cycle is unable to operate effectively because of low temperatures.

Transcription

1 Tree Physiology 24, Heron Publishing Victoria, Canada Seasonal changes in the xanthophyll cycle and antioxidants in sun-exposed and shaded parts of the crown of Cryptomeria japonica in relation to rhodoxanthin accumulation during cold acclimation QINGMIN HAN, 1,2 SHINICHIRO KATAHATA, 3 YOSHITAKA KAKUBARI 3 and YUZURU MUKAI 3 1 Department of Plant Ecology, Forestry and Forest Products Research Institute (FFPRI), Ibaraki , Japan 2 Corresponding author (qhan@ffpri.affrc.go.jp) 3 Faculty of Agriculture, University of Shizuoka, Shizuoka , Japan Received September 2, 2003; accepted October 31, 2003; published April 1, 2004 Summary Xanthophyll rhodoxanthin, which is present in sun-exposed needles of certain gymnosperms in winter, may have a photoprotective role during long-term cold acclimation. To examine how cold acclimation processes vary within tree crowns and to examine putative correlations between xanthophyll cycle pigments (VAZ), rhodoxanthin and the water water cycle in photoprotection, we monitored seasonal changes in the activities of two key antioxidant enzymes (ascorbate peroxidase (APX) and glutathione reductase (GR)), pigment composition and chlorophyll fluorescence parameters in sun and shade needles of crowns of the gymnosperm Cryptomeria japonica D. Don. Although APX and GR activities in both sun and shade needles were higher in winter than in summer when assayed at 20 C, differences between seasons were less pronounced when enzymatic activities in summer and winter were assayed at 20 and 5 C, respectively. These results suggest that increases in the potential activity of antioxidant enzymes in winter is an adaptation that helps counterbalance reductions in absolute enzyme activity caused by low temperature, and thus allows the photoprotective capacity of the water water cycle in C. japonica to be maintained at a roughly constant value throughout the year. In shade needles, the concentration of VAZ increased in winter, but no rhodoxanthin accumulated. Photosynthetic activity was maintained in winter. In sun needles, however, the electron transport rate (ETR) and photochemical quenching (q P ) decreased to their lowest values in December, just before the accumulation of rhodoxanthin, which coincided with the highest amount of VAZ. Changes in rhodoxanthin concentration mirrored changes in VAZ concentration from January to March. Winter values of ETR and q P were comparable with summer values after accumulation of rhodoxanthin, indicating that rhodoxanthin may play a more important role than the VAZ cycle in protecting the photosynthetic apparatus from photodamage in winter. Photosynthetic activity may be modulated, as a result of the interception of light by rhodoxanthin, to match the extent to which absorbed light energy can be utilized in winter when the VAZ cycle is unable to operate effectively because of low temperatures. Keywords: ascorbate peroxidase, electron transport rate, glutathione reductase, light, low temperature, non-photochemical quenching, photochemical efficiency of PSII, photoinhibition, water water cycle. Introduction The irradiance received by plants varies over a wide dynamic range in the natural environment. Therefore, light energy absorbed by plant leaves often exceeds the capacity of their photosynthetic system, which can suffer photo-oxidative damage as a result. Photo-oxidative damage is exacerbated by exposure to additional environmental stresses such as unfavorably low or high temperatures or water deficits. Plants acclimate to these stresses through various mechanisms to protect the photosynthetic apparatus against the deleterious effects of excess light absorption. Xanthophyll cycle-dependent energy dissipation is an important photoprotective process in the light-harvesting antenna of photosystem II (PSII) (Demmig et al. 1987, Gilmore 1997, Verhoeven et al. 1999). In this process, the formation of a ph gradient across the thylakoid membrane activates the de-epoxidation of violaxanthin (V ) to zeaxanthin (Z ) and antheraxanthin (A), facilitating the thermal dissipation of excess excitation energy (Demmig et al. 1987, Hager and Holocher 1994). Xanthophyll cycle-dependent energy dissipation down-regulates the photochemical efficiency of PSII. Plants adapted to high irradiances often have more of the xanthophyll cycle pigments (VAZ) than plants adapted to low irradiances, and the VAZ concentration is enhanced during cold acclimation (Logan et al. 1997, 1998, Adams et al. 1999, Niinemets et al. 1999, Schiefthaler et al. 1999). Another system that provides protection against the possible toxicity of reactive O 2 species is an integrated system of enzy-

