Yves Verhertbruggen

Axelle Bouder

Jacqueline Vigouroux

Camille Alvarado

Audrey Geairon

Fabienne Guillon

Mark D Wilkinson

Fabian Stritt

Markus Pauly

Mi Yeon Lee

Jenny C Mortimer

Henrik V Scheller

Rowan A C Mitchell

Catalin Voiniciuc

Luc Saulnier

Anne-Laure Chateigner-Boutin
email: anne-laure.chateigner-boutin@inrae.fr

The TaCslA12 gene expressed in the wheat grain endosperm synthesizes wheat-like mannan when expressed in yeast and Arabidopsis

Keywords: Wheat grain, developing endosperm, cell walls, mannan, heterologous expression alcohol insoluble residues, AKI: alkaline insoluble, AX: arabinoxylan, BIFC: bimolecular fluorescence complementation, Csl: cellulose synthase-like, °DAF: Celsius degrees per day after flowering, chromatography, GT: glycosyltransferase, HPAEC-PAD: high-performance anion-exchange chromatography coupled with pulsed electrochemical detection, MALDI: matrix-assisted laser desorption/ionization, MLG: mixed-linkage glucan, MS: mass spectrometry, MSR: mannan synthesis related, TM: triple mutant, TMD: transmembrane domain, TOF: time-of-flight, WT: wild type

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

Introduction

Wheat (Triticum aestivum) is a major crop cultivated worldwide to produce grains that feed humans and animals. The wheat grain is a dry fruit containing a single seed with an embryo and a specialized tissue, the endosperm, where storage compounds are stored to provide energy necessary for the germinating seedling. This endosperm tissue is also highly valuable for different industries including starch, bread, pasta, and brewing.

The cereal grain develops from the fertilized ovary and undergoes a precise program of development which has four stages: syncytium, cellularization, differentiation and maturation (reviewed in [START_REF] Evers | Cereal grain structure and development: some implications for quality[END_REF]). In the syncytium stage, the wheat endosperm consists of dividing nuclei and cytoplasm but is devoid of cell walls. The first cell walls are formed at the beginning of the cellularization stage and subsequently cells begin to undergo mitoses. Endosperm differentiation leads to the emergence of the central starchy endosperm (storage tissue), and a peripheral layer of aleurone cells, some of which face the ventral vascular bundle forming specialized transfer cells involved in nutrient and water uptake [START_REF] Xurun | Structural development of wheat nutrient transfer tissues and their relationships with filial tissues development[END_REF].

Cell wall polysaccharides comprise a minor fraction of the cereal starchy endosperm, typically about 5% dry weight in the mature seed. Wheat endosperm cell wall polysaccharides have been studied for more than 50 years, but very recently their composition and structure have been revised. The cell wall polysaccharide composition in mature wheat endosperm is now reported as 60-65% arabinoxylan (AX), 20-22% cellulose, 15% mixed-linkage glucan (MLG), 4-7% mannan with traces of pectin and xyloglucan [START_REF] Chateigner-Boutin | The wheat grain contains pectic domains exhibiting specific spatial and development-associated distribution[END_REF][START_REF] Gartaula | Quantitative structural organisation model for wheat endosperm cell walls: Cellulose as an important constituent[END_REF][START_REF] Verhertbruggen | Challenging the putative structure of mannan in wheat (Triticum aestivum) Endosperm[END_REF]. The structure and function of cereal AX and MLG have been well-characterized (for a review, see [START_REF] Saulnier | Cell wall deposition and metabolism in wheat grain[END_REF]), and more recently, we have elucidated the structure of wheat endosperm mannan [START_REF] Verhertbruggen | Challenging the putative structure of mannan in wheat (Triticum aestivum) Endosperm[END_REF].

Mannan-type polymers found in plant include: linear mannan, with a backbone exclusively made of β-1,4-linked mannose residues and no or little substitution, galactomannans, with the same backbone but harboring α-1,6-linked galactose substitutions, and glucomannans and galactoglucomannans with backbones made of mannose and glucose [7,[START_REF] Pauly | Hemicellulose biosynthesis[END_REF]. The structure of mannan influences its properties such as its crystallinity, solubility, viscosity, gel-forming and filmforming abilities, water absorptivity, and thickening (for a review, see [START_REF] Gómez | Manufacture and properties of glucomannans and glucomannooligosaccharides derived from konjac and other sources[END_REF]). Substitutions were suggested to prevent polymer aggregation [START_REF] Pitkänen | The effect of galactose side units and mannan chain length on the macromolecular characteristics of galactomannans[END_REF] or control interaction with other polymer [START_REF] Yu | The patterned structure of galactoglucomannan suggests it may bind to cellulose in seed mucilage[END_REF]. Thus, mannan structure both has an impact in planta and affects how extracted mannans are used in various industries. Wheat endosperm mannans are linear mannans that are weakly acetylated [START_REF] Verhertbruggen | Challenging the putative structure of mannan in wheat (Triticum aestivum) Endosperm[END_REF].

Linear mannans are usually insoluble as observed, for example, in ivory nuts [START_REF] Mdel | Softening-up mannan-rich cell walls[END_REF][START_REF] Grimaud | In vitro synthesis and crystallization of β-1,4-Mannan[END_REF], but backbone acetylation may confer solubility in aqueous solution [START_REF] Gómez | Manufacture and properties of glucomannans and glucomannooligosaccharides derived from konjac and other sources[END_REF].

Like most hemicelluloses, mannans are synthesized in the Golgi apparatus and then are secreted to the cell wall via plasma-membrane fused vesicles [START_REF] Northcote | A function of the Golgi apparatus in polysaccharide synthesis and transport in the root-cap cells of wheat[END_REF]. To date five families of proteins have been reported to be involved in mannan synthesis: carbohydrate active enzymes (CAZY; www.cazy.org) glycosyltransferase (GT) 2 cellulose synthase-like (Csl) family A [START_REF] Dhugga | Guar seed beta-mannan synthase is a member of the cellulose synthase super gene family[END_REF][START_REF] Liepman | The CELLULOSE SYNTHASE-LIKE A and CELLULOSE SYNTHASE-LIKE C families: recent advances and future perspectives[END_REF][START_REF] Goubet | AtCSLA7, a cellulose synthase-like putative glycosyltransferase, is important for pollen tube growth and embryogenesis in Arabidopsis[END_REF] and D [START_REF] Verhertbruggen | Mannan synthase activity in the CSLD family[END_REF][START_REF] Yin | The cooperative activities of CSLD2, CSLD3, and CSLD5 are required for normal Arabidopsis development[END_REF][START_REF] Yin | A survey of plant and algal genomes and transcriptomes reveals new insights into the evolution and function of the cellulose synthase superfamily[END_REF], the GT34 mannan galactosyltransferases [START_REF] Edwards | Transfer specificity of detergent-solubilized fenugreek galactomannan galactosyltransferase[END_REF][START_REF] Voiniciuc | MUCILAGE-RELATED10 produces galactoglucomannan that maintains pectin and cellulose architecture in arabidopsis seed mucilage[END_REF], the GT106 mannan synthesis related proteins (MSR) [START_REF] Wang | Identification of an additional protein involved in mannan biosynthesis[END_REF][START_REF] Voiniciuc | Mechanistic insights from plant heteromannan synthesis in yeast[END_REF] and the domain of unknown function (DUF) 231 / trichome birefringence like (TBL) family [START_REF] Zhong | Members of the DUF231 Family are O-acetyltransferases catalyzing 2-O-and 3-O-acetylation of mannan[END_REF]. Several CslA proteins from different plant species have been demonstrated to synthesize mannan and glucomannan in vitro after heterologous expression and purification from transgenic soybean cells, insect cells, or the yeast Pichia pastoris (named in the rest of the text as Pichia) [START_REF] Dhugga | Guar seed beta-mannan synthase is a member of the cellulose synthase super gene family[END_REF][START_REF] Liepman | Expression of cellulose synthase-like (Csl) genes in insect cells reveals that CslA family members encode mannan synthases[END_REF][START_REF] Gille | Oacetylation of Arabidopsis hemicellulose xyloglucan requires AXY4 or AXY4L, proteins with a TBL and DUF231 domain[END_REF]. Recently, Voiniciuc et al. [START_REF] Voiniciuc | Mechanistic insights from plant heteromannan synthesis in yeast[END_REF] engineered Pichia cells to produce and secrete β-1,4-mannan polysaccharides by expressing the Arabidopsis thaliana (named in the rest of the text as Arabidopsis) AtCslA2 alone, or glucomannan by co-expressing AtCslA2 and AtMSR1. Therefore, MSR proteins are cofactors modulating the structure of the mannan polymer synthesized by CslA enzymes, and different effects were observed depending on the CslA and MSR investigated [START_REF] Voiniciuc | Mechanistic insights from plant heteromannan synthesis in yeast[END_REF]. The overexpression of AtCslDs in tobacco has been shown to modulate the production of mannan [START_REF] Yin | The cooperative activities of CSLD2, CSLD3, and CSLD5 are required for normal Arabidopsis development[END_REF]. However, CslD proteins have also been reported to synthesize β-1,4-glucan [START_REF] Park | A role for CSLD3 during cell-wall synthesis in apical plasma membranes of tip-growing root-hair cells[END_REF][START_REF] Bak | Biochemical and genetic analysis identify CSLD3 as a beta-1,4-glucan synthase that functions during plant cell wall synthesis[END_REF]. Proteins containing the DUF 231 domain were shown to be responsible for mannan O-acetylation [START_REF] Zhong | Members of the DUF231 Family are O-acetyltransferases catalyzing 2-O-and 3-O-acetylation of mannan[END_REF]. Transcriptomic [START_REF] Pellny | Cell walls of developing wheat starchy endosperm: comparison of composition and RNA-Seq transcriptome[END_REF][START_REF] Nemeth | Down-regulation of the CSLF6 gene results in decreased (1,3;1,4)-beta-D-glucan in endosperm of wheat[END_REF][START_REF] Capron | Transcriptional profile analysis of E3 ligase and hormone-related genes expressed during wheat grain development[END_REF] and proteomic studies [START_REF] Suliman | Identification of glycosyltransferases involved in cell wall synthesis of wheat endosperm[END_REF] conducted on developing wheat grains have led to the identification of wheat proteins potentially involved in mannan metabolism, including several CslAs and CslDs. Acetyl esterase and glycosyl hydrolases that include mannanase, mannosidase, galactosidase and glucosidase are proteins involved in the hydrolysis and modification of mannan. Several of these proteins have been identified in cereal grains during germination [START_REF] Mdel | Softening-up mannan-rich cell walls[END_REF]. Although proteins might regulate the structure and the accumulation of mannan in the cereal endosperm, there is currently no evidence to support these hypotheses in wheat grain. No changes in the structure of mannan were observed in the developing endosperm of wheat [START_REF] Verhertbruggen | Challenging the putative structure of mannan in wheat (Triticum aestivum) Endosperm[END_REF] where no mannan-related glycosyl hydrolases have been formally identified [START_REF] Suliman | Identification of glycosyltransferases involved in cell wall synthesis of wheat endosperm[END_REF][START_REF] Cherkaoui | Cell wall proteome of wheat grain endosperm and outer layers at two key stages of early development[END_REF]. Heterologous expression [START_REF] Dhugga | Guar seed beta-mannan synthase is a member of the cellulose synthase super gene family[END_REF][START_REF] Voiniciuc | Mechanistic insights from plant heteromannan synthesis in yeast[END_REF] and mutant analysis [START_REF] Goubet | AtCSLA7, a cellulose synthase-like putative glycosyltransferase, is important for pollen tube growth and embryogenesis in Arabidopsis[END_REF] have greatly contributed to our knowledge of mannan metabolism and function in plants.

