Russian Journal of Plant Physiology, Vol. 50, No. 1, 2003, pp. 52–56. Translated from Fiziologiya Rastenii, Vol. 50, No. 1, 2003, pp. 59–64. Original Russian Text Copyright © 2003 by Maslova, Mamushina, Zubkova, Voitsekhovskaya.
Specific Features of Plastid Pigment Apparatus and Photosynthesis in the Leaves of Ephemeroid and Summer Plants as Related to Photoinhibition T. G. Maslova, N. S. Mamushina, E. K. Zubkova, and O. V. Voitsekhovskaya Komarov Botanical Institute, Russian Academy of Sciences, ul. Professora Popova 2, St. Petersburg, 197376 Russia; e-mail:
[email protected] Received May 31, 2001
Abstract—The state of the pigment apparatus and potential photosynthesis (PP) was compared in the leaves of plants falling into two ecological groups, ephemeroids (three species) and summer plants (two species). For the first time, the organization of the plastid pigment apparatus was investigated in ephemeroids using the data on chlorophyll and carotenoid distribution between the major photosynthetic pools. The molar ratio between xanthophylls and chlorophyll in the light-harvesting complex of plastids in the ephemeroids (0.5 to 0.6) considerably exceeded that in the summer plants (0.3–0.4). By using salicylaldoxime, an inhibitor of the reverse reaction of the violaxanthin cycle, we were able to calculate the active pool of violaxanthin on its way to zeaxanthin. This pool was shown to amount to 85% of the sum total of xanthophylls of the violaxanthin cycle in the ephemeroid leaf plastids as compared to 60% in the summer species. Thus, potentially, the photosynthetic apparatus in the ephemeroid leaves is better provided with the pigments essential for photoprotective function and for maintaining a high photosynthetic rate under early spring conditions. Under chilling temperatures of 5– 10°C and full insolation, PP in ephemeroids was as high as in the summer plants at 20°C. Key words: Ficaria verna - Corydalis solida - Anemone ranunculoides - Urtica dioica - Ranunculus acris ephemeroids - summer plants - potential photosynthesis - pigments - photoinhibition
INTRODUCTION Under natural conditions, high insolation often produces photoinhibition of photosynthesis aggravated when high solar radiation is combined with low temperature. In this case, only part of the light energy absorbed by chlorophyll is used in photosynthesis, and, as a result, there is an increasing danger of photodynamic damage to the photosynthetic apparatus and the whole cell [1]. To protect themselves against excess solar radiation, plants evolved several mechanisms, including those at the level of the plastid pigment complex, for survival within a wide range of irradiation and temperature conditions. Beginning in the 1980s, carotenoids, providing for both light harvesting and photoprotective functions, have been studied on a large and diverse scale [2, 3]. The transformations of carotenoids of the violaxanthin cycle, that is, the light-induced deepoxidation of violaxanthin into zeaxanthin, are particularly essential for protecting the photosynthetic apparatus under excessive solar radiation [4–6]. This conclusion was reached in the studies with plants grown under hothouse and field conditions and also with seven tropical species varying in their light requirements [7–9]. Our goal is to compare the peculiar characteristics of the photosynthetic apparatus, as related to photoinhiAbbreviations: LHC—light-harvesting complex; PP—potential photosynthesis; PS—photosystem.
bition, in the contrasting groups of boreal plant species. Summer plants (two species) and ephemeroids (three species) were the model plants. Ephemeroids grow and photosynthesize in early spring, when the combination of two factors, chilling temperatures and high insolation, promotes photoinhibition of photosynthesis. One would presume that the assimilating organs of the ephemeroid plants possess a specific system for protecting their photosynthetic apparatus. The objective of the present study was to assess the selected species in their natural habitats for the following indices of the leaf photosynthetic apparatus: (1) the potential photosynthesis (PP), (2) the total content of plastid pigments, (3) the ratio of chlorophyll to carotenoids in the light-harvesting complexes (LHC) and in the complexes of photosystems (PS), and (4) the quantitative evaluation of active violaxanthin pool as related to the total pool of the violaxanthin cycle components. The comparative analysis of these indices will help determine whether the mechanisms for protecting the photosynthetic apparatus against photodynamic damage differ in two contrasting plant groups and elucidate the potential role of the components of the violaxanthin cycle in such protection.
