Thierry Allario

Aude Tixier

Hosam Awad

Cédric Lemaire

Nicole Brunel-Michac

Eric Badel

Severien Tete

Jean-Louis Barigah

J.-L Julien

Pierre Peyret

Ewa J Mellerowicz

Cedric Lemaire

Nicole Brunel

Têtè S Barigah

Jean-Louis Julien

Herve Cochard

Stephane Herbette
email: stephane.herbette@uca.fr

PtxtPME1 and homogalacturonans influence xylem hydraulic properties in Poplar

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

Introduction

According to the cohesion-tension theory, sap flows from root to shoot in vascular plants through xylem conduits under negative pressure [START_REF] Tyree | The cohesion-tension theory of sap ascent. Current controversies[END_REF][START_REF] Steudle | The cohesion-tension mechanism and the acquisition of water by plant roots[END_REF]. This negative pressure makes the liquid sap metastable, with a risk of vaporization by cavitation inside conduits that considerably increase in plants during drought. Cavitation makes the conduits air-filled or embolized, and they can no longer transport water. During extreme drought, Abbreviations -4CL, 4-(hydroxy)cinnamoyl CoA ligase; AFM, atomic force microscopy; HG, homogalacturonan; PEMs, pectin methylesterases; PLC, percentage loss of xylem conductance; PM, pit membrane; RH, relative humidity; VC, vulnerability to cavitation; WAK, wall associated kinases.

extensive embolism blocks water transport leading to organ or plant death [START_REF] Barigah | Water stress-induced xylem hydraulic failure is a causal factor of tree mortality in beech and poplar[END_REF]. The resistance to drought-induced embolism is thus considered as an important adaptive trait for the woody species [START_REF] Brodribb | Hydraulic failure defines the recovery and point of death in water-stressed conifers[END_REF][START_REF] Choat | Global convergence in the vulnerability of forests to drought[END_REF][START_REF] Lens | Embolism resistance as a key mechanism to understand adaptive plant strategies[END_REF]. A global analysis on the relationship between vulnerability to cavitation (VC) and drought resistance pointed out the narrow hydraulic safety margin at which trees commonly operate [START_REF] Choat | Global convergence in the vulnerability of forests to drought[END_REF], inferring that research is needed on xylem VC under the current climate changes. Yet the genetic determinants of this critical trait are poorly understood.

In angiosperm species, sap flows through xylem vessels, which are formed by the perforated xylem vessel elements. To move from one vessel to another, the sap must flow via pits in the secondary cell wall. These pits represent an important limiting factor for the rate of water flow in the xylem [START_REF] Wheeler | Inter-vessel pitting and cavitation in woody Rosaceae and other vesselled plants: a basis for a safety versus efficiency trade-off in xylem transport[END_REF][START_REF] Choat | Direct measurements of intervessel pit membrane hydraulic resistance in two angiosperm tree species[END_REF]), but they are also barriers that block air passage between vessels [START_REF] Lens | Embolism resistance as a key mechanism to understand adaptive plant strategies[END_REF]. This ability of pits to prevent the passage of air bubbles is of key importance for VC. Indeed, drought-induced cavitation occurs as a result of air seeding through the pit membrane (PM) separating a sap-filled vessel under tension from an air-filled vessel at atmospheric pressure [START_REF] Cochard | Cavitation in trees[END_REF]). The VC is determined by several quantitative and qualitative pit characteristics, but their respective contribution is still in debate (Choat andPittermann 2009, Lens et al. 2013), which hinders the research on the genetic basis for VC. Considering variability in the porosity of PM and the occurrence of air-seeding in the largest PM pore, the probability of air-seeding into a conduit would increase in a stochastic fashion with its pit area [START_REF] Jarbeau | The mechanism of water-stress-induced embolism in two species of chaparral shrubs[END_REF][START_REF] Wheeler | Inter-vessel pitting and cavitation in woody Rosaceae and other vesselled plants: a basis for a safety versus efficiency trade-off in xylem transport[END_REF][START_REF] Christman | Testing the 'rare pit' hypothesis for xylem cavitation resistance in three species of Acer[END_REF]). This "rare pit" hypothesis is supported by a positive correlation between VC and the average area of pit connecting vessels based on average VC and pit area from around 80 species [START_REF] Wheeler | Inter-vessel pitting and cavitation in woody Rosaceae and other vesselled plants: a basis for a safety versus efficiency trade-off in xylem transport[END_REF][START_REF] Hacke | Scaling of angiosperm xylem structure with safety and efficiency[END_REF]. The vessel pitting is expected to be greater in xylem with short, highly connected and grouped vessels than in xylem with long and solitary vessels, supporting a role for the three-dimensional arrangement and connectivity of vessels in VC [START_REF] Choat | New insights into bordered pit structure and cavitation resistance in angiosperms and conifers[END_REF]. The "rare pit" hypothesis is not supported by other studies, suggesting the importance of qualitative pit features [START_REF] Lens | Testing hypotheses that link wood anatomy to cavitation resistance and hydraulic conductivity in the genus Acer[END_REF][START_REF] Scholz | The evolution and function of vessel and pit characters with respect to cavitation resistance across 10 Prunus species[END_REF]. Within angiosperms, a huge interspecific variation in the PM thickness and porosity exists, and the relationship between these two parameters indicates that thicker PMs have smaller pores and are more resistant to air-seeding [START_REF] Jansen | Morphological variation of intervessel pit membranes and implications to xylem function in angiosperms[END_REF]. The VC also depends on the mechanical behavior of the PM, especially its resistance to stretching that relies on the pit geometry [START_REF] Choat | Changes in pit membrane porosity due to deflection and stretching: the role of vestured pits[END_REF], 2008[START_REF] Sperry | Analysis of circular bordered pit function -I. Angiosperm vessels with homogenous pit membranes[END_REF], Tixier et al. 2014). PM porosity and mechanical properties are also tributary to their biochemistry, but we know little about this, despite recent insights on the biochemical composition [START_REF] Herbette | Immunolabelling of intervessel pits for polysaccharides and lignin helps in understanding their hydraulic properties in Populus tremula x alba[END_REF].

