Bastien G Dauphin

David Ropartz

Philippe Ranocha

Maxime Rouffle

Camille Carton

Aurélie Le Ru

Yves Martinez

Isabelle Fourquaux

Simon Ollivier

Jessica Mac-Bear

Pauline Trezel

Audrey Geairon

Elisabeth Jamet

Christophe Dunand

Jérôme Pelloux

Marie-Christine Ralet

Vincent Burlat
email: vincent.burlat@univ-tlse3.fr

TBL38 is an atypical homogalacturonan acetylesterase with a peculiar cell wall microdomain localization in Arabidopsis seed mucilage secretory cells

Keywords: Arabidopsis thaliana seed development, homogalacturonan acetylesterase, mucilage secretory cell, pectin maturation, plant cell wall microdomain remodeling

Plant cell walls are made of complex polysaccharidic/proteinaceous network whose biosynthesis and dynamics implicate several cell compartments and impact plant development.

The synthesis and remodeling of homogalacturonan pectins is associated with multiple developmental processes ranging from growth to response to biotic/abiotic stress. It encompasses Golgi-localized methylation and acetylation and subsequent demethylation and deacetylation in the cell wall. In the last decade, our comprehension of plant polysaccharides acetylation has increased significantly thanks to the study of the TRICHOME BIREFRINGENCE-LIKE (TBL) protein family. TBLs are mostly described as Golgi-localized

Introduction

Plant cells specifically design, synthesize and constantly remodel their cell wall (CW) to achieve proper cell shape and function along development to face environmental constraints.

Such dynamics lead to specific composition and properties of CW polymers. Accumulating evidence of chemical group modifications on particular polysaccharides involved in key developmental processes can be found in the literature. The importance of pectin methylesterification dynamics has been largely studied, particularly for homogalacturonans (HGs). In the Golgi apparatus, HGs are methylated on the COOH of galacturonic acids (GalA) through methyl-transferase activity [START_REF] Driouich | Golgi-mediated synthesis and secretion of matrix polysaccharides of the primary cell wall of higher plants[END_REF] and later undergo additional remodeling once in the CW. Methylated HG can be demethylesterified by pectin methyl esterases (PMEs) whose activity can be regulated by PME inhibitors (PMEIs). The joint activity of both enzyme categories is the source of many developmental roles [START_REF] Wolf | Homogalacturonan methyl-esterification and plant development[END_REF].

Additionally, O-acetylation of CW polymers such as pectins and hemicelluloses in the Golgi lumen has been described in multiple studies highlighting three groups of proteins involved in this process [START_REF] Gille | O-acetylation of plant cell wall polysaccharides[END_REF]. In Arabidopsis thaliana, (i) ALTERED XYLOGLUCAN 9 (AXY9) is encoded by a unique gene likely indirectly responsible for the non-specific acetylation of xyloglucan and xylan hemicelluloses [START_REF] Schultink | The role of the plant-specific ALTERED XYLOGLUCAN9 Protein in Arabidopsis cell wall polysaccharide O-Acetylation[END_REF] leading to smaller plants when mutated. (ii) The REDUCED WALL O-ACETYLATION (RWA) family has four members in A. thaliana which also participate in the O-acetylation machinery of xylans, mannans, xyloglucans and pectins [START_REF] Manabe | Loss-of-function mutation of REDUCED WALL ACETYLATION2 in Arabidopsis leads to reduced cell wall acetylation and increased resistance to Botrytis cinerea[END_REF][START_REF] Manabe | Reduced wall acetylation proteins play vital and distinct roles in cell wall O-acetylation in Arabidopsis[END_REF]. (iii) The TRICHOME BIREFRINGENCE-LIKE (TBL) family comprises 46 members distributed in four phylogenetic clades in A. thaliana that target specific CW polysaccharides (Figure 1; See Supplemental Table 1). Originally named based on the observed changes in the birefringence properties of a mutant of TRICHOME BIREFRINGENCE (TBR) [START_REF] Potikha | A mutant of Arabidopsis thaliana displaying altered patterns of cellulose deposition[END_REF], 21 TBLs have been characterized for their O-acetyltransferase activity specific for the xyloglucan backbone and side chains, xylans, mannans, rhamnogalacturonan I (RG-I) or HGs (Figure 1; Supplemental Table 1, reviewed in [START_REF] Gille | O-acetylation of plant cell wall polysaccharides[END_REF][START_REF] Pauly | New insights into wall polysaccharide O-acetylation[END_REF]. These activities are highly clade-specific since nine TBLs belonging to a similar clade displayed xylan-acetyltransferase activity (Figure 1, Supplemental Table 1).

Figure 1: The TBL family mostly comprises Golgi-localized acetyltransferases specifically acting on various CW polymers. The 46 members show high sequence homology and two conserved GDSL and DXXH motifs associated with esterase activities. Previous work showed that the esterase activity isnot targeted on CW polymers but rather allows the formation of an acyl-enzyme intermediate, and described multiple TBLs as participating in O-acetylation of a wide range of CW polymers in the Golgi with the notable exception of TBR which was positioned to the CW. Twenty one recombinant enzymatic activities (yellow), 13 fluorescent protein (FP) localization (green) and 6 CW proteome occurrence https://www.polebio.lrsv.ups-tlse.fr/WallProtDB/ (orange) were reported. http://aramemnon.uni-koeln.de/ predicts that the N-terminus hydrophobic domains either acts as a transmembrane (TM) domain (35 non-underlined proteins) or as a cleaved signal peptide (11 underlined proteins).

So far, each TBL or TBL phylogenetic cluster seems to specifically act on a CW polysaccharide which assumes various roles in development. The phylogenetic tree was adapted from [START_REF] Bischoff | TRICHOME BIREFRINGENCE and Its homolog AT5G01360 encode plantspecific DUF231 proteins required for cellulose biosynthesis in Arabidopsis[END_REF]. AE, acetylesterase; AT, acetyltransferase; PAE, pectin acetyl esterase; HG, homogalacturonan; RGI, rhamnogalacturonan I; XG, xyloglucan. See Supplemental Table 1 for detailed information and references. This study reports on TBL38 peculiar localization and activity. Mutations on particular TBLs led to (i) defect in CW structure and overall plant development as observed in tbr, tbl3, tbl29 and tbl37 [START_REF] Bischoff | TRICHOME BIREFRINGENCE and Its homolog AT5G01360 encode plantspecific DUF231 proteins required for cellulose biosynthesis in Arabidopsis[END_REF][START_REF] Lefebvre | ESKIMO1 disruption in Arabidopsis alters vascular tissue and impairs water transport[END_REF][START_REF] Sun | MYC2-activated TRICHOME BIREFRINGENCE-LIKE37 acetylates cell walls and enhances herbivore resistance[END_REF], and (ii) increased resistance to drought stress [START_REF] Stranne | TBL10 is required for O-acetylation of pectic rhamnogalacturonan-I in Arabidopsis thaliana[END_REF] or pathogen infection [START_REF] Chiniquy | PMR5, an acetylation protein at the intersection of pectin biosynthesis and defense against fungal pathogens[END_REF] in tbl10 and tbl44/pmr5, respectively (Supplemental Table 1). Like AXY9, TBLs have the conserved GDSL domain which is typically associated with esterase activity/lipase [START_REF] Akoh | GDSL family of serine esterases/lipases[END_REF][START_REF] Lai | Genome-wide analysis of GDSLtype esterases/lipases in Arabidopsis[END_REF], and a DXXH motif also found in a rhamnogalacturonan acetylesterase of Aspergillus aculeatus [START_REF] Molgaard | Crystal packing in two pH-dependent crystal forms of rhamnogalacturonan acetylesterase[END_REF]. The TBL acetylesterase activity allows the formation of an acyl-enzyme intermediate necessary for

the O-acetyltransferase activity targeting various CW polymers [START_REF] Gille | O-acetylation of plant cell wall polysaccharides[END_REF][START_REF] Lunin | Molecular mechanism of polysaccharide acetylation by the Arabidopsis xylan O-acetyltransferase XOAT1[END_REF] (Figure 1, Supplemental Table 1). Out of the 23 characterized TBLs, 12 have been localized to the Golgi alongside with AXY9 and RWAs, supporting their role as Oacetyltransferases (Figure 1, Supplemental Table 1). Surprisingly, TBR was localized to the CW [START_REF] Sinclair | Etiolated seedling development requires repression of photomorphogenesis by a small cell-wall-derived dark signal[END_REF]). Yet, although its precise activity is still unknown, an overall decrease in pectin acetylation was observed in tbr, suggesting an acetyltransferase activity that is surprising considering that the existence of a CW-localized acetyl donor is unclear [START_REF] Sinclair | Etiolated seedling development requires repression of photomorphogenesis by a small cell-wall-derived dark signal[END_REF]. In silico prediction of the targeting function of the N-terminal hydrophobic sequence sorted the 46 TBLs in two groups: 35 with a transmembrane anchoring domain, and 11 with a cleavable signal peptide. Six TBLs have been identified in CW-enriched fractions without clear relationship with the in silico targeting predictions (Supplemental Table 1). Overall, experimental evidence depicts TBLs as acetylesterases forming an acyl-enzyme intermediate to further perform an O-acetyltransferase activity targeted on various CW polymers during their synthesis in the Golgi lumen.