1021-4437/03/5001-0052$25.00 © 2003 MAIK “Nauka /Interperiodica”
SPECIFIC FEATURES OF PLASTID PIGMENT APPARATUS
53
Table 1. Pigment content and ratio in the leaves of ephemeroid and summer plants Chlorophyll a/b
Total carotenoids, mg/g fr wt
Chlorophyll a + b/total carotenoids
Violaxanthin + zeaxanthin, % of total carotenoids
1.0 ± 0.06 1.7 ± 0.09 1.8 ± 0.1
3.9 3.7 4.5
0.3 ± 0.01 0.4 ± 0.02 0.4 ± 0.02
4.0 4.2 5.0
22 31 21
2.4 ± 0.11 2.7 ± 0.13
3.5 3.5
0.4 ± 0.02 0.4 ± 0.02
6.7 6.2
20 23
RESULTS AND DISCUSSION The data on the contents and ratios of plastid pigments in the ephemeroid and summer plants are presented in Table 1. The total content of chlorophyll a + b in the ephemeroid leaves was 1.0–1.8 mg/g fr wt, the level characteristic of light-requiring plants. The summer species were collected in the open, yet they manifested higher chlorophyll contents. The two plant groups did not differ in their chlorophyll a/b ratios and in the total carotenoid contents. The ratio of chlorophyll RUSSIAN JOURNAL OF PLANT PHYSIOLOGY
Vol. 50
a + b to the total carotenoids was higher in the summer plants, because the leaves of the latter species were higher in chlorophyll. Table 2 presents the data describing pigment distribution between two major photosynthetic pools, LHC and the sum total of PSI and PSII. We calculated the relative chlorophyll content (as % of the total content) and the ratio between xanthophylls and chlorophyll in LHC. For PSI and PSII, we calculated the molar ratio between β-carotene and chlorophyll. The relative portions of chlorophyll in LHC of all plant species sampled in the open were quite similar (Table 2), whereas the species belonging to two different ecological groups differed in chlorophyll content (Table 1). Apparently, the relative portion of chlorophyll participating in light harvesting in the course of photosynthesis is a functiondependent index affected by the environmental conditions, particularly solar radiation, to a higher extent than the total chlorophyll content, an apparently genotype-dependent index. The molar ratios of chlorophyll Table 2. Pigment distribution between the major photosynthetic pools, LHC and PSI plus PSII, in plant species belonging to two ecological groups PSI and PSII
LHC
Plant species
Ephemeroids F. verna A. ranunculoides C. solida Summer plants U. dioica R. acris No. 1
2003
β-carotene, mol per 100 mol of chlorophyll
MATERIALS AND METHODS The study employed three species of ephemeroid plants (Ficaria verna Huds., Corydalis solida L., and Anemone ranunculoides L.) and two species of summer plants (Urtica dioica L. and Ranunculus acris L.). The latter species, as well as F. verna and A. ranunculoides, belong to the family Ranunculaceae. Plants were collected on the grounds of the Komarov Botanical Institute. The ephemeroids were studied in April and May, and the summer plants, in July. Mature leaves with the already developed photosynthetic apparatus were collected, fixed, and extracted with acetone [10]. The contents of chlorophyll a and b and total carotenoids were determined spectrophotometrically in leaf acetone extracts [11–14]. Carotenoids were separated by thin-layer chromatography [15]. The pigment distribution between two major photosynthetic pools, LHC and PSI plus PSII, was calculated [16–19]. To determine the proportion of the active violaxanthin pool in the total pool of the violaxanthin cycle carotenoids, we used a specific inhibitor salicylaldoxime at the concentration of 2.5 mM at 20°C [20, 21]. The same leaf samples were used to assess PP at 1% CO2 concentration and at the same specific radioactivity of 14CO2 + CO2 of 4.54 mCi/l [22]. The experiments were run in the open from 10 to 12 a.m. under the ambient insolation and temperature conditions. Illuminance was controlled by shading the photosynthetic chamber. Plants were exposed for 15 min. The experiments were run in triplicate through two field seasons. The tables present the mean values of biological replications.
