Global Environmental Change in the Ocean and on Land, Eds., M. Shiyomi et al., pp. 453Ð472. © by TERRAPUB, 2004.

Photosynthetic Characteristics of Mixed -Broadleaf Forests from to Stand

Takayoshi KOIKE1,2, Satoshi KITAOKA3, Tomoaki ICHIE1, Thomas T. LEI2* and Mitsutoshi KITAO2

1Hokkaido University Forests, FSC, Sapporo 060-0809, Japan 2Forestry and Forest Products Research Institute, Sapporo 062-8516, Japan 3Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan

Abstract. The phohotosynthetic characteristics of seedlings and in deciduous broad-leaved forests have been studied from the leaf to the stand level with the aim of improving canopy photosynthesis estimates from satellite data. The photosynthetic traits of canopy of seven deciduous broad- leaved trees were studied using a canopy tower. -specific patterns were found. In ash and maple, light saturated net photosynthetic rate (Psat) at midday in summer sunlight was detected at the sub-layer of the crown, but not at the top of the crown. The surface of the crown may have acted as a screen to reduce light to a level that is optimal for sub-layer leaves. In other species, such as birch and alder, the greatest Psat was observed at the canopy surface. Leaf senescence started from the inner part of the crown in alder and birch, but from within either the outer or top portion of the canopy in ash, basswood and maple. The chlorophyll-to-nitrogen ratio in leaves increased with decreasing photon flux density. There is a clear positive correlation between chlorophyll content and photosynthetic rate in individual species. Nitrogen concentration was positively correlated with light saturated photosynthesis in all species. To predict the photosynthetic capacity of the forest as a whole, the gas exchange capacity of seedlings invading the forest should be estimated. However, the photosynthetic capacity of deciduous tree seedlings regenerating in a gap or on the forest floor changes drastically with season. Moreover, there is significant year-to-year variation in photosynthetic rate at ambient CO2 concentration. This variation may be due to the diffusion resistance in a leaf, since it disappeared under CO2 saturation. The potential photosynthetic capacity of single leaves of deciduous broad-leaved trees can be estimated using a portable nitrogen detector in the field, and this can be scaled up to the stand level by monitoring the leaf nitrogen content of the canopy using satellite imagery to detect the nitrogen reflectance of the canopy.

Keywords: canopy photosynthesis, photoinhibition, chlorophyll and nitrogen, year-to-year variation, CO2 diffusion resistance

*Present address: Division of Industry, CSIRO, Narabri, NSW, Australia.

453 454 T. KOIKE et al.

1. INTRODUCTION Estimates of the canopy photosynthetic capacity of temperate forests provides information about their role in global carbon cycles, involving biomass production (Houghton, 1991; Wofsy et al., 1993; Bassow and Bazzaz, 1997, 1998). Biomass production and CO2 flux in a monoculture, even-aged forest has been estimated by modeling the interaction between the micro-environment and canopy photosynthetic activity (Waring et al., 1995); the latter can be found from satellite image data (Ehelinger and Field, 1993; Bazzaz, 1996). However, it is not easy to determine biomass production in mixed forests from satellite data, because different tree species have different photosynthetic capacities. Since canopy species have widely differing gas exchange capacities (Koike, 1988; Niinemets, 1997; Koike et al., 2001), an assessment of the photosynthetic capacity of each species from its leaves within the canopy would help to solve this problem. Previously, knowledge of the maximum photosynthetic rate of the exposed crown was believed to be sufficient (Saeki, 1959) to estimate the CO2 flux in a forest, in view of leaf nitrogen accumulation or translocation from older leaves to younger ones under full sunlight (Field, 1983; Hirose and Werger, 1987; Küppers, 1989; LeRoux et al., 1999; Schoettle and Smith, 1999). Shade leaves assign more foliage nitrogen to chlorophyll, so as to increase the efficiency of incident light capture at the forest floor (Osmond et al., 1980; Boardman, 1977; Kimura et al., 1998). Moreover, the positive correlation between photosynthetic capacity and leaf nitrogen content (Evans, 1989; Hikosaka and Terashima, 1995) suggests that the “big leaf” model (Kull and Kruijt, 1999) for describing canopy properties is too simplistic. Heterogeneity of species in a forest stand that includes gaps usually gives rise to complex dynamics of CO2 fixation by the trees and invasion of the gap by seedlings (Bassow and Bazzaz, 1998; Lambers et al., 1998). To assess more accurately the biomass production of the overall canopy may therefore necessitate a study of species-specific variations in leaf morphology, biochemistry and physiology of trees. The outer crown of canopy trees usually experiences strong light, together with high vapor pressure deficit, large temperature fluctuations, and periodic low CO2 concentration (Parker, 1995). Close to the canopy top, leaf water stress may be a factor that limits the photosynthetic functions of canopy trees (Hinckley et al., 1978; Horton and Hart, 1998), explaining the midday depression in photosynthesis (Wofsy et al., 1993; Holbrook and Lund, 1995; Bassow and Bazzaz, 1998; Hiromi et al., 1999). Light-demanding species can typically maintain high rates of photosynthesis in the upper canopy, but it is not clear that all canopy species exhibit their maximum rate of gas exchange capacity at all levels throughout the growing season. Moreover, many tree seedlings invade forest gaps, which may act as a large carbon sink during the growing season. It is therefore necessary to infer the photosynthetic capacity of canopy trees as well as these invading seedlings. The present work studies the capacity of leaf morphological and photosynthetic characteristics to adapt to the vertical foliage profile of overstory Photosynthetic Characteristics of Mixed Deciduous-Broadleaf Forests 455 trees in a secondary forest. Our objectives are: (1) to assess photosynthetic leaf variation along the canopy profile by measuring LMA (leaf mass per area = 1/ SLA, specific leaf area), nitrogen (N) and chlorophyll (Chl) concentrations, as well as light-saturated photosynthetic rates for leaves of seven tree species encountered in the stand; (2) to measure seasonal trends in the net photosynthetic rate (Psat) of seedlings regenerated in the forest and to explain the year-to-year variation in Psat; and (3) from those results, to describe more precisely the relation between canopy N content and leaf morphological and physiological variation of tall trees and their seedlings with changing light gradient in the forest canopy profile. This relation would then allow quantification of the carbon dynamics of a mixed forest canopy from remote sensing data.

