Martine Pastuglia

Dominique Roby

Christian Dumas

J Mark Cock

J Mark Cock

Rapid Induction by Wounding and Bacterial Infection of an S Gene Family Receptor-Like Kinase Gene in Brassica oleracea

come

INTRODUCTION

Cell surface receptors with an intrinsic protein kinase activity have been shown to play important roles in signal perception and transduction in animals. A number of similar genes predicted to encode receptor kinases have been identified in plants. In a few cases, protein kinase activity has been demonstrated, and the protein product has been shown to be anchored in the membrane [START_REF] Chang | The TMK1 gene from Arabidopsis codes for a protein with structural and biochemical characteristics of a receptor protein kinase[END_REF][START_REF] Goring | The S-locus receptor kinase gene in a self-incompatible Brassica napus line encodes a functional serine/threonine kinase[END_REF][START_REF] Mu | Characterization of a pollen-expressed receptor-like kinase gene of Petunia inflata and the activity of its encoded kinase[END_REF][START_REF] Delorme | Characterization of the S locus genes, SLG and SRK, of the Brassica S 3 haplotype: identification of a membrane-localized protein encoded by the S locus receptor kinase gene[END_REF][START_REF] Stein | SRK, the stigma-specific S locus receptor kinase of Brassica, is targeted to the plasma membrane in transgenic tobacco[END_REF]. Five different classes of plant receptor-like kinase have been identified, as defined by the predicted amino acid sequences of their extracellular domains. The first class consists of receptor-like kinases of the S gene family and is so designated because the members of this family share homology with the S locus glycoprotein (SLG) of Brassica [START_REF] Nasrallah | Signaling the arrest of pollen tube development in self-incompatible plants[END_REF]. This class includes the S locus receptor kinase (SRK) of Brassica [START_REF] Stein | Molecular cloning of a putative receptor protein kinase gene encoded at the selfincompatibility locus of Brassica oleracea[END_REF], ZmPKl of maize (Zea mays Protein Kinase 1; [START_REF] Walker | Relationship of a putative receptor protein kinase from maize to the S-locus glycoproteins of Brassica[END_REF], ARK1, ARK2, and ARK3 of Arabidopsis (ARK for Arabidopsis Receptor Kinase; [START_REF] Tobias | An Arabidopsis thaliana gene with sequence similarity to the S-locus receptor kinase of Brassica oleracea[END_REF][START_REF] Dwyer | A superfamily of S locus-related sequences in Arabidopsis: Diverse structures and expression patterns[END_REF], and OsPK10 of rice (OsPK10 for Oryza sativa Protein Kinase 10; [START_REF] Zhao | Molecular cloning and biochemical characterization of a receptor-like serine/threonine kinase from rice[END_REF]. The four other classes include receptor-like kinases predicted to possess extracellular domains containing leucine-rich repeats (e.g, TMKl for Transmembrane Kinase 1, PRKl for Pollen

Receptor Kinase 1, and Xa21; [START_REF] Chang | The TMK1 gene from Arabidopsis codes for a protein with structural and biochemical characteristics of a receptor protein kinase[END_REF][START_REF] Mu | Characterization of a pollen-expressed receptor-like kinase gene of Petunia inflata and the activity of its encoded kinase[END_REF][START_REF] Song | A receptor kinase-like protein encoded by the rice disease resistance gene, Xa21[END_REF], epidermal growth factor repeats (WAK1 for Wall Associated Kinase 1; [START_REF] Kohorn | An Arabidopsis serine/threonine kinase homologue with an epidermal growth factor repeat selected in yeast for its specificity for a thylakoid membrane protein[END_REF], a thaumatin-like domain (PR5K for Pathogenesis-Related 5 Kinase; [START_REF] Wang | The PR5K receptor protein kinase from Arabidopsis thaliana is structurally related to a family of plant defense proteins[END_REF], or a lectin-like domain (Ath.lecRK1 and LRK1 for Arabidopsis thaliana lectin Receptor Kinase 1 and Lectin Receptor Kinase 1 respectively; [START_REF] Hervé | Characterization of an Arabidopsis thaliana gene that defines a new class of putative plant receptor kinases with an extracellular lectin-like domain[END_REF][START_REF] Swarup | A new class of receptor-like protein kinase gene from Arabidopsis thaliana possessing a domain with similarity to plant lectin genes[END_REF]. Plant receptor-like kinase genes have been isolated from a wide range of both dicotyledonous and monocotyledonous plant species. They show markedly different patterns of expression, with some genes being expressed in specific reproductive tissues (PRK1 and SRK), others predominantly in vegetative tissues (ARK genes), whereas others show developmentally regulated expression in both floral and vegetative tissues (ZmPK1 and TMK1).

Xa21 and PRK1 are the only members of the plant receptor-like kinase family to have been unequivocally assigned a role. Rice transformation experiments have shown that Xa21 confers resistance to Xanthomonas oryzae pv oryzae race 6. Antisense inhibition of PRK1 expression in transgenic Petunia inflata has demonstrated that this gene is essential for postmeiotic development of pollen [START_REF] Lee | PRK1, a receptor-like kinase of Petunia inflata, is essential for postmeiotic development of pollen[END_REF]. Within the S gene sub-family of receptor-like kinases, SRK is thought to be involved in the pollen-pistil recognition step of the self-incompatibility response in Brassica [START_REF] Mccubbin | Molecular mechanisms of selfincompatibility[END_REF]. SRK's proposed role in self-incompatibility is based on its stigma-specific pattern of expression, its location at the S locus (which controls selfincompatibility), its polymorphic nature, and on the existence of self-compatible Brassica plants that also possess mutated SRK genes [START_REF] Goring | An S-receptor kinase gene in self-compatible Brassica napus has a 1 bp deletion[END_REF]Nasrallah et al., 1994a). Apart from XA21, PRK1 and SRK, very little information is available concerning the roles of receptor-like kinases in plants, in particular, the functions of other receptor-like kinases of the S gene family are not known.

It is likely that S gene family receptor-like kinases expressed in vegetative tissues are involved in cell-cell communication systems analogous to the self-incompatible pollen-pistil interaction, except that they probably regulate very different developmental or metabolic pathways. A detailed study using the b-glucuronidase (GUS) reporter gene fused to the promoters of ARK2 and ARK3 has shown that these receptor-like kinase genes exhibit highly specific patterns of temporal and tissue-specific regulation [START_REF] Dwyer | A superfamily of S locus-related sequences in Arabidopsis: Diverse structures and expression patterns[END_REF]. Based on these observations, these authors suggest that ARK2 and ARK3 may function in specific aspects of plant growth or development but that their specific role in the plant is as yet unknown. Another possible function of S gene family members in vegetative tissues is suggested by the numerous similarities between selfincompatibility and the plant's response to attack by a pathogen [START_REF] Hodgkin | Recognition in flowering plants: a comparison of the Brassica self-incompatibility system and pathogen interactions[END_REF]. In both phenomena, there exists genetically controlled recognition systems that allow one cell to recognize another, leading to the induction of the response: self-incompatibility occurs when the pollen and the papillae cell express the same S locus haplotype [START_REF] Nasrallah | Signaling the arrest of pollen tube development in self-incompatible plants[END_REF], whereas disease resistance in many cases depends on the pathogen expressing an avirulence (avr) gene which corresponds to a resistance gene present in the plant host [START_REF] Flor | Current status of the gene-for-gene concept[END_REF]. From a morphological point of view, the comparison with self-incompatibility is particularly vivid for a fungal pathogen where in both cases an elongated cell (a pollen tube or a germ tube) emerges from a spore-like structure (a pollen grain or a fungal spore) on the surface of the host plant and penetrates by growing within or between cell walls [START_REF] Dickinson | Simply a social disease?[END_REF].

In this study we show that a novel member of the S gene family encoding a receptor-like kinase is induced by a range of stimuli that induce plant defense genes. These stimuli include both wounding and bacterial attack. These results indicate that this gene may play a role in the response of the plant to mechanical and biological attacks.

RESULTS

Cloning and sequence analysis of the SFR2 receptor-like kinase gene.

A PCR probe, generated by amplification of genomic DNA sequences with oligonucleotides corresponding to conserved regions of the SLG 29 gene [START_REF] Trick | A homozygous S genotype of Brassica oleracea expresses two S-like genes[END_REF], was used to screen a leaf cDNA library. Five cDNAs hybridized with the probe, and these were shown to represent three different genes by restriction mapping and DNA sequencing. The complete cDNA sequence corresponding to one of these genes, designated SFR2 (for S gene Family Receptor 2), was reconstructed from two clones. One of the clones was not full length but included the poly (A) tail, and the second included the 5' untranslated region but lacked part of the 3' untranslated region due to a recombination event, which had apparently occurred during or after the construction of the library. Based on this sequence, SFR2 is predicted to encode a membrane-spanning, receptor-like kinase with a very similar structure to SRK, possessing an extracellular S domain, a single membrane-spanning domain, and an intracellular kinase domain.

The deduced amino acid sequences of both the S domain and the kinase domain of SFR2 (EMBL accession number X98520) was compared to those of several other members of the S gene family from both Brassica and Arabidopsis (Table 1). In all the pairwise comparisons, the kinase domains were more similar than the S domains but, in general, when the kinase domains were more closely related so were the S domains and visa versa. Interestingly, SFR2 was more similar to the ARK receptor-like kinases of Arabidopsis than to the Brassica receptor-like kinase SRK. It is possible that SFR2 may be the Brassica equivalent of one of the ARK genes. Two other receptor-like kinases from Arabidopsis, RLKl and RLK4, are more distantly related to SFR2 (Table l) as are four putative Brassica receptor-like kinase genes identified by [START_REF] Kumar | Sequence complexity of the S receptor kinase gene family in Brassica[END_REF]data not shown). In addition, an SFR2 S domain probe hybridized most strongly with genomic DNA fragments corresponding to the ARK genes in a genomic gel blot of Arabidopsis DNA (R. Swarup and J.M. Cock, unpublished results).

