Valerio Zaffalon

Sunil Kumar Mukherjee

Vanga Siva Reddy

Jeremy R Thompson

Mark Tepfer
email: mark.tepfer@versailles.inra.fr

A Survey of Geminiviruses and Associated Satellite DNAs in the Cotton-Growing Areas of Northwestern India

Keywords: Begomovirus, Cotton leafcurl disease, Burewala, India, betasatellite, alphasatellite

Severe symptoms of Cotton leaf curl disease (CLCuD) are caused by the association of a single-stranded circular DNA satellite (betasatellite) with a helper begomovirus. In this study we analyzed 40 leaf samples (primarily cotton with CLCuD symptoms and other plants growing close by) from four sites between New Delhi and the Pakistan/India border, using rolling circle amplification (RCA) and PCR. In total, the complete sequences of 12 different helper viruses, eight alphasatellites, and one betasatellite from five different plant species were obtained. A recombinant helper virusmolecule found in okra, a novel alphasatellite-related DNA from croton, and a circular DNA of 941 nucleotides isolated from clerodendrum are also described. This is the first report of the presence of both DNA components (Helper virus and betasatellite) associated with resistancebreaking CLCuD in India, and highlights the need for further work to combat its damage and spread.

Introduction

Geminiviruses and their associated satellites present modern agriculture with important challenges, inducing a variety of diseases in tropical and sub-tropical regions [25,30,34]. Economic losses caused by the geminiviral cotton leaf curl disease (CLCuD) are some of the most significant of any plant virus. In the Indian subcontinent it was first identified around the Pakistani city of Multan in 1986 [16], and has since spread rapidly; in 1991 in the Pakistani Punjab the disease affected 14,000 ha out of a total growing area of 2.5 million ha, and two years later had spread to affect an overall area of 202,000 ha, resulting in a reduction in cotton yield of 29% [7]. In the following years the disease continued to spread in the Punjab and to the surrounding Pakistani regions, and in 1993 was first reported across the border in India, in Sriganganagar, Rajasthan [19]. Between 1992 and 1997, the economic loss due to CLCuD in Pakistan was calculated to be about 5 billion US dollars [7]. The establishment of the disease was assumed to be principally due to the introduction of high yielding, highly CLCuD-susceptible varieties of cotton (Gossypium hirsutum), in place of the native, resistant cotton species G. arboreum, a problem only temporarily addressed by the development of new tolerant/resistant G. hirsutum varieties [START_REF] Akhtar | Comparison of resistance to cotton leaf curl disease (Multan/Burewala) in Gossypium hirsutum L. varieties and breeding lines[END_REF][START_REF] Ali | Breeding of cotton varieties for resistance to cotton leaf curl virus[END_REF]. Resistance subsequently

Materials and Methods

Samples

Leaves of symptomatic and asymptomatic plants were collected in Northwestern India on 17-18 th September 2007, in fields in or near the cities of Hisar and Sirsa (Haryana), Kumiharwala (a village in Muktsar district, Punjab) and Sriganganagar (Rajasthan) (Fig. 1, Table 1). Incidence of CLCuD symptoms was low in cotton fields in or near Hisar, and very high at Sirsa, Kumiharwala and Sriganganagar, where it was close to 100%. In Hisar, cotton, okra and croton samples were from an agricultural context, while the other species were sampled in a garden within the city. In Sirsa, samples were from the Central Cotton Research Institute, in Kumiharwala in and adjacent to cotton fields, and in Sriganganagar from the Agriculture Research Station. Samples were stored for 1-2 months at 4°C before being shipped by courier to Ca'Tron.

DNA extraction

The DNA was extracted using a modified version of the procedure described in Doyle and Doyle (1987) [12]. 100 mg of leaf tissue per sample were used for each extraction. The CTAB extraction solution was prepared adding 2.5% PVP-40 and using 2 M NaCl and 25mM EDTA. After the addition of isopropanol, the solution was centrifuges at 10,000g for 15 min and the pellet washed with 70% ethanol. The extracted nucleic acids were respuspended in water and left at 4°C overnight. The following day, the DNA was re-extracted using one volume of cold (4°C) 1:1 phenol (pH 8.0)/chloroform, in order to get a higher purity. Its quantity and quality were measured by fluorometry. Isolate code, host, and location of all samples analyzed in this study are shown in Table 1.

Comment citer ce document : Zaffalon, V., Mukherjee, S. K., Reddy, V. S., Thompson, J. R., Tepfer, M. (2012). A survey of geminiviruses and associated satellite DNAs in the cotton-growing areas of northwestern India.

Archives of Virology, 157 (3), 483-495. , DOI : 10.1007/s00705-011-1201-y

Rolling circle amplification (RCA) and cloning

The GE Healthcare Illustra™ TempliPhi™ 500 Amplification Kit was used, using 20 ng of total nucleic acid extraction. The amplified samples were digested by restriction endonucleases (REs) and separated by electrophoresis on a 1.2% agarose gel in TAE. The choice of RE depended on the circular DNA of interest, with a preference for a RE recognizing a single conserved sequence in related viral sequences found in the databases. The REs of choice therefore were BamHI, NcoI and SalI for the virus and AvaII for betasatellite, respectively. RCA digestion products were extracted and cloned into an appropriately RE-cut plasmid (either pBluescript SKII (Stratagene, La Jolla, CA) or pGEM-T Easy (Promega, Madison, WI)) using standard cloning procedures. Escherichia coli (DH5α) cells were then transformed by electroporation and plasmids extracted from resultant colonies.

PCR

Two primer pairs used were designed based on Clustal alignments (Vector NTI Advance TM 9 package (Invitrogen, Carlsbad, CA)) using available database begomoviral sequences and betasatellite associated sequences. For DNA-A, PCR was carried out with degenerate primers 302 (5'-TGTGARGGYCCWTGYAARGTYCA-3') and 424

(5'-CARRTMMRRTTCAAYHACAACMTVMGGA-3') to produce an amplicon of ~827 bp. For betasatellite, PCR was carried out with primers 419 (5'-CTACCCTCCCAGGGGTACACA-3') and 420 (5'-CACGTGTTGTCATGTTGGCTT-3') to produce an amplicon of ~614 bp. Both PCR products were cleaned and sequenced.

Computer software All sequences obtained were initially analyzed using the Vector NTI Advance TM 9 package. All phylograms were produced using PAUP* 4.0 [39] after selecting for the best nucleotide substitution method with jMODELTEST [32]. The software RDP3.27 [26] was used to analyze and detect recombination signals from DNA alignments implementing seven different methods. BLAST algorithms were used to search for sequence similarity in the GenBank (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and in the EBI databases (http://www.ebi.ac.uk/Tools/blast/). The algorithm of the NCBI Pairwise Sequence Comparison project (PASC) was used to classify the sequences taxonomically on the basis of sequence similarity [5].

Results

RCA-RFLP

RCA products were digested with AvaII, BamHI, NcoI, or SalI producing bands of different sizes (Full data set shown in Supplemental Table 1). In 14 cotton samples, out of a total of 23, no RCA product was obtained, which could be due either to the absence of circular DNA with the corresponding restriction site(s), or to the presence of contaminants that inhibited the formation of RCA products. Further RCA trials with diluted templates and repurified samples failed to generate restriction products (results not shown).

PCR

A PCR was considered positive if it yielded a detectable fragment of the expected size; in many cases, less intense fragments of larger and smaller sizes were also observed (results not shown). Out of a total of 40 samples tested, 36 and 35 were positive for the helper virus and betasatellite, respectively (Table 1). Only sample DhI produced no amplicons for either type of PCR. Samples 1a, 3n and HS1 were positive for betasatellite, yet negative for the helper virus; since betasatellite is dependent on a helper virus, the absence of detection of the latter is more likely due to a technical problem, rather than to the absence of the virus. Several samples (7, 10b, Cgc and RR1) were positive for the virus and negative for betasatellite, which could be absent in these samples.

Comment citer ce document : Zaffalon, V., Mukherjee, S. K., Reddy, V. S., Thompson, J. R., Tepfer, M. (2012). A survey of geminiviruses and associated satellite DNAs in the cotton-growing areas of northwestern India.

Archives of Virology, 157 (3), 483-495. , DOI : 10.1007/s00705-011-1201-y

Sequencing

To confirm their viral origin, all PCR products and cloned RCA fragments were sequenced. PCR amplicons were sequenced directly: 29 from helper virus amplifications and 33 from betasatellite amplifications, of which 17 and 24 yielded readable sequences, respectively. Only a subset of sequences was readable most probably because more than one related sequence was amplified per PCR. Of the RCA-derived fragments, 15 samples yielded 44 sequences. Those shown to be fulllength virus were subsequently aligned with all the geminivirus sequences in the database using the PASC algorithm [5] to determine to which viral species they belong. The main characteristics of all the sequences are shown in Table 2.

Phylogenetic analyses of the helper virus

Because of frequent observation of recombinants in begomoviruses, species attributions are made from full-length sequences, with 89% sequence identity as the species distinction cut-off (Fauquet et al. 2003). When a phylogenetic tree based on sequence similarity was generated using only the full-length sequences, all species were clearly distinguished (Fig. 2 and Supplemental Table 2). The most similar sequences in the databases were included to indicate species affiliation, and African cassava mosaic virus (ACMV, AF259894) was included as an outgroup. The relationship between Croton yellow vein mosaic virus (CYVMV) and Mesta yellow vein mosaic virus (MeYVMV) was further clarified by analysis of recombination sites, which clearly showed a tract of recombinant origin between nucleotide positions 450 and 1150 (Fig. 3). For virus and for betasatellite (see below), the sequences obtained by PCR and RCA from the same sample always grouped together, showing consistency in diagnosis (Figs 2b and4a).

Phylogenetic analyses of satellite DNA

Overall sequence similarity among betasatellites for the partial sequences obtained was high (Supplemental Table 4). In particular, all sequences obtained from croton, papaya, guar bean, and Tribulus terrestris (puncture vine), as well as six sequences from tolerant and one from a susceptible cotton plant (1a), were identical and correspond to that of a CLCuM betasatellite (DQ364230). The sequences analyzed could be seen to group somewhat according to the helper viruses CLCuRaV, CLCuBuV and CYVMV (Fig. 4a), although with only partial sequences any clear pattern in grouping will not be significant. For the alphasatellites three major clades can be distinguished (Fig. 4b). If, as for betasatellites, 83% identity is used as the cutoff for distinguishing species [27], clades I and II each correspond to a species, except for FN678901 and AM711115, which have 74-75% identity with the other members of clade I. Clade II is distinct from clade I, since its members have on the order of 60% identity with clade I. One croton sample (9c-RCA-a2-F) grouped with the more unusual satellite types of clade III, whose first member was described by Saunders et al (2002) from ageratum in Singapore, and more recently described members were from Oman [17], Venezuela [35] and Brazil [31]. Clade III is only distantly related to the other two clades, with inter-clade identity levels that are equivalent to those with the non-related outgroup sequence, Coconut foliar decay virus (CFDV). Clade III is also less homogeneous, since these satellites were associated with both monopartite and bipartite begomoviruses, and the level of intraclade sequence identity is below the 78% identity species cutoff. When the Rep protein of these satellites was compared with that of alphasatellites, nanoviruses and circoviruses, they clearly were on a separate branch from the three other groups [31,35].

