Xyloglucan exoglycosidases in the monocot model Brachypodium distachyon and the conservation of xyloglucan disassembly in angiosperms.

KEY MESSAGE: Brachypodium distachyon has a full set of exoglycosidases active on xyloglucan, including α-xylosidase, ß-galactosidase, soluble and membrane-bound ß-glucosidases and two α-fucosidases. However, unlike in Arabidopsis, both fucosidases are likely cytosolic. Xyloglucan is present in primary walls of all angiosperms. While in most groups it regulates cell wall extension, in Poaceae its role is still unclear. Five exoglycosidases participate in xyloglucan hydrolysis in Arabidopsis: α-xylosidase, ß-galactosidase, α-fucosidase, soluble ß-glucosidase and GPI-anchored ß-glucosidase. Mutants in the corresponding genes show alterations in xyloglucan composition. In this work putative orthologs in the model grass Brachypodium distachyon were tested for their ability to complement Arabidopsis mutants. Xylosidase and galactosidase mutants were complemented, respectively, by BdXYL1 (Bd2g02070) and BdBGAL1 (Bd2g56607). BdBGAL1, unlike other xyloglucan ß-galactosidases, is able to remove both galactoses from XLLG oligosaccharides. In addition, soluble ß-glucosidase BdBGLC1 (Bd1g08550) complemented a glucosidase mutant. Closely related BdBGLC2 (Bd2g51280), which has a putative GPI-anchor sequence, was found associated with the plasma membrane and only a truncated version without GPI-anchor complemented the mutant, proving that Brachypodium also has soluble and membrane-bound xyloglucan glucosidases. Both BdXFUC1 (Bd3g25226) and BdXFUC2 (Bd1g28366) can hydrolyze fucose from xyloglucan oligosaccharides but were unable to complement a fucosidase mutant. Fluorescent protein fusions of BdXFUC1 localized to the cytosol and both proteins lack a signal peptide. Signal peptides appear to have evolved only in some eudicot lineages of this family, like the one leading to Arabidopsis. These results could be explained if cytosolic xyloglucan α-fucosidases are the ancestral state in angiosperms, with fucosylated oligosaccharides transported across the plasma membrane.

Soluble and Membrane-Bound ß-Glucosidases Are Involved in Trimming the Xyloglucan Backbone.

In many flowering plants, xyloglucan is a major component of primary cell walls, where it plays an important role in growth regulation. Xyloglucan can be degraded by a suite of exoglycosidases that remove specific sugars. In this work, we show that the xyloglucan backbone, formed by (1→4)-linked ß-d-glucopyranosyl residues, can be attacked by two different Arabidopsis (Arabidopsis thaliana) ß-glucosidases from glycoside hydrolase family 3. While BGLC1 (At5g20950; for ß-glucosidase active against xyloglucan 1) is responsible for all or most of the soluble activity, BGLC3 (At5g04885) is usually a membrane-anchored protein. Mutations in these two genes, whether on their own or combined with mutations in other exoglycosidase genes, resulted in the accumulation of partially digested xyloglucan subunits, such as GXXG, GXLG, or GXFG. While a mutation in BGLC1 had significant effects on its own, lack of BGLC3 had only minor effects. On the other hand, double bglc1 bglc3 mutants revealed a synergistic interaction that supports a role for membrane-bound BGLC3 in xyloglucan metabolism. In addition, bglc1 bglc3 was complemented by overexpression of either BGLC1 or BGLC3 In overexpression lines, BGLC3 activity was concentrated in a microsome-enriched fraction but also was present in soluble form. Finally, both genes were generally expressed in the same cell types, although, in some cases, BGLC3 was expressed at earlier stages than BGLC1 We propose that functional specialization could explain the separate localization of both enzymes, as a membrane-bound ß-glucosidase could specifically digest soluble xyloglucan without affecting the wall-bound polymer.

AtBGAL10 is the main xyloglucan ß-galactosidase in Arabidopsis, and its absence results in unusual xyloglucan subunits and growth defects.