Initially, we screened genes involved in A. thaliana seed mucilage release with particular interest on candidates that could modify the esterification status of HGs. The mucilage is a polysaccharidic hydrogel synthesized by the seed mucilage secretory cells (MSCs) in the outer most cell layer of the seed coat of the so-called myxospermous species [START_REF] Francoz | Arabidopsis seed mucilage secretory cells: regulation and dynamics[END_REF][START_REF] Viudes | Myxospermy evolution in Brassicaceae: A highly complex and diverse trait with Arabidopsis as an uncommon model[END_REF][START_REF] Western | The sticky tale of seed coat mucilages: production, genetics, and role in seed germination and dispersal[END_REF]. During A. thaliana seed maturation, the HG methylesterification status is modified through the PECTIN METHYLESTERASE INHIBITOR6 6 (PMEI6) activity on an unknown PME in a CW microdomain at the top of the radial primary wall of MSCs, leading to a HG partially methylesterified pattern [START_REF] Francoz | Pectin demethylesterification generates platforms that anchor peroxidases to remodel plant cell wall domains[END_REF][START_REF] Saez-Aguayo | PECTIN METHYLESTERASE INHIBITOR6 promotes Arabidopsis mucilage release by limiting methylesterification of homogalacturonan in seed coat epidermal cells[END_REF]. This pattern acts as an anchoring platform for PEROXIDASE36 (PRX36) which then induces a local weakening and thinning of the CW microdomain [START_REF] Francoz | Pectin demethylesterification generates platforms that anchor peroxidases to remodel plant cell wall domains[END_REF][START_REF] Kunieda | Spatiotemporal secretion of PEROXIDASE36 is required for seed coat mucilage extrusion in Arabidopsis[END_REF]. After seed inhibition, the swelling mucilage breaks the pre-weakened CW microdomain and is extruded around the seed. We identified TBL38 (At1g29050) for its high co-expression level with PRX36 and PMEI6 during A. thaliana seed coat development. Based on the literature cited above and considering its coexpression with PMEI6, we first hypothesized TBL38 to be a Golgi localized protein acting as an acetyltransferase onto CW polymers possibly including HGs. Hereafter, we examined TBL38 localization, activity and putative role in MSCs and uncovered that TBL38 is an atypical CW-localized HG acetylesterase rather than a Golgi-localized acetyltransferase. Although

TBLs are not thought to influence the methylesterification status of CW polymers, we investigated the putative indirect role of TBL38-dependent HG O-deacetylation on the HG methylation status and on PRX36 anchoring, thus adding another layer of complexity on the fine tuning of HG remodeling during plant development.

Results

TBL38 is co-expressed with PRX36 and PMEI6 in mucilage secretory cells at intermediate developmental stages of seed development

Using the tissue-specific seed developmental kinetics GSE12404 dataset [START_REF] Belmonte | Comprehensive developmental profiles of gene activity in regions and subregions of the Arabidopsis seed[END_REF], we previously built a co-expression network centered on PRX36 in which PMEI6 came as the second hit [START_REF] Francoz | Pectin demethylesterification generates platforms that anchor peroxidases to remodel plant cell wall domains[END_REF]) (Supplemental Table 2). We noticed the high coexpression of TBL38 which was ranked 13 in this network with a strong specific seed coat expression at the linear cotyledon stage (Figure 2A-B; Supplemental Table 2). We filtered TBL expression data which was available for 34 out of the 46 TBLs, although only 23 were above the detection limit, and confirmed that TBL38 was likely to be the only TBL highly coexpressed with PRX36 and PMEI6 (Supplemental Table 2). The seed coat transcriptomic data encompasses five cell layers: ii1 (inner integument), ii1', ii2, oi1 (outer integument), and oi2, commonly addressed here as MSCs [START_REF] Debeaujon | Proanthocyanidin-accumulating cells in Arabidopsis testa: Regulation of differentiation and role in seed development[END_REF][START_REF] Francoz | Arabidopsis seed mucilage secretory cells: regulation and dynamics[END_REF]. Anti-sense probes showed highest purple signal in the outermost layer corresponding to MSCs between the early linear and early bending developmental stages. These signals should be subtracted from the overall background obtained on serial sections of the same seeds with the sense probe control and natural pigmentation. Bars: 200 µm.

Therefore, we determined MSC-specific TBL38 expression using in situ hybridization on tissue array paraffin sections encompassing the whole developmental kinetics of wild-type seeds.

TBL38 anti-sense probes displayed a clear purple/violet hybridization signal in MSCs, particularly between the early linear and early bending cotyledon developmental stages (Figure 2C). No such signal could be observed at earlier and later stages with the anti-sense probe and at all developmental stages with the sense probe used as negative control to account for the overall non-specific background and natural pigmentation. We could associate the specific TBL38 anti-sense probe signal to MSCs excluding the chalazal tissues, in agreement with -and refining-the transcriptomic data (Figure 2A-C).

TBL38-TagRFP is transiently localized to the PRX36-PMEI6 outer periclinal/radial cell wall microdomain

We generated a p35S::TBL38-TagRFP construct to transiently transform N. benthamiana leaves. The TBL38-TagRFP fluorescence did not colocalize with a Golgi-YFP marker, but rather displayed a cellular delineation (Figure 3A). Under plasmolysis conditions, we could clearly attribute the TBL38-TagRFP signal to the CW separating from the receding plasma membrane YFP marker signals (Figure 3A). We concluded that TBL38 is a CW-localized protein similarly to TBR and contrarily to the 12 other Golgi-localized TBLs [START_REF] Sinclair | Etiolated seedling development requires repression of photomorphogenesis by a small cell-wall-derived dark signal[END_REF] (Figure 1, Supplemental Table 1). We then moved on to assess protein localization in A. thaliana seed MSCs. As seed development advances, MSCs become mostly made of different CWs, namely the primary CW, the mucilage and the columella [START_REF] Francoz | Arabidopsis seed mucilage secretory cells: regulation and dynamics[END_REF].

Within MSCs, increasing evidence have positioned CW proteins in only one or two types of CW [START_REF] Francoz | Pectin demethylesterification generates platforms that anchor peroxidases to remodel plant cell wall domains[END_REF][START_REF] Mcgee | Assessing the utility of seed coatspecific promoters to engineer cell wall polysaccharide composition of mucilage[END_REF][START_REF] Sola | RUBY, a putative galactose oxidase, influences pectin properties and promotes cell-tocell adhesion in the seed coat epidermis of Arabidopsis[END_REF] or in sub-layer (microdomain) of one CW [START_REF] Dauphin | Cell-wall microdomain remodeling controls crucial developmental processes[END_REF][START_REF] Francoz | Pectin demethylesterification generates platforms that anchor peroxidases to remodel plant cell wall domains[END_REF]. TagRFP [START_REF] Francoz | Pectin demethylesterification generates platforms that anchor peroxidases to remodel plant cell wall domains[END_REF]. However, contrary to the previously observed stable localization of PRX36-TagRFP along seed development, TBL38-TagRFP fluorescence was delocalized to the future mucilage pocket at 8-12 DAP and eventually to the outer and inner margins of the mucilage-filled pocket at > 12 DAP. Sum intensity projections were done using similarly sized projection of 30 Z stacks with no singular edition. rcw, radial CW microdomain; m, mucilage pocket; im and om, inner and outer margins of mucilage pocket, respectively. Bars: 25 µm. (see Supplemental Figure 1 for full kinetics performed on three individual transformed lines). (C) αTag-RFP western blot performed using total protein extracts from developing seeds harvested at stages corresponding to the three sequential patterns of fluorescence shows that the band #2 corresponding to the TBL38-TagRFP fusion protein is only seen at the early stage when TBL38-TagRFP is localized to the CW microdomain. Non-specific bands #1 and #3 are also present in Col-0 samples (see Supplemental Figure 2 for full membrane visualization).

To localize TBL38 in A. thaliana MSCs, we generated a pPRX36:TBL38-TagRFP construct which was stably transformed in tbl38 plants. We localized TBL38-TagRFP to the radial primary CW of MSCs at the onset of pPRX36 activity at early developmental stages (Figure 3B; Supplemental Figure 1). Orthogonal projection allowed to refine TBL38-positonning at the top of the radial primary CW, corresponding to the CW microdomain encompassing PRX36 [START_REF] Francoz | Pectin demethylesterification generates platforms that anchor peroxidases to remodel plant cell wall domains[END_REF]. Intriguingly, while PRX36-TagRFP was stably accumulated at this location using the same promoter [START_REF] Francoz | Pectin demethylesterification generates platforms that anchor peroxidases to remodel plant cell wall domains[END_REF], TBL38-TagRFP appeared to be naturally delocalized to the future mucilage pocket and eventually pushed toward the inner and outer margins of the pocket when filled with mucilage after 12 DAP (Figure 3B; Supplemental Because its localization to the outer radial CW microdomain was strikingly similar to that of PRX36, we investigated TBL38 involvement in the above mentioned PRX36-PMEI6 molecular module and particularly its influence on HG modification.