In poplars, the intervessel PM is composed of cellulose microfibrils and contains epitopes of lignins, whereas pectins epitopes are only concentrated in the annulus, i.e. the border area of the PM [START_REF] Plavcova | Heterogeneous distribution of pectin epitopes and calcium in different pit types of four angiosperm species[END_REF][START_REF] Kim | Developmental localization of homogalacturonan and xyloglucan epitopes in pit membranes varies between pit types in two poplar species[END_REF][START_REF] Herbette | Immunolabelling of intervessel pits for polysaccharides and lignin helps in understanding their hydraulic properties in Populus tremula x alba[END_REF]. Homogalacturonans (HGs), which are the most abundant polymers of pectins, can be demethylesterified at the C-6 carboxyl [START_REF] Wolf | Homogalacturonan methyl-esterification and plant development[END_REF] to various degrees by pectin methylesterases (PMEs) [START_REF] Pelloux | New insights into pectin methylesterase structure and function[END_REF]. When HGs are demethylesterified in blocks, intermolecular Ca 2+ bonds are formed between pectins chains. As a result, the pectin mesh forms pectin gels that strengthen pectin structure, which impacts the mechanical and porosity properties of the wall matrix [START_REF] Willats | Pectin: cell biology and prospects for functional analysis[END_REF]. Although pectins epitopes are restricted to the edge of PM, larger pores for air seeding are frequently found at the periphery of the PM [START_REF] Hillabrand | Drought-induced xylem pit membrane damage in aspen and balsam poplar[END_REF]) and our previous studies argue for a critical role of pectins and their methylesterification pattern in VC (Herbette andCochard 2010, Dusotoit-Coucaud et al. 2014). Branches perfused with pectins hydrolyzing enzymes or with Ca 2+ chelators showed an increased VC. We hypothesized that the role of calcium in VC may be dependent on the HG content in the PM and its methylesterification pattern, and consequently on PME activity. By creating Ca 2+ bonds, PME activity would strengthen the pectin meshwork in the PM annulus. This would in turn protect pectins from hydrolysis during pit maturation, reduce pore sizes in PM and increase the annulus resistance to PM stretching. Moreover, PME activity is involved in mechanisms determining the cell dimensions in developing xylem [START_REF] Siedlecka | Pectin methyl esterase inhibits intrusive and symplastic cell growth in developing wood cells of populus[END_REF], and could thus affect xylem organization. We hypothesize that PME could monitor the three-dimensional arrangement and connectivity of vessels and thus the xylem hydraulic properties [START_REF] Choat | New insights into bordered pit structure and cavitation resistance in angiosperms and conifers[END_REF]. Hence, PMEs appear as good candidates for mediating hydraulic properties of xylem at different levels.

Plant functional genomics approach is a powerful tool that permits to characterize biological function of particular genes and to highlight gene interactions in biological processes. In this study, using transgenic aspen and poplar lines, we investigated the role of HG in the hydraulic properties of the PM and the role of a PME gene in the xylem hydraulic traits that relies both on the xylem vessel properties and on the pit properties. For the latter objective, we used the aspen transgenic lines that were either over-expressing or under-expressing PtxtPME1 under the 35SCAMV promoter (35S::PME1, [START_REF] Siedlecka | Pectin methyl esterase inhibits intrusive and symplastic cell growth in developing wood cells of populus[END_REF] and that were thus affected in HG methylesterification. For the former objective, we constructed the poplar transgenic lines overexpressing AnPGII gene coding for polygalacturonase (PG; E.C.4.2.2.2) [START_REF] Capodicasa | Targeted modification of homogalacturonan by transgenic expression of a fungal polygalacturonase alters plant growth[END_REF], which cleaves the 𝛼-1,4-glycosidic bond between the galacturonic acid units of HG, thus that were affected in HG chain length. To specifically target pit primary cell wall and avoid effects on xylem cell expansion, we expressed AnPGII under the control of the Ptxa4CL1 promoter. Populus 4CL1 is a gene encoding 4-(hydroxy)cinnamoyl CoA ligase (4CL) involved in monolignol biosynthesis, which is expressed specifically in developing xylem after xylem cell expansion [START_REF] Lu | Stable and specific expression of 4-coumarate:coenzyme a ligase gene (4CL1) driven by the xylem-specific Pto4CL1 promoter in the transgenic tobacco[END_REF].

Materials and methods

Plant material and culture conditions

The study was performed on transgenic Populus lines modified for expression of genes involved in pectin metabolism. Transgenic lines ectopically over-expressing (line 2B and 7) or under-expressing (by co-supppression, line 5) PtxtPME1 fused with 35S CAMV promoter were generated in Populus tremula × tremuloides clone T89 [START_REF] Siedlecka | Pectin methyl esterase inhibits intrusive and symplastic cell growth in developing wood cells of populus[END_REF]. The generation of the transgenic lines overexpressing the fungal polygalacturonase AnPGII fused the xylem-specific Ptxa4CL1 promoter [START_REF] Lu | Stable and specific expression of 4-coumarate:coenzyme a ligase gene (4CL1) driven by the xylem-specific Pto4CL1 promoter in the transgenic tobacco[END_REF] in Populus tremula × alba clone 717-1B4 is described in a further separate section.

All plants were multiplied in vitro on Murashige and Skoog medium. At 4 cm of height, plants were gradually acclimatized in hydroponic solution and grown in a climatic chamber with 16 h daylight (100 μmol m -2 s -1 ) at 22 ± 1 ∘ C (day) and 19 ± 1 ∘ C (night) with 70 ± 10% of relative humidity (RH). At 30 cm of height, plants were potted into 10-l pots filled with a commercial soil (40% black, 30% brown and 30% blond peat moss, pH 6.1, DUMONA-RN 75-3851 Arandon, Holland) and grown for at least 90 days in the greenhouse with 16 h daylight (350 μmol m -2 s -1 ) at 22 ± 1 ∘ C (day) and 19 ± 1 ∘ C (night) with 60 ± 20% RH. Water supply by dripping was controlled to get between 60 and 100% of field capacity.

We also used xylem material from Populus tremula × alba (clone 717-1B4) plants grown in 10 l pots under three contrasting water regimes, in a previous study [START_REF] Awad | Poplar vulnerability to xylem cavitation acclimates to drier soil conditions[END_REF]. A 10-cm long segment was collected above the one used for analysis of VC. This xylem material was used to investigate the expression of PtxaPME1.

RNA extraction and cDNA synthesis

Xylem tissue was isolated by peeling the bark. The xylem core was then immediately frozen in liquid nitrogen and stored at -80 ∘ C. Tissue was ground in liquid nitrogen and 2 g of ground xylem from each plant were pooled for RNA extraction. RNA was isolated using CTAB (Cetyl Trimethyl Ammonium Bromide) extraction buffer as described by [START_REF] Chang | Arabidopsis ethylene-response gene ETR1. Similarity of product to two-component regulators[END_REF] and was treated with DNase I. First strand cDNAs were synthesized using the Super Script™ III first strand synthesis system (Invitrogen, Paisley, UK) with an oligo-dT according to the manufacturer's instructions.

Generation of 4CL1::PGII poplar lines

The AnPGII cDNA sequence was previously fused with the sequence encoding a signal peptide of PGIP1 from Phaseolus vulgaris, and the resulting fused sequence was cloned in pBI121 [START_REF] Capodicasa | Targeted modification of homogalacturonan by transgenic expression of a fungal polygalacturonase alters plant growth[END_REF]). We amplified the gene fusion from a preparation of this construct using the primers 5 ′ -ATGACTCAATTCAATATCCCAG-3 ′ and 5 ′ -TAACAAGAGGCCACCGAA-3 ′ . The Ptxa4CL1 promoter sequence was amplified from genomic DNA of Populus tremula × alba using the primers 5 ′ -GACG GTGGCATGAACACAAAGC-3 ′ and 5 ′ -TGGCGAGTTTT AGGTGCTCT-3 ′ given in [START_REF] Lu | Stable and specific expression of 4-coumarate:coenzyme a ligase gene (4CL1) driven by the xylem-specific Pto4CL1 promoter in the transgenic tobacco[END_REF]. Finally, the polymerase chain reaction (PCR) products were inserted into PGEM-Teasy vector (Promega, Madison, WS), transformed into DH5𝛼 Escherichia coli strain, and they were sequenced on both strands.

