Je ´ro

ˆme Poli

Coralie Hoareau-Aveilla

Sandra Beaufort

Valentin Tilloy

Thomas Delerue

Jean-Pascal Capp

Jean-Luc Parrou
email: jean-luc.parrou@insa-toulouse.fr

Meeting report 10th Francophone Yeast Meeting 'Levures, Mode `les & Outils'

The sunny city of Toulouse in the south of France welcomed the 10th Edition of the Francophone Yeast Meeting 'Levures, Mode `les & Outils' (LMO) at the beginning of April 2012. Since its first editions in the early 90s, this conference has gathered together all francophone researchers who are devoting their passion to the same group of organisms: yeasts.

Between the first and tenth editions of this biannual rendezvous, yeasters were faced with an extraordinary revolution: Saccharomyces cerevisiae was indeed the first eukaryotic genome completely sequenced and released to the community, reviving its enormous potential for understanding life and opening the door to new approaches in biology. This revolution was immediately followed by ambitious programmes for sequencing the genomes of its distant cousins, e.g. the 'Genolevures' program held by a consortium of seven French laboratories, with the first aim of deciphering the different mechanisms of eukaryotic genome evolution over long periods of time. Taking advantage of the knowledge of these new genomes, researchers can now tackle much more easily relevant physiological studies in very different and distant yeasts, the diversity of which has once again been attractively represented at this 10th meeting. In addition to S. cerevisiae and Saccharomyces pombe still highly valued by researchers, many oral and written communications attested to the growing interest of our community for these 'less-conventional' yeasts. This explains why, in parallel with this genomic revolution, a mutation naturally occurred in the title of this meeting, with the recent emergence of three 's' and subsequent transition from 'Levure, Mode `le & Outil' to ''Levures, Mode `les & Outils'.

This latest edition of LMO therefore brought together 250 participants from fields that rarely interact in order to discuss the current status of their research interests. This massive participation certified that our community remains very active, but also that it is particularly fond of this appointment. It offers a panoramic view of the extraordinary potential of yeasts, that continue to be driving forces in many fields of biology, from basic research to biotechnological challenges in coming years. Topics therefore ranged from a convincing demonstration of the central role of the mediator complex in RNA-polymerase II-dependent transcription to the engineering of new yeast strains to meet emerging energy problems that humanity will have to face in the near future. This report highlights some of the latest findings presented at this meeting, where two of the sessions were held in memory of renowned French colleagues Barbara Winsor and Pierre Thuriaux, who sadly passed away on the eve of retirement.

1. Beverages and energy: from ancestral use of yeasts to cutting edge biotechnological challenges

The opening 'Physiology, Biotechnology & Bioprocess' session was of course devoted to S. cerevisiae, but for several years now, the singular properties of yeasts such as Kluveromyces lactis, Yarrowia lipolytica, Candida albicans and many other yeast species have been increasingly drawing the attention of researchers. Participants heard about yeasts responses to environmental stresses such as oxidative stress, acetic acid and temperature, exposure to heavy metals, CO 2 , nitrogen and lipid limitation and energy starvation. We even heard about viability of yeasts in response to ethanol, the main product of S. cerevisiae metabolism! These environmental conditions affect signalling, metabolic regulation, redox homeostasis and even cell viability, with all these effects investigated through classical or more modern global approaches. In addition to fundamental knowledge of life, these studies clearly stand as preliminary essential steps towards biotechnological applications and control of bioprocesses, such as production of molecules of interest in dedicated industrial environments. In this context, many of the works presented in this session, particularly those reported below, were jointly presented with industrial partners and therefore led to restrictions in data communication due to confidential constraints. The two aspects that are highlighted in this meeting report present, on the one hand, the use of yeasts for production of alternative and sustainable fuels, and on the other, yeasts as natural or engineered biocatalysts in the beverage industry.

Among the major industrial uses of yeast is production of ethanol as fuel from sugar cane in Brazil and from corn in the USA. But a second generation of sustainable biofuels produced by recombinant yeast strains is estimated to become the main transportation fuel over the coming decades. Current state-of-the-art progress in that field was nicely reviewed in the introductory conference of the meeting by Jack Pronk (Delft University of Technology, The Netherlands). In the context of sustainability, lignocellulosic biomass held the attention of researchers, since this material can be hydrolysed in a mixture of both pentose sugars and hexoses. Economically viable production of ethanol by yeast nevertheless requires efficient utilisation of this complex mix of sugars. Although most yeasts can ferment hexoses, this is not the case for sugars like xylose and arabinose, which can reach 35% of the total carbohydrate content. This situation becomes even more complicated when considering the significant amount of inhibitors in these lignocellulosic feedstocks, such as acetic acid that interferes with sugar assimilation. Jack Pronk showed that metabolic engineering made this fermentation possible by using, among others, expression of structural genes for the L- arabinose utilisation pathway of Lactobacillus plantarum into S. cerevisiae. Significant improvement in fermentation parameters was then achieved through repeated cycles of consecutive batch cultivation in media with different compositions. This evolutionary engineering strategy allowed rapid selection of an evolved strain exhibiting improved performance. It resulted in a 40% reduction in the time required to completely ferment glucoseexyloseearabinose mixtures with high ethanol yield (0.43 g/g of total sugar) [START_REF] Wisselink | Novel evolutionary engineering approach for accelerated utilization of glucose, xylose, and arabinose mixtures by engineered Saccharomyces cerevisiae strains[END_REF]. Julie Monthe ´ard (LISBP, Toulouse, France) also focused on xylose conversion into bioethanol using the alternative yeast Candida shehatae, which is able to naturally ferment xylose. The author successfully set up growth conditions to reach an average title of 40 g/l of ethanol, with 80 g/l of biomass dry weight without any loss of viability, which represents one of the best performances reported in the bibliography.

Still in the field of biofuels, development of new routes for hydrotreated lipid production is also being promoted. Nicolas Morin (MICALIS, Jouy-en-Josas, France) and Maud Babau (LISBP, Toulouse, France) showed that oleaginous yeasts such as Y. lipolytica and Rhodotorula glutinis could be used for lipid-based biofuels, including aeronautic applications in which specifications are drastically more restrictive than for car engines. By using a well-controlled fed-batch cultivation process, N. Morin integrated both quantitative physiological parameters and transcriptomic data to decrypt gene networks that control lipid synthesis during the transition from biomass formation to lipid accumulation in Y. lipolytica. Transcriptome profiling highlighted an early lipid accumulation phase, characterised by an increase in nitrogen metabolism together with strong repression of protein production and activity, followed by a late lipid accumulation phase characterised by the rerouting of carbon fluxes within cells [START_REF] Morin | Transcriptomic analyses during the transition from biomass production to lipid accumulation in the oleaginous yeast Yarrowia lipolytica[END_REF]. Through microbial engineering approaches, the LISBP team first demonstrated that R. glutinis could accumulate lipids up to 0.72 g/g of biomass with a yield of 0.22 g/g glucose, i.e. 95% of the theoretical maximal yield of this carbon source. This remarkable performance relative to lipid accumulation led M. Babau to study the possibility of growing R. glutinis using a mixture of both pentose sugars and hexoses. Despite a clear reduction in energetic and carbon yields while increasing the xylose/glucose ratio, they showed that xylose could be assimilated for biomass production, therefore confirming the possible use of this alternative yeast for lipid production from renewable non-food resources.

In addition to biofuels, yeast is of primary importance in oenology. In this particular applied context, alternatives to metabolic engineering approaches have been engaged to improve the fermentation potential of wine yeast strains. Bioprocess optimisation, evolutionary engineering and crossing approaches of parental yeasts that strongly diverge for one or more phenotypic properties (or traits) can be used for such purposes. Philippe Marullo (ISVV/SARCO, Bordeaux, France) presented data relative to the field of yeast breeding. His approach relies on heterosis, which represents the superiority of the hybrid in comparison to the progenitor strains for quantitative polygenic traits. While studying a total of 64 strains, both diploid S. cerevisiae and Saccharomyces uvarum parental strains and their resulting hybrids, he found many cases of punctual heterosis for either kinetic properties or fermentation products, including aromas. Interestingly, he noted that few interspecific hybrids showed high levels of ethyl esters. Another approach based on quantitative trait loci (QTL) analysis was highlighted by Jessica Noble (Lallemand R&D, Blagnac, France). Using functional complementation analysis, J. Noble identified loci implicated in the reduction of SO 2 , H 2 S and acetaldehyde, which are undesirable compounds in oenology. She also identified another locus that appeared to be determinant for fermentative performances under nitrogen starvation, which is one of the limiting parameters in wine fermentation. Claire Brice (SPO, Montpellier, France) illustrated in her poster the strategy developed to identify mechanisms underlying the diversity of nitrogen use in an oenological context. She tested three strains of S. cerevisiae with high nitrogen requirements and four strains with low nitrogen requirements, in both rich and poor nitrogen media. Transcriptome analysis highlighted that efficient de novo protein synthesis is of primary importance for strains with low nitrogen requirements, while it discarded the nitrogen storage and amino acid assimilation-based-hypotheses.

