Muriel M Thomas
email: muriel.thomas@jouy.inra.fr

Laura L Wrzosek

Leila Ben Yahia

M.-L Noordine

Christophe C Gitton

Didier D Chevret

Philippe P Langella

Camille Mayeur

Claire C Cherbuy

Francoise F Rul
email: franc¸oise.rul@jouy.inra.fr

Leila Ben-Yahia

Marie-Louise Noordine

Franc ¸oise Rul

Carbohydrate metabolism is essential for the colonization of streptococcus thermophilus in the digestive tract of gnotobiotic rats

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Streptoccocus thermophilus (S. thermophilus) is a dairy bacterium consumed by Humans for centuries. S. thermophilus is in the European Qualified Presumption of Safety list of food bacteria and is a generally recognized as a safe species (GRAS status); it has a long documented history of safe use in food and its genome is devoided of potential virulence functional genes [START_REF] Bolotin | Complete sequence and comparative genome analysis of the dairy bacterium Streptococcus thermophilus[END_REF][START_REF] Delorme | Safety assessment of dairy microorganisms: Streptococcus thermophilus[END_REF]. S. thermophilus is found in numerous cheeses and is one of the two bacteria in yogurt with its obligate partner Lactobacillus delbrueckii sp. bulgaricus (L. bulgaricus). Its high performance in fermenting milk and its resistance to elevated temperatures are two essential properties that accounted for a large utilization of strains of the species S. thermophilus in dairy industry. In addition, S. thermophilus is also recognized as a probiotic and, therefore, used to promote health [START_REF] Guarner | Should yoghurt cultures be considered probiotic?[END_REF][START_REF] Pagnini | Probiotics promote gut health through stimulation of epithelial innate immunity[END_REF][START_REF] Saavedra | Long-term consumption of infant formulas containing live probiotic bacteria: tolerance and safety[END_REF][START_REF] Veiga | Bifidobacterium animalis subsp. lactis fermented milk product reduces inflammation by altering a niche for colitogenic microbes[END_REF].

Recently, a general claim on yogurt on aiding lactose digestion has been accepted by the European Food Safety Authority. The latter considers that a cause and effect relationship is established between the consumption of live yogurt culture and improvement of lactose digestion in individuals with lactose mal-digestion (EFSA journal, 2010). In Humans, only a low proportion of population retains the capacity to degrade the lactose into adulthood, because the intestinal enzyme lactase-phlorizin hydrolase (more often called lactase or b-galactosidase) activity commonly starts decreasing after the first months or years of life [START_REF] Campbell | The molecular basis of lactose intolerance[END_REF][START_REF] Lomer | Review article: lactose intolerance in clinical practice-myths and realities[END_REF]. The benefits of yogurt are mainly linked to the metabolic capacities of L. bulgaricus and S. thermophilus that compensate the deficiency of the intestinal enzymes by their own b-galactosidase [START_REF] Drouault | Streptococcus thermophilus is able to produce a beta-galactosidase active during its transit in the digestive tract of germ-free mice[END_REF][START_REF] He | Effects of yogurt and bifidobacteria supplementation on the colonic microbiota in lactoseintolerant subjects[END_REF][START_REF] Pochart | Viable starter culture, betagalactosidase activity, and lactose in duodenum after yogurt ingestion in lactasedeficient humans[END_REF]. Our recent work brings concordant data about the preponderant role of metabolic adaptations of S. thermophilus in the gastrointestinal tract (GIT) of gnotobiotic rats. Previously, we have shown that S. thermophilus adapts its physiology to GIT by enhancing proteins devoted to carbohydrates metabolism [START_REF] Rul | Impact of the Metabolic Activity of Streptococcus thermophilus on the Colon Epithelium of Gnotobiotic Rats[END_REF]. It is likely that the overall glycolytic metabolic capacity of S. thermophilus is boosted in the GIT, thus explaining how the consumption of this viable microorganism may help the digestion of carbohydrates [START_REF] Drouault | Streptococcus thermophilus is able to produce a beta-galactosidase active during its transit in the digestive tract of germ-free mice[END_REF][START_REF] Rul | Impact of the Metabolic Activity of Streptococcus thermophilus on the Colon Epithelium of Gnotobiotic Rats[END_REF]. In yogurt at the end of fermentation process, there is a significant amount of lactose (4-5%) [START_REF] Lomer | Review article: lactose intolerance in clinical practice-myths and realities[END_REF][START_REF] Adolfsson | Yogurt and gut function[END_REF]. Thus, it could be hypothesized that residual lactose present in yogurt may be deleterious for lactose intolerant individuals; however, the inverse has been observed. The residual lactose in yogurt can be considered as a prebiotic for people with lactose mal-digestion [START_REF] Hertzler | How much lactose is low lactose?[END_REF][START_REF] Hertzler | Colonic adaptation to daily lactose feeding in lactose maldigesters reduces lactose intolerance[END_REF][START_REF] Szilagyi | Review article: lactose-a potential prebiotic[END_REF]. The main mechanisms involved are linked to the adaptation of microbiota activity/composition in the presence of lactose, as illustrating by an enhanced faecal b-galactosidase activity [START_REF] He | Effects of yogurt and bifidobacteria supplementation on the colonic microbiota in lactoseintolerant subjects[END_REF][START_REF] Hertzler | Colonic adaptation to daily lactose feeding in lactose maldigesters reduces lactose intolerance[END_REF]. Concordantly, in gnotobiotic rats, b-galactosidase activity of S. thermophilus is higher in the GIT after lactose supplementation [START_REF] Mater | Beta-galactosidase production by Streptococcus thermophilus is higher in the small intestine than in the caecum of human-microbiota-associated mice after lactose supplementation[END_REF]. Thus the lactose-related metabolism of viable bacteria present in a final dairy product, and their transit throughout the GIT, is central to better understanding the beneficial effects of fermented products on health [START_REF] Turpin | Lactobacilli as multifaceted probiotics with poorly disclosed molecular mechanisms[END_REF].

Although S. thermophilus can metabolize a number of different sugars (sucrose, glucose, fructose), lactose remains its favorite substrate. Lactose transport, metabolism, and regulation have been extensively studied in vitro by using different strains of S. thermophilus [START_REF] Van Den Bogaard | Control of lactose transport, beta-galactosidase activity, and glycolysis by CcpA in Streptococcus thermophilus: Evidence for carbon catabolite repression by a non-phosphoenolpyruvate-dependent phosphotransferase system sugar[END_REF][START_REF] Hols | New insights in the molecular biology and physiology of Streptococcus thermophilus revealed by comparative genomics[END_REF] and in regard to their genomic sequences [START_REF] Bolotin | Complete sequence and comparative genome analysis of the dairy bacterium Streptococcus thermophilus[END_REF][START_REF] Makarova | Comparative genomics of the lactic acid bacteria[END_REF]. In S. thermophilus, lactose is transported into the cell via the LacS permease which operates as a lactose/galactose antiporter or as a galactoside/H+ symporter [START_REF] Foucaud | Lactose transport system of Streptococcus thermophilus. Functional reconstitution of the protein and characterization of the kinetic mechanism of transport[END_REF]. LacS activity leads to the release of galactose into the extracellular medium after b-galactosidase has hydrolysed lactose into glucose and galactose. On the one hand, S. thermophilus is the archetype of lactose-adapted bacterium and on the other hand, we have observed that this specialized bacterium revealed a high adaptability of its carbohydrate metabolic pathways to the gut environment [START_REF] Rul | Impact of the Metabolic Activity of Streptococcus thermophilus on the Colon Epithelium of Gnotobiotic Rats[END_REF]. To this end, the objective of this work was to study the impact of lactose and lactose permease on S. thermophilus physiology in the GIT of gnotobiotic rats. We studied the sequenced strain LMD-9 for its colonization capacities, lactate production, proteomic profile and the related host responsiveness when animals received 4.5% of lactose that corresponded milk content of lactose. This work sheds new light on the lactoserelated metabolism of S. thermophilus in the GIT and the subsequent crosstalk with colonic epithelium. By comparing the wild-type S. thermophilus strain and a mutant strain inactivated in lactose permease LacS, we also studied the involvement of its transporter in the carbohydrate metabolism in vitro and in vivo.

Our results led us to propose a metabolic model of management of carbohydrates resources by S. thermophilus in vivo.

