Sebastian Wolf
email: sebastian.wolf@cos.uni-heidelberg.de

Dieuwertje Van Der Does

Friederike Ladwig

Carsten Sticht

Andreas Kolbeck

Ann-Kathrin Schurholz

Sebastian Augustin

Nana Keinath

Thomas Rausch

Steffen Greiner

Ann-Kathrin Schürholz

Karin Schumacher

Klaus Harter

Cyril Zipfel

Herman Höfte
email: herman.hofte@versailles.inra.fr.

Maarten J Chrispeels

A receptor-like protein mediates the response to

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.

The brassinosteroid (BR) signaling module is a central regulator of plant morphogenesis, as indicated by the large number of BRresponsive cell wall-related genes and the severe growth defects of BR mutants. Despite a detailed knowledge of the signaling components, the logic of this auto-/paracrine signaling module in growth control remains poorly understood. Recently, extensive cross-talk with other signaling pathways has been shown, suggesting that the outputs of BR signaling, such as gene-expression changes, are subject to complex control mechanisms. We previously provided evidence for a role of BR signaling in a feedback loop controlling the integrity of the cell wall. Here, we identify the first dedicated component of this feedback loop: a receptor-like protein (RLP44), which is essential for the compensatory triggering of BR signaling upon inhibition of pectin de-methylesterification in the cell wall. RLP44 is required for normal growth and stress responses and connects with the BR signaling pathway, presumably through a direct interaction with the regulatory receptor-like kinase BAK1. These findings corroborate a role for BR in controlling the sensitivity of a feedback signaling module involved in maintaining the physicochemical homeostasis of the cell wall during cell expansion.

brassinosteroids | cell wall integrity | pectin A ll organisms need to integrate outside information with in- trinsic cues through communication between the extracellular matrix and cell interior. This integration particularly applies to plants, the growth of which depends on biosynthesis, modification, and remodeling of their cell walls and needs to adapt to the widely varying environmental conditions inherent to a sessile lifestyle [START_REF] Cosgrove | Growth of the plant cell wall[END_REF][START_REF] Wolf | Growth control and cell wall signaling in plants[END_REF].

The wall of growing cells is composed of diverse classes of polysaccharides and proteins forming a complex and dynamic network able to withstand the high intracellular turgor pressure. Growth occurs when cell wall extensibility is selectively increased by the activity of cell wall-modifying enzymes, so that the walls yield to turgor pressure, resulting in an irreversible deformation [START_REF] Cosgrove | Growth of the plant cell wall[END_REF][START_REF] Wolf | Growth control and cell wall signaling in plants[END_REF]. The yielding of the cell wall needs to be precisely controlled to avoid rupture during growth. In addition, the abiotic and biotic environment can potentially change the cross-linking pattern of cell wall polymers and thus influence the extensibility of the cell wall. In this context, the state of the wall has to be constantly monitored and information has to be relayed to the cytosol to regulate cell wall biosynthesis and remodeling in response to developmental and environmental cues [START_REF] Wolf | Growth control and cell wall signaling in plants[END_REF]. Whereas cell wall integrity signaling has been extensively studied in yeast [START_REF] Levin | Cell wall integrity signaling in Saccharomyces cerevisiae[END_REF], in plants this process remains poorly understood [START_REF] Wolf | Growth control and cell wall signaling in plants[END_REF]. A small number of putative cell wall sensors have been described, including the pectinbinding wall-associated kinases [START_REF] Wagner | Wall-associated kinases are expressed throughout plant development and are required for cell expansion[END_REF][START_REF] Kohorn | The cell wall-associated kinases, WAKs, as pectin receptors[END_REF] and the receptor-like kinase THESEUS (6); however, very little is known about the signaling pathways involved. Here, we further investigated the previously identified function of brassinosteroid (BR) hormone signaling in maintaining cell wall integrity.

The well-characterized BR signaling module is initiated by the plasma membrane-bound receptor BRI1 and its coreceptor BAK1/SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK)3, which upon ligand binding transphosphorylate each other. A signal transduction cascade is initiated, starting with phosphorylation of receptor-like cytoplasmic kinases by BRI1 and which ultimately leads to transcriptional changes mediated by the transcription factors BZR1 and BES1/BZR2 (7-10). Among the transcriptional targets of BR signaling, cell wall-biosynthesis and -remodeling genes are overrepresented [START_REF] Sun | Integration of brassinosteroid signal transduction with the transcription network for plant growth regulation in Arabidopsis[END_REF], which is consistent with the fact that BR biosynthetic and BR-insensitive mutants show pronounced dwarfism mainly caused by reduced cell elongation. Accordingly, BR activation causes changes in cell wall enzyme activities, and induces hydration and remodeling of the cell wall [START_REF] Xie | Brassinosteroids can regulate cellulose biosynthesis by controlling the expression of CESA genes in Arabidopsis[END_REF][START_REF] Caesar | A fast brassinolide-regulated response pathway in the plasma membrane of Arabidopsis thaliana[END_REF][START_REF] Qu | Brassinosteroids regulate pectin methylesterase activity and AtPME41 expression in Arabidopsis under chilling stress[END_REF][START_REF] Wolf | Plant cell wall homeostasis is mediated by brassinosteroid feedback signaling[END_REF].

Intriguingly, in contrast to other hormones-which can act at a distance from their site of synthesis-genetic studies show that BRs seem to act more locally, on the same or adjacent cells [START_REF] Bishop | The tomato Dwarf gene isolated by heterologous transposon tagging encodes the first member of a new cytochrome P450 family[END_REF][START_REF] Savaldi-Goldstein | The epidermis both drives and restricts plant shoot growth[END_REF][START_REF] Fridman | Root growth is modulated by differential hormonal sensitivity in neighboring cells[END_REF]. Whereas long-distance signaling allows growth-rate modulation, for example in response to environmental cues, the logic of an autocrine/paracrine signaling module in growth control is

Significance

Plant growth and development depend on the biosynthesis and remodeling of the cell wall. To coordinate these two processes, surveillance mechanisms have evolved to monitor the state of the cell wall. The brassinosteroid (BR) hormone signaling pathway plays an essential role in growth control and regulates the expression of a plethora of cell wall-related genes. We have previously shown that feedback signaling from the wall can modulate the outputs of the BR pathway, ensuring cell wall homeostasis and integrity. Here, we identified a receptorlike protein (RLP44), which mediates the activation of BR signaling through direct interaction with the BR coreceptor BAK1. Thus, RLP44 integrates cell wall surveillance with hormone signaling to control cell wall integrity and growth.

not well understood. Our recent demonstration that a pathway exists, which activates BR hormone signaling upon cell wall perturbation [START_REF] Wolf | Plant cell wall homeostasis is mediated by brassinosteroid feedback signaling[END_REF] creates a new conceptual framework for the understanding of the role of BR signaling in growth control. In this view, the BR signaling module is recruited into a feedback loop regulating the physico-chemical homeostasis of the wall during the growth process. Here, we report the identification of the first dedicated component of this pathway: the receptor-like protein (RLP)44. RLP44 is required for normal growth and its expression is necessary and sufficient to activate BR signaling downstream of ligand perception. Moreover, RLP44 seems to integrate cell wall and BR signaling by interacting with the regulatory coreceptor BAK1.

Results

Identification of RLP44 as a Novel Modifier of BR Signaling. We have recently demonstrated that upon cell wall perturbation the BR signaling pathway is activated to initiate a compensatory response [START_REF] Wolf | Plant cell wall homeostasis is mediated by brassinosteroid feedback signaling[END_REF]. Thus, a pathway that relays information concerning cell wall alterations and consequences thereof to hormone signaling must exist (from here on referred to as "cell wall signaling"). To facilitate the identification of putative components involved in this pathway, we made use of plants that showed a strong BRmediated compensatory response to cell wall perturbation induced by the ectopic expression of a pectin methylesterase inhibitor (PMEIox) [START_REF] Wolf | Plant cell wall homeostasis is mediated by brassinosteroid feedback signaling[END_REF]. This response to reduced activity of pectin methylesterase (PME), which converts methylesterified pectin into (demethylesterified) pectate, includes growth retardation, root waving, agravitropism, organ fusion, and changes in gene expression [START_REF] Wolf | Plant cell wall homeostasis is mediated by brassinosteroid feedback signaling[END_REF] (see below). A forward genetic screen identified the recessive, extragenic suppressor mutant comfortably numb 2 (cnu2), which showed strong reduction of all observable BRmediated PMEIox responses (Fig. 1A and Fig. S1 A-G). Using map-based cloning, we identified a mutation associated with the cnu2 phenotype causing a premature stop codon in RLP44 (at3g49750) [START_REF] Wang | A genome-wide functional investigation into the roles of receptor-like proteins in Arabidopsis[END_REF], which encodes a receptor-like protein with a predicted signal peptide, a leucine-rich repeat (LRR)-type extracellular domain, a transmembrane region, and a short cytoplasmic domain (Fig. 1B). Plants homozygous for a T-DNA insertion in the same gene (rlp44-3) also suppressed the PMEIox phenotype (Fig. 1C and Fig. S1H). In addition, a GFP-tagged version of RLP44 (RLP44:GFP) was able to complement cnu2, as demonstrated by a PMEIox-like phenotype in transformant plants. Interestingly, some RLP44:GFP transformants even showed an enhanced PMEIox phenotype (Fig. 1A). Thus, loss of RLP44 function was causative for the suppression of PMEIox phenotypes in cnu2, whereas overexpression of RLP44 enhanced the compensatory response to PMEIox. Confocal laser-scanning analysis of RLP44:GFP in both the wild-type and the cnu2 background showed the presence of the fusion protein in the plasma membrane as well as in intracellular compartments (Fig. 1D and Fig. S1I).

