Fan Xu

Martine Gonneau

Elvina Faucher

Olivier Habrylo

Valérie Lefebvre

Jean-Marc Domon

Marjolaine Martin

Fabien Sénéchal

Alexis Peaucelle

Jérôme Pelloux

Herman Höfte
email: hermanus.hofte@inrae.fr

Biochemical characterization of Pectin Methylesterase Inhibitor 3 from Arabidopsis thaliana ☆

Keywords:

The de-methylesterification of the pectic polysaccharide homogalacturonan (HG) by pectin methylesterases (PMEs) is a critical step in the control of plant cell expansion and morphogenesis. Plants have large gene families encoding PMEs but also PME inhibitors (PMEIs) with differ in their biochemical properties. The Arabidopsis thaliana PECTIN METHYLESTERASE INHIBITOR 3 (PMEI3) gene is frequently used as a tool to manipulate pectin methylesterase activity in studies assessing its role in the control of morphogenesis. One limitation of these studies is that the exact biochemical activity of this protein has not yet been determined. In this manuscript we produced the protein in Pichia pastoris and characterized its activity in vitro. Like other PMEIs, PMEI3 inhibits PME activity at acidic pH in a variety of cell wall extracts and in purified PME preparations, but does not affect the much stronger PME activity at neutral pH. The protein is remarkable heat stable and shows higher activity against PME3 than against PME2, illustrating how different members of the large PMEI family can differ in their specificities towards PME targets. Finally, growing Arabidopsis thaliana seedlings in the presence of purified PMEI3 caused a dose-dependent inhibition of root growth associated with the overall inhibition of HG demethylesterification of the root surface. This suggests an essential in vivo role for PME activity at acidic pH in HG de-methylesterification and growth control. These results show that purified recombinant PMEI3 is a powerful tool to study the connection between pectin de-methylesterification and cell expansion.

Introduction

A central question in plant biology is how growth can occur despite the presence of strong cell walls [START_REF] Cosgrove | Growth of the plant cell wall[END_REF]. Cell walls of growing cells (i.e. primary cell walls) are highly dynamic polymer assemblies primarily consisting of cellulose, small amounts of structural proteins and the matrix polysaccharides hemicelluloses and pectins. Pectins are galacturonic acid (GalA) -containing polysaccharides among which the linear polymer HG is the most abundant matrix polysaccharide, for instance representing 20% of the total cell wall polysaccharides, in Arabidopsis thaliana leaves [START_REF] Zablackis | Characterization of the cell-wall polysaccharides of Arabidopsis thaliana leaves[END_REF] or even 50% in onion epidermis cells [START_REF] Wilson | Saccharide analysis of onion outer epidermal walls[END_REF]. In vivo studies show that the metabolism of HG plays a critical role in the control of cell expansion and plant morphogenesis [START_REF] Andres-Robin | Evidence for the regulation of gynoecium morphogenesis by ETTIN via cell wall dynamics[END_REF]Haas et al., 2020;[START_REF] Jobert | Auxin and pectin remodeling interplay during rootlet emergence in white lupin[END_REF][START_REF] Peaucelle | The Control of Growth Symmetry Breaking in the Arabidopsis Hypocotyl[END_REF][START_REF] Peaucelle | Arabidopsis Phyllotaxis Is Controlled by the Methyl-Esterification Status of Cell-Wall Pectins[END_REF][START_REF] Phyo | Gradients in wall mechanics and polysaccharides along growing inflorescence stems[END_REF][START_REF] Qi | Mechanical regulation of organ asymmetry in leaves[END_REF][START_REF] Stefanowicz | Plasmodiophora brassicae-Triggered Cell Enlargement and Loss of Cellular Integrity in Root Systems Are Mediated by Pectin Demethylation[END_REF][START_REF] Wachsman | Cell wall remodeling and vesicle trafficking mediate the root clock in <em> Arabidopsis</em&gt[END_REF], but the underlying mechanisms are still poorly understood.

HG is synthesized in a highly methylesterified form in Golgi bodies Abbreviations: HG, homogalacturonan; PME, pectin methylesterase; PMEI, PME inhibitor; GalA, galacturonic acid. ☆ Given his/her/their role as Editor, Herman Hofte had no involvement in the peer review of this article and has no access to information regarding its peer review. and delivered to the surface via secretory vesicles. In the cell wall, demethylesterification by PMEs (EC 3.1.1.11) uncovers the negatively charged carboxylic acid groups, which dramatically changes the physical properties of the individual polymers and polymer assemblies [START_REF] Atmodjo | Evolving views of pectin biosynthesis[END_REF][START_REF] Haas | The role of pectin phase separation in plant cell wall assembly and growth[END_REF]. In addition, depending on the methylesterification pattern, HG is more or less sensitive to enzymatic degradation by polygalacturonases and pectate lyases (Hocq et al., 2017). PMEs are part of large gene families (66 members in A. thaliana [START_REF] Wolf | Homogalacturonan Methyl-Esterification and Plant Development[END_REF]), with different expression patterns, but also different biochemical characteristics. For instance, most plant PMEs have processive activity, generating blockwise de-methylesterified HG, in contrast to characterized fungal PMEs, which are non-processive, generating more randomly de-methylesterified HG [START_REF] Wolf | Homogalacturonan Methyl-Esterification and Plant Development[END_REF]. PME activity is also strongly pH dependent, with all plant PMEs studied so far showing neutral or alkaline pH optima [START_REF] Hocq | The pH-dependent processivity of Arabidopsis AtPME2 can control cell wall mechanical properties[END_REF]. Interestingly, the pH also influences the mode of action of the enzymes. For instance, citrus PME and Arabidopsis thaliana PME2 activities are processive at pH 8 and non-processive at pH 5 [START_REF] Hocq | The pH-dependent processivity of Arabidopsis AtPME2 can control cell wall mechanical properties[END_REF]. The biological significance of this behavior, in particular in relation to growth control is not known. PME activity is also regulated by PMEIs, which are also encoded by large gene families in vascular plants (76 in A. thaliana [START_REF] Wolf | Homogalacturonan Methyl-Esterification and Plant Development[END_REF]). PMEIs inhibit PME activity by forming 1:1 complexes with them. A critical role for PME activity in the control of cell expansion was shown by the inducible overexpression of PME5 and PMEI3, which promoted and inhibited cell wall expansion respectively, where both treatments caused the loss of growth anisotropy [START_REF] Peaucelle | The Control of Growth Symmetry Breaking in the Arabidopsis Hypocotyl[END_REF]. PMEI3 overexpression also caused the inhibition of organ formation in the inflorescence meristem [START_REF] Peaucelle | Pectin-induced changes in cell wall mechanics underlie organ initiation in Arabidopsis[END_REF]. Similar observations were made in which PME5 and PMEI3 overexpression respectively caused an increased and reduced gynoecium length [START_REF] Andres-Robin | Evidence for the regulation of gynoecium morphogenesis by ETTIN via cell wall dynamics[END_REF], or reduced or increased cell expansion and loss of organ asymmetry in A. thaliana leaves [START_REF] Qi | Mechanical regulation of organ asymmetry in leaves[END_REF]. Finally, PMEI3 overexpression also led to the inhibition of lateral root formation [START_REF] Wachsman | Cell wall remodeling and vesicle trafficking mediate the root clock in <em> Arabidopsis</em&gt[END_REF]. Surprisingly, some studies revealed opposite effects of changing the degree of HG methylesterification on cell wall mechanics and expansion rate. Indeed, depending on the context, demethylesterification caused an increase or a decrease in wall elasticity and growth rate [START_REF] Peaucelle | The Control of Growth Symmetry Breaking in the Arabidopsis Hypocotyl[END_REF][START_REF] Phyo | Gradients in wall mechanics and polysaccharides along growing inflorescence stems[END_REF]. It has been proposed that these opposite effects may be related to the pHdependent, processive or non-processive nature of certain PMEs (Hocq et al., 2017). In this view, processive activity generates stretches of acidic GalA, which can form cooperative Ca 2+ crosslinks (the so-called egg boxes) and which would cause cell wall stiffening and growth inhibition. In contrast, randomly demethylated HG would form a more elastic Ca 2+ cross-linked network [START_REF] Vincent | Microrheological behaviour and nonlinear rheology of networks assembled from polysaccharides from the plant cell wall[END_REF]. An alternative view explaining PME-dependent growth was recently proposed based on the observation, using super-resolution optical microscopy, of oriented HG filaments in anticlinal cell walls of leaf pavement cells (Haas et al., 2020). In this view the de-methylesterification of HG leads to a volume increase of HG filaments, which would be a driver for cell expansion.

The above-mentioned studies used the inducible expression of the putative PMEI inhibitor PMEI3 (At5G20740) to manipulate pectin methylesterification. Despite its frequent use in vivo, the biochemical activity of PMEI3 has not been characterized so far. This knowledge is essential however, for the correct interpretation of the induced mechanical, cellular and macroscopic phenotypes. A few other PMEIs (PMEI4, 7 and 9) have been heterologously expressed and biochemically characterized (Hocq et al., 2017b;Senechal et al., 2015). This showed, for different PMEIs, pH-dependent (PMEI4, 7) or pH-independent (PMEI9) ineractions with target PMEs. In this report, we produced the PMEI3 protein in Pichia pastoris and characterized its activity and specificity. We show a pH-dependent inhibition of PME activity. Interestingly the inhibitory activity varied against different PME enzymes, showing the potential biochemical specialization of PME-PMEI pairs. Using purified PMEI3, we show that PME activity at acidic pH is critical for in vivo HG de-methylesterification and cell expansion in Arabidopsis roots.

Results

Expression in Pichia pastoris and purification of PMEI3

A codon-usage-optimized sequence of Arabidopsis thaliana PMEI3 (At5G20740), fused at its N-terminus to a secretion signal peptide and at its C-terminus to 6×His and c-MYC tags, was expressed in Pichia pastoris X33 (Fig. 1A). The mature protein could be detected in the culture supernatant using immunoblotting with anti-His antibodies (Fig. 1B). Upon His-tag affinity purification (Fig. S2A), a band of the expected molecular weight (~25kDa) was observed on Coomassie-stained SDS PAGE (Fig. 1C). The identification of the PMEI3 protein was verified using immunoblotting with anti-His antibodies (Fig. 1C) and LC-MS/MS on the band extracted from the gel, which detected in the tryptic digest 4 peptides matching PMEI3 (Fig. 1D).

pH-dependent inhibition of PME activities

We next characterized the inhibitory activity of PMEI3 on a number of PME preparations. The PME activity was first measured on commercial citrus pectin (Degree of Methylesterification [DM] 85%) at pH 7.5 using a colorimetric assay for methanol production on dilutions of the enzyme preparation (Fig. S1). Non-saturating dilutions were chosen for the PMEI assays (1/100 dilution for Orange PME and 1/10 dilution for flower and root PME preparations). The activity of purified PMEI3 was assayed using a gel diffusion assay at pH 5, 6.3, and 7.5, respectively. The activity was quantified by measuring the radius of the red halo (Fig. S2). We first tested the inhibitory activity of PMEI3 against orange PME. The results showed strong inhibition at pH 5 and decreasing inhibition from pH 6.3 to 7.5, respectively (Fig. 2A-C). Surprisingly, boiling the sample did not (at pH 5) or only slightly (at pH 6.3) reduce the PMEI3 activity. This indicates that this inhibitor is able to correctly refold upon heat denaturation. A similar pH-dependent inhibitory pattern was observed against PME preparations from roots (Fig. 2D-F) and inflorescences (Fig. 2G-I) respectively. Interestingly, the inhibitory activity was stronger on the extracts from roots than from flowers at both pH 5 and 6.3. In conclusion, PMEI3 showed a pHdependent inhibitory activity against one or more PME isoforms that are expressed in roots and flowers. The lower activity on flower extracts suggests that different PME isoforms may vary in their sensitivity to PMEI3 inhibition.

