Simultaneously measuring pulse-amplitude-modulated (PAM) chlorophyll fluorescence of leaves at wavelengths shorter and longer than 700 nm.

PAM fluorescence of leaves of cherry laurel (Prunus laurocerasus L.) was measured simultaneously in the spectral range below 700 nm (sw) and above 700 nm (lw). A high-sensitivity photodiode was employed to measure the low intensities of sw fluorescence. Photosystem II (PSII) performance was analyzed by the saturation pulse method during a light response curve with subsequent dark phase. The sw fluorescence was more variable, resulting in higher PSII photochemical yields compared to lw fluorescence. The variations between sw and lw data were explained by different levels of photosystem I (PSI) fluorescence: the contribution of PSI fluorescence to minimum fluorescence (F0) was calculated to be 14% at sw wavelengths and 45% at lw wavelengths. With the results obtained, the validity of an earlier method for the quantification of PSI fluorescence (Genty et al. in Photosynth Res 26:133-139, 1990, https://doi.org/10.1007/BF00047085 ) was reconsidered. After subtracting PSI fluorescence from all fluorescence levels, the maximum PSII photochemical yield (FV/FM) in the sw range was 0.862 and it was 0.883 in the lw range. The lower FV/FM at sw wavelengths was suggested to arise from inactive PSII reaction centers in the outermost leaf layers. Polyphasic fluorescence transients (OJIP or OI1I2P kinetics) were recorded simultaneously at sw and lw wavelengths: the slowest phase of the kinetics (IP or I2P) corresponded to 11% and 13% of total variable sw and lw fluorescence, respectively. The idea that this difference is due to variable PSI fluorescence is critically discussed. Potential future applications of simultaneously recording fluorescence in two spectral windows include studies of PSI non-photochemical quenching and state I-state II transitions, as well as measuring the fluorescence from pH-sensitive dyes simultaneously with chlorophyll fluorescence.

Linking chloroplast relocation to different responses of photosynthesis to blue and red radiation in low and high light-acclimated leaves of Arabidopsis thaliana (L.).

Low light (LL) and high light (HL)-acclimated plants of A. thaliana were exposed to blue (BB) or red (RR) light or to a mixture of blue and red light (BR) of incrementally increasing intensities. The light response of photosystem II was measured by pulse amplitude-modulated chlorophyll fluorescence and that of photosystem I by near infrared difference spectroscopy. The LL but not HL leaves exhibited blue light-specific responses which were assigned to relocation of chloroplasts from the dark to the light-avoidance arrangement. Blue light (BB and BR) decreased the minimum fluorescence ([Formula: see text]) more than RR light. This extra reduction of the [Formula: see text] was stronger than theoretically predicted for [Formula: see text] quenching by energy dissipation but actual measurement and theory agreed in RR treatments. The extra [Formula: see text] reduction was assigned to decreased light absorption of chloroplasts in the avoidance position. A maximum reduction of 30% was calculated. Increasing intensities of blue light affected the fluorescence parameters NPQ and qP to a lesser degree than red light. After correcting for the optical effects of chloroplast relocation, the NPQ responded similarly to blue and red light. The same correction method diminished the color-specific variations in qP but did not abolish it; thus strongly indicating the presence of another blue light effect which also moderates excitation pressure in PSII but cannot be ascribed to absorption variations. Only after RR exposure, a post-illumination overshoot of [Formula: see text] and fast oxidation of PSI electron acceptors occurred, thus, suggesting an electron flow from stromal reductants to the plastoquinone pool.

Linking chlorophyll a fluorescence to photosynthesis for remote sensing applications: mechanisms and challenges.