In growing cells, xyloglucan is thought to connect cellulose microfibrils and regulate their separation during wall extension. In Arabidopsis (Arabidopsis thaliana), a significant proportion of xyloglucan side chains contain ß-galactose linked to α-xylose at O2. In this work, we identified AtBGAL10 (At5g63810) as the gene responsible for the majority of ß-galactosidase activity against xyloglucan. Xyloglucan from bgal10 insertional mutants was found to contain a large proportion of unusual subunits, such as GLG and GLLG. These subunits were not detected in a bgal10 xyl1 double mutant, deficient in both ß-galactosidase and α-xylosidase. Xyloglucan from bgal10 xyl1 plants was enriched instead in XXLG/XLXG and XLLG subunits. In both cases, changes in xyloglucan composition were larger in the endoglucanase-accessible fraction. These results suggest that glycosidases acting on nonreducing ends digest large amounts of xyloglucan in wild-type plants, while plants deficient in any of these activities accumulate partly digested subunits. In both bgal10 and bgal10 xyl1, siliques and sepals were shorter, a phenotype that could be explained by an excess of nonreducing ends leading to a reinforced xyloglucan network. Additionally, AtBGAL10 expression was examined with a promoter-reporter construct. Expression was high in many cell types undergoing wall extension or remodeling, such as young stems, abscission zones, or developing vasculature, showing good correlation with α-xylosidase expression.

Regulation of secondary wall synthesis and cell death by NAC transcription factors in the monocot Brachypodium distachyon.

In several dicotyledonous species, NAC transcription factors act as master switches capable of turning on programmes of secondary cell-wall synthesis and cell death. This work used an oestradiol-inducible system to overexpress the NAC transcription factor BdSWN5 in the monocot model Brachypodium distachyon. This resulted in ectopic secondary cell-wall formation in both roots and shoots. Some of the genes upregulated in the process were a secondary cell-wall cellulose synthase (BdCESA4), a xylem-specific protease (BdXCP1) and an orthologue of AtMYB46 (BdMYB1). While activation of BdMYB1 may not be direct, this study showed that BdSWN5 is capable of transactivating the BdXCP1 promoter through two conserved binding sites. In the course of Brachypodium development, the BdXCP1 promoter was observed to be active in all types of differentiating tracheary elements. Together, these results suggest that Brachypodium SWNs can act as switches that turn on secondary cell-wall synthesis and programmed cell death.

MYB46 modulates disease susceptibility to Botrytis cinerea in Arabidopsis.

In this study, we show that the Arabidopsis (Arabidopsis thaliana) transcription factor MYB46, previously described to regulate secondary cell wall biosynthesis in the vascular tissue of the stem, is pivotal for mediating disease susceptibility to the fungal pathogen Botrytis cinerea. We identified MYB46 by its ability to bind to a new cis-element located in the 5' promoter region of the pathogen-induced Ep5C gene, which encodes a type III cell wall-bound peroxidase. We present genetic and molecular evidence indicating that MYB46 modulates the magnitude of Ep5C gene induction following pathogenic insults. Moreover, we demonstrate that different myb46 knockdown mutant plants exhibit increased disease resistance to B. cinerea, a phenotype that is accompanied by selective transcriptional reprogramming of a set of genes encoding cell wall proteins and enzymes, of which extracellular type III peroxidases are conspicuous. In essence, our results substantiate that defense-related signaling pathways and cell wall integrity are interconnected and that MYB46 likely functions as a disease susceptibility modulator to B. cinerea through the integration of cell wall remodeling and downstream activation of secondary lines of defense.

Lack of α-xylosidase activity in Arabidopsis alters xyloglucan composition and results in growth defects.

