Effects of natural intensities of visible and ultraviolet radiation on epidermal ultraviolet screening and photosynthesis in grape leaves

Abstract

Grape (Vitis vinifera cv Silvaner) vine plants were cultivated under shaded conditions in the absence of ultraviolet (UV) radiation in a greenhouse, and subsequently placed outdoors under three different light regimes for 7 d. Different light regimes were produced by filters transmitting natural radiation, or screening out the UV-B (280-315 nm), or screening out the UV-A (315-400 nm) and the UV-B spectral range. During exposure, synthesis of UV-screening phenolics in leaves was quantified using HPLC: All treatments increased concentrations of hydroxycinnamic acids but the rise was highest, reaching 230% of the initial value, when UV radiation was absent. In contrast, UV-B radiation specifically increased flavonoid concentrations resulting in more than a 10-fold increase. Transmittance in the UV of all extracted phenolics was lower than epidermal UV transmittance determined fluorimetrically, and the two parameters were curvilinearly related. It is suggested that curvilinearity results from different absorption properties of the homogeneously dissolved phenolics in extracts and of the non-homogeneous distribution of phenolics in the epidermis. UV-B-dependent inhibition of maximum photochemical yield of photosystem II (PSII), measured as variable fluorescence of dark-adapted leaves, recovered in parallel to the buildup of epidermal screening for UV-B radiation, suggesting that PSII is protected against UV-B damage by epidermal screening. However, UV-B inhibition of CO(2) assimilation rates was not diminished by efficient UV-B screening. We propose that protection of UV-B inactivation of PSII is observed because preceding damage is efficiently repaired while those factors determining UV-B inhibition of CO(2) assimilation recover more slowly.

Figure 1

Light conditions. A, Transmittance spectra of the teflon (1), polyester (2), and LEE 226 UV foils (3) used to modify natural sunlight to produce “vis + UVA + UVB,” “vis + UVA,” and “vis” regimes, respectively. B, Spectral irradiances under the foils, and also in the greenhouse (4, gray line).

Figure 2

Chromatography of phenolic compounds. HPLC analyses (unprocessed A₃₁₄ versus retention time) of methanolic extracts from a greenhouse-grown leaf (0:GH), and from leaves exposed for 7 d to vis, vis + UVA, or vis + UVA + UVB conditions designated “7:vis,” “7:vis + A,” and “7:vis + A + B,” respectively, are shown. Absorbance spectra representing the peaks labeled 1 through 10 are depicted in Figure 3. Std, The internal standard, quercetin.

Figure 3

Absorbance spectra of phenolic compounds. The absorbance spectra of chromatographically detected compounds, normalized to their long-wavelength maximum, are shown. Numbers identifying spectra refer to the peak numbers in Figure 2. The spectrum of compound 7 was indistinguishable from that of compound 8; the spectra of compounds 9 and 10 were also indistinguishable.

Figure 4

Concentration of phenolics during outdoor exposure. The figure depicts concentrations, normalized to leaf area, of the sum of cis- and trans-caffeoyl tartaric acid (A), and cis- and trans-coumaroyl tartaric acid (C) during exposure of grape leaves to different light conditions as defined in Figure 1. Data, normalized to leaf area, for quercetin (que1 + que2) and kaempferol (kae1 + kae2) are shown in B and D, respectively. que1 and que2 stand for HPLC peaks 7 and 8 (Fig. 2) representing different quercetin glycosides, and kae1 and kae2 correspond to peaks 9 and 10 (Fig. 2) representing different kaempferol glycosides. Here, and also in subsequent figures, triangles, squares, and diamonds symbolize data obtained under vis, vis + UVA and vis + UVA + UVB conditions, respectively. The white circles denote the initial data from greenhouse-grown vines measured immediately before outdoor exposure. For clarity, symbols of identical time intervals were slightly shifted relative to each other. Bars indicate ses of means (4 ≤ n ≤ 6). In the cases of small ses, bars are hidden by symbols. In A, vis data differed significantly those of the other treatments; in B and D, vis + UVA + UVB data differed significantly from those of the other treatments (see “Materials and Methods”).

Figure 5

Micrographs of a cross section from a grape leaf. The figure shows a light transmission image (A) and fluorescence images (B and C) either untreated (A and B) or ammonia treated (C) of the same cross section of a grape leaf acclimated to full sunlight. The presence of phenolic compounds in the upper epidermis is indicated by ammonia-enhanced green fluorescence (C).

Figure 6

Intensity of chlorophyll fluorescence excited by UV or blue-green light. Unprocessed chlorophyll fluorescence at the F₀ level excited by UV-B (F_UV-B), UV-A (F_UV-A), and blue-green radiation (F_BG) is depicted in A, B, and C, respectively. See Figure 4 for comments on symbols and error bars. In B, vis data differed significantly from those of the other treatments (see “Materials and Methods”).

Figure 7

Relation between epidermal screening and UV absorption of phenolics. Data of individual leaves are shown. In A and B, we plot epidermal transmittance for UV-A radiation (T_UV-A) against transmittance of extracted leaf phenolics at 360 nm (T₃₆₀), and epidermal UV-B transmittance (T_UV-B) against transmittance of extracted leaf phenolics at 314 nm (T₃₁₄), respectively (extracted phenolics correspond to peaks 1–10 in Fig. 2). In A and B, the bold line results from linear regression to solid circles, that is for T₃₆₀ > 3% and T₃₁₄ > 3%. In A and B, coefficients of determinations are r² = 0.874 and r² = 0.750, and equations resulting from regression analyses are T_UV-A = 11 + 0.72 × T₃₆₀ and T_UV-B = 8 + 1.22 × T₃₆₀, respectively. White circles (T₃₆₀ or T₃₁₄ < 3%) are also shown at amplified scales in inserts (ordinate, 0%–12%; abscissa, 0%–3%).

Figure 8

Photosynthetic parameters during exposure. Time courses of normalized variable chlorophyll fluorescence, F_v/F_m, of dark-adapted leaves and of light-saturated CO₂ assimilation rate (J_CO2) are shown in A and B, respectively. For both parameters, UV-B-dependent effects were calculated by subtracting mean values of vis + UVA + UVB minus mean values of vis + UVA conditions (C and D). See Figure 4 for comments on symbols and error bars. In A and B, vis + UVA + UVB data differed significantly from those of the other treatments (see “Materials and methods”).