Evolution and the genomics revolution

These later presentations naturally led us to the 'Genomes & Evolution' session, which was opened by Gianni Liti (IRCAN, Nice, France). G. Liti gave us a clear lecture on population genomics and complex traits in yeast, highlighting the advantages of yeast for linking natural genetic variations in a population to phenotypic variations in a given trait. The resequencing of a large collection of Saccharomyces strains isolated worldwide and characterised at the genomic and phenotypic levels both provided data on population structure and proved to be a powerful tool for identifying sequence variants accounting for phenotypic variations. In addition, the development of a new QTL mapping method and its immediate advantages were presented. This strategy, which involves several rounds of crosses, artificial selection and whole genome sequencing, enables measuring genome-wide changes in parental allele frequency, hence yielding a much higher resolution than previous genomic linkage studies [START_REF] Cubillos | Assessing the complex architecture of polygenic traits in diverged yeast populations[END_REF][START_REF] Parts | Revealing the genetic structure of a trait by sequencing a population under selection[END_REF]. The genes IRA1 and IRA2, encoding two Ras GTPase-activating proteins (GAP) that act as negative regulators of the RasecAMP signalling pathway, were validated as determinant QTLs for heat sensitivity.

The work reported by G. Liti was a perfect example of how the massive use of new sequencing methods has accelerated the production of genomic data, leading to conceptually new approaches, specifically relevant for genome evolution studies. However, the advent of new generation sequencing has also lowered the quality of the resulting data, especially in terms of assembling and annotation. Hence, general sites such as Genbank and EMBL now demand a set of meta-data, information on sequence quality and minimal annotation for publication of a new genome. In that context, Tiphaine Martin (LaBRI e Ge ´nolevures, Talence, France) gave a comprehensive presentation of these recent evolutions in the publication policies of sequencing data on public websites. Two examples of genome sequencing and analysis readily meeting these criteria were presented, both from biotechnologically relevant strains. In the first, Jean-Marc Daran (Delft University of Technology, The Netherlands) exposed the sequencing of the laboratory CEN.PK113-7D strain [START_REF] Nijkamp | De novo sequencing, assembly and analysis of the genome of the laboratory strain Saccharomyces cerevisiae CEN.PK113-7D, a model for modern industrial biotechnology[END_REF]. Numerous genomic variations have been identified compared to S288c, including copy number variations and, in particular, 6 duplicated clusters, some of them containing MAL genes (involved in maltose metabolism). Remarkably, genes encoding transcription factors and DNA binding proteins are overrepresented among sequences presenting single nucleotide variations and insertions/deletions. Moreover, the authors reported variations in the number of tandem repeats in genes like SNF11, involved in transcription efficiency. Genomic regions present only in the CEN.PK113-7D strain have also been identified, particularly the one on chromosome I that contains the first two genes of the biotin biogenesis pathway, enabling this strain to grow in the absence of biotin in the medium. Then, Virginie Galeote (SPO, Montpellier, France) exposed new results from sequencing of the commercial wine yeast strain EC1118. The genome of this strain contains 34 additional genes compared to S288c, clustered into three large regions that have been horizontally transferred from distant yeasts and may participate in its technological performances [START_REF] Novo | Eukaryote-to-eukaryote gene transfer events revealed by the genome sequence of the wine yeast Saccharomyces cerevisiae EC1118[END_REF]. Most of these genes are unknown, but some of them, encoding high-affinity fructose and oligopeptide transporters, could be implicated in oenological traits. V. Galeote and collaborators discovered that one of these DNA regions, originating from the wine contaminant Zygosaccharomyces bailii and recently transferred, is likely amplified in yeast genomes as an extrachromosomal circular DNA molecule which can integrate the Saccharomyces cerevisiae genome at different loci by non-homologous end joining (NHEJ) [START_REF] Galeote | Amplification of a Zygosaccharomyces bailii DNA segment in wine yeast genomes by extrachromosomal circular DNA formation[END_REF]. Indeed, karyotypic analysis revealed different possible locations and sometimes different copies of this region in other wine yeasts. Furthermore, gene organisation of these different fragments is variable and the breakpoint sequences show little or no homology, corresponding to insertions by NHEJ.

In addition to their intrinsic scientific interest for deciphering the genetic basis of physiological traits, these fine analyses of particular genomes allow development of new global approaches to searching for genes or gene networks of possible technological interest, such as that presented by Christian Brion (SPO, Montpellier, France). By analysing segregants from a S288c  EC1118 cross, he identified several interesting expression QTLs (eQTLs). One of them, triggered by the PDR8/HAP1 locus, is particularly relevant for its regulation of the PDR detoxification gene family, while another contains the oenological form of THI3 gene and contributes to increased expression of genes involved in the thiamine biogenesis pathway. Finally, a cluster of abnormal transcription on chromosome 16 revealed a translocated fragment associated with a higher fermentation rate at 70% of the fermentation process. From a similar perspective, but on a very different yeast model, Ste ´phanie Michely (MICALIS, Jouy-en-Josas, France) used comparative physiology and genome sequencing to investigate the basis of growth on hydrophobic substrates and lipid storage of Y. lipolytica and its eight closest species. Six complete genomes have been obtained and, together with physiological analysis, enabled identification of the POX family (encoding Acyl-CoA oxidases) as being associated with growth on oleic acid. Loss of POX4 or duplication of POX5, for instance, has been mentioned, and divergence of the POX4 sequence in one strain is linked to better growth on oleic acid.

Traffic and organelles: focus on mitochondrial ATP synthase

In the "traffic and organelles" session, mitochondria have been the object of several oral and poster presentations, mainly dealing with different aspects of mitochondrial physiology and metabolism. In particular, two talks were given on ATP synthase, studied from very different perspectives. Daniel Bre `thes (IBGC, Bordeaux, France) showed that the two ATP synthase subunits e and g, non-essential for enzyme activity, are required for both the supramolecular organisation of ATP synthase as well as for mitochondrial morphology and cristae formation. He also showed that those conserved in higher eukaryotes properties have significant consequences well beyond ATP synthase activity. Alexandre Martos (IBGC, Bordeaux, France) showed that evolutionary transfer of genes from the mitochondria to the nuclear genome requires modification of the gene product itself, but also adaptation of other cellular pathways allowing protein transfer to its final mitochondrial compartment. By using the mitochondrial ATP9 gene of S. cerevisiae as a case study, these authors successfully complemented the atp9 mutant through nuclear expression of the filamentous fungus Podospora anserina homologue, likely due to the low hydrophobicity of the first transmembrane segment of the protein. This hypothesis is strengthened by the inability to mature the S. cerevisiae ATP9 precursor protein, synthesised in the cytosol, by mitochondrial proteases, contrary to a hybrid ATP9 protein carrying the first transmembrane segment of P. anserina ATP9. Besides fundamental knowledge of mechanisms of gene transfer during evolution, their results augur well for future treatment of genetic diseases of mitochondrial origin.

Let's travel into the nucleus: from DNA organisation to transcription

Microorganisms are particularly exposed to fluctuating environments, but are able to swiftly adjust their behaviour according to their needs. How cells are able to sense these changes and subsequently activate specific regulatory pathways remains subject to debate. In eukaryotes, genomes are physically separated from other cellular components by the nuclear envelope (NE), enabling establishment of a distinct environment for DNA replication, gene expression and maintenance of genome integrity. Genome-wide, molecular and cellular approaches combined in different mutated backgrounds were used to unravel the complex regulation that underlies DNA-based processes in yeast.

The accurate replication of genomic DNA depends on sequential activation of hundreds of replication origins. During replicative stress induced by genotoxic agents, initiation at late origins is inhibited by the DNA replication checkpoint (DRC) (Tourrie `re and Pasero, 2007). Whether or not the DRC is also active during a normal S-phase is not known. Philippe Pasero (IGH, Montpellier, France) showed that the DRC delays the activation of late origins under normal growth conditions. He also reported that phosphorylated histone H2A, a mark of DRC activation, accumulates at transcribed regions immediately after passage of replication forks. These data suggest that the DRC detects interferences between replication and transcription in order to regulate S-phase progression. Replication forks are fragile structures that frequently encounter obstacles hindering their progression. How paused forks are maintained is still a matter of debate. Mireille Tittel-Elmer (University of Calgary, Canada) discovered that cohesins, a protein complex important for maintaining sister chromatid cohesion, are localised at sites of replication and spread along DNA as forks move. Moreover, Scc1p enrichment at sites of replication is dependent on the structural features of Rad50p and promotes fork restart. This finding suggests a new role for cohesin in the maintenance of replisome integrity.

Another level of regulation is provided by co-activators of transcription such as the Spt-Ada-Gcn5-acetyltransferase complex (SAGA), which regulates the expression of inducible genes in eukaryotes. Dominique Helmlinger (CRBM, Montpellier, France) found that the SAGA subunit Tra1 is not essential in S. pombe as compared to S. cerevisiae, enabling a functional study of this protein. He showed that rather than having a global role within SAGA, as other subunits do, Tra1 is involved in recruitment of SAGA at only a subset of SAGAdependent promoters and in transcriptional regulation of the same subset of genes in response to environmental stresses. Further findings on yeast transcription were presented by Jessie Colin (CGM, Gif-sur-Yvette, France), who showed results of a screen aimed at detecting new elements involved in RNAPII transcription termination. In the framework of this screen, J. Colin identified a new termination mechanism relying on a Myb-related transcription activator, Reb1p. This activator binds to an 8-nt conserved motif on DNA and elicits termination of a new class of non-coding unstable transcripts, providing a fail-safe termination mechanism for a number of mRNAs in vivo.

