ORIGINAL ARTICLE

Yusuke Onoda Æ Tadaki Hirose Æ Kouki Hikosaka

Effect of elevated CO2 levels on leaf starch, nitrogen and photosynthesisof plants growing at three natural CO2 springs in Japan

Received: 14 February 2006 / Accepted: 19 July 2006 / Published online: 13 September 2006� The Ecological Society of Japan 2006

Abstract Plant communities around natural CO2 springshave been exposed to elevated CO2 levels over manygenerations and give us a unique opportunity to inves-tigate the effects of long-term elevated CO2 levels onwild plants. We searched for natural CO2 springs in cooltemperate climate regions in Japan and found threesprings that were suitable for studying long-term re-sponses of plants to elevated levels of CO2: Ryuzin-numa, Yuno-kawa and Nyuu. At these CO2 springs, thesurrounding air was at high CO2 concentration with notoxic gas emissions throughout the growth season, andthere was natural vegetation around the springs. At eachsite, high-CO2 (HC) and low-CO2 (LC) plots wereestablished, and three dominant species at the shrublayers were used for physiological analyses. Althoughthe microenvironments were different among thesprings, dicotyledonous species growing at the HC plotstended to have more starch and less nitrogen per unitdry mass in the leaves than those growing at the LCplots. In contrast, monocotyledonous species growing inthe HC and LC plots had similar starch and nitrogenconcentrations. Photosynthetic rates at the mean growthCO2 concentration were higher in HC plants than LCplants, but photosynthetic rates at a common CO2

concentration were lower in HC plants. Efficiency ofwater and nitrogen use of leaves at growth CO2 con-centration was greatly increased in HC plants. These

results suggest that natural plants growing in elevatedCO2 levels under cool temperate climate conditions havedown-regulated their photosynthetic capacity but thatthey increased photosynthetic rates and resource useefficiencies due to the direct effect of elevated CO2 con-centration.

Keywords Down-regulation of photosynthesis ÆLong-term response to elevated CO2 Æ Photosyntheticnitrogen use efficiency Æ Water use efficiency ÆWild plants

Introduction

Over 20 years a number of studies have investigated theresponse of plants to elevated levels of CO2. Open-topchambers (OTC) and free-air CO2 enrichment (FACE)have been used to obtain a more realistic picture of plantresponses to elevated CO2 concentrations in naturalconditions. However, most results came from experi-ments that had run a maximum of 10 years (Ainsworthet al. 2003). Because many ecological processes (plant–plant interaction, microbe activity, decomposition andsoil condition) progress much slower and because thesechanges could also modulate plant responses (e.g., Luoand Reynolds 1999; Kohut 2003), there are still manyuncertainties as to how plants respond to long-term(>10 years) elevated levels of CO2. The vegetationaround natural CO2 springs is valuable, because it is theonly vegetation that has been exposed to high CO2

concentration for a very long time (Koch 1994; Migliettaet al. 1994; Kohut 2003).

Detailed field studies of natural CO2 springs werestarted in Italy in the early 1990s. Miglietta and Raschi(1993) and Miglietta et al. (1993a) described severalnatural CO2 springs where CO2 had been emitted fromvents in the Mediterranean climate. Studies on plantresponses to elevated CO2 levels have been carried out atCO2 springs with respect to photosynthesis (Jones et al.1995), photoinhibition (Stylinski et al. 2000), water

Y. Onoda Æ T. Hirose Æ K. HikosakaGraduate School of Life Sciences, Tohoku University,Aoba, Sendai, Japan

Y. Onoda (&)Section of Plant Ecology and Biodiversity,Institute of Environmental Biology,Utrecht University, P. O. Box 800.84,3508 TB Utrecht, The NetherlandsE-mail: y.onoda@bio.uu.nlTel.: +31-30-2516876Fax: +31-30-2536700

T. HiroseDepartment of International Agriculture Development,Tokyo University of Agriculture, Tokyo, Japan

Ecol Res (2007) 22: 475–484DOI 10.1007/s11284-006-0030-z

relations (Chaves et al. 1995; Jones et al. 1995; Tognettiet al. 1996, 1998, 1999), non-structural carbohydrate(Korner and Miglietta 1994), isoprene emission (Rap-parini et al. 2004; Scholefield et al. 2004), growth (Mi-glietta et al. 1993b), tree-ring expansion (Hattenschwileret al. 1997; Tognetti et al. 2000) and litter quality (Ga-hrooee 1998; Cotrufo et al. 1999). Although a number ofstudies has been done in Italy, only a few comparablestudies have been conducted in different climatic regions(e.g., New Zealand, Newton et al. 1996; Iceland, Cooket al. 1998; Venezuela, Fernandez et al. 1998; Slovenia,Vodnik et al. 2002). Differences in precipitation andtemperature may influence the effect of elevated CO2

levels on plants (Long 1991; Idso and Idso 1994; Drakeet al. 1997). Furthermore, interspecific variations in CO2

responses may be evident across plant communities thatwere established at different CO2 springs (Poorter 1993).Therefore, studies on natural CO2 springs in differentclimates are crucial to generalize plant response to long-term elevated concentrations of CO2.