2 610 HAN, KATAHATA, KAKUBARI AND MUKAI matic and non-enzymatic antioxidants that are concentrated in photosystem I (PSI) (Asada 1999). This system has been termed the water water cycle because the electron flow is from split water in PSII to water in PSI (Asada 1999). Three primary enzymes in this pathway are superoxide dismutase (SOD), which disproportionates superoxide to H 2 O 2 and O 2, ascorbate peroxidase (APX), which detoxifies H 2 O 2 to H 2 O, using ascorbate as a reductant, and glutathione reductase (GR), which reduces redox metabolites. In addition to scavenging reactive O 2 species and serving as a dissipative process, the water water cycle triggers the down-regulation of PSII by generating a ph gradient across the thylakoid membrane, and helps dissipate excess photon energy by modulating the status of the VAZ components (Osmond and Grace 1995, Makino et al. 2002). Activities of the antioxidant enzymes are elevated in some species during cold acclimation (Anderson et al. 1992, Krivosheeva et al. 1996, Fryer et al. 1998), but not in others (Polle et al. 1996, Logan et al. 1998). In addition to the xanthophyll cycle and water water cycle, rhodoxanthin plays an important photoprotective role in cold acclimation in certain gymnosperms, including Cryptomeria, Metasequoia, Taxodium, Chamaecyparis and Thuja (Ida 1981, Weger et al. 1993, Han and Mukai 1999, Han et al. 2003). Rhodoxanthin is a xanthophyll formed from the oxidation of zeaxanthin with manganese dioxide (Weedon 1965, Czeczuga 1987). It accumulates in lichens, some Bryophyta, numerous species of Pteridophyta, numerous gymnosperms and some angiosperms (Czeczuga 1987, Diaz et al. 1990). In gymnosperms, chloroplasts undergo a gradual change in color as autumn turns to winter, as well as a structural transformation into chromoplasts, in which reddish particles of rhodoxanthin accumulate (Ida 1981). Rhodoxanthin intercepts some of the light irradiating the needles and helps maintain an appropriate balance between light absorption, thermal dissipation and photosynthesis (Han et al. 2003). Rhodoxanthin is detected only in sun-exposed needles (Ida et al. 1991), suggesting that cold acclimation processes might differ between the sun-exposed and shaded parts of a tree crown. Rhodoxanthin, VAZ-dependent energy dissipation and the water water cycle may be integrated processes rather than three independent systems. However, no correlation between the concentrations of rhodoxanthin and VAZ and the activities of antioxidant enzymes has been documented during cold acclimation. In this study, we measured seasonal changes in pigment composition, chlorophyll fluorescence parameters, and two key antioxidant enzymes in sun-exposed and shaded crown parts of Cryptomeria japonica D. Don, a common evergreen coniferous afforestation species in Japan. Our main objective was to study cold acclimation processes and photoinhibition of the photosynthetic apparatus in needles receiving different irradiances and thus gain further insights into the correlation between rhodoxanthin, VAZ and antioxidant enzymes in photoprotective mechanisms. Materials and methods Plant materials Three 15-year-old C. japonica trees, grown in an open field at the University of Shizuoka (34 59 N, E), were selected for study. The trees were about 5.7 m tall in Current-year needles on sun-exposed and shaded branches orientated toward the south and north, respectively, were collected between August 1999 and April Needle samples were immediately frozen in liquid nitrogen in the field and stored at 80 C in the laboratory until analyzed. Mean annual precipitation and temperature near the site for the period were 2322 mm and 16.3 C, respectively (Japanese Bureau of Meteorology). In the same period, mean daily temperatures were about 26.8 and 7.0 C in August and February, respectively. Daily minimum temperatures ranged from 4.0 to 5.6 C in February 2000 (Figure 1). Rain fell throughout the study period (Figure 1). Light environment Photosynthetic photon flux (PPF) sensors (IKS-25, Koito, Yokohama, Japan) were placed near sample branches in the upper and lower crowns. All sensors faced upward and were calibrated against a PPF sensor (LI-190SA, Li-Cor, Lincoln, NE) before installation. Measurements of PPF were made every minute and recorded with a data logger (MES-901, Koito). Determination of pigment composition by HPLC Chlorophyll (Chl) concentrations were determined spectrophotometrically in acetone extracts (80%) as described by Arnon (1949). For quantification of carotenoids, acetone extracts were fractionated immediately on a high performance Figure 1. Seasonal courses of daily precipitation and daily maximum and minimum air temperatures from January 1999 to September TREE PHYSIOLOGY VOLUME 24, 2004

