Chlorophyll Fluorescence Excitation Research Articles

Photochemical activity in developing pea (Pisum sativum L.) cotyledons depends on the light transmittance of covering tissues and the spectral composition of light.

Many crops require not only leaf photosynthesis for their seed development but also the photochemical reactions that occur in the seeds. The purpose of this work was a comparative analysis of light transmittance and photochemical activity in the leaves of Pisum sativum L. and its pericarp, seed coat, and cotyledons at the early, middle, and late maturation stages. The spectral composition of light was measured using a spectroradiometer in the range of 390-760 nm. We assessed the light transmittance of plant tissues by placing the plant tissue between the light source and the spectroradiometer's sensor. PAM fluorometry was used to quantify the photochemical activity in plant tissues: this technique is handy for evaluating the efficiency of converting light energy into chemical energy through the analysis of the kinetics of chlorophyll fluorescence excitation and quenching. On average, a photochemically active green leaf of pea transmitted 15 % of solar radiation in the 390-760 nm, blue light was delayed entirely, and the transmitted red light never exceeded 5 %. Photochemically active radiation passing through the pericarp and coat and reaching the cotyledons at the early and middle seed maturation stages manifested a high proportion of green and far-red light; there was no blue light, and the percentage of red light was about 2 %. However, the cotyledons were photochemically active regardless of low irradiance and spectral ranges untypical of leaf photosynthesis. At the early and middle maturation stages, the maximum quantum yield of photosystem II (Fv/Fm) averaged 0.5 at the periphery of cotyledons and 0.3 at their center. Since the intensity of embryonic photochemical reactions significantly affects the efficiency of reserve nutrient accumulation, this parameter is a promising marker in pea breeding for seeds with improved nutritional qualities.

QTL Mapping of Wheat Stripe Rust Resistance in Relation to Epidermal Flavonoids Measured Using a Leaf‐Clip, Optical Sensor

Epidermal flavonoids have long been suggested as targets for selection in stress‐adaptive crop breeding, but routine selection is neither practicable nor cost effective because of the time‐consuming process involved in phenotyping. In this research, we found that the content of epidermal flavonoids, measured using the chlorophyll fluorescence excitation ratio method, was inversely related to stripe rust (Puccinia striiformis f. sp. tritici [Pst]) response in wheat (Triticum aestivum L.). We investigated the genetic basis by conducting quantitative trait locus (QTL) mapping with a 9K single nucleotide polymorphism (SNP) array in a cross of ‘Drysdale’ × ‘Waagan’ segregating for adult‐plant stripe rust resistance. Data from 3 yr of field experimental trials were analyzed, and heritability estimates for epidermal flavonoids varied from 0.16 to 0.47 across the experiment‐years. In contrast, heritability for stripe rust response varied from 0.79 to 0.84. Eleven QTL were identified for epidermal flavonoids, with a major locus on chromosome 4D, explaining between 19.5 and 47.3% of the phenotypic variation across years. Thirteen QTL were identified for stripe rust response, with the major locus explaining 68% of the phenotypic variation. Quantitative trait loci associated with epidermal flavonoids and stripe rust response were colocated on chromosomes 2B and 3B, but other genetic loci detected for the respective traits were independent. The study has established a genetic basis for epidermal flavonoids measured using the nondestructive, leaf‐clip sensor in wheat, which will encourage further research on the possible exploitation of the compound by breeding and selection.

Early optical detection of infection with brown rust in winter wheat by chlorophyll fluorescence excitation spectra

Puccinia recondita f. sp. tritici, the causative agent of brown rust disease is the reason for major yield losses in winter wheat plants (Triticum aestivum L.). Early detection of fungal infections would allow a specific fungicide application. Aim of the current study was to develop an automated and computer based device that differentiate infected and healthy plants non-invasively in an early stage of infection. To achieve the target an optical sensor device has been designed (“MultiDetExc”), which excites chlorophyll fluorescence in discrete wavelengths and detects the induced emissions. Wheat plants were infected artificially with brown rust in a climate chamber experiment to survey the capability of the instrument. The chlorophyll fluorescence excitation spectra of whole wheat plants were measured on several days after infection. As reference methods, HPLC and qPCR analysis were included in the current study to measure the polyphenol content of the leaves and the level of infection.The recently developed sensor device is an efficient technique to differentiate the infected and not infected wheat plants as soon as four days after inoculation. The measured fluorescence quotients correlate high positive with the polyphenol contents and the relative amount of fungal DNA. An untreated healthy control was measured parallelly, in order to associate the increased synthesis of polyphenols to the fungal infection definitely.