The Japan Archipelago is situated on the Pacificvolcanic belt and has at least 82 CO2 springs (Kimbara1992; Cook et al. 2000). The islands are influenced by theAsian monsoon climate, with relatively high rainfall andhumidity throughout the year, which is very differentfrom the Mediterranean climate. In this study we aimedto identify natural CO2 springs in Japan as a place tostudy plant responses to CO2 enrichment. We firstsearched CO2 springs that were suitable for biologicalresearch and described the CO2 environment to establishstudy sites. In contrast to the previous studies, whichused one or two springs, we used three CO2 springs,which overcame the general drawbacks of CO2 springstudy such as lack of real control and replication.

In this study we also studied leaf traits of plantsgrowing around natural CO2 springs. As CO2 is a sub-strate for photosynthesis, elevated CO2 concentrationenhances photosynthesis (e.g., Mott 1990). However,when plants grow under prolonged exposure to elevatedCO2 concentrations, this increase is often offset bydown-regulation of photosynthetic capacity (e.g., Longet al. 2004). Accumulation of non-structural carbohy-drates cause down-regulation by decreasing the amountand/or activity of photosynthetic proteins (Sheen 1990,Stitt and Krapp 1999). The extent of down-regulation is

sensitive to growing conditions (e.g., Arp 1991). As thegrowing conditions at CO2 springs would have beeninfluenced by ecosystem level feedbacks that occurredover a very long time (Luo and Reynolds 1999; Hiko-saka 2003), plants growing around CO2 springs can beused to test whether their CO2 responses are differentfrom those obtained in short-term growth experiments.We investigated how much starch was accumulated andto what extent photosynthesis down-regulated in plantsgrowing around three CO2 springs.

Materials and methods

A search for CO2 springs followed former geologicalinvestigations (Kimbara 1992; Tsurumi et al. 1999;Cook et al. 2000). We visited many springs and assessedwhether they could be used for biological research withrespect to the following aspects: (1) the CO2 concen-trations around the springs are continuously at highlevels, (2) toxic gasses for plants (e.g., H2S, SO2) are notemitted or are at levels too low to affect plant growth, (3)the springs are cold, (4) natural vegetation is abundantaround the CO2 spring. From preliminary surveys(1999–2002), three sites (Ryuzin-numa, Yuno-kawa andNyuu) had been found to be suitable for plant scienceresearch and are described in this paper.

Ryuzin-numa and Yuno-kawa are located at 570–575 m above sea level (a.s.l.), at the foot of MountHakkoda in Aomori Prefecture (Table 1; Fig. 1a). Thedistance between the two CO2 springs is 1.0 km,implying that they are likely to share the same source ofCO2. Nyuu is located at 1,000 m a.s.l., at the foot ofMount Gassan in Yamagata Prefecture, about 300 kmsouth of Ryuzin-numa and Yuno-kawa (Fig. 1a). TheseCO2 springs are situated in areas having a cool–tem-perate climate (Table 1), receiving high rainfall fromspring to autumn and a large amount of snow duringwinter. They are located in the National Parks, andnatural vegetation around the springs was generally wellpreserved (Fig. 1).

A CO2 concentration monitoring system wasconstructed with a portable infrared gas analyzer(LI-2820, Li-Cor, Lincoln, Nb., USA, full-scale20,000 lmol mol�1), a six-channel air sampling system

Table 1 Geographical co-ordinates, air pressure, mean air temperature, monthly mean rainfall from June to August in 2003, and theapproximate area where mean CO2 concentration was above 500 lmol mol�1 at three natural CO2 springs

CO2

springsLatitude–longitude Altitude

(m)Airpressure(kPa)

Meantemperaturea

(�C)

Rainfalla

(mm)HighCO2 area(m2)

Ryuzin-numa 40�41¢N–140�55¢E 575 95 17.1 186 >300Yuno-kawa 40�41¢N–140�55¢E 570 95 17.1 186 >400Nyuu 38�32¢N–139�59¢E 1,000 90 15.8 212 >600

aMeteorological data for Ryuzin-numa and Yuno-kawa are from the Sukayu meteorological station (a.s.l. 890 m) 7–8 km away and thosefor Nyuu are from the Hakkosawa meteorological station (a.s.l. 700 m) 3.5 km away. Temperature was adjusted for difference in altitudewith a lapse rate of 0.006�C m�1