2. MATERIALS AND METHODS

2.1 Study sites The canopy study site was located in the experimental forest of the Forestry and Forest Products Research Institute in Sapporo, Japan (42°58′ N, 141°23′ E, 160 m a.s.l.). The average precipitation is 1200 mm/year, and the maximum snow depth recorded was 2.6 m. The mean annual temperature is 6.1°C (max. 28°C; min. Ð16°C). We built a 24 m monitoring tower (with a canopy coverage of 5 × 5 m) in a mixed deciduous broad-leaved forest at a point where seven canopy tree species were accessible for measurements. We also planned to study the role played by understory tree seedlings in stand level photosynthesis, and we chose a discontinuous forest canopy with many invading seedlings. The soil in the research area is classified as brown forest soil mixed slightly with volcanic ash and charcoal; the site was burned in 1912. Our study of tree seedlings was conducted in three unmanaged larch stands (49 years old in 2003) mixed with several tree species (e.g. magnolia, kalopanax, ash, oak, Amur cork tree; Phellodendron amurense) at Tomakomai Experimental Forest (TOEF) in Hokkaido, Japan (mean annual temp. 6.5°C, precipitation ca. 1200 mm, snow depth not more than 50 cm; location: 42°40′ N, 141°36′ N, ca. 110 m a.s.l.). The soil at this study site also contains immature volcanic ash.

2.2 Plant species Leaf photosynthesis at various canopy profiles was determined for seven species: walnut (Juglans ailanthifolia; gap phase), alder (Alnus hirsuta; early successional), white birch (Betula platyphylla var. japonica; early successional), basswood (Tilia japonica; late successional), maple (Acer mono; late successional), elm (Ulmus davidiana var. japonica; mid successional) and ash (Fraxinus mandshurica var. japonica; gap phase). Other species near the tower but not measured include oak (var. crispula; mid-late successional), dogwood (Cornus contraversa; gap phase) and kalopanax (Kalopanax septemlobus; gap phase). The mean tree height was 18.5 m and the average diameter at breast height was 1.58 m (SD 0.12 m). 456 T. KOIKE et al.

To study seedlings regenerating in an unmanaged larch plantation, we examined seedlings of four successional tree species that had invaded a larch plantation. These were magnolia (Magnolia hyporeuca, a gap phase), carpinus (Carpinus cordata, late successional), cherry (Prunus ssiori, late successional) and oak (var. crispula, mid-late successional). Seedlings were analyzed phenologically (Kikuzawa, 1983) and by photosynthesis traits (Koike, 1988). The seedlings sampled were 70Ð120 cm high. Mature third, fourth or fifth leaves counted from the shoot tip were chosen (Koike, 1990) from four for measurement of gas exchange rates and leaf characteristics over three years. We counted the number of leaves per shoot of the trees and observed any changes in leaf color.

