny and shade branches. Beech leaf strips of ca 2 mm X 10 mm were cut from the detached leaves and fixed adaxially or abaxially on transparent paste film (tesa

7-1

L i

Figure 1. Schematic setup of the measurement of reflectance by Zeiss cytophotometer (abbreviations: M = microscope, LS = light source, 0 = objective, S = sample, PMT = photomultiplier tube, F = filter, CU = control unit, P = printer). The incident light beam at 45" (cp) is indicated by downward arrows, and the reflected beam at 0" with angle of view 0.49-7.85" (p) is indicated by lateral arrows.

film transparent, Beiersdorf, Germany) mounted on an object carrier in order to avoid any bending of the paradermal section due to drying as well as to minimize oxidative changes of the paradermal sections allowing only one side open to air (5). The transparent paste film used as a backing did not affect chloroplast fluorescence of beech leaves ( 5 ) and did not show any significant effects on the relative reflectances of beech leaves. Further, the effect of transparent paste film on relative reflectances of adaxial and abaxial surfaces of beech leaves was checked by comparison of the shapes of reflectance curves with those obtained from high-resolution reflectance spectroscopy (22).

Paradermal layer sections were cut using a sharp razor blade. Suc- cessive removal of tissue layers starting from either adaxial or abaxial epidermis of beech leaf strips gave paradermal sections of adaxial epidermis (front to rear side depth ca 0-10 pm), adaxial palisade parenchyma (upper cell layer, front to rear side depth ca l l - 60 pm), abaxial palisade parenchyma (lower cell layer, front to rear side depth ca 61-90 pm), spongy parenchyma (front to rear side depth ca 91-160 pm) and abaxial epidermis (front to rear side depth ca 161-170 pm). Although depths of cell/tissue types were measured by laser scanning microscopy and image analysis software using fluorescence images (see below), however, due to morpholog- ical and intraleaf variation the depth values in pm should be re- garded as typical distances from the front (adaxial) to the rear (abaxial) side of the leaf. Proper cutting of paradermal sections and focussing were checked by a microscope (model Zeiss MPM-200, Germany, see below) at a magnification of 20X. Comparison of the paradermal reflectances of spongy parenchyma from adaxial and abaxial sides after successive removal of paradermal layers of epidermis and palisade parenchyma from the adaxial side and of epidermis from the abaxial side, respectively, did not reveal any significant differences among them, i.e. depending on thickness (see Results).

In addition to the paradermal sections, thin transverse sections were cut from the same leaves and fixed on transparent paste film (see above). In order to assess the effects of thickness on the lateral reflective properties of tissues, thick (ca 40 pm thick) transverse sections of beech leaves were cut. Due to nearly doubling of lateral reflectances of epidermis and spongy parenchyma with doubling of thickness (ca 40 pm) using 5 pm measuring field diameter, only thin transverse sections (ca 20 pm thick) were used for our study. Thickness of transverse sections was checked by laser scanning microscopy and image analysis software (see below). Under the ex- perimental conditions, the paradermal as well as the transverse sections did not show any sign of oxidation or change of reflectance signal during measurement.

Cytophotomefric measurement of rejfectances. Figure 1 shows schematically the setup of measurement method employed in our studies. All the measurements were performed in the darkfield mode in the intensity range I: lo6 using an axioplan microscope photometer

500 Enamul Hoque and Guido Remus

loo c

Figure 2. Measurement of reflective light properties of tissue layers in beech leaves. (i) Schematic three dimensional diagram of the tissuekell layers (see below iid). Double arrows (dotted) indicate direction of cytophotometric measurement of reflectance (top double arrows-incident and reflected light beam for paradermal reflectance measurement using paradermal sections, lateral double arrows-incident and reflected light beam for lateral reflectance measurement using transverse sections); (ii) paradermal sections showing direction of reflectance measurement as indicated by upward arrow at 0", (a) palisade parenchyma without adaxial epidermis, but with spongy parenchyma and abaxial epidermis, (b) spongy parenchyma without adaxial epidermis and palisade parenchyma, but with abaxial epidermis, (c) spongy parenchyma without abaxial epidermis, but with palisade parenchyma and adaxial epidermis, (d) adaxial epidermis with all the tissue layers of intact leaf and (e) abaxial epidermis with all the tissue layers of intact leaf [abbreviations: pali, palisade parenchyma; spgy, spongy parenchyma; abxe, abaxial epidermis; adxe, adaxial epidermis).