476

(Meiwa Co. Ltd., Tokyo, Japan), a data logger (Li-1400,Li-Cor) and 12 V car batteries (38B19R, Nihon-Denki,Kyoto, Japan). Six sampling tubes were fixed 1 m abovethe ground at appropriate positions around the CO2

springs (see Fig. 1). CO2 concentrations were measuredevery second and logged as mean values every 30 s. Themonitoring was done for 2–4 days every month duringthe growing season (June–October). Hand-held CO2

monitors (Telaire 7001, TELAIRE, Goleta, Calif., USA,full-scale 10%) with data loggers (H08–007-02, Onset,Bourne, Mass., USA) were used for point-to-pointmeasurements and also for measurements at low CO2

areas. These CO2 analyzers were calibrated with a pureN2 and a CO2 standard gas (Tanuma-Sanso, Sendai,Japan) every month.

The concentrations of H2S and SO2 were measuredby H2S- and SO2 detecting tubes (no. 4LT and no. 5Lbfor H2S and SO2, respectively, Gastec, Ayase, Japan) atpoints where maximum concentrations were expected.

Relative light intensities around the CO2 springs weremeasured with light-sensitive films that fade and changetheir transmittance in proportion to the amount ofaccumulated light absorption (Opt leaf R-2D, Taisei-Kako, Tokyo, Japan) (Hikosaka et al. 2001). Films wereattached to plastic plates and placed horizontally at var-ious distances from the springs 1 m above the ground for3 days in July in 2002 (Ryuzin-numa) and in 2003 (Yuno-kawa and Nyuu). Films that were exposed to full lightintensity in open areas were used as a reference. The ex-tent of discoloration was measured at 560 nm with aspectrophotometer (UV-160A, Shimadzu, Kyoto, Japan).

At the three study sites, high CO2 (HC) and low CO2

(LC, close to ambient CO2) plots were established. Weselected LC plots by the following four criteria: (1) CO2

concentration was close to ambient at 370 lmol mol�1;(2) not far from the HC plot (to eliminate effects of anyunexpected environmental difference); (3) similar lightenvironment to HC plots; (4) some overlap of specieswith HC plot.2

To measure soil nutrients we sampled the surface soil(0–5 cm depth) of each plot in June 2003 (n=5). The soilwas sieved (2 mm wire mesh), and 10 g of the wet soilwas mixed with 50 ml of 2 M KCl. Nitrate and ammo-nium in the soil solution extract were determinedphotometrically with the Cataldo method and theindophenol blue method, respectively (Keeney andNelson 1982). The same soil solution was used for thedetermination of soil pH. The soil analysis was con-ducted again for Ryuzin-numa in August 2004 becauseof a possible error in the extraction procedure.

Physiological analysis

Three dominant species (see Table 3) at the shrub layerat each CO2 spring were used for analysis of leaf starch,nitrogen and carbon. Eight leaves were sampled fromabout 1 m above the ground surface (except for Plan-tago asiatica, 10 cm) at about noon from differentindividuals at HC and LC plots. The sampling was doneon 4 August 2002, 6 August 2003 and 28 August 2003for Ryuzin-numa, Yuno-kawa and Nyuu, respectively.

Ryuzin-numaYuno-kawaNyuu

(a)

Forest Herb & Shrub Reed Marsh Water Spring

10 m

LC

HC

HC

10 mLC

Track

1 2 345 6

10 m

HC

LCRoad

654321

Path

123456

(b) Ryuzin-numa

(c) Yuno-kawa (d) Nyuu

Fig. 1 Location of CO2 springsstudied (a) and vegetation atRyuzin-numa (b), Yuno-kawa(c) and Nyuu (d). HC (highCO2) and LC (low CO2,ambient CO2) plots wereestablished in the area withsimilar light climates. Numbers(1–6) in the figures indicate theplace at which CO2

concentrations were monitored

477

Leaves were brought to the laboratory in a cooledpolystyrene box (<5�C). Within 10 h of being sampled,eight disks of 1 cm diameter were punched out fromeach leaf. Four were weighed after being dried in anoven at 75�C for more than 3 days, and their totalnitrogen and carbon were determined with an NC ana-lyzer (NC-80, Shimadzu, Kyoto, Japan). The other fourleaf disks were frozen in liquid nitrogen and stored at�80�C until required for starch analysis.

The amount of starch was determined according toRufty and Huber (1983). Two disks from each leaf werehomogenized in liquid nitrogen and suspended in 1.5 mlof 80% ethanol. The suspension was heated at 80�C for30 min and centrifuged at 10,000 g for 10 min, and thesupernatant was removed. This extraction procedurewas repeated three times. The pellet was dried in an ovenfor 1 day, and then suspended in 1.0 ml of 0.2 M KOHand heated at 90�C for 30 min. After cooling, 0.2 ml of1.0 M acetic acid and 1.0 ml of amyloglucosidase(35 units ml�1 in 50 mM Na acetate buffer, pH 4.5,Rhizopus mold, Sigma, St Louis, USA) were added, andthe mixture was incubated at 55�C for 30 min. Thesuspension was centrifuged at 10,000 g for 10 min, andthe supernatant was used for determination of glucosewith the glucose B test Wako (Wako, Osaka, Japan).