In summary, RLP44 represents a new putative signaling component required for the BR-mediated response to PMEIoxinduced cell wall alterations.

RLP44 Is Required for Normal Growth and Stress Responses. We next assessed the role of RLP44 in normal plant development by studying the phenotype of the rlp44 cnu2 mutant separated from the PMEIox transgene. The mutant displayed a stunted growth phenotype under greenhouse conditions (Fig. 2A), indicating that RLP44 is required for normal growth. Compared with the control plants, rlp44 cnu2 rosette leaves had shorter petioles (Fig. S2A) and decreased rosette diameter (Fig. S2B). Impaired growth was also apparent for rapidly elongating dark-grown seedlings (Fig. S2C). In addition, rlp44 mutants were hypersensitive to stress generated by elevated concentrations of sucrose (Fig. 2B) or the presence of NaCl (Fig. S2D) in the medium, conditions that also aggravate the growth phenotype of BR mutants (Fig. S2 F andG) and, notably, that of mutants with altered cell wall composition or organization [START_REF] Benfey | Root development in Arabidopsis: Four mutants with dramatically altered root morphogenesis[END_REF][START_REF] Xu | Two leucine-rich repeat receptor kinases mediate signaling, linking cell wall biosynthesis and ACC synthase in Arabidopsis[END_REF]. In contrast, no difference in root growth was observed between wild-type and rlp44 cnu2 on medium supplemented with mannitol (Fig. S2E), ruling out purely osmotic effects. Consistent with the pleiotropic mutant phenotype, RLP44 has a broad expression spectrum (Fig. S3).

Loss of RLP44 Function Prevents Activation of BR Signaling by PMEI

Overexpression. In our previous study, we attributed the PMEIox phenotype primarily to enhanced BR signaling, which is expected to induce major changes in gene expression [START_REF] Wolf | Plant cell wall homeostasis is mediated by brassinosteroid feedback signaling[END_REF]. We therefore compared the transcriptome of the Col-0 wild-type, PMEIox, cnu2, and the cnu1 suppressor, which carries a hypomorphic allele of the BR receptor BRASSINOSTEROID INSENSITIVE1 (BRI1) ( 14) in a PMEIox background. In PMEIox, 1,018 genes were found to show significantly altered expression levels compared with the wild-type (553 up, 465 down). Interestingly, the vast majority of these transcripts reverted back to wild-type expression levels in both suppressor mutants, suggesting that RLP44 and BRI1 act in the same pathway (Fig. 3A and Dataset S1). Gene ontology (GO) enrichment analysis of the genes differentially regulated in PMEIox compared with the wild-type revealed BR metabolism to be among the significantly enriched categories (Fig. S4A), and many BR biosynthetic genes known to be under negative feedback control [START_REF] Sun | Integration of brassinosteroid signal transduction with the transcription network for plant growth regulation in Arabidopsis[END_REF] were down-regulated. This effect was alleviated or reversed in the suppressor mutants (Fig. S4B). The GO categories related to the cell wall were also enriched (Fig. S4 A and C), consistent with the cell wall changes observed in PMEIox [START_REF] Wolf | Plant cell wall homeostasis is mediated by brassinosteroid feedback signaling[END_REF].

To further analyze the gene-expression changes in PMEIox and the suppressor mutants, we visualized, by hierarchical cluster analysis, patterns of gene expression from the significantly enriched GO categories "response to organic substance" and "cell wall organization." Unsurprisingly, the suppressor mutants clustered with the wild-type as the majority of genes showed an expression similar to Col-0 (Fig. 3B and Fig. S4D). Notably, among the cell wall-related genes, three putative PMEs (which convert methylesterified pectin into pectate) and three putative enzymes (polygalacturonases) that degrade pectate (not pectin) were upand down-regulated, respectively, in PMEIox (Fig. S4D). This finding suggests that the initial lack of pectate (which is thought to play a role in the control of cell wall integrity through the formation of Ca 2+ cross-links) in the transformant may be compensated for by increased pectin de-methylesterification and decreased pectate turn-over (Fig. S4D) [START_REF] Wolf | Plant cell wall homeostasis is mediated by brassinosteroid feedback signaling[END_REF].

In summary, these results are consistent with enhanced BRsignaling being at least in part the underlying cause for the PMEIox phenotype and strongly suggest that in both cnu1 and cnu2 this is prevented by interference with an early step in the signaling cascade.

RLP44 Activates BR Signaling Downstream of Ligand Perception. We next investigated whether RLP44 is a constitutive member of the BR pathway itself. To this end we took advantage of the observation that both PMEIox and epi-brassinolide (BL) treatment render dark-grown seedlings agravitropic [START_REF] Wolf | Plant cell wall homeostasis is mediated by brassinosteroid feedback signaling[END_REF][START_REF] Gupta | Hypocotyl directional growth in Arabidopsis: A complex trait[END_REF]. Interestingly, whereas the rlp44 cnu2 loss-of-function mutant suppressed this phenotype in PMEIox (in the cnu2 mutant), it remained fully sensitive to the BL treatment (Fig. S5), indicating that RLP44 was not required for the response to exogenous BL. This finding was corroborated by the finding that the rlp44 cnu2 mutation did not further aggravate the phenotype of the weak BRI1 allele bri1 cnu1 [START_REF] Wolf | Plant cell wall homeostasis is mediated by brassinosteroid feedback signaling[END_REF] (see also Fig. S7A) and the analysis of RLP44 overexpression lines (RLP44ox). Indeed, RLP44ox showed a phenotype almost identical to that of BRI1 overexpressing plants (BRI1ox) [START_REF] Friedrichsen | Brassinosteroidinsensitive-1is a ubiquitously expressed leucine-rich repeat receptor serine/threonine kinase[END_REF] (i.e., elongated petioles and long, narrow, slightly epinastic leaf blades) (Fig. 3C). Accordingly, rosette diameter was increased similarly in BRI1ox and RLP44ox compared with the wild-type (Fig. 3C). In addition, expression levels of selected BR marker genes and genes reported to be BRI1oxresponsive [START_REF] Kim | Constitutive activation of stressinducible genes in a brassinosteroid-insensitive 1 (bri1) mutant results in higher tolerance to cold[END_REF] were similar in RLP44ox and BRI1ox seedlings (Fig. 3D). These observations, together with the transcriptome analysis of the loss-of-function mutants, indicate that RLP44, like BRI1, activates the BR signaling pathway. In sharp contrast to the similar behavior of BRI1ox and RLP44ox with respect to gene expression, they differed dramatically in their response to exogenous BRs. Upon depletion of endogenous BRs with the biosynthesis inhibitor propiconazole [START_REF] Hartwig | Propiconazole is a specific and accessible brassinosteroid (BR) biosynthesis inhibitor for Arabidopsis and maize[END_REF], root growth of BRI1ox was at least an order-of-magnitude more sensitive than the wildtype to both growth-promoting and growth-inhibiting doses of BL, whereas the RLP44ox response to BL was indistinguishable from that of the wild-type (Fig. 3E). Similar results were obtained with hypocotyl growth (Figs. S5B andS6). Thus, whereas overexpression of BRI1 strongly sensitized plants for BR, RLP44 overexpression activated the signaling pathway without increasing BR sensitivity. We next assessed whether RLP44 overexpression could rescue mutants of the BR receptors BRI1 and BAK1. Double-homozygous descendants of a cross between RLP44ox and bri1 cnu1 had a bri1 cnu1 -like phenotype, indicating that RLP44 overexpression cannot rescue BRI1 deficiency and that, in turn, activation of the BR pathway by RLP44 requires functional BRI1 (Fig. S7B). Because elevated expression of BR pathway components downstream of the receptor complex can typically rescue BR perception mutants [START_REF] Chung | Constitutive activation of brassinosteroid signaling in the Arabidopsis elongated-D/bak1 mutant[END_REF][START_REF] Kim | The CDG1 kinase mediates brassinosteroid signal transduction from BRI1 receptor kinase to BSU1 phosphatase and GSK3-like kinase BIN2[END_REF][START_REF] Tang | BSKs mediate signal transduction from the receptor kinase BRI1 in Arabidopsis[END_REF][START_REF] Nam | BRI1/BAK1, a receptor kinase pair mediating brassinosteroid signaling[END_REF][START_REF] Kim | Brassinosteroid signal transduction from cell-surface receptor kinases to nuclear transcription factors[END_REF], our results suggest that RLP44-mediated integration of cell wall and BR signaling occurs at or near the receptor complex. In contrast to the results with bri1 cnu1 , RLP44 overexpression was able to rescue the phenotype of the bak1-4 mutant (30, 31) (Fig. 3 F and G), possibly reflecting the different levels of genetic redundancy in the BRI1-like and BAK1/SERK gene families [START_REF] Fàbregas | The brassinosteroid insensitive1-like3 signalosome complex regulates Arabidopsis root development[END_REF][START_REF] Gou | Genetic evidence for an indispensable role of somatic embryogenesis receptor kinases in brassinosteroid signaling[END_REF]: BAK1/ SERK3 is partially redundant with SERK1 and SERK4, and a bri1 null-like phenotype is only observed in the absence of all three SERKs [START_REF] Gou | Genetic evidence for an indispensable role of somatic embryogenesis receptor kinases in brassinosteroid signaling[END_REF]. Thus, RLP44-mediated rescue of bak1-4, but not of bri1 cnu1 might be explained by a possible interaction of RLP44 with the two other redundant SERKs (see below).

