Availability of N and S affect nutrient acquisition efficiencies differently by Trifolium repens and Lolium perenne when grown in monoculture or in mixture

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Environmental and Experimental Botany 66 (2009) 309–316

Contents lists available at ScienceDirect

Environmental and Experimental Botany

journa l homepage: www.e lsev ier .com/ locate /envexpbot

vailability of N and S affect nutrient acquisition efficiencies differently byrifolium repens and Lolium perenne when grown in monoculture or in mixture

iphaine Talleca,b, Sylvain Diquéloua,b,∗, Jean-Christophe Avicea,b, Fabien Lesuffleura,b,ervane Lemauviel-Lavenanta,b, Jean-Bernard Cliqueta,b, Alain Ourrya,b

INRA, UMR950, F-14000 Caen, FranceUniversité de Caen Basse-Normandie, UMR950 Ecophysiologie Végétale Agronomie et nutrition N, C, S, F-14032 Caen cedex, France

r t i c l e i n f o

rticle history:eceived 12 September 2008eceived in revised form 12 January 2009ccepted 1 February 2009

eywords:olium perennerifolium repens:S availabilitiesand S yieldsand S recoveries

a b s t r a c t

Sulphur (S) deficiency is recognized as a limiting factor for crop production in many regions in the world. Ingrasslands, S availability has been shown to alter the biomass production of Trifolium repens and Loliumperenne and their specific interactions. To establish the role of N and S availabilities on the competi-tive interaction for these minerals by T. repens and L. perenne when grown together, two S rates (0 and30 kg S ha−1) combined with three N rates (0, 50 and 180 kg N ha−1) were investigated in a cut/regrowthexperiment over a period of 4 months under glasshouse conditions. N was applied as 15NH4

15NO3 todetermine their actual N fertilizer recovery in the harvested fraction of the shoot. S yields were used toestimate their apparent S fertilizer recovery. At final harvest, N reserves of T. repens stolons were ana-lyzed to estimate their implication in the regrowth process. In monoculture and in both cuts (1 and 2), Nbenefited both species by increasing their N and S yields. S benefited only T. repens. In mixture, at cut 1,

2 fixation L. perenne behaved as a better competitor than T. repens thanks to N, while at cut 2, T. repens dominatedthe community thanks to strong positive S effect. N recovery of L. perenne grown in mixture was greatlyimproved by S supply. For T. repens, S enhanced its ability to fix N2 and improved the accumulation ofsoluble proteins in its stolons. It is clear that the N:S ratio of soil may affect the functionality of grasslandplant communities and their structure. Results suggest that (i) the limitations in the availability of soilS could restrict leguminous species growth in high N soil conditions, and (ii) the modulation of S level

mod

could be used as a tool to

. Introduction

Sulphur (S) is an essential nutrient required for all living organ-sms. It is an essential constituent of cysteine, methionine, severalo-enzymes, thioredoxins, sulpholipids and proteins (Droux, 2004;ausch and Wachter, 2005; Hawkesford and De Kok, 2006). In theast, the requirement of S has been met by the presence of S inKP fertilizers and by the deposition of atmospheric S from indus-

rial pollution; thus S deficiency was rare. Nowadays, many regionsuffer from a fundamental shift in the S balance toward deficit inrops and grasslands (Mathot et al., 2008). One of the most impor-

ant reasons is the massive decline in the deposition of S fromtmospheric SO2 emissions (from 1600 kt S in 1980 to <219 kt S in006 in France) due to industrial pollution control policies. Thesehanges contribute to an increase of S deficiency in crops, which

∗ Corresponding author at: Université de Caen Basse-Normandie, UMR950 Eco-hysiologie Végétale Agronomie et nutrition N, C, S, F-14032 Caen cedex, France.el.: +33 231565598; fax: +33 231565360.

E-mail address: [email protected] (S. Diquélou).

098-8472/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.envexpbot.2009.02.002

ify the composition of grassland communities.© 2009 Elsevier B.V. All rights reserved.

may also reduce the use of other nutrients, particularly nitrogen(N). Several physiological studies have established regulatory inter-actions between N and S assimilation in plants like tobacco (Barneyand Bush, 1985) or oilseed rape (Kopriva et al., 2000; Kopriva andRennenberg, 2004; Hesse et al., 2004). S availability can regulateN use efficiency of plants and thus photosynthetic activity, growthand dry mass accumulation of crops since the accumulation of pho-toassimilates has a close relationship with N and S assimilation(Kopriva et al., 2000). However, these interactions remain largelyunknown for grassland species. S has been shown to be of consid-erable significance in grassland and hay production in Australia andin New Zealand (Walker et al., 1956; Sinclair et al., 1996). Fabaceaeappear to respond primarily to S, resulting in increasing yieldsregardless of N level (Gilbert and Robson, 1984a,b; Tallec et al.,2008a,b), whereas the response of ryegrass to S appears only athigh N availability (Brown et al., 2000; Tallec et al., 2008a,b).