Inhibitory activity and pH dependence of PMEI3 varies for different PME isoforms

To further investigate the specificity of PMEI3, we compared its activity against two heterologously expressed PME isoforms PME2 (At1g53830) [START_REF] Hocq | The pH-dependent processivity of Arabidopsis AtPME2 can control cell wall mechanical properties[END_REF] and PME3 (At3g14310) [START_REF] Guenin | Identification of pectin methylesterase 3 as a basic pectin methylesterase isoform involved in adventitious rooting in Arabidopsis thaliana[END_REF] (Figure 3). Both PME2 and PME3 are closely related isoforms with distinct expression patterns. PME3 is broadly expressed in roots and leaves (Fig. S3). Based on mutant analysis, PME3 represented around 30% of the PME activity in root extracts [START_REF] Guenin | Identification of pectin methylesterase 3 as a basic pectin methylesterase isoform involved in adventitious rooting in Arabidopsis thaliana[END_REF]. PME2 instead, has a more restricted expression pattern, with higher transcript levels in roots and flower petals (Fig. S4). In addition, mutant analysis showed that the contribution of PME2 to the total PME activity was only marginal at least in the organ investigated (dark-grown hypocotyl) [START_REF] Hocq | The pH-dependent processivity of Arabidopsis AtPME2 can control cell wall mechanical properties[END_REF]. The latter may reflect the epidermis-specific expression pattern of this gene as suggested by the cell type-specific RNA expression data [START_REF] Klepikova | A high resolution map of the Arabidopsis thaliana developmental transcriptome based on RNA-seq profiling[END_REF][START_REF] Wendrich | Vascular transcription factors guide plant epidermal responses to limiting phosphate conditions[END_REF] (Fig. S5). PME2 was produced in Pichia pastoris and purified using ion exchange chromatography [START_REF] Hocq | The pH-dependent processivity of Arabidopsis AtPME2 can control cell wall mechanical properties[END_REF]. PME3 fused to a C-terminal 6xHis-tag was transiently expressed in tobacco leaves and affinity purified (Senechal et al., 2015). Again, at pH 7.5, no PME inhibitory activity was observed against either one of the PMEs, with for the lower PMEI3 concentrations even a small but significant (t-test, P value = 0.008) PME3 promoting activity. Interestingly however, at pH 5, PMEI3 was at least 10-fold more active against PME3 than against PME2 (PME3 inhibition with 1.12 µM PMEI3 was comparable to PME2 inhibition with 11.2 µM PMEI3). In addition, at pH 6.3, PMEI3 was still active against PME3 but not against PME2. Together, these observations illustrate the high pH optimum for PMEI3 and its preference for PME3 over PME2.

In vivo effect of PME inhibition

We next assessed the in vivo effect of the inhibition of PME activity by supplementing purified PMEI3 to the growth medium of A. thaliana seedlings. The small molecule PME inhibitor epigallocatechin-3-gallate (EGCG) was used as a positive control. EGCG caused a dose-dependent reduction in the root length of 4-day-old seedlings, relative to the mock-treated control, with an IC 50 of around 55 µM (Fig. S6). Similarly, the root length of 3-day-old seedlings grown in the presence of PMEI3 was strongly reduced in a dose-dependent fashion (Fig. 4A,B) with an IC 50 of 0.16 µM. We next measured the amount of blockwise demethylesterified HG using the fluorescent probe COS 647 [START_REF] Mravec | Tracking developmentally regulated postsynthetic processing of homogalacturonan and chitin using reciprocal oligosaccharide probes[END_REF]. Interestingly the labeling was strongly reduced in roots grown in the presence of 0.2 or 0.6 µM PMEI3 relative to control roots (Fig. 4C,D). Increasing the sensitivity of the detection also showed a dose responsiveness for the inhibition of the fluorescent signal, with a stronger inhibition at 0.6 µM relative to 0.2 µM PMEI3. (0.6 µM sat and 0.2 µM sat, respectively in Fig. 4C,D). In summary, these results show that external supply of PMEI3, like the PME inhibitor EGCG, strongly inhibited processive PME activity and root growth. PMEI3 treatment thus mimics the effects of the inducible overexpression of the PMEI3 gene [START_REF] Peaucelle | The Control of Growth Symmetry Breaking in the Arabidopsis Hypocotyl[END_REF].

Discussion

Heterologously-expressed PMEI3 inhibited the activity of a variety of PME preparations. Interestingly, PMEI3 showed quantitative differences in its inhibitory activity, with higher activity against root PMEs and PME3 relative to flower and orange PMEs and PME2. PME3 is the major PME activity in vegetative tissues including roots, whereas PME2 transcripts are more abundant in flower petals. This may explain the higher sensitivity of root PMEs relative to flower PMEs for PMEI3. The comparison of the closely related 3D structures of PME2 and PME3 [START_REF] Hocq | The pH-dependent processivity of Arabidopsis AtPME2 can control cell wall mechanical properties[END_REF] may provide valuable information on the structural basis for these differences in specificity.

In vitro PMEI3 activity was strongly pH-dependent, with the highest inhibition at pH 5.5 and almost no or no inhibition at pH 7.5. Similar observations were made for PMEI4 and PMEI6 but not PMEI9, which also inhibited PME activity at pH 7.5 (Hocq et al., 2017b). PMEI3, like PMEI4 and 6, is therefore expected not to totally prevent PME activity but only to sharpen its pH optimum in vivo. Interestingly, the pH was recently shown to also affect the de-methylesterification patterns at least for orange PME and Arabidopsis thaliana PME2 [START_REF] Hocq | The pH-dependent processivity of Arabidopsis AtPME2 can control cell wall mechanical properties[END_REF]. Indeed, whereas both enzymes were processive at pH 7.5, generating blockwise demethylated HG, their activity became non-processive at pH 5.5. This suggests that the strong effect of PMEI3 overexpression on growth and development [START_REF] Andres-Robin | Evidence for the regulation of gynoecium morphogenesis by ETTIN via cell wall dynamics[END_REF][START_REF] Peaucelle | The Control of Growth Symmetry Breaking in the Arabidopsis Hypocotyl[END_REF][START_REF] Peaucelle | Pectin-induced changes in cell wall mechanics underlie organ initiation in Arabidopsis[END_REF][START_REF] Qi | Mechanical regulation of organ asymmetry in leaves[END_REF][START_REF] Wachsman | Cell wall remodeling and vesicle trafficking mediate the root clock in <em> Arabidopsis</em&gt[END_REF] might be related to a change in the HG methylation patterns, rather than the quantitative inhibition of PME activity. This is surprising given the strong overall reduction of COS 647 staining in PMEI3-treated seedling roots, and the fact that COS 647 selectively binds blockwise de-methylesterified HG, the product of processive PME activity (Fig. 4C,D) [START_REF] Mravec | Tracking developmentally regulated postsynthetic processing of homogalacturonan and chitin using reciprocal oligosaccharide probes[END_REF]. To explain this, it is possible that the activity in intact cell walls does not reflect the in vitro specificity on isolated wall extracts or purified PMEs. In this context it is interesting to note that the specificity of PME activity was shown to change when the enzyme was incorporated in pectin or pectin/cellulose gels [START_REF] Bonnin | Mobility of pectin methylesterase in pectin/cellulose gels is enhanced by the presence of cellulose and by its catalytic capacity[END_REF][START_REF] Vincent | Microrheological behaviour and nonlinear rheology of networks assembled from polysaccharides from the plant cell wall[END_REF]. Alternatively, the apparent inhibition of both non-processive and, the much stronger, processive PME activity in root cell walls by the external application of PMEI3 may suggest that non-processive PME activity at low pH might be an essential step to prime subsequent processive activity at a higher pH. It will be interesting to see whether PME activity at high pH is unable to attack fully methylesterified HG and, if so, to what extent such behavior may contribute to acid growth. It will also be interesting to investigate the effect on in vivo pectin methylesterification and plant development of PMEI9 overexpression, which is expected to suppress PME activity irrespective of the pH, and to what extent HG populations with different methylation patterns can have distinct in vivo functions. A striking example of a HG methylesterification pattern with a specific in vivo function was recently reported for the external cell wall of seed coat epidermis cells [START_REF] Francoz | Pectin Demethylesterification Generates Platforms that Anchor Peroxidases to Remodel Plant Cell Wall Domains[END_REF]. Indeed, a specific pattern, recognized by antibody LM20, and requiring the presence of PMEI6, appeared to recruit a peroxidase (PRX36) to a specific location, 4. In vivo activity of purified PMEI3. A,B: Representative photographs (A) and primary root lengths (B) of 3-day-old Arabidopsis thaliana seedlings. Curve is a second degree polynomal fit (y = 3,3692x 2 -3,6899x + 1,007; R 2 = 0,9947); IC50 = 0.16 µM. C,D: Representative photographs (C) and fluorescence quantification (D) of COS 647 -labeled roots. 0.2 µM sat and 0.6 µM sat refer to the same samples as for 0.2 µM and 0.6 µM but with an increased detection sensitivity. Two-day-old seedlings were transferred to 0; 0.2 µM or 0.63 µM PMEI3 and incubated for 24h before analysis. Error bars are SD (n > 30 for B and n=5 for D). *** t-test, P-value<0.001.

thus generating a brittle zone in the external cell wall facilitating cell wall rupture and mucilage release at this location during seed hydration.

Finally, we observed that the external supply of PMEI3 to the growth medium inhibited the growth of seedlings in a concentration dependent way paralleling the PME inhibitory activity, corroborating the observations that PMEI3 overexpression inhibits cell wall expansion [START_REF] Andres-Robin | Evidence for the regulation of gynoecium morphogenesis by ETTIN via cell wall dynamics[END_REF][START_REF] Peaucelle | The Control of Growth Symmetry Breaking in the Arabidopsis Hypocotyl[END_REF][START_REF] Peaucelle | Pectin-induced changes in cell wall mechanics underlie organ initiation in Arabidopsis[END_REF][START_REF] Qi | Mechanical regulation of organ asymmetry in leaves[END_REF][START_REF] Wachsman | Cell wall remodeling and vesicle trafficking mediate the root clock in <em> Arabidopsis</em&gt[END_REF]. The purified protein will be a valuable tool to study the in vivo dynamics of pectin metabolism and the developmental consequences of the manipulation of pectin methylesterification. Finally, it will be interesting to further explore the biochemical characteristics of other members of the large PME and PMEI families in plants.

Materials and methods

PMEI3 heterologous expression and purification

pPICZα B is a 3.6 kb vector used to express and secrete recombinant proteins in Pichia pastoris (ThermoFischer, Ref V19520). Recombinant proteins are expressed as fusions to an N-terminal peptide encoding the Saccharomyces cerevisiae α-factor secretion signal. The vector allows high-level, methanol-inducible expression of the gene of interest in Pichia, and can be used in Pichia strain X-33. A plant codon-optimized PMEI3 sequence (see supplementary data) was inserted into plasmid pPICZαB and transformed into E. coli strain TOP10. Transformants was selected on Low Salt LB plates containing 50 μg/mL Zeocin. Eight transformants for each were analyzed by restriction mapping or sequencing to confirm in-frame fusion of PMEI3 with the α-factor secretion signal and the C-terminal tag. The appropriate transformants were cultivated to collect more plasmid. Plasmids were purified using a DNA purification kit (NucleoBond PC 100/BAC kit, obtained from Machery-Nagel). The recombinant plasmids were purified and then linearized using the Pme1 restriction enzyme. Recombinant plasmids (at a concentration > 500 ng/μL) were transformed into Pichia pastoris strain X33. Transformed Pichia was plated onto YPDS medium containing 50 μg/mL of Zeocin.