(Zeiss model MPM-200, objective epiplan-neofluar 20X/0.50HD, Germany) and a microscope system processor (Zeiss model MSP- 20, Germany). For measurement, each microscopic paradermal and transverse section was divided into the following tissuekell zones: (1) adaxial (front) epidermis, characterized by thick-walled epidermal cells with negligible amounts of chloroplasts residing only in the sporadically occurring stomatal complex; (2) adaxial (upper) and (3) abaxial (lower) palisade parenchymatic cell layers, both characterized by high densities of compact elongated cells perpendicular to the leaf surface and by high intracellular densities of chloroplasts; (4) spongy parenchymatic tissues, characterized by large intercel- Mar spaces, low densities of loosely arranged spherical-irregular cells with high chloroplast contents and (5) abaxial (rear) epidermis, characterized by thick-walled epidermal cells with negligible amounts of chloroplasts residing only in the sporadically occurring stomatal complex as in adaxial epidermis (Fig. 2i). As all the par-

adermal sections retained either the lower or upper epidermis (Fig. 2ii), it should be borne in mind that the measured reflectances of paradermal sections do not always represent the upward light fluxes in the intact leaves on the corresponding depths and are compared in a restricted sense in order to show the trend. For example spongy parenchyma illuminated from adaxial side means the rest of a leaf without adaxial epidermis and palisade parenchyma (Fig. 2iib), while spongy parenchyma illuminated from abaxial side means the rest of a leaf without adaxial epidermis (Fig. Ziic).

Prior to reflectance measurements the protective glass cover was removed from the sections in order to avoid scattering due to glass. The excitation white light was generated by a halogen-glow lamp (12 V, 100 W) and was focused on the leaf paradermal or transverse sections at an incident angle of 45" for reflectances. The diameters of incident light beam or measuring fields (MF) were 5, 12.5, 31.5, 50 and 80 pm depending on the experiment (see below). Following excitation, reflectance signals at an angle of view 0.49 (80 pm MF- diameter) to 7.85" (5 pm MF-diameter) passing through respective interference filters [see below) were sensed by a photomultiplier (Hamamatsu R129, S-20 cathode, sensitivity range: 340-850 nm) at an angle of 0". The transmission maxima of the violet (spectral range: UV to blue), blue, green (spectral range: green to yellow), red and infrared interference filters (Schott, Germany) for parader- ma1 and transverse sections were located at 404, 453, 546, 658 and 782 nm with half band widths 49, 54, 47, 20 and 60 nm, respectively. Thus, the reflectances due to strong chlorophyll absorbances at 445 and 680 nm as well as due to weak chlorophyll absorbances at 550 nm and 780 nm could be measured.

In order to ensure sufficient measuring range, prior to each measurement in the violet, blue, green, red and infrared spectral ranges the measuring range of the photometer was calibrated using a reference homogenous PTFE white standard showing 98% absolute reflectance over the whole spectral range to its fourfold reflectance value (392% = 4 X 98%) (Zeiss, Germany). All the values minus dark current were recorded relative to the white standard and finally divided by four to obtain the corresponding normalized relative reflectance. Relative reflectance at a wavelength X for a particular tissue is defined as the ratio of reflectance of the tissue at a wavelength A and at a diameter (incident beam or MF-diameter) to the reflectance of the white standard at the same wavelength X and at the same MF-diameter (incident beam or MF-diameter). Thus, the measured relative reflectance is independent of instrumental effects of MF-diameter.

The reflectances of adaxial and abaxial surfaces were measured using MF of 80 pm diameter, whereas the paradermal and transverse sections were measured either using a MF of 5 pm diameter in order to detect the reflectances of chloroplasts or smaller structures, or using various MF in order to determine the relationship between reflectance and MF-diameter or between reflectance and number of chloroplasts.

Each cellular zone was sampled 50 times for a representative value and such measurement was repeated 15 times in order to give 15 representative values.

Microscopic imaging. For imaging, a Leitz Orthoplan microscope was used. Images were sensed at the first image plane at a magnification of 25 by a high-resolution charge-coupled device camera with ?4 inch detector size. Red, green and blue extracts were taken from each scene by inserting respective interference filters at the illumination path. The transmission maxima of the filters in use were located at 500, 550 and 680 nm with spectral half bandwidths at 20, 20 and 10 nm, respectively. The images were digitized and stored at a digital video frame memory (2). For color image photography, the intensity normalized and D/A converted signals of three color extracts are fed to a high-resolution slide-making machine to pro- duce true-color images. Using large-size true color photos, the densities of typical tissue/cell zones of the mesophyll were determined by planimetry.

Confocal laser scanning microscopy. The confocal laser scanning imaging was carried out in a Zeiss laser scanning microscope (LSM model LSM 10) equipped with a plan neofluar objective (20X). The monochromatic light of the mean excitation wavelength 488 nm was generated by an argon laser and was focused on the leaf adaxial (front) surface at an incident angle of 90" for chlorophyll excitation. Following excitation, chlorophyll fluorescence images (spatial res-


40 -

g 30 -

2 2 0 -

3 Ili 1 0 -

w 0 z

0

U W

0 -

0 0

8 8

-

300 400 500 600 700 800 900

WAVELENGTH (nm)

