T275 Whole oats effects on digestive system development in neonatal dairy calves. F. X. Suarez-Mena* 1 , A. J. Heinrichs 1 , T. M. Hill 2 , and C. M. Jones 1 , 1 The Pennsylvania State University, University Park, 2 Nurture Research Center, Provimi North America, Lewisburg, OH.

The objective of this experiment was to study effects of whole oats in starter grain on digestive system development of pre-weaned calves. Male Holstein calves (n = 8, 42.5 ± 2.0 kg BW at birth) were fitted with a rumen cannula in wk 2 of life. Calves were housed in individual pens in a heated facility; bedding was covered with landscape fabric to avoid any consumption of bedding. Water was offered free choice, and milk replacer was fed to 12% of birth BW. A fixed amount of starter (containing 25% oats either ground and in the pellet or whole) was offered daily based on average intakes of calves on similar milk replacer diets; orts were fed through the cannula. Calves were randomly assigned to all pelleted starter (P, n = 4) or pellets plus whole oats (O, n = 4). Rumen contents were sampled at -8, -4, 0, 2, 4, 8, and 12 h after grain feeding for pH determination at wk 3, 4, and 5 of age. At 35 ± 1 d of age calves were euthanized, and organs were harvested, emptied, rinsed, and weighed to gauge digestive organ development. Total starter intake (3 wk; 4,596 P vs. 4,449 O ± 111 g) was not different (P > 0.05). Digestive organ mass was analyzed as a percentage of BW at harvest. Reticulorumen (0.81 P vs. 0.81 O ± 0.05%) and liver (2.21 P vs. 2.25 O ± 0.08%) were not different (P > 0.05). Omasum (0.16 P vs. 0.19 O ± 0.01%) was greater (P < 0.05), and abomasum (0.40 P vs. 0.46 ± 0.02%) had a tendency to be greater (P < 0.10) for O. Rumen papillae length (0.67 P vs. 0.59 ± 0.05 mm), width (0.48 P, 0.45 O ± 0.02 mm), and rumen wall thickness (0.96 O vs. 0.90 O ± 0.09 mm) were not different (P > 0.05). Average rumen digesta pH at 3 wk of age (6.32 P vs. 6.22 O ± 0.16) was higher (P < 0.01) than at 4 (5.93 P vs. 5.85 O ± 0.16) and 5 (5.86 P vs. 5.74 O ± 0.16) wk; pH was not different (P > 0.05) between treatments. Whole oats had no effect on reticulorumen weight or rumen papillae size but increased omasum and abomasum weight. Lack of treatment effect on papillae may be related to calves eating grain for only 3 wk. Rumen digesta pH decreased with age likely as a consequence of greater intake of starch and digestible nutrients.

Key Words: calf, rumen development, oats

T276 Puberty attainment is affected by growth performance before 4 mo of age in dairy heifers. V. Lollivier 1,2 , F. Dessauge 1 , M. Boutinaud* 1 , and Y. Le Cozler 1,2 , 1 INRA UMR1348PEGASE, Saint Gilles, France, 2 Agrocampus Ouest, UMR1348 Pegase, Rennes, France.

In seasonal calving systems where heifers first calved at a young age, age at 1st insemination may be delayed for those born at the end of the calving period. A possible alternative is to increase growth intensity during the 1st year of rearing, and then, to reduce age at 1st calving (21 mo of age or less). Puberty should then occur at an early age for optimal fertility. A long-term experiment aiming at studying the effect of controlled growth intensity throughout feeding procedure is being conducted since 2009 including 60 to 70 Holstein heifers / year. In 2012, the effect of rearing intensity on puberty attainment was closely studied on 65 heifers born between August 26 and December 27, 2011. Animals born before December 1 were either fed a standard (SD) or an intensive (ID) diet from 0 to 6 mo of age, to reach 190 to 200 kg or 220 to 230 kg at 6 mo of age, respectively. Animals born after received ID until artificial insemination (AI). From 5.5 mo of age, blood samples were obtained from jugular vein every 10 d to determine plasma progesterone concentration. The plasma progesterone analyses were performed with AIA 360 robot. The intra-and inter-assay variabilities were 12.3 and 7.5%, respectively. Blood samples were collected until synchronization or AI. Single Anova analyses were performed through R Software. Puberty was detected on 56 animals, at an average age of 10.2 (±2.0) mo and 296 (±37) kg body weight (BW). Age varied between 6.2 and 14.4 mo, whereas BW was comprised between 224 and 369 kg. Because of huge ADG variations within feeding regimen and limited number of observations, no effect of SD or ID was noted on puberty onset. Puberty occurred earlier when average daily gain (ADG) before weaning (77 to 80 d of age on average) increased (9.7 vs. 10.8 mo when ADG varied from less than 700 g/d to 700 g/d or more respectively; P < 0.05). To a lesser extent, ADG from weaning to 4 mo of age also affected puberty onset. In agreement with previous published studies, this study indicates that enhanced growth program during lactating phase and/ or early stages of rearing may affect puberty attainment. Feeding an established volume of milk replacer (MR) solution without regard for dry matter (DM) by lowering the percentage solids is being done by producers. The objective of this study was to examine the effects of delivering a more dilute solution at the same volume on calf growth and performance. One hundred thirty-three (133) 3-10 d old Holstein calves with an average initial BW of 47.6 kg (SD = 2.21 kg) were shipped from Wisconsin to the Land O' Lakes Research Facility. Calves were randomly assigned according to BW and blood gamma globulin to one of 4 27% all milk protein/10% fat MR diets varying in dilution, MR DM, and volume amount. Days 8-49, treatments were as follows: High Solids, High DM, Low volume (HS/HDM/LV; 17.6% solids, 1.13 kg DM, 6.62 L solution/d); Medium Solids, High DM, Medium volume (MS/HDM/MV; 15% solids, 1.13 kg DM, 7.8 L solution/d); Medium Solids, Low DM, Low volume (MS/LDM/LV; 15% solids, 0.97 kg DM, 6.62 L solution/d); Low Solids, High DM, High volume (LS/HDM/ HV; 12.6% solids, 1.13 kg DM, 9.2 L solution/d). Days 1-7, calves were offered the same % solids and 72% of MR DM stated above, with volume amounts adjusted accordingly. Calves were fed MR twice per day. Offering of MR was reduced by half, and calves fed once per d, during the last wk. Calf starter (22% crude protein, as fed basis) was fed ad libitum throughout this 49 d trial. Data were analyzed by Mixed Procedures of SAS. While there were no statistical differences (P > 0.05) in total BW gain, starter feed intake, or feed:gain, calves fed the MS/ LDM/LV diet were numerically lower in total BW gain and numerically higher in starter feed intake when compared with calves on all other treatments. When considering offering a more dilute MR solution, it is important to deliver the same amount of MR DM to calves by offering more MR solution to support adequate growth of calves.

Key

  Words: heifer, puberty, onset T277 The effect of various dilutions, milk replacer dry matter, and volume amounts on calf growth and performance. T. J. Earleywine, B. L. Miller, W. S. Bowen Yoho*, and T. E. Johnson, Land O' Lakes, Inc., Webster City, IA.

Table 1 .

 1 Means in the same row not followed by a common letter differ (P < 0.05).

			MR Diet
		HS/HDM/	MS/HDM/	MS/LDM/	LS/HDM/
	Item 1	LV	MV	LV	HV	SE
	BW gain, kg	33.1	34.0	31.0	33.4	1.09
	MR intake
	(DM), kg	46.3 a	47.9 b	40.8 c	47.2 ab	0.380
	Starter intake
	(DM), kg	19.8	19.4	21.1	18.8	1.29
	Feed:Gain	2.05	2.00	2.01	2.01	0.040
	a-c

Key Words: calf, milk replacer, dilution


est destinée au dépôt

PhytoHUB: a new database dedicated to dietary phytochemicals and their human metabolites for nutritional metabolomics

Franck Giacomoni, Yann Fillatre, J.A Rothwell, Craig Knox, Roman Eisner, Daniel Cesaire, Mercedes Quintana, Blandine Comte, Estelle Pujos-Guillot,

Claudine Manach

To cite this version: The "food metabolome" comprises all metabolites present in biological fluids that are directly derived from the digestion of food. A large proportion of the food metabolome consists of phytochemical metabolites, which are products of intestinal and hepatic or microbial metabolism of molecules such as polyphenols, terpenoids and alkaloids. Identification of unknowns in metabolome profiles is a laborious step-by-step process and often a bottleneck in biomarker discovery. One major limitation for the interpretation of the food metabolome profiles is the incompleteness of existing databases with regard to phytochemical metabolites.

As part of the ANR PhenoMeNep project, we have designed an online database called PhytoHUB, dedicated to the study of the phytochemical part of the "food metabolome". The database will contain all phytochemicals present in edible plants and their known metabolites manually extracted from the literature. Since the metabolism of many phytochemicals has not been studied in humans, a list of predicted metabolites will be generated from expert knowledge of the metabolism of each phytochemical class and and analysis of precursor functional groups on precursor phytochemicals. Mass spectral data will be included from various sources: literature, other databases on plant phytochemicals and experimental data from our collaborative platforms.

Built with MySQL and Perl processing chains, an efficient relational design will underpin a powerful and intuitive web interface. For a queried monoisotopic mass or molecular formula, the database will return a list of possible metabolites, along with their physic-chemical properties, spectral data and possible dietary precursors linked to food sources. For a queried food, it will return a list of metabolites likely to be present in biofluids after consumption.