Transformants were selected and then cultivated in BMGY medium. After 12-h incubation at 37 • C with 250 rpm shaking, the OD 600 of the culture was measured. The recombinant Pichia was transferred into BMMY medium in a bigger volume (Table 7.2) for large-scale expression. The OD 600 was measured every 24 h to follow Pichia growth. In the meantime, 100% methanol was added to the medium to keep its final concentration at 0.5% (v/v). To make sure the recombinant proteins expressed correctly, the concentrated supernatant of the culture was tested using SDS-PAGE and/or Immunoblotting (details see below).

After 4-d expression, Pichia cells were removed by centrifugation at 5000 rpm for 5 min. To the protein-containing supernatant was added PBS buffer 10x (Eurobio, GAUPBS00-01), 400 mM NaCl, 5 mM imidazole and 500 μL cOmplete Hig-tag purification Resin (obtained from Roche, ref 05893682001). The mixture was gently shaken at 4 • C for 4 h in dark. The supernatant was removed by centrifugation at 8000 rpm for 5 min at 4 • C. The resin was washed by 5 ml of wash buffer 1 (PBS 1x with 400 mM NaCl and 5 mM imidazole), 5 ml of wash buffer 2 (PBS 1x with 400 mM NaCl and 30 mM imidazole) and eluted by elution buffer (PBS 1x with 400 mM NaCl and 400 mM imidazole) for 3 times (800 μL for each). The 3 elutes were collected and centrifuged at 8000 rpm for 30 min by using 3-kDa filter (Amicon Ultra-2 mL, UFC200324). The buffer change was performed using PD SpinTrap G-25 column. Final protein concentration was measured with a Bradford assay. The purified PMEI3 was frozen in liquid nitrogen and stored at -80 • C until use.

Cell lysis and immunoblotting

Pichia cells were pelleted, rapidly frozen by liquid nitrogen and then stored at -80 • C before use. Cells were resuspended in phosphate buffer pH 7.0 (2 mL for 1 mL pellet) containing 300 μl of protease inhibitor 1x (Constant systems Ltd. United Kingdom). Proteins were resolved using a 15% acrylamide/bisacrylamide SDS-PAGE (Table 7.3), with a running buffer composed of 25 mM Tris-base, 192 mM glycine, and 0.1% SDS at pH 8.7 (for Fig. 1B) or NuPage Bis/Tris 4-12 % acrylamide using MOPS running buffer (Fig. 1C). 13 μL of protein was added to 15 μL Laemmli buffer 2x and 2 μL of 1 M imidazole. The protein mix was heated at 95 • C for 20 min. 15 μL of protein mix was loaded into a well of a SDS-PAGE gel. After electrophoresis at 100 mA for 100 min, the gel was stained with Pageblue protein staining solution (Thermo Scientific, catalog no. 24620) and destained with distilled water for 6 h. After migration of proteins, the gel SDS-PAGE was immerged in cathode buffer (25 mM of Tris pH 9.4, 40mM of Glycine, 10% of ethanol) for 15 min. A piece of Hybond-P PVDF membrane (GE Healthcare, catalog no. RPN303F) was placed in 100% ethanol for 15 seconds and rinsed by water for 2 min. Then the membrane was incubated in anode buffer II (25 mM of Tris pH 10.4, 10% of ethanol) for 5 min. The membrane, gel, and 7 same-sized Whatmann papers (previously immerged in anode I, anode II and cathode buffers) were placed. The transfer "sandwich" was exposed to 25 V and 1 A for 30 mins by a Trans-Blot TURBO transfer system (Bio-Rad, catalog no. 170-4155). The membrane was incubated in 4.0% dry milk in PBS buffer, 0,1% Tween20 for 30 min and then incubated for 1 h at room temperature under shaking (50 rpm) in the same buffer containing 1/5000 of primary antibody raised against poly-His tag directly coupled with peroxidase (Sigma, catalog no. A7058). After washing 3 times by PBS 1x, 10 mins for each, 500 μL of DAB peroxidase substrate was added (Thermo Scientific, catalog no. 34002). Imaging was performed with a LAS-4000 according to the user manual. The concentration of proteins was determined according to Bradford using a BSA standard (1976).

Cell wall-enriched protein extraction

Fifty mg of frozen material of 10-day-old roots or 4-week-old flowers were ground with liquid nitrogen. One ml buffer (containing 50 mM Na 2 HPO 4 , 20 mM citric acid, 1 M NaCl, 0.01% Tween 20 at pH 7.0) was added. The solution was shaken at 250 rpm for 1 h. The extracts were clarified by centrifugation at 10000 rpm for 30 min at 4 • C. The supernatant was filtered using an Amicon ultracentrifuge filter 0.5 ml/10000 MWCO (Millipore, catalog no. UFC5010BK) in order to remove salts. Protein concentration was determined by the Bradford method (details see below).

Methanol colorimetric assay

Activity of AtPME2, AtPME3, citrus PME, and PMEs from flowers and roots were determined using a colorimetric microassay adapted from Klavons and Bennett (1986). The reaction solution contained 5 μL of purified protein, 85% methylesterified (DM85) Citrus pectin (Sigma P9561) at 1 mg/ml, 0.008 U of Pichia pastoris alcohol oxidase (Sigma A2404) and 50 mM sodium phosphate buffer (pH = 7.5) to a final volume of 100 μl. AtPME2, AtPME3, and citrus PME were prepared at 3 concentrations (1, 1/10 and 1/100 of purified proteins) and PMEs from flowers and roots at 2 concentrations (1 and 1/10). The mixture was incubated at 28 • C for 30 min. A first OD reading at 420 nm was done on a BioTek PowerWaveXS2 spectrometer. 100 μL of developing solution (2 mM ammonium acetate, 0.02 M pentane-2,4-dione, 0.05 M glacial acetic acid) was added and the solution was incubated at 68 • C for 15 min after which a second reading was done. A standard dilution series from 0 to 10 nmol of methanol was included in each batch. Results were expressed as nmol MeOH/min -1 .μL -1 of protein using the methanol standard curve.

Gel diffusion assay

DM85 Citrus pectin (Sigma P9561) was dissolved in 0.1 M citric acid and 0.2 M Na 2 HPO4 buffer at pH 5.0, 6.3 and 7.5 (Hocq et al. 2017b;

F. Xu et al. [START_REF] Sénéchal | Tuning of pectin methylesterification: PECTIN METHYLESTERASE INHIBITOR 7 modulates the processive activity of co-expressed PECTIN METHYLESTERASE 3 in a pH-dependent manner[END_REF]2017;[START_REF] Downie | A gel diffusion assay for quantification of pectin methylesterase activity[END_REF]. One percent (w/v) agarose was added to the pectin buffer and heated until the agarose had dissolved. The solution was cooled to 60 • C and transferred to a Petri dish (50 ml per dish) and allowed to solidify. Wells were punched in the gel with a 2-mm-diameter plastic sucker and the gel plug removed by aspiration. The gel was nicked and the orientation of the nick marked on the outside of the Petri dish with a marker. Ten μL of enzyme mixture was pipetted into each well. the wells were incubated at 37 • C for 16 h. After incubation, the gels were rinsed briefly with water and stained with 0.02% Ruthenium Red (Sigma) for 1h at room temperature. The dye was then poured off and the gel was rinsed with water again. Diameters of the red halos around each well were measured using the Fiji software.

Mass spectrometry

Each SDS-PAGE band was manually excised from the gels to be hydrolysed according to [START_REF] Shevchenko | Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels[END_REF]. Each sample was destained in a 40% ethanol-10% acetic acid solution (v/v) for 15 min and progressively dehydrated with 15 min incubations in first a 50% acetonitrile-25 mM ammonium bicarbonate solution and then in 100% acetonitrile. The dehydrated gel pieces were then reduced for 30 min with a 10 mM solution of DTT at 56 • C and alkylated for 45 min with 55 mM iodoacetamide. The samples were then dehydrated as above. For digestion, the samples were incubated overnight at 37 • C with 0.01 μg/ μL of trypsin (Promega, Madison, WI) and dissolved in 50 mM ammonium bicarbonate, pH 8. The tryptic fragments were extracted from the gel by centrifugation after incubation of 15 min in a 0.5% trifluoroacetic acid -50% acetonitrile (v/v) and in 100% acetonitrile solution, dried in a speed-vac, and reconstituted with 25 μL of a 2% acetonitrile -0.1% formic acid (v/v) buffer. HPLC was performed on a NanoLC-Ultra system (Eksigent). A 4 μL sample of the peptide solution was loaded at 7.5 μL min -1 on a precolumn cartridge (stationary phase: C18 Biosphere, 5 μm; column: 100 μm inner diameter, 2 cm; Nanoseparations) and desalted with 0.1% fluorhydric acid and 2% acetonitrile. After 3 min, the precolumn cartridge was connected to the separating PepMap C18 column (stationary phase: C18 Biosphere, 3 μm; column: 75 μm inner diameter, 150 mm; Nanoseparations). Buffers A and B respectively were prepared with 0.1% HCOOH in water, and with 0.1% HCOOH in acetonitrile. The peptide separation was achieved at 300 nL min-1 with a linear gradient: from minute 0 to 10, at 5 to 30% of B; from minute 10 to 12, at 30 to 95% of B; from minute 12 to 18, at 95% of B (the regeneration step); from minute 18 to 19, at 95 to 5% of B; from minute 19 to 20, at 5% of B (the equilibration step). Remaining percentages are those of buffer A. One run took 45 min. Eluted peptides were online analyzed with an LTQ XL ion trap (Thermo Electron) using a nanoelectrospray interface. Ionization (1.5 kV ionization potential) was performed with liquid junction and a non-coated capillary probe (10 μm inner diameter; New Objective). Peptide ions were analyzed using Xcalibur 2.07 with the following data-dependent acquisition steps: (1) full MS scan (mass-to-charge ratio (m/z) 350-1 400, centroid mode) and (2) MS/MS (isolation width 1m/z, qz = 0.25, activation time = 30 ms, and normalized collision energy = 35%; centroid mode). Step 2 was repeated for the three major ions detected in step 1. Dynamic exclusion was set to 45s.

A database search was performed with XTandem (version 10-12-01-1) (http://www.thegpm.org/TANDEM/). Enzymatic cleavage was defined as a trypsin digestion with one possible miscleavage. Regarding protein modifications, Cys carboxyamidomethylation was set to static. As potential modifications, Met oxidation, Nter acetylation, Nter deamidation on Gln, Nter deamidation on carbamidomethylated Cys, Nter neutral loss on Glu were set. Precursor mass and fragment mass tolerance were 2.0 and 0.8, respectively. A refinement search was added with similar parameters. The Araport11_genes.201606 database and a contaminant database (trypsin, keratins) were used for peptides identification. Identified peptides were filtered and grouped using Xtandem according to: (1) A minimum of two different peptides required with an E-value smaller than 0.05, (2) a protein log(E-value) (calculated as the product of unique peptide E-values) smaller than -4.

Growth inhibition assays

Arabidopsis seedlings were grown for three days on vertical petridishes on ½ MS medium (Duchefa, reference M0221) with 1.2% phytoagar (Duchefa, reference: P1003) at 22 • C, 60% relative humidity, 50 µmol.m -2 .s -1 white light (light source LED) with 18h-8h day-night cycle, before transfer to microtiter plates (4 seedlings per well) containing 100 µl of liquid ½ MS medium supplemented or not with PMEI3 or the PMEI inhibitor epigallocatechin-3-gallate (EGCG, Sigma-Aldrich) as a positive control. The root length was scored 14h later. Statistics were performed using the R statistics platforms. Significance (α = 0.05) was assessed by analysis of variance (ANOVA ; parametric) using linear mixed-effect models followed by Tukey HSD.

Fig. 1 .

 1 Fig. 1. Expression in Pichia pastoris and purification of PMEI3. A. Scheme of the expression construct, B. Immunoblot with anti-His antibodies of 2 culture supernatants (#1 and #2) and positive control (PC) of purified PMEI3 . C. Coomassie stained gel (left) anti-His immunoblot (right) of purified protein. D. PMEI3matching tryptic fragments (in red) from the purified protein identified by MS-MS.

Fig. 2 .