In conclusion, our results show that RLP44 is not a constitutive component of the BR signaling pathway itself but sensitizes this pathway in response to cell wall-derived cues.

RLP44 Is Associated with the BR Receptor Complex. To investigate how RLP44 might activate BR signaling, we tested candidate proteins for interaction with RLP44 using bimolecular fluorescence complementation assays (BiFC). Coexpression in Nicotiana benthamiana leaves of RLP44 fused to the C-terminal portion of the yellow fluorescent protein (nYFP) and BAK1 fused to nYFP gave rise to YFP fluorescence, suggesting that RLP44 interacts with BAK1 (Fig. 4A). Interestingly, the BAK1:nYFP/RLP44:cYFP signal was consistently stronger than what was observed with the BRI1/BAK1 combination used as a positive control (Fig. 4A) [START_REF] Nam | BRI1/BAK1, a receptor kinase pair mediating brassinosteroid signaling[END_REF][START_REF] Schwessinger | Phosphorylation-dependent differential regulation of plant growth, cell death, and innate immunity by the regulatory receptor-like kinase BAK1[END_REF], whereas control infiltrations of RLP44 and FLS2, a membranebound pathogen-associated molecular pattern receptor [START_REF] Gómez-Gómez | FLS2: An LRR receptor-like kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis[END_REF], did not result in detectable fluorescence (Fig. S8A). Interaction of BAK1 and RLP44 was independently confirmed using FRET-FLIM in N. benthamiana (Fig. S8B) and the mating-based Split-Ubiqitin system in yeast (36) (Fig. S8C). In addition, coimmunoprecipitation experiments showed that both BAK1 and BRI1 were present in immunoprecipitates of RLP44:GFP from a line in a wild-type background and from a complemented cnu2 line (Fig. 4B). Consistent with the hypothesis that RLP44 exerts its effect at the level of the BR receptor complex, RLP44 overexpression was not able to rescue the subtle phenotype of bsk3-1 (Fig. S8D), which is mutated in one of the immediate downstream targets of BRI1 [START_REF] Tang | BSKs mediate signal transduction from the receptor kinase BRI1 in Arabidopsis[END_REF]. In addition, we noticed that RLP44 is phosphorylated, presumably in its cytoplasmic tail, and the extent of phosphorylation is affected in a PMEIox background, substantiating our assumption that RLP44 is involved in cell wall-responsive signaling (Fig. 4C). In summary, our data indicate that RLP44 recruits the BR signaling module in response to PMEIox-induced cell wall perturbation at least in part through a direct interaction with BAK1 (Fig. 4D).

Discussion

A major question in plant biology is how extrinsic cues are integrated with development to generate the phenotypic plasticity required for sessile organisms challenged by changing environments. It is assumed that cell wall surveillance is an important cellular self-perception mechanism for the control of growth and development. In addition, environmental cues, such as the presence of pathogens or neighboring cells, could be sensed by their effect on wall properties and wall integrity. Importantly, information from these cell wall surveillance pathways has to be integrated with intracellular growth regulation and defense pathways to mediate an adequate response, which includes-but is not limited to-feedback regulation of cell wall modification. In the present study, we have identified RLP44 as a positive regulator of BR signaling strength upon perturbation of the cell wall, as indicated by loss-of-function analyses, transcriptome comparison, and the effects of RLP44 overexpression. Interaction of RLP44 with the core BR signaling component BAK1 likely represents the convergence point of cell wall and BR signaling, whereas phosphorylation of RLP44 may play a role in the transduction of cell wall-derived signals.

At present, it is unclear whether the pathway harboring RLP44 is directly involved in cell wall sensing. It is conceivable that abundance or modification of cell wall polymers is monitored directly; for example, by physical interaction with the extracellular domain of RLP44, perhaps in a way similar to that of the pectin-binding wall-associated kinases [START_REF] Wagner | Wall-associated kinases are expressed throughout plant development and are required for cell expansion[END_REF][START_REF] Kohorn | The cell wall-associated kinases, WAKs, as pectin receptors[END_REF]. Although there is as of yet no evidence supporting this hypothesis, it is noteworthy that at least one LRR protein, the polygalacturonase inhibitor protein, can interact with de-methylesterified pectin in the wall [START_REF] Spadoni | Polygalacturonase-inhibiting protein interacts with pectin through a binding site formed by four clustered residues of arginine and lysine[END_REF]. Importantly, the abundance of this particular cell wall polymer is affected in the PMEIox line used as genetic background for the suppressor screen, which identified RLP44 [START_REF] Wolf | Plant cell wall homeostasis is mediated by brassinosteroid feedback signaling[END_REF]. However, it is also possible that changes in pectin are sensed by a different mechanism; in this scenario, RLP44 could be a downstream component, receiving information from unknown interaction partners or signaling molecules. Alternatively, derived properties, such as membrane tension, could be the relevant parameter under surveillance [START_REF] Monshausen | Feeling green: Mechanosensing in plants[END_REF][START_REF] Haswell | Two MscS homologs provide mechanosensitive channel activities in the Arabidopsis root[END_REF]. Again, in this scenario RLP44 could either be directly involved in sensing (e.g., as a part of a mechanosensor) or receive signals from upstream components. Clearly, more work is needed to distinguish between these possibilities. Here, we focused on the integration of BR and cell wall signaling. We revealed a possible mechanism with the identification of RLP44 as a modifier of BR signaling, which interacts with the regulatory receptor-like kinase BAK1. Possibly, interaction with RLP44 could favor BAK1-BRI1 interaction or stabilize the interactions in the signaling complex with downstream components, such as receptor-like cytoplasmic kinases [START_REF] Kim | The CDG1 kinase mediates brassinosteroid signal transduction from BRI1 receptor kinase to BSU1 phosphatase and GSK3-like kinase BIN2[END_REF][START_REF] Tang | BSKs mediate signal transduction from the receptor kinase BRI1 in Arabidopsis[END_REF][START_REF] Sreeramulu | BSKs are partially redundant positive regulators of brassinosteroid signaling in Arabidopsis[END_REF].

Interestingly, our results show that RLP44 is not a core BR signaling component but rather provides a lateral input modulating the signaling strength of the BR pathway. Indeed, whereas bri1 mutation suppressed the comparable phenotype triggered by either the inhibition of PME activity (in PMEIox) or the exogenous application of BL, rlp44 mutations likewise suppressed the PMEIox phenotype but instead remained fully sensitive to BL. Similarly, RLP44ox phenocopied BRI1ox, but whereas BRI1ox was hypersensitive to BL, RLP44ox showed BL sensitivity indistinguishable from that of the wild-type.

Signal integration and cross-talk with other signaling pathways has been demonstrated for the BR signaling pathway before. For example, light and gibberellic acid signaling are known to converge with the BR pathway at the level of the transcription factors [START_REF] Bai | Brassinosteroid, gibberellin and phytochrome impinge on a common transcription module in Arabidopsis[END_REF][START_REF] Gallego-Bartolomé | Molecular mechanism for the interaction between gibberellin and brassinosteroid signaling pathways in Arabidopsis[END_REF], whereas cross-talk with stomatal development appears to occur more upstream in the pathway [START_REF] Kim | Brassinosteroid regulates stomatal development by GSK3-mediated inhibition of a MAPK pathway[END_REF][START_REF] Gudesblat | SPEECHLESS integrates brassinosteroid and stomata signalling pathways[END_REF]. Cell wall-mediated feedback activation of BR signaling could help to fine-tune expression of cell wall biosynthetic and remodeling genes to ensure cell wall homeostasis during cell expansion and in response to extrinsic cues, such as environmental conditions that affect the physical properties of the cell wall. In accordance with this, RLP44, perhaps with other members of the large RLP gene family [START_REF] Wolf | Plant cell wall homeostasis is mediated by brassinosteroid feedback signaling[END_REF], is required for normal growth and the response to stress conditions.

