SEARCH

SEARCH BY CITATION

Keywords:

  • chromatography;
  • crop plant;
  • epidermis;
  • flavonoid;
  • fluorescence microscopy;
  • fluorometry;
  • hydroxycinnamic acid;
  • model;
  • sieve effect;
  • UV radiation

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

A recent review of climate patterns in Southern Germany has suggested significant increases in ultraviolet (UV) radiation due to decreases in cloud coverage and in cloud frequency which compound the effects of stratospheric ozone depletion. Whether such UV radiation increases result in UV damage of higher plant leaves depends partly on the capacity of UV-absorbing hydroxycinnamic acids and flavonoids located in the plant epidermis to screen out UV radiation. Epidermal UV screening is most often assessed from UV absorbance of whole-leaf extracts but in the present work, this method is critically examined. In grapevine (Vitis vinifera L.), hydroxycinnamic acid as well as mono-hydroxylated and ortho-dihydroxylated flavonoid concentrations increased in parallel with fluorometrically detected adaxial epidermal UV absorbance but only the latter class of flavonoids was associated with epidermal UV absorbance in barley (Hordeum vulgare L). For both species, curvilinear relationships between epidermal and total phenolic UV absorbance were established: initial slopes of the curves differed markedly between species. Modelling suggested that curvilinearity arises from UV-transparent epidermal areas located between vacuoles which are particularly UV-absorbing due to high levels of phenolics. The species-dependent differences were related to allocation of high amounts of phenolics in the mesophyll and abaxial epidermis in barley but not in grapevine. Both factors, optical heterogeneity and variable distribution of phenolics, severely restrict the use of phenolic absorbance to estimate true epidermal screening.


INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Decreased stratospheric ozone in recent decades resulting in increased UV-B radiation (280–315 nm) at the Earth's surface has been associated with the release of chlorofluorocarbons which is now being brought under control by the Montreal Protocol (Solomon 2004). Model calculations have predicted that with adherence to this Protocol, UV-B radiation in Central Europe will decline in 2050 to intensities encountered in 1970 (Reuder, Dameris & Koepke 2001). The model assumes that clouds, which are known to affect doses on ground of the entire natural UV range (280–400 nm), remain unchanged (Koepke 2000). A recent review of climate conditions from 1968 to 2001 in Southern Germany, however, has indicated that cloud frequency and cloud cover showed a clear tendency to decrease during May to August resulting in much higher increases in UV radiation than expected from stratospheric ozone depletion alone (Trepte & Winkler 2004). If this trend persists, the beneficial effects of ozone recovery on UV-B radiation may be offset by future increases in UV loads during summer months not only in Southern Germany but also in other areas experiencing similar atmospheric changes. Importantly, decreased UV screening by clouds will also increase UV-A radiation (315–400 nm) and might cause reductions in plant productivity as photosystem II is markedly inhibited by increased UV-A radiation (Turcsányi & Vass 2000; Nayak et al. 2003; Pfündel 2003).

The potential of UV radiation to damage photosystem II and other photosynthetic compounds (Hollósy 2002) essentially depends on UV intensities reaching the leaf mesophyll. In many higher plants, UV radiation is screened out by UV-absorbing phenylpropanoids accumulated in the epidermis (Caldwell, Robberecht & Flint 1983; Tevini, Braun & Fieser 1991; Bornman & Teramura 1993; Jordan 1996). These phenolics can be classified into the mostly UV-B absorbing hydroxycinnamic acids, having a C6-C3 carbon skeleton, and flavonoids, exhibiting a C15 backbone and broader absorbance including also the UV-A (Cockell & Knowland 1999; Winkel-Shirley 2001). Exact information on the capacity of epidermal UV screening is vitally important not only to predict productivity of current crop plants under future climate scenarios but also to assist crop plant breeders to select cultivars with efficient UV shielding.

Epidermal screening is generally estimated from UV absorbance of extracted leaf phenolics. Often, leaf phenolics are not confined to the epidermis but occur in considerable concentrations in the mesophyll tissue (Effertz & Weissenböck 1976; Strack, Meurer & Weissenböck 1982; Liu, Gitz & McClure 1995; Burchard, Bilger & Weissenböck 2000; Rozema et al. 2002; Semerdjieva et al. 2003). Naturally, these mesophyll phenolics are ineffective in epidermal UV screening but contribute to UV absorbance of leaf extracts. In fact, by comparing extracted absorbance with direct measurements of epidermal UV screening using a fibre-optic microprobe, it has been clearly shown that absorbance by extracted phenolics does not always correlate with epidermal screening capacity (Liakoura, Bornman & Karabourniotis 2003).

In addition to the fibre-optic method, fluorometry has been established to quantify authentic epidermal screening (Sheahan 1996; Bilger et al. 1997; Barnes et al. 2000; Burchard et al. 2000; Mazza et al. 2000; Markstädter et al. 2001; Ounis et al. 2001; Cerovic et al. 2002). By employing the latter technique, the present work compares UV absorbance of the epidermis with UV absorbance of leaf phenolics with the aim of defining factors which interfere with correlations between epidermal screening and phenolic absorbance.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Plants

Two-year-old-grafted vines (Vitis vinifera cv. Silvaner) were grown in a shaded glasshouse as described in Kolb et al. (2003). Only fully developed leaves, of insertion levels 3–6, counted from the bottom to the top, were investigated except for Fig. 1a, which includes data from differently exposed leaves from field-grown Silvaner cultivars. In addition, in our shaded glasshouse, two cultivars of barley (Hordeum vulgare cv. Ricarda and cv. IPZ 24727) were grown from seeds provided by the Landesanstalt für Landwirtschaft (Freising, Germany) planted in 20-cm-diameter pots containing Torfsubstrat Cultural F (Euflor, Munich, Germany); however, no further distinction will be made between these cultivars which behaved identically in our experiments; the plants were watered daily and fertilized weekly using Flory 3 (Euflor). Only fully expanded F-1 leaves (the leaves below flag leaves) of plants about 8 weeks old were examined. In the greenhouse, UV radiation was virtually absent, and visible light intensity was 15% of the outdoor intensity. The concentrations of phenolics were lowest in greenhouse-grown plants but increased during outdoor exposure for periods up to 8 d: outside the greenhouse, the plants were exposed to different radiation conditions as defined in Kolb et al. (2001).