 2 Fig. 2. pH-sensitive inhibition of PME activity by PMEI3.Relative PME activity measured at pH 5 (A,D,G); 6.3 (B,E,H) and 7.5 (CF,I) in the presence of 0; 1.12 µM; 2.25 µM; 11.24 µM and boiled 11.24 µM PMEI3 in PME preparations from orange (A-C), Arabidopsis thaliana roots (D-F) or flowers (G-I). PME activity is expressed as % of the control without PMEI3. Error bars are SD (n=3) and stars refer to the P-value of the t-tests on the comparison with the 0 control or for the boiled samples for the comparison with non-boiled 11.24 µM samples. *:<0.05, **:<0.01, ***:<0.001 , ns : not significant.

Fig. 3 .

 3 Fig. 3. Specific PMEI3 activity against purified PMEs. Relative PME activity measured in purified PME3 (A-C) and PME2 (D-F) preparations. at pH 5 (A-D); 6.3 (B-E) and 7.5 (C-F) in the presence of 0; 1.12 µM; 2.25 µM; 11.24 µM and boiled 11.24 µM PMEI3. PME activity is expressed as % of the control without PMEI3. Error bars are standard deviations (n=3) and stars refer to the P-value of the t-tests on the comparison with the 0 control or for the boiled samples for the comparison with non-boiled 11.24 µM samples. *:<0.05, **:<0.01, ***:<0.001, ns : not significant.

Fig.

  Fig.4. In vivo activity of purified PMEI3. A,B: Representative photographs (A) and primary root lengths (B) of 3-day-old Arabidopsis thaliana seedlings. Curve is a second degree polynomal fit (y = 3,3692x 2 -3,6899x + 1,007; R 2 = 0,9947); IC50 = 0.16 µM. C,D: Representative photographs (C) and fluorescence quantification (D) of COS 647 -labeled roots. 0.2 µM sat and 0.6 µM sat refer to the same samples as for 0.2 µM and 0.6 µM but with an increased detection sensitivity. Two-day-old seedlings were transferred to 0; 0.2 µM or 0.63 µM PMEI3 and incubated for 24h before analysis. Error bars are SD (n > 30 for B and n=5 for D). *** t-test, P-value<0.001.

Acknowledgements

Gladys Cloarec is thanked for assistance in the use of the confocal microscope. Jozef Mravec is thanked for kindly providing the COS 647 probe. The work was financed by ANR projects "Pectosign" and "Homeowall" and the CSC PhD fellowship Nr 201506300059 to FX. The IJPB benefits from the support of Saclay Plant Sciences-SPS (ANR-17-EUR-0007). This work has benefited from the support of IJPB's Plant Observatory technological platforms.

COS 647

labeling of roots

Seedlings were fixed for 10 min in 10% acetic acid and 3.8% formaldehyde in "2F4 buffer" (Haas et al., 2020) and neutralized overnight in 10 mM NH 4 Cl in "2F4 buffer". After washing with 25 mM MES buffer pH5.7, the samples were incubated in the same buffer for 10 min with 1/ 500 COS 647 [START_REF] Mravec | Tracking developmentally regulated postsynthetic processing of homogalacturonan and chitin using reciprocal oligosaccharide probes[END_REF] and, following washing, imaged with a confocal (Zeiss 710). COS 647 was kindly provided by Jozef Mravec (University of Copenhagen, DK).

CRediT authorship contribution statement

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi. org/10.1016/j.tcsw.2022.100080.


Nathalie Bouré

Alexis Peaucelle

Magali Goussot

Bernard Adroher

Ludivine Soubigou-Taconnat

Néro Borrega

Eric Biot

Zakia Tariq

Marie-Laure Martin-Magniette

Véronique Pautot

Nathalie Bouré1

Neŕo Borrega

Veŕonique Pautot

Patrick Laufs

Nicolas Arnaud
email: nicolas.arnaud@inrae.fr

A cell wall-associated gene network shapes leaf boundary domains

Keywords: Arabidopsis, Boundaries, Morphogenesis, Growth Handling

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

INTRODUCTION

Boundaries act both as frontiers to separate adjacent tissues or organs and as organizing centers providing positional clues to control the fate of neighboring cells [START_REF] Dahmann | Boundary formation and maintenance in tissue development[END_REF][START_REF] Irvine | Boundaries in development: formation and function[END_REF]. Thus, boundary domains are required to pattern developing organs correctly. For instance, in animals defects in boundaries lead to developmental abnormalities, including impaired wing or brain development [START_REF] Dahmann | Boundary formation and maintenance in tissue development[END_REF]. In contrast with the determinate development occurring in animals, plants continuously form new aerial growth axes separated from the shoot apical meristem (SAM) to build their architecture. These new growth axes can either produce new branches or give rise to specialized lateral organs, such as leaves or flowers. Independently of their fate, all lateral organs are separated from the meristem by boundary domains, which delimitate cell territories and orchestrate their development [START_REF] Tasaka | Morphogenesis and patterning at the organ boundaries in the higher plant shoot apex[END_REF]. Despite decades of efforts to decipher their functions, plant boundary domains remain an elusive population of cells for which little information is available.

The patterning and maintenance of boundary domains rely on the activity of the CUP-SHAPED COTYLEDON (CUC) transcription factors, which belong to the NAC transcription factor family [START_REF] Aida | Genes involved in organ separation in Arabidopsis: an analysis of the cup-shaped cotyledon mutant[END_REF][START_REF] Vroemen | The CUP-SHAPED COTYLEDON3 gene is required for boundary and shoot meristem formation in arabidopsis[END_REF]. There are three CUC genes in Arabidopsis: CUC1, CUC2 and CUC3. CUC1 and CUC2 mRNA but not CUC3 are targeted by the microRNA MIR164 [START_REF] Laufs | MicroRNA regulation of the CUC genes is required for boundary size control in Arabidopsis meristems[END_REF]. The CUC transcription factors regulate both shoot meristem formation [START_REF] Aida | Shoot apical meristem and cotyledon formation during Arabidopsis embryogenesis: interaction among the CUP-SHAPED COTYLEDON and SHOOT MERISTEMLESS genes[END_REF] and correct organ separation in various developmental contexts [START_REF] Aida | Genes involved in organ separation in Arabidopsis: an analysis of the cup-shaped cotyledon mutant[END_REF][START_REF] Burian | The CUP-SHAPED COTYLEDON2 and 3 genes have a post-meristematic effect on Arabidopsis thaliana phyllotaxis[END_REF][START_REF] Gonçalves | A conserved role for CUP-SHAPED COTYLEDON genes during ovule development[END_REF]. Accordingly, CUC2 and CUC3 are key regulators of leaf shape through their roles on leaf margin development [START_REF] Blein | A conserved molecular framework for compound leaf development[END_REF][START_REF] Hasson | Evolution and diverse roles of the CUP-SHAPED COTYLEDON genes in Arabidopsis leaf development[END_REF][START_REF] Nikovics | The balance between the MIR164A and CUC2 genes controls leaf margin serration in Arabidopsis[END_REF], CUC1 being not expressed during leaf development [START_REF] Nikovics | The balance between the MIR164A and CUC2 genes controls leaf margin serration in Arabidopsis[END_REF]. During leaf development, CUC2 and CUC3 define boundary domains at the leaf margincalled sinusesallowing differential growth to shape the leaf. At the cellular level, the coordinated activity of CUC2/3 transcription factors suppresses growth locally and has a positive effect at a distance on the initiation and maintenance of high growth rate probably via a mechanism involving auxin [START_REF] Bilsborough | Model for the regulation of Arabidopsis thaliana leaf margin development[END_REF]. The CUC2 transcription factor acts through the activation of CUC3 and KLU (also known as CYP78A5), encoding a cytochrome P450, which serves as a molecular relay, and through modulation of auxin signaling pathway [START_REF] Maugarny-Cales | Dissecting the pathways coordinating patterning and growth by plant boundary domains[END_REF]. Acting downstream of CUC2, CUC3 maintains reduced growth of the boundary domains via the control of cell growth through unknown molecular mechanisms [START_REF] Serra | Spatiotemporal control of cell growth by CUC3 shapes leaf margins[END_REF]. Accordingly, cuc2 loss-of-function mutants fail to initiate teeth and in cuc3 loss-of-function mutants teeth growth is not maintained [START_REF] Hasson | Evolution and diverse roles of the CUP-SHAPED COTYLEDON genes in Arabidopsis leaf development[END_REF]. Several hormonal pathways impinge on boundary domain establishment [START_REF] Hepworth | Beyond the divide: boundaries for patterning and stem cell regulation in plants[END_REF]. For instance, brassinosteroids (BRs) have been shown to antagonize boundary domain formation through the downregulation of CUC genes [START_REF] Gendron | Brassinosteroids regulate organ boundary formation in the shoot apical meristem of Arabidopsis[END_REF]. Low BR levels are maintained within boundary domains by the activation of BAS1, a cytochrome P450 involved in BR catabolism, thus leading to the reduced growth of boundary domains [START_REF] Bell | Arabidopsis LATERAL ORGAN BOUNDARIES negatively regulates brassinosteroid accumulation to limit growth in organ boundaries[END_REF]. Auxin also plays a fundamental role during boundary domain establishment, exemplified by its implication in leaf serration development [START_REF] Bilsborough | Model for the regulation of Arabidopsis thaliana leaf margin development[END_REF]. Other regulatory molecules have recently emerged as important regulators of boundary domains. The EPF/EPFL secreted peptides and the ERECTA family receptors contribute to boundary domain formation both during leaf development and ovule initiation, probably through modulation of auxin responses [START_REF] Kawamoto | A peptide pair coordinates regular ovule initiation patterns with seed number and fruit size[END_REF][START_REF] Kosentka | EPFL signals in the boundary region of the SAM restrict its size and promote leaf initiation[END_REF][START_REF] Tameshige | A secreted peptide and its receptors shape the auxin response pattern and leaf margin morphogenesis[END_REF].

In an attempt to identify new actors of boundary domains, we previously performed a genetic suppressor screen of a line overexpressing CUC2 and identified MUR1, coding for a GDP-Dmannose 4,6-dehydratase involved in GDP-L-fucose production. More specifically, we showed that L-fucose contributes to boundary domain establishment in various developmental contexts [START_REF] Gonçalves | GDP-L-fucose is required for boundary definition in plants[END_REF]. Fucose is a hexose incorporated into xyloglucans, rhamnogalacturonan II and arabinogalactans in plant cell walls [START_REF] O'neill | Requirement of borate cross-linking of cell wall rhamnogalacturonan II for Arabidopsis growth[END_REF][START_REF] Van Hengel | Fucosylated arabinogalactan-proteins are required for full root cell elongation in arabidopsis[END_REF] and added to proteins through the activity of specific fucosyltransferases [START_REF] Strasser | Plant protein glycosylation[END_REF].