It has been reported that the presence of mannan is ubiquitous in the cell walls of plant tissues

where it has a structural function. It is widely accepted that mannans and glucomannans are highly conserved throughout plant evolution [7,[START_REF] Mdel | Softening-up mannan-rich cell walls[END_REF]. Moreover, several plant species develop specialized tissues where mannan is stored as reserves of energy. This is the case of the corm of Amorphophallus konjac (konjac) which contains 49-60% of glucomannan [START_REF] Li | Grain-size effect on the structure and antiobesity activity of konjac flour[END_REF] and of the seed endosperm of several Fabaceae species such as Cyamopsis tetragonoloba (guar) or Cassia grandis that contains 50% galactomannans [START_REF] Joshi | Cassia grandis Linn. f. seed galactomannan: structural and crystallographical studies[END_REF][START_REF] Buckeridge | Seed cell wall storage polysaccharides: models to understand cell wall biosynthesis and degradation[END_REF][START_REF] Moreira | An overview of mannan structure and mannan-degrading enzyme systems[END_REF]. In Arabidopsis, mannan has been shown to have a fundamental role as certain mutant plants lacking mannan or glucomannan are either barely viable or embryo lethal [START_REF] Goubet | AtCSLA7, a cellulose synthase-like putative glycosyltransferase, is important for pollen tube growth and embryogenesis in Arabidopsis[END_REF][START_REF] Yin | The cooperative activities of CSLD2, CSLD3, and CSLD5 are required for normal Arabidopsis development[END_REF]. Furthermore, it has been suggested that mannan is involved in zygotic embryogenesis, oligosaccharide signaling and cell differentiation [START_REF] Liepman | The CELLULOSE SYNTHASE-LIKE A and CELLULOSE SYNTHASE-LIKE C families: recent advances and future perspectives[END_REF].

Here, we investigated the biosynthesis and function of wheat mannan, a polysaccharide that is synthesized and deposited in the cell walls of the developing wheat grain, especially in the endosperm tissue.

Material and methods

Plant material

Hexaploid (AA,BB,DD) wheat (Triticum aestivum L.) cv Recital were grown in pots in controlled glasshouses in INRAE Le Rheu (France) under natural day light. Individual spikes were tagged at flowering. Development stages were assessed by the thermal time method, by calculating the cumulated average temperature per day elapsed since flowering (°Celsius per day after flowering, °DAF). The two basal grains of the spikelets located in the middle of the spikes were harvested at different stages of development.

Arabidopsis thaliana Columbia-0 (Col-0) and triple mutant (TM) csla2/csla3/csla9 [START_REF] Goubet | AtCSLA7, a cellulose synthase-like putative glycosyltransferase, is important for pollen tube growth and embryogenesis in Arabidopsis[END_REF] seeds were sown on soil and grown as previously described [START_REF] Liang | A screening method to identify efficient sgRNAs in Arabidopsis, used in conjunction with cell-specific lignin reduction[END_REF]. Tobacco plants (Nicotiana tabacum and Nicotiana benthamiana) were cultivated as in [START_REF] Suliman | Identification of glycosyltransferases involved in cell wall synthesis of wheat endosperm[END_REF].

Immunolabeling and histological staining

Grain samples were fixed and embedded as described in [START_REF] Chateigner-Boutin | The wheat grain contains pectic domains exhibiting specific spatial and development-associated distribution[END_REF]. Cross-sections were made in the equatorial region of the grains using an ultramicrotome (UC7, Leica) and the sections were stained using toluidine blue-O as described in [START_REF] Chateigner-Boutin | Developing Pericarp of Maize: A Model to Study Arabinoxylan Synthesis and Feruloylation[END_REF]. Other sections were used for immunolabeling experiments where the procedure was performed as described in [START_REF] Chateigner-Boutin | The wheat grain contains pectic domains exhibiting specific spatial and development-associated distribution[END_REF]. The antibodies used were LM21 and LM22 (dilution 1:5; [START_REF] Marcus | Restricted access of proteins to mannan polysaccharides in intact plant cell walls[END_REF]), which recognize mannan polysaccharides and oligosaccharides (DP2-DP5). Whereas LM21 displays a wide recognition of mannans, gluco-and galactomannans, LM22 does not detect galactomannans.

Sequence-based identification of the wheat candidate genes

The tentative full-length sequences of TaCslD2, TaCslA7 and TaCslA12 were originally obtained by assembling wheat EST contigs, wheat Chinese Spring genomic contigs, and RNAseq sequences [START_REF] Pellny | Cell walls of developing wheat starchy endosperm: comparison of composition and RNA-Seq transcriptome[END_REF]. The sequence assemblies were compared to nucleotide sequences contained in the NCBI database using the BlastN sequence alignment program (restricted to Poaceae), and aligned to the best hits from Hordeum vulgare and Brachypodium distachyon.

The cDNA sequences were then isolated from the developing endosperm of wheat grain (cv Recital). RNA isolation, cDNA synthesis and PCR were performed as described in [START_REF] Suliman | Identification of glycosyltransferases involved in cell wall synthesis of wheat endosperm[END_REF]. Total RNA was isolated from manually dissected endosperm of wheat grains at 250°DAF using the phenolchloroform method [START_REF] Suliman | Identification of glycosyltransferases involved in cell wall synthesis of wheat endosperm[END_REF]. RNA was treated twice with DNase (Qiagen) and then purified through the RNeasy MinElute Cleanup kit (Qiagen), following the manufacturer's recommendations. Reverse transcription was carried out with 2 μg of total RNA primed with random hexamers using the Transcriptor First Strand cDNA synthesis kit (Roche). cDNA was amplified by PCR using a high fidelity Q5 DNA polymerase (New England Biolabs) with the primers listed in Table S1. PCR products were cloned into pCR-BluntII-Topo vector (Thermofisher) and sequenced.

In silico analysis of the wheat sequences

Sequences were translated in silico using the ExPASy Translate tool (https://web.expasy.org/translate/). Nucleotide and protein sequences were aligned using Clustal Omega (1.2.4) and Multalin (http://multalin.toulouse.inra.fr/multalin/). Transmembrane domains were predicted using TMHMM 2.0 (http://www.cbs.dtu.dk/services/TMHMM/). Signal peptide and cleavage site prediction was obtained with TargetP 1.1 (http://www.cbs.dtu.dk/services/TargetP/) for Plant. The expression profiles of the targeted genes were obtained from microarray experiments [START_REF] Nemeth | Down-regulation of the CSLF6 gene results in decreased (1,3;1,4)-beta-D-glucan in endosperm of wheat[END_REF][START_REF] Capron | Transcriptional profile analysis of E3 ligase and hormone-related genes expressed during wheat grain development[END_REF], RNAseq experiments [START_REF] Pellny | Cell walls of developing wheat starchy endosperm: comparison of composition and RNA-Seq transcriptome[END_REF] and from data available at the PlaNet expression database (http://aranet.mpimp-golm.mpg.de/index.html) [START_REF] Mutwil | PlaNet: combined sequence and expression comparisons across plant networks derived from seven species[END_REF] and at www.wheat-expression.com [START_REF] Ramírez-González | Consortium IWGS, The transcriptional landscape of polyploid wheat[END_REF][START_REF] Borrill | expVIP: a customizable RNA-seq data analysis and visualization platform[END_REF]. The cDNA sequences were also aligned against the recently released gene models for wheat Chinese Spring (IWGSC 2018) (Ensembl Wheat release 47 -April 2020) available at https://plants.ensembl.org/Triticum_aestivum/Info/Index.

Expression of wheat genes in tobacco plants

Wheat cDNAs were amplified using primers containing attB sequences for gateway recombination (Table S1). For subcellular localization studies, the binary pK7FWG2 and pK7RWG2 vectors containing the 35S promoter of cauliflower mosaic virus (CaMV) and the eGFP (enhanced green fluorescent protein) or mRFP (monomeric red fluorescent protein) respectively were used to generate C-terminal fusions. The inserted genes were TaCslD2, TaCslA7 and TaCslA12 and the Golgi marker, sialyltransferase (ST; [START_REF] Sparkes | Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants[END_REF]), and the endoplasmic reticulum (ER) marker, dihydrosphingosine phosphate lyase 1 (DPL1; [START_REF] Nishikawa | Degradation of sphingoid long-chain base 1-phosphates (LCB-1Ps): functional characterization and expression of AtDPL1 encoding LCB-1P lyase involved in the dehydration stress response in Arabidopsis[END_REF]). Co-transformation experiments were conducted to observe co-localization. For bimolecular fluorescence complementation (BIFC) experiments, the vectors pBIFP-1 and pBIFP-4 containing the N-terminal part and C-terminal parts of the fluorescent eYFP (enhanced yellow fluorescent protein) respectively were used to generate C-terminal fusions [START_REF] Azimzadeh | Arabidopsis TONNEAU1 proteins are essential for preprophase band formation and interact with centrin[END_REF]. Interacting MADS-box transcription factors DEFICIENS and GLOBOSA [START_REF] Schwarz-Sommer | Characterization of the Antirrhinum floral homeotic MADS-box gene deficiens: evidence for DNA binding and autoregulation of its persistent expression throughout flower development[END_REF] were used as positive controls. Agroinfiltration of tobacco leaves and observations using a confocal microscope were carried out as described in [START_REF] Suliman | Identification of glycosyltransferases involved in cell wall synthesis of wheat endosperm[END_REF]. N. tabacum was used for subcellular localization studies and N. benthamiana transformed with the silencing inhibitor P19 was used for BIFC experiments as in [START_REF] Azimzadeh | Arabidopsis TONNEAU1 proteins are essential for preprophase band formation and interact with centrin[END_REF].

Expression of wheat genes in Pichia pastoris

The expression of CslA genes in Pichia was performed essentially as described in [START_REF] Voiniciuc | Mechanistic insights from plant heteromannan synthesis in yeast[END_REF].

Plant cDNAs were cloned using the EasySelect Pichia Expression Kit (Invitrogen), according to the manufacturer's manual. The cDNA sequences, including stop codons, were inserted using restriction enzymes in the multiple cloning site of the pPICZ B vector (Invitrogen) and transformed in Escherichia coli TOP10F' cells for propagation. The primers used to amplify the genes of interest are listed in Table S1. The resulting pPICZ B + CslA plasmids were verified by colony PCR and sequencing using the vector-and gene-specific primers listed in Table S1. Linearized plasmid DNA (150 ng) was used to transform Pichia X-33 cells by electroporation [START_REF] Lin-Cereghino | Condensed protocol for competent cell preparation and transformation of the methylotrophic yeast Pichia pastoris[END_REF].

Transgenes were integrated into the pAOX1 region of the Pichia genome via homologous recombination, and verified by yeast colony PCR.

Multiple independent Pichia transformed colonies were cultured in 24-well plates for around 60 h and the pAOX1 promoter was then induced by the addition of methanol as described in [START_REF] Voiniciuc | Mechanistic insights from plant heteromannan synthesis in yeast[END_REF].

After 24 h of methanol induction, Pichia cells were transferred to 2 mL tubes and collected by centrifugation. Alcohol Insoluble residue (AIR) containing yeast wall material was obtained and mannosylated proteins were removed with 1 M NaOH for 60 min at 75°C and 1,400 rpm in a thermomixer (Eppendorf) as described in [START_REF] Voiniciuc | Mechanistic insights from plant heteromannan synthesis in yeast[END_REF]. Alkaline-insoluble (AKI) polysaccharides were pelleted at 20,000 g for 2 min and washed with water. The resulting AKI material was resuspended in 600 µL of water by mixing at 30 Hz for 2 min 30 s using a ball mill, and used immediately for enzymatic digestion or stored at 4°C. The monosaccharide composition of the AKI polymers was determined via highperformance anion-exchange chromatography coupled with pulsed electrochemical detection (HPAEC-PAD), as previously described [START_REF] Voiniciuc | MUCILAGE-RELATED10 produces galactoglucomannan that maintains pectin and cellulose architecture in arabidopsis seed mucilage[END_REF][START_REF] Voiniciuc | Analysis of monosaccharides in total mucilage extractable from Arabidopsis seeds[END_REF], on a 940 Professional IC Vario ONE/ChS/PP/LPG instrument (Metrohm) equipped with a CarboPac PA20 guard and analytical column.