Figure 3. Paradermal reflectances at adaxial and abaxial surfaces of intact sun (0, adaxial side, Fig. 2iid; 0, abaxial side, Fig. 2iie) and shade (A, adaxial side, Fig. 2iid; V, abaxial side, Fig. 2iie) leaves depending on spectral bands. Reflectances were measured using 80 pm measuring fields. Due to low standard deviations, vertical error bars cannot be shown. For better illustration, the course of the mean reflectance is shown by a solid nonlinear regression line even for the discrete spectral bands.

olution in the confocal mode 0.2-2 pm; depth contrast 0.2 p,m) were sensed at an emission wavelength of 658 2 20 nm using a photomultiplier. As the confocal LSM allows correct transformation of object points to image points, the fluorescence objects could be observed by confocal LSM at a high resolution and depth contrast. Repeated measurements gave digital smoothing of the values. The spatially resolved structures were digitized in real time and stored at a video frame store (5). They were then made available for the digital analysis by superimposition of measuring fields.

RESULTS AND DISCUSSION Paradermal reflective properties of various tissues of sun and shade leaves

The reflectances of adaxial and abaxial sides of intact sun and shade leaves using 80 km incident beam are depicted in Fig. 3. Irrespective of leaf morphology, the abaxial side reflected stronger than the adaxial side. In the sun leaf, the difference of reflectances between abaxial (rear) and adaxial (front) sides was especially high for the green band (546 nm) of chlorophyll reflectance and for the red band (658 nm) of chlorophyll absorbance. The results are in agreement with those obtained by high-resolution reflectance spectroscopy data of adaxial and abaxial surfaces of beech leaves (22).

In the shade leaf (Fig. 3), the difference of reflectances of abaxial and adaxial sides was especially high for the green band of chlorophyll reflectance and the violet to blue band (404-453 nm) of chlorophyll absorbance instead of the red band (658 nm) of sun leaf. Although the shade leaf lacks one layer of palisade parenchyma cells as compared to the sun leaf, the reflectance of shade leaf illuminated from the adaxial side (Fig. 2iid) increased only in the infrared range. Whereas the reflectance of the shade leaf illuminated from the abaxial side (Fig. 2iie) increased, as expected, in all the spectral bands as compared to those of sun leaves. Thus, the attenuance of reflective light in the violet to red bands (404-

50

0

I I I I I r 1 300 400 500 600 700 800 900

WAVELENGTH (nm)

Figure 4. Paradermal reflectances of palisade parenchyma without adaxial epidermis, but with spongy parenchyma plus abaxial epidermis (A; Fig. 2iia), and of spongy parenchyma either without adaxial epidermis, hut with abaxial epidermis (0; Fig. 2iib) or without abaxial epidermis, but with palisade parenchyma plus adaxial epidermis (0; Fig. 2iic) using 80 pm measuring fields. Due to low standard deviations, vertical error bars cannot be shown. For better illustration, the course of the mean reflectance is shown by a solid nonlinear regression line even for the discrete spectral bands.

658 nm) occurred in the shade leaf as compared to that of sun leaf, if the leaf is illuminated from the adaxial side.

One should, however, bear in mind that any measured flux is a sum of light reflected from the illuminated surface and remitted from the depth of the leaf, the second item being influenced by absorption, remission and transmission properties of all leaf layers. Thus, successive removal of strong absorptive tissue layers of leaves would mean increment of reflectances. Indeed, it was found that the paradermal reflectances of leaves enhanced with successive removal of tissue layers to maximum values in spongy parenchyma with abaxial epidermis (Figs. 2iib, 3 and 4). Thus, the reflectances of intact sun leaves at adaxial surfaces (see Fig. 2iid) were lower than those of palisade parenchyma with spongy parenchyma and abaxial epidermis (Figs. 3 and 4; see Fig. 2iia), and the reflectances at palisade parenchyma with spongy parenchyma and abaxial epidermis (Fig. 2iia) were lower than those at spongy parenchyma with abaxial epidermis, especially in the green and red bands. The parader- ma1 reflectances at the abaxial epidermis of the intact leaf were slightly lower than those in the spongy parenchyma with abaxial epidermis. Terashima and Saeki (23) also reported larger reflectances in spongy tissues than those in palisade tissues of Camellia leaves. Assuming the parader- ma1 transmission of light for a definite wavelength constant, the increasing pattern of paradermal reflectances with successive removal of tissue layers would mean a decreasing pattern of light absorbances in the Fagus mesophyll. In this connection, it is worth mentioning that Terashima and Saeki (24) demonstrated a decreasing pattern of light absorbances for Camellia mesophyll with increasing depth. Paradermal reflectances at 404 and 658 nm (strong absorption) and at 546 and 782 nm (weak absorption) showed a strong and weak increment of paradermal reflectances with successive


removal of paradermal tissue layers up to spongy parenchyma, respectively (Figs. 3 and 4).