PhytoHUB will be the first publicly accessible database to collate information on phytochemical metabolites from a metabolomics standpoint, and should facilitate identification of unknowns in non-targeted profiling. A first version of the database is planned for 2013.

ANR Phenomenep ALIA-2010-007 / Conseil Regional Auvergne-FEDER post-doc grant (YF)

02749856 P12-11 PhytoHUB: A NEW DATABASE DEDICATED TO DIETARY PHYTOCHEMICALS AND THEIR HUMAN METABOLITES FOR NUTRITIONAL METABOLOMICS

	Franck Giacomoni 1 , Yoann Fillatre Cesaire 1 , Mercedes Quintana 1 , Blandine Comte 1 , J.A. Rothwell 1 , Craig Knox 1 , Estelle Pujos-Guillot 2 , Roman Eisner 1 , Claudine Manach 2 , Daniel 1
	1 Human Nutrition Unit, UMR 1019 INRA / University of Auvergne, Clermont-Ferrand, France, Siliflo Inc, Edmonton, Canada	2 In

Franck Giacomoni, Yann Fillatre, J.A Rothwell, Craig Knox, Roman Eisner, et al.. PhytoHUB: a new database dedicated to dietary phytochemicals and their human metabolites for nutritional metabolomics. 9. Annual conference of the Metabolomics Society, Jul 2013, Glasgow, United

Kingdom. 2013. hal-


Cet exemple fait apparaître deux comportements thermoacoustiques distincts :

une différence de température peut engendrer un son • un son peut servir au transport de chaleur. • Considérer qu'une différence de température puisse servir à transporter de la chaleur n'est pas qu'une association d'idées mais une nouvelle idée ! Il s'agit par exemple de faire du froid avec du chaud via une onde. Vous obtenez alors un réfrigérateur thermoacoustique.

Où sonna l'heure de la thermoacoustique

Une onde sonore, ou son, est formée par de petites fluctuations de pression se déplaçant dans un milieu fluide ou solide. En se déplaçant, la membrane d'un haut-parleur ou un piston actionné par une bielle comprime puis détend un petit volume de fluide (un paquet de molécules de gaz) contre un volume voisin qui à son tour en comprime puis détend un autre… et ainsi de suite jusqu'à nos oreilles. Or un gaz comprimé s'échauffe alors qu'un gaz détendu se refroidit. Chaque compression et détente d'un volume de gaz s'accompagne donc d'une élévation puis d'une baisse de température (illustration ci-dessus).

L'interaction du son et de la chaleur fut montrée pour la première fois il y a moins de deux siècles par le français Simon Laplace (1749-1827). Comprenant que le son en se déplaçant modifiait la température de l'air, il corrigea les calculs de sa vitesse réalisés un siècle auparavant par Isaac Newton (1642-1727). Depuis des siècles, les souffleurs de verre ont pu observer la naissance d'un "chant" au sein des tubes froids dont l'extrémité était chauffée par le verre en fusion. Là se présente l'autre manifestation de la thermoacoustique : la génération d'une onde acoustique au sein d'un résonateur par l'application d'un gradient de température. Ce phénomène fut décrit pour la première fois, et de façon expérimentale, en

Volume de fluide soumis à une onde acoustique. Le fluide s'échauffe lors des phases de compression et se refroidit lors des phases de détente tout en se déplaçant en effectuant de petits allers et retours autour d'une position moyenne.

Où l'homme eut vent de la musique thermoacoustique

La première théorie est attribuée à Lord Rayleigh qui montra l'importance de l'existence d'un décalage temporel entre la pression et la température, base de l'effet thermoacoustique. Il claironna ainsi haut et fort la possibilité de soutenir un son en plaçant judicieusement une source de chaleur dans un tube. Mais l'ère de la thermoacoustique moderne fut ouverte un siècle plus tard par Nicolas Rott qui le premier mit en équations dans les années soixante le phénomène des ondes de Taconis. Au début des années quatre-vingt, Weathley du Los Alamos Laboratory (E-U) expérimenta le processus inverse : la génération d'une différence de température (un gradient de température) par l'application d'une onde acoustique sur une paroi. Ouvrant la voie vers des systèmes de type pompe à chaleur, ses travaux firent suite aux études menées par Gifford et Longworth (1963) qui employèrent des ondes à très basses fréquences (quelques Hertz) pour la production de froid dans des systèmes nommés tubes à gaz pulsé.

Où la musique rafraichît sa bière

Cette équipe offrit également, par l'intermédiaire d'un de ses membres les plus éminents : Greg Swift, une description détaillée de la thermoacoustique. A travers l'étude d'un réfrigérateur thermoacoustique, le "beercooler", il établit en 1988 une théorie linéaire de ce phénomène, présentant l'importance du rapport des capacités thermiques entre le fluide dans lequel se propage l'onde acoustique et la paroi solide.

Mais comment ça marche ?

Comme nous l'avons dit, une onde de pression comprime des volumes de gaz voisins les uns contre les autres. Elle les fait également osciller autour d'une position moyenne. Ainsi, ils se retrouvent comprimés et chauds d'un côté de cette position à la fin d'une moitié de cycle et détendus et froids de l'autre côté de cette position à la fin du cycle (illustration page précédente). Ceci peut continuer longtemps sans que rien ne se passe. Mais si on introduit un milieu solide, une plaque métallique par exemple, celui-ci va jouer le rôle d'accumulateur ou de ralentisseur thermique (illustration ci-dessus).

Ainsi à chaque phase de compression, le gaz échauffé donne de la chaleur à la paroi alors que lors des phases de détente il lui en prend. Avec du recul et en considérant l'ensemble de ces petits volumes élémentaires, on voit apparaître un transport de chaleur d'un bout à l'autre de la paroi. C'est ce transport de chaleur qui va créer une différence de température aux extrémités de cette plaque. Il suffit alors de mettre en bout de plaque des échangeurs de chaleur afin de pouvoir prendre de la chaleur d'un milieu donné (bière, sorbet ou gaz) pour le refroidir ou au contraire en donner (air, vapeur d'eau) pour le réchauffer.

Petits gestes grands effets

Reste que si avoir du coffre est un bon début, cela ne suffit pas. Car les fluctuations de pression (quelques dizaines de Pascal) émises par votre voix engendreront des variations de température de l'ordre de quelques dix-millièmes de degrés (0,0001°C) ! Hurler à tue-tête dans votre salon ne vous permettra donc pas de sortir anorak et ski, ambiance Absolut Vodka ® . Autant dire que les variations de pression doivent être conséquentes ; c'est du gros son qu'il nous faut : de l'ordre du bar (100 000 Pascals). Un tube résonant dans lequel le son

Couplage : la thermoacoustique sens dessus dessous

Reste que si le principe thermoacoustique est relativement simple, trouver la bonne source sonore reste un problème. Car la technologie actuelle des haut-parleurs n'est pas suffisante pour répondre de manière satisfaisante au besoin de cette technologie. Il faut avoir recours à un système plus puissant comme un moteur à piston, avec les difficultés technologiques que cela implique (non contamination du système par de l'huile, hautes fréquences mécaniques…). Cependant la thermoacoustique trouve là encore une parade puisque ce même principe, appliqué à l'envers, permet de créer une onde à partir d'une source de chaleur.

Il devient alors possible, par couplage d'un système de génération d'onde avec un système de pompe à chaleur tous deux thermoacoustiques de faire du froid avec du chaud ou du encore plus chaud avec du déjà chaud. Résumons : une source de chaleur permet de produire une onde qui à son tour va être utilisée pour faire du froid ou du chaud (illustration ci-dessous). Vers le succès on chauffe !

Faites le neuf

Mais rien de nouveau sous le soleil me direz-vous ; tout cela reste du réchauffé. D'une part, la source de chaleur est au libre choix de l'utilisateur : combustion de gaz ou de déchets, rayonnement solaire, etc. D'autre part, le système fonctionne sans aucune pièce mobile. Il est donc d'une manière générale extrêmement fiable et ne nécessite que très peu de maintenance. Autre facteur important en ces temps de prise de conscience de l'environnement, ces systèmes utilisent dans leurs tubes des gaz inertes pour l'environnement tel que l'hélium. Finis donc les affreux CFC pollueurs. D'autant plus que ces derniers, produisant du froid par changement de phase vapeur-liquide, ne permettaient d'atteindre qu'un seul niveau de température ; la régulation de la température s'effectuant par mise en route ou arrêt du système. Les systèmes thermoacoustiques ont cet avantage que la puissance thermique produite peut être modifiée et régulée en fonction de la puissance mise en entrée pour produire l'onde acoustique. De plus, il est possible de descendre à n'importe quelle température (cela peut nécessiter un ou plusieurs éléments en série) en fonction de la puissance mise en jeu.

Réfrigérateur thermoacoustique ; un générateur d'onde thermoacoustique (gauche) utilise de la chaleur pour produire une onde qui permettra à son tour de pomper de la chaleur pour faire du froid (droite).

Empilement de plaques planes utilisé dans un système thermoacoustique.

Applications

Aujourd'hui, la reconnaissance des enjeux économiques, écologiques de la thermoacoustique, ainsi que les nouveaux moyens d'analyse liés à l'informatique, font croître très rapidement les investigations dans ce domaine. La thermoacoustique pourrait également trouver un avenir commun avec les accélérateurs de nouvelle génération, pour lesquels elle userait des ses talents de fiabilité, de niveau de température variable afin par exemple de refroidir des détecteurs même en environnement contraignant.

