Oecologia 9 Springer-Verlag1986

Oecologia (Berlin) (1986) 71:6-11

Seasonal changes in photosynthetic characteristics of Pachysandra terminalis (Buxaceae), an evergreen woodland chamaephyte, in the cool temperate regions of Japan F. Yoshie 1 and S. Kawano 2

1 The Institute of Low Temperature Science, Hokkaido University, Sapporo 060, Japan 2 Department of Botany, Faculty of Science, Kyoto University, Kyoto 606, Japan

Summary. Seasonal changes in photosynthetic capacity, and photosynthetic responses to intercellular CO2 concentration and irradiance were investigated under laboratory conditions on intact leaves of Pachysandra terminalis. Photosynthetic capacity and stomatal conductance under saturating light intensity and constant water vapor pressure deficit showed almost the same seasonal trend. They increased from early June just after the expansion of leaves, reached the maximum in late-Septemer, and then decreased to winter. In over-wintering leaves they recovered and increased immediately after snow-melting, reached a first maximum in late April, and then decreased to early July in response to the reduction of light intensity on the forest floor. Thereafter, they increased from mid August, reached a second maximum in late September, and then decreased to winter. The parallel changes of photosynthesis and stomatal conductance indicate a more or less constant intercellular COz concentration throughout the year. The calculated values of relative stomatal limitation of photosynthesis were nearly constant throughout the year, irrespective of leaf age. The results indicate that the seasonal changes in light-saturated photosynthetic capacity are not due to a change of stomatal conductance, but to a change in the photosynthetic capacity of mesophyll. Indeed, carboxylation efficiency assessed by the inital slope of the Ci-photosynthesis curve changed in proportion to seasonal changes of the photosynthetic capacity in both current-year and over-wintered leaves. High photosynthetic capacity in current-year leaves as compared with one-year-old leaves was also due to the high photosynthetic capacity of mesophyll. Nevertheless, stomatal conductance changed in proportion to photosynthetic capacity, indicating that stomatal conductance is regulated by the mesophyll photosynthetic capacity such that the intercellular CO2 concentrations are maintained constant. The quantum yield also changed seasonally parallel with that in the photosynthetic capacity. Key words: Photosynthesis- Leaf conductance

internal CO2 - Seasonal course

Mesophyll

Pachysandra

In Pachysandra terminalis and several other evergreen woodland herbs which are representative members of the * Contribution No. 2893 from the Institute of Low Temperature Science Offprint requests to. F. Yoshie

summergreen broad-leaved forests in southern cool temperate regions of Japan, leaves that had over-wintered show a considerable summer decline in light-saturated photosynthetic capacity (Pc), and this was considered to be an adaptation to drastic summer reduction of the light intensity on the deciduous forest floor due to the canopy closure (Kawano and Masuda 1979; Kawano et al. 1983). Furthermore, these evergreen herbs rarely showed a winter depression of Pc (Kawano and Masuda 1979; Kawano etal. 1983). However, little is known about the annual course of Pc in woodland evergreens growing in the northern cool temperate region of their geographical ranges. A major difference in the woodland habitat conditions between northern and southern cool temperate regions would be winter air temperatures. For instance, mean air temperature of December before snowfall in Toyama where Pc of Pachysandra terminalis in the southern cool temperate region was obtained (Kawano et al. 1983) decreased to around 5~ C, while that in Sapporo, in the northern cool temperate region, where the present study was carried out, usually decreased to around - 2 ~ C. It is, therefore, expected that in northern cool temperate regions where severe winter low temperatures are prevailing, northern populations of the evergreen woodland herbs may exhibit a marked decrease in the Pc in winter, different from the behaviours of those which occur in southern cool temperate regions. Likewise, they probably show the summer decline in Pc of over-wintered leaves, because summer reduction of light intensity on the deciduous forest floor is a common phenomenon in the cool temperate regions as well. Changes in Pc are considered to be caused by either a change of stomatal conductance or a change in the photosynthetic capacity of mesophyll, or both (Bj6rkman 1981). On the other hand, Schulze and Hall (1982) reported that maximum photosynthetic capacity versus stomatal conductance gave a similar slope independent of the preconditions within a C3 species, and thus a species showed rather constant intercellular CO2 concentration (Ci). In the temperate forest region, Yoshie (1986) reported that under saturation light intensity and constant deficit of water vapor pressure, intercellular CO2 concentrations were nearly constant in the foliage leaves of a single species, irrespective of the leaf age or season in plants, including Pachysandra terminalis. A constant Ci strongly suggests that the changes in Pc are not due to a change in the stomatal conductance, i.e., a change of CO2 diffusion from leaf surface to mesophyll, but to a change in the photosynthetic capacity of the mesophyll.