Quantification of light screening by anthocyanins in leaves of Berberis thunbergii.

Up to 40% of incident light was screened in red Berberis leaves in vivo by anthocyanins, resulting also in up to 40% reduction of light-limited photosynthesis. The biological function of anthocyanins in leaves has been strongly discussed, but the hypothesis of a screening function is favored by most authors. For an evaluation of the function as photoprotective pigments, a quantification of their screening of the mesophyll is important. Here, chlorophyll fluorescence excitation of leaves of a red and a green variety of Berberis thunbergii was used to estimate the extent of screening by anthocyanins at 545nm and over the whole photosynthetically active wavelength range. Growth at high light (430µmolm-2s-1) resulted in 90% screening at 545nm corresponding to 40-50% screening over the whole wavelength range, depending on the light source. The concomitant reduction of photosynthetic quantum yield was of the same size as the calculated reduction of light reaching the chloroplasts. The induction of anthocyanins in the red variety also enhanced the epoxidation state of the violaxanthin cycle under growth conditions, indicating that red leaves were suffering less from excessive irradiance. Pool sizes of violaxanthin cycle carotenoids indicated a shade acclimation of the light harvesting complexes in red leaves. The observed reduction of internal light in anthocyanic leaves has by necessity a photoprotective effect.

The use of in vivo microscopy to image the cnidarian stress response in three octocoral species

AbstractEnergy‐converting symbionts and organelles may be central to the cnidarian stress response. Stress may damage photochemistry in the endosymbiotic dinoflagellate Symbiodinium spp., leaving photosystems I and II relatively reduced and increasing reactive oxygen species (ROS). Alternatively, ROS may emanate from mitochondria of either the host or the symbiont, or both. These sources of ROS can be distinguished by using microscopy to examine the effects of light on stressed cnidarians incubated in the dark with a fluorescent, ROS‐detecting probe. Experiments were carried out with three species of alcyonacean octocoral, Phenganax parrini, Sarcothelia sp., and Sympodium sp. After incubation of colonies for 1 h at elevated temperature, imaging and illumination (excitation 450–490 nm, emission 515–565 nm) were begun simultaneously. Formation of ROS largely corresponded to the onset of illumination. On the other hand, chlorophyll fluorescence (excitation 530–580 nm, emission 620–690 nm) did not conform to this pattern. This difference is consistent with the expected rates of reaction. Remarkably, treatment with the inhibitor 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea (DCMU) resulted in dramatically higher levels of light‐induced ROS. Chlorophyll fluorescence was higher in the DCMU treatment but not significantly so. By controlling for variation between individual symbionts, however, DCMU produced significantly greater levels of chlorophyll fluorescence, indicating the expected greater reduction of photosystem II. A brief exposure to light and thermal stress produced a similar effect in all three species. In addition to ROS being initiated by light, these results indicate that: (1) a brief period of stress shifts photosystem redox state toward reduction, (2) photosystem II can donate electrons to oxygen when blocked with DCMU, and (3) chlorophyll fluorescence is highly variable among individuals of Symbiodinium. Imaging of individual symbionts in hospite thus provides a powerful method for understanding the initial steps of the cnidarian stress response.

Lettuce flavonoids screening and phenotyping by chlorophyll fluorescence excitation ratio.