Recently, SPINDLY (SPY) has been described as a Ofucosyltransferase able to target REPRESSOR OF GA (RGA), a negative regulator of the gibberellin (GA) signaling pathway from the DELLA family [START_REF] Zentella | The arabidopsis O-fucosyltransferase SPINDLY activates nuclear growth repressor DELLA[END_REF], as well as PSEUDO-RESPONSE REGULATOR 5 (PRR5), a core circadian clock component [START_REF] Wang | Nuclear Localized O -Fucosyltransferase SPY facilitates PRR5 proteolysis to fine-tune the pace of arabidopsis circadian clock[END_REF]. spy loss-of-function mutants were originally identified in a genetic screen for plantlet resistant to the GA biosynthesis inhibitor paclobutrazol [START_REF] Jacobsen | Mutations at the SPINDLY locus of Arabidopsis alter gibberellin signal transduction[END_REF]. This mutant displays constitutive GA phenotypes, suggesting that SPY negatively regulates the GA signaling pathway [START_REF] Jacobsen | SPINDLY, a tetratricopeptide repeat protein involved in gibberellin signal transduction in Arabidopsis[END_REF]. The implication that SPY may directly O-fucosylate RGA provides a molecular framework for the function of SPY in the GA signaling pathway. However, as spy mutants do not completely resemble wild-type (WT) plants treated with GA [START_REF] Swain | Altered expression of SPINDLY affects gibberellin response and plant development 1[END_REF], it is likely that SPY also acts through GA-independent pathways. This has been recently shown during root development where SPY regulates root hair patterning in a GAindependent pathway [START_REF] Mutanwad | The Arabidopsis O-fucosyltransferase SPINDLY regulates root hair patterning independently of gibberellin signaling[END_REF]. Accordingly, SPY has been proposed to positively regulate cytokinin (CK) signaling, highlighting a central role in the regulation of GA/CK crosstalk throughout plant development [START_REF] Greenboim-Wainberg | Cross talk between Gibberellin and Cytokinin : the arabidopsis GA response inhibitor SPINDLY plays a positive role in cytokinin signaling[END_REF]. This GA/CK hormonal crosstalk is instrumental in the maintenance of KNOX (Class I KNOTTED1-like homeobox)-dependent meristematic activity [START_REF] Hay | The gibberellin pathway mediates KNOTTED1-type homeobox function in plants with different body plans[END_REF][START_REF] Jasinski | KNOX action in arabidopsis is mediated by coordinate regulation of cytokinin and Gibberellin activities[END_REF], which implies that SPY has a crucial role during SAM development and/or maintenance. In addition to this function in the SAM, several reports show that spy mutants have altered leaf development with little or no serrations at their margins [START_REF] Greenboim-Wainberg | Cross talk between Gibberellin and Cytokinin : the arabidopsis GA response inhibitor SPINDLY plays a positive role in cytokinin signaling[END_REF][START_REF] Maymon | Cytosolic activity of SPINDLY implies the existence of a DELLA-independent gibberellin-response pathway[END_REF], but detailed analysis of the function of SPY in these boundary domains development is lacking.

Here, we investigated the role of SPY in leaf development. SPY is required to maintain restricted growth of the sinus cells. Furthermore, our genetic analysis suggests that SPY acts redundantly with CUC2 and CUC3 to control boundary domain development. At the molecular level, both SPY and CUC2 regulate a common set of genes controlling cell wall properties. Accordingly, we show that CUC2 represses cell growth independently of CUC3, possibly via modifications of cell wall mechanics. Together, these data provide a molecular framework for the role of SPY during boundary domain development whereby SPY and CUC2 act through a common pathway, and reveal a concealed growth repressive function for CUC2 involving cell expansion.

RESULTS

SPY regulates leaf morphogenesis

Several spy mutant alleles [START_REF] Greenboim-Wainberg | Cross talk between Gibberellin and Cytokinin : the arabidopsis GA response inhibitor SPINDLY plays a positive role in cytokinin signaling[END_REF][START_REF] Maymon | Cytosolic activity of SPINDLY implies the existence of a DELLA-independent gibberellin-response pathway[END_REF][START_REF] Steiner | The arabidopsis O -linked Nacetylglucosamine transferase SPINDLY interacts with class I TCPs to facilitate cytokinin responses in leaves and flowers[END_REF] have been reported to result in leaves with little or no serration, but this leaf phenotype was never fully characterized. Therefore, we quantified the leaf shape of the loss-of-function spy-3 mutant. The spy-3 mutant contains a G>A transition changing a glycine to a serine residue at position 593 of the SPY protein, resulting in a non-functional protein [START_REF] Jacobsen | SPINDLY, a tetratricopeptide repeat protein involved in gibberellin signal transduction in Arabidopsis[END_REF] (Fig. S1). spy-3 mutant and WT mature leaves displayed similar blade lengths but their widths, and hence their blade area, were smaller in spy-3 (Fig. S2A-C). Both morphometrics and dissection index (DI) calculations, global descriptors of leaf complexity [START_REF] Gonçalves | GDP-L-fucose is required for boundary definition in plants[END_REF], showed that spy-3 leaves are smoother than WT leaves (Fig. 1A-C). To investigate further, we measured the shape of the second tooth (as the ratio between tooth height and tooth width) in this dataset, revealing that WT serrations are pointier than spy-3 serrations (Fig. 1D). Two additional alleles, namely spy-22 and spy-23 (Figs S1 andS3), had smoother leaves than the WT with less-pronounced serrations, ruling out the possibility of spy-3-specific bias on leaf morphology. Furthermore, a spy-22 mutant expressing the pSPY::SPY-FLAG construct had a restored WT leaf shape phenotype (Fig. S3). Taken together, our results show that SPY is involved in the development of leaf serrations.

2

SPY is required for growth serration maintenance As mature leaf shape results from the sum of processes occurring at different developmental times, it is important to access growth kinetic data of the spindly mutant to conclude about the precise roles of SPY during leaf shape development. To do this, we reconstructed detailed developmental trajectories of spy-3 lossof-function mutant leaves using MorphoLeaf software from a set of leaves from ranks 11, 12 and 13. As both the leaf initiation rate and the leaf growth rates are comparable between spy-3 and the WT up to 6 mm length (Fig. S4), we chose to limit our morphometric analysis to this early stage and use leaf blade length as a proxy for leaf developmental stage. Tooth 1 height was drastically reduced in spy-3 from early stages and never reached WT values (Fig. 2A), whereas tooth 1 width was not modified at early developmental stages (up to 3 mm) (Fig. 2B), resulting in sharper teeth in the WT (Fig. 2C). Together, our data suggest that SPY is involved in teeth growth maintenance. As maintenance of tooth growth is associated with the definition of the leaf boundary domain at the sinus [START_REF] Hasson | Evolution and diverse roles of the CUP-SHAPED COTYLEDON genes in Arabidopsis leaf development[END_REF][START_REF] Maugarny-Cales | Dissecting the pathways coordinating patterning and growth by plant boundary domains[END_REF], we analyzed sinus angle as a local parameter related to local growth repression at the sinus. Although the evolution of the sinus angle for the distal sinus of tooth 1 throughout its development had comparable dynamics both in spy-3 and in WT, the spy-3 sinus angle was always less pronounced than the WT sinus angle (Fig. 2D). These data suggest that the alteration of leaf shape may partially result from local defects in boundary domain definition in the spy-3 mutant.

SPY is required to inhibit sinus cell growth during leaf development

During leaf development, spatial differences in cell growth rate sustain tooth outgrowths [START_REF] Serra | Spatiotemporal control of cell growth by CUC3 shapes leaf margins[END_REF], which are integrated at the leaf level leading to final leaf shape. As sinus angle was altered in the spy-3 mutant compared with the WT, we set out to analyze the sinus at the cellular level. The distribution of cell surface in 3D acquisitions for tooth 1 of leaves from ranks 11, 12 and 13 was hence measured in both genotypes (Fig. 2E). We focused on the first distal sinus to limit bias due to the mechanical constraints of the previous tooth outgrowth. The shape of the tooth and the depth of the sinus were different between spy-3 and WT in these 3D acquisitions, which confirmed the data from the 2D developmental kinetics. In order to assess the size of the sinus cells specifically, we analyzed the Gaussian curvature of the 3D projected surface. Sinuses were identified as surface areas that exhibited a negative Gaussian curvature (Fig. S5) (Serra and Perrotrechenmann, 2020) and the surfaces of sinus-specific cells were measured from independent leaves. Sinus cell surfaces were then plotted according to tooth width for both spy-3 and WT leaves (Fig. 2F). For teeth 1 up to 150 µm wide, sinus cell sizes of early leaf primordia were not significantly different between spy-3 and the WT. Later, for teeth ranging from 150 to 250 µm and 250 to 500 µm, sinuses of spy-3 mutant leaves were composed of larger cells than those in WT. Our data show that SPY is required to maintain restricted sinus cell growth at late stages of tooth development. Interestingly, the cuc3-105 loss-of-function mutant has been described to have bigger cells at the sinus due to local release of cell growth [START_REF] Serra | Spatiotemporal control of cell growth by CUC3 shapes leaf margins[END_REF]. Thus, spy-3 and cuc3-105 mutants display very similar sinus cell phenotypes, suggesting that SPY and CUC transcription factors have similar roles in sinus cell development. This leads to the question of whether they function through a common pathway to coordinate growth restriction of sinus cells.

SPY, CUC2 and CUC3 act redundantly during boundary domain development

To check whether SPY acts in a CUC-dependent pathway to define boundaries, we first analyzed CUC/SPY genetic interactions using CUC2g-m4, a mutated version of CUC2 with altered MIR164-target site, leading to a local overexpression of CUC2 mRNA and very serrated leaves [START_REF] Maugarny-Cales | Dissecting the pathways coordinating patterning and growth by plant boundary domains[END_REF][START_REF] Nikovics | The balance between the MIR164A and CUC2 genes controls leaf margin serration in Arabidopsis[END_REF]. CUC3, which acts downstream of CUC2, has already been shown to reduce leaf serration of the CUC2g-m4 line [START_REF] Hasson | Evolution and diverse roles of the CUP-SHAPED COTYLEDON genes in Arabidopsis leaf development[END_REF]. CUC2 overexpression leaf phenotypes (measured by DI calculations) were suppressed in spy-3 and cuc3-105 backgrounds (Fig. 3A). Furthermore, we used the mir164a loss-of-function mutant as an alternative way of increasing CUC2 levels and found that the over-serrated leaf phenotype of mir164a was suppressed by the spy-3 mutation (Fig. 3A). Together, these genetic data suggest that CUC2 requires SPY or CUC3 activity to control leaf serration development.

Given that spy3 and cuc3-105 mutants have very similar phenotypes both at the organ and at the cellular scales, and that they both suppress the CUC2 highly serrated leaves phenotype, we hypothesize that CUC3 and SPY act in the same genetic pathway to control serration development. To test this idea, we generated a double spy3 cuc3-105 mutant and analyzed its leaf developmental trajectory (Fig. 3B). As CUC3 maintains tooth growth by locally inhibiting the growth of sinus cells, we used tooth height of the first tooth as a proxy of CUC3 activity. Strikingly, when the tooth height of the first tooth was measured for different tooth width classes, cuc3-105 and spy-3 displayed comparable quantitative phenotypes, whereas the two mutations together had an additive effect on tooth height, suggesting that they act independently on leaf shape (Fig. 3B). Accordingly, spy-3 and cuc3-105 mutations also had an additive effect on the over-serrated CUC2gm-4 phenotype (Fig. 3A). These data imply that alternative routes exist to restrict growth at the sinus independently of CUC3. In order to decipher the relative contribution of SPY, CUC2 and CUC3 to boundary cell growth, we decided to analyze their roles during cotyledon rather than during leaf development because no serrations are initiated when CUC2 activity is altered. Both double mutants spy-3 cuc3-105 and spy-3 cuc2-1 showed stronger cotyledon fusion phenotypes compared with the corresponding simple mutants showing that SPY acts redundantly with CUC2 and CUC3 to define boundaries (Table 1, Fig. S6). This result indicates that SPY acts in different developmental contexts and suggests that SPY contributes more generally to the definition of developmental boundary domains.

SPY and CUC2 act through a common molecular network to restrict sinus cell growth

Our genetic analysis suggests that CUC2, CUC3 and SPY redundantly restrict boundary domain growth. As we saw a local growth defect in the spy-3 mutant, we first tested whether SPY was expressed together with the CUC genes within the leaf boundary domains. We used the pSPY::SPY-GFP reporter line crossed with either pCUC3::CFP or pCUC2::RFP to monitor simultaneously SPY localization and CUC gene expression patterns. Although SPY is broadly expressed in leaf epidermis at early developmental stages, it overlapped with CUC2 and CUC3 within leaf boundary domain cells (Fig. S7). In addition, we show that even though CUC2 levels vary greatly between cuc2-1 mutant, the WT and the CUC2gm-4 line, the expression levels of SPY do not change, suggesting that SPY expression is not regulated by CUC2 (Fig. S8).