image

Figure 1. Adaxial UV-A absorbance versus abaxial UV-A absorbance of grape (a) and barley leaves (b) Epidermal UV-A absorbance was measured using a portable modulated UV-A chlorophyll fluorometer; only leaf areas in which the adaxial side coincided with the upward directed side were measured, and so turned leaf areas which occurred frequently in barley leaves were excluded. Straight lines were obtained by regression analyses which both yielded P-values < 0.001 (SE, standard error of slope; R2, coefficient of determination).

Download figure to PowerPoint

Data were recorded during late evening hours. Determination of epidermal transmittance in the UV-A range, using a portable modulated UV-A chlorophyll fluorometer (UV-A-PAM fluorometer; Gademann Messgeräte, Würzburg, Germany; see below), was carried out directly at the experimental site. For measurements of epidermal transmittance in the UV-A and UV-B with another modulated chlorophyll fluorometer (Xe-PAM fluorometer; Walz, Effeltrich, Germany; see below), leaves were transferred into the laboratory in a dark, moist container where discs were punched out for fluorometry and, then frozen in liquid nitrogen and stored at −80 °C until chromatographic analysis  of  phenolic  compounds.  To  minimize  any  effects  of longitudinal gradients in barley (Wagner, Gilbert & Wilhelm  2003)  only  the  most  exposed  area  in  the  middle of the leaf was probed.

Epidermal UV absorbance

Epidermal UV transmittance was determined using a modulated chlorophyll fluorometer (Xe-PAM fluorometer, Walz) as described earlier (Bilger et al. 1997; Kolb et al. 2001; Markstädter et al. 2001). Chlorophyll fluorescence at the F0 level was elicited by UV-B (FUV-B: 314 nm, bandwidth 24 nm), by UV-A (FUV-A: 360 nm, bandwidth 28 nm) or by blue-green radiation (FBG: 490 nm, bandwidth 165 nm), and the fluorescence at wavelengths above 690 nm was measured. Transmittance of UV-B and UV-A, denoted TUV-B and TUV-A, respectively, was estimated according to

  • TUV-B = (FUV-B/FBG)·(FMes,UV-B/FMes,BG)−1
  • TUV-A = (FUV-A/FBG)·(FMes,UV-A/FMes,BG)−1

where FMes,UV-B, FMes,UV-A, and FMes,BG corresponds to the fluorescence from non-screened mesophyll tissue excited by UV-B, UV-A or blue-green light, respectively. Because preparation of epidermis-free mesophyll was not possible in either species, FMes values were measured with greenhouse-grown Pisum sativum L., mutant Argenteum, which possesses a loosely attached leaf epidermis. Transmittance was transformed into absorbance according to the Beer–Lambert law.

The low intensities of FUV-B or FUV-A in the presence of efficient epidermal UV shielding are difficult to measure accurately because, firstly, stray UV radiation could add significantly to the signal detected by our Xe-PAM fluorometer, and, secondly, incomplete absorption of visible radiation by our UV excitation filters could result in chlorophyll fluorescence elicited by visible radiation which might contribute notably to low FUV-B or FUV-A. The latter appears unimportant as a foil that absorbs virtually all UV but transmits visible radiation (Lee 226 UV; FFL-Rieger, Munich, Germany; transmittance spectrum in Kolb et al. 2001) placed before leaf discs of varying origins resulted in epidermal transmittance very close to zero (TUV-B = 0.0036, SE = 0.0019, n = 25; TUV-A = 0.0017, SE = 0.0009, n = 25): for these calculations, background signals caused by stray radiation by the foil alone were subtracted from the original FUV-B or FUV-A signals.

Generally, some stray light was detected by our Xe-PAM fluorometer as the plain sample holder yielded minor signals for all three excitation bands. In our earlier work, the latter signals were routinely subtracted from the respective signals from leaf discs prior to data handling (Kolb et al. 2001, 2003). Here, we estimated that the stray UV radiation of the leaf discs corresponded to 160 and 150% of that of the plain sample holder for UV-B and UV-A excitation, respectively, by measuring grape leaves exhibiting highly efficient epidermal UV screening in comparison with the plain sample holder in our Xe-PAM fluorometer with emission filters removed. In the present work, these newly established background signals were subtracted from fluorescence signals prior to calculations of transmittance.

Epidermal UV-A transmittance was also estimated using a newly developed portable modulated UV-A chlorophyll fluorometer (UV-A-PAM fluorometer; Gademann Messgeräte) which excites F0 chlorophyll fluorescence by diodes emitting radiation at 375 nm (10 nm bandwidth) and at 470 nm (25 nm bandwidth). Fluorescence was detected at wavelengths above 650 nm. Here, the ratio of fluorescence excited at 375 and 470 nm was used to estimate UV screening and FMES was derived from a fluorescent standard foil as described by Krause et al. (2003). In both species, UV-A transmittances measured by the UV-A chlorophyll fluorometer and the Xe-PAM fluorometer were linearly related to each other (not shown).