Environmentally induced variation and the genotypic differences in flavonoid and phenolic content in lettuce can be reliably detected using the appropriate parameters derived from the records of rapid non-invasive fluorescence technique. The chlorophyll fluorescence excitation ratio method was designed as a rapid and non-invasive tool to estimate the content of UV-absorbing phenolic compounds in plants. Using this technique, we have assessed the dynamics of accumulation of flavonoids related to developmental changes and environmental effects. Moreover, we have tested appropriateness of the method to identify the genotypic differences and fluctuations in total phenolics and flavonoid content in lettuce. Six green and two red genotypes of lettuce (Lactuca sativa L.) grown in pots were exposed to two different environments for 50days: direct sunlight (UV-exposed) and greenhouse conditions (low UV). The indices based on the measurements of chlorophyll fluorescence after red, green and UV excitation indicated increase of the content of UV-absorbing compounds and anthocyanins in the epidermis of lettuce leaves. In similar, the biochemical analyses performed at the end of the experiment confirmed significantly higher total phenolic and flavonoid content in lettuce plants exposed to direct sun compared to greenhouse conditions and in red compared to green genotypes. As the correlation between the standard fluorescence indices and the biochemical records was negatively influenced by the presence of red genotypes, we proposed the use of a new parameter named Modified Flavonoid Index (MFI) taking into an account both absorbance changes due to flavonol and anthocyanin content, for which the correlation with flavonoid and phenolic content was relatively good. Thus, our results confirmed that the fluorescence excitation ratio method is useful for identifying the major differences in phenolic and flavonoid content in lettuce plants and it can be used for high-throughput pre-screening and phenotyping of leafy vegetables in research and breeding applications towards improvement of vegetable health effects.

A simple indicator for non-destructive estimation of the violaxanthin cycle pigment content in leaves.

The photosynthetic apparatus of higher plants acclimates to irradiance. Among the features which are changing is the pool size of the pigments belonging to the violaxanthin cycle, in which zeaxanthin is formed. In high light grown leaves, the violaxanthin cycle pool size is up to five times larger than in low light. The changes are reversible on a time scale of several days. Since it has been published that violaxanthin cycle pigments do not transfer absorbed energy to chlorophyll, we hypothesized that excitation of chlorophyll fluorescence in the blue spectral region may be reduced in high light-acclimated leaves. Fluorescence excitation spectra of leaves of the Arabidopsis thaliana tt3 mutant showed strong differences between high and low light-acclimated plants from 430 to 520nm. The resulting difference spectrum was similar to carotenoids but shifted by about 20nm to higher wavelengths. A good correlation was observed between the fluorescence excitation ratio F 470/F 660 and the violaxanthin cycle pool size when leaves were acclimated to a range of irradiances. In parallel to the decline of F 470/F 660 with high light acclimation also the quantum yield of photosynthetic oxygen evolution in blue light decreased. The data confirm that violaxanthin cycle carotenoids do not transfer absorbed light to chlorophyll. It is proposed to use the ratio F 470/F 660 as an indicator for the light acclimation status of the chloroplasts in a leaf.

Spectral analysis on origination of the bands at 437 nm and 475.5 nm of chlorophyll fluorescence excitation spectrum in Arabidopsis chloroplasts.

Chlorophyll fluorescence has been often used as an intrinsic optical molecular probe to study photosynthesis. In this study, the origin of bands at 437 and 475.5 nm in the chlorophyll fluorescence excitation spectrum for emission at 685 nm in Arabidopsis chloroplasts was investigated using various optical analysis methods. The results revealed that this fluorescence excitation spectrum was related to the absorption characteristics of pigment molecules in PSII complexes. Moreover, the excitation band centred at 475.5 nm had a blue shift, but the excitation band at 437 nm changed relatively less due to induction of non-photochemical quenching (NPQ). Furthermore, fluorescence emission spectra showed that this blue shift occurred when excitation energy transfer from both chlorophyll b (Chl b) and carotenoids (Cars) to chlorophyll a (Chl a) was blocked. These results demonstrate that the excitation band at 437 nm was mainly contributed by Chl a, while the excitation band at 475.5 nm was mainly contributed by Chl b and Cars. The chlorophyll fluorescence excitation spectrum, therefore, could serve as a useful tool to describe specific characteristics of light absorption and energy transfer between light-harvesting pigments.