For enzymatic characterization, AKI polymers were incubated with 1 U of endo-1,4-βmannanase (Megazyme, E-BMABC) for 30 min at 40°C and 1,000 rpm in a thermomixer (Eppendorf). Afterwards, the samples were centrifuged for 2 min at 20,000 g, and 100 µL of the supernatant was used to quantify the amount of sugar released. Total hexose sugars were quantified using the anthrone assay [START_REF] Foster | Comprehensive compositional analysis of plant cell walls (lignocellulosic biomass) Part II: Carbohydrates[END_REF].

Glycosidic linkages in Pichia material were analyzed by gas chromatography-mass spectrometry (GC-MS) following derivatization of partially methylated alditol acetates as described in [START_REF] Ciucanu | A simple and rapid method for the permethylation of carbohydrates[END_REF], with modifications as described in [START_REF] Ciucanu | Per-O-methylation reaction for structural analysis of carbohydrates by mass spectrometry[END_REF]. Carbohydrates were separated using a 5977A Series GC-MS system (Agilent) equipped with a SP-2380 capillary column (Supelco).

Carbohydrate peaks were annotated based on their retention time and ion spectra relative to standards.

Expression of wheat genes in Arabidopsis thaliana

The vector used for Arabidopsis transformation derived from the pIpKB1 vector [START_REF] Himmelbach | A set of modular binary vectors for transformation of cereals[END_REF]. The nucleotide sequence of the IRX5 promoter (AtCesA4) was amplified from the pTKan-pIRX5-GW [START_REF] Eudes | Biosynthesis and incorporation of side-chain-truncated lignin monomers to reduce lignin polymerization and enhance saccharification[END_REF] using the primers SpeI_AtCesa4For and AtCesa4_AscIRev (Table S1) and introduced by ligation after restriction using SpeI and AscI into the promoter cloning site of the pIpKB1 vector.

The Arabidopsis triple mutant (TM) csla2/csla3/csla9 [START_REF] Goubet | AtCSLA7, a cellulose synthase-like putative glycosyltransferase, is important for pollen tube growth and embryogenesis in Arabidopsis[END_REF] was grown as described above, and transformed using the floral dip method [START_REF] Clough | Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana[END_REF]. The resulting seeds were surface sterilized and selected in Murashige and Skoog (MS) medium containing 25 µg/mL hygromycin. Seedlings from six transformed lines were then transferred to soil.

Seeds from Col-0 (WT) and TM were sown on MS medium without hygromycin and grown in the same conditions as the transformed lines. The genotype of the background line and the presence of the wheat gene was verified by extracting DNA from the lines using the fast dipstick protocol [START_REF] Zou | Nucleic acid purification from plants, animals and microbes in under 30 seconds[END_REF] and PCR performed using the primers listed in Table S1.

Inflorescence stems of mature plants (basal 15 cm) were harvested and stored at -80°C.

The stems were weighed, cut and incubated in 96% ethanol at 65°C for 30 min. The ethanol was removed and the stems were washed several times with 96% ethanol until the liquid stayed transparent. The samples were oven-dried at 40°C and then ground with liquid nitrogen in a freezer miller to obtain AIR. AIR samples were hydrolyzed in 26N H2SO4 for 30 min at 25°C and then with 2N H2SO4 at 100°C for 2 h. Individual monosaccharides were then converted into alditol acetates [START_REF] Englyst | Improved method of measurement of dietary fiber as non-starch polysaccharides in plant foods[END_REF] and analyzed by gas liquid chromatography as previously described [START_REF] Yacoubi | Water-soluble fractions obtained by enzymatic treatment of wheat grains promote short chain fatty acids production by broiler cecal microbiota[END_REF]. The values were obtained from technical duplicate.

The mannan structure of Arabidopsis stems was studied using enzymatic fingerprinting with a recombinant endo-1,4-β-mannanase from Cellvibrio japonicus (Megazyme, E-BMACJ) as described in [START_REF] Verhertbruggen | Challenging the putative structure of mannan in wheat (Triticum aestivum) Endosperm[END_REF]. Briefly, 15 mg of AIR were suspended in 1 mL of distilled water and incubated at 40°C overnight with 40 U. mL -1 mannanase. After centrifugation, the supernatant was collected and heated for 10 min at 95°C. 200 µL was mixed with 400 µL 96% ethanol and incubated at 4°C for 4 h. After centrifugation at 13,000 g, the supernatant was collected and 200 µL of supernatant was mixed with 1.8 mL of water and filtered through a 0.45 µm filter. The oligosaccharides were analyzed by HPAEC-PAD with a Dionex Carbo-Pac PA1 column (4×250mm, Thermo Fisher Scientific) at 30°C eluted at 1 mL. min -1 using a linear gradient of sodium acetate from 0 to 0.1 M in 0.1 M NaOH for 30 min. They were also analyzed by matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF) mass spectrometry (MS) as described in [START_REF] Verhertbruggen | Challenging the putative structure of mannan in wheat (Triticum aestivum) Endosperm[END_REF]. The samples (1 μL) were deposited and covered by the matrix (2,5-dihydroxybenzoic acid and N,N-dimethylaniline; 1 μL) on a polished steel MALDI target plate. MALDI measurements were then performed on a rapifleX MALDI-TOF spectrometer (Bruker) operated with positive polarity in a reflectron mode, and spectra were acquired in the range of 500-2500 m/z.

Results

Mannan is deposited in the cell walls of the developing endosperm from the cellularization stage

The study of cell wall polymers of wheat grain revealed the presence of mannosecontaining polysaccharides in the dry mature grain and in the developing grain [START_REF] Verhertbruggen | Challenging the putative structure of mannan in wheat (Triticum aestivum) Endosperm[END_REF][START_REF] Mares | Studies on wheat endosperm. I. Chemical composition and ultrastructure of the cell walls[END_REF][START_REF] Chateigner-Boutin | Endomembrane proteomics reveals putative enzymes involved in cell wall metabolism in wheat grain outer layers[END_REF][START_REF] Palmer | Comparative in situ analyses of cell wall matrix polysaccharide dynamics in developing rice and wheat grain[END_REF].

Immunolabeling experiments demonstrated that mannan epitopes are predominantly detected in the walls of the wheat grain endosperm and less in the maternal outer tissues of the grain [START_REF] Pellny | Cell walls of developing wheat starchy endosperm: comparison of composition and RNA-Seq transcriptome[END_REF][START_REF] Palmer | Comparative in situ analyses of cell wall matrix polysaccharide dynamics in developing rice and wheat grain[END_REF]. This heterogeneity was not as apparent in the grain tissues of rice [START_REF] Palmer | Comparative in situ analyses of cell wall matrix polysaccharide dynamics in developing rice and wheat grain[END_REF] and Brachypodium distachyon [START_REF] Francin-Allami | Spatial and temporal distribution of cell wall polysaccharides during grain development of Brachypodium distachyon[END_REF]. To improve our knowledge of the physiological functions of mannan in cereal grains, we conducted immunolabeling experiments with antibodies targeting mannan epitopes on sections of wheat grains harvested at different stages of development. The aim was to refine the kinetics of mannan deposition in wheat grain and particularly to monitor its first appearance in the walls of the developing endosperm.

Two antibodies, LM21 and LM22, are commercially available to detect mannan (www.plantprobes.net). The LM21 antibody has been shown to bind to a wider range of mannan polysaccharides than the LM22 antibody [START_REF] Marcus | Restricted access of proteins to mannan polysaccharides in intact plant cell walls[END_REF]. In the Arabidopsis stem, mannan was barely detected when using LM22 and strongly detected with the LM21 antibody [START_REF] Kim | Immunolocalization of hemicelluloses in Arabidopsis thaliana stem. Part II: Mannan deposition is regulated by phase of development and its patterns of temporal and spatial distribution differ between cell types[END_REF]. We have observed a similar result when using both antibodies on sections of developing wheat grains. Only a weak signal of fluorescence was observed in the endosperm cell walls with the LM22 antibody (Fig. S1A). By contrast, mannan was abundantly detected when using LM21 (Fig. S1B). Therefore, in all of the following experiments, we used the LM21 antibody. No mannan was detected in transverse sections of the ovary at anthesis or of grains harvested at 75°DAF, a very early stage of development that occurs within the first four days after flowering. As shown in our bright-field micrographs, it is worth to note that at 75°DAF cell walls are already present in the developing endosperm (Fig. 1). The LM21 mannan epitope was detected in the endosperm and in some cells of the maternal tissues from wheat grain harvested at 90°DAF (Fig. 1). The maternal cells labeled with LM21 are located in the nucellar projection in the crease region (Fig. 1). In the developing endosperm cells at 90°DAF, the LM21 epitope was strongly detected in the walls of the first cell layers bordering the endosperm cavity, and a gradient of signal intensity was observed from the ventral region of the endosperm (crease region) to the dorsal region of the endosperm (Fig. 1). At this developmental stage, signs of endosperm cell division are visible (see the bright-field micrographs). At subsequent stages, the LM21 antibody labels the walls of the endosperm until maturity (Fig. S1B).

Identification of mannan-related genes expressed in wheat grain endosperm

Since mannan is deposited during development in the nucellar projection and endosperm (Fig. 1), tissues that are important for seed filling, it could impact grain quality. We therefore decided to isolate and study mannan-related genes expressed in wheat grain. Transcriptomic [START_REF] Pellny | Cell walls of developing wheat starchy endosperm: comparison of composition and RNA-Seq transcriptome[END_REF][START_REF] Nemeth | Down-regulation of the CSLF6 gene results in decreased (1,3;1,4)-beta-D-glucan in endosperm of wheat[END_REF][START_REF] Capron | Transcriptional profile analysis of E3 ligase and hormone-related genes expressed during wheat grain development[END_REF] and proteomic studies [START_REF] Suliman | Identification of glycosyltransferases involved in cell wall synthesis of wheat endosperm[END_REF] conducted on developing wheat grain and isolated endosperms have led to the identification of wheat genes potentially involved in mannan metabolism. Our initial bioinformatics analysis of the expression profile of these genes in different organs was performed in 2013 using data from [START_REF] Pellny | Cell walls of developing wheat starchy endosperm: comparison of composition and RNA-Seq transcriptome[END_REF][START_REF] Nemeth | Down-regulation of the CSLF6 gene results in decreased (1,3;1,4)-beta-D-glucan in endosperm of wheat[END_REF][START_REF] Capron | Transcriptional profile analysis of E3 ligase and hormone-related genes expressed during wheat grain development[END_REF] and from the expression platform PlaNet (http://aranet.mpimp-golm.mpg.de/index.html) (Table 1 and Fig. S2). These studies suggested that TaCslD2, TaCslA7, and TaCslA12 were predominantly expressed in the developing endosperm of wheat and were therefore further characterized. More recent expression data (www.wheat-expression.com) revealed that while TaCslA12 really appears to be specifically and highly expressed in the wheat developing grain, bothTaCslA7 and TaCslD2 have significant expression in other tissues in addition to endosperm (Fig. S2).

