- Email: [email protected]
Responses of the photosynthetic apparatus to winter conditions in broadleaved evergreen trees growing in warm temperate regions of Japan
Plant Physiology and Biochemistry 86 (2015) 147e154
Contents lists available at ScienceDirect
Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy
Research article
Responses of the photosynthetic apparatus to winter conditions in broadleaved evergreen trees growing in warm temperate regions of Japan Chizuru Tanaka a, Takashi Nakano b, Jun-ya Yamazaki a, *, Emiko Maruta a a b
Department of Biology, Faculty of Science, Toho University, Miyama 2-2-1, Funabashi, Chiba 274-8510, Japan Mount Fuji Research Institute, Yamanashi Prefectural Government (MFRI), Kenmarubi 5597-1, Kamiyoshida, Fujiyoshida City, Yamanashi 403-0005, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 September 2014 Accepted 2 December 2014 Available online 3 December 2014
Photosynthetic characteristics of two broadleaved evergreen trees, Quercus myrsinaefolia and Machilus thunbergii, were compared in autumn and winter. The irradiance was similar in both seasons, but the air temperature was lower in winter. Under the winter conditions, net photosynthesis under natural sunlight (Anet) in both species dropped to 4 mmol CO2 m2 s1, and the quantum yield of photosystem II (PSII) photochemistry in dark-adapted leaves (Fv/Fm) also dropped to 0.60. In both species the maximum carboxylation rates of Rubisco (Vcmax) decreased, and the amount of Rubisco increased in winter. A decline in chlorophyll (Chl) concentration and an increase in the Chl a/b ratio in winter resulted in a reduction in the size of the light-harvesting antennae. From measurements of Chl a fluorescence parameters, both the relative fraction and the energy flux rates of thermal dissipation through other nonphotochemical processes were markedly elevated in winter. The results indicate that the photosynthetic apparatus in broadleaved evergreen species in warm temperate regions responds to winter through regulatory mechanisms involving the downregulation of light-harvesting and photosynthesis coupled with increased photoprotective thermal energy dissipation to minimize photodamage in winter. These mechanisms aid a quick restart of photosynthesis without the development of new leaves in the following spring. © 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Broadleaved evergreens Energy flux Energy partition Quercus myrsinaefolia Machilus thunbergii
1. Introduction
Abbreviations: Amax, the potential photosynthetic rate; Anet, the net photosynthesis under natural sunlight; Chl, Chlorophyll; F, steady-state fluorescence; Fm, Fo and Fv, the maximum, minimum and variable fluorescence yields, respectively; Fm0 , Fo0 and Fv0, the maximum, minimum and variable fluorescence yields, respectively, during energization; Fv/Fm, the quantum yield of PSII photochemistry in darkadapted leaves; gs, stomatal conductance; JNPQ, energy flux rate of NPQ associated with the xanthophyll cycle; JONP, energy flux rate of non-photochemical processes not associated with the xanthophyll cycle; JPSII, energy flux rate of PSII photochemistry; NPQ, non-photochemical quenching of chlorophyll fluorescence; PPFD, photosynthetic photon flux density; PSI and PSII, photosystem I and photosystem II, respectively; qL, the fraction of open PSII reaction centers based on the lake model; TBARS, thiobarbituric acid reaction substance; Vcmax, the maximum carboxylation rates of Rubisco; 4, the apparent quantum yield of CO2 fixation on an illuminatedlight basis; FNPQ, the quantum yield of non-photochemical quenching associated with the xanthophyll cycle; FONP, the quantum yield of non-photochemical processes not associated with the xanthophyll cycle; FPSII, the quantum yield of PSII photochemistry. * Corresponding author. E-mail address: [email protected] (J.-y. Yamazaki). http://dx.doi.org/10.1016/j.plaphy.2014.12.002 0981-9428/© 2014 Elsevier Masson SAS. All rights reserved.
Broadleaved evergreen species grown in the monsoonal region originated in warm regions and spread from central Japan westwards across a large belt of southern China, including Taiwan, to the southern side of the Himalayan mountains (Ohsawa, 1990). The species growing in this region have large, very thick, often shiny leaves. In this region, the climate features high levels of precipitation and high temperatures in summer, but is somewhat xeric with low temperatures in winter. The trees endemic to this area are confined exclusively to oceanic, mild winter areas such as those in Japan (Sakai, 1975). Quercus species, which are distributed from the southern end to 38 N in Japan, are the major canopy components of warm temperate broadleaved evergreen forests. The distribution of Machilus overlaps with that of Quercus, and the Machilus species observed in warmer coastal regions are exposed to warm currents (the Black Current on the Pacific Ocean side and the Tsushima Current on the Japan Sea side) that reach as far north as the Tohoku
148
C. Tanaka et al. / Plant Physiology and Biochemistry 86 (2015) 147e154
district (below 40 N), which is the northern limit of the Machilus species. There have been two major findings with respect to the determinants of the distribution of broadleaved evergreen trees. Sakai (1975) determined the freezing tolerance of these trees on the basis of the freezing temperatures in the stems, leaves, and buds, and concluded that cold tolerance is the main determinant of the distribution of broadleaved evergreen trees. Taneda and Tateno (2005) reported that evergreen broadleaved trees with large-diameter vessels were significantly more vulnerable to freezeethaw embolism than were evergreen conifers with small-diameter tracheids. This result suggested that the cross-sectional diameter of the xylem elements is one of the important determinants of the distribution of evergreen woody species. Photosynthetic activities are lower at low temperatures in winter than at optimal or high temperatures in the primary growing season. Evergreen conifers growing under harsh winter conditions in subalpine regions exhibit downregulation in both capacity of photosynthesis and efficiency of photosystem (PS) II (Zarter et al., 2006a, 2006b; Yamazaki et al., 2003, 2007). In contrast, those at lower altitudes may or may not exhibit downregulation of photosynthetic capacity in winter (Adams et al., 2004; Zarter et al., 2006a) but invariably experience downregulation of PSII efficiency (Adams and Demmig-Adams, 1994; Adams et al., 1995, 2004; Ebbert et al., 2005; Zarter et al., 2006a). Broadleaved evergreen trees show lowered photosynthetic activity in winter (Adams and Demmig-Adams, 1995; Adams et al., 1995; Verhoeven et al., 1996). In addition, given that broadleaved trees continue to perform photosynthesis under direct sunlight throughout winter, winter photosynthesis may exceed that in the growing season and account for a large part of the annual photosynthesis (Miyazawa and Kikuzawa, 2005). Winter stress induces suppression of the photochemical effi€ ciency of PSII and the CalvineBenson-cycle enzymes (Oquist and Huner, 2003). Excessive photons reduce molecular oxygen, resulting in the production of harmful active oxygen species and severe photoinhibition of photosynthesis (Ottander et al., 1995; Yamazaki et al., 2007). The photosynthetic apparatus of overwintering evergreen trees must accordingly develop protective mechanisms against these stresses to survive the winter and recover their capacity for photosynthesis by the following spring (Ottander et al., 1995; Miyazawa and Kikuzawa, 2005; Yamazaki et al., 2003, 2007). However, in winter, broadleaved evergreen species in warm temperate regions of East Asia are exposed to milder winter conditions than those in boreal and continental climate regions. Under mild winter conditions, the PSII complexes are not degraded but phosphorylated, owing to the fast recovery of overall photosynthesis when the climate becomes warm (Ebbert et al., 2005; Demmig-Adams and Adams, 2006; Verhoeven et al., 2009). Studies of xanthophyll cycle-related energy dissipation from antennae attached to the PSII complexes in winter have been performed (Adams et al., 1995, 2004; Yamazaki et al., 2011). We previously reported the involvement of thermal dissipation and changes in chlorophyll (Chl) forms in overwintering broadleaved evergreen species (Yamazaki et al., 2011). The present study, by comparing leaves in autumn with those in winter, attempts to elucidate in further detail how mechanisms of thermal energy dissipation are involved in the protection of the photosynthetic apparatus in overwintering broadleaved evergreens in warm temperate regions of Japan.