PGIP1-AnPGII and Ptxa4CL1 promoter sequences were cloned into gateway vectors (Gateway ® Cloning, Invitrogen, Carlsbad, CA). Entry clones, resulting from the recombination of AnPGII or Ptxa4CL1 promoter sequences in donor vectors, were transfected into E. coli by heat shock. Transformed E. coli (One Shot ® Mach1™ T1 R, ThermoFisher, Waltham, MA) were grown on selective medium with kanamycin and targeted gene incorporation was verified by PCR and sequencing. Entry clone with Ptxa4CL1 promoter and entry clone containing PGIP1:AnPGII hybrid cDNA sequence were recombined in pK7WG destination vector. The recombined pK7WG vector was transferred to poplar by Agrobacterium-mediated transformation as described in [START_REF] Leple | Transgenic poplars: expression of chimeric genes using four different constructs[END_REF]. Kanamycin-resistant lines were selected and clonally propagated in vitro. Transgene insertions were checked in selected lines by PCR as described by [START_REF] Mai | Contrasting strategies to cope with chilling stress among clones of a tropical tree, Hevea brasiliensis[END_REF].

Expression analysis

Expression levels of PtxtPME1 were assessed by quantitative real time PCR (qPCR) in xylem from poplar plants grown under three contrasting water regimes [START_REF] Awad | Poplar vulnerability to xylem cavitation acclimates to drier soil conditions[END_REF]). The single strand cDNA was used as a template in qPCR as described in [START_REF] Mai | Contrasting strategies to cope with chilling stress among clones of a tropical tree, Hevea brasiliensis[END_REF]. qPCRs were carried out in a iCycler iQ™ (Bio-Rad, Hercules, CA), using platinum Taq DNA polymerase (Invitrogen, Paisley, UK), SYBR Green (Sigma, St. Louis, MO; 1:1000 in 25% dimethyl sulfoxide), and the primers 5 ′ -GAGATGGTTAGGGAGG-3 ′ and 5'-AATTACAGTTCGCCGAG-3 ′ . The program was as follows: 95 ∘ C for 5 min, 40 cycles of 94 ∘ C for 15 s, 55 ∘ C for 20 s and 72 ∘ C for 30 s, followed by a melting curve analysis to check the specificity of fragment amplification. The cDNA expression was normalized according to 18S rRNA expression [START_REF] Nualpun | Molecular cloning of a new cDNA and expression of 3-hydroxy-3-menthylglutaryl-CoA synthase gene from Hevea brasiliensis[END_REF]. The method to calculate the relative expression ratio is described by [START_REF] Pfaffl | A new mathematical model for relative quantification in real-time RT-PCR[END_REF].

Expression of AnPGII was checked by semiquantitative RT-PCR in 4CL1::PGII lines and WT plants grown for 2 months in the greenhouse in order to allow wood to be developed. PCR was performed on the first strand cDNA with the primers 5 ′ -ATGACTCAATTCAATATCCCAG-3 ′ and 5 ′ -AGTAGAG CATTTCGCCTTGC-3 ′ . The program was as follows: 95 ∘ C for 2 min, 30 cycles of 94 ∘ C for 30 s, 60 ∘ C for 20 s and 72 ∘ C for 30 s. To check for similar quantity of cDNA, we also amplified the 18S rRNA cDNA as a control with the same conditions.

Leaf water potential and growth measurements

Midday leaf water potential (Ψ md ) was measured for each plant on two mature leaves per plant with a pressure bomb (PMS instrument, Albany, OR) at solar noon just before harvesting plants for hydraulic measurements. Plant size and stem diameter were measured, respectively, with a meter tape and a caliper.

Native embolism and specific conductivity

Entire stem of each plant was defoliated, harvested, wrapped in moist paper, sealed in humid black plastic bag and stored at 4 ∘ C for hydraulic measurements at the laboratory (within 2 days). Native embolism and specific conductivity were measured using XYL'EM ® (Bronkhorst, Montigny-les-Cormeilles, France), as described by Herbette and Cochard (2010). The xylem hydraulic conductivity was measured on 0.35 m long stem segments prepared from the upper part of the plant. We checked that there was no open vessel at both ends of segments using the low pressure air injection test [START_REF] Ewers | Techniques for measuring vessel lengths and diameters in stems of woody plants[END_REF]. Then, we measured the xylem area on cross sections prepared from both ends. The xylem-specific conductivity of the sample was determined by dividing the hydraulic conductivity by the mean xylem area, taking then into consideration differences in xylem cross-sectional area.

Vulnerability curves

Vulnerability to xylem cavitation was measured on 0.38 m long stem segments located just below the part used for specific conductivity measurement using the Cavitron technique [START_REF] Cochard | A technique for measuring xylem hydraulic conductance under high negative pressures[END_REF][START_REF] Cochard | Evaluation of a new centrifuge technique for rapid generation of xylem vulnerability curves[END_REF]. Four to eight samples, each from a different individual tree, were measured for each transgenic line. The technique uses a centrifugal force that increases the water tension in the xylem segment, and the resulting loss of hydraulic conductance is measured at the same time. The curve of percentage loss of xylem conductance (PLC) vs xylem water tension represents the sample's vulnerability curve. Xylem pressure (P) was first set to a reference pressure (-0.5 MPa) and the maximal conductance (K max ) of the sample was determined. The xylem pressure was then set to a more negative value for 3 min and the conductance (K) of the sample was remeasured. The PLC of the sample was then computed as:

PLC = 100 × ( 1 -K∕K max ) (1)
The procedure was repeated for more negative pressures (with -0.25 or -0.5 MPa step increments) until PLC reached at least 90%. The PLC curves were fitted using the following sigmoid function:

PLC = 100∕ ( 1 + e S(P-P50)∕25 ) (2)
where P 50 is the xylem pressure causing 50% loss of conductance and s is the slope of the curve at this point [START_REF] Pammenter | A mathematical and statistical analysis of the curves illustrating vulnerability of xylem to cavitation[END_REF].

To test for the role of calcium in VC in transgenic plants, stem segments were infiltrated a 10 mM sodium phosphate solution at pH 10 as described in Herbette and Cochard (2010) before Cavitron analyses.

Light and transmission electron microscopy

For light and electron microscopy, we only used fresh samples to avoid any modification of the PM structure as reported [START_REF] Li | Intervessel pit membrane thickness as a key determinant of embolism resistance in angiosperm xylem[END_REF][START_REF] Zhang | Bordered pits in xylem of vesselless angiosperms and their possible misinterpretation as perforation plates[END_REF]. Samples were prepared below the basal part of stem segments that were used to measure VC, as described in [START_REF] Herbette | Immunolabelling of intervessel pits for polysaccharides and lignin helps in understanding their hydraulic properties in Populus tremula x alba[END_REF].