Materials and Methods

Map-Based Cloning. To approximate the position of the cnu2 mutation, 23 single sequence-length polymorphism markers distributed throughout the genome were assayed (details available on request) in a population of 50 F 2 individuals originating from a cross of cnu2 with nontransgenic plants of the Landsberg erecta ecotype. Only plants showing a wild-type-like appearance in combination with BASTA resistance (resistance derived from PMEIox transgene cassette) were selected. Strong bias was observed for the Columbia-specific band of markers CER456071 and CER470172 located at the bottom of chromosome 3 (see Table S1 for oligo sequences). CER470172 and S18811 were identified as flanking markers and 219 plants from the mapping population were analyzed with additional markers F2K15, CER470169, and T16K5. Candidate genes in the remaining interval between CER470169 and T16K5 were amplified and the PCR products were sequenced to identify the mutation.

Confocal Laser-Scanning Microscopy Analysis. Laser lines of 488 nm (GFP, FM4-64) or 514 nm (YFP) on a Zeiss LSM 510 Meta and Zeiss LSM 710 microscope systems equipped with 25× and 63× water immersion objectives were used for excitation. Fluorescence was recorded between 530 and 600 nm for YFP and between 490 and 525 nm for GFP. FM4-64 fluorescence was recorded between 600 nm and 720 nm. Images were analyzed with Zeiss Zen software and Fiji. Images were processed with Fiji and Adobe Photoshop CS for figure preparation.

Protein-Protein Interaction Analysis. Coimmunoprecipitation [START_REF] Albrecht | Brassinosteroids inhibit pathogen-associated molecular pattern-triggered immune signaling independent of the receptor kinase BAK1[END_REF], matingbased split-ubiquitin system [START_REF] Grefen | The determination of protein-protein interactions by the mating-based split-ubiquitin system (mbSUS)[END_REF], and FRET-FLIM [START_REF] Veerabagu | The Arabidopsis B-type response regulator 18 homomerizes and positively regulates cytokinin responses[END_REF] were essentially performed as described. Details can be found in SI Material and Methods.

Expression Analysis. Microarray data reported in this study were deposited at the National Center for Biotechnology Information Gene Expression Omnibus with the accession number GSE48596. Detailed experimental procedures can be found in SI Material and Methods.

Fig. 1 .

 1 Fig. 1. Identification of RLP44, a receptor-like protein involved in cell wall signaling. (A) Wild-type-like phenotype of the suppressor mutant cnu2 compared with the parental line, PMEIox, in which cell wall alterations elicit a BR-mediated response resulting in a severe growth phenotype. Expression of RLP44:GFP in cnu2 reconstitutes or enhances the PMEIox phenotype. (B) Overview of map-based cloning procedure: 219 F2 plants showing the cnu2 phenotype derived from a cross of cnu2 and nontransgenic plants of the Landsberg erecta ecotype were analyzed with PCR-based markers. Sequencing of genes in the mapping interval revealed a C-to-T mutation, leading to a premature stop codon in RLP44 (at3g49750). CD, short, cytosolic domain; gray shading, LRR repeats; SP, signal peptide; TM, transmembrane domain;. (C) Knock-out of RLP44 in PMEIox through T-DNA insertion reinstates the cnu2 phenotype, confirming that RLP44 lossof-function is causative for suppression of PMEIox. (D) RLP44:GFP localizes to the plasma membrane in addition to intracellular punctate structures, which are partially colabeled by the endocytic tracer dye FM4-64. (Scale bars, 10 μm.)

Fig. 2 .

 2 Fig. 2. RLP44 is required for normal growth. (A) Morphological phenotype of young rlp44 cnu2 mutant plants compared with wild-type, PMEIox, and cnu2 (Left). Adult plants (Right) show reduced height compared with wildtype. (B) rlp44 mutant plants are hypersensitive toward elevated concentrations of sucrose in the medium. Bars indicate mean hypocotyl length of 7-d-old seedlings ± SD (n > 14). Asterisks indicate significance as determined by Student t test with P < 0.001.

Fig. 3 .

 3 Fig. 3. RLP44 activates BR signaling. (A) Venn diagram analysis of genes with significantly altered expression compared with the Col-0 wild-type in PMEIox, cnu1 and cnu2, respectively. Of the 1,018 genes differentially expressed in PMEIox, 906 are reverted in cnu2 and 942 are reverted in cnu1. The expression of only 23 genes (including the PMEIox transgene) is altered in all three genotypes (Upper). Approximately 90% of genes reverted in cnu2 are shared with cnu1, indicating that the underlying mutated genes act in the same pathway. Microarray analysis was performed on 4-d-old dark-grown seedlings. (B) Hierarchical clustering analysis of genes of the GO category "response to organic substance" differentially expressed in PMEIox. (C) Strong overexpression of RLP44 phenocopied BRI1 overexpressing plants with long petioles (quantification indicated by numbers, millimeter ± SD, n >15; P < 0.001 for both comparisons between Col-0 and transgenics according to a Student t test; differences between the transgenic lines are nonsignificant) and narrow, twisted leaf blades, suggesting that RLP44 can activate the BR signaling pathway similar to BRI1. (D) The expression of BR-regulated genes (19) is similar in the RLP44ox and BRI1ox lines in 5-d-old light-grown seedlings. Bars denote means of three biological replicates ± 95% confidence interval, n = 3. Asterisks indicate statistical significance as determined by Student t test with P < 0.05. (E) Both BRI1ox and RLP44ox are resistant toward depletion of endogenous BRs but only BRI1ox is hypersensitive to application of BL. Bars indicate mean root length of 5-d-old seedlings ± SD (n > 8). Data were analyzed using one-way ANOVA. Asterisks indicate significance with P < 0.001 as determined by Tukey's test. Note that significance is only indicated for comparisons within each treatment. (F and G) Expression of RLP44 rescues the bak1-4 mutant phenotype. (G) Leaf length measurements indicate significant differences between bak1-4 and both bak1-4 RLP44ox and Col-0, but not between Col-0 and bak1-4 RLP44ox. Asterisks indicate significance as determined by Student t test with P < 0.001.

Fig. 4 .

 4 Fig.[START_REF] Wagner | Wall-associated kinases are expressed throughout plant development and are required for cell expansion[END_REF]. RLP44 interacts with BAK1 to activate BR signaling. (A) BiFC analysis of molecular interaction between RLP44 and BAK1. YFP fluorescence is observed when RLP44:cYFP is coexpressed with BAK1: nYFP. No YFP fluorescence is detected in the control infiltration of BRI1/RLP44, BAK1:nYFP with cYFP, RLP44:cYFP with nYFP, and RLP44:cYFP and BAK1: nYFP alone, respectively. (Magnification: A, 76×.) (B) Coimmunoprecipitation reveals that both BAK1 and BRI1 are physically associated with RLP44 in RLP44:GFP overexpressing lines. Treatment with epi-BL for 90 min (40) had no effect on complex formation. PM:GFP, GFP directed to the plasma membrane by fusion with Lti6b. (C ) RLP44 is phosphorylated at one or more serine residues, presumably in its cytoplasmic tail, and this posttranslational modification is responsive to cell wall status. (D) Model of BR pathway activation by RLP44 upon cell wall perturbation. RLP44 is not part of BR signaling itself, but can activate the pathway laterally by interacting with the receptor complex. Transcriptional regulation is indicated by blue lines.

| www.pnas.org/cgi/doi/10.1073/pnas.1322979111 Wolf et al.

| www.pnas.org/cgi/doi/10.1073/pnas.1322979111 Wolf et al.

| www.pnas.org/cgi/doi/10.1073/pnas.1322979111 Wolf et al.

ACKNOWLEDGMENTS. We thank Heike Steininger for technical assistance. This study was supported in part by Deutsche Forschungsgemeinschaft HA 2146/18-1 (to K.H.) and "Agence Nationale pour la Recherche" Project BLAN08-1_332764 (to H.H.). S.W. was a recipient of Postdoctoral Fellowship WO 1660/1-1 from the Deutsche Forschungsgemeinschaft and is supported by the Emmy Noether Programme (WO 1660/2-1), C.Z. received funding from The Gatsby Charitable Foundation, and D.v.d.D. is a recipient of a long-term postdoctoral fellowship from the European Molecular Biology Organization.