CO2 response curves of photosynthesis were deter-mined on one dominant species from each spring(Polygonum sachalinense in Ryuzin-numa and Yuno-kawa, and Plantago asiatica in Nyuu) on 2–3 July, 4–6August and 26–27 August 2003 for Ryuzin-numa,Yuno-kawa and Nyuu, respectively (n=8 at each plot).At Ryuzin-numa and Yuno-kawa, photosynthesis wasmeasured on attached, fully expanded young leaves withan open gas-exchange system (LI-6400, Li-Cor). AtNyuu, fully expanded young leaves were cut at the pet-iole and used for the measurement in the laboratory.During measurements, leaf temperature was kept at20�C, and the vapor pressure deficit in the chamber at<1.0 kPa. Photosynthetic photon flux density (PPFD)was set at a saturating light intensity for plants at eachspring (1,200 lmol m�2 s�1, 800 lmol m�2 s�1,1,200 lmol m�2 s�1 for plants at Ryuzin-numa, Yuno-kawa and Nyuu, respectively).

We analyzed the CO2 response curve of photosyn-thesis in accordance with the model of Farquhar et al.(1980) to determine the maximum RuBP carboxylationrate (Vcmax) and the maximum electron transport ratedriving RuBP regeneration (Jmax). Michaelis–Mentenconstants of Rubisco (ribulose-1,5-bisphosphate car-boxylase/oxygenase) and the CO2 compensation point inthe absence of day respiration followed Bernacchi et al.(2001). Day respiration was assumed to be 0.02 of Vcmax

(von Caemmerer 2000).Water use efficiency (WUE) was defined as the pho-

tosynthetic rate at the mean growth CO2 concentration(Agrowth) divided by the transpiration rate. Similarly,photosynthetic N use efficiency (PNUE) was defined asAgrowth divided by leaf nitrogen concentration per unitarea (Narea).

Statistical tests were performed with STATVIEWv. 5.0 (SAS Institute, Cary, Ind., USA). The effects ofCO2 (HC and LC) and sites (Ryuzin-numa, Yuno-kawaand Nyuu) on leaf characteristics were evaluated byanalysis of variance (ANOVA). The t -test was used totest the difference between HC and LC.

In the present study the genus name is used when aplant name is abbreviated, because some species havethe same initial for their genus name, which would makea confusion of the species if the genus names wereabbreviated.

Results

Site characteristics

Ryuzin-numa, which is characterized by the presence ofa pond (Fig. 1b), was a semi-open space surrounded bya birch–oak forest with more than 20 species of shrubsand herbs. A gradient of CO2 concentrations was ob-served along a distance from the vent. Mean daytimeCO2 was about 680 lmol mol�1 (at 95 kPa) at thenearest monitoring point (Fig. 2) and 375 lmol mol�1

at 50 m from the vent. There was no clear seasonal trendin CO2 concentrations during the growth season,(Fig. 2). Diurnally, CO2 concentration was, at most,twice as high in the night as in the day. CO2 concen-tration was generally higher near the surface and grad-ually decreased with height, but it was still higher thanambient CO2 at 5 m above the ground (data not shown).The area with [CO2] above 500 lmol mol�1 wasapproximately 300 m2. Near the CO2 springs, Polygo-num sachalinense, Sasa kurilensis, Hydrangea paniculataand Calamagrostis matsumurae were abundant. Lyco-podium obscurum and Lycopodium clavatum (lycophytes)that are rare at the foot of Mount Hakkoda were alsoobserved.

Yuno-kawa is located in a gentle valley area withmature beech–oak forests. The CO2 concentration herewas relatively stable and stayed at around530 lmol mol�1 (at 95 kPa) (Fig. 2). Low wind speedsdue to the dense forest and depressed topography mighthave reduced CO2 fluctuations. The vertical gradient of[CO2] was fairly small from 0.1 m to 5 m above ground,and no diurnal change in CO2 concentration was ob-served (data not shown). CO2 concentration was highalong the stream and decreased gradually with distancefrom the springs but was still higher than the ambientCO2 300 m downstream from the spring. The approxi-mate area where mean [CO2] was above 500 lmol mol�1

was greater than 400 m2. About 20 shade-tolerant spe-cies grew around the springs, of which Tiarella po-lyphylla, Sanicula chinensism, Sasa and Polygonum wererelatively abundant.

