PLANT BIOPHYSICS - Y. I. Posudin - 2004

II. TRANSPORT PROCESSES IN THE SOIL-PLANT-ATMOSPHERE SYSTEM

9. ENERGY TRANSPORT

9.2. PRACTICAL APPLICATIONS OF RADIATION TRANSPORT

Class="center">9.2.1. Spectroscopic monitoring of plants and vegetation canopies

Vegetation canopy is a complex concept that encompasses the soil along with the plant ensemble and the near-surface atmospheric layer. Controlling the state of such a system requires The Development of advanced, modern monitoring Methods that provide accurate and comprehensive information on all stages of plant development and The impact of various natural and anthropogenic factors.

Promising approaches for addressing these tasks include optical and laser spectroscopy Methods based on the interaction of optical radiation with matter. Optical spectroscopy is founded on recording how changes in this radiation depend on the light wavelength. Let us consider the Basic principles and potential Structure/179.html">Practical Applications OF spectroscopic tools for laboratory and field monitoring of individual plants and vegetation canopies as a whole. Modern Methods of spectroscopic monitoring of vegetation canopies are based on recording and analyzing radiation reflected from the canopy or chlorophyll fluorescence emission.

9.2.2. Reflectance Spectroscopy

Reflection mechanisms. There are several theories attempting to explain the mechanisms of optical radiation reflection from a leaf surface based on geometric optics laws. These are founded on METABOLISM/2.html">THE CONCEPT OF independent light rays that propagate rectilinearly in an optically uniform medium while undergoing refraction and reflection at the boundaries between media with different optical properties.

For instance, one theory [Willstatter and Stoli, 1928] assumes the dominant role of total internal reflection of optical radiation at The Cell-air interface. Assuming a cell refractive index of 1.5, the critical angle determining total internal reflection is 41.8°. This process occurs primarily in the spongy mesophyll region, which contains numerous randomly oriented Cells (Fig. 9.3). The palisade parenchyma (a leaf tissue containing cylindrical parenchymal cells rich in Chloroplasts and oriented perpendicularly to the leaf surface) plays a less significant role in optical radiation propagation.

Fig. 9.3. Total internal reflection within leaf structures.

Another hypothesis suggests the occurrence of diffuse reflection of optical radiation on cellular microfibrils, which serve as the primary Structural elements of palisade Tissues [Sinclair, 1973]. Each microfibril has a diameter of about 10 nm and a length of 10 µm; all of them are randomly oriented (Fig. 9.4).

Fig. 9.4. Light reflection from cellular microfibrils (explanation in text).

The actual mechanism of optical radiation propagation inside a leaf is considerably more complex [Kumar and Silva, 1973]. First, optical radiation can be reflected from cell walls when the angle of incidence does not exceed the critical angle; second, radiation scattering also occurs due to inhomogeneities in the internal cellular environment. The propagation pattern of optical radiation is influenced by Changes in the refractive index of internal leaf components, accompanied by corresponding alterations in the refraction trajectory at the interfaces of "Cell wall-air", "air-cell wall", "cell wall-protoplasm", "protoplasm-cell wall", "cell wall-chloroplast", "chloroplast-cell wall", "chloroplast-protoplasm", and "protoplasm-chloroplast" (Fig. 9.5).

Diffuse reflection

Fig. 9.5. Scheme of radiation scattering by inhomogeneities in the internal cellular environment (explanation in text).

Modern understandings of the mechanisms of optical radiation reflection from a leaf are based on the premise that both diffuse and specular reflection take place. The Nature of reflection depends on the light wavelength, angle of incidence, leaf surface structure, presence of leaf hairs, epicuticular wax, nutritional status, and environmental factors.

Reflective properties of an individual leaf. Reflectance r is defined as The ratio of the intensity of optical radiation I reflected from a given surface of an ideal diffuser in a given direction to the intensity of radiation L incident on that surface:

The reflectance spectrum—that is, the dependence of green leaf reflectance r on the light wavelength—is shown in Fig. 9.6. It features three main regions: 500–750 nm, where plant pigments such as chlorophylls a and b, as well as carotenoids, xanthophylls, and anthocyanins, absorb light; 0.75–1.35 µm, characterized by a high reflectance level due to internal leaf structures (particularly Cellulose); and 1.35–2.50 µm, a region dominated by intense Water absorption with maxima at 1.45 and 1.95 µm. Thus, the reflectance spectrum of an individual leaf is distinguished by a reflectance maximum at 550 nm in the visible spectral region, a broad band at 0.75–1.35 µm, and reflectance maxima at 1.65 and 2.20 µm in the near-infrared region.