Ana L Martínez-López

Elizabeth Carvajal
email: ecarvajal@ciad.mx

Millan Valérie

Micard Agustín

Rascón-Chu Francisco Brown-Bojorquez

Norberto Sotelo-Cruz

Yolanda L López-Franco

Jaime Lizardi-Mendoza

Elizabeth Carvajal-Millan

Valérie Micard
email: valerie.micard@supagro.fr

Agustín Rascón- Chu

Francisco Brown-Bojorquez

In vitro degradation of covalently cross-linked arabinoxylan hydrogels by Bifidobacteria

Keywords: cross-linked arabinoxylans, gels, biodegradability, bifidobacteria, fermentation

Covalently crosslinked arabinoxylans (AX) gels with different structure were obtained AX gels were biodegradable by mixture of two Bifidobacterium Changes in microstructure of AX gels allowed different degradation profiles AX gels could be used as microflora-activated system for colon delivery

Introduction

Polysaccharide-based hydrogels have been extensively studied for their potential as colonspecific drug delivery systems due to their chemical and three-dimensional structures, biocompatibility properties and swelling [START_REF] Peppas | Hydrogels and drug delivery[END_REF][START_REF] Sinha | Polysaccharides in colon-specific drug delivery[END_REF]. However, most of these hydrogels are susceptible to changes according to the low gastrointestinal pH or the duration of transit time, thus leading to premature drug release. For these reasons, some polysaccharides such chitosan, alginate and pectin have been chemically modified or cross-linked or combined with hydrophobic polymers for use in the development of systems that are specifically biodegradable by colonic bacteria [START_REF] Brøndsted | Crosslinked dextran -a new capsule material for colon targeting of drugs[END_REF][START_REF] Chambin | Colon-specific drug delivery: Influence of solution reticulation properties upon pectin beads performance[END_REF][START_REF] Tozaki | Chitosan capsules for colon-specific drug delivery: improvement of insulin absorption from the rat colon[END_REF] Comment citer ce document : Martínez-López, A. L., Carvajal-Millan, E. (Auteur de correspondance), Micard, V., Rascón-Chu, A., Brown-Bojorquez, F., Sotelo-Cruz, N., López-Franco, Y. L., Lizardi-Mendoza, J. ( 2016). In vitro degradation of covalently cross-linked arabinoxylan hydrogels by Bifidobacteria.

Carbohydrate Polymers,. DOI : 10.1016/j.carbpol.2016.02.031

Page 4 of 27

A c c e p t e d M a n u s c r i p t 4 1997). Covalent cross-linking has been used to enhance the mechanical strength and chemical stability of the hydrogels. However, the chemical cross-linking agents used are often toxic compounds that require harsh reaction conditions. Another alternative is to use polysaccharides, which can form covalent hydrogels via enzymatic cross-linking.

Biological enzymes can catalyze polysaccharide cross-linking into gels via strong covalent bonds under mild conditions in the absence of organic solvents [START_REF] Figueroa-Espinoza | Attempt to cross-link feruloylated arabinoxylans and proteins with a fungal laccase[END_REF][START_REF] Micard | Oxidative gelation of sugar-beet pectins: use of laccases and hydration properties of the cross-linked pectins[END_REF]. Unlike most polysaccharides, arabinoxylans (AX) can form covalent hydrogels via enzymatic cross-linking of ferulic acid esterified to the polysaccharide (Izydorczyk & Biliaderi, 1995;[START_REF] Vansteenkiste | Oxidative gelation of feruloylated arabinoxylan as affected by protein. Influence on protein enzymatic hydrolysis[END_REF]. Covalently cross-linked AX gels are thermo-irreversible [START_REF] Carvajal-Millan | Storage stability of laccase induced arabinoxylan gels[END_REF], generally present an absence of pH or electrolyte susceptibility and exhibit no syneresis after long periods of storage [START_REF] Izydorczyk | Cereal arabinoxylans: advances in structure and physicochemical properties[END_REF].

AX are non-starch polysaccharides that resist digestion and absorption in the human small intestine and are fermented in the large intestine by microbiota in the colon, especially

Bacteroides and Bifidobacterium [START_REF] Hopkins | Degradation of cross-linked and non-cross-linked arabinoxylans by the intestinal microbiota in children[END_REF][START_REF] Hughes | In vitro fermentation by human fecal microflora of wheat arabinoxylans[END_REF]. The basic structure of AX consists of a linear backbone chain of xylose units containing arabinose substituents attached through O-2 and/or O-3 [START_REF] Izydorczyk | Cereal arabinoxylans: advances in structure and physicochemical properties[END_REF]. Some of the arabinose residues are ester-linked to ferulic acid (FA) through (O)-5 [START_REF] Smith | Occurrence and nature of ferulic acid substitution of cell-wall polysaccharides in graminaceous plants[END_REF]. FA oxidation by enzymatic free radical generating agents (laccase or peroxidase/H 2 O 2 ) allows for the coupling of AX chains through the formation of FA dimers and trimers (di-FA, tri-FA) as covalent cross-linking structures [START_REF] Carvajal-Millan | Arabinoxylan gels: impact of the feruloylation degree on their structure and properties[END_REF][START_REF] Izydorczyk | Cereal arabinoxylans: advances in structure and physicochemical properties[END_REF]. Recent studies have demonstrated that AX gels formed via oxidative cross-linking can be used for the controlled release of model proteins (Carvajal-

Comment citer ce document : Martínez-López, A. L., Carvajal-Millan, E. (Auteur de correspondance), Micard, V., Rascón-Chu, A., Brown-Bojorquez, F., Sotelo-Cruz, N [START_REF] Carvajal-Millan | Arabinoxylan/protein gels: Structural, rheological and controlled release properties[END_REF], methylxanthine [START_REF] Iravani | Physical characterization of arabinoxylan powder and its hydrogel containing a methyl xanthine[END_REF], diclofenac sodium [START_REF] Iqbal | Evaluation of hotwater extracted arabinoxylans from ispaghula seeds as drug carriers[END_REF]), lycopene (Hernández-Espinoza, Piñón-Muñiz, Rascón-Chu, Santana-Rodríguez, & Carvajal-Millan, 2012) and probiotic bacteria [START_REF] Morales-Ortega | Entrapment of probiotics in water extractable arabinoxylan gels: rheological and microstructural characterization[END_REF].

The degradation of the AX structure results from the activity of different bacterial enzymes, which mainly include endo-xylanases, beta-xylosidases, reducing end xylose-releasing exooligoxylanases and alpha-L-arabinofuranosidases [START_REF] Pollet | In vitro fermentation of arabinoxylan oligosaccharides and low molecular mass arabinoxylans with different structural properties from wheat (Triticum aestivum L.) bran and psyllium (Plantago ovata Forsk) seed husk[END_REF]. The mechanism of AX degradation depends on the degree of polymerization, crosslinking and branching present in the AX structure [START_REF] Hughes | In vitro fermentation by human fecal microflora of wheat arabinoxylans[END_REF][START_REF] Rose | Structural differences among alkalisoluble arabinoxylans from maize (Zea mays), rice (Oryza sativa), and wheat (Triticum aestivum) brans influence human fecal fermentation profiles[END_REF].

Notably, bifidobacteria comprise up to 25% of the cultivable gut microflora, with

Bifidobacterium longum and Bifidobacterium adolescentis predominating in adults.

Mixtures of these strains have been associated with the complete fermentation of low molecular weight arabinoxylans [START_REF] Pastell | In vitro fermentation of arabinoxylan-derived carbohydrates by bifidobacteria and mixed fecal microbiota[END_REF]. Though the fermentation properties of arabinoxylans have been researched extensively, only a few studies have reported the degradation of the covalently cross-linked arabinoxylan network, and these studies did not report the structural features, cross-linking density and mechanical properties of the gel [START_REF] Hopkins | Degradation of cross-linked and non-cross-linked arabinoxylans by the intestinal microbiota in children[END_REF]. These structural parameters of AX matrix gel could influence the ability of colonic microflora to degrade polysaccharides. In the present study, the ability of a mixture of Bifidobacterium longum and Bifidobacterium adolescentis to degrade covalent arabinoxylan hydrogels with different swelling and structural parameters was examined.

Experimental

Comment citer ce document : Martínez-López, A. L., Carvajal-Millan, E. (Auteur de correspondance), Micard, V., Rascón-Chu, A., Brown-Bojorquez, F., Sotelo-Cruz, N

Preparation of MBAX gels

MBAX solutions at 4% (MBAX-4) and 6% (MBAX-6) (w/v) were prepared in 0.1 M acetate buffer (pH 5.5). The buffer was filtered through a 0.2 µm filter to prevent microbial contamination. Laccase (1.675 nkat/mg of MBAX) was used as a cross-linking agent. The gels were allowed to develop for 6 h at 25 ºC.

Rheological properties

The rheological tests were performed by small amplitude oscillatory shearing using a strain-controlled rheometer (Discovery HR-2 rheometer, TA Instruments, New Castle, DE, USA) as reported by [START_REF] Vansteenkiste | Oxidative gelation of feruloylated arabinoxylan as affected by protein. Influence on protein enzymatic hydrolysis[END_REF]. MBAX gelation was studied for 6 h at 25 °C. All measurements were carried out at a frequency of 0.25 Hz and 5% strain (linearity range of viscoelastic behavior). The mechanical spectra of gels were obtained by conducting a frequency sweep from 0.01 to 100 Hz at 25 °C. The FA, di-FA and tri-FA contents in the MBAX gels were quantified by RP-HPLC after a de-esterification step as described by [START_REF] Vansteenkiste | Oxidative gelation of feruloylated arabinoxylan as affected by protein. Influence on protein enzymatic hydrolysis[END_REF]. An Alltima (Alltech, Deerfield, IL, USA) C18 column (250 × 4.6 mm) and a photodiode array detector Waters 996 (Millipore Co., Milford, MA, USA) were used. Detection was followed by UV absorbance at 320 nm.

