ISSN 10214437, Russian Journal of Plant Physiology, 2011, Vol. 58, No. 4, pp. 674–680. © Pleiades Publishing, Ltd., 2011. Original Russian Text © A.E. Solovchenko, O.B. Chivkunova, 2011, published in Fiziologiya Rastenii, 2011, Vol. 58, No. 4, pp. 582–589.
RESEARCH PAPERS
Physiological Role of Anthocyanin Accumulation in Common Hazel Juvenile Leaves A. E. Solovchenko and O. B. Chivkunova Faculty of Biology, Moscow State University, Moscow, 119951 Russia; email:
[email protected] Received December 24, 2010
Abstract—Common hazel (Corylus avellana L., Fusca rubra Dipp.) juvenile leaves from the periphery of the canopy and thus subjected to high fluxes of solar radiation are characterized by red coloration due to antho cyanin accumulation disappearing in mature leaves. To elucidate the physiological role of anthocyanin accu mulation, the interrelations between anthocyanin content, a degree of attenuation by the pigments of the light reaching the photosynthetic apparatus (PSA), and PSA tolerance to photoinhibition in C. avellana juve nile leaves were studied. Absorption spectra were calculated taking into account the light losses due to reflec tion by the leaf. The analysis of the spectra showed that, in red common hazel leaves accumulating high amounts of anthocyanins in the vacuoles of the upper and lower epidermal cells, up to 95% of visible radiation entering the leaf blade was absorbed by these pigments. The rate of the linear electron transport (ETR) in the chloroplast electron transport chain (ETC) was closely correlated with the anthocyanin content (r2 = 0.87). In red leaves, the saturation of ETR dependence on irradiance was observed at the higher values of PAR than in green leaves. In red juvenile leaves, this value was close to that in mature green leaves tolerant to high light. There were no differences between red and green leaves in the level of nonphotochemical quenching, the content of violaxanthin cycle pigments, a degree of their deepoxidation under natural illumination and at irradiation with high PAR fluxes. Basing on the data obtained, one may conclude that anthocyanins in C. avellana juvenile leaves serve PSA photoprotection, preventing injury of immature PSA with excessive fluxes of PAR. Keywords: Corylus avellana, juvenile leaves, anthocyanins, violaxanthin cycle, reflection spectra, photoadap tation, photoinhibition. DOI: 10.1134/S1021443711040157
INTRODUCTION Anthocyanins (Anc) are a specific group of water soluble pigments of phenolic nature found in higher plants [1]; they absorb far UV (UVC) and green visi ble light (520–530 nm) [2]. Since UVC is almost absent from solar radiation near the Earth surface, only the long wave maximum of anthocyanin absorp tion is of substantial importance for green plant phys iology. Anthocyanins fulfill many functions in plants, including pollinator attraction, protection against low abovezero temperatures, drought, and excessive inso lation [1, 3, 4]. Anc accumulation is often developed as a result of high insolation on the background of low abovezero temperatures [5], drought, soil salinity, and other unfavorable factor action [1, 3]. Therefore Abbreviations: Anc—anthocyanins; Ant—antheraxanthin; Car— carotenoids; Chl—chlorophyll(s); DE—degree of violaxanthin deepoxidation; ETR—the rate of electron linear transport in chloroplast electron transport chain; ETRmax—the maximum value of ETR; PAR—photosynthetically active radiation; PS— photosystem; PSA—photosynthetic apparatus; Vio—violaxan thin; VXC—violaxanthin cycle; Zea—zeaxanthin.