The mucilage release and the anchoring of PRX36 are not significantly modified in tbl38

Mutation in either PRX36 or PMEI6 results in impaired mucilage release because the primary CW microdomain improperly brakes up upon seed imbibition [START_REF] Francoz | Pectin demethylesterification generates platforms that anchor peroxidases to remodel plant cell wall domains[END_REF][START_REF] Kunieda | Spatiotemporal secretion of PEROXIDASE36 is required for seed coat mucilage extrusion in Arabidopsis[END_REF][START_REF] Saez-Aguayo | PECTIN METHYLESTERASE INHIBITOR6 promotes Arabidopsis mucilage release by limiting methylesterification of homogalacturonan in seed coat epidermal cells[END_REF]. Because TBL38 and PRX36 gene products have similar spatio-temporal expression and positioning, we assessed whether mucilage release was affected in mutated seeds. We isolated a homozygous KO line for tbl38 (Supplemental Figure 3) and used it for comparison with Col-0 wild type seeds using ruthenium red staining of adherent mucilage. Visually, we did not see any differences between WT and tbl38 seeds (Supplemental Figure 4A), contrarily to impaired mucilage previously observed in prx36 or pmei6 [START_REF] Francoz | Pectin demethylesterification generates platforms that anchor peroxidases to remodel plant cell wall domains[END_REF][START_REF] Kunieda | Spatiotemporal secretion of PEROXIDASE36 is required for seed coat mucilage extrusion in Arabidopsis[END_REF][START_REF] Saez-Aguayo | PECTIN METHYLESTERASE INHIBITOR6 promotes Arabidopsis mucilage release by limiting methylesterification of homogalacturonan in seed coat epidermal cells[END_REF]. Quantification of adherent mucilage area and circularity on > 1,000 seeds confirmed the similarity of the patterns of Col-0 and tbl38 (Supplemental Figure 4B). We did not see any obvious change in mucilage staining intensity as sometimes observed in mutated background responsible for mucilage modification [START_REF] Turbant | PME58 plays a role in pectin distribution during seed coat mucilage extrusion through homogalacturonan modification[END_REF].

Since PRX36 anchoring to a specific HG platform and subsequent CW weakening are required for proper mucilage release, we investigated PRX36 positioning in tbl38. For this, we used the αPRX36 antibody on paraffin sections of developing seeds from Col-0 or tbl38. Anti-PRX36

similarly labeled the top of the radial primary CW in Col-0 and tbl38 at all developmental stages (Supplemental Figure 4C). tbl38 did not show the loss of signal previously observed in prx36

or pmei6 [START_REF] Francoz | Pectin demethylesterification generates platforms that anchor peroxidases to remodel plant cell wall domains[END_REF]. To further validate this result, we generated a pPRX36::PRX36-TagRFP construct, which we expressed in prx36 and tbl38 backgrounds.

Observation of the PRX36-TagRFP signal was made on developing seeds aged from 6-to-12 DAP, a period which encompasses the onset of the PRX36 promoter activity up to the last stage of the clearly visible PRX36-TagRFP fluorescence signal [START_REF] Francoz | Pectin demethylesterification generates platforms that anchor peroxidases to remodel plant cell wall domains[END_REF]. PRX36-TagRFP showed very similar signal localization at the top of the radial primary CW in both tbl38 and prx36 (Supplemental Figure 4D). Again, no delocalization of the signal was observed in tbl38 contrary to the PRX36-TagRFP delocalization in the mucilage pocket previously observed in pmei6 [START_REF] Francoz | Pectin demethylesterification generates platforms that anchor peroxidases to remodel plant cell wall domains[END_REF].

So far, the particular PMEI6-specific HG methylation pattern that exists at the top of the radial primary CW linked proper mucilage release with PRX36 anchoring [START_REF] Francoz | Pectin demethylesterification generates platforms that anchor peroxidases to remodel plant cell wall domains[END_REF].

Our results did not show such a relationship between TBL38, mucilage release and PRX36

positioning, suggesting that no changes in the HG methylation pattern occur in tbl38. However, TBL38 could be involved in acetylation of CW polymers similarly to other described TBLs.

Because changes in HG acetylation could lead to different HG methylesterification status, possibly through indirect modulation of PME activity or hindering the dimerization of pectin chains [START_REF] Kohn | Binding of calcium ions to acetyl derivatives of pectin[END_REF][START_REF] Ralet | Polyelectrolyte behaviour and calcium binding properties of sugar beet pectins differing in their degrees of methylation and acetylation[END_REF], we first studied the HG methylation pattern in tbl38.

The partially methylated homogalacturonan JIM7 epitope corresponding to the PRX36-PMEI6 CW microdomain is maintained in tbl38, while the supposed similar LM20 epitope is surprisingly lost

The specific HG platform enabling PRX36 anchoring, is generated through the inhibition of an unknown PME by PMEI6 leading to a partially methylesterified pattern in HGs at the top of the radial primary CW. This molecular pattern could be labeled by both JIM7 and LM20, two monoclonal antibodies routinely used to characterize partially methylesterified HGs, and these labelings were lost in pmei6 [START_REF] Francoz | Pectin demethylesterification generates platforms that anchor peroxidases to remodel plant cell wall domains[END_REF]. Thus, we incubated JIM7 and LM20 on paraffin sections of developing seeds from tbl38 and Col-0.

As a control, JIM7 signals were correctly positioned at the top of the radial primary CW in Col-0 developmental kinetics, particularly as MSC development advanced to later stages (Figure 4A). Very similar JIM7 labeling was observed in tbl38 (Figure 4A) contrary to the loss of signal previously observed in pmei6 [START_REF] Francoz | Pectin demethylesterification generates platforms that anchor peroxidases to remodel plant cell wall domains[END_REF]. A similar pattern was observed with LM20 along the developmental kinetics of the Col-0 control genotype (Figure 4B).

However, the LM20 labeling was intriguingly lost in tbl38 (Figure 4B). This was related to the tbl38 mutation since LM20 labeling was restored along the developmental kinetics of tbl38 plants complemented with a pPRX36::TBL38-TagRFP construct (Figure 4B). While both antibodies are generally used in parallel to similarly label partially methylesterified HG, their partially characterized epitopes may be somehow different [START_REF] Clausen | Synthetic methyl hexagalacturonate hapten inhibitors of antihomogalacturonan monoclonal antibodies LM7, JIM5 and JIM7[END_REF][START_REF] Verhertbruggen | An extended set of monoclonal antibodies to pectic homogalacturonan[END_REF]. Overall, we concluded that tbl38 mutation resulted in lack of the LM20 signal, but not of that of JIM7. Yet, it was still unclear if this could be due to putative changes in the acetylation of HGs which may prevent LM20 binding through steric hindrance, or to changes in HG methylation as indirect effect of acetylation. Regardless, we sought to investigate TBL38 biochemical function in MSCs.

TBL38 displays an atypical acetylesterase activity towards CW polymers

We took advantage of MSC surface accessibility to generate a MSC surface-enriched CW abrasion powder from dry seeds using home-made abrasion columns (see Methods). The resulting homogeneous powder (about 1 mg per 50 mg seeds) was collected through a nylon mesh and subsequently analyzed (Supplemental Figure 5A-F). We confirmed that the abrasion process was restricted to the MSC surface as MSC CW autofluorescence was strongly reduced but could still be barely observed in abraded seeds (Supplemental Figure 5G-H) which released residual amount of adherent mucilage when imbibed (Supplemental Figure 5I-J). Although the overall MSC surface morphology was affected, we could still see the base of broken columella and radial CW (Supplemental Figure 5M-N). We successfully recovered a surface-abraded powder containing the top of the radial primary wall, considering the loss of LM20 signal in Col-0 abraded seeds (Supplemental Figure 5K-L). We validated the method since JIM7 and LM20 dot blot signal intensities in pmei6 were both about 5 % as compared to

Col-0 (Figure 5A,B), in agreement with the previously observed loss of JIM7 and LM20 immunofluorescence signals in development kinetics of pmei6 MSCs [START_REF] Francoz | Pectin demethylesterification generates platforms that anchor peroxidases to remodel plant cell wall domains[END_REF].

Again, in agreement with the present immunofluorescence study on development kinetics (Figure 4), the JIM7 signal intensity from dry seed surface abraded fractions was very similar in all genotypes except pmei6, since tbl38 and three complemented lines all showed 91-95 % of Col-0 intensity (Figure 5A). However, the LM20 signal intensity in tbl38 was only 4 % of that in Col-0 control while the three complemented lines showed a strong -though partial-signal restoration with 66-79 % of Col-0 intensity (Figure 5B).

