Epidermal Flavonols Research Articles

Non-photochemical quenching in natural accessions of Arabidopsis thaliana during cold acclimation

Cold acclimation, initiated by non-freezing low temperatures and light, is a natural strategy for increasing plant survival even at sub-zero temperatures. However, it remains unclear how the non-photochemical quenching processes, which are crucial for excessive light energy dissipation, are modulated during cold acclimation. We compared the effects of two weeks of acclimation to sub-optimal temperatures, at 10 °C (AC10) and 4 °C (AC4), with non-acclimated (NAC) Arabidopsis thaliana natural accessions grown at 21 °C, on their growth (rosette area), biochemistry (chlorophylls and epidermal flavonols), and physiology (CO2 assimilation rate, and quantum yields of photochemical and non-photochemical quenching processes). AC10 reduced rosette area in all (six) accessions, while chlorophylls and CO2 assimilation rate (Asat) decreased only in three accessions and it had no effect on maximum quantum yield (Fv/Fm). However, AC4 significantly decreased rosette area, chlorophylls, and Fv/Fm, in all accessions. Both AC10 and AC4 treatments increased the accumulation of epidermal flavonols in all accessions. In AC4 accessions, we found an increase in additional non-regulatory NPQ, Φf,d, and a decrease in the fraction of excitation energy used by PSII photochemistry, ΦPSII. A similar irradiance resulted in a marginal difference in regulatory NPQ, Φnpq, among NAC and AC10 or AC4 plants; however, AC10 plants have more energy-dependent fastest NPQ, ΦqE, whereas AC4 predominates state transition quenching, ΦqT. These variations in dissipation of absorbed light energy, when combined with reduced chlorophylls and accumulated flavonols, help to reduce the risk of photoinhibition in plants during cold periods. These findings provide new insights into how suboptimal temperature acclimation affects the regulation of NPQ molecular mechanisms in Arabidopsis thaliana natural accessions.

Effects of Trichoderma harzianum on Photosynthetic Characteristics and Fruit Quality of Tomato Plants.

The beneficial role of fungi from the Trichoderma genus and its secondary metabolites in promoting plant growth, uptake and use efficiency of macronutrients and oligo/micro-nutrients, activation of plant secondary metabolism and plant protection from diseases makes it interesting for application in environmentally friendly agriculture. However, the literature data on the effect of Trichoderma inoculation on tomato fruit quality is scarce. Commercially used tomato cultivars were chosen in combination with indigenous Trichodrema species previously characterized on molecular and biochemical level, to investigate the effect of Trichoderma on photosynthetic characteristics and fruit quality of plants grown in organic system of production. Examined cultivars differed in the majority of examined parameters. Response of cultivar Gružanski zlatni to Trichoderma application was more significant. As a consequence of increased epidermal flavonols and decreased chlorophyll, the nitrogen balance index in leaves has decreased, indicating a shift from primary to secondary metabolism. The quality of its fruit was altered in the sense of increased total flavonoids content, decreased starch, increased Bioaccumulation Index (BI) for Fe and Cr, and decreased BI for heavy metals Ni and Pb. Higher expression of swolenin gene in tomato roots of more responsive tomato cultivar indicates better root colonization, which correlates with observed positive effects of Trichodrema.

Ultraviolet-B exposure and exogenous hydrogen peroxide application lead to cross-tolerance toward drought in Nicotiana tabacum L.

Acclimation of plants to water deficit involves biochemical and physiological adjustments. Here, we studied how ultraviolet (UV)-B exposure and exogenously applied hydrogen peroxide (H2 O2 ) potentiates drought tolerance in tobacco (Nicotiana tabacum L. cv. xanthi nc). Separate and combined applications for 14 days of 1.75 kJ m-2 day-1 UV-B radiation and 0.2 mM H2 O2 were assessed. Both factors, individually and combined, resulted in inhibition of growth. Furthermore, the combined treatment led to the most compacted plants. UV-B- and UV-B + H2 O2 -treated plants increased total antioxidant capacity and foliar epidermal flavonol index. H2 O2 - and UV-B + H2 O2 -pre-treated plants showed cross-tolerance to a subsequent 7-day moderate drought treatment, which was assessed as the absence of negative impact on growth, leaf wilting, and leaf relative water content. Plant responses to the pre-treatment were notably different: (1) H2 O2 increased the activity of catalase (EC 1.11.1.6), phenylalanine ammonia lyase (EC 4.3.1.5), and peroxidase activities (EC 1.11.1.7), and (2) the combined treatment induced epidermal flavonols which were key to drought tolerance. We report synergistic effects of UV-B and H2 O2 on transcription accumulation of UV RESISTANCE LOCUS 8, NAC DOMAIN PROTEIN 13 (NAC13), and BRI1-EMS-SUPPRESSOR 1 (BES1). Our data demonstrate a pre-treatment-dependent response to drought for NAC13, BES1, and CHALCONE SYNTHASE transcript accumulation. This study highlights the potential of combining UV-B and H2 O2 to improve drought tolerance which could become a useful tool to reduce water use.