White clover (Trifolium repens L.) and perennial ryegrass (Loliumperenne L.) are two pasture-dominant species. These species areconsidered to be keys to modern high yielding forage-based agri-cultural systems. The persistence of an adequate level of cloverin mixture is of major interest in the maintenance of grass/clover

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10 T. Tallec et al. / Environmental and

ssociation. The N:S ratio has been proved to modulate relative L.erenne/T. repens abundance in mixtures, and Tallec et al. (2008a)roposed that S fertilizer could be used as a tool to control T.epens abundance in cut grass systems, especially in N-rich envi-onments. In agreement with the findings of Walker and Adams1958), the results of Tallec et al. (2008a) suggest that S shoulde taken into account when considering the group of elements forhich L. perenne competes intensely when grown with T. repens. Theeffect on species balance and on T. repens persistence is poorly

ocumented, in contrast to the N effect. Indeed, T. repens and L.erenne have contrasting responses to soil mineral N availability.yegrass acquires most of its N from soil mineral N, whereas cloveran supplement uptake from the soil with N acquired by fixationf atmospheric nitrogen (N2). Incompatibility of clover persistenceith N fertilization has been frequently reported (Soussana andrregui, 1995). The persistence of clover depends largely on itsbility to regrow after the removal of some of its photosyntheticurface. Because defoliation after cutting causes a marked reduc-ion in acquisition from symbiotic fixation of N2 (strong decreasef nitrogenase activity) and/or mineral N uptake (Ourry et al., 1994;or a review on the specific effect of defoliation, see Volenec et al.,996), the mobilisation of endogenous organic N reserves is cru-ial to meet the N requirements of new shoots regrowth during therst days following cutting (Ourry et al., 1989; Meuriot et al., 2004).oluble proteins are one of the most important soluble N fractionn the main storage organs (stolons and roots) of T. repens (Corret al., 1996). In perennial forage legumes such as T. repens (Corre etl., 1996) or Medicago sativa (Avice et al., 1997; Justes et al., 2002),pecific soluble proteins called vegetative storage proteins (VSP)re extensively mobilised to meet the N requirements of new shootrowth in spring or after cutting. Additionally, the storage level ofoluble proteins as well as VSP is influenced by several environ-ental factors, such as N availability, temperature or photoperiod

Corre et al., 1996; Goulas et al., 2001; Noquet et al., 2003; Dhont etl., 2006), while the effect of S availability remains unknown. One ofhe major known factors affecting the accumulation of soluble pro-eins in forage leguminous is the mineral N availability (Meuriot etl., 2003). In contrast, S, to our knowledge, has never been investi-ated with respect to VSP despite the fact that the metabolism ofhese elements is closely linked. As S has a positive effect on the pro-uction of clovers (Walker et al., 1956; Gilbert and Robson, 1984a,b;inclair et al., 1996), and particularly in the regrowth phases (Tallect al., 2008a), this element may play an important role in the sur-ival of T. repens in mixtures, via a regulation from N-assimilationAnderson, 1990) to N accumulation in perennial organs. Therefore,s previously reported for N availability, the level of mineral S couldave an impact on the accumulation of soluble proteins (includingSP) in perennial organs.

As the application of S has been shown to increase L. perenneroduction in pot experiment (Tallec et al., 2008a) and its N usefficiency in a field experiment (Brown et al., 2000), especially atigh N, we hypothesis that higher dry matter yields of grass achiev-ble with added S may be associated with greater mineral N uptake.he second hypothesis is that S has a related physiological contri-ution to the competitive abilities and persistence of T. repens inommunity, in increasing N acquisition efficiency and storage. Toest our hypotheses, we analyzed the impact of different N:S initialvailabilities on (i) the nutrient status (N and S yields) of L. perennend T. repens submitted to cutting; (ii) the plant capacity to takep S and N, the latter being determined by 15N labelling and (iii)he quantitative and qualitative changes of the soluble proteins in

tolons of T. repens. The first two points were analyzed at establish-ent phase (cut 1), where N was potentially ample available, and

t regrowth phase (cut 2), where N limitation was exacerbated. Theast point was determined at the end of the experiment (cut 3). Thehird hypothesis is that both species response is altered by species

mental Botany 66 (2009) 309–316

neighbouring. To test it, we considered monoculture and mixtureof both species.