Light microscopy

Semi-thin (2 μm) cross sections were stained with polychromatic Toluidine blue dye and were observed with an optical microscope (Zeiss Axioplan 2, Zeiss, Jena, Germany). Pictures were taken and analyzed using IMAGEJ software [START_REF] Schneider | NIH image to ImageJ: 25 years of image analysis[END_REF]. Vessels were isolated by automatic segmentation and we measured their frequency (F v , number of vessel per wood area, mm -2 ), their diameters (D v , μm), the fraction of wood area they occupied (the total lumen area per wood area, A v ), the solitary vessel index (S v , ratio of total number of solitary vessels to total vessel groupings) and the contact fraction (F c = fraction of the total vessel wall perimeter in contact with another vessel). Analyses were performed on four plants per line, each plant represented by 33 to 64 cross sections for vessels parameters.

P b = 1/3*E/(1 -v 2 )*T m 3 /(D p 2 -D a 2 ) 2 *L p 𝜖 - Maximum PM strain at P b 𝜀 maxPb = 𝜀 (r = 0) =-(3/8)*([1 -v 2 ]/E)*(R 2 /T m 2 )*P b

Transmission electron microscopy

Ultra-thin (70 nm) cross sections were prepared and observed as described in [START_REF] Dusotoit-Coucaud | Hydrolase treatments help unravel the function of intervessel pits in xylem hydraulics[END_REF].

Aperture length (D a ), pit chamber depth (L p ), pit diameter (D p ) and PM thickness (T m ) were measured as described in [START_REF] Tixier | Modelling the mechanical behaviour of pit membranes in bordered pits with respect to cavitation resistance in angiosperms[END_REF], Table 1, Fig. 1). These measurements were performed on images with pits showing two apertures, and neither shrinkage nor stretching of the PM as these modifications can disturb the PM structure [START_REF] Li | Intervessel pit membrane thickness as a key determinant of embolism resistance in angiosperm xylem[END_REF][START_REF] Zhang | Bordered pits in xylem of vesselless angiosperms and their possible misinterpretation as perforation plates[END_REF]). On the same images, the primary wall thickness (T pw ) was measured between adjacent vessels, near the pits. Analyses were performed on four plants per line, each plant represented by 27 to 66 pits for pit parameters.

Mechanical model of PM deflection

The flexural rigidity of the membrane (D), the pressure needed to deflect the membrane against the pit aperture (P b ) and the mechanical strain of the PM beyond the aspiration pressure (𝜀 b ) were estimated using the mechanical model developed and described in [START_REF] Tixier | Modelling the mechanical behaviour of pit membranes in bordered pits with respect to cavitation resistance in angiosperms[END_REF].

Input and output variables of this model are listed in Table 1.

Results

Expression of 4CL1::PGII in poplar increases VC

Five 4CL1::PGII transgenic lines (3.1, 5.1, 10.1, 11.1 and 12.1) showed an AnPGII expression in xylem tissues by RT-PCR (Fig. 2). Line 10.1 exhibited the highest relative transcript levels, lines 3.1 and 11.1 were intermediate, whereas lines 12.1 and 5.1 were the lowest expressing lines. Although the Populus 4CL1 promoter offers a developmental control of over-expression, which is limited to late stages of xylogenesis, this restriction makes the detection of enzymatic activity in the protein extract impossible [START_REF] Lu | Stable and specific expression of 4-coumarate:coenzyme a ligase gene (4CL1) driven by the xylem-specific Pto4CL1 promoter in the transgenic tobacco[END_REF]).

No differences in height growth, leaf water potential, native embolism or specific hydraulic conductivity between 4CL1::PGII lines and wild-type (WT) plants were observed; however, xylem pressure causing 50% of loss of hydraulic conductance (P 50 ) was significantly higher (less negative) in the transgenic lines (Table 2). Xylem vulnerability curves that were used to calculate P 50 are presented in Fig. 3B. These curves clearly show that the transgenic lines exhibit significantly higher loss of conductance over a large spectrum of xylem pressures than the WT plants. This indicates that the expression of polygalacturonase during late stages of xylogenesis caused an increase in xylem VC.

Xylem hydraulic traits in 35S::PME1 transgenic lines

To investigate the role of PME in the hydraulic properties of xylem, we used the 35S::PME1 expressing aspen (lines 7, 2B and 5). Unlike the 4CL1::PGII expressing plants, the over-or under-expression of PtxtPME1 induced a reduction in height growth (Table 2). Leaf water potential and native embolism was not altered in transgenic plants when compared to WT plants (Table 2). However, the specific hydraulic conductivities were lower in transgenic lines both over-and under-expressing PtxtPME1 than in WT plants (Table 2). Moreover, stems from 35S::PME1 lines 2B and 5 were significantly less vulnerable to cavitation than WT (P 50 values in Table 2). Line 7 showed intermediate P 50 values (Table 2), which is consistent with a lower change in the degree of methylesterification in this line and in the xylem structure [START_REF] Siedlecka | Pectin methyl esterase inhibits intrusive and symplastic cell growth in developing wood cells of populus[END_REF]. The lower vulnerability of the 35S::PME1 lines compared to WT can be clearly seen by analyzing the P 50 values and the vulnerability curves over an entire spectrum of xylem pressure (Table 2, Fig. 3A). Thus, with regard to the xylem vulnerability, 35S::PME1 and 4CL1::PGII transgenic lines behave in the opposite way, the former is less vulnerable, while the latter is more vulnerable to hydraulic loss under water stress. The low levels in native embolism and lack of difference between the transgenic lines and the respective WT (Table 2) exclude an effect of the native embolism on the VC measurements. If a set of plants have higher level of embolism, then the vulnerability curves would be plotted on the remaining functional vessels, which are the most resistant, resulting in an artificial decrease in VC. It was thus essential to observe similar levels of native embolism across genotypes in order to decipher true differences from artificial shifts in vulnerability curves. To test for the role of pectin-bound calcium in the transgenic lines, the stem segments were infiltrated with a sodium phosphate solution that removes calcium from the wall before Cavitron analyses (Fig. 4). No modification was observed for P 50 values between treated and mock-treated branches in each tested line, suggesting that the calcium removal did not affect their VC.

Although this was not the objective of this study, and 4CL1::PGII lines and 35S::PME1 lines cannot be compared since they were grown and analyzed separately, their respective WT plants showed difference for their VC (Fig. 3) especially for their P 50 values (Table 2).

Xylem structural traits in 35S::PME1 and 4CL1::PGII transgenic lines

To understand how PtxtPME1 and AnPGII could modify the xylem hydraulic traits, we investigated several anatomical traits of the xylem and the pit structures in the respective transgenic lines (Table 3). We found a relationship between PtxtPME1 expression level and vessel frequency (F v ) or vessel diameter (D v ). Indeed, the vessel density increased with PtxtPME1 expression whereas the mean vessel diameter followed an opposite trend. In addition, line 5 had higher solitary vessel index (S v ) and lower contact fraction (F c ) compared to other lines. In contrast, the anatomical parameters of the vessels of the 4CL1::PGII lines were not significantly different from WT.