The cDNA sequence of wheat TaCslD2, TaCslA7 and TaCslA12 was obtained from RNA isolated from wheat developing endosperm (cv Recital). The sequences were first aligned with the available sequences of related species to verify their coverage. Notably TaCslA7, TaCslA12 and proteins contain the conserved sequences among polymerizing beta-glycosyltransferases (aspartyl residues as well as the motif QXXRW (Gln-X-X-Arg-Trp) (Fig. S3A) and the DxD motif of GT (the DXD motif in the catalytic site interacts with the phosphates of the NDP-sugar via a metal ion [START_REF] Fulton | Crystal structure of a UDP-glucosespecific glycosyltransferase from a Mycobacterium species[END_REF]). However, when compared to other CslA proteins and in particular to the mannan synthase (ManS) CslA of guar, konjac and Arabidopsis (Fig. S3A), we realized that the TaCslA7 sequence was longer in the N-terminal region. That region is not conserved among CslAs.

Prediction tools for subcellular location and signal peptide prediction were applied. Putative cleavage sites were found using TargetP for TaCslA7, TaCsl12 and AkCslA3 (Fig. S3A). Thus, CslA polypeptides might contain N-terminal peptides cleaved in the mature proteins. This would explain why Dhugga et al. [START_REF] Dhugga | Guar seed beta-mannan synthase is a member of the cellulose synthase super gene family[END_REF] and Liepman et al. [START_REF] Liepman | Expression of cellulose synthase-like (Csl) genes in insect cells reveals that CslA family members encode mannan synthases[END_REF] observed that CslA proteins displayed an apparent lower molecular weight than predicted from the corresponding peptide sequence upon migration in gels. N-terminal cleavage of TaCslA12 and TaCslA7 would remove one transmembrane domain (TMD). The TMD prediction program TMHMM predicted that the long sequence between the N-terminal TMD and the four C-terminal TMDs, which contains the active site would be located in the Golgi lumen (inside) with or without cleavage (Fig. S3B The cloned TaCslD2 cDNA sequence was used to identify the best Blast hits in Chinese Spring wheat. Two very close sequences were identified in chromosome 7 and 4, each in only one genome. Best hits were TraesCS7D02G029700.1 (genome D) and TraesCS4A02G456300.1 (genome A) (Table 1). The protein sequence (Fig. S3C) contained the conserved sequence for polymerizing beta-glycosyltransferases QXXRW (Gln-X-X-Arg-Trp) and the DxD motif, TMDs, and it bears a Zinc finger domain (IPR013083) which could be involved in protein-protein interaction.

The heterologous expression of wheat TaCslA12-B leads to the production of wheat-like mannan

We attempted to produce knocked-out or down-regulated wheat lines for the TaCslA7, TaCslA12 and TaCslD2 genes. Regardless of the strategy trialled, these attempts were unsuccessful ( [START_REF] Nemeth | Down-regulation of the CSLF6 gene results in decreased (1,3;1,4)-beta-D-glucan in endosperm of wheat[END_REF] and data not shown). Therefore, as an alternative approach to study these genes, we expressed the wheat mannan-related genes in heterologous systems.

Heterologous expression in tobacco leaf

To verify the subcellular localization of the proteins, the coding sequence was cloned in fusion with a fluorescent reporter gene and introduced into tobacco by leaf agroinfiltration. Fusion proteins were detected for the three constructs by confocal microscopy. TaCslA7 and TaCslA12 fusion proteins were observed as small fluorescent dots which co-localized with the Golgi marker sialyltransferase (Fig. 2). These results indicate that TaCslA7 and TaCslA12 are localized in the Golgi apparatus. This is in accordance with previous results for CslA proteins from other species and with the fact that mannan biosynthesis occurs in the Golgi apparatus [START_REF] Dhugga | Guar seed beta-mannan synthase is a member of the cellulose synthase super gene family[END_REF]. However, the TaCslD2 fusion protein was observed as a network and co-localized with the ER marker. No Golgi signal was detected for TaCslD2 alone when fused to either GFP or RFP as C-terminal or Nterminal fusion. A co-transformation experiment was conducted with TaCslD2 and TaCslA12. We observed that the subcellular localization differs when the fusion proteins were expressed alone or together in tobacco leaf. Fluorescent signals for both TaCslD2 and TaCslA12 were seen as both dots and networks, with partial overlays of the signals (Fig. 2). One potential explanation for the changed localization is that the proteins interact. To test this, BIFC experiments were carried out.

TaCslD2 and TaCslA12 and positive controls were fused in their C-terminal region to both N-and C-terminal regions of the split fluorescent YFP and introduced into tobacco leaves. Fluorescence signals marking the formation of a full functional YFP by close co-localization of the tested partners were only obtained for the positive controls (Fig. S4). No significant signal was observed when TaCslD2 and TaCslA12 were co-infiltrated. Moreover, BIFC experiments were also conducted with either TaCslD2 or TaCslA12 infiltrated as two fusion proteins with N-and Cterminal regions of the YFP. No significant signal indicative of homomultimeric interactions was observed (Fig. S4).

Heterologous expression in the yeast Pichia pastoris

Recently the yeast Pichia showed its great utility as a system for testing the heterologous activity of plant mannan synthesis genes [START_REF] Voiniciuc | Mechanistic insights from plant heteromannan synthesis in yeast[END_REF]. We took advantage of this system and transformed Pichia with the TaCslA7 and TaCslA12 coding sequences. Pichia expressing the AkCslA3 glucomannan synthase from konjac [START_REF] Voiniciuc | Mechanistic insights from plant heteromannan synthesis in yeast[END_REF][START_REF] Gille | Oacetylation of Arabidopsis hemicellulose xyloglucan requires AXY4 or AXY4L, proteins with a TBL and DUF231 domain[END_REF] was used as positive control and yeast containing no heterologous enzyme (empty vector) served as negative control. After culture and methanol induction, alkaline-insoluble polysaccharides were isolated and screened for the presence of mannan using an endo-β-1,4-mannanase. The colorimetric anthrone assay showed that the content of hexose sugars released by mannanase digestion significantly increased in Pichia expressing AkCslA3 or TaCslA12 (Fig. 3). Unlike TaCslA12 and AkCslA3, the TaCslA7 construct was not sufficient for mannan production in Pichia. Permethylation experiments identified an enrichment in 1,4-mannosyl linkages in the cell wall polymers of both the AkCslA3 and the TaCslA12 yeast strains. However, the 1,4-glucosyl linkage content was only significantly increased by the expression of AkCslA3 (Fig. 3). These assays suggest that, when expressed alone in P. pastoris, the wheat CslA12 gene produced β-1,4-mannan chains that do not contain glucose moieties. This is consistent with the mannan structure found in the cell walls of the mature and developing wheat endosperm [START_REF] Verhertbruggen | Challenging the putative structure of mannan in wheat (Triticum aestivum) Endosperm[END_REF].

Heterologous expression in a triple mutant of Arabidopsis devoid of mannan in its stem

The wheat TaCslA7_D and A12_B cDNAs were also expressed in Arabidopsis. The coding sequences were introduced in a triple mutant (TM) csla2/csla3/csla9 which lacks mannan in the cell wall of its stem [START_REF] Goubet | AtCSLA7, a cellulose synthase-like putative glycosyltransferase, is important for pollen tube growth and embryogenesis in Arabidopsis[END_REF] but grows phenotypically normally unlike the embryo-lethal csla7 mutants [START_REF] Goubet | AtCSLA7, a cellulose synthase-like putative glycosyltransferase, is important for pollen tube growth and embryogenesis in Arabidopsis[END_REF]. The wheat gene was introduced under the control of the promoter of AtCesA4/irx5, which targets the expression in the secondary cell walls of the stem [START_REF] Eudes | Biosynthesis and incorporation of side-chain-truncated lignin monomers to reduce lignin polymerization and enhance saccharification[END_REF]. The strong 35S promoter was avoided so as to not disturb embryogenesis and seedling development. Selection of transformed lines by growth on hygromycin and PCR targeting the wheat insert led to the identification of several independent transformed lines for TaCslA12 but none were recovered for TaCslA7. No phenotypic differences were noticed between Col-0 (WT), TM and the transformed TM lines.

Alcohol insoluble polysaccharides were isolated and analyzed by chemical hydrolysis, alditol acetate derivatization and GC analysis to study the cell wall composition of the mature stems. The mannose content of WT stems reached about 3.7% of total neutral sugars and no mannose was detected in the TM line. In two independent TM lines transformed with the irx5::TaCslA12 construct (71.1 and 72.1), mannose was detected and reached a percentage of total neutral sugars similar to that of the WT (Table 2). Thus, the TaCslA12_B gene is functional in Arabidopsis stem and leads to the production of mannan.

Alcohol insoluble polysaccharides of WT, TM and TM transformed with the proirx5::TaCslA12 (71.1 and 72.1) were incubated with a recombinant endo-1,4-β-mannanase and the released oligosaccharides were compared by HPAEC profiling. Several peaks were obtained in the WT sample not treated with endo-mannanase, and hence those peaks were considered non-specific to the mannanase hydrolysis (Fig. 4). Commercial mannose, manno-oligosaccharides (DP2 to DP6) and endo-mannanase-treated glucomannan from konjac were used as standards to identify the peaks resulting from the action of the endo-mannanase. The profile of the mannanasetreated TM contained only small peaks, among which two had similar retention time to mannose and mannobiose. This was also observed in the chromatogram of the untreated WT (Fig. 4). The profile of the mannanase-treated WT samples and that of the lines 71.1 and 72.1 contain peaks that can be attributed to mannose and manno-oligosaccharides (DP2 to at least DP4). The profile of konjac and WT Arabidopsis samples contains additional glucomannan-specific peaks, that are not present in the profile of the complemented lines 71.1 and 72.1 (Fig. 4). Arabidopsis stems contain glucomannan in their secondary walls [START_REF] Handford | Localisation and characterisation of cell wall mannan polysaccharides in Arabidopsis thaliana[END_REF]. These results suggest that the TaCslA12 protein introduced into Arabidopsis produced mannan but not glucomannan, and this is in agreement with the result obtained in Pichia. Differences in peak area were observed. For example, most manno-oligosaccharides of small DP showed proportionally larger peaks in the 71.1 and 72.1 samples as compared to the WT. Mass spectrometry experiments were conducted to monitor mannan acetylation in WT and in complemented lines. Indeed, one possible explanation for the lower signal for manno-oligosaccharides of small DP could be that mannans in WT Arabidopsis are less easily hydrolyzed by the mannanase due to a higher level of acetylation. Fig. 5 shows the mass profile of the oligosaccharides generated by the endo-mannanase from stems of a WT and a complemented line (72.1). In the m/z range investigated, the detected oligosaccharides were oligo-hexose of DP3 (Hex-DP3+K + m/z 543.14) to DP15 (Hex-DP15+K + m/z 2487.77). The mass profiles identified more oligo-hexose of higher DP in WT when compared to the complemented line. Typical mass spectrometric signatures of acetylation (addition of m/z 42.01 and its multiple) were present. While O-acetylation was found in line 72.1, extensive acetylation was revealed in the WT sample. Interestingly very high signals were found in WT for Hex-DP5+K + + 2 acetyl-substituents (m/z 951.25) and Hex-DP6+K + + 2 acetyl-substituents (m/z 1113.28).

Discussion

Recently, we have shown that mannan in wheat grain endosperm are relatively short chains of β-1,4-linked mannose residues that are weakly acetylated. We have also observed that the structure of mannan does not vary during grain development [START_REF] Verhertbruggen | Challenging the putative structure of mannan in wheat (Triticum aestivum) Endosperm[END_REF]. To improve our knowledge of this polysaccharide in wheat, the deposition pattern of mannan, the cell machinery involved, and the physiological function and importance of wheat endosperm mannan were investigated.

