Holy Ravalason
email: holy.ravalason@esil.univmed.fr

Sacha Grisel

Didier Chevret

Anne Favel

Jean-Guy Berrin

Jean-Claude Sigoillot

Isabelle Herpoël-Gimbert

Fusarium verticillioides secretome as a source of auxiliary enzymes to enhance saccharification of wheat straw

Keywords:

Fusarium verticillioides secretome as a source of auxiliary enzymes to enhance saccharification of wheat straw

Second generation bioethanol produced from various lignocellulosic materials, such as wood, agricultural or forestry residues, is one of the most promising renewable energies. Wheat straw is a feedstock of particular interest since it represents a potentially large and readily available agricultural residue resource of low value. Current costs of enzymatic saccharification are among the largest contributors to the overall cost of lignocellulosic ethanol production. A major challenge is the development of an efficient and economically viable hydrolysis process step [START_REF] Margeot | New improvements for lignocellulosic ethanol[END_REF].

The filamentous fungus Trichoderma reesei remains one of the most effective producers of cellulases. For the conversion of cellulose into glucose, three types of cellulolytic enzymes are secreted by T. reesei: endoglucanases (EG, EC 3.2.1.4), cellobiohydrolases (CBH, EC 3.2.1.91) and b-glucosidases (BGL,EC 3.2.1.21). Compared to other filamentous fungi, this model fungus was shown to be lacking many hemicellulase and pectinase families [START_REF] Martinez | Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina)[END_REF]. In order to overcome the recalcitrant structure of lignocellulose and release the locked polysaccharides, enzymes altering the interaction between lignin, hemicellulose and cellulose or those involved in degradation of hemicellulose and lignin are required. Previous studies demonstrated that supplementation of cellulases with various hemicellulases or other polysaccharide-and ligninmodifying enzymes can have a beneficial effect on enzymatic hydrolysis, presumably by improving cellulose accessibility [START_REF] Couturier | Podospora anserina hemicellulases potentiate the Trichoderma reesei secretome for saccharification of lignocellulosic biomass[END_REF][START_REF] Gao | Hemicellulases and auxiliary enzymes for improved conversion of lignocellulosic biomass to monosaccharides[END_REF].

Many filamentous fungi gain nutrition from the breakdown and the decay of plant biomass and therefore are particularly interesting producers of cell wall-degrading enzymes (CWDEs) including cellulases, hemicellulases, ligninases and pectinases ( [START_REF] Van Den Brink | Fungal enzyme sets for plant polysaccharide degradation[END_REF]. Exploration of fungal biodiversity for additional auxiliary enzymes that could be added to T. reesei cellulases preparations to improve biomass degradation is of great interest. With the increasing availability of fungal genome sequences and the development of proteomic methods, it has become possible to identify the secreted enzyme pools involved in lignocellulosic biomass degradation. Several descriptive and/or differential analyses of secreted proteins by fungi grown on lignocellulose and related carbon sources have been reported (for a review see [START_REF] Bouws | Fungal secretomes-nature's toolbox for white biotechnology[END_REF]). However, only a few studies have highlighted the relationships between distribution and prevalence Comment citer ce document : Ravalason, H. (Auteur de correspondance), Grisel, S., Chevret, D., Favel, A., Berrin, J.-G., Sigoillot, J.-C., Herpoël-Gimbert, I. (2012). Fusarium verticillioides secretome as a source of auxiliary enzymes to enhance saccharification of wheat straw. [START_REF] Ravalason | Secretome analysis of Phanerochaete chrysosporium strain CIRM-BRFM41 grown on softwood[END_REF][START_REF] Sharma | Evaluation of glycosyl hydrolases in the secretome of Aspergillus fumigatus and saccharification of alkali-treated rice straw[END_REF][START_REF] Couturier | Post-genomic analyses of fungal lignocellulosic biomass degradation reveal the unexpected potential of the plant pathogen Ustilago maydis[END_REF].

In the present study, the secretome of Fusarium verticillioides, an ascomycete filamentous fungus well-known as a corn pathogen, was demonstrated to enhance the saccharification of wheat straw in supplementation to commercial cellulases. Taking advantage of the availability of genomic data for this fungus [START_REF] Ma | Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium[END_REF], the secretome was analyzed and it is discussed how the CWDEs found in the F. verticillioides secretome increase the hydrolysis efficiency of T. reesei cellulases.

Methods

Raw material

Dried wheat straw (Triticum aestivum var. Samuraï,) was obtained from ARD (Pomacle, France) and reduced to particles of 1-2 mm using an analytical mill (IKA A11, IKA-Werke, Staufen, Germany). The composition of untreated wheat straw was 35.6% cellulose, 22.6% xylan, 1.5% galactan, 3.3% arabinan and 17.2% lignin (on a dry weight basis).

Fungal strain

F. verticillioides strain 7600 (obtained from the Fungal Genetics Stock Center, FGSC, Kansas City, USA) was maintained in the ''Centre International de Ressources Microbiennes'' culture collection dedicated to filamentous fungi of biotechnological interest (CIRM-CF; http://www.inra.fr/crb-cirm/), National Institute of Agricultural Research (INRA), Marseille, France. It was selected from a panel of fungal strains previously screened for their capacity to improve the hydrolysis of wheat straw in combination with the commercial cellulases GC220 (Genencor Danisco, NY, USA; data not shown).

Growth conditions

F. verticillioides was grown in a medium containing (g per liter), autoclaved fraction of maize bran (AFMB) [START_REF] Bonnin | Aspergillus niger I-1472 and Pycnoporus cinnabarinus MUCL39533, selected for the biotransformation of ferulic acid to vanillin, are also able to produce cell wall polysaccharide-degrading enzymes and feruloyl esterases[END_REF] [START_REF] Tatum | Biochemical mutant strains of Neurospora produced by physical and chemical treatment[END_REF]. Cultures were carried out in 500-mL baffled flasks, containing 100 mL medium. Five-millimeters malt agar disks of mycelium from a Petri dish (4 per flask) ground in a FastPrep-24 sample grinder (Q-BIOgene, Irvine, CA) at 4 m/s for 1 min in 1 mL of sterilised purified water (MilliQ, Millipore) were used as inoculums. Incubation took place at 30 °C in a rotary shaker (Infors AG, Switzerland) at 110 rpm. After 7 days, the culture was stopped and the culture medium (secretome) filtered through a 0.45 lm-pore size PVDF membrane (Millipore, Guyancourt, France) and stored at 4 °C until use.

Enzymatic hydrolysis of wheat straw

The crude secretome of F. verticillioides grown on AFMB was used for wheat straw hydrolysis in the presence of T. reesei cellulases. Experiments were carried out in 50-mL screw-cap tubes containing the reaction mixture including 1 g of ground wheat straw (dry weight) and 40 mL fungal secretome adjusted at pH 4.8 with HCl (2 M). The suspension was further supplemented with 6.2 FPU/g substrate (dry weight) of commercial cellulases GC220 from T. reesei and 22.6 U/g substrate (dry weight) of b-glucosidase Novozyme SP188 from Aspergillus niger (Novozymes, Bagsvaerd, Danemark). Commercial enzyme loadings refer to enzymatic activities measured at 45 °C and in a citrate-phosphate buffer (pH 4.8) (see Section 2.5). Tetracycline (12.5 g/L) and cycloheximide (10 g/L) were added to prevent microbial contamination. Enzymatic hydrolysis was performed at 45 °C and 110 rpm in a rotary shaker (Infors AG, Switzerland) for 72 h. Control experiments were carried out under the same conditions using the commercial enzymes and secretome individually, and enzymes without substrate.