Fig. 9.6. Reflectance spectrum of a green leaf.

Reflectance spectroscopy methods. All Methods of Plant reflectance spectroscopy can be divided into laboratory methods, near-field methods (where measurements are taken at a short distance from the plant object), and remote sensing methods [Posudin, 1998, 2000, 2003].

Laboratory methods involve studying leaf reflectance spectra using spectrophotometers equipped with an integrating sphere and a BaSO₄ plate used as a standard.

Near-field methods are based on measuring the reflectance spectrum of a plant canopy under field conditions. A typical instrument for measuring canopy reflectance characteristics in the near-field mode is shown in Fig. 9.7. The measurement range of the instrument, which contains 864 channels, is 400-2500 nm.

Fig. 9.7. Typical instrument for measuring canopy reflectance characteristics in the near-field mode: 1 - window, 2 - screen, 3 - motor, 4 - filter, 5 - CCD detector, 6 - diffraction grating, 7 - information Processing system, 8 - computer, 9 - recording system.

Remote sensing methods are based on The Use of multispectral scanners, whose operating principle consists in recording the spectral reflectance of plant canopies in specific spectral Regions of the visible and infrared spectrum (0.3-14 µm). These regions can be either broad (about 0.2 µm) or narrow (less than 0.01 µm). Multispectral scanning instruments mounted on satellites make it possible to obtain data with a spatial resolution of about 10 m while scanning areas ranging from 60 to 185 km in size. THE PRINCIPLE OF remote sensing using a multispectral scanner is illustrated in Fig. 9.8. The advantage of multispectral scanners is their ability to utilize narrow spectral bands and acquire data in digital form. Multispectral scanners are used for analyzing the Earth's surface, plant canopies, cartography, soil moisture determination, estimation of plant biomass, snow cover, and impassable areas.

Fig. 9.8. Remote sensing of the Earth's surface using a satellite:

a - operating principle of a multispectral scanner; b - scanning system: 1 - motor, 2 - scanner, 3 - monochromator, 4 - mirror, 5 - slit.

Reflectance vegetation indices. To establish functional relationships between the vegetation characteristics of plants under stress and their reflectance parameters, it is advisable to use so-called spectral vegetation indices, which represent the sum, difference, or ratio of spectral parameters determined at specific analytical wavelengths. Let us consider the reflectance vegetation indices used in the spectroscopic monitoring of plants and plant canopies.

Ratio Vegetation Index (RVI) is the ratio of canopy reflectance in the near-infrared (NIR) region to that in the red (RED) region of the spectrum:

Here, NIR corresponds to the 750-1359 nm region and RED to 600-700 nm.

Normalized Difference Vegetation Index (NDVI) is defined as follows:

The advantage of this index is its nearly linear dependence on The amount of plant biomass.

Perpendicular Vegetation Index (PVI), proposed to eliminate The Influence of soil Background, is equal to:

where a and b are constants.

Other spectral indices also exist.

Influence of various factors on leaf reflectance. The reflectance characteristics of an individual leaf are affected by such factors as the level of pigmentation (for example, high chlorophyll concentrations correspond to lower reflectance values), THE POSITION OF the leaf on a particular tier of the plant (older leaves exhibit higher reflectance), and the side of the leaf (the upper side contains more chlorophyll than the lower side). In addition, leaf reflectance is influenced by various stress conditions associated with dehydration (water deficit), extreme temperatures, nutrient deficiencies, and ozone excess.

Reflectance Properties of the plant canopy. A plant canopy can be represented as a multilayer system (Fig. 9.9). Each leaf reflects approximately 50% and transmits approximately 50% of optical radiation. Furthermore, the interaction of the radiation that has passed through the first layer with the second layer also results in splitting the optical radiation into two parts; the process continues with each subsequent layer. Consequently, the effective reflection of optical radiation from a plant canopy differs fundamentally from the situation observed in a single leaf.

Fig. 9.9. Plant canopy as a multilayer system.