The NE bridges the cytoplasm to the nucleoplasm through the nuclear pores and integral NE proteins, allowing for regulation of cellular events. Hugo Bretes (IJM, Paris, France) focused on Pml39p, a protein anchored to the nuclear pore complex by the NE myosin-like proteins Mlp1p and Mlp2p. Together with Mlp1p and Ulp1p, Pml39p is required for proper export of messenger ribonucleoparticles (mRNPs) resulting from intron-containing genes, thus preventing inappropriate expression of mRNAs derived from these genes. New roles for the NE in chromosome positioning were also presented by Maya Spichal (Institut Pasteur, France) who showed that the outer NE protein Csm4p might have an effect on telomere localisation and dynamics through its interaction with currently unidentified components of the inner NE. Telomere homeostasis is mainly regulated by the well-known telomerase enzyme. Using high-resolution microscopy, Franck Gallardo (LBME, Toulouse, France) demonstrated that telomerase-recruitment clusters (T-recs) are formed on telomeres only at the end of the S-phase. This technique could be used to screen for telomerase activity in cancerous cells [START_REF] Gallardo | Live cell imaging of telomerase RNA dynamics reveals cell cycle-dependent clustering of telomerase at elongating telomeres[END_REF].

RNA life, from synthesis to translation

The yeast is an easy genetic tool allowing researchers to delve deeper into the understanding of complex mechanisms, particularly relevant for many aspects of the RNA life cycle that were developed in the 'RNA, Ribosome and Translation' session. DEAD-box RNA helicases are ubiquitous proteins involved in virtually all processes involving RNA. They are RNA-dependent ATPases and ATP-dependent RNA binding proteins, but they display only weak helicase activity when assayed in vitro; this makes it difficult to characterise their specificity, regulation or target identification. Meriem Senissar and Agne `s Le Saux (IBPC, Paris, France) presented some of the data they recently obtained concerning the essential DEAD-box family member Ded1p. This protein is homologous to the human DDX3 and it is one of the most active helicases in vitro. However, it shows no specificity in vitro, indicating that in vivo its specificity is likely due to cofactors that need to be identified. They showed that Ded1p interacts directly with the translation initiation factors eIF4G and eIF4E, present on the 7-methylguanosine CAP of mRNAs, and with Pab1p, which is a polyA binding protein present at the 3 0end of mRNA. They also established that some of these interactions stimulate the ATPase activity of Ded1p in vitro. They showed that Ded1p is associated with mRNPs containing eIF4G, eIF4E and Pab1p, but that it is not associated with polysomes. This demonstrates that Ded1p has an affinity for mRNA. The identification of Ded1p partners and substrates should contribute to characterisation of its function and lead to better understanding of the role of RNA helicases in the cell.

Splicing is an important regulatory step in the mRNA life cycle. It can be affected either in cis, by the mRNA splice sites, or in trans, by regulatory proteins involved in the splicing event. The spliceosome is an RNAeprotein structure composed of five small nuclear ribonucleoproteins (snRNP) associated with Sm proteins. The biogenesis of snRNPs is a complex process mediated by the methylosome and the survival motor neuron (SMN) complex. In his presentation, Adrien Barbarossa (IGMM, Montpellier, France) described the identification of an S. pombe ICln homologue. Mutations into spICln lead to growth and splicing defects. He noted that some introns with suboptimal polypyrimidine tracts are inefficiently spliced in the Dicln mutant.

Ribosomes are RNAeprotein macromolecular complexes that require a plethora of factors for their synthesis. During the last two decades, a large number of genome-wide strategies have been employed to identify trans-acting factors involved in ribosome biogenesis. This gave rise to the discovery of not less than 200 factors, including proteins and small nucleolar ribonucleoprotein particles (snoRNPs) implicated at different steps of ribosomal RNA precursor (pre-rRNA) maturation and ribosome assembly. The challenge now is to determine the precise function of these 200 factors. Nearly all of the ribosome biogenesis process was covered during this 10th LMO meeting, including the study of the early nucleolar pre-90S, the pre-60S and the late cytoplasmic pre-40S particles, work mainly done in the 'Laboratoire de Biologie Mole ´culaire Eucaryote' (LBME, Toulouse, France), directed by Miche `le Caizergues-Ferrer. The very first steps in ribosome assembly were investigated through characterisation of the Utp23p and Kri1p proteins. Indeed, Coralie Hoareau-Aveilla (LBME) showed that these two proteins interact specifically and in a mutually exclusive manner with the snR30-snoRNP. In addition, she reported evidence suggesting that Utp23p plays a crucial role in early pre-ribosome conformation changes to promote snR30 release from the pre-ribosomal particles [START_REF] Hoareau-Aveilla | Utp23p is required for dissociation of snR30 small nucleolar RNP from preribosomal particles[END_REF]. To the already long list of early trans-acting factors taking part in the small subunit (SSU) processome, Elodie Choque (LBME) added a new member: the Efg1p protein. This nucleolar and non-essential protein is associated in vivo with the 35S, 23 pre-rRNAs and the U3 snoRNA. Furthermore, yeast cells depleted for Efg1p cultured at 37 C showed defective 18S rRNA production. Indeed, E. Choque showed that Efg1p is required for 35S pre-rRNA early cleavages at sites A0, A1 and A2. These cleavages are known to take place very early during the processing pathway of the 35S pre-rRNA and to be essential for the elimination of the 5 0 external (5 0 ETS) and internal (ITS1) transcribed sequences surrounding the 18S rRNA sequence. The talk given by Yanling Chen (LBME) discussed early pre-60S ribosome biogenesis. Using immunoprecipitation experiments, Yan-ling showed that the Gno1p protein, previously reported to interact with the helicase Prp43p, is associated with pre-60S ribosomal particles and is required for their production. In addition, using in vitro enzymatic tests, she demonstrated that PinX1, the human homologue of Gno1p, stimulates the helicase activity of Prp43p. Thus, it was proposed that Prp43p, implicated in several steps of ribosome biogenesis, is specifically regulated by Gno1p in the pre-60S particle maturation pathway. Late pre-40S biogenesis in humans was broached by Kamila Baumas (LBME), who identified RioK3 as a new member of the RIO kinase family involved in rRNA 18S production. She showed that RioK3 is associated in the cytoplasm with pre-40S particles and seems to display a cytoplasmic function required for correct maturation of the 21S pre-rRNA [START_REF] Baumas | Human RioK3 is a novel component of cytoplasmic pre-40S pre-ribosomal particles[END_REF].

Finally, Yuri Motorin (AREMS, Nancy, France) presented a structureefunction analysis of the Nop2 protein, a putative m 5 C:methyltransferase implicated in maturation of the 25S rRNA. Using various functional complementation experiments, he showed that the N-terminal part of Nop2p is important for viability and nucleolar localisation. Thanks to bisulfite RNA sequencing he discovered new methylation sites of the 25S rRNA that he directly linked to Nop2p activity.

In this 'RNA, Ribosome and Translation' session, another important aspect of ribosome life was presented: the regulation of ribosome function, and more particularly translational frameshifting. Oliver Namy and Isabelle Hatin (IGM, Orsay, France) reported their recent results on the [PSI(þ)] prion protein corresponding to an aggregated conformation of the translation release factor eRF3. They showed that expression of [PSI(þ)] stimulates a þ1 frameshift event during translation of OAZ1 mRNA, which encodes a negative regulator of cellular polyamines. This leads to enhancement of antizyme production and, as a direct consequence, a decrease in the polyamine level. To go further, genome-wide approaches such as ribosome profiling were employed to identify a more general role for [PSI(þ)] in translation regulation. This ongoing work possibly points out new and unexpected functions of the prion [PSI(þ)]. In turn, Sandra Blanchet (IGM, Orsay, France) showed that the PSI(þ)] strain can be used as a tool to study how natural tRNA suppressors are selected during stop codon readthrough. Using a reporter gene harbouring a premature stop codon expressed in the PSI(þ)] strain, she was able to precisely measure the selection rate of each possible tRNA suppressor and incorporation of the corresponding amino acid. This study is of particular importance for the treatment of human pathologies caused by the presence of stop codons in critical genes such as the P53 gene in cancers and the DMD gene in Duchenne muscular dystrophy. Indeed, depending on its nature, the incorporated amino acid may differentially affect the function of the mutated protein.

mRNA level regulation does not always come from where it is expected. Indeed Damien Hermand (LGM, Namur, Belgium) explained that mRNA abundance could come from the codon content that affects translation efficiency. The elongator complex is responsible for the 5methoxycarbonylmethyl-2-thiouridine modification of the wobble uridine present in the tRNA. The lack of tRNA modification due to elongator mutants leads to various phenotypes such as mitosis delay or cytokinesis failure due to inefficient translation. D. Hermand found that mRNAs that contain more AAA codons than AAG are less translated in mutants of the elongator complex. He highlighted a new mechanism by which the codon content, coupled with the tRNA modifications by elongator, regulates the protein level through translation efficiency.