The work described here has revealed new information about cell wall formation during cereal grain development. Our immunolabeling assays suggest that mannan is involved in the early development of endosperm cells as it is first detected in their walls during the cellularization stage. However, mannan was absent in the first cell walls formed in the endosperm. At 75°DAF, prior to the occurrence of mannan, immunolabeling experiments have shown that the endosperm cell walls contain callose and mixed-linkage glucans [START_REF] Philippe | Arabinoxylan and (1-->3),(1-->4)-beta-glucan deposition in cell walls during wheat endosperm development[END_REF]. Arabinoxylan, the main polysaccharide of mature endosperm cell walls, is detected from 90°DAF in the grain cell walls, and appears visible from 150°DAF in the endosperm cell walls [69]. The precise order of deposition of the different cell wall polymers is likely important for the correct establishment of the endosperm cell walls and possibly for the functional differentiation of the endosperm cell types (aleurone cells, transfer cells, starchy endosperm).

To study the impact of mannan in wheat endosperm, we targeted members of the GT2 CslD and CslA families that were demonstrated to be related to mannan synthesis in other species [START_REF] Dhugga | Guar seed beta-mannan synthase is a member of the cellulose synthase super gene family[END_REF][START_REF] Verhertbruggen | Mannan synthase activity in the CSLD family[END_REF]. While the activity of CslDs is still under debate, it has been firmly demonstrated that CslA proteins synthesize linear mannan and glucomannan. The targeted genes were well expressed in the developing wheat endosperm as revealed by previous transcriptomic and proteomic studies [START_REF] Suliman | Identification of glycosyltransferases involved in cell wall synthesis of wheat endosperm[END_REF][START_REF] Pellny | Cell walls of developing wheat starchy endosperm: comparison of composition and RNA-Seq transcriptome[END_REF]. We attempted to obtain wheat mutants and transformants with altered mannan content in their endosperm, but our efforts have been unsuccessful.

We therefore studied the selected wheat Csl genes heterologously in several systems. In tobacco leaves as C-terminal GFP fusion protein, we found that TaCslD2 was localized in the ER when expressed alone. This does not support a direct role in mannan synthesis. However, different subcellular localizations have been observed for other CslD proteins: ER, Golgi and plasma membrane [START_REF] Park | A role for CSLD3 during cell-wall synthesis in apical plasma membranes of tip-growing root-hair cells[END_REF][START_REF] Bernal | Functional analysis of the cellulose synthase-like genes CSLD1, CSLD2, and CSLD4 in tip-growing Arabidopsis cells[END_REF][START_REF] Qi | Two poplar cellulose synthase-like D genes, PdCSLD5 and PdCSLD6, are functionally conserved with Arabidopsis CSLD3[END_REF]. These studies showed that the observed localization varied for the same CslD protein depending on the cell type or fusion type (C-terminal or N-terminal fusion). Moreover, while several studies have demonstrated that CslD proteins have an impact on mannan content [START_REF] Yin | The cooperative activities of CSLD2, CSLD3, and CSLD5 are required for normal Arabidopsis development[END_REF][START_REF] Hu | Cotton CSLD3 restores cell elongation and cell wall integrity mainly by enhancing primary cellulose production in the Arabidopsis cesa6 mutant[END_REF], there is an accumulation of evidence that suggests that this may be indirect.

Meanwhile, many studies suggest a role for CslD proteins in the synthesis of cellulose [START_REF] Park | A role for CSLD3 during cell-wall synthesis in apical plasma membranes of tip-growing root-hair cells[END_REF][START_REF] Bak | Biochemical and genetic analysis identify CSLD3 as a beta-1,4-glucan synthase that functions during plant cell wall synthesis[END_REF][START_REF] Qi | Two poplar cellulose synthase-like D genes, PdCSLD5 and PdCSLD6, are functionally conserved with Arabidopsis CSLD3[END_REF][START_REF] Hu | AtCSLD3 and GhCSLD3 mediate root growth and cell elongation downstream of the ethylene response pathway in Arabidopsis[END_REF][START_REF] Hu | Cotton CSLD3 restores cell elongation and cell wall integrity mainly by enhancing primary cellulose production in the Arabidopsis cesa6 mutant[END_REF].

TaCslA7 and TaCslA12 were located in the Golgi as expected for mannan synthases. The TaCslA7 protein fused with the GFP was expressed and targeted to the expected subcellular location. However, the same coding sequence transformed into Pichia pastoris was not able to produce mannan polymers in the yeast wall. Numerous reasons could explain this discrepancy.

The long, uncleaved TaCslA7 might not fold correctly hindering its activity. Alternatively, Voiniciuc et al. [START_REF] Voiniciuc | Mechanistic insights from plant heteromannan synthesis in yeast[END_REF] showed that some CslA proteins such as the Coffea canephora (Cc) MANS1 required a CcMSR1 cofactor to synthesize mannan in Pichia. It is thus possible that TaCslA7 requires a wheat cofactor in Pichia. It is interesting to note that the ortholog of AtMSR1 in Chinese Spring wheat is expressed in the same tissues as TaCslA7 (Fig. S2).

In Pichia, TaCslA12_B expressed alone produced a wheat-like linear β-1,4-mannan. In Arabidopsis TM depleted in glucomannan, the introduction of TaCslA12_B under the control of a promoter targeting stem tissues restored the WT mannose level. The structure of the mannan polysaccharides incorporated in cell walls is apparently different from that of the WT Arabidopsis glucomannan as revealed by enzymatic fingerprinting. The structure is also slightly different from that obtained from mature wheat endosperm since endo-mannanase fingerprinting produced profiles with only mannose, mannobiose and traces of mannotriose [START_REF] Verhertbruggen | Challenging the putative structure of mannan in wheat (Triticum aestivum) Endosperm[END_REF]. Mass spectrometry profiling showed that the mannan produced by TaCslA12_B in Arabidopsis stems are significantly acetylated but to a lower extent than the WT Arabidopsis glucomannan. Therefore, it seems that the Arabidopsis glucomannan acetyltransferase is versatile enough to acetylate linear mannan. In wheat endosperm, traces of acetylation were detected in mannan and associated with hexotriose [START_REF] Verhertbruggen | Challenging the putative structure of mannan in wheat (Triticum aestivum) Endosperm[END_REF]. The TaCslA12_B mannan in Arabidopsis therefore appears to be more acetylated than the wheat endosperm mannan. The remaining mannotriose and larger DP oligosaccharides likely reflect the recalcitrance to hydrolysis of acetylated mannooligosaccharides.

Many questions remain unanswered. First concerning the GT2-Csl superfamily and the mannan biosynthesis machinery. Several genome-wide studies of GT2 families have been conducted over the last decades in Arabidopsis [START_REF] Richmond | The cellulose synthase superfamily[END_REF], rice [START_REF] Wang | Expression profiling and integrative analysis of the CESA/CSL superfamily in rice[END_REF], wheat [START_REF] Kaur | Genome-wide analysis of the cellulose synthaselike (Csl) gene family in bread wheat (Triticum aestivum L.)[END_REF] and recently in 46 species [START_REF] Little | Revised phylogeny of the cellulose synthase gene superfamily: insights into cell wall evolution[END_REF]. Functions have been assigned to many Csl families in hemicellulose backbone synthesis. However, a recent publication revealed that not all Csl proteins are involved in cell wall synthesis. An ER localized Csl protein was shown to glucuronidate specialized metabolites (triterpenoid saponins) [START_REF] Jozwiak | Plant terpenoid metabolism co-opts a component of the cell wall biosynthesis machinery[END_REF]. This example shows that caution should be exercised before predicting the function of a protein on the basis of its sequence homology and that experimental evidence is needed.

Csl families are multigene families. In addition, many genes exist as variants. This is particularly true for polyploid species such as bread wheat. Kaur et al. [START_REF] Kaur | Genome-wide analysis of the cellulose synthaselike (Csl) gene family in bread wheat (Triticum aestivum L.)[END_REF] identified 108 genes in the wheat Csl families, each with two to three homeoalleles (subgenomes A, B, D) and for many of them 2 to 3 splice variants. Some of the genes we studied here were not annotated in Kaur et al. [START_REF] Kaur | Genome-wide analysis of the cellulose synthaselike (Csl) gene family in bread wheat (Triticum aestivum L.)[END_REF], and therefore the total number of Csl genes will increase as improved wheat genomes and transcriptomes become available. It is not clear why plant genomes contain so many Csl genes even within the same Csl family since to date the characterized members of the same Csl family have shown similar enzymatic activities. For the CslA family: 9 genes are present in the genome of Arabidopsis and 9 in rice [START_REF] Wang | Expression profiling and integrative analysis of the CESA/CSL superfamily in rice[END_REF]. According to the Ensembl wheat (Chinese Spring wheat genome release 47 April 2020), CslA genes have 52 paralogue sequences (16 paralogues for A genome, 18 paralogues for B genome, 18 paralogues for D genome). After elimination of closely related CslC family members, the number is reduced to 35 (12 genes, Table 1). Kaur et al. [START_REF] Kaur | Genome-wide analysis of the cellulose synthaselike (Csl) gene family in bread wheat (Triticum aestivum L.)[END_REF] explored publicly available expression data for wheat Csl genes in which only some of these genes would show tissue specific expression in wheat. One would expect some organ and/or tissue even cell type specificity and also developmental regulations. Transcriptomic and proteomic experiments with better spatial and temporal resolution are required for wheat to elucidate this question.

In the developing wheat endosperm, previous experiments showed that five CslA genes are expressed and that TaCslA12 and A7 are the most highly expressed [START_REF] Pellny | Cell walls of developing wheat starchy endosperm: comparison of composition and RNA-Seq transcriptome[END_REF]. Mining transcriptome databases, TaCslA12 was found to be the only wheat CslA gene specifically expressed in the wheat grain (Fig. S2). One explanation for producing 5 CslA proteins in the same tissue is that several CslA proteins might form homo/heteromultimeric complexes as this has been observed for example for the biosynthesis complex involved in cellulose, xylan, xyloglucan, homogalacturonan and arabinan synthesis [79,[START_REF] Harholt | ARAD proteins associated with pectic Arabinan biosynthesis form complexes when transiently overexpressed in planta[END_REF][START_REF] Chou | Protein-Protein Interactions Among xyloglucan-synthesizing enzymes and formation of golgi-localized multiprotein complexes[END_REF][START_REF] Jiang | Composition, assembly, and trafficking of a wheat xylan synthase complex[END_REF][START_REF] Hill | The Arabidopsis cellulose synthase complex: a proposed hexamer of CESA trimers in an equimolar stoichiometry[END_REF]. This is a point to further investigate for CslAs. Our data show that at least TaCslA12 can function on its own in Pichia. Our BIFC experiments failed to reveal homodimeric complex. One can envision that even if TaCslA12 has the ability of acting alone (as a monomer or an homomultimeric complex) in mannan synthesis, it might act more efficiently in heteromeric complexes to increase production rate or chain length as described for other GTs in Amos and Mohnen [START_REF] Amos | Critical review of plant cell wall matrix polysaccharide glycosyltransferase activities verified by heterologous protein expression[END_REF].

Another explanation for producing several CslA proteins in a same tissue would be that these CslAs might synthesize different types of mannan. That would be in accordance with the expression in the wheat grain of an orthologue of AtMSR1, which is involved in the synthesis of glucomannan [START_REF] Voiniciuc | Mechanistic insights from plant heteromannan synthesis in yeast[END_REF]. However, only linear mannan with no glucose and no substitution (other than acetylation) were detected in wheat endosperm [START_REF] Verhertbruggen | Challenging the putative structure of mannan in wheat (Triticum aestivum) Endosperm[END_REF]. Therefore, our data suggest that all wheat endosperm CslAs synthesize linear mannan.