Structural features of full-length helper virus molecules

Genome structure analysis showed that all the virus molecules contain ORFs AV1, AV2, AC1, AC3 and either one of the two putative AC5 ORFs (Suppl. Fig. 1a). The ToLCNDV molecule extracted from bottle gourd sample 4 had an extra ORF, named AV3, which is not present in the other molecules. Only the CLCuBuV molecules found in cotton samples L2 and L4K lacked a complete AC2 ORF, which encodes the transcriptional activator protein, TrAP, and may also function as an RNAi suppressor. This is also the case in the CLCuBuV reference sequence (AM421522). An alignment was performed using the three sequences found in this study and the CLCuBuV sequence (GQ247893), which was the most similar sequence present in the databases. The two single mutations at positions 1487 and 1499 (C to A and T to G, corresponding to positions 1489-1501 in CLCuBuV) created two in-frame stop codons, which shorten the ORF from 405 to 105 bp and would reduce the TrAP protein to a polypeptide of 35 aa, instead of 135.

It was suspected that molecule 10c-RCA-A1-F was the product of recombination between geminiviral DNAs, a fact confirmed with the RDP 3.27 package [26] of sequence 10c-RCA-A1-F and the reference sequences of MeYVMV (FJ159262) and Bhendi yellow vein mosaic virus (BYVMV) (EU589392) (Fig. 5) Two breakpoints were detected, at nucleotide positions 1108 and 1458 (encompassing the AC3 ORF (1459-1058)), with P-values of seven methods ranging from 4.39 x 10 -04 to 1.58 x 10 -11 (Supplemental Table 6). Using the BLAST algorithm a PASC value of 87.4% was obtained for 10c-RCA-A1-F with the nearest sequence, MeYVMV, therefore suggesting that in fact this virus molecule might be a putative new species.

Structural features of full-length satellite DNA sequences

Eight full-length alphasatellite-related sequences from six samples were found using RCA (Tables 1 and2). All had the typical structure of an alphasatellite: a single ORF (coding for a replicationassociated protein) and an A-rich region immediately downstream (Suppl. Fig. 1b).

Only one full-length betasatellite sequence (L2-RCA-b1-F) was obtained in this study, and was associated with CLCuBuV (Suppl. Fig. 1c). However, another circular betasatellite sequence (LG-RCA-b1-r) was only slightly shorter than L2-RCA-b1-F (1228 nts, instead of 1354 nts), and in it the region immediately upstream of the beta C1 ORF, located between nucleotide 507 and nucleotide 874, was replaced by an inverted repeat of 242 nucleotides (nts 263 to 505), which would encode most of the βC1 ORF (Suppl. Fig. 1d). The resultant molecule has an entire beta C1 ORF on the complementary sense and an almost completely duplicated beta C1 ORF on the virion sense. This is similar to another betasatellite found in the database (Acc. No. EU384591.

In order to determine whether 9c-RCA-a2 was a full-length circular DNA, PCR (not shown) with primers binding in the region flanking the BamHI restriction site was performed. The PCR confirmed that the molecule was circular, but that it was 24 nucleotides longer than the cloned RCA molecule, due to the presence of an additional BamHI site. Despite the low similarity to other alphasatellites, this molecule shared the same genomic structure (a single ORF in the virion sense and an A-rich region immediately downstream) and the same nona-nucleotide sequence at the putative origin of replication (TAGTATT^AC) as other alphasatellites.

Defective virus and satellite DNA molecules

RCA produced several DNA molecules of both alphasatellite (9S1-RCA-a4-D; 9S1-RCA-a5-D) and helper virus (10a1-RCA-A2-D; 10a1-RCA-A3-D; L2-RCA-A2-D; LG-RCA-A3-D) that, although the origin of replication was intact, contained a deletion in the central region. PCR using specific primers flanking the putative deleted region confirmed that the deletions were not simply artefacts (not shown). The organization of the defective virus molecules was similar, since they lacked the central region, with only certain ORFs intact, namely AV2, AV1, AC1, AC4, AC5 (Suppl. Fig. 1a). The sizes of some of the molecules were about one quarter (LG-RCA-A3-D), one third (L2-RCA-A2-D) or half (alphasatellites: 9S1-RCA-a4-D, 9S1-RCA-a5-D) of that of a fulllength molecule. Molecule L2-RCA-A2-D had two deletions (confirmed also by PCR) (at positions 195-1811 and 2023-2307), and as a result, only three ORFs, a partial AV2 sequence, a partial AC1 sequence and an ORF composed of the first part of AC4 and the end of AC1.

There were three RCA-derived molecules that were helper virus recombinants, either with sequences of unknown origin (10a1-RCA-A6-r), or with viral sequences of anotherorigin (L2-RCA-A3-r, 9S1-RCA-a6-r). Molecule L2-RCA-A3-r (Suppl. Fig. 1e) was a recombinant between virus and a betasatellite, the latter being an insert in the central region. Molecule 9S1-RCA-a6-r (Suppl.

Comment citer ce document : Zaffalon, V., Mukherjee, S. K., Reddy, V. S., Thompson, J. R., Tepfer, M. (2012). A survey of geminiviruses and associated satellite DNAs in the cotton-growing areas of northwestern India.

Archives of Virology, 157 (3), 483-495. , DOI : 10.1007/s00705-011-1201-y 1f), which we initially classified as an alphasatellite, is more correctly a recombinant virus sequence containing the origins of replication of both helper virus and alphasatellite.

Virus-host specificity

In general, there was a correlation between the virus species and the host species. CYVMV was detected only in croton and guar bean; the cotton leaf curl viruses were only observed in cotton; and the tomato leaf curl viruses were only in bottle gourd, cucumber, papaya and congress grass. Clerodendrum golden mosaic China virus is a bipartite geminivirus, the virus sequence of which was found only in the clerodendrum sample. Plants infected with more than one virus species were rare, the exceptions being okra samples 10a1 and 10c which were infected with MeYVMV and BYVMV, and croton sample 9c which was infected with CYVMV and BYVMV (and Tomato leaf curl Patna virus (ToLCPaV) as a partial sequence). It is of note that in one cotton sample, 9S1, three full-length alphasatellite sequences were detected, along with two defective sequences and a recombinant molecule between an alphasatellite and a CLCuV.

Discussion

The principal findings of this study are the following: 1) confirmation that CLCuBuV and associated betasatellite are present in Indian cotton fields, 2) none of the geminiviruses infecting cotton were found associated with nearby crops or weeds, 3) associated betasatellites were hostpromiscuous, 4) the identification of a novel alphasatellite-related molecule. Additionally, we have demonstrated that RCA and PCR are complementary techniques (in particular when in this case RCA yields -most likely because of reduced sample quality -false negatives), and together can form a robust approach to circular DNA virus and satellite characterization.

The presence of CLCuBuV molecules signifies that this species has spread from its origin in Pakistan [4]. [24]; many of the plants from Kumiharwala and Sriganganagar, the sites closest to Pakistan, also harbor it. To our knowledge this is the first published report of the presence of this virus in India.

Sequence analysis of the CLCuBuV isolates highlights a notable truncation in the C2 gene, a homologue of the TrAP protein of bipartite begomoviruses, caused by the presence of an opal and amber stop codon. The C2 protein is multifunctional, up-regulating virion-sense gene transcription and host gene expression, while down-regulating suppression of gene silencing and the hypersensitive response [13-14, 38, 41]. More specifically, cell death elicited by the AV2 protein was shown to be suppressed by the AC2 protein [29]. The persistence of a virus with a truncated form of a gene that is clearly implicated in the modulation of plant antiviral defences is surprising, although the potential leakiness of the opal and amber stop codons [37], may point to a possible mode of gene regulation rather than complete truncation. The C-terminus functions as a transcriptional activator [10], which may or may not be functionally linked to suppression of gene silencing [41]. It may be significant that the nuclear localization signal 28 RRRR 31 is just upstream of the stop codons, signifying a possible retention of gene regulatory function, although one of the residues (30) in two of isolates is a lysine instead of a arginine, a change that might affect disease phenotype [10]. Identification of a truncated TrAP gene in Pakistani CLCuV isolates has also been recently reported [4]. In that study, they showed that CLCuBuV, aside from being the only virus present in resistant cotton, possessed a C2 gene containing three mutations; facts that point possibly to more prolonged selection in favor of this virus.

One of the epidemiological aspects of CLCuD that would be useful to address is how the virus survives after cropping; it is not seed-borne and is known to have malvaceous and solanaeous hosts, including ageratum, cotton, hibiscus, okra, tobacco, and tomato. Other sources could also include weeds and cotton rattoons [7]. Although there was a limited number of cultivated crops and weeds examined in this study in none did we find viruses similar to those found in cotton, therefore suggesting a degree of host specialization, although it has been shown that the same species of begomoviruses that naturally infect okra are also found in cotton [43]. Similarly, in Burkina Faso, Comment citer ce document : Zaffalon, V., Mukherjee, S. K., Reddy, V. S., Thompson, J. R., Tepfer, M. (2012). A survey of geminiviruses and associated satellite DNAs in the cotton-growing areas of northwestern India.

Archives . Thus, while our results do not exclude the possibility transfer of CLCuV between hosts, they do not provide evidence that it is the norm.

In contrast, the picture obtained from the betasatellites points to a less selective, more promiscuous distribution. These findings are in line with previous work where seven begomoviruses associated with CLCuD have been found associated with a single betasatellite [8,23], and Cotton leaf curl Multan betasatellite (CLCuMB) was found with yellow vein mosaic disease in mesta [11]. In our study, sequences of CLCuV-associated betasatellite were found in croton, guar bean, papaya and Tribulus.

All the defective molecules identified here are similar to those described previously; they contain the intergenic region and part of the AC1 gene, and are accompanied by full-length helper virus [21]. These molecules may attenuate symptoms, as has been shown for East African cassava mosaic Cameroon virus [6], and they could also serve as templates for recombination allowing for rapid repair of degenerate viral sequences or the creation of novel combinations of viral genes. Of the 12 full-length virus molecules sequenced in this work, only one was a novel recombinant, and its structure was the same as that of other begomoviruses described [20].

All the full-length molecules characterized here were generated by RCA, which is proving to be an invaluable tool for studying geminiviruses [15]. Our failure to obtain clonable amounts of amplified DNA from specific cotton samples by RCA, while confirming the presence of such molecules by PCR, highlights the usefulness of applying both methods for virus characterization in particular when because of less than optimal sample quality RCA yields false negatives. Cotton is a difficult material for extraction of nucleic acids, and this problem might have limited the efficiency of RCA in our lab. With RCA-negative samples, even when the extracted nucleic acids were diluted to avoid the possible harmful effects of contaminating substances [15], the RCA experiments remained unsuccessful. Our results clearly show that, at least for detection purposes, a good set of PCR primers is sufficient although in most cases the partial sequence produced cannot be used to conclusively identify the DNA component.