As our genetic analysis shows that CUC2 activity requires SPY, we next investigated how this translates at the molecular level.

Previous transcriptomic analysis identified genes differentially expressed in spy-3 compared with WT [START_REF] Qin | SPINDLY, a negative regulator of gibberellic acid signaling, is involved in the plant abiotic[END_REF]. To identify CUC2 downstream elements, we performed transcriptomic profiling on whole seedlings of an activated CUC2 dexamethasone (DEX)-inducible line. Among the differentially expressed genes, we found that about 20% of the genes that were upregulated in the spy-3 mutant were downregulated upon CUC2 induction (Table S3). Indeed, we identified 2569 genes that were downregulated 6 h after CUC2 induction (FDR<0.05) and, of these, 100 were upregulated in the spy-3 mutant, which represents a significant proportion of the 493 genes upregulated in total in the spy-3 mutant (hypergeometric test, P=6.06E-14) (Fig. 4A). Gene ontology analysis performed using this set of 100 genes revealed an enrichment in genes related to plant-type cell wall [GO:0009505, enrichment 9.65, raw P=4.31E-05 (Fisher exact test), FDR=4.45E-02] with a function related to cell wall organization and biogenesis [GO:0071554, enrichment 7.38, raw P=1.13E-06 (Fisher exact test), FDR=6.75E03]. Among these genes, we identified several genes coding for xyloglucan endotransglucosylase/hydrolases (XTH4, XTH15, XTH18 and XTH19), arabinogalactan proteins (AGP4, AGP7, AGP9 and AGP12), as well as two genes coding for expansin-like proteins (EXLA1 and EXLA2). These genes contribute to cell wall loosening. Indeed, XTH18 and XTH19 were both previously shown to be involved in the control of hypocotyl growth, as overexpressing lines for XTH18 and XTH19 both promoted hypocotyl growth in the dark [START_REF] Miedes | Xyloglucan endotransglucosylase/hydrolase (XTH) overexpression affects growth and cell wall mechanics in etiolated Arabidopsis hypocotyls[END_REF]. In addition, AGP4, AGP7, AGP9 and AGP12 were identified in a large-scale gene expression pattern study on fast-growing seedlings as robust markers of growth [START_REF] Kohnen | Neighbor detection induces organ-specific transcriptomes, revealing patterns underlying hypocotyl-specific growth[END_REF]. Similar observations for XTH proteins, EXLA2 overexpression is able to increase growth in hypocotyls grown in the dark [START_REF] Boron | Over-expression of AtEXLA2 alters etiolated arabidopsis hypocotyl growth[END_REF]. In addition, a biomechanical analysis of an EXLA2-overexpressing line showed that cell wall resistance was decreased in the hypocotyl, suggesting that EXLA2 may modify the cell wall organization and composition [START_REF] Boron | Over-expression of AtEXLA2 alters etiolated arabidopsis hypocotyl growth[END_REF].

Importantly, we have independently measured mRNA levels within leaf margins using laser-assisted microdissection in both WT and the CUC2gm-4 line followed by transcriptomic analysis by RNA sequencing (RNA-seq). Our data confirm, first, that the cell wall remodeling genes identified so far are expressed at the leaf margin of WT plants, and then, that among these genes, AGP12, EXLA2, XTH18 and XTH19 had significantly reduced mRNA levels in CUC2gm-4 leaf margins (Table S1). Therefore, the combined analysis of our transcriptomic study suggests that cell wallremodeling enzymes are the functional elements acting downstream of CUC2 and SPY.

CUC2 represses cell expansion independently of CUC3

To test whether CUC2 overexpression does indeed inhibit cell expansion, we analyzed the effect of CUC2 ectopic activation upon DEX treatment during the elongation of hypocotyls grown in the dark, which results mostly from cell elongation rather than [START_REF] Gendreau | Cellular basis of hypocotyl growth in Arabidopsis thaliana[END_REF]. First, we independently validated that the expression level of EXLA1 and EXLA2, two cell wall-remodeling genes, was inhibited upon CUC2 activation in this system model in accordance with our transcriptomic data (Fig. 4B). We then analyzed the effect of CUC2 activation on cell behavior and tissue phenotype. Under CUC-activation conditions, hypocotyls grown in the dark were significantly shorter and exhibited smaller hypocotyl epidermal cells compared with noninduced conditions (Fig. 4B,C). These results are in agreement with the reduced levels of expression of genes linked with cell wall plasticity after CUC2 induction. Taken together, our data suggest that overexpression of the transcription factor CUC2 is sufficient to repress a set of genes that have functions relating to cell wall loosening, providing a plausible molecular framework for the activity of CUC2.

The activity of CUC2 is mediated by CUC3, which acts as a molecular relay [START_REF] Maugarny-Cales | Dissecting the pathways coordinating patterning and growth by plant boundary domains[END_REF]. As CUC2 and CUC3 redundantly control boundary domain development, it is possible that CUC2 alters cell elongation independently of CUC3. As no serrations are initiated in a cuc2 loss-of-function mutant, this hypothesis has been difficult to test during leaf development. Here, we have the opportunity to test whether the CUC2-dependent growth repression function that we have highlighted with the DEX-induced CUC2 line in dark-grown hypocotyl depends on CUC3. When CUC2 is induced by DEX in absence of CUC3, dark-grown hypocotyls are shorter with smaller epidermal cells than those in non-induced conditions (Fig. 5A,B), showing that CUC2 acts independently of CUC3 on cell elongation in the dark-grown hypocotyl model. In order to check whether this reduction of growth is associated with changes in cell wall-related gene expression, we monitored their mRNA accumulation upon CUC2 induction in dark-grown hypocotyls. In a cuc2-3 cuc3-105 mutant background, CUC2 induction is sufficient to drastically reduce the accumulation of XTH18, XTH19, EXLA1 and EXLA2 mRNA, showing that CUC2 inhibits their expression independently of CUC3 (Fig. 5C). These results suggest that changes in cell wall gene expression triggered by CUC2 may counteract cell expansion.

Cell wall mechanics at the leaf margin

Our molecular data support a role for CUC2 in the control of cell wall properties. In order to check whether this is also the case in the organs where CUC2 is expressed, we used atomic force microscopy (AFM) to measure the cell wall stiffness of sinus cells (where CUC2 is expressed) and compare with the cell wall stiffness of tooth cells (where CUC2 is not expressed) (Fig. S9). In young leaf primordia, when the tooth starts to emerge, as shown by topographical images obtained using AFM, cells of the margins do not display different sizes between sinus and tooth domains in WT (Fig. 6A,B). Yet, sinus and tooth show a differential stiffness, cell sinus walls being stiffer than the cell tooth walls (Fig. 6A,B,E). This is consistent with both the expression pattern of CUC2 and our molecular data showing that CUC2 inhibits the expression of genes known to promote cell wall loosening. To validate these data, we quantified the Apparent Young's modulus (Ea) to evaluate the elasticity of the leaf margin tissue in another set of experiments analyzing cell wall stiffness in 12 young dissected leaves of the WT. This showed once again that sinus domains are consistently stiffer than tooth domains (Fig. 6F). As sinus and tooth definition trigger local topographical changes, it is possible that the changes in cell wall stiffness observed at the sinus may be reinforced by mechanical feedback. To test whether CUC2 can promote cell wall stiffness independently of topographical tissue changes, we ectopically expressed CUC2 in dark-grown hypocotyls using the DEX-inducible p35S:CUC2-GR line. In this experiment, no tissue deformation occurred in the hypocotyl, but we still measured stiffer cell walls when CUC2 was overexpressed (Fig. S10), suggesting that CUC2 expression is sufficient to trigger changes in cell wall stiffness. Together, these results provide a mechanical framework for the development of boundary domains, which is in agreement with the quantitative description of leaf margin development in WT and observations in mutants harboring the null allele cuc2-1, in which local growth repression at the sinus preceding outgrowth of the tooth is specifically lost [START_REF] Biot | Multiscale quantification of morphodynamics: MorphoLeaf software for 2D shape analysis[END_REF].

As CUC2 is required to initiate teeth, it is difficult to use cuc2 loss of function to assess whether CUC2-dependent inhibition of cell expansion occurs also at the leaf margin. The spy-3 mutant initiates teeth but their growth is not maintained owing to local cellular changes at the sinus during teeth development. spy-3 therefore uncouples CUC2 functions as SPY leads to repression of the expression of a common set of genes with CUC2 that are related to the cell wall remodeling. We therefore used the spy-3 mutant to check whether the reduced expression of cell wall genes acting downstream of CUC2 is sufficient to change the mechanical properties of the cell wall at the leaf margin. spy-3 mutant young leaf primordia showed a reduction of the differential stiffness between tooth and sinus observed in the WT (Fig. 6C-E). We confirmed this by quantifying the Apparent Young's modulus (Ea) in another set of experiments analyzing cell wall stiffness in seven young spy-3 dissected leaves (Fig. 6F). Although we highlight a significant difference between the sinus and tooth Ea values in spy-3 leaves, differential stiffness was reduced compared with the sinus and tooth Ea values of the WT. These data support the idea that the set of genes commonly downregulated by CUC2 and SPY may impact cell wall stiffness at the leaf margin. Furthermore, it is important to note that stiffness changes precede the sinus cell morphological changes we observed in the spy-3 mutant and occur at early stages of leaf development, thus providing a mechanical framework for the subsequent differential growth.

DISCUSSION

Plant development and architecture rely on the iterative production of lateral organs separated from the stem cell pool by boundary domains. Here, through the characterization of leaf margin phenotypes of spy mutants at multiple scales, we highlight a role for SPY in maintaining local growth repression of sinus cells redundantly with CUC transcription factors. Furthermore, we present evidence that SPY and CUC2 act through a common molecular network involving the reduction of the expression of genes associated with cell wall loosening. Accordingly, using hypocotyls grown in the dark together with a CUC2-inducible system, we show that CUC2 restricts cell expansion. This mode of action is supported by the fact that teeth and sinuses of young leaf primordia display differential cell wall stiffness: sinuses, where CUC2 is expressed, show more rigid cell walls. Accordingly, cell wall stiffness is lower in the sinuses of spy-3 leaves compared with the WT. Our data support a model whereby CUC2 can inhibit cell expansion independently of CUC3 acting through the repression of cell wall-relaxing genes.

SPY is a component of the boundary domain network

Here, we have shown that spy mutants display boundary domain defects during leaf development, resulting in leaves with reduced serrations. Leaf development is a complex and integrated process, which results from both global and local changes throughout developmental time. Therefore, we cannot rule out the possibility that the local changes we observed in the spy-3 mutant may result from growth rate changes at the whole-organ level. Indeed, GA is involved in the proliferation/differentiation switch that occurs during leaf development. In Arabidopsis, DELLAs have been shown to increase the transcript levels of KIP-RELATED PROTEIN 2 (KRP2), SIAMESE (SIM) and SIM RELATED 1 and 2 (SMR1 and SMR2), which are all involved in inhibition of cell cycle progression [START_REF] Achard | Gibberellin signaling controls cell proliferation rate in Arabidopsis[END_REF]. SPY has been shown to activate RGA [START_REF] Zentella | The arabidopsis O-fucosyltransferase SPINDLY activates nuclear growth repressor DELLA[END_REF], and in spy mutants, cell cycle progression is less restricted, triggering a faster differentiation of the leaf blade. In addition, GA20ox1 overexpression results in increased levels of bioactive GA and the corresponding lines display large leaves with more and larger cells [START_REF] Gonzalez | Increased leaf size: different means to an end[END_REF]. Together, these results suggest that GA levels control both cell expansion and cell proliferation in leaves. In addition to its role in GA signaling repression, former studies indicated that SPY has a role in CK signaling promotion [START_REF] Greenboim-Wainberg | Cross talk between Gibberellin and Cytokinin : the arabidopsis GA response inhibitor SPINDLY plays a positive role in cytokinin signaling[END_REF][START_REF] Steiner | The arabidopsis O -linked Nacetylglucosamine transferase SPINDLY interacts with class I TCPs to facilitate cytokinin responses in leaves and flowers[END_REF]. However, it was shown that CK promotes cell proliferation and that reduced CK levels lead to a decrease in cell divisions and consequently to smaller organs [START_REF] Holst | Enhanced cytokinin degradation in leaf primordia of transgenic Arabidopsis plants reduces leaf size and shoot organ primordia formation[END_REF]. Moreover, a recent study revealed that a CK/GA balance is responsible for leaf complexity in tomato plants as it controls the morphogenesis/differentiation switch [START_REF] Israeli | Coordinating the morphogenesis-differentiation balance by tweaking the cytokinin-gibberellin equilibrium[END_REF]. Hence, it is also possible that CK signaling is partially impaired in the spy-3 8 mutant and, as a consequence, modifies the serration growth dynamics. Although we cannot exclude the possibility that SPY impacts global leaf development in multiple ways, we have seen that the spy-3 mutation results in local changes in cell size, which indicate a SPY function in boundary definition. Accordingly, SPY acts redundantly with CUC2 and CUC3 to promote cotyledon separation, which is a developmental process in which cell expansion and cell division mostly do not occur simultaneously.