Investigations on the primary wall thickness and on the intervessel pit structure using transmission electron microscopy analysis were carried out on the transgenic lines that displayed the greatest difference for P 50 with WT (35S::PME1 -lines 2B and 5 and 4CL1::PGIIline 5.1). First, the line 5 showed a thinner primary wall compared to WT (Table 3), while no difference was observed for the other lines. At the pit level, there 3). The lines 2B and 5 showed smaller D p , smaller L p but larger D a and T m . The line 5.1 showed bigger pits than WT plants, with significantly higher values for every parameter measured.

To evaluate the impact of these structural pit modifications, the PM mechanical behavior was estimated using these structural parameters and the mechanical model developed by [START_REF] Tixier | Modelling the mechanical behaviour of pit membranes in bordered pits with respect to cavitation resistance in angiosperms[END_REF]. The maximum PM strain (𝜀 max ) as a function of pressure was different between the 35S::PME1 lines and WT plants, especially with a lower slope of strain for the line 2B (Fig. 5A). The behavior of line 5 was opposite. Thus, the PM was stiffer in the line overexpressing PME1 and less stiff in the line with suppressed PME1 expression, compared to WT. In contrast to these pronounced effects in the 35S::PME1 lines, the mechanical behavior of PM in line 5.1 overexpressing 4CL1::PGII was not affected (Fig. 5B), because the overall pit relative geometry was not altered in this line compared to WT despite the overall increase in size of the pit parameters (Table 3). Important PM mechanical parameters such as experienced strains for a given pressure and pressure required to deflect the PM against pit borders (P b ) were similar (Fig. 5B).

Expression of PME1 in poplars grown under contrasted water regimes

Since expression of PME1 affected several key variables related to xylem hydraulic conductivity in transgenic plants, we were asking if PME1 could have a role in determining xylem hydraulic properties under drought in relation to the level of acclimation to water stress. Therefore, we subjected WT poplar to different water regimes, which resulted in differences in their VC [START_REF] Awad | Poplar vulnerability to xylem cavitation acclimates to drier soil conditions[END_REF], and analyzed the expression level of PtxaPME1 in the xylem of these plants. The PtxaPME1 expression was found to be reduced in poplars grown under drier conditions and displaying lower VC (Fig. 6).

Discussion

We conducted two distinct experiments to investigate the role of HGs in PM hydraulics and the role of a PME gene in the hydraulic properties of the xylem. The respective WT for the 4CL::PGII and 35S::PME1 transgenic lines show differences for P 50 (Table 2) and for some structural traits of xylem vessels or pits (Table 3). This can be explained by their different genetic backgrounds [START_REF] Cochard | Xylem vulnerability to cavitation varies among poplar and willow clones and correlates with yield[END_REF], and by different growth conditions as the VC has been reported to depend on growth conditions (Harvey and Van Den Driessche 1999, Awad et al. 2010, Herbette et al. 2010). The trees were grown in greenhouse, which does not allow an exact reproducibility of conditions between distinct experiments. Such differences between the respective WT did not allow comparing the 35S::PME1 lines with the 4CL::PGII lines.

Role of pectins in PM hydraulic properties

Hydraulic conductivity is a complex trait that is of key importance for plant survival and productivity. Here, we attempted to dissect some of the underlying mechanisms involved in regulation of this trait, using transgenic plants. 4CL::PGII transgenics allowed questioning the importance of HGs in the PM for hydraulic conductivity without its confounding effects on xylem cell growth. The results demonstrated the importance of intact HG in the PM -the transgenic lines all exhibited significant increases vulnerability when fungal polygalacturonase is expressed in fully grown xylem cells.

Investigating the role of HG in PM is a tricky task since the pectin epitopes were only detected in the annulus, a restricted area of the PM [START_REF] Plavcova | Heterogeneous distribution of pectin epitopes and calcium in different pit types of four angiosperm species[END_REF]Hacke 2011, Herbette et al. 2015). When infiltrating the stems with a sodium phosphate solution that precipitates calcium from the wall (Herbette and Cochard 2010), the VC of the lines WT and 2B was not modified as compared to mock-treated stems (Fig. 4), suggesting that pectin-bound calcium does not influence the PM hydraulic properties. This is not surprising since calcium removal did not affect the VC for other hygrophilous species such as Betula and Salix (Herbette and Cochard 2010). Experiments with stem infiltration with a pectin-hydrolyzing enzyme support a role for pectins in VC [START_REF] Dusotoit-Coucaud | Hydrolase treatments help unravel the function of intervessel pits in xylem hydraulics[END_REF], but there was no evidence that it was the pectins located in the PM. Here, the greater VC in 4CL1::PGII plants support a role in VC for PM-located pectins. Many changes were observed in their pit structure (Table 3), but they did not Table 3. Anatomical parameters of xylem vessels and their intervessel pits in the transgenic plants 35S::PME1 and 4CL1::PGII. Vessel parameters were investigated using light microscopy while pit parameters were investigated using electron microscopy. Each value represents the mean ± SE from four plants per line, with 33 to 64 cross sections per plant for vessels parameters and 27 to 66 pits per plant for pit parameters. For each parameter, values not followed by the same letter differ at P < 0.05 (one-way ANOVA followed by Tukey' test or Student's t-test). Vessel parameters: A result in a change in mechanical behavior of the PM as modeled from pit structure (Fig. 5). We assume that AnPGII partly hydrolyzed the pectins that are highly concentrated in the PM annulus [START_REF] Herbette | Immunolabelling of intervessel pits for polysaccharides and lignin helps in understanding their hydraulic properties in Populus tremula x alba[END_REF].

Although the role of this annulus is unclear, it probably ensures the mechanical resistance of PM to strain [START_REF] Plavcova | Heterogeneous distribution of pectin epitopes and calcium in different pit types of four angiosperm species[END_REF]Hacke 2011, Herbette et al. 2015). Thus, weakening the PM by pectin hydrolysis would increase the VC. Moreover, it could also increase PM porosity in the region of annulus which would increase air-seeding.

Unfortunately, measuring the PM porosity is a tricky task, especially for investigating the effect of pectin hydrolysis on pore size. A major concern in such analyses using electron microscopy is the alteration of ultrastructural features during sample preparation [START_REF] Choat | Pit membrane porosity and water stress-induced cavitation in four co-existing dry rainforest tree species[END_REF][START_REF] Pesacreta | Atomic force microscopy of the intervessel pit membrane in the stem of Sapium sebiferum (Euphorbiaceae)[END_REF], Jansen et al. 2008). For scanning or transmission electronic microscopy, sample preparation involves dehydration of tissue (replacing water with ethanol then with resin) that alters subtle features such as pit porosity and the highly hydrated pectin network. Such preparation artifacts would explain the discrepancy between the observed and calculated pore size for air seeding [START_REF] Jansen | Morphological variation of intervessel pit membranes and implications to xylem function in angiosperms[END_REF]). In addition, pits observed under electron microscopy are in a relaxed state, whereas PM pores enlarge by stretching when they undergo a pressure difference across conduits [START_REF] Choat | Changes in pit membrane porosity due to deflection and stretching: the role of vestured pits[END_REF][START_REF] Sperry | Analysis of circular bordered pit function -I. Angiosperm vessels with homogenous pit membranes[END_REF], Tixier et al. 2014).