Chlorophyll a fluorescence (ChlF) has been used for decades to study the organization, functioning, and physiology of photosynthesis at the leaf and subcellular levels. ChlF is now measurable from remote sensing platforms. This provides a new optical means to track photosynthesis and gross primary productivity of terrestrial ecosystems. Importantly, the spatiotemporal and methodological context of the new applications is dramatically different compared with most of the available ChlF literature, which raises a number of important considerations. Although we have a good mechanistic understanding of the processes that control the ChlF signal over the short term, the seasonal link between ChlF and photosynthesis remains obscure. Additionally, while the current understanding of in vivo ChlF is based on pulse amplitude-modulated (PAM) measurements, remote sensing applications are based on the measurement of the passive solar-induced chlorophyll fluorescence (SIF), which entails important differences and new challenges that remain to be solved. In this review we introduce and revisit the physical, physiological, and methodological factors that control the leaf-level ChlF signal in the context of the new remote sensing applications. Specifically, we present the basis of photosynthetic acclimation and its optical signals, we introduce the physical and physiological basis of ChlF from the molecular to the leaf level and beyond, and we introduce and compare PAM and SIF methodology. Finally, we evaluate and identify the challenges that still remain to be answered in order to consolidate our mechanistic understanding of the remotely sensed SIF signal.

Deriving fluorometer-specific values of relative PSI fluorescence intensity from quenching of F(0) fluorescence in leaves of Arabidopsis thaliana and Zea mays.

The effect of stepwise increments of red light intensities on pulse-amplitude modulated (PAM) chlorophyll (Chl) fluorescence from leaves of A. thaliana and Z. mays was investigated. Minimum and maximum fluorescence were measured before illumination (F(0) and F(M), respectively) and at the end of each light step (F'(0) and F'(M), respectively). Calculated F'(0) values derived from F(0), F(M) and F'(M) fluorescence according to Oxborough and Baker (1997) were lower than the corresponding measured F'(0) values. Based on the concept that calculated F'(0) values are under-estimated because the underlying theory ignores PSI fluorescence, a method was devised to gain relative PSI fluorescence intensities from differences between calculated and measured F'(0). This method yields fluorometer-specific PSI data as its input data (F(0), F(M), F'(0) and F'(M)) depend solely on the spectral properties of the fluorometer used. Under the present conditions, the PSI contribution to F (0) fluorescence was 0.24 in A. thaliana and it was independent on the light acclimation status; the corresponding value was 0.50 in Z. mays. Correction for PSI fluorescence affected Z. mays most: the linear relationship between PSI and PSII photochemical yields was clearly shifted toward the one-to-one proportionality line and maximum electron transport was increased by 50 %. Further, correction for PSI fluorescence increased the PSII reaction center-specific parameter, 1/F(0) - 1/F(M), up to 50 % in A. thaliana and up to 400 % in Z. mays.

Deriving room temperature excitation spectra for photosystem I and photosystem II fluorescence in intact leaves from the dependence of FV/FM on excitation wavelength.

The F(0) and F(M) level fluorescence from a wild-type barley, a Chl b-less mutant barley, and a maize leaf was determined from 430 to 685 nm at 10 nm intervals using pulse amplitude-modulated (PAM) fluorimetry. Variable wavelengths of the pulsed excitation light were achieved by passing the broadband emission of a Xe flash lamp through a birefringent tunable optical filter. For the three leaf types, spectra of F(V)/F(M) (=(F(M) - F(0))/F (M)) have been derived: within each of the three spectra of F(V)/F(M), statistically meaningful variations were detected. Also, at distinct wavelength regions, the (V)/F(M) differed significantly between leaf types. From spectra of F(V)/F (M), excitation spectra of PS I and PS II fluorescence were calculated using a model that considers PS I fluorescence to be constant but variable PS II fluorescence. The photosystem spectra suggest that LHC II absorption results in high values of F(V)/F(M) between 470 and 490 nm in the two wild-type leaves but the absence of LHC II in the Chl b-less mutant barley leaf decreases the F(V)/F(M) at these wavelengths. All three leaves exhibited low values of F(V)/F(M) around 520 nm which was tentatively ascribed to light absorption by PS I-associated carotenoids. In the 550-650 nm region, the F(V)/F(M) in the maize leaf was lower than in the barley wild-type leaf which is explained with higher light absorption by PS I in maize, which is a NADP-ME C(4) species, than in barley, a C(3) species. Finally, low values of F(V)/F(M) at 685 in maize leaf and in the Chl b-less mutant barley leaf are in agreement with preferential PS I absorption at this wavelength. The potential use of spectra of the F(V)/F(M) ratio to derive information on spectral absorption properties of PS I and PS II is discussed.