Samples were taken from the reaction mixture and the monosaccharides released after hydrolysis were analyzed by High Performance Anion Exchange Chromatography (HPAEC) coupled with PAD (ICS3000; Dionex, Sunnyvale, CA) equipped with a Carbo-Pac PA-1 analytical column (250 by 4 mm) as described by [START_REF] Couturier | Podospora anserina hemicellulases potentiate the Trichoderma reesei secretome for saccharification of lignocellulosic biomass[END_REF]. Enzymatic reactions were stopped by the addition of 18 mM NaOH before injection (5 lL) into the HPAEC system. Elution (1 mL/min) was carried out in 18 mM NaOH. Calibration curves were plotted using glucose, xylose, arabinose, galactose and cellobiose standards (Sigma-Aldrich, Lyon, France) and response factors were calculated (Chromeleon program, Dionex). All assays were carried out in triplicate. The concentration of reducing sugars in the hydrolysates was analyzed with the dinitrosalicylic acid method using glucose as a standard, as described by [START_REF] Navarro | Automated assay for screening the enzymatic release of reducing sugars from micronized biomass[END_REF].

Enzyme activity assays and protein determination

Unless otherwise indicated, all enzymatic activities were measured in citrate-phosphate buffer (50 mM, pH 4.8) and at 45 °C.

Overall cellulase activity was determined using filter paper (FP). Whatman No. 1 filter paper disks (6 mm diameter) were incubated with the enzyme solutions for 2 h, in a final reaction volume of 150 lL. The reducing sugars released were measured using the dinitrosalicylic acid method with glucose as the standard. One unit of filter paper activity was defined as the amount of protein that released 1 lmol of reducing sugar per min.

The activities of enzymes towards carboxymethylcellulose (CMC), microcrystalline cellulose (avicel PH-101), low viscosity wheat arabinoxylan, insoluble wheat arabinoxylan, birchwood xylan, citrus pectin, sugar beet arabinan and larch arabinogalactan were measured by using the dinitrosalicylic acid method, as described by [START_REF] Couturier | Post-genomic analyses of fungal lignocellulosic biomass degradation reveal the unexpected potential of the plant pathogen Ustilago maydis[END_REF]. Briefly, 20 lL of enzymes were mixed with 100 lL of substrates at 1% concentration and incubated for 1 h. The reaction was terminated by the addition of dinitrosalicylic acid reagent (1% w/v) and boiled for 10 min. The absorbance at 540 nm was measured relative to a glucose standard curve. One unit of enzyme activity was defined as the amount of protein that released 1 lmol of reducing sugar per min.

b-Glucosidase, b-xylosidase, a-arabinofuranosidase and agalactosidase activities were assayed by monitoring the release of p-nitrophenol from p-nitrophenyl-b-D-glucopyranoside (pNP-Glu), p-nitrophenyl-b-D-xylopyranoside (pNP-X), p-nitrophenyl-a-L-arabinofuranoside (pNP-A), and p-nitrophenyl-a-D-galactopyranoside (pNP-Gal), respectively, as described by [START_REF] Couturier | Post-genomic analyses of fungal lignocellulosic biomass degradation reveal the unexpected potential of the plant pathogen Ustilago maydis[END_REF]. Briefly, 20 lL of enzymes were mixed with 100 lL of substrate (1 mM) and incubated for 20 min. The reaction was stopped by adding 130 lL of 1 M sodium carbonate (pH 11.5), and the release of 4-nitrophenol was quantified at 410 nm using a 4-nitrophenol standard curve. One unit of enzyme activity was defined as the amount of protein that released 1 lmol of p-nitrophenol per min. Laccase and cellobiose dehydrogenase (CDH) activities in the culture supernatant were assayed as described in [START_REF] Sigoillot | Lignocellulolytic and hemicellulolytic system of Pycnoporus cinnabarinus: isolation and characterization of a cellobiose dehydrogenase and a new xylanase[END_REF]. One unit of enzyme activity was defined as the amount of protein that oxidizes 1 lmol of substrate per min.

Lignin and manganese peroxidase activities were determined spectrophotometrically as described by [START_REF] Ravalason | Secretome analysis of Phanerochaete chrysosporium strain CIRM-BRFM41 grown on softwood[END_REF]. One unit of enzyme activity was equivalent to 1 lmol of product formed per min Protein concentration was determined by using the BioRad protein assay kit with bovine serum albumin as the standard (BioRad, Marnes-la-Coquette, France).

All the experiments were performed in triplicate and the standard deviation was lower than 10% of the mean.

Identification of proteins by LC-MS/MS analysis

Proteins (40 lg) were separated by one-dimensional (1D) electrophoresis (Precasted Tris-Glycine 10% SDS-PAGE, BioRad, Marnes-la-Coquette, France) and stained with Coomassie blue (BioRad, Marnes-la-Coquette, France). Each 1D electrophoresis lane was cut into 24 pieces of gel (2 mm in width) and protein identification was performed using PAPPSO platform facilities (http://pappso.inra.fr). In-gel digestion was carried out with the Progest system (Genomic Solution) according to a standard trypsinolysis protocol. Gel pieces were first washed twice with 50% (v/v) acetonitrile, 25 mM ammonium carbonate in water. Each sample was supplemented with 10 lL of 10 mM DTT and incubated for 1 h at 56 °C. After cooling, the supernatant was removed and the samples were incubated in the presence of 10 lL of 55 mM iodoacetamide at room temperature in the dark. Gel plugs were washed with acetonitrile and then dried in a vacuum speed concentrator. Digestion was performed for 6 h at 37 °C with 100 ng of modified trypsin (Promega) dissolved in 25 mM NH 4 CO 3 . Tryptic peptides were first extracted with 50% (v/v) acetonitrile, 0.5% trifluoroacetic acid in water, and then with pure acetonitrile. Both peptide extracts were pooled, dried in a vacuum speed concentrator and suspended in 25 lL of 2% (v/v) acetonitrile, 0.08% (v/v) trifluoroacetic acid in water.

LC-MS/MS analysis was performed using the method described by [START_REF] Couturier | Post-genomic analyses of fungal lignocellulosic biomass degradation reveal the unexpected potential of the plant pathogen Ustilago maydis[END_REF]. The raw mass data obtained were first converted to mzXML format with the ReAdW software (http:// tools.proteomecenter.org/software.php). Protein identification was performed querying MS/MS data against the F. verticilloides 7600 protein database (Fusarium Comparative Sequencing Project, Broad Institute of Harvard and MIT (http://www.broadinstitute.org/), 2011.01.28) together with an in-house contaminant database, using the X!Tandem software (X!Tandem Tornado 2008.02.01.3, http://www.thegpm.org) with the following parameters: one trypsin missed cleavage allowed, alkylation of cysteine and conditional oxidation of methionine, precursor and fragment ion set at 10 ppm and 0.5 Da, respectively. A refined search was added with similar parameters except that semitryptic peptides and possible N-terminal acetylation of proteins were searched. All peptides matched with an E-value lower than 0.01 were parsed with X!Tandem pipeline software (http://pappso.inra.fr/bioinfo/ xtandempipeline/). Proteins identified with at least two unique peptides and a log (E-value) lower than 4 were validated.

Results and discussion

Effect of F. verticillioides secretome supplementation on the hydrolysis of wheat straw