Meanwhile the enzymatic role of CslD proteins is still under debate. In several species (Arabidopsis, rice and maize for instance), characterized mutants or overexpression lines altered in CslD genes exhibit strong phenotypes: root hair defects [START_REF] Kim | OsCSLD1, a cellulose synthase-like D1 gene, is required for root hair morphogenesis in rice[END_REF], defect in pollen tube growth [START_REF] Bernal | Functional analysis of the cellulose synthase-like genes CSLD1, CSLD2, and CSLD4 in tip-growing Arabidopsis cells[END_REF], dwarfism, reduced organ size [START_REF] Gu | Arabidopsis CSLD5 Functions in Cell Plate Formation in a Cell Cycle-Dependent Manner[END_REF], and lethality [START_REF] Yin | The cooperative activities of CSLD2, CSLD3, and CSLD5 are required for normal Arabidopsis development[END_REF]. According to Gu et al. [START_REF] Gu | Arabidopsis CSLD5 Functions in Cell Plate Formation in a Cell Cycle-Dependent Manner[END_REF] and Li et al. [START_REF] Li | Cellulose synthase-like D1 controls organ size in maize[END_REF],

CslDs would participate in the biosynthesis of the cell plate during cell division thus having a role in controlling organ size. Possible implication of CslDs in mannan and in cellulose synthesis were reported [START_REF] Verhertbruggen | Mannan synthase activity in the CSLD family[END_REF][START_REF] Park | A role for CSLD3 during cell-wall synthesis in apical plasma membranes of tip-growing root-hair cells[END_REF][START_REF] Bak | Biochemical and genetic analysis identify CSLD3 as a beta-1,4-glucan synthase that functions during plant cell wall synthesis[END_REF]. The CslD family is the Csl family phylogenetically closest to the CESA family, which are the catalytic proteins producing cellulose in cellulose synthase complexes [START_REF] Richmond | The cellulose synthase superfamily[END_REF]. Several csld mutants exhibit decrease in cellulose content [START_REF] Luan | Sun, OsCD1 encodes a putative member of the cellulose synthase-like D sub-family and is essential for rice plant architecture and growth[END_REF][START_REF] Douchkov | The barley (Hordeum vulgare) cellulose synthase-like D2 gene (HvCslD2) mediates penetration resistance to host-adapted and nonhost isolates of the powdery mildew fungus[END_REF], chimeric AtCslD3 protein containing AtCESA6 catalytic domain complemented the root hair defect of the csld3 mutant [START_REF] Park | A role for CSLD3 during cell-wall synthesis in apical plasma membranes of tip-growing root-hair cells[END_REF]. The Arabidopsis csld2/csld3/csld5 mutants lacked the immunolabeling of mannan epitopes found in WT interfascicular fibers [START_REF] Verhertbruggen | Mannan synthase activity in the CSLD family[END_REF]. In addition, tobacco microsomes isolated from plants expressing Arabidopsis CslDs incorporate more GDP- 14 C-mannose into alcohol-insoluble polymers than the control untransformed plants [START_REF] Verhertbruggen | Mannan synthase activity in the CSLD family[END_REF][START_REF] Yin | The cooperative activities of CSLD2, CSLD3, and CSLD5 are required for normal Arabidopsis development[END_REF].

Several studies have revealed concomitant expression of CslD and CslA proteins in tissues producing mannans. This is the case for the wheat endosperm here, for the early developing cotton fiber before accumulation of cellulose [START_REF] Hernandez-Gomez | Heteromannan and heteroxylan cell wall polysaccharides display different dynamics during the elongation and secondary cell wall deposition phases of cotton fiber cell development[END_REF] and for the developing seed endosperm of guar conducted during the massive accumulation of galactomannan [START_REF] Hu | Cotton CSLD3 restores cell elongation and cell wall integrity mainly by enhancing primary cellulose production in the Arabidopsis cesa6 mutant[END_REF][START_REF] Chaudhury | Elucidation of galactomannan biosynthesis pathway genes through transcriptome sequencing of seeds collected at different developmental stages of commercially important indian varieties of cluster bean (Cyamopsis tetragonoloba L.)[END_REF]. Here we showed that heterologous co-expression of TaCslD2 and TaCslA12 in tobacco leaves resulted in the colocalization of both proteins. This raises the possibility that these CslDs might act in close cooperation with CslAs.

Mannans are widespread in plant cell walls. In some species, mannan is the main hemicellulose component of cell walls. This is the case for ferns such as Adiantum raddianum [START_REF] Silva | Cell wall polysaccharides from fern leaves: Evidence for a mannan-rich Type III cell wall in Adiantum raddianum[END_REF] and the wood of gymnosperms [START_REF] Pauly | Plant cell wall polymers as precursors for biofuels[END_REF]. Several physiological functions were attributed to mannans (reviewed in [START_REF] Liepman | The CELLULOSE SYNTHASE-LIKE A and CELLULOSE SYNTHASE-LIKE C families: recent advances and future perspectives[END_REF]). In some species, mannan is stored as reserves of energy and water [START_REF] Buckeridge | Seed cell wall storage polysaccharides: models to understand cell wall biosynthesis and degradation[END_REF]. In fern roots, mannans are deposited to limit pathogen attacks [START_REF] Leroux | Ultrastructure and composition of cell wall appositions in the roots of Asplenium (Polypodiales)[END_REF]. Other functions were reported such as to increase tissue rigidity of seeds in tomato [START_REF] Buckeridge | Seed cell wall storage polysaccharides: models to understand cell wall biosynthesis and degradation[END_REF], a function in signaling (galactoglucomannan oligosaccharides) [START_REF] Benova-Kakosova | Galactoglucomannans increase cell population density and alter the protoxylem/metaxylem tracheary element ratio in xylogenic cultures of zinnia[END_REF] and, in Arabidopsis, mannan has been suggested to function in trichome adhesion [START_REF] Marks | A new method for isolating large quantities of Arabidopsis trichomes for transcriptome, cell wall and other types of analyses[END_REF], pollen tube growth [START_REF] Goubet | AtCSLA7, a cellulose synthase-like putative glycosyltransferase, is important for pollen tube growth and embryogenesis in Arabidopsis[END_REF], and embryogenesis [START_REF] Goubet | AtCSLA7, a cellulose synthase-like putative glycosyltransferase, is important for pollen tube growth and embryogenesis in Arabidopsis[END_REF]. These functions depend on the fine structure of mannan and likely on its abundance. It is unclear what the physiological advantage is for wheat to produce a few percent of mannan molecules in the cell walls of its endosperm. What is the relevance of the programmed deposition order of polysaccharides in the first cell walls of the developing endosperm? And what about the relevance of the observed polarity between the ventral and the dorsal region of the endosperm? Could mannan be involved in cell differentiation? In Arabidopsis, mannan has been shown to have a fundamental role in seed development since the Arabidopsis csla7 mutant is embryo-lethal [START_REF] Goubet | AtCSLA7, a cellulose synthase-like putative glycosyltransferase, is important for pollen tube growth and embryogenesis in Arabidopsis[END_REF]. Further studies will be required to address these questions.

Wheat mutants or variants may produce useful additional information. So far, we failed to obtain such mutants but there is no doubt that, in the future, mutagenesis and CRISPR technologies or overexpression will help to better understand the functions of mannan in wheat grain and other economically important seeds.

Conclusion

Mannans were detected in cell walls of the developing grain endosperm from early stages of development but not since the first appearance of endosperm cell walls. TaCslA12 a gene preferentially expressed in the developing wheat endosperm encodes a protein located in the Golgi in plant cells where mannan synthesis occurs and, when expressed heterologously leads to the production of β-1,4-mannan in yeast and in the stem of an Arabidopsis mutant lacking glucomannan. Further studies will be needed in order to demonstrate if mannan is also essential for early cell development and grain development in wheat.

Tables:

Table 1 : Wheat CslA and CslD genes.

In red genes studied here.

CsLD2-RFP RE-GFP

TaCslD2

  sequences were aligned to barley (Hordeum vulgare) AK359952.1, AK376872.1 and BAJ93918.1, respectively. An open reading frame was identified for each sequence and was amplified for subsequent cloning. We analyzed the sequences of the TaCslD2, TaCslA7 and TaCslA12 cDNA we originally obtained and we compared them to the coding sequences of the wheat gene models that were recently released (IWGSC 2018, release 47, April 2020). The protein sequences derived from TaCslA12 and TaCslA7 cDNAs in our study and those predicted for the Chinese Spring wheat cultivar TraesCS2B02G560100.1 and TraesCS2D02G163000.2, respectively, are almost identical. The cloned cDNAs are encoded by the B genome for TaCslA12 and the D genome for TaCslA7. TaCslA12_B and TaCslA7_D

  ). According to the gene model in Ensembl wheat (release 47 April 2020), there are 2 splicing variants for TaCslA7_D. TraesCS2D02G163000.2 encodes a protein of 581 amino acids and TraesCS2D02G163000.1 encodes a protein of 353 amino acids, lacks an exon in N-terminus and therefore contains only the four C-terminal TMDs. Homoeologous sequences were identified in the Chinese Spring genome for TaCslA12 (TaCslA12_A: TraesCS2A02G530900 and D: TraesCS2D02G532800) and TaCslA7 (TaCslA7_A: TraesCS2A02G157100 and B: TraesCS2B02G182700).

FiguresFig. 1 .

 1 Figures

Fig. 2 .

 2 Fig. 2. Subcellular localization of TaCslA7, TaCslA12 and TaCslD2. Agroinfiltration of Nicotiana tabacum leaves with TaCslA7 or TaCslA12 or TaCslD2 fused with a fluorescent protein and co-transformation with the Golgi marker (sialyltransferase (ST)) or ER marker dihydrosphingosine phosphate lyase 1 (DPL1) fused with a fluorescent protein. Left panels: images showing fluorescence associated to the GFP, middle panel; fluorescence associated to the RFP and right panels: merge images with both signals showing co-localization. Bottom : coexpression TaCslA12-GFP/TaCslD2-RFP (two independent assays). The arrows point to colocalization. Scale bar: 10 µm.

Fig. 3 .

 3 Fig. 3. Analysis of Pichia pastoris lines expressing plant CslA genes. A Enzymatic quantification of mannan in Pichia wall. Pichia cell wall Alkaline-Insoluble (AKI) polysaccharides were treated with β-1,4-mannanase. Percentage of hexose sugars solubilized were quantified using the anthrone assay. Data show the mean + SD of three biological replicates. Different letters denote significant changes based on one-way ANOVA with post-hoc Tukey HSD Test (P < 0.01). B Glycosidic linkage analysis of Pichia wall AKI. Values represent the percentage of total carbohydrate area detected. Ak -Amorphophallus konjac. Data show the mean + SD of three biological replicates. The other glycosidic linkages are shown in Supplementary Fig.S5. For linkage, different letters denote significant changes based on one-way ANOVA with post-hoc Tukey HSD Test (P < 0.01).

Fig. 4 .

 4 Fig. 4. Analysis of Arabidopsis thaliana lines expressing wheat CslA genes: HPAEC profiling. Chromatograms showing elution profiles of mannose, mannooligosaccharides (MOS), mannanase treated konjac glucomannan and alcohol insoluble residues obtained from Arabidopsis stems. The resulting mannose and oligosaccharides were separated by HPAEC on a Carbo-Pac PA1 column. The same amount of alcohol insoluble residues was used for WT, the csld2/csld3/csld9 (TM) mutant and TM transformed with proIrx5::TaCslA12 (71.1 and 72.1). M1: mannose; M2: mannobiose; M3: mannotriose; M4: mannotetraose; M5: mannopentaose; M6: mannohexaose; GM: glucomannan.

Fig. 5 .

 5 Fig. 5. Mass spectrometry profiles of alcohol insoluble residues obtained from Arabidopsis stems treated with endo-mannanase. The same amount of alcohol insoluble residues was used for WT and the csld2/csld3/csld9 mutant transformed with proIrx5::TaCslA12 (72.1). Arrows: indicate a mass of 162 corresponding to anhydro-hexose. Red stars: indicate the number of acetyl groups on oligo-hexoses.

Fig. 5 .