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The aim of this study is thus to uncover seasonal changes in the photosynthetic characteristics of Pachysandra terrninalis growing in the northern cool temperate region compared with those in the southern cool temperate region in relation to environmental factors on the woodland floor. Material and methods

Pachysandra terminalis Sieb. et Zucc. (Buxaceae) is a prostrate evergreen chamaephyte distributed mainly in the deciduous broad-leaved forests of the cool temperate region. Pachysandra terminalis usually has pseudo-verticillate leaves persisting for 3 to 4 years on an ascending stem. The ascending stem over 3 to 4-years old gradually becomes prostrate and finally becomes a rhizome with root. Samples for gas exchange measurement were collected in the spring of 1982 in the deciduous broad-leaved forests in Miyanomori, Sapporo, Hokkaido in the northern cool temperate region of Japan. Aerial shoots were cut at the base and potted with humid soil for rooting and vegetative reproduction. The potted plants were cultivated in deciduous broadleaved forests on the Hokkaido University campus, Sapporo (6 km east of the sampling site), until the gas exchange measurements were made. The potted plants sprouted new shoots slightly later than the plants at the native habitats in 1982 due to transplanting effects. After 1983 they showed the same phenological performance as they do in their natural habitat, in the time course of leaf-sprouting to maturation and the duration of leaf-persistence, time of flowering and fruit maturation, and so on. In addition, Pachysandra terminalis transplanted to the cultivation site has reproduced actively and showed the same phenological performance during last 10 years. Seasonal change in the relative light intensity at the forest floor of the cultivation site was very similar to that in the sampling site. Relative light intensity began to decrease in early May and dropped to a few

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Fig. t. Seasonal changes in photosynthetic capacity and intercellular CO2 concentration under saturation PFD. Symbols are current-year (e), one-year-old (o), and two-yearold (zx)leaves. Dotted line indicates the mean Ci (228 gbar) of all points measured. The measuring conditions are given in the text percent of unshaded levels from mid June to early September and then increased to late November. Thus, the behavior of the experimental plants is similar to that in their natural habitat, and they are cultivated under conditions which are representative of their natural environments. The water potential of Pachysandra terminalis as well as many woodland herbs was usually from - 0 . 5 to 3.0 bar in the morning, and decreased from - 5 to - 8 bar in midday during the growing season both at the natural and the cultivation sites. The experimental plants were, therefore, fully watered 12 h before measurements to obtain the photosynthetic capacity in the morning at the natural habitat. Photosynthesis and transpiration of intact leaves were measured simultaneously with an open system gas analysis apparatus in 1984 and 1985. Some of the pseudo-verticillate leaves were cut off to avoid shading the sample leaves. An assimilation chamber (2,450 cm 3) was equipped with a fan (5 cm diameter), 3 copper-constantan thermocouples (0.10 mm diameter), and transparent nylon threads stretched across the chamber to hold the pseudo-verticillate leaves horizontal. The leaves in the chamber were irradiated with two metal halide lamps (DR 400/TL, Toshiba Co., Tokyo). In all gas exchange measurements, the leaf temperature, monitored by 2 thermocouples, was controlled at 20.0 +_0.3 ~ C. Pachysandra terminalis changed its photosynthetic temperature-optimum seasonally from 12~ to 23 ~ C, but the reduction of Pc at 20~ was very small, with a max. reduction of c.a. 15 % in winter (Yoshie unpublished data). Thus, the photosynthetic characteristics obtained in this study are regarded to be nearly the same as those in the natural habitat. The root remperatures were controlled at 18.5_+0.5 ~ C. The annual course of gas exchange was measured under a saturation PFD (400-700 nm) of 500 lamol m - 2 s - t , normal ambient CO2 concentration of 332_+ 15 gbar, and water vapor pressure deficit from the leaf to ambient air of 9.22_+0.81 rob. Pho-