In order to evaluated the potential of F. verticilloides secretome to improve the enzymatic hydrolysis of untreated wheat straw, the fungal strain was grown on a medium containing Autoclaved Fraction of Maize Bran (AFMB) as carbon supplement. AFMB is constituted of soluble and highly substituted arabinoxylan that is known to promote the production of CWDEs [START_REF] Bonnin | Aspergillus niger I-1472 and Pycnoporus cinnabarinus MUCL39533, selected for the biotransformation of ferulic acid to vanillin, are also able to produce cell wall polysaccharide-degrading enzymes and feruloyl esterases[END_REF][START_REF] Couturier | Post-genomic analyses of fungal lignocellulosic biomass degradation reveal the unexpected potential of the plant pathogen Ustilago maydis[END_REF]. The effect of the resulting fungal secretome on wheat straw saccharification was studied by determining the kinetics of glucose, xylose, arabinose and reducing sugars released during a 72 h hydrolysis (Fig. 1). All enzymatic systems were supplemented with b-glucosidase (Novozyme SP188) to prevent end-product inhibition from cellobiose accumulation for efficient and extensive hydrolysis. Cellulose to glucose conversion yield reached 24% with GC220 alone (Fig. 1A). This value is in accordance with those of previous studies on enzymatic hydrolysis of untreated wheat straw [START_REF] Rémond | Combination of ammonia and xylanase pretreatments: impact on enzymatic xylan and cellulose recovery from wheat straw[END_REF]. Although lower amounts of glucose were released with F. verticillioides secretome alone, supplementation of GC220 with the secretome resulted in a 24% increase in cellulose conversion, indicating a synergism between both enzyme systems. A more striking effect was observed for xylose and arabinose release, as the addition of F. verticilloides secretome to GC220 led to an 88% increase in xylan conversion (Fig. 1B) and a 68% increase in arabinan conversion (Fig. 1C). According to these results, the highest release of reducing sugars was obtained using the combination of F. verticilloides secretome and GC220, compared to any of the single enzymes systems (Fig. 1D). It should be noted that the total amount of reducing sugars released was higher than the sum of the individual monosaccharides glucose, xylose and arabinose (Table 1). These findings suggest the release of other sugars from wheat straw, either in monomeric or oligomeric form. The impact of protein loading on the hydrolysis efficiency was also evaluated (Table 1). Interestingly, proteins from F. verticillioides crude secretome supplementing GC220 accounted for only approximately 20% of the total protein load. Furthermore, by using a twofold protein load for the F. verticillioides secretome, no significant additional benefits to cellulose and xylan conversion were observed. An increase in GC220 load did not impact glucose release and only led to a slight increase in xylose, arabinose and reducing sugars.

To characterize the enzymatic system produced by F. verticillioides in comparison to GC220 and Novozyme SP188, cellulose-, hemicellulose-, and pectin-degrading enzymes activities were measured using commercial substrates. Results were expressed as activity loading used for enzymatic hydrolysis in relation to wheat straw dry weight (Table 2). Cellulase activity on filter paper, Avicel PH101 and CMC arose mainly from GC220, and b-glucosidase activity was similar in both GC220 and the F. verticillioides secretome. As expected, Novozyme SP188 contained mainly b-glucosidase activity. Furthermore, the main auxiliary activities measured in GC220 were xylan-related activities, while the F. verticillioides secretome accounted for a broader set of enzymes, including other hemicellulases and pectinases. It should be pointed out that a-arabinofuranosidase, b-xylosidase and pectin-related activities were in significantly lower amounts in the commercial cocktails than in the F. verticillioides secretome.

Due to the low amount of cellulolytic activities of the F. verticillioides secretome, saccharification enhancement was not thought to be due to cellulolytic enzymes. Likewise, doubling the commercial cellulases loading did not result in an increase in cellulose conversion (Table 1). Since no cellobiose was detected in the hydrolysis products (data not shown), b-glucosidase activity was not a limiting factor for cellulose conversion. Consequently, further addition of b-glucosidase, provided by the F. verticillioides secretome, was not thought to result in improved hydrolysis. Saccharification improvement might therefore be explained by the action of enzymes not directly targeting the cellulose backbone, such as hemicellulases. It is well known that one of the bottlenecks in enzymatic saccharification is the accessibility of cellulose adversely affected by hemicelluloses acting as a physical barrier [START_REF] Alvira | Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: a review[END_REF]. Wheat straw hemicellulose consists mainly of arabinoxylan substituted by a-L-arabinofuranose, 4-O-methylglucuronic acid and acetyl groups [START_REF] Sun | Extraction and characterization of original lignin and hemicelluloses from wheat straw[END_REF] 1). However, the xylose release was twofold higher when GC220 was supplemented with F. verticillioides secretome, whereas xylanase activity was threefold lower than that provided by GC220. In addi-tion, when doubling the GC220 loading, xylose and reducing sugars release remained lower than that obtained by applying GC220 in combination with the F. verticillioides secretome (Table 1). Moreover, the highest release of arabinose was obtained using a combination of enzymatic systems, which might be correlated to the high a-arabinofuranosidase activity present in the secretome.

These results suggest that the activities related to the boosting effect might arise from more efficient or different xylanases and a-

Identification of CWDEs secreted by F. verticillioides

Most of the studies on F. verticillioides have been devoted to the understanding of mycotoxin biosynthesis, pathogenesis of fungalplant interactions and the development of new fungal control strategies [START_REF] Brown | Fusarium genomic resources: tools to limit crop diseases and mycotoxin contamination[END_REF]. Here, this fungus was demonstrated to produce extracellular enzymes able to improve sugar conversion by T. reesei cellulases. Plant pathogenic fungi, in particular fungi belonging to the genus Fusarium, have already been shown to represent potent sources of novel enzymes for efficient lignocellulose degradation. Indeed, they are able to secrete a large number and diversity of CWDEs involved in the breakdown of plant cell walls and plant tissue colonization [START_REF] Phalip | Plant cell wall degradation with a powerful Fusarium graminearum enzymatic arsenal[END_REF][START_REF] King | Arsenal of plant cell wall degrading enzymes reflects host preference among plant pathogenic fungi[END_REF][START_REF] Couturier | Post-genomic analyses of fungal lignocellulosic biomass degradation reveal the unexpected potential of the plant pathogen Ustilago maydis[END_REF]. In order to provide a qualitative global analysis of the F. verticillioides secretome, especially of proteins involved in biomass degradation, a proteomic approach was adopted. Using one-dimensional electrophoresis followed by LC-MS/MS, 166 proteins were detected and identified with high confidence. Since only 17% of the proteins detected in the secretome were annotated in the F. verticillioides genome database, BlastP was used to assign putative functions by homology searches against all protein sequences in the NCBI nr database (http:// www.ncbi.nlm.nih.gov/blast) using an e-value threshold of e-40. These analyses led to the identification of 151 proteins representing a 90% identification rate. Of these, 57 proteins (34%) were identified as putative plant CWDEs. The remaining 94 identified proteins included proteins putatively involved in (i) proteolysis and amino-acid metabolism (23%), (ii) fungal cell-wall biogenesis and biodegradation (11%), (iii) lipid metabolism (4%) and (iv) defense and stress response (2%) (Fig. 2). Unsurprisingly, the closest relatives in the databases were mainly proteins from fungi phylogenetically related to F. verticillioides, such as Fusarium graminearum, Fusarium oxysporum and Nectria haematoccoca ( Fusarium solani). Similarities were high, since the large majority of proteins displayed more than 70% identity with F. verticillioides proteins.

The plant CWDEs identified in the F. verticillioides secretome included cellulose-, hemicellulose-, pectin-, lignin-degrading and other carbohydrate-modifying enzymes (Table 3). Families were assigned using the CAZy database (http://www.cazy.org; [START_REF] Cantarel | The carbohydrate-active enzymes database (CAZy): an expert resource for glycogenomics[END_REF]. Forty-seven glycoside hydrolases (GH) classified in 27 families, two carbohydrate esterases (CE) and one polysaccharide lyase (PL) were identified. Seven proteins harbored a carbohydrate-binding module (CBM) from families CBM1, CBM4, CBM6 and CBM9 that target the plant cell wall. Putative CWDEs were also detected, including proteins of unknown function containing a CBM, or proteins for which no clear putative function could be assigned. The number of proteins corresponding to characterized enzymes from F. verticillioides was relatively small, i.e., one GH28 b a-Arabinofuranosidases can also release arabinose residues from pectin. [START_REF] Raiola | A single amino acid substitution in highly similar endo-PGs from Fusarium verticillioides and related Fusarium species affects PGIP inhibition[END_REF], two GH15 glucoamylases (ABY89280 and ABY89281; [START_REF] Bluhm | Involvement of ZFR1 of Fusarium verticillioides in kernel colonization and the regulation of FST1, a putative sugar transporter gene required for fumonisin biosynthesis on maize kernels[END_REF] and one GH13 a-amylase (AAZ73168; [START_REF] Bluhm | Amylopectin induces fumonisin B1 production by Fusarium verticillioides during colonization of maize kernels[END_REF]. Interestingly, some of the proteins identified, most particularly xylanases, b-xylosidases and a-arabinofuranosidases, were closely related to enzymes detected in secretome studies of F. graminearum grown on hop and wheat [START_REF] Phalip | Diversity of the exoproteome of Fusarium graminearum grown on plant cell wall[END_REF][START_REF] Paper | Comparative proteomics of extracellular proteins in vitro and in planta from the pathogenic fungus Fusarium graminearum[END_REF].

