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Logo of annbotAboutAuthor GuidelinesEditorial BoardAnnals of Botany
 
Ann Bot. 2009 September; 104(4): 621–633.
Published online 2009 June 27. doi:  10.1093/aob/mcp144
PMCID: PMC2729627

Developmental changes in spatial distribution of in vivo fluorescence and epidermal UV absorbance over Quercus petraea leaves

Abstract

Background and Aims

Epidermal phenolic compounds (mainly flavonoids) constitute a vital screen that protects the leaf from damage by natural ultraviolet (UV) radiation. The effectiveness of epidermal UV-screening depends on leaf anatomy, the content of UV-screening compounds and their spatial uniformity over the leaf area. To investigate in vivo the spatial pattern of the epidermal UV-screen during leaf development, a fluorescence imaging method was developed to map the epidermal UV-absorbance at a microscopic scale. This study was done on oak (Quercus petraea) leaves that were used as a model of woody dicotyledonous leaves.

Methods

The leaf development of 2-year-old trees, grown outdoors, was monitored, at a macroscopic scale, by in vivo measurements of chlorophyll content per unit area and epidermal UV-absorbance using two optical leaf-clip meters. The distribution of pigments within leaves was assessed in vivo spectroscopically. The microscopic images of UV-induced fluorescence and UV-absorbance acquired in vivo during leaf development were interpreted from spectral characteristics of leaves.

Key Results

At a macroscopic scale, epidermal UV-absorbance was high on the upper leaf side during leaf development, while it increased on the lower leaf side during leaf expansion and reached the adaxial value at maturity. At a microscopic scale, in immature leaves, for both leaf sides, the spatial distribution of epidermal UV-absorbance was heterogeneous, with a pattern depending on the flavonoid content of vacuoles in developing epidermal cells. At maturity, epidermal UV-absorbance was uniform.

Conclusions

The spatial pattern of epidermal UV-screen over the area of oak leaves is related to leaf anatomy during development. In vivo spectroscopy and fluorescence imaging of the leaf surface showed the distribution of pigments within the leaf and hence can provide a tool to monitor optically the leaf development in nature.

Key words: Blue-green fluorescence, chlorophyll fluorescence, epidermis, flavonoids, leaf development, microscopic imaging, polyphenols, Quercus petraea

INTRODUCTION

In the field, epidermal phenolic compounds constitute a vital screen that protects leaves from damage by natural ultraviolet (UV) radiation (Caldwell et al., 1983). The effectiveness of epidermal UV-screening depends not only on leaf anatomy and content of UV-screening pigments, but also on their uniformity over the leaf area (Day et al., 1993). During leaf development, epidermal thickening and the accumulation of phenolic compounds increase the effectiveness of epidermal UV-screening (DeLucia et al., 1992; Ruhland and Day, 1996). However, the changes in spatial distribution of epidermal UV-screening over the leaf area during leaf development have not been investigated before, although they may have important consequences on the protection of developing leaves against UV light.

To investigate epidermal UV-absorbance in the field, researchers have developed portable leaf-clips, like Dualex and UV-A PAM fluorimeters, which were described earlier (Bilger et al., 2001; Goulas et al., 2004; Pfündel et al., 2007). These fluorimeters measure the UV absorbance of the leaf epidermis by double excitation of chlorophyll a fluorescence (ChlF; all abbreviations are defined in Appendix I), according to Bilger et al. (1997), using UV (375 nm) and visible (650 nm, in the case of Dualex) light. Epidermal UV-absorbance is determined from the UV/red ChlF excitation ratio (FER). Red light is not absorbed by the epidermis and fully penetrates into the mesophyll where it excits chlorophyll. Therefore, red-excited ChlF can serve as a reference signal to which UV-excited ChlF can be related. Red excitation light is also not absorbed by anthocyanins (Pfündel et al., 2007) that often occur in immature foliage (Hughes et al., 2007). On the other hand, absorbance at 375 nm is mainly due to soluble flavonoids in epidermal vacuoles (Cerovic et al., 2002). Actually, these fluorimeters measure a mean epidermal UV-absorbance over a 5-mm-diameter leaf area and therefore do not spatially resolve the epidermal UV-screening over the leaf area. Thus, multispectral fluorescence imaging has been developed to visualize, non-invasively, the epidermal UV-absorbance over the whole leaf area by using the concept of double excitation of ChlF (Lenk and Buschmann, 2006; Lenk et al., 2007). This macroscopic imaging method has to be complemented by microscopic observations to spatially resolve UV-absorbance at the scale of epidermal cells and to investigate the spatial pattern of flavonoid accumulation in epidermal vacuoles in developing leaves.