In vivo assessing flavonols in white grape berries (Vitis vinifera L. cv. Pinot Blanc) of different degrees of ripeness using chlorophyll fluorescence imaging.

Exposed and non-exposed halves of field-grown berries of the white grapevine Vitis vinifera L. cv. Pinot Blanc at various stages of ripeness were analysed using chlorophyll fluorescence imaging. The stage of ripeness was classified by the total sugar concentration which ranged between 120 and 300 g L-1 for the different berries but was similar in the exposed and the non-exposed half of individual berries. Fluorescence was excited in the UV-A and the blue spectral region and detected at red as well as far-red wavelengths. At both emission ranges, UV-excited fluorescence was weak and required correction for the contribution of small false signals. After correction, in vivo UV screening by berry skins was derived from the ratio of UV-A to blue-excited fluorescence intensities, and a relationship between in vivo UV screening and flavonol quantity was established: the quantity of flavonols was determined by spectral analysis of extracted phenolics. Significantly high flavonol concentrations, and effective in vivo UV screening, were detected in most exposed half-berries at sugar concentrations higher than 200 g L-1 but not in non-exposed samples. This suggests that radiation-exposure conditions determine flavonol synthesis. Based on the absence of flavonol accumulation in exposed half-berries with sugar concentrations smaller than 200 g L-1, however, it is suggested that berries need to arrive at an advanced stage of ripeness before responding to radiation-exposure by synthesising large amounts of UV-protecting flavonols. Chlorophyll degradation, which was followed by blue-excited intensities of far-red fluorescence, progressed in parallel with increasing sugar content suggesting that chlorophyll degradation is associated with berry ripening. In addition, exposure to sunlight appeared to slightly stimulate chlorophyll decay.

Investigating UV screening in leaves by two different types of portable UV fluorimeter reveals in vivo screening by anthocyanins and carotenoids.

Two portable instruments, designed to evaluate epidermal UV screening in leaves, were compared: the Dualex and the UV-A-PAM fluorimeter. Both instruments excite chlorophyll fluorescence at the same UV wavelengths but reference excitation is in the red and the blue spectral range in the former and the latter fluorimeter, respectively. When analyzing green leaves, general agreement of the data is obtained with the two instruments. In the presence of anthocyanins, the UV-A-PAM fluorimeter provided higher estimates for epidermal UV transmittance than the Dualex fluorimeter, which was attributed to absorption of blue excitation light by anthocyanins. By comparing data from the instruments, anthocyanin-dependent transmittance of 50% was determined in abaxial sides of some autumn leaves, and also in abaxial sides of tropical shade plants. Further, with leaves of chlorophyll b-less mutants of H. vulgare, unusually high epidermal UV transmittance was detected but this was attributed to the lack of chlorophyll b absorption and, in addition, to absorption of blue radiation by xanthophylls which are not functionally connected to photosystems.

Multispectral fluorescence and reflectance imaging at the leaf level and its possible applications.

Images taken at different spectral bands are increasingly used for characterizing plants and their health status. In contrast to conventional point measurements, imaging detects the distribution and quantity of signals and thus improves the interpretation of fluorescence and reflectance signatures. In multispectral fluorescence and reflectance set-ups, images are separately acquired for the fluorescence in the blue, green, red, and far red, as well as for the reflectance in the green and in the near infrared regions. In addition, 'reference' colour images are taken with an RGB (red, green, blue) camera. Examples of imaging for the detection of photosynthetic activity, UV screening caused by UV-absorbing substances, fruit quality, leaf tissue structure, and disease symptoms are introduced. Subsequently, the different instrumentations used for multispectral fluorescence and reflectance imaging of leaves and fruits are discussed. Various types of irradiation and excitation light sources, detectors, and components for image acquisition and image processing are outlined. The acquired images (or image sequences) can be analysed either directly for each spectral range (wherein they were captured) or after calculating ratios of the different spectral bands. This analysis can be carried out for different regions of interest selected manually or (semi)-automatically. Fluorescence and reflectance imaging in different spectral bands represents a promising tool for non-destructive plant monitoring and a 'road' to a broad range of identification tasks.