It should be noted that proteomic data were generally in agreement with the enzymatic activities detected in the F. verticillioides secretome. Concerning lignin-degrading enzymes, Fusarium species, including F. verticillioides, were shown to secrete lignin peroxidase, manganese peroxidase and laccase [START_REF] Pant | Enhanced production of ligninolytic enzymes and decolorization of molasses distillery wastewater by fungi under solid state fermentation[END_REF][START_REF] Li | Screening of a fungus capable of powerful and selective delignification on wheat straw[END_REF]. Only a low level of laccase activity was detected in the secretome (data not shown), which could match to a peptide corresponding to a putative multicopper oxidase (protein accession number FVEG_12358 in the F. verticillioides genome database).

The presence of some of the enzymes detected could explain the efficiency of the F. verticillioides secretome to potentiate T. reesei cellulase cocktail. Hence, even if T. reesei is considered as an efficient biomass degrader, genome analysis surprisingly revealed the presence of a relatively low diversity of carbohydrate-active enzymes, especially when compared to phytopathogenic fungi [START_REF] Martinez | Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina)[END_REF]. In particular T. reesei was shown to have only a limited number of hemicellulases encoding-genes. Among them only 5 xylanases, belonging to GH10 and GH11 families, and a limited number of b-xylosidases and a-arabinofuranosidases found in GH3, GH43, GH54 and GH62 families are represented. In comparison, the F. verticillioides secretome analysis revealed the presence of a great diversity of GH families assigned to hemicellulases, representing the most numerous class of the CWDEs detected (40% of the proteins). Among them, eight members of the GH43 family, including putative xylanases, b-xylosidases and a-arabinofuranosidases, were identified, while only two GH43 are represented in T. reesei. Furthermore, the detection of eight xylanases (from GH10, GH11, GH30 and GH43 families), five bxylosidases (from GH30, GH39 and GH43 families), and one member of almost all a-arabinofuranosidases GH families (GH43, GH51, GH54 and GH62) in F. verticillioides secretome strongly suggested a better ability for this fungus to degrade hemicellulose than T. reesei. These results correlate with the fact that very few hemicellulose-degrading enzymes are generally detected in T. reesei secretomes or commercial enzyme preparations [START_REF] Herpoël-Gimbert | Comparative secretome analyses of two Trichoderma reesei RUT-C30 and CL847 hypersecretory strains[END_REF][START_REF] Nagendran | Reduced genomic potential for secreted plant cell-wall-degrading enzymes in the ectomycorrhizal fungus Amanita bisporigera, based on the secretome of Trichoderma reesei[END_REF][START_REF] Couturier | Post-genomic analyses of fungal lignocellulosic biomass degradation reveal the unexpected potential of the plant pathogen Ustilago maydis[END_REF]. Furthermore, wheat straw xylan conversion yield with GC220 remained quite low despite its relatively high xylanase activity. Therefore, as suggested by both enzymatic activities and proteomic data, it is likely that F. verticillioides a-arabinofuranosidases and b-xylosidases, acting synergistically with xylanases, were partially responsible for the hydrolysis improvement. These auxiliary enzymes have already been shown to increase the efficiency of lignocellulosic biomass degradation [START_REF] Couturier | Podospora anserina hemicellulases potentiate the Trichoderma reesei secretome for saccharification of lignocellulosic biomass[END_REF][START_REF] Gao | Hemicellulases and auxiliary enzymes for improved conversion of lignocellulosic biomass to monosaccharides[END_REF]. Besides hemicellulases, a set of pectinases were also detected in the F. verticillioides secretome. It is worth noting that among the enzymes detected, two GH93 exo-a-1,5-arabinanases, one PL4 rhamnogalacturonan lyase and one CE8 pectin methyl esterase are absent from T. reesei genome. It is well known that T. reesei has only a few pectin-degrading enzymes, mainly represented by four GH28 polygalacturonases and one GH105 unsaturated rhamnogalacturonyl hydrolase, indicating that this fungus is more adapted to degrade decaying biomass than intact or living plants [START_REF] Kubicek | Comparative genome sequence analysis underscores mycoparasitism as the ancestral life style of Trichoderma[END_REF]. In contrast, plant pathogenic fungi are known to produce many pectinases, considered as key virulence factors. In particular, wheat infection with Fusaria is characterized by the production of pectin-degrading enzymes as main CWDEs in the early stage of plant invasion [START_REF] Kang | Ultrastructural and cytochemical studies on cellulose, xylan and pectin degradation in wheat spikes infected by Fusarium culmorum[END_REF][START_REF] Tomassini | Characterization and expression of Fusarium graminearum endo-polygalacturonases in vitro and during wheat infection[END_REF]. Pectinases are thought to promote plant cell wall penetration and tissue colonization during the infection process. The presence of pectin, even in small amounts, acts as a glue for other polysaccharides and influences cell wall plasticity. It has been shown that reduction of pectin in several plant tissues (including wheat) improved saccharification yields [START_REF] Lionetti | Engineering the cell wall by reducing demethyl-esterified homogalacturonan improves saccharification of plant tissues for bioconversion[END_REF]. Furthermore, pectinase supplementation to commercial cellulase and b-glucosidase preparations has been reported to significantly enhance the hydrolysis of wheat straw [START_REF] Saha | Dilute acid pretreatment, enzymatic saccharification, and fermentation of wheat straw to ethanol[END_REF]. The present data suggest that pectin-degrading enzymes from the F. verticillioides secretome participate in the depolymerisation of pectin, leading to the weakening of the surrounding cell wall and thus revealing other plant cell wall polysaccharides.

The increase in sugar conversion when the T. reesei enzymatic cocktail was supplemented with the F. verticillioides secretome could therefore be explained by the presence of CWDEs absent in T. reesei, and/or by a greater diversity of enzymes targeting plant polysaccharides, particularly arabinoxylans and pectin.

Conclusion

The addition of crude F. verticillioides secretome improves the hydrolytic efficiency of the commercial cellulases cocktail GC220 from T. reesei on untreated wheat straw. The F. verticillioides secretome contains other and/or more active CWDEs than GC220, as revealed by its lignocellulolytic enzymes activity pattern in relation

  Holly H. Ravalason, Sacha S. Grisel, Didier D. Chevret, Anne A. Favel, Jean-Guy J.-G. Berrin, Jean-Claude J.-C. Sigoillot, Isabelle Gimbert Herpoël-Gimbert To cite this version: Holly H. Ravalason, Sacha S. Grisel, Didier D. Chevret, Anne A. Favel, Jean-Guy J.-G. Berrin, et al.. Fusarium verticillioides secretome as a source of auxiliary enzymes to enhance saccharification of wheat straw. Bioresource Technology, 2012, 114, pp.589-596. 10.1016/j.biortech.2012.03.009. hal-1. Introduction

  Comment citer ce document : Ravalason, H. (Auteur de correspondance), Grisel, S., Chevret, D., Favel, A., Berrin, J.-G., Sigoillot, J.-C., Herpoël-Gimbert, I. (2012). Fusarium verticillioides secretome as a source of auxiliary enzymes to enhance saccharification of wheat straw. Bioresource Technology, 114, 589-596. DOI : 10.1016/j.biortech.2012.03.009 Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Version définitive du manuscrit publiée dans / Final version of the manuscript published in : Bioresource Technology (2012), Vol. 114, p. 589-596, DOI: 10.1016/j.biortech.2012.03.009 Journal homepage: http://www.elsevier.com/locate/biortech

Fig. 1 .

 1 Fig. 1. Enzymatic hydrolysis of wheat straw. Time course of cellulose conversion (A), xylan conversion (B), arabinan conversion (C) and reducing sugar release (D) by F. verticillioides extracellular enzymes (4), T. reesei commercial cellulases (j) and both used in combination (N). Polysaccharides conversion yields are expressed as the percentage of maximal theoretical yields. Assays were performed with F. verticillioides crude secretome and/or 6.2 FPU/g substrate commercial cellulases (GC220), supplemented with 22.6 U/g substrate commercial b-glucosidase (Novozyme SP188), for 72 h at pH 4.8 and 45 °C. All experiments were conducted in triplicate, and the error bars indicate the standard deviations.