It is also important to note that, although SPY and CUCs are commonly expressed within leaf boundary domain cells, SPY expression in not restricted to these domains. This observation suggests that sinus-localized defects in the spy-3 mutant may reflect different activity of SPY depending on the tissue where it is expressed and/or the possibility that leaf cells do not respond uniformly to SPY activity alterations.

SPY and CUC2 act through a common cell wall-related molecular network

At the organ level, the origin of serrations has long been debated [START_REF] Bilsborough | Model for the regulation of Arabidopsis thaliana leaf margin development[END_REF][START_REF] Kawamura | Mechanisms of leaf tooth formation in Arabidopsis[END_REF][START_REF] Nikovics | The balance between the MIR164A and CUC2 genes controls leaf margin serration in Arabidopsis[END_REF]. Recently, morphometrics was used to reconstruct leaf developmental growth trajectories of the WT and cuc2-1 loss-offunction mutants, which do not initiate serrations [START_REF] Biot | Multiscale quantification of morphodynamics: MorphoLeaf software for 2D shape analysis[END_REF]. This work shows that local growth repression arises first at the sinus of young WT leaf primordia, where CUC2 is expressed, then, subsequently, outgrowth appears at a distance. As neither growth repression nor teeth initiation was observed in the cuc2-1 mutant, it was concluded that CUC2 is a key regulator for the coordination of cellular processes leading to the development of serrations. Our work provides a molecular framework for the growth repression function of CUC2. Indeed, we present evidence that CUC2 and SPY downregulate a set of common genes involved in cell wall loosening. Accordingly, we demonstrate that CUC2 inhibits cell elongation and that this cellular mode of action is accompanied with downregulation of transcripts encoding expansin-like and xyloglucan endotransglucosylases/hydrolases, which have been reported to be sufficient to promote hypocotyl cell elongation when overexpressed [START_REF] Boron | Over-expression of AtEXLA2 alters etiolated arabidopsis hypocotyl growth[END_REF][START_REF] Miedes | Xyloglucan endotransglucosylase/hydrolase (XTH) overexpression affects growth and cell wall mechanics in etiolated Arabidopsis hypocotyls[END_REF]. Unfortunately, no growth phenotypes have been reported for lossof-function mutants for these genes, probably because of the high level of redundancy or the deleterious effects of multiple pleiotropic mutations.

Additionally, we demonstrate here that CUC2 can inhibit cell expansion independently of CUC3. Our current model for CUC2 activity states that CUC2 is expressed early during leaf development and triggers serration development through CUC3 and KLUH, which act as molecular relays [START_REF] Maugarny-Cales | Dissecting the pathways coordinating patterning and growth by plant boundary domains[END_REF]. Accordingly, CUC3 has been shown to inhibit cell expansion of sinus cells, hence contributing to shaping of the leaf [START_REF] Serra | Spatiotemporal control of cell growth by CUC3 shapes leaf margins[END_REF]. Here, we completed this model by adding another route for the growth repression of sinus cells. As CUC2 can regulate cell expansion independently of CUC3, it is probable that CUC2 contributes also to the local growth repression process per se. This reveals an entangled role for CUC2 in coordinating patterning and cell growth to define boundary domains.

Cell wall mechanics at the leaf margin

Our data show that sinuses and teeth display differential cell wall stiffness even at early stages of leaf development. This differential stiffness is reduced in the spy-3 mutant, in which sinus cell walls resemble teeth cell walls. Here, the spy-3 mutant allows us to differentiate CUC2 functions as SPY and CUC2 act on a common molecular network related to cell wall loosening. Our work reveals the contribution of cell wall mechanics to morphogenesis: local cell wall parameters will greatly impinge on the growth of the whole organ. Moreover, CUC3 has been shown to act downstream of CUC2 [START_REF] Maugarny-Cales | Dissecting the pathways coordinating patterning and growth by plant boundary domains[END_REF], and its expression is promoted by mechanical stresses [START_REF] Fal | Interplay between miRNA regulation and mechanical stress for CUC gene expression at the shoot apical meristem[END_REF]. It is therefore tempting to propose a model in which CUC2 activity induces mechanical stress at the margins, which could then trigger CUC3 expression to serve as a relay for local growth repression. Further experiments will be needed to provide a comprehensive view of the integration of hormonal, genetic and mechanical stress in leaf development and boundary domain development in general.

MATERIALS AND METHODS

Plant material and growth conditions

All plants used in this study were in the Columbia (Col-0) background except the cuc2-1 mutant which was originally obtained in the Landsberg (Ler-0) background and back-crossed five times to Col-0. For morphometric analysis, seeds were immersed in distilled water for 2 days in the dark at 4°C before sowing on soil. Then, plants were grown under short-day conditions [6 h day (21°C, hygrometry 65%, light 120 µM/m 2 /s), 1 h dusk (21°C, hygrometry 65%, light 80 µM/m 2 /s), 16 h night (18°C, hygrometry 65%, dark conditions), 1 h dawn (19°C, hygrometry 65%, light 80 µM/m 2 /s)]. For in vitro cultures, seeds were sown on Arabidopsis medium [START_REF] Gonçalves | GDP-L-fucose is required for boundary definition in plants[END_REF], stratified for 48 h in the dark at 4°C then transferred to long-day conditions (21°C, 16 h day/8 h night, light 50 µM/m 2 /s).

Morphometric analysis

For morphometric analysis of mature leaves, leaves from ranks 11, 12 and 13 from 6-week-old plants were harvested and glued on a paper sheet prior to scanning using a Perfection V800 Photo scanner (Epson) at 1600 dpi. For morphometric analysis of developing leaves, young leaf primordia (ranks 11, 12 and 13) were dissected using a stereomicroscope throughout development starting at day 22 after sowing. Leaves were mounted between a slide and a coverslip in a buffer containing 10 mM Tris HCl, pH 8.5, and 0.01% (v/v) Triton X-100 and imaged using an Axio Zoom.V16 microscope (Carl Zeiss Microscopy). Depending on the developmental stage imaged, either the chlorophyll fluorescent signal or the brightfield signal were collected. Leaf silhouettes and measurements were obtained using MorphoLeaf software, which allows semi-automatic leaf segmentation and the extraction of relevant biological parameters [START_REF] Biot | Multiscale quantification of morphodynamics: MorphoLeaf software for 2D shape analysis[END_REF]. Output data analysis, statistics and plots were generated using R software (http://www.R-project.org/) and the graphics package ggplot2. For all boxplots, the lower box limit represents the 25th percentile (Q1) and the upper box limit represents the 75th percentile (Q3); the median (Q2) is represented by the centre line; whiskers indicate the minimum and maximum values in the data [respectively, Q1-1.5*interquartile range (IQR) and Q3+1.5*IQR].

Confocal imaging

For quantification of cellular parameters, we used the pPDF1::mCitrine-KA1 line [START_REF] Stanislas | A phosphoinositide map at the shoot apical meristem in Arabidopsis thaliana[END_REF] in order to visualize the plasma membrane in the leaf epidermis. Col-0 and spy-3 plants (26-31 days old) containing the pPDF1::mCitrine-KA1 construct were grown under short-day conditions prior to dissecting, mounting in a buffer containing 10 mM Tris HCl, pH 8.5, and 0.01% (v/v) Triton X-100 and direct imaging with a Leica SP5 inverted microscope (Leica Microsystems). Samples were excited using a 514 nm laser and fluorescence was collected with a hybrid detector at between 569 and 611 nm. TIF images were rotated using the TransformJ plugin of ImageJ. Subsequent cell segmentations, cell curvature and cell surface area measurements were then obtained using MorphoGraphX (MGX) [START_REF] De Reuille | MorphoGraphX: a platform for quantifying morphogenesis in 4D[END_REF] software (http://www.mpipz.mpg.de/ MorphoGraphX/). Cells corresponding to tooth sinus were identified as cells displaying a fully negative signal when projecting a 15 µm-neighboring Gaussian curvature [START_REF] Serra | Spatiotemporal control of cell growth by CUC3 shapes leaf margins[END_REF]. The pSPY::SPY-GFP, pCUC3::CFP and pCUC2::RFP reporter lines were imaged with a Leica SP5 inverted microscope (Leica Microsystems). GFP was excited using a 488 nm laser and fluorescence was collected with a hybrid detector at between 500 and 530 nm. RFP was excited at 561 nm and detected with a PMT detector within 570 and 635 nm. CFP was excited at 458 nm and detected with a PMT detector within 460 and 475 nm.

Transcriptomic analysis RNA samples

For RNA-seq assays, p35S:CUC2-GR seedlings were grown in liquid Arabidopsis medium with constant shaking. After 10 days of growth under constant light, seedlings were treated with DEX or mock solution for 6 h and then snap-frozen in liquid nitrogen. DEX (Sigma-Aldrich, D1756) was dissolved in 100% ethanol and used at a final concentration of 10 µM. Total RNA extraction was performed with the mirVana extraction kit (Ambion) following the manufacturer's recommendations.

Leaf margins from WT and the CUC2g-m4 line grown for 3 weeks in shortday conditions were microdissected in triplicate with the ZEISS PALM MicroBeam using a Fluar 5×/0.25 M27 objective. Young leaves (≈1-2 mm long) from ranks 6 and 7 were dissected, placed onto MMI membrane slides and microdissected leaf margins (defined as proximal teeth without differentiated trichomes) were collected in Zeiss adhesive caps. For every biological replicate, approximately ten leaf margins were collected. Total RNA extraction was performed using the Arcturus PicoPure RNA Isolation Kit (Thermo Fisher Scientific), following the manufacturer's instructions and RNA quality was controlled using the Agilent RNA 6000 Pico Kit.

RNA-seq libraries

RNA-seq libraries were constructed by the POPS platform (IPS2) using the TruSeq non-stranded mRNA library prep kit (Illumina) according to the supplier's instructions. The libraries were sequenced in paired-end reads (PE, 2×100 bases) on an Illumina HiSeq 2000 to generate a mean of 30 million of PE reads per sample. A quality control process removed PE reads with Qscore<20, length<30 bases and ribosomal reads.