A solution to these methodological problems is provided by a study employing atomic force microscopy (AFM), in which PMs were examined in non-dried and dried states [START_REF] Pesacreta | Atomic force microscopy of the intervessel pit membrane in the stem of Sapium sebiferum (Euphorbiaceae)[END_REF]). However, this technique is not suitable for measurement series on PMs because it is difficult to access this tiny structure.

Role of PME1 in xylem hydraulic properties

Previous studies indicated that PME1 is a dominant PME in developing wood, expressed mainly in the early and late xylem cell expansion phase, and mediating xylem cell expansion [START_REF] Hertzberg | A transcriptional roadmap to wood formation[END_REF][START_REF] Pelloux | New insights into pectin methylesterase structure and function[END_REF][START_REF] Siedlecka | Pectin methyl esterase inhibits intrusive and symplastic cell growth in developing wood cells of populus[END_REF]). Here, we report that PtxtPME1 mRNA accumulation is reduced in xylem formed under dry conditions and with a low VC (Fig. 6).

An under-expression of PtxtPME1 in transgenic plants also induced a lower xylem VC (Fig. 3). These results suggest that PtxtPME1 might indeed be involved in regulation of wood hydraulic properties and that the action of PME1 can be important at many levels: anatomy, chemistry and mechanical properties of PM, and PM porosity. Since the VC is determined by several quantitative and qualitative structural traits, there are many putative ways for PtxtPME1 to influence the wood hydraulic properties.

Besides, under-expression and ectopic over-expression of PtxtPME1 induced different structural modifications that were associated with decrease in VC and specific hydraulic conductivity (Table 2). What happens to the primary cell wall when the methylester pattern changes are not obvious for explaining the effect on hydraulic properties in 35S::PME1 transgenic poplars. Only the line 5 showed a weak reduction of the primary wall thickness, but this did not result in a reduction of the PM thickness (Table 3) since the opposite trend was observed. The molecular mechanisms occurring in 35S::PME1 transgenic poplars are not known, but previous studies put the light on a role for HG methylesterification in a wall-dependent cell signaling that regulate developmental processes, especially through wall-associated kinases (WAK) pathway [START_REF] Kohorn | Cell wall-associated kinases and pectin perception[END_REF]. Considering this role, we thus propose a functional understanding of the relationship between the PtxtPME1 expression level and the xylem hydraulic properties. For the xylem of the line 2B, the decrease in the specific hydraulic conductivity can be related to the vessels' diameter. Such vessel diameter reduction in the line 2B agrees with a role of PtxtPME1 in inhibiting cell growth of vessel elements [START_REF] Siedlecka | Pectin methyl esterase inhibits intrusive and symplastic cell growth in developing wood cells of populus[END_REF]). The higher PME activity in the line 2B was also shown to stabilize the pectin networks [START_REF] Siedlecka | Pectin methyl esterase inhibits intrusive and symplastic cell growth in developing wood cells of populus[END_REF]). Consequently, the hydrolysis of the PM would be hindered during PM maturation leading to a thicker PM in the line 2B compared to WT plants. This thicker PM may contribute to both the decrease in conductivity, and to the decrease in VC, by increasing the pressure required for air-seeding [START_REF] Jansen | Morphological variation of intervessel pit membranes and implications to xylem function in angiosperms[END_REF]. This is consistent with the fact that a thicker PM drastically reduces the mechanical strain the PM experiences [START_REF] Tixier | Modelling the mechanical behaviour of pit membranes in bordered pits with respect to cavitation resistance in angiosperms[END_REF]. Increase in PtxtPME1 activity affects the overall pit geometry with changes in PM and aperture diameters (D p and D a , respectively). To test the impact of these changes in VC, these pit parameters have been integrated in the mechanical model proposed by [START_REF] Tixier | Modelling the mechanical behaviour of pit membranes in bordered pits with respect to cavitation resistance in angiosperms[END_REF]. PM of the line 2B appeared to experience lower maximum strain than the PM of the WT at equal pressure, which may result in a decrease of VC, as highlighted between species [START_REF] Tixier | Modelling the mechanical behaviour of pit membranes in bordered pits with respect to cavitation resistance in angiosperms[END_REF]).

In the line 5, the lower PtxtPME1 expression level induced an increase in vessel diameter that suggests that the decrease in hydraulic conductivity is not related to an increase in lumen resistance, but rather to an increase in end wall resistance. First, the vessel grouping parameters, i.e. the contact fraction (F c ) and the solitary vessel index (S v ), are significantly lower and higher in the line 5 compared to WT plants. These parameters quantify the actual overlap between neighboring vessels and thus allow estimating the vessel connectivity. F c was shown to be strongly correlated to VC and hydraulic conductivity in a study carried out on Acer species [START_REF] Lens | Testing hypotheses that link wood anatomy to cavitation resistance and hydraulic conductivity in the genus Acer[END_REF], while S v was measured and found to be negatively correlated to VC across different Prunus species [START_REF] Scholz | The evolution and function of vessel and pit characters with respect to cavitation resistance across 10 Prunus species[END_REF]. The changes in F c and S v would thus support the decreases in hydraulic conductivity and VC in the line 5. Moreover, the lower pit diameter (D p ) and pit chamber depth (L p ) values in the line 5, when compared to WT, can also contribute in reducing the conductivity. The pit diameter is directly influencing the area allowing the sap flow between vessel, while L p influences the water flow inside the pit chamber (Sperry andHacke 2004, Schulte et al. 2015). A recent study reported an oxidative stress and tylose formation in the wood of the line 5 [START_REF] Lesniewska | Defense responses in aspen with altered pectin methylesterase activity reveal the hormonal inducers of tyloses[END_REF]. Such ingrowths into the lumen of xylem vessels would contribute to the decrease hydraulic conductance in the line 5, but they cannot explain a lower VC. The oxidative stress in wood would also affect the wall metabolism, including the lignification process [START_REF] Mader | Role of peroxidase in lignification of tobacco cells: I. Oxidation of nicotinamide adenine dinucleotide and formation of hydrogen peroxide by cell wall peroxidases[END_REF], and would be an explanation for the structural changes in wood observed for this line.

In addition to providing support for the importance of HGs in PM for hydraulic conductivity, and identifying PME1 as a genetic component of hydraulic properties, our study contributes to the understanding of trades-off in xylem functions. We confirm the supposed role of structural traits in the trade-off between safe xylem (low VC) and efficient xylem (high conductivity), especially T m , F c and L p [START_REF] Lens | Testing hypotheses that link wood anatomy to cavitation resistance and hydraulic conductivity in the genus Acer[END_REF].