  Glu: p-nitrophenyl-b-D-glucopyranoside. pNP-X: p-nitrophenyl-b-D-xylopyranoside. pNP-A: p-nitrophenyl-a-L-arabinofuranoside. pNP-Gal: p-nitrophenyl-a-Dgalactopyranoside.

Fig. 2 .

 2 Fig. 2. Functional classification of the 166 proteins detected in F. verticillioides secretome grown on autoclaved fraction of maize bran.

589-596, DOI: 10.1016/j.biortech.2012.03.009 Journal homepage: http://www.elsevier.com/locate/biortech

Bioresource Technology, 114, 589-596. DOI : 10.1016/j.biortech.2012.03.009 Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Version définitive du manuscrit publiée dans / Final version of the manuscript published in : Bioresource Technology (2012), Vol. 114, p. of plant CWDEs and efficiency of lignocellulose breakdown

2012), Vol. 114, p. 589-596, DOI: 10.1016/j.biortech.2012.03.009 Journal homepage: http://www.elsevier.com/locate/biortech

. Complete arabinoxylan hydrolysis therefore requires xylanase and b-xylosidase activities for backbone biodegradation, but also auxiliary Comment citer ce document : Ravalason, H. (Auteur de correspondance), Grisel, S., Chevret, D., Favel, A., Berrin, J.-G., Sigoillot, J.-C., Herpoël-Gimbert, I. (2012). Fusarium verticillioides secretome as a source of auxiliary enzymes to enhance saccharification of wheat straw. Bioresource Technology, 114, 589-596. DOI : 10.1016/j.biortech.2012.03.009 Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Version définitive du manuscrit publiée dans / Final version of the manuscript published in : Bioresource Technology (enzymes such as a-arabinofuranosidases, a-glucuronidases and acetyl xylan esterases acting cooperatively to release side chains residues (Van den Brink and de Vries, 2011). The high xylanase activities measured in GC220 could explain that 9.3% of xylan was converted to xylose after 72 h of hydrolysis (Table

Table 1 Glucose

 1

	Glucose	Xylose	Arabinose	Reducing sugars

, xylose, arabinose and reducing sugars released after 72 h hydrolysis of wheat straw by F. verticillioides crude secretome and commercial enzyme preparations.

Enzymes and protein loading (mg/g wheat straw)

Sugars released (mg/g wheat straw)

Comment citer ce document : Ravalason, H. (Auteur de correspondance), Grisel, S., Chevret, D., Favel, A., Berrin, J.-G., Sigoillot, J.-C., Herpoël-Gimbert, I. (2012). Fusarium verticillioides secretome as a source of auxiliary enzymes to enhance saccharification of wheat straw. Bioresource Technology, 114, 589-596. DOI : 10.1016/j.biortech.2012.03.009 Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Version définitive du manuscrit publiée dans / Final version of the manuscript published in : Bioresource Technology (2012), Vol. 114, p.

589-596, DOI: 10.1016/j.biortech.2012.03.009 Journal homepage: http://www.elsevier.com/locate/biortech arabinofuranosidases

than those found in GC220, and/or from other auxiliary hemicellulolytic enzymes absent or different in this commercial cocktail.

Table 2

 2 CWDE activities (per g dry matter) of F. verticillioides crude secretome and commercial enzyme preparations used in wheat straw hydrolysis experiments.

	Enzymes	Cellulose-degrading		Xylan-degrading enzymes (U/g wheat straw)			Pectin-degrading enzymes (U/g wheat straw)
		enzymes (U/g wheat straw)
		FP	Avicel CMC pNP-	Birch	Wheat xylan	Wheat xylan	pNP-	pNP-	Pectin Arabinan Arabinogalactan pNP-
					Glu a	Xylan	(soluble)	(insoluble)	X a	A a,b				Gal a
	F. verticillioides	0.1 0.0	0.7	36.1	39.4	55.9	12.0	2.2	5.6	19.8	2.2	4.6	7.2
	secretome
	Commercial	6.2	7.5	51.8	32.9
	enzymes	0.2	0.0	0.0	22.6
	GC220
	Novozyme
	SP188

Table 3

 3 CWDE identified in F. verticillioides secretome grown on autoclaved fraction of maize bran. Comment citer ce document : Ravalason, H. (Auteur de correspondance), Grisel, S., Chevret, D., Favel, A., Berrin, J.-G., Sigoillot, J.-C., Herpoël-Gimbert, I. (2012). Fusarium verticillioides secretome as a source of auxiliary enzymes to enhance saccharification of wheat straw. Bioresource Technology, 114, 589-596. DOI : 10.1016/j.biortech.2012.03.009 Version définitive du manuscrit publiée dans / Final version of the manuscript published in :