High performance liquid chromatography

One grape leaf disc (1.3 cm diameter) frozen in liquid nitrogen was reduced to a fine frozen suspension in a 5-mL Teflon sample flask of a Mikro-Dismembrator II equipped with an agate grinding ball (B. Braun Melsungen, Melsungen, Germany), which had both been immersed in liquid nitrogen together with 250 µL of extraction medium (50% (v/v) aqueous methanol containing 0.01% (w/v) phosphoric acid and 30 µg mL−1 quercetin as internal standard). Subsequently, the frozen suspension was thawed, centrifuged and the supernatant collected. Pellets were extracted twice more at room temperature with 250 µL of extraction medium. The extract was clarified by further centrifugation before analysis on an 1100 Series chromatograph which includes an 1040 M diode array detector (Agilent Technologies, Waldbronn, Germany). Basically, the extraction procedure for grapevine was also employed for barley leaf discs with minor deviations: barley leaf discs had a diameter of only 1 cm, and they were lyophilized after fluorometry, and extracted four times with 250 µL of extraction medium.

Separation of phenolics was performed on a 5-µm particle LiChrospher-100 RP18 column of 250 mm length and 4.6 mm inner diameter thermostatically controlled at 20 °C (Knauer, Berlin, Germany) using a flow rate of 1 mL min−1. The injection volume was 10 µL. Elution of grape leaf phenolics started with a linear decrease of solvent A [0.01% (w/v) H3PO4] from 80% (v/v) to 66% (v/v) with solvent B [methanol: 0.1% (w/v) H3PO4 (9 : 1, v/v)] over a period of 7 min, followed by isocratic elution for 5 min. A decrease to 56% (v/v) of solvent A then occurred within 2 min, to 40% (v/v) during a further 18 min, and to 35% during another 3 min. Finally, 100% solvent B was reached during a 2-min gradient followed by isocratic elution for 5 min. Starting conditions were restored during a 1-min gradient followed by column equilibration for 7 min.

Barley phenolics were eluted using the same solvents but a different elution routine. After the first 12 min, conducted as above, solvent A was subsequently decreased to 60% (v/v) during 6 min followed by isocratic elution for 5 min. During the following 25 min, solvent A was further decreased to 35% (v/v). Finally, 100% B (v/v) was reached within 1 min followed by isocratic elution for 5 min. Starting conditions were restored during a 2-min gradient followed by column equilibration for 7 min. High performance liquid chromatography (HPLC) grade solvents (Fluka, Deisenhofen, Germany) were used. Isoorientin and saponarin were purchased from Extrasynthèse (Genay Cedex, France) and ferulic acid from Fluka. with the exception of the chromatographic traces (Fig. 2a & b), all absorbance values were normalized to leaf area. For data handling, SigmaPlot scientific graphing software (SPSS, Munich, Germany) was used.

image

Figure 2. Chromatograms of grapevine (a) and barley leaf phenolics (b) and absorbance spectra of the predominant chromatographic peaks (c and d, respectively). Chromatograms were detected by absorbance at 314 nm and normalized to the highest peak. Identification of grape leaf phenolics is based on earlier results (Kolb et al. 2001, 2003), phenolics in barley are classified by comparison with published retention behaviour and spectral properties. Chromatogram peak labels: A1 to A4, apigenin derivatives; HCA, hydroxycinnamic acids; K1 and K2, kaempferol derivatives; L1 to L5, luteolin derivatives; Q1 and Q2, quercetin derivatives. Absorbance spectra of K1 and K2 (denoted ‘K1,2’ in panel c) and those of Q1 and Q2 (denoted ‘Q1,2’ in panel c) were similar. Additional labels in panels c and d: caf, trans-caffeic acid tartrate (main peak at 6 min in a); cou, trans-coumaric acid tartrate (main peak at 9 min in a); hca, main HCA in barley leaves (eluting at 10 min in b).

Download figure to PowerPoint

RESULTS AND DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Epidermal UV screening and UV absorbance by phenolics

Dissimilar relationships between adaxial and abaxial UV-screening were observed in grape and barley leaves using a modulated UV-A chlorophyll fluorometer: in grape leaves, different radiation treatments produced large variations in epidermal screening but, generally, adaxial UV-A absorbance was twice the corresponding abaxial value (see slope of regression line in Fig. 1a). In contrast, variable UV-A absorbance in barley was similar on both sides of the leaf (Fig. 1b).

Different concentrations of epidermal phenolics are probably involved in the variations in UV screening reported in Fig. 1. To test this, we measured adaxial UV screening in leaf discs using a modulated chlorophyll fluorometer (Xe-PAM) and, subsequently, examined phenolics extracted from these discs by HPLC. Elution profiles were established routinely by absorbance at 314 nm which is the wavelength of the transmission maximum of the UV-B excitation window of our Xe-PAM fluorometer. The substances giving rise to major peaks in chromatograms of grapevine phenolics (Fig. 2a) have been identified earlier (Kolb et al. 2001, 2003). Compounds eluting at retention times below 12 min are hydroxycinnamic acid derivatives with the predominant compound being trans-caffeoyl-tartaric acid, whereas those eluting above 24 min, such as peaks Q1 and Q2, correspond to derivatives of quercetin, a B-ring ortho-di-hydroxylated flavonol, whereas K1 and K2 represent derivatives of kaempferol, a B-ring mono-hydroxylated flavonol.

Phenolics from barley leaves (Fig. 2b) were classified by their retention behaviour and UV absorbance characteristics (Fig. 2d). A distinct group of compounds eluting with short retention times (less than 14 min) possessed spectral properties of hydroxycinnamic acid derivatives from barley leaves (Reuber, Bornman & Weissenböck 1996; Reuber et al. 1997). Further, the conspicuous elution peaks at 7 and 10 min exhibited the absorbance spectrum of pure ferulic acid (see spectrum ‘hca’ in Fig. 2d). Obviously, the group of early eluting compounds are hydroxycinnamic acid derivatives.