The results of this study have unequivocally demonstrated the presence of the resistancebreaking CLCuBuV in India, a fact that can be in part explained by the virus' initial rapid spread from its source in Pakistan, assisted by wind conditions that would favor the movement of Bemisia tabaci, and the nature of geography of the border region. Recent work in breeding has so far not provided a complete solution to this threat to cotton cultivation [START_REF] Akhtar | Comparison of resistance to cotton leaf curl disease (Multan/Burewala) in Gossypium hirsutum L. varieties and breeding lines[END_REF], therefore highlighting the urgent need for a more concerted effort to understand the ecology of CLCuD-associated viruses and their control.

Comment citer ce document : Zaffalon, V., Mukherjee, S. K., Reddy, V. S., Thompson, J. R., Tepfer, M. (2012). A survey of geminiviruses and associated satellite DNAs in the cotton-growing areas of northwestern India.

Archives Comment citer ce document : Zaffalon, V., Mukherjee, S. K., Reddy, V. S., Thompson, J. R., Tepfer, M. (2012). A survey of geminiviruses and associated satellite DNAs in the cotton-growing areas of northwestern India.

Archives The length of the sequence is displayed along with the most similar sequence found using blastn tool against GenBank database sequences: the accession number of the best hit, along with the coverage and the identities of the alignment and the score of PASC alignment. A dash in the accession number column means that the sequence was identical to another submitted sequence. GdSA -

Gossypium davidsonii symptomless alphasatellite, ClGMV -Clerodendron yellow mosaic virus

Comment citer ce document : Zaffalon, V., Mukherjee, S. K., Reddy, V. S., Thompson, J. R., Tepfer, M. (2012). A survey of geminiviruses and associated satellite DNAs in the cotton-growing areas of northwestern India.

Archives LG PCR B1 100 100

ACMV 10c-RCA-A2-F FJ179370 10a1-RCA-A1-F EU589392 AM501481 3d-RCA-A1-F GQ220850 DQ191160 10c-RCA-A1-F FJ159262 AM421522 L2-RCA-A1-F L4K-RCA-A1-F AJ507777 9c-RCA-A2-F 8-RCA-A1-F 9b-RCA-A1-F 9c-RCA-A1-F Ku-RCA-A1-F FJ514798 AM286434 AY939926
LG-RCA-b1-r

Comment citer ce document : Zaffalon, V., Mukherjee, S. K., Reddy, V. S., Thompson, J. R., Tepfer, M. (2012). A survey of geminiviruses and associated satellite DNAs in the cotton-growing areas of northwestern India.

Archives of Virology, 157 (3), 483-495. , DOI : 10.1007/s00705-011-1201-y

Fig. 1 .

 1 Fig. 1. Plant symptoms and sampling locations. Cotton and croton plants in a field in Northwestern India. (a) a healthy cotton plant (left) and a cotton plant affected by leaf curl disease (right). (b) a typical Northwestern Indian cotton field environment with infected croton (foreground) growing next to a cotton field. (c) a healthy croton plant (left) and a croton plant affected by yellow vein mosaic disease (right). (d) map of the sampling region. The cities where the samples were collected are shown along with the cities of Multan (Pakistan), where CLCuD was originally detected, and Burewala from which currently the most dominant species takes its name.

Fig. 2 .Fig. 3 .Fig. 4 .

 234 Fig. 2. Phylogenetic trees of helper virus sequences (a) full-length sequences and (b) 492-498 nts in the AV1 ORF.Trees were obtained obtained using PAUP*4.0 (neighbor-joining) (Swofford, 1993) after selecting for the best nucleotide substitution model with jModeltest (Posada, 2008). Bootstrap values were calculated from 100 iterations. Branches with bootstrap values less than 70 were collapsed. Bar: number of substitutions per nucleotide. Virus species in bold. ACMV was used as outgroup. TLCuNDV -Tomato leaf curl New Delhi virus, CGMV -Clerodendrum golden mosaic China virus, ToLCKV -Tomato leaf curl Karnataka virus

  Version définitive du manuscrit publié dans / Final version of the manuscript published in : Archives of Virology, 2012, 157, 483-495. DOI: 10.1007/s00705-011-1201-y. The original publication is availalble at http://link.springer.com.

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  Version définitive du manuscrit publié dans / Final version of the manuscript published in : Archives of Virology, 2012, 157, 483-495. DOI: 10.1007/s00705-011-1201-y. The original publication is availalble at http://link.springer.com.

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  Version définitive du manuscrit publié dans / Final version of the manuscript published in : Archives of Virology, 2012, 157, 483-495. DOI: 10.1007/s00705-011-1201-y. The original publication is availalble at http://link.springer.com.

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  Version définitive du manuscrit publié dans / Final version of the manuscript published in : Archives of Virology, 2012, 157, 483-495. DOI: 10.1007/s00705-011-1201-y. The original publication is availalble at http://link.springer.com.Okra leaf curl diseased plants were infected by cotton leaf curl Gezira virus [40], while yellow vein disease of Digera arvensis, a common weed, was found associated with CLCuRaV[28]

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	Manuscrit d'auteur / Author manuscript	4. Amrao L, Oxford, pp 342-348. 6. Behjatnia SA, Dry IB, Rezaian MA (2007) Characterization and transient replication of tomato leaf curl virus defective DNAs. Arch Virol 152:1127-1138 7. Briddon RW, Markham PG (2000) Cotton leaf curl virus disease. Virus Res 71:151-159 8. Briddon RW, Mansoor S, Bedford ID, Pinner MS, Saunders K, Stanley J, Zafar Y, Malik KA, Markham PG (2001) Identification of dna components required for induction of cotton leaf curl disease. Virology 285:234-243 9. Briddon RW, Stanley J (2006) Subviral agents associated with plant single-stranded DNA viruses. Virology 344:198-210 10. Chowda-Reddy RV, Dong W, Felton C, Ryman D, Ballard K, Fondong VN (2009) Characterization of the cassava geminivirus transcription activation protein putative nuclear localization signal. Virus Res 145:270-278 11. Das S,
	Manuscrit d'auteur / Author manuscript
	Manuscrit d'auteur / Author manuscript	8

of Virology, 157 (3), 483-495. , DOI : 10.1007/s00705-011-1201-y

Table 1 . Symptoms and presence/absence of geminiviral DNAs in samples analysed

 1 Version définitive du manuscrit publié dans / Final version of the manuscript published in : Archives of Virology, 2012, 157, 483-495. DOI: 10.1007/s00705-011-1201-y. The original publication is availalble at http://link.springer.com. Version définitive du manuscrit publié dans / Final version of the manuscript published in : Archives of Virology, 2012, 157, 483-495. DOI: 10.1007/s00705-011-1201-y. The original publication is availalble at http://link.springer.com.

	15

of Virology, 157 (3), 483-495. , DOI : 10.1007/s00705-011-1201-y not all tests); DNAs detected by RCA, (A, DNA-A; α, alphasatellite; β, betasatellite; d, other molecule; +, RCA product observed, sequence not obtained; n.a., sample not amplified). Highlighted PCR or RCA products were sequenced.

Comment citer ce document : Zaffalon, V., Mukherjee, S. K., Reddy, V. S., Thompson, J. R., Tepfer, M. (2012). A survey of geminiviruses and associated satellite DNAs in the cotton-growing areas of northwestern India.

Archives of Virology, 157 (3), 483-495. , DOI : 10.1007/s00705-011-1201-y

Table 2 . Characteristics of viral sequences detected by PCR and RCA.

 2 Version définitive du manuscrit publié dans / Final version of the manuscript published in : Archives of Virology, 2012, 157, 483-495. DOI: 10.1007/s00705-011-1201-y. The original publication is availalble at http://link.springer.com. Version définitive du manuscrit publié dans / Final version of the manuscript published in : Archives of Virology, 2012, 157, 483-495. DOI: 10.1007/s00705-011-1201-y. The original publication is availalble at http://link.springer.com.

	Sample	Host plant	Sequence name	Accession	Length	Species assignment	Closest hit	Accession	Coverage	Max.	PASC
				number				number of	(%)	identity	(%)
								closest hit		(%)
	9a	Croton	9a-PCR-A1	FN645895	586	n.a.	CYVMV	AJ507777.1	100	99
			9a-RCA-A1-p	FN645896	1814	n.a.	ToLCPaV	EU862323.1	100	87
			9a-RCA-a1-F	FN658709	1372	n.a.	GdSA	EU384652.1	88	91
			9a-PCR-B1	-	499	n.a.	CLCuMuB	DQ364230.1	100	100
	9b	Croton	9b-PCR-A1	FN645897	676	n.a.	CYVMV	AJ507777.1	99	99
			9b-RCA-A1-F	FN645898	2760	CYVMV	n.a.	AJ507777.1			95.6
			9b-PCR-B1	-	546	n.a.	CLCuMuB	DQ364230.1	100	99
	9c	Croton	9c-PCR-A1	FN645900	742	n.a.	CYVMV	AJ507777.1	100	99
			9c-RCA-A1-F	FN645901	2749	CYVMV	n.a.	AJ507777.1	100	95
			9c-RCA-A2-F	FN645902	2750	CYVMV	n.a.	AJ507777.1	98	95
			9c-RCA-A3-p	FN645903	1558	n.a.	BYVMV	AJ002453.1	100	96
			9c-RCA-a1-p	FN658710	1214	n.a.	Alphasatellite	AJ512958.1	90	97
			9c-RCA-a2-F	FN658711	1347	n.a.	AYVV alphasatellite	AJ416153.1	31	67
	4	Bottle gourd	4-PCR-A1	FN645904	675	n.a.	ToLCNDV	AY939926.1	100	96
			4-RCA-A1-F	FN645905	2737	ToLCNDV	n.a.	AY939926.1			89.8
			4-RCA-A2-p	FN645906	701	n.a.	ToLCNDV	DQ629102.1	100	96
	7	Clerodendrum 7-PCR-A1	FN645907	739	n.a.	ClGMV	EF408037.1	100	99
			7-RCA-A1-p	FN645908	527	n.a.	ClGMV	EF408037.1	100	99
			7-RCA-d1-F	FN658712	941	n.a.	No similarity found
	1a	Cotton	1a-PCR-B1	-	394	n.a.	CLCuMuB	DQ364230.1	100	100
	1b	Cotton	1b-PCR-A1	FN645909	663	n.a.	CLCuBuV	AM421522.1	100	99
			1b-PCR-B1	FN658713	284	n.a.	CLCuMuB	AY795608.2	100	98
	3n	Cotton	3n-PCR-B1	-	535	n.a.	CLCuMuB	DQ364230.1	100	100
	3b	Cotton	3b-PCR-A1	FN645910	521	n.a.	CLCuMuV	DQ191160.1	100	98
			3b-PCR-B1	FN658714	232	n.a.	CLCuMuB	AY795608.2	100	98
	3d	Cotton	3d-PCR-A1	FN645911	664	n.a.	CLCuRaV	GQ220850.1	100	100
			3d-RCA-A1-F	FN645912	2753	CLCuRaV	n.a.	AM501481.1			99.6
			3d-PCR-B1	FN658715	269	n.a.	CLCuMuB	AY820772.1	100	100
	5	Cucumber	5-PCR-A1	FN645913	655	n.a.	ToLCNDV	AM286434.1	100	98
	8	Guar bean	8-PCR-A1	FN645914	639	n.a.	CYVMV	AJ507777.1	99	97
			8-RCA-A1-F	FN645915	2755	CYVMV	n.a.	AJ507777.1			94.7
			8-RCA-A2-p	FN645916	754	n.a.	ToLCNDV	AY428769.1	100	98
					+ 650
			8-PCR-B1	-	400	n.a.	CLCuMuB	DQ364230.1	100	100

Comment citer ce document : Zaffalon, V., Mukherjee, S. K., Reddy, V. S., Thompson, J. R., Tepfer, M. (2012). A survey of geminiviruses and associated satellite DNAs in the cotton-growing areas of northwestern India.