Nyuu is located on a gentle hill-slope facing west andis covered by a species-rich beech-oak forest. CO2

emissions at this site were the largest among the threeCO2 springs, and a large area with high CO2 levels was

478

maintained (Table 2). The stream of CO2-rich water alsoresulted in an adjacent open area having high CO2 levels.CO2 concentration in this open area fluctuated, due tovarying wind speeds, but it was higher than that of theambient CO2 with a gradual reduction with distancefrom the vent (Fig. 2). A diurnal change in CO2 con-centration was not observed. CO2 concentration de-creased gradually with height, but it was still higher thanambient at 5 m above the ground surface (data notshown). Under the forest, some species such as Sasa and

Hydrangea grew. In the open place with high CO2 con-centration, more than 30 species of herbs and shrubswere found. Phragmites australis was a dominantspecies, especially in swampy areas, and its heightappeared to be dependent on CO2 concentration. Alonga small path (Fig. 1d), where vegetation was annuallymown, Plantago asiatica dominated.

At all the study sites, H2S and SO2 were not detected(<0.1 lmol mol�1) even at points where maximumconcentrations were expected. Furthermore, we did notperceive the smell of H2S at any of the springs, whichindicates that the concentration was lower than0.03 lmol mol�1.

At each site, HC and LC plots were established withrespect to the CO2 concentration, distance, light envi-ronment and plant species (Fig. 1). CO2 concentrationsat LC plots were found to be close to ambient CO2 (c.370 lmol mol�1) by monthly measurements in thegrowing season. The relative light intensity (as comparedwith open place) was similar between HC and LC plots(Table 2). Soil pH was similar for the HC and LC plot atall springs. Also, there was no significant difference insoil ammonium content between HC and LC plot in allsprings (P>0.05, Table 2). No nitrate was detected(<5 lg g�1), probably because of the lower soil pH(Table 2).

Leaf traits

Leaves of the dicotyledonous species (Hydrangea,Polygonum, Plantago and Tiarella) growing at HC plotstended to accumulate more starch than those growing atLC plots (Table 3). Monocotyledonous species (Sasaand Phragmites) responded less to elevated CO2 con-centrations and accumulated smaller amounts of starchthan the dicotyledonous species did. Plants growing atYuno-kawa were heavily shaded, and their concentra-tions of starch were less than those at other springs, butpositive effects of elevated CO2 levels were evident in thedicotyledonous species (Polygonum and Tiarella).

The response of leaf mass per area (LMA) to elevatedconcentrations of CO2 was not consistent among spe-cies: LMA increased in Polygonum and Plantago, de-creased in Sasa but did not change in Hydrangea,Tiarella and Phragmites (Table 3). Starch-free LMAresponded similarly to LMA. In the case of Polygonumand Plantago, the increase in starch-free LMA with

JuneJulyAugust

1 2 3 4 5 6

(a) Ryuzin-numa

(b)

(c)

Yuno-kawa0

250

500

Nyuu0

500

1000

0

250

500

750

1000

SeptemberOctoberAverage

Monitoring points

CO

2 co

ncen

trat

ion

(µm

ol m

ol-1

)

Fig. 2 CO2 concentration at a height of 1 m from the soil level atRyuzin-numa (a), Yuno-kawa (b) and Nyuu (c). CO2 concentra-tions were measured at six plots (1–6) in Fig. 1. Each pointindicates the daytime average for 2–4 days. There were no data forJune and July in Nyuu because of technical problems

Table 2 Microenvironments ofthe study plots. The locationsare presented in Fig. 1. Valuesare means ± 1 SD (for soil pHand NH4

+, n=5). ND notdetected (fewer than 5 lg g�1)

aMeasured at height 1 m fromthe ground

CO2 springs Plot(CO2, lmol mol�1)

Relative lightintensity (%)a

Soil pH Soil NH4+

(lg g�1)Soil NO3

�

(lg g�1)

Ryuzin-numa HC (670) 30–50 3.6 ± 0.1 4.5 ± 4.8 NDLC (375) 30–60 3.1 ± 0.3 5.4 ± 1.7 ND

Yuno-kawa HC (530) 2–10 4.1 ± 0.4 10.3 ± 4.9 NDLC (370) 2–10 4.1 ± 0.4 5.8 ± 1.0 ND

Nyuu HC (760) 75–100 3.9 ± 0.3 9.4 ± 10.0 NDLC (370) 75–100 4.2 ± 0.1 6.7 ± 2.0 ND

479

elevated CO2 was smaller than the increase in LMA dueto substantial starch accumulation (Table 3).