In contrast to these hydroxycinnamic acid derivatives, all major compounds eluting beyond 16 min (Fig. 2b) showed an absorbance maximum at around 270 nm and another in the UV-A spectral region (Fig. 2d). This is consistent with the spectral characteristics of flavones which represent the predominant class of flavonoids in barley (Reuber et al. 1997). Barley flavones can be most simply grouped into the B-ring mono-hydroxylated apigenin derivatives, and the B-ring ortho-dihydroxylated luteolin derivatives (Fröst, Harborne & King 1977; Nørbæk, Brandt & Kondo 2000).

We observed two principal types of absorbance spectra of flavones (see Fig. 2d): one category showed a single peak at 270 nm and a long-wavelength maximum at 340 nm, whereas the other exhibited peaks at 255, 270 and 350 nm. It is known, that B-ring-3′,4′ dioxygenated flavones usually exhibit two short-wavelength absorbance maxima but not the mono-oxygenated ones, and, further, that increasing the oxidation of the B-ring in flavones results in a bathochromic shift of the UV-A band (Mabry, Markham & Thomas 1970). Hence, based on absorbance spectra, most of our flavones (see Fig. 2b) could be unequivocally classified as apigenin derivatives (peaks A1 to A4) and luteolin derivatives (L1 to L3). In fact, spectra of L1 to L3 were almost identical to the isoorientin spectrum (luteolin 6-C-glucoside) whereas absorbance spectra and retention behaviour of A1 was identical to saponarin (apigenin 6-C-glucosyl-7-O-glucoside). Compounds L4 and L5 were only tentatively assigned as luteolin derivatives because their UV-A spectra did not match the criteria of B-ring-3′,4′ dioxygenated flavones.

We investigated to what degree variations in UV-shielding of adaxial epidermis can be explained by variations in leaf phenolics by plotting fluorometrically detected UV-B and UV-A absorbance against absorbance of phenolics at 314 and 360 nm, respectively (Fig. 3). Association between data was estimated using linear regression analysis. In grape and in barley leaves, concentrations of the different B-ring ortho-dihydroxylated, and also of the different mono-hydroxylated flavonoids, varied roughly in parallel (data not shown). Therefore, these two groups of compounds were considered separately (Fig. 3e–h).

image

Figure 3. The four left-handed panels show data from grapevine, other panels represent results from barley. Fluorometrically detected UV-B (AUV-B) and UV-A absorbance (AUV-A) of adaxial leaf sides is plotted against absorbance at 314 nm (A314; panels a, c, e and g) and 360 nm (A360; panels b, d, f and h), respectively, of extracted hydroxycinnamic acids (‘HCA’; panels a to d) and flavonoids (panels e to h). The A314 and A360 are derived from chromatography (see Fig. 2) and are normalized to represent the absorbance that phenolics in the entire leaf blade would have if they were homogeneously dissolved. In panels e and f, the sum of absorbances of the two main kaempferol peaks (K1 and K2 denoted ‘K1,2’) and the two main quercetin peaks (Q1 and Q2 denoted ‘Q1,2’) are considered separately. Likewise, panels g and h treat the sum of absorbances of apigenin (A1 to A4 denoted ‘A1-4’) and luteolin derivatives independently (L1 to L5 denoted ‘L1-5’). Linear regression analyses were applied to all 12 data sets but regression lines are only shown for R2 > 0.1.

Download figure to PowerPoint

Linear regression supports the view that hydroxycinnamic acids are involved in variable epidermal UV-B screening in grape leaves but clearly not in barley leaves (Fig. 3a–d). This is consistent with earlier work reporting conflicting data on the importance of hydroxycinnamic acids for UV-B screening in different species (Landry, Chapple & Last 1995; Sheahan 1996; Burchard et al. 2000; Markstädter et al. 2001).

Kaempferol and quercetin derivatives in grape leaves but only luteolin derivatives (and not apigenin derivatives) in barley leaves seem to play a role in variable UV-B screening (Fig. 3e–h). In both species, increased epidermal UV-screening was most effectively induced by UV exposure (data not shown). Therefore, the specific increase in luteolin derivatives observed in efficiently screening barley leaves (Fig. 3g & h) corresponds to increased ratios of ortho-dihydroxylated to mono-hydroxylated flavonoids under UV radiation in other species (Markham et al. 1998a, b; Ryan et al. 1998). This supports the notion that UV-induced flavonoid accumulation also increases the leaf's antioxidative capacity because the dihydroxlated flavonoids exhibit higher radical scavenging activity than the mono-hydroxylated compounds (Torel, Cillard & Cillard 1986; Husain, Cillard & Cillard 1987; De Beer et al. 2002; Yamasaki, Sakihama & Ikehara 2003). It remains unexplained why a build-up of high UV screening occurred without a shift to flavonoids with increased B-ring oxidation in grape leaves (Fig. 3e & f) and also in grape berries (Kolb et al. 2003).

The UV absorbance of hydroxycinnamic acids in grape leaves and of apigenin derivatives in barley leaves was always clearly above zero under all experimental conditions (Fig. 3) in agreement with the recent proposal that UV-absorbing compounds in leaves always remain above a certain threshold even under low levels of UV radiation (Liakoura et al. 2003). The present work additionally suggests that, dependent on species, hydroxycinnamic acids or flavonoids can be engaged in maintaining this threshold concentration of UV-B absorbing compounds. The nature of this species-dependency awaits further clarification.

Relationship between epidermal UV absorbance and UV absorbance by phenolics

The previous section demonstrated that different classes of phenolics are involved in variable UV absorbance of the adaxial epidermis in grape and barley leaves. This prompted us to search for species-dependent differences in the relationships between total UV absorbance of leaf phenolics and UV screening of adaxial epidermis. For grape and barley leaves, total phenolic absorbance was derived from the sum of absorbance of all peaks labelled in Fig. 2a and b, respectively: in fact, these peaks represented at least 92% but generally more that 95% of chromatographically determined UV absorbance. This holds for both 314 and 360 nm as the HPLC detection wavelengths; the latter wavelength coincides with the maximum of the excitation window of our Xe-PAM fluorometer used to determine epidermal UV-A screening.