2. Materials and methods

2.1. Greenhouse experiment

Ryegrass (L. perenne cv. Bravo) and white clover (T. repens cv.Huia) were cultivated in a greenhouse pots experiment in mono-culture and in mixture from March to July 2005 (Tallec et al.,2008a). Pots consisted in a polyvinyl-chloride (PVC) containers(soil volume: 1755 cm3, basal diameter: 10.5 cm, top diameter:14 cm, height: 15.5 cm) filled with a homogeneous and nutrient-poor sieved soil (2 mm mesh). It was a silty-clay soil (clay content:36.7%, silt content: 41.3%), and the organic matter content was 4.2%.The total N and S contents of the soil was respectively 0.318% and0.1%. Three weeks of after germination (Day 0 of the experiment),eight seedlings per pot were established in a regular pattern. Potscontained either monocultures of the two species or mixtures offour seedlings of each species per pot. T. repens plants were inocu-lated with a standard mixture of Rhizobium trifolii T354 known tosupport N2 fixation.

2.2. Treatments

Six fertility levels were imposed at Day 0 by combining three Nlevels with two S levels. S was applied as calcium sulphate (CaSO4)at a rate of 0 (LS, low S availability) and 30 kg S ha−1 (HS, high Savailability: 46 mg S per pot; fertilization level suggested for cere-als: Withers et al., 1997). N was applied as ammonium nitrate at 0(LN, low N availability), 50 kg N ha−1 (IN, intermediate N availabil-ity: 77 mg N per pot; conventional local fertilization of permanentgrasslands) and 180 kg N ha−1 (HN, high N availability: 277 mg N perpot; local fertilization of intensive sown grasslands), labelled with5% atom 15N excess to quantify N recovery by each species. A basalmix of potassium (150 kg K ha−1:231 mg K per pot) and phosphorus(60 kg P ha−1:92 mg P per pot) as K2HPO4 was applied to each pot,at the same time. Pots receiving no CaSO4 were supplemented withCaCl2, so that each pot received the same amount of Ca. Four potswere used as replicates for each treatment, and the pots, placed insaucers, were moved twice a week to avoid any positional effect.The soil moisture content in each pot was kept at ±25% (relative todry weight (DW)) by weighing the pots once a week and wateringthem four times a week during the experiment. Air temperaturewas kept at 20/16 ± 2 ◦C (day/night). Plants were grown under nat-ural light until transfer to pots (Day 0), and then supplemented byartificial illumination (400 W high-pressure sodium lamps, Phyto-claude) providing 300 �mol m−2 s−1 PAR (photosynthetically activeradiation) at plant height with a 16/8 h photoperiod.

2.3. Plant chemical analyses

Three cuts were harvested at 5 cm plant height for chemicalanalyses of the total above-ground matter: 42 days (cut 1), 84 days(cut 2) and 126 days (cut 3) after planting. At the end of the exper-iment (cut 3), stolons of T. repens were separated and an aliquot of450 mg of fresh matter was stored at −80 ◦C until further analysis ofsoluble protein concentration and electrophoresis. The fresh mate-rial was dried at 65 ◦C for 48 h for the determination of dry weightand then finely ground for analysis of mineral concentration. N and

S contents as a % of DW and 15N abundance were estimated usingan isotope ratio mass spectrometer (IRMS, Isoprime, GV Instru-ment). Natural 15N abundance (0.3663 ± 0.0004%) of atmosphericN2 was used as a reference for 15N analysis. In the above-groundplant biomass, the quantity of N derived from fertilizer (NDFF) was

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alculated by isotope mass balance as:

DFF (%) = E15Nplant

E15Nfert× 100

here E15N is the 15N atom % excess of plant (Nplant) or of fertilizerNfert).

The N and S fertilizer recoveries (Nrecovery and Srecovery, respec-ively as % of fertilizer applied) in the harvested fraction of shootsere calculated per plant as:

recovery = Nplant × NDFFNfert

here Nplant is the N content in the harvested above-groundiomass as mg per plant, Nfert is the amount of N fertilizer applied

ig. 1. Shoot N content and N derived from fertilizer (NDFF) in Lolium perenne grown inonoculture (E and G) and in mixture (F and H), at cuts 1 and 2, along the N and S grad

xis); histograms: NDFF (N derived from fertilizer expressed as percentage of shoot N conignificant difference; *P ≤ 0.05.

mental Botany 66 (2009) 309–316 311

as mg per pot:

Srecovery = Splant.HS − Splant.LS

Sfert× 100

where Splant.HS is the S content in the harvested above-groundbiomass at high S level as mg per plant; Splant.LS, the S content inthe harvested above-ground biomass at low S level (non-S fertilizedpot) as mg per plant; Sfert the amount of S fertilizer applied as mgper pot.