Results

Colonization and metabolic activity of S. thermophilus in the GIT and effects on colon epithelium Germ free rats were orally inoculated with 6610 8 CFU of S. thermophilus LMD-9 and received lactose (4.5% wt/vol) in the drinking water (Ino-LMD9 +lac ) (Fig. 1). S. thermophilus was present along the GIT with an increasing amount of bacteria from the ileum to the colon (Fig. 1A). The maximum level of colonization for S. thermophilus occurred within the first week after gavage and this was also observed with another strain of S. thermophilus, LMG18311 strain (Ino-LMG18311 +lac ) (Fig. 1B). This rapid and high level of colonization was in sharp contrast with the progressive and lower final colonization observed when S. thermophilus was inoculated in rats that did not receive lactose (Fig. 1C) and [START_REF] Rul | Impact of the Metabolic Activity of Streptococcus thermophilus on the Colon Epithelium of Gnotobiotic Rats[END_REF].

L-lactate -an end product of S. thermophilus metabolismaccumulated at a 49.965.7 mM concentration in Ino-LMD9 +lac caecal content, that represented 20 fold higher L-lactate than in GF +lac (2.561.5 mM). D-lactate was not detected in all groups of rats assessed. In parallel to this luminal L-lactate accumulation, a significant (p,0.05) acidification was noted with a luminal pH of 6.560.1 in Ino-LMD9 +lac when compared to 7.460.5 in GF +lac . In Ino-LMG18311 +lac , 55.864.2 mM L-lactate accumulated in caecal content. Thus, S. thermophilus -either LMD-9 or LMG18311 strain -when colonizing the GIT was metabolically active by producing a high amount of L-lactate.

The global structure of Ino-LMD9 +lac epithelium was analysed by HES staining (Fig. 2A). The mean depth of colon crypt in Ino-LMD9 +lac was 20669 mm that was not statistically different from values obtained with Ino-LMG18311 +lac (22164 mm), GF +lac (204617 mm) and GF [START_REF] Cherbuy | Microbiota matures colonic epithelium through a coordinated induction of cell cycle-related proteins in gnotobiotic rat[END_REF]. As illustrated, colon epithelium of Ino-LMD9 +lac was characterized by large and slack crypts and by the presence of crypts which seems to be ready to split (crypt fission). This global aspect of epithelium was reminiscent of our previous observations with various mono-associated and germ free rats [START_REF] Cherbuy | Microbiota matures colonic epithelium through a coordinated induction of cell cycle-related proteins in gnotobiotic rat[END_REF]. We have previously proposed that lactate production from the metabolic activity of S. thermophilus stimulates an increases in monocarboxylic acid transporters mRNAs (SLC16A1 and SLC5A8) and a lactate-sensitive protein involved in the cell cycle arrest (p27 kip1 ) in colon [START_REF] Rul | Impact of the Metabolic Activity of Streptococcus thermophilus on the Colon Epithelium of Gnotobiotic Rats[END_REF]. SLC16A1 and SLC5A8 mRNAs were respectively 1.8 (60.05) and 1.6 (60.3) induced in Ino-LMD9 +lac (n = 5) relative to GF +lac (n = 3). p27 kip1 protein was 1.8 (60.03) induced in Ino-LMD9 +lac in comparison with GF +lac (Fig. 2B), thus confirming that lactate may serve as a biological signal to communicate with host epithelium.

The sugar utilisation of S. thermophilus is preferentially induced in vivo

We compared the proteomic profiles of S. thermophilus, by bidimensional electrophoresis, after growth in milk (inoculum solution) and after 30 days residence in the GIT (faeces of Ino-LMD9 +lac ) (Figure S1). Sixty-three proteins displayed different abundances between the 2 conditions: 25 were up-regulated (induction from 2 to 12 fold) and 38 were down-regulated (repression from 2 to 10 fold) in faeces compared to milk (Table 1).

Down-regulated proteins were mainly involved in transcription, translation, nitrogen metabolism (AA biosynthesis, peptidases), and purine and pyrimidine metabolisms. Thus, the S. thermophilus population was maintained at a high level (10 9 CFU/g) in the GIT even with a low expression level of these pathways, whereas its growth in milk required a high expression and availability of these enzymes.

Sixteen out of twenty-five proteins up-regulated in Ino-LMD9 +lac faeces, compared to milk alone, belong to the carbohydrate metabolism (Figure S1 and Table 1). Of these 16 proteins, five were glycolytic enzymes (PfkA, Fba, GapA1, Eno, Pyk) while the others were ''upstream'' enzymes, (leading to the formation of compounds feeding the glycolytic pathway) and ''downstream'' enzymes (dissipating the ultimate product of glycolysis, pyruvate). Interestingly, enzymes potentially involved in metabolism of glycogen (GlgP, MalQ), sucrose (PtsK, ScrB), galactose (GalT, GalE), maltose (MalQ) or in polysaccharide synthesis (GalE) were significantly more abundant in the GIT than in milk (Figure S1). In comparison with a culture in milk, S. thermophilus +lac diversified its carbon source utilization by inducing the metabolism of alternative sugars in the GIT.

The presence of lactose strengthens the sugar utilisation in vivo

To study the effect of lactose on metabolic pathway of S. thermophilus, we compared two sets of data: i) Primarily listing the proteins differentially expressed in the Ino-LMD (without lactose) digestive tract versus milk and ii) a second listing of proteins differentially expressed in the Ino-LMD9 +lac digestive tract versus milk (Fig. 3). As soon as S. thermophilus was growing in the digestive tract, a large amount of proteins were down-regulated (38 and 41, in presence or absence of lactose, respectively). Thirty four of these Figure 1. Colonization of S. thermophilus in mono-associated rats. One ml of S. thermophilus LMD-9 strain (6610 8 CFU) was inoculated in germ free rats thus leading to Ino-LMD9 +lac rats, which received water with lactose (4.5% wt/vol). Counts of S. thermophilus were obtained by plating on M17 agar lactose the luminal content from ileum, caecum, colon, faeces of Ino-LMD9 +lac (n = 11) rats 30 days after the inoculation. B) Log 10 CFU/g of S. thermophilus LMD-9 and LMG18311 counted from faecal samples all along the experiment for each mono-associated rat. The colonization curves obtained with LMG18311 and LMD-9 were not significantly different (p = 0.4). C) Comparison of colonization profile of LMD-9 when rats received lactose (Ino-LMD9 +lac ) or not [Ino-LMD9, data from our previous work [START_REF] Rul | Impact of the Metabolic Activity of Streptococcus thermophilus on the Colon Epithelium of Gnotobiotic Rats[END_REF]]. Each column symbolized one rat and X, %, e, n represented means of S. thermophilus LMD-9 counts obtained from faecal samples recovered, respectively, during the first, second, third and fourth week. doi:10.1371/journal.pone.0028789.g001 intestine-repressed proteins that were both repressed in presence or absence of lactose (Fig. 3) were involved in staple functions as transcription and translation. These results confirmed that a low level of these proteins was sufficient to allow S. thermophilus to grow in the GIT and establish a metabolically active population.

Of the intestine-boosted proteins, 7 were involved in glycolysis and over-expressed either in the presence or in the absence of lactose (Fig. 3). General-(DnaK, GroEL) or oxidative-stress proteins (SodA) as well as the elongation factor Tuf -also potentially involved in adhesion/colonization-were more abundant in the GIT than in milk suggesting that they are related to environmental signal responses [START_REF] Pochart | Viable starter culture, betagalactosidase activity, and lactose in duodenum after yogurt ingestion in lactasedeficient humans[END_REF].

In the presence of lactose, 9 supplementary proteins involved in carbohydrate metabolism were over-expressed in the digestive tract (Fig. 3). The presence of lactose allowed S. thermophilus to enhance and diversify the metabolism of diverse carbohydrate sources, potentially explaining how the population of S. thermophilus overwhelms 10 fold the implanted population in absence of lactose (Fig. 1C). Based on the colonization curve, the proteomic signature and the resultant lactate end-product, the mobilization of the carbohydrate metabolism is determinant and limiting step for the colonization, the growth and the activity of S. thermophilus in vivo (Fig. 4).

Effect of lactose permease LacS inactivation on the S. thermophilus metabolism

In S. thermophilus, lactose is transported into the cell via the LacS permease which operates mainly as a lactose/galactose antiporter. Considering the central role of S. thermophilus carbohydrates metabolism in vivo, a negative mutant for the lacS gene was generated by inserting a kanamycine resistance cassette in the lacS gene. The resulting DlacS strain was validated in vitro, since it did not grow in the presence of lactose in contrast to the wild-type LMD-9 strain (Fig. 5A). In the presence of glucose, sucrose or fructose, DlacS and the wild-type LMD-9 strains displayed similar growth rates. In vitro, the inactivation of LacS permease impaired the growth in presence of lactose, while preserving the capacity of utilising other carbon sources.