De par le monde, de nombreuses équipes se lancent actuellement dans des divers projets, témoignant d'un intérêt croissant pour cette technologie. Les développements sont aussi intéressants que nombreux et la compétition s'accroît. Alors un grand avenir pour la thermoacoustique ? Pour les trente prochaines années nous l'espérons.

  «thermo acoustique» qualifie les phénomènes faisant interagir la propagation d'une onde acoustique et des effets thermiques, c'est-à-dire son et chaleur. C'est le cas par exemple lorsqu'un bruit est généré dans un pot catalytique, peu après le démarrage d'une voiture. Ces effets, souvent gênants peuvent être parfois dévastateurs. Ainsi, dans le domaine de la cryogénie, les ondes dites de Taconis sont engendrées lorsqu'un tube est partiellement immergé dans un bain d'hélium liquide. La différence de température entre la partie immergée dans le liquide (froid) et celle qui reste à «l'hélium libre», produit alors un son dans ce tube. L'aspect néfaste est que cette production de son s'accompagne d'un transport de chaleur en direction du bain. Cet apport peut être important et entraîner une vaporisation désastreuse du bain. Le côté positif est que le son émis peut servir à indiquer la présence du bain d'hélium liquide lorsqu'on y plonge l'extrémité d'un tube. C'est le principe de la sonde de Taconis qui permet d'indiquer un niveau de liquide d'hélium.

  1977 par Putman et Denis, grâce à l'excitation d'un tuyau d'orgue (ouvert aux deux bouts), au moyen d'une flamme à hydrogène judicieusement placée à l'intérieur. Mais les études les plus célèbres sont sans doute celles réalisées par Sondhauss, à partir de 1850 et par Rijke (1859), avec un tube résonant quart d'onde (c'est-à-dire une des deux extrémités ouverte), une flamme placée à l'intérieur. Expériences pouvant éclairer le son entendu par Wayne et Chuck… « Lorsque la lumière de ce soleil rouge disparut à l'horizon, Wayne craqua une allumette et titilla la mèche de notre petite lampe à pétrole. La lumière éclaboussa la terrasse. Me laissant bercer par la douceur d'une clarté retrouvée, un petit bruit de narine me parvint aux oreilles ; mais Wayne ne dormait pas et se grattait la barbe. Puis s'amplifiant, la note continua quelques minutes, comme sortie d'une flûte de Pan. Pourtant, pas un poncho au fond de ce Far West. Encore un des mystères de l'Ouest. » Encore plus mystérieux est que cette note aurait peut-être pu leur permettre de boire une bière bien fraîche en plein désert. Mais quel rapport entre la mélodie d'une flûte de Pan et une bière bien fraîche ? C'est que rafraîchir une bière avec une flûte de Pan, c'est le Pérou ! Et à l'IPN, nous aimerions y parvenir via la thermoacoustique.

  Un nouveau pas fut enfin franchi dans les années quatre-vingt par Hofler de la même équipe de Los Alamos qui réalisa le premier un générateur d'onde thermoacoustique à l'aide d'un tube quart d'onde et de l'emploi d'un solide poreux d'une grande importance : le régénérateur.

  Propagation d'une onde acoustique le long d'une paroi solide. Lors des phases de compression et de détente, de la chaleur est échangée avec la paroi conduisant à une différence de température aux extrémités de celle-ci.

  alors nécessaire. Il permet d'accroître les effets acoustiques de la même manière qu'un bruit de bouche est amplifié dans un didgeridoo. Mettre au préalable le gaz sous pression (quelques dizaines de bars) permet de décupler les variations de pression. Enfin, pour augmenter la puissance thermique transportée il faut accroître la surface d'interaction entre le fluide et le solide. Ceci conduit à augmenter le nombre de parois amenant à des empilements de plaques planes parallèles (photo ci-dessus) ou à l'utilisation de milieux poreux.

  D'autant que les applications sont nombreuses et portent aussi bien sur des marchés très ouverts que sur des niches technologiques. Ainsi, certains s'intéressent au refroidissement des serveurs utilisés en télécommunication, à celui des détecteurs embarqués dans le domaine spatial. D'autres s'occupent de récupérer et promouvoir la chaleur perdue dans les processus industriels… Et ça n'est pas du pipeau. L'équipe de Steven Garret de la Penn State University a, par exemple, réalisé en 2003 le premier prototype de congélateur industriel pour la société Ben & Jerry en utilisant un moteur linéaire comme source acoustique. Le procédé thermoacoustique peut également être utilisé pour la liquéfaction du gaz naturel. L'avantage d'un tel système dans ce domaine est d'utiliser une source de chaleur pour mettre en oeuvre un processus de réfrigération. Ainsi, la combustion d'une petite partie du volume de gaz naturel considéré permet de liquéfier ou de maintenir à l'état liquide le reste du volume. Sont donc concernées des applications de plusieurs centaines de kilowatts de chaleur extraite correspondant à des mégawatts de puissance en source chaude. Avec presque trente ans d'expérience dans le domaine et après avoir envoyé un réfrigérateur thermoacoustique dans l'espace en collaboration avec la NASA, l'équipe du Los Alamos laboratory dirigée par Greg Swift tente de développer des systèmes de forte puissance pour des applications gazières. Sur le chemin du succès, cette équipe possède déjà des bancs expérimentaux impressionnants (photos ci-contre) Plus modestement, l'IPN, en collaboration avec le LIMSI qui depuis une dizaine d'années développe des activités de recherche en thermoacoustique, réalise un système destiné à être un démonstrateur de liquéfaction de gaz naturel (photos ci-contre). Financé par le CNRS, le Conseil Général de l'Essonne, Oséo (ANVAR), les Universités de Paris 6 et 11, il doit permettre la démonstration de la faisabilité d'une telle technologie à une échelle intermédiaire entre le banc expérimental de recherche fondamentale et le prototype industriel ; l'objectif de puissance thermique extraite est ici de 2kW à une température de 120K. Après deux ans de calculs et de fabrication, le générateur thermoacoustique d'onde a été monté au bâtiment 109 fin 2005 et est actuellement en cours d'essai (page suivante, photos en haut). Concept américain et prototype d'unités embarquées de production de Gaz Naturel Liquéfié. D é m o n s t r a t e u en association avec la société Hekyom et l'Institut Français du Pétrole, l'IPN a développé et réalisé un banc expérimental d'amplification thermoacoustique qui est en service au bâtiment 103 (photo ci-dessus en bas). Son objectif est de générer puis amplifier une onde au moyen de la technologie acoustique afin d'obtenir une source thermique a fort rendement. Une première étape vient donc d'être franchie en doublant la puissance acoustique produite par un générateur simple en utilisant un deuxième étage amplificateur.

  de l'IPN en cours de montage et installé au bâtiment 109. Banc d'amplification thermoacoustique d'onde au bâtiment 103. Contacts : Patxi Duthil duthil@ipno.in2p3.fr


Christophe Billette
email: billette@bordeaux.inra.fr

Thierry Gibard

Marie Foulongne-Oriol

Jean-Michel Savoie

ORIGIN OF LACCASE GENE STRUCTURAL DIVERSITY IN EDIBLE MUSHROOMS

Keywords: Laccase phylogeny, laccase sub-families, intron position, Agaricus bisporus, basidiomycetes, protein evolution

Laccase genes have been found in fungi, plants, insects and bacteria. In Basidiomycetes, the number of laccase genes ranges from 0 to 17. The role of these genes is not well known. It seems to be important in fungal interaction, development, melanine synthesis, human and plant pathogenesis, [ectomycorrhizal association and nutrition of the fungi. Their role as ligninmodifying enzymes is controversial. Laccase phylogeny already published is not congruent with species phylogeny. Phylogeny of gene is generally deduced from analyse of aligned sequences of nucleotides or amino acids. Presence of indels, position of introns can give complementary information. To better understand the origin and evolution of Basidiomycete laccase genes, we studied the structure of diverse multi copper oxidase genes in different phylum of Eukaryotes, in basidomycetes and in Agaricus bisporus. A mutation has been conserved in all basidiomycete laccases and constitutes a molecular synapomorphy for basidiomycete laccase genes. At least three main laccase subfamilies (A, B and C) are present in Polyporales and Agaricales. In A. bisporus the 4 new laccase genes we have sequenced (lcc3-lcc6) belong to a new sub-family for this species. Cladogram of laccase genes from edible mushrooms built with Dollo parsimony of intron position is congruent with dendrograms built with homologous amino acid sequences, but is not identical. Some consequences on the evolution of laccase genes and their diversity are discussed.

INTRODUCTION

Laccase are polyphenol oxydase belonging to two protein families that are involved in various systems of interactions between fungi and their biotic and abiotic environment [START_REF] Kues | Multiple Multi-Copper Oxidase Gene Families in Basidiomycetes-What for?[END_REF][START_REF] Savoie | Variability in brown line formation and extracellular laccase production during interaction between white-rot basidiomycetes and Trichoderma harzianum biotype Th2[END_REF][START_REF] Kilaru | The laccase multi-gene family in Coprinopsis cinerea has seventeen different members that divide into two distinct subfamilies[END_REF][START_REF] Courty | Phylogenetic analysis, genomic organization, and expression analysis of multi-copper oxidases in the ectomycorrhizal basidiomycete Laccaria bicolor[END_REF]. The multi copper oxidase family (mco) is the large one and contains also other enzymes as ferroxidase, ascorbate oxidase, ferroxidase-laccase [START_REF] Kues | Multiple Multi-Copper Oxidase Gene Families in Basidiomycetes-What for?[END_REF]. The second family, recently discovered, contains proteins with the DUF152 domain, present in a hypothetical Bacteroides of the bovine rumen and in Eschericia coli [START_REF] Savoie | Variability in brown line formation and extracellular laccase production during interaction between white-rot basidiomycetes and Trichoderma harzianum biotype Th2[END_REF].