Singlet oxygen, flavonols and photoinhibition in green and senescing silver birch leaves

Key messageDecreased absorptance and increased singlet oxygen production may cause photoinhibition of both PSII and PSI in birch leaves during autumn senescence; however, photosynthetic electron transfer stays functional until late senescence.During autumn senescence, deciduous trees degrade chlorophyll and may synthesize flavonols. We measured photosynthetic parameters, epidermal flavonols, singlet oxygen production in vivo and photoinhibition of the photosystems (PSII and PSI) from green and senescing silver birch (Betula pendula) leaves. Chlorophyll a fluorescence and P700 absorbance measurements showed that the amounts of both photosystems decreased throughout autumn senescence, but the remaining PSII units stayed functional until ~ 90% of leaf chlorophyll was degraded. An increase in the chlorophyll a to b ratio, a decrease in > 700 nm absorbance and a blue shift of the PSI fluorescence peak at 77 K suggest that light-harvesting complex I was first degraded during senescence, followed by light-harvesting complex II and finally the photosystems. Senescing leaves produced more singlet oxygen than green leaves, possibly because low light absorption by senescing leaves allows high flux of incident light per photosystem. Senescing leaves also induced less non-photochemical quenching, which may contribute to increased singlet oxygen production. Faster photoinhibition of both photosystems in senescing than in green leaves, under high light, was most probably caused by low absorption of light and rapid singlet oxygen production. However, senescing leaves maintained the capacity to recover from photoinhibition of PSII. Amounts of epidermal flavonols and singlet oxygen correlated neither in green nor in senescing leaves of silver birch. Moreover, Arabidopsis thaliana mutants, incapable of synthesizing flavonols, were not more susceptible to photoinhibition of PSII or PSI than wild type plants; screening of chlorophyll absorption by flavonols was, however, small in A. thaliana. These results suggest that flavonols do not protect against photoinhibition or singlet oxygen production in chloroplasts.

Effects of low temperature on photoinhibition and singlet oxygen production in four natural accessions of Arabidopsis

Main conclusionsLow temperature decreases PSII damage in vivo, confirming earlier in vitro results. Susceptibility to photoinhibition differs among Arabidopsis accessions and moderately decreases after 2-week cold-treatment. Flavonols may alleviate photoinhibition.The rate of light-induced inactivation of photosystem II (PSII) at 22 and 4 °C was measured from natural accessions of Arabidopsis thaliana (Rschew, Tenela, Columbia-0, Coimbra) grown under optimal conditions (21 °C), and at 4 °C from plants shifted to 4 °C for 2 weeks. Measurements were done in the absence and presence of lincomycin (to block repair). PSII activity was assayed with the chlorophyll a fluorescence parameter Fv/Fm and with light-saturated rate of oxygen evolution using a quinone acceptor. When grown at 21 °C, Rschew was the most tolerant to photoinhibition and Coimbra the least. Damage to PSII, judged from fitting the decrease in oxygen evolution or Fv/Fm to a first-order equation, proceeded more slowly or equally at 4 than at 22 °C. The 2-week cold-treatment decreased photoinhibition at 4 °C consistently in Columbia-0 and Coimbra, whereas in Rschew and Tenela the results depended on the method used to assay photoinhibition. The rate of singlet oxygen production by isolated thylakoid membranes, measured with histidine, stayed the same or slightly decreased with decreasing temperature. On the other hand, measurements of singlet oxygen from leaves with Singlet Oxygen Sensor Green suggest that in vivo more singlet oxygen is produced at 4 °C. Under high light, the PSII electron acceptor QA was more reduced at 4 than at 22 °C. Singlet oxygen production, in vitro or in vivo, did not decrease due to the cold-treatment. Epidermal flavonols increased during the cold-treatment and, in Columbia-0 and Coimbra, the amount correlated with photoinhibition tolerance.