Anc are often called “stress pigments” [3]. Recently, the importance of Anc for protection of ageing leaves during retranslocation of photoassimilates and valu able compounds from the leaf blade to wintering organs during preparation of deciduous plants for the period of dormancy was established [6]. However, a comprehensive hypothesis about the mechanism of Anc protective action is lacking so far. Anc were shown to function in vivo as antioxidants [7, 8] and osmopro tectants [3]. Actually, Anc seem to be polyfunctional compounds. Available experimental data indicate that these pigments function predominantly as optical bar riers, i.e., as internal filters attenuating excessive PAR [9, 10]. Transient anthocyanin pigmentation appearing in leaves and stems of many plant species under unfavor able conditions [11], especially in juvenile period [12, 13], attracts a great attention. Such pigmentation dis appears after juvenile period or the cessation of the action of the stressor. Various explanations of physiological role of tran sient anthocyanin pigmentation in juvenile leaves were suggested. One of them is a protection of immature
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MATERIALS AND METHODS Plant material. Juvenile leaves of common hazel (Corylus avellana L., Fusca rubra Dipp.) grown in the open near the Faculty of Biology (Moscow State Uni versity) were used. Sampling was performed in spring (from April 15 to May 15) of 2005–2010 in 10:00 a.m., i.e., when direct sunlight does not fall on the leaves. For experiments, 20–25 undamaged juvenile leaves (from the 3rd to 5th leaves on the shoot) no less than 5 cm in width were taken (80 tested samples in total). On the basis of reflectance in the green region of the spectrum, the leaves with high (> 40 nmol/cm2, red leaves) and low (< 15 nmol/cm2, green leaves) content of Ant, close content of chlorophyll (Chl), an absorp tion and reflection in the red region spectra similar in shape and amplitude were selected (Fig. 1). In some experiments, mature leaves were also analyzed; they were collected in the last decade of July. All samples were analyzed within 1 h after harvesting. Pigment extraction and analysis. Total contents of Chl, carotenoids (Car), and Anc in leaf extracts pre pared by the Folch [14] method with our modification were determined spectrophotometrically [14, 15] using a Hitachi 15020 spectrophotometer (Japan). The content of individual carotenoids was analyzed by HPLC using the Lichrospher RP C18 column and elution with a solvent gradient as described in [16]. The degree of deepoxidation (DE) of violaxanthin (Vio) was calculated taking into account the content of antheraxanthin (Ant) and zeaxanthin (Zea): DE = (0.5 Ant + Zea)/(Vio + Ant + Zea) [17]. Condition of irradiation. To assess induction of the violaxanthin cycle (VXC), leaves were irradiated for 1 min through 5cm water filter. Dia projector with a KGM 150/24 halogen lamp (150 W, Svetotekhnika, Saransk, Russia) served a source of visible light. PAR flux density on the leaf surface, as measured with a LI 250A light meter (LICOR Biosciences, United States), equaled 2500 W/m2 PAR. Spectral measurements. The absorption spectra of pigments and also the spectra of transmission and reflection of intact leaves were recorded with a Hitachi RUSSIAN JOURNAL OF PLANT PHYSIOLOGY
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photosynthetic apparatus (PSA) against damage by solar radiation, especially in spring when sharp changes of temperature on the background of strong sunlight occur [1]. However, the current amount of data about Anc is insufficient to establish a single hypothesis about the mechanism of their photoprotec tive action. In this connection and to elucidate physi ological role of Anc in juvenile leaves, we studied dependences between Anc accumulation, changes in their optical properties, and PSA tolerance to photo inhibition. Common hazel was chosen as a model spe cies for this study because juvenile (but not mature) leaves of this plant have a pronounced anthocyanin pigmentation.
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Fig. 1. Typical spectra of optical density for green (1) and red (2) common hazel juvenile leaves calculated taking into account reflected light, their difference spectrum (3), and the efficiency of attenuation by anthocyanins of the light entering the leaf blade (4, right scale).
15020 spectrophotometer supplied with 150mm integrating sphere. The spectra of leaf optical density were calculated taking into account light losses because of its reflection by the leaf blade according to the following equation: I ( λ ) ⎞ = log ⎛ 100 – R ( λ)⎞ A ( λ ) = – log ⎛ , ⎝ T(λ) ⎠ ⎝ I 0 ( λ )⎠
(1)
where I0(λ) is the spectrum of radiation entering the leaf blade; I(λ) is the spectrum of radiation attenuated by the leaf blade; R(λ) is the reflection spectrum; and T(λ) is the transmission spectrum. The relative proportion of PAR absorbed by Anc in red leaves as compared to green leaves was calculated as follows: ΔA R – G ( λ ) = A R ( λ ) – A G ( λ ),
(2)
or in percent: ΔA [ R – G ]% ( λ ) = 10
{ 2 – [ AR ( λ ) – AG ( λ ) ] }
,
(3)
where A R ( λ ) and A G ( λ ) are A ( λ ) spectra for red and green leaf, respectively. Light microscopy. Wet mount preparations of C. avellana leaf transverse sections were analyzed with a Zeiss Axioscope photomicroscope equipped with an MRc digital camera (Carl Zeiss, Germany). Measurements of Chl fluorescence parameters. The ground and the maximum levels of Chl fluorescence in dark (F0, Fm) and lightadapted ( F 0' , F m' ) leaves as well as the curves of Chl fluorescence induction were No. 4
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6 8 Fig. 2. Scheme of transverse section of the red juvenile leaf blade. (1) Cuticle; (2) upper epidermis; (3) lower epidermis; (4) palisade mesophyll; (5) spongy mesophyll; (6) vacuoles (Anccontaining vacuoles are marked in black); (7) inter cellular space; (8) stoma; (9) chloroplasts.