Laccases of scomycetes and basidiomycetes form two clades belonging to the mco family. Laccases have the ability to oxidize a wide range of substrates [START_REF] Hoegger | Phylogenetic comparison and classification of laccase and related multicopper oxidase protein sequences[END_REF]. Some laccases play a role in sclerotization (insects), in lignification process (LMCO of plantes) but there roles in basidiomycetes are not well elucidated (see Kues and Ruhl for review [START_REF] Kues | Multiple Multi-Copper Oxidase Gene Families in Basidiomycetes-What for?[END_REF]). During a long time, an important role of basidiomycete laccases has been attributed to delignification, but it is also contested [START_REF] Lundell | Lignin-modifying enzymes in filamentous basidiomycetesecological, functional and phylogenetic review[END_REF]. Most of fungal laccases are secreted and some of them seem to play a role in recycling recalcitrant organic matter and in the detoxification of natural environments [START_REF] Qasemian | Halotolerant laccases from Chaetomium sp., Xylogone sphaerospora and Coprinopsis sp. isolated from a Mediterranean coastal area: potential to transform polycyclic aromatic hydrocarbons[END_REF]. Some are involved in ectomycorrhizal association [START_REF] Courty | Phylogenetic analysis, genomic organization, and expression analysis of multi-copper oxidases in the ectomycorrhizal basidiomycete Laccaria bicolor[END_REF], in plant pathogenesis [START_REF] Hoegger | Phylogenetic comparison and classification of laccase and related multicopper oxidase protein sequences[END_REF] or in fungal-fungal interaction [START_REF] Savoie | Variability in brown line formation and extracellular laccase production during interaction between white-rot basidiomycetes and Trichoderma harzianum biotype Th2[END_REF][START_REF] Savoie | Oxidative processes in Agaricus bisporus dry bubbles[END_REF]. For instance, to improve A. bisporus strains for their resistance to Leucanicillium fungicola, one possible track is to better understand the mechanism of interaction of the two fungi. Thus, laccase genes appeared to be good candidates. At the beginning of this work, two laccase genes, lcc1 and lcc2, were known [START_REF] Perry | Identification of two laccase genes in the cultivated mushroom Agaricus bisporus[END_REF]. We have discovered and annotated 4 new laccase genes in A. bisporus.

Intron positions are not suitable information for phylogenetic analysis at long evolution distances [START_REF] Rogozin | Remarkable interkingdom conservation of intron positions and massive, lineage-specific intron loss and gain in eukaryotic evolution[END_REF]. Nevertheless, Kilaru et al. [START_REF] Kilaru | The laccase multi-gene family in Coprinopsis cinerea has seventeen different members that divide into two distinct subfamilies[END_REF] on Coprinopsis cinerea laccases, Tavares et al. [START_REF] Tavares | Organization and structural evolution of four multigene families in Arabidopsis thaliana: AtLCAD, AtLGT, AtMYST and AtHD-GL2[END_REF] and Boulet et al. [START_REF] Boudet | Evolution of intron/exon structure of DEAD helicase family genes in Arabidopsis, Caenorhabditis, and Drosophila[END_REF] on Arabidopsis, Caenorhabditis and Drosophila and Matheny et al. [START_REF] Matheny | Contributions of rpb2 and tef1 to the phylogeny of mushrooms and allies (Basidiomycota, Fungi)[END_REF] for basidiomycota phylogeny, showed that they could be very useful in phylogeny in complement of analyses based on protein or nucleotide sequences. Moreover, the intron positions could help to elucidate general mechanisms of gene family evolution. Indeed intron presence/absence is a relatively very slowly evolving character [START_REF] Irimia | Spliceosomal introns as tools for genomic and evolutionary analysis[END_REF] and selective forces acting on it differ to those acting on protein sequences.

The study of intron positions and protein structure would help to go further in the origin and evolution of basidiomycete laccases. That also may help us to deduce putative laccase functions. We supposed that mco family is a very old family that has experienced several intron gain and loss waves. We also supposed that no parallel intron insertion has occurred in laccase genes. In the present work, we compared the phylogeny of basidiomycetes species using laccase genes, based on protein sequence or on intron positions. We discovered insertion of 5 amino acids present in all basidiomycetes laccases but absent in all other mco. We also discovered a Lentinula edodes gene with presence or absence of three introns depending of the strain. Consequences for the evolution of laccase genes and their diversity are discussed.

MATERIALS AND METHODS

Sequences used. Sequences used for phylogeny analyses (Table 1) were obtained from GenBank (http://www.ncbi.nlm.nih.gov/), Joint Genome Institute (http://genome.jgipsf.org/programs/fungi/index.jsf) and Broad Institute (http://www.broadinstitute.org/scientificcommunity/data). For phylogenetic analyses, we had chosen all published genomic DNA Laccase sequences to have intron positions. The translated coding sequences of these genes were used for the amino acid phylogeny.

Two Polyporus laccase genes and three different C. cinerea laccase genes (two belonging from two different well defined clades of subfamily A and lcc8) were used as controls in the analyses. Three ferroxidase gene sequences have been used as out group.

Identification of new A. bisporus genes. Agaricus bisporus strain U1-7 (having the same mating type allele than H93) is a homocaryotic strain coming from a protoplast of the cultivar U1 [START_REF] Callac | Morphological, genetic, and interfertility analyses reveal a novel, tetrasporic variety of Agaricus bisporus from the sonoran desert of California[END_REF][START_REF] Kerrigan | The heterothallic life-cycle of Agaricus bisporus var burnettii and the inheritance of its tetrasporic trait[END_REF]. DNA extraction was made with the Nucleon Phytopure extraction kit (GE Healthcare) from lyophilised mycelium. The lcc2 cDNA sequence [START_REF] Perry | Identification of two laccase genes in the cultivated mushroom Agaricus bisporus[END_REF] was used to generate primers and to amplify and sequence the gene. Inverse PCR [START_REF] Ochman | Genetic Applications of an Inverse Polymerase Chain-Reaction[END_REF] was used to obtain the 3' end of the gene. One (or more) restriction enzyme was chosen for inverse PCR, cutting the known sequence. Primers were constructed with Primer 3 on the 3' side of the restriction site, present on the already known sequence, to orientate inverse PCR. When AK7/AK8 primers from D'Souza et al. [START_REF] D'souza | Isolation of laccase gene specific sequences from white rot and brown rot fungi by PCR[END_REF] were used to amplify lcc1 and lcc2, two bands were obtained in electrophoresis. Lcc1 and lcc2 corresponded to the same band. So, the second band was expected to be another laccase gene (renamed hereafter as lcc3). This bans had been sequenced. To obtain a larger part of the gene, a new degenerated primer (AK9 : TGRCARTGGARGAACCAKGG) was constructed on part of a simplified l4 signature of laccase sequences (P-W-F-(LF)-H-C-H). The totality of the gene was obtained by inverse PCR in both 3' and 5' directions. Two clones sequenced from a genomic library available in our lab corresponded to partial laccase genes sequences. By inverse PCR we obtained the totality of these new genes (lcc4 and lcc5). Lcc6 was obtained from an extra band during one of these inverse PCRs. Amino acid phylogeny. The phylogenetic analysis of laccase sequences was performed on the Phylogeny website platform (www.phylogeny.fr). Sequences were aligned with MUSCLE (v3.7) with default settings. After alignment, ambiguous regions (i.e. containing gaps and/or poorly aligned) were removed with Gblocks (v0.91b) with default settings. The phylogenetic tree was reconstructed using the maximum likelihood method implemented in the PhyML program (v3.0 aLRT) with default settings. Reliability for internal branch was assessed using the bootstrapping method (500 bootstrap replicates). Graphical representations of the phylogenetic tree were performed with TreeDyn (v198.3).

Intron position phylogeny.

In GenBank, intron positions are indicated on gDNA sequences and protein sequence is deduced by translating CDS. The positions of introns of the encoding gene were determined on aligned protein sequences by an annotation on a BioEdit file [20]. The convention presented in Fig. 1 for schematizing the exact position of the intron on each codon.

W F L H F L TTG TTC CTC CAT a b W F L H F L TTG TTC CTC CAT W F W F L H L H F L F L TTG TTC CTC CAT TTG TTC CTC CAT a b

Dollo phylogeny.

A matrix was built with all intron positions in all sequences. Paup was used to perform the Dollo parsimony analysis [START_REF] Swofford | PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Met hods)[END_REF]. One thousand bootstrap replicates were used. We made the approximation that the ancestor gene had no intron at all.

Analysis of insertions in laccase genes.

T-Coffee (Phylogeny.fr) was used to align sequences. The alignment were visualised with Jalview tool.