Phenolic Acids Content

Swelling and structural parameters

The MBAX-4 and MBAX-6 were allowed to swell as described by [START_REF] Carvajal-Millan | Arabinoxylan gels: impact of the feruloylation degree on their structure and properties[END_REF]. The equilibrium swelling was reached when the weight of the samples changed by no more than 3% (0.06 g). The swelling ratio (q) was calculated as follows:

q = (Ws -W MBAX )/W MBAX ,
where Ws is the weight of swollen gels and W MBAX is the weight of the MBAX-4 or MBAX-6 (w/v) gels. From the swelling measurements, the molecular weight between two cross-links (M c ), the cross-linking density (ρ c ) and the mesh size (ξ) values of the MBAX-4

and MBAX-6 gels were calculated as reported by [START_REF] Carvajal-Millan | Impact of the structure of arabinoxylan gels on their rheological and protein transport properties[END_REF], using the model of [START_REF] Flory | Statistical Mechanics of Cross-Linked Polymer Networks I. Rubberlike Elasticity[END_REF] modified by [START_REF] Peppas | Poly(vinyl alcohol) hydrogels: Reinforcement of radiation-crosslinked networks by crystallization[END_REF] for gels in which the cross-links are introduced in solution.

Scanning electron microscopy

The microstructure of the MBAX-4 and MBAX-6 gels and surface morphology of the VA, USA). The bacteria were maintained at -80 °C in glycerol stock solutions. The bacterial suspensions were thawed, and the bacteria were revived by culturing in the basal media as described previously by [START_REF] Hughes | In vitro fermentation by human fecal microflora of wheat arabinoxylans[END_REF]. The strains were incubated in the broth under anaerobic conditions at 37 °C until an optical density of one (OD= 1) was reached. Afterward, they were used as an inoculum for growth studies on MBAX gels.

In vitro fermentation

For the fermentation analysis, the MBAX-4 and MBAX-6 gels were formed in Hungate

Fermentation product analysis

Before analysis, at time 0 of fermentation, one milliliter of each MBAX gel was treated with 0.5 mL 0.5 N NaOH for 15 min at 25°C to disrupt the gels by releasing the phenolic bridges. The pH was then immediately adjusted to 7 to prevent MBAX chain degradation.

The samples for the other fermentation time points were only centrifuged (15,000 g, 4 °C The SCFA in the samples was quantified using external calibration curves of acetic, propionic and butyric acids, and the results were expressed in µg/mL.

Statistical analysis

The results are expressed as the means ± Standard Deviation (S.D.) from three replicates.

The significance of differences was determined by analysis of variance (ANOVA) (OriginPro software, version 8.6. Originlab Corporation, USA) followed by Tukey's test and the results were considered to be statistically significant when p< 0.05.

Results and discussion

Features of MBAX gels

Rheological properties and covalent cross-links

The formation of gel networks was monitored by storage (G') and loss (G'') modulus changes in 4 and 6% (w/v) AX solutions undergoing oxidative gelation by laccase (1.675 nkat/mg of MBAX) (Fig. 1a). The gelation profile vs. time exhibited an initial increase of the G' modulus, followed by a plateau region resulting from the sol-gel transition. By increasing the MBAX concentration, the gelation time (time G' = G'', i.e., tan delta= 1) decreased and the gelation rate (rate of G' development) increased. This relationship could be associated with a higher density of polysaccharide chains enhancing the probability of contact between ferulic acid free radicals, which is an essential requirement for the in an approximately two-fold increase in the value of G' (Fig. 1a). These results were in agreement with data reported previously by [START_REF] Rattan | Structure and rheological behaviour of arabinoxylans from canadian brad wheat flours[END_REF], who found linear relationships between G' (plateau values) and polymer concentration in samples of wheat AX. This G' vs. polymer concentration trend has also been observed in chemically crosslinked alginate gels [START_REF] Choudhary | Rheology and nanostructure of hydrophobically modified alginate (HMA) gels and solutions[END_REF] and dextran-lactate gels cross-linked by stereocomplex formation [START_REF] De | Biodegradable hydrogels based on stereocomplex formation between lactic acid oligomers grafted to dextran[END_REF]. The mechanical strength of the matrix is known to have a direct bearing on drug delivery characteristics [START_REF] Coviello | A new polysaccharidic gel matrix for drug delivery: preparation and mechanical properties[END_REF][START_REF] Woolfson | Rheological, mechanical and membrane penetration properties of novel dual drug systems for percutaneous delivery[END_REF]. The mechanical spectra of the The extent of covalent cross-linking in the MBAX gels was determined by the content of total di-FA and tri-FA after 6 h of gelation (Table 1). The amounts of di-FA and tri-FA produced during gelation never compensated for the loss of FA monomer. Indeed, at the end of gelation, 80 and 70% of the FA initially present in MBAX was oxidized; by and MBAX-4, respectively. Nevertheless, the tan δ (G''/G') values confirmed the formation of an elastic covalent system for both gels (Fig. 1a). These behaviors in MBAX gels have been previously reported by several authors [START_REF] Carvajal-Millan | Maize bran gum: Extraction, characterization and functional properties[END_REF][START_REF] Lapierre | The phenolic fraction of maize bran: evidence for lignin-heteroxylan association[END_REF][START_REF] Niño-Medina | Maize processing waste water arabinoxylans: Gelling capability and cross-linking content[END_REF]. These authors attributed such phenomena to the formation of higher ferulate cross-linking structures unreleased by mild alkaline hydrolysis and/or to the participation of lignin residues coupled with FA monomers in the formation of MBAX gels. At the end of gelation, the 8-5' form (especially benzofuran) contributed approximately 68 -79% of the total di-FA in MBAX-4 and MBAX-6, whereas the 5-5' and 8-O-4' forms contributed 22 -12% and 10 -9%, respectively. The predominance of 8-5' di-FA was also observed in wheat flour AX treated with a peroxidase/H 2 O 2 system or laccase [START_REF] Carvajal-Millan | Arabinoxylan gels: impact of the feruloylation degree on their structure and properties[END_REF]Figueroa-Espinosa et al., 1999). Nevertheless, the 5-5' di-FA structure has been reported to be predominant in AX gels from maize bran [START_REF] Carvajal-Millan | Maize bran gum: Extraction, characterization and functional properties[END_REF][START_REF] Niño-Medina | Maize processing waste water arabinoxylans: Gelling capability and cross-linking content[END_REF]. In a previous study, [START_REF] Hatfield | Modelling the feasibility of intramolecular dehydrodiferulate formation in grass walls[END_REF] reported that only 5-5' di-FA can be formed intramolecularly in arabinoxylans, i.e., on the same polysaccharide chains. In this study, the greater proportion of the 5-5' di-FA formed in the MBAX-4 gel (22%) compared with the MBAX-6 gel (12%) might have indicated that in MBAX-4, the intrachain bonds were favored and thus the polymer network showed decreasing continuity.

G'= 4 2 1 Pa G' M B AX -6 G' 'M B A X -6 G' M B AX -4 G' 'M B A X -4 G', G'' (
These results may partly explain some differences in the mechanical properties of the gels. d Mesh size.

Swelling and structure of gels

The swelling ratio (q) of gels is an important parameter that describes the amount of water stored within the gel network. The swelling behavior of the MBAX gels at 4 and 6% (w/v) was assessed in water for 15 h at 25 °C, with the equilibrium being reached between 8 and 6 h in MBAX-4 and MBAX-6, respectively. The q value (in g water/g of MBAX dry weight) in the MBAX gels decreased from 18 to 9 when the MBAX concentration in the gel increased from 4 to 6% (w/v) (Table 1). The q value found for the MBAX-4 gel was similar to the value reported for maize bran AX gels (3.5% (w/v), q= 20) (Berlanga-Reyes, Carvajal-Millan, Caire Juvera, et al., 2009). The lower swelling ratio value registered at the highest MBAX concentration in the gel (MBAX-6) may have been related to the more compact polymeric structure that limited the water absorption [START_REF] Meyvis | Influence of the degradation mechanism of hydrogels on their elastic and swelling properties during degradation[END_REF][START_REF] Ross-Murphy | Polysaccharide strong and weak gels[END_REF]. A similar effect of polysaccharide concentrations on the swelling capacity of a gel has been reported for waterextractable AX (0.5 to 2 % (w/v), q values ranging from 231 to 69) or sugar beet pectin (2 [START_REF] Carvajal-Millan | Impact of the structure of arabinoxylan gels on their rheological and protein transport properties[END_REF][START_REF] Micard | Oxidative gelation of sugar-beet pectins: use of laccases and hydration properties of the cross-linked pectins[END_REF].

