REGULAR ARTICLE

Interactions between elevated CO2 and N2-fixationdetermine soybean yield—a test usinga non-nodulated mutant

Shimpei Oikawa & Kay-May Miyagi & Kouki Hikosaka & Masumi Okada &

Toshinori Matsunami & Makie Kokubun & Toshihiko Kinugasa & Tadaki Hirose

Received: 19 May 2009 /Accepted: 1 October 2009 /Published online: 14 October 2009# Springer Science + Business Media B.V. 2009

Abstract Elevated CO2 increases seed productionmore in plant species that form a symbiotic associa-tion with N2–fixing bacteria than in species withoutsuch association. We studied the mechanism of theincrease of seed production with elevated CO2 usingnodulated soybean (Glycine max cv. Enrei) and itsnon-nodulated isogenic line (cv. En1282). Increase inseed production with elevated CO2 was observed innodulated Enrei but was not in non-nodulated En1282.The increase in seed production in Enrei was explainedby the increase in the rate of dry mass production

during the reproductive period. This increase wasassociated with the increase in N assimilation in thereproductive period and the seed N concentration thatremained the same as that at ambient CO2. Dry massproduction and nitrogen assimilation did not increasein the vegetative phase in both lines. These resultsaccorded with the amount of nodules in Enrei thatincreased at elevated CO2 especially after flowering.We conclude that the increase in N assimilation in thereproductive period would be the key for increasingsoybean yield in the future high-CO2 world.

Plant Soil (2010) 330:163–172DOI 10.1007/s11104-009-0189-5

Responsible Editor: Alfonso Escudero.

S. OikawaCenter for Bioresource Field Science,Kyoto Institute of Technology,Saga, Kyoto 616-8354, Japan

K.-M. Miyagi :K. HikosakaGraduate School of Life Sciences,Tohoku University,Sendai, Miyagi 980-8578, Japan

M. OkadaFaculty of Agriculture,Iwate University,Morioka, Iwate 020-8550, Japan

T. MatsunamiAkita Prefectural Agriculture,Forestry and Fisheries Research Center,Yuuwa, Akita 010-1231, Japan

M. KokubunGraduate School of Agricultural Science,Tohoku University,Sendai, Miyagi 981-8555, Japan

T. KinugasaFaculty of Agriculture, Tottori University,Koyama-Minami, Tottori 680-8553, Japan

T. HiroseDepartment of International Agricultural Development,Tokyo University of Agriculture,Setagaya, Tokyo 156-8502, Japan

Present Address:S. Oikawa (*)Department of Plant Biology, University of Illinois,Urbana, IL 61801, USAe-mail: oikawaDX@gmail.com

Keywords Glycine max . Elevated carbon dioxideconcentration . Non-nodulated isogenic line .

Symbiosis . Seed production . Nitrogen

Introduction

Influence of elevated carbon dioxide (CO2) concen-tration on seed crop will be critical to future foodsupply for humans. Seed production, however, mightnot increase at elevated CO2 unless nitrogen (N)uptake increased simultaneously (Kinugasa et al.2003; Hirose et al. 2005). This may be particularlytrue in plants that have a high seed N concentration,where the availability of N limits seed developmenteven though the availability of photosynthates mayincrease at elevated CO2 (Ainsworth et al. 2004;Rogers et al. 2004; Hikosaka et al. 2005). Nitrogen-fixing plants would have an advantage in this respect,because N2-fixation is stimulated by the increasingsupply of photosynthate from the host plant atelevated CO2. Hence it has been hypothesized thatplant species that form a symbiotic association withN2–fixing bacteria exhibit a significant increase inseed production at elevated CO2 (Hardy and Havelka1976; Soussana and Hartwig 1996; Ainsworth et al.2008). There are a number of studies that support thishypothesis. Elevated CO2 increased dry mass produc-tion more in legumes than in other species (e.g. Navaset al. 1997; Lüscher et al. 1998; Kimball et al. 2002).Miyagi et al. (2007) showed that the stimulation ofseed production by elevated CO2 was greater inlegumes than non-legumes. N2-fixation also playsan important role for maintaining seed quality atelevated CO2. Meta-analyses showed that elevatedCO2 decreased seed N or protein concentration inmany species, but to a lesser extent in legumes(Jablonski et al. 2002; Taub et al. 2008). There were,however, also studies that did not support thishypothesis. For example, a meta-analysis of free-airCO2 enrichment (FACE) experiments showed that thestimulation of plant growth was smaller in legumesthan such trees as aspen and loblolly pine (Ainsworthand Long 2005). Ziska and Bunce (2007) compilingstudies from FACE experiments and enclosure studiesshowed that relative stimulation of yield at elevatedCO2 was highest in wheat, followed by soybean andrice. West et al. (2005) found that the influence ofelevated CO2 on N2-fixation and growth of legumes