RESULTS AND DISCUSSION

Obtention of lcc2 gDNA sequence and identification of four new laccase genes in A. bisporus. The complete genomic sequences of lcc2, lcc3, lcc4 and lcc5 and the partial sequence of lcc6 have been obtained (GenBank accession number : FJ838791, EU580106, FJ392313, FJ617019 and FJ878811 respectively). It is not surprising to find at least six laccase genes in this species as seventeen have been found in C. cinerea [START_REF] Kilaru | The laccase multi-gene family in Coprinopsis cinerea has seventeen different members that divide into two distinct subfamilies[END_REF] which is in the same Agaricoid clade. The sequences of the six laccase genes of A. bisporus were compared between each other. Intron positions in lcc2 were the same as in lcc1 except for the 3' UTR intron 15 which has not been found in lcc2. The 3' end of lcc4 is still hypothetical, an intron could permit the prolongation of the protein (lcc4bis end: CPIWDSEPNFVKHAATMILDPLINFAFGPIFPVYIL LRFPTSLICMKPNAFH). Sequencing the mRNA will be necessary to control the end of the protein. The study of intron positions of these genes revealed the presence of a new laccase subfamily in A. bisporus (Fig. 2). This has led us to compare the intron positions of numerous laccase genes and especially from C. cinerea as two laccase sub-families had been found in this species [START_REF] Kilaru | The laccase multi-gene family in Coprinopsis cinerea has seventeen different members that divide into two distinct subfamilies[END_REF]. To better understand the phylogenetic relationships of these genes we have build and compared a phylogeny based on amino acid homologous sequences and a phylogeny based on the conservation of intron positions. Congruence but not identity between protein sequences and intron positions based phylogeny. Fig. 3A shows the maximum likelihood phylogeny of 38 protein sequences of laccases from edible mushrooms. The ferroxidase genes added as outgroup represented a clade.

A second clade was composed by laccases of the subfamily B identified by Kilaru [START_REF] Kilaru | The laccase multi-gene family in Coprinopsis cinerea has seventeen different members that divide into two distinct subfamilies[END_REF]. In a third clade (renamed hereafter as "clade C"), there were the large subunit of heterodimeric proteins encoded by A. bisporus lcc1, lcc2 and P.ostreatus poxa3 cluster. This clade wais confirmed by the analysis of intron position (Fig. 3B). We propose to name the proteins belonging to clade C "laccase subfamily C". In a fourth clade ("clade A"), we found the three control proteins from C. cinerea belonging to subfamily A [START_REF] Kilaru | The laccase multi-gene family in Coprinopsis cinerea has seventeen different members that divide into two distinct subfamilies[END_REF]. The proteins contained in clade A were renamed "laccase subfamily A". Clade A corresponds to subfamily 1 in Kues and Ruhl [START_REF] Kues | Multiple Multi-Copper Oxidase Gene Families in Basidiomycetes-What for?[END_REF], clade B and C to subfamily 2. Here clade A and C clustered, with good bootstrap values. The two most divergent proteins found in this last clade were from Polyporales. Even if the bootstrap values were very low, it was the first time that Polyporales laccases were placed at a basal position of the clade A in a phylogenic tree of laccases belonging to Polyporales and Agaricales. With this positioning, laccase phylogeny is congruent with the phylogeny of species and the origin of laccase subfamily A should be anterior to the divergence of Polyporales and Agaricales. In this phylogenetic tree A. bisporus laccases are divided into two clades, A and C. All the new A. bisporus laccases belong to a subfamily unknown before in this species. Agaricus bisporus laccases of clade A formed a specific clade. This suggests that these four genes appeared by duplication events after the divergence between the ancestor of A. bisporus and the ancestor of the other species of Agaricoid clade VI (ie. C. cinerea and L. bicolor). These laccases clustered, but with very low bootstrap, with C. cinerea lcc7 encoded protein. Pleurotus ostreatus laccases were divided into three groups, two in clade A and one in clade C. Laccaria bicolor laccases were divided into two groups of clade A as previously shown [START_REF] Courty | Phylogenetic analysis, genomic organization, and expression analysis of multi-copper oxidases in the ectomycorrhizal basidiomycete Laccaria bicolor[END_REF]. Lentinula edodes laccases were divided into three groups of clade A. The bootstrap values for the three clades A, B and C wre very high, 91, 99 and 100 respectively. A laccase of clade A had more similarity to any another laccase of this clade that to a laccase of clade B or C. However Polyporales and Agaricales laccases were in clade A and only Agaricales laccases were in clade B and C. As phylogenic studies had demonstrated that Polyporales order was not nested inside Agaricales order [START_REF] Garcia-Sandoval | Molecular phylogenetics of the Gloeophyllales and relative ages of clades of Agaricomycotina producing a brown rot[END_REF], we can deduce that the separation between these three clades was anterior to the divergence between Agaricales and Polyporales. In the recent genome sequence of a Russulale, Heterobasidion annosum (http://genome.jgipsf.org/Hetan2/Hetan2.home.html), a putative laccase sequence belonging to subfamily C have been found. This also indicates that the origin of subfamily C might be anterior to the divergence of Russulales and Agaricales. Wahleithner et al. [START_REF] Wahleithner | The identification and characterization of four laccases from the plant pathogenic fungus Rhizoctonia solani[END_REF] demonstrated the presence of large insertions in three of the four Thanateforus cucumeris laccases, suggesting the existence of other old sub-families in Cantharellales.

Fig. 3B shows one out the eight most parsimonious trees obtained with the Dollo parsimony of intron positions of the laccase genes used for the phylogenetic tree of protein presented in Fig. 3A. Two proteins for which gDNA was not available, R. microsporus lcc1, L. edodes lcc4 and one that has no intron, S. cereviciae Fet3, are absent of this second tree. In the 37 genes 112 intron positions were identified. The intron-late theory seems to be more acceptable than the intron-early one supposing that there were 112 different introns in an ancestral laccase gene. Moreover, as we have studied new laccase genes and thus found new intron positions, we may expected to find other new intron positions with the future finding of new laccase genes. Stajich and al. [START_REF] Stajich | Comparative genomic analysis of fungal genomes reveals intronrich ancestors[END_REF] stated that an invasion by splicosomal introns took place at the time of fungus-animal ancestor, followed by recurrent intron loss occurring in all fungal clades. In this study we considered that the ancestor of all laccase genes had no intron at all; this constitutes for us the plesiomorphic character. Then introns had invaded laccase genes bit by bit, and then some introns disappeared after gene duplication and after speciation. This phenomenon is well analysed by the Dollo parsimony model in which convergences are excluded but reversions are admitted [START_REF] Darlu | La Reconstruction phylogénétique[END_REF].

The phylogenetic tree of Fig. 3B is globally congruent with the one of Fig. 3A. The ferroxidase clade positioned as outgroup with a bootstrap value of 96%. These two genes share only three intron positions between each other but they share no position with laccase genes. The three principal laccase clades A, B and C, are present, with bootstrap values of 63 and 100% for clade A and B, but a low value for clade C. This was explained by the presence, in others of the eight most parsimonious trees, of clades in which P. ostreatus poxa3 clustered with the clade A. But two intron positions are common to clade C and absent to clade A: n°42 and 44 (Fig. 1). All laccase subfamily A genes from Agaricoid clade VI (A. bisporus, C. cinerea and L. bicolor) clustered together in Fig. 3A but not in Fig. 3B. Subfamily A laccases of A. bisporus had identical intron positions except for lcc4 where a supplementary intron position was proposed (lcc4bis). These A. bisporus laccase genes clustered with C. cinerea lcc7 as in Fig. 3A but also with Cci_lcc8 which was joined here to Cci_lcc7 and with Lbi_lcc4. All these genes possess intron positions 7 and 11 (see Kilaru et al. [START_REF] Kilaru | The laccase multi-gene family in Coprinopsis cinerea has seventeen different members that divide into two distinct subfamilies[END_REF] and Ahlawat and Billette [START_REF] Ahlawat | Positioning of introns in different laccase genes, a relevant tool for solving phylogenetic position ambiguity of Volvariella volvacea laccase genes[END_REF] for nomenclature of introns). Here we see an important divergence between the results of the two analyses (Fig. 3) for the position of Cci_lcc8 and Lbi_lcc4, but Lbi_lcc4 clustered with Cci_ lcc7 as previously found in a Neighbour joining analysis done by Courty et al. [4]. This similarity reinforces the position of Lbi_lcc4 in the same clade as Cci_lcc7 and Abi_lcc3-4-5-6. Another clade with a bootstrap value of 87% was formed by Cci_lcc3 and five L. bicolor genes. This clade was congruent in the two analyses, except for the position of Lbi_lcc4. All the genes of this clade possess intron position 6 and 10. Positions of Lbi_lcc6-7-8 were different in the two analyses. In that case, intron position analysis was not efficient as these genes possess neither intron positions 6,7,10 and 11 which allowed distinguishing the two clades described above. We can name these two clades "the clade of Cci_lcc7" and "the clade of Cci_lcc3". Volvariella volvacea lcc3, studied by Ahlawat and Billette [START_REF] Ahlawat | Positioning of introns in different laccase genes, a relevant tool for solving phylogenetic position ambiguity of Volvariella volvacea laccase genes[END_REF], also possess intron positions 6 and 10 and belong probably to the Cci_lcc3 clade. So the divergence between these two clades is probably older that Agaricoid clade VI.