The DlacS strain was then challenged in vivo for its capacity to colonize the GIT in germ-free rats receiving 4.5% lactose (Ino-DlacS +lac rats). The final level of colonization was identical with DlacS and LMD-9 strains, with 1.5 10 9 CFU/g (Fig. 5B). Concordantly, the final amount of faecal lactate was identical in both lots of rats (3060.5 mM in Ino-DlacS +lac versus 34.167 mM in Ino-LMD9 +lac ). However, the kinetic of colonisation was altered with DlacS strain in the first days after inoculation as the maximal colonization DlacS being delayed in comparison with the wild-type strain. In concordance with this delayed colonisation, the L-lactate was undetectable in the first days after inoculation in Ino-DlacS +lac faeces, whereas it was already high (41612 mM) in faeces of Ino-LMD9 +lac . These results indicated that LacS permease inactivation delayed the colonisation of S. thermophilus and suggested that an adaptation process occurred during the first 15 days after inoculation.

Discussion

Our study demonstrates that the presence of lactose enhanced the fermentative activity of S. thermophilus leading to higher level of luminal lactate (63.5 caecal lactate in Ino-LMD9 +lac compared to Ino-LMD9 [START_REF] Rul | Impact of the Metabolic Activity of Streptococcus thermophilus on the Colon Epithelium of Gnotobiotic Rats[END_REF]) which subsequent acts to modulate the host epithelium. Therefore, activation of enzymes involved in carbohydrate metabolism constitutes the metabolic signature of S. thermophilus in the GIT and allows the dialogue with colon epithelium. Lactose boosted the S. thermophilus carbohydrates metabolism that is already high in the digestive tract. S. thermophilus, probably because of its intensive use in dairy industry, Table 1. Functional distribution of S. thermophilus LMD-9 proteins whose abundance changed between the milk inoculum used for gavage and the faeces of Ino-LMD9 +lac rats (2-DE analysis).

Functional category (number of proteins)

Range of modulation has evolved by a specialization to lactose degradation with a genome that is considered as ''poor'', having lost many genes. The sugar metabolism appears here to have key functions in adaptation to the GIT environment since life cycle of S. thermophilus in the GIT relies on carbon metabolism. Recently, it has been shown that the efficiency of carbohydrates consumption by bifidobacteria correlated with their ability to protect host against infection [START_REF] Fukuda | Bifidobacteria can protect from enteropathogenic infection through production of acetate[END_REF]. From indigenous gut microbiota to sub-abundant transient species, the carbohydrate metabolism is central for bacterial colonization, activity and subsequent dialogue with host [START_REF] Chang | Carbon nutrition of Escherichia coli in the mouse intestine[END_REF][START_REF] Denou | Identification of genes associated with the long-gut-persistence phenotype of the probiotic Lactobacillus johnsonii strain NCC533 using a combination of genomics and transcriptome analysis[END_REF][START_REF] Marco | Convergence in probiotic Lactobacillus gut-adaptive responses in humans and mice[END_REF][START_REF] Marco | Lifestyle of Lactobacillus plantarum in the mouse caecum[END_REF][START_REF] Roy | Proteomic investigation of the adaptation of Lactococcus lactis to the mouse digestive tract[END_REF]. Although S. thermophilus is rather specialized in the degradation of simple sugars and belongs to numerically minor group of microbiota [START_REF] Qin | A human gut microbial gene catalogue established by metagenomic sequencing[END_REF], its carbohydrate metabolism is also prevalent in regulating its own adaptation in GIT and its dialogue with host.

The present work confirmed our previous data stating that lactate produced in Ino-LMD9 by S. thermophilus induced both mRNAs SLC16A1/SLC5A8 monocarboxylic transporters and a cell-cycle arrest protein p27 kip1 [START_REF] Rul | Impact of the Metabolic Activity of Streptococcus thermophilus on the Colon Epithelium of Gnotobiotic Rats[END_REF]. The levels of induction between Ino-LMD9 +lac and GF +lac were not statistically different to that obtained between Ino-LMD9 and GF [START_REF] Rul | Impact of the Metabolic Activity of Streptococcus thermophilus on the Colon Epithelium of Gnotobiotic Rats[END_REF], indicating that the 3.7 fold highest content of lactate in Ino-LMD9 +lac (compared to Ino-LMD9) did not correlate with higher stimulation of epithelial transporters and p27 kip1 . All these results highlight the homeostatic responsiveness of the epithelium, as we have previously observed after intestinal resection [START_REF] Joly | Morphological adaptation with preserved proliferation/transporter content in the colon of patients with short bowel syndrome[END_REF]. In monoassociated rats, it is likely that a small portion of lactate was shuttled inside colonic cells, while the remaining luminal lactate was excreted in faeces (49.965.7 mM caecum versus 34.167 mM in faeces). In such models, the major proportion of lactate was excreted, because of the absence of other bacteria that normally inhabit the gut and metabolize lactate. In healthy human adults, lactate produced by gut microbiota is nearly undetectable in faecal samples since it is absorbed by host and also consumed by lactateutilizing bacteria to produce mainly butyrate [START_REF] Duncan | Reduced dietary intake of carbohydrates by obese subjects results in decreased concentrations of butyrate and butyrate-producing bacteria in feces[END_REF][START_REF] Hove | Colonic lactate metabolism and D-lactic acidosis[END_REF][START_REF] Hove | Lactate and pH in faeces from patients with colonic adenomas or cancer[END_REF][START_REF] Munoz-Tamayo | Kinetic modelling of lactate utilization and butyrate production by key human colonic bacterial species[END_REF]. Therefore, one can suppose that, in a context of a complex microbiota, S. thermophilus could contribute to microbial ecosystem by favoring lactate-utilizing enzymes or inhibiting low-pH sensitive bacteria [START_REF] Veiga | Bifidobacterium animalis subsp. lactis fermented milk product reduces inflammation by altering a niche for colitogenic microbes[END_REF].

Our study presents data on S. thermophilus physiology and demonstrates the flexibility of this bacterium to adapt to digestive environmental constraints and its capacity to diversify the use of alternative sugars. In absence of lactose, S. thermophilus develops a capacity to use alternative sugars in vivo ant this suggests that similar mechanisms occurred in the absence of a functional LacS permease. The presence of lactose enhanced LMD-9 colonization in the GIT, probably by favoring the use of diverse sugars in the GIT via the induction of metabolisms of galactose, sucrose, Cytoplasmic extracts of S. thermophilus LMD-9 after passage through the GIT (ino-LMD9 +lac rats) were compared with those obtained in milk culture (inoculum for gavage). Data obtained in absence of lactose (Ino-LMD9) were previously described [START_REF] Rul | Impact of the Metabolic Activity of Streptococcus thermophilus on the Colon Epithelium of Gnotobiotic Rats[END_REF]. doi:10.1371/journal.pone.0028789.g003 maltose, and glycogen. Each metabolic pathway feeds glycolysis, suggesting that S. thermophilus metabolism converges to ATP production in the GIT. This also indicates that most of the sugar routes predicted by genome analysis, and so far not yet studied, are probably functional in S. thermophilus LMD-9. DlacS strain needed about 15 days to stably and maximally implant and thus possibly to set up a metabolic response by using alternative sugars and/or by favoring the entrance of lactose by other unknown transporters. By studying the physiology of S. thermophilus in the digestive tract, we also propose that the protein MalQ may be involved in glycogen synthesis rather than in maltose metabolism. In absence of lactose, MalQ was induced in gut and we hypothesized that S. thermophilus stocked glycogen in order to face limiting environmental conditions [START_REF] Wilson | Regulation of glycogen metabolism in yeast and bacteria[END_REF]. In presence of lactose, both MalQ and Figure 4. Model of carbohydrate metabolism of S. thermophilus in the digestive tract. Carbohydrate metabolism in S. thermophilus LMD-9 after its passage through the GIT, A) in presence (Ino-LMD9 +lac ) or B) in absence of lactose (Ino-LMD9). Up-regulated enzymes in GIT compared to milk are grey shaded. We constructed this model from the present proteomic data and from predictions of metabolism pathways of LMD-9 strain genome. doi:10.1371/journal.pone.0028789.g004 GlgP [responsible for glycogen breakdown in bacteria [START_REF] Alonso-Casajus | Glycogen phosphorylase, the product of the glgP Gene, catalyzes glycogen breakdown by removing glucose units from the nonreducing ends in Escherichia coli[END_REF]] were induced, suggesting that equilibrium between anabolism and catabolism occurs in S. thermophilus. A similar simultaneous glycogen synthesis and degradation have been described in Corynebacterium glutamicum where glycogen is constantly recycled [START_REF] Seibold | The glgX gene product of Corynebacterium glutamicum is required for glycogen degradation and for fast adaptation to hyperosmotic stress[END_REF]. All our observations show that MalQ is not involved in maltose metabolism, in concordant with the fact that no MalE transporter is present in the genome of LMD-9 and that genes malQ and glgP (encoding potentially for a glycogen phosphorylase) are an operon in LMD-9. Glycogen could be of prime importance in the colonization, adaptation and survival of S. thermophilus within the GIT, as previously observed for other microorganisms, when present in starvation conditions [START_REF] Wilson | Regulation of glycogen metabolism in yeast and bacteria[END_REF] or during transition between nutrient-rich-and nutrient-poor environments [START_REF] Bourassa | Glycogen contributes to the environmental persistence and transmission of Vibrio cholerae[END_REF].