2.4. Extraction and separation by SDS-PAGE of soluble proteins

from stolons of T. repens

Frozen stolon samples (450 mg fresh weight) were ground in amortar with liquid nitrogen and extracted in citrate Na-phosphatebuffer (20 mM citrate and 160 mM Na2HPO4, pH 6.8) in the pres-

monoculture (A and C) and in mixture (B and D) and in Trifolium repens grown inients. Lines: shoot N content (right vertical axis; note the changed scales for thistent; left vertical axis). Bars are mean ± S.E. (n = 4). Two different letters indicate a

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nce of 150 mg PVPP (polyvinylpolypyrrolidone). The homogenateas centrifuged at 12,000 × g, 4 ◦C for 1 h. The resulting super-atant was used for determination of total soluble proteins byrotein-dye staining (Bradford, 1976) using bovine serum albumins standard. For detection of total soluble proteins, 3 �g (oneolume of each triplicate mixed) were prepared in 2× Laemmliysis buffer (Laemli, 1970) containing �-mercaptoethanol (5%, v/v).DS-PAGE was performed as described by Laemli (1970) using a.5% polyacrylamide (w/v) stacking gel and a 15% polyacrylamidew/v) resolving gel. Equal amounts of protein (3 �g) were loadednto the gel. Gels were stained with the silver staining procedureescribed by Blum et al. (1987). Gels were scanned with the ProX-RESS 2D proteomic Imaging System (PerkinElmer) and analyzedsing the Millipore BioImage computerized image analysis systemy measurement of integrated intensity. The molecular massf protein bands were estimated by comparison with standardarkers of known molecular mass.

.5. Data analysis

Data were analyzed using two-way analysis of variance (ANOVA)nd significantly different means between treatments were sep-rated with the Tukey’s multiple range test (P ≤ 0.05). Whenariables did not satisfy normality due to culture effects, the non-arametric Kruskall–Wallis test was used to test N effect, S effectnd treatment effect (N + S) as it does not test the N × S interac-ion. When H-values were significant, data were subjected to Mood

edian test to determine which means differed significantly.

. Results

Only data for the first cut (establishment phase) and the secondut (regrowth after cutting) are presented, because results from thehird cut followed the same trend as the second.

able 1ffect of different N and S levels on the mean N % in the harvested fraction of the shoot D

Lolium perenne

Monoculture Mixture

LS HS LS

ut 1 LN 2.68a 2.34a 2.93AB

IN 4.13ab 3.97b 2.30A

HN 6.75c 6.16c 3.81BC

ut 2 LN 1.03a 0.94a 1.09A

IN 1.00a 0.96a 1.13A

HN 1.32b 1.05a 1.56C

wo different lower case letters indicate a significant difference between treatment inifferent upper case letters indicate a significant difference between treatment in mixtureut; *P ≤ 0.05.

able 2ffect of different N and S levels on the mean actual N recovery (N fertilizer recovery in terenne and Trifolium repens, at cuts 1 and 2.

Lolium perenne

Monoculture Mixture

LS HS LS

ut 1 IN 7.04NS 7.04NS 7.72A

HN 6.81NS 7.53NS 7.75A

ut 2 IN 0.84a 0.80a 1.05A

HN 1.07b 1.04b 1.67C

wo different lower case letters indicate a significant difference between treatment inifferent upper case letters indicate a significant difference between treatment in mixtureut; *P ≤ 0.05.

mental Botany 66 (2009) 309–316

3.1. N yield and uptake

For L. perenne, shoot N content and N derived from 15N labelledfertilizer (NDFF (%), Fig. 1) were increased by N fertilization, irre-spective of culture condition or cuts (monoculture versus mixture;Fig. 1A and C versus Fig. 1B and D). N effect on % N depended on cutand S level. At cut 1, N enhanced % N (Table 1). At cut 2, it signifi-cantly increased % N at LS only, in both monoculture and mixture. Sfertilization slightly increase shoot N content or NDFF of L. perenneonly in HN monoculture at cut 1 (by 1.2, LS versus HS, Fig. 1A) andin LN mixture at cut 2 (LS versus HS, Fig. 1D). Between cut 1 (Fig. 1Aand B) and cut 2 (Fig. 1C and D), while NDFF slightly decreased,shoot N content decreased more drastically, but remained higherby about twofold in mixture than in monoculture (Fig. 1D versusFig. 1C). Percent N in the shoot of L. perenne at cut 2 was alsohigher when grown with T. repens (left side of Table 1). More-over, high S increased % N in the shoot of L. perenne when grownas mixture whereas it decreased it when grown as monoculture(Table 1).