Tissue-specific expression of the SFR2 gene.

RNA blot analysis was used to look at the abundance of SFR2 transcripts in a number of floral and vegetative tissues (Figure 1). A 320-bp, 3' untranslated region probe was used for these experiments. This probe was shown to be gene-specific by hybridization to a gel blot of B. oleracea genomic DNA digested with several restriction enzymes (data not shown). The SFR2 probe detected a major transcript of 2.8-kb and two, less abundant transcripts of 1.3-and 1.6-kb. The size of the 2.8-kb transcript is consistent with it being the full-length, fully-spliced transcript corresponding to the SFR2 cDNA. The 1.3-and 1.6-kb bands may represent alternative transcripts of the SFR2 gene generated by alternative splicing. Multiple transcripts have been reported for the closely related SRK gene in Brassica oleracea [START_REF] Stein | Molecular cloning of a putative receptor protein kinase gene encoded at the selfincompatibility locus of Brassica oleracea[END_REF][START_REF] Delorme | Characterization of the S locus genes, SLG and SRK, of the Brassica S 3 haplotype: identification of a membrane-localized protein encoded by the S locus receptor kinase gene[END_REF]. The 2.8-kb transcript accumulated in a developmentally regulated, tissue-specific manner and was most abundant in flower buds. mRNA levels were at their highest when the buds were 8 mm in length (Figure 1A). When the individual organs of the flower were analyzed, SFR2 mRNA was most abundant in anthers from 5-to 7-mm-long flower buds. There was also an accumulation of SFR2 mRNA in stigmas but at a later stage, with the highest levels being detected in stigmas from open flowers (Figure 1B). No SFR2 mRNA was detected in petals, sepals, or ovary tissue at the open flower stage, but a very low abundance of SFR2 mRNA was detected in the seeds throughout the development of the silique. In vegetative tissues, SFR2 mRNA was not detected in 3-day-old seedlings but was present both in leaves of field-cultivated plants and in roots of 3-week-old plants, with the highest level being in the leaves (Figure 1C).

Accumulation of SFR2 mRNA in response to wounding and bacterial infection

To determine whether the expression of SFR2 could be influenced by an external stimulus, leaves of B. oleracea plants were wounded and SFR2 mRNA abundance was assayed using RNA blots. Figure 2 shows that SFR2 transcripts began to accumulate between 15 and 30 min after wounding, reaching a maximum within 2 hr. SFR2 mRNA, therefore, accumulated rapidly and transiently in wounded leaves.

The kinetics of accumulation of SFR2 mRNA after mechanical wounding was compared with that of PAL mRNA, which is known to be accumulated early during the defense response. PAL is a well characterized defense gene that encodes the first enzyme of the phenylpropanoid pathway, phenylalanine ammonia-lyase. SFR2 and PAL mRNAs were shown to start accumulating at approximately the same time after the wound stimulus (Figure 2).

An experiment was then carried out to determine whether SFR2 mRNA also accumulated in response to invasion of the plant by a bacterial pathogen. B. oleracea leaves were infiltrated with two strains of Xanthomonas campestris pv campestris, one wild type (pathogenic) and the second mutated in the hrp gene cluster, which is required both for pathogenicity in the plant host and for the induction of the hypersensitive (HR) response in non-host plants [START_REF] Arlat | Xanthomonas campestris contains a cluster of hrp genes related to the larger hrp cluster of Pseudomonas solanacearum[END_REF]. The hrp mutant strain elicited no visible symptoms. Water was also infiltrated as a control. Figures 3A and3B show that SFR2 mRNA accumulated in response to both bacterial strains. At the highest concentration of wild type bacteria SFR2 mRNA had already started to accumulate 1 hr after infiltration and by 6 hr was 66-fold more abundant than in the control. Again, the accumulation of SFR2 mRNA was transient and, at the highest concentration of bacteria, mRNA abundance had returned to the same level as in the control after 48 hr. Accumulation of SFR2 mRNA occurred later when a lower concentration of bacteria were infiltrated into the leaves. No significant accumulation of SFR2 mRNA was seen when the leaves were infiltrated with water alone (Figure 3C), indicating that the mRNA accumulation induced by the bacteria was not due to the leaf being wounded during infiltration. Hybridizations with phenylalanine ammonia-lyase (PAL) and chitinase probes demonstrated that, like the SFR2 gene, these two defense-related genes were induced in leaves infiltrated with either the pathogenic (8004) or the non-pathogenic (8004::A2) strain. Similar results have been reported for these two genes in bean after infiltration with a Pseudomonas syringae pv tabacci hrp mutant [START_REF] Jakobek | Generalized induction of defense responses in bean is not correlated with the induction of the hypersensitive reaction[END_REF]. In their study, Jakobek and Lindgren also reported that a saprophytic bacterium such as Escherichia coli was capable of inducing an accumulation of PAL and chitinase mRNAs. We therefore tested whether E. coli was able to induce SFR2 mRNA accumulation. Figure 4 shows that SFR2 mRNA accumulated in B. oleracea leaves infiltrated with E. coli.

Several plant-derived chemicals have been shown to induce some of the responses that follow wounding and/or invasion by a pathogen. Three of these molecules, salicylic acid (SA), abscisic acid (ABA) and methyl jasmonate (MeJA), were infiltrated into B. oleracea leaves, and changes in SFR2 mRNA abundance were analyzed (Figure 5). A rapid accumulation of SFR2 mRNA, peaking between 3 and 6 hr after treatment, was observed in response to 1 mM SA. Recent studies have suggested that SA intervenes in the plant's defense response by inhibiting a catalase (the SA binding protein), leading to an increased H 2 O 2 levels and to the activation of defense-related genes [START_REF] Chen | Active oxygen species in the induction of plant systemic acquired resistance by salicylic acid[END_REF]. Using a range of SA analogs, a correlation has been demonstrated between the ability to bind SA binding protein and inhibit its catalase activity and the effect on pathogenesis-related protein induction. We tested the activity of two analogs, 4hydroxybenzoic acid and 4-aminosalicylic acid, that are unable to bind the catalase [START_REF] Chen | Active oxygen species in the induction of plant systemic acquired resistance by salicylic acid[END_REF][START_REF] Sanchez-Casas | A salicylic acid-binding activity and a salicylic acid-inhibitable catalase are present in a variety of plant species[END_REF]. Neither analog induced a significant accumulation of SFR2 mRNA or chitinase mRNA in B. oleracea leaves compared with the control treatments (data not shown).

In contrast to the response to SA, no significant accumulation of SFR2 mRNA was seen following infiltration of ABA or MeJA. In order to confirm that the infiltrated tissue was correctly treated with these chemicals, radiolabelled probes corresponding to genes known to be induced in the presence of MeJA (Atvsp for Arabidopsis thaliana vegetative storage protein; [START_REF] Berger | Arabidopsis thaliana Atvsp is homologous to soybean VspA and VspB, genes encoding vegetative storage protein acid phosphatases, and is regulated by methyl jasmonate, wounding, sugars, light and phosphate[END_REF] or ABA (AtDi21 for Arabidopsis thaliana Drought-induced 21; [START_REF] Gosti | Abscisic aciddependent and -independent regulation of gene expression by progressive drought in Arabidopsis thaliana[END_REF] were hybridized with the RNA gel blots corresponding to the middle and the lower panels of Figure 5 respectively. In both cases mRNA accumulated following treatment (data not shown).

Analysis of the SFR2 promoter region and the expression of an SFR2::uidA reporter gene fusion in transgenic tobacco A 320-bp fragment corresponding to the 3' untranslated region of the SFR2 cDNA was used as a probe to isolate clones carrying the SFR2 gene from a genomic library. The DNA sequence was determined for a region of 1480 bp upstream of the ATG initiation codon (Figure 6). There was no obvious similarity between the SFR2 promoter and those of other members of the S gene family, in particular, no homology was detected with the 5 conserved "boxes" present in the promoter regions of SRK, SLG, and SLRI (SLR1 for S Locus Related 1; [START_REF] Dzelzkalns | Distinct cis-acting elements direct pistil-specific and pollen-specific activity of the Brassica S locus glycoprotein gene promoter[END_REF][START_REF] Delorme | Characterization of the S locus genes, SLG and SRK, of the Brassica S 3 haplotype: identification of a membrane-localized protein encoded by the S locus receptor kinase gene[END_REF]. However, a number of sequences with significant similarity to previously characterized elements were identified including elements identified in many inducible genes, such as the SA-responsive element TCA-1, the elicitor-responsive element ELI box l, and the wound-responsive elements WUN and PI WUN (Figure 6).

To determine whether the SFR2 promoter is able to direct inducible gene expression, a transcriptional fusion between the SFR2 promoter and the uidA bacterial reporter gene (which encodes GUS) was introduced into tobacco by transformation, and the leaves of transgenic plants were subjected to a range of stimuli. Three different strains of Ralstonia (Pseudomonas) solanacearum were locally infiltrated into undetached leaves. Strain K60 caused the typical lethal wilting disease. Strain GMI1000 induced the development of an HR on tobacco leaves within 18-24 hr after infiltration. No symptoms were observed with strain Dhrp, which carries a deletion in the hrp gene cluster required for elicitation of the HR response in tobacco [START_REF] Boucher | Transposon mutagenesis of Pseudomonas solanacearum: isolation of Tn5-induced avirulent mutants[END_REF].