measured using an FP100 pulse fluorimeter (Photon Systems Instruments, Czech Republic). The levels of F0 and Fm were recorded in the leaves darkadapted for 30 min. Maximum quantum yield of PSII, Qy was cal culated as follows: Qy = (Fm – F0)/Fm = Fv/Fm; the level of nonphotochemical quenching (NPQ), as Fm/( F m' – 1) [18]. The rate of linear electron transport (ETR) at a given irradiation was calculated as Qy × PPFD × 0.5 ×Abs, where 0.5 is the factor taking into account a requirement of two light quanta for trans port of a single electron, PPFD is a photosynthetic photon flux density, and Abs is a coefficient of the inci dent light absorption by the leaf [19]. Figures present means (n = 80) and their standard errors, unless otherwise indicated. RESULTS Juvenile leaves with a high Anc content (>40 nmol/cm2) (such leaves will be further referred to as ‘red’ leaves) were mainly positioned at the periphery of the canopy subjected to the highest flux of direct solar radiation (1500 μE/(m2 s) PAR on the leaf blade surface at solar zenith). In the inner part of the canopy the leaves with low Anc content (< 15 nmol/cm2) pre vailed (such leaves will be further referred to as ‘green’) and the intensity of solar radiation, largely diffuse, was 45 times lower (300–400 μE/(m2 s) PAR).
According to light microscopy (Fig. 2), Anc in C. avellana juvenile leaves were localized predomi nantly in the vacuoles of epidermal cells, whereas in palisade and spongy mesophyll cells with Antcon taining vacuoles were only sporadically noted. Radiation Attenuation by Anthocyanins in C. avellana Leaves To assess a potential photoprotective effect of Anc in vivo, optical properties of leaves differing in Anc content but close in Chl content were compared. In the red region of the green leaf optical density spectra calculated taking into account reflected light A G ( λ ) (Fig. 1, curve 1),the maximum at 678 nm was deter mined by a combined absorption by Chl a and Chl b, whereas, in the bluegreen region, the 437 nm maxi mum and a shoulder were determined by a combined absorption by Chl and Car. In the red region, spectra of red and green leaves, A R ( λ ) and A G ( λ ), were close in their shapes and amplitudes; at the shorter wavelength, red leaves absorbed much stronger than green ones (cf. curves 1 and 2 in Fig. 1). Correspondingly, difference spectra A R – G ( λ ) displayed weak maxima in the red region, whereas the maxima with a high amplitude (1.0–1.5 OD units, curve 3 in Fig. 1) were found in the green region (λmax = 550 nm). The comparison of spectra A R ( λ ) and A G ( λ ), as well as difference spectra A [ R – G ]% ( λ ) (Fig. 1, curve 4) showed that, in red common hazel leaves, Anc absorbed up to 90–95% of the light enter ing the leaf blade (in the band of Anc absorption max imum in vivo, 550 nm). Parameters of Chl Fluorescence in Leaves Differing in Anthocyanin Content The functioning of PSA in common hazel juvenile leaves differing in Anc content was characterized using Chl fluorescence measurements (Figs. 3–6). A com parison of Chl fluorescence induction in green and red leaves showed that, in red leaves, the amplitude of the curve was 2–3 times lower than in green leaves (Fig. 3, curves 1 and 2). The same curves normalized to the highest intensity values did not differ significantly (Fig. 3, curves 1' and 2'). Differences in the highest quantum efficiency of PSII (0.63 ± 0.08 and 0.65 ± 0.05 in red and green leaves, respectively) and in the level of nonphotochemical quenching of Chl fluores cence (2.37 ± 0.53 and 1.87 ± 0.58 in red and green leaves, respectively) were insignificant. The analysis of light curves within the range of 1– 1000 μE/(m2 s) PAR (Fig. 4) showed that, in green leaves, the saturation of ETR in chloroplast ETC on the level of PAR occurred at 70 μE/(m2 s), and a fur ther increase in irradiation resulted in the suppression of electron transport in chloroplast ETC (Fig. 4a,
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Fig. 3. Chlorophyll fluorescence induction in green (1) and red (2) juvenile common hazel leaves before (1 and 2, left ordinate) and after (1' and 2', right ordinate) normalizing to the maximum value of fluorescence intensity.