For grape and barley leaves, UV-A and UV-B absorbance of the adaxial epidermis as a function of phenolic absorbance at 360 and 314 nm, respectively, is shown in Fig. 4. In all four plots, epidermal absorbance was considerably smaller than the corresponding phenolic absorbance, except for the smallest values, and curvilinear relationships were observed. Curvilinear behaviour was already apparent in Fig. 3 although these data were analysed using linear regression; for example, outermost data points of absorbance by quercetin derivatives versus epidermal UV-A absorbance in grape leaves fell below the regression line but the greater proportion of central points resided above the line (Fig. 3f). Curvilinear relations differed markedly between species: initial slopes determined by linear regression for A314 or A360 < 2 were about 0.45 for grape leaves (Fig. 4a & c) but 0.25 for barley leaves (Fig. 4b & d).

image

Figure 4. Fluorometrically determined UV absorbance of adaxial sides of grape (a and c) and barley leaves (b and d) as a function of total UV absorbance of all major extractable phenolics. In (a) and (b), epidermal UV-A absorbance is plotted versus phenolic absorbance at 360 nm; epidermal UV-B screening versus phenolic absorbance at 314 nm is depicted in (c) and (d). Curved lines correspond to the best fit of our model function (Eqn 4) to the data. Parameters of these curves are summarized in Fig. 5.

Download figure to PowerPoint

As stated in Material and Methods, stray UV radiation from our leaf samples could have influenced calculations of epidermal transmittance particularly when true epidermal UV screening results in low intensities of FUV-B or FUV-A. Hence, curved relationships in Fig. 4 could have been the result of measuring artefacts. However, in the present study we corrected for stray radiation by employing a method which obtains realistic estimates for background signals in Xe-PAM measurements (see Material and Methods). Further, curvilinear relationships obtained with grape leaves were still apparent when the background signals from an aluminum foil, which certainly are higher than those from leaf samples, were subtracted from FUV-B and FUV-A measurements (not shown). Therefore, it seems unlikely that the curvilinear behaviour of data in Fig. 4 originates from instrumental artifacts, and we looked for a model function which explains these relationships by taking into account differences in the optical properties of phenolics in the leaf and in solution.

Pre-requisites for the model are fibre-optical measurements demonstrating that the major part of the leaf's UV screen is located within the epidermis (Day, Vogelmann & DeLucia 1992; Cen & Bornman 1993; Day, Martin & Vogelmann 1993; Olsson, Veit & Bornman 2000; Liakoura et al. 2003). That phenolics are involved in variable epidermal UV screening is suggested by Fig. 3. Further, it is known that phenolics in the epidermis of leaves of many flowering plants are accumulated in vacuoles (Caldwell et al. 1983; Weissenböck et al. 1984; Day et al. 1993; Hutzler et al. 1998; Kolb et al. 2001). Therefore, variable epidermal UV screening can be linked to variable concentrations of phenolics in epidermal vacuoles causing variable UV transmittance of these organelles which we denote as TUVv.

The lowest absorbance of the adaxial epidermis is observed for lowest absorbance of extracted phenolics (Fig. 4). In this case, our conception requires that TUVv equals zero and minimum concentrations of UV-absorbing phenolics are present in vacuoles. We assume for lowest epidermal absorbance that transmittance of the epidermal area including vacuoules (aVc) does not deviate markedly from transmittance of the remaining epidermal area (1 −aVc), and consider the epidermis as a homogeneous optical layer characterized by basal transmittance TUVbasal. Considering variable UV screening, the product of TUVv and TUVbasal determines epidermal transmittance of aVc; but, TUVbasal alone describes transmittance of (1 − aVc). Transmittance of the entire epidermis, TUV, corresponds to the sum of (aVc·TUVv·TUVbasal) plus [(1 − aVcTUVbasal]

  • image(1)

Replacing transmittance by absorbance terms yields

  • image(2)

We further define the absorbance of extracted phenolics, AEX.UV, which includes the absorbance of phenolics that are present when epidermal screening is minimal, AEX.UVbasal, plus variable phenolic absorbance, AUVv. Note that AEX.UVbasal solely results from extracted phenolics but AUVbasal is composed of soluble and insoluble phenolics as well as other epidermal constituents absorbing in the UV. We assume that during our relatively short exposure intervals, changes in AUVbasal are negligible in comparison with changes in variable absorbance of the adaxial epidermal vacuole (AUVv). As explained above, AUVv results from variable phenolic absorbance located in these vacuoles. Extraction distributes the vacuole-confined absorbance homogeneously and, thus, dilutes AUVv by the factor aVc. Figure 1 indicates that phenolic absorbance in abaxial sides change in parallel to that of the adaxial side. Hence, variable phenolics from adaxial side (aVc·AUVv) is multiplied by (1 + f) to obtain the entire variable absorbance in the extract. More generally, the f-value is defined as the phenolic absorbance not involved in adaxial UV screening but varying parallel to phenolic absorbance active in adaxial UV screening, expressed relative to phenolic absorbance active in adaxial UV screening. One can formulate

  • image(3)

Combining Eqns 2 and 3 results in a model function that describes the four data sets in Fig. 4, where the AUV corresponds to ordinates and the AEX.UV to the abscissas

  • image(4)

For each species and UV range, the unknown parameters AUVbasal and AEX.UVbasal were assessed from means of the five lowest values of epidermal absorbance and absorbance of extracted phenolics, respectively. In all cases, these minimum values originated from the group of plants grown under shaded and practically UV-free growth conditions. Using the mean values for AEX.UVbasal and AUVbasal, the free parameters, aVc and f, were estimated by fitting our model function to the four data sets in Fig. 4. Solid lines in Fig. 4 represent best fits of theory to experiment: corresponding parameters are depicted in Fig. 5.