While transgenic approach is a powerful mean to test for gene function, there are limitations due to strong and ectopic gene expression. In addition to the transgenic plants investigated here, we also used transgenic plants over-expressing PtxtPL1-27 [START_REF] Biswal | Aspen pectate lyase PtxtPL1-27 mobilizes matrix polysaccharides from woody tissues and improves saccharification yield[END_REF]), a pectate lyase that degrades HG, and is expressed at the onset of secondary wall formation during xylem differentiation. Despite a severe growth reduction, we did not observe changes in xylem hydraulic properties (Appendix S1), which can be explained by compensatory effects occurring early during xylogenesis. Previously, Arabidopsis polygalacturonase mutants were found to develop an inflorescence stem with a greater VC than WT plants [START_REF] Tixier | Arabidopsis thaliana as a model species for xylem hydraulics: does size matter?[END_REF], highlighting a role for this gene and pectins in VC. However, how this gene affects the xylem hydraulic has not been clarified. We thus produced the 4CL1::PGII transgenic plants using the promoter Ptxa4CL1 that allows a late and a xylem-specific transgene expression. In addition to target the transgene expression, this expression pattern reduces the likelihood of inducing side effects with compensatory effects during xylogenesis. This is supported by a normal growth associated to changes in VC in 4Cl1-PGII plants. However, the AnPGII expression is limited in time and space, and it is thus extremely challenging to detect increase in PG activity [START_REF] Lu | Stable and specific expression of 4-coumarate:coenzyme a ligase gene (4CL1) driven by the xylem-specific Pto4CL1 promoter in the transgenic tobacco[END_REF], that could weaken the effect on the phenotype. Untangling the genetic basis of VC is an extremely intricate process requiring meticulous genotyping and novel minute phenotyping techniques.

To conclude, our study demonstrates a role for HGs in the xylem hydraulic properties through their effect on the characteristics of vessels and intervessel pits, while the presence of pectins in PM and their contributions to hydraulic properties are debated in the literature. Moreover, it identifies the xylem-specific PME1 gene as a genetic component of the xylem hydraulic properties, and it puts in light the complexity of the genetic control of these hydraulic properties, as PME1 would influence them in different ways.

Fig. 1 .

 1 Fig. 1. A transverse section of a bordered pit as observed using TEM. Black arrows show the measured pit dimensions (Table 1): D a , pit aperture diameter; D p , pit membrane diameter; L p , pit chamber depth; T m , pit membrane thickness. Scale bar = 2 μm.

Fig. 2 .

 2 Fig. 2. Expression of AnPGII in the 4CL1::PGII transgenic plants. Transcript accumulation was analyzed by RT-PCR as described in the section Materials and methods, then the PCR products were separated on agarose gels. The name of the line is indicated below each lane, and the name of the target gene is indicated to the right of the panel. 18S rRNA was used as the loading control. This result is representative of three replicates.

Fig. 3 .

 3 Fig. 3. Xylem vulnerability curves of stems from the transgenic plants expressing 35S::PME1 (A) or 4CL1::PGII (B). The percentage loss of conductance with decreasing xylem water potential is represented for the 35S::PME1 and 4CL1::PGII lines (closed symbols) and their corresponding WT plants (T89 and 717-1B4, respectively, open symbols). Curves represent the fitted data according to equation from Pammenter and Van Der Willigen (1998, see section materials and methods). Each data point represents the mean value from five to nine samples and bars represent standard errors. For each pressure point, values from transgenic plants significantly different from wild-type plants are marked of an asterisk (Student's t-test, P < 0.05).

Fig. 4 .

 4 Fig. 4. Effect of calcium removal on the xylem VC of stems from the transgenic plants expressing 35S::PME1. The xylem pressure inducing 50% of PLC (P 50 ) is deduced from vulnerability curves of stems perfused ( ) or not perfused ( ) with a sodium phosphate solution chelating calcium. The analyses were performed on the transgenic lines 2B and 7 and on the WT plants (T89). Data are mean values from three to nine samples and bars represent standard errors. No significant difference was found between treatments in each genotype (Student's t-test, P < 0.05).

  v , fraction of area occupied by vessels; D v , vessel diameter; F c , contact fraction; F v , vessel frequency per mm 2 ; S v , solitary vessel index. Pit parameters: D a , pit aperture diameter; D p , pit diameter; L p , pit chamber depth; T m , PM thickness. Pit parameters are illustrated in Fig. 1.

Fig. 5 .

 5 Fig. 5. Maximum PM strain (𝜀 max ) plotted against pressure for the transgenic plants expressing 35S::PME1 (A) or 4CL1::PGII (B). The 𝜀 max was calculated using input values of pit parameters measured in Table3for the lines expressing 35S::PME1 (line 2B and 5, dotted and dashed lines, respectively), the line 4CL1::PGII 5.1 (dotted line) and their respective WT plants (T89 and 717-1B4, respectively, continuous line). Black dots represent the value of membrane maximum strain reached for P 50 mean value in each line.

Fig. 6 .

 6 Fig. 6. RT-qPCR analysis of the relative abundance of the PtxaPME1 transcripts under different water regimes in WT plants. Relative transcript abundance was expressed as the ratio of gene expression level in moderately water stressed (𝜓 pd = -0.32) or severe water-stressed plants (𝜓 pd = -0.60) compared with plants grown under optimal water regime (𝜓 pd = -0.19). Data are means values from three replicates and bars represent standard deviations. For each water condition, mean values of predawn water potentials (𝜓 pd ) measured on plants prior to RT-qPCR analysis were indicated, with standard deviations in brackets.

Table 1 .

 1 List of parameters used in modeling the PM mechanical behavior with symbols, units, definitions and equations.

		Symbol	Unit	Definition	Equation
	Constants	E	Pa	Young's modulus of PM
		V	-	Poisson's ratio = 0.3
	Input variables	D p	μm	Pit membrane diameter
		L p	μm	Distance from the PM surface to the
				inner pit aperture
		D a	μm	Pit aperture diameter
		T m	μm	PM thickness
	Output variables	D	MPa nm 3	PM flexural rigidity	D = (E*T m	3 )/12(1 -v 2 )
		P b	MPa	Pressure required to deflect the
				membrane against the aperture

Table 2 .

 2 Plant height and physiological parameters in stems and leaves related to water transport in transgenic plants 4CL1::PGII and 35S::PME1 as compared to wild-type. Values represent means (±SE) from 6 to 10 plants per line. For each parameter, values not followed by the same letter differ at P

	50 , the xylem pressure
	followed by Tukey's test). WT, wild-type plants; P
	< 0.05 (one-way ANOVA

p , D a and T m (Table

Acknowledgements -The authors thank Christelle Boisselet, Pierre Conchon and Romain Souchal (PIAF laboratory) for their technical assistance, and Christelle Blavignac and Claire Szczepaniak from the CICS platform (Clermont-Ferrand, France) for running the TEM observations. We gratefully acknowledge the gift of the PGIP1 -AnPGII fused construct from Professor Felice Cervone

Author contributions

T.A., A.T. and H.A. performed most of the experiments, analyzed the data and wrote some parts of the manuscript; C.L. reviewed thoroughly the manuscript; N.B. contributed and supervised the microscope analyses; T.B. contributed and supervised the ecophysiological analyses; J.-L.J. and P.P. help in defining the research project; E.M. provided the PME transgenic plants and helped for the transgenic plant analyses; H.C. contributed in analyzing the ecophysiological data; S.H. supervised the whole research project, especially the students (A.T., T.A., C.L. and H.A.), and contributed to most of the analyses, and wrote most part of the paper.