Terashima and Saeki (23) suggested a change of spectral composition of light for Camellia leaves with depth. In all the tissues of Fagus sun leaves, the paradermal reflectances in the green (546 nm) and far-redinfrared bands (782 nm) also dominated. In addition to these spectral bands, the reflectances of photosynthetic spectral zones 404 2 49 nm and 658 2 20 nm were strongly enhanced in the spongy tissues (Fig. 4; see Fig. 2iib). Thus, the change of spectral composition of light with depth also takes place in Fagus leaves, whereby in addition to green and far-red light as observed for Camellia leaves (23) the photosynthetic blue and red light was especially enriched in the spongy parenchyma of Fagus leaves. Thus, the high attenuance (apparent absorbance) of light by spongy parenchyma as suggested by Ter- ashima and Saeki (23) for Camellia leaves is required to utilize the photosynthetic light by the low-density of chloroplasts per unit area in spongy tissues of Fagus leaves. The high density of chloroplasts per unit area increased the absorbance of light in palisade tissues and lowered the parader- ma1 reflectance relative to that in the spongy parenchyma (Fig. 4), although the attenuance of light by palisade tissues was lower than that by spongy tissues (23). The high attenuance of light by spongy parenchyma could be partly due to the detour or random pathlength statistics effect (1416).

The paradermal reflectances of spongy parenchyma of a sun leaf measured from the adaxial or abaxial side after removal of adaxial epidermis and palisade parenchyma or abaxial epidermis, respectively, did not show major differences in the blue, green and red bands. The difference of reflectances in the palisade (see Fig. 2iia) and spongy (see Fig. 2iib) parenchyma was created by the enhanced absorbance of light in the palisade parenchyma as well as by the enhanced reflectances of especially green and red light in the spongy parenchyma. Thus, the detour effect (14,15) due to multiple scattering and absorbances (see above) seems to play an important role in the enhancement of reflectances by the spongy parenchyma.

Relationship between paradermal reflectances and MF- diameter

With increasing MF-diameters, the reflectances increased nonlinearly in all the spectral ranges for palisade (Fig. 2iia) and spongy (Fig. 2iib) parenchyma (Fig. 5). Due to the quadratic relationship of area with diameter, the nonlinear relationship of reflectance with MF-diameter could be easily interpreted. All the deviations of the MF-diameter-dependent reflectance curves from the theoretical curve y = (n/4)X2 with y = reflectance (%, log-scale) and x = MF-diameter (Fm; see legend to Fig. 5 ) are mainly due to the detour effect and the sieve effect, whereby the effects increase up to saturation with increasing MF-diameter. At lower MF-diameters, the deviations of the reflectances from the theoretical values could be due to the smaller cellular components. The greater is the difference of the reflectance curve for a particular wavelength depending on MF-diameter from the theoretical curve, the stronger is the absorbance of light due to the detour effect. The less is the difference of the reflectance curve for a particular wavelength from the theoretical curve,

100

h 10

W 0 z I 5 1 e

0

7 PALISADE PARENCHYMA

A

I 1 - I 1.-

0 10 20 30 40 50 60 70 80 90

MF-DIAMETER (pm)

SPONGY PARENCHYMA

I , , ,

0 10 20 30 40 50 60 70 80 90

MF-DIAMETER (p)

Figure 5. Relationship between paradermal reflectances (%; y-axis, logarithmic scale) and MF-diameters (pm; x-axis, linear scale) in palisade parenchyma with spongy parenchyma plus abaxial epidermis (top; see Fig. 2iia) and spongy parenchyma with abaxial epidermis (bottom; see Fig. 2iib): The parabolic relationships in palisade parenchyma with spongy parenchyma plus abaxial epidermis could be described by the following regression equations, V-782 nm y = -0.92781 + 0.07036~ - 0 .000484~~ (r2 = 0.98), L . 5 4 6 nm y = - 1.38920 + 0.06855~ - 0 . 0 0 0 4 6 8 ~ ~ (r2 = 0.98), A-658 nm y = -1.88710 + 0.06954~ - 0 . 0 0 0 4 8 3 ~ ~ (r2 = 0.98) and 0- 404 nm y = -1.68789 + 0.03844~ - 0.000193~~ (rz = 0.98), respectively. A proportionally calculated theoretical reflectance curve with maximum 98% reflectance for d 4 X (MF-diameter = 80)* dependent only on MF-diameter is indicated by the symbol + . The parabolic relationships in spongy parenchyma with abaxial epidermis could be described by the following regression equations, 7-782 nm y = - 1.022705 + 0.068288~ - 0 .000464~~ (r2 = 0.98), U-546 nm y = -1.78565 + 0.08982~ - 0.000650~~ (r2 = 0.98), A-658 nm y = -1.52158 + 0.07177~ - 0 . 0 0 0 4 9 3 ~ ~ (r2 = 0.99) and 0 4 0 4 nm y = -1.82743 + 0.07654~ - 0 .000544~~ (r2 = 0.98), respectively. A proportionally calculated theoretical reflectance curve with maximum 98% reflectance for d4 X (MF-diameter = 80)2 dependent only on MF-diameter is indicated by the symbol 0 . Due to low standard deviations, vertical error bars cannot be shown.

the greater is the reflectance of light due to the sieve effect. Apparently, absorption of blue and red light by chloroplast- protein complexes was additionally enhanced by the detour effect with a concomitant reduction of the reflectance of palisade parenchyma (Fig. 2iia). The diffuse reflectances (6,7) of spongy parenchyma for all the spectral bands could be due to the detour effect (14,15,17) and the sieve effect (15- 17). The multiple scattering due to the detour effect (14-16) and the low absorbance of nonphotosynthetic light (see below) in the palisade parenchyma (Fig. 2iia) apparently led


40

?--.