Mass-based leaf N concentration (Nmass) tended todecrease with elevated CO2 in plants at Ryuzin-numaand Nyuu (Table 3). Plants growing at Yuno-kawa wereheavily shaded and did not reduce leaf N concentration,and Tiarella had even higher Nmass at the HC plot. TheCO2 response of Nmass in the monocotyledonous specieswas small compared with that of the dicotyledonousspecies. Nitrogen concentration per unit structural mass(starch-free Nmass) was still lower in HC plants than inLC plants for most of the species showing low leaf Nconcentrations. This suggests that the reduction in leafN concentration with elevated CO2 level could be as-cribed not only to dilution by starch accumulationbut also to different allocation patterns of N in the plant.The response of leaf N concentration per unit area(Narea) to elevated CO2 levels varied among species,due to the various CO2 responses of LMA (Narea=Nmass·LMA). Differences in leaf carbon concentrationbetween the HC and LC were fairly small and were notsignificant in most species except for Polygonum inYuno-kawa, which increased by 2.4% under elevatedCO2 concentrations.

Photosynthetic rates at the mean growth CO2 con-centration (Agrowth; 670 lmol mol�1, 530 lmol mol�1

and 760 lmol mol�1 for plants at HC plots of Ryuzin-numa, Yuno-kawa and Nyuu, respectively, and370 lmol mol�1 for plants at all LC plots) were con-sistently higher in plants growing in HC than in LC plots(Fig. 3a). Stomatal conductance at the mean growthCO2 was generally lower in plants at HC plots (Fig. 3b).The maximum RuBP carboxylation rate (Vcmax) andmaximum electron transport rate (Jmax) were consis-tently lower in plants at HC plots, although the differ-ences were not always significant (Fig. 3c, d).

The higher Agrowth and lower stomatal conductancein plants growing at the HC plot led to significantlyhigher instantaneous WUE (Fig. 4a). Similarly, PNUEwas significantly higher in plants growing at HC plots(Fig. 4b). Vcmax/Narea, which indicates fraction of leafnitrogen in Rubisco, was not significantly different be-tween the two plots (Fig. 4c).

Discussion

The present study described three CO2 springs in Ja-pan. Although no scientific study on the history ofthese CO2 springs was available, examination of oldpublic documents suggested a long history of CO2

emissions at these springs. Both Ryuzin-numa andYuno-kawa were described in a document produced byAomori City (Anonymous 1953), in which these springswere presumed to have existed for more than a hundredyears. Information on Nyuu is rather limited, but theadjacent hot spring (500 m from Nyuu) has been re-garded as a sacred place by the Yudonosan Shrine forT

able

3Leafcharacteristics

ofplantsgrowingattheHCandLCplotsofthreenaturalCO

2springs.M

monocotyledonous,D

dicotyledonous,Starchconcentration,LMAleafmass

per

area,st-freeLMAstarch-freeLMA,N

massN

concentration,st-freeN

starch-freemass

base

Nconcentration,N

areaN

concentrationper

unitareaandcarbonconcentrationare

shown.

Values

are

means±

1SD

(n=

7–8).Significance

(t-test):nsnotsignificant,+

P<

0.1,*P<

0.05,**P<

0.01,***P<

0.001

Name

Species(m

onocotyledonous

ordicotyledonous)

Site

Starch(%

)LMA

(gm�2)

st-freeLMA

(gm�2)

Nmass(%

)st-freeN

(%)

Narea(g

m�2)

C(%

)

Ryuzin-numa

Hydrangea

paniculata

(D)

LC

3.50±

1.05

44.5

±9.4

42.9

±8.6

2.90±

0.31

3.01±

0.32

1.28±

0.24

48.4

±1.2

HC

6.18±

0.83*

43.8

±6.5

ns

41.2

±6.5

ns

2.20±

0.40**

2.35±

0.41**

0.95±

0.11**

47.4

±0.7

ns

Polygonum

sachalinense

(D)

LC

4.36±

1.03

35.3

±4.8

33.8

±4.6

3.80±

0.80

4.00±

0.80

1.31±

0.18

50.8

±1.5

HC

12.0

±4.17***

44.1

±7.1

*39.2

±2.1

+2.92±

1.03+

3.28±

1.08ns

1.23±

0.44ns

49.7

±1.2

ns

Sasa

kurilensis(M

)LC

1.07±

0.28

60.6

±3.5

60.0

±3.5

2.67±

0.26

2.70±

0.26

1.61±

0.14

45.8

±0.9

HC

2.02±

1.10*

54.8

±5.3

*53.7

±5.3

*2.42±

0.20+

2.47±

0.20+

1.33±

0.37**

46.5

±1.3

ns

Yuno-kawa

Polygonum

sachalinense

(D)