Comment
The cross-sectional microstructures of the MBAX-4 and MBAX-6 gels before swelling were observed by scanning electron microscopy (SEM). Cross-sectional SEM images of the MBAX gels are shown in Fig. 2. Both gels showed a three-dimensional and heterogeneous network structure formed by the aggregation of nodular structures into clusters. MBAX-6 (Fig. 2b) presented a more compact microstructure in comparison with the geometries and irregular pores sizes of the MBAX-4 structure (Fig. 2a). These results are consistent with the structural parameters calculated from the equilibrium swelling test. The molecular weight between two cross-links (Mc), the cross-linking density (ρ c ) and the mesh size (ξ) values for MBAX-6 and MBAX-4 are presented in Table 1. When the MBAX concentration increased in the gel from 4 to 6%, Mc and ξ decreased, leading to an increase in the cross-linking density ρ c . Similar Mc, ρ c and ξ values have been reported in 3 to 5 % AX gels (Berlanga-Reyes, Carvajal-Millan, Caire Juvera, et al., 2009;[START_REF] Carvajal-Millan | Impact of the structure of arabinoxylan gels on their rheological and protein transport properties[END_REF]. The structural parameters are important in determining the physical properties of the gels, including their mechanical strength, the diffusivity of the released molecule, and their degradability [START_REF] Hoffman | Hydrogels for biomedical applications[END_REF]. In hydrogels for drugs and protein delivery or cell encapsulation, a typical ξ range of 5 to 300 nm has been reported for the swollen state [START_REF] Carvajal-Millan | Arabinoxylan/protein gels: Structural, rheological and controlled release properties[END_REF][START_REF] Choh | Facile synthesis and characterization of disulfide-cross-linked hyaluronic acid hydrogels for protein delivery and cell encapsulation[END_REF][START_REF] Choudhary | Rheology and nanostructure of hydrophobically modified alginate (HMA) gels and solutions[END_REF].

Degradation of MBAX gels

The surface structures of degraded MBAX-4 and MBAX-6 hydrogels were studied by SEM and are presented in Fig. 3. At least two independent replicates of each sample were analyzed for their surface morphologies, and similar results were obtained for the replicates. Fig. 3a-b show the surface structures of MBAX-4 and MBA-6, respectively, after 18 h of incubation under the same conditions as those used for fermentation but without the bacterial strains. The changes in the surface morphology of the MBAX-4 and MBAX-6 hydrogels were observed as a function of their exposure time to Bifidobacterium culture (a mixture of Bifidobacterium longum and Bifidobacterium adolescentis). SEM images taken after 18 h and 36 h of incubation with the Bifidobacterium culture showed the extent of the gel morphological changes (Fig. 3c-f). For both samples, the undegraded MBAX gels presented relatively smooth and continuous surface structures without holes.

The degraded gels lost this structure and exhibited multiple cavities in their microstructure, which increased in size and number with the time of incubation due to the enzymatic activity of B. longum and B. adolescentis. However, at 18 h of incubation, differences in the size of the cavities were observed between the MBAX gels (Fig. 3d vs. Fig. 3c). At 0 h of 1 and Fig. 2), which may have reduced the accessibility of the gel to enzymes and thus slowed down its degradation. Nonetheless, at 36 h of incubation, the structure of both MBAX gels appeared to be collapsed rather than porous, as shown in Fig. 3e-f. Therefore, it is probable that after an initial surface erosion, the enzymes diffused into the polymeric network and thus caused the degradation of the most internal sites. Similar results were obtained for chemically crosslinked dextran gels that were degraded by microbial enzymes [START_REF] Brøndsted | Crosslinked dextran -a new capsule material for colon targeting of drugs[END_REF]. arabinoxylans [START_REF] Pastell | In vitro fermentation of arabinoxylan-derived carbohydrates by bifidobacteria and mixed fecal microbiota[END_REF][START_REF] Wang | In vitro fermentation of xylooligosaccharides from wheat bran insoluble dietary fiber by Bifidobacteria[END_REF]. The ungelified MBAX showed over 80% of molecular weight loss after 18 h incubation and Mw distributions of less than 50 kDa. This pattern was similar for different times of incubation of ungelified MBAX (Fig 4a). Fig. 4c shows that MBAX-6 was slowly degraded and had a Mw distribution of less than 50 kDa after 72 h of incubation, whereas MBAX-4 presented this pattern after 36 h of incubation (Fig. 4b). However, in the first 18 h of incubation, a fraction of both gels had Mw greater than 110 kDa. [START_REF] Hopkins | Degradation of cross-linked and non-cross-linked arabinoxylans by the intestinal microbiota in children[END_REF] reported that the breakdown of the xylan backbone in crosslinked AX was more resistant to degradation compared with the uncrosslinked AX due to the restricted access of xylanolytic enzymes.

Thus, the degradation of the gel in the early hours of incubation was restricted by the ability of the enzymes to access their target sites. hydrolyzed by bacterial xylanases, as reflected the increase in the A/X ratio. Hence, the MBAX solution, MBAX-4, and MBAX-6 were degraded by a debranching mechanism [START_REF] Rose | Structural differences among alkalisoluble arabinoxylans from maize (Zea mays), rice (Oryza sativa), and wheat (Triticum aestivum) brans influence human fecal fermentation profiles[END_REF]. The MBAX-4 and MBAX-6 gels showed different slopes for the linear decrease of the A/X ratio, in which the A/X ratio decreased much more rapidly in the MBAX-4 gel than in the MBAX-6 gel. This difference was mainly related to the low average mesh size (Table 1), compact microstructure (Fig. 2) and probably higher content of intrachain di-FA in MBAX-6 compared to MBAX-4. [START_REF] Rosa | Effects of disintegration on in vitro fermentation and conversion patterns of wheat aleurone in a metabolical colon model[END_REF] observed an effect of the pore size on the in vitro fermentation of wheat aleurone with a high content of insoluble AX. Moreover, [START_REF] Park | Investigation of the degradation-retarding effect caused by the low swelling capacity of a novel hyaluronic acid filler developed by solid-phase crosslinking technology[END_REF] reported that the main factor causing the high resistance to degradation of hyaluronic acid gels is the low swelling ratio resulting from a high concentration of polysaccharides and a high degree of crosslinking. Hence, the slow removal of the arabinose side chains from MBAX-6 may have been due to its more compact structure, which slowed down enzymes penetration.

longum, B. adolescentis).

The results above are consistent with the production of short-chain fatty acids registered during the in vitro fermentation by the Bifidobacterium mix (Fig. 6). All three samples produced significantly higher concentrations of total SCFA in comparison to the control cultures at different times (MBAX-free substrate). The SCFA production increased with the time that the MBAX-4 and MBAX-6 gels were fermented. In contrast, the SCFA production in the ungelified MBAX remained constant from 36 to 72 h. Ungelified MBAX produced a significantly (p < 0.05) higher amount of SCFA in comparison to the MBAX gels at all fermentation times. At 18 h, both MBAX gels showed no significant differences in the amount of SCFA produced. However, at 36 and 72 h, MBAX-6 presented a lower amount of SCFA than MBAX-4. The total SCFA decreased significantly when the degree of cross-linking increased in the MBAX gels. Acetic acid was the main acid produced, followed by propionic and butyric acids. The ungelified MBAX and MBAX-4 produced more acetic and propionic acids than MBAX-6, while butyric acid production was not significantly different among the three fermented samples. The SCFA profiles resulting from the fermentation of the ungelified MBAX and MBAX gels were similar to the one reported for alkali-soluble arabinoxylans from maize [START_REF] Rose | Structural differences among alkalisoluble arabinoxylans from maize (Zea mays), rice (Oryza sativa), and wheat (Triticum aestivum) brans influence human fecal fermentation profiles[END_REF]. These results

indicate that a certain portion of the AX chains released during the degradation of MBAX gels was fermentable and that carbohydrates were efficiently converted to SCFA by the Bifidobacterium mix.

  tubes and autoclaved at 121 °C for 15 min. MBAX without gelation was used as a positive control. The MBAX and MBAX gels were mixed with autoclaved basal media to give a final concentration of 5 g/L (w/v) MBAX as a sole carbon source. 100 µL Oxyrase (Oxyrase Inc. Mansfield, OH, USA) was added as the broth to remove oxygen from the microenvironment of the test tube. The tubes were sealed anaerobically by flushing the headspace with carbon dioxide and placed at 4 °C overnight. These Hungate tubes were inoculated with a 1% (v/v) mixture of two bifidobacteria species (B. adolescentis and B. longum; ratio 1:1) and incubated in the dark without shaking at 37 °C for 18, 36 and 72 h. Fermentations were carried out in triplicate, and duplicate blank tubes containing no Comment citer ce document : Martínez-López, A. L., Carvajal-Millan, E. (Auteur de correspondance), Micard, V., Rascón-Chu, A., Brown-Bojorquez, F., Sotelo-Cruz, N., López-Franco, Y. L., Lizardi-Mendoza, J. (2016). In vitro degradation of covalently cross-linked arabinoxylan hydrogels by Bifidobacteria. Carbohydrate Polymers, 144 (76-82). DOI : 10.1016/j.carbpol.2016.02for each time point. The OD (650 nm) was measured at 18, 36 and 72 h after inoculation. The pH was measured at the end of each different growth period, and random samples were examined microscopically to exclude contamination by the bacterial cultures.