Low levels of ultraviolet-B radiation from fluorescent tubes induce an efficient flavonoid synthesis in Lollo Rosso lettuce without negative impact on growth

Plants grown in Norwegian greenhouses during winter synthesise low amounts of anthocyanins and flavonols (colourless flavonoids). Higher content of anthocyanins enhances the red colour in lettuce and improves the external quality and marketability of the product. The flavonoids may also benefit consumer health due to their anti-oxidative and presumed anti-carcinogenic effects. The effects of ultraviolet-B (UV-B) radiation given by UV-B tubes (Q-panel UV 313) and by standard cool white fluorescent tubes (Philips Master TL-D 36W/840) on flavonol and anthocyanin synthesis in lettuce cv. Lollo Rosso (Carmoli) were studied. The aim was to increase the content of flavonoids without reducing the fresh weight of the plant. Cool white fluorescent tubes induced high amounts of flavonoids in the plants during a five-day experimental period, with no growth reduction. The highest doses of UV-B from the UV-B tubes resulted in higher flavonoid synthesis, but with some growth reduction. The increase in flavonoids was highest in the first two to three days. The kinetics of flavonoid accumulation were measured with the chlorophyll fluorescence excitation ratio (FER) method using a portable multiple wavelength fluorometer. The accumulation of flavonoids can be rapidly and frequently measured with this non-destructive FER method. The FER method can easily be used by cultivators to get an estimate of the flavonol and anthocyanin content that may contribute to the nutritional value and marketability.

Characterisation of the Polyphenol Content in the Kiwifruit (Actinidia deliciosa) Exocarp for the Calibration of a Fruit‐sorting Optical Sensor

Kiwifruit contains high amounts of anti-oxidants beneficial to health. Its quality is influenced by ripening time, genotype, cultivation techniques, climate and storage conditions after harvest. The aim of the present study was to characterise the phenolic content by HPLC methods and to evaluate the performance of a portable optical sensor (Multiplex 3), for in vivo non-destructive phenolic compound assessment in kiwifruits. Kiwifruits peel extracts were characterised by reverse-phase (RP) HPLC with diode-array detector (DAD) and electrospray ionisation (ESI) with MS using the Zorbax SB-Aq. column from Agilent. The fluorimetric sensor method is based on the screening of fruit chlorophyll fluorescence excitation and allows the UV absorbance of intact fruit skin to be measured. The flavonol index, FLAV, was calculated as log(FRF(R)/FRF(UV)), where FRF(R) and FRF(UV) are the chlorophyll fluorescence excited with red and UV light. Hydroxycinnamic acids, procyanidins, and quercetin glycosides were the main polyphenol classes detected by HPLC-DAD-ESI/MS in the kiwifruit skin. A good linear regression (R² = 0.88) was found between the fluorimetric sensor FLAV index and flavonol chromatographic analysis of the fruits. The FLAV index was able to detect the higher content of flavonols in sun-exposed fruits with respect to mid-shaded and shaded ones in accordance with the destructive analysis. The fluorimetric sensor represents a rapid and non-invasive tool to: (i) monitor the flavonol accumulation in kiwifruit and to assess its quality concerning the healthy anti-oxidant properties; (ii) evaluate the effect of environmental and agronomical factors related to the fruit quality; and (iii) select fruits with the largest flavonol content, and consequently less susceptible to pathogen attack, in order to improve their storage durability.

Non-destructive optical monitoring of grape maturation by proximal sensing.

A new, commercial, fluorescence-based optical sensor for plant constituent assessment was recently introduced. This sensor, called the Multiplex® (FORCE-A, Orsay, France), was used to monitor grape maturation by specifically monitoring anthocyanin accumulation. We derived the empirical anthocyanin content calibration curves for Champagne red grape cultivars, and we also propose a general model for the influence of the proportion of red berries, skin anthocyanin content and berry size on Multiplex® indices. The Multiplex® was used on both berry samples in the laboratory and on intact clusters in the vineyard. We found that the inverted and log-transformed far-red fluorescence signal called the FERARI index, although sensitive to sample size and distance, is potentially the most widely applicable. The more robust indices, based on chlorophyll fluorescence excitation ratios, showed three ranges of dependence on anthocyanin content. We found that up to 0.16 mg cm−2, equivalent to approximately 0.6 mg g−1, all indices increase with accumulation of skin anthocyanin content. Excitation ratio-based indices decrease with anthocyanin accumulation beyond 0.27 mg cm−2. We showed that the Multiplex® can be advantageously used in vineyards on intact clusters for the non-destructive assessment of anthocyanin content of vine blocks and can now be tested on other fruits and vegetables based on the same model.