2. Materials and methods 2.1. Study site and plant materials All measurements were performed on the campus of Toho University, Funabashi, Japan (latitude 35 410 N, longitude 140 20 E; altitude 20 m), located in a warm temperate region. Two evergreen species [Quercus myrsinaefolia (Fagaceae) and Machilus thunbergii (Lauraceae)] were used. The heights of the Machilus and Quercus trees included in the survey were approximately 8 and 10 m, respectively, and their diameters at breast height were approximately 30 and 20 cm, respectively. Photosynthetic photon flux density (PPFD) was measured with a photon sensor (IKS-27; Koito Co., Japan) mounted on a horizontal plane with respect to the earth's surface, and the data obtained were recorded by a data logger (KADEC-UP; KONA system, Japan) at 10-min intervals. Maximum and minimum air temperatures were 31.1 C (October 5, 2007)/2.2 C (February 18, 2008) and 29.6 C (October 12, 2013)/ 3.1 C (January 11, 2014), obtained from an automated meteorological data acquisition system (AMeDAS) at Funabashi Meteorological Observatory (latitude 35 420 N, longitude 140 20 E, altitude: 28 m) provided by the Japan Meteorological Agency (JMA; http:// www.jma.go.jp/). Between the gap of the two experiments (2007e2014) there were no environmental and characteristic differences such as light conditions, the features of the campus, and current year's leaf characteristics at the study site. In addition, the significant climate changes were not observed from the data obtained from the JMA about the gap of multiple years between the two experiments (2007e2014). Hereafter, the data of November 13, 2007 and October 26, 2013 will be referred to as autumn data, and the data of February 1, 2008 and February 7, 2014 as winter data. 2.2. Measurements of photosynthetic parameters Photosynthetic parameters were measured on sun-exposed current-year leaves on November 13, 2007 and February 1, 2008. Under field conditions, net photosynthesis (Anet) of the leaves was measured between 11:00 and 14:00 (local time) with a portable gas-exchange analyzer (LI-6400; LI-COR, Inc., USA) attached to a transparent standard leaf chamber (6400-08; LI-COR, Inc.). The potential photosynthetic rate (Amax), the apparent quantum yield of CO2 fixation on an illuminated-light basis (4), and the potential rate of carboxylation by Rubisco (Vcmax) were measured under ambient temperature with an LI-6400 attached to an LED light and CO2 concentration control unit (6400-02B; LI-COR, Inc.). For the determination of Amax, the net photosynthetic rates at 10 stepwise actinic light intensity intervals from 0 to 1500 mmol photons m2 s1 were measured under a cuvette CO2 concentration of 370 mmol mol1. It took 3e4 min to obtain a stable photosynthetic rates at each reduction in the LED intensity. Using these data, Amax was calculated by the equation of Johnson and Thornley (1984). The value of 4 was estimated by linear regression at PPFD <100 mmol m2 s1. For the determination of Vcmax, the net photosynthetic rate was measured at 10 stepwise cuvette CO2 concentrations varying 0e2000 mmol mol1 under an illuminated irradiance fixed at 1200 mmol photons m2 s1 (at which the photosynthetic rates reached saturation). It took 4e5 min to obtain a stable photosynthetic rate at each increase in the cuvette CO2 concentration. Assuming that, at a low partial pressure of intercellular CO2, the assimilation of CO2 was limited solely by the amount, activity, and kinetic properties of Rubisco, Vcmax was calculated by the method of Farquhar et al. (1980). The stomatal conductance at saturating PPFD and a CO2 concentration of 370 mmol mol1 (gs, mol m2 s1) was measured concomitantly as photosynthesis under a controlled environment.
C. Tanaka et al. / Plant Physiology and Biochemistry 86 (2015) 147e154
149
2.3. Chl fluorescence measurements
2.5. Determination of Chl concentration
Chl fluorescence parameters were measured on October 26, 2013 and February 7, 2014. Under field conditions, the potential quantum yield of PSII in the dark-adapted leaves (Fv/Fm) 2 h after sunset was measured with a Chl fluorometer (Mini-PAM; Walz, Germany). There was no statistical difference between values measured at predawn and those measured after sunset. Fv/Fm was calculated as (Fm Fo)/Fm, where Fo, Fm, and Fv represent minimum, maximum, and variable fluorescence yields, respectively. Fo was measured by switching on a modulated measuring light at 0.6 kHz. Fm was determined by firing the saturation pulse at 20 kHz with an 800-ms pulse of 8000 mmol photons m2 s1 of halogen light.
Leaves were punched with a cork borer (1.75 cm2) for assays of the Chl concentration. Punched leaf discs were immediately frozen in liquid nitrogen and stored in a freezer at 70 C until use. Chl were extracted with 80% (v/v) aqueous acetone and spectrophotometrically determined following the method of Porra et al. (1989).
2.4. Estimation of fractions of energy allocation and the energy flux of photochemistry and thermal energy dissipation The energy allocations of absorbed light energy in PSII were measured under room temperature with a PAM-2100 fluorometer (Walz) connected to a computer with acquisition software (PamWin 1.24; Walz). The energy allocation in PSII was estimated by the model of Yamazaki et al. (2011), which is derived from Kramer's lake model (2004). The allocation can be estimated from the equation of each of the three energy-consuming fractions as follows:
FPSII ¼
FONP ¼
0 F Fm 0 Fm
(1)
2.6. Determination of Rubisco contents Leaf discs (1.75 cm2) were punched from the central part of the lamina and stored at 70 C until use. The discs were frozen in liquid nitrogen and powdered with a chilled pestle and mortar in homogenization buffer containing 100 mM TriseHCl (pH 8.0), 10 mM NaCl, 5 mM MgCl2, 6 M urea, 5 mM monoiodoacetic acid, 5 mM EDTA, 5% (w/v) SDS, and 1 mM dithiothreitol. The homogenate was centrifuged at 10,000 g for 15 min at 10 C. The supernatant was added to 5% (v/v) 2-mercaptoethanol and 0.01% (v/v) bromophenol blue and then subjected to SDS-PAGE using 12.5% acrylamide gel and the buffer system of Laemmli (1970). The gels were stained with 0.1% (w/v) Coomassie Brilliant Blue R-250 (Wako Pure Chemicals, Osaka, Japan) and after destaining, were scanned with a flatbed scanner. Rubisco large subunit was quantified with ImageJ software (Version 1.46; NIH, http://rsbweb.nih.gov/ij/). A calibration curve for the determination of Rubisco contents was constructed from the BSA standard. A linear relationship between BSA and Rubisco contents was confirmed. 2.7. Lipid peroxidation
1 NPQ þ 1 þ qL
Fm Fo
FNPQ ¼ 1 FPSII FONP ;
(2)
1 (3)
where the SterneVolmer non-photochemical quenching (NPQ) and the fraction of open PSII reaction centers based on the lake model 0 Þ 1 and ðF 0 F=F 0 F 0 Þ$ðF 0 =FÞ, (qL) were calculated as ðFm =Fm m m o o respectively. Consequently, the sum of all quantum yields for photochemistry and the dissipative processes of the energy absorbed by PSII is expressed as FPSII þ FNPQ þ FONP ¼ 1. FPSII is the fraction of the efficient quantum yield of PSII photochemistry (Genty et al., 1989). FNPQ is the quantum yield of NPQ associated with xanthophyll-cycle-dependent energy dissipation. FONP is the quantum yield of the other non-photochemical processes (ONP) and reflects basal and dark quenching processes. Fluorescence pa0 , F 0 , and F 0 ¼ F 0 F 0 are maximum, minimum, and rameters Fm o v m o variable fluorescences during energization, and F is the steady-state fluorescence. Fo0 was determined as Fo/(Fv/Fm þ Fo/Fm0 ) from the equation of Oxborough and Baker (1997). The energy flux rates of PSII photochemistry (JPSII), those of NPQdependent energy dissipation (JNPQ), and those of other nonphotochemical processes (JONP) were measured under the same conditions and with the same apparatus. Leaves that had been dark-adapted for 30 min were exposed to stepwise sequences of halogen light (0e1500 mmol photons m2 s1). It took 40 s to obtain the stable rates at each increase in the halogen light. Assuming that PSII and PSI absorb equal amounts of light, JPSII, JNPQ, and JONP were estimated by analogy to the equation of Ishida et al. (2014): JPSII ¼ FPSII PPFD 0.5 a; JNPQ ¼ FNPQ PPFD 0.5 a; JONP ¼ FONP PPFD 0.5 a. a represents leaf absorbance estimated from the Chl concentration (Evans, 1993). The energy flux rates were fitted by the equations of Eilers and Peeters (1988).
To evaluate the extent of lipid peroxidation, the thiobarbituric acid (TBA) reaction was used following the method of Heath and Packer (1968), a useful technique for detecting lipid peroxidation as TBA-reactive substances (TBARSs). Leaves were frozen in liquid nitrogen and powdered with a chilled pestle and mortar. The powdered leaves were suspended in 0.3 M trichloroacetic acid (TCA) on ice, and the residue of the suspension was rinsed into a centrifuge tube with 1.5 ml of 0.3 M TCA. Supernatant (1.5 ml) was added to the same volume of 0.35 M TBA dissolved in 1.2 M TCA, and the mixture was heated at 95 C for 25 min. The colored mixture obtained was measured at 440 nm in addition to 532 and 600 nm, and each sample had a reference without TBA. The TBARS concentration was calculated from the equation of Hodges et al. (1999), and the millimolar extinction coefficient used was 155 mM1 cm1 (Heath and Packer, 1968). 2.8. Statistical analysis Statistical differences between autumn and winter samples were evaluated by Student's t-test with KaleidaGraph 4.5 software. The regression curves of the energy fluxes were drawn using the same software. 3. Results This study was performed in 2007/2008 and 2013/2014. The daily mean maximum and minimum temperatures from October 2007 to March 2008 were 13.6 C and 5.1 C, respectively, and those from October 2013 to March 2014 were 13.9 C and 5.2 C, respectively. The lowest temperatures were recorded on February 18, 2008 (2.2 C; Fig. 1a) and January 11, 2014 (3.1 C; Fig. 1b). Fig. 2a and b shows typical diurnal changes of PPFD on clear sunny days on November 4, 2007 and February 7, 2008, respectively. The maximum PPFD reached approximately
150
C. Tanaka et al. / Plant Physiology and Biochemistry 86 (2015) 147e154
Fig. 1. Comparison of air temperatures from September to March (a) in 2007/2008 with those (b) in 2013/2014 on the Toho University campus. Solid and dashed lines represent daily maximum and minimum temperatures, respectively.
Fig. 2. Daily courses of photosynthetic photon flux density (PPFD) (a) on November 4, 2007 (autumn) and (b) February 7, 2008 (winter). A photon sensor was mounted on a horizontal plane with respect to the earth's surface.