Three intron positions are present in two to four P. ostreatus laccase subfamily A genes and are absent in all other genes. Consequently, in Fig. 3B, contrary to Fig. 3A,P. ostreatus laccase subfamily A genes clustered together (bootstraps value 58%) and outside the cluster formed by Agaricoid clade VI laccase subfamily A genes. This is more congruent with the phylogenetic tree of the species proposed by Matheny [START_REF] Matheny | Major clades of Agaricales: a multilocus phylogenetic overview[END_REF] or Binder [START_REF] Binder | Amylocorticiales ord. nov and Jaapiales ord. nov.: Early diverging clades of Agaricomycetidae dominated by corticioid forms[END_REF] than all already published laccase phylogenetic trees. In Fig. 3B, all Marasmioid (L. edodes and F. velutipes) genes were outside the clade formed by P. ostreatus and Agaricoid laccase subfamily A. This is congruent with Binder's phylogeny of species [START_REF] Binder | Amylocorticiales ord. nov and Jaapiales ord. nov.: Early diverging clades of Agaricomycetidae dominated by corticioid forms[END_REF] unlike the corresponding part of the tree in Fig. 3A and laccase trees published earlier.

Moreover L. edodes laccase genes were in two clades. In one of them there are F. velutipes and P. rudis (Polyporales) laccase genes. Four intron positions are shared by P. rudis lac A and F. velutipes laccase, three of them are shared with L. edodes. As they are present in Agaricales and Polyporales, these positions constitute a plesiomorphic (ancestral) character for these two orders. The position of P. rudis lacA inside a clade of Agaricales laccase genes is a problem. This clad emight be constituted by genes that evolved very little. Te presence of few and mostly ancestral introns in Pru_lacA (12 introns) and Fve_laccase (9 introns) might have resulted in a wrong place of Pru_lacA in the phylogenetic tree, unlike in Fig. 3A. This problem is not resolved here.

Identification of a conserved, basidiomycete specifique laccase mutation. Fig. 4 shows a difference of 5 amino acid positions between aligned sequences of basidiomycete laccases and other multicopper oxidases. Very early divergent multi copper oxidases (mco) such as plants LMCO, insect laccases, archae or bacterial mco and two sub-unit mco don't possess most of these 5 amino acids positions. Two exceptions have been found among 240 mco sequences examined, a mco from the Ustilaginomycotina Malassezia globosa (XP_001729535) with a much bigger insertion at the same place and a mco from Rhizopus oryzae (RO3G_07290.3 at the Broad institute www.broadinstitute.org/annotation/genome/rhizopus_oryzae/MultiHome.html) that has the same 5 amino acid position as basidiomycete laccases, but with no apparent homology. that results probably from an independent mutation. All basidiomycetes bona fide laccases actually published possess these 5 amino acids positions with some substitutions and none of ascomycete laccase actually known possess them. Consequently the absence of these five amino acids positions is the plesiomorphic state of this character and the presence of these 5 amino acids positions is a synapomorphy for all bona fide basidiomycete laccases. Neither the exact position of the insertion nor the exact number of amino acids inserted (4 or 5) were already known.

The presence of these 5 amino acids results from an insertion of 15 bp in the common ancestor of all Basidiomycetes possessing a laccase gene sensu stricto, i.e. all homobasidiomycetes clades. This putative insertion, as it is present in Thanatephorus cucumeris, took place before the divergence between chantarelloid fungi, Auriculariales and all other homobasidiomycetes [START_REF] Matheny | Contributions of rpb2 and tef1 to the phylogeny of mushrooms and allies (Basidiomycota, Fungi)[END_REF]. It is the first time that a structural difference is demonstrated between basidiomycete laccases and all the other laccases, and especially ascomycetes laccases. Some parts of the laccase sequences contain variable indels depending of the gene in the same species or depending of the species: e.g. the substrate binding loops. Nevertheless it's the first time that a difference is detected at the level of a whole fungal clade.

To our knowledge, these amino acid positions have never been described as important for laccase catalytic process, neither for substrate affinity. If this mutation has been conserved in all homobasidiomycetes it can result from the neutral evolution and the genetic drift or it gives a selective advantage. Which type of advantage it may give (consequence on the catalytic site, on the substrate affinity) will have to be looked for. Consequence of this mutation on the structure and function of the protein has to be elucidated. Consequence on the evolution of the protein is also interesting: has the mutation given rise to accelerate evolution in other parts of the protein? The origin and date of the mutation should be elucidated.

The consequence of this mutation, if there is one, on the lignin degradation or on the detoxification of its by-products by basidiomycetes, may give us a new explanation for the different fitness of ascomycetes and basidiomycetes. This discovery could also have an interest for biotechnological applications: e.g. to improve ascomycete laccases. Many hypotheses have now to be tested. Sequence names consist of species code (first letter of genus and first two letters of species name), GenBank protein accession number and protein name (when necessary, laccase is abbreviated to lac and the number used for the gene is added, ferroxidase to Fer, diphenol oxidase to diphO, ascorbate oxidase to AO, copper oxidase to copO, multi copper oxidase to mco) when it exist or gene name. For species name see Table 1 First time discovery of the presence and absence of three introns in a gene in the same species. We compared some L. edodes laccase protein sequences from various origins and available in Genbank database (AAF13037 [START_REF] Zhao | Characterization, molecular cloning, and differential expression analysis of laccase genes from the edible mushroom Lentinula edodes[END_REF], AAF13038 [START_REF] Zhao | Characterization, molecular cloning, and differential expression analysis of laccase genes from the edible mushroom Lentinula edodes[END_REF], BAB83132, BAB84355, BAB83133, AAT99286, ACR24356). These sequences were initially described as different laccases. We observed that all these sequences are probably encoded by different alleles of the same gene lac1 published first by Zhao et al. [START_REF] Zhao | Characterization, molecular cloning, and differential expression analysis of laccase genes from the edible mushroom Lentinula edodes[END_REF]. However, we observed some differences between the first published sequences [START_REF] Zhao | Characterization, molecular cloning, and differential expression analysis of laccase genes from the edible mushroom Lentinula edodes[END_REF] and the GenBank sequences. Moreover these GenBank releases were without intron sequences and, probably introns have not been correctly spliced during the annotation. ACR24356 has 99% identity with BAB84355 at the protein level (2 / 533 amino acid differences), but the encoding gene has 3 introns less (10 instead of 13), the three last introns (positions 14, 15 and 16 in Fig. 2). Moreover, BAB83133 don't have the three last introns but had a mutation in intron 14 that leads to disappearance of part of it and to transformation of another part to an exonic sequence, this resulted to a frame shift leading to an early stop codon ( The conservation of the synteny of the flanking genes or the all genome sequencing might confirm that these are alleles of the same gene lac1. It is the first time, to our knowledge, that the presence and absence of three introns are found in one gene in the same species. Presence and absence of one intron in the same species have been found very rarely within a living organism [START_REF] Llopart | Intron presence-absence polymorphism in Drosophila driven by positive Darwinian selection[END_REF][START_REF] Omilian | Intron presence-absence polymorphisms in Daphnia[END_REF].

In addition to the interest of this L. edodes gene for his splicing variants, it might be very instructive to study it in different strains, as it is an opportunity to study the mechanism of the putative linked insertion or deletion of three introns in a gene. This could help to better understand the mechanism of appearance or disappearance of splicosomal introns which are still not well understood.

CONCLUSION

The identification of a new laccase sub-family (A) in addition to laccase sub-family C in A. bisporus, as in P. ostreatus, has led us to study intron positions and protein structure of mushroom laccases. Phylogeny based on intron positions compared to phylogeny of species could give complementary information to that based on amino acid sequences. We propose the existence of at least three very old laccase sub-families (A, B and C) in Agaricales and Polyporales in addition of Cantharellales laccase sub-families. The specific laccase roles in each sub family are no yet known but clade specific laccase functions will probably be find in the future. The expression of lcc1, lcc2 and lcc3 have been studied by Largeteau et al. [START_REF] Largeteau | Expression of phenol oxidase and heat-shock genes during the development of Agaricus bisporus fruiting bodies, healthy and infected by Lecanicillium fungicola[END_REF] to find if one of them was more expressed on sporophores contaminated by Leucanicillium fungicola. None of these genes where over expressed under this condition compared to healthy sporophores. Comparative position of each laccase sub-family on chromosomes and all genome sequences may give confirmations of the phylogenetic hypotheses proposed here, as well as new data. A five amino acid insertion was probably at the origin of all basidiomycetes bona fide laccases. We identified a L. edodes gene with presence or absence of 3 introns depending of the allele. Studies of intron positions and comparative analysis of protein structure in laccase subfamily in relation to species phylogeny may generate many original data on the evolution of this protein and gene family and also on evolution of introns. New hypotheses have been proposed and are now to be tested.

Figure 1 :

 1 Figure 1: Convention used on BioEdit (version 7.0.5.3) to schematize intron position in a gene on the corresponding protein sequence. a DNA sequence with three arrows indicating three possible positions of introns in the codons, b corresponding protein sequence with arrows and diamond overlapping two amino acid letters to indicate intron position in the encoding gene.

Figure 2 :

 2 Figure 2: Relative positions of introns in different laccases genes showing the presence of two subfamilies in Agaricus bisporus. Nomenclature of introns 2 to 35 [3], 36 to 44 (A. bisporus lcc1 and lcc2), 45 to 53 (P. ostreatus poxa3).