GUS activity was measured at different times after infiltration with the bacteria. Figure 7 shows that there was a low level of induction of expression of the gene fusion when the leaves were infiltrated with water but that gene expression significantly increased in the presence of bacteria. Strains Dhrp, K60, and GMI1000 all consistently induced expression of the gene fusion. The SFR2 gene was therefore shown to respond to bacteria from two different genera, Xanthomonas and Ralstonia.

Histochemical detection of GUS was carried out with leaves of transgenic plants locally infiltrated with the three R. solanacearum strains (Figure 8). GUS activity was first detected in the infiltrated area 20 hr after infiltration with either the K60 or the Dhrp isolate. In the case of the GMI1000 isolate, GUS activity was first detected 12 hr after inoculation. After 18 hr, an HR developed, and GUS activity was restricted to cells surrounding the necrotic area.

The SFR2:uidA gene fusion was induced by infiltration with SA but not with ABA nor with MeJA (data not shown). This result indicates that the SFR2:uidA gene fusion was regulated in a similar manner in tobacco to the endogenous SFR2 gene in B. oleracea.

DISCUSSION

In this study, we characterize the SFR2 gene that encodes a putative receptor kinase of the S gene family in B. oleracea. The structure of the predicted protein is similar to that of other, previously described, members of the family in that it consists of an S domain, similar to the S locus glycoprotein, and a kinase domain separated by a hydrophobic domain that is predicted to span the membrane. Sequence comparison (Table 1) indicates that SFR2 is more closely related to the ARK family of receptor-like kinases in Arabidopsis than to other known receptor-like kinases from Brassica. The highest amino acid similarity was to ARK3, although the expression pattern of SFR2 more closely resembles those of ARKI and ARK2, which are both expressed most strongly in the leaves.

Several treatments, including wounding, bacterial infection, and SA application, were shown to induce an accumulation of SFR2 mRNA (Figures 2,3,4,5,7 and 8). The response to SA was highly specific; SRF2 mRNA did not accumulate in response to inactive analogs. There was no change in SFR2 mRNA abundance in response to infiltration with MeJA, a substance that has been implicated in the signal transduction pathway leading to the induction of wound-induced genes, such as those encoding proteinase inhibitors [START_REF] Farmer | Octadecanoid precursors of jasmonic acid activate the synthesis of wound-inducible proteinase inhibitors[END_REF]. Although the expression of many woundinduced genes is stimulated by MeJA there are exceptions [START_REF] Farmer | Fatty acid signalling in plants and their associated microorganisms[END_REF] indicating that wounding induces gene expression by at least two different signal transduction pathways.

The accumulation of SFR2 mRNA in response to a stimulus was always transient, and mRNA abundance had returned to a basal level 6 to 24 hr after treatment, depending on the stimulus. The inducibility of SFR2 in response to the stimuli tested is typical of a gene involved in plant defense and is consistent with the presence of sequences in the SFR2 promoter region with similarity to regulatory elements implicated in the response to SA, elicitors, and wounding (Figure 6). Broad-response defense genes such as PAL and chitinase showed similar patterns of expression to SFR2. Transcripts corresponding to all three of these genes accumulated in response to wounding and to infiltration with Xanthomonas strains which either caused disease or elicited no visible symptoms (Figures 2 and3).

Analysis of the spatial pattern of expression using an SFR2::uidA reporter gene fusion in transgenic tobacco infiltrated with different strains of R. solanacearum showed that gene induction was initially restricted to the infiltrated region of the leaf (Figure 8). Later, in response to strain GMI1000, which caused an HR, expression extended slightly beyond the infiltrated area and corresponded to a region immediately surrounding the dying cells. In the case of the compatible interaction, it is likely that distal cells respond to the pathogen as the disease progresses, although this was not observed in the experiment carried out here that focused on the early stages of the infection process. Again, the pattern of expression observed with the gene fusion is typical of defense genes [START_REF] Graham | Cellular coordination of molecular responses in plant defenses[END_REF]. The SFR2::uidA reporter gene fusion responded to three different strains of R. solanacearum strains causing an HR response, disease, or no visible symptoms (Figures 7 and8). Similarily, in B. oleracea, SFR2 mRNA accumulated in the presence of both pathogenic and non-pathogenic strains of X. c. campestris (Figure 3) and in the presence of a saprophytic bacterium, E. coli (Figure 4) but not in response to infiltration with water. These data suggest that SFR2 is induced by a wide range of bacteria and that it does not respond uniquely to pathogenic bacteria. Similar results have been reported for PAL and chitinase genes in bean [START_REF] Jakobek | Generalized induction of defense responses in bean is not correlated with the induction of the hypersensitive reaction[END_REF].

There is increasing evidence that protein kinases play a role in the plant's response both to wounding and to attack by pathogens. For example, there is evidence that a serine/threonine protein phosphorylation cascade constitutes part of the signal transduction pathway which leads to resistance of tomato plants to P. syringae pv tabaci strains carrying the avrPto avirulence gene [START_REF] Martin | Map-based cloning of a protein kinase gene conferring disease resistance in tomato[END_REF][START_REF] Zhou | The tomato gene Pti1 encodes a serine/threonine kinase that is phosphorylated by Pto and is involved in the hypersensitive response[END_REF]. Moreover, protein kinases resembling mammalian mitogen-activated protein (MAP) kinases have been shown to be rapidly activated (in less than 5 min) following wounding [START_REF] Usami | Cutting activates a 46-kilodalton protein kinase in plants[END_REF] or elicitor treatment [START_REF] Suzuki | Transient activation and tyrosine phosphorylation of a protein kinase in tobacco cells treated with a fungal elicitor[END_REF]. mRNA corresponding to a MAP kinase homolog from tobacco has been shown to accumulate within one minute of wounding [START_REF] Seo | Tobacco MAP kinase: a possible mediator in wound signal transduction pathways[END_REF]. In mammals, recent evidence indicates that the MAP kinase pathway can mediate signal transduction from serine/threonine kinase receptors [START_REF] Yamaguchi | Identification of a member of the MAPKKK family as a potential mediator of TGF-b signal transduction[END_REF], although this connection has yet to be demonstrated in plants. Two other members of the receptor-like kinase supergene family have also been implicated in the defense response in plants: Xa21, which was identified as a resistance gene [START_REF] Song | A receptor kinase-like protein encoded by the rice disease resistance gene, Xa21[END_REF], and PR5K, which is predicted to encode a receptor-like protein kinase with an extracellular domain related to a family of plant defense proteins [START_REF] Wang | The PR5K receptor protein kinase from Arabidopsis thaliana is structurally related to a family of plant defense proteins[END_REF].

The data presented here conclusively demonstrate that SFR2 is inducible. It has recently been reported [START_REF] Sasseen | Expression of RLK4, a receptor protein kinase in Arabidopsis thaliana[END_REF] that RLK4, an S gene family receptor-like kinase gene from Arabidopsis, is induced in the presence of auxin but, to our knowledge, there is no evidence that any of the other members of the receptor-like kinase superfamily, including PR5K and Xa21 (or any of the more distantly related resistance genes; [START_REF] Staskawicz | Molecular genetics of plant disease resistance[END_REF] are inducible. However, inducible gene expression has been observed for receptor kinases in animals. For example, both the epidermal growth factor receptor and the platelet-derived growth factor receptors are up-regulated by their ligands [START_REF] Clark | Epidermal growth factor regulates the expression of its own receptor[END_REF][START_REF] Eriksson | Induction of platelet-derived growth factor a-and b-receptor mRNA and protein by platelet-derived growth factor BB[END_REF]. In both cases, however, mRNA accumulation seems to be primarily the result of post-transcriptional regulation whereas, in the case of SFR2, the inducibility of a GUS reporter gene fusion indicates a transcriptional control. Transcriptional activation has been reported for the interleukin 2 receptor [START_REF] Depper | Interleukin 2 (IL-2) augments transcription of the IL-2 receptor gene[END_REF]. Mammalian receptor kinases have been shown to be upregulated transiently following wounding [START_REF] Antoniades | Injury induces in vivo expression of the platelet-derived growth factor (PDGF) and PDGF receptor mRNAs in skin epithelial cells and PDGF mRNA in connective tissue fibroblasts[END_REF][START_REF] Wenczak | Epidermal growth factor receptor distribution in burn wounds[END_REF]. These receptors are thought to contribute in a number of ways to wound healing, for example by allowing normally quiescent cells to respond to growth factors by proliferating to repair the wound.

SFR2 mRNA accumulated rapidly in response to the various treatments tested, for example mRNA was already detected 15 to 30 min after wounding. The rapidity of SFR2 induction was comparible to that of the early defense gene PAL (Figures 2 and3). Nonetheless, assuming that SFR2 functions as a receptor, there is likely to be a significant lag between the initiation of SFR2 induction and any response mediated by signal transduction via de novo synthesized SFR2. Hence, SFR2 may not be involved in the initial steps of the defense response but rather be implicated at a slightly later stage. Alternatively, it is possible that SFR2 may function in the initial stages of the defense response if low levels of SFR2 protein are present in the tissue before it is challenged. In such tissues, SFR2 mRNA accumulation following wounding could then serve to amplify signal transduction via SFR2. Interestingly, a low level of SFR2 mRNA was detected in leaves of field grown plants (Figure 1).