So far, all the described TBLs were either directly characterized as acetyltransferases or associated with lower acetylation content of CW polymers in mutated backgrounds (Supplemental Table 1). Therefore, we quantified the acetylation of CW polymers using the surface-enriched CW abrasion powders. Surprisingly, the mean amount of released acetic acid in tbl38 was 199 % of that in Col-0 and the level dropped back to 117-153 % of that of Col-0 in the three complemented lines (Figure 5C). This suggested that TBL38 activity was akin to an acetylesterase rather than the previously described acetyltransferase of other characterized TBLs. Because the increased acetylation level in tbl38 could be achieved through acetylation of multiple polymers, we sought to identify TBL38 substrates in these fractions.

TBL38 is an homogalacturonan acetylesterase

Surface-enriched CW abrasion powders were enzymatically hydrolyzed with a pectin lyase (PL) or a polygalacturonase (PG), displaying specificity toward highly and lowly methylesterified HGs, respectively. The resulting oligogalacturonates (OGAs) from Col-0, tbl38 as well as pmei6 and the complemented line PTR3.5 used as additional controls, have then been analyzed by mass spectrometry, which allowed to determine the degree of polymerization (DP), degree of methylation (DM) and degree of acetylation (DA). Technical repeats showed the low standard deviation of the intensities of identified species validating the approach despite the low amount of biological material (Supplemental Table 3A). We first used a PL having a high affinity for HG with a high DM. The intensity of the identified OGA species was summed for the identified OGA species of same DA and DP regardless the DM, or same DM and DP regardless the DA (Supplemental Table 3B) and for each genotype, the results were plotted as DA vs DP (Figure 6A-D), or DM vs DP (Figure 6E-H). We observed a clear qualitative HG acetylation phenotype in tbl38 as compared to Col-0 and pmei6 (Figure 6A-C) and a qualitative complementation of PTR3.5 (Figure 6D). This was in agreement and refined the high acetylation phenotype observed in tbl38 with the enzymatic assay (Figure 5C) since new OGA species of higher DA appeared in tbl38 as compared to the three other genotypes. This strongly suggests that TBL38 could be an HG acetylesterase. Conversely, a quantitative HG methylation phenotype was observed for pmei6 as compared to Col-0, tbl38 and PTR3.5 (Figure 6E-H).

The same analysis and data processing were performed with PG which has an affinity for lowly methylated HG (Figure 6I-N; Supplemental Table 4A- Supplemental Table 4A) vs those of high DM species identified with PL (Figure 6A-H; Supplemental Table 3B). This could reflect the nature of the major HGs in MSCs (low DM and low DA) and/or be related to a possible lower activity of PL towards HGs with relatively high DA [START_REF] Zeuner | Comparative characterization of Aspergillus pectin lyases by discriminative substrate degradation profiling[END_REF]. To strengthen the HG high acetylation phenotype of tbl38, we additionally compared Col-0 and tbl38 rhamnogalacturonate acetylation profiles to see whether TBL38 could also target the abundant RGI present in seed mucilage (Figure 6O-P; Supplemental Table 5A). No increased acetylation phenotype occurred in tbl38 for RGI since the very strong intensity of the identified RGI species that were non-acetylated had similar profiles in both Col-0 and tbl38. This result demonstrates the specificity of the acetylation phenotype of tbl38 towards HGs rather than RGI.

Finally, we produced recombinant TBL38 in Pichia pastoris (Figure 7A) and tested its acetyl esterase activity in vitro towards three acetylated substrates. Albeit low, we detected acetyl esterase activity using acetylated pectins from sugar beet and to a lesser extent with the generic acetylated substrate triacetin, while no significant activity could be detected using acetylated xylans (Figure 7B). highlighted in red and blue, respectively. The amino acids participating to the putative catalytic triad S136-H360-D357 appear in bold underlined, in red, green and blue, respectively. L139 in TBL38 underlined in yellow replaces R219 in TBL29/ESK1/XOAT1 that was demonstrated to be necessary for acetyltransferase activity [START_REF] Lunin | Molecular mechanism of polysaccharide acetylation by the Arabidopsis xylan O-acetyltransferase XOAT1[END_REF]. (D) The TBL38 structural model proposed in this study is a Phyre2 prediction [START_REF] Kelley | The Phyre2 web portal for protein modeling, prediction and analysis[END_REF] with the highest ranking score (Confidence: 100.0%; Coverage: 85%). The template was the crystallographic structure (X-Ray diffraction, 1.85 Å) of A. thaliana TBL29 (At3g55990; PDB 6CCI; Phyred2 c6cciA) [START_REF] Lunin | Molecular mechanism of polysaccharide acetylation by the Arabidopsis xylan O-acetyltransferase XOAT1[END_REF]. (E)

Zoom on the putative catalytic triad S136-H360-D357 originally identified in Ser proteases [START_REF] Dodson | Catalytic triads and their relatives[END_REF], and necessary for the acyl-enzyme formation and acetyltransferase activity of TBL29 [START_REF] Lunin | Molecular mechanism of polysaccharide acetylation by the Arabidopsis xylan O-acetyltransferase XOAT1[END_REF]. (F and G) structure and zoom on the catalytic triad of TBL29 for comparison [START_REF] Lunin | Molecular mechanism of polysaccharide acetylation by the Arabidopsis xylan O-acetyltransferase XOAT1[END_REF]. Note that R219 necessary for acetyltransferase activity in TBL29 [START_REF] Lunin | Molecular mechanism of polysaccharide acetylation by the Arabidopsis xylan O-acetyltransferase XOAT1[END_REF] is replaced by L139 in TBL38.

Discussion

TBL38 is atypically positioned in the PRX36/PMEI6 cell wall microdomain of MSCs with a transient pattern Originally, we searched for genes involved in the PMEI6-JIM7/LM20 HG epitopes-PRX36 molecular module [START_REF] Francoz | Pectin demethylesterification generates platforms that anchor peroxidases to remodel plant cell wall domains[END_REF] with a particular interest in HG remodeling enzymes.

Using spatio-temporal co-expression with PRX36, we selected TBL38 for its putative role in HG acetylation. Previously described TBLs are mostly anchored to the Golgi membrane (Supplemental Table 1) with the exception of TBR [START_REF] Sinclair | Etiolated seedling development requires repression of photomorphogenesis by a small cell-wall-derived dark signal[END_REF]. Although the authors did not refute the possibility of some TBR-GFP signal in the Golgi, they concluded that TBR was a CW protein. The Golgi experimental localization matches with the N-terminal transmembrane domain prediction for 10 TBLs (TBL03,27,28,29,30,31,32,33,34,35) while the Golgi-localized TBL37 has a predicted cleavable signal peptide (akin to a secreted CW protein), and conversely, TBR that has been localized to the CW has a predicted N-terminal transmembrane domain (Supplemental Table 1). Six TBLs including TBL38 have been identified in CW proteomes but these proteins did not match perfectly to the targeting prediction: there is a good match with cleavable signal peptide prediction for TBL9, 38, 39 and 45, but a bad match with transmembrane domain prediction for TBL15 and TBL40

(Supplemental Table 1). Here, we clearly localized TBL38-TagRFP in the CW of tobacco leaves or A. thaliana MSCs (Figure 3) in agreement with TBL38 presence in a CW proteome and its predicted cleavable signal peptide (Supplemental Table 1). In MSCs, we showed that this targeting is restricted to the CW microdomain harboring the PMEI6-JIM7/LM20 HG epitopes-PRX36 molecular module with an intriguing transient localization pattern (6)(7)(8).

The localization to the CW microdomain occurs as soon as the fusion protein is produced just after the onset of PRX36 promoter activity. We chose this promoter considering the strong coexpression of PRX36 and TBL38 and because it worked well with stable localization in various CW microdomains in previous studies [START_REF] Francoz | Pectin demethylesterification generates platforms that anchor peroxidases to remodel plant cell wall domains[END_REF][START_REF] Kunieda | Spatiotemporal secretion of PEROXIDASE36 is required for seed coat mucilage extrusion in Arabidopsis[END_REF][START_REF] Mcgee | Assessing the utility of seed coatspecific promoters to engineer cell wall polysaccharide composition of mucilage[END_REF][START_REF] Sola | RUBY, a putative galactose oxidase, influences pectin properties and promotes cell-tocell adhesion in the seed coat epidermis of Arabidopsis[END_REF]. The physiological relevance of the transient localization pattern of TBL38-TagRFP is attested by the fact that the PTR lines displayed functional complementation in our different assays discussed hereafter. Then, the early delocalization of the fusion protein could be related to the loss of the putative anchoring motif (acetylated HG) as soon as TBL38 performed its acetylesterase activity, leading to degradation of the protein.