Xanthophylls, mol per 100 mol of chlorophyll
Ephemeroids F. verna A. ranunculoides C. solida Summer plants U. dioica R. acris
Chlorophyll a + b, mg/g fr wt
Chlorophyll a + b, % of total content
Plant species
45 47 40
68 62 58
17 17 14
52 49
32 41
14 13
54
MASLOVA et al.
Plant species
Ephemeroids F. verna
A. ranunculoides C. solida Summer plants U. dioica R. acris
Illuminance, klx
Table 3. Zeaxanthin content in leaves treated with 2.5 M salicylaldoxime Zeaxanthin, % of the total content of xanthophylls of the violaxanthin cycle untreated
salicylaldoxime
0 5 10 20 70 80 100 0 100 0 100
tr. 37 32 35 44 68 69 tr. 61 n.d. 65
n.d. 58 63 67 68 68 68 n.d. 79 n.d. 85
0 100 0 100
n.d. 54 n.d. 47
n.d. 59 n.d. 57
Notes: n.d.—below measurable level, tr.—traces.
to xanthophylls in LHC of ephemeroid leaves considerably exceeded those in the summer plants: 60–70 versus 30–40 mol xanthophylls per 100 mol chlorophyll. The two groups of plant species did not notably differ as to the molar ratios of β-carotene to chlorophyll in PSI and PSII. Previously, we observed similarly high molar ratios of xanthophylls to chlorophyll (50–60 mol xanthophylls per 100 mol chlorophyll) in LHC of species growing under extremely high-mountain and tundra conditions [19]. To compare, the corresponding index in the arboreal species in the Leningrad oblast was 20–30 mol comparable to the summer plants described in this communication, and the highest value of this index, up to 90 mol, was found in some highmountain plants of the Eastern Pamirs, where plants strive under extremely hard growth conditions: high solar radiation and low temperature [19]. It follows that the ephemeroids, similar to the Arctic and high-mountain plants, are potentially better provided with the pigments necessary for protecting their photosynthetic apparatus against the environmental conditions that could probably cause photoinhibition. Having in mind the photoprotective function of LHC, with 60–90 mol xanthophylls per 100 mol chlorophyll, we will discuss in more detail two xanthophylls, violaxanthin and zeaxanthin, which are the components of
the violaxanthin cycle. Currently, zeaxanthin is seen as the major agent protecting the photosynthetic apparatus against photodynamic damage under adverse environmental conditions, e.g., excessive insolation. The protective role depends on the zeaxanthin capacity to capture from excited chlorophyll surplus energy, which is not consumed by photosynthesis. This energy is further dissipated into heat [23–25]. Special studies demonstrated that among all xanthophylls, only zeaxanthin and β-carotene can accept the energy from excited chlorophyll [26]. In darkness, leaves are typically devoid of zeaxanthin, and it is accumulated in the light. However, violaxanthin is not completely transformed into zeaxanthin even under favorable conditions, and, on the basis of this fact, Sapozhnikov [27] presumed, as early as in 1969, that the initial violaxanthin pool is heterogeneous [28, 29]. Zeaxanthin is derived from socalled active violaxanthin. To assess the latter quantitatively, one may determine the relative content of zeaxanthin in the total pool of violaxanthin and zeaxanthin using an inhibitor of the reverse reaction of the violaxanthin cycle. Such an inhibitor, salicylaldoxime, when used at the concentration of 2.5 mM, inactivates copper-containing oxidases, including epoxidase instrumental in the zeaxanthin to violaxanthin transformation in the course of the violaxanthin cycle [20, 21]. When this reaction is inhibited, zeaxanthin is accumulated. We studied in considerable detail zeaxanthin formation from active violaxanthin in F. verna leaves in the range of illuminance levels and at 100 klx in the leaves of all other plant species. Under low light, the content of zeaxanthin in untreated F. verna leaves was lower than in the presence of salicylaldoxime (Table 3). Only 32–37% of the total xanthophylls from the violaxanthin pool turned into zeaxanthin, and the inhibitor considerably increased zeaxanthin accumulation (over 