6. It's all about control: key elements in cell cycle progression and establishment of cell polarity Several new and fascinating results were presented during the sessions devoted to 'Cell cycle, cytoskeleton and polarity'. The importance of the fission yeast S. pombe as a model for cell division was here again demonstrated, with two talks on microtubule organisation and one on the contractile ring. First, Phong Tran (Institut Curie, Paris, France) in his introductory lecture attempted to answer the question: "How to build a bipolar mitotic spindle?" He described a screen for spindle-assembly-defective mutants and the identification of a new protein, Sad2p, involved in initiation of mitotic spindle bipolarity. Then, Yannick Gachet (LBCMCP, Toulouse, France) described the development of a powerful mathematical model of chromosome segregation which enables predicting defects in segregation and chromosome attachment to microtubules in different mutants and conditions. Finally, Anne Paoletti (Institut Curie, Paris, France) gave a presentation on contractile ring assembly. A previous work from the author showed that Pom1p kinase concentrates at cell ends, so its concentration decreases in the middle of the growing cell, hence allowing cell division [START_REF] Moseley | A spatial gradient coordinates cell size and mitotic entry in fission yeast[END_REF]. A. Paoletti now reported that Pom1p inhibits Mid1p/Anilin, a major factor in division plane definition and cell size control during division. Pom1p also inhibits kinases Nim1p, Cdr1p and Cdr2p, three inhibitors of Wee1p. Pom1p phosphorylation sites on Cdr2p have been mapped and it is suggested that they influence Cdr2peMid1p interactions and Cdr2p kinase activity.

The budding yeast S. cerevisiae was nevertheless the model used in several other cell-cycle-related presentations. In his talk, Damien Laporte (IBGC, Bordeaux, France) demonstrated that S. cerevisiae quiescent cells are distinct from G1-arrested cells. Indeed, they identified G 2 budding cells that are bona fide quiescent and that display characteristic structures of quiescent cells, such as actin bodies and proteasome storage granules (PSGs). Remarkably, carbon starvation is sufficient to trigger entry into quiescence, including for cells in G 2 /M phase [START_REF] Laporte | Metabolic status rather than cell cycle signals control quiescence entry and exit[END_REF]. Subsequently, glucose alone can activate mobilisation of the actin bodies and re-entry into proliferation, but it has to be internalised, enter glycolysis and be metabolised at least until pyruvate. The subtlety of proliferation and growth control by nutritional signals was also illustrated by Gregory Bonfils (University of Fribourg, Switzerland). He showed that leucyl-tRNA synthetase Cdc60p controls TORC1, an essential regulator of eukaryotic cell growth, via the EGO complex [START_REF] Bonfils | Leucyl-tRNA synthetase controls TORC1 via the EGO complex[END_REF]. In this work, a leucine-dependent physical interaction between Cdc60p and the TORC1 regulators GTPase Gtr1p was identified. This interaction, necessary and sufficient for TORC1 activation by leucine, is mediated by the amino-acid-editing domain CP1 (connective peptide 1) of Cdc60p, and does not require functionally independent amino acylation activity of Cdc60p. The authors proposed a model in which the conformational change in CP1, resulting from its engagement in editing mischarged tRNA Leu , disrupts the Cdc60peGtr1p interaction and consequently downregulates TORC1.

Another elegant study conducted on S. cerevisiae was presented by Fabrice Caudron (IBC, ETH Zu ¨rich, Switzerland), who consensually received the jury's Nikon prize for the quality of his photographs. In the absence of an actual mating partner, haploid cells adapt to low concentrations of pheromones and restart budding. Once they have escaped from pheromone arrest, mother cells maintain this memory and go on budding. The molecular details of this behaviour have been described and are shown to depend on a new mechanism of cytoplasmic epigenetics.

Acronyms of the laboratories

AREMS

  Biochimie et Ge ´ne ´tique Cellulaires IBPC Institut de Biologie Physico-Chimique IGH Institut de Ge ´ne ´tique Humaine IGMM Institut de Ge ´ne ´tique Mole ´culaire de Montpellier IGM, Orsay Institut de Ge ´ne ´tique et Microbiologie IJM Institut Jacques Monod IRCAN Institut de Recherche sur le Cancer et le Vieillissement ISVV Institut Supe ´rieur de la Vigne et du Vin LABRI Laboratoire Bordelais de Recherche en Informatique LBCMCP Laboratoire de Biologie Cellulaire et Mole ´culaire du Contro ˆle de la Prolife ´ration LBME Laboratoire de Biologie Mole ´culaire Eucaryote LGM Laboratoire de Ge ´ne ´tique Mole ´culaire LISBP Laboratoire d'Inge ´nierie des Syste `mes Biologiques et des Proce ´de ´s MICALIS Microbiologie de l'Alimentation au Service de la Sante ´humaine SPO Sciences Pour l'Oenologie

		ARN-RNP, structure fonction-maturation, enzy-
		mologie mole ´culaire et structurale
	CGM	Centre de Ge ´ne ´tique Mole ´culaire
	CRBM	Centre	de	Recherche	de	Biochimie
		Macromole ´culaire
	IBC	Institute of Biochemistry
	IBGC	Institut de


Lucas Moriniere

Alexandre Burlet

Perrine Portier

Céline Lavire

Xavier Nesme

Franck Bertolla

Back to basics : Rethinking the diversity and pathogeny of the causal agent of Bacterial Leaf Spot of Lettuce

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

Back to basics :

Rethinking the diversity and pathogeny of the causal agent of Bacterial Leaf Spot of Lettuce Bacterial Leaf Spot of Lettuce (BLSL) is a major threat to lettuce fields worldwide due to the lack of knowledge on its causal agent, Xanthomonas hortorum pv. vitians (Xhv). Moreover, this disease seems to have emerged since the last few years in the Rhône-Alpes region, a major area of lettuce production in France.

Characterization of accurate genetic diversity and taxonomy is a necessary step preliminary to epidemiological studies. However, genetic diversity of Xhv has been described only on northern-american strains so far and its taxonomy remains unclear [START_REF] Fayette | Multilocus sequence typing of strains of bacterial spot of lettuce collected in the United States[END_REF]. Indeed, this pathogen is currently divided in two groups : group B includes all the Xhv strains except for the pathotype strain (CFBP2538). This last strain is registered as X. citri pv. vitians and considered as group A.

Furthermore, the pathway this pathogen follows to invade the plant and its histological localization during infection have not been yet fully described [START_REF] Barak | Movement of Xanthomonas campestris pv. vitians in the stems of lettuce and seed contamination[END_REF]. Such parameters are determinant to understand the symptomatology and can be valuable to ensure the pathovar homogeneity [START_REF] Hugouvieux | Entry of Xanthomonas campestris pv. campestris into hydathodes of Arabidopsis thaliana leaves: a system for studying early infection events in bacterial pathogenesis[END_REF] .

BLSL on a Leaf Lettuce (Rhône-Alpes, summer 2016)

Objectives of this study

Investigate the genetic diversity of strains from various origins and their phylogenetic relatedness with other pathovars and close Xanthomonas species.

Search for potential virulence variation and cross these phenotypic data with phylogenetic analyses to redefine the limits of the vitians pathovar. As expected, the pathotype strain X. citri pv. vitians CFBP2538 differs from the other Xhv strains and had been shown to be non-pathogenic on lettuce. Thus, a more suitable pathotype strain should be described.

Conclusions

  Characterize the entry gate(s) of Xhv in planta. . vitians • Maximum Likelihood phylogeny (GTR + γ + I) on 1640 positions (1000 bootstraps, branches condensed if supported by < 60 % bootstraps) Systemic spreading via the vascular system • Disease Index on a 5-point scale, based on number of spots and lesions size (on Oakleaf lettuce cv. Kirinia, adapted from Bull & Koike 2005) observations of blue final reaction product accumulation in leaf tissues (X-gluc substrate overnight bath at 37°C followed by chlorophyll extraction in 70% ethanol bath at 80°C)

Lucas MORINIERE 1 , Alexandre BURLET 2 , Perrine PORTIER 3 , Céline LAVIRE 1 , Xavier NESME 1 , Franck BERTOLLA 1 1

  Ecologie Microbienne Lyon, UMR CNRS 5557 INRA 1418, Université de Lyon, Villeurbanne Cedex, France 2 Station d'Expérimentation Rhône-Alpes Information Légumes SERAIL, Brindas, France 3 IRHS, INRA, AGROCAMPUS-Ouest, Université d'Angers, SFR4207 QUASAV, CIRM-CFBP, 42 rue Georges Morel, 49071 Beaucouzé cedex, France

  Xhv taxa without any visible influence of geographical origins or time of isolation. These results are consistent with a recent similar study on northern-American strains by Fayette et al. in 2016, suggesting that these three phylotypes are prevalent worldwide.