Supporting Information

Additional Supporting Information may be found in the online version of this article:


Abdelmalek Mouazer

Karima Sedki

Rosy Tsopra

Jean-Baptiste Lamy

Visualization of drug interactions for supporting medication review

Keywords: Visual analytic, Medication review, Drug interactions

The number of elderly patients with multimorbidities has considerably increased since recent years. These patients are often polymedicated and at higher risk of safety incidents due to polypharmacy. To reduce polypharmacy, one solution is Medication review (MR). MR aimed at optimizing drug treatments, is unfortunately not very frequent in practice. Indeed, consulting the properties of 5-20 drugs in parallel is a cognitively complex task. It is therefore necessary to develop software for supporting MR. The existing tools only list alerts concerning drugs and their interactions. The objective of our work is to facilitate the pharmacist's access to the medical knowledge necessary for drug interactions. Using visual analytics, we propose an interactive tool that synthesizes information on drug interactions. It shows an overview of drug treatment and make it visually accessible by the pharmacist to facilitate MR.

Introduction

The number of elderly patients with multimorbidities has considerably increased since recent years. These patients are often polymedicated (i.e. they receive 5 drugs or more) and are consequently at higher risk of safety incidents due to polypharmacy. Polypharmacy is both a public health and an economic issue.

To reduce polypharmacy, one solution is medication reviews (MR). A MR is "a structured interview with the patient, carried out by the pharmacist in collaboration with the general practitioner (GP) with the aim of optimizing patient care" [START_REF] Masson | Le bilan de médication en soins primaires chez les plus de 65 ans[END_REF]. In many countries, including France, MR is reimbursed by health insurances.

However, the simultaneous consultation of the properties of 5-20 drugs is a long, difficult and cognitively complex task. This often discourages pharmacists, and in practice few MR are performed. It is therefore necessary to develop software for supporting MR.

Current systems for MR display a flat list of all drug interactions; when interactions are numerous, the list occupies multiple pages and it is tedious to read and to summarize because the user has to scroll continuously.Other systems offer a tablebased approach to summarize drug information [START_REF] Koskela | User perspectives on an electronic decision-support tool performing comprehensive medication reviews -a focus group study with physicians and nurses[END_REF], but it becomes visually less readable as the number of drugs and drug properties increases. In a previous work [START_REF] Lamy | Using visual analytics for presenting comparative information on new drugs[END_REF], we showed that visual analytics can help to present drug properties when comparing similar drugs.

The objective of this work is to propose an interactive tool relying on visual analytics for providing an at-a-glance overview of all interactions of the drugs taken by a given patient, in order to facilitate the pharmacist's access to the necessary medical knowledge during MR.

Method

To carry out this project, we exploited the Thériaque drug database (http://www.theriaque.org/), one of the four accredited databases in France, to extract drug interactions and contraindications. In our approach, we were inspired by the approach of A.Galopin for the medication management of poly-pathological patients [START_REF] Séroussi | Using Therapeutic Circles to Visualize Guideline-Based Therapeutic Recommendations for Patients with Multiple Chronic Conditions: A Case Study with GO-DSS on Hypertension, Type 2 Diabetes, and Dyslipidemia[END_REF]. In the context of prescription support, the method he proposed consisted in representing recommended drug association using "therapeutic circles": candidate drugs are represented by small circles, organized in a large circle. Then, recommended bitherapies are represented by edges connecting two drugs. We adapted this idea by presenting the drugs taken by the patient, with edges representing the drug-drug interactions. In addition, we represented drug-disease interactions by the color of the drug circles.

For implementation, we worked with Python and the Django framework for the development of a web application. We generated vector images (SVG) for the visualization of the analysis of drug interactions.

Results

Figure 1 shows the proposed interface showing drug-drug and drug-disease interactions.

First, the drugs are represented by small circles. The first letters of the drug name are displayed in the circle; the full name appears by moving the mouse over the drug. The system detects interactions between the patient's condition and the drug. According to the level of risk, it colors the circles of the drugs according to a gradient of color ranging from yellow for the drugs presenting a minor risk to red for the contraindicated drugs. The other circles are green. Second, the system detects all drug-drug interactions and represents each interaction by an edge connecting the two drugs involved. These edges are colored according to the four levels of interaction with a gradient of colors ranging from yellow (for minor interactions) to red (for major interactions). In addition, the system offers the user the possibility of accessing the explanatory text of interactions and contraindications in an interactive way by clicking on the segment representing the interaction or on the drug presenting an alert.

In Figure 1, one can immediately identify two drug-disease interactions, and see that there are many (15) drug-drug interactions, 5 of them being of moderate seriousness. The visualization allows identifying at a glance the drugs with the highest number of interactions (here, Oxomemazine) and those involved in the most serious interactions (Bisoprolol). The pharmacist may decide to reevaluate the risk-benefit balance of these drugs, and suggest to stop them or reduce their posology.

Discussion and conclusion

The system we propose provides an at-a-glance overview of drug-drug and drugdisease interactions of all drug in a given drug order. It allows information to be accessed quickly in a concise and efficient manner.

This type of representation in a circle has the advantage of synthesizing interactions well. It is effective in displaying all interactions and thus makes it possible to visually detect the most risky and potentially dangerous drugs, e.g. those with the highest number of interactions and the most severe ones. In some circumstances, the drug database detects interactions that are not relevant, for example: (a) the interaction is linked to the dose while this dose is not reached (e.g. aspirin -furosemide); (b) the interaction is limited to a clinical condition that the patient does not have (such as pregnancy); (c) the contraindications are purposely violated by the prescribers for very specific reasons. However, these problems are well-known in drug alert systems, and are not specific to the proposed visual tool.

In the future, the proposed interface could be improved as follows: (a) Drugs could be grouped or arranged according to their indication or their prescriber (e.g. cardiologist, endocrinologist, etc.), this will make it possible to detect interactions between drugs from different prescribers. (b) Interactions could be grouped according to the potential risk for facilitating the risk assessment. (c) Interactions could be filtered to keep only the most relevant according to the patient profile and the dose of the drugs. (d) Drugs could be ordered in order to avoid intersecting edges. (e) The system can be enriched with proposals of therapeutic alternatives. (f) The visualization could also list the foods that may interact with the treatment. (g) Inappropriate medicines for the elderly could also be detected and highlighted in the interface.

Another perspective of this work is the evaluation of the proposed interface in controlled and/or clinical conditions with pharmacists.

Figure 1 .

 1 Figure 1. Screenshot of the proposed visual interface showing 11 drugs, 2 drug-disease interactions and 15 drug-drug interactions.

Corresponding Author: malikmouazer@gmail.com.

Acknowledgment

Funding was obtained from the "Agence Nationale de Sécurité du Médicament et des Produits de santé (ANSM)". APP 2016 -RaMiPA Project (Raisonner pour Mieux Prescrire les Antibiotiques or Reasoning for better antibiotic prescription).