Archives of Virology, 157 (3), 483-495. , DOI : 10.1007/s00705-011-1201-y

Comment citer ce document : Zaffalon, V., Mukherjee, S. K., Reddy, V. S., Thompson, J. R., Tepfer, M. (2012). A survey of geminiviruses and associated satellite DNAs in the cotton-growing areas of northwestern India.

Archives of Virology, 157 (3), 483-495. , DOI : 10.1007/s00705-011-1201-y

Table 1 .

 1 of Virology, 157 (3), 483-495. , DOI : 10.1007/s00705-011-1201-y Manuscrit d'auteur / Author manuscript Fragment lengths of restricted RCA products. RCA products of all samples were digested with restriction enzymes. Fragment lengths were calculated by the Biorad Quantity One ® software and rounded to the nearest 50 bases. Fragments in parentheses were cloned together. A more concise version of this table showing only those fragment lengths cloned and sequenced is shown in table 2. Comment citer ce document : Zaffalon, V., Mukherjee, S. K., Reddy, V. S., Thompson, J. R., Tepfer, M. (2012). A survey of geminiviruses and associated satellite DNAs in the cotton-growing areas of northwestern India. Archives of Virology, 157 (3), 483-495. , DOI : 10.1007/s00705-011-1201-y Version définitive du manuscrit publié dans / Final version of the manuscript published in : Archives of Virology, 2012, 157, 483-495. DOI: 10.1007/s00705-011-1201-y. The original publication is availalble at http://link.springer.com. Comment citer ce document : Zaffalon, V., Mukherjee, S. K., Reddy, V. S., Thompson, J. R., Tepfer, M. (2012). A survey of geminiviruses and associated satellite DNAs in the cotton-growing areas of northwestern India. Archives of Virology, 157 (3), 483-495. , DOI : 10.1007/s00705-011-1201-y Version définitive du manuscrit publié dans / Final version of the manuscript published in : Archives of Virology, 2012, 157, 483-495. DOI: 10.1007/s00705-011-1201-y. The original publication is availalble at http://link.springer.com.

	Manuscrit d'auteur / Author manuscript	Supplemental Host species 9a Croton 9b Croton 9c Croton 4 Bottle Gourd 7 Clerodendrum 1a Cotton	AvaII 1750 1550 1300 1050 900 400 200 1450 800 650 350 250 1550 900 850 600 450 250 1300 900 850 750 600 250 1050 800 500 450 N.A.	BamHI 2700 1200 700 600 N.P. NcoI 2600 1150 700 650 250 N.P. 2750 1300 1200 800 N.P. 2750 2100 N.P. 2100 (1350 1100) 850 300 N.P. N.A. N.P.	SalI 2800 2750 2700 2700 900 N.P.
		1b	Cotton	1300 900	1650 1250	N.P.	N.P.
		3n	Cotton		2850	N.P.	N.P.
		3d	Cotton	1350 1150 900 550	2750 1550 1350 1150	N.P.	2800 1400
	Manuscrit d'auteur / Author manuscript	3b 5 8 10a 10b 10c 6 HS2 Cotton Cotton Cucumber Guar Bean Okra Okra Okra Papaya HS1 Cotton	400 N.A. 1200 900 650 1550 1190 900 1250 1000 900 750 600 450 1500 1200 950 850 800 650 600 350 150 50 1250 1050 900 1050 950 800 700 650 550 500 200 N.A. 1000 600	700 N.A. N.A. 2750 2100 1350 700 N.P. N.P. N.P. 2650 (1550 1350) 1200 N.P. 2700 (1650 1450) 1300 N.P. 2700 (1550 1350) 1150 N.P. N.A. N.P. N.A. N.A. N.A. N.P.	N.P. N.P. 2650 1600 1050 1700 1150 1650 1050 N.P. N.A. N.P.
		Ku	Croton	1550 1300 900 800	2750	N.P.	2650
				400
		Cg	Congress grass 1450	N.A.	N.P.	N.P.
		Cgc	Congress grass 1250 1100	2900	N.P.	N.P.
		CR	Cotton	N.A.	N.A.	N.P.	N.P.
		L2	Cotton	1350 1050 850 550	1600 1200	2550	2500 1250
				350 300	900 650
		L4K Cotton	1400 1050 850 550	1650 1250	2450	2600 1250
				200
		L5K Cotton	N.A.	N.A.	N.A.	N.A.
		Lc	Cotton	N.A.	N.A.	N.P.	N.P.
		LG	Cotton	1250 1000 900 750	1600 (1350 1200)	N.P.	2900 1400
				600 500	650
		130	Cotton	N.A.	N.A.	N.P.	N.P.
		22	Cotton	N.A.	2800	N.P.	N.P.
		FR1 Cotton	N.A.	N.A.	N.P.	N.P.

Table 2 .

 2 Percent nucleotide identities between full-length DNA-A sequences analyzed

Table 3 .

 3 Version définitive du manuscrit publié dans / Final version of the manuscript published in : Archives of Virology, 2012, 157, 483-495. DOI: 10.1007/s00705-011-1201-y. The original publication is availalble at http://link.springer.com. Percent nucleotide identities between partial DNA-A sequences analyzed.Comment citer ce document : Zaffalon, V., Mukherjee, S. K., Reddy, V. S., Thompson, J. R., Tepfer, M. (2012). A survey of geminiviruses and associated satellite DNAs in the cotton-growing areas of northwestern India.Version définitive du manuscrit publié dans / Final version of the manuscript published in : Archives of Virology, 2012, 157, 483-495. DOI: 10.1007/s00705-011-1201-y. The original publication is availalble at http://link.springer.com.

	4-RCA-A1-F	EF408037

Comment citer ce document : Zaffalon, V., Mukherjee, S. K., Reddy, V. S., Thompson, J. R., Tepfer, M. (2012). A survey of geminiviruses and associated satellite DNAs in the cotton-growing areas of northwestern India. Archives of Virology, 157 (3), 483-495. , DOI : 10.1007/s00705-011-1201-y Archives of Virology, 157 (3), 483-495. , DOI : 10.1007/s00705-011-1201-y

Table 4 .

 4 Percent nucleotide identities between partial betasatellite sequences analyzed.

	DQ364230	AY795604	HS1 PCR B1	HS2 PCR B1	AJ316035	L5K PCR B1	3d PCR B1	AY820772	EU384591	HYVMDaSat	AM084379	L4K PCR B1	RS1 PCR B1	L2-RCA-b1-F	L2 PCR B1	LG PCR B1	LG-RCA-b1-r	RS2-PCR B1
	DQ364230	98 99 99 96 94 85 85 78 56 96 93 92 93 93 93 93 92
	AY795604		98 98 97 94 84 84 78 56 94 91 90 91 91 91 91 91
	HS1 PCR B1			100 96 94 85 85 78 56 95 92 91 92 92 92 92 92
	HS2 PCR B1				96 94 85 85 78 55 94 92 91 92 92 92 92 91
	AJ316035					97 83 83 78 55 93 90 89 90 90 90 90 90
	L5K PCR B1						84 84 80 56 91 89 88 89 89 89 89 89
	3d PCR B1							100 85 54 81 84 83 84 84 84 84 84
	AY820772								85 54 81 84 83 84 84 84 84 84
	EU384591									54 76 77 77 77 77 77 77 77
	HYVMDaSat										56 56 57 57 57 56 56 57
	AM084379											96 95 96 96 96 96 95
	L4K PCR B1												99 100 100 100 99
	PCR B1													98 98 98 98 98
	L2-RCA-b1-F														100 100 100 100
	L2 PCR B1															100 100 100

Table 5 .

 5 Version définitive du manuscrit publié dans / Final version of the manuscript published in : Archives of Virology, 2012, 157, 483-495. DOI: 10.1007/s00705-011-1201-y. The original publication is availalble at http://link.springer.com. Percent nucleotide identities between full-length alphasatellite sequences analyzed.Comment citer ce document : Zaffalon, V., Mukherjee, S. K., Reddy, V. S., Thompson, J. R., Tepfer, M. (2012). A survey of geminiviruses and associated satellite DNAs in the cotton-growing areas of northwestern India.Version définitive du manuscrit publié dans / Final version of the manuscript published in : Archives of Virology, 2012, 157, 483-495. DOI: 10.1007/s00705-011-1201-y. The original publication is availalble at http://link.springer.com.

	24

Archives of Virology, 157 (3), 483-495. , DOI : 10.1007/s00705-011-1201-y

Table 6 .

 6 The P-values measured by the different methods used by RDP 3.27 for the recombination event at nucleotide positions 1108 and 1458 resulting in molecule 10c-RCA-A1-F, shown in Fig.8.

	Method	P-value
	RDP	1.68 x 10 -11
	GENECONV 4.39 x 10 -04
	Bootscan	1.70 x 10 -09
	Maxchi	4.03 x 10 -09
	Chimaera	4.33 x 10 -10
	SiScan	1.58 x 10 -11
	PhylPro	Ns
	LARD	Ns
	3Seq	1.78 x 10 -09

Acknowledgements

Many thanks to Dr. Dilip Mongia, CICR Sirsa, Prof. Narayan Rishi, CCS Agriculture University, Hissar for assistance in the field, and to Mirco Boem, Laura Chiappetta, Slavica Matic, Marco Morroni, Arianna Friscina and Amit Bhardwaj for technical support and sound advice. This work was funded in part by Fondazione Cassamarca. Financial support of ICGEB is acknowledged.