LC

0.97±

0.48

23.9

±2.3

23.6

±2.3

4.56±

0.33

4.61±

0.32

1.09±

0.10

49.2

±0.7

HC

1.97±

0.85*

27.6

±4.1

*27.0

±3.8

+4.53±

0.35ns

4.63±

0.35ns

1.24±

0.15*

51.6

±1.0

***

Sasa

kurilensis(M

)LC

0.43±

0.21

51.5

±4.7

51.2

±4.8

2.51±

0.24

2.52±

0.24

1.29±

0.11

47.9

±0.6

HC

0.39±

0.04ns

45.1

±2.5

**

45.0

±2.5

**

2.58±

0.24ns

2.58±

0.24ns

1.16±

0.10*

48.0

±0.6

ns

Tiarellapolyphylla(M

)LC

0.29±

0.20

20.5

±1.6

20.5

±1.6

3.15±

0.34

3.16±

0.34

0.64±

0.04

46.3

±1.0

HC

0.79±

0.35**

21.4

±4.4

ns

21.3

±4.3

ns

4.80±

0.53***

4.84±

0.52***

1.01±

0.12***

45.3

±1.4

ns

Nyuu

Hydrangea

paniculata

(D)

LC

4.64±

2.56

45.5

±10.1

43.6

±10.9

2.99±

0.60

3.14±

0.68

1.33±

0.26

47.8

±1.9

HC

6.19±

7.27ns

48.7

±8.9

ns

45.2

±5.7

ns

2.95±

0.50ns

3.14±

0.42ns

1.41±

0.24ns

47.5

±1.2

ns

Phragmites

australis(M

)LC

1.41±

0.40

55.0

±4.6

55.1

±4.3

3.13±

0.25

3.18±

0.26

1.75±

0.20

47.5

±0.5

HC

1.28±

0.20ns

55.0

±4.5

ns

54.3

±4.5

ns

2.87±

0.29ns

2.91±

0.30ns

1.57±

0.11+

47.8

±0.5

ns

Plantagoasiatica

(D)

LC

1.11±

0.48

26.1

±3.2

25.8

±3.2

2.78±

0.48

2.81±

0.48

0.72±

0.08

43.3

±1.4

HC

6.96±

3.54***

30.1

±5.4

+27.9

±4.0

ns

1.97±

0.38**

2.11±

0.35**

0.58±

0.05***

42.1

±0.8

+

480

more than 300 years, and local people affirmed thatNyuu Cold Spring has existed for more than 150 years.All this information suggests that the three CO2 springshave existed for more than a 100 years, and, thus, wecan assume that the plants growing around the springshave been subject to elevated CO2 levels for long time.

One of the biggest concerns in biological study withnatural CO2 springs is a possible effect of other gasses,especially H2S and SO2, which can be emitted from thesprings (Miglietta and Raschi 1993; Koch 1994). H2S isa phytotoxic gas and can significantly affect plantgrowth when the concentration is higher than0.03 lmol mol�1 (Miglietta et al. 1993a). Some ItalianCO2 springs, which have been often used in biologicalstudy, were found to be contaminated by these gasses(Grill et al. 2004). At our study sites we did not detectH2S and SO2, even at the spring points where themaximum concentrations were expected, suggesting thatthe plants in our study sites were free from those phy-totoxic gases.

Most of the HC plants had higher amount of starch,which is consistent with the findings in many previousstudies (e.g., Drake et al. 1997, Long et al. 2004). Kornerand Miglietta (1994) also found that herbaceous plantsgrowing at Italian CO2 springs had, on average, 47%higher total non-structural carbohydrate (of which 70–80% was starch). Although carbohydrate accumulationis sensitive to other environmental factors, such as lightand soil nutrient (e.g., Wong 1990), the general trend ofhigher starch accumulation in HC plants proves that thisresponse is one of the most typical responses to highlevels of CO2. On the other hand, there was a consid-erable difference in starch accumulation among specieseven at the same CO2 spring (Table 3). These results

suggest an importance of species-specific change, whichcould influence plant–herbivore interactions (e.g.,Coviella and Trumble 1999). The dicotyledonous speciestended to have larger responses than monocotyledonousspecies (Table 3). This may be explained by differenttypes of carbohydrate storage: the dicotyledonous spe-cies in this study tended to accumulate starch, whereasthe monocotyledonous species accumulated sucrose (cf.Nakano et al. 1998).

The Agrowth was 21–58% higher in 1.5–2 times higherCO2 concentration (Fig. 3a), which was more or less in asimilar range to that of plants in Italian CO2 springs(26–69%, Blaschke et al. 2001; 36–77%, Stylinski et al.2000). The value also accords with that in literature re-views of elevated CO2 experiments (e.g., 33%, Wandet al. 1999; 31%, Ainsworth and Long 2005). Whenphotosynthetic rates were compared at a common CO2

concentration, they were on average 16% lower at theHC plot, as indicated by Vcmax (Fig. 3c) which wassimilar to the meta-analysis data of the FACE study(13%, Ainsworth and Long 2005). The good agreementof the value between FACE and CO2 springs study leadsus to speculate that the long-term feedback effect onphotosynthesis at the ecosystem level was not large.However, this study used only a part of CO2 springs; alarger scale analysis of soil environments and plant re-sponses would be required to obtain a robust conclu-sion.