The measurement of 15N labelling in the harvested fraction ofshoots allowed the estimation of actual N recovery (Table 2). Fiftyto 60% of applied N fertilizer (calculated from N recovery of eightplants in monoculture and from four plants of each species in mix-ture) was recovered at the first cut, and then less than 10% atthe second cut. Increasing N availability modified N recovery of L.perenne only at cut 2: N recovery was 1.3 times higher at HN thanat IN in both monoculture and mixture (left side of Table 2). Veryclearly, S fertilization strongly increased N recovery for L. perenneonly when grown with T. repens, regardless of the N level (IN or HN)

or cut. As a result, N recovery found in mixture always exceededthat found in monoculture, especially at HS (Table 2).

T. repens, due to its capacity to fix atmospheric N, showed a morecomplex pattern than L. perenne between cuts 1 and 2. At cut 1 andwhatever the culture type, increasing N improved T. repens shoot N

W per plant of Lolium perenne and Trifolium repens, at cuts 1 and 2.

Trifolium repens

Monoculture Mixture

HS LS HS LS HS

2.36A 3.69a 3.64a 3.26A 3.73B

2.62AB 3.79a 4.06b 3.05A 3.44A

4.12C 5.51c 5.36c 4.68C 4.61C

1.41C 1.79a 3.40c 1.96A 3.48B

1.29BC 1.52a 3.36c 2.10A 3.48B

1.47C 1.67a 3.45c 1.98A 3.39B

monoculture (four replicates; eight plants of each species per replicate) and two(four replicates; four plants of each species per replicate) within each species and

he harvested fraction of the shoot as % of 15N fertilizer applied) per plant of Lolium

Trifolium repens

Monoculture Mixture

HS LS HS LS HS

9.32B 6.74b 6.17b 3.24A 5.05B

9.60B 4.91a 6.50b 5.11B 4.42AB

1.39B 0.49a 0.96b 0.41A 0.69AB

1.94D 0.96b 1.67c 1.38C 0.85BC

monoculture (four replicates; eight plants of each species per replicate) and two(four replicates; four plants of each species per replicate) within each species and

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Fig. 2. Shoot S content of Lolium perenne grown in monoculture (A and C) and inmixture (B and D) and of Trifolium repens grown in monoculture (E and G) and in

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ontent, NDFF (from 20% to 30% under IN to 60% under HN; Fig. 1End F), and % N (right side of Table 1). At cut 2, for which a limi-ation of N availability was expected, N increased shoot N contentnly in HS monoculture (Fig. 1G), whereas it was reduced in HSixture (Fig. 1H). Shoot N content at LS remained similar what-

ver the N level, suggesting in this case a deficiency for S that wasot found for L. perenne (Fig. 1C and D). The response of T. repens

n terms of % N (Table 1) further supported the previous results,s % N in the shoot was (i) decreased between cuts 1 and 2, (ii)trongly increased between low S and high S at cut 2, (iii) slightlyecreased by L. perenne competition at cut 1, while (iv) an oppositerend was noticed at cut 2 (higher % N in mixture). Overall, N yieldf T. repens was increased by more than twofold between LS and HSFig. 1G and H), showing its highest competitive ability under LNvailability if sufficient S level was available. In contrast, S signifi-antly decreased NDFF, especially in HN treatment in both cultureypes at cut 2 (Fig. 1G and H).

The efficiency with which T. repens used N from fertilizer waslways lower than that found for L. perenne. At cut 1, N altered Necovery of T. repens in LS treatment, decreasing it in monocul-ure, but increasing it in mixture (right side of Table 2). At cut 2,

increased N recovery in all treatments. As previously found for. perenne in mixture, S generally increased N recovery of T. repenshatever the cut, except in HN mixture (Table 2).

.2. S yield and uptake

The shoot S content of L. perenne (Fig. 2A–D) increased with Supply and was even higher when plants received N fertilization.his general trend was independent of cut or culture type (mono-ulture, Fig. 2A and C; mixture, Fig. 2B and D). Accordingly, % S inhe shoot of L. perenne (left side of Table 3), was increased by twoo fivefold by S supply. Percent S in the shoot was not affected by Nevel at the first cut, but was lower at the second cut at HN.