Our present work sheds new light on the established association of S. thermophilus and lactose by revealing that lactose enhanced S. thermophilus kinetics, level of colonization and fermentative activity in the GIT. Our work suggests that a food product containing both a live S. thermophilus and lactose would favor the colonization and fermentative activity of S. thermophilus in vivo. In this context, our results follow the rationale of previous clinical observations and the nutritional assertion that the consumption of yogurt (containing lactose) could alleviate the symptoms of lactose intolerance.

Materials and Methods

Bacterial strains, media, and inoculating samples

The strains S. thermophilus LMD-9 (ATCC BAA-491, USA), and LMG18311 (BCCM collection, Belgium) were used. Stock cultures of S. thermophilus LMD-9 and LMG18311 were prepared in reconstituted 10% (wt/vol) Nilac skim milk (NIZO, Ede, the Netherlands) as previously described [START_REF] Herve-Jimenez | Physiology of Streptococcus thermophilus during the late stage of milk fermentation with special regard to sulfur amino-acid metabolism[END_REF]. S. thermophilus monocultures were obtained by inoculating Nilac milk with 10 6 CFU/ ml of stock cultures and incubated at 42uC until pH 5.4-5.5. One ml of culture was used for rat gavage and the remaining aliquots were frozen in liquid nitrogen and stored at 220uC until protein extraction. The cultures were enumerated a posteriori by plating appropriate dilutions on M17 agar lactose (10 g/L) for S. thermophilus. After 16 h (S. thermophilus) incubation at 42uC under anaerobiosis (Anaerocult A, Merck, Darmstadt, Germany), colonies were counted.

m max determination

Cultures (n = 3) of S. thermophilus strains were performed in M17 supplemented with 10 g/L lactose, glucose, sucrose or fructose; the apparent growth rate (m max ) was defined as the maximum slope of semi-logarithmic representation of growth curves assessed by O. D. 650 nm measurements.

Insertional inactivation of S. thermophilus LMD-9 lacS gene

The kanamycin cassette of the plasmid pKa 2000 was PCRamplified using the Phusion high fidelity DNA polymerase with AphA3-F ( 59 CCAGCGAACCATTTGA 39 ) and AphA3-R ( 59 GTTGCGGATGTACTTCAG 39 ) primers. The 1489 bp DNA fragments flanking the lacS gene were PCR-amplified using the Phusion DNA polymerase, the LMD-9 DNA as a template, and primers LacS-up ( 59 TATGTGCCTGCCAGTCCA 39 )/Kana-up-R ( 59 AGGGGTCCCGAGCGCCTACGAGGAATTTGTATC-GATGACCTTTAGATTTTTCCAT 39 ) for the upstream fragment and primers LacS-down ( 59 AACTGGAACGACTTCA-AC 39 )/Kana-down ( 59 CTTACCTATCACCTCAAATGGTTC-GCTGGGTTTATCGTAACTAATTCAGAAAAA 39 ) for the downstream fragment. The 39 end of the upstream generated fragment contained a sequence complementary to the 59 end of the kana cassette whereas the 59 end of the downstream generated fragment contained a sequence complementary to the 39 end of the cassette. This allowed joining of these three fragments subsequent to Taq Phusion PCR using primers LacS-up and LacS-down. After purification with a QIAquick PCR purification kit, 500 ng of the resulting 3.4 kb fragment was further used to transform LMD-9 natural competent cells as described by Gardan et al. [START_REF] Gardan | The oligopeptide transport system is essential for the development of natural competence in Streptococcus thermophilus strain LMD-9[END_REF]. Transformants were selected on M17Glu plates with kanamyin and were then checked by PCR using oligonucleotides LacS-up and KanaR. Finally sequencing of the flanking regions was performed to ensure that no unwanted mutations were introduced.

Animals and experimental design

All procedures were carried out in accordance with European and French guidelines for the care and use of laboratory animals. Permission 78-123 is a permit number dedicated to M. Thomas. MICALIS (Microbiologie de l'Alimentation au Service de la Sante ´) review board specifically approved this study. At the age of 2 months, germ-free (GF) rats (male, Fisher 344) were inoculated either with S. thermophilus LMD-9 (Ino-LMD9 +lac , n = 11) or S. thermophilus LMG18311 (Ino-LMG18311 +lac , n = 8). 1 ml of a culture of S. thermophilus in Nilac milk (5610 8 CFU/ml) was transferred to GF rats by oral gavage. As a control, GF +lac rats were also inoculated with 1 ml of sterile Nilac milk (without bacteria). Following gavage, rats received water enriched with lactose (4.5%wt/vol). GF and mono-associated rats were housed in sterile Plexiglas isolators (Inge ´nia, Vitry-sur-Seine, France). All groups of rats received the same standard diet (UAR), which was sterilized by gamma irradiation. Twice a week S. thermophilus was enumerated by plating serial dilutions of the faeces on M17 lactose agar. All rats were euthanized at three months old, 30 days after gavage.

S. thermophilus DlacS strain was inoculated in GF rats to obtain Ino-DlacS +lac rats. The DlacS inoculum was grown in M17+glucose and bacteria were enumerated by plating serial dilutions of the faeces on M17 glucose agar supplemented with kanamycine (1 mg/ml). Ino-DlacS +lac (n = 5) rats drank 4.5% lactose-enriched water. The presence of kanamycin cassette disrupting the lacS gene was checked by PCR in faeces of Ino-DlacS +lac . Ino-DlacS +lac rats were euthanized 18 days after gavage.

Rats were anesthetized with isoflurane and tissues were recovered. The colon was immediately used, for colonic epithelial cell isolation or for histological procedures as it has been described in Cherbuy et al. [START_REF] Cherbuy | Expression of mitochondrial HMGCoA synthase and glutaminase in the colonic mucosa is modulated by bacterial species[END_REF][START_REF] Cherbuy | Effect of germfree state on the capacities of isolated rat colonocytes to metabolize nbutyrate, glucose, and glutamine[END_REF].

Western blot analysis

Colonic proteins from isolated epithelial cells were used for Western blot analysis as previously described [START_REF] Cherbuy | Microbiota matures colonic epithelium through a coordinated induction of cell cycle-related proteins in gnotobiotic rat[END_REF] by using a denaturing (SDS)-polyacrylamide gel. Proteins were analysed using anti-p27 kip1 (Santa Cruz Biotechnology; 1/500). GAPDH was used as loading control. Signals imprinted on autoradiography films were quantified by scanning densitometry of the autoradiograph using Biovision 1000 and logiciel bio1D (Vilber Lourmat, France).

Dosage of D-and L-lactates

D-and L-lactates were measured in caecal contents and faeces with the Biosentec D/L lactic acid enzymatic kits according to the manufacturer instructions (Biosentec, Toulouse, France) as also described in Rul et al. [START_REF] Rul | Impact of the Metabolic Activity of Streptococcus thermophilus on the Colon Epithelium of Gnotobiotic Rats[END_REF].

Histology analysis

Colon samples were cut into 2 cm sections, fixed in 4% paraformaldehyde (4 hours, room temperature), dehydrated and embedded in paraffin according to standard histological protocols. Four micrometer sections were mounted on SuperFrostH Plus slides. Slides were stained with Hematoxylin-Eosin-Safran (HES) for histological analysis. Crypt depths were determined with NDP.view software (Hamamatsu). Only U shaped longitudinally cut crypts with open lumina along the crypt axis were analysed. Results were the mean obtained by analysis at least 20 crypts per rat (Ino-LMD9 +lac , n = 5; Ino-LMG18311 +lac , n = 3).

RNA isolation and quantitative RT-PCR analysis

Total RNAs were extracted from colon epithelial cells of GF +lac (n = 3), and Ino-LMD9 +lac (n = 5) by the guanidinium thiocyanate method. The slc16a1 and slc5a8 mRNA quantification was performed as we have previously described [START_REF] Rul | Impact of the Metabolic Activity of Streptococcus thermophilus on the Colon Epithelium of Gnotobiotic Rats[END_REF].