Influence of various factors on canopy reflectance. The reflectance properties of a plant canopy depend on canopy geometry (leaf area and orientation, number of leaf layers) and the type of plants forming the canopy. In addition, meteorological and climatic conditions, solar elevation angle, presence of clouds, dust, aerosols, and atmospheric pollution, as well as soil type and spectral properties, and agrochemical field treatments exert a significant influence. As a result, the reflectance spectrum of a plant canopy is characterized by a more pronounced reflection band within the 750-1350 nm range compared to the reflectance spectrum of an individual leaf.

9.2.3. Fluorescence Spectroscopy

Mechanisms of fluorescence. The conversion of solar light energy into the chemical energy of plant tissues lies at The Heart of Photosynthesis. This process includes such stages as Light absorption by a pigment molecule, excitation energy transfer, and Chemical Reactions within the PSII photosystem. The de-excitation of absorbed light energy is accompanied by heat dissipation and the emission of light in the form of chlorophyll fluorescence. Fluorescence is a process accompanied by a transition from the singlet state back to the ground state. Notably, this process exceeds thermal radiation in rate and lasts significantly longer than a single period of light oscillation. Fluorescence consists of the excitation of a substance molecule (a fluorophore capable of fluorescing at a specific wavelength) and the subsequent emission of light at a longer wavelength. It has been established that approximately 2–5% of excitation energy is converted into emitted energy by chlorophyll. The relationship between chlorophyll fluorescence and the overall photosynthetic process is quite complex; however, it should be noted that recording chlorophyll fluorescence in green plant leaves can be used to assess plant status under the influence of various abiotic and anthropogenic factors in both laboratory and field conditions [Techniques..., 1986].

Fluorescence properties of an individual leaf. The fluorescence spectrum of a green leaf is characterized by maxima at 440–450 nm (blue region) and 685–690 nm (red region). The fluorescence spectra of certain plants also exhibit a shoulder at 520–530 nm (green region).

According to current understanding, chlorophyll a is responsible for fluorescence in the red region of the spectrum. Fluorescence in the blue region is associated with chemical compounds such as chlorogenic acid, caffeic acid, Coumarins (aesculin and scopoletin), and stilbenes (i-stilbene, rhaponticin). Fluorophores in the green spectrum region may include the alkaloid berberine and quercetin. THE CONTRIBUTION OF riboflavin, NADP, and phyllohydroquinone to blue and green region fluorescence can be considered negligible.

Methods of fluorescence spectroscopy. Fluorimetry of intact leaves makes it possible to analyze how the shape and intensity of emission spectra depend on chlorophyll fluorescence excitation (Fig. 9.10). A disadvantage of this method is the lengthy recording process, during which certain changes induced by chlorophyll fluorescence kinetics may occur within the leaf.

Fig. 9.10. Chlorophyll fluorescence emission spectrum of an intact leaf.

Recording chlorophyll fluorescence induction makes it possible to observe the temporal kinetics of fluorescence intensity in a dark-adapted green leaf. The core principle is that when a green leaf kept in the dark for 15–20 minutes is illuminated, its chlorophyll fluorescence exhibits induction kinetics (known as the Kautsky effect [Kautsky, Hirsch, 1931]). Two distinct intervals can be identified in this temporal behavior of chlorophyll fluorescence: a rapid rise in fluorescence to a maximum value within 100–500 ms, followed by a slow decline to a steady-state level over 3–5 minutes. Fluorescence induction can be explained by the uncoupling of Photosystems I and II in the dark and the transition of the photosynthetic apparatus from state I to state II upon illumination. A typical induction curve is shown in Fig. 9.11, and a schematic diagram of a dual-wavelength fluorimeter is presented in Fig. 9.12. The method of direct green sample fluorescence induction recording is not without certain drawbacks, such as the dependence of the recorded signal on excitation light intensity and Interference from ambient light.

Fig. 9.11. Typical induction curve.

Fig. 9.12. Schematic diagram of a dual-wavelength fluorimeter: 1 – He-Ne laser, 2 – optical fiber, 3 – leaf, 4 – 690 nm filter, 5 – 740 nm filter, 6 – photodiodes, 7 – recording system.

Optical multichannel analysis (OMA) is based on the simultaneous recording of fluorescence in the blue, green, and red spectral regions using a polychromator with a diffraction grating and a linear diode array detector (containing up to 512 elements). Chlorophyll fluorescence is excited by ultraviolet laser radiation. Rather than sequentially recording fluorescence spectra, such a system captures fluorescence intensity across all wavelengths simultaneously. Consequently, chlorophyll fluorescence induction can be neglected when using this method. The layout of an OMA system is shown in Fig. 9.13.