TBL38 is a HG acetylesterase

So far, all described TBLs have been associated with a Golgi-localized acetyltransferase activity, either on hemicelluloses or pectins (Figure 1; Supplemental Table 1). Therefore, the question arose on whether the CW-localization of TBL38 could lead to similar activity. Yet, both acetic acid quantification and mass spectrometry analysis, as well as in vitro enzymatic assays on rTBL38 indicate that TBL38 is an acetylesterase targeting acetylated HGs as opposed to other TBLs (Figures 567). Sequence analysis of the TBL family highlighted the conservation of two characteristic domains: the GDS(L) and DXXH motifs commonly found in esterases [START_REF] Akoh | GDSL family of serine esterases/lipases[END_REF][START_REF] Molgaard | Crystal packing in two pH-dependent crystal forms of rhamnogalacturonan acetylesterase[END_REF]. Crystallography analysis of TBL29/ESK1/XOAT1 pointed to the spatial reunion of these two domains forming the conserved Ser-His-Asp catalytic site, Ser coming from GDS(L) and Asp and His coming from DXXH [START_REF] Lunin | Molecular mechanism of polysaccharide acetylation by the Arabidopsis xylan O-acetyltransferase XOAT1[END_REF]. TBL29 displayed an xylan acetyl transferase activity involving the formation of an acyl-enzyme intermediate on Ser129 [START_REF] Lunin | Molecular mechanism of polysaccharide acetylation by the Arabidopsis xylan O-acetyltransferase XOAT1[END_REF]. Therefore, the acetyltransferase activity of TBL29 necessitated a generic acetylesterase activity on both the acetyl donor and the acyl-enzyme intermediate. Based on the crystallography structure of TBL29, we built an in silico model of TBL38 to determine the position of the above mentioned amino acids. The predicted catalytic site was correctly positioned in a putative polysaccharide binding groove (Figure 7D,E). Interestingly, in TBL29, the nearby R219 (Figure 7F-G) was

shown to stabilize the catalysis [START_REF] Lunin | Molecular mechanism of polysaccharide acetylation by the Arabidopsis xylan O-acetyltransferase XOAT1[END_REF] as R219A site mutation led to loss of transfer of acetyl groups for the acetyl transferase activity without loss of the acetylesterase activity necessary for the acyl-enzyme formation [START_REF] Lunin | Molecular mechanism of polysaccharide acetylation by the Arabidopsis xylan O-acetyltransferase XOAT1[END_REF]. TBL multiple alignment [START_REF] Lunin | Molecular mechanism of polysaccharide acetylation by the Arabidopsis xylan O-acetyltransferase XOAT1[END_REF] shows that 9 TBLs, including TBL38, do not have this particular Arg residue: TBL12 (E135), TBL30 (Y156), TBL37 (L143), TBL38 (L139) (Figure 7C-E), TBL39 (T125), TBL40 (L123), TBL41 (L112), TBL42 (N115), TBL43 (L122). All the other TBLs have an Arg except TBL44/PMR5 which possess another polar positive residue at this position (K145)

and an acetyltransferase activity [START_REF] Chiniquy | PMR5, an acetylation protein at the intersection of pectin biosynthesis and defense against fungal pathogens[END_REF]. Among these 9 TBLs with no polar positive residue at this position, 4 correspond to the 6 TBLs identified in CW proteomes (Supplemental Table 1). It is therefore tempting to postulate that the absence of this residue could fit with the absence of a (Golgi-localized) acetyltransferase activity and the presence of a (CW-localized) acetylesterase activity as for TBL38. However, this cannot be the sole determinant since TBL37 and TBL30/XOAT3 both display a Golgi-localized acetyltransferase activity [START_REF] Sun | MYC2-activated TRICHOME BIREFRINGENCE-LIKE37 acetylates cell walls and enhances herbivore resistance[END_REF][START_REF] Zhong | Regiospecific acetylation of xylan is mediated by a group of DUF231-containing O-acetyltransferases[END_REF]. The characterization of TBL12, TBL39-43 could help in deciphering the importance of this residue at this specific position. Since we could not identify in TBL38 a particular motif or conformation which could explain such opposite esterase vs transferase activity, we propose that the opposite activity of TBL38 is primarily related to its CW localization. Indeed, TBL-dependent polysaccharide acetylation is done in two consecutive steps: hydrolysis of the acetyl group from the donor as evidence by the formation of the Acyl-enzyme intermediate, followed by transfer of the O-acetyl moiety to its polysaccharide target. To our knowledge, neither acetyl-CoA nor another putative acetyl donor is present in the CW, which could prevent the CW-localized TBL38 to complete the successive steps of the transferase reaction, the enzyme could then rather function as a direct acetylesterase on acetylated HGs. It should be noted that an esterase activity targeted on CW polymers was not observed for TBR despite its CW localization. tbr was associated with lower acetylation level of HGs (the opposite phenotype of tbl38), possibly through TBR-mediated protection of HG from pectin acetylesterases [START_REF] Sinclair | Etiolated seedling development requires repression of photomorphogenesis by a small cell-wall-derived dark signal[END_REF]. Although is it unclear how such a mechanism could play out, it is difficult to explain the differences between the measured acetylation levels. Characterization of additional TBLs with regards to subcellular localization and acetylesterase activity is required for a better understanding of TBL roles.

HG fine tuning of esterification is affected in tbl38

Although the control of HG acetylation and methylation is achieved by different proteins, proof of the direct and indirect interplay between both chemical groups was given. Indeed, HG acetylation could inhibit PME activity by steric hindrance [START_REF] Celus | Influence of pectin structural properties on interactions with divalent cations and its associated functionalities[END_REF][START_REF] Kohn | Binding of calcium ions to acetyl derivatives of pectin[END_REF]. Additionally, methyl ester groups are preferentially added on non-acetylated galacturonic acid moieties, which may explain the overall low level of HG acetylation observed in our results with Col-0 and in most plants [START_REF] Pérez | The three-dimensional structures of the pectic polysaccharides[END_REF][START_REF] Ralet | Mapping sugar beet pectin acetylation pattern[END_REF], (Figures 6 and7). Although we did not quantify HG methylesterification, the total number of identified species was particularly low in tbl38, when using a PL that has high affinity for highly methylesterified HGs (Figure 6). Even if we cannot exclude the possibility that the acetyl groups in tbl38 negatively impact the PL activity, this could be associated with reduced HG methylesterification in tbl38 considering the lack of LM20 labeling in tbl38. It should be noted, however, that the combined use of PG/PL might not give the full representation of HG species, since randomly methylated HG of DM around 50% are mediocre substrates for both PL and PG [START_REF] Ralet | Innovative enzymatic approach to resolve homogalacturonans based on their methylesterification pattern[END_REF]. Additionally, JIM7 antibody still labels tbl38 seeds suggesting that the lack of LM20 binding might rather be due to steric hindrance caused by HG acetylation, rather than changes in the pattern of methylesterification. Therefore, LM20 could display specificity for partially methylesterified and non-acetylated HGs, while JIM7 would be specific for partially methylesterified HGs regardless of the acetylation status. Interestingly, nanofibrils of HGs could be observed with LM20 but not with JIM7 in epidermis anticlinal CW from pavement cells [START_REF] Haas | Pectin homogalacturonan nanofilament expansion drives morphogenesis in plant epidermal cells[END_REF][START_REF] Haas | The role of pectin phase separation in plant cell wall assembly and growth[END_REF]. Considering the proposed different specificities of the two antibodies, one interpretation would be that the reduced steric hindrance and/or altered HG gelling properties [START_REF] Ralet | Polyelectrolyte behaviour and calcium binding properties of sugar beet pectins differing in their degrees of methylation and acetylation[END_REF] related to the absence of acetyl groups would be necessary for proper assembly of HGs in nanofibrils. Evaluation of PME activity in tbl38

developing MSCs could give clues as to TBL38 indirect influence on HG methylesterification.

Regardless of the DM, our results confirmed increased amounts of HG acetylation in tbl38 seed surface extracts. Contrary to methylesterification, acetylation does not influence the charge density and therefore the cation binding capacity of HGs [START_REF] Dronnet | Characterisation and selectivity of divalent metal ions binding by citrus and sugar beet pectins[END_REF][START_REF] Ralet | Polyelectrolyte behaviour and calcium binding properties of sugar beet pectins differing in their degrees of methylation and acetylation[END_REF]. However, experimental evidences have consistently associated highly acetylated HGs with reduced affinity to cations binding such as Ca 2+ [START_REF] Celus | Influence of pectin structural properties on interactions with divalent cations and its associated functionalities[END_REF][START_REF] Ralet | Polyelectrolyte behaviour and calcium binding properties of sugar beet pectins differing in their degrees of methylation and acetylation[END_REF].

Conceptually, this could be due to direct influence of acetylation on conformation, access or cations binding properties, but also indirect effect on the PME activity as stated above [START_REF] Oosterveld | Effect of enzymatic deacetylation on gelation of sugar beet pectin in the presence of calcium[END_REF][START_REF] Renard | Acetylation and methylation of homogalacturonans -2: Effect on ion-binding properties and conformations[END_REF].