Bacterial protein extraction, comparative bi-dimensional (2-DE) protein analysis and image analysis

Cytoplasmic proteins were extracted from bacteria as previously described [START_REF] Rul | Impact of the Metabolic Activity of Streptococcus thermophilus on the Colon Epithelium of Gnotobiotic Rats[END_REF] either from milk cultures (n = 3) or faecal samples (5 g of frozen faeces from 3 Ino-LMD9 +lac rats). A volume of cytosolic fraction corresponding to 250 mg of proteins was treated as previously described [START_REF] Derzelle | Proteome analysis of Streptococcus thermophilus grown in milk reveals pyruvate formate-lyase as the major upregulated protein[END_REF]. Bi-dimensional (2-DE) protein analysis and image analysis were performed according to Rul et al. [START_REF] Rul | Impact of the Metabolic Activity of Streptococcus thermophilus on the Colon Epithelium of Gnotobiotic Rats[END_REF]. MS analyses were performed using a Voyager-DE-STR (Applied Biosystems, Framingham, USA) on the PAPPSO proteomic platform (http://pappso.inra.fr). The proteins were identified using MS-FIT (http://prospector.ucsf.edu).

Statistical analysis

Results are presented as means 6 SE for the number of animals indicated. Comparisons of group data between different batches of rats were performed using one-way analysis of variance (ANOVA) followed by Tukey's student range test where appropriate. Significance was for P value lower than 0.05. Statistical analysis was performed using the JMPH software (version 7, SAS institute INC).

Figure 2 .

 2 Figure 2. Colon epithelium of Ino-LMD9 +lac . A) Representative photomicrograph represents a slide of a colon crypt from Ino-LMD9 +lac and GF +lac stained with HES. The arrow represented crypt fission. B) Representative Western blot detected p27 kip1 and a loading control (GAPDH). The total proteins of colon epithelium were from Ino-LMD9 +lac (n = 5) and GF +lac (n = 2). doi:10.1371/journal.pone.0028789.g002

  regulated proteins in the digestive tract -Transcription/translation: 17 -Diverse: 9 -Nitrogen metabolism: 6 -Nucleotide metabolism: 5 -Carbohydrate metabolism: .1371/journal.pone.0028789.t001

Figure 3 .

 3 Figure 3. Comparative proteomic analysis (2-DE) of S. thermophilus LMD-9.Cytoplasmic extracts of S. thermophilus LMD-9 after passage through the GIT (ino-LMD9 +lac rats) were compared with those obtained in milk culture (inoculum for gavage). Data obtained in absence of lactose (Ino-LMD9) were previously described[START_REF] Rul | Impact of the Metabolic Activity of Streptococcus thermophilus on the Colon Epithelium of Gnotobiotic Rats[END_REF]. doi:10.1371/journal.pone.0028789.g003

Figure 5 .

 5 Figure 5. S. thermophilus DlacS characterization. A) in vitro: growth of the wild-type (&) and the DlacS (%) strains in M17 in presence of different sugars. B) in vivo: enumeration of viable wild-type (m) and DlacS (N) strains isolated from faeces of Ino-LMD9 +lac (n = 5) and Ino-DlacS +lac (n = 5), respectively. doi:10.1371/journal.pone.0028789.g005

PLoS ONE | www.plosone.org 1 December 2011 | Volume 6 | Issue 12 | e28789

PLoS ONE | www.plosone.org

December 2011 | Volume 6 | Issue 12 | e28789

December 2011 | Volume 6 | Issue | e28789

Acknowledgments

We thank Sylvie Miquel and Neil Dufton for fruitful discussions and critical reading of the manuscript. The authors would like to thank the team of animal facilities (ANAXEM team, Micalis) and Stephan Bouet for technical assistance.

This work was financially supported by Syndifrais-CNIEL (Syndicat National des Fabricants de Produits Frais/Centre National Interprofessionnel de l'Economie Laitie `re) (Paris, France) (http://www.cniel.com/quifait/Syndicat/SYNDIFRAI/syndifr.html). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding has been received for this study.

Supporting Information


Elisabeth E Chevreau
email: elisabeth.chevreau@angers.inra.fr

Fabrice Dupuis

Jean-Paul P Taglioni

Sophie Sourice

Raphael R Cournol

C Deswartes

A Bersegeay

Julie Descombin

Myriam Siegwart

Karine K Loridon

Effect of ectopic expression of the eutypine detoxifying gene Vr-ERE in transgenic apple plants

Keywords: aldehyde reductase, apple, eutypine, genetic engineering, Malus x domestica 6-Benzyladenine BD: benzaldehyde CaMV :Cauliflower mosaic virus Conyl: coniferylaldehyde Decyl: decylaldehyde 3FBD: 3-fluoro-benzaldehyde GUS :ß-Glucuronidase IBA :Indole-3-butyric acid 2HBD: 2-hydroxy-benzaldehyde 3HBD: 3hydroxy-benzaldehyde 4HBD: 4-hydroxy-benzaldehyde 3MBD: 3-metoxy-benzaldehyde

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Current apple transformation methods are based on the use of selectable marker genes that confer resistance to antibiotics as a selection pressure [START_REF] Brown | Malus x domestica Apple[END_REF].

However, public concern about food safety of genetically engineered plants and potential horizontal flow of antibiotic resistance genes requires new alternative strategies. A few reports have indicated the feasibility of alternative selection pressure for apple transformation.

The bialaphos resistance gene (bar) enabled the production of transgenic apple using 1 mg/l bialaphos as a selection pressure in Malus prunifolia Asami [START_REF] Ogasawara | Introduction of a herbicide resistant gene into an apple rootstock with Agrobacterium tumefaciens[END_REF]

and in

Elstar and Holsteiner Cox [START_REF] Szankowski | Transformation of apple (Malus domestica Borkh.) with the stilbene synthase gene from grapevine (Vitis vinifera L.) and a PGIP gene from kiwi (Actinidia deliciosa)[END_REF]. Even though several bar-containing crop plants have received approval for sale, the use of an herbicide-resistance marker does not answer all the public concerns about environmental issues. Two teams have reported the transformation of apple using the phosphomannose isomerase gene as a selectable marker and mannose (1 to 2.5 g/l) as a selection pressure [START_REF] Zhu | The use of mannose as a selection agent in transformation of the apple rootstock M26 via Agrobacterium tumefaciens[END_REF][START_REF] Degenhardt | The use of phosphomannoseisomerase/mannose selection system to recover transgenic apple plants[END_REF].

However, all the above-cited methods rely on the use of non-plant genes. A major trend in current biotechnological projects is the development of the cisgenic or intragenic approaches.

These technologies aim at transforming plants with native expression cassettes only, to finetune the activity and/or specificity of target genes, without any introduction of non-plant DNA [START_REF] Schouten | Cisgenesis and intragenesis, sisters in innovative plant breeding[END_REF]. In line with this objective, we decided to search for an alternative selection system for apple, based on a plant gene.

Eutypine is a toxin produced by Eutypa lata, the causal agent of eutypa dieback of grapevine [START_REF] Amborabé | Effects of eutypine, a toxin from Eutypa lata, on plant cell plasma membrane: possible subsequent implications in disease development[END_REF]. Eutypine is detoxified in many plant species, among which tissues of mung bean (Vigna radiata) exhibited the highest detoxification activity.

E

Version définitive du manuscrit publié dans / Final version of the manuscript published in : Plant Cell, Tissue and Organ Culture, 2011, vol.106, no.1, 161-168. DOI: 10.1007 Purification of the eutypine reductase enzyme from mung bean led to the identification of an eutypine detoxifying gene (Vr-ERE) encoding an NADPH-dependent aldehyde reductase, which converts eutypine into the corresponding alcohol, eutypinol, a non-toxic form of the toxin [START_REF] Guillen | 'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript 16 reductase gene from Vigna radiata confers resistance to the grapevine fungal toxin eutypine[END_REF]. The Vr-ERE protein is a monomeric NADPH-dependent oxidoreductase with a molecular mass of 36 kDa that exhibits broad substrate specificity for various natural or synthetic aldehydes [START_REF] Colrat | Purification and characterization of a NADPH-dependent aldehyde reductase from mung bean that detoxifies eutypine, a toxin from Eutypa lata[END_REF]. The precise physiological function of this enzyme in mung bean, a non-host plant of Eutypa lata, is still unclear.

Transgenic rootstock grapevine 110 Richter Vitis berlandieri x V. rupestris was successfully transformed with the Vr-ERE gene under the control of the 35S promoter, using kanamycin as selective agent. Three clones constitutively expressing Vr-ERE demonstrated enhanced resistance to eutypine at the in vitro stage [START_REF] Legrand | Constitutive expression of Vr-ERE gene in transformed grapevines confers resistance to eutypine, a toxin from Eutypa lata[END_REF].