 5 Fig.3.

Expression wheat endosperm RNAseq (days post flowering) Pellny 2012 Name prot size AA Ensembl 47 April 2020 best blast hit

				Ortholog Ensembl rice	Nemeth 2010	rice ortholog	LOC_ rice	wheat EST grain	Affymetrix probe set	10	14	17	21	28	sum	rank
	TaCslA12	528	TraesCS2B02G560100.1	Os09g0572500	A12	OsCslA12	Os09g39920	10	Ta.28315.1.S1_at	63	88	31	19	33	811	14
									TaAffx.78802.1.S1_at	9
	TaCslA7	581	TraesCS2D02G163000.2	Os07g0630900	A7	OsCslA7	Os07g43710	6	TaAffx.30043.1.S1_at	19	13	19	11	125	768	15
			(2) TraesCS2B02G182700						TaAffx.95239.1.A1_at	8	5	1	9
	TaCslA1	518	TraesCS6B02G197200.1	Os02g0192500	A1	OsCslA1	Os02g09930	4	TaAffx.42425.1.S1_at	7	1	8	2	14	32	120
									Ta.10065.1.S1_at
	TaCslA3	547	TraesCS7B02G093900	Os06g0230100	A3	OsCslA3	Os06g12460	3	TaAffx.9510.1.S1_at	20	22	27	11	11	91	87
									Ta.15893.1.S1_at
	TaCslA2	528	TraesCS6B02G135100	Os10g0406400	A2	OsCslA2	Os10g26630			21	21	20	19	16	98	83
	TaCslA9	515	TraesCS7B02G295800	Os06g0625700	A9	OsCslA9	Os06g42020	1
	TaCslA11	408	TraesCS3B02G054500	Os08g0434632	A11	OsCslA11	Os08g33740
		571	TraesCS3B02G558400 (2)	Os08g0434632
		524	TraesCS7B02G494000	Os09g0572500
		522	TraesCS7B02G296000	x
		534	TraesCS3B02G540800	Os02g0744650
		519	TraesCS7B02G296100	x
	TaCslD1	993	TraesCS7A02G331500		cslD1	OsCslD1	Os10g42750	8	Ta.9118.1.S1_at	15	6	5	2	4	32	121
	TaCslD2	1188	TraesCS7D02G029700.1		cslD2	OsCslD2	Os06g02180	5	Ta.9118.1.S1	17	78	39	42	52	386	24
			(very close paralog							6
			TraesCS4A02g456300.1)						TaAffx.105670.1.S1
					cslD4	OsCslD4	Os12g36890	5	TaAffx.118567.1.A1

Table 2 .

 2 Individual neutral sugars composition of cell walls for the mature stems of Arabidopsis lines (% individual neutral sugar in Alcohol Insoluble Residue)

	Sugar/sample WT1	WT2	TM1	TM2	71.1	72.1
	Rhamnose	1.25 +/-0.15	1.29 +/-0.08	1.54 +/-0.11	1.30 +/-0.05	1.50 +/-0.05	1.17 +/-0.01
	Fucose	0.32 +/-0.12	0.30 +/-0.09	0.41 +/-0.00	0.35 +/-0.10	0.25 +/-0.05	0.33 +/-0.04
	Arabinose	2.90 +/-0.28	2.89 +/-0.27	3.04 +/-0.03	3.57 +/-0.41	2.72 +/-0.07	3.36 +/-0.13
	Xylose	27.96 +/-0.54 28.36 +/-0.02 29.37 +/-0.27 31.16 +/-0.44 28.30 +/-1.09 28.62 +/-0.01
	Glucose	60.67 +/-0.42 60.46 +/-0.12	61.90 +/-0.12 60.05 +/-0.81 61.31 +/-1.18 59.45 +/-0.33
	Galactose	2.94 +/-0.58	3.18 +/-0.24	3.74 +/-0.52	3.58 +/-0.20	2.49 +/-0.04	3.15 +/-0.09
	Mannose	3.97 +/-0.45	3.52 +/-0.19	0.00 +/-00	0.00 +/-00	3.44 +/-0.07	3.91 +/-0.05

WT: Wild type Arabidopsis (Col0) WT1 and WT2 are two different plants; TM: triple mutant csld2/csld3/csld5 TM1 and TM2 are two different plants; 71 and 72: two independent lines corresponding to the TM transformed with proIrx5::TaCslA12. Average values and standard deviation from technical replicates.

Acknowledgments

The authors acknowledge JC Helleisen and B. Rolland (INRAE Le Rheu) for growing wheat, L.

Helary for technical help, M. Fanuel for training on the rapifleX and M. Francin-Allami for providing the BIFC vectors.

Funding

YV received funding from the European Union through the 7th Framework Program for research and the Horizon 2020 Research and Innovation Programme under the Marie Skłodowska-Curie [grant agreement No FP7-267196-MSCA-COFUND-AgreenSkills] and [H2020708329-MSCA-IF-2015]. The Maldi-Tof MS experiment was conducted using the equipment of the BIBS facility in Nantes (UR1268 BIA, IBiSA, Phenome-Emphasis-FR [grant number ANR-11-INBS-0012]). 22 The mannan research at the Institute for Plant Cell Biology and Biotechnology, Düsseldorf, is funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany's Excellence Strategy -EXC 2048/1 -[Project ID: 390686111] and Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) [grant PA 1366/1-1]. C.V. is supported by the DFG Research Grant [Project ID: 414353267]. Work by the DOE Joint BioEnergy Institute (http://www.jbei.org) was supported by the U. S. Department of Energy, Office of Science, Office of Biological and Environmental Research, through contract [DE-AC02-05CH11231] between Lawrence Berkeley National Laboratory and the U. S. Department of Energy.


Anne-Marie Lagadec
email: anne-marie.lagadec@chu-brest.fr

9 regards croisés La mobilisation des étudiants en santé durant la crise sanitaire

Keywords: adaptation, apprentissage, Covid-19, étudiant en santé, expérience

Les étudiants en santé ont vécu une expérience inédite au cours de la crise sanitaire. Ceux qui connaissaient déjà l'hôpital ont éprouvé leur capacité d'adaptation à une situation exceptionnelle. Ceux qui débutaient dans le métier du soin ont été immergés de façon brutale au coeur d'une activité hospitalière sous tension. Témoignage de quatre d'entre eux.

Je prends contact avec les infirmiers et aides-soignants qui vont constituer cette équipe et travailler en garde de douze heures, jour et nuit. Je dois admettre que je suis un peu anxieuse. La situation est inédite et quand j'accueille ces professionnels, je perçois bien leur inquiétude. Il faut faire preuve de sérénité mais aussi d'écoute et d'attention. Le fait de dépendre d'un établissement important a certainement facilité la logistique. Néanmoins, cela a demandé une certaine agilité dans les organisations.

Pour les professionnels de santé, prendre soin des résidents devient une priorité absolue, malgré l'incertitude qu'engendre cette La gestion du matériel dans un contexte de pénurie nationale m'apparaît comme la problématique majeure. La temporalité pour la construction des modes opératoires et leur mise en application est très rapide et change constamment. Dans ces conditions, il est difficile, pour le cadre, de rester crédible auprès des équipes de soins, de mettre en oeuvre tous les changements et de les argumenter. Encadré flottant « Une mobilisation sans faille » « Comme pour le reste de la population, tout est allé très vite en mars. Le vendredi, nous étions en stage dans nos services et trois jours plus tard, nous nous retrouvions tous confinés chez nous. Le plus dur à ce moment-là, c'est que la société se séparait en deux. D'un côté, il y avait ceux qui agissaient et changeaient le cours des choses. De l'autre, il y avait le monde auquel on m'avait demandé d'appartenir, celui qui devait rester chez lui. Après deux semaines, le doyen nous a offert la possibilité de changer cet état de fait. Je me suis donc retrouvé aide-soignant de nuit, de 21 heures à 7 heures, dans un service où tous les patients et une partie des soignants souffraient de la coronavirus disease 2019 . Place à une mobilisation sans faille. Les cadres m'ont prévenu que, dans ce service, les patients décèdent parfois et souffrent toujours. Je m'habille avec toutes les protections nécessaires et je commence mon tour. Un patient sonne au fond du couloir, l'autre aide-soignante me demande de répondre pendant qu'elle prend les constantes. Je rentre dans la chambre, le patient a les yeux fermés. Il soupire longuement, sa poitrine se soulève, puis plus rien. Sa mort était annoncée pour la nuit. Il ne sera pas réanimé. Il est 21 h 30 et je comprends que ce que je vais voir pendant un mois n'est pas du domaine du normal, même pour l'hôpital. Nous avons préparé le défunt, nous l'avons placé dans des draps puis dans un sac. Sa femme n'est pas venue ce soir-là. J'ai scellé le sac. Je suis donc la dernière personne qui a vu son visage. Je me suis arrêté deux minutes et me suis demandé comment on pouvait accepter de vivre cela tous les jours. Dans le même temps, je me rends compte que ce décès n'arrête ni les aides-soignants, ni les infirmiers. Tout de suite après, un autre patient appelle, puis un autre. En reprenant mon rôle et en faisant ce qui est attendu de moi, je comprends que si je m'arrête, personne ne le fera à ma place. Les patients seraient ainsi seuls face à leur souffrance. À la fin de cette expérience, de nombreuses questions se posent vis-à-vis de ce que nous avons vécu pendant l'épidémie. Compte tenu du désarroi et de la solitude de ces patients qui se savaient condamnés, était-il nécessaire d'imposer un isolement si strict ? Peut-être. Les malades ne pouvaient même pas être préparés selon les rituels avant d'être mis dans de simples sacs. Il ne s'agit pas de juger ce qui a été fait mais de s'interroger. Le système social français n'a pas apporté les réponses que l'on pouvait attendre. La France n'est pas un bon élève en Europe en matière de Covid-19. Nous devons changer cela. » Pablo Martin-Moro, étudiant en 3 e année de médecine Encadré flottant « Une proximité indispensable » « Durant ma formation à l'IFCS de Rouen et alors que j'effectuais le stage "fonction d'encadrement", la pandémie de Covid-19 s'est fortement accélérée en France. Après une semaine, nos stages ont été suspendus afin que nous puissions être mis à la disposition de nos établissements employeurs. J'ai donc été rapidement redéployée dans les secteurs en tension au sein du groupement hospitalier de territoire Sud Yvelines. La situation sanitaire dans cette région était devenue très préoccupante. Avec une demande d'hospitalisations de patients croissante, une réorganisation régionale des services de soins a été mise en oeuvre et les effectifs ont été remaniés. J'ai ainsi effectué un retour partiel dans les soins en tant qu'infirmière. Engagée dans une formation en management, avec des objectifs à remplir, il m'est apparu difficile de maintenir un positionnement clair face à des équipes très sollicitées compte tenu des événements. Confrontée aux difficultés pratiques des soignants dans leur travail, j'ai souvent été prise à partie. Le fait de naviguer entre la fonction soignante et celle d'encadrement a constitué un défi : il fallait pouvoir établir un lien entre des visions différentes de cette gestion de crise. Néanmoins, cette position intermédiaire entre équipe et hiérarchie m'a permis d'observer de très près les soignants en action. J'ai pu développer mes capacités d'écoute, me rendre disponible afin de favoriser la mise en place d'un climat propice à la régulation et à la communication pour assurer la prise en charge des patients. J'ai été témoin de nombreux dysfonctionnements. La gestion difficile des émotions et un sentiment d'insécurité chez les soignants résultaient, entre autres, des problèmes d'approvisionnement en matériel. C'est sans doute ce qui m'a le plus marquée. Le caractère inédit de l'épidémie a provoqué inquiétude et perte de repères. Le challenge quotidien a été de rester dans la proximité pour discerner les problématiques, les anticiper quand c'était possible et tenter d'y répondre afin de faciliter le travail des soignants. À la fin de mon stage, il a fallu que je me remobilise. En effet, l'organisation de la formation cadre a été modifiée, le stage de fin d'année a été placé à la suite du stage "fonction d'encadrement". J'ai ainsi été sur le terrain de la crise pendant près de deux mois. Dans la seconde partie de ce stage, j'ai eu du mal à me sentir encore étudiante car j'avais été missionnée en faisant fonction de cadre de santé. Cependant, cela m'a permis d'approfondir certaines compétences managériales. J'ai développé mes capacités relationnelles pour coordonner l'action de tous les collaborateurs, ce qui a mis en exergue le rôle de médiateur et de relais de l'information du cadre de santé. J'ai pu clairement identifier l'importance des temps d'échanges pour revenir sur les phases difficiles et "désamorcer'' le stress ambiant. L'équipe a ainsi exprimé ses inquiétudes et ses interrogations. L'information régulière sur la situation et son traitement au niveau de l'établissement ont fait naître une nouvelle dynamique de travail collectif. Plus que jamais, la proximité du cadre de santé et la gestion de ses propres émotions se révèlent indispensables. Elles sont essentielles pour observer, écouter, analyser et ainsi garantir une continuité des soins de qualité, même dans un contexte de crise sanitaire inédit. » Pauline Prosperi, étudiante cadre de santé à l'IFCS de Rouen (76), promotion 2019-2020 Encadré flottant « Une période historique » « Le 16 mars 2020, je reçois mon affectation pour renforcer le service de réanimation de l'hôpital dont je dépends. J'éprouve à la fois de l'excitation, à l'idée d'aller sur le terrain, et une certaine inquiétude. Durant mon parcours professionnel, j'ai pu travailler en service de chirurgie ou de bloc opératoire mais je n'ai pas encore eu l'occasion d'oeuvrer dans cette spécialité. À mon arrivée, l'équipe est contente d'accueillir des personnes supplémentaires pour alléger un peu la charge de travail. J'ai l'impression de devoir tout réapprendre. Alors qu'habituellement, la période d'intégration d'une infirmière en service de réanimation est d'un mois, je ne suis "doublée" que pour une journée. J'essaie néanmoins d'enregistrer toutes les informations que l'on me transmet, considérant que je dois être autonome rapidement : dès le lendemain, on m'attribuera la responsabilité de deux, voire trois patients.