curve 1). In red leaves with high content of Anc (Fig. 4a, curve 2), the saturation of ETR dependence on PAR occurred at the higher PAR values (110– 130 μE/(m2 s) PAR). Light curves built for mature leaves with low Anc content (< 2 nmol/cm2) collected in the second decade of July (Fig. 4a, curve 3) were close to those for red leaves. Red and green leaves differed markedly in the maximum values of ETR (ETRmax): 22 and 12 μmol/s, respectively (Fig. 4b). The values of ETRmax in mature leaves were close to those in juvenile red leaves. One should note the tight correlation of ETRmax and Anc content in red leaves; in green leaves, such correlation was not observed (Fig. 5). Composition of Carotenoids in Red and Green Leaves The chromatographic analysis of pigments showed that βcarotene, lutein, and violaxanthin were the pre dominant Car in common hazel leaves differing in Anc content (Fig. 6). Small but significant differences in carotenetoxanthophyll ratio were observed between red and green leaves (1.66 ± 0.03 and 1.84 ± 0.02, respectively). Regardless of Anc content, juvenile leaves were characterized by a large pool of xantho phylls involved in the functioning of VXC; the size of the pool was close in both leaf groups (43.13 ± 1.17 and 44.25 ± 1.58 for green and red leaves, respectively). Under natural illumination, the analyzed leaves dif RUSSIAN JOURNAL OF PLANT PHYSIOLOGY
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fered in the degree of violaxanthin deepoxidation (DE). In red leaves with high Anc content subjected to strong direct sun light DE was ~7%, whereas in green leaves, DE was approximately twice higher (Fig. 7) in spite of their positioning within the depth of the canopy where they were subjected to much lower PAR fluxes. Irradiation with high PAR (2500 W/m2) lead to a similar increase in DE in both leaf groups (up to 50%). DISCUSSION Anc are known to function as efficient scavengers of radicals in vivo [3, 20, 21]. However, it is difficult to explain Anc photoprotective effect in vivo predomi nantly by their antioxidant action, especially in the case of C. avellama. Thus, Anc are characterized by vacuolar localization in the cell (Fig. 2, see also [1, 10]). At the same time, the sites of ROS generation, which mediate plant photooxidative damages under high light, are situated mainly in chloroplasts [22, 23]. Certainly, one cannot rule out completely a possibility of neutral ROS diffusion, such as 1O2, H2O2, or •OH, into other cell compartments including the vacuole. Nevertheless, due to their high reactivity and appro priate substrate (e.g. lipids of thylakoid membranes containing polyunsaturated fatty acids) proximity, it is far more likely for the ROS generated under irradia tion with high light to attack the components of PSA No. 4
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Fig. 5. Dependence of the highest rate of linear electron transport in chloroplast ETC on the content of Anc in green (䊐) and red (䊏) common hazel juvenile leaves.
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Fig. 4. Dependence of the rate of linear electron transport in chloroplast ETC on PAR intensity for green (1) and red (2) juvenile as well as green mature (3) common hazel leaves (a) and the maximum ETR in the chloroplast ETC in juvenile common hazel leaves differing in the Anc con tent (b).
and chloroplast membranes neighboring the sites of ROS generation [22, 23] before reaching Anc mole cules localized in vacuoles. Furthermore, in many plant species [9. 13], including C. avellana, Anc are localized in juvenile leaves in the vacuoles of epider mal cells (Fig. 2) devoid of green plastids and less vul nerable to photooxidative damage. On the other hand, the vacuoles of mesophyll cells, containing chloro plasts and more vulnerable to photooxidative damage, do not essentially contain Anc in the juvenile leaves.