3 PIGMENT COMPOSITION AND ANTIOXIDANTS WITHIN CRYPTOMERIA JAPONICA CROWNS 611 liquid chromatography (HPLC) system (LC-10AD, Shimadzu, Kyoto, Japan) equipped with a Wakosil ODS 5 µm C 18 column (4.6 mm i.d. 250 mm; Wako, Osaka, Japan) as described by Gilmore and Yamamoto (1991) and modified by Han et al. (2003). Six replicates were analyzed for each pigment. Pigment concentrations were expressed on an area basis. Projected leaf areas were measured with an image-analysis system (DIAS, Delta-T, Cambridge, U.K.) in which a video camera records images of needles and transfers them in digital format to a computer. Measurements of chlorophyll fluorescence We determined dark-adapted photochemical efficiency of PSII (F v /F m ) in situ 2 h after sunset with a chlorophyll fluorometer (Mini-Pam, Walz, Effeltrich, Germany). On the next clear day, photochemical efficiency of PSII in the same needles was measured at noon under natural environmental conditions. Six to 12 replicates were analyzed for chlorophyll fluorescence. Chlorophyll fluorescence parameters were calculated as described by van Kooten and Snel (1990), with the exception that minimal fluorescence of light-adapted foliage (F 0 ) was not measured, but calculated from the expression: F 0 =F 0 /(F v /F m + F 0 /F m ) (Oxborough and Baker 1997), where F 0 and F m are minimal and maximal fluorescence of dark-adapted foliage, respectively, F v is variable fluorescence and calculated as F m F 0, and F m is maximal fluorescence of light-adapted foliage. Electron transport rates (ETR) were calculated as the quantum yield of PSII ((F m F s )/F m ) multiplied by leaf absorptance and 0.5, assuming that the fraction of absorbed photons was distributed equally between PSI and PSII (Krall and Edwards 1992). Leaf absorptance was calculated from Chl concentration as described by Evans (1993) because the morphological characteristics of needles make it technically difficult to measure absorptance directly (Han et al. 2003). Results Light environment Figure 2 shows representative diurnal courses of PPF for both sun-exposed and shaded crown parts on a clear day in winter. In shaded crown parts, PPF was about µmol m 2 s 1 during most of the daytime (Figure 2). Sunflecks reached shaded crown parts when the temperature was relatively high during daytime. Daily integrated PPF values were 25.7 and 2.3 mol m 2 day 1 in sun-exposed and shaded crown parts, respectively. Differences in pigment composition between sun and shade needles Both sun and shade needles were still immature in August (Figure 3a). Sun needles always contained less Chl than shade needles. The Chl concentration decreased in both needle types during the winter. The Chl a/b ratio was higher in sun needles than in shade needles until November, but then decreased to similar values as in shade needles in the following months (Figure 3b). Sun needles started to accumulate rhodoxanthin in January (Figure 3c). The concentration of rhodoxanthin peaked in February, decreased significantly in March and fell to zero in April. In contrast, shade needles remained green over the entire winter, and no rhodoxanthin was detected in them (Figure 3c). The concentration of VAZ increased in both sun and shade needles in winter (Figure 3d) and reached a maximum in shade needles in February. In contrast, the VAZ concentration in sun needles reached a maximum in December, then decreased in January and February in parallel with the accumulation of rhodoxanthin (Figure 3c). The VAZ concentration increased again in March when most of the rhodoxanthin disappeared. Antioxidant enzyme assays Ascorbate peroxidase (APX) and glutathione reductase (GR) were extracted and their activities measured spectrophotometrically at both 20 and 5 C in a temperature-controlled cuvette as described by Han and Mukai (1999). Total protein content in the enzyme extracts was measured according to Bradford (1976) using a protein assay rapid kit (Wako). Three to five replicates were used for the antioxidant analyses. Data analyses Multiple comparisons between sun and shade needles in each season were carried out by the Student s t-test, assuming the results to be statistically significant when P < 0.05, unless indicated otherwise. Data were also subjected to the Student s t-test to determine statistically significant seasonal differences within a crown position (P < 0.05). Figure 2. Diurnal variations in photosynthetic photon flux (PPF) in sun-exposed (thin line) and shaded (thick line) crowns of Cryptomeria japonica on a representative clear day in February TREE PHYSIOLOGY ONLINE at