Effect of Trichoderma harzianum on morpho-physiological parameters and metal uptake of tomato plants

In this study we have investigated the effect of T. harzianum on growth, content of chlorophyll and epidermal flavonols and metal distribution in tomato plants. Trichoderma strain, isolated from the A horizon (5-30 cm) of agricultural soil used in organic production, was applied near the root in the sixth leaf development phase of tomato. Tomato plants were grown in a growth cabinet up to the stage of 10 leaves. Content of chlorophyll (Chl), epidermal flavonols (Flav) and antocyanins (Ant) were measured in vivo non-destructively. The concentration of Cd, Co, Cu, Cr, Fe, Mn, Ni, Se, and Zn was measured in different parts of tomato plants by ICP-OES method. Results have shown that Trichoderma application positively affected growth of tomato plants, and significantly decreased nitrogen balance index. In Trichoderma treated plants the contents of Cr and Ni were significantly reduced in roots, while content of Cd tended to decrease in all plant parts, significantly in roots. Presented results indicate that investigated isolate is worthwhile testing for plant growth promotion in field conditions, taking in account different supply of macro and micronutrients.

Combined effects of drought and high temperature on photosynthetic characteristics in four winter wheat genotypes

Terrestrial ecosystems are expected to experience more intense and longer drought and heat-waves in the future. How these environmental factors and their interaction influence photosynthetic activity and water use efficiency remains, however, an open question. Since the photosynthetic activity determines yield response, we investigated gas-exchange and chlorophyll fluorescence traits of flag leaves in four winter wheat cultivars, including two genotypes widely grown in Central Europe and two genotypes considered as drought tolerant. Pot-grown plants were cultivated under natural field conditions until anthesis (DC 61). Subsequently, the plants were exposed to a set of temperature regimes with daily maxima of 26–41 °C (temperature treatment) and maximum soil water holding capacity above 70% and below 30% (drought treatment) using laboratory growth chambers. Primary photochemical reactions after 7 and 14 days of acclimation, measured as maximum quantum yield of photosystem II photochemistry and total chlorophyll content, showed typical interactions of temperature and water availability resulting in an amplified response under combined influence of drought and temperatures above 35 °C. In contrast, drought and temperature treatment had only minor effects on content of epidermal flavonols. A dominant effect of drought over temperature on stomatal conductance (GSmax) was observed. Although substantial genotype-specific responses were found, reduced stomatal conductance resulted in significant decrease in light-saturated rates of CO2 assimilation (Amax) in all genotypes studied. The GSmax–Amax relationship, however, revealed limitation of CO2 uptake by other, non-stomatal processes at temperatures above 32 °C, particularly in the sensitive genotypes. Strong interaction of combined drought and temperature treatments was found on water use efficiency (WUE). Decline in WUE with increasing temperature was steeper in water-deficit than well-watered plants of all genotypes studied. Our results thus document a strong interactive effect of elevated temperature and drought on photosynthetic carbon uptake. Detected thresholds of sensitivity to combined drought and heat stress will contribute to improved modelling of wheat growth and production under expected future climate conditions.

Flavonols in broccoli (Brassica oleracea L. var. italica) flower buds as affected by postharvest temperature and radiation treatments

The aim of this study was to evaluate the effects of postharvest temperature, light and UV-B irradiation on the flavonol content in broccoli using a multi-level design simulating commercial storage conditions from harvest until consumer purchase. The flavonol content in broccoli flower buds was examined during pre-storage and storage of broccoli heads, representing refrigerated transport with wholesale distribution and retail, respectively. Broccoli heads were pre-stored for four or seven days at 0 or 4°C in the dark and then stored for three days at 10 or 18°C. During storage, the broccoli heads were exposed for 12h per day to three different levels of visible light (13, 19 or 25μmolm−2s−1) or a combination of visible light (19μmolm−2s−1) and UV-B irradiation (0.23Wm−2), or they were stored in the dark. The contents of flavonols (quercetin, kaempferol and isorhamnetin), measured by HPLC as aglycons in flower bud extracts, were not significantly influenced by pre-storage period, pre-storage temperature or storage temperature. However, the interaction between pre-storage period and pre-storage temperature was found significant for flavonol contents during storage, with higher contents observed for broccoli pre-stored shorter (4d) at 4°C and longer (7d) at 0°C. The levels of epidermal flavonols, monitored with non-destructive repeated measurements of the broccoli heads during storage, were observed to increase with the duration of pre-storage and storage. General linear model analysis revealed that the radiation treatments significantly affected both quercetin content and epidermal flavonol levels in broccoli flower buds during storage, with the highest levels observed after a combination of visible light and UV-B irradiation treatment. However, the outcome of that postharvest treatment was influenced by a combination of factors including pre-storage period, pre-storage temperature and storage temperature. For one group of broccoli heads, that were pre-stored for four days at 4°C and exposed to visible light with UV-B irradiation during storage at 10°C, a significant 3.5-fold increase in the level of all flavonols was observed as compared with after pre-storage. The non-destructive measurements demonstrated that accumulation of epidermal flavonols upon UV-B exposure was initiated earlier in broccoli heads when stored at higher temperature (18°C).