image

Figure 5. Parameters of the four model functions (solid lines in Fig. 4). ‘UV-A’ and ‘UV-B’ refers to curves in the UV-A (Fig. 4a & b) and the UV-B spectral range (Fig. 4c & d), respectively. Data for grape leaves are shown as open and those for barley leaves as closed bars. (a) and (b) depict basal absorbance of extracted phenolics and basal absorbance of adaxial epidermis, respectively, obtained with leaves grown in a shaded greenhouse and in the virtual absence of UV radiation (error bars: standard error, n = 5). Other panels depict parameter estimates obtained by fitting our model function to experimental data; error bars represent standard errors calculated by the curve fitting program. (c) Relative area of adaxial epidermis enclosing vacuoles. (d) UV absorbance of extracted phenolics which are not involved in adaxial UV screening but vary parallel to UV absorbance of phenolics active in adaxial UV screening, expressed relative to UV absorbance of the adaxially screening phenolics.

Download figure to PowerPoint

Basal epidermal UV absorbance was always higher than 0.25 (Fig. 5b). In comparison, we calculated from the transmittance spectrum of a methanol-extracted epidermis from Vicia faba (Markstädter et al. 2001) values of 0.07 and 0.01 at 314 and 360 nm, respectively, using the absorbance at 490 nm as the baseline (the 490 nm represents the wavelength position of maximum blue-green excitation of our Xe-PAM fluorometer). The much higher UV screening of the intact epidermis is consistent with presence of some UV-absorbing phenolics in the epidermis even in the absence of UV stress (Liakoura et al. 2003).

Direct comparison of basal absorbance of the epidermis (Fig. 5b) with basal absorbance of extracted phenolics (Fig. 5a) is difficult because the former also includes contributions by insoluble phenolics and non-phenolic factors. However, in grapevine, basal epidermal screening in the UV-B was higher than in the UV-A, and this pattern was paralleled by basal absorbance of extracted phenolics. The higher UV–B absorbance in these extracts is consistent with considerable amounts of the preferably UV-B-absorbing (Fig. 2c) hydroxycinnamic acids in grape leaves exhibiting basal epidermal UV screening (Fig. 3a). That basal epidermal screening in the UV-B was higher than in the UV-A is therefore well explained by localization of a major part of hydroxycinnamic acids in the adaxial epidermis. Similar basal epidermal screening for both UV spectral ranges in barley leaves (Fig. 5b) agrees with the presence of apigenin derivatives in the epidermis (Fig. 3), which absorb in the UV-A as well as in the UV-B spectral region (Fig. 2d).

When variable UV screening is considered, it is important to realize that our model function (Eqn 4) describes a straight line for aVc = 1; that is when spaces between strongly UV-absorbing vacuoles are negligibly narrow. Fitting theory to experiment, however, always yielded curvilinear relations with estimates for aVc of around 0.9 (Fig. 5c). These data agree with Day et al. (1993) who have demonstrated that the epidermis of outdoor-grown leaves behave as an inhomogeneous UV filter exhibiting an ‘optical sieve effect’ due to UV-transparent gaps between highly UV-absorbing vacuoles.

Unlike aVc, the f-value differs considerably between species (Fig. 5d): In grape leaves, the f-value was 0.75 and 0.5 for the UV-A and UV-B range, respectively; but the comparable values were 2.3 and 1.7 in barley leaves. In grape leaves, adaxial UV-A absorbance was about twice as high as the abaxial one (Fig. 1a) which corresponds to an f-value of 0.5. In comparison, similar epidermal UV-A absorbance of both barley leaf sides results in an f-value of approximately 1 (Fig. 1b). Therefore, in grape leaves, the theoretical f-values were 150 and 100% in the UV-A and UV-B range, respectively, of the f-value derived from Fig. 1, but the equivalent percentages were 230 and 170% in barley. An obvious explanation for the markedly higher deviations of theory from experiment in barley is that a considerable fraction of variable phenolic absorbance is located in the barley leaf mesophyll but not in the grapevine leaf mesophyll. This agrees well with Liu et al. (1995) who deduced from their data that epidermal and mesophyll flavonoids in barley increased in parallel in response to artificial UV-B radiation. Evidently, different allocation patterns of phenolics gave rise to the much different initial slopes of the relationships between adaxial UV absorbance and UV absorbance of leaf phenolics observed for grape and barley leaves (Fig. 4).

CONCLUSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

To study to what degree UV screening by the adaxial epidermis can be predicted from the UV absorbance of extracted phenolics, we have chosen two economically important crop species: grapevine exhibiting bifacial and barley exhibiting equifacial leaf anatomy. Analysing the relations between the two measuring parameters suggested that areas containing vacuoles contributed roughly 90% to total area of the epidermis. If radiation stimuli provoke loading of these vacuoles with UV-absorbing phenolics, epidermal UV screening is enhanced. Because of relatively UV-transparent areas between vacuoles, total epidermal UV absorbance is lower than UV absorbance of vacuoles alone. This results in an increasing over-estimation of true epidermal UV absorbance with increasing absorbance of extracted phenolics. In both species therefore optical heterogeneity of the epidermis interferes severely with correlation between epidermal UV absorbance and phenolic absorbance. In addition, interspecific differences in allocation of phenolics were detected. In barley leaves, but not in grape leaves, most of variable phenolic absorbance appears to be located in the mesophyll tissue and the abaxial epidermis. Consequently, comparison of epidermal screening between species based on phenolic absorbance can be highly unreliable if the distribution pattern of phenolics is unknown. In conclusion, the use of phenolic absorbance as a measure of the capacity of leaf's UV screening requires both consideration of epidermal optical properties and of the distribution of phenolics within the leaf to define the relevant error margins for each individual species when using this method.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

For providing us with grapevine plants, we are grateful to Josef Herrmann, Landesanstalt für Wein- und Gartenbau, Veitshöchheim, Germany. We are grateful to Dr A. Meister for important comments and Dr R. J. Porra for help in preparing the manuscript. We thank Professor Markus Riederer for continuous support throughout this study. This work was supported by a grant from the state of Bavaria (BayForUV program).