The interpretation of multispectral fluorescence images of the leaf surface is based on the in vivo estimation of the nature and location of fluorophores and UV-absorbing compounds within leaves by using fluorescence emission and excitation spectra (Buschmann and Lichtenthaler, 1998; Cerovic et al., 1999; Cerovic et al., 2002; Lenk and Buschmann, 2006). Under UV-excitation, dicotyledonous green leaves emit blue-green fluorescence (400–630 nm; Cerovic et al., 1999), mainly from hydroxycinnamic acids bound to cell walls and cuticle (Hartley and Harris, 1981; Lang et al., 1991, 1994) and red (630–700 nm) and far-red (700–800 nm) fluorescence from chlorophyll a in the mesophyll (for reviews, see Buschmann and Lichtenthaler, 1998; Cerovic et al., 2002). Fluorescence emission spectra have been characterized for mature and senescent dicotyledonous leaves (Lang and Lichtenthaler, 1991; Subhash et al., 1999), and used to interpret the difference of the spatial pattern of fluorescence between adaxial (upper) and abaxial (lower) leaf surfaces (Lang et al., 1994; Lenk and Buschmann, 2006). However, early processes of leaf development have not been studied. They result in chlorophyll synthesis (Niinemets et al., 2004) and flavonoid accumulation at emergence (Salminen et al., 2004), while epidermal and mesophyll cells are dividing and expanding (Avery, 1932). All these changes may strongly affect the spatial pattern of UV-induced fluorescence emission, ChlF excitation and therefore epidermal UV-absorbance on both leaf sides.

In this study, oak was used as a model of woody dicotyledonous species from temperate forest, since their leaves have typical anatomy that is bifacial, heterobaric and hypostomatous (Esau, 1953). The developmental variations in chlorophyll content (Gond et al., 1999) and in leaf phenolic compounds (Covelo and Gaillardo, 2001; Salminen et al., 2004) in field-grown trees are known for this genus. Quercetin derivatives, together with those of kaempferol, represent the dominant flavonoids in oak leaves (Bate-Smith, 1962; Salminen et al., 2004).

Therefore, the aims of this study were (a) to characterize the in vivo fluorescence emission and UV-absorption of oak leaves and to investigate the spatial pattern of UV-induced visible fluorescence during leaf development, (b) to map the epidermal UV-absorbance in immature and mature leaves using the method of the double ChlF excitation at a microscopic scale, and (c) to investigate the relationship between anatomical development and optically assessed biochemical development of leaves.

MATERIALS AND METHODS

Plant material and experimental design

Sessile oaks [Quercus petraea (Matt.) Liebl.] were grown outdoors in pots in commercial nurseries at Alençon (48°25′50″N, 00°05′35″E). In December 2005, 2-year-old trees about 1·5 m tall were planted in 90-L pots containing a sand/compost mixture (50/50, v/v) without the addition of fertilizer. The pots were spaced 2 m apart in an open field on the campus of the University of Paris-Sud (48°42′N, 02°10′E, France, at an elevation of 65 m). The pots were wrapped in plastic bags to exclude rainfall. The trees were watered once a week (12 L of water per pot, corresponding to the field capacity). One tree among the 70 trees used for other experiments (Maunoury-Danger, 2007), was used for this study. Bud burst took place at day of year (DOY) 105 (mid-April 2007).

Eight DOY were chosen during the growth for sampling, corresponding to key developmental stages, according to Maunoury-Danger (2007). At each sampling date, two leaves per tree were harvested from a south-west-facing branch at around 1100 h and kept in a Petri dish in a humid atmosphere under ambient low light. Chlorophyll and epidermal phenolic compound contents were optically assessed using portable leaf-clip devices, the SPAD chlorophyll meter (hereafter referred to as SPAD) and the Dualex fluorimeter (hereafter referred to as Dualex), respectively, just before recording the excitation and emission spectra. Then, the leaves were stored overnight at low temperature and examined microscopically the following day at an imaging facility (Imaging of Dynamic Processes in Cell and Developmental Biology, Institut Jacques Monod, Paris, France).

Optical measurements of content of chlorophyll and epidermal phenolic compounds in leaves

Chlorophyll content per unit area was estimated with a SPAD-502 chlorophyll meter (Minolta, Carrière-sur-Seine, France). Five measurements were taken on the leaf, at different places, with the SPAD light emitters at the adaxial surface, according to Cartelat et al. (2005). Phenolic compounds per unit area were estimated at approximately the same location as the SPAD readings using the Dualex fluorimeter (FORCE-A, Orsay, France). In oak leaves, the Dualex mainly detected flavonols that absorb at 375 nm. Ten Dualex readings (DA375) were taken per leaf, five per leaf side. The DA375 values of each side were summed to estimate the total content of phenolic compounds in the leaf epidermis (Cartelat et al., 2005). Relative SPAD and Dualex values were used.