	F.	Total number	Number	Predicted protein function	Predicted	Predicted	Closest informative homolog (BlastP)
	verticillioides	of spectra	of unique		family b	MW (kDa)/
	ORF a	detected	peptides			predicted pI
							GenBank	Organism	E value
							Accession
							number
	Cellulose-degrading enzymes
	FVEG_05521	196	25	b-1,4-Glucosidase	GH3	97/5.3	XP386781	F. graminearum	0
	FVEG_13391	147	23	b-1,4-Glucosidase	GH3	65/5.2	XP003052401	N. haematococca	0
	FVEG_09772	16	10	b-1,4-Glucosidase	GH1	70/5.0	XP003049770	N. haematococca	0
	FVEG_04722	16	6	Endo-b-1,4-glucanase	GH61	41/5.2	XP386263	F. graminearum	5.00E-157
	FVEG_13055	9	8	b-1,4-Glucosidase	GH3	69/4.8	XP003043120	N. haematococca	0
	FVEG_01037	5	4	Endo-b-1,4-glucanase	GH7	55/5.7	AAA65586	F. oxysporum	0
	FVEG_12423	5	5	Endo-b-1,4-glucanase	GH5 -CBM1	50/5.6	XP391242	F. graminearum	0
	FVEG_08890	4	3	b-1,4-Glucosidase	GH3	88/5.3	XP003043563	N. haematococca	0
	FVEG_08825	2	2	Endo-b-1,4-glucanase	GH6 -CBM1	49/5.3	AAA65585	F. oxysporum	0
	Xylan-degrading enzymes
	FVEG_09861	86	25	b-1,4-Xylosidase	GH3	72/6.6	XP388169	F. graminearum	0
	FVEG_07702	58	11	b-1,4-Xylosidase	GH39	49/7.1	XP381924	F. graminearum	2.00E-165
	FVEG_13578	46	9	Endo-b-1,4-xylanase	GH30	52/6.1	ADG62369	Bispora sp.	6.00E-141
	FVEG_08605	40	9	Endo-b-1,4-xylanase	GH43	63/8.0	AAV98256	F. graminearum	0
	FVEG_08824	39	12	a-Glucuronidase	GH67	94/7.3	CAA92949	T. reesei	0
	FVEG_08345	16	7	b-1,4-Xylosidase	GH43	56/8.8	XP387871	F. graminearum	0
	FVEG_12502	14	8	Endo-b-1,4-xylanase	GH10	39/8.9	AAV98254	F. graminearum	6.00E-167
	FVEG_10625	9	7	b-1,4-Xylosidase	GH43	37/7.5	XP383179	F. graminearum	3.00E-164
	FVEG_10098	8	5	Endo-b-1,4-xylanase	GH43	46/5.6	XP384081	F. graminearum	9.00E-167
	FVEG_13426	7	4	b-1,4-xylosidase	GH43	40/7.7	XP388169	F. graminearum	3.00E-134
	FVEG_08344	6	3	Endo-b-1,4-xylanase	GH43 -CBM6	49/5.8	AAT84252	F. graminearum	0
	FVEG_08828	4	2	Endo-b-1,4-xylanase	GH11	25/9.0	XP003050975	F. graminearum	1.00E-95
	FVEG_13343	3	2	Endo-b-1,4-xylanase	GH10	36/8.9	AAC06240.1	F. oxysporum	0
	FVEG_13553	3	2	Endo-b-1,4-xylanase	GH43	40/4.6	XP384081	F. graminearum	3.00E-101
	FVEG_11825	2	2		GH10	26/6.5	XP391434	F. graminearum	4.00E-105
	Pectin-degrading enzymes
	FVEG_13077	174	29	a-Galactopyranosidase/b-	GH27	59/4.9	BAH10648	F. oxysporum	0
				arabinopyranosidase
	FVEG_12289	69	15	a-Galactopyranosidase/b-	GH27	63/8.8	BAH10649	F. oxysporum	0
				arabinopyranosidase
	FVEG_12299	46	15	Endo-b-1,6-galactanase	GH5	41/5.1	BAF42338	F. oxysporum	0
	FVEG_13262	45	14	Exo-a-1,5-arabinanase	GH93	41/6.7	CAJ83998	F. oxysporum	0
	FVEG_07518	15	9	a-Galactosidase	GH27	45/7.6	BAJ23877	F. oxysporum	0
	FVEG_08342	14	4	D-4,5-Unsaturated-	GH88	42/4.4	XP383319	F. graminearum	3.00E-162
				glucuronyl
				hydrolase
	FVEG_04751	6	2	Rhamnogalacturonan lyase	PL4	75/6.2	XP003050105	N. haematoccoca	0
	FVEG_12766	6	2	Pectin methyl esterase	CE8	42/8.4	XP003041606	N. haematoccoca	1.00E-168
	FVEG_10735	4	2	Exo-a-1,5-arabinanase	GH93	43/8.8	CAX17703	F. graminearum	0
	FVEG_13516	4	2	Endo-a-1,4-	GH28	39/6.1	ABQ11230	F. verticillioides	0
				polygalacturonase
	FVEG_12744	3	2	Exo-a-1,4-	GH28	51/8.1	BAE97083	F. oxysporum	0
				polygalacturonase
	Xylan/pectin-degrading enzymes
	FVEG_12952	65	15	a-Arabinofuranosidase	GH54	51/5.9	BAG80559	F. oxysporum	0
	FVEG_05689	33	14	a-Arabinofuranosidase	GH51	73/8.3	XP003045662	N. haematoccoca	0
	FVEG_13328	12	5	a-Arabinofuranosidase	GH43	49/5.5	BAG80558	F. oxysporum	0
	FVEG_12666	2	2	a-Arabinofuranosidase	GH62	35/8.4	AAV66346	F. graminearum	2.00E-143
	Lignin and xylan/lignin degrading enzymes
	FVEG_10901	41	14	Feruloyl esterase	CE1	61/7.5	CAC83933	A. niger	2.00E-116
	FVEG_01031	16	4	Superoxide dismutase		28/5.5	XP_380752	F. graminearum	9.00E-105
	Xyloglucan-degrading enzymes
	FVEG_12553	159	26	a-Fucosidase	GH29	65/5.2	CBX99860	L. maculans	9.00E-180
	FVEG_12189	96	23	a-Fucosidase	GH29	67/5.3	XP391430	F. graminearum	0
	Starch-degrading enzymes
	FVEG_13849	109	27	Glucoamylase	GH15 -CBM20	63/8.8	ABY89281	F. verticillioides	0
	FVEG_13659	106	31	a-Glucosidase	GH31	116/5.8	XM003040737	N. haematococca	0
	FVEG_04947	51	14	Glucoamylase	GH15 -CBM20	69/5.6	ABY89280	F. verticillioides	0
	FVEG_12957	27	11	a-Amylase	GH13	51/5.4	AAZ73168	F. verticillioides	0
	Other carbohydrate-modifying enzymes
	FVEG_07688	50	13	Galactose mutarotase	GH32	44/5.7	XP382005	F. graminearum	0
	FVEG_11933	5	5	b-Fructofuranosidase		57/4.7	XP003037462	S. commune	3.00E-167
	FVEG_01938	3	3	Transaldolase		36/5.2	XP388899	F. graminearum	9.00E-176
	FVEG_12482	2	2	Glucose/sorbosone		45/4.7	XP384041	F. graminearum	0
				dehydrogenase

Bioresource Technology (2012), Vol. 114, p. 589-596, DOI: 10.1016/j.biortech.2012.03.009 Journal homepage: http://www.elsevier.com/locate/biortech

endo-polygalacturonase (GenBank Accession No. ABQ11230;

Table 3

 3 (continued) ORF number provided by the Broad Institute F. verticillioides database. b Families were assigned according to the CAZy database. GH, glycoside hydrolase; PL, polysaccharide lyase; CE, carbohydrate esterase; CBM, carbohydrate binding module.

	F.	Total number	Number	Predicted protein function	Predicted	Predicted	Closest informative homolog (BlastP)
	verticillioides	of spectra	of unique		family b	MW (kDa)/
	ORF a	detected	peptides			predicted pI
							GenBank	Organism	E value
							Accession
							number
	Putative cell-wall modifying enzymes
	FVEG_06728	48	7	b-1,4-Glucosidase/	GH3	88/4.9	XP003050575	N. haematoccoca	0
				avenacinase
	FVEG_12856	19	5	Glycoside hydrolase	Related to GH61	37/9.6	XP003047765	N. haematoccoca	9.00E-120
	FVEG_12107	8	3	Hypothetical protein	CBM9	25/5.3	XP388097	F. graminearum	2.00E-100
	FVEG_03820	8	4	Related to ice-nucleation	GH16	45/4.8	AAQ14297	F. acuminatum	3.00E-176
				protein
	FVEG_10100	7	3	Hypothetical protein	CBM4	28/4.3	XP380274	F. graminearum	1.00E-48
	FVEG_10606	4	2	b-Glucuronidase/	GH79	50/5.7	XP003040950	N. haematoccoca	0
				heparanase

a

H. Ravalason et al. / Bioresource Technology 114 (2012) 589-596

Acknowledgements

This study was supported by the Futurol project. Holy Ravalason is grateful to the Futurol project for her postdoctoral fellowship.


Lionel Bombrun

Ivan Pétillot

Gabriel Vasile

Michel Gay

Emmanuel Trouvé

Philippe Bolon

Jean-Marie Nicolas
email: nicolas@tsi.enst.fr

Tania Landes
email: tania.landes@insa-strasbourg.fr

Multi-date ERS tandem interferogram analysis: application to alpine glaciers

Temperate glaciers are an indicator of the local effects of global climate change. For economical and security reasons in the surrounding areas, the monitoring of those geophysical objects is being a necessity. SAR data are expected to provide dense measurements of physical parameters which are necessary to detect significant changes and to constrain glacier flow models. In this paper, five descending one-day ERS-1/2 tandem interferometric data pairs from July 1995 to April 1996 are studied in the Chamonix Mont-Blanc area (French Alps). This multi-temporal interferogram series is used to analyse the coherence levels and fringe patterns over nine glaciers. Moreover, when the coherence is sufficient, Differential SAR Interferometry (D-InSAR) processing are applicable to derive a three-dimensional (3-D) velocity fields. An expert knowledge and a ten years measurements analysis of glacier flow are used to fix the line of sight (LOS) displacement offset. Finally, an analysis of the wrapped phase difference between interferograms is exposed to detect the presence of residual topographic fringes and to characterize the surface flow field evolution.

I. INTRODUCTION

Temperate glaciers are an indicator of the local effects of global climate change. For economical and security reasons in the surrounding areas, the monitoring of those geophysical objects is being a necessity. Compared to sparse terrestrial ground measurements, Synthetic Aperture Radar (SAR) allows regular acquisitions on mountainous areas. SAR data are expected to provide dense measurements of physical parameters which are necessary to detect significant changes and to constrain glacier flow models. For example, InSAR techniques can be applied to measure glacier surface flow fields which can reach several decimeters per day in the French Alps. Spaceborne data from ERS-1/2 tandem mission have been successfully used to derive velocity fields [START_REF] Mattar | Validation of alpine glacier velocity measurements using ERS tandem-mission SAR data[END_REF], mainly during the cold season because of the strong temporal decorrelation in summer [START_REF] Trouvé | Combining airborne photographs and spaceborne SAR data to monitor temperate glaciers. potentials and limits[END_REF].