2.3 Measurement of photosynthetic capacity The photosynthetic photon flux density (PPFD) at different canopy heights (0.5, 2, 4, 10, 14, 18 and 24 m) was recorded continuously recorded using quantum sensors (“solar monitor”, Kyokko Trade Co., Tokyo, Japan). The relative PPFD (rPPFD) was determined as the ratio of PPFD at an open site to the PPFD at a chosen place in the forest (multiplied by 100 to convert to a percentage). The PPDF was expressed as the integrated value over approximately one month. The CO2 concentration at these heights was measured using an infra-red gas analyzer (ZDF, Fuji Elect. Co., Tokyo, Japan) with a 6-channel auto-sampler (DAIWA Air Regulation Co. Ltd., Sapporo, Japan). The photosynthetic light response curve was measured using an open system portable IRGA (ADC H3 and H4, UK). Other portable IRGAs (LI-6200 and LI-6400, Lincoln, Nebraska, USA) were used in determining the maximum photosynthetic rates of individual leaves; at saturated PPFD this was 600 µmolámÐ2sÐ1 for late successional species, and µ Ð2 Ð1 1000 molám s for early successional species, where the ambient CO2 level was ca. 350 ppm and VPD ca. 2.4 kPa. For measurements on seedlings, light and the CO2 saturated photosynthetic rate at 1500 ppm (Pmax) was determined using a CO2 injection bomb of the LiCor system.

2.4 Analysis of leaf characteristics Leaf area was determined using an area meter (LI-300, LiCor, NB, USA) following the photosynthesis measurements. The leaf was then dried for 24 hrs at 65°C for dry mass determination and chemical analysis. The leaf area and dry mass were used to calculate the specific leaf area (SLA = 1/LMA) of individual leaves. Leaf chlorophyll content was determined using the DMSO (Dimethyl sulfoxide) extraction method (Barnes et al., 1992; Shinano et al., 1996). Chl a and b, and total chlorophyll (Chl a + b) were calculated from the absorbance (A) at two wavelengths using the well-known equations (subscript denotes wavelength in Ð1 × × × nm) (unit ugáml ): Chl a = 14.85 A664.9 Ð 5.14 A648.2, Chl b = 25.48 A648.2 × × × Ð 7.36 A664.9, Chl a + b = 7.49 A664.9 + 20.3 A648.2, where Chl b is usually the main component of light harvesting chlorophyll protein (LHCP), so that the amount of this pigment is significant for the capacity of the leaf to accommodate Photosynthetic Characteristics of Mixed Deciduous-Broadleaf Forests 457 to the shade condition (Kura-Hotta et al., 1987; Lei et al., 1996). Content of chlorophyll is expressed as the unit of (mgámÐ2) by way of SLA. The leaf nitrogen concentration and C/N ratio were determined using a N/C analyzer (NC-900, Shimadzu, Kyoto, Japan).

3. LEAF PHENOLOGY AND MICRO-ENVIRONMENT IN A FOREST STAND

3.1 Leaf phenology Canopy trees such as alder, birch and elm begin the unfolding of their leaves in early May. Of the seven tree species examined, all but the maple initiated leaf unfolding from the lower part of the crown. In birch, late leaves developed about 2 weeks after the flush of early leaves. Ash leaves were flushed two weeks later than birch, in early June, and leaves continued to emerge until early July. Alder started leaf shedding in mid July; the shed leaves were still green. In all other species except ash, leaf senescence (yellowing of leaves) began around late September; all leaves were shed by late October except in alder and birch, which were bare by early November with the onset of frost. Senescence began in ash much earlier than in the other canopy trees, and also more rapidly so that all leaves were shed by mid-September. Alder maintained some green leaves at the canopy top until early November. Similar trends in leaf phenology and growth have been found in a northeast U.S. Forest (Bicknell, 1982). Sapling leaves flushed at almost the same time as canopy trees. However, leaf shedding in saplings began one to two weeks later than in canopy trees. Magnolia, a gap phase species, flushed four or five leaves per shoot around late May, and shed its leaves at the end of September. Cherry is a late successional tree species and flushed four or five leaves per shoot at the end of April, keeping its leaves until mid-October. Carpinus flushed its leaves in mid May; it had five leaves per shoot, and these leaves remained green until they were killed by frost at the end of October. Oak flushed 4Ð5 leaves in mid May, and kept its leaves until mid September, gradually shedding them in late October. Variation of the leaf phenological pattern from year to year was clear in magnolia. In 1999 and 2001, bud break and leaf expansion of magnolia started in mid May, with a similar leaf phenology pattern. However, bud break and leaf expansion began 7 days later in 2000. Similar trends of year-to-year variation were found in other species.