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tosynthetic response curves to P F D were obtained under ambient CO2 concentration o f 335 _+ 14 gbar and water vapor pressure deficit of 8.96+_0.86 mb. Ci-photosynthesis curves, obtained by increasing ambient CO2 concentration at 100 to 150 gbar steps, were measured under a saturation P F D of 500 g m o l m - 2 s - 1 and water vapor pressure deficit of 8.85 + 0.79 mb. Carbon dioxide concentrations were determined with an infrared gas analyser (VIA-300, Horiba Co., Tokyo). All gas exchange measurements were made as soon as possible after the plants reached steady-state conditions, to clarify the photosynthetic performance of the naturally growing plants: It usually takes 2, 5 and 8 h to measure the seasonal change in Pc, and photosynthetic response to P F D and Ci, respectively. Strictly speaking the experimental plants in winter did not reach steady-state conditions, but the increase of photosynthesis and stomatal conductance during the experimental procedure was very small and negligible. Water vapour pressures were determined with a thermocouple psychrometer as described by Slatyer and Bierhuizen (1964), with s o m e modifications. Ci was calculated by the equation of Farquhar et al (1978). Stomatal conductance to CO2 transfer was evaluated according to Gaastra (1959) using an H 2 0 : C O 2 diffusion ratio of 1.6 (Jarvis 1971). Relative stomatal limitation of photosynthesis was calculated according to the equation of Farquhar and Sharkey (1982), by using the photosynthetic response curve to Ci.

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Fig. 3. Seasonal changes in the photosynthetic response curve to intercellular COz concentration of mesophyll in one-year-old leaves. The measuring conditions are given in the text. Different symbols refer to different individuals. Mean carboxylation efficiency (C. E., gmol m 2 s-1 gbar-1) calculated from the lienar regression of the initial slope, is indicated. The arrows indicate the Ci at which the actual photosynthesis (A) was estimated for calculation of relative stomatal limitation of photosynthesis (ls)

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Fig. 5. Seasonal changes in the photosynthetic response curve to incident quantum flux density in one-year-old leaves. The measuring conditions are given in the text. Different symbols refer to different individuals. The mean quantum yield (~2~, molCOz mol quanta-1) calculated from the initial slope is indicated trend as that of Pc in all current-year, one-year-old and two-year-old leaves, as represented in Fig. 2. A good correlation between Pc and the stomatal conductance resulted in a more or less constant Ci throughout the year, irrespective of leaf age (Fig. 1) and consistent with the previous study (Yoshie 1986).

Responses of photosynthesis to intercellular C02 concentration and quantum flux density Results

Annual course in gas exchange under saturation quantum flux density The Pc of current-year leaves increased from early June just after leaf-unfolding and reached its maximum value in late September (Fig. 1). Pc then gradually decreased from early October to late December in response to the decrease in air and soil temperatures. Pachysandra terminalis wintered under snow cover during late Decembr to early April. One-year-old leaves recovered and rapidly increased their Pc immediately after snow-melt, reaching a first maximum value in late April. Pc then decreased from early May to early July in accordance with the marked reduction in the light intensity on the forest floor. Pc recovered again from mid August and increased to early October harmoniously with gradual increase of the light intensity. Thereafter, Pc decreased to winter. Two-year-old leaves showed the same seasonal trend on Pc as that of one-year-old leaves, although the former had lower Pc values throughout the year. Stomatal conductance showed almost the same seasonal