						Moreover, phylotype in the Phenotypically, Xhv seems able to we described three penetrate the leaf indistinctly through
						hydathodes	and	stomata	causing
						vascular and parenchyma infection.
						However, stomatal penetration and
			Consequently the	parenchyma	infection	should	be
	vitians	pathovar	appears	to	be	confirmed by microscopy experiments.
	monophyletic, all strains sharing the
	same	direct	common	ancestor.

Acknowledgments

• Laurène Mirabel for her invaluable technical assistance • Elise Lacroix from Greenhouse platform • Marion Fischer-Le Saux for her advises and suggestions • Cindy Morris for her welcome at the INRA station of Montfavet and our very inspirational conversation


Amélie Quiquerez
email: amelie.quiquerez@u-bourgogne.fr

Emmanuel Chevigny

Pascal Allemand

Pierre Curmi

Christophe Petit

Philippe Grandjean

Assessing the impact of soil surface characteristics on vineyard erosion from very high spatial resolution aerial images (Côte de Beaune, Burgundy, France)

Keywords:

We describe a new method to map intra-plot soil surface heterogeneities at a 5 cm spatial resolution. Our approach unites aerial image classification acquired at very high spatial resolution (VHSR) with local soil sampling. VHSR aerial image processing, based on image classification, allows precise mapping of the spatial distribution of soil surfaces; soil sampling defines soil typology by physical and chemical characteristics. This method has been applied to a plot area located on the hillslopes of Burgundy vineyards (Monthelie, France), where decennial erosion data were already available, in order to assess the effect of soil surface characteristics and slope angle on erosion intensity and localisation. From this method, four classes of radiance were distinguished and interpreted as four soil surface state classes (SSC), defining specific areas within the studied plot. These SSCs have been characterised by their grain-size distribution, their organic carbon, calcium carbonate, and total nitrogen contents. By allowing soil surface states to be mapped at five centimetre resolution, this approach provides novel insights into the characterisation of soil patterns and into erosion analysis on cultivated hillslopes. Our work shows that the spatial distribution of soil erosion is related to the local slope steepness but also to the spatial distribution of stoniness that results from water and tillage erosion processes.

Introduction

Although vineyards cover an only small portion of the Earth's surface, they provide an excellent example of an erosive land-use context. Vineyards undergo substantial soil loss, ten to thousand tonnes per hectare per year, in comparison with other types of agricultural land [START_REF] Boardman | Soil erosion in Europe: major processes, causes and consequences[END_REF][START_REF] Brenot | Erosion rates and sediment budgets in vineyards at 1-m resolution based on stock unearthing (Burgundy, France)[END_REF][START_REF] Cerdan | Rates and spatial variations of soil erosion in Europe: a study based on erosion plot data[END_REF][START_REF] Hooke | Human impacts on fluvial systems in the Mediterranean region[END_REF][START_REF] Krause | Multi-parameter finger printing of sediment deposition in a small gullied catchment in SE Australia[END_REF][START_REF] Bissonnais | L'érosion hydrique des sols en France[END_REF][START_REF] Paroissien | A regional-scale study of multidecennial erosion of vineyard fields using vine-stock unearthing-burying measurements[END_REF]. They epitomise a situation where soil sustainability is threatened, leading in some cases to major economic and social problems (Le [START_REF] Bissonnais | L'érosion hydrique des sols en France[END_REF][START_REF] Martínez-Casanovas | On-site effects of concentrated flow erosion in vineyard fields: some economic implications[END_REF][START_REF] Martínez-Casasnovas | The cost of soil erosion in vineyard fields in the Penedès-Anoia Region (NE Spain)[END_REF].

Several studies have already shown the importance of relief (slope length and mean slope angle) both in the morphology of initiated erosive structures and in the volumes involved in soil erosion [START_REF] Fox | The relationship of soil loss by interrill erosion to slope gradient[END_REF][START_REF] Hooke | Human impacts on fluvial systems in the Mediterranean region[END_REF][START_REF] Martínez-Casanovas | On-site effects of concentrated flow erosion in vineyard fields: some economic implications[END_REF][START_REF] Montgomery | A physically based model for the topographic control on shallow landsliding[END_REF][START_REF] Quiquerez | Soil degradation caused by a highintensity rainfall event: Implications for medium-term soil sustainability in Burgundian vineyards[END_REF][START_REF] Wischmeier | Predicting rainfall-erosion losses: a guide to conservation planning[END_REF]. At metre scale, however, the erosion rate sometimes exhibits intra-plot heterogeneity, which cannot be explained by mean slope angle and slope length of the plot [START_REF] Brenot | Erosion rates and sediment budgets in vineyards at 1-m resolution based on stock unearthing (Burgundy, France)[END_REF][START_REF] Cerdan | Rates and spatial variations of soil erosion in Europe: a study based on erosion plot data[END_REF]. As soil surface characteristics (soil cover, grain size, texture etc. …) evolve spatially at the metre scale [START_REF] Corbane | Remote sensing of soil surface characteristics from a multiscale classification approach[END_REF], they too can influence hydric properties and erosion rates [START_REF] Arnaez | Factors affecting runoff and erosion under simulated rainfall in Mediterranean vineyards[END_REF][START_REF] Auzet | Concentrated flow erosion in cultivated catchments: influence of soil surface state[END_REF][START_REF] Auzet | Soil surface characteristics: dynamics and impact on soil erosion[END_REF][START_REF] Auzet | Surface characterisation for soil erosion forecasting[END_REF]. Agricultural practices may also affect soil variability and erosion rates. For example, tillage may induce some translocation and dispersion of the plough layer [START_REF] Armand | Runoff generation related to intra-field soil surface characteristics variability, application to conservation tillage context[END_REF][START_REF] Blavet | Effect of land use and management on the early stages of soil water erosion in French Mediterranean vineyards[END_REF][START_REF] Govers | The role of tillage in soil redistribution on hillslopes[END_REF][START_REF] Govers | Tillage erosion and translocation: emergence of a new paradigm in soil erosion research[END_REF]Poesen et al., 1994;[START_REF] Schumacher | Modeling spatial variation in productivity due to tillage and water erosion[END_REF].

Intra-plot soil surface variability may be investigated from soil surface mapping [START_REF] Mulder | The use of remote sensing in soil and terrain mapping-a review[END_REF]. Soil surface mapping often uses conventional soil surveys and laboratory analyses that are precise but time-consuming and extremely costly because of the number of measurements needed for soil surface mapping at a high spatial resolution [START_REF] Mulder | The use of remote sensing in soil and terrain mapping-a review[END_REF][START_REF] Webster | Statistical Methods in Soil and Land Resource Survey[END_REF]. Conversely, other methods, using remote sensing techniques that have emerged over the last decades, are recognised as efficient methods to examine soil and landform relationships [START_REF] Martin | Capturing spatial variability of soil and litter properties in a forest stand by landform segmentation procedures[END_REF][START_REF] Mulder | The use of remote sensing in soil and terrain mapping-a review[END_REF]. Remote sensing techniques have successfully been employed for soil and terrain mapping at a global or catchment scale [START_REF] Irvin | Fuzzy and isodata classification of landform elements from digital terrain data in Pleasant Valley[END_REF][START_REF] Martin | Capturing spatial variability of soil and litter properties in a forest stand by landform segmentation procedures[END_REF] and are expected to contribute to Digital Soil Mapping as an auxiliary data source [START_REF] Lagacherie | Digital soil mapping: an introductory perspective[END_REF][START_REF] Mcbratney | On digital soil mapping[END_REF] (see [START_REF] Mulder | The use of remote sensing in soil and terrain mapping-a review[END_REF] for a review). Aerial or satellite imagery has also demonstrated their usefulness when studying the relationships between hydrological runoff, sediment erosion and soil surface characteristics [START_REF] King | The application of remote sensing data to monitoring and modelling of soil erosion[END_REF], evaluating surface roughness [START_REF] Baghdadi | Retrieving surface roughness and soil moisture from synthetic aperture radar (SAR) data using neural networks[END_REF], or assessing intra-plot soil surface variability by estimating soil surface reflectance [START_REF] Corbane | Reconnaissance des états de surface en milieu cultivé méditerranéen par télédétection optique à très haute résolution spatiale[END_REF][START_REF] Corbane | Remote sensing of soil surface characteristics from a multiscale classification approach[END_REF][START_REF] Soto | Mise en relation de la cartographie du rendement avec la distribution spatiale de l'état de surface du sol observée par télédétection[END_REF][START_REF] Wassenaar | Soil surface infiltration capacity classification based on the bi-directional reflectance distribution function sampled by aerial photographs. The case of vineyards in a Mediterranean area[END_REF].