5 30 w 0 Z

2 y 20 J U W IY

10

PALISADE PARENCHYMA

v 6

A 6 v

A I

1 - I a d - 400 450 500 550 600 650 700 750 800

WAVELENGTH (nm) 35

SPONGY PARENCHYMA

301

0 5L 0

nl 0

Q 1

400 450 500 550 600 650 700 750

WAVELENGTH (nm)

Figure 6. Paradermal reflectance curves of palisade parenchyma with spongy parenchyma plus ahaxial epidermis (0-5, B--12.5, A-31.5, V-50, and +-SO pm MF-diameter; see Fig. 2iia) and spongy parenchyma with ahaxial epidermis (0-5, 0-12.5, A- 3 1 S, V-50 and 0 -80 pm MF-diameter; see Fig. 2iih) for various MF-diameters depending on spectral hands. Due to low standard deviations, vertical error bars cannot he shown.

to the increase of reflectances in the infrared (782 nm) and green (546 nm) spectral bands with increasing MF-diameters (Fig. 5). Larger differences between reflectance curves in palisade parenchyma (Fig. 2iia) than those in spongy parenchyma (Fig. 2iib) indicated comparatively stronger influenc- es of detour effect on absorption of photosynthetic light and reflectance of nonphotosynthetic light in the palisade parenchyma.

The reflectance curves of palisade and spongy parenchyma depending on wavelengths as depicted in Fig. 6 show that the typical reflectance curve of beech leaf (22) could be obtained by averaging diffuse reflectance signals from MF- diameters larger than or equal to 3 1.5 pm. For MF-diameters below 3 1 .5 pm, the proportion of diffuse reflectance at mean reflectance was suggested to decrease and the mean reflectance was mainly due to the optical properties of cells/& sues. Apparently, due to the low absorbance of red spectral band per unit area by low-density spongy parenchyma (ratio tissue area : total transverse section area = 0.5; typical chlo-

Figure 7. Chlorophyll fluorescence image (inverse-video represen- tation: high intensity white fluorescence pixels represented as black pixels) of paradermal chloroplasts of beech leaves seen at the adaxial surface. The circles indicate superimposition of various MF for the chloroplast count, e.g. interior circle below 0.63 and interior circle below 1.6 indicate 50 pm and 80 pm MF-diameter, respectively.

roplast countlcell: 4-5) as compared to palisade parenchyma (ratio tissue area : total transverse section area = 0.9; typical chloroplast countlcell: 1 l), the ratio of reflectance at 680 nm to reflectance at 782 nm decreased.

Relationship between paradermal reflectances and chloroplast count

Figure 7 shows the paradermal chloroplast fluorescence image taken by confocal LSM with various measuring field diameters superimposed. Thus, the count of chloroplasts in each MF could be determined with acceptable accuracy.

As the area increases parabolically with increasing MF- diameters, as expected, the count of chloroplasts averaged from areas increased parabolically (Fig. 8). Due to the quadratic relationship of the paradermal reflectance with MF- diameter (Fig. 5), the paradermal reflectances of adaxial palisade parenchyma (Fig. 2iia) increased linearly with the count of chloroplasts, strongly for green (546 nm) and infrared (782 nm) spectral bands (Fig. 8). The absorbance of photosynthetically active light in the violet (404 ? 49 nm, Fig. 8) to blue (paradermal reflectance at 453 nm for palisade, Fig. 2iia: 3.81 % and for spongy parenchyma, Fig. 2iib: 4.57%; data not shown in Fig. 8) and red spectral (658 ? 20 nm) zones reduced strongly the respective reflectances. The shape of the light gradients within Cucurbitu cotyledons was related to the amount of absorption by the tissues. At 730 nm, where there was little absorption, the calculated gradient was approximately linear, whereas at other wavelengths of strong absorbances such as 450 nm, it decreased exponentially with depth (21). Thus, in paradermal palisade tissues (Fig. 2iia) the chlorophyll density-related absorbance effect should be considered for the interpretation of experi- mental results.