Low stomatal conductance and high WUE are oftenobserved in plants grown in elevated CO2 concentrations(Drake et al. 1997). At Italian CO2 springs, plants savedwater consumption under elevated CO2 levels and sus-tained a longer photosynthetically active period in a dryseason (Jones et al. 1995; Hattenschwiler et al. 1997).

Pol

ygon

um

Yun

o-ka

wa

Pol

ygon

um

Ryu

zin-

num

a

Pla

ntag

o

Nyu

u

Pol

ygon

um

Yun

o-ka

wa

Pol

ygon

um

Ryu

zin-

num

a

(c)

30

60

90

(d)

00

40

80

120

160

(a)

0

5

10

15

20

25(b)

0

0.1

0.2

0.3

0.4

0.5+45% *

+21% +

+58% ***

-25%ns

-41%* -27%+

-17%ns

-11%ns

-20%*

-12%ns

-11%ns

-18%*

CO2 P < 0.001Site P < 0.001C x S P = 0.063

CO2 P = 0.007Site P < 0.001C x S P = 0.632

CO2 P = 0.013Site P < 0.001C x S P = 0.880

CO2 P = 0.019Site P < 0.001C x S P = 0.525

J max

(µm

ol m

-2 s

-1)

g s (m

ol m

-2 s

-1)

Agr

owth

(µm

ol m

-2 s

-1)

Vcm

ax (

µmol

m-2

s-1

)

Pla

ntag

o

Nyu

u

Fig. 3 Light-saturated rate ofphotosynthesis at mean growthCO2 concentration (Agrowth) (a),stomatal conductance at meangrowth CO2 concentration (gs)(b), the maximum rate of RuBPcarboxylation (Vcmax) (c), andthe maximum rate of electrontransport driving RuBPregeneration (Jmax) (d) of adominant species growing inboth high CO2 (closed columns)and low CO2 (ambient CO2,open columns) plots at each CO2

spring (Ryuzin-numa. Yuno-kawa and Nyuu).Mean ± 1 SD (n=8).Significance levels (t-test):***P<0.001,*P<0.05, + P<0.1, ns P>0.1

481

Reduction in stomatal conductance with elevated CO2

concentration was also observed in humid climate re-gions like Japan (Fig. 3b). This may be explained by themechanism of the plant to keep a constant ratio ofintercellular CO2 to air CO2 concentration (Wong et al.1979; Drake et al. 1997). Furthermore, as stomatalconductance is associated with leaf temperature throughheat dissipation (Kimball et al. 1995), the lower con-ductance may ameliorate the low temperature stress inplants growing in cool climates.

Conclusions

We described three CO2 springs, where wild plants havebeen exposed to CO2 enrichment for hundreds of yearswithout the effects of sulfur gases. The CO2 responsesin leaf traits observed in the present study were quali-

tatively not different from those in previous experi-mental studies. However, there was a large differencesin CO2 response in starch accumulation among species,even within the same CO2 spring, suggesting that theresponse was quantitatively species-specific. Althoughdown-regulation of photosynthesis was observed athigh CO2 plots, the photosynthetic rate at growth CO2

concentration was stimulated as a result of the directeffect of high CO2 levels. Similar results between FACEand CO2 spring studies suggest that the feedback effecton photosynthesis at an ecosystem level would not belarge.

Acknowledgments The landowners of CO2 springs (Hakkoda-On-sen Co., Tashiro Bokuya-Chikusan Kumiai and Yudonosanshrine) kindly gave us a permission to use their land for this study.We thank Makoto Tsurumi (Hirosaki Univ.) and Atsuki Uchida(Nippon Geophysical Prospecting Co.) for geological informationof Ryuzin-numa and Yuno-kawa, and Taku Noro (Ministry ofLand, Infrastructure and Transport) for climate data at Hakkos-awa meteorological station. We also thank Mark Lieffering (Ag-Research, NZ) for his comments on the draft manuscript, KoujiYonekura (Tohoku Univ.) for sorting plant species, and YukoYasumura, Hisashi Tsujisawa and Riichi Oguchi (Tohoku Univ.)for their technical assistance. Noriyuki Osada (Tohoku Univ.)kindly provided his data of soil nutrients and also gave us con-structive comments on the draft manuscript. This study was partlysupported by grants-in-aid from JSPS for Young Research Fellows(Y.O.), and from the Japan Ministry of Education, Culture, Sports,Science and Technology to K.H. and T.H.

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