The apparent S recovery, calculated from the balance of S recov-red in the harvested above-ground biomass of plants with (HS) orithout (LS) S fertilization is given in Table 4. S recovery of L. perenne

lways increased along the N gradient whatever the cut and the cul-ure type, being of the same magnitude in both monoculture and

ixture.For T. repens, at both cuts, S supply increased shoot S content

Fig. 2E–H) which, in the case of monoculture only, was even higherhen N was applied (Fig. 2E and G). At cut 2 HN supply reduced

hoot S content in mixture (Fig. 2H). The % S in the shoot of T. repensas increased by S supply (right side of Table 3), but did not reach

alues as high as those found for L. perenne. Increasing N availabilitylightly increased the % S of T. repens, but only at cut 1 (Table 3).oreover the % S decreased between cuts 1 and 2.The apparent S recovery of T. repens (Table 4) was lower at cut 1

han that of L. perenne, but similar or higher at cut 2. When grown

mixture (F and H) at cuts 1 and 2 under low (dashed lines) and high (solid lines)S availability along the N gradient level. Bars are mean ± S.E. (n = 4). Two differentletters indicate a significant difference; *P ≤ 0.05.

able 3ffect of different N and S levels on the mean S% in the harvested fraction of the shoot DW per plant of Lolium perenne and Trifolium repens, at cuts 1 and 2.

Lolium perenne Trifolium repens

Monoculture Mixture Monoculture Mixture

LS HS LS HS LS HS LS HS

ut 1 LN 0.23a 0.40b 0.25B 0.36C 0.16a 0.18ab 0.14A 0.22D

IN 0.15a 0.38b 0.17A 0.40C 0.17ab 0.23c 0.14A 0.20C

HN 0.12a 0.36b 0.17A 0.36C 0.20bc 0.25c 0.16B 0.26E

ut 2 LN 0.14c 0.43e 0.13C 0.49F 0.06b 0.14c 0.07A 0.16B

IN 0.10b 0.44e 0.09B 0.34E 0.04a 0.14c 0.07A 0.16B

HN 0.05a 0.25d 0.05A 0.19D 0.05ab 0.14c 0.06A 0.16B

wo different lower case letters indicate a significant difference between treatment in monoculture (four replicates; eight plants of each species per replicate) and twoifferent upper case letters indicate a significant difference between treatment in mixture (four replicates; four plants of each species per replicate) within each species andut; *P ≤ 0.05.

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Table 4Effect of different N and S levels on the mean apparent S recovery (S fertilizer recov-ery in the harvested fraction of the shoot as % of S fertilizer applied) per plant ofLolium perenne and Trifolium repens, at cuts 1 and 2.

Lolium perenne Trifolium repens

Monoculture Mixture Monoculture Mixture

Cut 1 LN 1.72a 1.06NS 0.34a 0.50NS

IN 3.00b 3.19NS 0.50a 0.82NS

HN 4.16b 3.67NS 1.52b 0.57NS

Cut 2 LN 1.19a 1.64A 1.43a 4.08B

IN 1.89b 2.18AB 2.10b 4.81B

HN 3.09c 2.47B 2.57b 2.98A

Tmame

iire

3r

awiedup

aawuSt

Fa

wo different lower case letters indicate a significant difference between treat-ent in monoculture (four replicates; eight plants of each species per replicate)

nd two different upper case letters indicate a significant difference between treat-ent in mixture (four replicates; four plants of each species per replicate) within

ach species and cut; *P ≤ 0.05.

n monoculture, N supply increased S recovery, while when grownn mixture, at cut 2, high N decreased it (Table 4). In mixture, Secovery of T. repens reached higher values than in monoculture,xcept at high N level.

.3. Dry matter and changes in soluble proteins in stolons of T.epens

The stolon growth of T. repens, estimated by dry weight (Fig. 3And B) as well as soluble protein concentration (Fig. 3C and D)ere unaffected by N supply (except a slight increase of stolon DW

n HS monoculture; Fig. 3A). However, S supply had a significantffect. S increased stolon DW on average by 40% (Fig. 3A and B) andoubled soluble protein concentration (Fig. 3C and D). Similar val-es were found whether grown in monoculture or mixture with L.erenne.