was positive, neutral or negative, depending onspecies. Differences in growth form and life historyamong species as well as in experimental conditionsconfounded a test of the significance of the symbioticN2-fixation.

Isogenic lines lacking the ability to form nodulesmay provide a straightforward test of the significanceof N2-fixation to the CO2-response of plant reproduc-tion within a common genetic background. Severalauthors have studied the growth of non-nodulatedlines at elevated CO2. Nakamura et al. (1999)observed that plant dry mass increased at elevatedCO2 in a nodulated soybean (Glycine max) but did notin the non-nodulated isogenic line. Similarly, drymass and N per plant increased at elevated CO2 innodulated lucerne (Medicago sativa) while they didnot in a less-nodulated isogenic line (Lüscher et al.2000). Ainsworth et al. (2004) found that the light-saturated rate of leaf photosynthesis was stimulatedby elevated CO2 in a nodulated cultivar of soybeanbut was not in the non-nodulated isogenic line.Matsunami et al. (2009) observed that dry massincreased at elevated CO2 both in a nodulated and ina non-nodulated soybean. To our knowledge, how-ever, there have been no studies that compared seedproduction in terms of both quantity (dry mass) andquality (nitrogen concentration) between nodulatedand non-nodulated lines at elevated CO2 nor studiesthat analyzed the effect on the vegetative andreproductive growth separately and the effect of theformer on the latter. In soybean, the foliar ureide andthe total amino acid contents (putatively indicating thenodule activity) increased sharply after flowering(Rogers et al. 2006), suggesting that the presence ofN sink (i.e. seeds) accelerated the N assimilation. Thisimplies that elevated CO2 enhances N assimilationand dry mass growth more in the reproductive than inthe vegetative phase.

To identify the key processes for CO2-inducedincrease in seed production in soybean, in the presentstudy we analysed seed production (Y) as the productof the rate of dry mass production during thereproductive phase (G), the fraction of biomassallocated to seed (U) and the duration of thereproductive phase (T) (Shitaka and Hirose 1998).

Y ¼ G� U � T ð1ÞIn an annual herb, Xanthium canadense, Kinugasa

et al. (2003) found that CO2 elevation increased G

164 Plant Soil (2010) 330:163–172

and decreased U at a high N availability, while it didnot alter G and U under at a low N availability. T wasnot affected in any case. Likewise, the effect ofelevated CO2 on seed N (YN) was analysed as theproduct of the rate of N assimilation during thereproductive phase (A), the fraction of N allocated toseed (UN) and T (Miyagi et al. 2007).

YN ¼ A� UN � T ð2ÞMiyagi et al. (2007) found in annual species that

CO2 elevation increased G and A but decreased U andUN. These results suggest that the increase in seednitrogen and seed dry mass at elevated CO2 is primarilycaused by the increase in the dry mass production andthe N assimilation in the reproductive phase.