Light-induced accumulation of ortho-dihydroxylated flavonoids as non-destructively monitored by chlorophyll fluorescence excitation techniques

Chlorophyll fluorescence excitation techniques have been used in plant science for more than one decade to non-destructively estimate phenolic compounds in the epidermal cell layer. These techniques have been used here to evaluate the effect of different light intensities and spectral quality on the accumulation of ortho-dihydroxylated flavonoids in Phyllirea latifolia L., Myrtus communis L. and Ligustrum vulgare L. In a first experiment, chlorophyll fluorescence excitation spectra were measured (with a double arm optical fiber bundle connected to a spectrofluorimeter) on the adaxial and abaxial leaf surfaces of container-grown P. latifolia and M. communis exposed to 20% or 100% full sunlight. Differences in epidermal absorption spectra (referred to as epidermal absorption spectra throughout the paper) were then calculated from the relative chlorophyll fluorescence excitation spectra. This allowed comparing the content of UV-absorbing compounds between differentially irradiated leaves as well as between adaxial and abaxial epidermal layers. The absorption spectra were characterized by a band centered at 360–380 nm, which was greater in sun than in shade leaves and in the adaxial than in the abaxial surfaces, irrespective of species. Based upon HPLC-DAD and HPLC–MS analyses of leaf extracts and UV-spectral features of individual flavonoids, we conclude that quercetin, luteolin and myricetin derivatives were responsible for the observed light-induced changes in the spectral features of examined tissues. In a second experiment, we grew L. vulgare potted plants at 30% or 85% full sunlight in the presence or in the absence of UV radiation. We measured the absorbance characteristics at 370 nm of three leaf-pairs located in the apical portion of each shoot, using a portable fluorimetric sensor, the Multiplex ®. This allowed estimating, non-destructively, an index closely related to the concentration of epidermal flavonols and flavones having an ortho-dihydroxyl substitution in the B-ring. This index was greater in leaves growing at 85% than at 30% sunlight, irrespective of UV irradiance. When plants acclimated for 3 weeks to 30% sunlight, in the presence or in the absence of UV irradiance, were transferred to 85% sunlight, the flavonoid index exponentially increased, reaching a maximum within 10 days. On the whole, our experiments conclusively show that light-responsive flavonoids are mostly the dihydroxy B-ring-substituted structures. These flavonoids are the most effective, among the wide array of flavonoid structures, in preventing the generation and scavenging reactive oxygen species. As a consequence, we suggest that flavonoids do not merely serve as UV-screening agents, but behave as ROS-detoxifying agents in the mechanisms of photoprotection.

INFLUENCE OF IRRIGATION AND NITROGEN FERTILIZATION ON BROCCOLI POLYPHENOLICS CONCENTRATION

Polyphenolics are phytochemical compounds that may be beneficial or toxic to humans. They are found in fruits and vegetables known for their health promoting characteristics. These carbon-based secondary metabolites have roles such as protection against ultraviolet radiation, free radicals, oxidative stress, herbivores and pathogens. Their synthesis in plant material is therefore linked to both biotic and abiotic stress sources. Under nitrogen stress, for instance, an increase in polyphenolics concentration has been reported. This behaviour has been exploited in the design and marketing of the Dualex, a new diagnostic instrument aimed at establishing the in-season requirements of crops for nitrogen (N) fertilizer. The Dualex can estimate leaf polyphenolics concentrations from the measurement of ultraviolet (UV, 375 nm) absorption of the leaf epidermis by the double excitation of chlorophyll fluorescence. The value of the UV absorbance is directly connected to the amount of leaf polyphenolics. The objective of this study was to determine if irrigation and nitrogen fertilization can influence broccoli polyphenolics concentrations. A broccoli experimental field was established in 2005 at the Agriculture and Agri-Food Canada experimental farm of L'Acadie (Quebec). The crop was fertilized with either 0, 105, 135, 165, 195 or 225 kg N/ha and provided or not with supplementary irrigation after crop establishment. Four Dualex measurement campaigns were made, from 26 to 47 days after planting, as well as one laboratory estimation of polyphenolics. The absence of irrigation increased significantly polyphenolics concentrations on the two last dates (by 36 and 35%, respectively). The increase in fertilizer N was related to a linear or curvilinear decrease in polyphenolics. At the last date, unfertilized plots had 86 and 16% higher polyphenolics than fully fertilized ones, in irrigated and non-irriguated plots, respectively. Yields of marketable heads increased in a curvilinear matter with N rate, with a tendency to plateau at high rates. The percent of hollow stem was increased at high N rates, only under the irrigated treatment. Plots placed under conditions to produce the highest possible polyphenolics content were however the least productive ones, from a fresh market standpoint. Since these stresses are impacting crop growth and development, there is a clear antagonism between the production of polyphenolics from broccoli, on one hand, and fresh market production, on the other hand. The Dualex has the potential to establish broccoli N requirements by its capacity of detecting polyphenolics concentration in leaves but irrigation may interfere in the diagnosis.