SDS-PAGE electrophoresis revealed slight changes in relativebundance of some proteins along the N and S gradients. The VSP

t 17.3 kDa was slightly accumulated for HS only in monoculture,hatever N level. In mixture, the abundance of RubisCO small sub-nit (SSU) at 15 kDa increased along the N gradient whatever thelevel. This could be related to a high photosynthetic rate of this

issue resulting from L. perenne competition.

ig. 3. Stolon soluble protein concentration (in mg/g dry weight) and stolon dry weight olong the N and S gradients. Bars are mean ± S.E. (n = 4). Two different letters indicate a si

mental Botany 66 (2009) 309–316

4. Discussion

The work described here focused on nutrient capture and relatedcompetitive interactions between two important grassland species,L. perenne and T. repens, under variable levels of N and S. Althoughother mechanisms may be involved to explain competition betweenthese species, amongst which, competition for light is of primeimportance, our work was conducted over a relatively short termand included cutting, which may reduce this kind of competition.The growth period was divided in two successive phases: establish-ment of plants with different and potentially ample availabilities ofN and S, and, following cutting, a regrowth period during which Ndeficiency may exacerbate the competition for nutrients betweenthe grass and the legume. Analysis of plant N and S recoveriesin monoculture and in mixture was then used to identify specificeffects of competition.

During the establishment phase, L. perenne benefited fromincreasing N availability as expected (e.g. Jones et al., 1973;Marschner, 1986; McKenzie, 1996). T. repens followed the sametrend in monoculture. Such effects can be seen through theincreased amount of N content in the harvested fraction of shootsand through the highest values of N derived from fertilizer. How-ever, in contrast to T. repens, L. perenne shoot N content, NDFF andN recovery were not affected by S supply in monoculture. This pro-vides evidences that the increase in its biomass, noticed by Tallec etal. (2008a) in the same growth condition, at high N, was not relied toan improvement of its capacity to take up N fertilizer with increas-ing S availability. S supply acted in promoting a higher dilution ofN in plant tissues: N% DW of harvested above-ground biomass wasalways lower at high S, this was significant in high N treatmentduring regrowth phase, while shoot S content and S% increased.This could be explained by Freney et al. (1978) and Gilbert et al.(1997) findings on wheat and barley whose growth was reduced andamides accumulated when a lack of S for protein synthesis occurred.On the contrary, in mixture N recovery of L. perenne was greater with

S supply. That was probably due to a better availability of N fertilizerfor L. perenne than in monoculture as N2 fixation by four T. repensindividuals occured and reduced intraspecific competitiveness formineral N. Mechanisms of S effect on the enhancement of L. perennegrowth so depend on N availability. First, S allowed higher rate of

f Trifolium repens grown in monoculture (A and C) and in mixture (B and D) at cut 3gnificant difference; *P ≤ 0.05.

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Acknowledgments

T. Tallec et al. / Environmental and

rotein synthesis, then, if sufficient soil N is available, N uptake isncreased. These results validate our first hypothesis, stating that

application contributes to greater mineral N uptake than with-ut S. In both monoculture and mixture, S recovery was also highlyncreased by N supply. Our results highlight the occurrence of closeelationships between N and S metabolisms in grassland speciesnd the co-regulation of their uptake, then their assimilation forrotein synthesis.

During the regrowth period, a very strong positive effect of Supply was found with T. repens. S reduced its NDFF, even at high, while its N content in the harvested fraction of the shoot signif-

cantly increased. From these results, it appears clear that S supplyead to a lower inhibition of N2 fixation by mineral N. Indeed, at low–low N 100% of T. repens shoot N content depended on fixation orther minor pools (low soil N reserves, N remobilisation from otherlant organs), while these proportions decreased to 97% and 70%t intermediate and high N respectively. This negative N effect onlover had been described by numerous authors (Macduff et al.,996; Soussana et al., 2002). They explained this phenomenon bydecrease in nodule number and dry weight and by a direct effectf nitrate on nitrogenase activity. However, high S supply allowedhe percent of N derived from other pools than fertilizer to reach8% and 93% of total shoot N content at intermediate N and highrespectively. These results emphasized DeBoer and Duke (1982)

ndings on M. sativa which showed that S deprivation restored Nield whereas fixation was decreased under low S. However, atigh N, the beneficial effect of S on shoot N content of T. repensas lower than at intermediate N availability, demonstrating that,

ven if fixation process was restored, its efficiency was still low-red by N. Indeed, at high N and S availabilities, T. repens shoot Sontent significantly fell, compared to other N levels. This indirecteffect validates our second hypothesis, stating that S applica-

ion contributes to greater atmospheric N acquisition efficiencyy T. repens. This appears to be the main mechanisms involvedn the large increase in biomass production reported by Tallec etl. (2008a). Different physiological mechanisms may explain thiseffect. Scherer and Lange (1996) and Habtemichial et al. (2007),

ound that S fertilization increased nodule number and nodule fresheight of many legume species (M. sativa, Trifolium pratense, Vicia

aba and Pisum sativum), especially by enhancing root growth. Varint al. (2009) demonstrated that at the same time, the proportion ofodules containing leghaemoglobin might increase. Another regu-

atory process may be suggested: Janssen and Vitosh (1974) showedhat S limitation triggers an increase in amino-acid content in plantissue, which may subsequently down-regulate N2 fixation.