4 612 HAN, KATAHATA, KAKUBARI AND MUKAI Figure 3. Seasonal changes in (a) concentration of total chlorophyll (Chl a + b), (b) Chl a/b ratio, (c) concentration of rhodoxanthin, (d) concentration of xanthophyll cycle pigments (VAZ) and (e) ratio of Z + A to VAZ (DPS) at noon (solid lines) and night (dashed lines) in sun ( ) and shade ( ) needles of Cryptomeria japonica. Values are means ± SE of 3 6 needles. Abbreviations: V = violaxanthin; A = antheraxanthin; Z = zeaxanthin; and DPS = de-epoxidation state. Sun needles contained higher concentrations of VAZ than shade needles during most of the study period, except in February, when expressed on a Chl basis (Figures 3a and 3d). The de-epoxidation state (DPS; ratio of Z + A to VAZ) at noon on sunny days remained relatively stable (at about 0.6) in sun needles over the entire study period (Figure 3e). Most of the A and Z in sun needles was re-epoxidized to V in August during the night, and consequently DPS values fell. However, the concentration of retained A and Z increased gradually in the winter, and consequently, DPS values rose. In shade needles, less V was de-epoxidized to A and Z at noon and so the value of DPS was lower than 0.1 in most seasons. Changes in chlorophyll fluorescence parameters The mean value of F v /F m in sun needles remained high (above 0.78) until November, gradually decreased to its lowest value (0.60) in February (Figure 4a), then returned to close to summer values in March and April. In contrast, in shade needles, F v /F m remained higher than 0.78 throughout the entire experimental period. There were no significant differences in F v /F m between sun and shade needles in the summer (P > 0.2). However, differences in F v /F m between sun and shade needles became significant from November onward throughout the winter (P < 0.002). The photochemical efficiency of opened PSII centers (F v /F m ) measured at noon on clear days had similar values to F v /F m throughout the study period in shade needles, but remained higher until December in sun needles (Figure 4b). In sun needles, minimum photochemical quenching (q P )occurred in December, but recovered to summer values in January and February (Figure 4c), and non-photochemical quenching (NPQ) increased significantly in the winter (Figure 4d). Changes in both q P and NPQ were less pronounced in shade needles than in sun needles during the experiment (Figures 4c and 4d). Electron transport rate decreased in both sun and shade needles in winter (Figure 4e). Reasoning that this reduction may be caused by seasonal changes in incident PPF (Figure 4f), we analyzed the relationship between absorbed PPF and ETR and found a linear relationship between the two variables in shade needles (Figure 5, inset). The ETR was still linearly correlated to PPF when data from sun needles that lacked rhodoxanthin were considered, except in December (Figure 5, solid line). Changes in activities of antioxidant enzymes Activities of APX and GR were higher in both sun and shade needles in winter than in summer when measured at the same temperatures (Figure 6); however, these seasonal differences in activities were less pronounced when the enzymes were assayed at 20 C in summer and at 5 C in winter needles. In TREE PHYSIOLOGY VOLUME 24, 2004