100, , , , , I , I I , I

10 t /

40

0

0 0 0 0

W 50 V

V

0 0 B U

0 A 0 30 0

300 400 500 600 700 800 900

WAVELENGTH (nm)

Figure 9. Lateral reflectance curves of adaxial epidermis (O), adaxial palisade parenchyma (O), abaxial palisade parenchyma (A), spongy parenchyma focused on cells (V), spongy parenchyma focused on intercellular spaces ( 0 ) and abaxial epidermis (0) using transverse sections (thickness ca 20 pm) depending on spectral bands. The reflectances of definite tissuekell layers were measured using 5 pm measuring fields. High reflectances of epidermal tissues on transverse sections, especially in the infrared range, are apparently due to the nonabsorbance of infrared light and high opacity of lateral epidermal tissues to infrared light.

A

0 I I'

0 10 20 30 40 50 60 70 80 90

CHLOROPLASTS (COUNT)

Figure 8. Top-Parabolic relationship between chloroplast count (y) and MF-diameter (x = pm). The parabolic relationship could be described by the regression equation, y = 0.33270 - 0.02175~ + 0 . 0 1 2 9 3 ~ ~ (r2 = 1.00). Vertical bars indicate standard deviations. Bottom-linear relationships between paradermal reflectances of palisade parenchyma with spongy parenchyma plus abaxial epidermis (y; see Fig. 2iia) and chloroplast count (x). The linear relationships could be described by the following regression lines, V- 782 nm y = 1.23694 + 2.14817~ (r2 = 1.00). W-546 nm y = 0.34132 + 0.67331~ (r2 = 1.00), A-658 nm y = 0.26750 + 0.20479~ (r2 = 1.00) and 0 4 0 4 nm y = 0.09654 + 0.06756~ (r2 = 1.00), respectively. Due to low standard deviations, vertical error bars cannot be shown.

Lateral reflective light gradient within a beech leaf

The reflectances of the adaxial epidermis, adaxial palisade parenchyma, abaxial palisade parenchyma, spongy parenchyma and the abaxial epidermis depending on wavelengths for transverse sections are shown in Fig. 9. Over all the spectral bands, the reflectance of the upper and lower cell layers of palisade parenchyma tissues of a sun leaf were similar. The reflectances of spongy parenchyma were higher than those of the palisade parenchyma. The reflectances of the spongy parenchyma were mainly due to the cellular structures and cell contents (Fig. 9). Over the whole spectral range (404-782 nm), the reflectances of the cells were higher than those of the intercellular region, whereby the greatest difference was shown in the green band of chlorophyll reflectance. The reflectances of abaxial epidermis were slightly lower than those of the adaxial epidermis in the spectral

range 404-546 nm but significantly higher than those of the adaxial epidermis in the spectral range 658-782 nm indicat- ing lower absorbance and enhanced scattering of red and infrared light by the abaxial epidermis. The minimum of reflectance at 658 nm as observed also for adaxial and to a less extent for abaxial epidermis could be due to the occur- rence of negligible amounts of chloroplasts in the stomata1 complexes (see Materials and Methods).

The reflectances across various tissue layers of transverse sections showed an inverse relationship with the count of chloroplasts (Fig. 10). The changes of reflectances depending on distance of tissue layers from the adaxial epidermal surface up to the abaxial epidermis (see figure legends for relationship between distance and tissuekell zone) took the shape of a parabola irrespective of wavelengths, as shown in Fig. 10 and showed typical shape of reflectance curves for all the corresponding tissuekell zones (Fig. 9). The low- est reflectances of violet light (404 nm) observed for the adaxial and abaxial epidermis indicated the absorptive screening function of the epidermis for the violet portion of sunlight. The protection of beech leaves from violet and ultraviolet (UVB) light might be mediated by the phenolic substances and pigments located in the epidermis (25). The reflectances of red light were similar to those of the blue band in the adaxial epidermis and the upper layer of palisade parenchyma but higher in the lower layer of palisade parenchyma, in the intracellular spongy parenchyma and especially higher in the abaxial epidermis. The reflectance curves of infrared light showed the highest values across all the tissues. In the adaxial epidermis, its value was similar to that of the green band, but higher than that of the upper and lower layer of palisade parenchyma, intracellular spongy tissue and abaxial epidermis. The reflective green light was


p 1 0 - z 3 0 2 8 - v) k (I)

4 6 - 0 u:

I 0

a

E: 4 -

I

i 1

E E

wavelengths of strong absorbances (Fig. 10) supports the view that the attenuance of light in mesophyll tissues could be expressed by Beer’s law for wavelengths of strong absorbances (23) .