1200 mmol photons m2 s1 both in autumn and winter. The peak irradiance was determined from a sensor mounted on a horizontal plane with respect to the earth's surface. Measurement of net photosynthesis (Anet) for the two species in autumn and winter under field conditions revealed that the level of photosynthesis remained high even in autumn and was low in winter (Fig. 3a). The mean value of Fv/Fm remained high (approximately 0.80) in autumn and dropped to 0.60 in winter (Fig. 3b). The parameters of photosynthesis in the two species in autumn and winter under controlled environmental conditions are shown in Fig. 3cef. In Q. myrsinaefolia and M. thunbergii, the values of Amax (Fig. 3c) and Vcmax (Fig. 3d) were lower in winter than in autumn, and the apparent quantum yield of photosynthesis (4) (Fig. 3e) was significantly lower in winter than in autumn. The stomatal conductance (gs, mol H2O m2 s1) showed patterns of change similar to those of Amax in both species (Fig. 3f). The Chl concentrations in Q. myrsinaefolia and M. thunbergii showed approximately 45% and 20% decreases in winter compared with those in autumn, respectively (Fig. 4A). The Chl a/b ratio in both species markedly increased in winter (Fig. 4B). In contrast, the amount of Rubisco on a leaf area basis of the two species increased in winter (Fig. 4C). The TBARS concentration showed no difference between autumn and winter (Fig. 4D).
In both species, increases in the proportion of FPSII led to decreases in those of FONP (Fig. 5). In autumn, the proportions of FPSII and FNPQ were high, whereas in winter, the proportion of FONP was twice as high as that in autumn (Fig. 5). The estimated energy flux of photochemistry (JPSII) in the two species was approximately the same (50 mmol photons processed m2 s1) in autumn, but decreased by approximately 40% in both species in winter compared with that in autumn (Fig. 6AeD). The photon flux dissipated thermally through NPQ associated with the xanthophyll cycle (JNPQ), which was proportional to the increase in PPFD, reached maximum PPFD levels at 1400 mmol photons m2 s1. These levels were approximately seven and six times the numbers used in photochemistry in Quercus and Machilus, respectively, in autumn. However, the levels decreased by 10% and 7% in Quercus and Machilus, respectively, in winter compared with those in autumn. The photon flux dissipated thermally through ONP (JONP) reached maximum PPFD levels at 1400 mmol photons m2 s1, approximately twice the numbers those used in photochemistry in both species in autumn. However, the flux in winter was twice as high as that in autumn (Fig. 6AeD).
C. Tanaka et al. / Plant Physiology and Biochemistry 86 (2015) 147e154
151
Fig. 3. Variation in photosynthetic characteristics in Quercus myrsinaefolia and Machilus thunbergii. (a) Assimilation rate under natural sunlight (Anet); (b), maximal quantum yield of PSII in the dark-adapted leaves (Fv/Fm); (c) maximum assimilation rate (Amax) estimated from the equation of Johnson and Thornley (1984); (d) maximum carboxylation rate (Vcmax) estimated from the equation of Farquhar et al. (1980); (e) apparent quantum yield (4) estimated from the initial slope in the low PPFD region (0e100 mmol photons m2 s1); (f) stomatal conductance (gs) measured under natural sunlight. Open and closed bars represent autumn and winter, respectively. Bars represent SD (n ¼ 3e5). The difference between autumn and winter was tested using Student's t-test. Significance levels: *, P < 0.1; **, P < 0.01; ***, P < 0.001; NS, not significant.
4. Discussion Given that photosynthesis in winter contributes to the annual production of overwintering evergreens (Saeki and Nomoto, 1958), winter photosynthesis is particularly important for broadleaved evergreens (Miyazawa and Kikuzawa, 2005). However, low temperature and direct sunlight in winter create stress for over€ wintering evergreen species (Oquist and Huner, 2003; Yamazaki et al., 2007). Although broadleaved evergreen species grown in Japan are exposed to milder winter conditions than those grown in boreal and inland regions, the entire photosynthetic machinery is suppressed under winter conditions. Although the air temperature was low in winter, the irradiance received on the leaves was similar between autumn and winter (Figs. 1 and 2), indicating that the photosynthetic apparatus experiences more irradiance stress in winter than in autumn.
Under winter conditions, decreases in the photochemical efficiency of PSII (Fv/Fm, Fig. 3b) are regulatory and respond to decreases in downstream processes such as Anet (Fig. 3a), Amax (Fig. 3c), the potential rate of carboxylation by Rubisco (Vcmax) € (Fig. 3d) and the stomatal conductance (Fig. 3f) (Oquist and Huner, 2003; Miyazawa and Kikuzawa, 2005). In contrast, the amount of Rubisco showed a significant increase in winter in both species (Fig. 4D). These results suggest that a reduced electron sink in the CalvineBenson cycle would feed back to light harvesting and energy partitioning (Adams et al., 2013), to which the plants can respond by increasing FONP (see below). Depression of the Fv/Fm ratio (Fig. 3b) indicates that winter stress had direct effects on the primary photochemistry of PSII and suggests that the water-splitting complexes were inactivated in this period (Yamazaki et al., 2007). However, all water-splitting systems did not malfunction in broadleaved evergreen species, in contrast
152
C. Tanaka et al. / Plant Physiology and Biochemistry 86 (2015) 147e154
Fig. 4. Variation in leaf properties in Quercus myrsinaefolia and Machilus thunbergii. (A) Total Chl concentration; (B) Chl a/b ratio (molar ratio); (C) Rubisco contents expressed on a leaf area basis; (D) MDA contents expressed on a leaf area basis. Open and closed bars represent autumn and winter, respectively. Bars represent SD (n ¼ 5e6). The difference between autumn and winter was tested using Student's t-test. Significance levels: *, P < 0.1; **, P < 0.01; ***, P < 0.001; NS, not significant.
Fig. 5. Variation in the allocation of absorbed light energy in PSII in (a) Quercus myrsinaefolia and (b) Machilus thunbergii leaves. The fractions were calculated as FPSII ¼ (Fm0 F)/Fm0 , the fraction of light energy that is consumed by photochemistry; FNPQ ¼ 1 FPSII FONP, the fraction of the energy dissipation associated with NPQ; FONP ¼ 1/NPQ þ 1 þ qL[(Fm/ Fo) 1], the fraction of the energy dissipated through other processes.
to overwintering conifers grown in the subalpine region (Yamazaki et al., 2007). Elevation of pool size of xanthophyll-cycle pigments and daytime de-epoxidation state of xanthophyll-cycle pigments were found in the evergreens in the period from the autumn to the spring season, indicating the presence of sustained high levels of zeaxanthin and antheraxanthin and a partial downregulation of photochemical capacity with enhanced thermal dissipation through the xanthophyll cycle in winter (Adams and DemmigAdams, 1995; Verhoeven et al., 1996; Yamazaki et al., 2011). These findings indicate that zeaxanthin becomes nocturnally retained,
and typically locked in an engaged state, so that thermal energy dissipation can become instantly engaged when the sun rises over a frozen landscape at dawn (Adams and Demmig-Adams 1994; Adams et al., 1995; Verhoeven et al., 1996; Zarter et al., 2006a, 2006b). Under the sink-limited conditions of winter, photosynthesis is downregulated, including the selective removal of the watersplitting complex and D1-protein and the sustained engagement of zeaxanthin in thermal energy dissipation (Adams et al., 2004, 2006; 2013). Although the Asada scheme and the cyclic electron flow around PSI serve as a safety valve for the excess electrons
C. Tanaka et al. / Plant Physiology and Biochemistry 86 (2015) 147e154
153
Fig. 6. Estimated energy fluxes of photochemistry and thermal energy dissipation in Quercus myrsinaefolia and Machilus thunbergii leaves. Open circles, closed circles, and closed squares represent the energy fluxes of FPSII (JPSII), FNPQ (JNPQ), and FONP (JONP), respectively. Left (A, C) and right (B, D) panels represent energy fluxes in autumn and winter, respectively. Top (A, B) and bottom (C, D) panels represent leaves of Q. myrsinaefolia and M. thunbergii, respectively. All regression lines showed high coefficients (r2 0.9). Bars represent SD (n ¼ 5e6).
(Asada, 1999; Miyake, 2010), these pathways are expected to be irrelevant to winter stress (data not shown). In pine needles, the amount of the light-harvesting Chl a/bprotein complex is relatively resistant to winter stress, facilitating rapid recovery of photosynthesis in spring (Monson et al., 2005; Zarter et al., 2006b). In the present study, although the decline of the Chl concentration in winter (Fig. 4A) caused a reduction of light absorption, this response is explained by the acclimation of the photosynthetic apparatus to winter conditions (Yamazaki et al., 2007). In contrast, an increase in the Chl a/b ratio was observed (Fig. 4B). These results indicate a decline in the size of lightharvesting antennae, leading to a reduction in light use efficiency in the initial slope estimated from 4 in winter (Fig. 3e) (Okada and Katoh, 1998; Yamazaki et al., 1999). The leaf level regulation as well as the thylakoid level regulation in winter needs further investigation. In this study, the model of Kramer et al. (2004), which takes into account the predicted presence of connectivity, was used for the determination of the absorbed light energy allocation within PSII. There is now debate over which model, the isolated puddle model or the lake model, is valid. Several studies have reported the presence of connectivity between PSII units (Kramer et al., 2004; Stirbet, 2013). It is thus appropriate to estimate the thermal dissipation processes using the lake model. The proportions of FONP were higher in winter than in autumn, indicating a shift from FNPQ to FONP. Moreover, JNPQ was approximately the same in autumn and winter, whereas JONP was twice as high in winter as that in autumn
(Fig. 6). This finding would explain why the excess energy was efficiently quenched before it reached the PSII reaction centers observed in the conifers (Yokota et al., 2008). Furthermore, the concentration of TBARS, an index of lipid peroxidation, showed no difference between autumn and winter (Fig. 4D). This result indicates that attacks by active oxygen species are mitigated by primary photoprotective responses such as thermal energy dissipation (Yamazaki et al., 2007). In conclusion, this study reveals that the photosynthetic apparatus in winter in broadleaved evergreens grown in warm temperate regions is partly protected by a shift from FNPQ to FONP. Under such winter conditions, to minimize the influence of excessive light energy, the photosynthetic apparatus in overwintering leaves subjected to direct sunlight and low temperatures in warm temperate regions in Japan regulates temperatureindependent physical processes, so that thermal energy dissipation acts to divert excess excitation energy harmlessly to heat. Consequently, these responses contribute to a quick restart of photosynthesis without the development of new leaves in the following spring.