Figure 3 :

 3 Figure 3: A-Maximum likelihood (ML) tree of edible mushroom laccases. B-One of the eight most parsimonious trees obtained with the Dollo parsimony of intron position of edible mushrooms laccase genes. Blue bare indicate Agaricoid clade VI laccase subfamily A genes. The scale at the bottom indicates a unique event: insertion or deletion of an intron. Bootstrap values are from 500 replicates in A, 1000 in B. The vertical bars indicate the positions of A. bisporus laccases. Lcc1-2 =lcc1, lcc2; pox1-2-4 = pox1, pox2, pox4. P. ostreatus gene nomenclature corresponds to: Pox1(LACC9), Pox2 (LACC10), Pox3 (LACC4), Pox4 (LACC1), Pox5 (LACC11), PoxA1b (LACC6) and Poxa3 (LACC2)

Figure 4 :

 4 Figure 4 : Alignement of partial sequences of basidiomycete laccases and other multi copper oxidases showing the position of a putative insertion of 5 amino acids in the first ones (grey line) just before the L1 signature of laccases (black line).

  or see below. Basidiomycetes laccases sub-family A -Abi_lac3, Cci_lac3, Led_lac1, Pru_lacA, sub-family C -Pos_poxa3, sub-family B -Cci_lac16, Cantharellales sub-family -Tcu_lac2 (Thanatephorus cucumeris), ferroxidase -Lbic_Fer, Apo_lac1, Sce_Fet3, ascomycete laccases -Fox_lcc4 (Fusarium oxysporum), Pan_Lac2 (Podospora anserina) ferroxidaselaccase -Fne_lac1(Filobasidiella neoformans), Fne_diphO, Pch_mco1 (Phanerochaete chrysosporium), Uma (Ustilago maydis), insect laccase -Dme_isoA (Drosophila melanogaster), nematode multi copper oxidase Cel (Caenorhabditis elegans), fungal pigment -Afu_brown (Aspergillus fumigatus), ascorbate oxidase -Acr_asom (Acremonium sp.), Cpe_AO (Cucurbita pepo), Plant LMCO -Ath_LMCO (Arabidopsis thaliana), Archae mco -Nma_mco (Nitrosopumilus maritimus), Bacteria copper resistance protein -Psy_CopA (Pseudomonas syringae), Bacteria copper oxidase -Pae_copO (Paenibacillus sp.), two subunit fungal mco -AMA_mco (Allomyces macrogynus http://www.broadinstitute.org/annotation/genome/multicellularity_project/MultiHome.html)

Fig

Figure 5 :

 5 Figure 5 : Alignment of 3 Lentinula edodes lac1 alleles showing two types of mutations affecting introns. In a is presented the BAB83133 protein sequence on the top line (NPGP … RRNG). A mutation in intron 14 leads to disappearance of part of it and to transformation of another part to an exonic sequence. The resulting frame shift stops the protein very early (blue segment). The three first lines correspond to the three reading frames obtained with the gDNA sequence present in the fourth line (Artemis software). In b ACR24356 sequence. In c, BAB84355 sequence with the position of the three introns sharing the gene coding for this sequence.

  Transformation of part of intron 14 to an exonic sequence[START_REF] Matheny | Contributions of rpb2 and tef1 to the phylogeny of mushrooms and allies (Basidiomycota, Fungi)[END_REF] 15 16

Table 1 :

 1 Gene list, intron numbers and GenBank entries of fungal laccases and ferroxidases used in the two phylogenetic analyses.

	Enzymes	Code	clades	Organism	gDNA accession number and region	Protein accession number	Gene name	Number of introns
	Laccases		Agaricales
		Abi	Agaricoid clade (VI) Agaricus bisporus	L10664	AAC18877	lcc1
					FJ838791	ACZ06558	lcc2
					EU580106	ACE73659	lcc3
					FJ392313	ACR19861	lcc4
					FJ617019	ACU52699	lcc5
					FJ878811	ACZ57763	lcc6
					NW_001889910
		Lbi		Laccaria bicolor	<486085..>488315	XP_001886681	lcc1
					NW_001889940
					<72836..>75100	XP_001889567	lcc2
					NW_001889873
					complement(109817..
					112096)	XP_001874989	lcc3
					NW_001889884
					194127..196383	ACN49091	lcc4
					DS547137
					<116239..>118647	EDR01587	lcc5
					NW_001889885
					complement(429758..
					431860)	XP_001881925	lcc6
					NW_001889885
					complement(432396..
					434513)	XP_001881926	lcc7
					NW_001889885
					complement(435301..
					437493)	XP_001881927	lcc8
					NW_001889938
					<142966..145306	XP_001889429	lcc9
		Cci		Coprinopsis cinerea	BK004113	DAA04508	lcc3
					BK004118	DAA04513	lcc8	8
					BK004117	DAA04512	lcc7
					BK004126	DAA04521	lcc16
					BK004127	DAA04522	lcc17
		Pos	Pluteoid clade (II) Pleurotus ostreatus	AJ344434	CAC69853	poxa3
					Z22591	CAA80305	pox1
					Z49075	Q12739	pox2
					FM202669	CAR48257	pox3
					FM202670	CAR48258	pox4
					AJ005017	CAA06291	poxA1b
					FM202671	PSEUDO: CAR48259	pox5
			Marasmioid clade (IV)
		Led		Lentinula edodes	AB055157	BAB84354	lcc1
					AB055158	BAB84355	lcc2=lac1
							=lac1
							without 3
					FJ473386	ACR24356	introns
					AB055159	BAB84356	lcc3
					AB543788	BAJ12091	lcc5
					AB543787	BAJ12090	lcc6
					-	BAH80447	lcc4	nd
		Fve		Flammulina velutipes	AY485826	AAR82931	laccase	9
		Pru	Polyporales	Panus rudis	AY839935	AAW28932	lacA
				Rigidoporus microporus	-	CAE81289	lcc1	nd
		Rmi		(Fomes lignosus)
	Ferroxidases Lbi		Laccaria bicolor	NW_001889910	XP_001886726	lcc11	9
					complement(<222411..
					>224748)
		Apo		Auricularia polytricha	AY616035	AAT73205	lac1
		sce		Saccharomyces cerevisiae	L25090	AAA64929	Fet3	0

Proceedings of the 7 th International Conference on Mushroom Biology and Mushroom Products (ICMBMP7) 2011Section: Physiology and Development

Proceedings of the 7 th International Conference on Mushroom Biology and Mushroom Products (ICMBMP7) 2011

ACKNOWLEDGMENT:

Serge Moukha taught us inverse PCR technique. Many students helped to obtain the complete new Agaricus laccase sequences: Florie Fauché, Emilie Lespinasse, Katia Lehn, Melissa Elie.


M Boutinaud

N Isaka

A Deflandre

E Gandemer

P.-G Marnet

F Des- Sauge

V Lollivier

Keywords: dairy cow, milk quality, free amino acids W293 Prolactin-inhibitor cabergoline hastened the mammary involution during drying-off in dairy cows

published or not. The documents may come

Prolactin-inhibitor cabergoline hastened the mammary involution during drying-off in dairy cows

Marion Boutinaud, N. Isaka, A. Deflandre, Eva Gandemer,

In ruminants, the early phase of drying-off is a period of mammary gland involution that is marked by the cessation of prolactin (PRL) release. The analysis of the changes of mammary secretion composition can provide valuable information about the speed of the mammary involution. To assess the effect of PRL inhibition by cabergoline on mammary gland involution, 14 Holstein dairy cows were injected with a single i.m. administration of 5.6 mg cabergoline (n = 7) or placebo (n = 7) at the first day of drying-off (D0). Mammary biopsy samples were collected one week before drying-off (D-6), at D1 and at D8 and used for RNA extraction and RT-PCR analyses. Mammary secretion samples were collected using a teat-cannula once during lactation (D-6) and at D1, D2, D3, D4, D8 and D14 after the drying-off. The mammary secretion samples were used for milk fat, lactose, true protein, a-lactalbumin, lactoferrin and citrate analysis. The decrease in lactose content of mammary secretions seemed to be faster in cabergoline treated cows compared to controls, demonstrated by lower levels of lactose in cabergoline treated cows already by D1 than in control cows (P < 0.05). Cabergoline treatment tended to increase fat content at D3 after drying-off (P < 0.10), whereas an increase in fat content was only observed at D4 in the control group. The rise of lactoferrin was significant starting at D4 in the cabergoline treated cows whereas it only happened at D8 in controls, and overall there was a tendency towards greater lactoferrin content in cabergoline treated cows (P = 0.10). Cabergoline did not seem to alter citrate content. However, the decrease in lactoferrin/citrate ratio happened faster in cabergoline treated cows compared to controls on D1 (P = 0.01). No significant effects of cabergoline treatment were observed both in true protein and in a-lactalbumin contents in mammary secretions or in a-lactalbumin and k-casein mRNA levels in mammary tissues. These changes in lactose, lactoferrin, lactoferrin/citrate ratio and fat, indicate that cabergoline treatment was efficient to hasten the mammary gland involution without affecting milk synthesis in the mammary tissue Key Words: cow, drying-off, prolactin W294 Effects of omitting the dry period on plasma progesterone and prolactin during lactogenesis and on colostrum IgG content in dairy cows. R. S. Zbinden 1 , H. A. van Dorland 1 , G. Remmelink 3 , B. Kemp 2 , A. T. M. van Knegsel 2 , and R. M. Bruckmaier* 1 , 1 Veterinary Physiology, Vetsuisse Faculty University of Bern, Bern, Switzerland, 2 Adaptation Physiology Group, Wageningen University, Wageningen, the Netherlands, 3 Livestock Research, Wageningen University and Research Centre, Lelystad, the Netherlands.