5 PIGMENT COMPOSITION AND ANTIOXIDANTS WITHIN CRYPTOMERIA JAPONICA CROWNS 613 Figure 4. Seasonal changes in (a) photochemical efficiency of photosystem II (PSII) measured at night (F v /F m ), (b) photochemical efficiency of opened PSII centers (F v /F m ), (c) photochemical quenching (q P ), (d) non-photochemical quenching (NPQ) and (e) electron transport rate (ETR) measured at incident photosynthetic photon flux (PPF) at noon (f ) for sun ( ) and shade ( ) needles of Cryptomeria japonica. Values are means ± SE of 6 12 needles. summer, APX activity was higher in sun needles than in shade needles (Figure 6a), but both needle types had similar APX activities in winter. The GR activities in sun and shade needles did not differ significantly in most months (Figure 6c). Discussion We examined seasonal changes in pigment composition, chlorophyll fluorescence parameters, and activities of two key antioxidant enzymes in sun and shade needles of C. japonica crowns. In winter, rhodoxanthin accumulated in sun needles, but not in shade needles (Figure 3c), indicating that high irradiances are essential to induce rhodoxanthin synthesis. Rhodoxanthin concentration was highest when mean daily temperature was less than 8.4 C in C. japonica and 13.4 C in Taxodium distichum (L.) Rich., indicating that low temperature is as important as high irradiance to stimulate rhodoxanthin synthesis (Ida et al. 1991). It is known that oxidative stress plays a key role in the induction of chromoplast carotenoid synthesis and in the transformation of chloroplasts into chromoplasts (Bouvier et al. 1998). The interaction between light and temperature increased the excitation pressure in PSII (1 q P ; Figure 4c) and thereby induced rhodoxanthin synthesis in C. japonica. There was a linear relationship between PPF and ETR in green needles (both sun and shade) on all occasions tested, ex- Figure 5. Linear relationship between absorbed photosynthetic photon flux (PPF) and electron transport rate (ETR) in sun ( ) and shade ( ) needles in seasons without rhodoxanthin (r 2 = 0.984, P < 0.001). Arrows indicate changes in ETR in rhodoxanthin-containing needles when light interception by rhodoxanthin was ( ), or was not ( ), taken into account in calculating ETR. The numbers beside the open circles refer to the months in which the measurements were taken.the inset shows the same correlations when only shade needles were considered (r 2 = 0.995, P < 0.001). TREE PHYSIOLOGY ONLINE at

6 614 HAN, KATAHATA, KAKUBARI AND MUKAI Figure 6. Seasonal changes in activities of (a, b) ascorbate peroxidase (APX; µmol min 1 mg 1 protein) and (c, d) glutathione reductase (GR; µmol min 1 mg 1 protein) in sun ( ) and shade ( ) needles of Cryptomeria japonica measured at controlled temperatures of (a, c) 20 C and (b, d) 5 C. Values are means ± SE of 3 5 needles. cept in sun needles in December (Figure 5), indicating that photosynthetic activity was maintained in shade needles in winter. Reddish particles of rhodoxanthin are localized in chromoplasts that develop from chloroplasts during coloration in winter (Ida 1981, Ida et al. 1991). Chromoplasts are often observed in mesophyll cells that also contain empty and colorless plastids. Furthermore, the number of reddish particles within chromoplasts decreases from the sunny side of a leaf to the shady side (Ida et al. 1991). Thus, more rhodoxanthin than Chl is located closer to the surface of the sunny side of a leaf. In addition, assuming that the specific absorption properties of rhodoxanthin are similar to those of Chl (mole for mole), rhodoxanthin should intercept about 12% of the incident light in February (Figures 3a and 3c). Recalculated ETR values for the rhodoxanthin-containing needles adjusted to account for this absorption would be consistent with the previously mentioned linear relationship, shown by the triangles in Figure 5. The lowest value of ETR was observed in December, just before the accumulation of rhodoxanthin, coinciding with the highest concentration of VAZ (Figure 3d) and the lowest value of q P (Figure 4c). The accumulation of rhodoxanthin resulted in the high values of q P observed in January and February. These results indicate that rhodoxanthin has a photoprotective role. By comparing the light response curves of both photosynthesis and chlorophyll fluorescence, Han et al. (2003) found that NPQ and photosynthetic rate at saturated PPF were similar in summer and winter, but quantum yield of CO 2 fixation decreased in winter. Taken together, our results suggest that rhodoxanthin acts as a sunscreen rather than an energy quencher. Photosynthetic activity may be modulated, as a result of the interception of light by rhodoxanthin, to match the extent to which absorbed light energy can be utilized in winter. Antioxidant enzyme activities were higher in winter than in summer, when measurements were made at 20 C (Figure 6), which is consistent with previous reports (Anderson et al. 1992, Krivosheeva et al. 1996, Fryer et al. 1998). However, the cited studies only compared enzymatic activities at room temperature, and thus overestimated their actual