Increased HG acetylation does not influence PRX36 anchoring

Our results have identified TBL38 as a CW localized HG acetylesterase with increased HG acetylation level in tbl38 and no obvious effect on PRX36 positioning, despite the colocalization of TBL38 and PRX36 in the same CW microdomain. Previous work has demonstrated that PRX36 binding to HG was dependent on both methyl and Ca 2+ as the PRX36-TagRFP localization was severely impacted in pmei6 or using EDTA, respectively [START_REF] Francoz | Pectin demethylesterification generates platforms that anchor peroxidases to remodel plant cell wall domains[END_REF]. To better understand the absence of PRX36 delocalization in tbl38, we investigated the implication of acetylated HGs on PRX36 docking using an in silico model based on PRX53 crystallographic data [START_REF] Oosterveld | Effect of enzymatic deacetylation on gelation of sugar beet pectin in the presence of calcium[END_REF]. The previous in silico docking study tested the five OGAs of DP6 used to define JIM7 specificity [START_REF] Clausen | Synthetic methyl hexagalacturonate hapten inhibitors of antihomogalacturonan monoclonal antibodies LM7, JIM5 and JIM7[END_REF][START_REF] Francoz | Pectin demethylesterification generates platforms that anchor peroxidases to remodel plant cell wall domains[END_REF].

It turned out that the three OGAs (so-called Clausen 3, 4 and to a lesser extend 5) that were the best recognized by JIM7 could be used for in silico docking on a PRX36 valley that was validated by site-directed mutagenesis [START_REF] Francoz | Pectin demethylesterification generates platforms that anchor peroxidases to remodel plant cell wall domains[END_REF]. Here, we first randomly screened in silico PRX36 anchoring on 124 OGAs representing all the combinations of methylesterification from DP2 to DP6 (Supplemental Table 6). The in silico models consisted in nine poses of decreasing affinity (increasing energy level). To rationalize the distribution of these poses on the protein surface, we systematically measured the root mean square deviation (RMSD) showing the distance between the best pose and the eight other poses. We integrated the sum of energy levels and sum of RMSD for each OGA as a proxy of affinity and distances (Supplemental Table 6; see Methods). The 124 OGAs could then be sorted in a rationalethough of course imperfect manner-for their ability to bind to the PRX36 valley. This strongly reinforced the previous docking experiments made with a priori with Clausen1-5 OGAs [START_REF] Francoz | Pectin demethylesterification generates platforms that anchor peroxidases to remodel plant cell wall domains[END_REF] since Clausen 4 and Clausen 3 ranked 1 and 2 (Supplemental Figure 6; Supplemental Table 6). Finally, we tested the impact on in silico docking of the addition of 2-O or 3-O acetyl on the non-methylated galacturonic acids of Clausen 3 and 4 (Supplemental Figure 6). The addition of acetyl on hit number 2 (Clausen 3) strongly impaired PRX36 docking capacity, which does not fit with the observed results (no obvious modification of PRX36

anchoring and no mucilage release phenotype in tbl38). However, the addition of acetyl on hit number 1 (Clausen 4) did not have a strong impact on the docking capacity (Supplemental In light of our results, we conclude that TBL38 acts as an HG acetylesterase in the PMEI6-JIM7/LM20 HG epitopes-PRX36 CW microdomain with no effect on PRX36 anchoring observed in tbl38 contrarily to pmei6. The uncommon acetylesterase activity could be related to the localization to the CW were acetyl-CoA is absent. We could hypothesize that TBL38

could have an acetyltransferase activity if artificially targeted in a different environment such as the Golgi considering the presence of the canonical amino acids required for acetyltransferase activity. It remains to be understood why evolution has selected acetyltransferase-capable protein to deacetylate HGs instead of traditionally described PAEs, though in A. thaliana, no PAE have yet been linked to HG deacetylation. We were not able to associate the increased HG acetylation observed in tbl38 to any clear developmental phenotype.

However, considering the localization of this process to a remote CW microdomain, additional tools will be required to search for subtle developmental phenotypes, similarly to the development of the new original abrasive method allowing the characterization of the biochemical phenotype. Beyond this case study, this work paves the way for further understanding of the role of fine tuning of complex CW polysaccharides.

Methods

Plant material and growth conditions

A. thaliana mutants were ordered from the NASC for tbl38: SAIL_34_C03 (https://arabidopsis.info/; Supplemental Figure 3), or were previously available for prx36

(SAIL_194_G03 [START_REF] Kunieda | Spatiotemporal secretion of PEROXIDASE36 is required for seed coat mucilage extrusion in Arabidopsis[END_REF]) and pmei6 (SM_3.19557 (Saez-Aguayo et al., 2013)).

Homozygous lines for tbl38 were identified by PCR (Supplemental Figure 3; Supplemental Table 7). The knock out status of tbl38 was determined by RT-PCR (Supplemental Figure 3; Supplemental Table 7). A. thaliana culture was performed as previously described [START_REF] Francoz | Pectin demethylesterification generates platforms that anchor peroxidases to remodel plant cell wall domains[END_REF].

Nicotiana benthamiana were cultivated in a growth chamber under a 16 h day / 8 h night cycle at 23°C (Neon (86.90 µmol/m/s-1) / 22°C upon 70 % humidity. They were transplanted after 14 days and fertilized each week.

Transcriptomic data mining

PRX36, PMEI6 and TBL38 seed-specific expression profiles were obtained using the seed data source of eFP browser (http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi?dataSource=Seed).

Tissue-specific seed development transcriptomic data [START_REF] Belmonte | Comprehensive developmental profiles of gene activity in regions and subregions of the Arabidopsis seed[END_REF] was used to build the PRX36 co-expression network [START_REF] Francoz | Pectin demethylesterification generates platforms that anchor peroxidases to remodel plant cell wall domains[END_REF]. The TBL family [START_REF] Bischoff | TRICHOME BIREFRINGENCE and Its homolog AT5G01360 encode plantspecific DUF231 proteins required for cellulose biosynthesis in Arabidopsis[END_REF] was filtered out.

Bioinformatic analysis of the TBL family

Previous TBL38 phylogeny [START_REF] Bischoff | TRICHOME BIREFRINGENCE and Its homolog AT5G01360 encode plantspecific DUF231 proteins required for cellulose biosynthesis in Arabidopsis[END_REF] was used. The occurrence of TBLs in cell wall proteomes was assessed using http://www.polebio.lrsv.ups-tlse.fr/WallProtDB/. The topological prediction of the N-terminal hydrophobic sequence as being a transmembrane domain or a signal peptide was determined using http://aramemnon.uni-koeln.de/.

Ruthenium Red Mucilage Release Test, Image Analysis and Statistical Analysis

High-throughput adherent mucilage release semi-quantitative phenotyping used the ruthenium red (Supplemental Table 8) staining method previously described allowing to calculate adherent mucilage area and circularity [START_REF] Francoz | Pectin demethylesterification generates platforms that anchor peroxidases to remodel plant cell wall domains[END_REF]. The images were analyzed using ImageJ 1.8 (https://imagej.nih.gov/ij/) without edition of native images using an updated ImageJ script that facilitates cleaning of the data and that allows their automatic storage (see Supplemental Methods for more details).

TagRFP reporting constructs, plant transformation and selection

The primers used for vector construction are listed in Supplemental GoldenGate generated constructs are detailed in Supplemental Table 11. Level 1 plasmid was pICH47811 "pL1V-R2"pL1V-R2 vector [START_REF] Weber | Assembly of designer TAL effectors by Golden Gate cloning[END_REF]. Level 2 plasmid was EC15027 "pL2V-HYG" with pICH471744 "pL1M-ELE-2" as a linker-containing plasmid (gift from Dr Pierre-Marc Delaux, LRSV, Auzeville-Tolosane, France). Every construct was checked by restriction analysis and sequencing prior to its transfer into Agrobacterium tumefaciens GV3101::pMP90 strain [START_REF] Koncz | The promoter of TL-DNA GENE 5 controls the tissue-specific expression of chimeric genes carried by a novel type of Agrobacterium binary vector[END_REF]. The transformed bacteria were grown in the presence of 15 µg.mL -1 gentamycin, 50 µg.mL -1 rifampicin and 25 µg.mL -1 hygromycin. A. Three independent homozygous transformed plant lines were studied for each construct.

The pro35S::TBL38-TagRFP construct was transiently co-transformed with either G-yb CD3-966 sialyltransferase-YFP Golgi marker or pm-yb-CD3-1006 aquaporin-YFP plasma membrane marker [START_REF] Nelson | A multicolored set of in vivo organelle markers for colocalization studies in Arabidopsis and other plants[END_REF] in 30-day old N. benthamiana leaves. The final inoculum consisted in a mix of various A. tumefaciens lines: 10 % (v/v) subcellular YFP-marker line, 80% (v/v) fusion protein-Tag-RFP line of interest, and 10 % (v/v) P19 silencing inhibitorcontaining vector line. Leaf infiltration was done on the abaxial face through the stomata with a 1 mL syringe and pieces of leaves were mounted and analyzed under confocal microscopy 48 h-post agro-infiltration.

Arabidopsis silique fixation, paraffin tissue array embedding and microtomy

The protocol was as previously described [START_REF] Francoz | Pectin demethylesterification generates platforms that anchor peroxidases to remodel plant cell wall domains[END_REF], with minor improvement using biopsy foam enabling handling more samples (see Supplemental Methods for more details).

In situ RNA hybridization

In situ RNA hybridization experiments were performed as described (Francoz et al., 2019a;[START_REF] Francoz | Complementarity of medium-throughput in situ RNA hybridization and tissue-specific transcriptomics: case study of Arabidopsis seed development kinetics[END_REF]. More details are provided in Supplemental Methods.

