The fluorescence of plants is a strong indicator of their photosynthetic efficiency as information on the energy transfer processes that occur in plants when exposed to light can be obtained through the spectral characteristics and intensity of fluorescence. Fluorescence can also be used to investigate the effect of environmental factors such as light and temperature on the photochemistry of plants.
Photosystems I and II (PSI and PSII) are protein complexes contained within the chloroplasts whose function is to drive the photosynthetic chain. They are composed of pigments which, when excited with light, initiate a chain of redox reactions which ultimately result in the generation of chemical energy. Chlorophylls and carotenoids are the principal photosynthetic pigments in green plants. Carotenoids are accessory pigments to chlorophylls, which are light-responsive electron donors vital to the photosynthesis chain. Figure 1 illustrates a schematic of the process.
Figure 1. Simplified schematic of the photosynthesis process (Z scheme). P680 and P700 are the chlorophyll centers in photosystems I and II, respectively.
Although there is significant spectral overlap between PSI and PSII, they vary in their fluorescence and absorption transitions. Inside a leaf, the emission spectra of isolated photosystems varie due to interactions with the surrounding matrix. The main PSII emission transitions occur at 685 and 740 nm, where the latter is a side band caused due to vibrational sublevels. PSI emission occurs as a broad band at 720 nm, which is considerably lower in intensity.1 PSII, which is the most widely investigated photosystem, is the principal contributor to fluorescence at room temperature.
The fluorescence of plant leaves is usually investigated using pulse-modulated measurements, which can be overly complicated. Instead, a simpler method is continuous fluorescence analysis, which offers valuable photochemical information.2 It is also possible to use continuous fluorometers for complementary measurements of quantum yields with an integrating sphere.3
When compared to chlorophyll in solution, plant leaves exhibit a considerably lower fluoresence quantum yield. In vivo, the excitation light is converted into either excess heat or photosynthesis. Not only the relative absorbance of the distinct photosynthetic pigments but also the balance between fluorescence, photosynthesis, and non-photochemical quenching can be affected by environmental factors.
Characterization of the absorption of the leaf, besides the fluorescence emission, is essential to obtain a complete picture of the photosynthesis process. This can be achieved with the help of an integrating sphere; but this is not a widely used method and most scientists use an assumed value for absorbance.4 Moreover, low-temperature investigations in leaves are a better procedure for analyzing the effect of quenching on the electron transport chain.5 Essentially, comparisons are made between the behavior at room temperature and at 77 K.
This article presents temperature-dependent measurements of absolute fluorescence quantum yield of perennial leaves from 77 to 300 K. An FLS1000 Photoluminescence Spectrometer including a Cryosphere accessory has been used to obtain the steady-state and quantum yield data. The Edinburgh Instruments Cryosphere enables exact characterization of the variations in quantum yield with temperature, taking into account any variations in absorption that are dependent on temperature.
Investigations of two perennial leaves were performed: viburnum tinus (Sample 1) and holly (Sample 2). The leaf was cut into a square of 1 cm x 1 cm, washed with distilled water, and positioned in an FLS1000 Photoluminescence Spectrometer. A front-face solid sample holder (N-J03) and an Xe2 continuous xenon lamp were used for obtaining the emission spectra at room temperature.
The Edinburgh Instruments Cryosphere accessory was used to carry out the quantum yield measurements, which are shown in Figure 2. The Cryosphere includes a liquid nitrogen cryostat enclosed within an integrating sphere. A 1 cm x 1 cm square of PTFE was used as the reference to perform the direct method of quantum yield measurement was employed. The excitation region of the spectrum was measured without detector saturation effects by positioning a neutral density filter (OD 2) in the emission path.
Figure 2. Variable temperature sample holder (left) and side view of Edinburgh Instruments Cryosphere accessory (right).