60%). A similar level was observed in untreated leaves when illuminance was increased to 80–100 klx, indicating that, at high irradiation, the pool of active violaxanthin is completely realized. It is noteworthy that, at high insolation of 100 klx, the ephemeroids produce more zeaxanthin than the summer plants: up to 85% of the total xanthophylls from the violaxanthin pool as compared to 60% in the latter group of plant species. Under natural conditions, high solar radiation, especially when combined with low temperature, enhances the danger of photoinhibiting photosynthesis. In this case, especially important is the size of the total pool of xanthophylls from the violaxanthin cycle and the amount of zeaxanthin formed from the active violaxanthin, the latter being considerably larger in the ephemeroids than in the summer plants (Table 3). Because of that, the photosynthetic apparatus of the ephemeroids, as compared to the summer plants, is probably better equipped for potential defense against high insolation by rapid transformation of violaxanthin into zeaxanthin. Some support to this hypothesis comes from the data obtained by measuring PP in these ecologically
RUSSIAN JOURNAL OF PLANT PHYSIOLOGY
Vol. 50
No. 1
2003
SPECIFIC FEATURES OF PLASTID PIGMENT APPARATUS
55
Table 4. Potential photosynthesis as related to illuminance and temperature Rate of photosynthesis, mg CO2 /(g dry wt h) Plant species
Ephemeroids F. verna C. solida A. ranunculoides Summer plants U. dioica R. acris
Temperature, °C
illuminance, klx 10
20
40
70
100
10 10 10
30.0 ± 2.6 15.0 ± 2.3 20.0 ± 3.1
42.0 ± 3.2 24.0 ± 2.8 –
82.0 ± 3.4 50.0 ± 3.5 30.0 ± 2.8
112.0 ± 4.0 100.0 ± 3.6 69.0 ± 3.9
154.0 ± 4.1 145.0 ± 3.8 128.0 ± 4.2
20 20
12.0 ± 2.0 11.0 ± 1.8
16.0 ± 2.0 18.0 ± 1.8
35.0 ± 2.4 50.0 ± 3.1
47.0 ± 2.7 76.0 ± 2.4
121.0 ± 4.1 91.0 ± 3.7
different plant groups. Experiments involving various ambient temperatures and levels of illuminance were performed with the same leaf samples that were employed in the pigment studies (see Table 3). The information value of PP index was frequently discussed by Zalenskii [30], and here we will emphasize that this index characterizes both the capacity of the Calvin cycle functioning and the coupled activity of the photosystems providing the energy equivalents for light metabolism in mesophyll cells. The results of PP measurements are shown in Table 4. The common feature of the two plant groups is unsaturated photosynthesis even at the maximum insolation, meaning that, under our experimental conditions, nothing limited the process. Yet the two plant groups considerably differed in their rates of photosynthesis. Thus, in the ephemeroids, PP at low illuminance of 10 klx and low temperature of 5–10°C reached 30 mg CO2/g dry wt, whereas in the summer plants, this characteristic was lower at the same illuminance and a temperature of 20°C. At the maximum solar radiation of 100 klx, PP in the ephemeroid leaves at 5–10°C was high, 128– 154 mg CO2/(g dry wt h), and in the summer plants, a similar PP was attained only at 20°C. Apparently, neither the primary photosynthetic reactions nor the Calvin cycle activities are limited in ephemeroids at chilling temperatures. Previously, leaves of the same two species, an ephemeroid A. ranunculoides and a summer plant R. acris, were used for comparative studies of 14C metabolism and PP at 70 klx as depending on temperature [31]. In anemone, PP at 5°C was practically similar to that in buttercup at 10°C, and, at 10– 20°C, PP values in the ephemeroid exceeded twofold that of the summer plant. In addition, we demonstrated that the rate of 14C incorporation into the carbohydrate metabolic channel stayed high even at chilling temperatures. One should emphasize that carbohydrate synthesis in the course of photosynthesis is an energy-consuming process [32]. In this case, this process predominates and boosts PP in A. ranunculoides leaves to a higher value than in R. acris. RUSSIAN JOURNAL OF PLANT PHYSIOLOGY
Vol. 50