Omitting the dry period represents a strategy that may reduce metabolic stress in early lactating cows. A drawback of continuously milking is discussed to be a compromised colostrum quality, although insufficient studies have been done to confirm this. The objective of this study was to evaluate the effects of omitting the dry period on key hormone patterns during lactogenesis and the IgG content of colostrum in periparturient dairy cows. Twenty Holstein-Friesian dairy cows were randomly assigned to 2 experimental groups (0 or 60 d dry period; DRY0 or DRY60, respectively). Milk yields were recorded daily and plasma concentrations of progesterone and prolactin were determined from d 5 pre-until d 3 postpartum. Milk samples were collected for analysis of IgG concentration from d 7 pre-until d 3 postpartum for DRY0, and from d 0 until d 3 postpartum for DRY60 cows. Data were analyzed with a mixed model procedure including dry period length, day and their interaction as fixed effects. Plasma prolactin did not differ between DRY0 and DRY60 and started to increase significantly from one day before calving in both groups (P < 0.05). Progesterone dropped prepartum and followed a similar pattern in both groups, but was significantly lower on one day before calving in DRY60 compared with DRY0. This may point to a stronger decrease in progesterone concentration for DRY0 and could imply a faster calving process after the progesterone drop. IgG concentration started to increase from d 6 prepartum in DRY0 cows. From calving up to d 3 postpartum, IgG level decreased, but no difference was observed between the groups. However, calculated IgG mass was significantly higher for DRY60 compared with DRY0 (P < 0.05) across the study time, due to the higher milk yield in DRY60 (21.8 versus 13.5 kg/d on d 0; P < 0.05). In conclusion, the endocrine profiles supporting lactogenesis remained unaffected, and the colostrum quality was not compromised by omitting the dry period in dairy cows. It can be speculated that milking related oxytocin releases during the peripartal period induced an increased labor activity and faster birth. Peripheral circadian rhythms are well described in liver and adipose tissue and S14 expression follows a circadian rhythm. The objectives of the current study were to characterize the mammary circadian rhythm of wild-type (WT) mice and investigate the role of S14 in the rhythm of milk fat synthesis. Wild-type and S14 null mice were euthanized on d 14 of lactation (0600, 1200, 1800, or 2400 h; n = 6 per time point per genotype) and dam mammary tissue and pup stomach milk clots were collected. Dam intake and body weight and litter gain were recorded twice per day. Data were analyzed by ANOVA with genotype, time, and the interaction of genotype and time as fixed effects and second fit to a cosine function with a 24 h period for rhythm analysis. Wild-type mice consumed more feed during the dark than the light phase (66% vs. 34%; P < 0.01). Total intake and eating patterns were maintained in S14 null. Litters gained 22% more during daytime compared with nighttime in both WT and S14 null (P < 0.05). Pup milk clot fat concentration followed a circadian rhythm and the amplitude and phase were decreased and delayed by 18.7% and 15.6 h in S14 null mice (P < 0.05). In S14 null mice, the amplitude and phase of 16-carbon FA were decreased by 66% and advanced by 2.9 h, compared with WT (P < 0.05). In the mammary gland, expression of the core clock genes (CLOCK, BMAL1, CRY1&2, and PER1&2), lipogenic regulators (SREBP1c and S14), and lipogenic enzymes (FASN and SCD1) followed a circadian rhythm in

Key

  Words: colostrum, immunoglobulin, dry period W295 Characterization of mammary circadian rhythms of wild-type C57BL/6J mice and the role of thyroid hormone responsive spot 14 (S14) in circadian regulation of milk fat synthesis. L. Ma*, Y. Ying, A. Clarke, P. Bartell, and K. J. Harvatine, Penn State University, University Park.


Joffre R Cochard
email: nico09.martin@gmail.com

Deciphering 's'marts from 'r'ubbish VCs : going further the matter of letters?

Introduction

 Vulnerability curve (VC, Figure1), the relationship between the percent loss conductivity (PLC) and the xylem potential, describes plant vulnerability to drought induced cavitation, a crucial trait that correlates with several plant functions and adaptative strategies. CAV produced clear vessel length artifact as VC were systematically r-shaped. PS-PLC produced a gradient of VC shapes from r to s : The shape of VCs likely depends on the sample feature at the point of air injection. Our result suggests that intermediates VC that do not fall into r or s shapes may also misestimate true vulnerability to cavitation because of length ). Among the VC published, an apparent dichotomy exists [1] (i.e. Figure 1) :  s-shaped curves present safety margins consistent with field measurements of xylem water potential.  r-shaped curves are suspected to be the result of an measurement artifact that would overestimate embolism rate when the sample length is shorter than vessels length; these curves have mostly been reported for long vessels species such as ring porous trees [1] .  This possible vessel length artifact in the VC estimates is still debated [1,2,3] but may have serious implications.

Results & Discussion

To ensure an accurate measurement of the VC it has been recommended to check the shape of the curve, to cross-validate different methods, and to check if the points of lower xylem potential (or higher pressure) match the points of higher PLC.  BD-PLC produced consistent s-shaped VC, with a P50~4.7 Mpa in agreement with minimum water potential observed in the field [4] . (Fig. 3B).  CAV produced inconsistent r-shaped VC, using 2 different sample lengths below maximum vessel length, in accordance with the hypothesis of a vessel length artifact (Fig. 3D).  PS-PLC produced neither 'r' nor 's' shaped VCs but, VC were linear when all points were averaged (Fig3. C). Surprisingly, longer samples (25cm) were less resistant and less S-shaped than shorter ones (15cm). Additionally a very important variability in both the VC' shape and the P50 (-2  -8MPa) was observed (Fig. 3C). Such variability could be explained by the diameter of the sample (Fig. 4), suggesting that the VC depends on local vessel feature likely in the area of influence of the sleeve.

R²=0.3*

 Additional supports for a vessel length artifact for VCs build using the CAV and the PS-PLC methods come from the distribution of PLC along the sample after decreasing artificially water potential to -4 Mpa (Fig. 5). Whereas PLC should be maximum in the points of minimum pressure, it shows a decreasing pattern. The decrease is steeper for the CAV than for the PS-PLC method.

  Assessing the validity of 3 methods of VC construction in the long vessel species Quercus ilex  Plant materials size and sampling : All measurements were performed on current year Quercus ilex resprouts sampled at full maturity. Resprouts present 2 advantages :  They form long and straight samples (>1.20 m) greater than the maximal vessel length we measured (ca. 70 to 90 cm).  VC construction methods : The Bench Dehydration-PLC (BD-PLC, Fig. 2A) consists in letting large sampled branches dehydrate freely in the air to mimic natural cavitation. PLC is assessed by measuring gravimetrically the current conductivity and maximum conductivity of the sample. This is considered as a standard as it mimics field conditions. It was applied on resprouts exceeding the maximal vessel length (1.20m long).

  In the Pressure Sleeve-PLC (PS-PLC , Fig.2B) positive pressures are applied artificially to a branch sample to mimic the negative pressure of the sap. PLC was assessed gravimetrically. Curves were constructed using samples from 15 to 25 cm length. The Cavitron (CAV, Fig.2C) uses centrifugation forces to mimic the negative pressure of the sap and generate embolism. In this method conductivity can be assess during the spinning procedure by creating a pressure gradient that allows a water flow during the centrifugation. Curves were constructed using 2 different rotors (28 & 42 cm length) For CAV & PS-PLC, Additional measurements of PLC distribution along the sample at -4MPa were performed.  They experience lower water deficit (<1.5MPa) than adult tree (~-4.5MPa) thus limiting previous embolism and subsequent cavitation fatigue.  Several techniques have been developed to build VC, differing in the way embolism is induced and PLC is assessed (see Mat. & Met.

Figure 1 (

 1 Figure 1 (From Cochard et al., 2010) : r'shaped curves (interupted line) show an exponential rise and levelling-off. S'shaped (continuous line) have ecolligically meaningfull safety margin (grey area)

Figure 3 :

 3 Figure 3: VC build from the 3 different methods using Q. ilex resprouts. (A) Fitted sigmoid function using the data points from each method. (B) Cavitron using 2 sample size. (C) Pressure sleeve air injection (PS-PLC) method using 2 sample size, the insert shows curves fitted individually for each sample. (D) Bench drying method (BD-PLC) using samples larger than 1 meter (vessel length did not exceed). Error bars are standard errors.

Figure 5 :

 5 Figure5: Distribution of PLC of both side of the point where water potential is supposed to be the lowest for a pressure of -4MPa exerted with the Ps-PLC or th CAV method. For the Air Injection method, the « air injection point » where the sleeve is placed, corresponds to the point were pressure is maximum. For the cavitron, the middle of the sample match the point of lower potential. Sample length were between 28 and 50 cm. Error bars are standard errors (n=6).

Figure 4 :

 4 Figure 4: P50 of VC measured using the PS-PLC as a function of the diameter sample.

Figure 2 :

 2 Figure 2: Outline of the 3 methods used to build VCs in this study. (A) The standard bench drying method. (B) The pressure sleeve air injection method. (C) The Cavitron method.

Acknowledgement: We warmly thank Eric Badel for kindly accepting to bring the poster to the meeting at the last minute….

PLC(%)