Photosynthesis as a function of Ci at saturation light intensity showed a linear response to Ci up to ca. 200 gbar and then became curvilinear in both current-year leaves (data are not shown) and one-year-old leaves (Fig. 3) in every season tested. The carboxylation efficiency assessed by the linear regression of the initial slope in Ci-photosynthesis curve was correlated with R u B P carboxylase-oxygenase activity (yon Caemmerer and Farquhar 1981; Farquhar and Sharkey 1982). In current-year leaves the carboxylation efficiency increased from mid-summer to mid-autumn and then decreased in winter. In one-year-old leaves the carboxylation efficiencies were higher in spring and autumn than in summer and winter. The seasonal changes in the carboxylation efficiency were in good agreement with those in Pc in both current-year and one-year-old leaves. This was demonstrated in Fig. 4, showing a clear correlation between the carboxylation efficiency and Pc. Relative stomatal limitation of photosynthesis (ls) was calculated from the following equation proposed by Farquhar and Sharkey (1982): l s = ( A o - A ) / A o , where A o is a photosynthetic rate which would occur if stomata1 resistance to CO2 diffusion is zero,

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Fig. 6. Relationship between light-saturated photosynthesis and quantum yield. The symbols and the lines are the same as shown in Fig. 4 and A is the actual photosynthetic rate. The photosynthetic rate at a Ci of 330 pbar in the photosynthetic response curves to Ci was used as Ao in this study. The actual photosynthetic rate (A) was estimated by the net photosynthesis at mean Ci of each month calculated from Fig. 1 and indicated by arrows in Fig. 3. The calculated mean value of relative stomatal limitation was nearly constant throughout the year irrespective of leaf age and ranged from 28% to 3]% (Table 1). Net photosynthesis saturated at PFD near 200 gmol m - 2 s 1 both in current-year leaves (data are not shown) and in one-year-old leaves (Fig. 5) in every season tested. The shape of the PFD-photosynthesis curve in all leaves is similar to the shade-leaf type in its general pattern with low saturation PFD, compensation PFD, and dark respiration rates throughout the year. Quantum yield was calculated from the inital slope of net photosynthesis versus incident PFD (up to 50 gmol quanta m -2 s 1). The quantum yield was low in immature leaves in mid-summer, and became high in autumn and then decreased in winter in current-year leaves. In one-year-old leaves the quantum yield was high in spring and autumn and were low in summer and winter. The quantum yield changed harmoniously with that of Pc and thus a conspicuous correlation was detected between these two variables (Fig. 6). Little differnce in Ci and in relative stomatal limitation was found among different aged leaves (Fig. 1, Table 1). Both carboxylation efficiency and quantum yield were higher in current-year leaves than in one-year-old leaves in the same season, except for the immature expanding leaves.

Discussion A marked winter depression in Pc in the northern cool temperate region compared with that in the southern cool