We investigated the role of N2-fixation in seedproduction of soybean at elevated CO2 with anodulated cultivar of soybean and its non-nodulatedisogenic line to test the following hypothesis: (1) Nassimilation and consequently growth and seedproduction increase at elevated CO2 in the nodulatedbut to a lesser extent in the non-nodulated cultivar. (2)Increase in seed production at elevated CO2 is causedby an enhancement of dry mass production and the Nassimilation in the reproductive phase, not in thevegetative phase. (3) Decline in seed N concentrationat elevated CO2 is small in the nodulated but large inthe non-nodulated cultivar. (4) The increase in growthis larger in reproductive mass than in vegetative massin the nodulated cultivar where N2-fixation is greaterin the reproductive than the vegetative phase.

Materials and methods

Experimental design

The experiment was conducted in 2004 with the‘Gradiotron’ system of the National AgriculturalResearch Center for Tohoku Region, Morioka, Japan(39°44′N, 141°7′E) for CO2 treatment. It consisted oftwo adjacent greenhouse chambers, each 6 m wide, 30m long and 3 m high; one controlled at ambient CO2

concentration (LC) and the other at 200 μmol mol−1

above the ambient (HC). The two chambers were thesame in dimension and designed to have the samemicroclimate excepts CO2 concentration (see Okada etal. 2000 for detailed description). Air was continuouslyventilated to control the temperature in the chamber.

CO2 gas was released at the air inlet of the elevatedCO2 chamber and the gas emission rate was regulatedin proportion to the ventilation rate. Mean CO2

concentration that was recorded at 0.5-hour intervalsthrough the experiment was 384.7±9.1 μmol mol−1

(mean±s.d.) in LC and 593.0±42.0 μmol mol−1 inHC. The large variation in HC was caused byunexpected failures of the CO2 control line thatoccurred for 10 days in total, resulting in over-fumigation of CO2 or no additional CO2 supply. Inthe chamber, there was a consistent temperaturegradient along the long axis, due to the air ventilationin longitudinal direction. Plants were located at the lowtemperature zone near the air inlet of the chambers toavoid unexpected heat stresses on plants. Mean airtemperature at the center of the experimental block was22.7°C in July, 22.6°C in August, 21.2°C in Septemberand 17.3°C in October. Since our experimental standextended 3 m along the temperature gradient, there wasa temperature difference of 1.3°C between two ends ofthe stand consistently throughout the growing season.We assumed that this difference in temperature had nosignificant effect on our results because we rotated potswithin the stand and sampled plants randomly from thestand. Temperature difference between LC and HC wassmaller than 0.1°C. No additional light was provided.

We used a nodulated cultivar of soybean (Glycinemax cv. Enrei) and the non-nodulated isogenic line(G. max cv. En1282, Francisco and Akao 1993), bothof which show a determinate growth habit. Seedswere sown in a nursery filled with vermiculite on 27June 2004 and seedlings were transplanted individu-ally into 1/5000-a plastic pots (4.0 L) filled with soil(andosol) on 1 July 2004. The soil was collected froma rice paddy and dried in a greenhouse for 1 year.Each pot was supplied with 0.3 g N as NH4

+, 1.0 g Pas P2O5 and 1.0 g K as K2O at transplantation,according to the regional standard agronomic practice.Bradyrhizobium japonicum was inoculated to all potsof Enrei and En1282 at transplantation. These potswere arranged tightly on the ground (32.7 plants m−2

ground area) to establish four stands (2 cultivars x 2CO2 treatments). A total of 90 plants were allotted toeach stand. Shade-cloths with 70% light transmissionwere put on the sides of stands, where no plantobstructed solar beam penetrating through the sides.Top of the shade-cloths was shifted according toheight growth. Water was added every day and potswere rotated within each stand every 10 days.

Plant Soil (2010) 330:163–172 165

Initiation of the reproductive phase was defined ineach stand when 50% of individuals flowered.

Biomass production and nitrogen assimilation

At about 1-month intervals (8 July, 6 August,8 September and 15 October), five plants wererandomly harvested per stand and separated intoleaves, stems, pods, roots and nodules. Roots andnodules were carefully washed free of soil. Dry masswas determined after drying in an oven at 70°C for48 h. Dried samples were ground and the N con-centration was determined with an NC analyzer(Sumigraph NC-80, Sumika-Bunseki Center, Osaka,Japan). The amount of N was calculated by multiply-ing dry mass by its N concentration.