Noninvasive evaluation of the degree of ripeness in grape berries (vitis vinifera L. Cv. Bacchus and silvaner) by chlorophyll fluorescence.

The use of chlorophyll fluorescence measurements to noninvasively evaluate degrees of ripeness was investigated in berries at various stages of ripening from two white grapevine cultivars (Vitis vinifera L. Cv. Bacchus and Silvaner). Berries were characterized by diameter, weight, and density and by concentrations of fructose, glucose, sucrose, and total sugars, as well as fructose/glucose ratios, and also by chlorophyll fluorescence at F(0) and F(M) levels and the fluorescence ratio F(V)/F(M). Pearson product moment correlation analysis on data from both cultivars revealed clear negative associations between F(0) and concentrations of fructose, glucose, and total sugars, and fructose/glucose ratios (correlation coefficient < -0.89). Curvilinear trend-lines were established for plots of F(0) versus concentrations of fructose, glucose, and total sugars, but a linear relationship between F(0) and fructose/glucose ratios was found: the corresponding coefficients of determination were always >0.82. Therefore, chlorophyll fluorescence measurements are well-suited to determine noninvasively sugar accumulation in grape berries during ripening.

Action of UV and visible radiation on chlorophyll fluorescence from dark-adapted grape leaves (Vitis vinifera L.).

Grapevine plants (Vitis vinifera L. cv. Silvaner) were cultivated under shaded conditions in the absence of UV radiation in a greenhouse, and subsequently placed outdoors under filters transmitting natural radiation, or screening out the UV-B (280 to 315 nm), or screening out the UV-A (315 to 400 nm) and the UV-B spectral range. All conditions decreased maximum chlorophyll fluorescence (F(M)) and increased minimum chlorophyll fluorescence (F(0)) from dark-adapted leaves; however, with increasing UV, F(M) quenching was stimulated but increases in F(0) were reduced. The F(V)/F(M) ratio (where F(V)=F(M)-F(0)) was clearly reduced by visible radiation (VIS): UV-B caused a moderate extra-reduction in F(V)/F(M). Exposure of leaves (V. vinifera L. cv. Bacchus) to UV or VIS lamps quenched the F(M) to similar extents; further, UV-B doses comparable to the field, quenched F(0). A model was developed to describe how natural radiation intensities affect PS II and thereby change leaf fluorescence. Fitting theory to experiment was successful when the same F(M) yield for UV- and VIS-inactivated PS II was assumed, and for lower F(0) yields of UV- than for VIS-inactivated PS II. It is deduced, that natural UV can produce inactivated PS II exhibiting relatively high F(V)/F(M). The presence of UV-inactivated PS II is difficult to detect by measuring F(V)/F(M) in leaves. Hence, relative concentrations of intact PS II during outdoor exposure were derived from F(M). These concentrations, but not F(V)/F(M), correlated reasonably well with CO(2) gas exchange measurements. Consequently, PS II inhibition by natural UV could be a main factor for UV inhibition of photosynthesis.

UV screening by phenolics in berries of grapevine (Vitis vinifera).

The role of phenolics in UV-screening was investigated in berries of a white grape cultivar (Vitis vinifera L. cv. Bacchus). Fluorescence microscopy revealed accumulation of phenolics in the skin of berries and, by high performance liquid chromatography and mass spectrometry, flavonols and hydroxycinnamic acids were identified as the main groups of UV-absorbing phenolics. Relationships between natural radiation and the synthesis of phenolics were studied in plants that were cultivated in the absence of UV radiation in a greenhouse before outdoor exposure to three different light regimes: the entire solar spectrum, the solar spectrum minus UV-B radiation and only visible radiation. During six days of exposure, flavonol synthesis was significantly stimulated by natural UV, in particular UV-B, but concentrations of hydroxycinnamic acids decreased under all conditions. Direct comparison of fluorimetrically-determined skin absorbance with absorbance of extracted flavonols or hydroxycinnamic acids suggested that acclimation of UV screening depends almost exclusively on flavonol synthesis. While increased flavonol levels resulted in efficient UV-A shielding, UV-B shielding was incomplete, probably due to decreased levels of the UV-B-absorbing hydroxycinnamic acids during exposure.