, 5 min

 5 ) to remove the bacteria. The changes in the molecular weight distribution of the MBAX and MBAX gels at different times of fermentation (0, 18, 36, 72 h) were determined by SE-HPLC at 38 ºC using a TSKGel G5000PWXL column (7.8 x 300 mm) in combination with a PWX-guard column (6 x 40 mm) (Tosoh Bioscience, Stuttgart, Germany). Molecular weights were estimated after universal calibration with pullulans as standards (P50 to P800). Isocratic elution was conducted at 0.6 mL/min with 0.01 M LiNO 3 filtered through 0.2 µm. Differential refractometry was used for detection.Residual carbohydrates in the freeze-dried fermentation residues and monosaccharides in the supernatant of the centrifuged samples were quantified by RP-HPLC as described by[START_REF] Carvajal-Millan | Maize bran gum: Extraction, characterization and functional properties[END_REF]. The main monosaccharides of MBAX (arabinose, xylose, galactose and glucose) were used as standards.Short-chain fatty acids (SCFA) were analyzed based on[START_REF] Pastell | In vitro fermentation of arabinoxylan-derived carbohydrates by bifidobacteria and mixed fecal microbiota[END_REF]. The SCFA analysis was carried out using GC (Clarus 580, PerkinElmer, Waltham, MA, USA) with a flame ionization detector and a capillarity Comment citer ce document : Martínez-López, A. L., Carvajal-Millan, E. (Auteur de correspondance), Micard, V., Rascón-Chu, A., Brown-Bojorquez, F., Sotelo-Cruz, N., López-Franco, Y. L., Lizardi-Mendoza, J. (2016). In vitro degradation of covalently cross-linked arabinoxylan hydrogels by Bifidobacteria. Carbohydrate Polymers, 144 (76-82). DOI : 10.1016/j.carbpol.2016.02.031 Page 10 of 27 A c c e p t e d M a n u s c r i p t 10 column (Elite-FFAP 30 m x 0.50 mm I.D; film thickness, 1 µm).

  establishment of a three-dimensional AX network[START_REF] Carvajal-Millan | Impact of the structure of arabinoxylan gels on their rheological and protein transport properties[END_REF][START_REF] Rattan | Structure and rheological behaviour of arabinoxylans from canadian brad wheat flours[END_REF]. The G' values (215 Pa) of the Comment citer ce document : Martínez-López, A. L., Carvajal-Millan, E. (Auteur de correspondance), Micard, V., Rascón-Chu, A., Brown-Bojorquez, F., Sotelo-Cruz, N., López-Franco, Y. L., Lizardi-Mendoza, J. (2016). In vitro degradation of covalently cross-linked arabinoxylan hydrogels by Bifidobacteria. Carbohydrate Polymers, 144 (76-82). DOI : 10.1016/j.carbpol.2016.02.031 Page 11 of 27 A c c e p t e d M a n u s c r i p t 11MBAX gel at 4% (w/v) were higher than those reported for laccase-induced maize bran gel (20 Pa) or waste water arabinoxylans gel (2 Pa) at the same concentration, though MBAX-4 had a similar initial FA content to both of these gels (0.23 and 0.37 µg/mg AX, respectively)[START_REF] Hernández-Espinoza | Lycopene/arabinoxylan gels: rheological and controlled release characteristics[END_REF][START_REF] Niño-Medina | Maize processing waste water arabinoxylans: Gelling capability and cross-linking content[END_REF]. Such behaviors might have their origin in the structural and conformational characteristics of AX molecules. The increase in the MBAX concentration from 4 to 6% (w/v) in the gel resulted

MBAX- 4

 4 and MBAX-6 gels after 6 h of gelation are shown in Fig.1b. Though the MBAX-6 gel exhibited a solid-like behavior with G' > G'' and a plateau towards the low-frequency range (0.01 to 10 Hz), G' decreased steeply with increasing frequency (beyond 40 Hz). This behavior is typical of strong gels that present rupture and fail at large deformations. The MBAX-4 gel showed a linear G' independent of the frequency of oscillation and a G'' that was drastically lower than G' and linearly dependent on the frequency[START_REF] Ross-Murphy | Polysaccharide strong and weak gels[END_REF]. The dependence of G'' on the frequency has been related to the participation of physical entanglement interactions in the maturation of the gel and the various levels of connectivity and junction zones of disparate lengths and strengths within the three-dimensional network[START_REF] Kasapis | Phase separation in biopolymer gels: a low-to high-solid exploration of structural morphology and functionality[END_REF][START_REF] Picout | Rheology of biopolymer solutions and gels[END_REF]. Hence, theseComment citer ce document : Martínez-López, A. L., Carvajal-Millan, E. (Auteur de correspondance), Micard, V., Rascón-Chu, A., Brown-Bojorquez, F., Sotelo-Cruz, N., López-Franco, Y. L., Lizardi-Mendoza, J. (2016). In vitro degradation of covalently cross-linked arabinoxylan hydrogels by Bifidobacteria. Carbohydrate Polymers, 144 (76-82). DOI : 10.1016/j.carbpol.2016.02attributed to the covalent cross-linking distribution and the physical entanglement of AX chains in MBAX gels as a function of AX concentration. Similar mechanical spectra have been previously reported for strong gels with potential uses as carriers of proteins and drugs systems[START_REF] Choh | Facile synthesis and characterization of disulfide-cross-linked hyaluronic acid hydrogels for protein delivery and cell encapsulation[END_REF][START_REF] Choudhary | Rheology and nanostructure of hydrophobically modified alginate (HMA) gels and solutions[END_REF][START_REF] De | Biodegradable hydrogels based on stereocomplex formation between lactic acid oligomers grafted to dextran[END_REF].

Fig. 1 .

 1 Fig. 1. (a) Monitoring the storage (G') and loss (G'') modulus of MBAX solution at 4 and

  Comment citer ce document : Martínez-López, A. L., Carvajal-Millan, E. (Auteur de correspondance), Micard, V., Rascón-Chu, A., Brown-Bojorquez, F., Sotelo-Cruz, N., López-Franco, Y. L., Lizardi-Mendoza, J. (2016). In vitro degradation of covalently cross-linked arabinoxylan hydrogels by Bifidobacteria. Carbohydrate Polymers, 144 (76-82). DOI : 10.1016/j.carbpol.2016.0232 and 34% was recovered as a combination of di-FA and tri-FA in MBAX-6

  citer ce document : Martínez-López, A. L., Carvajal-Millan, E. (Auteur de correspondance), Micard, V., Rascón-Chu, A., Brown-Bojorquez, F., Sotelo-Cruz, N., López-Franco, Y. L., Lizardi-Mendoza, J. (2016). In vitro degradation of covalently cross-linked arabinoxylan hydrogels by Bifidobacteria. Carbohydrate Polymers, 144 (76-82). DOI : 10.1016/j.carbpol.2016.02w/v), q values ranging from 120 to 35) after treatment with laccase (Carvajal-

Fig. 2 .

 2 Fig. 2. SEM image showing the microstructures of cross-sectional MBAX-4 (a) and

Fig 3 .

 3 Fig 3. Scanning electron micrographs showing the surface morphologies of covalently

Fig. 4 .

 4 Fig. 4. HPSEC elution profiles of hydrolysis products of (a) ungelified MBAX, (b) MBAX-

Fig. 5 .

 5 Fig. 5. Changes in Arabinose/Xylose ratio during in vitro fermentation of ungelified

Fig. 6 .

 6 Fig. 6. Total short-chain fatty acid (SCFA) production during fermentation of ungelified

Table 1 .

 1 Swelling and structural parameters of MBAX gels.

	Page 14 of 27

Comment citer ce document : Martínez-López, A. L., Carvajal-Millan, E. (Auteur de correspondance), Micard, V., Rascón-Chu, A., Brown-Bojorquez, F., Sotelo-Cruz, N., López-Franco, Y. L., Lizardi-Mendoza, J. (2016). In vitro degradation of covalently cross-linked arabinoxylan hydrogels by Bifidobacteria. Carbohydrate Polymers, 144 (76-82). DOI : 10.1016/j.carbpol.2016.02.031

  Comment citer ce document : Martínez-López, A. L., Carvajal-Millan, E. (Auteur de correspondance), Micard, V., Rascón-Chu,

A., Brown-Bojorquez, F., Sotelo-Cruz, N., López-Franco, Y. L., Lizardi-Mendoza, J. (

2016

). In vitro degradation of covalently cross-linked arabinoxylan hydrogels by Bifidobacteria. Carbohydrate Polymers, 144 (76-82). DOI : 10.1016/j.carbpol.2016.02.031 Page 19 of 27

Acknowledgements

This research was supported by Fondo Sectorial de Investigación en Salud y Seguridad Social SSA/IMSS/ISSSTE-CONACYT, México (grant 179746 to E. Carvajal-Millán). The authors are pleased to acknowledge Aurélie Putois (INRA, Montpellier, France) for determining the phenolic acids and Alma C. Campa-Mada (CIAD) for the technical assistance.