Spectroscopy

ChlF emission and excitation spectra were recorded on a Cary Eclipse spectrofluorimeter (Varian Inc., Les Ulis, France) as described (Bidel et al., 2007). ChlF excitation spectra were scanned from 300 to 700 nm for an emission wavelength of 750 nm, corresponding to the far-red maximum of ChlF emission in intact leaves (Cerovic et al., 1999). Emission spectra were acquired under three excitation wavelengths, UV (365 nm), blue (460 nm) and red (628 nm). All the spectra were from the same median position on the blade between the major veins on both leaf sides. Spectra were measured on 6-mm-diameter areas of the leaf surface at room temperature (around 20°C) under dim light. Leaves were acclimated to these conditions before measurements. Spectra were fully corrected for excitation efficiency and detection response of the spectrofluorimeter and expressed in quinine sulphate-equivalent units (QSEU) to allow a quantitative comparison among spectra (Cerovic et al., 1999). One QSEU corresponds to the fluorescence of 1 pmol ml−1 of quinine sulphate in a 1 cm layer of 0·105 mol l−1 perchloric acid in water (1 pmol cm−2), excited at 347·5 nm and emitted at 450 nm under identical measuring conditions. The spectra were smoothed according to Cerovic et al. (2002).

The absorbance difference spectra of both leaf sides and between different DOY were obtained from the logarithm of the ratio of the ChlF excitation spectra (logFER) (Cerovic et al., 2002). Smoothed spectra were normalized prior to logFER calculations, so that the integral between 640 and 660 nm equals one, allowing comparison of logFER spectra between samples, but only when UV-induced ChlF was above 25 QSEU (Bidel et al., 2007).

Fluorescence microscopy

Microscopic images were taken from the same leaf area used for fluorescence spectra. Half of the area was mounted with the adaxial side exposed to the excitation light, and half with the abaxial side exposed to the excitation light. Another area of the leaf, from a symmetrical position on the blade with respect to the main vein, was used as an experimental replicate (four replicates per DOY). Samples were mounted in water with Tween 20 (0·5%, v/v) (Sigma, St Louis, USA). Images of fluorescence were made with an inverted epifluorescence microscope (Axiovert 200, Zeiss, Göttingen, Germany) with a monochrome cooled CCD camera (AxioCam MRm, Zeiss, Germany) and a colour cooled CCD camera (AxioCam HRc, Zeiss, Germany). A 100-W mercury vapour lamp (HBO, Zeiss, Germany) provided the excitation light. Leaf fluorescence was imaged using four filter sets described in the Appendix II. Images of 1388 × 1040 pixel size corresponded to an area of 1·7 × 1·3 and 0·22 × 0·17 mm2 at ×5 and ×40 magnification (Plan-Neofluar quartz objectives, Zeiss), respectively. Monochrome and colour images were stored at 12-bit and 14-bit resolution, respectively. Experiments were automated using Axio Vision software (AxioVision 4·4, Zeiss, Germany) and images were processed using Image J 1·36b (National Institutes of Health, USA).

Imaging of UV-absorbance

Microscopic images were made on a fluorescence standard (0·5 × 0·5 cm2 piece of plastic filter, Urban blue, Rosco, Sydenham, London, UK) and samples of two types: leaf samples and artificial screens (0·5 × 0·5 cm2; Lee Filters, Andover, UK) superimposed on a 0·5 × 0·5 cm2 fluorescence standard. All samples were mounted as described. Twelve Lee filters were selected to cover UV-absorbance from 0·2 to 2·4, corresponding to the Dualex range. With these artificial screens, different methods of UV-absorbance estimation could be compared (Pfündel et al., 2007). In this study, Dualex and microscopic imaging were compared.

Images of epidermal UV-absorbance at 365 nm (MA365) were computed according to the logFER method (Cerovic et al., 2002). At a given magnification, two images of far-red ChlF were successively acquired, one excited by red light using the first filter set (628MFfar-red), the other excited by UV light using the second filter set (365MFfar-red). These images were from the fluorescence standard and the samples. All images were corrected for optical heterogeneity. A map of MA365 was computed pixel by pixel, at 32-bit resolution, by taking into account the differences in time exposure (t) between images of 628MFfar-red and 365MFfar-red according to:

equation image
1

with

equation image
2

To compare MA365 with DA375 values, the FER of the standard was divided by 100·1, where 0·1 was the Dualex measurement on the fluorescence standard (the absorbance difference between 628 and 375 nm). The FER of the artificial screens was corrected for transmission of plastic filters at 628 nm. Frequency distribution histograms were computed from images of MA365 with 32-bit resolution.