To conclude, a comparative study of the leaf photosynthetic apparatus in ecologically diverse plants elucidated the specific adaptation characteristics of photosynthesis. The ephemeroids exemplified high photosynthetic rates even at the combined adverse conditions, such as high insolation and relatively low temperatures, which would theoretically result in photoinhibition. However, due to the high relative content of carotenoids in LHC, including the expanded pool of active violaxanthin, the photosynthetic apparatus in ephemeroid leaves manages to avoid photodynamic damage under these conditions, both at the cellular level and at the level of photosynthetic membranes. REFERENCES 1. Krause, G.H., Photoinhibition Induced by Low Temperatures, Oxford: Bios Sci. Publ., 1994, pp. 331–348. 2. Siefermann-Harms, D., Carotenoids in Photosynthesis: 1. Location in Photosynthetic Membranes and LightHarversting Function, Biochim. Biophys. Acta, 1985, vol. 811, pp. 325–355. 3. Siefermann-Harms, D., The Light-Harvesting and Protective Function of Carotenoids in Photosynthetic Membranes, Physiol. Plant., 1987, vol. 69, pp. 561–568. 4. Sapozhnikov, D.I., Investigation of the Violaxanthin Cycle, Pure Appl. Chem., 1973, vol. 35, pp. 47–61. 5. Hager, A., Die reversiblen lichtabhängigen Xanthophyllumwanddlungen im Chloroplasten, Ber. Dtsch. Bot. Ges., 1975, vol. 88, pp. 27–44. 6. Pfundel, E. and Bilger, W., Regulation and Possible Function of the Violaxanthin Cycle, Photosynth. Res., 1994, vol. 42, pp. 89–109. 7. Demmig-Adams, B., Winter, K., Kruger, A., and Czygan, F.C., Zeaxanthin Synthesis Energy Dissipation and Photoprotection of Photosystem II at Chilling Temperatures, Plant Physiol., 1989, vol. 90, pp. 894–898. 8. Demmig-Adams, B., Carotenoids and Photoprotection in Plants: A Role for the Xanthophyll Zeaxanthin, Biochim. Biophys. Acta, 1990, vol. 1020, pp. 1–24. 9. Demmig-Adams, B., Adams III, W.W., The Xanthophyll Cycle, Protein Turnover and the High Light Tolerance of No. 1
2003
56
11. 12.
13.
Sun-Acclimated Leaves, Plant Physiol., 1993, vol. 103, pp. 1413–1420. Sapozhnikov, D.I., Maslova, T.G., Popova, O.F., Popova, I.A., and Koroleva, O.Ya., The Method of Leaf Fixation and Preservation for Quantification of Pigments in Plastids, Bot. Zh. (Leningrad), 1978, vol. 63, pp. 1586–1592. Shlyk, A.A., Spectrophotometric Determination of Chlorophylls a and b, Biokhimiya, 1968, vol. 33, pp. 275–285. Lichtenthaler, H.K. and Wellburn, A.R., Determination of Total Carotenoids and Chlorophylls a and b of Leaf Extracts in Different Solvents, Biochem. Soc. Trans., 1983, vol. 11, pp. 591–592. Sesták, Z., Chlorophylls and Carotenoids during Leaf Ontogeny, Photosynthesis during Leaf Development, Sesták, Z., Ed., Praha: Academia, 1985, pp. 76–106. Maslova, T.G., Popova, I.A., and Popova, O.F., Critical Evaluation of Spectrophotometric Quantification of Carotenoids, Fiziol. Rast. (Moscow), 1986, vol. 33, pp. 615–619 (Sov. Plant Physiol., Engl. Transl.). Kornyushenko, G.A. and Sapozhnikov, D.I., Thin-Layer Chromatography of Carotenoids in Green Leaves, Tr. Vses. Inst. Rastenievod. (Leningrad), 1969, vol. 40, pp. 181–192. Thornber, J.P., Chlorophyll–Proteins: Light-Harvesting and Reaction Center Components of Plants, Annu. Rev. Plant Physiol., 1975, vol. 26, pp. 127–158. Alberte, R.S., McClure, P.R., and Thornber, J.P., Photosynthesis in Trees: Organization of Chlorophyll and Photosynthetic Unit Size in Isolated Gymnosperm Chloroplasts, Plant Physiol., 1976, vol. 58, pp. 341–344. Lichtenthaler, H.K., Chlorophylls and Carotenoids: Pigments of Photosynthetic Biomembranes, Methods Enzymol., 1987, vol. 148, pp. 350–382. Maslova, T.G. and Popova, I.A., Adaptive Properties of the Plant Pigment Systems, Photosynthetica, 1993, vol. 29, pp. 195–203. Popova, I.A., Ryzhova, E.F., and Sapozhnikov, D.I., Some Properties of the Reaction of Violaxanthin Deepoxidation, Dokl. Akad. Nauk SSSR, 1971, vol. 201, pp. 494–496. Maslova, T.G., Popova, I.A., Kornyushenko, G.A., and Koroleva, O.Ya., Violaxanthin Cycle in Photosynthesis: History and Current Concept, Fiziol. Rast. (Moscow),
22.