performance in the low-temperature (winter) environment (Grace and Logan 1996). By taking the actual growth temperature into account, we found that antioxidant enzyme activities in winter, measured at 5 C, did not differ from their respective levels in summer, measured at 20 C. These results suggest that increases in the potential activities of antioxidant enzymes in winter counterbalance decreases in the absolute activities caused by low temperature and, thus, the photoprotective capacity provided by the water water cycle appears to be maintained constant throughout the entire season in C. japonica. The concentration of VAZ in shade needles also increased in the winter, although V was seldom de-epoxidized to A and Z (Figure 3e), and xanthophyll-dependent thermal energy dissipation increased less in winter in shade needles than in sun needles (Figure 4d). The increase in VAZ concentration might be an adaptive measure allowing energy dissipation during sunflecks in winter, compensating for the lower activity of de-epoxidase at low temperature (Eskling and Åkerlund 1998). The rapid modulation of energy dissipation activity in response to sunflecks via A + Z occurs through changes in ph across the thylakoid membrane, leading to rapid engagement and disengagement of A + Z in dissipation (Logan et al. 1997, Adams et al. 1999). The enhanced capacity of the water water cycle might facilitate the formation of the ph across the thylakoid membrane required for dissipation (Asada 1999, Makino et al. 2002). Increased amounts of both VAZ and antioxidant enzyme activities completely protected the photosynthetic apparatus of shade needles in C. japonica (see inset in Figure 5). We conclude that, even in a single tree crown, photo- TREE PHYSIOLOGY VOLUME 24, 2004

7 PIGMENT COMPOSITION AND ANTIOXIDANTS WITHIN CRYPTOMERIA JAPONICA CROWNS 615 synthetic acclimation to low temperatures in winter may depend on the light environment. Shade needles are likely to be source-limited, because of the low irradiances they receive, and so maintain photosynthetic capacity. Excess light absorption was prevented by increased amounts of VAZ and antioxidant enzyme activities. In contrast, in sun needles, photoinhibition appeared to trigger the accumulation of rhodoxanthin. Photosynthetic activity may be modulated, as a result of light interception by rhodoxanthin, to match the extent to which absorbed light energy can be utilized in winter. Rhodoxanthin might maintain a better balance between light absorption, photosynthesis and the thermal dissipation of excess light energy when the VAZ cycle is unable to operate effectively because of low temperatures. Acknowledgments This research was supported by a Grant-in-Aid for Scientific Research (No. B ) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. References Adams, III, W.W., B. Demmig-Adams, B.A. Logan, D.H. Barker and C.B. Osmond Rapid changes in xanthophyll cycle-dependent energy dissipation and photosystem II efficiency in two vines, Stephania japonica and Smilax australis, growing in the understory of an open Eucalyptus forest. Plant Cell Environ. 22: Anderson, J.V., B.I. Chevone and J.L. Hess Seasonal variation in the antioxidant system of eastern white pine needles. Plant Physiol. 98: Arnon, D.I Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24:1 15. Asada, K The water water cycle in chloroplasts: scavenging of active oxygens and dissipation of excess photons. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50: Bouvier, F., R.A. Backhaus and B. Camara Induction and control of chromoplast-specific carotenoid genes by oxidative stress. J. Biol. Chem. 273:30,651 30,659. Bradford, M.M A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: Czeczuga, B Different rhodoxanthin contents in the leaves of gymnosperms grown under various light intensities. Biochem. Syst. Ecol. 15: Demmig, B., K. Winter, A. Krüger and F.-C. Czygan Photoinhibition and zeaxanthin formation in intact leaves. A possible role of the xanthophyll cycle in the dissipation of excess light energy. Plant Physiol. 84: Diaz, M., E. Ball and U. Lüttge Stress-induced accumulation of the xanthophyll rhodoxanthin in leaves of Aloe vera. Plant Physiol. Biochem. 28: Eskling, M. and H.-E. Åkerlund Changes in the quantities of violaxanthin de-epoxidase, xanthophylls and ascorbate in spinach upon shift from low to high light. Photosynth. Res. 57: Evans, J.R Photosynthetic acclimation and nitrogen partitioning within a Lucerne canopy. II. Stability through time and comparison with a theoretical optimum. Aust. J. Plant Physiol. 