Bioinformatic analyses

Filtered PE reads were mapped using Bowtie 2 [START_REF] Langmead | Fast gapped-read alignment with Bowtie 2[END_REF] with the -local option against the Arabidopsis thaliana transcriptome. The 33,602 genes were extracted from TAIR v10 database; 95% of PE reads were associated with one gene unambiguously, 2% were removed for multihits. Genes with <1 read per million in at least half of the samples were discarded. The resulting raw count matrix was fed into edgeR [START_REF] Robinson | edgeR: a Bioconductor package for differential expression analysis of digital gene expression data[END_REF] for differential expression testing by fitting a negative binomial generalized log-linear model (GLM) including a condition factor and a replicate factor to the TMM-normalized read counts for each gene. We performed pairwise comparisons of each of the DEX-treated condition to the control condition. The distribution of the resulting P-values followed the quality criterion described by [START_REF] Rigaill | Synthetic data sets for the identification of key ingredients for RNA-seq differential analysis[END_REF]. Genes with an adjusted P-value (FDR, Benjamini-Hochberg adjustment) below 0.05 were considered as differentially expressed.

Dark-grown hypocotyl measurements

For dark-grown hypocotyl experiments, seeds were surface-sterilized and subsequently dispatched on 1% agar (w/v) Arabidopsis media with 10 µM DEX (Sigma-Aldrich, D1756) or mock treatment. After 48 h in the dark at 4°C, plates were transferred to a growth chamber for 6 h (21°C, light 50 µM/ m 2 /s) before being placed vertically in the dark at 21°C. Plates were scanned after 72 h of dark growth using a Perfection V800 Photo scanner (Epson) at 1600 dpi. Hypocotyl sizes were measured using the NeuronJ plugin from Fiji and data were analyzed using R software (http://www.R-project.org/).

Expression data

Total RNA was isolated using the RNeasy Plant Mini Kit (QIAGEN) following the manufacturer's recommendation for plant tissue. Reverse transcription was performed using RevertAid H Minus M-MuLV Reverse transcriptase (Fermentas/Thermo Fisher Scientific) followed by a RNase H treatment was performed for 20 min 37°C to eliminate DNA-RNA duplexes. Real-time PCR analysis was performed on a 384-well QuantStudio™ 5 Real-Time PCR System, using the SsoAdvanced Universal SYBR Green Supermix with the following PCR conditions: 95°C 3 min; (95°C 15 s; 63°C 30 s)×40 cycles. Raw data were analyzed using QuantStudio Design and Analysis software v2.2. Primers used for real-time PCR analysis are available in Table S2. Expression data were normalized using the ΔΔCt method using at least two independent reference genes.

AFM

For mechanical characterization of the leaf cell wall, WT and spy-3 plants were grown on soil in short-day conditions. About 100-to 200 µm-long young leaf primordia from rank higher than 11 were hand-dissected under a stereomicroscope and collected. For mechanical characterization of darkgrown hypocotyl cell wall of plants overexpressing CUC2, 48-h-old seedlings from the p35S:CUC2-GR line and WT grown in the dark on 1 µM DEX were collected. Samples were fixed in low melting agarose with blade facing the AFM tip following the protocol described by [START_REF] Peaucelle | AFM-based mapping of the elastic properties of cell walls: at tissue, cellular, and subcellular resolutions[END_REF]. Samples were plasmolysed by immersion in 10% (m/v) sorbitol. An AFM cantilever loaded with a 1 μm diameter tip was used in these measurements and scanned 100 μm×30 μm areas with a fixed force leading to a maximum indentation value of 800 nm with a speed of 40 µms -1 . The measurement of the rigidity constant was performed only on the second cantiliever used as a reference tip as described by [START_REF] Peaucelle | AFM-based mapping of the elastic properties of cell walls: at tissue, cellular, and subcellular resolutions[END_REF]. The relative stiffness was used as a proxy of cell wall stiffness, which is relative to the rigidity constant of the cantilever used. Apparent Young's modulus was determined by a Hertzian indentation model on each indentation point. Cell topography was reconstructed using the height at each point of contact. Data were analyzed and maps were plotted using MATLAB.

Fig. 1 .

 1 Fig. 1. Morphometric analysis of spy-3 mature leaf shape. (A) WT and spy-3 mutant rosette from plants grown in short-day conditions for 6 weeks. Representative silhouettes from mature leaves from ranks 11-13 are also represented. Scale bars: 1 cm. (B) Mean shape of mature leaves of WT and spy-3 mutant. Scale bar: 1 cm. (C) Quantification of alpha-hull normalized dissection index for WT and spy-3 mature leaves. (D) Quantification of the shape of the second tooth from WT and spy-3 mature leaves. (B-D) WT (n=19) and spy-3 (n=20) 6-week-old leaves grown in short-day conditions, ranks 11-13. ***P<0.0001 (one-tailed unpaired Student's t-test).

Fig. 2 .

 2 Fig. 2. Developmental kinetics and cell size quantification during spy-3 serration development. (A) First tooth height plotted against blade length for WT and spy-3. (B) First tooth width plotted against blade length for WT and spy-3. (C) Tooth shape of the first tooth, calculated as tooth height over tooth width plotted against blade length for WT and spy-3. (A-C) Leaf ranks 11-13 dissected at different stages of development from WT (n=190) and spy-3 (n=194) plants grown in short-day conditions were used. Each tooth is represented by a dot, and a LOESS curve is shown for visual interpretation. (D) Mean first sinus angle measured in short-day-grown WT (n=190) and spy-3 (n=194) and plotted against blade length. For statistical analysis, data were split into 250 µm-wide classes. **P<0.01, ***P<0.001, ****P<0.0001 (one-tailed unpaired Student's t-test). (E) Representative cell area heatmaps for WT and spy-3 from the first tooth sinus cells. Arrowheads indicate the crease defining the first apical sinus on each tooth. Scale bars: 50 µm. (F) Projected surface quantification from the first tooth sinus cells plotted against tooth width. WT (n=17), spy-3 (n=6) for 0-150 µm; WT (n=37), spy-3 (n=30) for 150-250 µm; WT (n=22), spy-3 (n=28) for 250-500 µm. ***P<0.001 (one-tailed unpaired Student's t-test). ns, not significant.

Fig. 3 .

 3 Fig.3. CUC and SPY genetic interactions during leaf development. (A) Quantification of alpha-hull-normalized dissection index for WT (n=17), CUC2g-m4 (n=14), cuc3-105 (n=15), CUC2g-m4 cuc3-105 (n=16), spy-3 (n=18), CUC2g-m4 spy-3 (n=16), CUC2g-m4 spy-3 cuc3-105 (n=17), spy-3 cuc3-105 (n=17), mir164a (n=15) and spy-3 mir164a (n=16) mature leaves of ranks 11-13. A representative leaf silhouette is shown for each genotype analyzed. Scale bar: 1 cm. (B) Analysis of CUC3 and SPY genetic interactions during leaf development. First tooth height from WT (n=190), spy-3 (n=194), cuc3-105 (n=213) and spy-3 cuc3-105 (n=191) plotted against 50 µm-wide first tooth width classes. Short-day-grown plants from 22 to 50 days after sowing were used. Different colored letters represent statistical significance in one-way ANOVA analysis performed within each class for every genotype (colored letters correspond to the genotype analyzed), followed by Tukey comparison test (P<0.05).

Fig. 4 .

 4 Fig. 4. CUC2 inhibition of darkinduced hypocotyl elongation is associated with downregulation of cell wall-relaxing genes. (A) A total of 2569 genes were downregulated in DEX-induced p35S:CUC2-GR line; 493 genes upregulated in spy-3 mutant compared with WT were identified. Pvalue below the Venn diagram corresponds to hypergeometric probability (N total At genes =33,602) (overenrichment based on the cumulative distribution function of the hypergeometric distribution). (B) Expression level of EXLA1 and EXLA2 in a p35S:CUC2-GR cuc2-3 line grown in the dark for 72 h in vitro, treated either with mock treatment or with 10 µM DEX. Each dot represents a biological RNA sample. EXLA1 and EXLA2 transcript levels were measured by real-time quantitative RT-PCR normalized to EF1ALPHA and ACTIN 2. *P<0.05 (one-tailed unpaired Student's t-test). (C,D) Representative phenotypes (C) and corresponding hypocotyl length quantification (D) in p35S:CUC2-GR cuc2-3 line and cuc2-3 control grown in the dark for 72 h in vitro, treated either with mock treatment or with 10 µM DEX.

Fig. 5 .

 5 Fig. 5. CUC2 inhibits dark-induced cell elongation independently of CUC3. (A,B) Hypocotyl length quantification (A) and hypocotyl cell length quantification (B) in p35S:CUC2-GR cuc2-3 cuc3-105 line and cuc2-3 cuc3-105 control grown in the dark for 72 h in vitro, treated either with mock treatment or with 10 µM DEX. (C) Expression levels of EXLA1, EXLA2, XTH18 and XTH19 in a p35S:CUC2-GR cuc2-3 line grown in the dark for 72 h in vitro, treated either with mock treatment or with 10 µM DEX. Each dot represents a biological RNA sample. Transcript levels were measured by real-time quantitative RT-PCR normalized to EF1ALPHA and ACTIN 2. ***P<0.001 (one-tailed unpaired Student's t-test).

Fig. 6 .

 6 Fig. 6. Mechanics at the leaf margin of WT and spy-3 mutant. (A,C) Representative maps of relative stiffness (arbitrary units, AU) measured on the middle domain of an approximately 150 µm-long growing primordium (rank 11) from WT (A) and spy-3 (C) plants grown in short-day conditions. Each pixel represents the relative stiffness calculated from a single force-indentation curve. Apico-basal polarity is indicated. Position of tooth (T) as well as sinus (S) are indicated. Scale bars: 10 µm. (B,D) Projection of relative stiffness (from A,C) on measured leaf topography for WT (B) and spy-3 (D). (E) Quantification of relative stiffness cell walls of the tooth (T) and the sinus (S). Each point represents a measured pixel. (F) Apparent Young's modulus (Pa) measured on transversal cell walls of WT teeth (n=11) and sinuses (n=11) and spy-3 teeth (n=7) and sinuses (n=7). **P<0.01, ***P<0.001 (one-tailed unpaired Student's t-test).

Table 1 .

 1 Quantification of cotyledon fusion defects in cuc2-1, cuc3-105 and spy-3 mutant combinations

			Phenotype		Total seedlings
		Normal	Weak	Mild	Strong
	Genotype	(%)	(%)	(%)	(%)	n
	Col-0	99.37	0	0.63	0	315
	cuc3-105	99.52	0	0	0.48	631
	spy-3	100	0	0	0	317
	spy-3 cuc3-105	82.79	4.66	10.39	2.17	645
	cuc2-1	100	0	0	0	320
	spy-3 cuc2-1	82.57	6.59	10.52	0.31	637
						5

Acknowledgements

We thank Utpal Nath (Indian Institute of Science Bangalore) for the gift of spy-3 seeds; Doris Lucyshyn (BOKU, Vienna, Austria) for sharing with us the spy-22 and spy-23 mutants, the pSPY::SPY-FLAG spy-22 and the pSPY::SPY-GFP lines; and Ben Scheres (Wageningen University, the Netherlands) for the gift of the p35S: CUC2-GR line. We thank Genoscope -Centre National de Sequençage (Evry, France) for allowing us privileged access to perform sequencing. We thank Antoine Nicolas for discussions and critical reading of the manuscript. The IJPB benefits from the support of the Labex Saclay Plant Sciences-SPS (ANR-10-LABX-0040-SPS).

Funding

This work was supported by the Agence National de la Recherche grants LEAFNET (ANR-12-PDOC-0003) and CHARMFUL (ANR-11-BSV2-0005) and by an Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement (INRAE) grant MorphEAT (AAP BAP 2020).

Data availability

RNA-seq data have been deposited in the Gene Expression Omnibus database under accession number GSE184530. All steps of the experiment, from growth conditions to bioinformatic analyses, are detailed in POPS database, CATdb [START_REF] Gagnot | CATdb: a public access to Arabidopsis transcriptome data from the URGV-CATMA platform[END_REF]; http://tools.ips2.u-psud.fr/CATdb/project.html?project_id=491).

Peer review history

The peer review history is available online at https://journals.biologists.com/dev/ article-lookup/doi/10.1242/dev.200359.

Competing interests

The authors declare no competing or financial interests.

Author contributions

Conceptualization