3.2 Vertical gradient of light and CO2 Seasonal change in the rPPFD coincided with leaf phenology and the maturation of tree leaves (Fig. 1A). The level of irradiation at the canopy top increased from its mid-April value of around 20 MJámÐ2dayÐ1, peaked at June at around 23 MJámÐ2dayÐ1, then decreased to a value of 5 MJámÐ2dayÐ1 in late October. After leaf unfolding, rPPFD decreased sharply from 72% to 49% at 18 m from the ground, where the canopy top was located, and from 32% to 13% at 10 m where the foliage of sub-canopy trees was situated. After yellowing of the leaves in most canopy trees and completion of leaf shedding in ash, rPPFD 458 T. KOIKE et al.

Fig. 1. Levels of light-rPPFD (A) and ambient CO2 (B) levels within the forest canopy profile. Morning and Night correspond to approximately 10:30 and 20:30 at JST (adapted from Koike et al., 2001).

increased from 49% to 85% at 18 m, and from 13% to 21% at 10 m. In summer there were large variations in CO2 concentration across the vertical canopy profile and diurnally near the ground (Fig. 1B). The CO2 concentration ranged from 320Ð350 ppm at the upper canopy (at 14Ð16 m) and 405Ð560 ppm near the ground (at 0.5Ð4 m). The lowest concentration of ambient CO2 was observed during the day and the highest at night. The largest variation in CO2 concentration was found in the lower canopy. Before and after the summer, CO2 variation in the stand was relatively smaller between the top of the canopy and near the ground as compared with mid-summer when photosynthetic activity of plants is high. Similar variation in CO2 was also found in an unmanaged larch stand (Kitaoka et al., 2001) and an oak-hornbeam forest in central Europe (Elias et al., 1989).

3.3 Seasonal changes in light environment of forest floor Within a larch forest, seasonal changes of rPPFD near gaps and on the forest floor showed drastic changes with the dynamics of the top canopy over three growing periods (Fig. 2). Before canopy closure, rPPFD averaged 40Ð60% over three years near the gap and also on the forest floor. Near gaps, rPPFD decreased rapidly from May to June, then remained at ca. 18% until late September. Larch needles are shed in the latter part of October, causing rPPFD to increase rapidly. On the forest floor, rPPFD diminished with canopy closure, and maintained a low value of ca. 10% until October. In 2000, the reduction in rPPFD to ca. 10% was more rapid than in 1999 and 2001, in which years it was ca. 20% at that time. The rate of foliar development was strongly affected by climatic factors and especially the ambient temperature; the hot spring in 2000 probably accelerated the rapid formation of the canopy cover. Photosynthetic Characteristics of Mixed Deciduous-Broadleaf Forests 459

Autumn Shedding Autumn Shedding Bud Break Leafly period coloring needles Bud Break Leafly period coloring needles phenology Larch needle Forest floor Forest gap 80 60 1999 1999 40 2001 2001 2000 20 2000 0 Relative PPFD (%) MJ JASO MJ JASO Month

Fig. 2. Seasonal changes in rPPFD at forest floor (left) and gap (right) of a larch plantation accompanied by canopy phenology of larch trees. There is a large yearly difference in the rPPFD, which may be related to ambient temperature. Canopy closure is accelerated by high temperature (after Kitaoka and Koike, 2004).

ABC

Alder

Birch

Elm

Maple

Ash Tree heightTree (m) Basswood

Walnut

0 50 100 150 0 0.1 0.2 0.3 0 5 10 15 Leaf area (cm2) Laef thickness (mm) Leaf mass area (g m-2)

Fig. 3. Variation in leaf characteristics of seven tree species within the canopy profiles. A. Leaf area of all species. B. Leaf thickness. C. Leaf mass area (LMA). All measurements were taken in mid August (adopted from Koike et al., 2001).

4. CANOPY PHOTOSYNTHETIC CHARACTERISTICS

4.1 Leaf functional changes along a forest profile Individual leaf area increased gradually from the canopy top to ground level during late July to early September (Fig. 3A). This trend was stronger for basswood and elm, and weaker for walnut and birch. Except for walnut, leaf thickness decreased in accordance with rPPFD down through the canopy profile. 460 T. KOIKE et al.

25 June Aug . Oct

20 Alder

Birch 15 Elm

Maple 10 Ash

Basswood Tree heightTree (m) 5 Walnut

0 0 200 400 600 800 0 200 400 600 800 0 200 400 600 800 Chlorophyll (mg m-2)

Fig. 4. Seasonal changes in the vertical profile of total chlorophyll (a + b) content in leaves of the seven deciduous species.