Photosynthetic response of mountain grassland species to drought stress is affected by UV-induced accumulation of epidermal flavonols

Interactive effects of drought and ultraviolet (UV) radiation on CO2 assimilation rate and accumulation of epidermal flavonols were studied in two herbs (Hypericum maculatum and Rumex obtusifolius) and two grasses (Agrostis capillaris and Holcus mollis) under field conditions of Beskydy Mts. The main objective of this study was to evaluate a protective role of epidermal flavonols against drought stress. Experimental shelters were used to manipulate amount of incident precipitation and UV radiation intensity for 12 weeks (May–July). The light-saturated CO2 assimilation rate (Amax) and the content of epidermal flavonols were regularly measured at intervals of 2–3 weeks during the entire period. Drought-induced reduction of Amax was enhanced by high UV intensity in R. obtusifolius, whereas in other species UV radiation reduced a negative effect of drought. Generally, drought increased flavonol content in leaves as compared to non-stressed plants. Similarly, high UV intensities increased the flavonol content in control plants, but not in drought-stressed ones. Regression analysis between the flavonol content and Amax revealed a positive correlation especially for A. capillaris and R. obtusifolius. In both species, a separation of these relationships was observed as the result of water availability. Thus lower Amax were observed at the same flavonol content in drought-stressed plants when compared to control, well-watered counterparts. We conclude that UV-induced accumulation of epidermal flavonols can alleviate negative impacts of summer drought on photosynthesis, particularly in species with slower ontogeny like H. maculatum and A. capillaris.

Dynamics of flavonol accumulation in leaf tissues under different UV-B regimes in Centella asiatica (Apiaceae).

A cumulative effect of UV-B doses on epidermal flavonol accumulation was observed during the first week of a time course study in Centella asiatica (Apiaceae). However, once flavonol levels had peaked, additional accumulation was possible only if higher daily UV-B irradiances were applied. We aimed to understand the dynamics of flavonol accumulation in leaf tissues using non-destructive spectroscopy and HPLC-mass spectrometry. When leaves that had grown without UV-B were given brief daily exposures to low-irradiance UV-B, they accumulated flavonols, predominantly kaempferol-3-O-β-D-glucuronopyranoside and quercetin-3-O-β-D-glucuronopyranoside, in their exposed epidermis, reaching a plateau after 7days. More prolonged UV-B exposures or higher doses eventually augmented flavonol concentrations even in non-exposed tissues. If UV-B irradiance was subsequently reduced, leaves appeared to lose their ability to accumulate further flavonols in their epidermis even if the duration of daily exposure was increased. A higher irradiance level was then necessary to further increase flavonol accumulation. When subsequently acclimated to higher UV-B irradiances, mature leaves accumulated less flavonols than did developing ones. Our study suggests that levels of epidermal flavonols in leaves are governed primarily by UV-B irradiance rather than by duration of exposure.