The fact that a variety of phytotoxic compounds containing an aldehyde group can act as substrates for the Vr-ERE enzyme opens the possibility to select one of them in order to use Vr-ERE as an alternative selection system for plant transformation. As a first step of our project, we report here the effects of overexpressing the Vr-ERE in transgenic apple plants.

MATERIAL AND METHODS

Plant and bacterial material

The apple variety used in this study, Greensleeves, was selected from a cross between James Grieve and Golden Delicious; this variety is known as very easy to transform since the pioneer work of [START_REF] James | Genetic transformation of apple (Malus pumila Mill.) using disarmed Ti-binary vector[END_REF].

The bacterial strain used in the transformation experiments was Agrobacterium tumefaciens strain EHA105 containing the ternary plasmid pBBR1MCS-5 with a constitutive [START_REF] Van Der Fits | The ternary transformation system : constitutive virG on a compatible plasmid dramatically increases Agrobacteriummediated plant transformation[END_REF]. The Vr-ERE gene from the pGA-Vr-ERE binary plasmid (kindly provided by Dr. Roustan, INRA Toulouse) was placed under the control of a duplicated cauliflower mosaic virus (CaMV) 35S promoter [START_REF] Kay | Duplication of CaMV 35S promoter sequences creates a strong enhancer for plant genes[END_REF] and cloned in a pCAMBIA2301 plasmid, to generate the binary vector pCambiaVr-ERE-GUS (Figure 1-A), which was used for all the transformation experiments.

In vitro culture, regeneration and transformation

Proliferating shoot cultures were established on Murashige and Skoog (MS) (1962) medium supplemented with 0.5 mg/l 6-benzyladenine (BA) and 0.1 mg/l 3-indolebutyric acid (IBA). The youngest leaves of 4-week-old shoots were placed with the adaxial side down on a regeneration medium consisting of MS medium containing 5 mg/l thidiazuron (TDZ), 0.5 mg/l IBA, solidified with 0.3% gelrite, and supplemented with varying concentrations of aldehyde reductase substrates. Explants were incubated in the dark and, after one month, subcultured on a similar medium, still in the dark. Callus formation, extent of necrosis, rate of regeneration and number of buds per explant were assessed after two months.

Transformation experiments were conducted as previously described on apple [START_REF] Norelli | Leaf wounding increases efficiency of Agrobacterium-mediated transformation of apple[END_REF]. In brief, the youngest leaves of 4-week-old in vitro shoots were crushed with nontraumatic forceps, and then immersed in the inoculum prepared at a concentration of 10 9 bacteria/ml for five minutes. Leaves were then blotted and plated on cocultivation medium for three days in the dark. Leaves were then wounded transversely with a scalpel and plated adaxial side down on regeneration medium containing cefotaxime at 200 mg/l, timentin at 100 mg/l and the appropriate selection agent (kanamycin or aldehydes). The explants were transferred to fresh medium every month for six months and kept in the dark.

Selected transgenic clones were rooted by auxin treatment (IBA 3 mg/l for 7 days) followed by transfer to hormone-free medium. Acclimatization was performed in greenhouse,

Ploidy level assessment

The ploidy level of transgenic clones and controls was estimated by flow cytometry.

Nuclei were isolated from in vitro leaves by manual chopping with a razor blade into the buffer [START_REF] Brown | Flow cytometry of nuclei for ploidy and cell cycle analysis[END_REF] with 2% (v/v) 4',6 diamidino-2-phenylindole (Cheminex) followed by filtration through 20 µm nylon mesh and analyzed by the cytometer (Cell analyzer II; Partec, Münster, Germany). Pea leaf nuclei were used as internal reference.

Molecular analyses of transgenic clones

The presence of the Vr-ERE gene was confirmed by PCR using specific oligonucleotides (F1 : forward, 5'-TTCTTCTCGAACGCGGCTAC-3' and R1 : reverse, 5'-GTCTGCGGATCTTTGGCATC-3') after DNA extraction from leaves from in vitro and greenhouse-grown plants using the DNeasy® Plant Mini Kit (Qiagen).

For real-time PCR analysis (Q-PCR), RNA was isolated from young leaves from greenhouse-grown plants (2 biological repeats) using the NucleoSpin® RNA Plant kit (Macherey-Nagel) according to the manufacturer's recommendations. Nucleic acids were quantified (Nanodrop Technologies Inc., Wilmington, USA) and their quality was checked by electrophoresis on agarose gel. 2 µg RNA were denatured for 10 min at 70 °C with 0.5 µg of oligo(dT)15 (Promega) and then subjected to reverse transcription with 200 U of MMLV-RT (Promega), 0.5 mM of each dNTP in a final volume of 30 µl for 1 h at 42 °C. Absence of genomic DNA in the cDNA was checked by PCR with the reverse primer R1 (as described above) and a forward primer in the sequence of the 35S promoter (F2 : 5'-CCTCGTGGGTGGGGGTCC-3'. an Opticon Monitor (Bio-RAD). The amount of plant RNA in each sample was normalized using the Actin gene as reference. The relative expression level was calculated according to [START_REF] Pfaffl | A new mathematical model for relative quantification in real-time RT-PCR[END_REF], using the background signal observed in the Greensleeves control as calibrator.

The standard error was calculated from four repeats per sample (2 replicates from independent RNA extraction x 2 technical repeats).

For western-blot analysis, 1.5 g of young leaves from greenhouse-grown plants was ground with 5 ml of extraction buffer (Tris-HCl 100 mM, pH 7.5, 2.5 mM dithiothreitol, 2.5% polyvinylpolypyrrolidone). After centrifugation at 13000 g for 20 min at 4°C, the supernatant was immediately used or stored at -80 °C. Protein content was determined using a bicinchonic acid dye reagent (Sigma) and bovine serum albumin as standard. Aliquots (20 μg) of protein extract from control and transgenic clones in Laemmli buffer were separated on 12% SDStricine polyacrylamide (w/v) gel according to the discontinuous procedure of [START_REF] Schägger | Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in range from 1 to 100 kDa[END_REF]

Measurement of aldehyde reductase activity

Proteins were extracted as it is described for western-blot analysis. Aldehyde reductase activity was assessed spectrophotometrically by measuring the rate of enzyme-dependent decrease of NADPH absorption at 340 nm for 16 minutes, as described previously [START_REF] Colrat | Purification and characterization of a NADPH-dependent aldehyde reductase from mung bean that detoxifies eutypine, a toxin from Eutypa lata[END_REF]. Briefly, the reaction mixture consisted of 200 mM Na 2 HPO 4 / 100 mM citric acid buffer, pH6.5, 80 µM NADPH, 50 µg/ml of protein extract and substrate at a concentration of 10 times the K M value of each substrate as defined in [START_REF] Colrat | Purification and characterization of a NADPH-dependent aldehyde reductase from mung bean that detoxifies eutypine, a toxin from Eutypa lata[END_REF]. Thirteen substrates were tested: benzaldehyde (BD), 2-hydroxy-benzaldehyde (2HBD), 3-hydroxy-benzaldehyde (3HBD), 4-hydroxy-benzaldehyde (4HBD), 3-metoxy-benzaldehyde (3MBD), 4-metoxybenzaldehyde (4MBD), 3-nitro-benzaldehyde (3NBD), 4-nitro-benzaldehyde (4NBD), 3fluoro-benzaldehyde (3FBD), 4-pyridoxy-benzaldehyde (4PBD), tolylaldehyde (Tolyl), decylaldehyde (Decyl), coniferylaldehyde (Conyl). A control without protein extract was used to evaluate the rate of NADPH degradation. Assays were performed in quadruplicate, on a single extract for each transgenic clone.

Statistical analyses

Statistical analyses -including ANOVA and Dunnett's test to compare the rate of bud regeneration -performed with SAS 9.1 software (SAS institute, Cary, N.C.) Western blot analysis of proteins extracted from leaves of Vr-ERE transgenic clones from Greensleeves, using antibodies raised against the Vr-ERE protein, revealed a 36 kDa protein corresponding to the expected size of the Vr-ERE protein (Figure 3). This band was absent in the Greensleeves control (GLC), and strongly detected in a protein extract of mung bean (Vigna radiata). This band was clearly detected in seven transgenic clones. The band was absent or below the detection threshold in clone GL3. In addition, a non-specific band of about 26 kDa was detected in all samples.