SFR2 mRNA also accumulated in anthers and stigmas at specific developmental stages (Figure 1). Developmentally regulated expression has been reported for many defense genes, including pathogenesis-related genes [START_REF] Castresana | Tissue-specific and pathogen-induced regulation of a Nicotiana plumbaginifolia b-1,3-glucanase gene[END_REF][START_REF] Vogeli-Lange | Evidence for a role of b-1,3glucanase in dicot seed germination[END_REF], genes involved in phenylpropanoid metabolism [START_REF] Liang | Developmental and environmental regulation of a phenylalanine ammonia-lyase-bglucuronidase gene fusion in transgenic tobacco plants[END_REF][START_REF] Ohl | Functional properties of a phenylalanine ammonia-lyase promoter from Arabidopsis[END_REF]) and a member of the msr (multiple stimulus response) gene family [START_REF] Gough | Developmental and pathogen-induced activation of an msr gene, str 246C, from tobacco involves multiple regulatory elements[END_REF]. All of these genes are strongly expressed during floral development. As has been suggested for these other defense genes, is possible that SFR2 has a defense function at these specific, development stages. Alternatively, SFR2 may control the expression of genes that are required both for defense and during development.

Evidence is accumulating of a role for members of the S gene family in a wide range of processes within the plant, including pollen-pistil interactions [START_REF] Stein | Molecular cloning of a putative receptor protein kinase gene encoded at the selfincompatibility locus of Brassica oleracea[END_REF][START_REF] Nasrallah | Signaling the arrest of pollen tube development in self-incompatible plants[END_REF], plant defense (this work), and development [START_REF] Dwyer | A superfamily of S locus-related sequences in Arabidopsis: Diverse structures and expression patterns[END_REF]. These data suggest a model in which an ancestral recognition system has been adapted and diversified to undertake multiple roles in the plant. Preliminary investigations indicate that the S gene family includes a large number of receptor-like kinase genes, at least in Brassica [START_REF] Kumar | Sequence complexity of the S receptor kinase gene family in Brassica[END_REF]. Further characterization and functional analysis of the different members of this family is expected to provide valuable insight into the mechanisms of cell-cell communication in plants.

METHODS

Plant material

The Brassica oleracea var acephala S 3 homozygous line has been described previously [START_REF] Delorme | Characterization of the S locus genes, SLG and SRK, of the Brassica S 3 haplotype: identification of a membrane-localized protein encoded by the S locus receptor kinase gene[END_REF]. Five hundred thousand plaque-forming units of the primary library were screened with this probe, and hybridizing clones were plaque purified. The SFR2 gene was isolated from a genomic library constructed with DNA of the same line [START_REF] Delorme | Characterization of the S locus genes, SLG and SRK, of the Brassica S 3 haplotype: identification of a membrane-localized protein encoded by the S locus receptor kinase gene[END_REF].

Sequencing was carried out by the dideoxynucleotide chain termination method [START_REF] Sanger | DNA sequencing with chain termination inhibitors[END_REF] using the Sequenase system (version 2.0; U.S. Biochemicals) and customsynthesized oligonucleotides. Sequence data was analyzed using Lasergene sequence analysis software (DNASTAR, London, UK). The transcription start site of SFR2 was determined by RNase protection mapping (Guardian RNase protection assay kit; Clontech, Palo Alto, CA).

Wounding and infiltration of bacteria and inducers into B. oleracea leaves

For the following experiments, B. oleracea plants were grown in a growth chamber at 22°C with 16-hr-light period. Each experiment was repeated at least twice. For the wounding experiments, fully expanded leaves were wounded by rubbing the undersides with abrasive paper. Whole leaves were harvested for RNA extraction. In the bacterial infections, one strain of Escherichia coli (DH5a) and two strains of Xanthomonas campestris pv campestris were used for infections, one wild type (8004) and the second carrying a Tn5 transposon insertion in the hrp gene cluster (8004::A2), which results in a hrp phenotype [START_REF] Arlat | Xanthomonas campestris contains a cluster of hrp genes related to the larger hrp cluster of Pseudomonas solanacearum[END_REF]. Pathogenicity tests were carried out by infecting leaves in which a small notch had been cut at the end of a vein [START_REF] Gough | Cloning of two endoglucanase genes of Xanthomonas campestris pv. campestris: analysis of the role of the major endoglucanase in pathogenesis[END_REF]. With the X. c. campestris wild-type strain, symptoms appeared after 5 days and later spread to the rest of the leaf. No symptoms were observed following infection with the X. c. campestris 8004::A2 or E. coli DH5a. The effect of bacterial strains on SFR2 expression was assessed by infiltrating, using a syringe, bacterial suspensions of 10 7 , 10 8 , or 10 9 cells mL -l into an area of approximately 2 to 3 cm 2 of the leaf through a small hole made with a needle. The infiltrated area was marked with a pen, and only this part of the leaf was harvested for RNA extraction. Analysis of the growth of the two strains in the infiltrated area over a four day period showed that, although the pathogenic strain 8004 had multiplied by a factor of 10 3 , strain 8004::A2, which produced no visible symptoms, had multiplied only 10-fold. Salicylic acid (SA), SA analogs, abscisic acid (ABA), and methyl jasmonate (MeJA) were purchased from Sigma or Aldrich and infiltrated into leaves by the same procedure used for the bacterial suspensions. Control infiltrations were carried out using the carrying solution minus the chemical inducer. This was 5 mM phosphate buffer, pH 7, for SA and SA analogs, 5 mM phosphate buffer, pH 7, 0.053% ethanol for ABA, and water for MeJA.

RNA gel blot analysis

RNA was extracted from a range of tissues. The developmental stage of anthers was determined by fluorescence microscopy observation of 4',6-diamidino-2-phenylindolestained microspores and approximately correlated with bud length. Buds of less than 3 mm contained tetrads of microspores, 5-mm buds contained free uninucleate microspores, 5-to 7-mm buds contained both uninucleate and binucleate microspores, 8mm buds contained binucleate microspores, and 9-to 10-mm buds contained trinucleate microspores while open flowers contained mature pollen grains. Total RNA was extracted by the method of [START_REF] Jackson | Influence of ionic strength, pH and chelation of different metals on isolation of polyribosomes from tobacco leaves[END_REF]. Twenty-five micrograms per lane of total RNA was separated on formaldehyde gels and stained with ethidium bromide to check that equal amounts of RNA had been loaded. The RNA was then transferred to nylon filters for hybridization. Equal transfer was controlled by visualizing RNA with ethidium bromide. DNA probes were prepared using a random priming DNA labeling kit (Boehringer Mannheim). Filters were prehybridized and hybridized at 42°C in 50% formamide, 6 x SSC (1 x SSC is 0.15M NaCl, 0.015M sodium citrate), 0.5% SDS, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin, and herring sperm DNA at 100 µg mL -1 buffer. Filters were washed twice for 20 min in 0.1 x SSC and 0.1% SDS at 50°C. Autoradiographs were scanned for densitometry using an Arcus scanner (Agfa, St. Quentin-en-Yvelines, France), and the data were analyzed with Scan Analysis software (Biosoft, Cambridge, UK).

Construction of an SFR2::uidA reporter gene fusion, plant transformation, and analysis of ß-glucuronidase activity.

A 1520-kb DNA fragment containing the SFR2 promoter region was generated by polymerase chain reaction amplification with two specific oligonucleotides: 5'-GGTGTCGACTTGTGTTCTTGTTTTTTC-3' and 5'-GTCAAGCTTACTACCACCCGACC-3'. Restriction sites (SalI and HindIII) incorporated into the ends of the oligonucleotide sequences were used to insert the SFR2 promoter upstream of the uidA reporter gene in the binary vector pBI101 [START_REF] Jefferson | GUS gene fusions: b-Glucuronidase as a sensitive and versatile gene fusion marker in higher plants[END_REF], and the construct was transferred into Agrobacterium tumefaciens LBA4404. Tobacco plants (Nicotiana tabacum cv Xanthi) were transformed by the leaf disc method [START_REF] Horsh | A simple and general method for transferring genes into plants[END_REF], and transformants were selected on Murashige and Skoog (MS) medium [START_REF] Murashige | A revised medium for rapid growth and bioassays with tobacco tissue cultures[END_REF]) containing kanamycin at 200 µg mL -1 and cefotaxime at 350 µg mL -1 . Transgenic plants (T 1 ) were self-fertilized, and seeds (T 2 ) were collected and sown on MS medium containing kanamycin (400 µg mL -1 ). Seedlings were grown in vitro on MS medium for 4 weeks and then transferred to soil in a growth chamber at 25°C with a 16-hr-light period. The infection experiments were performed on 8-week-old kanamycin-resistant T 2 plants, with two plants per experimental condition. The bacterial suspensions were infiltrated at 10 8 cells mL -1 into tobacco leaves by the syringe infiltration procedure [START_REF] Pontier | hsr203J, a tobacco gene whose activation is rapid, highly localized and specific for incompatible plant/ pathogen interactions[END_REF]. Three independent transformants were analyzed, and a similar pattern of expression was observed for all three transformants. All R. solanacearum strains were provided by C. Boucher [START_REF] Boucher | Transposon mutagenesis of Pseudomonas solanacearum: isolation of Tn5-induced avirulent mutants[END_REF] and have been described previously [START_REF] Pontier | hsr203J, a tobacco gene whose activation is rapid, highly localized and specific for incompatible plant/ pathogen interactions[END_REF]. GUS activity was assayed fluorometrically or histochemically on fresh tissue using either 4-methylumbelliferyl b-D-glucuronide (Sigma) or X-gluc (5-bromo-4-chloro-3-indolyl-b-D-glucuronide, Clontech), respectively, as the substrate [START_REF] Jefferson | GUS gene fusions: b-Glucuronidase as a sensitive and versatile gene fusion marker in higher plants[END_REF]. All experiments were repeated at least twice. (C) Seeds from siliques that were 1 to 2, 6 to 7 or 7 to 8 cm long, 3-day-old seedlings, leaves from field cultivated plants, and root tissue from 3-week-old plants.