Our approach combines sampling and remote sensing techniques at very high spatial resolution (VHSR), i.e. at a five centimetre spatial scale, to highlight fine spatial distribution of soil surface heterogeneities. This is an intermediate scale, between the regional scale of Digital Soil Mapping and limited local observations. The capacity and efficiency of remote sensing techniques to derive soil information have been proven at the regional scale, and are now applied in this study at plot scale. Our approach is applied in Burgundy vineyards where decennial erosion data are already available at a square metre scale [START_REF] Brenot | Quantification de la dynamique sédimentaire en contexte anthropisé[END_REF][START_REF] Brenot | Erosion rates and sediment budgets in vineyards at 1-m resolution based on stock unearthing (Burgundy, France)[END_REF], to assess the influence of soil surface heterogeneity and slope angle on erosion intensity in the Monthelie vineyards. In the following sections, the plot characteristics (soil surface topography and soil erosion data) and aerial image classification will be presented. The results of soil mapping will be combined with an intra-plot soil description allowing soil types to be defined by physical and chemical characteristics (stoniness, grain-size distribution, organic carbon, calcium carbonate, and total nitrogen content). Finally, soil surface topography, topsoil types and soil erosion data will be compared to assess their effects on erosion.

Material and methods

2.1. The study area 2.1.1. Description of the study site and plot characteristics

The study site is located on the hillslopes of the Monthelie vineyards, in the "Côte de Beaune" area (Burgundy, France) (Fig. 1A). The hillslopes, shaped by the Bressan rifting, form the eastern border of the Burgundy plateau. Vine-growing is attested on this fault-scarp relief since the Middle Ages [START_REF] Garcia | Le vignoble galloromain de Gevrey-Chambertin «Au-dessus-de Bergis», Côte-d'Or[END_REF]. The hillslopes are overlain by a silty-clayey calcaric cambisol (World reference base for soil resources, 2006) that develops on Jurassic marls, where white limestones contrast with the dark matrix of the soil (Figs. 1B,2A). The plot is bordered upslope by the limestone plateau. Downslope, the plot is bounded by a rill collecting water and sediment. Auger holes performed on the plot reveal that the soils are superficial and range in thickness from 30 cm upslope to 50 cm downslope. The landscape is characterised by a vine monoculture where the plot limits, i.e. paths and walls, form the only discontinuities on the hillslopes [START_REF] Garrier | Le phylloxéra. Une guerre de trente ans[END_REF]. Vine stocks are planted 1 m apart, in rows 1 m apart; chemical weeding produces non-grassed rows; tillage in the inter-row is performed once or twice a year. The tillage is superficial and the inter-row roughness induced by the straddle-tractor traffic is minimal. These soil management practices and the slope-oriented vine rows are all factors which greatly increase erosion.

Topographic data

The studied plot (833.880 m, 6.657.500 m; Lambert 93 projection) is located on the northern part of the Combe Danay valley, on the upper part of the hillslope. The plot, corresponds to a total area of 9500 m 2 (Fig. 2A). A digital elevation model (DEM) was obtained from a topographic survey with a laser theodolite (resolution of 15 m, horizontal and vertical precision b 1 cm). Slopes vary regularly from 1°t o 13°from a planar to concave morphology (Fig. 2B). Three main slope classes were distinguished: the steepest slopes (N9°) are located in the south-western part of the plot (covering 24% of the plot), the central part is characterised by moderate slopes (5 to 9°, covering 67% of the plot), whilst gentle slopes (less than 5°) are found in the upper part of the plot (covering 9% of the plot). In this plot, vines were planted in 1982 with the rows in the steepest slope direction.

Erosion data

A 30-year average erosion map was performed at a square metre scale, from more than 9000 stock unearthing measurements (SUM method, see [START_REF] Brenot | Quantification de la dynamique sédimentaire en contexte anthropisé[END_REF][START_REF] Brenot | Erosion rates and sediment budgets in vineyards at 1-m resolution based on stock unearthing (Burgundy, France)[END_REF] (Fig. 3A). The principle of soil loss measurement is based on considering the unearthing of the stock located on the vine plants as a passive marker of soil surface vertical displacement since the year of plantation. Plantation practice, regimented by regional by-laws demands that the scion-graft union be located 1 cm above the soil surface when vines are planted [START_REF] Galet | Precis de viticulture[END_REF], in order to prevent contact between the scion and the soil. During the vine's growth, the stock develops its own roots that are concentrated at the lower part of the stock whilst vine shoots develop from the scion [START_REF] Brenot | Erosion rates and sediment budgets in vineyards at 1-m resolution based on stock unearthing (Burgundy, France)[END_REF]. The stock exposure is interpreted as reflecting the dynamics of soil aggradation/ablation, considering the scion-graft limit to be a marker of the initial topography. Plantation legislation imposes a density of 10,000 vines per hectare which allows the quantification of soil erosion at 1 metre resolution (Fig. 3B). Errors in unearthing measurements are related essentially to the vine plantation and to the downward shifting of the graft limit, which may underestimate the true soil lowering [START_REF] Brenot | Erosion rates and sediment budgets in vineyards at 1-m resolution based on stock unearthing (Burgundy, France)[END_REF]. An error margin of ±1 cm was added to the SUM to take into account the measurement error at the date of plantation [START_REF] Brenot | Erosion rates and sediment budgets in vineyards at 1-m resolution based on stock unearthing (Burgundy, France)[END_REF]. The erosion class distribution is presented in Fig. 3C. Each pixel represents a measurement value, which is given in centimetre (Fig. 3D). The analysis of value distribution shows that all data are negative. This means that the entire plot is affected by erosion on a 30 year period. The mean erosion is about -2.7 cm (± 1 cm) with values ranging from -1 (±1 cm) to -14 cm (±1 cm). Null erosion values (erosion value ≤ 1 cm) represent 30% of total measurements. Forty per cent of the values of the dataset are associated with low erosion (1 cm b erosion value ≤ 3 cm). Moderate (3 cm b erosion value ≤ 5 cm) and high erosion values (erosion value N5 cm) are obtained for about 19% and 9% of the measurements, respectively.

Topsoil mapping from very high spatial resolution (VHSR) image processing

The VHSR images were acquired in April 2010 by the unmanned helicopter DRELIO (Université Lyon 1-Université de Bretagne Occidentale, France) [START_REF] Delacourt | DRELIO: an unmanned helicopter for imaging coastal areas[END_REF] at 5 cm spatial resolution (Fig. 2A). The helicopter is equipped with a reflex digital camera (Nikon D200, 10 MegaPixels) with a 35 mm equivalent focal length (chip size 24 mm), an automatic piloting system and an onboard GPS giving the geographic position during the flight. Images were acquired at a constant 70 m flying altitude to avoid geometrical deformation and during early spring, to avoid leaf and plant cover. The camera was set to "manual" with a velocity acquisition of 1/4000 s and a constant opening (f/4.5). The images were saved in 12-bit raw mode to preserve the maximum of radiance information. The duration of a flight was less than 30 min and the flight took place near solar midday. Illumination conditions were thus nearly constant and no substantial radiance differences have been observed from one image to the next.

Image pre-processing consists in the construction of a mosaic from 30 selected images. The images were first georeferenced using control points from a differential GPS, and then pseudo-orthorectified. The artefacts obstructing soil information (roads, agricultural paths, vinestocks and their shadows) were masked by segmentation before image analysis. Images of the soil surface were classified using an unsupervised ISODATA method (i.e. "Iterative Self Organizing Data Analysis Technique") [START_REF] Hall | Statistical Methods for Digital Computers[END_REF] that is frequently applied to map landform or to examine soil-landform relationships in various land uses [START_REF] Adediran | Computer-assisted discrimination of morphological units on north-central Crete (Greece) by applying multivariate statistics to local relief gradients[END_REF][START_REF] Burrough | High-resolution landform classification using fuzzy k-means[END_REF][START_REF] Irvin | Fuzzy and isodata classification of landform elements from digital terrain data in Pleasant Valley[END_REF]. The algorithm uses an iterative statistical approach to cluster pixels into homogeneous areas of similar characteristics (i.e. into classes), without any a priori knowledge of their spatial distribution. The user defines a minimum and a maximum number of possible classes. The pixels are affected to a given class using a minimum Euclidian distance technique by an iterative computation. The result is independent of the interpretation of the image by the user.

In the study site, we merely set between two and eight the number of soil classes in the plots. This range of values implies that more than two different radiance values should be identified, which is justified by the presence of white limestones and dark soil matrix. This also signifies that fewer than eight soil classes may be defined. This value was never reached after iterative processing, validating a posteriori this choice. Finally, to allow the comparison between soil surface distribution and erosion measurements, a low-pass filter (Gaussian blurring) on a 5 * 5 kernel grid (25 cm * 25 cm) has been applied on the classified images to smooth any local superficial heterogeneity present at a scale less than 1 m 2 . The obtained images have then been resampled at a 1-m resolution using a median filter.

Topsoil characteristics from laboratory analyses

Local observations and analyses were performed to describe the soil surface states defined by image processing (Fig. 4). Soil sampling sites were selected within the spatial distribution of the classes described above. For each class, five random samples were collected in order to characterise the physical and chemical properties of the soil surface state classes defined by image processing. For each sample, we performed field observations and laboratory analyses to define stoniness and grain-size distribution according to the techniques of the French Association for Normalisation (AFNOR). These samples were used to quantify the total organic carbon, calcium carbonate, and total nitrogen contents. All results are presented in Fig. 6 and in Table 1.