Contributions C.T. and J.Y. wrote the manuscript. C.T. and J.Y. performed experiments. C.T., J.Y., T.N. and E.M. designed this study.
154
C. Tanaka et al. / Plant Physiology and Biochemistry 86 (2015) 147e154
Acknowledgment We thank Kyoko Kamata and the all members of our laboratory for their assistance and suggestion throughout this work. Valuable comments raised by anonymous reviewers and handling editor are heartily acknowledged. We also thank Enago (http://www.enago. jp) for the English language review. References Adams III, W.W., Demmig-Adams, B., 1994. Carotenoid composition and down regulation of photosystem II in three conifer species during the winter. Physiol. Plant. 92, 451e458. Adams III, W.W., Demmig-Adams, B., 1995. The xanthophyll cycle and sustained thermal energy dissipation activity in Vinca minor and Euonymus kiautschovicus in winter. Plant Cell Environ. 18, 117e127. Adams III, W.W., Demmig-Adams, B., Verhoeven, A.S., Barker, D.H., 1995. ‘Photoinhibition’ during winter stress: involvement of sustained xanthophyll cycledependent energy dissipation. Aust. J. Plant Physiol. 22, 261e276. Adams III, W.W., Muller, O., Cohu, C.M., Demmig-Adams, B., 2013. May photoinhibition be a consequence, rather than a cause, of limited plant productivity? Photosynth. Res. 117, 31e44. Adams III, W.W., Zarter, C.R., Ebbert, V., Demmig-Adams, B., 2004. Photoprotective strategies of overwintering evergreens. BioScience 54, 41e49. Adams III, W.W., Zarter, C.R., Mueh, K.E., Amiard, V., Demmig-Adams, B., 2006. Energy dissipation and photoinhibition: a continuum of photoprotection. In: Demmig-Adams, B., Adams III, W.W., Mattoo, A.K. (Eds.), Photoprotection, Photoinhibition, Gene Regulation, and Environment, Advances in Photosynthesis and Respiration, vol. 21. Springer, Dordrecht, pp. 49e64. Asada, K., 1999. The waterewater cycle in chloroplasts: scavenging of active oxygens and dissipation of excess photons. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 601e639. Demmig-Adams, B., Adams III, W.W., 2006. Photoprotection in an ecological context: the remarkable complexity of thermal energy dissipation. New Phytol. 172, 11e21. Ebbert, V., Adams III, W.W., Mattoo, K.K., Sokolenko, A., Demmig-Adams, B., 2005. Upregulation of a PSII core protein phosphatase inhibitor and sustained D1 phosphorylation in zeaxanthin-retaining, photoinhibited needles of overwintering Douglas fir. Plant Cell Environ. 28, 232e240. Eilers, P.H.C., Peeters, J.C.H., 1988. A model for the relationship between light intensity and the rate of photosynthesis in phytoplankton. Ecol. Model. 42, 199e215. Evans, J.R., 1993. Photosynthetic acclimation and nitrogen partitioning within a lucerne canopy. II Stability through time and comparison with a theoretical optimum. Aust. J. Plant Physiol. 20, 69e82. Farquhar, G.D., Caemmerer, S., Berry, J.A., 1980. A biochemical model of photosynthetic CO2 assimilation in leaves of C3 species. Planta 149, 78e90. Genty, B., Briantais, J.M., Baker, N.R., 1989. The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochim. Biophys. Acta 99, 87e92. Heath, R.L., Packer, L., 1968. Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation. Arch. Biochem. Biophys. 125, 189e198. Hodges, D.M., DeLong, J.M., Forney, C.F., Prange, R.K., 1999. Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. Planta 207, 604e611. Ishida, S., Uebayashi, N., Tazoe, Y., Ikeuchi, M., Homma, K., Sato, F., Endo, T., 2014. Diurnal and developmental changes in energy allocation of absorbed light at PSII in field-grown rice. Plant Cell Physiol. 55, 171e182. Johnson, I.R., Thornley, J.H.M., 1984. A model of instantaneous and daily canopy photosynthesis. J. Theor. Biol. 107, 531e545. Kramer, D.M., Johnson, G., Kiirats, O., Edwards, G.E., 2004. New fluorescence parameters for the determination of QA redox state and excitation energy fluxes. Photosynth. Res. 79, 209e218. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680e685.
Miyake, C., 2010. Alternative electron flows (water-water cycle and cyclic electron flow around PSI) in photosynthesis: molecular mechanisms and physiological functions. Plant Cell Physiol. 51, 1951e1963. Miyazawa, Y., Kikuzawa, K., 2005. Winter photosynthesis by saplings of evergreen broadleaved trees in a deciduous temperate forest. New Phytol. 165, 857e866. Monson, R.K., Sparks, J.P., Rosenstiel, T.N., Scott-Denton, L.E., Huxman, T.E., Harley, P.C., Turnipseed, A.A., Burns, S.P., Backlund, B., Hu, J., 2005. Climatic influences on net ecosystem CO2 exchange during the transition from wintertime carbon source to springtime carbon sink in a high-elevation, subalpine forest. Oecologia 146, 130e147. Ohsawa, M., 1990. An interpretation of latitudinal patterns of forest limits in south and east Asian mountains. J. Ecol. 78, 326e339. Okada, K., Katoh, S., 1998. Two long-term effects of light that control the stability of proteins related to photosynthesis during senescence of rice leaves. Plant Cell Physiol. 39, 394e404. € Oquist, G., Huner, N.P.A., 2003. Photosynthesis of overwintering evergreen plants. Annu. Rev. Plant Biol. 54, 329e355. € Ottander, C., Campbell, D., Oquist, G., 1995. Seasonal changes in photosystem II organisation and pigment composition in Pinus sylvestris. Planta 197, 176e183. Oxborough, K., Baker, N.R., 1997. Resolving chlorophyll fluorescence images photosynthetic efficiency into photochemical and non-photochemical components-calculation of qP and Fv0 /Fm0 without measuring Fo0 . Photosynth. Res. 54, 135e142. Porra, R.J., Thompson, W.A., Kriedemann, P.E., 1989. Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophyll a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim. Biophys. Acta 975, 384e394. Saeki, T., Nomoto, N., 1958. On the seasonal change of photosynthetic activity of some deciduous and evergreen broadleaf trees. Bot. Mag. Tokyo 71, 235e241. Sakai, A., 1975. Freezing resistance of evergreen and deciduous broad-leaf trees in Japan with special reference to their distributions. Jpn. J. Ecol. 25, 101e111 (in Japanese). Stirbet, A., 2013. Excitonic connectivity between photosystem II units: what is it, and how to measure it? Photosynth. Res. 116, 189e214. Taneda, H., Tateno, M., 2005. Hydraulic conductivity, photosynthesis and leaf water balance in six evergreen woody species from fall to winter. Tree Physiol. 25, 299e306. Verhoeven, A.S., Adams III, W.W., Demmig-Adams, B., 1996. Close relationship between the state of the xanthophyll cycle pigments and photosystem II efficiency during recovery from winter stress. Physiol. Plant. 96, 567e576. Verhoeven, A.S., Osmolak, A., Morales, P., Crow, J., 2009. Seasonal changes in abundance and phosphorylation status of photosynthetic proteins in eastern white pine and balsam fir. Tree Physiol. 29, 361e374. Yamazaki, J., Kamimura, Y., Okada, M., Sugimura, Y., 1999. Changes in photosynthetic characteristics and photosystem stoichiometries in the lower leaves in rice seedlings. Plant Sci. 148, 155e163. Yamazaki, J., Kamata, K., Maruta, E., 2011. Seasonal changes in the excess energy dissipation from photosystem II antennae in overwintering evergreen broadleaved trees Quercus myrsinaefolia and Machilus thunbergii. J. Photochem. Photobiol. 104B, 348e356. Yamazaki, J., Ohashi, A., Hashimoto, Y., Negishi, E., Kumagai, S., Kubo, T., Oikawa, T., Maruta, E., Kamimura, Y., 2003. Effects of high light and low temperature during harsh winter on needle photodamage of Abies mariesii growing at the forest limit on Mt. Norikura in central Japan. Plant Sci. 165, 257e264. Yamazaki, J., Tsuchiya, S., Nagano, S., Maruta, E., 2007. Photoprotective mechanisms against winter stresses in the needles of Abies mariesii grown at the tree line on Mt. Norikura in Central Japan. Photosynthetica 45, 547e554. Yokota, M., Akimoto, S., Tanaka, A., 2008. Seasonal changes of excitation energy transfer and thylakoid stacking in the evergreen tree Taxus cuspidate: how does it divert excess energy from photosynthetic reaction center? Biochim. Biophys. Acta 1777, 379e387. Zarter, C.R., Adams III, W.W., Ebbert, V., Cuthbertson, D., Adamska, I., DemmigAdams, B., 2006a. Winter downregulation of intrinsic photosynthetic capacity coupled with up-regulation of Elip-like proteins and persistent energy dissipation in a subalpine forest. New Phytol. 172, 272e282. Zarter, C.R., Demmig-Adams, B., Ebbert, V., Adamska, I., Adams III, W.W., 2006b. Photosynthetic capacity and light harvesting efficiency during the winter-tospring transition in subalpine conifers. New Phytol. 172, 283e292.