Xyloglucan is the main hemicellulose in the primary cell walls of most seed plants and is thought to play a role in regulating the separation of cellulose microfibrils during growth. Xylose side chains block the degradation of the backbone, and α-xylosidase activity is necessary to remove them. Two Arabidopsis (Arabidopsis thaliana) mutant lines with insertions in the α-xylosidase gene AtXYL1 were characterized in this work. Both lines showed a reduction to undetectable levels of α-xylosidase activity against xyloglucan oligosaccharides. This reduction resulted in the accumulation of XXXG and XXLG in the liquid growth medium of Atxyl1 seedlings. The presence of XXLG suggests that it is a poor substrate for xyloglucan ß-galactosidase. In addition, the polymeric xyloglucan of Atxyl1 lines was found to be enriched in XXLG subunits, with a concomitant decrease in XXFG and XLFG. This change can be explained by extensive exoglycosidase activity at the nonreducing ends of xyloglucan chains. These enzymes could thus have a larger role than previously thought in the metabolism of xyloglucan. Finally, Atxyl1 lines showed a reduced ability to control the anisotropic growth pattern of different organs, pointing to the importance of xyloglucan in this process. The promoter of AtXYL1 was shown to direct expression to many different organs and cell types undergoing cell wall modifications, including trichomes, vasculature, stomata, and elongating anther filaments.

Potential role for purple acid phosphatase in the dephosphorylation of wall proteins in tobacco cells.

It is not yet known whether dephosphorylation of proteins catalyzed by phosphatases occurs in the apoplastic space. In this study, we found that tobacco (Nicotiana tabacum) purple acid phosphatase could dephosphorylate the phosphoryl residues of three apoplastic proteins, two of which were identified as alpha-xylosidase and beta-glucosidase. The dephosphorylation and phosphorylation of recombinant alpha-xylosidase resulted in a decrease and an increase in its activity, respectively, when xyloglucan heptasaccharide was used as a substrate. Attempted overexpression of the tobacco purple acid phosphatase NtPAP12 in tobacco cells not only decreased the activity levels of the glycosidases but also increased levels of xyloglucan oligosaccharides and cello-oligosaccharides in the apoplast during the exponential phase. We suggest that purple acid phosphatase controls the activity of alpha-xylosidase and beta-glucosidase, which are responsible for the degradation of xyloglucan oligosaccharides and cello-oligosaccharides in the cell walls.

The overexpression of AtPrx37, an apoplastic peroxidase, reduces growth in Arabidopsis.

Understanding peroxidase function in plants is difficult because of the lack of substrate specificity, the high number of genes and their diversity in structure. In the present study, the relative expression of 22 genes coding putative peroxidases (E.C 1.11.1.x) in Arabidopsis was studied. The relative expression of AtPrx37 showed a correlation with the cessation of growth in rosette leaves as well as with the growth capacity along the flower stem. Using AtPrx37::GUS construction, its expression was associated with the vascular bundles. Furthermore, the overexpression of AtPrx37 under the control of CaMV 35S promoter rendered a dwarf phenotype with smaller plants and delayed development. The plants overexpressing AtPrx37 also showed an increase in the amount of esterified phenolic material associated with their walls. A role in the growth cessation and phenolic cross-linking during lignin deposition is postulated.

Analysis of Xyloglucan Composition in Arabidopsis Leaves.

Xyloglucan is one of the main components of the primary cell wall in most species of plants. This protocol describes a method to analyze the composition of the enzyme-accessible and enzyme-inaccessible fractions of xyloglucan in the model species Arabidopsis thaliana. It is based on digestion with an endoglucanase that attacks unsubstituted glucose residues in the backbone. The identities and relative amounts of released xyloglucan fragments are then determined using MALDI-TOF mass spectrometry.

Apoplastic glycosidases active against xyloglucan oligosaccharides of Arabidopsis thaliana.

All four glycanases necessary for the degradation of xyloglucan oligosaccharides (alpha-fucosidase, alpha-xylosidase, beta-galactosidase and beta-glucosidase) were found in the apoplastic fluid of Arabidopsis thaliana. These activities acted cooperatively on xyloglucan oligosaccharides (XLFG), leading to the sequential formation of XXFG, XXLG, XXXG, GXXG and XXG, as identified by matrix-assisted laser desorption ionization time of flight (MALDI-TOF). AtFXG1 (At1g67830) and AtXYL1 (At1g68560) had been previously identified as the Arabidopsis genes coding for alpha-fucosidase and alpha-xylosidase, respectively. As for the genes coding for beta-galactosidase activity, we identified in phylogenetic trees 12 candidates from family 35 of glycoside hydrolases. Similarly, four genes from family 3 were selected as possible beta-glucosidases active on xyloglucan. The expression level of all the selected genes was studied in different plant regions (young and mature rosette leaves, apical and basal region of the inflorescence stem, roots, flower and siliques) using quantitative real-time reverse transcription-PCR. The expression patterns were very diverse as well as their relationship with growth rates, showing a very complex situation. This could lead to highly varying proportions of the different xyloglucan oligosaccharides in different plant regions and developmental stages.