Five plants per stand were randomly selected andmarked for more frequent monitoring of pod dry massand pod N. The pod length was measured for all podsat about 10-day intervals. Pod emergence was definedat the time when it reached 10 mm in length. Drymass and N in pods of the monitor plants wereestimated non-destructively from the length with theallometric relationships determined for plants thatwere used for neither monitoring nor destructiveharvesting (data not shown). These allometric relation-ships differed significantly between cultivars, treat-ments and dates of census. When the pods brownedand dried up, they were harvested and separated seedsfrom the capsule (the final harvest). Their dry massand N concentration were determined.

All dead leaves and aborted pods (litter) producedbetween harvests were collected and put together.After drying in an oven, the dry mass and the Nconcentration were determined. Loss of dry mass andN per plant was calculated from those data divided bythe total number of plants that existed at collection.Dry mass production per plant was calculated as thesum of the increase in standing plant dry mass and thedry mass having lost. Nitrogen assimilation per plantwas the sum of the increase in standing plant N andthe N having lost. The amount of N in the seed at thestart of experiment was not subtracted in the calcula-tion of N assimilation, since it was much smallerthan the N assimilated by a plant during the lifetime(0.021±0.003 g N per seed [mean ± s.d.] in Enrei and0.006±0.001 g N per seed in En1282). For eachsampling date, five plants were arranged according tothe order of their total plant dry mass and the increase

or decrease in dry mass production and N assimilationduring time intervals was calculated pairwise for theplants in the same order (Kinugasa et al. 2003). Weassumed that there was no biomass and N loss in theroots during the experiment.

Statistics

To test the effects of CO2 and cultivars on dry massand N of organs, we applied the generalized linearmodel procedure comprising an identity link functionand a gamma distribution, followed by the likelihoodratio test. “Cultivar” and “CO2” were added as factorsinto the models. Likelihood ratio was calculated for 5plants per treatment (n=5). Computations wereperformed with a statistical package R ver. 2.5.0(R development Core Team 2006).

Results

Dry mass and nitrogen in leaves, stems and roots

Plants increased their height continuously until mid-September. Lateral branch formation initiated in earlyAugust and continued until early September. Up to8 lateral branches were produced per plant. Newleaves were produced at the tip of the main stem andbranches and a total of ca. 27 leaves were producedper plant. The number of leaves was not significantlydifferent between cultivars and between CO2 treat-ments (data not shown). Flowering was observed inmore than 50% of the plants on 11 August and inall plants on 18 August. There were no significantdifferences in the flowering time between cultivarsand between CO2 treatments.

In nodulated Enrei, the peak leaf mass was largerin elevated than ambient CO2 (Fig. 1a) while in non-nodulated En1282, the difference between elevatedand ambient CO2 was not significant (Fig. 1b). Thepeak stem mass (Fig. 1c, d) and the peak root mass(Fig. 1e, f) increased with elevated CO2 in bothcultivars. Leaf N per plant did not change withelevated CO2 in either cultivar throughout growth(Fig. 1g, h). Peak stem N increased marginally withelevated CO2 in Enrei (Fig. 1i; P=0.097) while inEn1282, the difference was not significant (Fig. 1j).Peak root N increased with elevated CO2 in bothcultivars (Fig. 1k, l).

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Dry mass and nitrogen in seeds

Pod (seeds + capsule) mass per plant increased withelevated CO2 in Enrei (Fig. 2a) but did not in En1282(Fig. 2b). In Enrei, seed mass per plant (seed produc-tion) at the final harvest was 1.36 times greater at HCthan at LC, while capsule mass per plant was not affectedby CO2 (Table 1). In En1282, both seed and capsulemass did not differ between HC and LC (Table 1).

