CSIRO PUBLISHING
Functional Plant Biology, 2003, 30, 265–270
www.publish.csiro.au/journals/fpb
Exposed red (anthocyanic) leaves of Quercus coccifera display shade characteristics Yiannis ManetasA,B, Yiola PetropoulouA, George K. PsarasA and Antonia DriniaA ADepartment
of Biology, Laboratory of Plant Physiology, Section of Plant Biology, University of Patras, Patras 265 00, Greece. BCorresponding author; e-mail:
[email protected]
Abstract. Young leaves in some plants are transiently red due to the presence of anthocyanins, which disappear upon maturation. We investigated the hypothesis that light attenuation by anthocyanins may lead to a shade acclimation of the photosynthetic machinery in red leaves. We took advantage of the intra-species variation in anthocyanin levels of young, exposed leaves of Quercus coccifera. Thus, photosynthetic and photoprotective characteristics were compared in young green and red leaves of the same age, sampled from the corresponding phenotypes occupying the same habitat. Red leaves displayed several shade attributes like thinner laminae, lower Chl a/b ratios and lower levels of the xanthophyll cycle components and β-carotene. In addition, although both leaf kinds had the same area based levels of chlorophylls, these pigments were excluded from the sub-epidermic anthocyanic cell layers, leading to a further reduction of effective mesophyll thickness and an increase in chlorophyll density. Accordingly, red leaves had higher absolute chlorophyll fluorescence signals. In spite of these apparent shade characters, red leaves were less prone to photoinhibition under mild laboratory conditions and displayed slightly but significantly higher PS II photochemical efficiencies at pre-dawn in the field. No differences in all the above measured parameters were found in mature green leaves of the two phenotypes. The results confirm the light acclimation hypothesis and are also compatible with a photoprotective function of anthocyanins. Keywords: chlorophyll fluorescence, photoinhibition, Quercus coccifera, shade character, xanthophylls.
Introduction Anthocyanins are flavonoids with modifications in the number and orientation of hydroxyl groups which shift their absorbance maxima within the visible part of the spectrum. As such, they are responsible (together with other pigments) for the blue, yellow and red colours and tints of flowers and fruits, providing optical guides facilitating pollination and seed dispersal. Yet, low levels of anthocyanins are present in leaves as well, and in some rare cases their concentrations are sufficient to mask the green chlorophyll colour. More common is the transient appearance of redness during the early phases of leaf development and during senescence, while the mature leaf is green. Although the functional significance of flower and fruit anthocyanins is clear, their possible role(s) in leaves is rather obscure. In the past, leaf anthocyanins have been correlated with resistance against a variety of biotic or abiotic agents like fungi, herbivores, drought, cold and excess radiation, both UV-B and visible (see recent reviews by Chalker-Scott 1999; Hoch et al. 2001; Steyn et al. 2002). Concerning photoprotection, one may argue that a compound preventing photosynthetically active radiation (PAR) from reaching the chloroplasts would have not been selected, unless visible © CSIRO 2003
radiation occasionally becomes inhibitory. There are various circumstances for high light to be inhibitory. Whenever the available light energy exceeds that which can be used for CO2 assimilation (i.e. under cold, drought or mineral deficiencies), photo-oxidative conditions may be developed, necessitating morphological and/or biochemical adjustments for the avoidance or harmless dissipation of excess excitation energy (Smirnoff 1993; Long et al. 1994). Dissipation of this energy as heat engages the interconversions of the xanthophyll cycle components (DemmigAdams et al. 1996). It happens that the above environmental parameters (cold, drought, mineral deficiency) are also inductive for anthocyanin accumulation (Chalker-Scott 1999; Steyn et al. 2002), leading to the assumption that anthocyanins may protect from photoinhibition. Apart from environmental stress, the unbalance principle between light absorption and utilisation can be applied in young leaves as well, since photosynthetic rates in these leaves are low (see Sestak 1985 inter alia) and CO2 assimilation is limited compared with photosynthetic O2 evolution (Miranda et al. 1981; Sestak 1985). The hypothesis for a photoprotective role of anthocyanins in leaves has been addressed several times, with
10.1071/FP02226
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conflicting results. A correlation between tolerance to photoinhibition and anthocyanin accumulation was suggested in some studies (Gould et al. 1995; Krol et al. 1995; Mendez et al. 1999; Smillie and Hetherington 1999; Field et al. 2001; Manetas et al. 2002) but not in others (Burger and Edwards 1996; Dodd et al. 1998; Gould et al. 2000). In red leaves, one may additionally postulate that the attenuation of visible light by anthocyanins could compensate for the need of a strong photodissipative capacity and lead to carotenoid compositions characterising a shade leaf or inducing other shade characters (Anderson 1986; Thayer and Björkman 1990). The shade acclimation hypothesis for anthocyanic leaves was experimentally approached only recently with mature leaves of the canopy tree Quintinia serrata (Gould et al. 2002b). In that study, a comparison of the cardinal points of light saturation curves of photosynthesis between red and green phenotypes indicated shade characters in the former. However, shade acclimation of a leaf is characterised by a whole syndrome of attributes (Anderson 1986; Larcher 1995), which should be compared in order to strengthen the above hypothesis. This was the aim of our study. Preliminary field observations revealed that individuals of the Mediterranean evergreen sclerophyll Quercus coccifera, growing under the same environmental conditions, can be distinguished on the basis of the colour of young, exposed leaves, which become invariably green upon maturation. Therefore, we proceeded in a comparative study of some photosynthetic characteristics in young green and red leaves.