There was a linear correlation between the mean value of MA365 and the DA375 values from the 12 Lee filters, with the ×40 objective (MA365 = 0·99 × DA375 − 0·036, r2 = 0·98, P < 0·0001, n = 50) and the ×5 objective (MA365 = 0·46 × DA375 + 0·080, r2 = 0·97, P < 0·0001, n = 41). Using the ×5 objective, the mean value of MA365 underestimated the DA375 value because 365MFfar-red is overestimated by scattering light (S). The decreasing exponential relationship between the mean value of 365MFfar-red and the DA375 values provided an estimate of S (mean 365MFfar-red = 996 + 4522 × exp−1·79*DA375 with S = 996 ± 58, n = 46; the corresponding regression between 0 and 1·5 unit of absorbance being log(mean 365MFfar-red) = 3·66–0·41 × DA375, r2 = 0·967, P < 0·0001, n = 42). Therefore, a mean S value was subtracted from 365MFfar-red obtained for both samples and fluorescence standard using the ×5 objective. The S value was 1000 for a 12-bit image.

Fluorescence images were also acquired on transverse leaf sections using the first filter set. Sections were cut manually with a razor blade and observed as previously described.

RESULTS

Developmental patterns of SPAD and Dualex values

Figure 1 shows the variation of SPAD, summed DA375 and adaxial and abaxial DA375 values during leaf development. Three main periods could be defined: (1) an early spring period (until DOY 155 corresponding to late May), during which leaves contained large concentrations of anthocyanins and expanded rapidly, then thickened and stiffened; SPAD value increased; adaxial and abaxial DA375 values decreased slightly and increased markedly, respectively; (2) a late spring period (from DOY 155 to DOY 184, corresponding to June), during which leaves were green and fully expanded; SPAD value was more or less constant and both adaxial and abaxial DA375 values were similar and high; (3) a summer period (from DOY 184 to DOY 265, corresponding to July to late September), during which leaves were ageing then senescing; SPAD value decreased; DA375 values remained stable.

Fig. 1.
Developmental variation of SPAD values (A), and DA375 summed values and adaxial and abaxial DA375 values (B) measured on leaves. Dotted lines at DOY 155 and 183 show the transition from immature to mature leaves and from mature to ageing leaves, respectively. ...

Developmental variation of absorbance difference spectra

Absorbance difference spectra of leaves were measured via the logFER parameter, which corresponds to the algebraic difference between spectra. Figure 2A shows the difference between each one of the spectra taken at different DOY to that taken at DOY 109, of the abaxial surface of leaves. Note that UV-induced ChlF from the adaxial leaf side was below the threshold required for accurately calculating the logFER. The logFER value at 365 and 375 nm corresponds to the maximum absorption of band I flavonols (Cerovic et al., 2002; Bidel et al., 2007). As shown in Fig. 1B, logFER values increased from DOY 109 to DOY 155, showing an increase in flavonol content per unit area. Thereafter, logFER values remained stable. LogFER values around 550 nm were negative from DOY 123 onward, indicating an early decrease in anthocyanin content. In the blue-green spectral domain (450–530 nm), the shape of the spectra revealed the presence of carotenoids that increased apparently at DOY 265, due to chlorophyll breakdown during senescence (Fig. 1A).

Fig. 2.
Absorbance difference spectra obtained from LogFER between each DOY indicated and DOY 109, which was taken as a reference, for the abaxial side (A), and from LogFER between adaxial and abaxial sides of leaves at different DOY (B). The emission wavelength ...

Figure 2B shows the difference in absorbance, estimated via the logFER parameter, between adaxial and abaxial leaf surfaces throughout leaf development. Note that the logFER spectra could not be calculated for wavelengths below 385–390 nm, because UV-induced ChlF from the adaxial leaf side was below 25 QSEU. Around 390–400 nm, logFER values decreased from DOY 109 to DOY 123, but were stable until DOY 205. At DOY 109, the positive values of logFER around 530 nm suggested higher adaxial anthocyanin content. Thereafter, absorbance at the green waveband was similar on both leaf surfaces. The positive values of logFER around 450–530 nm at each DOY indicated a higher carotenoid content on the adaxial than on the abaxial side.

Developmental variation of fluorescence emission spectra of leaves

To investigate further the nature and the location of leaf fluorophores and absorbing compounds within developing leaves, the fluorescence emission spectra of leaves were recorded using UV, blue and red excitation light. The absorption profile within leaves of UV, blue and red lights is different, depending on the relative location and content of the main pigments, that is UV-absorbing compounds, carotenoids and chlorophyll (Louis et al., 2006; Buschmann, 2007).