In the first part of this paper, five descending one-day ERS-1/2 tandem interferometric data pairs from July 1995 to April 1996 are studied in the Chamonix Mont-Blanc area (French Alps). This multi-temporal interferogram series is used to analyse the coherence levels and fringe patterns over nine glaciers. Like coherence is a criteria of interferometric processing performance, an analysis of multi-date coherence in glaciers and non-glaciers areas is exposed. A survey concerning the altitude influence on coherence preservation is achieved.

Next, when the coherence is sufficient, Differential Interferometry SAR processing is applicable to derive a 3-D velocity field. However, several difficulties have to be overcame. Due to the high topography, most of the Chamonix Valley glaciers are visible only on descending pass. Since, one pass interferometric phase provides the 3-D surface displacement projected on the SAR line of sight axis, only one velocity component is available. Moreover the phase unwrapping step provides over each glaciers a displacement field with an unknown offset. An expert knowledge and a ten years measurements analysis of glacier flow allow the detection of areas where the velocity is stationary during different seasons. Annual insitu measurements are taken into account to fix this LOS displacement offset. A displacement profile is shown and a comparison with differential GPS measurements is exposed.

Finally, an analysis of the wrapped phase difference between interferograms is proposed. A method based on a comparison of the fringes with the perpendicular baseline is described. It determines if residual topographic fringes are correctly removed or not in the interferogrmas. Therefore, Digital Terrain Model (DTM) errors can be retrieved by this analysis. Furthermore, when topographic fringes are correctly removed, a comparison of multi-date interferometric phases allows a characterization of surface flow field evolution.

II. MULTI-TEMPORAL COHERENCE ANALYSIS

Five descending one-day ERS-1/2 tandem interferometric pairs from July 1995 to April 1996 are processed with the Repeat Orbit Interferometry Package (ROI-PAC) software from the Jet Propulsion Laboratory (JPL). This processor takes the precise orbits from the Delft University, the Netherlands, into account to remove the orbital fringes [START_REF] Scharoo | Precise orbit determination and gravity field improvement for the ERS satellites[END_REF]. Moreover, a DTM is used by ROI-PAC software to estimate the topographic fringe component and remove it from the interferogram. After the ROI-PAC topographic and orbital fringe removal, an adaptive neighborhood filter has been applied to re-estimate the interferometric phase and coherence [START_REF] Vasile | General adaptive neighborhood technique for improving SAR interferometric coherence estimation[END_REF].

A. Multi-date coherence analysis

To investigate the potential of ERS-1/2 tandem interferometry all along the year, an analysis of the interferometric coherence from July 1995 to April 1996 is proposed.

Among the five interferometric couples shown in Fig. 1, the highest coherence is observed in March 1996. Indeed, almost 45% of the Argentière glacier has a coherence greater than 0.5 (see table II). The couple of March has the smallest perpendicular baseline (B ⊥ = 9m) (see table I). Moreover, the interferograms acquired in winter show a better coherence than those acquired in the other seasons. Compared to March, the 31 Dec.95/1 Jan.96 interferogram shows a loss of coherence. This loss is observed on glacier (2%) and non glacier (7%) areas. The large perpendicular baseline (B ⊥ = 208m) causes a significant volume decorrelation, especially in the valley forested areas. In warmer seasons (October and April), the coherence is preserved only on the upper parts of the glaciers. Indeed, for low altitudes, the higher temperature leads to a relevant change of the glacier surface state and causes a loss of the interferometric coherence. For the same reason, during the hot season, coherence is not preserved on the whole surface of the nine observed glaciers.

B. Influence of altitude on the interferometric coherence

In April 1996, coherence is preserved only on the upper part of the Argentière glacier. 35% of the pixels have a coherence greater than 0.5, whereas on the Mer de Glace/Leschaux glacier, only 1% of the pixels preserve the coherence. As the altitude is higher on the top of Argentière glacier than on the Mer de Glace/Leschaux glacier, coherence preservation may be dependent on glacier elevation. To investigate this relation, a Digital Terrain Model (DTM) has been used to build a mask of visibility. This mask determines which regions are not visible (foldover, shadow) on the ERS-1/2 images. Then the transformation from ground geometry to the SAR slant A low coherence can be observed in December 1995 (green line) due to the large baseline of this interferometric pair. Although coherence is well preserved on July 1995 (black line) on non glaciers areas, on the Argentière glacier, the significant change of the glacier surface state in summer does not allow a coherence preservation. Note that in October 1995, March and April 1996, coherence increases with altitude on the Argentière glacier. The higher the glacier is, the more coherence is preserved. This relation cannot be established on non glacier areas because coherence varies a lot, with a strong effect of the slope on high altitudes areas.

III. 3-D SURFACE DISPLACEMENT

When the coherence is sufficient, glacier displacement can be monitored by Differential SAR Interferometry processing. Three-dimensional (3-D) velocity fields can be derived with ERS 1-day interferograms from October 1995 to April 1996 on most of the glaciers of the studied area.

The phase unwrapping step provides over each glaciers a displacement field with an unknown offset. To fix this constant, a common technique is to find a part of the glacier where the displacement is assume to be zero. Unfortunately, such assumption cannot be done on the Argentière glacier due to the quite steep slope.

An expert knowledge and a ten year measurement analysis of glacier flow allow the detection of areas where the velocity is stationary during different seasons. Fig. 3(b) shows the annual displacement of the Argentière glacier from 1994 to 2004 at 4 altitudes. Note that at 2700m above sea level (asl) (profile7 on Fig. 3(a)) the displacement is the same every year. Moreover an expert knowledge confirms that the displacement in this part of the glacier is stationary during different seasons. Consequently, an annual in situ measurements at 2700m asl can be used to fix the one-day LOS displacement offset.

Since, one pass interferometric phase provides the 3-D surface displacement flow projected on the SAR LOS axis, only one velocity component is available. Thus, two common assumptions are made to retrieve the 3-D surface displacement flow [START_REF] Joughin | Interferometric Estimation of Three-Dimensional Ice-Flow Using Ascending and Descending Passes[END_REF]:

• a flow parallel to the glacier surface.

• a flow in the direction of maximum averaged downhill slope. Fig. 3(c) shows a longitudinal displacement profile on the Argentière glacier. The first cross is the only point used to fix the LOS offset. Note that near the reference point, the 1day displacement is stationary. There isn't much variation on the displacement profile from October 1995 to April 1996. Annual in-situ measurements are in good agreements with the displacements obtained by interferometry. On the bottom of the Argentère glacier, the 1-day displacement varies more with the date of acquisition. The fluctuations between March and April 1996 velocities seems to confirm that the hypothesis of a stationary area would not be valid in the lower part of this glacier.

IV. ANALYSIS OF THE WRAPPED PHASE DIFFERENCE BETWEEN DIFFERENTIAL INTERFEROGRAMS

In this section, a study of the temporal variation of D-InSAR displacement fields is proposed. This analysis was achieved to determine if all non-displacement fringes are removed from the interferograms. If Φ i and Φ j denote two one-day interferograms acquired at date (i, i + 1) and (j, j + 1) respectively. The wrapped phase difference between interferograms ∆Φ i-j is defined as:

∆Φ i-j = [Φ i -Φ j ] modulo 2π (1)
The two main sources of possible residual fringes in ∆Φ i-j are topographic fringes and atmospheric perturbations. First, an algorithm is proposed to detect the presence of residual fringes in ∆Φ i-j . Next, a comparison of fringes pattern is achieved to determine if atmospheric fringes can be neglected. Finally, if non-displacement (topographic, tropospheric, ...) fringes are correctly removed or negligible in each oneday interferogram phase, ∆Φ i-j allows a characterization of surface flow field evolution.