Nathalie Poupin

Ce ´cile Bos

Jean-Franc ¸ois Huneau

Daniel Tome ´1

He ´le `ne Fouillet
email: helene.fouillet@agroparistech.fr

The Nature of the Dietary Protein Impacts the Tissue-to-Diet 15 N Discrimination Factors in Laboratory Rats

Due to the existence of isotope effects on some metabolic pathways of amino acid and protein metabolism, animal tissues are 15 N-enriched relative to their dietary nitrogen sources and this 15 N enrichment varies among different tissues and metabolic pools. The magnitude of the tissue-to-diet discrimination (D 15 N) has also been shown to depend on dietary factors. Since dietary protein sources affect amino acid and protein metabolism, we hypothesized that they would impact this discrimination factor, with selective effects at the tissue level. To test this hypothesis, we investigated in rats the influence of a milk or soy protein-based diet on D 15 N in various nitrogen fractions (urea, protein and non-protein fractions) of blood and tissues, focusing on visceral tissues. Regardless of the diet, the different protein fractions of blood and tissues were generally 15 N-enriched relative to their non-protein fraction and to the diet (D 15 N.0), with large variations in the D 15 N between tissue proteins. D 15 N values were markedly lower in tissue proteins of rats fed milk proteins compared to those fed soy proteins, in all sampled tissues except in the intestine, and the amplitude of D 15 N differences between diets differed between tissues. Both between-tissue and between-diet D 15 N differences are probably related to modulations of the relative orientation of dietary and endogenous amino acids in the different metabolic pathways. More specifically, the smaller D 15 N values observed in tissue proteins with milk than soy dietary protein may be due to a slightly more direct channeling of dietary amino acids for tissue protein renewal and to a lower recycling of amino acids through fractionating pathways. In conclusion, the present data indicate that natural D 15 N of tissue are sensitive markers of the specific subtle regional modifications of the protein and amino acid metabolism induced by the protein dietary source.

Introduction

In ecological studies, naturally-occurring stable isotope signatures of animals have been extensively used to track diets and reconstruct trophic relationships within food webs [START_REF] Caut | Discrimination factors (Delta N-15 and Delta C-13) in an omnivorous consumer: effect of diet isotopic ratio[END_REF][START_REF] Dalerum | Resolving temporal variation in vertebrate diets using naturally occurring stable isotopes[END_REF][START_REF] Hobson | Tracing origins and migration of wildlife using stable isotopes: a review[END_REF]. Animals are generally 15 N-enriched over their diet, with an average discrimination factor (D 15 N, the difference in 15 N natural abundance between body and diet) of 3.4% [START_REF] Deniro | Influence of diet on the distribution of nitrogen isotopes in animals[END_REF], which varies among tissues and metabolic pools [START_REF] Deniro | Influence of diet on the distribution of nitrogen isotopes in animals[END_REF][START_REF] Caut | Variation in discrimination factors (Delta N-15 and Delta C-13): the effect of diet isotopic values and applications for diet reconstruction[END_REF][START_REF] Vanderklift | Sources of variation in consumer-diet delta N-15 enrichment: a meta-analysis[END_REF]. This discrimination originates from the existence of kinetic isotope effects in metabolic pathways involving the bond-breaking or synthesis of nitrogenous compounds, during which the molecules that contain the lighter nitrogen isotope ( 14 N) are preferentially utilized to the detriment of the same molecules containing the heavier isotope ( 15 N). When located at a metabolic branch point, such kinetic isotope effects induce a preferential flow of 14 N into the products generated by the reaction with the larger isotope effect, which results in an isotopic fractionation between the metabolic pools produced by the competing reactions, as reflected by a difference in D 15 N of these pools [START_REF] Hayes | Fractionation of carbon and hydrogen isotopes in biosynthetic processes[END_REF]. More specifically, deamination and transamination enzymes are likely to preferentially convert amino groups containing 14 N [START_REF] Gaebler | Isotope effects in metabolism of 14N and 15N from unlabeled dietary proteins[END_REF][START_REF] Macko | Kinetic fractionation of stable nitrogen isotopes during amino-acid transamination[END_REF], which is thought to result in the urinary excretion of 15 N-depleted end products and the incorporation of residual 15 N-enriched amino acids into the body [START_REF] Gannes | Natural abundance variations in stable isotopes and their potential uses in animal physiological ecology[END_REF][START_REF] Steele | Fractionation of nitrogen isotopes by animals -a further complication to use of variations in natural abundance of n-15 for tracer studies[END_REF] in line with the body-to-diet positive D 15 N. Interestingly, there is a growing body of evidence that the magnitude of D 15 N vary between individuals depending on their specific nutritional, physiological or pathological conditions. For instance, the hair 15 N natural abundance values are good markers of animal protein consumption [START_REF] Petzke | Carbon and nitrogen stable isotopic composition of hair protein and amino acids can be used as biomarkers for animal-derived dietary protein intake in humans[END_REF] and vary with pregnancy [START_REF] Fuller | Nitrogen balance and delta N-15: why you're not what you eat during pregnancy[END_REF], nutritional stress [START_REF] Fuller | Nitrogen balance and delta N-15: why you're not what you eat during nutritional stress[END_REF], anorexia [START_REF] Mekota | Serial analysis of stable nitrogen and carbon isotopes in hair: monitoring starvation and recovery phases of patients suffering from anorexia nervosa[END_REF] and cirrhosis [START_REF] Petzke | Nitrogen isotopic composition in hair protein is different in liver cirrhotic patients[END_REF] in humans. More specifically, D 15 N values in some animal tissues have been shown to vary with protein intake, balance or starvation [START_REF] Vanderklift | Sources of variation in consumer-diet delta N-15 enrichment: a meta-analysis[END_REF][START_REF] Fuller | Nitrogen balance and delta N-15: why you're not what you eat during nutritional stress[END_REF][START_REF] Mekota | Serial analysis of stable nitrogen and carbon isotopes in hair: monitoring starvation and recovery phases of patients suffering from anorexia nervosa[END_REF][START_REF] Del Rio | Mass-balance models for animal isotopic ecology[END_REF][START_REF] Barboza | Body protein stores and isotopic indicators of N balance in female reindeer (Rangifer tarandus) during winter[END_REF][START_REF] Ponsard | Should growing and adult animals fed on the same diet show different delta N-15 values?[END_REF] as well as with dietary protein source and quality [START_REF] Vanderklift | Sources of variation in consumer-diet delta N-15 enrichment: a meta-analysis[END_REF][START_REF] Robbins | The effect of dietary protein quality on nitrogen isotope discrimination in mammals and birds[END_REF][START_REF] Vander Zanden | Variation in delta N-15 and delta C-13 trophic fractionation: Implications for aquatic food web studies[END_REF]. However, because the latter studies were meta-analyses of literature data over different species from studies where the quantitative protein intake was not controlled for, there is a large risk of confusion between the specific effects of the qualitative and quantitative aspects of protein intake. Indeed, a difference in the dietary protein source is often associated with a difference in the level of protein intake and these two factors may affect D 15 N values [START_REF] Del Rio | Isotopic ecology ten years after a call for more laboratory experiments[END_REF]. Accordingly, dedicated experimental investigations under controlled nutritional conditions are required to examine the changes in the pattern of isotopic discrimination between the animal tissues and the diet that specifically relate to the nature and the quality of the dietary protein source.

The magnitude of D 15 N has been proposed to reflect the efficiency of dietary protein assimilation and the importance of amino acid recycling [START_REF] Del Rio | Mass-balance models for animal isotopic ecology[END_REF][START_REF] Gaye-Siessegger | Individual protein balance strongly influences delta N-15 and delta C-13 values in Nile tilapia, Oreochromis niloticus[END_REF][START_REF] Waters-Rist | The Effect of Growth on Stable Nitrogen Isotope Ratios in Subadult Bone Collagen[END_REF], which both vary with the dietary protein source and quality [START_REF] Deutz | Infusion of soy and casein protein meals affects interorgan amino acid metabolism and urea kinetics differently in pigs[END_REF][START_REF] Fouillet | Absorption kinetics are a key factor regulating postprandial protein metabolism in response to qualitative and quantitative variations in protein intake[END_REF][START_REF] Morens | Increasing habitual protein intake accentuates differences in postprandial dietary nitrogen utilization between protein sources in humans[END_REF]. In the present work, we hypothesized that dietary protein of different composition and nutritional characteristics would result in different discrimination factors in nitrogen pools. Since different dietary proteins may differentially impact amino acid metabolism in tissues [START_REF] Deutz | Infusion of soy and casein protein meals affects interorgan amino acid metabolism and urea kinetics differently in pigs[END_REF][START_REF] Fouillet | Peripheral and splanchnic metabolism of dietary nitrogen are differently affected by the protein source in humans as assessed by compartmental modeling[END_REF], we also hypothesized that the magnitude of the impact of the dietary protein source on nitrogen discrimination factors might vary between tissues. Most studies reporting variations in natural 15 N abundances depending on nutritional or physiological conditions have focused on pools that can be easily sampled, such as blood or hair [START_REF] Petzke | Carbon and nitrogen stable isotopic composition of hair protein and amino acids can be used as biomarkers for animal-derived dietary protein intake in humans[END_REF][START_REF] Fuller | Nitrogen balance and delta N-15: why you're not what you eat during pregnancy[END_REF][START_REF] Fuller | Nitrogen balance and delta N-15: why you're not what you eat during nutritional stress[END_REF][START_REF] Mekota | Serial analysis of stable nitrogen and carbon isotopes in hair: monitoring starvation and recovery phases of patients suffering from anorexia nervosa[END_REF][START_REF] Petzke | Nitrogen isotopic composition in hair protein is different in liver cirrhotic patients[END_REF][START_REF] Barboza | Body protein stores and isotopic indicators of N balance in female reindeer (Rangifer tarandus) during winter[END_REF][START_REF] Robbins | The effect of dietary protein quality on nitrogen isotope discrimination in mammals and birds[END_REF][START_REF] O'brien | Red blood cell delta N-15: a novel biomarker of dietary eicosapentaenoic acid and docosahexaenoic acid intake[END_REF]. Therefore, the data are fragmented, and the potential isotopic variations in other active metabolic pools have seldom been described, while such variations may interestingly indicate underlying changes in amino acid and protein metabolism. In this context, multiple simultaneous D 15 N measurements taken from different tissues may trace complex changes in inter-organ protein metabolism. In this study, we determined the isotopic nitrogen composition of a large set of pools in rats after their adaptation to two dietary protein sources (milk and soy proteins) differing slightly in their amino acid composition and their effect on the tissue protein metabolism [START_REF] Deutz | Infusion of soy and casein protein meals affects interorgan amino acid metabolism and urea kinetics differently in pigs[END_REF][START_REF] Fouillet | Peripheral and splanchnic metabolism of dietary nitrogen are differently affected by the protein source in humans as assessed by compartmental modeling[END_REF].

Results

Body composition, tissue mass and nitrogen content

The two experimental groups receiving a diet with either milk protein (MP) or soy protein (SP) had similar growth rates and tissue masses (Table 1). Nitrogen contents of the protein (PF) and non-protein (nPF) fractions of tissues at the end of the 3-wk experimentation period were similar between groups (Table S1), except for a lower nitrogen content in the nPF of the total muscle mass (237%) in the rats fed with SP compared to MP. None of the total nitrogen content of tissues was impacted by the diet (Table S1). No differences were found in nitrogen content of plasma proteins and urea between groups (Table S1).