Nondestructive Diagnostic Test for Nitrogen Nutrition of Grapevine (Vitis viniferaL.) Based on Dualex Leaf-Clip Measurements in the Field

Crop nitrogen status is a major issue for crop yield and quality. It is usually assessed by destructive leaf or petiole tissue analysis. A quantitative nondestructive optical estimation of N sufficiency would be a great leap forward toward precision crop management. We therefore calibrated three optical indices against leaf nitrogen content: chlorophyll (Chl), epidermal flavonols, and the nitrogen balance index (NBI), which is the ratio of the former two indices. NBI was the best estimator of leaf N content measured by the Dumas or Kjeldahl method with a root-mean-square error smaller than 2 mg of N g(-1) dry weight, followed by Chl (3 mg g(-1)) and flavonols (4 mg g(-1)). This allowed us to propose the threshold values for the Dualex optical indices that characterize nitrogen supply to grapevines: the first is the threshold below which N supply to the vine can be considered deficient, and the second is the threshold above which N supply is excessive. For a putative optimal N content of 30 mg g(-1) < x < 40 mg g(-1), these thresholds are 30 μg cm(-2) < x < 40 μg cm(-2) for Chl and 11 < x < 18 for NBI at flowering. At bunch closure, for N thresholds of 22 < x < 32, Chl is 29 < x < 37 and NBI is 8 < x < 11, in respective units. These values should be verified and refined in the future for various growth regions and cultivars using the specified protocol. The sample size should be 36-60 leaves from a fixed node position, preferably node no. 5 from the tip of the shoot. An alternative to the use of the NBI would be to discard leaves that are not light exposed by checking their flavonol content and to deduce the N sufficiency directly from the Chl values.

Ultraviolet and photosynthetically active radiation can both induce photoprotective capacity allowing barley to overcome high radiation stress

The main objective of this study was to determine the effects of acclimation to ultraviolet (UV) and photosynthetically active radiation (PAR) on photoprotective mechanisms in barley leaves. Barley plants were acclimated for 7 days under three combinations of high or low UV and PAR treatments ([UV−PAR−], [UV−PAR+], [UV+PAR+]). Subsequently, plants were exposed to short-term high radiation stress (HRS; defined by high intensities of PAR – 1000 μmol m−2 s−1, UV-A – 10 W m−2 and UV-B 2 W m−2 for 4 h), to test their photoprotective capacity. The barley variety sensitive to photooxidative stress (Barke) had low constitutive flavonoid content compared to the resistant variety (Bonus) under low UV and PAR intensities. The accumulation of lutonarin and 3-feruloylquinic acid, but not of saponarin, was greatly enhanced by high PAR and further increased by UV exposure. Acclimation of plants to both high UV and PAR intensities also increased the total pool of xanthophyll-cycle pigments (VAZ). Subsequent exposure to HRS revealed that prior acclimation to UV and PAR was able to ameliorate the negative consequences of HRS on photosynthesis. Both total contents of epidermal flavonols and the total pool of VAZ were closely correlated with small reductions in light-saturated CO2 assimilation rate and maximum quantum yield of photosystem II photochemistry caused by HRS. Based on these results, we conclude that growth under high PAR can substantially increase the photoprotective capacity of barley plants compared with plants grown under low PAR. However, additional UV radiation is necessary to fully induce photoprotective mechanisms in the variety Barke. This study demonstrates that UV-exposure can lead to enhanced photoprotective capacity and can contribute to the induction of tolerance to high radiation stress in barley.

High blue light improves acclimation and photosynthetic recovery of pepper plants exposed to UV stress

Intensity of photosynthetic active radiation (PAR) plays an important role in the acclimation of plants to UV radiation. Thereby, specific morphological and physiological characteristics influenced by high irradiance are also affected by blue light. With this background we conducted two experiments to evaluate the impact of light intensity and the relevance of blue light for the acclimation of pepper plants to UV. In this context we hypothesized that higher amount of blue light in the PAR spectrum significantly improves the plant acclimation and recovery to UV radiation. Our results demonstrate that UV stressed plants cultivated either under the higher light intensity (PAR 300 μmol m−2 s−1) or under higher amount of blue light (62%) show better photosynthetic performance (i.e., higher photosynthetic rate (Pn), higher maximal photochemical efficiency of PSII (Fv/Fm) and lower non-photochemical quenching (NPQ)) than UV stressed plants grown under lower light intensity (PAR 100 μmol m−2 s−1) or lower amount of blue light (30%). Contents of chlorophyll a and b, as well as carotenoids, had a stronger decrease due to UV in those plants cultivated either under the lower light intensity or under the lower amount of blue light. In contrast, plants grown either under 300 μmol m−2 s−1 or 62% blue light accumulated more epidermal flavonols. Analogous to the well described effects of high PAR intensity, we demonstrate here that high amount of blue light triggers specific biochemical and physiological processes resulting in better acclimation and recovery of plants to UV stress.