Stoniness and grain-size distribution analysis

Samples were collected in the 0-10 cm soil layer in the inter-row over a 0.25 m 2 surface to determine soil stoniness and grain-size distribution. Stoniness (N 2 mm fraction) is obtained by weighing and sieving on undispersed material using dryness agitation. The moist soil residue was weighed and oven-dried for 24 h (40 °C) to determine the dry weight. A grain-size distribution was obtained using sieve sizes for the 250 μm-2 mm fraction (coarse sand), the 63 μm-250 μm (medium and fine sand), and the fraction lower than 63 μm (silts and clays) (French norm NF P18-560).

Total carbon, calcium carbonate, nitrogen and organic carbon contents

The total calcium carbonate content was estimated using the Bernard calcimeter (international norm NF ISO 10693). Total carbon and nitrogen contents were determined by pyrolysis (980 °C) under oxidising conditions and dosage of CO 2 and N 2 released by gas chromatography, following the standard norm NF ISO 10694. The organic carbon content was obtained from this value by correcting from the total carbonate carbon (NF ISO 10963).

Results

Soil surface classification from VHSR

Fig. 4A andB illustrates respectively the soil surface classification at 5 cm and 1 m resolution. The image classification accentuates soil radiance differences detectable in the natural mosaic between bright surfaces covered by stones and dark surface dominated by the siltyclayey matrix (Fig. 2A).

Four classes of radiance were distinguished and interpreted as four soil surface state classes (SSC), defining specific areas within the studied plots (Figs. 4 and5). The SSC1 (35% of the total surface) and the SSC2 (38% of the surface) are essentially located in the central part of the plot and are defined by bright soil surface states with high radiance values. The SSC3 (16% of the surface) and SSC4 classes (11% of the surface) are observed in the upper eastern part and in the lower part of the plot. They correspond to darker soil surfaces, defined by lower radiance values.

Soil surface characteristics

Physico-chemical characteristics of soil surface classes (SSCs) are presented in Table 1. Total carbonate content ranging between 128‰ and 144‰ was not a discriminating factor between SSCs. Organic carbon and nitrogen contents varied from 3.5% to 4.7%, and 0.20% to 0.30% respectively. SSC3 and SSC4 displayed a high variability in organic carbon and nitrogen contents, whilst the range of variations remained limited for SSC1 and SSC2. Stoniness was high and decreased from SSC1 (77%) to SSC4 (56%). This trend was anti-correlated to the sand and fine silty-clayey fractions. SSCs displayed low intra-class variability in the stoniness, but also in the silty-clayey fraction, with the exception of SSC3.

SSC1 showed a very high stoniness (77%), a very low silty-clayey fraction (18%), and a total organic carbon content of 4.1%. The range of variation was limited in both the physical and chemical properties. SSC2 was similar to SSC1 but displayed a lower stoniness (70%), a higher silty-clayey fraction (24%), a higher organic carbon and total nitrogen contents (4.7% and 0.30% respectively). SSC3 showed the highest values of calcium carbonate (144‰), displaying moderate stoniness, high silty-clayey fraction, lower organic carbon and nitrogen contents, and a higher C/N ratio (26.2), in comparison to those of SSC1 and SSC2. SSC4 was similar to SSC3 but contains lower carbonate content (131‰), higher organic carbon content (4.1%) and a lower variability than that of SSC3.

Box plot (Fig. 6) highlighted some differences between SSCs in stoniness, silty-clayey fraction, organic carbon and total nitrogen contents. Therefore, these analyses showed that each soil surface class identified from image processing was representative of soil surface characteristics defined by specific chemical and physical properties.

This new approach makes it possible to assess the spatial relation between soil surface characteristics (Fig. 4), slopes (Fig. 2B) and erosion (Fig. 3). All the data were collected in a GIS database. The comparisons between slope, soil and erosion maps have been synthesised in Figs. 7 and8.

Soil surface characteristics, slope and erosion

Slope and erosion

At the plot scale, spatial and statistical analyses of the slope and erosion data (Figs. 2B and3D) showed that the erosion values were higher in the south-western part of the plot, located on steep slopes (mean value of 2.8 cm ± 1.7) than in the upper part, located on gentle slopes (mean value of 2.2 cm ± 1.5) (p b 0.0001 Mann-Whitney test).

Fig. 7A illustrates the distribution of erosion for each slope class. The surfaces covering the same slope values were not identical. Therefore, the erosion data distribution had been normalised over the surface according the moderate slope class that is the most representative, in order to evaluate the importance of the slope effect on erosion (Fig. 7B). Null to very low erosion values (SUM ≤ 1 cm) were found dominantly in the gentler slopes (slopes b 5°) whilst low erosion values (1 b SUM ≤ 3 cm) are almost slope-independent. Conversely, moderate to high unearthing values (SUM ≥4 cm) are dominantly found on moderate and steep slopes. More than 85% very high values (SUM ≥8 cm) are located on moderate and steep slopes (slopes N9°).

Spatial relation between SSC and erosion

The soil surface distribution (Fig. 4) has been compared to the erosion map (Fig. 3) to study the spatial relationships between the intensity and localisation of erosion and the topsoil characteristics. Table 2 showed that SSC1 and SSC2, that contains a high stoniness, are associated to lower mean values (mean SUM of 2.5 cm and 2.6 cm respectively) than those for SSC3 and SSC4 (2.9 cm and 3 cm respectively) (p b 0.0001, Mann-Whitney test). Fig. 7C presented the distribution of SSC classes for each SUM class. Values have been normalised over the surface covered by each SSC to allow comparison between data (Fig. 7D). Very low values (SUM ≤1 cm) are more commonly found in SSC1 and SSC2 (stony soil surface) located in the central part of the plot, than in SSC3 and SSC4 (more silty-clayey soils). Low erosion classes (1 b SUM ≤ 3 cm), which are slope-independent, are also uniformly distributed across the plot, independently to the SSCs (approximately 40% for each SSC). Fig. 8 showed that very high erosion values (SUM N5 cm) were preferentially associated with low stoniness surface states encountered in SSC3, SSC4 located in the south-western part and on the northern border of the plot. In this plot, the pattern of erosion seemed to be related to the spatial distribution of soil surface states, and specifically to the stoniness. Fig. 9 shows that low to moderate erosion (SUM ≤ 5 cm) are associated to high radiance values induced by a white stone surface cover. Conversely, in the south-western part of the plot, soils that are rich in fine fraction are more easily eroded, and display a lower radiance (Fig. 9 for SUM ≥8 cm).

Discussion

In vineyard contexts, erosion is controlled by interactions and feedback between natural and anthropogenic processes [START_REF] Boardman | Soil erosion in Europe: major processes, causes and consequences[END_REF]. Several studies have demonstrated the importance of water-induced erosion in contributing to sediment transfer at the hillslope scale [START_REF] Hooke | Human impacts on fluvial systems in the Mediterranean region[END_REF][START_REF] Martínez-Casanovas | On-site effects of concentrated flow erosion in vineyard fields: some economic implications[END_REF][START_REF] Quiquerez | Soil degradation caused by a highintensity rainfall event: Implications for medium-term soil sustainability in Burgundian vineyards[END_REF]. Other studies have highlighted the role of anthropogenic factors, i.e. tillage and agricultural management, on the spatial distribution of erosion [START_REF] Armand | Runoff generation related to intra-field soil surface characteristics variability, application to conservation tillage context[END_REF][START_REF] Blavet | Effect of land use and management on the early stages of soil water erosion in French Mediterranean vineyards[END_REF][START_REF] Chartin | Classification and mapping of anthropogenic landforms on cultivated hillslopes using DEMs and soil thickness data-example from the SW Parisian Basin, France[END_REF][START_REF] Kosmas | The effect of land use on runoff and soil erosion rates under Mediterranean conditions[END_REF][START_REF] Paroissien | A regional-scale study of multidecennial erosion of vineyard fields using vine-stock unearthing-burying measurements[END_REF][START_REF] Schumacher | Modeling spatial variation in productivity due to tillage and water erosion[END_REF]. Others have shown that sediment dynamics on hillslope may Fig. 6. Stoniness, sand and silty-clayey fractions, organic matter content, total nitrogen content and C/N content for each of the SSC classes defined by image analysis.e also be related to former plot boundaries in agricultural landscapes at catchment scale [START_REF] Hooke | Human impacts on fluvial systems in the Mediterranean region[END_REF][START_REF] Jordan | Historical land use changes and their impact on sediment fluxes in the Balaton basin (Hungary)[END_REF][START_REF] Szilassi | Impacts of historical land use changes on erosion and agricultural soil properties in the Kali Basin at Lake Balaton, Hungary[END_REF].

In the Monthelie vineyard plot, comparisons between soil surface distribution and erosion maps show that low and moderate erosion areas are preferentially located in the central part of the plot, where stony SSC1 and SSC2 prevail. Conversely, high erosion values are located in the south-western part of the plot, which is less stony, and where there is a break in slope. We suggest that these SSCs reflect the action of hydric erosion, present-day management practices and, possibly a land use evolution over time.