The sizes of SFR2 transcripts are indicated on the right. Fully expanded leaves were wounded by rubbing the undersides with abrasive paper. RNA was prepared at different times after treatment (0 to 4 hr), and an RNA blot was probed either with the SFR2 probe (SFR2) or a PAL10 probe (PAL; [START_REF] Dong | Induction of Arabidopsis defense genes by virulent and avirulent Pseudomonas syringae strains and by a cloned avirulence gene[END_REF]. (A) and (B) Fully expanded leaves infiltrated with either wild type (8004 strain) or hrp mutant (8004::A2 strain) bacteria suspended at a concentration of 10 7 , 10 8 , and 10 9 cells mL -1 . RNA was prepared from the infiltrated tissue at different times after infection (0 to 48 hr) and an RNA blot was probed with the SFR2 probe, a PAL10 probe (PAL), and a basic chitinase probe (CHT; [START_REF] Rasmussen | Cloning and characterization of a pathogen-induced chitinase in Brassica napus[END_REF]. (C) Fully expanded leaves infiltrated with water alone. Time in hours is indicated above the gels. Fully expanded leaves were infiltrated with 1 mM SA, 100 mM ABA or 50 mM MeJa.

RNA was prepared at different times after treatment (0 to 24 hr), and RNA blots were probed with the SFR2 probe. Control plants (C) were treated with the carrying solution minus the inducer (5 mM phosphate buffer, pH 7, for SA, 5 mM phosphate buffer, pH 7, 0.053% ethanol for ABA, and water for MeJa) and tissue was harvested after 6 hr. Numbering is relative to the first base of the ATG codon, which is shown in italics.

The transcription start site, as determined by ribonuclease protection analysis, is indicated with a bent arrow. Motifs with significant similarity to previously identified cis acting elements are underlined or overlined. These include CAAT and TATA boxes, an SA-response element (TCA-1, [START_REF] Goldsbrough | Salicylic acid-inducible binding of a tobacco nuclear protein to a 10 bp sequence which is highly conserved amongst stress-inducible genes[END_REF], an elicitor-responsive element (ELI box 3, Ohl et al., 1990), wound-responsive elements (WUN, [START_REF] Matton | Identification of cis-acting elements involved in the regulation of the pathogenesis-related gene STH-2 in potato[END_REF]PI WUN, Palm et al., 1990), a MeJaresponsive element (box 1, [START_REF] Mason | Identification of a methyl jasmonate-responsive domain in the soybean vspB promoter[END_REF], the heat shock element (HSE, [START_REF] Gurley | Transcriptional regulation of the heat-shock response: a plant perspective[END_REF]Key, 1991), a G-box element (G box, Nagao et al., 1993), an auxin-responsive element (TGA-1, Nagao et al., 1193), and a gibberelin-responsive element (GARE, [START_REF] Sutliff | Gibberellin treatment stimulates nuclear factor binding to the gibberellin response complex in a barley a-amylase promoter[END_REF]. A short region of near identity to part of the Heat Shock Protein (HSP) 27 promoter (Hsp27) is also underlined. Also shown is a region of the promoter where four sequences (indicated by arrows) are repeated directly downstream. The EMBL accession number for the SFR2 promoter region is X98521. 57.767.467.379.618.325.9SRK 6 67.856.152.959.316.521.4ARK178.268.677.269.715.922.6ARK277.867.392.968.717.4 20.3ARK383.468.879.480.917.723.8RLK125.425.125.725.728.615.4RLK426.426.427.528.527.035.3 a

The results of the comparisons are expressed as percentage similarity which is calculated as follows: 100 times the number of matched amino acids divided by the sum of the length in amino acids of the aligned region, plus the number of gaps introduced to optimize the alignment.

b Above the diagonal: similarity between pairs of S domains; below the diagonal: similarity between pairs of kinase domains. SRK 6 is from B. oleracea (S 6 haplotype; [START_REF] Stein | Molecular cloning of a putative receptor protein kinase gene encoded at the selfincompatibility locus of Brassica oleracea[END_REF], and ARK1, ARK2, ARK3 [START_REF] Dwyer | A superfamily of S locus-related sequences in Arabidopsis: Diverse structures and expression patterns[END_REF][START_REF] Tobias | An Arabidopsis thaliana gene with sequence similarity to the S-locus receptor kinase of Brassica oleracea[END_REF], RLK1 and RLK4 are from Arabidopsis [START_REF] Walker | Receptor-like protein kinase genes of Arabidopsis thaliana[END_REF].

  extracted from leaves of field cultivated B. oleracea plants was reverse transcribed and the resulting cDNA was cloned into l Zap II (Stratagene, La Jolla, CA). A polymerase chain reaction probe was generated by amplification of genomic DNA sequences of the S 3 homozygous B. ol. var acephala line with two oligonucleotides c o r r e s p o n d i n g t o c o n s e r v e d r e g i o n s o f S L G 2 9 ( 5 ' -CAGAGATGAAACTGGGTTACGACC-3' and 5'-AAATCACACAACCCGTCCC-3').

Figure 1 .

 1 Figure 1. Tissue-specific and developmental regulation of SFR2 mRNA accumulation in B. oleracea. RNA gel blot analysis of SFR2 mRNA in a range of tissues at different stages of development. (A) Flower buds of different lengths (given in mm) and stigma, anther, ovary, petal and sepal from open flowers.

Figure 2 .

 2 Figure 2. Wound-inducible accumulation of SFR2 and PAL mRNA in B. oleracea leaves.Fully expanded leaves were wounded by rubbing the undersides with abrasive paper. RNA was prepared at different times after treatment (0 to 4 hr), and an RNA blot was probed either with the SFR2 probe (SFR2) or a PAL10 probe (PAL;[START_REF] Dong | Induction of Arabidopsis defense genes by virulent and avirulent Pseudomonas syringae strains and by a cloned avirulence gene[END_REF].

Figure 3 .

 3 Figure 3. Induction of SFR2, PAL and chitinase mRNA accumulation following infection of B. oleracea leaves with the bacterial pathogen X. c. campestris.(A) and (B) Fully expanded leaves infiltrated with either wild type (8004 strain) or hrp mutant (8004::A2 strain) bacteria suspended at a concentration of 10 7 , 10 8 , and 10 9 cells mL -1 . RNA was prepared from the infiltrated tissue at different times after infection (0 to 48 hr) and an RNA blot was probed with the SFR2 probe, a PAL10 probe (PAL), and a basic chitinase probe (CHT;[START_REF] Rasmussen | Cloning and characterization of a pathogen-induced chitinase in Brassica napus[END_REF]. (C) Fully expanded leaves infiltrated with water alone. Time in hours is indicated above the gels.

Figure 4 .

 4 Figure 4. SFR2 mRNA accumulation in B. oleracea leaves infiltrated with the saprophytic bacterium E. coli. Fully expanded leaves were infiltrated with E. coli strain DH5a at a concentration of 5x10 8 cells mL -1 or with water alone.

Figure 5 .

 5 Figure 5. Effects of chemical inducers on SFR2 mRNA abundance.Fully expanded leaves were infiltrated with 1 mM SA, 100 mM ABA or 50 mM MeJa. RNA was prepared at different times after treatment (0 to 24 hr), and RNA blots were probed with the SFR2 probe. Control plants (C) were treated with the carrying solution minus the inducer (5 mM phosphate buffer, pH 7, for SA, 5 mM phosphate buffer, pH 7, 0.053% ethanol for ABA, and water for MeJa) and tissue was harvested after 6 hr.

Figure 6 .

 6 Figure 6. Nucleotide sequence of the promoter region of SFR2.Numbering is relative to the first base of the ATG codon, which is shown in italics. The transcription start site, as determined by ribonuclease protection analysis, is indicated with a bent arrow. Motifs with significant similarity to previously identified cis acting elements are underlined or overlined. These include CAAT and TATA boxes, an SA-response element (TCA-1,[START_REF] Goldsbrough | Salicylic acid-inducible binding of a tobacco nuclear protein to a 10 bp sequence which is highly conserved amongst stress-inducible genes[END_REF], an elicitor-responsive element(ELI box 3, Ohl et al., 1990), wound-responsive elements (WUN,[START_REF] Matton | Identification of cis-acting elements involved in the regulation of the pathogenesis-related gene STH-2 in potato[END_REF] PI WUN, Palm et al., 1990), a MeJaresponsive element (box 1,[START_REF] Mason | Identification of a methyl jasmonate-responsive domain in the soybean vspB promoter[END_REF], the heat shock element (HSE,[START_REF] Gurley | Transcriptional regulation of the heat-shock response: a plant perspective[END_REF] Key, 1991), a G-box element (G box, Nagao et al., 1993), an auxin-

Figure 7 .

 7 Figure 7. Time course of SFR2 promoter activation after bacterial infection in transgenic tobacco plants. GUS activity was analyzed in locally-infiltrated leaves with water or with three different isolates of R. solanacearum.: Dhrp (no visible symptoms), K60 (disease symptoms) or GMI1000 (HR).

Figure 8 .

 8 Figure 8. Histochemical localization of GUS gene expression in leaves after bacterial infiltration of transgenic tobacco plants. Tobacco leaves were locally-infiltrated with water (A) or with one of three R. solanacearum strains: (B) Dhrp (no visible symptoms), (C) K60 (disease symptoms) or (D) GMI1000 (HR response). Black line indicated the borders of the infiltrated area.

TABLETable 1 .