Immunofluorescence on tissue-array sections

We used our previously described protocol [START_REF] Francoz | Pectin demethylesterification generates platforms that anchor peroxidases to remodel plant cell wall domains[END_REF] with anti-PRX36 primary antibodies and goat-anti rabbit-A488 secondary antibodies (Supplemental Table 8) hybridized on serial sections of Col-0 and tbl38 paraffin-embedded seed developmental kinetics. Serial sections of Col-0, tbl38, PTR2.4 and PTR3.5 developmental kinetics were similarly labeled with JIM7 or LM20 primary antibodies followed by goat anti-rat A488 secondary antibodies (Supplemental Table 8). More details are provided in Supplemental Methods.

Confocal spinning disk microscopy of seed development kinetics of A. thaliana stable lines

Developing siliques taken from plants stably expressing the different TagRFP constructs in various genetic backgrounds were dissected and the replums containing the seeds were mounted under a coverslip in distilled water. Images were taken with a PLAN APO 20x/0.75 dry objective using the confocal spinning disk microscope from Perkin Elmer driven by the Volocity 6.3.0 software and equipped with a Yokogama CSU-X1 scan head, two EmCCD Hamamatsu C9100-13 cameras (Hamamatsu, Hamamatsu City, Japan) and a 580 nm beam splitter to separate dual staining on the two cameras as previously shown [START_REF] Francoz | Pectin demethylesterification generates platforms that anchor peroxidases to remodel plant cell wall domains[END_REF].

Details of image processing are provided in the Supplemental Methods.

protocol of incubation with JIM7 and LM20 primary antibodies followed by goat anti-rabbit IgG-AP secondary antibodies as well as the semi-quantitative analysis of the results are provided in Supplemental methods.

Acetyl group deesterification and acetic acid semi-quantitative analysis from dry seed

MSC abrasive fractions

The acetylation of cell wall polymers in MSC surface wall abrasive samples was analyzed directly in the collection tube following adaptation of a previously described method [START_REF] Stranne | TBL10 is required for O-acetylation of pectic rhamnogalacturonan-I in Arabidopsis thaliana[END_REF] using the K-ACET enzymatic Acetic Acid Assay Kit (Megazyme). The detailed protocol is provided in Supplemental Methods.

Structural oligosaccharide composition of dry seed MSC abrasive fractions by IP-RP-UHPC-MS analysis

MSC surface wall powders were resuspended in 1 mL distilled water for 1 h. An aliquot (450 µL) was mixed with 50 µL of 500 mM sodium acetate buffer pH 5.0 and polysaccharides were hydrolyzed for 24 h at 40°C under 500 rpm shaking with either a pectin lyase (PL) specific for methylesterified HGs [START_REF] Ralet | Innovative enzymatic approach to resolve homogalacturonans based on their methylesterification pattern[END_REF], a polygalacturonase (PG) [START_REF] Safran | Plant polygalacturonase structures specify enzyme dynamics and processivities to fine-tune cell wall pectins[END_REF] specific for non-methylesterified HGs or a rhamnogalacturonase (RG, Swiss-Prot Q00018, provided by Novozymes, Copenhagen, Denmark) specific for the RGI backbone [START_REF] Ralet | Monoclonal antibodies to rhamnogalacturonan I backbone[END_REF].

Resulting OGA digests were filtered on 0.45 µm and analyzed by IP-RP-UHPC-MS.

Acquisitions were performed on a Select Series Cyclic IMS (Waters, Wilmslow, UK) coupled with a UHPLC system (Acquity H-Class Plus, Waters, Manchester, UK). The chromatographic separations were performed on an hypersil GOLD TM (100 mm × 1 mm, packed with 1.9 μm porosity particles; Thermo-Fisher Scientific, Bremen, Germany). A ternary gradient was used Data were recorded with the Quartz software (Waters embedded software, release 5) and processed using Mass Lynx 4.2 (Waters) and MzMine software [START_REF] Pluskal | MZmine 2: Modular framework for processing, visualizing, and analyzing mass spectrometry-based molecular profile data[END_REF]. MzMine was used to produce a database of annotated structures (m/z; retention time; intensity). The identified species (charge states and anomers were merged) were annotated according to their degree of polymerization (DP), degree of methylesterification (DM) and degree of acetylation (DA). The data was analyzed using Microsoft Excel.

Production of recombinant TBL38 in Pichia pastoris and enzymatic activity assays

The coding sequence of TBL38 (Q8VY22) was codon-optimized for Pichia pastoris and synthesized without signal peptide in frame with His-tag in pPCIZ-αB by ProteoGenix (Schiltigheim, France). rTBL38 was produced in P. pastoris following the previously described protocol [START_REF] Lemaire | Three novel rhamnogalacturonan Ipectins degrading enzymes from Aspergillus aculeatinus: Biochemical characterization and application potential[END_REF]. Activity assays of rTBL38 was performed with the acetic acid assay kit (K-ACETRM, Megazyme) using the activity buffer at 40°C and with three different acetylated substrates: 100 mM Triacetine, 10 mg.ml -1 xylan (24 % acetylation), and 10 mg.mL - 1 sugar beet pectins (31 % acetylation). The detailed protocol is provided in Supplemental Methods.

In silico model and docking simulations

Homology models for PRX36 and TBL38 (UniProt accession number Q9SD46 and Q8VY22, respectively) were built with the Phyre2 server [START_REF] Kelley | The Phyre2 web portal for protein modeling, prediction and analysis[END_REF]. For PRX36, the template was the crystallographic structure (X-Ray diffraction, 1.45 A ) of A. thaliana PRX53 (At5g06720; Protein Data Bank no.1PA2) [START_REF] Ostergaard | Arabidopsis ATP A2 peroxidase. Expression and high-resolution structure of a plant peroxidase with implications for lignification[END_REF]. For TBL38, the template was the crystallographic structure (X-Ray diffraction, 1.85 Å) of A. thaliana TBL29/ESK1/XOAT1 [START_REF] Lunin | Molecular mechanism of polysaccharide acetylation by the Arabidopsis xylan O-acetyltransferase XOAT1[END_REF]. For comparison with the TBL38 model, the TBL29 structure was drawn as well using the 6cci pdb file [START_REF] Lunin | Molecular mechanism of polysaccharide acetylation by the Arabidopsis xylan O-acetyltransferase XOAT1[END_REF] visualized and analyzed with Swiss-PdbViewer (http://www.expasy.org/spdbv/) [START_REF] Guex | SWISS-MODEL and the Swiss-PdbViewer: An environment for comparative protein modeling[END_REF].

For PRX36 docking experiments, the -D-(1-4) polygalacturonic acid structural model (Braccini et al., 1999) was retrieved from the Glyco3D portal [START_REF] Perez | Glyco3D: a portal for structural glycosciences[END_REF] http://glyco3d.cermav.cnrs.fr/mol.php?type=polysaccharide&molecule=2504). It was modified to initially build the five hexagalacturonates models used to establish JIM7 specificity [START_REF] Clausen | Synthetic methyl hexagalacturonate hapten inhibitors of antihomogalacturonan monoclonal antibodies LM7, JIM5 and JIM7[END_REF]. This selection was extended to the 124 oligogalacturonates (OGAs) from DP2 to DP6 covering all the theoretical combination of methylation (64 OGAs of DP6 + 32

OGAs of DP5 + 16 OGAs of DP4 + 8 OGAs of DP3 +4 OGAs of DP2). AutoDock Tools [START_REF] Morris | AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility[END_REF] and AutoDock Vina [START_REF] Trott | Software news and update AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading[END_REF] were used for simulating the binding of the 124 OGAs to PRX36 within a search box encompassing the whole target protein enabling to recover the nine best poses for each OGA. Details on data processing are provided in Supplemental Methods.

Figure 3 :

 3 Figure 3: TBL38 is a cell wall protein transiently localized to the radial primary CW microdomain of MSCs. (A) Confocal microscopy visualization of both TBL38-TagRFP and a Golgi-YFP marker transiently overexpressed in N. benthamiana leaves indicates that TBL38 is not a Golgi-localized protein. Under plasmolysis, the TBL38-TagRFP signal and the plasma membrane-YFP marker signal are separated, indicating the localization of TBL38-TagRFP to the CW. First row images are extracted from a maximum projection of 17 stacks. Second row images are a representative view of a single confocal plan. Bars: 10 µm. (B) Confocal spinning disk observations of TBL38-TagRFP in A. thaliana developing seeds. At the onset of pPRX36 activity (Francoz et al., 2019b), TBL38-TagRFP fluorescence was positioned to the radial primary CW of MSCs. Orthogonal projection refined this localization to the CW microdomain consisting in the top of the radial primary CW similarly to PRX36-

FigureFigure 2 ).