The UV-induced blue-green fluorescence, 365SF420–630, increased with DOY, in both leaf sides (Fig. 3A, B), therefore it was not affected by the accumulation of UV-absorbing flavonoids in the epidermis. This suggests that 365SF420–630 originated mostly from the leaf surface. The shape of the emission spectra changed during leaf development. This suggests that the nature of blue-green fluorophores changed and this could reveal that 365SF420–630 emanated also from the mesophyll where carotenoids and chlorophyll reabsorbed it. On the abaxial side, a background of wide-band fluorescence or stray light in this spectral domain increased with DOY and might be due to cuticle thickening, or enlargement of intercellular spaces in spongy parenchyma. UV-induced red (365SF680) and far-red (365SF740) ChlF were low on the adaxial side and decreased with DOY on the abaxial side (Fig. 3A, B), suggesting the accumulation of UV-absorbing compounds, as shown in Figs 1B and 2A. Under blue excitation light, the fluorescence emitted between 500 and 630 nm (470SF530–630) was low, but increased with DOY, especially at DOY 265, during chlorophyll breakdown (Fig. 3C, D). Hence, the corresponding fluorophores could be in the mesophyll. The ChlF emission peaks, 460SF680, 460SF740, 628SF680 and 628SF740, were very close between DOY 123 and 184, suggesting a similar chlorophyll distribution within the mesophyll (Fig. 3C–F). At DOY 265, the 460SF740 and 628SF740 decreased, especially on the adaxial side, since the chlorophyll breakdown decreased the ChlF reabsorption. The difference in the shape of the spectra between the adaxial (Fig. 3A, C, E) and the abaxial (Fig. 3B, D, F) surface is clearly related to the dorso-ventral asymmetry characteristic of dicotyledonous leaves (Lang and Lichtenthaler, 1991; Louis et al., 2006; Buschmann, 2007) and especially showed that chlorophyll and carotenoid density was higher in palisade parenchyma than in spongy parenchyma from DOY 123 onwards.

Fig. 3.
Fluorescence emission spectra acquired on adaxial (A, C, E) and abaxial (C, D, F) sides of leaves at DOY 123, 184 and 265. The excitation wavelength was set at 365 nm (A, B), 460 nm (C, D) and 628 nm (E, F). In (C) and (D), the inserts show the detail ...

Anatomical origin of UV-induced visible fluorescence throughout development

Images of leaf transverse sections show that blue-green fluorophores were apparent in cell walls, abaxial trichomes and the adaxial cuticle from DOY 123 onwards, once the leaf was fully expanded and began to thicken and stiffen (Fig. 4). The final thickness of the leaf was reached at DOY 184.

Fig. 4.
Colour fluorescence images of leaf transverse sections at DOY 109 (A), 123 (B) and 184 (C). The fluorescence was excited at 365 nm, using the first filter set (cf. Appendix II).

Images of UV-induced visible fluorescence over each leaf side show the minor leaf venation delimiting the small areas of mesophyll, termed areoles (Fig. 5). The ChlF emission was patchy in areoles at DOY 123 (Fig. 5A, E) on both leaf sides and at DOY 155 on the abaxial side (Fig. 5F), suggesting heterogeneous accumulation of UV-screening compounds at the surface of immature leaves. From DOY 184 onwards, ChlF emission became more uniformly low (Fig. 5C–H). At DOY 265, some clusters of bright blue fluorescing epidermal cells appeared on the adaxial side (Fig. 5D, insert) and may have contributed towards the increase in 365SF420–630 (Fig. 3A). On the abaxial side, blue fluorescence emanated from stomata, trichomes and veins (Fig. 5H).

Fig. 5.
Colour fluorescence images of adaxial (A–D) and abaxial (E–H) sides of leaves at DOY 123 (A, E), 155 (B, F), 184 (C, G) and 265 (D, H). The fluorescence was excited at 365 nm, using the first filter set (cf. Appendix II). Images were taken ...

Map of epidermal UV-absorbance in immature and mature leaves

Figures 6 and and77 show images of 628MFfar-red, 365MFfar-red, the calculated image of MA365 and the corresponding frequency distribution for both sides of immature and mature leaves.

Fig. 6.
Fluorescence images (628MFfar-red and 365MFfar-red), UV absorbance images (MA365) and frequency distributions of MA365 of a leaf at DOY 109. Adaxial (A–D) and abaxial (E–H) sides of the leaf were imaged with a ×5 objective. 365 ...
Fig. 7.
Fluorescence images (628MFfar-red and 365MFfar-red), UV absorbance images (MA365) and frequency distributions of MA365 of a leaf at DOY 184. Adaxial (A–D) and abaxial (E–H) sides of the same leaf were imaged with a ×5 objective. ...