 1 Comparison of the percent amino acid similarity a between the sequence of SFR2 and those of selected receptor-like kinases of the S gene family b .

	SFR2SRK 6 ARK1 ARK2 ARK3 RLK1 RLK4SFR2

ACKNOWLEDGMENTS

We thank Drs. Michael J. Daniels (John Innes Centre, Norwich, UK), Matthieu Arlat, and Christian Boucher (INRA-CNRS, Toulouse, France) for supplying bacterial strains, Drs. Fred Ausubel (Harvard Medical School, Cambridge, MA), Henriette Giese (Risø National Laboratory, Roskilde, Denmark), Jérome Giraudat (CNRS, Gif-sur-Yvette, France) and José-Sanchez Serrano (Centro Nacional de Biotecnologia, Madrid, Spain) for supplying DNA probes, Drs. Charles Manceau, Bernard Fritig and Serge Kaufmann for helpful discussions, and Richard Blanc and Hervé Leyral for technical assistance. M.P. and J.M.C. are members of the Institut National de la Recherche Agronomique, D.R. is a member of the Centre National de la Recherche Scientifique.


B Lawson

K Aguir

T Fiorido

V Martini-Laithier

R Bouchakour

Ch Reynard-Carette

M Bendahan

Skin Alcohol perspiration Measurements Using MOX Sensors

Keywords: metal Oxide sensor, gas sensor, ethanol detection, perspiration, transdermal alcohol

The aim of this study is to develop the first system based on chemical MOX sensors for monitoring ethanol on the skin after consumption. Thus, non-invasive measurements to monitor alcohol concentration and their correlation with blood and breathe Alcohol become possible. Transdermal alcohol emissions by perspiration have been investigated during clinical

trials to demonstrate the relevance of this method. First, three commercial MOX sensors were used after calibration in respect of the thermodynamic conditions of the skin surface. Then, six volunteers have been selected for clinical trials with two cohorts of BAC target: 0.5 g/l and 0.8 g/l, and the three different sensors have been integrated in a wristband. We observed that the skin emits ethanol concentrations and we found a consistent correspondence between the kinetics of blood, breath and perspiration. MOX sensors can reliably estimate whether a drinker consumed a low or significant amount of alcohol. After validating this method, we developed our sensors based on tin dioxide thin sensitive layers with thickness of 50nm.

Sensitive properties of the films to ethanol were studied in the conditions of the skin surface. The sensor shows a higher response to ethanol (r = 7.8 for 50 ppm) with relative humidity of 50% and a lower consumption (55 mW) than commercial sensors (MICS 5524: 65 mW).

A C C E P T E D M

A N U S C R I P T

Introduction

Alcohol is one of the most important factors of avoidable death across the world, and represents a high socio economic cost due to the alcohol related incidents and health concerns (Alcoholism, alcoholic cirrhosis, cancer of upper aero digestive tracts, etc…), which continue to increase [START_REF] Castillo-Manzano | An assement of the effects of alcohol consumption and prevention policies on traffic fatality rates in the enlarged EU. Time for zero alcohol tolerance[END_REF][START_REF] Rahman | Chapter Six -Recent advances in nicotinic receptor signaling alcohol abuse and alcoholism[END_REF][START_REF] Rundio | Understanding Alcoholism[END_REF]. Currently, the most commonly used alcohol detection methods are the analysis of biological fluids such as blood, breath, or urine [START_REF] Thungon | Advances in developing rapid, reliable and portable detection systems for alcohol[END_REF][START_REF] Verster | The breathtaking thruth about breath alcohol readings of zero[END_REF][START_REF] Drummond-Lage | Correlation between blood alcohol concentratration (BAC), breath alcohol concentration (BrAC) and physchomotor evaluation in a clinical monitored study of alcohol intake in Brazil[END_REF]. However, these detection techniques have a limit of efficiency by not allowing continuous monitoring or passive detection of the alcohol concentration in a non-invasive way. There is a growing need to monitor indirectly the blood alcohol level of people who have jobs at risks, or for abstention from alcohol for medical or legal reasons [START_REF] Chan | Smart wearable systems: current status and future challenges[END_REF][START_REF] Kim | Noninvasive alcohol monitoring using a wearable tattoo-Based iontophoreticbiosensing system[END_REF].

Breathalyzers are widely used to determine BAC (Blood Alcohol Concentration) indirectly, thanks to breath sample collection. Henry's law allow to estimate BAC with a factor of 2100 comparing to BrAC (Breath Alcohol Concentration), but its results can be alter by humidity, temperature, and individuals traits or by environmental factor [START_REF] Adrian | The measurement of breath alcohol concentration and the inherent physiology of the lung[END_REF]. One of the research areas currently being investigated, is the detection of trace of alcohol on biological fluids existing around the skin [START_REF] Venugopal | Clinical evaluation of a novel interstitial fluid sensor system for remote continuous alcohol monitoring[END_REF][START_REF] Vinu Mohana | Continuous minimallyinvasive alcohol monitoring using microneedle sensor arrays[END_REF][START_REF] Gamella | A novel non-invasive electrochemical biosensing device for in situ determination of the alcohol content in blood by monitoring[END_REF][START_REF] Swift | Direct measurement of alcohol and metabolites[END_REF].

The interstitial fluid located just below the skin (around 500 µm of the stratum corneum) have an ionic composition similar to blood, and has been recently studied for alcohol monitoring.

The device is composed of an assembly of pyramidal structures with micro-needles incorporating Pt and Ag wires, each with a micro cavity opening and integrating alcohol

A C C E P T E D M A N U S C R I P T
oxidase enzyme for the detection of ethanol in the interstitial fluid. However this device is invasive even if it is very low scale [START_REF] Venugopal | Clinical evaluation of a novel interstitial fluid sensor system for remote continuous alcohol monitoring[END_REF][START_REF] Sawdon | Capillary dynamics and the interstitial fluid-lymphatic system[END_REF][START_REF] Thiele | Relationship between skin temperature and the insensible perspiration of the human skin[END_REF].

Skin is considered as the largest organ in the body with around 1.5 m 2 surface area in adults and the water loss (insensible perspiration) of the human skin shows a wide variations between 3 and 25 µg H20 cm -2 min -1 on the forearm [START_REF] Thiele | Relationship between skin temperature and the insensible perspiration of the human skin[END_REF]. Thus, since several decades the increasing of noninvasive means for human lives safety, created great interest in research field of volatile organic compounds (VOCs) detection in human body for wearable sensor applications. The literature reported up to 400 VOCs emitted by human skin including ethanol. Indeed, alcohol consumption generates the increase of ethanol concentration emitted by skin [START_REF] Mochalski | Emission rates of selected volatile organic compounds from skin of healthy volunteers[END_REF]. After ethanol ingestion, it is metabolized principally by the liver and a little part is removed in pure state by exhaled air or breath and also through the skin in proportion of 1 % of consumed alcohol reported by Nyman and Palmlov [START_REF] Marques | Evaluating Transdermal Alcohol Measuring Devices[END_REF][START_REF] Anderson | The kinetics of transdermal ethanol exchange[END_REF].

Recent works used iontophoresis and electrochemical biosensing methods for determining the BAC by monitoring ethanol concentration in sweat (sensible perspiration) induced by pilocarpine via iontophoresis and amperometric detection of ethanol [START_REF] Gamella | A novel non-invasive electrochemical biosensing device for in situ determination of the alcohol content in blood by monitoring[END_REF]. The biosensor is composed by alcohol-oxidase enzyme and the Prussian blue electrode transducer. We notice the correlation between the BAC and the values measured by the biosensor, however the device is combined with a bulky non-wearable iontophoretic system, and required calibrating the devices [START_REF] Kim | Noninvasive alcohol monitoring using a wearable tattoo-Based iontophoreticbiosensing system[END_REF]. Besides other electrochemical biosensors based on iontophoresis and amperometric detection of ethanol on a wearable temporary-tattoo platform have been developed to detect alcohol on sweat [START_REF] Kim | Noninvasive alcohol monitoring using a wearable tattoo-Based iontophoreticbiosensing system[END_REF][START_REF] Gamella | A novel non-invasive electrochemical biosensing device for in situ determination of the alcohol content in blood by monitoring[END_REF]. Skin emitted also insensible perspiration which enable to detect trace of alcohol and correlate with Blood Alcohol Concentration. Indeed, water is lost from the skin in two ways: constant, not visible, unnoticeable sweat in vapor phase from epithelial surface (insensible perspiration)

A C C E P T E D M A N U S C R I P T
and sweat in liquid phase secreted by sweat glands [START_REF] Marques | Evaluating Transdermal Alcohol Measuring Devices[END_REF][START_REF] Anderson | The kinetics of transdermal ethanol exchange[END_REF]. Thus, insensible perspiration has been studied in literature and allowed the development of both wearable devices (SCRAM and WrisTAS) for measuring traces of alcohol on the skin [START_REF] Anderson | The kinetics of transdermal ethanol exchange[END_REF][START_REF] Hawtworne | Transdermal alcohol measurement: a review of the literature[END_REF][START_REF] Widmark | Principles and Applications of Medico Legal Alcohol Determination[END_REF][START_REF] Dubowski | Absorption, distribution and elimination of alcohol: Highway safety aspects[END_REF]. The SCRAM is the ankle bracelet currently used in USA by judicial system for monitoring alcohol consumption by measuring ethanol in insensible perspiration. These devices are composed by electrochemical sensors, are wearable and provide a reliable transdermal alcohol concentration. However they are bulky and need regularly recalibration [START_REF] Marques | Evaluating Transdermal Alcohol Measuring Devices[END_REF][START_REF] Hawtworne | Transdermal alcohol measurement: a review of the literature[END_REF].