Elevated CO2 decreased the seed N concentrationin En1282 but did not in Enrei (Table 1). Capsule Nconcentration was not affected by CO2 in either cultivar(Table 1). In Enrei, seed N per plant at the final harvestwas 1.31 times greater at HC than at LC, while inEn1282, it did not differ between HC and LC (Table 1).Capsule N per plant was not affected by CO2 in bothcultivars. In Enrei, pod N per plant increased withelevated CO2 (Fig. 2c), owing to the greater seed N perplant at HC than at LC. In En1282, pod N per plant didnot change at elevated CO2 (Fig. 2d).

Dry mass production and nitrogen assimilation

In Enrei, dry mass production was greater at HC thanat LC in the reproductive phase, but was not in thevegetative phase (Fig. 3a). Dry mass production ofEn1282 was not significantly influenced by CO2 bothin the vegetative and in the reproductive phase(Fig. 3b). In the vegetative phase, nitrogen assimila-tion of Enrei did not increase with elevated CO2 butin the reproductive phase, it increased (Fig. 3c). InEn1282, elevated CO2 had no significant effect onN assimilation both in the vegetative and in thereproductive phase (Fig. 3d).

Determinants of seed production

Seed production was analyzed as the product of therate of biomass production during the reproductivephase (G), the fraction of biomass allocated toreproduction (U) and the duration of the reproductive

Fig. 1 Dry mass of leaf (a, b), stem (c, d) and root (e, f), andnitrogen in leaf (g, h), stem (i, j) and root (k, l) of nodulatedsoybean (Glycine max cv. Enrei; left panels) and the non-nodulated isogenic line (G. max cv. En1282; right panels)

grown at ambient- (open circles) and elevated CO2 (closedcircles). Symbols represent the mean and standard deviation(n=5). Significant pairwise linear contrasts between treatmentswithin the same date are denoted by asterisks (*, P<0.05)

Plant Soil (2010) 330:163–172 167

period (T) (Eq. 1). In Enrei, G was higher at HC thanat LC (Table 2, Fig. 3a) while in En1282, it was notsignificantly altered by elevated CO2 (Table 2,Fig. 3b). U was greater in Enrei than in En1282 butthere was no effect of CO2 elevation (Table 2). Nosignificant difference was found in T between cul-tivars and CO2 treatments. T averaged over cultivarsand treatments was 72 days. These results indicatethat the increase in seed production by CO2 elevationfound in Enrei (Table 1) was caused primarily by theincrease in G.

Seed N was analyzed as the product of the rateof N assimilation during the reproductive phase (A),the fraction of N allocated to reproduction (UN) andT (Eq. 2). In Enrei, A was higher at HC than at LC(Table 2, Fig. 3c). A of En1282 was not influencedby CO2 (Table 2, Fig. 3d). In Enrei, UN was notinfluenced by CO2 elevation but it decreased inEn1282 (Table 2). These results indicate that theincrease in seed N with CO2 elevation found inEnrei (Table 1) was caused primarily by the increasein A.

Nodule dry mass and nitrogen

In Enrei, nodule mass per plant increased sharplyfrom mid-August through to September (Fig. 4a).This period corresponded to the time of flowering andthe early stages of pod filling of the plants (Fig. 2).Peak nodule mass was greater at HC than at LC. InEn1282, the amount of nodules was extremely smalland no significant stimulation by elevated CO2 wasobserved at any harvest date (Fig. 4b). Low Nconcentration of nodules in En1282 (Fig. 4d) thatwas comparable to that of roots suggests little noduleactivity in this cultivar.