Our work shows that the pattern of erosion of this plot is inversely correlated to the spatial distribution of the stone percentage at the metre scale. The paved soil surface, rich in stones, protects from the splash effect, delays runoff processes and thus reduces soil erosion [START_REF] Martínez-Zavala | Effect of rock fragment cover on interrill soil erosion from bare soils in Western Andalusia, Spain[END_REF][START_REF] Martinez-Zavala | Relationships between rock fragment cover and soil hydrological response in a Mediterranean environment[END_REF][START_REF] Poesen | Rock fragments in top soils: significance and processes[END_REF][START_REF] Puigdefabregas | The Rambla Honda field site: interactions of soil land vegetation along a catena in semi-arid Southeast Spain[END_REF]. In the central part of the plot, where erosion is limited, the high stoniness and the superficial pavement observed in SSC1 and SSC2 may have resulted from the selective and progressive removal of fine particles by overland flows during repeated high intensity rainfall events [START_REF] Poesen | Rock fragments in top soils: significance and processes[END_REF][START_REF] Poesen | The hydrological response of soil surfaces to rainfall as affected by cover and position of rock fragments in the top layer[END_REF]. Indeed, in Burgundian vineyards, intense rainfall events are known to mobilise and transport particles. Conversely, repeated tillage operations may cause the enrichment of the rock-fragment fraction in the topsoil [START_REF] Follain | The effects of redistribution processes on rock fragment variability within a vineyard topsoil in Mediterranean France[END_REF]Govers et al., 1994, 1999;[START_REF] Poesen | Patterns of rock fragment cover generated by tillage erosion[END_REF]. Here, SSC1 can hardly be attributed to water erosion alone, since slope gradient is minimal in the upper part of the plot, suggesting a medium to long-term combined water and tillagecontrolled erosion. Identical surface soil degradation in upper slope positions has been observed in various parts of the European Mediterranean belt in cultivated areas [START_REF] Cerdan | Rates and spatial variations of soil erosion in Europe: a study based on erosion plot data[END_REF][START_REF] Poesen | Rock fragments in top soils: significance and processes[END_REF][START_REF] Schumacher | Modeling spatial variation in productivity due to tillage and water erosion[END_REF]. This degradation is however complete, since the observed pavement suggests that SSC1 will evolve no further, unless superficial tillage is performed once or twice a year.

Our work also shows that there is a global control of the topography over the localisation and intensity of the erosion operating over the plot. In the south-western part of the plot, where a break in slope is observable, the erosion rates are high and the SSCs are characterised by low stoniness. Auger holes performed in the steep slope area show that no rock fragments have been found deeper in the soil profile. The soils remain superficial since they range in thickness from 30 cm upslope to 50 cm downslope. The steep slope suggests water-dominated erosion in this area, illustrating the superficial dynamics currently operating on these hillslopes. Fine-grained particles are easily mobilised during each high intensity rainfall event. They are gradually transferred downslope and may be temporarily stored, even on these steep slopes. The evolution from upslope topsoils that are depleted in fine particles to enriched downslope soils may reflect these short-term hillslope dynamics. In the medium to long term, such soil erosion can lead to a modification of these SSCs, in quantitative and qualitative ways, since upslope fine particles are exported out of the plot. Evidence of such hillslope dynamics can be illustrated by the soil characteristics in the SSC2 and SSC3 classes, which present intermediate values between those of SSC1 and SSC4, in terms of stoniness, sand and silty-clayey fractions. Fig. 4B shows that the soil surface grades more or less gradually from one soil class to another.

If sediment dynamics may contribute to the enrichment of soils in fine particles in the south-western part of the plot, the examination of a 1953 aerial photograph (Fig. 10) suggests that the current pattern of soil distribution may have been also influenced by a land-use change in the medium term. The 1953 aerial photograph shows that the upslope part of the plot was occupied by a wasteland, whilst the downslope area was covered by forest. The limit between the wasteland and the forest matches the break in slope, between the moderate and steep slope areas (Fig. 10). Therefore, it is possible that the forest cover before 1982 had played a role in the location and preservation of the enriched soils observed in the southern-part of the slope.

Finally, our study shows that analysis of VHSR aerial images is a nonintrusive, quickly-performed method that it makes possible to generate full, detailed views of the soil surface. The classification map created from VHSR aerial images can be considered as an efficient qualitative indicator of soil surface distribution if it is combined with on-site quantitative soil analysis to define soil surface characteristics. Our combined observations based on very high spatial resolution suggest that the current pattern of soil distribution and erosion reflect the effects of geomorphological processes but also of present-day tillage and probably of a land-use change in the medium term.

These high resolution observations provide new insights to explain intra-plot spatial variability by integrating spatial, temporal, local and continuous data. Such maps can be helpful to assess the role of soil distribution in soil degradation processes and forecasting. Such an approach could be also very useful to optimise management practices for precision viticulture.

Conclusions

This paper presents a simple, highly efficient approach to map soil surface heterogeneities at a five centimetre resolution in Burgundy vineyards. This approach relies on the combination between very high resolution aerial data at the plot/hillslope scale and local soil observations. VHSR aerial image processing allows precise mapping of the spatial distribution of soil surfaces; soil sampling defines soil typology by physical and chemical characteristics. The combination of on-site soil data and VHSR aerial photos permits production of continuous and accurate spatial distribution maps at a very fine spatial resolution. By allowing the visualisation of soil distribution at very high spatial resolution, this approach offers new insights and possibilities for documenting soil patterns and for exploring and predicting soil evolution through space and time on hillslopes, which can be important for agricultural planning.

Fig. 1 .

 1 Fig. 1. (A) Location of the study area in the Monthelie vineyard, in the Côte de Beaune area (Burgundy, France) and (B) geology of the study area.

Fig. 2 .

 2 Fig. 2. (A) VHSR mosaic of the plot obtained from the unmanned DRELIO helicopter at a 5 cm spatial resolution. (B) Slope map illustrating three slope classes: gentle (b5°), moderate (5 to 9°) and steep slopes (N9°)et al. / Catena 116 (2014) 163-172

Fig. 3 .

 3 Fig. 3. (A) SUM method for calculation of erosion rate at a square metre scale (Brenot et al., 2008 modified). Graft union limit shows initial topography and stock unearthing indicates soil level changes. (B) Rows and vines spaced 1 m allow measuring erosion at a square metre scale. (C) Histogram representing SUM data distribution. (D) Unearthing erosion map of the plot. Yellow indicates null to very low erosion (≤1 cm) whereas blue indicates very high erosion (≥8 cm).

Fig. 4 .

 4 Fig. 4. (A) Classification from aerial image at 5 cm resolution, (B) median filter was applied to allow comparison between the classification map and the erosion map performed at 1-m resolution. On each map, four classes have been determined on the basis on radiance values. The soil sampling locations are defined by the spatial distribution of these classes.

Fig. 5 .

 5 Fig. 5. Photographs of the four SSC recognised by image analyses. Stoniness decreases from SSC1 to SSC4.

Fig. 7 .

 7 Fig. 7. (A) Distribution of SUM classes as a function of slope classes; (B) distribution of SUM classes normalised over the surface covered; (C) distribution of SUM classes as a function of SSC; (D) distribution of SUM classes normalised over the surface covered.

Fig. 8 .

 8 Fig. 8. Spatial distribution of very high erosion values (SUM ≥6 cm) (A) over the VHSR aerial image, (B) over the topsoil classification map.

Fig. 9 .

 9 Fig. 9. Distribution of SUM classes as a function of raster value. Very high erosion values (SUM ≥8 cm) are related to lower raster values than low to moderate erosion values (SUM ≤5 cm).

Fig. 10 .

 10 Fig. 10. The 1953 aerial photography highlights the presence of a forest in the downslope part of the plot whereas the upslope part was occupied by a wasteland. The limit matches the break in slope, between the moderate and steep slope areas.1

Table 1

 1 Mean and standard deviation (between brackets) values for each SSC.

		Stoniness	Sands	Silts and clays	Total carbonate content	Organic carbon content	Total nitrogen content	C/N ratio
		%	%	%	‰	%	%
	SSC1	77 (5)	5 (1)	18 (4)	128 (17)	4.1 (0.3)	0.25 (0.02)	21.3 (2.5)
	SSC2	70 (5)	6 (3)	24 (3)	129 (30)	4.7 (0.4)	0.30 (0.02)	22.0 (2.8)
	SSC3	58 (10)	7 (3)	34 (9)	144 (62)	3.5 (1.1)	0.20 (0.07)	26.2 (5.6)
	SSC4	56 (7)	8 (1)	35 (7)	131 (35)	4.1 (0.6)	0.27 (0.07)	22.8 (6.4)

Table 2

 2 Mean SUM and standard deviation values for each SSCs.

		SUM (cm)
		Mean	Standard deviation
	SSC1	2.5	1.7
	SSC2	2.6	1.7
	SSC3	2.9	1.8
	SSC4	3.0	1.9

Acknowledgements

This work was financially supported by the Burgundy regional council (CRB) and the inter-professional bureau of Burgundy vines (BIVB). Thanks to C. Chateau-Smith for proof-reading the English text. The detailed comments of the anonymous reviewers were greatly appreciated and helped to improve the manuscript.