In this document we will discuss a first wearable metal oxide gas sensors which enable noninvasive alcohol monitoring on the skin. Our work consisted in the development of Metal OXide (MOX) sensor capable to detect ethanol in insensible perspiration. The metal oxide gas sensors are interesting because of their small size, low power consumption, continuous and noninvasive measurements, good sensitivity and short response time and can be integrated in embedded and autonomous device [START_REF] Liu | A survey on gas sensing technology[END_REF][START_REF] Kim | Highly sensitive and selective gas sensors using p-type oxide semiconductors: overview[END_REF][START_REF] Yan | Synthesis of SnO2-ZnO heterostructured nanofibers for enhanced ethanol gas-sensing performance[END_REF].

In the first stage, we monitored continuously transdermal alcohol concentration (TAC) with commercial MOX microsensors and we correlated these data with blood (BAC) and breath (BrAC) alcohol concentration, during clinical tests. With the aim to perform the clinical trials, we selected 3 commercial MOX microsensors with good sensitivity to ethanol and we integrated them in electronic bracelet.

After demonstrating that the ethanol is emitted through the skin in an amount sufficient to be detected by the MOX sensors, we developed the first MOX multisensors for ethanol monitoring through the skin, more sensitive and with the possibility of selective detection.

The sensitive material of SnO2 thin film were deposited by RF reactive magnetron sputtering on new transducers developed at the IM2NP Laboratory. MOX Sensor properties will be investigated under ethanol exposure with interfering as humidity.

A C C E P T E D

M A N U S C R I P T 2. Experiment

2.1.Experimental Setup

An innovative and automated sensor calibration system was designed and built thanks to the APPRISE (Personal Assistant of Health Prevention) project from Aix-Marseille University (Foundation A*MIDEX) in order to test and study the sensor performances under ethanol exposures. The experimental setup (Figure 1) was divided into three parts:

-Generation and control bench of ethanol, acetone and humidity vapors. Ethanol vapor is generated by permeation system with different stages of dilutions via several automated mass flow controllers (Figure 1a). Ethanol concentrations have been studied between 1 ppm and 100 ppm with relative humidity of 50 % -An environmental chamber with temperature control and an integrated test cell made in our laboratory to characterize and calibrate the sensors in the presence of various ethanol concentrations (Figure 1b) -A control and data acquisition systems (Figure 1c).

The gas flow rate is fixed at 250 sccm. Ethanol vapor is carried in the test chamber placed in the environmental chamber at 33 °C which corresponds to the average temperature of the skin surface. The sensor resistances are measured directly by three Keithley 2450 sourcemeters with a constant current biased sensor.

A C C E P T E D M A N U S C R I P T
At a concentrations of 80 and 100 ppm, we observe two response levels, probably due to the sensors saturation, hence the non-repeatability of the measurements.

2.3.Clinical trials 2.3.1.Subjects and criteria

All protocols were approved by the Ethics Committee and the National Agency for Safety of Medicines and health Products (ANSM) and conducted according to Good Clinical Practices and all subjects gave written informed consent before starting the trials. A total of six Caucasian healthy adult alcohol drinkers (Alcohol consumption between 3 to 14 standard glasses/week) composed of three men and three women were recruited and financially compensated for their participation. Participants were recruited according to several inclusion criteria. These characteristics included for example the age (between 22 and 28), a current or past psychiatric disorder history, substance dependence history (no smoker or <5 cig/day), a body mass index between 19 to 28 kg/m 2 . Demographic characteristics are given in table 2.

2.3.2.Trial description

Trial lasted one day for each participant. Subjects were asked to fast after midnight on the day of trial and it was confirmed via self-report on the morning of trial. Subjects' urine and breath samples were collected to perform drug, alcohol and pregnancy tests immediately upon arrival at the pharmacology center of the Timone hospital at 7: 00 am. Then the subjects took a breakfast low in lipid and fitted 2 wristbands, the first integrated MOX gas sensors and the other wristband included humidity and temperature sensors. The wristbands were placed on the subjects wrist so the sensors were almost directly facing the skin (about 2 mm). Volunteers began alcohol administration 30 min after putting the wristbands on, allowing them to create a base line of MOX sensors. Alcohol consumption lasted for 10 to 15 min before we began the breath and blood sample collection to determine the Breath Alcohol Consumption (BrAC) and

A C C E P T E D M A N U S C R I P T
the Blood Alcohol Consumption (BAC), while MOX sensor measured alcohol emitted through the skin by insensible perspiration (TAC). A meal was provided four hours after alcohol administration. The test ended ten hours after alcohol consumption and the subjects BAC controlled at 0.0 g/l for at least 3h minimum. Then the wristbands were removed. The Subjects confirmed they were in total sobriety before leaving.

2.3.3.Alcohol administration protocol

The alcohol was given orally, which is the most described protocol found in literature and which corresponds to the usual practice of alcohol ingestion. During the trial two different targets of BAC will be studied: BAC of 0.5 g/l and 0.8 g/l, in compliance with the limits laid down by French legislation. The main alcohol beverages consumed by each subject consisted of a strong alcohol at 40 % (Vodka) mixed with a fruit juice. The amount of alcohol consumed by each subject was calculated using the Widmark formula which is widely used but not absolute and therefore may have some variability [START_REF] Widmark | Principles and Applications of Medico Legal Alcohol Determination[END_REF]. The final volume of beverage was between 300 to 400 ml, depending on the subjects' corpulence. Transdermal alcohol concentration were performed thanks to wristband which integrated a circuit of MOX gas sensors (TGS 8100, MICS 5524, CCS801) on a PCB included in little cavity with a size of 0.5 cm 3 . The cavity is opened on the top of the wrist skin surface. The mobile test bench (Figure 3) and this specific wristband (Figure 4) have been especially designed for clinical trials. MOX gas sensors were polarized with a constant current (I) and the conductance variation were measured by three Keithley 2450 sourcemeters continuously all the time of clinical trial for each subjects. In this study we will present only the response of the central sensor. A thickness of 50nm of the sensitive layer was measured by Dektak-6 profilometer. In fact, the thickness of the sensitive layers plays a major role in improving the sensitivity of the sensors. The choice of a thickness of 50 nm follows from previous studies and literature [START_REF] Aguir | Role of the active layer thickness on the sensitivity of WO3 gas sensors[END_REF]. During the deposition process a residual gas pressure and the Ar/O2 mixture were fixed respectively at 22 µbar and 40 %/60 %. Finally an annealing under dry air at 500 °C for 12 hours was done in order to improve the film oxidation, the nano-crystallization, and the stability of the sensitive layer.

.Blood Alcohol Concentration

After collecting and analyzing blood samples from each subjects, we observed that only the subjects n°6 (Male) and n°1 (Female) actually achieved the expected blood alcohol levels, see figure 6. We focused the results on the subjects N ° 1 and N ° 6, because it is for these subjects that the measurement protocol was the most successful and which reached the blood alcohol concentration expected for (respectively 0.5 g / l and 0.8 g / l), following ingestion of the alcohol dose. (peak BAC: 0.8 g/L). Transdermal Alcohol Concentration is based on MICS sensor conductance value. The first measurements were not very satisfactory as the sensors already detected the ethanol in the ambient air coming from the glass before consumption. Also, we had to improve the measurement protocol by isolating the sensor from the environment of the room by improving the tightness of the bracelet to be sure only the ethanol obtained by perspiration will be detected.

After drinking alcohol, blood tests and breath analyses showed a rapid rise in blood alcohol levels to a maximum of about 30 min for subject N°1 and 45 min for subject N°6. Regarding subject N°6, it takes about 6h to stop traces of alcohol in the blood and in the breath. In the same way we will note an increase in the conductance of the MOX sensors, followed by a slow decrease in both cases. This variation can therefore be correlated with the concentration of alcohol in the blood. These results show that the quantity of ethanol evaporated from the skin is sufficient quantity to be detected by MOX sensors by perspiration and the blood alcohol level (BAC) can be estimated after sensors calibration. We can also observed that the TAC alcohol curve (G) is right shifted from the BrAC and BAC alcohol curves, with a peak delay of about 80 minutes and took longer to reach zero. This behavior of the sensor responses is certainly due to the metabolism of the human body at the skin level. The same kinetic behavior was observed in alcohol elimination by urine [START_REF] Batt | Adsorption, distribution and elimination of alcohol[END_REF].

A C C E P T E D M A N U S C R I P T
After demonstrating that the alcohol emitted by the skin after consumption can be detected by the MOX sensors and can be correlated with the blood alcohol Concentration, we will develop our MOX sensors based on SnO2 sensitive layer, that will be more sensitive to ethanol, with a more stable baseline and with lower power consumption than the current sensors.

3.2.SnO2 Gas sensor results

Working temperature

Gas sensors were characterized using the ethanol test bench previously described in section "experimental setup 2.1". Figure 9 illustrates the sensor dependence to the temperature using different concentrations (10 and 50 ppm) of ethanol with relative humidity of 0% and a gas flow rate of 250 sccm. The responses increase with temperature up to 280°C and seems to be decreasing after this temperature. It is well known that the response has a maximum for a given temperature. Indeed the increase of the sensitivity can be attributed to the fact that the thermal energy supplied is high enough to overcome the activation energy barrier to enable the redox reaction and a significant increase in electron concentration induced from the sensing reaction. Here we had chosen to operate at 280 °C which represents our optimal temperature. Therefore we set the operating temperature at 280 °C, the power required to reach this temperature is 55 mW.