Contents lists available at ScienceDirect
Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy
Research article
Responses of the photosynthetic apparatus to winter conditions in broadleaved evergreen trees growing in warm temperate regions of Japan Chizuru Tanaka a, Takashi Nakano b, Jun-ya Yamazaki a, *, Emiko Maruta a a b
Department of Biology, Faculty of Science, Toho University, Miyama 2-2-1, Funabashi, Chiba 274-8510, Japan Mount Fuji Research Institute, Yamanashi Prefectural Government (MFRI), Kenmarubi 5597-1, Kamiyoshida, Fujiyoshida City, Yamanashi 403-0005, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 September 2014 Accepted 2 December 2014 Available online 3 December 2014
Photosynthetic characteristics of two broadleaved evergreen trees, Quercus myrsinaefolia and Machilus thunbergii, were compared in autumn and winter. The irradiance was similar in both seasons, but the air temperature was lower in winter. Under the winter conditions, net photosynthesis under natural sunlight (Anet) in both species dropped to 4 mmol CO2 m2 s1, and the quantum yield of photosystem II (PSII) photochemistry in dark-adapted leaves (Fv/Fm) also dropped to 0.60. In both species the maximum carboxylation rates of Rubisco (Vcmax) decreased, and the amount of Rubisco increased in winter. A decline in chlorophyll (Chl) concentration and an increase in the Chl a/b ratio in winter resulted in a reduction in the size of the light-harvesting antennae. From measurements of Chl a fluorescence parameters, both the relative fraction and the energy flux rates of thermal dissipation through other nonphotochemical processes were markedly elevated in winter. The results indicate that the photosynthetic apparatus in broadleaved evergreen species in warm temperate regions responds to winter through regulatory mechanisms involving the downregulation of light-harvesting and photosynthesis coupled with increased photoprotective thermal energy dissipation to minimize photodamage in winter. These mechanisms aid a quick restart of photosynthesis without the development of new leaves in the following spring. © 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Broadleaved evergreens Energy flux Energy partition Quercus myrsinaefolia Machilus thunbergii
1. Introduction
Abbreviations: Amax, the potential photosynthetic rate; Anet, the net photosynthesis under natural sunlight; Chl, Chlorophyll; F, steady-state fluorescence; Fm, Fo and Fv, the maximum, minimum and variable fluorescence yields, respectively; Fm0 , Fo0 and Fv0, the maximum, minimum and variable fluorescence yields, respectively, during energization; Fv/Fm, the quantum yield of PSII photochemistry in darkadapted leaves; gs, stomatal conductance; JNPQ, energy flux rate of NPQ associated with the xanthophyll cycle; JONP, energy flux rate of non-photochemical processes not associated with the xanthophyll cycle; JPSII, energy flux rate of PSII photochemistry; NPQ, non-photochemical quenching of chlorophyll fluorescence; PPFD, photosynthetic photon flux density; PSI and PSII, photosystem I and photosystem II, respectively; qL, the fraction of open PSII reaction centers based on the lake model; TBARS, thiobarbituric acid reaction substance; Vcmax, the maximum carboxylation rates of Rubisco; 4, the apparent quantum yield of CO2 fixation on an illuminatedlight basis; FNPQ, the quantum yield of non-photochemical quenching associated with the xanthophyll cycle; FONP, the quantum yield of non-photochemical processes not associated with the xanthophyll cycle; FPSII, the quantum yield of PSII photochemistry. * Corresponding author. E-mail address: [email protected] (J.-y. Yamazaki). http://dx.doi.org/10.1016/j.plaphy.2014.12.002 0981-9428/© 2014 Elsevier Masson SAS. All rights reserved.
Broadleaved evergreen species grown in the monsoonal region originated in warm regions and spread from central Japan westwards across a large belt of southern China, including Taiwan, to the southern side of the Himalayan mountains (Ohsawa, 1990). The species growing in this region have large, very thick, often shiny leaves. In this region, the climate features high levels of precipitation and high temperatures in summer, but is somewhat xeric with low temperatures in winter. The trees endemic to this area are confined exclusively to oceanic, mild winter areas such as those in Japan (Sakai, 1975). Quercus species, which are distributed from the southern end to 38 N in Japan, are the major canopy components of warm temperate broadleaved evergreen forests. The distribution of Machilus overlaps with that of Quercus, and the Machilus species observed in warmer coastal regions are exposed to warm currents (the Black Current on the Pacific Ocean side and the Tsushima Current on the Japan Sea side) that reach as far north as the Tohoku
148
C. Tanaka et al. / Plant Physiology and Biochemistry 86 (2015) 147e154
district (below 40 N), which is the northern limit of the Machilus species. There have been two major findings with respect to the determinants of the distribution of broadleaved evergreen trees. Sakai (1975) determined the freezing tolerance of these trees on the basis of the freezing temperatures in the stems, leaves, and buds, and concluded that cold tolerance is the main determinant of the distribution of broadleaved evergreen trees. Taneda and Tateno (2005) reported that evergreen broadleaved trees with large-diameter vessels were significantly more vulnerable to freezeethaw embolism than were evergreen conifers with small-diameter tracheids. This result suggested that the cross-sectional diameter of the xylem elements is one of the important determinants of the distribution of evergreen woody species. Photosynthetic activities are lower at low temperatures in winter than at optimal or high temperatures in the primary growing season. Evergreen conifers growing under harsh winter conditions in subalpine regions exhibit downregulation in both capacity of photosynthesis and efficiency of photosystem (PS) II (Zarter et al., 2006a, 2006b; Yamazaki et al., 2003, 2007). In contrast, those at lower altitudes may or may not exhibit downregulation of photosynthetic capacity in winter (Adams et al., 2004; Zarter et al., 2006a) but invariably experience downregulation of PSII efficiency (Adams and Demmig-Adams, 1994; Adams et al., 1995, 2004; Ebbert et al., 2005; Zarter et al., 2006a). Broadleaved evergreen trees show lowered photosynthetic activity in winter (Adams and Demmig-Adams, 1995; Adams et al., 1995; Verhoeven et al., 1996). In addition, given that broadleaved trees continue to perform photosynthesis under direct sunlight throughout winter, winter photosynthesis may exceed that in the growing season and account for a large part of the annual photosynthesis (Miyazawa and Kikuzawa, 2005). Winter stress induces suppression of the photochemical effi€ ciency of PSII and the CalvineBenson-cycle enzymes (Oquist and Huner, 2003). Excessive photons reduce molecular oxygen, resulting in the production of harmful active oxygen species and severe photoinhibition of photosynthesis (Ottander et al., 1995; Yamazaki et al., 2007). The photosynthetic apparatus of overwintering evergreen trees must accordingly develop protective mechanisms against these stresses to survive the winter and recover their capacity for photosynthesis by the following spring (Ottander et al., 1995; Miyazawa and Kikuzawa, 2005; Yamazaki et al., 2003, 2007). However, in winter, broadleaved evergreen species in warm temperate regions of East Asia are exposed to milder winter conditions than those in boreal and continental climate regions. Under mild winter conditions, the PSII complexes are not degraded but phosphorylated, owing to the fast recovery of overall photosynthesis when the climate becomes warm (Ebbert et al., 2005; Demmig-Adams and Adams, 2006; Verhoeven et al., 2009). Studies of xanthophyll cycle-related energy dissipation from antennae attached to the PSII complexes in winter have been performed (Adams et al., 1995, 2004; Yamazaki et al., 2011). We previously reported the involvement of thermal dissipation and changes in chlorophyll (Chl) forms in overwintering broadleaved evergreen species (Yamazaki et al., 2011). The present study, by comparing leaves in autumn with those in winter, attempts to elucidate in further detail how mechanisms of thermal energy dissipation are involved in the protection of the photosynthetic apparatus in overwintering broadleaved evergreens in warm temperate regions of Japan.