AtFXG1, an Arabidopsis gene encoding alpha-L-fucosidase active against fucosylated xyloglucan oligosaccharides.

An alpha-L-fucosidase (EC 3.2.1.51) able to release the t-fucosyl residue from the side chain of xyloglucan oligosaccharides has been detected in the leaves of Arabidopsis plants. Moreover, an alpha-L-fucosidase with similar substrate specificity was purified from cabbage (Brassica oleracea) leaves to render a single band on SDS-PAGE. Two peptide sequences were obtained from this protein band, and they were used to identify an Arabidopsis gene coding for an alpha-fucosidase that we propose to call AtFXG1. In addition, an Arabidopsis gene with homology with known alpha-L-fucosidases has been also found, and we proposed to name it as AtFUC1. Both AtFXG1 and ATFUC1 were heterologously expressed in Pichia pastoris cells and the alpha-L-fucosidase activities secreted to the culture medium. The alpha-L-fucosidase encoded by AtFXG1 was active against the oligosaccharides from xyloglucan XXFG as well as against 2'-fucosyl-lactitol but not against p-nitrophenyl-alpha-L-fucopyranoside. However, the AtFUC1 heterologously expressed was active only against 2'-fucosyl-lactitol. Thus, the former must be related to xyloglucan metabolism.

Changes in alpha-xylosidase during intact and auxin-induced growth of pine hypocotyls.

A complete cDNA from Pinus pinaster Aiton, potentially coding for an alpha-xylosidase able to remove the xylose residue from xyloglucan oligosaccharides, has been cloned. Its sequence was homologous to previously published alpha-xylosidase genes from Arabidopsis and nasturtium. The protein also showed the two signature regions of family 31 of glycosyl hydrolases. The gene expression level was quantified by competitive RT-PCR, under different growth conditions, throughout seedling development, in different regions along the hypocotyls and in auxin-treated hypocotyl segments, and related with growth capacity and alpha-xylosidase activity. A role of alpha-xylosidase in regulating the level of xyloglucan oligosaccharides within the apoplast is proposed. The action of an alpha-xylosidase removing the xylose residue, would make possible the action of a beta-glucosidase deblocking the xyloglucan oligosaccharide degradation and it could serve as a control point for the regulation of the apoplastic levels of xyloglucan oligosaccharides.

Role of apoplastic ascorbate and hydrogen peroxide in the control of cell growth in pine hypocotyls.

The growth cessation of plant axis has been related with the formation of diphenyl bridges among the pectic components of the cell wall caused by the action of apoplastic peroxidases using hydrogen peroxide as electron acceptor. The formation of diphenyl bridges is prevented by the presence of ascorbate in the apoplastic fluid which acts as a hydrogen peroxide scavenger. The current work focuses on the role of the apoplastic ascorbate and hydrogen peroxide in the cell growth. The addition of hydrogen peroxide caused an inhibition of the auxin-induced growth as well as a significant decrease in the cell wall creep induced by acid-pH solutions. The hydrogen peroxide content in apoplastic fluid increased with the hypocotyl age and along the hypocotyl axis of 10-day-old pine seedlings, as the growth capacity decreased. On the other hand, the ascorbate content in the apoplastic fluid decreased with the hypocotyl age and along the hypocotyl axis of 10-day-old seedlings. A very significant correlation between the hydrogen peroxide apoplastic level and the growth rate as well as between the ascorbate/hydrogen peroxide molar ratio and the growth rate of hypocotyls have been found suggesting that the redox state is the main factor controlling the cell wall stiffening mechanism and thus growth in pine hypocotyls.