Light-dependent changes in leaf thickness were larger for elm and ash and smaller for walnut (Fig. 3B). Parallel with changes in leaf thickness, there was an increase in SLA (=1/LMA) with decreasing rPPFD (Fig. 3C). Just before the start of autumn coloration, LMA decreased in all species except alder; alder leaves do not change color before shedding. Similar patterns were reported in a deciduous broadleaved forest in central Europe (Niinemets et al., 1998). The chlorophyll content (Chl) of ash, birch and elm was highest in mid- canopy (15 m) or at the position of the shady crown of the top canopy (Fig. 4). Except for October, Chl was generally highest in the sub-layer just below the canopy. Chl for alder and walnut were highest in their canopy top leaves. The missing values for canopy top leaves of ash and walnut in October indicate that these leaves had already been shed. Alder maintained a markedly high Chl value even in the latter part of the growing season. In other species, though there was an overall decrease in Chl within the canopy profile, the pattern of Chl distribution did not change throughout the season. This result was also found by Elias and Masarovicova (1980). Chl b reveals the acclimation traits of leaves to shady environment, since it is the main component of light harvesting Chl protein (LHCP). In all species, Chl b was maintained at a stable value throughout the growing season. An increase in nitrogen allocation to Chl is normally a form of adaptation to shady conditions (e.g. Koike et al., 1997; Kimura et al., 1998; Koike et al., 2001). The Chl/N ratio in August showed an increase with decreasing rPPFD in all species (data not shown). In October, there was a great scatter in the Chl/N ratio across species because of the onset of leaf yellowing.

4.2 Vertical pattern of photosynthetic function

Variation in the light-saturated photosynthetic rate (Psat) at ambient CO2 within the vertical canopy profile was insignificant for ash, basswood and elm in Photosynthetic Characteristics of Mixed Deciduous-Broadleaf Forests 461

June Aug . Oct .

Alder

Birch

Elm

Maple

Ash

Basswood

Tree height (m) Tree Walnut

0 5 10 15 0 5 10 15 0 5 10 15

Psat (µmol m-2s-1)

Fig. 5. Seasonal and vertical changes in light saturated photosynthetic rate (Psat) of the seven deciduous species in a forest stand. In June and August, leaves at the canopy surface in alder, birch and elm show lower Psat than leaves at the inner part of the same canopy (adopted from Koike et al., 2001).

June and October (Fig. 5). In August, except for walnut, Psat at the canopy top was lower than for leaves located 3 m below the top. In walnut, Psat within the crown varied little between sun and shade leaves. By October, the onset of senescence resulted in a substantial decline in photosynthetic rates in all species compared to mid-season values. For ash, elm and walnut, the reduced lower light saturated photosynthetic rate at the canopy top was maintained. A large difference between sun and shade leaves has also been found for seedlings native to Illinois (Bazzaz and Carlson, 1982; Koike, 1986). The difference in Psat between sun and shade leaves within the crown was greater for early successional species, such as alder and birch, and smaller for late or mid successional species such as maple and basswood. For early successional species, the Net photosynthetic rate (Pn) saturated at around 1000Ð1200 µmolá mÐ2sÐ1 for sun leaves at the canopy top, and at 800Ð900 µmolámÐ2sÐ1 for shade leaves lower in the crown. The saturation PPFD was much lower in late successional species, at 400Ð500 µmolámÐ2sÐ1 for both sun and shade leaves. Mid successional species (elm) or gap phase species (walnut and ash) had light requirements intermediate between early and late successional species (basswood and maple). In particular, Psat of sun leaves of ash, walnut and basswood showed signs of photoinhibition, such that Psat decreased with increasing PPFD above the saturation level. Maple, perhaps because of its thin leaves, is highly susceptible to strong light (Kitao et al., 2000; Kitaoka et al., 2001). A stratified acclimation would therefore be expected within the canopy profile, differing across species with differing patterns of canopy development. For example, the leaf senescence pattern of a tree crown is closely linked to the growth strategy of the species. There is a clear contrast in the progression of autumn coloration between species, which start from the inner part of a crown 462 T. KOIKE et al.

(early successional species) and from the outer surface of a crown (late successional species) (Koike, 1990, Kozlowski et al., 1991). The pattern of late season nitrogen translocation between senescence types would then have an effect on shade acclimation and seasonal biomass production. In future it would be helpful to integrate these variations over space (canopy profile) and time (across season) to scale up from the leaf to the canopy level.