2. Materials and methods 2.1. Study site and plant materials All measurements were performed on the campus of Toho University, Funabashi, Japan (latitude 35 410 N, longitude 140 20 E; altitude 20 m), located in a warm temperate region. Two evergreen species [Quercus myrsinaefolia (Fagaceae) and Machilus thunbergii (Lauraceae)] were used. The heights of the Machilus and Quercus trees included in the survey were approximately 8 and 10 m, respectively, and their diameters at breast height were approximately 30 and 20 cm, respectively. Photosynthetic photon flux density (PPFD) was measured with a photon sensor (IKS-27; Koito Co., Japan) mounted on a horizontal plane with respect to the earth's surface, and the data obtained were recorded by a data logger (KADEC-UP; KONA system, Japan) at 10-min intervals. Maximum and minimum air temperatures were 31.1 C (October 5, 2007)/2.2 C (February 18, 2008) and 29.6 C (October 12, 2013)/ 3.1 C (January 11, 2014), obtained from an automated meteorological data acquisition system (AMeDAS) at Funabashi Meteorological Observatory (latitude 35 420 N, longitude 140 20 E, altitude: 28 m) provided by the Japan Meteorological Agency (JMA; http:// www.jma.go.jp/). Between the gap of the two experiments (2007e2014) there were no environmental and characteristic differences such as light conditions, the features of the campus, and current year's leaf characteristics at the study site. In addition, the significant climate changes were not observed from the data obtained from the JMA about the gap of multiple years between the two experiments (2007e2014). Hereafter, the data of November 13, 2007 and October 26, 2013 will be referred to as autumn data, and the data of February 1, 2008 and February 7, 2014 as winter data. 2.2. Measurements of photosynthetic parameters Photosynthetic parameters were measured on sun-exposed current-year leaves on November 13, 2007 and February 1, 2008. Under field conditions, net photosynthesis (Anet) of the leaves was measured between 11:00 and 14:00 (local time) with a portable gas-exchange analyzer (LI-6400; LI-COR, Inc., USA) attached to a transparent standard leaf chamber (6400-08; LI-COR, Inc.). The potential photosynthetic rate (Amax), the apparent quantum yield of CO2 fixation on an illuminated-light basis (4), and the potential rate of carboxylation by Rubisco (Vcmax) were measured under ambient temperature with an LI-6400 attached to an LED light and CO2 concentration control unit (6400-02B; LI-COR, Inc.). For the determination of Amax, the net photosynthetic rates at 10 stepwise actinic light intensity intervals from 0 to 1500 mmol photons m2 s1 were measured under a cuvette CO2 concentration of 370 mmol mol1. It took 3e4 min to obtain a stable photosynthetic rates at each reduction in the LED intensity. Using these data, Amax was calculated by the equation of Johnson and Thornley (1984). The value of 4 was estimated by linear regression at PPFD <100 mmol m2 s1. For the determination of Vcmax, the net photosynthetic rate was measured at 10 stepwise cuvette CO2 concentrations varying 0e2000 mmol mol1 under an illuminated irradiance fixed at 1200 mmol photons m2 s1 (at which the photosynthetic rates reached saturation). It took 4e5 min to obtain a stable photosynthetic rate at each increase in the cuvette CO2 concentration. Assuming that, at a low partial pressure of intercellular CO2, the assimilation of CO2 was limited solely by the amount, activity, and kinetic properties of Rubisco, Vcmax was calculated by the method of Farquhar et al. (1980). The stomatal conductance at saturating PPFD and a CO2 concentration of 370 mmol mol1 (gs, mol m2 s1) was measured concomitantly as photosynthesis under a controlled environment.
C. Tanaka et al. / Plant Physiology and Biochemistry 86 (2015) 147e154
149
2.3. Chl fluorescence measurements
2.5. Determination of Chl concentration
Chl fluorescence parameters were measured on October 26, 2013 and February 7, 2014. Under field conditions, the potential quantum yield of PSII in the dark-adapted leaves (Fv/Fm) 2 h after sunset was measured with a Chl fluorometer (Mini-PAM; Walz, Germany). There was no statistical difference between values measured at predawn and those measured after sunset. Fv/Fm was calculated as (Fm Fo)/Fm, where Fo, Fm, and Fv represent minimum, maximum, and variable fluorescence yields, respectively. Fo was measured by switching on a modulated measuring light at 0.6 kHz. Fm was determined by firing the saturation pulse at 20 kHz with an 800-ms pulse of 8000 mmol photons m2 s1 of halogen light.
Leaves were punched with a cork borer (1.75 cm2) for assays of the Chl concentration. Punched leaf discs were immediately frozen in liquid nitrogen and stored in a freezer at 70 C until use. Chl were extracted with 80% (v/v) aqueous acetone and spectrophotometrically determined following the method of Porra et al. (1989).
2.4. Estimation of fractions of energy allocation and the energy flux of photochemistry and thermal energy dissipation The energy allocations of absorbed light energy in PSII were measured under room temperature with a PAM-2100 fluorometer (Walz) connected to a computer with acquisition software (PamWin 1.24; Walz). The energy allocation in PSII was estimated by the model of Yamazaki et al. (2011), which is derived from Kramer's lake model (2004). The allocation can be estimated from the equation of each of the three energy-consuming fractions as follows:
FPSII ¼
FONP ¼
0 F Fm 0 Fm
(1)
2.6. Determination of Rubisco contents Leaf discs (1.75 cm2) were punched from the central part of the lamina and stored at 70 C until use. The discs were frozen in liquid nitrogen and powdered with a chilled pestle and mortar in homogenization buffer containing 100 mM TriseHCl (pH 8.0), 10 mM NaCl, 5 mM MgCl2, 6 M urea, 5 mM monoiodoacetic acid, 5 mM EDTA, 5% (w/v) SDS, and 1 mM dithiothreitol. The homogenate was centrifuged at 10,000 g for 15 min at 10 C. The supernatant was added to 5% (v/v) 2-mercaptoethanol and 0.01% (v/v) bromophenol blue and then subjected to SDS-PAGE using 12.5% acrylamide gel and the buffer system of Laemmli (1970). The gels were stained with 0.1% (w/v) Coomassie Brilliant Blue R-250 (Wako Pure Chemicals, Osaka, Japan) and after destaining, were scanned with a flatbed scanner. Rubisco large subunit was quantified with ImageJ software (Version 1.46; NIH, http://rsbweb.nih.gov/ij/). A calibration curve for the determination of Rubisco contents was constructed from the BSA standard. A linear relationship between BSA and Rubisco contents was confirmed. 2.7. Lipid peroxidation
1 NPQ þ 1 þ qL
Fm Fo
FNPQ ¼ 1 FPSII FONP ;
(2)
1 (3)
where the SterneVolmer non-photochemical quenching (NPQ) and the fraction of open PSII reaction centers based on the lake model 0 Þ 1 and ðF 0 F=F 0 F 0 Þ$ðF 0 =FÞ, (qL) were calculated as ðFm =Fm m m o o respectively. Consequently, the sum of all quantum yields for photochemistry and the dissipative processes of the energy absorbed by PSII is expressed as FPSII þ FNPQ þ FONP ¼ 1. FPSII is the fraction of the efficient quantum yield of PSII photochemistry (Genty et al., 1989). FNPQ is the quantum yield of NPQ associated with xanthophyll-cycle-dependent energy dissipation. FONP is the quantum yield of the other non-photochemical processes (ONP) and reflects basal and dark quenching processes. Fluorescence pa0 , F 0 , and F 0 ¼ F 0 F 0 are maximum, minimum, and rameters Fm o v m o variable fluorescences during energization, and F is the steady-state fluorescence. Fo0 was determined as Fo/(Fv/Fm þ Fo/Fm0 ) from the equation of Oxborough and Baker (1997). The energy flux rates of PSII photochemistry (JPSII), those of NPQdependent energy dissipation (JNPQ), and those of other nonphotochemical processes (JONP) were measured under the same conditions and with the same apparatus. Leaves that had been dark-adapted for 30 min were exposed to stepwise sequences of halogen light (0e1500 mmol photons m2 s1). It took 40 s to obtain the stable rates at each increase in the halogen light. Assuming that PSII and PSI absorb equal amounts of light, JPSII, JNPQ, and JONP were estimated by analogy to the equation of Ishida et al. (2014): JPSII ¼ FPSII PPFD 0.5 a; JNPQ ¼ FNPQ PPFD 0.5 a; JONP ¼ FONP PPFD 0.5 a. a represents leaf absorbance estimated from the Chl concentration (Evans, 1993). The energy flux rates were fitted by the equations of Eilers and Peeters (1988).
To evaluate the extent of lipid peroxidation, the thiobarbituric acid (TBA) reaction was used following the method of Heath and Packer (1968), a useful technique for detecting lipid peroxidation as TBA-reactive substances (TBARSs). Leaves were frozen in liquid nitrogen and powdered with a chilled pestle and mortar. The powdered leaves were suspended in 0.3 M trichloroacetic acid (TCA) on ice, and the residue of the suspension was rinsed into a centrifuge tube with 1.5 ml of 0.3 M TCA. Supernatant (1.5 ml) was added to the same volume of 0.35 M TBA dissolved in 1.2 M TCA, and the mixture was heated at 95 C for 25 min. The colored mixture obtained was measured at 440 nm in addition to 532 and 600 nm, and each sample had a reference without TBA. The TBARS concentration was calculated from the equation of Hodges et al. (1999), and the millimolar extinction coefficient used was 155 mM1 cm1 (Heath and Packer, 1968). 2.8. Statistical analysis Statistical differences between autumn and winter samples were evaluated by Student's t-test with KaleidaGraph 4.5 software. The regression curves of the energy fluxes were drawn using the same software. 3. Results This study was performed in 2007/2008 and 2013/2014. The daily mean maximum and minimum temperatures from October 2007 to March 2008 were 13.6 C and 5.1 C, respectively, and those from October 2013 to March 2014 were 13.9 C and 5.2 C, respectively. The lowest temperatures were recorded on February 18, 2008 (2.2 C; Fig. 1a) and January 11, 2014 (3.1 C; Fig. 1b). Fig. 2a and b shows typical diurnal changes of PPFD on clear sunny days on November 4, 2007 and February 7, 2008, respectively. The maximum PPFD reached approximately
150
C. Tanaka et al. / Plant Physiology and Biochemistry 86 (2015) 147e154
Fig. 1. Comparison of air temperatures from September to March (a) in 2007/2008 with those (b) in 2013/2014 on the Toho University campus. Solid and dashed lines represent daily maximum and minimum temperatures, respectively.
Fig. 2. Daily courses of photosynthetic photon flux density (PPFD) (a) on November 4, 2007 (autumn) and (b) February 7, 2008 (winter). A photon sensor was mounted on a horizontal plane with respect to the earth's surface.