Effect of anthocyanins, carotenoids, and flavonols on chlorophyll fluorescence excitation spectra in apple fruit: signature analysis, assessment, modelling, and relevance to photoprotection

Whole apple fruit (Malus domestica Borkh.) widely differing in pigment content and composition has been examined by recording its chlorophyll fluorescence excitation and diffuse reflection spectra in the visible and near UV regions. Spectral bands sensitive to the pigment concentration have been identified, and linear models for non-destructive assessment of anthocyanins, carotenoids, and flavonols via chlorophyll fluorescence measurements are put forward. The adaptation of apple fruit to high light stress involves accumulation of these protective pigments, which absorb solar radiation in broad spectral ranges extending from UV to the green and, in anthocyanin-containing cultivars, to the red regions of the spectrum. In ripening apples the protective effect in the blue region could be attributed to extrathylakoid carotenoids. A simple model, which allows the simulation of chlorophyll fluorescence excitation spectra in the visible range and a quantitative evaluation of competitive absorption by anthocyanins, carotenoids, and flavonols, is described. Evidence is presented to support the view that anthocyanins, carotenoids, and flavonols play, in fruit with low-to-moderate pigment content, the role of internal traps (insofar as they compete with chlorophylls for the absorption of incident light in specific spectral bands), affecting thereby the shape of the chlorophyll fluorescence excitation spectrum.

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

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

Can dual chlorophyll fluorescence excitation be used to assess the variation in the content of UV-absorbing phenolic compounds in leaves of temperate tree species along a light gradient?

The present study assesses light-induced variations in phenolic compounds in leaves of saplings of two co-occurring temperate species (Acer platanoides L., and Fraxinus excelsior L.) along a light gradient using a new non-invasive optical method (Dualex). The Dualex-derived UV absorbance of leaf epidermis (the sum of the adaxial and abaxial faces, AUV) increased significantly with increasing light in both species. AUV values were correlated with absorbance of the leaf extract at 305 nm and 375 nm (A305 and A375) in both species with similar slopes for both species. However, a large difference in intercept was observed between the two species when A305 was regressed against AUV. Similarly, AUV values were well correlated with the amount of phenolics in the leaf extracts assessed by the Folin-Ciocalteu method, but slopes were significantly different for the two species. Thus, the UV-A epidermal transmittance, despite being a reliable indicator of the UV-screening capacity of the leaf epidermis, cannot be used for any quantitative estimate of UV-B screening capacity or of energetic requirement for leaf construction without a species-specific calibration.

Assessment of Anthocyanins in Grape (Vitis viniferaL.) Berries Using a Noninvasive Chlorophyll Fluorescence Method