In immature leaves, at DOY 109, MA365 of the adaxial side was heterogeneous, with patchy areoles at a distance from the main vein (Fig. 6). Areoles covered 58 % of the imaged leaf area with a mean MA365 of 0·93 ± 0·08 (Fig. 6C). MA365 was approx. 0·8 in veins. The patchy pattern of MA365 images was related to that of 365MFfar-red (Fig. 6B). In contrast, the 628MFfar-red of areoles was uniform and was the highest along the main vein, where the mesophyll was thickest. The frequency distribution of MA365 was uni-modal, with a shoulder for low values of MA365 that corresponded to veins and patches in areoles (Fig. 6D). On the abaxial side, the heterogeneous distribution of MA365 was related to the leaf venation (Fig. 6G). MA365 was lower in areoles than in minor veins and in the perivascular area of the main veins. This pattern was related to that of 365MFfar-red (Fig. 6F), whereas 628MFfar-red was more uniform (Fig. 6E). Mean MA365 in areoles was 0·73 ± 0·03, and areoles covered 52 % of the area imaged. MA365 was approx. 0·8 in the veins. The frequency distribution of MA365 was large but uni-modal, therefore the difference in MA365 between veins and areoles was slight (Fig. 6H).

In mature leaves, at DOY 184, on the adaxial side, images of 628MFfar-red and 365MFfar-red were almost uniform in areoles (Fig. 7A, B). MA365 was uniformly high in areoles and low in veins (Fig. 7C). Mean MA365 in areoles was 1·1 ± 0·04. Areoles covered 71 % of the leaf area considered. The frequency distribution of MA365 was uni-modal, with a large skew to the left that corresponded to veins (approx. 0·9; Fig. 7D). On the abaxial side, images of 628MFfar-red, 365MFfar-red and MA365 showed patches in areoles that corresponded to stomata (Fig. 7E–G). Mean MA365 in areoles was 0·93 ± 0·05. Areoles covered 59 % of the surface imaged (Fig. 7G) and corresponded to the left shoulder of the frequency distribution, the main peak of which was due to veins (approx. 1·04; Fig. 7H). Note that, in Fig. 7, the MA365 was not the true one for epidermal cells situated above the veins because, in these areas, the cells below the epidermis did not contain chlorophyll (see Fig. 4C). The slight ChlF detected in vein areas (Fig. 7A, B, E and F) could be a diffuse one emanating from the surrounding mesophyll. Hence, the relatively high MA365 in veins compared with areoles (Fig. 7G) that contrasted with the pattern observed on the adaxial side (Fig. 7C) suggests that the adaxial 365MFfar-red in vein areas was enhanced by diffuse ChlF emanating from the palisade parenchyma.

The heterogeneous spatial pattern of MA365 at DOY 109 (Fig. 6) was related to the UV-absorbance of individual epidermal cells, for both leaf sides (Figs 8 and and9).9). On the adaxial side, at DOY 109, the value of MA365 was around 1·2 and 1·3 in low and high UV-screening cells, respectively (Fig. 8A, insert). At DOY 123, the patchiness continued to characterize epidermal cells, enlarged by 2-fold as the leaf grew (Fig. 8B). At DOY 184, MA365 was uniformly distributed over all epidermal cells, with a high mean value of 1·4 ± 0·02 (Fig. 8C), the maximum values being in vacuoles. On the abaxial side at DOY 109 (Fig. 9A), mean MA365 was 1·1 ± 0·02 and 0·96 ± 0·02 in veins and areoles, respectively. The value of MA365 varied according to the cell vacuolation. Small and undifferentiated epidermal cells, that seemed to result from a recent mitosis, contained several small vacuoles exhibiting a relatively low value of MA365 (Fig. 9D, upper arrow). They juxtaposed large epidermal cells containing a large vacuole exhibiting a relatively high MA365 value (Fig. 9D, lower arrow). A high value of MA365 was also observed in the vacuole of differentiating stomata (Fig. 9E, upper arrow). At DOY 123 and 184 (Fig. 9B, C and F), areoles were enlarged but the corresponding MA365 remained lower than in veins. In differentiated stomata, chloroplasts appeared as dark spots showing no UV-screening of the excitation light responsible for stomatal ChlF emission (Fig. 9F).