Même si je me présente à l'ensemble de l'équipe en tant qu'étudiante infirmière anesthésiste diplômée d'État (EIADE), je comprends vite que, pour mes collègues, je suis avant tout une infirmière et non une EIADE. Cela ne me gêne pas. Notre but est le même : prendre soin des patients et permettre la continuité des soins, en toute humilité.

Je suis une soignante avant tout et j'estime qu'il est de mon devoir de servir au plus près des patients. Je veux me sentir utile. La moindre amélioration de l'état de santé d'un malade m'encourage à poursuivre dans cette voie. Les conditions de travail sont certes difficiles mais les

sur le nombre de cas et veulent savoir si elles seront averties au cas où leur proche serait contaminé. Les médecins font preuve d'une grande transparence. Les visites auprès des résidents non contaminés sont extrêmement compliquées, voire impossibles, l'accès à la résidence étant limité. Heureusement, les moyens technologiques permettent les

  Nous avons eu à déplorer le décès d'un résident Covid+. Le protocole à appliquer dans ce contexte est particulier. Ce jour-là, l'aide-soignant de l'équipe dédiée "vit" son premier décès. Je les accompagne et leur offre mon réconfort. En fin de journée, avec le chef de pôle et la cadre supérieure, nous nous entretenons avec eux. Elles ont toujours été en soutien et je les remercie pour leur investissement. La solidarité entre tous les professionnels, quel que soit le niveau hiérarchique,

	est bien réelle.
	Il n'existe pas d'autres cas de résidents contaminés. Nous arrivons à contenir ce virus avec
	énormément d'attention et de multiples réorganisations. Le confinement en chambre est compliqué
	pour les résidents. Les familles sont inquiètes. Elles font entièrement confiance aux équipes mais
	s'interrogent conversations vidéo avec les familles, ce qui permet d'entretenir un lien précieux. Un peu plus d'un
	mois après, la situation est toujours stable.
	Cette expérience face à une situation inédite m'aura énormément appris. La solidarité et le soutien
	ont été exceptionnels et nous mesurons la chance de vivre dans une région qui a été épargnée. »
	Estelle Corolleur,
	étudiante cadre de santé, IFCS, centre hospitalier régional universitaire de Brest (29)

Dochead dossier Sous-dochead Les soignants face à la Covid-


Pascale Pascale Piednoir

Ulrich Clarac

Amélie Rolle

Sylvaine Bastian

Gaelle Gruel

Frédéric Martino

Hossein Mehdaoui

Marc Valette

Sébastien Breurec

MD Michel Carles
email: carles.michel@gmail.com

Ameĺie Rolle

Fredeŕic Martino

Sebastien Breurec

Spontaneous community-acquired bacterial meningitis in adults admitted to the intensive care units in the Caribbean French West Indies: Unusual prevalence of Klebsiella pneumonia

Keywords:

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

Text -Word count : 825

Community-acquired bacterial meningitis (CABM) are rare in Western countries with an incidence of 0.9 per 100 000 per year, associated with unfavorable outcome in approximatively 40% of cases [START_REF] Bijlsma | Community-acquired bacterial meningitis in adults in the Netherlands, 2006-14: a prospective cohort study[END_REF]. Most common involved pathogens are Streptococcus pneumoniae and Neisseria meningitidis, including CABM requiring an intensive care unit (ICU) admission [START_REF] Bijlsma | Community-acquired bacterial meningitis in adults in the Netherlands, 2006-14: a prospective cohort study[END_REF]. Guadeloupe and Martinique, two French overseas islands, located in the Caribbean (about 800 000 inhabitants, mostly Caribbean and European) were considered as very high-resource countries in 2013 (http://hdr.undp.org/). Very few data are available about CABM epidemiology in this area [START_REF] Peŕez | Community acquired bacterial meningitis in Cuba: a follow up of a decade[END_REF]. We report here the clinical and microbiological characteristics, and outcome of spontaneous CABM in adults admitted in the ICU of two University Hospitals from Caribbean French West Indies.

Over a 6 year-period (January 2012 to December 2017), all patients over 18 years admitted for a diagnosis of CABM with a positive cerebrospinal fluid culture were retrospectively included. No post neurosurgical nor device-associated meningitis were included. API biochemical galleries (API, BioMerieuxTM, Marcy l'Etoile, France) were used for bacterial identification. Antibacterial drug susceptibility was determined using the disc diffusion method, according to the 2018 guidelines of CA-SFM/EUCAST (http://www.sfmmicrobiologie.org). Detection of iuc (coding for aerobactin) and rmpA (upregulator of capsule expression) genes, and capsule serotyping of Klebsiella pneumionae isolates were performed as previously described (3). A standardized specific questionnaire was fulfilled to get demographic, clinical and biological data, and outcome.

Chi-2 or Fisher's exact tests (if required) and Student's t test were used to compare categorical and continuous variables, respectively. A p-value under 0.05 was significant. The ethical committee of the University Hospital in Guadeloupe approved the protocol (registration number: n° A1_18_06-01_MEN).

After exclusion of 7 patients because alternative diagnosis or lack of microbiological confirmation, a total of 25 patients with culture-proven bacterial meningitis were included.

General data including underlying comorbidities for 17 (68%) patients are reported in the Table . Main involved bacteria were S. pneumoniae (n=7, 28%), K. pneumoniae (n=6, 24%), S. aureus (n=5, 20%) and E. coli (n=3, 12%). One patient displayed a mixed infection with E. coli and Streptococcus spp. No difference has been shown regarding the bacterial epidemiology between the two hospitals (p>0.05). Suppurative complications of the CABM-related bacteria occurred in 4 patients: a cerebral abscess in two patients (due to S. aureus and a combination of E. coli and Streptococcus spp., respectively), a K. pneumoniae liver abscess in one patient and a S. aureus pulmonary abscess in another one. Moreover, an endocarditis was diagnosed for 4 out of the 5 S. aureus CABM. The overall mortality was 52% (13 patients).

According to previous data (1,2), S. pneumoniae was the most frequent pathogen (28%) in our study. Unexpectedly for CABM, we described a high prevalence of CABM-associated K. pneumoniae (24%), mostly described as one of the main CABM-related pathogens in Asia [START_REF] Moon | Changing etiology of community-acquired bacterial meningitis in adults: a nationwide multicenter study in Korea[END_REF][START_REF] Lee | Clinical features and in vitro antimicrobial susceptibilities of community-acquired Klebsiella pneumoniae meningitis in Taiwan[END_REF]. In Taiwan, K. pneumoniae was even the first involved pathogen in adult CABM with prevalence rates up to 50% [START_REF] Lien | Epidemiologic trend of adult bacterial meningitis in southern Taiwan (2006-2015)[END_REF]. The identification of Gram negative bacillary and S. aureus among the leading cause of bacterial adult's CABM is unusual and several hypothesis could be considered: specific local epidemiology in the Caribbean with high frequency of K. pneumonia-related infections (reservoir issue), the role of the introduction of conjugated vaccines for S. pneumoniae and Neisseria meningitidis as suggested elsewhere [START_REF] Castelblanco | Epidemiology of bacterial meningitis in the USA from 1997 to 2010: a population-based observational study[END_REF].

The emergence of severe community-acquired hypervirulent K. pneumoniae (CA-HvKp) disease has been reported during the past three decades, usually pyogenic liver abscess, but also meningitis, brain abscess or opthalmitis, initially in Asia and more recently in Western countries [START_REF] Shon | Hypervirulent (hypermucoviscous) Klebsiella pneumoniae: a new and dangerous breed[END_REF]. Out of the 4 isolates investigated, all harbored rmpa and iuc genes. These two genes coding for an increased expression of capsular material and an increased efficiency of iron-acquisition, respectively, are two major hvKp-specific virulence factors [START_REF] Shon | Hypervirulent (hypermucoviscous) Klebsiella pneumoniae: a new and dangerous breed[END_REF]. In addition, all but one (K2) were assigned to the K1 capsular serotype. This serotype is strongly associated with the Sequence Type 23, the most commonly genetic background recovered worldwide associated to CA-HvKp [START_REF] Lam | Population genomics of hypervirulent Klebsiella pneumoniae clonal-group 23 reveals early emergence and rapid global dissemination[END_REF]. We did not found any significant difference of the clinical presentation or outcome between patients with K. pneumoniae CABM versus others (after exclusion of those displaying S. aureus endocarditis), related in part to the small sample size (p>0.05) (Table 1). We noticed that 2 of the 4 K. pneumonia-infected patients have predisposing risk factors of CA-HvKp infection in our population (8), i.e. diabetes mellitus and alcoholism. Taking into account that diabetes and alcoholism are overrepresented public health issues in the Caribbean [START_REF] Carrere | Diabetes mellitus and obesity in the French Caribbean: A special vulnerability for women?[END_REF], a survey of such infections (CA-HvKp) is urgently needed.

The mortality is high (around 50%) with a short delay after admission. In term of antimicrobial resistance, a recent rise of extended spectrum beta-lactamase -and carbapenemaseproducing HvKp has been reported. In our study, all isolates were susceptible to all antimicrobials tested except for ampicillin and ticarcillin. Altogether, our data suggests the need for comprehensive epidemiologic studies focused on CA-HvKp infections in the Caribbean. A better understanding of environmental niches that act as reservoirs of these isolates may enable preventive interventions.

Acknowledgments : none

Conflict of interest : none

Funding Source : Institut Pasteur de Guadeloupe Ethical approval : The ethical committee of the University Hospital in Guadeloupe approved the protocol (registration number: n° A1_18_06-01_MEN).