Fig. 9.13. Optical multichannel analyzer: a – analyzer layout: 1 – UV laser, 2 – leaf, 3 – window, 4 – diffraction grating, 5 – diode array, 6 – optical multichannel system, 7 – amplifier, 8 – recording system, 9 – trigger; b – simultaneously recorded chlorophyll fluorescence emission spectra of a green leaf.

Pulse-amplitude modulation fluorimetry, or "PAM fluorimetry" [Schreiber et al., 1986], involves estimating the ground fluorescence level F_o, maximum fluorescence F_m, as well as the photochemical (qP) and non-photochemical (qN) quenching coefficients. Electron transport along the Electron Transport Chain is accompanied by the decrease (quenching) of chlorophyll fluorescence. This reduction occurs due to The oxidation of the acceptor, which is a complex of pheophytin and Quinones. When the acceptor is oxidized via electron transfer to NADP and ultimately to CO₂, fluorescence decreases. This process is termed "photochemical quenching" and is characterized by the photochemical quenching coefficient qP. Concurrently, other quenching mechanisms of a non-chemical nature—known as "non-photochemical quenching"—take place and are characterized by the non-photochemical quenching coefficient qN. The primary quenching processes include energy-dependent quenching associated with the proton gradient induced across the thylakoid membrane, and photoinhibition-induced quenching caused by excess irradiation. Thus, fluorescence is a complementary process to photochemical and thermal pathways: the fluorescence yield increases as energy consumption by photochemical reactions or heat dissipation decreases.

The instrument implementing pulse-amplitude modulation fluorimetry is equipped with a light-emitting diode (LED) operating at an emission wavelength of 655 nm, generating measuring pulses at a frequency ranging from 600 Hz to 20 kHz with an intensity of 0.1 µmol·m^-2·s^-1 PAR. Additionally, the fluorimeter features a halogen lamp with a filter, used to provide actinic radiation (up to 5000 µmol·m^-2·s^-1) and saturation flashes (up to 15000 µmol·m^-2·s^-1). The operational principle of amplitude-modulated fluorimetry relies on exciting fluorescence with a series of light pulses. The intensity of these pulses is too low to drive photosynthesis, yet sufficient to stimulate a fluorescence signal corresponding to the initial baseline level F₀. The moment a "measuring" pulse is applied is designated by the letter A for dark-adapted samples and A' for light-adapted samples (Fig. 9.14). Subsequently, the sample is illuminated with an intense light pulse that causes "saturation" of Reaction Centers and drives them to a closed state. Closed reaction centers gradually reopen and participate in photochemical reactions. In this case, fluorescence intensity rises to the maximum level F_m for dark-adapted samples and to F_m' for samples exposed to actinic light (points B and B', Fig. 9.14). Saturation pulses repeated every 10 s completely reduce the primary acceptor Q_A of PSII. As a result, The electron transport chain between the Two Photosystems is rapidly interrupted. Furthermore, due to the saturating pulse, photochemical fluorescence quenching drops to zero, whereas non-photochemical quenching persists.

Fig. 9.14. Principle of amplitude-modulated fluorimetry: A and A’ denote the timing of the "measuring" pulse application for dark-adapted and light-adapted samples, respectively; B and B’ represent the moments of sample illumination with a saturation pulse.

Fluorescence leaf imaging was performed using a system utilizing a Q-switched Nd:YAG laser as the fluorescence excitation source. An expanded laser beam illuminates the leaf. The emitted fluorescence is directed into an imaging system comprising an interference filter, a lens, an image intensifier, and a computer-interfaced video camera (Fig. 9.15). This setup allows researchers to record the fluorescence field across the entire leaf surface and measure fluorescence intensity profiles longitudinally and transversely.

Fig. 9.15. Fluorescence imaging system: 1 – He-Ne laser, 2 – modulator, 3 – leaf, 4 – lens, 5 – spectrometer, 6 – CCD detector, 7 – computer.

A laser spectrofluorimeter for near-field measurement of plant canopy fluorescence consists of a dye laser as the excitation source (pumped by a 308 nm XeCl excimer laser), a telescope, and a multichannel spectral analyzer. The dye laser beam is directed onto the plant canopy under study. The resulting fluorescence is collected via the telescope and fed into a multichannel analyzer equipped with a diffraction grating and a detector array.