Fig. 8.
Images of MA365 of the leaf adaxial side at DOY 109 (A), 123 (B) and 184 (C). Black and white pixels correspond to 0·6 and 1·1 (A) or 1·1 and 1·5 (insert of A, and B, C), respectively. Images were taken with ×5 ...
Fig. 9.
Images of MA365 of the leaf abaxial side at DOY 109 (A, D, E), 123 (B) and 184 (C, F). Black and white pixels correspond to 0·8 and 1·2 (A), 0·5 and 1·0 (B, C, F) or 0·9 and 1·1 (D, E), respectively. Images ...

DISCUSSION

The results show heterogeneous epidermal UV-absorbance in immature leaves (DOY 109 and 123). This was not due to a heterogeneous distribution of chlorophyll since images of 628MFfar-red and images of reflectance (not shown) were uniform in areoles. Therefore lower 365MFfar-red in areoles was associated with higher epidermal UV-absorbance. Furthermore, the MA365 heterogeneity was not induced by Tween 20, which is non-ionic and non-toxic. Moreover, the refractive index of Tween 20 is 1·47, which is close to that of cell walls (1·42; Vogelmann et al., 1996).

MA365 values were not strictly similar to DA375 values, despite corrections by S, inter-calibration and the measurement of far-red ChlF that was not reabsorbed (Buschmann, 2007). However, DA375 and MA365 were not obtained by using exactly the same excitation wavelengths and the DA375 value was a mean over a larger area than the mean MA365 (see Appendix I); therefore, depending on the pattern of heterogeneity, DA375 and mean MA365 values could be different. Actually, the complexity of the leaf optical properties (cf. Kim et al., 2001) and the inherent limits of the logFER method (Agati et al., 2005; Kolb and Pfündel, 2005; Lenk et al., 2007) still restricted the fully quantitative correspondence between macroscopic and microscopic measurements of UV-absorbance over the leaf area. Nevertheless, there was a general accordance between the developmental pattern of DA375 (Fig. 1B), spectroscopy (Figs 2 and and3A3A and B), fluorescence imaging (Fig. 5) and UV-absorbance mapping (Figs 669). The spatial pattern of UV-induced fluorescence images (Fig. 5) was in accordance with the MA365 pattern acquired at ×5 (with S correction, Figs 6 and and7)7) and ×40 (no S correction, Figs 8 and and9)9) magnifications. Therefore, images of MA365 accurately depicted the spatial pattern of epidermal absorbance at 365 nm.

In immature leaves (until DOY 155), anthocyanins decreased as leaves expanded, as previously reported (Coley and Barone, 1996; Hughes et al., 2007); the abaxial flavonoid content per unit area increased, indicating a gradual acclimation of leaves to natural UV light. Adaxial and abaxial epidermises were not uniform UV-screens for the developing mesophyll. The accumulation of flavonoids in the vacuole varied from cell to cell and reflected the asynchronous development of epidermal cells (Avery, 1932). The spatial pattern of MA365 on the abaxial side suggested that epidermal cells situated above veins accumulated the first flavonoids. Immature leaves are sinks, importing sugar through phloem from tree storage to synthesize soluble phenolic compounds (Kleiner et al., 1999). Therefore, the spatial distribution of MA365 might reflect micro sink–source relationships. The epidermal cells close to the importing area might accumulate flavonoids in vacuoles earlier than distant epidermal cells. A strong dorso-ventral asymmetry in pigment distribution appeared early within developing leaves (DOY 109) since photoprotective pigments (epidermal flavonoids, anthocyanins and carotenoids) were mainly accumulated on the adaxial side, i.e. the one mostly exposed to visible and UV light. Blue-green fluorophores were not apparent at DOY 109 (see Fig. 6) and were accumulated from DOY 123 onwards, mainly on abaxial sides, which bore photoprotective trichomes (Karabourniotis et al., 1995).

In mature leaves (from DOY 155 to DOY 184), adaxial and abaxial epidermal UV-absorbance were similar, high and uniform among areoles (except for stomata). Therefore, both epidermises constituted a uniform UV-screen for the mesophyll, and leaves were uniformly acclimated to natural UV. The increase of 365SF420–630 from DOY 123 onwards, suggested an accumulation of hydroxycinnamic acids bound to cuticle and cell walls and the lignification of the xylem in veins and bundle sheath extensions (Wiermann, 1981). This could provide not only an additional UV-screen at the leaf surface (Day et al., 1993), but also structural support to the leaf lamina and protection against biotic attacks (Coley and Barone, 1996). Therefore, the 365SF420–630 could reflect the toughing of leaves. The dorso-ventral asymmetry of 365SF420–630 was also shown in beech leaves (Lang and Lichtenthaler, 1991) and could result from higher amount of hydroxycinnamic acids on the abaxial side (Bidel et al., 2007) as well as a lower reabsorption of blue fluorescence by abaxial mesophyll (Lang and Lichtenthaler, 1991).