5. PHOTOSYNTHESIS OF TREE SEEDLINGS IN A FOREST FLOOR AND GAP

5.1 Specific pattern of leaf photosynthesis Measurement of light photosynthetic curves for magnolia (gap phase species) showed that Psat was 6.0 µmolámÐ2sÐ1, saturating at a value of around 1000 µmolámÐ2sÐ1, which is higher than for the other species (Fig. 6). Psat for carpinus and cherry (late successional) were both 2.5 µmolámÐ2sÐ1, saturating at around 500 µmolámÐ2sÐ1. In oak (mid-late successional), Psat was 4.0 µmolámÐ2sÐ1, saturating at around 1000 µmolámÐ2sÐ1. Based on measurements made over three years, tree seedlings that invade a larch plantation are well adapted to the light heterogeneity found in the stand across seasons. Adaptations that enhanced survival in this environment included longer leaf lifespan, higher Chl content, better photosynthetic traits in low light, and re-translocation of leaf nitrogen before leaf fall. These functional adaptations agree well with successional traits of trees in cool temperate broad-leaved forests (Bazzaz, 1979; Küppers, 1984; Koike, 1988; Jurik et al., 1988). Late successional

10 Magnolia Oak 1999 8 2000 6

) 2001 -1 s

-2 4 m 2 mol 0 6 Psat ( Cherry Carpinus 4 2 0 MJ JASO MJ JASO Month

Fig. 6. Seasonal trends in Psat in four seedlings regenerated in the forest floor of an unmanaged forest. There were large seasonal changes with species and from year to year (Kitaoka and Koike, 2004). Photosynthetic Characteristics of Mixed Deciduous-Broadleaf Forests 463 species such as cherry and carpinus maintained a low value of their light saturated photosynthetic rate for long times, whereas magnolia, which is a gap phase species, showed a high photosynthetic rate for a short time. Light use traits of oak were intermediate between magnolia and carpinus (data not shown). On the forest floor, understory seedlings must utilize light incident during the spring leafless period of the canopy trees (Harrington et al., 1989; Lei and Koike, 1998a, b; Seiwa, 1998). Almost 30% of the photosynthetic production in understory plants can take place during early spring in deciduous broad-leaved forests (Harrington et al., 1989). Growth of most seedlings in forests is dependent on sunflecks (Chazdon, 1988; Pearcy, 1988). The cherry flushed its leaves before canopy closure, and was able to exploit the light environment efficiently. In contrast, carpinus maintained its leaves after the overstory trees had shed their leaves until frost came. The green leaves in this late season may allow a longer photosynthetic period even though carpinus has lower re-translocation of leaf nitrogen in the autumn. It is well documented that these photosynthetic characteristics allow undergrowth seedlings to exploit the low light environment in forest stands, as a result of adaptive leaf phenology between seedling and canopy trees (Augspurger and Bartlett, 2003). Seasonal trends in Psat showed species-specific patterns during the three years of the study (Fig. 7). Magnolia showed clear seasonal changes in Psat, which increased gradually from June to August and then gradually fell toward late September. Cherry and carpinus showed no marked seasonal changes in Psat, which remained constant throughout the growing season. Oak displayed a pattern of seasonal change intermediate between that of magnolia, and cherry or carpinus. )

-1 6 s -2 Magnolia m

Oak

mol 4

Cherry 2 Carpinus

0

-2 Net photosynthetic rates ( 0 500 1000 1500 2000 Photosynthetic photon flux density ( mol m-2s-1)

Fig. 7. The light dependent net photosynthetic rate of four tree seedlings in an unmanaged forest. All measurements were made during mid- to late-August. Magnolia and oak in the gap show higher Psat at higher PPFD, and cherry and carpinus at the forest floor show lower Psat at lower PPFD (Kitaoka, unpublished data). 464 T. KOIKE et al.

For oak, increased gradually from June to August, then remained constant until the end of September and decreased in October. The maximum photosynthetic rate of all four species fluctuated from year to year. Nitrogen allocation patterns also indicate tolerance to shade (Bazzaz, 1996; Niinemets and Tenhunen, 1997). In the magnolia and cherry, the Chl/N ratio increased slightly with canopy closure. This observation suggests that both species can exploit the resulting shady environment, and might utilize both sunny and shady light environments. In magnolia, the increase in the Chl/N ratio in September is partly due to leaf senescence. Chl protein complexes are more difficult to decompose than other photosynthetic proteins (Larcher, 2003). As the leaves wither, other photosynthetic nitrogen compounds decompose and remobilize to the plant body. The leaf Chl/N ratio should therefore increase, as was observed in the late growing season (data not shown).