RESULTS

Manuscrit

Aldehyde reductase activity in the Vr-ERE transgenic apple plants Determination of the eutypine reductase activity due to the expression of the Vr-ERE transgene could not be performed directly with eutypine as a substrate, as this compound was not available. However, the previous studies of Guilen et al. (1998) and [START_REF] Colrat | Purification and characterization of a NADPH-dependent aldehyde reductase from mung bean that detoxifies eutypine, a toxin from Eutypa lata[END_REF] demonstrated that Vr-ERE has a broad range of substrates including various aromatic and aliphatic aldehydes. In the first step, aldehyde reductase activity was determined using protein extracts from leaves of greenhouse-grown plants of three apple cultivars Greensleeves, Ariane and Galaxy and five substrates of Vr-ERE (Figure 4). Enzymatic activities varied between 2 and 14 nkatal.mg-1 protein, which is higher than the value reported for the grapevine 110

Richter tested with eutypine by [START_REF] Legrand | Constitutive expression of Vr-ERE gene in transformed grapevines confers resistance to eutypine, a toxin from Eutypa lata[END_REF]. Our results indicated marked variability between apple genotypes, Galaxy having the highest average activity, followed by Ariane, then Greensleeves. Furthermore, an interaction between genotype and substrate was detected.

In the second step, detailed aldehyde reductase enzymatic activity measurements, using 13 different substrates of Vr-ERE, were performed on eight apple Vr-ERE transgenic clones from Greensleeves. A summary of the results is given in substrates, and only a few positive or negative differences in one to three substrates.

However, the two clones (GL11 and GL12) expressed significantly more activity than the control with the large majority of substrates. However, even for these two clones, the highest aldehyde reductase activity was only about 12 nkatal.mg-1 protein, which is significantly higher than the activity of the non-transgenic control Greensleeves, but within the range of variation of activity measured in the other non-transgenic apple cultivars Galaxy and Ariane (Figure 4).

Tolerance to aldehydes in the Vr-ERE transgenic apple plants Tolerance to aldehydes in Greensleeves transgenic clones expressing the Vr-ERE transgene was assessed during micropropagation and during adventitious bud regeneration experiments, using several different substrates of Vr-ERE. Toxicity of several aldehyde compounds (BD, 3FBD, 3MBD, 3NBD, 4PBD) was observed on the Greensleeves control clone, at doses around 1 mM. However, none of these experiments revealed a significant increase in tolerance in all the transgenic clones tested, compared to the Greensleeves control.

As an example, the results obtained with clones GL11 and GL12, the two clones that expressed significantly higher aldehyde reductase activity with a majority of substrates, are given in Tables 2 and3. No difference in tolerance to a range of 3NBD concentrations was detected during micropropagation, and all explants died on 1.5 mM 3NBD (Table 2).

Similarly, during regeneration experiments from leaves of these two clones necrosis was very similar to that induced on control leaves by concentrations of 3MBD (0.5 to 1.5 mM) and 3FBD (0.25 to 1 mM). The regeneration potential of these two clones on a medium without any selection pressure was lower than in the control, and regeneration was totally inhibited at 3).

DISCUSSION

Because stable transformation of plant cells usually occurs at low frequencies, selection is a necessity to recover transformants of many plant species. So far, the nptII selectable marker conferring resistance to kanamycin has been widely used in many crops.

This antibiotic is widely dispersed in nature and has very limited therapeutic use (Miki and McHugh, 2004). Consequently the rationale for the development of new selection systems is to improve a general public perception and acceptability. Among the alternative systems currently being developed are positive selection systems such as mannose selection [START_REF] Wallbraun | Phosphomannoseisomerase (pmi) gene as a selectable marker for Agrobacterium-mediated transformation of rapeseed[END_REF], and site-specific recombination systems that generate marker-free plants [START_REF] Thirukkumaran | Isopentenyl transferase gene expression offers the positive selection of marker-free transgenic plant of Kalanchoe blossfeldiana[END_REF][START_REF] Khan | Rol (root loci) gene as a positive selection marker to produce marker-free Petunia hybrida[END_REF]. Most of the alternative systems studied on apple are based on bacterial genes (bar, PMI). Currently the presence of coding sequences of bacterial origin is a concern for the safety of transgenic plants. The Vr-ERE gene, from

Vigna radiata is a natural detoxification system of plant origin. It thus appears to be an attractive alternative for apple transformation. The purpose of the present work is to study the effects of overexpression of the Vr-ERE gene in apple transgenic plants, and thus to evaluate the feasibility of using Vr-ERE as a selectable marker.

Our results have demonstrated that the production of viable transgenic apple plants expressing the Vr-ERE gene is possible using kanamycin selection. Transformation efficiency in this experiment was in the same range as previously reported transformation efficiencies obtained in the same laboratory, on cvs. Galaxy and Ariane [START_REF] Faize | Chitinases of Trichoderma atroviridae induce scab resistance and some metabolic changes in two cultivars of apple[END_REF][START_REF] Faize | Expression of wheat puroindoline-b reduces scab susceptibility in transgenic apple (Malus x domestica Borkh.)[END_REF]. Our Several positive selection systems based on toxic metabolites intermediates have been developed for the production of transgenic plants. However, none of these alternative selectable marker genes has yet progressed to the level of the major antibiotic or herbicide marker genes (Miki and McHugh, 2004). Our work with transgenic apple plants overexpressing the detoxifying gene Vr-ERE illustrates some of the difficulties in developing an alternative selection pressure. Further studies will be needed to develop an efficient selection system based on a plant gene. The following criteria can be proposed: 1) narrow substrate specificity of the detoxifying gene, 2) high toxicity of the chosen substrate in the target species, 3) limited similarity with endogenous genes of the target species, 4) limited modification of the transgenic plant endogenous metabolism, 5) absence of composition or growth abnormalities in the transgenic plants.

E

Version définitive du manuscrit publié dans / Final version of the manuscript published in : Plant Cell, Tissue and Organ Culture, 2011, vol.106, no.1, 161-168. DOI: 10.1007

B

Ladder GLC Plasm. GL1 GL3 GL4 GL5 GL6 GL7 GL10 GL11 GL12 GL13 GL14 GL16 GL17 water 220 bp

  /s11240-010-9904-Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript 4

  rooted plants in a peat/perlite mixture and progressively decreasing the relative humidity.

  PCR reactions were performed in duplicate using 3 µl of retrotranscription (RT) product in a final volume of 15 µl containing 1X IQ SYBR Green Supermix (Bio-RAD), and 0.2 µM of each primer. Primers for amplification of Vr-ERE were the same as for PCR analysis (F1 and R1). Primers for the reference gene Actin (accession CV151413. Amplifications were performed using an Opticon 4RealTime PCR detector (Bio-RAD) as follows: 95°C for 3 min; 40 X [95 °C for 15 s; 62 °C for 1 min]. The amplification specificity was verified by a final dissociation curve ranging from 60 to 95 °C. Amplification and dissociation curves were monitored and analyzed with

  d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript 9Transformation experiments with the Vr-ERE transgene Production of transgenic plants constitutively expressing Vr-ERE was attempted on the apple cultivar Greensleeves using kanamycin as selection pressure. In total, 156 buds were regenerated from a total of 410 leaf explants. Only one regenerating bud per leaf was subsequently micropropagated under the light, to insure that only independent transformation events were analyzed. Seventeen buds developed into plantlets under kanamycin selection pressure. Ploidy level determined by flow cytometry revealed only diploid clones among the regenerants. All diploid clones that survived on kanamycin were checked for the presence of the Vr-ERE gene by PCR analysis (Figure1-B). Unlike the control, a specific fragment of the expected size (220 bp) was amplified from the genomic DNA extracted from all transgenic clones except one (GL6). The final transformation efficiency was 3.9% of the inoculated explants producing a diploid transgenic clone.Eight Vr-ERE transgenic clones from Greensleeves were rooted and acclimatized in the greenhouse with an average survival rate of 95%. Growth of these clones was monitored for seven months (Figure2-A). The length and morphology of the shoots in most clones were similar to those in the control. Two clones (GL1 and GL5) were slightly shorter than the control. All the subsequent analyses were performed on this sample of eight transgenic clones.Level of expression of the Vr-ERE transgene in transgenic apple plantsReal-time PCR was used to quantify Vr-ERE mRNA in Greensleeves transgenic clones growing in the greenhouse. Only background signal was obtained for the nontransgenic control clone, whereas all transgenic clones revealed varying levels of expression of Vr-ERE (Figure 2-B). According to these results, the tested transgenic clones were separated into three groups: one clone was a low expressor (GL3), three clones were high Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript 10 expressors (GL5, 7 and 11), the remaining four clones were intermediate (GL1, 10, 12 and 13).