Comparison of paradermal and lateral reflectances

The spongy parenchyma (Fig. 2iib) contributed considerably to the magnitude of paradermal reflectances (Fig. 4). The intrinsic optical properties (mainly reflectances and absorbance of light) of leaf tissues determine the vertical light gradient in the paradermal and the horizontal light gradient in the lateral direction. In the paradermal and lateral direc-

0 0 20 40 60 80 100 120 140 160 160 200

DISTANCE OF TISSUE LAYERS (pm)

70 - I 60

I tions the dispersion and absorbance of photosynthetic light

0 20 40 60 80 100 120 140 160 180 200

DISTANCE OF TISSUE LAYERS (pm)

Figure 10. Top-parabolic relationship between chloroplast count (y) and typical distance of definite tissuekell layers of transverse sections (ca 20 pm thick) from adaxial surface (see legend in Fig. 9, and below for tissue/cell layers). The relationship between chloroplast count and typical distance of tissue/cell layers (x. in pm; see below) can be described by the regression equation y = -2.04420 + 0.27035~ - 0 . 0 0 1 5 0 ~ ~ (r2 = 0.94), whereby x = typical distance of definite tissue/cell layers. Bottom-parabolic relationships between lateral reflectances (y, in %) and typical distance of definite tissuekell layers (x = pm). The parabolic relationships could be described by the following quadratic regressions, 0-782 nm y = 337.90904 - 4 .46653~ + 0 . 0 2 4 5 0 ~ ~ (r2 = 0.92), A--546 nm y = 318.10205 ~ 4.63972~ + 0 . 0 2 4 7 0 ~ ~ (r2 = 0.96), 0-658 nm y = 196.74872 - 4.05727~ + 0 . 0 2 3 9 2 ~ ~ (r2 = 0.83), 0 4 5 3 nm y = 202.7201 - 4.06940~ + 0 . 0 2 1 2 7 ~ ~ (r2 = 0.96) and 0 4 0 4 nm y = 158.75336 - 3.11756~ + 0 . 0 1 6 2 6 ~ ~ (r2 = 0.96), respectively. The reflectances of definite tissuekell layers (0-10 pm: adaxial epidermis, 11-60 pm: adaxial palisade parenchyma, 61-90 pm: abaxial palisade parenchyma, 9 1-1 60 pm: spongy parenchyma, 161-170 pm: abaxial epidermis; see Materials and Methods) were measured using 5 pm MF.

especially more intensive than that of the red light across all the tissues except the abaxial epidermis. It should be noted that the nonphotosynthetic green light was absorbed to some extent by the mesophyll.

Thus, the absorbance of laterally transmitted andor re- fracted monochromatic light by chloroplasts/chlorophyll molecules plays a decisive role for the lateral light gradient within a leaf. The nonlinear decreasing pattern of reflectances with increasing depth up to spongy parenchyma for

I

(blue and red light band) by mesophyll tissues, especially by palisade parenchyma, and reflectances of nonphotosynthetic light by mesophyll, especially by spongy parenchyma, mainly establish the vertical and horizontal light gradient in the three dimensional space of leaves.

Terashima and Inoue (26) reported the intraleaf vertical gradient in the photosynthetic properties of Camellia chloroplasts. As photosynthesis requires light, the intraleaf vertical light gradient is an obvious prerequisite for the intraleaf vertical gradient in the photosynthetic properties of Camellia chloroplasts. Thus, depending on intraleaf light gradient an intraleaf vertical and horizontal gradient in the photosynthetic properties of Fagus chloroplasts could be expected.

CONCLUSIONS The reflective light gradient between adaxial and abaxial sides is created by the intrinsic optical properties and optical effects of adaxial, abaxial epidermis and mesophyll tissues. The scattering of light within the leaf is highly complex. The macroscopic observations of the scattering phenomena (1,6,7,16) as well as the enhancement of reflectance by the detour effect (1 5,16) and the increase of reflectances by lowering the efficiency of the absorbers by the sieve effect (15,16) could be well described either by the multiple-scattering wave theories or the theory of radiative transfer based on two-flux treatment (Kubelka-Munk theory) and multiflux treatment (16,19) and could be true for paradermal reflectances of Fagus leaf tissues. The reflectance and the absorbance of light in the spongy parenchyma is enhanced by multiple scattering. However, selective absorbances of scattered light (see above) at wavelengths of strong absorbances by chloroplasts/chlorophyll-protein complexes and multiple scattering by chloroplasts at wavelengths of weak absorbances significantly determine the magnitude of paradermal reflectances in the tissuekell layers of Fagus leaves. Simi- larly, at wavelengths of strong absorbances, e.g. 450 and 680 nm, light was strongly absorbed in Medicago leaves and 90% was removed by the initial 50 pm of the leaflet, which corresponds to the passage through an epidermal cell and one of a palisade cell in the Medicago leaflet (21).

In all the spectral ranges, the chlorophyll molecules of chloroplasts absorb light apparently so strongly that the lateral reflectance values in the mesophyll tissues decreased below the corresponding values of the epidermal tissues (Fig. 10). Our results support, therefore, the view that intraleaf lateral reflectances are mainly determined by selective absorbances of light by chlorophyll-protein complexes of


chloroplasts in the mesophyll at wavelengths of strong absorbances (blue and red bands) and by multiple scattering (1,6,16) especially at wavelengths of weak absorbances, e.g. green and far-redinfrared bands.