Anthocyanins (Anths) in grape (Vitis vinifera L.) berries harvested at véraison from Pinot Noir and Pinot Meunier cultivars were assessed nondestructively by measuring chlorophyll fluorescence (ChlF) excitation spectra. With increasing Anth content, less excitation light was transmitted to the deeper Chl layers, and thus the ChlF signal decreased proportionally. By applying Beer-Lambert's law, the logarithm of the ratio between the fluorescence excitation spectra (log FER) from a green and a red berry gave the in vivo absorption spectrum of Anths, which peaked at about 540 nm. Absolute quantitative nondestructive determination of Anths for each berry was obtained by the log FER calculated for two excitation wavelengths, 540 and 635 nm (absorbed and not-absorbed by Anths, respectively) of ChlF at 685 nm. Over a range of skin colors going from green to purple, the relationship between the log [ChlF(635)/ChlF(540)] and the Anth concentration of berry extracts was fairly well fitted (r 2 = 0.92) using a power function. Reflectance spectra on the same berry samples were also measured, and Anth reflectance indices, which were originally developed for apples and table grapes, were derived. The log FER Anth index was superior to the reflectance-ratio-based index, but was as good as the color index for red grapes (CIRG) calculated from the whole visible reflectance spectrum. The proposed log FER method, applied by means of suitable portable devices, may represent a new, rapid, and noninvasive tool for the assessment of grape phenolic maturity in vineyards.

Color of illumination during growth affects LHCII chiral macroaggregates in pea plant leaves

To determine whether the color of illumination under which plants are grown, affects the structure of photosynthetic antennae, pea plants were grown under either blue-enriched, red-enriched, or white light. Carotenoid content of isolated chloroplasts was found to be insensitive to the color of illumination during growth, while chlorophyll a/ b ratio in chloroplasts isolated from young illuminated leaves showed susceptibility to color. Color of illumination affects the LHCII chiral macroaggregates in intact leaves and isolated chloroplasts, providing light-induced alteration of the handedness of the LHCII chiral macroaggregate, as measured with circular dichroism and circularly polarized luminescence. The susceptibility of handedness to current illumination (red light excitation of chlorophyll fluorescence) is dependent on the color under which the plants were grown, and was maximal for the red-enriched illumination. We propose the existence of a long-term (growth period) color memory, which influences the susceptibility of the handedness of LHCII chiral macroaggregates to current light.

Quantitative and qualitative evaluation of phytoplankton communities by trichromatic chlorophyll fluorescence excitation with special focus on cyanobacteria

We present results that were obtained with a newly developed fluorometer, the 'PhytoSensor'. They are based on multi-wavelength excitation of chlorophyll fluorescence to detect the phytoplankton biomass and to identify main taxons (among cyanobacteria, green and brown microalgae). A method to evaluate the photosynthetic potential of the phytoplankton was established. Attention was focused on the measurement of the cyanobacterial biomass. A modelling to distinguish between the two spectral groups (blue and red) of cyanobacteria as a function of their pigments and physiological status is proposed. The main innovation of the device results in the recording of the fluorescence induction kinetics of the phytoplankton to confirm and refine the evaluation of the taxonomic composition. The PhytoSensor abilities were compared with pigment analysis, commercial fluorometers, particle and microscopic counting and identification. The PhytoSensor has been used with success to monitor the dynamics of phytoplankton in drinking-water supply reservoirs in Southeast Asia.

A comparison of photosynthetic apparatus of the detached leaves of the resurrection plant Boea hygrometrica with its non-tolerant relative Chirita heterotrichia in response to dehydration and rehydration

Comparisons of the water contents in detached leaves of Boea hygrometrica (Bunge) R. Br ., and other members of the family Gesneriaceae have shown that B. hygrometrica can tolerate an extraordinarily high degree of water loss and restore its life upon rehydration. We have investigated the effects of dehydration on photosynthetic function in B. hygrometrica in comparison with its non-resurrection relative Chirita heterotrichia Merr. Studies on the net photosynthetic rate, chlorophyll fluorescence emission and excitation spectra, and the kinetics of chlorophyll fluorescence induction revealed that the effects of dehydration on both species were similar insofar as photosystem II was more sensitive to dehydration than photosystem I. However, B. hygrometrica responded more rapidly than C. heterotrichia. Although photosynthetic activity could be restored in both species upon rehydration after 2 days of dehydration, only B. hygrometrica recovered when the dehydration period was extended to 8 days. In contrast to C. heterotrichia leaves, in which the levels of carotenoids decreased rapidly during desiccation, carotenoid content actually increased in B. hygrometrica leaves upon dehydration and returned to the normal level after rehydration. Non-denaturing electrophoresis revealed that the thylakoid pigment–protein complexes of B. hygrometrica were highly stable during desiccation and rehydration, whereas those of C. heterotrichia were totally destroyed after 8 days of desiccation.