In ageing and subsequent senescing leaves (from DOY 184 to DOY 265), adaxial and abaxial epidermal UV-absorbance remained high and uniform (see Fig. 5). Therefore the mesophyll was protected against UV light during chlorophyll breakdown. The increase in 365SF420–630 at DOY 265 might result from the decrease in reabsorption by carotenoids and chlorophylls (Lichtenthaler, 1987; Lang and Lichtenthaler, 1991) and the synthesis of blue-green fluorescent phytoalexins (Cerovic et al., 1999) in response to the colonization of the leaf surface by microorganisms, which might be facilitated by a loss of cuticle hydrophobicity (Bringe et al., 2006). The increase of 470SF530–630 at DOY 265 might be related to local cell necrosis (Bussotti et al., 2005).

In conclusion, spectroscopy and fluorescence imaging allowed an in vivo assessment of changes in oak leaf structure and pigments throughout development. 365SF450 and 365SFfar-red excitation increased and decreased with leaf age, respectively, in sessile oak, in beech (Lang and Lichtenthaler, 1991), tobacco (Subhash et al., 1999) and wheat (Meyer et al., 2003), species that exhibit different phytochemistry, anatomy and growth patterns. Therefore, these parameters can provide a signature for in vivo phenology monitoring. Microscopic imaging of epidermal UV-absorbance complements the macroscopic imaging (Lenk and Buschmann, 2006) and is a promising tool for investigating further the genetic and environmental controls of flavonoids accumulation and compartmentation in epidermal cells. This is a prerequisite for an effective use of epidermal UV-absorbance and fluorescence parameters in the field to monitor non-invasively the physiological stage of the plant using portable multispectral fluorimeters (Cerovic et al., 2008).

ACKNOWLEDGEMENTS

This work was supported by the French National Centre for Scientific Research (CNRS). We thank Gwendal Latouche (ESE, UMR 8079, CNRS, France) for assistance in choosing the filter set and for helpful discussion, Christophe Chamot (Plate-forme de Recherche ‘Imagerie des processus dynamiques en biologie cellulaire et biologie du développement’, Institut Jacques Monod, INSERM, CNRS, France) for technical assistance. We are grateful to Jean-Marc Ducruet (ESE, UMR 8079, CNRS, France) and Marine Le Moigne (FORCE-A, France) for helpful comments on the manuscript. American Journal Experts (Durham, USA) reviewed the paper for English usage.

We dedicate this study to the memory of the late Nicolae Moise, an exceptional and friendly researcher without whom this study, like many others, would not have been possible.

APPENDIX I

Abbreviations and usefulness of the parameters acquired using the different optical methods

AbbreviationDefinitionUsefulness for the experiment
ChlFChlorophyll fluorescenceChlF excitation is used to compute the logFER
logFERLogarithm of the fluorescence excitation ratio (Cerovic et al., 2002)Parameter providing the absorbance difference spectra between leaf ages or leaf sides
Basic parameter used to measure the epidermal UV-absorbance from double excitation of ChlF by UV and red light using the Dualex fluorimeter or microscopic imaging device
DA375Epidermal UV-absorbance at 375 nm measured with Dualex fluorimeter on intact leaves (Goulas et al., 2004)In vivo macroscopic estimation of the mean epidermal UV-absorbance over a 5-mm-diameter leaf surface (20 mm2)
MA365Microscopic image of epidermal UV-absorbance at 365 nmIn vivo visualization of the spatial pattern of epidermal UV-absorbance over a 2·2 mm2 leaf area (×5 magnification)
λexcMFλemMicroscopic image of fluorescence intensity, excited at wavelength λexc and emitted at wavelength λemBasic parameters used to compute MA365 using the logFER method
λexcSFλemFluorescence intensity, excited at wavelength λexc and emitted at wavelength λem, determined from fluorescence emission spectraSpectroscopic parameters acquired in vivo on a 6-mm-diameter leaf surface and used to assess the leaf fluorescence properties during development and to interpret fluorescence images

APPENDIX II

Description of the four filter sets used for microscopic fluorescence imaging

No. of the filters setExcitation filtersDichroic beamsplittersEmission filters
Set 1628/40 (Semrock, Rochester, USA)650DRLP02 (Semrock)Long pass RG695 (2·5 mm thick, Schott, Vanves, France)
Set 2365HT25 (Semrock)390DRLP02 (Omega, Brattleboro, USA)Long pass RG695 (2·5 mm thick, Schott)
Set 3330WB80 (Semrock)DCLP400 (Omega)Long pass LP400 (Omega)
Set 4330WB80 (Semrock)390DRLP02 (Omega)450WB80 (Omega)

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