1200 mmol photons m2 s1 both in autumn and winter. The peak irradiance was determined from a sensor mounted on a horizontal plane with respect to the earth's surface. Measurement of net photosynthesis (Anet) for the two species in autumn and winter under field conditions revealed that the level of photosynthesis remained high even in autumn and was low in winter (Fig. 3a). The mean value of Fv/Fm remained high (approximately 0.80) in autumn and dropped to 0.60 in winter (Fig. 3b). The parameters of photosynthesis in the two species in autumn and winter under controlled environmental conditions are shown in Fig. 3cef. In Q. myrsinaefolia and M. thunbergii, the values of Amax (Fig. 3c) and Vcmax (Fig. 3d) were lower in winter than in autumn, and the apparent quantum yield of photosynthesis (4) (Fig. 3e) was significantly lower in winter than in autumn. The stomatal conductance (gs, mol H2O m2 s1) showed patterns of change similar to those of Amax in both species (Fig. 3f). The Chl concentrations in Q. myrsinaefolia and M. thunbergii showed approximately 45% and 20% decreases in winter compared with those in autumn, respectively (Fig. 4A). The Chl a/b ratio in both species markedly increased in winter (Fig. 4B). In contrast, the amount of Rubisco on a leaf area basis of the two species increased in winter (Fig. 4C). The TBARS concentration showed no difference between autumn and winter (Fig. 4D).
In both species, increases in the proportion of FPSII led to decreases in those of FONP (Fig. 5). In autumn, the proportions of FPSII and FNPQ were high, whereas in winter, the proportion of FONP was twice as high as that in autumn (Fig. 5). The estimated energy flux of photochemistry (JPSII) in the two species was approximately the same (50 mmol photons processed m2 s1) in autumn, but decreased by approximately 40% in both species in winter compared with that in autumn (Fig. 6AeD). The photon flux dissipated thermally through NPQ associated with the xanthophyll cycle (JNPQ), which was proportional to the increase in PPFD, reached maximum PPFD levels at 1400 mmol photons m2 s1. These levels were approximately seven and six times the numbers used in photochemistry in Quercus and Machilus, respectively, in autumn. However, the levels decreased by 10% and 7% in Quercus and Machilus, respectively, in winter compared with those in autumn. The photon flux dissipated thermally through ONP (JONP) reached maximum PPFD levels at 1400 mmol photons m2 s1, approximately twice the numbers those used in photochemistry in both species in autumn. However, the flux in winter was twice as high as that in autumn (Fig. 6AeD).
C. Tanaka et al. / Plant Physiology and Biochemistry 86 (2015) 147e154
151
Fig. 3. Variation in photosynthetic characteristics in Quercus myrsinaefolia and Machilus thunbergii. (a) Assimilation rate under natural sunlight (Anet); (b), maximal quantum yield of PSII in the dark-adapted leaves (Fv/Fm); (c) maximum assimilation rate (Amax) estimated from the equation of Johnson and Thornley (1984); (d) maximum carboxylation rate (Vcmax) estimated from the equation of Farquhar et al. (1980); (e) apparent quantum yield (4) estimated from the initial slope in the low PPFD region (0e100 mmol photons m2 s1); (f) stomatal conductance (gs) measured under natural sunlight. Open and closed bars represent autumn and winter, respectively. Bars represent SD (n ¼ 3e5). The difference between autumn and winter was tested using Student's t-test. Significance levels: *, P < 0.1; **, P < 0.01; ***, P < 0.001; NS, not significant.
4. Discussion Given that photosynthesis in winter contributes to the annual production of overwintering evergreens (Saeki and Nomoto, 1958), winter photosynthesis is particularly important for broadleaved evergreens (Miyazawa and Kikuzawa, 2005). However, low temperature and direct sunlight in winter create stress for over€ wintering evergreen species (Oquist and Huner, 2003; Yamazaki et al., 2007). Although broadleaved evergreen species grown in Japan are exposed to milder winter conditions than those grown in boreal and inland regions, the entire photosynthetic machinery is suppressed under winter conditions. Although the air temperature was low in winter, the irradiance received on the leaves was similar between autumn and winter (Figs. 1 and 2), indicating that the photosynthetic apparatus experiences more irradiance stress in winter than in autumn.
Under winter conditions, decreases in the photochemical efficiency of PSII (Fv/Fm, Fig. 3b) are regulatory and respond to decreases in downstream processes such as Anet (Fig. 3a), Amax (Fig. 3c), the potential rate of carboxylation by Rubisco (Vcmax) € (Fig. 3d) and the stomatal conductance (Fig. 3f) (Oquist and Huner, 2003; Miyazawa and Kikuzawa, 2005). In contrast, the amount of Rubisco showed a significant increase in winter in both species (Fig. 4D). These results suggest that a reduced electron sink in the CalvineBenson cycle would feed back to light harvesting and energy partitioning (Adams et al., 2013), to which the plants can respond by increasing FONP (see below). Depression of the Fv/Fm ratio (Fig. 3b) indicates that winter stress had direct effects on the primary photochemistry of PSII and suggests that the water-splitting complexes were inactivated in this period (Yamazaki et al., 2007). However, all water-splitting systems did not malfunction in broadleaved evergreen species, in contrast
152
C. Tanaka et al. / Plant Physiology and Biochemistry 86 (2015) 147e154
Fig. 4. Variation in leaf properties in Quercus myrsinaefolia and Machilus thunbergii. (A) Total Chl concentration; (B) Chl a/b ratio (molar ratio); (C) Rubisco contents expressed on a leaf area basis; (D) MDA contents expressed on a leaf area basis. Open and closed bars represent autumn and winter, respectively. Bars represent SD (n ¼ 5e6). The difference between autumn and winter was tested using Student's t-test. Significance levels: *, P < 0.1; **, P < 0.01; ***, P < 0.001; NS, not significant.
Fig. 5. Variation in the allocation of absorbed light energy in PSII in (a) Quercus myrsinaefolia and (b) Machilus thunbergii leaves. The fractions were calculated as FPSII ¼ (Fm0 F)/Fm0 , the fraction of light energy that is consumed by photochemistry; FNPQ ¼ 1 FPSII FONP, the fraction of the energy dissipation associated with NPQ; FONP ¼ 1/NPQ þ 1 þ qL[(Fm/ Fo) 1], the fraction of the energy dissipated through other processes.
to overwintering conifers grown in the subalpine region (Yamazaki et al., 2007). Elevation of pool size of xanthophyll-cycle pigments and daytime de-epoxidation state of xanthophyll-cycle pigments were found in the evergreens in the period from the autumn to the spring season, indicating the presence of sustained high levels of zeaxanthin and antheraxanthin and a partial downregulation of photochemical capacity with enhanced thermal dissipation through the xanthophyll cycle in winter (Adams and DemmigAdams, 1995; Verhoeven et al., 1996; Yamazaki et al., 2011). These findings indicate that zeaxanthin becomes nocturnally retained,
and typically locked in an engaged state, so that thermal energy dissipation can become instantly engaged when the sun rises over a frozen landscape at dawn (Adams and Demmig-Adams 1994; Adams et al., 1995; Verhoeven et al., 1996; Zarter et al., 2006a, 2006b). Under the sink-limited conditions of winter, photosynthesis is downregulated, including the selective removal of the watersplitting complex and D1-protein and the sustained engagement of zeaxanthin in thermal energy dissipation (Adams et al., 2004, 2006; 2013). Although the Asada scheme and the cyclic electron flow around PSI serve as a safety valve for the excess electrons
C. Tanaka et al. / Plant Physiology and Biochemistry 86 (2015) 147e154
153
Fig. 6. Estimated energy fluxes of photochemistry and thermal energy dissipation in Quercus myrsinaefolia and Machilus thunbergii leaves. Open circles, closed circles, and closed squares represent the energy fluxes of FPSII (JPSII), FNPQ (JNPQ), and FONP (JONP), respectively. Left (A, C) and right (B, D) panels represent energy fluxes in autumn and winter, respectively. Top (A, B) and bottom (C, D) panels represent leaves of Q. myrsinaefolia and M. thunbergii, respectively. All regression lines showed high coefficients (r2 0.9). Bars represent SD (n ¼ 5e6).
(Asada, 1999; Miyake, 2010), these pathways are expected to be irrelevant to winter stress (data not shown). In pine needles, the amount of the light-harvesting Chl a/bprotein complex is relatively resistant to winter stress, facilitating rapid recovery of photosynthesis in spring (Monson et al., 2005; Zarter et al., 2006b). In the present study, although the decline of the Chl concentration in winter (Fig. 4A) caused a reduction of light absorption, this response is explained by the acclimation of the photosynthetic apparatus to winter conditions (Yamazaki et al., 2007). In contrast, an increase in the Chl a/b ratio was observed (Fig. 4B). These results indicate a decline in the size of lightharvesting antennae, leading to a reduction in light use efficiency in the initial slope estimated from 4 in winter (Fig. 3e) (Okada and Katoh, 1998; Yamazaki et al., 1999). The leaf level regulation as well as the thylakoid level regulation in winter needs further investigation. In this study, the model of Kramer et al. (2004), which takes into account the predicted presence of connectivity, was used for the determination of the absorbed light energy allocation within PSII. There is now debate over which model, the isolated puddle model or the lake model, is valid. Several studies have reported the presence of connectivity between PSII units (Kramer et al., 2004; Stirbet, 2013). It is thus appropriate to estimate the thermal dissipation processes using the lake model. The proportions of FONP were higher in winter than in autumn, indicating a shift from FNPQ to FONP. Moreover, JNPQ was approximately the same in autumn and winter, whereas JONP was twice as high in winter as that in autumn
(Fig. 6). This finding would explain why the excess energy was efficiently quenched before it reached the PSII reaction centers observed in the conifers (Yokota et al., 2008). Furthermore, the concentration of TBARS, an index of lipid peroxidation, showed no difference between autumn and winter (Fig. 4D). This result indicates that attacks by active oxygen species are mitigated by primary photoprotective responses such as thermal energy dissipation (Yamazaki et al., 2007). In conclusion, this study reveals that the photosynthetic apparatus in winter in broadleaved evergreens grown in warm temperate regions is partly protected by a shift from FNPQ to FONP. Under such winter conditions, to minimize the influence of excessive light energy, the photosynthetic apparatus in overwintering leaves subjected to direct sunlight and low temperatures in warm temperate regions in Japan regulates temperatureindependent physical processes, so that thermal energy dissipation acts to divert excess excitation energy harmlessly to heat. Consequently, these responses contribute to a quick restart of photosynthesis without the development of new leaves in the following spring.