5.2 Year-to-year variation in photosynthetic rates A linear relation was found between leaf nitrogen content and Psat (detected at light saturation with 360 ppm) both per unit area (Fig. 8) and unit mass (Kitaoka and Koike, 2004) for each species in each year. However, the gradient of the linear relation was different for each species. The correlation between leaf nitrogen and Psat was clearer based on leaf dry mass. Variation across years was larger in magnolia and oak than in cherry and carpinus. There was a strong positive correlation between leaf nitrogen content and Pmax (detected by saturated light and CO2 at 1500 ppm) for each species (Fig. 9). In general, Psat of deciduous broadleaved trees is closely correlated with LMA (e.g. Koike, 1988). Although there were no marked differences in LMA in each of the three years in magnolia and oak, LMA was slightly larger in cherry and carpinus in, 1999 than in 2000 or 2001 throughout the growing season. What

10 Magnolia Oak

8 2001 ) 1999 -1 6

s

-2

m 4

↔ 2 2000

mol

µ

( 0 6

Psat Cherry Carpinus 4 2 0 0 0.5 1.0 1.5 2.0 0 0.5 1.0 1.5 2.0 Leaf nitrogen content (g↔m-2)

Fig. 8. Relation between leaf nitrogen content and Psat in four species during three years. Different symbols show the value determined in situ for different years (adapted from Kitaoka and Koike, 2004). Photosynthetic Characteristics of Mixed Deciduous-Broadleaf Forests 465 factors influence the relation between Psat and leaf N in a species? Photosynthesis in individual leaves is determined by: stomatal conductance (e.g. Schulze and Hall, 1982), CO2 diffusion, nitrogen allocation of photosynthetic proteins, and the kinetics of Rubisco (Field and Mooney, 1986; Larcher, 2003). LMA is representative of leaf morphological and physiological activity (Ellsworth and Reich, 1993; Reich et al., 1995, 1998, 1999; Evans and Caemmerer, 1996; Evans and Poorter, 2001). Integration of photosynthetic parameters with LMA is one of the tools scaling from leaf to canopy (Reich et al., 1995; Waring et al., 1995). LMA (=1/SLA) is strongly affected by several factors during leaf expansion (Tichá, 1985; Tardieu et al., 1999). It has been reported that leaf growth rate is an exponential function of the maximum air temperature in crop plants (Biscoe and Gallagher, 1977) and birches (Koike, 1995). Our results suggest that cherry and carpinus had larger LMA in the hottest and driest year (1999) than in the other two years of the study. LMA is determined by leaf thickness and density (Niinemets, 1999, 2001). Niinemets (2001) has also reported that leaf thickness and LMA correlate positively with the mean monthly air temperature and solar radiation during leaf expansion, but that leaf densities (dry mass per leaf volume) are negatively correlated with precipitation, so that the dry year of 1999 should have small sized leaves with high density. Differences in precipitation during leaf expansion might therefore affect the leaf density in our study, explaining the larger LMA of cherry and carpinus in 1999. The photosynthesis-nitrogen relationship might also be strongly influenced by these climatic factors, especially temperature. Consequently, the large differences in air temperature and

20 Magnolia Oak 15 )

-1 10 s -2

m 5

mol 0 20 Cherry1999 Carpinus

Pmax ( 15 2000 2001 10

5

0 0 0.5 1 1.5 2 0 0.5 1 1.5 2 Leaf nitrogen content (g m-2)

Fig. 9. Positive correlation between leaf nitrogen content and Pmax (measured at light and CO2 saturated condition). There was no yearly variation between leaf nitrogen content and Pmax (adopted from Kitaoka and Koike, 2004). 466 T. KOIKE et al. precipitation in the leaf development period between 1999 and the other years may underlie the differences observed in leaf photosynthetic rate, via changes in LMA and leaf physiology. Of course, stomatal conductance correlates positively with Pn (Schulze and Hall, 1992), which is affected by several biochemical processes. Moreover, biochemical factors and nitrogen allocation would also be influenced by climatic conditions, especially temperature (Makino et al., 1994). Consequently, diffusion resistance in a leaf seems to be essential in regulating CO2 (Hinckley et al., 1978; Björkman, 1981; Evans and Caemmerer, 1996; Terashima et al., 2001).

6. ESTIMATION OF PHOTOSYNTHESIS WITH A PORTABLE NITROGEN DETECTOR In previous sections, we described a strong positive correlation between leaf nitrogen content and the potential photosynthetic capacity. Figure 10 shows the relation between leaf nitrogen content and Psat in canopy trees and seedlings in deciduous forests. There are two regression lines between these. A steep slope was found for leaves of the sunny crown and seedlings grown at forest gaps, and a gentle slope for leaves of the shady crown and seedlings in forest floor. The former leaves had high nitrogen use efficiency (NUE), and the latter group had lower NUE. A shallow slope relation was also reported in leaves located in shady conditions of a peach crown (DeJong et al., 1989). Some leaves with high nitrogen content showed lower Pn because they suffered from photoinhibition in summer (Koike et al., 2001). The distribution of nitrogen throughout a leaf is considered to be essential for maximizing the photosynthetic rate.