Sampled leaves from each individual were extracted separately. To facilitate extraction, the leaves were frozen in the mortar by adding a small volume of liquid nitrogen. Pigment extraction was performed in dim light by grinding the frozen samples in the mortar with 100% purified acetone in the presence of a small amount of CaCO 3. The extract was centrifuged at 5000 g for 10 min at 2°C and the supernatant was further cleared by passing through a 0.45 µm filter. Chlorophylls were measured spectrophotometrically, using a Shimadzu UV-160A double beam spectrophotometer (Shimadzu Deutschland GmbH, Duisburg, Germany), and concentration was estimated according to the equations of Lichtenthaler and Wellburn (1983). Carotenoid separation was performed with a Shimadzu LC-10 AD HPL chromatograph, equipped with a non-endcapped Zorbax ODS (4.6 × 250 mm) column (Rockland Technologies, Inc., Chadds Ford, PA) and calibrated against purified β-carotene (Sigma Chemical, St. Louis, MO) and freshly prepared carotenoids by TLC, as previously described (Kyparissis et al. 1995). Development was performed isocratically at 1 mL min–1 (20 min with acetonitrile:methanol, 85:15 v/v, and 20 min methanol:ethyl acetate, 68:32 v/v), according to Thayer and Björkman (1990). Pigments were detected by measuring absorbance at 445 nm, using a Shimadzu SPD-10A UV-VIS detector and peak areas were integrated by a Shimadzu C-R6A Chromatopac. For anthocyanin determination, an aliquot of the acetone extract was acidified to 1% v/v HCl and absorbance was scanned at 400–700 nm. The peak anthocyanin absorbance (530 nm) was corrected for the contribution of chlorophyll pigments at this wavelength (Lindoo and Caldwell 1978) and normalised to 1 mL of extract and 1 cm2 of leaf surface.
Materials and methods
In vivo chlorophyll fluorescence
Plant material and sampling
For fluorescence induction curves (Kautsky curves) in fully darkadapted (whole night) leaves, a time resolving system (Plant Efficiency Analyser (PEA), Hansatech, King’s Lynn, UK) was used, with exciting red light set at 1500 µmol m–2 s–1. Thus F0, Fm and Fv (as Fm – F0) were measured. F0 (initial fluorescence) is due to excited chlorophyll molecules in the antennae of PS II before the excitation energy reaches the reaction centres and Fm is the maximum fluorescence when all PS II reaction centres are closed after a saturating light pulse. Fv /Fm indicates the maximum PS II photochemical efficiency and its value for a healthy leaf is about 0.8 (Schreiber et al. 1995).
The evergreen sclerophyll Quercus coccifera L. is a characteristic member of the Mediterranean macchia vegetation. Individuals within this species can be distinguished by the intensity of redness in their young, developing leaves, while mature leaves are invariably green. New leaves burst during the spring (early April to mid-May) at the top of older branches, i.e. they are fully exposed to direct solar radiation. In the present investigation, plants whose young leaves are at the two ends of the colour gradient (either dark red or green) were used. Sixteen individuals (eight red and eight green) growing wild side by side in a typically Mediterranean, small sunlit site (200 × 200 m) in the neighbourhood of the Patras University Campus were tagged and used throughout this study. For all measurements and experiments, as indicated in the legends of figures and tables, a number of exposed, south-facing leaves were selected from each individual late in the afternoon, put in air-tight plastic bags containing a moist filter paper, left in the dark at room temperature all night and analysed next morning. Care was taken to use leaves of comparable physiological age. In preliminary trials it was found that in both red and green phenotypes the final attained size of mature leaves was the same and that same-sized young leaves had the same chlorophyll levels. Thus, criteria for leaf selection were the similarity of their dimensions and chlorophyll contents. Chlorophyll levels were assessed in the field nondestructively before harvest from the readings of a Minolta SPAD–502 portable Chl meter (Spectrum Technologies Inc., Plainfield, IL, USA). The credibility of this instrument in the measurement of chlorophyll content of red leaves has been previously confirmed (Manetas et al.
1998). In general we used young leaves having attained approximately 10% of their final size and 25% of their mature chlorophyll content. All measurements on young leaves were performed during spring of 2001 (April–May), while mature green leaves from the same individuals were analysed during fall of 2001 (October). Photosynthetic pigments
Photoinhibitory treatment Leaf discs were cut from pre-darkened (whole night) leaves and their Fv /Fm determined. They were subsequently put into covered petri dishes on the top of moistened filter paper and illuminated with 1600 µmol m–2 s–1 PAR measured with a LI-COR Li-190 quantum sensor (LI-Cor, Lincoln, NE, USA) through a 400W Osram quartzhalogen bulb in a thermostated (20 ± 1°C), double-walled glass chamber. Since the illumination field showed a ±5% variation in PAR, the petri dishes were rotated every 15 min to avoid site effects. After 1 h under these conditions the discs were transferred to the dark at room temperature. Fv /Fm was measured after 0.5, 1 and 19 h relaxation time. Microscopy Fresh leaves were freehand cross-sectioned and examined under a Zeiss Axioplan microscope with epifluorescence optics (HBO-50W mercury lamp), equipped with a UV-G365 excitation filter set (G365
Are red leaves shade leaves?
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Table 1.
Leaf and photosynthetically effective mesophyll (i.e. chlorophyll containing) thickness of red and green young leaves of of Q. coccifera Data are means ± s.d. from the number of individuals indicated in parentheses, with three leaves measured per individual. In the case of photosynthetically-effective mesophyll thickness, three sections were measured per leaf Parameter measured Leaf thickness (µm) Photosynthetically-effective mesophyll (µm)
Red
Green
F
P
183 ± 12 (20) 131 ± 13 (8)
197 ± 16 (20) 173 ± 12 (8)
10.263 44.37
0.003