Discussion

Elevated CO2 increased seed production and seed N innodulated soybean (cv. Enrei) but did not in its non-nodulated isogenic line (cv. En1282) (Table 1). Stim-ulation of seed production in Enrei at elevated CO2

was caused by the increase in dry mass production in

Fig. 2 Pod (seeds + capsule) dry mass (a, b) and nitrogen (c, d)of nodulated soybean (Glycine max cv. Enrei; left panels) andthe non-nodulated isogenic line (G. max cv. En1282; rightpanels) grown at ambient- (open circles) and elevated CO2

concentration (closed circles). Symbols represent the mean andstandard deviation (n=5). Significant pairwise linear contrastsbetween treatments within the same date are denoted byasterisks (*, P<0.05)

Fig. 3 Dry mass production and nitrogen assimilation ofnodulated soybean (Glycine max cv. Enrei; left panels) andthe non-nodulated isogenic line (G. max cv. En1282; rightpanels) grown at ambient- (open columns) and elevated CO2

concentration (closed columns) in the vegetative and thereproductive phase. Mean and the standard deviation (n=5).Significant differences between the value from ambient andelevated CO2 concentration are denoted by asterisks (***,P<0.001)

168 Plant Soil (2010) 330:163–172

the reproductive period (G) but not by the fraction ofbiomass allocated to reproduction (U) and the durationof the reproductive phase (T) (Eq. 1, Table 2). This isin agreement with Miyagi et al. (2007) who found thatthe increase in seed production observed in legumesand non-legumes at elevated CO2 was due to theirhigher G. Thus G was the primary determinant of seedproduction in both legumes and non-legumes atelevated CO2. The increased seed N at HC in nodulatedEnrei (Table 1) resulted from the increase in Nassimilation in the reproductive period (A) but not fromthe fraction of N allocated to seed (UN) and T (Eq. 2,Table 2). Other legumes also increased A at elevatedCO2 due to increased nodule activity at elevated CO2

(Hungate et al. 1999; Lüscher et al. 2000). Increase in Aobserved in non-legumes was due to the increased rootsize and/or root activity by elevated CO2. (Larigauderieet al. 1994; Norby 1994; BassiriRad et al. 1997; Kim etal. 2001; Miyagi et al. 2007).

In nodulated Enrei, dry mass production and Nassimilation increased at elevated CO2 in the repro-ductive phase but did not in the vegetative phase

(Fig. 3a, c). These results accord with the observationthat the amount of nodules in Enrei increased sharplyin the reproductive phase (Fig. 4a). In field-grownEnrei, the N2-fixing activity determined as the ureideconcentration in sap was highest 20 days afterflowering (Takahashi 2005). Morgan et al. (2005)reported a larger stimulation of growth in thereproductive than the vegetative phase in soybean.In a non-nodulated cultivar of soybean (G. max cv.Lee), on the other hand, growth enhancement withCO2 elevation observed in the early stage of plantgrowth diminished toward the end of the growthperiod including reproduction (Cure et al. 1988). Suchdiminishment through the life span was often found innon-legumes (Ackerly and Bazzaz 1995; Kim et al.2001). Kinugasa et al. (2003) showed that elevatedCO2 increased vegetative growth, but did not seedproduction in an annual Xanthium canadense. Theyascribed it to limited N availability for seed production(see also Hirose et al. 2005). The present studyshowed that nodule activity enabled soybean to assim-ilate more N in the reproductive period, and conse-quently led to a larger seed production at elevated CO2.

Table 1 Dry mass, nitrogen concentration and nitrogen ofseeds and the capsule of nodulated soybean (Glycine max cv.Enrei) and the non-nodulated isogenic line (G. max cv. En1282)grown at ambient (LC) and elevated CO2 (HC) at the finalharvest (21 October)

Cultivar CO2 Seeds Capsule

Dry mass (g DM plant−1)

Enrei (nodulated) LC 13.48 (2.36)b 4.59 (0.99)a

HC 18.32 (0.29)a 5.43 (0.32)a

En1282 (non-nodulated) LC 8.20 (1.94)c 3.19 (0.77)b

HC 8.90 (2.68)c 3.56 (1.12)b

Nitrogen concentration (%)

Enrei (nodulated) LC 7.57 (0.38)a 0.58 (0.02)a

HC 7.34 (0.17)a 0.57 (0.04)a

En1282 (non-nodulated) LC 4.21 (0.12)b 0.38 (0.05)b

HC 3.70 (0.11)c 0.43 (0.02)b

Nitrogen (g N plant−1)