Further, S also enhanced T. repens ability to store N and to colo-ize space. Our results show that S increased stolons growth, thenolonization ability, as stolons are the means by which T. repensersists and colonizes in grassland (Chapman, 1987). This might

mply a long-term competitive advantage compared to individualsrowing in poor S soils. S may also induce a high competitive advan-age during regrowth after cutting. Indeed, initial S supply stronglynhanced the accumulation of soluble proteins in stolons. This posi-ive effect validates our second hypothesis highlighting that S inputlso modifies N allocation to storage organs. This increase was cor-elated with the enhancement of N2 fixation. Among these solubleroteins, VSP are considered as crucial (Volenec et al., 1996; Corret al., 1996; Goulas et al., 2001) for regrowth potential and sub-equent photosynthesis process. However, S availability did notlearly affect specifically their contents, but it does not exclude thatoluble protein in general may be able to be remobilised to sup-

ort the growth of new tissues after cutting as already shown forther species (Loualhia et al., 1999). This S effect should be partic-larly crucial under high N condition, to allow T. repens persistence

n intensive grasslands as S appears to be seriously implied in theegrowth process of T. repens.

mental Botany 66 (2009) 309–316 315

During the establishment period, L. perenne clearly behaved asa better competitor than T. repens. This can be illustrated by highershoot N and S contents and higher N and S recoveries being fur-ther increased with higher N and S availabilities. The actual N andapparent S recoveries were higher in L. perenne than in T. repens. Thisillustrates the competitive ability of L. perenne for nutrient capturewhen its growth is not limited by one of them and projects thisspecies as the best performer. Ryegrass species have a deeper anda better prospective branched root system during the first growthstage than legumes, and can probably absorb more S at the expenseof the latter, especially under HN availability as proved by Gilbertand Robson (1984c) with the annual species Lolium rigidum andTrifolium subterraneum.

During the regrowth period, the reduction in N availability, asillustrated by the strong decrease in the N recoveries, changed theratio of mineral resources, which became more favourable for T.repens. In such cases, T. repens dominated mixtures (Tallec et al.,2008a). When nutrient acquisition efficiency is considered, it seemsclear that T. repens, when being in competition with L. perenne,strongly increased its S recovery. Similarly, when N2 fixation by T.repens was increased by S fertilization, more mineral N was avail-able for L. perenne which maximized its N recovery, probably dueto a lower competition of T. repens which derived most of its Nfrom N2 fixation. In addition, in low N mixture, S supply increasedthe shoot N content of L. perenne whereas it remained the samein monoculture. In this latter case, the S effect may also be linkedwith the high performance of T. repens, whose N nutrition dependedmostly on N2 fixation. As already discussed, fixation was stronglyenhanced by S. L. perenne could then benefit from higher N trans-fer from T. repens. Thus, L. perenne could benefit from an indirectS effect via T. repens. As stated in our third hypothesis, the typeof neighbouring can alter the capacity of plants to acquire nutri-ents.

5. Conclusion

The overall findings indicate that S fertilization may lead toimprove N acquisition efficiency. In temporary grassland, L. perenneand T. repens have to compete with neighbours of their own andother species to colonize gaps in the sward canopy. Their successin colonizing horizontal and vertical space depends partly on rapiddry matter accumulation and subsequently on nutrient uptake abil-ities and mobilisation. A significant effect of S was achieved on the Nrecovery of L. perenne while it greatly increased N yield of T. repens,by stimulating the N2 fixation process. The uptake of both N and Sand their assimilation seemed to be mutually regulated denotingthe existence of a strong link between both metabolic pathways.Moreover, our research study highlighted previous results showingthat S improved the capacity of T. repens to persist in N-rich envi-ronments, by enhancing particularly the accumulation of solubleproteins in stolons, which may be used for both stolon growth andshoot development after cutting. Without S, T. repens was a poorperformer. Finally, since T. repens brings N into the soil, that bene-fits other species, S inputs should be recognized as a managementtool and integrated in fertilizers in order to minimize unfavourableeffects of N fertilization like the disappearance of legumes in inten-sive grassland.

We gratefully thank MP Bataillé, P Beauclair, J Bonnefoy, RSegura and AF Ameline for help with the experimental workand T Gordon and E Personeni helpful for comments on themanuscript.

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