REFERENCES

1. Tucker, C.J. and M.W. Garratt (1977) Leaf optical system mod- eled as a stochastic process. Appl. Opr. 16, 635-642.

2. Hoque, E., P.J.S. Hutzler and H.K. Seidlitz (1988) Relationship between discoloration and histological changes in leaves of trees affected by forest decline. Remote Sens. Environ. 26, 171-184.

3. Hoque, E., P.J.S. Hutzler and H. Hiendl (1990) Studies on reflective features of Norway spruce and their possible applica- tions in remote sensing of forest damage. Toxicol. Environ. Chem. 27, 209-215.

4. Hoque, E., P.J.S. Hutzler and H. Hiendl (1992) Reflectance, colour, and histological features as parameters for early assessment of forest damages. Can. J . Remote Sens. 18, 98-104.

5. Hoque, E. and G. Remus (1994) Native and atrazine-induced fluorescence of chloroplasts from palisade and spongy parenchyma of beech (Fagus sylvatica L.) leaves. Remote Sens. En- viron. 47, 77-86.

6. Sinclair, T.R., M.M. Schreiber and R.M. Hoffer (1973) Diffuse reflectance hypothesis for the pathway of solar radiation through leaves. Agron. J . 65, 276-283.

7. Willstatter, R. and A. Stoll (1918) Untersuchungen uber die Assimilation der Kohlensaure, pp. 122-1 27. Springer Verlag, Berlin.

8. Kubelka, P. and F. Munk (1931) Ein Beitrag zur Optik der Far- banstriche. 2. technische Physik l la , 593-601.

9. Allen, W.A., H.W. Gausman and A.J. Richardson (1973) Will- statter-Stoll theory of leaf reflectance evaluated by ray tracing.

10. Kumar, R. and L. Silva (1973) Light ray tracing through a leaf

1 1 . Gausmann, H.W. (1977) Reflectance of leaf components. Re-

12. Allen, W.A. and A.J. Richardson (1968) Interaction of light with

13. Luedeker, W. (1989) Reflektionsspektroskopie an griinen Blat-

Appl. Opt. 12, 2448-2453.

transverse section. Appl. Opt. 12, 2950-2954.

mote Sens. Environ. 6, 1-9.

a plant canopy. J. Opt. SOC. Am. 58, 1023-1028.

tern. Diplom Thesis, Faculty of Physics, University of Olden- burg, FRG.

14. Kok, B. (1948) A critical consideration of the quantum yield of Chlorella photosynthesis. Enzymologia 13, 1-56.

15. Fukshansky, L. (1981) Optical properties of plants. In Plants and the Day Light Spectrum (Edited by H. Smith), pp. 21-40. Academic Press, London.

16. Fukshansky, L., A. Martinez v. Remisowsky, J. McClendon, A. Ritterbusch, T. Richter and H. Mohr (1993) Absorption spectra of leaves corrected for scattering and distributional error: a radiative transfer and absorption statistics treatment. Photochem. Phorobiol. 57, 538-555.

17. Duysens, L.N.M. (1956) The flattening of the absorption spectrum of suspensions as compared to that of solutions. Biochim. Biophys. Acta 19, 1-12.

18. Riihle, W. and A. Wild (1979) The intensification of absorbance changes in leaves by light dispersion. Differences between high- light and low-light leaves. Planta 146, 551-557.

19. Martinez v. Remisowsky, A,, J.H. McClendon and L. Fukshan- sky (1992) Estimation of the optical parameters and light gradients in leaves: multi-flux versus two-flux treatment. Photo- chem. Photobiol. 55, 857-865.

20. Born, M. (1965) Optik, pp. 373-375. Springer Verlag, Berlin. 21. Vogelmann, T.C. (1989) Penetration of light into plants. Pho-

tochem. Photobiol. 50, 895-902. 22. Hoque, E. and P.J.S. Hutzler (1992) Spectral blue-shift of red

edge monitors damage class of beech trees. Remote Sens. En- viron. 39, 81-84.

23. Terashima, I. and T. Saeki (1983) Light environment within a leaf I. Optical properties of paradermal sections of Camellia leaves with special reference to differences in the optical properties of palisade and spongy tissues. Plant Cell Physiol. 24, 1493-1501.

24. Terashima, I. and T. Saeki (1985) A new model for leaf photosynthesis incorporating the gradients of light environment and of photosynthetic properties of chloroplasts within a leaf. Ann. Bot. 56, 489-499.

25. Lovelock, C.E., B.F. Clough and I.E. Woodrow (1992) Distri- bution and accumulation of ultraviolet-radiation-absorbing com- pounds in leaves of tropical mangroves. Planta 188, 143-154.

26. Terashima, I. and Y. Inoue (1984) Comparative photosynthetic properties of palisade tissue chloroplasts and spongy tissue chloroplasts of Camellia japonica L.: functional adjustment of the photosynthetic apparatus to light environment within a leaf. Plant Cell Physiol. 25, 555-563.

Documents

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