Variations in D 15 N among tissues and proteins

Using the 15 N natural abundances (d 15 N) measured in each sampled pool (Table S2), nitrogen stable isotope discrimination values (D 15 N, the difference in d 15 N between the pool under consideration and the diet) were estimated for the fast turning-over pools that had likely reached, or almost reached, their isotopic equilibrium with the diet at the end of the 3-wk dietary period, and D 15 N values were calculated at different levels (whole visceral area, each individual tissue and each nitrogen fraction in a given tissue and in plasma). . At the tissue level, D 15 N values were positive and differed greatly among tissues, with liver having the highest 15 N abundance among the sampled visceral tissues for both groups (Table 2). The D 15 N values of tissues were generally close to the D 15 N values of their PF, because of the relative amounts of nitrogen in tissue fractions. The D 15 N of PF exhibited significant inter-tissues differences (Fig. 1A) and ranged from +1.35% (60.25%) in colon to +3.81% (60.18%) in plasma in the MP group, and from +2.24% (60.39%) in small intestine (SI) mucosa to +4.61% (60.24%) in plasma in the SP group. For both groups, the plasma proteins (liver exported proteins, measured in the vena cava) had the highest D 15 N values, and the liver constitutive proteins had D 15 N values lower than those of plasma proteins but higher than those of the other visceral proteins (all Ps,0.05, Tables S3 &S4).

Effect of diet on D 15 N in tissues and proteins D 15 N also varied relative to the protein source. At the tissue level, liver, kidney and colon were more enriched in 15 N in rats fed the SP diet than in rats fed the MP diet, resulting in a higher overall visceral D 15 N (3.53%60.14%and 2.58%60.11% with SP and MP respectively, Table 2). At the protein level, D 15 N values were also higher with SP than MP diet in the PF of all tissues except SI mucosa (Fig. 1A). These differences between the two diets also varied between tissues. In colon proteins, the D 15 N values were ,160% higher in the SP group than in the MP group (3.52% and 1.35%, respectively), ,60% higher in kidney proteins (2.75% and 1.73%, respectively) and stomach proteins (3.39% and 2.05%, respectively), ,40% higher in liver constitutive proteins (4.14% and 2.98%, respectively) and ,20% higher in liver-exported plasma proteins (4.61% and 3.81%, respectively).

Variations in D 15 N between nitrogen fractions within plasma and tissues Furthermore, D 15 N values differed between nitrogen fractions within plasma (PF vs. urea), and also within tissues (PF vs. nPF). Whatever the diet, plasma urea was depleted in 15 N relative to the diet and relative to the plasma PF, with no diet effect on the D 15 N values of plasma urea (21.3661.66% and 21.6161.22% in MP and SP groups, respectively). Plasma urea was also 15 N-depleted relative to the liver nPF, its precursor pool (Fig. 1B,C). In tissues, the PF were always enriched in 15 N relative to the diets (Fig. 1A) whereas the nPF had either higher, similar or lower 15 N abundances compared to the diet depending on both tissue and diet (Fig. 1B). In tissues, the PF was generally enriched in 15 N relative to the nPF (Table 2), except in some tissues were there was no difference between fractions (namely, liver for both MP and SP groups and SI mucosa for the SP group only). Differences in 15 N abundance between PF and nPF varied from tissue to tissue and between diets (Table 2): they were higher for the SP diet than for the MP diet in colon, kidneys and stomach, whereas they were lower in plasma and SI mucosa.

Discussion

In this study, we investigated the specific impact of the dietary protein quality (milk vs. soy protein) on the nitrogen isotopic signatures in a large set of tissues and pools of rats under controlled conditions, i.e. under similar conditions of quantitative protein intake, growth and body composition between groups. The discrimination values (D 15 N) were calculated in the fast turningover pools (visceral tissues and plasma) being at, or close to, their isotopic equilibrium with the diet at the end of the 3-wk experimental period. We showed that D 15 N vary greatly among pools and are clearly impacted by the nature of the dietary protein in most of the pools. The observed D 15 N differences between diets likely result from subtle modulations of the protein and amino acid metabolism induced by the differences in amino acid composition and quality between milk and soy proteins.

Variations in the discrimination values among nitrogen fractions within tissues and plasma

Contrary to ecological studies, which have generally reported D 15 N values for plasma or tissues analyzed as a whole, we here specifically measured the natural 15 N abundance in different nitrogen fractions of tissues (PF and nPF) and plasma (PF, nPF and urea).

Firstly, our results of D 15 N values for the tissue protein fractions, which represent the main part of tissue nitrogen, confirm that tissues, and more specifically tissue proteins, are generally 15 Nenriched relative to the diet, with a range of D 15 N values (from 1.4% to 4.6%) in line with literature values reported for whole tissues in rodents [START_REF] Caut | Discrimination factors (Delta N-15 and Delta C-13) in an omnivorous consumer: effect of diet isotopic ratio[END_REF][START_REF] Deniro | Influence of diet on the distribution of nitrogen isotopes in animals[END_REF][START_REF] Gaebler | Isotope effects in metabolism of 14N and 15N from unlabeled dietary proteins[END_REF][START_REF] Ambrose | Controlled diet and climate experiments on nitrogen isotope ratios of rats[END_REF][START_REF] Arneson | Carbon, nitrogen, and sulfur diet-tissue discrimination in mouse tissues[END_REF][START_REF] Demots | Tissue-diet discrimination factors and turnover of stable carbon and nitrogen isotopes in white-footed mice (Peromyscus leucopus)[END_REF][START_REF] Kurle | Interpreting temporal variation in omnivore foraging ecology via stable isotope modelling[END_REF][START_REF] Robbins | The impact of protein quality on stable nitrogen isotope ratio discrimination and assimilated diet estimation[END_REF][START_REF] Yoneyama | Variations of natural carbon-13 and nitrogen-15 abundances in rat tissues and their correlation[END_REF][START_REF] Macavoy | Correlation of metabolism with tissue carbon and nitrogen turnover rate in small mammals[END_REF]. Also, we showed that, for a given tissue, D 15 N values varied between its nitrogen fractions. PF were generally significantly 15 N-enriched compared to nPF, which may be explained by an isotope effect along one or several of the metabolic pathways within tissues, such as metabolic interconversions of amino acids, protein synthesis or protein breakdown.

D 15 N values also varied between the different nitrogen fractions of plasma, with plasma urea being 15 N-depleted compared to plasma proteins. Plasma urea was also 15 N-depleted compared to the liver nPF, i.e. its main precursor pool, confirming the preferential removal of 15 N-depleted liver amino acids for urea production because of the isotope effect associated to deamination [START_REF] Gaebler | Isotope effects in metabolism of 14N and 15N from unlabeled dietary proteins[END_REF]. Nitrogen metabolism in the liver is a branched pathway, with precursor amino acids (liver nPF) being concurrently used for catabolic (urea production) or anabolic (synthesis of liver and plasma proteins) purposes. The isotopic fractionation observed between the end-products of each branch (i.e., 15 N-depletion of plasma urea relative to hepatic-synthesized proteins) suggests that the amino acids that are not directed toward deamination but used for protein synthesis become 15 N-enriched, and thereby contribute to the 15 N-enrichment of the hepatic-synthesized proteins. The magnitude of this fractionation is expected to depend both on the difference between the isotope effects associated to each of the hepatic metabolic pathways and on their relative activity [START_REF] Schoeller | Isotope fractionation: Why aren't we what we eat?[END_REF][START_REF] Sick | Amino acid utilization and isotope discrimination of amino nitrogen in nitrogen metabolism of rat liver in vivo[END_REF]. Although it is a common hypothesis in the literature that deamination is involved in the 15 N enrichment of body tissues [START_REF] Sponheimer | An experimental study of nitrogen flux in llamas: is N-14 preferentially excreted?[END_REF], this had not been demonstrated yet and our study is the first to actually report D 15 N values for the metabolic pools of the hepatic branched pathway.

Variations in the discrimination values among body proteins

We observed significant differences in D 15 N values among body proteins. For instance, whatever the dietary protein source, the plasma proteins (which mostly originate from synthesis in the liver) had the highest 15 N abundance compared to all other sampled proteins including the liver constitutive proteins, and the liver proteins had a higher 15 N abundance than the other sampled proteins. Consistent observations of higher D 15 N values in plasma than liver [START_REF] Caut | Variation in discrimination factors (Delta N-15 and Delta C-13): the effect of diet isotopic values and applications for diet reconstruction[END_REF][START_REF] Roth | Stable carbon and nitrogen isotopic fractionation between diet and tissue of captive red fox: implications for dietary reconstruction[END_REF] and in liver than kidney [START_REF] Deniro | Influence of diet on the distribution of nitrogen isotopes in animals[END_REF][START_REF] Yoneyama | Variations of natural carbon-13 and nitrogen-15 abundances in rat tissues and their correlation[END_REF] were already reported in the literature.

There is probably no simple mechanism that can solely explain the observed differences between tissues D 15 N. The isotopic signatures observed in the nitrogen pools of tissues are actually the result of the relative inward and outward fluxes of nitrogen isotopes in the many different pathways involved in the metabolism of proteins and amino acids (protein synthesis, protein degradation, non-protein metabolism, deamination, transamination, transport…), some of which being known to be associated with an isotope fractionating effect [START_REF] Macko | Kinetic fractionation of stable nitrogen isotopes during amino-acid transamination[END_REF][START_REF] Seila | Kinetic isotope effect analysis of the ribosomal peptidyl transferase reaction[END_REF][START_REF] Silfer | Kinetic fractionation of stable carbon and nitrogen isotopes during peptide-bond hydrolysis -experimental-evidence and geochemical implications[END_REF]. Therefore, variations in D 15 N among body proteins may arise from differences in the number and intensity of these metabolic processes (for instance, greater recycling of amino acids from and for tissue protein turnover) and are probably associated with difference in tissues metabolic rates, as previously suggested to explain the greater 15 N abundance in the liver [START_REF] Arneson | Carbon, nitrogen, and sulfur diet-tissue discrimination in mouse tissues[END_REF][START_REF] Demots | Tissue-diet discrimination factors and turnover of stable carbon and nitrogen isotopes in white-footed mice (Peromyscus leucopus)[END_REF]. Notably, the observed differences in D 15 N between tissues cannot be solely ascribed to differences in tissues turnover rates, since the reported D 15 N values of the different body proteins did not rank the same way as their known turnover rate and this ranking greatly varied with the protein source. In addition, the observed differences in isotopic composition between the tissue proteins may be linked to their relative composition in amino acids, which exhibit different isotopic abundances [START_REF] Gaebler | Isotope effects in metabolism of 14N and 15N from unlabeled dietary proteins[END_REF]. Further investigations are needed to test and delimit the role of these possible mechanisms in producing D 15 N differences between the tissue proteins.