Ecologically relevant UV-B dose combined with high PAR intensity distinctly affect plant growth and accumulation of secondary metabolites in leaves of Centella asiatica L. Urban

We investigated the effects of environmentally relevant dose of ultraviolet (UV)-B and photosynthetic active radiation (PAR) on saponin accumulation in leaves on the example of Centella asiatica L. Urban. For this purpose, plants were exposed to one of four light regimes i.e., two PAR intensities with or without UV-B radiation. The experiment was conducted in technically complex sun simulators under almost natural irradiance and climatic conditions. As observed, UV-B radiation increased herb and leaf production as well as the content of epidermal flavonols, which was monitored by non-destructive fluorescence measurements. Specific fluorescence indices also indicate an increase in the content of anthocyanins under high PAR; this increase was likewise observed for the saponin concentrations. In contrast, UV-B radiation had no distinct effects on saponin and sapogenin concentrations. Our findings suggest that besides flavonoids, also saponins were accumulated under high PAR protecting the plant from oxidative damage. Furthermore, glycosylation of sapogenins seems to be important either for the protective function and/or for compartmentalization of the compounds. Moreover, our study revealed that younger leaves contain higher amounts of saponins, while in older leaves the sapogenins were the most abundant constituents. Concluding, our results proof that ambient dose of UV-B and high PAR intensity distinctly affect the accumulation of flavonoids and saponins, enabling the plant tissue to adapt to the light conditions.

Estimation of flavonoid and centelloside accumulation in leaves of Centella asiatica L. Urban by multiparametric fluorescence measurements

Recently, we have shown the relevance of nitrogen (N), phosphorus (P) and potassium (K) supply levels for resource partitioning between primary and secondary metabolism, and the concentration of centellosides in Centella asiatica L. Urban leaves. So far, no efforts have been made to investigate the effects of mineral supply on flavonoid concentrations in this species. Here, we aimed to examine the accumulation of centellosides in C. asiatica leaves in vivo by means of non-destructive fluorescence measurements using products of the secondary metabolism, particularly the epidermal flavonols and anthocyanins, as reference. For this purpose we conducted three discrete experiments in a greenhouse having N, P and K levels as experimental factors. Our results reveal that flavonoid and anthocyanin accumulation is affected by N, P and K fertigation in the same way as the centelloside accumulation. More precisely, limitations in plant growth were accompanied by higher flavonoid and anthocyanin concentrations, confirming the proposed trade-off between the plant's primary and secondary metabolism. The fluorescence-based flavonol (FLAV) and anthocyanin (ANTH_RG) indices correlated fairly with flavonoid and especially with anthocyanin concentrations. Moreover, centellosides were positively correlated with the FLAV and ANTH_RG indices, and with the BFRR_UV index, which is considered as universal ‘stress-indicator’. Thus, here we indicate for the first time, that the fluorescence-based indices FLAV, ANTH_RG as well as BFRR_UV enable the monitoring of flavonoid and centelloside concentrations in leaves of C. asiatica. Our results support and highlight the significant potential for further development and application of fluorescence-based sensors in ecophysiological research as well as in the production of medicinal plants.

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.

Functional role of anthocyanins in the leaves of Quintinia serrata A. Cunn.

The protective functions that have been ascribed to anthocyanins in leaves can be performed as effectively by a number of other compounds. The possibility that anthocyanins accumulate most abundantly in leaves deficient in other phytoprotective pigments has been tested. Pigment concentrations and their histological distribution were surveyed for a sample of 1000 leaves from a forest population of Quintinia serrata, which displays natural polymorphism in leaf colour. Eight leaf phenotypes were recognized according to their patterns of red coloration. Anthocyanins were observed in almost all combinations of every leaf tissue, but were most commonly located in the vacuoles of photosynthetic cells. Red leaves contained two anthocyanins (Cy-3-glc and Cy-3-gal), epicuticular flavones, epidermal flavonols, hydroxycinnamic acids, chlorophylls, and carotenoids. Green leaves lacked anthocyanins, but had otherwise similar pigment profiles. Foliar anthocyanin levels varied significantly between branches and among trees, but were not correlated to concentrations of other pigments. Anthocyanins were most abundant in older leaves on trees under canopies with south-facing gaps. These data indicate that anthocyanins are associated with photosynthesis, but do not serve an auxiliary phytoprotective role. They may serve to protect shade-adapted chloroplasts from brief exposure to high intensity sunflecks.