A. Residual topographic fringes

Fig. 4 shows the wrapped phase difference between interferograms on the Tour, Trient and Saleina glaciers at three dates. Residual fringes can be observed on the Trient glacier situated on the North East of the image. If all non-displacement fringes are removed from the interferograms, it means that the Trient glacier displacement is not stationary all along the year. The displacement changes a lot from October to April. In order to see if these fringes correspond to displacement variation or residual fringes, we propose to make a comparison between the residual fringes observed on the Trient glacier and the perpendicular baseline differences.

Let Φ i topo be the interferometric terms at date i corresponding to topographic fringes, ie: fringes due to height variation ∆z over the studied area. Φ i topo can be expressed as a function of the orthogonal baseline at date i (B i ⊥ ) by the following equation as:

Φ i topo = 4π λ B i ⊥ R 1 sin θ ∆z = 2π e i a ∆z (2)
with the altitude of ambiguity defined as:

e i a = λ 2 R 1 sin θ B i ⊥ (3)
Next, the equivalent orthogonal baseline ∆B ij ⊥ can be defined as the difference of the two orthogonal baseline at dates i and j. Similarly, the equivalent altitude of ambiguity ∆e ij a can be defined by Eq. ( 5):

∆B ij ⊥ = B i ⊥ -B j ⊥ ( 4
)
∆e ij a = λ 2 R 1 sin θ B i ⊥ -B j ⊥ = 1 1 e i a -1 e j a ( 5
)
The equivalent topographic terms ∆Φ ij topo in the wrapped phase difference between interferograms at dates i and j can be expressed by:

∆Φ ij topo = 4π λ |B i ⊥ -B j ⊥ | R 1 sin θ ∆z (6) = 2π ∆e ij a ∆z (7)
Let N ij (X, Y ) be the number of fringes observed on the interferogram difference ∆Φ i-j between two points X and Y and ∆z(X, Y ) a height difference between X and Y . N ij (X, Y ) is assumed to be positive if the fringe varies from the white to black color. The hypothesis that the observed residual fringes are linked to the topographic variations yields to:

∆Φ ij topo = 2πN ij (X, Y ) (8)
and by combining Eq. (7) and Eq. (8) to:

∆z(X, Y ) = N ij (X, Y )∆e ij a (9)
This allows us to test this hypothesis by checking if the product N ij (X, Y )∆e ij a is constant over the different interferograms pairs (i,j).

N ij (X, Y )∆e ij a = C te (X, Y ) ∀i, j (10)
In this case the constant C te (X, Y ) can be interpreted as a height difference ∆z(X, Y ) which is not correctly removed in the D-InSAR processing and probably due to DTM errors. In other words, the equivalent altitude of ambiguity ∆e ij a multiplied by the number of fringes observed is an indicator of the presence of residual topographic fringes.

The proposed method is applied on the four Tandem ERS-1/2 couples from October 1995 to April 1996. The following ERS parameters are used:

• R 1 = 790 km • λ = 5.6 cm • θ = 23°T
able III shows the equivalent orthogonal baseline and the equivalent altitude of ambiguity for the six different pairs of interferograms where coherence is preserved on glaciers. We can observe that the parameter N ∆e a is unchanged for all the wrapped phase difference between interferograms on the Trient glacier. Therefore, according to Eq. ( 9) and results in

B. Atmospheric perturbations

A second possible source of variation of D-InSAR fringe pattern is the presence of atmospheric perturbations. Fig. 5 shows the wrapped phase difference between interferograms over the Mer de Glace -Leschaux and Argentière glaciers. As no residual fringe pattern are observable on the wrapped phase difference between interferograms, the studied glaciers are apparently not affected by global atmospheric changes [START_REF] Trouvé | Combining airborne photographs and spaceborne SAR data to monitor temperate glaciers. potentials and limits[END_REF]. Moreover, the glacier surfaces are rather small compared to a full ERS image and the path delay variation between due to saturation water vapor pressure less important with winter temperature (close or below 0 degree in this area). Accordingly, we can conclude that at first order, atmospheric perturbations are negligible compared to glacier displacement..

Once the topographic fringes are removed and atmospheric fringes neglected, ∆Φ i-j allows a characterization of surface flow field time evolution. Note that for two consecutive interferograms pair (April and March in Fig. 5(a)), the wrapped phase difference is closed to zero and slightly increases with the time interval of the two interferograms (using March-December Fig. 5(b) and March-October Fig. 5(c) interferograms). Moreover, no residual fringes can be observed at the stationary area on Argentière glacier define on section III. It seems that at 2700m asl, the displacement is the same during the seasons.

Nevertheless, a precise characterization of the flow field evolution can be done only if topographic fringes are correctly removed from the interferograms and atmospheric fringes totally negligible. As topographic fringes are directly related with the altitude of the glacier and the glacier surface varies with time, a DEM at the date of acquisition should be used. Moreover, meteorological in situ measurements should be acquired at the date of the satellite acquisition to determine if interferograms are affected by tropospheric perturbations.

V. CONCLUSION

A multi-date ERS tandem interferogram analysis was carried out over glaciers and non glaciers areas in the Alps. This study shows when InSAR processings can be used to monitor temperate glaciers. A comparison of interferometric coherence preservation as a function of altitude was exposed. Thanks to the knowledge of a stable area over the Argentière glacier, a The continuous GPS will provide one day displacement at the date of the SAR acquisition, which will be useful to fix precisely the SAR LOS offset. No more a priori knowledge (stationary area) will be necessary. Moreover, to exploit the phase difference between differential interferograms as an indicator of the velocity field evolution, the variation of atmospheric perturbations must be negligible compare to the variation of displacement. This observation cannot be done with the only information provided by ERS interferograms. The continuous GPS should allow us to remove the small atmospheric effects from the interferograms and to use the phase difference between interferograms to characterize the evolution of the displacement field.

Fig. 1 .

 1 Fig. 1. ERS-1/2 tandem interferograms over the Mer de Glace -Leschaux and Argentière glaciers area (1024×1024 pixels). Coherence filtered by amplitude driven adaptive neighborhood. (a) 9/10 Jul.95, (b) 22/23 Oct.95, (c) 31 Dec.95/1 Jan.96, (d) 10/11 Mar.96, (e) 14/15 Apr.96

Fig. 2 .

 2 Fig. 2. Influence of altitude on multi-date ERS-1/2 tandem interferometric coherence. (a) on the Argentière glacier, (b) on non glacier areas

Fig. 3 .

 3 Fig. 3. Analysis of Argentière glacier displacement: (a) Displacement velocity field (red arrows), location of the annual measurements presented in Fig. 3(b)) and position of the longitudinal profile (blue line) presented in Fig. 3(c) , (b) Comparison of annual displacement from 1994 to 2004 at 4 altitudes, (c) Longitudinal velocity profile from the upper part (2900m asl) up to profile 4 (2300m asl)

Fig. 4 .

 4 Fig. 4. Wrapped phase difference between interferograms on the Tour, Trient and Saleina glaciers: (a) ∆Φ April-March , (b) ∆Φ April-December , (c) ∆Φ April-October

Fig. 5 .

 5 Fig. 5. ERS-1/2 wrapped phase difference between interferograms over the Mer de Glace -Leschaux and Argentière glaciers area. (a) ∆Φ April-March , (b) ∆Φ March-December , (c) ∆Φ March-October

TABLE II PERCENTAGE

 II OF PIXELS HAVING A COHERENCE ≥ 0.5

		glacier name
	date of acquisition	Mer de Glace Leschaux	Argentière	non glacier areas
	July 95	5.33	0.70	38.65
	October 95	27.75	37.17	39.28
	December 95	2.45	1.49	7.34
	March 96	35.05	44.94	32.20
	April 96	1.37	34.90	9.41

ACKNOWLEDGMENT

The authors wish to thank ESA for the ERS images provided through category 1 projects number 1088 and 3525, the GdR ISIS CACHANT for their support to the change detection community. They would like to thank the OSUG and the LGGE (http://www-lgge.ujfgrenoble.fr/ServiceObs/SiteWebPOG/) and the GLACIOCLIM project for providing annual in-situ measurements on the Chamonix valley glaciers.