Contributions C.T. and J.Y. wrote the manuscript. C.T. and J.Y. performed experiments. C.T., J.Y., T.N. and E.M. designed this study.
154
C. Tanaka et al. / Plant Physiology and Biochemistry 86 (2015) 147e154
Acknowledgment We thank Kyoko Kamata and the all members of our laboratory for their assistance and suggestion throughout this work. Valuable comments raised by anonymous reviewers and handling editor are heartily acknowledged. We also thank Enago (http://www.enago. jp) for the English language review. References Adams III, W.W., Demmig-Adams, B., 1994. Carotenoid composition and down regulation of photosystem II in three conifer species during the winter. Physiol. Plant. 92, 451e458. Adams III, W.W., Demmig-Adams, B., 1995. The xanthophyll cycle and sustained thermal energy dissipation activity in Vinca minor and Euonymus kiautschovicus in winter. Plant Cell Environ. 18, 117e127. Adams III, W.W., Demmig-Adams, B., Verhoeven, A.S., Barker, D.H., 1995. ‘Photoinhibition’ during winter stress: involvement of sustained xanthophyll cycledependent energy dissipation. Aust. J. Plant Physiol. 22, 261e276. Adams III, W.W., Muller, O., Cohu, C.M., Demmig-Adams, B., 2013. May photoinhibition be a consequence, rather than a cause, of limited plant productivity? Photosynth. Res. 117, 31e44. Adams III, W.W., Zarter, C.R., Ebbert, V., Demmig-Adams, B., 2004. Photoprotective strategies of overwintering evergreens. BioScience 54, 41e49. Adams III, W.W., Zarter, C.R., Mueh, K.E., Amiard, V., Demmig-Adams, B., 2006. Energy dissipation and photoinhibition: a continuum of photoprotection. In: Demmig-Adams, B., Adams III, W.W., Mattoo, A.K. (Eds.), Photoprotection, Photoinhibition, Gene Regulation, and Environment, Advances in Photosynthesis and Respiration, vol. 21. Springer, Dordrecht, pp. 49e64. Asada, K., 1999. The waterewater cycle in chloroplasts: scavenging of active oxygens and dissipation of excess photons. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 601e639. Demmig-Adams, B., Adams III, W.W., 2006. Photoprotection in an ecological context: the remarkable complexity of thermal energy dissipation. New Phytol. 172, 11e21. Ebbert, V., Adams III, W.W., Mattoo, K.K., Sokolenko, A., Demmig-Adams, B., 2005. Upregulation of a PSII core protein phosphatase inhibitor and sustained D1 phosphorylation in zeaxanthin-retaining, photoinhibited needles of overwintering Douglas fir. Plant Cell Environ. 28, 232e240. Eilers, P.H.C., Peeters, J.C.H., 1988. A model for the relationship between light intensity and the rate of photosynthesis in phytoplankton. Ecol. Model. 42, 199e215. Evans, J.R., 1993. Photosynthetic acclimation and nitrogen partitioning within a lucerne canopy. II Stability through time and comparison with a theoretical optimum. Aust. J. Plant Physiol. 20, 69e82. Farquhar, G.D., Caemmerer, S., Berry, J.A., 1980. A biochemical model of photosynthetic CO2 assimilation in leaves of C3 species. Planta 149, 78e90. Genty, B., Briantais, J.M., Baker, N.R., 1989. The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochim. Biophys. Acta 99, 87e92. Heath, R.L., Packer, L., 1968. Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation. Arch. Biochem. Biophys. 125, 189e198. Hodges, D.M., DeLong, J.M., Forney, C.F., Prange, R.K., 1999. Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. Planta 207, 604e611. Ishida, S., Uebayashi, N., Tazoe, Y., Ikeuchi, M., Homma, K., Sato, F., Endo, T., 2014. Diurnal and developmental changes in energy allocation of absorbed light at PSII in field-grown rice. Plant Cell Physiol. 55, 171e182. Johnson, I.R., Thornley, J.H.M., 1984. A model of instantaneous and daily canopy photosynthesis. J. Theor. Biol. 107, 531e545. Kramer, D.M., Johnson, G., Kiirats, O., Edwards, G.E., 2004. New fluorescence parameters for the determination of QA redox state and excitation energy fluxes. Photosynth. Res. 79, 209e218. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680e685.
Miyake, C., 2010. Alternative electron flows (water-water cycle and cyclic electron flow around PSI) in photosynthesis: molecular mechanisms and physiological functions. Plant Cell Physiol. 51, 1951e1963. Miyazawa, Y., Kikuzawa, K., 2005. Winter photosynthesis by saplings of evergreen broadleaved trees in a deciduous temperate forest. New Phytol. 165, 857e866. Monson, R.K., Sparks, J.P., Rosenstiel, T.N., Scott-Denton, L.E., Huxman, T.E., Harley, P.C., Turnipseed, A.A., Burns, S.P., Backlund, B., Hu, J., 2005. Climatic influences on net ecosystem CO2 exchange during the transition from wintertime carbon source to springtime carbon sink in a high-elevation, subalpine forest. Oecologia 146, 130e147. Ohsawa, M., 1990. An interpretation of latitudinal patterns of forest limits in south and east Asian mountains. J. Ecol. 78, 326e339. Okada, K., Katoh, S., 1998. Two long-term effects of light that control the stability of proteins related to photosynthesis during senescence of rice leaves. Plant Cell Physiol. 39, 394e404. € Oquist, G., Huner, N.P.A., 2003. Photosynthesis of overwintering evergreen plants. Annu. Rev. Plant Biol. 54, 329e355. € Ottander, C., Campbell, D., Oquist, G., 1995. Seasonal changes in photosystem II organisation and pigment composition in Pinus sylvestris. Planta 197, 176e183. Oxborough, K., Baker, N.R., 1997. Resolving chlorophyll fluorescence images photosynthetic efficiency into photochemical and non-photochemical components-calculation of qP and Fv0 /Fm0 without measuring Fo0 . Photosynth. Res. 54, 135e142. Porra, R.J., Thompson, W.A., Kriedemann, P.E., 1989. Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophyll a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim. Biophys. Acta 975, 384e394. Saeki, T., Nomoto, N., 1958. On the seasonal change of photosynthetic activity of some deciduous and evergreen broadleaf trees. Bot. Mag. Tokyo 71, 235e241. Sakai, A., 1975. Freezing resistance of evergreen and deciduous broad-leaf trees in Japan with special reference to their distributions. Jpn. J. Ecol. 25, 101e111 (in Japanese). Stirbet, A., 2013. Excitonic connectivity between photosystem II units: what is it, and how to measure it? Photosynth. Res. 116, 189e214. Taneda, H., Tateno, M., 2005. Hydraulic conductivity, photosynthesis and leaf water balance in six evergreen woody species from fall to winter. Tree Physiol. 25, 299e306. Verhoeven, A.S., Adams III, W.W., Demmig-Adams, B., 1996. Close relationship between the state of the xanthophyll cycle pigments and photosystem II efficiency during recovery from winter stress. Physiol. Plant. 96, 567e576. Verhoeven, A.S., Osmolak, A., Morales, P., Crow, J., 2009. Seasonal changes in abundance and phosphorylation status of photosynthetic proteins in eastern white pine and balsam fir. Tree Physiol. 29, 361e374. Yamazaki, J., Kamimura, Y., Okada, M., Sugimura, Y., 1999. Changes in photosynthetic characteristics and photosystem stoichiometries in the lower leaves in rice seedlings. Plant Sci. 148, 155e163. Yamazaki, J., Kamata, K., Maruta, E., 2011. Seasonal changes in the excess energy dissipation from photosystem II antennae in overwintering evergreen broadleaved trees Quercus myrsinaefolia and Machilus thunbergii. J. Photochem. Photobiol. 104B, 348e356. Yamazaki, J., Ohashi, A., Hashimoto, Y., Negishi, E., Kumagai, S., Kubo, T., Oikawa, T., Maruta, E., Kamimura, Y., 2003. Effects of high light and low temperature during harsh winter on needle photodamage of Abies mariesii growing at the forest limit on Mt. Norikura in central Japan. Plant Sci. 165, 257e264. Yamazaki, J., Tsuchiya, S., Nagano, S., Maruta, E., 2007. Photoprotective mechanisms against winter stresses in the needles of Abies mariesii grown at the tree line on Mt. Norikura in Central Japan. Photosynthetica 45, 547e554. Yokota, M., Akimoto, S., Tanaka, A., 2008. Seasonal changes of excitation energy transfer and thylakoid stacking in the evergreen tree Taxus cuspidate: how does it divert excess energy from photosynthetic reaction center? Biochim. Biophys. Acta 1777, 379e387. Zarter, C.R., Adams III, W.W., Ebbert, V., Cuthbertson, D., Adamska, I., DemmigAdams, B., 2006a. Winter downregulation of intrinsic photosynthetic capacity coupled with up-regulation of Elip-like proteins and persistent energy dissipation in a subalpine forest. New Phytol. 172, 272e282. Zarter, C.R., Demmig-Adams, B., Ebbert, V., Adamska, I., Adams III, W.W., 2006b. Photosynthetic capacity and light harvesting efficiency during the winter-tospring transition in subalpine conifers. New Phytol. 172, 283e292.