  and 3FBD lower than the inhibitory concentrations for the control leaves (Table

  transgenic plants also indicate that there is no general deleterious effect of the overexpression of Vr-ERE in apple, whereas in grape, overexpression of Vr-ERE had a clear morphological effect, with loss of apical dominance(A. Bouquet, personal communication).RT-PCR analyses indicated that the Vr-ERE transgene was expressed at variable RNA level in Greensleeves transgenic clones. Western blot analyses revealed normal translation of Vr-ERE mRNAs in most Greensleeves transgenic clones, but marked variability among clones regarding the amount of detectable protein. A precise correlation between mRNA and protein levels of expression could not be calculated, but the general trend of the results indicates coherence between these two evaluations of transgene expression. In particular, no Vr-ERE protein signal was detected in clone GL3, which also had the lowest mRNA level.Detailed aldehyde reductase enzymatic activity measurements, using 13 different substrates of Vr-ERE, revealed only a very limited increase in aldehyde reductase activity conferred by the expression of the Vr-ERE transgene. Furthermore, micropropagation and regeneration experiments revealed no increase of tolerance to aldehydes in Greensleeves transgenic clones. This low efficiency of Vr-ERE expression in transgenic apple clones may have the following explanation. The precise function of Vr-ERE in its donor organism (Vigna radiata) is still unknown. A search in protein sequence databases indicated that the Vr-ERE protein presents significant similarities to enzymes of two secondary metabolic pathways: lignin with Cinnamyl alcohol dehydrogenase (CAD) and Cinammoyl-CoA reductase (CCR), and flavonoid with dihydroflavonol-4-reductase (DFR)(Guillen et al. 1988). Amino acid similarity with the Malus domestica CCR is 71%. Because of this similarity to endogenous Malus sequences, a certain degree of silencing of the transgene can be expected. However, partial silencing of Vr-ERE in transgenic plants cannot be solely responsible for the lack of tolerance to toxic aldehyde compounds, since the presence of the Vr-ERE transgenic protein E Version définitive du manuscrit publié dans / Final version of the manuscript published in : Plant Cell, Tissue and OrganCulture, 2011, vol.106, no.1, 161-168. DOI: 10.1007/s11240-010-9904-Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript 14 was clearly demonstrated by western-blot analysis. Preliminary transformation experiments on cultivars Galaxy and Ariane using BD as a selection pressure indicated insufficient selection in favor of transgenic cells (data non shown).

Figure 1 :

 1 Figure1: A) Schematic structure of binary vector pCambiaVr-ERE-GUS, prepared by cloning a "double35S-Vr-ERE" cassette at the HindIII site of pCambia2301. LB: left border; t-35S: CaMV35S terminator; nptII: neomycin phosphotransferase II gene; 35S: CaMV35S promoter; Vr-ERE: Vigna radiata eutypine reductase gene; GUS: intron-containing uidA gene; t-nos: nopaline synthase terminator; RB: right border. Primers for PCR and RT-PCR : R1 : reverse 1, F1 and F2 : forward 1 and 2. B) PCR analysis of Vr-ERE transgenic Greensleeves clones. Gel electrophoresis of Vr-ERE PCR products with the expected size of 220 bp. Ladder: 100 pb; GLC: Greensleeves control; Plasm: pCambiaVr-ERE-GUS; GL1 to 17: transgenic Greensleeves clones.

Figure 2 :

 2 Figure2: A) Growth of Vr-ERE transgenic clones from Greensleeves (GL), 7 months after greenhouse acclimatization. Clone GL6 is transgenic (presence of nptII transgene) but with no integration of the Vr-ERE transgene. Bars are the mean of 3 to 32 shoots per clone ± confidence interval at α = 5%. *: transgenic clones differed significantly from the GL control at p<0.05 according to Dunnett's test. B) 4: Determination of transcription of Vr-ERE in transgenic clones of Greensleeves (GL1 to GL13) by real-time PCR. GLC: Greensleeves control. Bars are the mean of 4 repeats (2 independent RNA extractions x 2 technical repeats) ± confidence interval at α = 5%.

Figure 3 :

 3 Figure3: Western blot analysis of proteins from Greensleeves transgenic clones (GL1 to GL13). GLC: Greensleeves control, V.r.: Vigna radiata control.

Figure 4 :

 4 Figure 4: Enzymatic activities measured with 5 different substrates of Vr-ERE, on control clones of Ariane, Galaxy and Greensleeves. Each bar is the mean of 5 replicates ± confidence interval at α = 5%.

Figure 1 E

 1 Figure 1

  Version définitive du manuscrit publié dans / Final version of the manuscript published in : Plant Cell, Tissue and OrganCulture, 2011, vol.106, no.1, 161-168. DOI: 10.1007/s11240-010-9904-

	Amersham) using horseradish peroxidase-labeled secondary antibody, according to the
	manufacturer's instructions.
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. After electrophoresis, proteins were blotted onto Hybond C nitrocellulose membrane (Amersham) by passive transfer. Polyclonal rabbit anti-Vr-ERE antiserum was used. Vr-ERE was detected with the enhanced chemiluminescence detection system (ECL, E

Table 1 .

 1 Most of the apple

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Table 1 :

 1 Summary of enzymatic activity measurements of Vr-ERE transgenic clones tested with 13 substrates of Vr-ERE ERE transgenic clones from Greensleeves. Each clone was measured in quadruplicate. +, ++, +++: clones whose activity significantly higher than in the Greensleeves control, according to Dunnett's test at p<0.1, 0.05 and 0.01 respectively. -, --, ---: clones whose activity was significantly lower than in the Greensleeves control, according to Dunnett's test at p<0.1, 0.05 and 0.01 respectively; n.s. clones whose activity was not significantly different from that in the Greensleeves control; n.t.: non tested.

	Génotype	BD	List of tested Vr-ERE substrates 2HBD 3HBD 4HBD 3MBD 4MBD 3NBD 4NBD 3FBD 4PBD Tolyl Decyl Conyl
	GL1	n.s.	n.s.	n.s.	n.s.	+++	n.s.	+	n.s.	n.s.	n.s.	++	-	n.s.
	GL3	n.s.	n.s.	n.t.	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	++	n.s.	n.s.	n.s.
	GL5	n.s.	n.s.	n.s.	n.s.	n.s.	---	n.s.	-	n.s.	n.s.	--	++	n.s.
	GL7	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	+++	n.s.
	GL10	+++	n.s.	n.s.	n.s.	n.s.	n.s.	+++	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.
	GL11	+++	+++	n.s.	+++	++	+++	+++	+++	+++	+++	n.s.	+++	n.s.
	GL12	++	+++	++	n.s.	+++	n.s.	++	+++	n.s.	+++	+++	+++	n.s.
	GL13	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	n.t.
	GL1 to GL13: apple Vr-

Table 2 :

 2 Effect of 3-Nitro-benzaldehyde (3NBD) on micropropagation of Greensleeves Vr-ERE transgenic clones (GL11, GL12) and control (GLC)

	Treatment	Dose	% shoot survival after 1 month
		(mM)	GLC	GL11	GL12
		0	100	100	100
		0.5	68	50	60
	3NBD	0.75	60	45	55
		1	25	3	5
		1.5	0	0	0
	Data were collected after 1 month on micropropagation medium, on 2 replicates of 20 shoots per treatment.

Table 3 :

 3 Effect of 3-Methyl-benzaldehyde (3MBD) and 3-Fluoro-benzaldehyde (3FBD) on regeneration from leaves of Greensleeves Vr-ERE transgenic clones (GL11, GL12) and control (GLC)

	Treatment	Dose (mM)	Necrosis score (scale from 0 to 3) GLC GL11 GL12	% regenerating leaves GLC GL11 GL12
		0	0	0	0	83.5	46.0	68
	3MBD	0.5 1	0 2.59	0.37 3.00	1.12 3.00	23.0 0	1.5 0	5.0 0
		1.5	3.00	3.00	3.00	0	0	0
		0	0	0	0	70.0	53.3	11.7
		0.25	0	0	0	41.7	38.3	8.3
	3FBD	0.5	0	0	0	15.0	0	6.7
		0.75	1.73	2.63	0.98	5.0	0	0
		1	2.40	3.00	2.4	8.3	0	0
	Data were collected after 2 months on regeneration medium, on 2 replicates of 30 leaves per treatment.

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		A
							R1	F1 F2
	Manuscrit d'auteur / Author manuscript	LB	t	NPTII	35S	t	Vr-ERE	35Sx2	35S	GUS	t	RB
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Acknowledgements

This work was partly funded by a grant N° 062906338 from the French Ministry of Industry, for the collaborative project "Amélioration variétale fruits à pépins", conducted within the "Pôle de Compétitivité Végépolys". The authors wish to thank Dr. J.P. Roustan (ENSA Toulouse) for the gift of the pGA-Vr-ERE plasmid and Vr-ERE antibody and Dr. A. Bouquet (INRA Montpellier) for the gift of transgenic grapevine material containing Vr-ERE.

The ternary plasmid pBBR1MCS-5 was kindly provided by Dr Memelink (Clusius Laboratory, Leiden). The authors wish to thank Daphne Goodfellow (traductor) for her correction of the English manuscript.