temperate region (Kawano et al. 1983), is probably due to severe winter temperatures and secondary induced water stress. This was supported by the study on Cryptomeria japonica D. Don, an evergreen phanerophyte, in which winter depression of the photosynthetic capacity and stomatal conductance was partially removed by increasing soil temperature (Hashimoto and Suzaki 1978). Pc of Pachysandra terminalis was considerably higher in the northern cool temperate region than in the southern cool temperate region during the growing season except for mid-summer. The higher Pc in the northern cool temperate region may compensate for a shorter growing season, i.e., about two months shorter than in the southern cool temperate region. This is in accord with the general trend that net photosynthesis of young leaves is negatively related to leaf life span in deciduous forest herbs (Chabot and Hicks 1982). A more or less constant Ci and the relative stomatal limitation of photosynthesis in Pachysandra terrninalis leaves throughout the year indicate that the seasonal changes in Pc, including the characteristic summer decline and winter depression, were not due to a change of stomatal conductance. The seasonal change in Pc was, therefore, due to changes in the photosynthetic capacity of mesophyll. Indeed, high correlations between Pc and carboxylation efficiency were detected in both current-year and one-year-old leaves. High Pc in current-year leaves as compared with one-year-old leaves in the same season was also due to the high carboxylation efficiency. The carboxylation efficiency is a very important factor, since the Pc measured under normal ambient COz concentration was plotted on the nearly linear portion of Ci-photosynthesis curves. This may be supported by the fact that in Pachysandra terminalis leaves from the southern region of its range the content of Fraction-I-protein per unit leaf area, which is synonymous with RuBP carboxylase-oxygenase, changed seasonally parallel to that of net photosynthesis (Kawano et al. unpublished). The importance of carboxylation efficiency as a regulating factor on the seasonal change in Pc obtained in this study is in acordance with the study of Quercus coccifera (Tenhunen et al. 1984b), and is comparable to the study of the diurnal change in Quercus coccifera (Tenhunen et al. 1985), Quercus suber (Tenhunen et al. 1984a; Tenhunen et al. 1985) and Arbutus unedo (Lange et al. 1985), all measured at the natural habitats in the mediterranean region. On the other hand, stomatal conductance changed in parallel with the photosynthetic capacity of mesophyll. This suggests that a mechanism of stomatal regulation by the mesophyll photosynthetic capacity maintaining constant Ci and proposed by Wong et al. (1979), is unchanged and maintained throughout the year, irrespective of leaf age. A similar seasonal constancy of Ci under saturation PFD was found from diverse temperate elements, such as spring ephemerals, and from evergreen, wintergreen and summergreen species (Yoshie 1986). Conversely, this strongly suggests that seasonal changes in Pc of woodland herbs in this region are due to the photosynthetic capacity of mesophyll, just as was found from Pachysandra terminalis in this study. The shape of the PFD-photosynthesis curve in all leaves of Pachysandra terminalis is similar to the shade-leaf type in its general pattern. When the shade plants were exposed to high light intensity, they showed photoinhibition with decreasing light-saturated photosynthesis and photochemi-

11 cal efficiency in general (see Bj6rkman 1981). However, in the prevernal period when direct and continuous solar radiation illuminates the deciduous forest floor, wintered leaves of Pachysandra terminalis do not show any photoinhibition at all, but rather an acclimation to a high irradiance with increasing carboxylation and photochemical efficiencies of mesphoyll. Conversely, the photosynthetic capacity declined in summer in accordance with the drastic reduction of irradiance on the forest floor. These seasonal photosynthetic responses are, therefore, considered to be characteristics of the w o o d l a n d evergreens in response to the seasonally-changing light regime. O n the other hand, it has n o t been clarified whether these seasonally simultaneous changes in Pc, carboxylation efficiency a n d q u a n t u m yield are due to the environmental constraints, as found in the mediterranean sclerophyllous shrubs ( T e n h u n e n etal. 1984b), or are intrinsic rhythms in consequence of an adaptation to woodland environments. F u r t h e r studies on the cause of the seasonal changes are clearly needed.

Acknowledgements. We express our hearty thanks to Dr. M. J. Lechowicz of McGill University for his critical reading of the manuscript and to Prof. S. Yoshida of Hokkaido University for his helpful discussion throughout the study. We also express our gratitude to Prof. O.L. Lange of Universit/it Wfirzburg for his invaluable comments on the manuscript. Thanks are also due to Dr. Y. Sakagami, Dr. K. Takahashi, and Dr. Y. Fujimura of Hokkaido Branch, Forestry and Forest Products Research Institute for their helpful discussion and for their kind permission to conduct the preliminary experiments of this work in their laboratory.