3.2.2.SnO2 sensors sensitive properties to ethanol

The sensors were exposed to different concentration of ethanol. Ethanol concentrations have been studied between 1 ppm and 100 ppm with relative humidity of 50 % during 30 s. The gas flow rate is fixed at 250 sccm with a temperature of 33 °C. The sensors were polarized at 1 V and the power consumption was fixed at 55 mW for the three sensors, which corresponds to the optimum working temperature around 280 °C. Figure 10 shows the SnO2 typical sensor response to ethanol. It can be seen that the sensor shows a high sensitivity, a baseline stability and fast response and recovery. These results can be attributed to the transducer used and to the sensitive layer properties. In fact, in metal oxide ethanol sensors as SnO2 sensors, the interaction between ethanol molecules and lattice oxygen O0 x can be described by the reaction (in Kröger-Vink) as below [START_REF] Liu | A Highly Sensitive and Fast-Responding SnO2 Sensor Fabricated by Combustion Chemical Vapor Deposition[END_REF]:

6O0 x + CH3CH2OH(ads) = 2CO2 (g) + 3H2O (g) + 6V0 + 12e -
In the same way, the interaction between ethanol molecules and adsorbed species as O2 -and O2 2-can be described as:

CH3CH2OH (ads) + 3O2 -(ads) = 2CO2 (g) + 3H2O(vap) +3e - CH3CH2OH (ads) + 3O2 2-(ads) = 2CO2 (g) + 3H2O(vap) +6e -
As a result, the conductivity of the sensor will increase with the ethanol concentration due to the electrons produced by these reactions, as SnO2 is an n-type semiconductor. with a relative humidity of 50%. It's easy to see that our sensor is more sensitive than the commercial sensors tested and therefore will have a better detection of the transdermal alcohol on the surface of the skin.

However the SnO2 sensors can be more or less sensitive to ethanol and interference gases.

The reactivity between gases and the sensitive layer is a key part of the selectivity.

Conclusion and perspectives

This study reports real possibilities to detect ethanol vapor through perspiration across the skin using MOX sensors. The results confirm that the two concentrations of ethanol absorbed by the volunteers (0.5 g/l and 0.8 g/l) can be discriminated. So, we have seen that it is possible to monitor non-invasively the blood alcohol levels. The responses were slightly shifted in time due to the body metabolism processes. The delay between BAC peak and TrAC peak has varied between 25 and 80 min. To improve MOX sensor sensitivity, we developed metal oxide sensor based on SnO2 sensitive nanostructured material. The sensors characterization have demonstrated a high response to ethanol and a fast response and recovery times compared to commercial sensors. These sensors should allow a better detection of alcohol on the skin surface. Additional studies will improve the selectivity of the sensors by the use of all three sensors of our chip.

Acknowledgment:

We wish to thank AMU-AMIDEX for their financial support. We also thanks the "Centre de Pharmacologie Clinique et d'Evaluations Thérapeutiques (CPCET)" of La Timone

Figure 1 . 2 . 2 .Table1:

 122 Figure 1. Experimental setup for ethanol microsensors characterization and calibration: Vapor generation (a), Test chamber (b) and Data acquisition system (c).

Figure 2 .

 2 Figure 2. Comparison of commercial sensor responses under different ethanol concentrations

4 .

 4 Sample and Data collectionBlood samples were collected to determine and monitor the alcohol pharmacokinetics appending on the amount ingested. The Blood samples were taken at precise time: T15' (15 minutes after the end of consumption), T30', T45', T60', T1h30, T2h, T2h30, T3h, T4h, T6h, T8h, T10h. The total amount of blood was 84ml. The determination of BAC in the plasma was performed by gas chromatography (GC) coupled to an Agilent 6850 Flame Ionization Detector (FID). The chromatographic column is a Macherey-Nagel capillary column OPTIMA WAX 30m-0.25mm-0.25µm.The Breath Alcohol concentration were measured at: T15'(15 minutes after the end of consumption), 30',45', 60', 1h30, 2h, 2h30, 3h, 4h, 6h, 8h, 10h using Alcotest® 7410 Plus from Dräger.

Figure 3 .

 3 Figure 3. Mobile test bench for clinical trials

Figure 4 . 2 . 4 . 1 .

 4241 Figure 4. Bracelet with the three MOX sensors 2.4.SnO2 sensors for transdermal ethanol monitoring

Figure 5 Figure 5 . 3 .

 553 Figure 5 shows the XRD patterns of SnO2 thin film. The diffraction peaks show tetragonal rutile

Figure 6 .

 6 Figure 6. Blood Alcohol Concentration Peaks by GC-FID analysis according to clinical trial subjects

2 .

 2 Comparison of BAC, BrAC, TACThe sensor conductance variations have been carried out continuously during the clinical trials. Figures7 and 8illustrate the evolution of Blood, Breath and Transdermal Alcohol Concentrations as function of time respectively for Subjects N°1 (peak BAC: 0.5 g/l) and N°6

Figure 7 .

 7 Figure 7. Comparison of BAC, BrAC, and TAC for subject N°1

Figure 8 .

 8 Figure 8. Comparison of BAC, BrAC, and TAC for subject N°6

Figure 9 .

 9 Figure 9. SnO2 Sensor response versus temperature for ethanol concentrations with RH of 0%

Figure 10 .

 10 Figure 10. Sensor response for ethanol concentrations, from 1 ppm to 100 ppm

  Otherwise our SnO2 sensor response was compared to commercial sensors, used during the selection of commercial MOX sensors (TGS 8100, MICS 5524, CCS 803, CCS 801, TGS 2620, TGS 2602) for clinical trials, see figure 11.

Figure 11 .our

 11 Figure 11. Comparison of sensors response to ethanol

Bruno

  Lawson is a PhD student since 2015 at the microsensors team of IM2NP of Aix Marseille University and CNRS, France. He started his research on chemical microsensors for gas and vapor detection of transdermal alcohol on the skin after consumption. He got a Master degree in microelectronic detection systems at Aix Marseille University after doing internship on study of gas sensors for air quality control in the same team from March to July 2015. He was graduated in license of applied Physics and electronics in 2012 from the Catholic University of Togo. Currently, his research activity is focused on chemical sensor with highly sensitive and selective for ethanol toward medical diagnosis. Khalifa Aguir is a Professor at Aix Marseille University, France. He was awarhjded his Doctorat d'Etat ès Sciences degree from Paul Sabatier University, Toulouse 1987. He was during the 16 years the head of Microsensors Group at the Institute of Materials Microelectronic Nanosciences of Provence (IM2NP-CNRS) at Aix-Marseille University, Marseille. His principal research interests are directed towards metal -oxide thin films gas microsensors, selectivity, signal treatments and multivariable analysis, modeling, flexible current sensors. He is the author of over 200 publications and international conferences. Referee of several scientific journals, he has conducted numerous expertise and he is a member of scientific committees of international conferences. Tomas Fiorido is a Research Engineer at Aix-Marseille University, France. He received his PhD from Paul Cezanne University (France) in2009. The research performed within the framework of this thesis concerned the realization and the characterization of new gas, light and temperature sensors based on polymer material layered over flexible supports. Currently, his research activities are focused on the realization organic or inorganic thin films for sensors, on the engineering of gas microsensors, smoke detector and on the miniature calorimeter for nuclear. Virginie Laithier is a lecturer at Aix-Marseille University, France. She received her PhD in 2012 at the Aix Marseille University. Since 2008, she has been with the Sensors Group at the Institute of Materials, Microelectronics and Nanoscience of Provence (IM2NP). The research performed within the framework of her thesis concerned thermal simulations and microfluidic gas sensor fabrication and characterization. Her current work involves gas sensors microtechnologies. Rachid Bouchakour is a Professor at Aix Marseille University, France. He was awarded his PHD in Physics (1988) and Habilitation à Diriger les Recherches (1995) from Paris 6 University. He was Director of the Institute of Materials Microelectronic Nanosciences of Provence (IM2NP-CNRS) at Aix-Marseille University, Marseille, from 2006 to 2015, Research Director of Polytech 'Marseille, from 2007 to 2012 and many other directions on the teaching side. He has conducted numerous expertise in various French organizations ANR, ANRT, MSTP and AERES. His principal research interests are directed towards microelectronics, physics of semiconductor devices, modeling and simulation, reliability of silicon technologies, nonvolatile innovative memories. He is the author of over 190 publications, international conferences, patents, books and he is a member of scientific committees of IEEE international conferences. Christelle Reynard Carette is Assistant professor at Aix-Marseille University, France. She is currently the Co-head of the Microsensors Team at the Institute of Materials Microelectronic Nanosciences of Provence. She received her PhD in 2001 and HDR in 2011 at the Aix Marseille University. Her activity field is dedicated to instrumentation, measurement, metrology,

Table 2 :

 2 Subject demographic characteristics and parameters of alcohol consumption

	Subject	1	2	3	4	5	6
	sex	Female	Male	Female	Male	Female	Male
	Age (years)	25.5	24.5	26.0	25.0	23	24.5
	Weight (Kg)	54	70	47	75	58	77
	Target (BAC)	0.5	0.5	0.5	0.5	0.8	0.8
	Alcohol consumed (ml)	47.0	54.0	73.0	79.0	83.5	129.0

Marc Bendahan is a Professor at Aix-Marseille University, France. He is currently the head of the Microsensors Team at the Institute of Materials Microelectronic Nanosciences of Provence and Lecturer at the Institute of Technology. He was awarded his PhD degree from AMU in 1996 with a thesis on shape memory alloys thin films for micro-actuator applications.

He is specialized in thin films preparation and characterization for applications in microsystems. His principal research interest is now directed towards the conception, realization and electrical characterization of microsensors: gas microsensors based on inorganic metal-oxides and organic materials, flexible temperature and pressure sensors.