Enrei (nodulated) LC 1.02 (0.18)b 0.027 (0.006)a

HC 1.34 (0.02)a 0.031 (0.002)a

En1282 (non-nodulated) LC 0.34 (0.08)c 0.012 (0.003)b

HC 0.33 (0.10)c 0.015 (0.005)b

Mean and the standard deviation in parentheses

Values within a given column designated by different letters aresignificantly different at 0.05 level

Fig. 4 Nodule dry mass (a, b) and nodule nitrogen concentra-tion (c, d) of nodulated soybean (Glycine max cv. Enrei; leftpanels) and the non-nodulated isogenic line (G. max cv.En1282; right panels) grown at ambient- (open circles) andelevated CO2 concentration (closed circles). Mean and thestandard deviations (n=5). Significant pairwise linear contrastsbetween treatments within the same date are denoted byasterisks (***, P<0.001; **, P<0.01)

Plant Soil (2010) 330:163–172 169

Nitrogen comes from N2-fixation as well asmineral N uptake in nodulated Enrei. We did notdistinguish mineral N uptake and N2-fixation in Enreibut mineral N uptake was unlikely to be significantlydifferent between in LC and in HC. This is because Nassimilation was not significantly different betweenLC and HC in non-nodulated En1282, whose N issupplied from mineral N in soil, despite the increasedroot dry mass at elevated CO2 (Fig. 1f). The totalamount of N assimilation was 0.60±0.08 g N plant−1

(mean±s.d.) in LC and 0.68±0.11 g N plant−1 in HCin non-nodulated En1282 and 1.52±0.09 g N plant−1

in LC and 1.80±0.14 g N plant−1 in HC in nodulatedEnrei. There was thus a difference between the CO2

treatments in Enrei (P<0.001, likelihood ratio test)but was not in En1282 (P=0.23). If nodulated Enreitook up the same amount of mineral N from soil asnon-nodulated En1282, the difference in N assimila-tion between Enrei and En1282 should be the Nderived from N2-fixation: 0.88±0.09 g N plant−1 in LCand 1.16±0.14 g N plant−1 in HC. [These valueswould be a minimal estimation for N2-fixation becausenodulated Enrei that had a smaller root mass than non-nodulated En1282 did (P<0.05; Fig. 1e, f) might haveabsorbed smaller amounts of mineral N in soil thanEn1282.] The ratio of N derived from N2-fixation tototal plant N was higher in HC (0.64±0.03) than in LC(0.58±0.02) (P<0.001, likelihood ratio test). Thus thesymbiotic N2-fixation conducted to the increase in Nassimilation at elevated CO2 in nodulated Enrei.

The bacterial symbiont greatly contributed tomaintaining seed quality in nodulated soybean atelevated CO2. Seed N concentration decreased atelevated CO2 in non-nodulated En1282 but did not in

nodulated Enrei (Table 1). Jablonski et al. (2002) whoanalyzed more than 150 studies concluded that seed Nconcentration was not influenced by CO2 in soybean,while it decreased in most of the other crops and wildherbs. Similarly, Taub et al. (2008) showed that seedprotein concentration was not influenced by elevatedCO2 in soybean while it decreased in rice, barley andwheat. Comparing non-nodulated soybean with theparent nodulated cultivar, we demonstrated that seedN concentration was maintained by symbiotic associ-ation. Seed protein concentration of soybean rangesfrom 31.5 to 38.2%, being higher than that of theother seed crops (Sinclair and de Wit 1975). Thelarger nodule mass at elevated CO2 (Fig. 4) corrob-orates the increase in N2-fixation to meet the Ndemand that increased at elevated CO2.