Variations in the discrimination values in body proteins according to the dietary protein source

The dietary protein source strongly affected the nitrogen isotopic discrimination between body proteins and diet, with higher D 15 N in rats fed soy than milk proteins for all reported proteins except that of the SI mucosa. Higher D 15 N values have already been reported in liver of rats fed with plant vs. animal protein-based diets [START_REF] Caut | Discrimination factors (Delta N-15 and Delta C-13) in an omnivorous consumer: effect of diet isotopic ratio[END_REF][START_REF] Gaebler | Isotope effects in metabolism of 14N and 15N from unlabeled dietary proteins[END_REF]. More specifically, higher D 15 N values were observed in liver [START_REF] Gaebler | Isotope effects in metabolism of 14N and 15N from unlabeled dietary proteins[END_REF] and in plasma and jejunum proteins [START_REF] Sick | Amino acid utilization and isotope discrimination of amino nitrogen in nitrogen metabolism of rat liver in vivo[END_REF] of rats fed with soy vs. milk protein-based diets. We here show that this effect is a general feature, inasmuch as it is consistent over all the sampled visceral protein pools, except the small intestine. The amplitude of D 15 N differences between diets varied among tissues, suggesting that the protein metabolism of tissues may be differentially impacted by the dietary protein source.

The differences in amino acid composition between these two dietary proteins, which induce subtle modulations in their metabolic fate and in the endogenous protein metabolism, certainly explain the higher D 15 N values in rats fed soy than milk proteins. Studies in humans [START_REF] Fouillet | Absorption kinetics are a key factor regulating postprandial protein metabolism in response to qualitative and quantitative variations in protein intake[END_REF][START_REF] Morens | Increasing habitual protein intake accentuates differences in postprandial dietary nitrogen utilization between protein sources in humans[END_REF][START_REF] Fouillet | Peripheral and splanchnic metabolism of dietary nitrogen are differently affected by the protein source in humans as assessed by compartmental modeling[END_REF] and animals [START_REF] Deutz | Infusion of soy and casein protein meals affects interorgan amino acid metabolism and urea kinetics differently in pigs[END_REF][START_REF] Lohrke | Activation of skeletal muscle protein breakdown following consumption of soyabean protein in pigs[END_REF] have shown that the soy and milk proteins exhibit different overall and regional rates of utilization, the soy protein being less used for postprandial protein anabolism (i.e., lower net protein utilization) than the milk protein. Indeed, in the case of the soy protein, which contains a lower amount of indispensable amino acids, dietary amino acids are more deaminated and a larger proportion of amino acids incorporated in tissue proteins probably originate from endogenous amino acids that have been transaminated or recycled from protein degradation. Because these metabolic pathways are considered as a source of isotopic fractionation [START_REF] Gaebler | Isotope effects in metabolism of 14N and 15N from unlabeled dietary proteins[END_REF][START_REF] Silfer | Kinetic fractionation of stable carbon and nitrogen isotopes during peptide-bond hydrolysis -experimental-evidence and geochemical implications[END_REF], the higher nitrogen isotopic discrimination with soy protein confirms the existence of these additional steps in the utilization of soy protein. It can therefore be hypothesized that the best a dietary protein source matches the metabolic demand for indispensable amino acids, the larger is the proportion of dietary amino acids that are utilized for body protein synthesis and, as a result, the closer is the isotopic composition of the tissues to that of the diet. The fact that, in our study, we generally observed higher D 15 N values in body proteins of SP-fed rats than MP-fed rats therefore strongly suggests that nitrogen isotopic discrimination increases when the dietary protein efficiency of deposition decreases. This interestingly agrees with the hypothesis of a negative relationship between D 15 N and the net protein utilization of the diet protein source that was suggested in the literature from a set of fragmented data in ecological studies [START_REF] Vanderklift | Sources of variation in consumer-diet delta N-15 enrichment: a meta-analysis[END_REF][START_REF] Robbins | The effect of dietary protein quality on nitrogen isotope discrimination in mammals and birds[END_REF][START_REF] Vander Zanden | Variation in delta N-15 and delta C-13 trophic fractionation: Implications for aquatic food web studies[END_REF][START_REF] Gaye-Siessegger | Individual protein balance strongly influences delta N-15 and delta C-13 values in Nile tilapia, Oreochromis niloticus[END_REF], where however, several important factors of variation (amount of nitrogen intake, growth rate, physiological condition, etc.) were not controlled for. By contrast, the present study assessed the impact of the dietary protein source on isotopic composition without any potential bias from variations in the protein intake, body composition or tissue mass (Table 1 andS1). Indeed, despite their differences in amino acids composition, the milk and soy proteins were provided in sufficient amounts in the diet (20% of energy) to satisfy the requirements for indispensable amino acids, as shown by the similar growth and tissue protein accretion in the two dietary groups. The existence of a relation between D 15 N and dietary protein deposition efficiency is of significant importance because it indicates that the natural nitrogen isotopic enrichment pattern of tissues could be used as an indicator the local anabolic use of dietary protein, and thus complements the data regarding total protein metabolism as obtained by classical tracer studies [START_REF] Fouillet | Approaches to quantifying protein metabolism in response to nutrient ingestion[END_REF][START_REF] Smith | The measurement of tissue protein turnover[END_REF].

A limitation of this study is that the experimental diet was not provided over a long enough period of time to allow the slowturning over, peripheral tissues to reach their isotopic equilibrium with the diet, so that we were only able to investigate the effect of the dietary protein source on tissue-to-diet discrimination values in the fast-turning over tissues, i.e. visceral organs and plasma. Indeed, the rate at which a tissue incorporates the isotopic signature of a diet varies among tissues and is slower for tissues with slow protein turnover rates and/or low growth rates [START_REF] Del Rio | Isotopic ecology ten years after a call for more laboratory experiments[END_REF]. For instance, more than 6 months are required to ensure nitrogen isotope equilibrium in muscle or fur in adult, non-growing rats [START_REF] Kurle | Interpreting temporal variation in omnivore foraging ecology via stable isotope modelling[END_REF]. We therefore did not calculate the D 15 N in peripheral tissues as, after 3 wk, these values would still be impacted by the initial isotopic state, so that differences between diets in such D 15 N values could not be solely attributed to a diet effect on protein metabolism and would thus be difficult to interpret. By contrast, in fast-turning over pools like liver or plasma, D 15 N reaches a plateau after 2 weeks [START_REF] Gaebler | Isotope effects in metabolism of 14N and 15N from unlabeled dietary proteins[END_REF][START_REF] Macavoy | Growth versus metabolic tissue replacment in mouse tissues[END_REF] or is near equilibrium after 3 weeks [START_REF] Kurle | Interpreting temporal variation in omnivore foraging ecology via stable isotope modelling[END_REF], as shown by data from diet-shift experiments in rodents. Concerning the other fast-turning over tissues like kidney, we found very few data of isotopic incorporation rates in the literature and the reported values often vary among studies according to the experimental conditions and growth rates [START_REF] Kurle | Interpreting temporal variation in omnivore foraging ecology via stable isotope modelling[END_REF][START_REF] Arneson | Metabolic protein replacement drives tissue turnover in adult mice[END_REF]. In the present study, we believe that all the visceral tissues were very close to their isotopic equilibrium with the diet after 3-wk, given their high protein turnover rates and the high growth rate of the rats, which should have shortened the equilibration time [START_REF] Ponsard | Should growing and adult animals fed on the same diet show different delta N-15 values?[END_REF]. Longer-term studies would be of interest to investigate, on a wider range of tissues, the effect of the dietary protein source that we here evidenced on visceral tissues.

In conclusion, the 15 N discrimination values varied among tissues, between the nitrogen fractions (PF, nPF and urea) and according to the dietary protein source. We showed consistently higher D 15 N values in virtually all visceral proteins of rats fed soy than in those fed milk proteins and interpreted this higher discrimination with soy than milk proteins as resulting from a lower dietary protein anabolic use and higher endogenous protein mobilization and recycling of amino acids. In view of these results, nitrogen stable isotope signatures of body proteins appear as highly sensitive markers of the delicate nutritional modulations of protein and amino acid metabolism. As far as we know, our work is the first demonstration that subtle tissue-specific modulations of protein metabolism induced by different dietary proteins and without any detectable effect on tissue composition actually leave a marked selective isotopic footprint in tissues. This novel approach, based on the investigation of the isotopic signatures of metabolic pools at natural abundance level, opens new, exciting perspectives to better characterize and understand the amino acid and protein metabolism. Some interesting mass-balance models have been developed to understand natural variations in D 15 N at the level of the whole organism or its lean mass [START_REF] Del Rio | Mass-balance models for animal isotopic ecology[END_REF][START_REF] Ponsard | Should growing and adult animals fed on the same diet show different delta N-15 values?[END_REF][START_REF] Balter | Box-modeling of N-15/N-14 in mammals[END_REF], and further modeling studies are still needed to mechanistically understand inter-tissue and inter-fraction variations in nitrogen isotopic composition.

Materials and Methods

Ethics Statement

Experiments were carried out in accordance with the recommendations in the NIH Guide for the Care and Use of Laboratory Animals. The protocol was approved by the Ethics Committee for Animal Experiments (COMETHEA) of the Jouy-en-Josas INRA centre and AgroParisTech (Approval Number 11/015).

Animals and diets

Male Wistar Rats (n = 18), initially weighing 191 g, were purchased from Harlan (France) and housed in a temperatureregulated room (2262uC) on a 12-h light-dark cycle (dark period from 9:00 to 21:00). Before the beginning of the experiment, the rats were adapted to the laboratory conditions for one wk with free access to a commercial laboratory diet (d 15 N<4%). Animals were then randomly divided into two groups and assigned to receive one of the two experimental diets. Initial body weights were similar for the two groups.

The two diets were isoenergetic, identical in their nutrient composition (20% of energy as protein, 30% as lipids and 50% as carbohydrates) and met the requirements for growth. They differed in their protein source, either milk protein (MP, n = 9) or soy protein (SP, n = 9) (Table 3). Diets were prepared by the UPAE (Unite ´de Pre ´paration des Re ´gimes Expe ´rimentaux, French National Institut of Agronomic Research, INRA, Jouy en Josas, France).

Experimental protocol and sampling procedures

Following the 1-wk adaptation period, the rats were fed one of the two experimental diets for 3 wk. Rats were given free access to food from 9:00 to 19:00 (during the dark period) and had free access to water throughout the day. Body weight was measured twice weekly. At the end of week #3 (day 21 or 22), all the rats were killed after an overnight fast.

The rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (100 mg/kg of BW, a dose which is too small to significantly affect the measured isotopic signatures based on the mode of administration of this anesthetic and on distribution kinetics of barbiturates in tissues and plasma of rats [START_REF] Blakey | Quantitative structure-pharmacokinetics relationships: I. Development of a whole-body physiologically based model to characterize changes in pharmacokinetics across a homologous series of barbiturates in the rat[END_REF]). The abdomen was opened and approximately 5 ml of blood were quickly withdrawn from the vena cava. The animals were then killed by rupture of the vena cava and aorta. The liver, small intestine (SI), stomach, colon, kidneys, gastrocnemius and soleus muscles were rapidly dissected out, rinsed, weighed and snap frozen in liquid nitrogen. The SI was scraped to collect the mucosa, which was frozen separately. Hair and skin were sampled. All samples were stored at 220uC until analysis.

Samples preparation

In each tissue, the protein fraction (PF) and the non-protein fraction (nPF) were isolated for separate determination of nitrogen content and isotopic ratios in each fraction. Frozen tissues were pulverized with a mortar and pestle cooled in liquid nitrogen and precipitated by adding 700 mL of 5-sulfosalicylic acid (10%) per 100 mg of tissue. After centrifugation (2,500 g, 4uC, 15 min), the supernatant was extracted and the pellet was rinsed twice with 700 mL of 5-sulfosalicylic acid (10%). The soluble fraction (i.e., nPF containing the free amino acids) and the insoluble fraction (i.e., PF containing the protein-bound amino acids) were then freeze-dried.

In plasma, the different nitrogen fractions (PF, nPF and urea) were also separated. PF was isolated by precipitation with sulfosalicylic acid (200 mL, 1 g/mL). After 1-h storage at 4uC and centrifugation (2,000 g, 4uC, 20 min), the pellet was rinsed with 1 mL sulfosalicylic acid (1 g/mL), centrifugated again and freeze-dried. The extracted soluble fraction -containing nPF and urea-was neutralized and transferred onto 0.5 mL cation exchange resin (Dowex AG50X8, Biorad, Marnes-la-coquette, France) to bind the ammonium released from urea hydrolysis (8 mL urease for 2 h at 30uC). The ammonium-loaded resin was washed and kept at 4uC until analysis, while the urea-depleted supernatant, containing amino acids and peptides, was collected and filtered to eliminate peptides larger than 3 kDa (Amicon Ultra-4, Ultracel 3k, Millipore, Carrigtwohill, Ireland) and isolate nPF. Before isotopic determination, urea-derived-ammonia was eluted from the resins by the addition of KHSO 4 (2.5 mol/L).

Elemental analysis and isotopic determinations

The natural stable isotope ratios of nitrogen were determined in plasma urea and in PF and nPF of tissues and plasma using an isotope-ratio mass spectrometer (Isoprime, VG Instruments, Manchester, UK) coupled to an elemental analyzer (EA 3000, Eurovector, Italy). Standards were included in every run to correct for the possible variations in raw values measured by the mass spectrometer. Results were expressed using the delta notation according to the following equation:

d 15 N~½(R sample =R standard )-1|1000,
where R sample and R standard are the nitrogen isotope ratio of the heavier isotope to the lighter isotope ( 15 N/ 14 N) for the sample being analyzed and the internationally defined standard (atmospheric N 2 , R standard = 0.0036765), respectively, and d is the delta notation in parts per 1000 or per mill (%) relative to the standard.

The total nitrogen content of the tissues PF and nPF and of the plasma PF were determined using the elemental analyzer (EA 3000, Eurovector, Italy), with atropine as standard. Urea concentrations in plasma were determined with a commercial kit using an enzymatic method (Urea kit S-1000, Biomerieux, Craponne, France).

Calculations and statistics

Tissue samples were weighed before and after freeze-drying to estimate tissue dry matter. The nitrogen content of each sampled tissue fraction (PF or nPF) was calculated on the basis of the following equation:

N (mmol)~1000|(%N=100|%DM=100|m)=14,
where %N and %DM (%) are respectively the nitrogen and the dry matter percentages measured in the sample, and m (g) is the total mass of the tissue. Total muscle and skin masses were not measured, but were estimated as 45% [START_REF] Morens | A high-protein meal exceeds anabolic and catabolic capacities in rats adapted to a normal protein diet[END_REF] and 15% [START_REF] Lacroix | A longterm high-protein diet markedly reduces adipose tissue without major side effects in Wistar male rats[END_REF] of total body mass, respectively.

The nitrogen content of plasma PF and nPF was calculated as the product of the nitrogen concentration in the fraction and the plasma volume, estimated as 3.5% of body mass [START_REF] Waynforth | Experimental and surgical technique in the rat[END_REF]. The nitrogen concentration in the plasma nPF was estimated using aminoacidemia values obtained by our group in similar conditions (3.1 mmol/L i.e., 4.4 mmol/L of nitrogen equivalents) [START_REF] Morens | Daily delivery of dietary nitrogen to the periphery is stable in rats adapted to increased protein intake[END_REF]. The nitrogen content of the plasma urea pool (N urea , mmol) was assessed on the basis of the plasma urea concentration (C urea , mmol/L of urea nitrogen), its volume of distribution (i.e., total body water, estimated as 62.3% of the body mass, BM, Kg) [START_REF] Boutry | Rapeseed and milk protein exhibit a similar overall nutritional value but marked difference in postprandial regional nitrogen utilization in rats[END_REF], and a correction factor of 92% which represents the water content of the blood: N urea ~Curea |0,623|BM=0,92 [START_REF] Sharp | The laboratory rat The Laboratory Animal Pocket Reference Series[END_REF].

Isotopic nitrogen discrimination between a pool and the diet was described as the difference in d 15 N using the D notation, where D 15 N = d 15 N pool 2d 15 N diet (D 15 N.0 indicates a greater abundance in 15 N in the pool relative to the diet). As MP and SP diets had different natural isotopic ratios (d 15 N diet were 7.7% for MP and 1.7% for SP), D 15 N is used to compare the isotopic composition of tissues of animals fed different diets.

D 15 N values were calculated in the fast turning-over pools having likely reached, or almost reached, their isotopic equilibrium with the diet at the end of the 3-wk experimental period (plasma urea and each of the PF and nPF of visceral tissues and plasma). For each visceral tissue and plasma, an overall D 15 N value was calculated as the weighted average of the isotopic compositions of its different nitrogen fractions, as D 15 N~P m i |D 15 N i = P m i , where m i is the amount of nitrogen in the fraction i and D i is the isotopic discrimination between the fraction i and the diet. Likewise, a pooled visceral D 15 N was calculated as the weighted average of the isotopic compositions of the PF and nPF of liver, SI mucosa, stomach, kidneys and colon.

Results are expressed as means 6 SD. The effects of diet (MP versus SP) and tissue and their interaction were analyzed by a two-

Figure 1 .

 1 Figure 1. Nitrogen isotopic discrimination values (D 15 N) among the sampled tissues of rats. D 15 N of (A) tissues protein fractions (d 15 N protein fraction 2d 15 N diet ), (B) tissue non-protein fractions (d 15 N non-protein fraction 2d 15 N diet ) and (C) plasma urea (d 15 N plasma urea 2d 15 N diet ), of rats fed the MP (black bars) and the SP (grey bars) diets. Values are means 6 SD. *Effect of the dietary protein source in a given tissue (Post hoc tests with Tukey adjustments, P,0.001). All values are significantly different from 0 (Student t-tests, P,0.05) except for the non-protein fractions of SI mucosa and kidneys for the MP group. MP, Milk Protein; SP, Soy Protein; SI, Small Intestine. doi:10.1371/journal.pone.0028046.g001

Table 1 .

 1 Body weight, tissue masses and plasma variables in rats fed a milk protein-or a soy protein-based diet for 3 wk.

		Milk Protein diet Soy Protein diet
		(n = 9)	(n = 9)
	Initial body weight (g)	23467	23466
	Final body weight (g)	361617	356617
	Tissue mass (g/100 g BW)
	Stomach	0.4060.03	0.3760.02
	Small intestine mucosa	0.7160.14	0.7560.20
	Liver	2.9860.12	2.9960.26
	Kidneys	0.6560.02	0.6360.04
	Gastrocnemius muscle	0.3560.02	0.3660.02
	Soleus Muscle	0.0860.01	0.0860.01
	Plasma proteins (g/L)	52.463.5	55.363.8
	Plasma urea (mmol/L)	6.660.4	6.261.2

Values are means 6 SD. BW, Body Weight. There was no effect of the dietary protein source on tissue masses (Post hoc tests with Bonferroni adjustments, P,0.05). doi:10.1371/journal.pone.0028046.t001

Table 2 .

 2 Variations in nitrogen isotopic discrimination values among the different tissues and between nitrogen fractions within tissues for rats fed a milk protein-or a soy protein-based diet for 3 wk.

		Milk Protein diet		Soy Protein diet
		(n = 9)		(n = 9)
		D 15 N	D 15 N PF -D 15 N nPF	D 15 N	D 15 N PF -D 15 N nPF
		%
	Liver	3.0060.16 a	20.1861.80	4.1360.10 a *	0.3161.34
	Small intestine mucosa	2.0360.39 b	2.1260.50	2.2360.40 c	0.1360.55*
	Stomach	1.8660.75 b	1.3261.00	2.4160.35 c	10.9961.05*
	Kidneys	1.5960.22 bc	1.8860.33	2.0460.34 c *	7.1061.44*
	Colon	1.2360.27 c	1.9960.50	3.2960.19 b *	4.1060.67*
	Overall (visceral area)	2.5860.11	1.0560.

Table 3 .

 3 Composition of diets. :10.1371/journal.pone.0028046.t003way analysis of variance (Proc GLM, SAS 9.1, SAS Institute, Cary, NC, USA). Post hoc tests with Tukey or Bonferroni adjustments were used for multiple comparisons between tissues and dietary groups (MP versus SP). P values#0.05 were considered significant.

	Component	Milk Protein Diet Soy Protein Diet
		(n = 9)	(n = 9)
		g/kg DM
	Total milk protein	199.3
	Soy protein		199.3
	Cornstarch	428.2	433.7
	Sucrose	33.6	45.8
	Lactose	17.4
	Soybean oil	127.7	132.9
	Dairy fat	5.0
	Cellulose	52.8	53.6
	AIN-93M-MX mineral mixture	36.9	37.5
	AIN-93-VX vitamin mixture	10.6	10.7
	Choline	2.4	2.5
	DM, Dry Matter.

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PLoS ONE | www.plosone.org

November 2011 | Volume 6 | Issue 11 | e28046

Acknowledgments

This work was presented in part at the Experimental Biology Meeting in Anaheim, CA, U.S.A, April 24-28, 2010 (Abstract 740.6, published in FASEB J.). We thank the reviewers for their insightful remarks and suggestions, which helped to improve the quality of the manuscript.

Nathalie Poupin is supported by a doctoral fellowship from the French Ministry for Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have read the journal's policy and have the following conflicts: One of the authors (Daniel Tome ´) is a member of PLoS ONE academic editorial board.

Supporting Information

Table S1 Nitrogen composition of tissues and plasma of rats fed a milk protein-or a soy protein-based diet for 3 wk. (DOC)