References Bj6rkman O (1981) Responses to different quantum flux densities. In : Lange OL, Nobel PS, Osmond CB, Ziegler H (eds) Encyclopedia of Plant Physiology, New Series Vol 12B. Physiological Plant Ecology I. Response to the Physical Environment. Springer, Berlin Heidelberg New York, pp 57-107 Caemmerer S von, Farquhar GD (1981) Some relationship between the biochemistry of photosynthesis and the gas exchange of leaves. Planta 153:376-387 Chabot BF, Hicks DJ (1982) The ecology of leaf life spans. Ann Rev Ecol Syst 13:229 259 Farquhar GD, Sharkey TD (1982) Stomatal conductance and photosynthesis. Ann Rev Plant Physiol 33:317-345 Farquhar GD, Dubbe DR, Raschke K (1978) Gain of the feedback loop involving carbon dioxide and stomata. Theory and measurement. Plant Physiol 62:406-412 Gaastra P (1959) Photosynthesis of crop plants as influenced by

light, carbon dioxide, temperature, and stomatal diffusion resistance. Meded Landbouwhogeschool, Wageningen 59:1-68 Hashimoto R, Suzaki T (1978) Resistance to carbon dioxide transfer in leaves of Cryptomeriajaponica in winter, and influences of soil temperature on them. J Jap For Soc 60:24-30 Jarvis PG (1971) The estimation of resistance to carbon dioxide transfer. In : Sestak Z, Catsky J, Jarvis PG (eds) Plant photosynthetic Production - Manual and Methods, Dr. Jung NV Publishers, The Hargue p 566 Kawano S, Masuda J (1979) The productive and reproductive biology of flowering plants VI. Assimilation behavior and reproductive allocation of Coptisjaponica (Thunb.) Makino (Ranunculaceae). J Coll Lib Arts Toyama Univ (Nat Sci) 12:49-63 Kawano S, Masuda J, Takasu H, Yoshie F (1983) The productive and reproductive biology of flowering plants. XI. Assimilation behavior of several evergreen temperate woodland plants and its evolutionary-ecological implications. J Coll Lib Arts Toyama Univ (Nat Sci) 16:31-65 Lange OL, Tenhunen JD, Beyschlag W (1985) Effects of humudity during diurnal courses on the CO2- and light-saturated rate of net COz uptake in the sclerophyllous leaves of Arbutus unedo. Oecologia (Berlin) 67:301 304 Schulze ED, Hall AE (1982) Stomatal responses, water loss and CO2 assimilation rates of plants in contrasting environments. In: Lange OL, Nobel PS, Osmond CB, Ziegler H (eds) Encyclopedia of Plant Physiology, New Series, Vol 12B. Physiological Plant Ecology II. Water Relations and Carbon Assimilation. Springer, Berlin Heidelberg New York, pp 181-230 Slatyer RO, Bierhiuzen JF (J964) A differential psychrometer for continuous measurements of transpiration. Plant Physiol 39:1051-1056 Tenhunen JD, Lange OL, Gebel J, Beyschlag W, Weber JA (1984a) Changes in photosynthetic capacity, carboxylation efficiency, and CO2 compensation point associcated with midday stomatal closure and midday depression of net COz exchange of leaves of Quercus suber. Planta 162:193-203 Tenhunen JD, Meister HP, Caldwell MM, Lange OL (1984 b) Environmental constraints on productivity of the mediterranean sclerophyll shrub Quercus coccifera. Options Mediterrraneennes Ciheamiamz 84:33-53 Tenhunen JD, Lange OL, Harley PC, Beyschlag W, Meyer A (1985) Limitations due to water stress on leaf net photosynthesis of Quercus coccifera in the Portuguese evergreen scrub. Oecologia (Berlin) 67: 23-30 Wong SC, Cowan IR, Farquhar GD (1979) Stomatal conductance correlates with photosynthetic capacity. Nature 282:424-426 Yoshie F (1986) Intercellular CO2 concentration and water-use efficiency of temperate plants with different life-forms and from different microhabitats. Oecologia (Berlin) 68 : 370-374 Received May 22, 1986