In the vegetative phase, across two CO2 levels, drymass production and N assimilation were greater innon-nodulated En1282 than in nodulated Enrei (Fig. 3a, b). Since a large amount of carbohydrates isconsumed by nodules (Minchin et al. 1981), plantgrowth might have been retarded by the noduleactivity in nodulated Enrei. On the other hand,Nakamura et al. (1999) found that the dry massproduction during the vegetative phase was greater inthe nodulated than in the non-nodulated cultivar.This result is different from ours: the dry massproduction was greater in non-nodulated En1282 thanin nodulated Enrei (Fig. 3a, b). The difference may beexplained by the difference in the experimental set-upincluding the level of basal fertilizers and applied CO2

concentration. The lower the level of basal fertilizers,the higher the contribution of N2-faxation would be(Streeter and Wong 1988; Hardarson and Atkins

Table 2 Determinants of seed dry mass and seed nitrogen in nodulated soybean (Glycine max cv. Enrei) and the non-nodulatedisogenic line (G. max cv. En1282) grown at ambient and elevated CO2 concentration: dry mass production during reproductive phase(G), fraction of dry mass allocated to seeds (U), N assimilation during the reproductive phase (A), and fraction of N allocated to seeds(UN)

Cultivar CO2 G U A UN

(g DM day−1) (g DM g−1 DM) (g N day−1) (g N g−1 N)

Enrei (nodulated) LC 0.55 (0.03)b 0.34 (0.05)a 0.017 (0.001)b 0.81 (0.12)b

HC 0.68 (0.05)a 0.37 (0.02)a 0.022 (0.002)a 0.82 (0.08)b

En1282 (non-nodulated) LC 0.45 (0.05)c 0.27 (0.03)b 0.004 (0.001)c 1.15 (0.25)a

HC 0.54 (0.07)bc 0.23 (0.05)b 0.005 (0.001)c 0.82 (0.16)b

Mean and the standard deviation (n=5) in parenthesis

Values within a given column designated by different letters are significantly different at 0.05 level

Table 2 Determinants of seed dry mass and seed nitrogen innodulated soybean (Glycine max cv. Enrei) and the non-nodulated isogenic line (G. max cv. En1282) grown at ambientand elevated CO2 concentration: dry mass production during

reproductive phase (G), fraction of dry mass allocated to seeds(U), N assimilation during the reproductive phase (A), andfraction of N allocated to seeds (UN)

170 Plant Soil (2010) 330:163–172

2003). If basal fertilizer were limited, nodulatedcultivars would respond to elevated CO2 more thannon-nodulated cultivars. The CO2 level was 700 μmolmol−1 in Nakamura et al. (1999) that was higher thanthat in the present study, 593 μmol mol−1. A meta-analysis showed that dry mass production of shootand root at elevated CO2 increased more when higherCO2 concentrations were employed (Ainsworth et al.2002). The higher CO2 concentration in Nakamura etal. (1999) also might have increased biomass produc-tion of nodulated soybean more than in our treatment.

Several studies showed that the increase in Nassimilation with elevated CO2 was smaller in theplant that absorbed only mineral N from soil than thatfixed N2 symbiotically (Soussana and Hartwig 1996;Lüscher et al. 1998). Symbiotic bacteria consume partof photosynthates that would otherwise be used forplant growth. However, an increase in dry mass andseed production due to increased N availability morethan compensated the dry mass used for symbioticassociation, and the increase would be substantialparticularly at elevated CO2. With a nodulatedcultivar of soybean and its non-nodulated isogenicline, we showed that plant species with symbioticassociation with N2-fixing bacteria would better beable to adapt to the future high-CO2 environment thanthose without symbiotic association. We also showedthat the increase in N assimilation in the reproductiveperiod would be the key for increasing soybean yield.

Acknowledgements We thank Kazumasa Ishikawa, ChihoKamiyama and Yosuke Matsumoto of Tohoku University, andMeguru Inoue, Teruo Saito, Yukichi Satoh and other membersof NARCT for technical assistance. We are also grateful toShoichiro Akao for allowing us to use En1282. David Lawlorprovided valuable comments on an earlier version of this paper.This work was supported in part by Grant-in-aid from the JapanMinistry of Education, Science and Culture.

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