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  • Barnes P.W., Searles P.S., Ballaré C.L., Ryel R.J. & Caldwell M.M. (2000) Non-invasive measurements of leaf epidermal transmittance of UV radiation using chlorophyll fluorescence: field and laboratory studies. Physiologia Plantarum 109, 274283.
  • Bilger W., Veit M., Schreiber L. & Schreiber U. (1997) Measurement of leaf epidermal transmittance of UV radiation by chlorophyll fluorescence. Physiologia Plantarum 101, 754763.
  • Bornman J.F. & Teramura A.H. (1993) Effects of ultraviolet-B radiation on terrestrial plants. In Environmental UV Photobiology (ed. A.R.Young), pp. 427471. Plenum Press, New York, USA.
  • Burchard P., Bilger W. & Weissenböck G. (2000) Contribution of hydroxycinnamates and flavonoids to epidermal shielding of UV-A and UV-B radiation in developing rye primary leaves as assessed by ultraviolet-induced chlorophyll fluorescence measurements. Plant, Cell and Environment 23, 13731380.
  • Caldwell M.M., Robberecht R. & Flint S.D. (1983) Internal filters: Prospects for UV-acclimation in higher plants. Physiologia Plantarum 58, 445450.
  • Cen Y.P. & Bornman J. (1993) The effect of exposure to enhanced UV-B radiation on the penetration of monochromatic and polychromatic UV-B radiation in leaves of Brassica napus. Physiologia Plantarum 87, 249255.
  • Cerovic Z.G., Ounis A., Cartelat A., Latouche G., Goulas Y., Meyer S. & Moya I. (2002) The use of chlorophyll fluorescence excitation spectra for the non-destructive in situ assessment of UV-absorbing compounds in leaves. Plant, Cell and Environment 25, 16631676.
  • Cockell C.S. & Knowland J. (1999) Ultraviolet radiation screening compounds. Biological Reviews of the Cambridge Philosophical Society 74, 311345.
  • Day T.A., Martin G. & Vogelmann T.C. (1993) Penetration of UV-B radiation in foliage: evidence that the epidermis behaves as a non-uniform filter. Plant, Cell and Environment 16, 735741.
  • Day T.A., Vogelmann T.C. & DeLucia E.H. (1992) Are some plant life forms more effective than others in screening out ultraviolet-B radiation? Oecologia 92, 513519.
  • De Beer D., Joubert E., Gelderblom W.C.A. & Manley M. (2002) Phenolic compounds: a review of their possible role as in vivo antioxidants of wine. South African Journal of Enology and Viticulture 23, 4861.
  • Effertz B. & Weissenböck G. (1976) Dynamics of flavonoid accumulation in primary leaf tissues of Avena sativa. Berichte der Deutschen Botanischen Gesellschaft 89, 473481.
  • Fröst S., Harborne J.B. & King L. (1977) Identification of the flavonoids in 5 chemical races of cultivated barley. Hereditas 85, 163168.
  • Hollósy F. (2002) Effects of ultraviolet radiation on plant cells. Micron 33, 179197.
  • Husain S.R., Cillard J. & Cillard P. (1987) Hydroxyl radical scavenging activity of flavonoids. Phytochemistry 26, 24892492.
  • Hutzler P., Fischbach R., Heller W., Jungblut T.P., Reuber S., Schmitz R., Veit M., Weissenböck G. & Schnitzler J.P. (1998) Tissue localization of phenolic compounds in plants by confocal laser scanning microscopy. Journal of Experimental Botany 49, 953965.
  • Jordan B.R. (1996) The effects of ultraviolet-B radiation on plants: a molecular perspective. Advances in Botanical Research 22, 97162.
  • Koepke P. (2000) UV-Strahlung an der Erdoberfläche. In Handbuch der Umweltveränderungen und Ökotoxilogie, Vol. 1B: Atmosphäre (ed. R.Guderian), pp. 299331. Springer, Berlin, Germany.
  • Kolb C.A., Käser M.A., Kopecký J., Zotz G., Riederer M. & Pfündel E.E. (2001) Effects of natural intensities of visible and UV radiation on epidermal UV-screening and photosynthesis in grape leaves (Vitis vinifera cv. Silvaner). Plant Physiology 127, 863875.
  • Kolb C.A., Kopecký J., Riederer M. & Pfündel E.E. (2003) UV screening by phenolics in berries of grapevine (Vitis vinifera). Functional Plant Biology 30, 11771186.
  • Krause G.H., Gallé A., Gademann R. & Winter K. (2003) Capacity of protection against ultraviolet radiation in sun and shade leaves of tropical forest plants. Functional Plant Biology 30, 533542.
  • Landry L.G., Chapple C.C.S. & Last R.L. (1995) Arabidopsis mutants lacking phenolic sunscreens exhibit enhanced ultraviolet-B injury and oxidative damage. Plant Physiology 109, 11591166.
  • Liakoura V., Bornman J.F. & Karabourniotis G. (2003) The ability of abaxial and adaxial epidermis of sun and shade leaves to attenuate UV-A and UV-B radiation in relation to the UV absorbing capacity of the whole leaf methanolic extracts. Physiologia Plantarum 117, 3343.
  • Liu L., Gitz D.C. III & McClure J.W. (1995) Effects of UV-B on flavonoids, ferulic acid, growth and photosynthesis in barley primary leaves. Physiologia Plantarum 93, 725733.
  • Mabry T.J., Markham K.R. & Thomas M.B. (1970) The Systematic Identification of Flavonoids. Springer Verlag, Berlin-Heidelberg-New York.
  • Markham K.R., Ryan K.G., Bloor S.J. & Mitchell K.A. (1998a) An increase in the luteolin: apigenin ratio in Marchantia polymorpha on UV-B enhancement. Phytochemistry 48, 791794.