23. 24. 25.
^
10.
MASLOVA et al.
^
14.
15.
16. 17.
18. 19. 20.
21.
26.
27.
28. 29.
30.
31.
32.
1996, vol. 43, pp. 437–449 (Russ. J. Plant Physiol., Engl. Transl.). Voznesenskii, V.L., Zalenskii, O.V., and Semikhatova, O.A., Metody issledovaniya fotosinteza i dykhaniya rastenii (Methods for Studying Photosynthesis and Respiration in Plants), Moscow: Nauka, 1965. Mathis, P.A. and Kleo, Y., The Triplet State of β-Carotene and of Analog Polyenes of Different Length, Photochem. Photobiol., 1973, vol. 18, pp. 343–346. Krinsky, N.I., Carotenoid Protection against Oxidation, Pure Appl. Chem., 1979, vol. 51, pp. 649–660. Demmig-Adams, B., Adams III, W.W., Carotenoid Composition in Sun and Shade Leaves of Plants with Different Life Forms, Plant Cell Environ., 1992, vol. 15, pp. 411–419. Phillip, D., Ruban, S., Horton, P., Asato, H., and Yound, A.J., Carotenoid S1 Energy Level and Quenching in LHC II b, Photosynth. Res. Suppl., Abstracts, Int. 10th, Photosynth. Congr., Montpelliers (France), 1995, p. 84. Sapozhnikov, D.I. and Kornyushenko, G.A., Heterogeneity of Violaxanthin in Pea Leaves, Fiziol. Rast. (Moscow), 1969, vol. 16, pp. 1038–1041 (Sov. Plant Physiol., Engl. Transl.). Siefermann-Harms, D., Evidence for a Heterogeneous Organization of Violaxanthin in Thylakoid Membranes, Photochem. Photobiol., 1984, vol. 40, pp. 507–512. Denise, P., Molnar, P., Toth, G., and Young, A., LightInduced Formation of 13-cis-Violaxanthin in Leaves of Hordeum vulgare, J. Photochem. Photobiol., 1999, vol. 49, pp. 89–95. Zalenskii, O.V., Ekologo-fiziologicheskie aspekty izucheniya fotosinteza, 37-e Timiryazevskoe chtenie (Ecological and Physiological Aspects of Studying Photosynthesis, the 37th Timiryazev Lecture), Leningrad: Nauka, 1976. Mamushina, N.S. and Zubkova, E.K., Effect of Temperature on Potential Photosynthesis and Photosynthetic Carbon Metabolism in C3 Plants with Different Seasonal Pattern of Development, Fiziol. Rast. (Moscow), 1996, vol. 43, pp. 360–366 (Russ. J. Plant Physiol., Engl. Transl.). Kelly, Y.J., Latzko, E., and Gibbs, M., Regulatory Aspects of Photosynthetic Carbon Metabolism, Annu. Rev. Plant. Physiol., 1976, vol. 27, pp. 181–205.
RUSSIAN JOURNAL OF PLANT PHYSIOLOGY
Vol. 50
No. 1
2003