20: Fryer, M.J., J.R. Andrews, K. Oxborough, D.A. Blowers and N.R. Baker Relationship between CO 2 assimilation, photosynthetic electron transport, and active O 2 metabolism in leaves of maize in the field during periods of low temperature. Plant Physiol. 116: Gilmore, A.M Mechanistic aspects of xanthophyll cycle-dependent photoprotection in higher plant chloroplasts and leaves. Physiol. Plant. 99: Gilmore, A.M. and H.Y. Yamamoto Resolution of lutein and zeaxanthin using a non-endcapped, lightly carbon-loaded C 18 high-performance liquid chromatographic column. J. Chromat. 543: Grace, S.C. and B.A. Logan Acclimation of foliar antioxidant systems to growth irradiance in three broad-leaved evergreen species. Plant Physiol. 112: Hager, A. and K. Holocher Localization of the xanthophyll-cycle enzyme violaxanthin de-epoxidase within the thylakoid lumen and abolition of its mobility by a (light-dependent) ph decrease. Planta 192: Han, Q. and Y. Mukai Cold acclimation and photoinhibition of photosynthesis accompanied by needle color changes in Cryptomeria japonica during the winter. J. For. Res. 4: Han, Q., K. Shinohara, Y. Kakubari and Y. Mukai Photoprotective role of rhodoxanthin during cold acclimation in Cryptomeria japonica. Plant Cell Environ. 26: Ida, K Eco-physiological studies on the response of taxodiaceous conifers to shading, with special reference to the behaviour of leaf pigments. I. Distribution of carotenoids in green and autumnal reddish brown leaves of gymnosperms. Bot. Mag. Tokyo 94: Ida, K., F. Saito and S. Takeda Isomers of rhodoxanthin in reddish brown leaves of gymnosperms and effect of daylight intensity on the contents of pigments during autumnal coloration. Bot. Mag. Tokyo 104: Krall, J.P. and G.E. Edwards Relationship between photosystem II activity and CO 2 fixation in leaves. Physiol. Plant. 86: Krivosheeva, A., D.-L. Tao, C. Ottander, G. Wingsle, S.L. Dube and G. Öquist Cold acclimation and photoinhibition of photosynthesis in Scots pine. Planta 200: Logan, B.A., D.H. Barker, W.W. Adams, III and B. Demmig-Adams The response of xanthophyll cycle-dependent energy dissipation in Alocasia brisbanensis to sunflecks in a subtropical rainforest. Aust. J. Plant Physiol. 24: Logan, B.A., S.C. Grace, W.W. Adams, III and B. Demmig-Adams Seasonal differences in xanthophyll cycle characteristics and antioxidants in Mahonia repens growing in different light environments. Oecologia 116:9 17. Makino, A., C. Miyake and A. Yokota Physiological functions of the water water cycle (Mehler reaction) and the cyclic electron flow around PSI in rice leaves. Plant Cell Physiol. 43: Niinemets, Ü., W. Bilger, O. Kull and J.D. Tenhunen Responses of foliar photosynthetic electron transport, pigment stoichiometry, and stomatal conductance to interacting environmental factors in a mixed species forest canopy. Tree Physiol. 19: Osmond, C.B. and S.C. Grace Perspectives on photoinhibition and photorespiration in the field: quintessential inefficiencies of the light and dark reactions of photosynthesis? J. Exp. Bot. 46: Oxborough, K. and N.R. Baker Resolving chlorophyll a fluorescence images of photosynthetic efficiency into photochemical and non-photochemical components calculation of q P and F v /F m without measuring F 0. Photosynth. Res. 54: TREE PHYSIOLOGY ONLINE at

8 616 HAN, KATAHATA, KAKUBARI AND MUKAI Polle, A., W. Kröniger and H. Rennenberg Seasonal fluctuations of ascorbate-related enzymes: acute and delayed effects of late frost in spring on antioxidative systems in needles of Norway spruce (Picea abies L.). Plant Cell Physiol. 37: Schiefthaler, U., A.W. Russell, H.R. Bolhàr-Nordenkampf and C. Critchley Photoregulation and photodamage in Schefflera arboricola leaves adapted to different light environments. Aust. J. Plant Physiol. 26: van Kooten, O. and J.F.H. Snel The use of chlorophyll fluorescence nomenclature in plant stress physiology. Photosynth. Res. 25: Verhoeven, A.S., W.W. Adams, III, B. Demmig-Adams, R. Croce and R. Bassi Xanthophyll cycle pigment localization and dynamics during exposure to low temperatures and light stress in Vinca major. Plant Physiol. 120: Weedon, B.C.L Chemistry of the carotenoids. In Chemistry and Biochemistry of Plant Pigments. Ed. T.W. Goodwin. Academic Press, London, pp Weger, H.G., S.N. Silim and R.D. Guy Photosynthetic acclimation to low temperature by western red cedar seedlings. Plant Cell Environ. 16: TREE PHYSIOLOGY VOLUME 24, 2004