  • Markham K.R., Tanner G.J., Caasi-Lit M., Whitecross M.I., Nayudu M. & Mitchell K.A. (1998b) Possible protective role for 3′,4′-dihydroxyflavones induced by enhanced UV-B in a UV-tolerant rice cultivar. Phytochemistry 49, 19131919.
  • Markstädter C., Queck I., Baumeister J., Riederer M., Schreiber U. & Bilger W. (2001) Epidermal transmittance of leaves of Vicia faba for UV radiation as determined by two different methods. Photosynthesis Research 67, 1725.
  • Mazza C.A., Boccalandro H.E., Giordano C.V., Battista D., Scopel A.L. & Ballaré C.L. (2000) Functional significance and induction by solar radiation of ultraviolet-absorbing sunscreens in field-grown soybean crops. Plant Physiology 122, 117126.
  • Nayak L., Biswal B., Ramaswamy N.K., Iyer R.K., Nair J.S. & Biswal U.C. (2003) Ultraviolet-A induced changes in photosystem II of thylakoids: effects of senescence and high growth temperature. Journal of Photochemistry and Photobiology B: Biology 70, 5965.
  • Nørbæk R., Brandt K. & Kondo T. (2000) Identification of flavone C-glycosides including a new flavonoid chromophore from barley leaves (Hordeum vulgare L.) by improved NMR techniques. Journal of Agricultural and Food Chemistry 48, 17031707.
  • Olsson L.C., Veit M. & Bornman J.F. (2000) Epidermal transmittance and phenolic composition in leaves of atrazine-tolerant and atrazine-sensitive cultivars of Brassica napus grown under enhanced UV-B radiation. Physiologia Plantarum 107, 259266.
  • Ounis A., Cerovic Z.G., Briantais J.M. & Moya I. (2001) Dual-excitation FLIDAR for the estimation of epidermal UV absorption in leaves and canopies. Remote Sensing of Environment 76, 3348.
  • Pfündel E.E. (2003) Action of UV and visible radiation on chlorophyll fluorescence from dark-adapted grape leaves (Vitis vinifera L.) Photosynthesis Research 75, 2939.
  • Reuber S., Bornman J.F. & Weissenböck G. (1996) A flavonoid mutant of barley (Hordeum vulgare L.) exhibits increased sensitivity to UV-B radiation in the primary leaf. Plant, Cell and Environment 19, 593601.
  • Reuber S., Jende-Strid B., Wray V. & Weissenböck G. (1997) Accumulation of the chalcone isosalipurposide in primary leaves of barley flavonoid mutants indicates a defective chalcone isomerase. Physiologia Plantarum 101, 827832.
  • Reuder J., Dameris M. & Koepke P. (2001) Future UV radiation in Central Europe modelled from ozone scenarios. Journal of Photochemistry and Photobiology B: Biology 61, 94105.
  • Rozema J., Björn L.O., Bornman J.F., et al. (2002) The role of UV-B radiation in aquatic and terrestrial ecosystems-an experimental and functional analysis of the evolution of UV-absorbing compounds. Journal of Photochemistry and Photobiology B: Biology 66, 212.
  • Ryan K.G., Markham K.R., Bloor S.J., Bradley J.M., Mitchell K.A. & Jordan B.R. (1998) UVB radiation induced increase in quercetin: kaempferol ratio in wild-type and transgenic lines of Petunia. Photochemistry and Photobiology 68, 323330.
  • Semerdjieva S.I., Sheffield E., Phoenix G.K., Gwynn-Jones D., Callaghan T.V. & Johnson N. (2003) Contrasting strategies for UV-B screening in sub-Arctic dwarf shrubs. Plant, Cell and Environment 26, 957964.
  • Sheahan J.J. (1996) Sinapate esters provide greater UV-B attenuation than flavonoids in Arabidopsis thaliana (Brassicaceae). American Journal of Botany 83, 679686.
  • Solomon S. (2004) The hole truth. What's news (and what's not) about the ozone hole. Nature 427, 289291.
  • Strack D., Meurer B. & Weissenböck G. (1982) Tissue specific kinetics of flavonoid accumulation in primary leaves of rye (). Zeitschrift für Pflanzenphysiologie 108, 131142.
  • Tevini M., Braun J. & Fieser G. (1991) The protective function of the epidermal layer of rye seedlings against ultraviolet-B radiation. Photochemistry and Photobiology 53, 329333.
  • Torel J., Cillard J. & Cillard P. (1986) Antioxidant activity of flavonoids and reactivity with peroxy radical. Phytochemistry 25, 383386.
  • Trepte S. & Winkler P. (2004) Reconstruction of erythemal UV irradiance and dose at Hohenpeissenberg (1968–2001) considering trends of total ozone, cloudiness and turbidity. Theoretical and Applied Climatology 77, 159171.
  • Turcsányi E. & Vass I. (2000) Inhibition of photosynthetic electron transport by UV-A radiation targets the photosystem II complex. Photochemistry and Photobiology 72, 513520.
  • Wagner H., Gilbert M. & Wilhelm C. (2003) Longitudinal leaf gradients of UV-absorbing pigments in barley (Hordeum vulgare). Physiologia Plantarum 117, 383391.
  • Weissenböck G., Schnabl H., Sachs G., Elbert C. & Heller F.-O. (1984) Flavonol content of guard cell and mesophyll cell protoplasts isolated from Vicia faba leaves. Physiologia Plantarum 62, 356362.
  • Winkel-Shirley B. (2001) Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiology 126, 485493.
  • Yamasaki H., Sakihama Y. & Ikehara N. (2003) Flavonoid-peroxidase reaction as a detoxification mechanism of plant cells against H2O2. Plant Physiology 115, 14051412.