Taking into account the circumstances noted above, it was suggested that, in juvenile C. avellana leaves, Anc attenuate excessive solar radiation and protect PSA against photooxidative damage. To verify this suggestion, a comparative analysis of common hazel leaf optical properties was performed. It was shown earlier that in the leaves of a number of plant species Anc and Chl absorb light independently [13] and that Lambert–Beer absorption law is obeyed in the wide range of Anc concentrations [24]. This allowed us to apply difference spectroscopy for the comparative analysis of the leaf optical properties. It should be noted that a comparison of pigment extract spectra gives only limited indication of the spectroscopy of these compounds in vivo and, thus, of potential efficiency of photoprotection they provide [10]. In this connection and according to the approach we have suggested earlier [9, 13], we compared spec tral characteristics of intact leaves differing in Anc content but close in the content of Chl, the main pho tosensitizer of ROS generation in photosynthesizing cells [23]. For the analysis, the spectra of leaf optical density were calculated taking into account the reflected radiation after the formula (1). This method allows a more precise characterization of the part of incident light that enters the leaf blade [13]. As expected, in leaves with close Chl content, light absorption in the red turned out to be close as well (Fig. 1, curves 1 and 2). Correspondingly, the ampli tude of difference spectra in this region was low (Fig. 1,
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Fig. 6, Carotenoid composition in red (a) and green (b) common hazel juvenile leaves. Neo—neoxanthin; Vio—violaxanthin; Ant–antheraxan thin; Lut—lutein; Zea—zeaxanthin; βCar—βcaro tene.
curve 3). The analysis of the spectra of leaves with high Anc (Fig. 1, curve 2) and their comparison with spec tra of leaves with low Anc content (Fig. 1, curve 1) showed that in vivo Anc actively absorb light in the 400–650 nm range. We estimate that in C. avellana Anc are capable of absorbing, on an average, 60–70% of radiation entering the leaf blade in this range and up to 90–95% of such radiation in the band of Anc absorption maximum (Fig. 1, curve 4). This is of importance for the realization of Anc photoprotective effect because, in the absence of these pigments, radi ation within this range is absorbed by the photosyn thetic pigments (Chl and Car). An additional argu ment for efficient Anc functioning as “internal filters” attenuation light attaining chloroplasts is a decrease in the intensity of Chl fluorescence excited by bluegreen light observed in the presence of Anc and proportional to the content of these pigments [9]. It should be noted that the complex system of defense mechanisms against PSA injury functions in plant cells and tissues [22, 23, 25], and its state should be taken into account in the analysis of photoprotec tive mechanisms. A comparison of PSA functional parameters by the analysis of the curves of Chl fluores cence induction and calculation of parameters derived on their basis, which characterize the state of reaction centers and pools of quinone electron transporters [19] did not reveal substantial differences between green and red leaves (cf. curves 1' and 2' in Fig. 3). There was no significant difference in the maximum quantum yield of PSII and the level of nonphoto chemical quenching of Chl fluorescence as well. This agrees well with the results obtained for the pool of xanthophylls, the participants of VXC: both red and green leaves contained a sizeable pool of these pig ments; at the same time, irradiation with PAR fluxes sufficiently high for VXC induction in the presence of Anc resulted in approximately similar growth of DE in RUSSIAN JOURNAL OF PLANT PHYSIOLOGY
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Fig. 7. The degree of deepoxidation (DE) of violaxanthin in green (1, 3) and red (2, 4) common hazel juvenile leaves at the highest natural illumination (1, 2, sun at its Zenith) and after strong artificial irradiation (2500 W/m2 PAR) for 1 min (3, 4).
all leaf groups (Fig. 7). All these data allow a conclu sion that the tested C. avellana leaves are characterized by approximately similar level of development of pho toprotective mechanism based on heat dissipation of excessive light energy absorbed (VXC). At the same time, red and green leaves of common hazel differed substantially in the level of saturating irradiance for the dependence of ETR on PAR flux density as well as in ETRmax values (Figs. 4–6). The analysis of light curves showed that, at the close levels of VXC induction, in the presence of photoprotective pigments PSA of juvenile leaves was by 2–3 times more tolerant to photoinhibition by high PAR fluxes (Figs. 4, 5). It is important to note that the degree of this tolerance was proportional to the content of Anc (Fig. 6). It should be also noted that tolerance to pho toinhibition of red juvenile leaves was close to that of mature leaves devoid of Anc but featuring wellformed PSA (Fig. 4, curve 3). Collectively, the data obtained in this work suggest that the most likely role of Anc in juvenile C. avellana leaves is the attenuation of the excessive solar radiation and, as a consequence, protection of PSA against pho tooxidative damage. An additional comparative study ing of the enzymatic system of ROS elimination related to the presence of lightscreening pigments is required for the ultimate conclusion about predomi nant Anc role in photoprotection of common hazel leaf. No. 4
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ACKNOWLEDGMENTS This work was supported by the Russian Founda tion for Basic Research, project no. 090400419a.
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