Prior to the flow of liquid N2, the samples were positioned in the Cryosphere holder and placed under a vacuum of 10−5 mbar with the help of a turbomolecular pump. The Fluoracle software was used to control the temperature.
The room temperature emission spectra of Sample 1 obtained in 90° configuration and in the Cryosphere is illustrated in Figure 3. In line with the expectations, chlorophyll fluorescence occurred in the 650–850 nm range. In agreement with earlier measurements of fluorescence in plant leaves, the 680 nm transition was found to be weaker than the 740 nm band.6
The reason for this is the reabsorption of PSII fluorescence: the PSII absorption band extends up to 740 nm, making the observed intensity to be lower than anticipated at λ < 740 nm. Furthermore, the geometry of the measurement system governs the self-absorption effect. In the Cryosphere, the emitted light is reflected back to the sample, leading to an increase in reabsorption. The normalized spectra are in agreement above 740 nm, the region which is not affected by self-absorption.
Figure 3. Sample 1 emission spectra acquired at room temperature in front-face sample holder (blue) and in Cryosphere (red, reference background subtracted). The spectra are normalized at 745 nm. Front-face measurement conditions: λex = 460 nm, Δλex = 2 nm, Δλem = 2 nm, step size = 2 nm, dwell time = 0.1 s. Cryosphere measurement conditions: λex = 465 nm, Δλex = 7 nm, Δλem = 4 nm, step size = 0.5 nm, dwell time = 1 s.
There are variations in the shape of the chlorophyll emission bands at 77 K, and in specific cases, maxima can be further differentiated. Apart from the 685 and 740 nm maxima, two bands occur at intermediate wavelengths.2 Emission spectra from Samples 1 and 2 recorded at 77 K are illustrated in Figure 4.
Both the spectra clearly present bands that originate from PSII; however, for Sample 1, a third maximum appears at 696 nm. This band does not appear at room temperature and indicates distinctive distributions of photosynthetic pigments in the samples.1
Figure 4. Peak-normalized 77 K emission spectra from Sample 1 (blue) and Sample 2 (red), recorded in the Cryosphere. Measurement conditions: λex = 465 nm, Δλex = 7 nm, Δλem = 4 nm, step size = 0.5 nm, dwell time = 1 s.
The photosynthetic electron chain is suppressed by cold temperatures, thereby increasing the quantum yield of fluorescence. Figure 5 illustrates this effect, where emission spectra from Sample 2 at different temperatures are shown. As reabsorption has a high influence on the 684 nm transition, it seems to be independent of temperature. Since the scattering peaks and the emission spectra were simultaneously recorded in the Cryosphere, the absolute quantum yield of fluorescence can be calculated using the Fluoracle software package.
The result is illustrated in Figure 6 and indicates a well-defined trend which agrees well with analyses of isolated reaction centers.7
Figure 5. Emission spectra from Sample 2 acquired in Cryosphere at varying temperatures (indicated in legend). Measurement conditions: λex = 465 nm, Δλex = 7 nm, Δλem = 4 nm, step size = 0.5 nm, dwell time = 1 s.
Figure 6. Absolute photoluminescence quantum yield of Sample 2 obtained from the experimental data in Figure 5. The scattering region of the spectrum and the reference sample were measured under the same conditions.
The photochemistry and composition of plant leaves can be analyzed by studying their fluorescence at cryogenic temperatures. The higher sensitivity of the FLS1000 spectrometer enables accurate measurement of the comparatively low fluorescence quantum yield values.
Increases in temperature leads to decrease in the quantum yield, from 5% at 77 to 1% at 300 K. Characterization of this decreasing trend can be easily achieved using the Edinburgh Instruments Cryosphere accessory.
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 E. G. Andrizhiyevskaya, A, Chojnicka, J. A. Bautista, B. A. Diner, R. van Grondelle, and J. P. Dekker. Origin of the F685 and F695 fluorescence in Photosystem II, Photosynthesis Research 84, 173–180 (2005)
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