 2 Figure1).

Figure 4 :

 4 Figure 4: LM20 but not JIM7 labeling is lost in tbl38. (A) In Col-0, JIM7 labeling of partially methylesterified HG is visible as early as 6 DAP at the MSC surface and is restricted to the top of the radial primary CW microdomain of more mature MSCs (7-10 DAP). A very similar labeling pattern occurs in tbl38 as well as in two complemented lines (tbl38 transformed with pPRX36::TBL38-TagRFP). (B) LM20 was previously thought to bind to similar epitopes as JIM7 in MSCs since both epitopes were lost in pmei6 (Francoz et al., 2019b). However, contrary to JIM7, the LM20 labeling at the top of the radial primary CW microdomain was lost in tbl38 and restored in the two tested complemented lines. 40 x scans of fluorescence and brightfield channels were merged. The same parameters were used to analyze each image which are a representative of n > 20 MSCs at each developmental stage. Additional stages positioned between the staged ranks are shown. Bars: 25µm.

Figure 5 :Figure 6 :

 56 Figure 5: Dry seed surface enriched fraction shows a decreased LM20 epitope signal intensity and an increased acetic acid content in tbl38 compared to Col-0, and the wild type phenotype is restored in complemented lines. JIM7 (A) and LM20 (B) immuno dot blot of dry seed surface CW-enriched fraction from Col-0, pmei6, tbl38 and three tbl38 complemented lines expressing pPRX36:TBL38-TagRFP (PTR2.4, 3.5 and 5.2). On top of the panels are shown screen shots of individual dots from the same nitrocellulose membrane. On the bottom of the panel are shown box plot representations of the data analysis. (C) Box plot representation of the acetic acid content released following acid hydrolysis of dry seed surface CW enriched fraction from Col-0, tbl38 and three tbl38 complemented lines expressing pPRX36::TBL38-TagRFP (PTR2.4, 3.5 and 5.2). Three biological replicates each with at least two technical replicates were analyzed. Letters above the bars show the statistical distribution of the results using ANOVA and TUKEY HSD tests.

  D). The DA vs DP profiles of Col-0, tbl38 and pmei6 were qualitatively similar though a slight increase of species of DA > 0 occurred for tbl38 (Figure 6I-K; Supplemental Table 4A-D. The DM vs DP plots showed a quantitative phenotype of pmei6 as compared to Col-0 and tbl38 in agreement with the PMEI activity of PMEI6 (Saez-Aguayo et al., 2013) (Figure 6L-N). We also noticed an about tenfold higher intensity of OGA species of low DM identified with PG (Figure 6I-N;

Figure 7 :

 7 Figure 7: Pectin acetyl esterase activity of recombinant TBL38 and molecular modeling of TBL38 displaying its putative catalytic triad and substrate binding pocket. (A) SDS-PAGE showing the production of recombinant TBL38 (rTBL38) in Pichia pastoris. (B) In vitro enzymatic activity on acetylated substrates. The activity of the purified rTBL38 (100 mM imidazole fraction) was assayed using triacetin, acetylated xylans and sugar beet pectins as substrates and the ACET enzymatic acetic acid assay kit. Boiled samples were used as negative control, mean values  SD, *, p-value < 0.05. (C) Amino acid sequence of TBL38 (At1g29050). The predicted signal peptide (https://services.healthtech.dtu.dk/services/SignalP-6.0/) is underlined. The amino acids in italics correspond to the 15% of the protein not covered by the molecular modeling performed in (D). GDSL and DxxS tetrads that are highly conserved among the 46 TBR/TBLs including TBL38[START_REF] Bischoff | TRICHOME BIREFRINGENCE and Its homolog AT5G01360 encode plantspecific DUF231 proteins required for cellulose biosynthesis in Arabidopsis[END_REF]

Figure 6 ).

 6 Figure 6). In turn, this is consistent with the immunolocalization of αPRX36 and confocal imaging of PRX36-TagRFP in tbl38 background (Supplemental Figure 4) which did not show any obvious mislocalization of PRX36, suggesting that the Clausen 4 model might have a higher affinity towards PRX36 in muro.

  thaliana plants were transformed by floral-dipping[START_REF] Clough | Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana[END_REF] or spraying flower buds with an Agromix containing 0.05 % (v/v) Silwet L-77 (De Sangosse 2000235).

  proPRX36::PRX36-TagRFP construct was transformed in prx36 and tbl38 plants. The proPRX36::TBL38-TagRFP construct was transformed in tbl38 plants. These last complemented lines were referred as PTR2.4, PTR3.5 and PTR5.2 in the manuscript. Seeds were selected on Murashige and Skoog (MS) medium containing 25 µg.mL -1 hygromycin.

(

  A: Milli-Q water, B: 100% methanol, and C: 20 mM heptylammonium formate, pH 6), from 2 % to 25 % of solvent B for 10 min, then up to 73 % at 23.5 min and maintained at 73 % for 4 min. Percentage of solvent C was kept constant at 25%. The flow rate was 0.175 mL min -1 , and the column was heated to 45°C. Spectra were recorded in positive electrospray ionization (ESI) ionization mode in the m/z range 150-2000, with the TOF operating in the V-mode. The source parameters were as follows: capillary voltage 2.8 kV; cone voltage 120 V; source temperature 100°C; desolvation temperature 350°C; desolvation gas 350 L/h; and nebulization gas 6 bar.

Figure 2: TBL38 is co-expressed with PRX36 and PMEI6 in the seed coat and transcript localization of TBL38 is tied to the mucilage secretory cells (outer integument

2). (A) eFP browser pictogram displaying the similarity in the spatiotemporal expression of PRX36, PMEI6 and TBL38 in A. thaliana seed coat. Absolute tissuespecific expression level is represented in color variation from yellow (lowest) to red (highest). Adapted from http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi. (B) PRX36 co-expression network using the same dataset highlighting the similar profiles of PRX36, PMEI6 and TBL38 (see Supplemental Table

2

for details). (C) In situ hybridization of dig-labeled RNA probes targeting TBL38 on paraffin sections of developing wild-type seeds.

Table 7 .

 7 Full length cDNA clones and custom-ordered DNA plasmids used as template DNA for further cloning are listed in Supplemental

Table 9 .

 9 Level 0 GoldenGate generated constructs are listed in Supplemental

Table 10 .

 10 Level 1 GoldenGate plasmids assembled in pL1V-R2 vector and finalized Level 2

Acknowledgments

We thank the Paul Sabatier Toulouse 3 University and the Centre National de la Recherche Scientifique (CNRS) for granting this work. This work was specifically supported by the French National Research Agency project ''MicroWall'' (ANR-18-CE20-0007). It was also supported by the French Laboratory of Excellence project ''TULIP'' (ANR-10-LABX-41; ANR-11-IDEX-0002-02). We are grateful to Dr. H. North (IJPB, Versailles, France) and I.-H. Nishimura (Kyoto University, Japan) for providing seeds and to Dr. P.-M. Delaux (LRSV, Auzeville-Tolosane, France) for the gift of GoldenGate plasmids and helpful discussions about Golden Gate cloning.

Confocal microscopy of leaves of N. benthamiana transient transformants

Transiently transformed N. benthamiana leaves were observed 48 h post agroinfiltration using an upright confocal laser scanning microscope (LEICA SP8) with a 40 x apochromatic water immersion lens. TagRFP and YFP fluorescences were imaged with the following settings: excitation: 561 nm / emission: 582-622 nm; excitation 514 nm / emission: 527-530 nm, respectively. Z stacks (maximum intensity) were performed using ImageJ.

SDS-PAGE and anti-TagRFP western-blots from tbl38 complemented lines (PTR)

The TagRFP fluorescence patterns of PTR lines were monitored using spinning disk confocal microscopy along developmental kinetics of seeds from staged floral stem. The seeds that displayed the early (6-8 DAP), medium (8-12 DAP) and late (> 12 DAP) fluorescence patterns were carefully extracted from the dissected siliques (3-4 siliques per fluorescence pattern) and pooled in separated 2 mL tubes with a metallic grind ball before freezing in liquid nitrogen.

Total proteins were extracted and analyzed by western blot as previously described [START_REF] Francoz | Pectin demethylesterification generates platforms that anchor peroxidases to remodel plant cell wall domains[END_REF], using anti-TagRFP primary and anti-rabbit-AP secondary antibodies (Supplemental Table 8) with minor modifications as detailed in Supplemental Methods.

Dry seed MSC surface wall abrasive fraction

Abrasion columns were a home-made design allowing for homogenous dry seed MSC surface wall abrasive enrichment using plastic columns and collector tubes from the GeneJET Plasmid Miniprep Kit (Thermo Scientific K0502). The preparation of the columns, the abrasion procedure and the characterization of the efficiency are described in detail in Supplemental Methods.

Immuno dotblot and semi-quantitative analysis from dry seed MSC abrasive fractions

Powder resulting of MSC surface abrasion was chemically extracted in the collector tube with extraction buffer (80 μL of 5 mM sodium acetate pH 4.6 containing 1 μL.mL -1 of plant protease inhibitor cocktail (Sigma P9599) per mg of powder). The collector tube with cap and a metallic grind ball was shaken at 250 rpm during 1 h at 4°C to ensure proper extraction. After a 5 min centrifugation at 14,400 rpm, the supernatant was recovered, diluted (1 μL in 50 μL milliQ- preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.