15 Sunny crown Shade crown Sunny crown (photoinhibition) )

-1 Forest gap s

-2 10 Forest floor m mol 5 Psat (

0 0 123 Leaf nitrogen content (g m-2)

Fig. 10. Relation between leaf nitrogen content (N) and Psat in canopy trees and seedlings. There are two regression lines between N and Psat; a steep slope is found for sunny crown and seedlings at gap, and a shallow slope is found for shady crown and seedlings at the forest floor. Photosynthetic Characteristics of Mixed Deciduous-Broadleaf Forests 467

Photosynthetic production of plant communities is usually estimated from the activities of canopy surface (Saeki, 1959). To scale up from leaf to canopy level, it is necessary to have reliable foliage parameters that relate directly to photosynthetic function which are independent of species, i.e. leaf nitrogen content of canopy surface. We therefore introduced a nitrogen meter for non- destructive continuous measurement of the nitrogen condition of the forest canopy. A portable, non-destructive type of nitrogen meter (Agriexpert PPW- 3000) was tried out on several leaves sampled from ten forest species (Ichie et al., 2002). Also investigated was the potential relation between leaf nitrogen, chlorophyll content and the readings taken with the Agriexpert and a chlorophyll meter (SPAD-502). The principal idea behind Agriexpert is the same as for SPAD (a portable chlorophyll detector). Agriexpert detects at the peak of 560 (leaf reflectance or permeation), 660 (chlorophyll), 880 (protein with methyl group), 950 nm (water absorption as baseline) (Ichie et al., 2002). A significant positive correlation was found between the readings of the Agriexpert and the nitrogen content derived using the N/C Analyzer in the same leaves; the correlation between leaf chlorophyll content and the Agriexpert was positive but weaker. Psat was estimated from the readings of the Agriexpert because of the strong correlation between the readings of Agriexpert and nitrogen content in the leaf (Fig. 11). The same relation was found for the significant positive correlation between the actual chlorophyll content and the SPAD readings. It is necessary to check whether Agriexpert is useful for each species because the limitation of openness of the detection part of the actual detector inhibit for thick leaves. Moreover, the nitrogen and chlorophyll distribution in leaves is not

8 Carpinus )

-1 Oak s 6 -2 m

mol 4 Psat ( 2

0 2 2.4 2.8 3.2 3.6 4 Reading value of Agriexpert

Fig. 11. Correlation between the reading of the Agriexpert (nitrogen detector) and Psat (after Ichie et al., 2002). For carpinus: Y = Ð4.281 + 3.1267X, r2 = 0.94, and oak: Y = Ð18.833 + 10.602X, r2 = 0.77. 468 T. KOIKE et al. uniform, but varies with the stage of leaf development and location of the plant canopy (Sesták,ˇ 1985). When using the Agriexpert it may therefore be necessary to consider not only the location of the leaves sampled, but also their light condition and age, and sample across all of these variables. In conclusion, we have found large differences in leaf phenology, leaf photosynthetic characteristics and leaf functional traits (e.g. LMA, N, Chl, Chl b, Chl/N ratio) across tree species in a mixed forest. There were also large differences in the leaf phenology of understory seedlings, leading to significant yearly variations in leaf photosynthetic capacity (Kitaoka and Koike, 2004). Though there is stomatal limitation (Schulze and Hall, 1982), the year-to-year variation in the leaf photosynthetic rate may be attributed to CO2 diffusion resistance mediated by LMA, since no yearly fluctuation was found in Pmax (detected at light and CO2 saturation) or leaf nitrogen concentration. A specific relation was found between leaf nitrogen concentration and Pmax. This will permit extrapolation of the potential capacity of photosynthesis from a leaf to the stand from continuous measurement of the canopy nitrogen content. The midday depression with photoinhibition must of course be allowed for (Long et al., 1994; Kitao et al., 2000). Based on these data, it might be possible to scale up from canopy photosynthesis to biome productivity using satellite images (e.g. Waring et al., 1995, Nakaji et al., 2003).

Acknowledgements—This research is sponsored in part by the Ministry of Education, Sports, Culture, Science & Technology (MEXT) of Japan through the Special Coordination Fund “GCMAPS” program. (GCMAPS means Global carbon cycle and related mapping based on satellite imagery) and by JSPS (Basic research B) grant to T. Koike. Thanks are also due to two anonymous reviewers for their constructive comments and suggestions.

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T. Koike (e-mail: [email protected]), S. Kitaoka, T. Ichie, T. T. Lei and M. Kitao