Fluorescence indices. Certain fluorescence parameters can serve as indices for quantifying stress-induced changes in plants. When using spectrofluorimetry, such an index can be expressed as the ratio F(690)/F(740), where F(690) and F(740) represent fluorescence intensities at 690 nm and 740 nm, respectively (Fig. 9.16). For fluorescence induction measurements, the vitality index Rfd = f_d/f_s is used—measured at two wavelengths, Rfd(690) and Rfd(740)—along with the stress adaptation index A_p = 1 - [Rfd(740) + 1]/[Rfd(690) + 1], where f_d = f_m — f_s represents the fluorescence decrease, f_m is the maximum fluorescence, and f_s is the steady-state fluorescence (see Fig. 9.11).

Fig. 9.16. Chlorophyll fluorescence emission spectra with maxima at 690 nm and 740 nm: 1, 2 — upper and lower sides of the leaf, respectively.

The photosynthetic activity of dark-adapted samples is evaluated using PAM fluorometry via the optimal quantum yield where F_v/F_m is the ratio of variable fluorescence F_v = F_m — F₀ to maximum fluorescence F_m, corresponding to closed PSII reaction centers; F₀ is initial fluorescence corresponding to open Photosystem II (PSII) reaction centers. Additionally, it is possible to determine the coefficients of photochemical and non-photochemical and quenching for dark-adapted samples, where F_m’ is the maximum fluorescence intensity of the illuminated sample, and F is the fluorescence intensity at a given moment in time.

Valuable information can also be obtained without dark adaptation of the sample. When the sample is illuminated with high-intensity light, the PSII reaction center participates in light absorption, energy capture, and electron transfer — all processes that take a certain time during which the reaction center remains "closed." The more reaction centers are closed, the lower the photosynthetic efficiency.

To quantify photosynthetic efficiency, parameters such as the effective quantum yield of photosynthesis and the relative electron transport rate are used, where PAR is the photon flux density of photosynthetically active radiation, measured in μmol·m²·s^-1.

Important parameters characterizing the photosynthetic process include photosynthetic efficiency, defined as the linear slope of the relative electron transport rate ETR versus actinic light intensity I (the slope φ_max of the ETR = f(I) dependence is informative here), as well as photosynthetic capacity, which corresponds to the maximum value of the relative electron transport rate ETR_max at the saturation of the ETR = f(I) curve. A typical light curve is shown in Fig. 9.17. Here, 1 and 2 are different levels of light curve saturation corresponding to different values of the sample's photosynthetic capacity; A is the area between the linear portion of the ETR = f(I) curve extrapolated to intersect the saturation level and the curve itself, which corresponds to the amount of absorbed light that could theoretically be used for photosynthesis but is lost due to internal processes occurring in the photosynthetic apparatus; B is the area between two ETR = f(I) curves recorded at optimal and excessive light intensities, corresponding to energy losses due to photoinhibition; C is the area corresponding to the absorbed light energy reduced as a result of photoinhibition.

Fig. 9.17. Typical light curve (explanation in text).

Effect of various factors on the fluorescence properties of an individual leaf. The shape of the leaf fluorescence emission spectrum is affected by: chlorophyll content, leaf development stage, side and segment of the leaf, its age, plant canopy tier, mechanical damage, dehydration, extreme temperatures, nitrogen deficiency, nutrient imbalance, and pollution.

Fluorescence properties of plant canopies. Plant canopy fluorescence under natural conditions differs somewhat from that observed at the individual leaf level. First, one should note the inhomogeneous distribution of chlorophyll among plants. This heterogeneity is related to varying leaf age and, consequently, unequal rates of photosynthesis, pigment concentrations, the dependence of plant physiological status on natural lighting conditions, agrochemical applications, water and Temperature stresses, time of day, and season. Experimental data indicate that the fluorescence emission spectrum is characterized by a 25% reduction in the blue spectrum maximum and an almost complete suppression of the red maxima upon transition from an individual leaf to the plant canopy, provided the excitation wavelength is 337 nm. When excited at a wavelength of 632 nm, an intense increase in fluorescence is observed in the red region of the spectrum. This can be explained by the fact that ultraviolet radiation does not penetrate the epidermis and fails to reach the mesophyll cells.

Effect of various factors on plant canopy fluorescence. Fluorescence spectroscopy makes it possible to record and analyze the impact of various natural and anthropogenic stresses on plant canopies, specifically: agrochemicals, dehydration, mechanical damage, temperature, nitrogen deficiency, biosphere pollution, etc.