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| Plant Physiol. 2008 May; 147(1): 63–77. doi: 10.1104/pp.107.110023. | PMCID: PMC2330305 |
Copyright © 2008, American Society of Plant Biologists Cell Wall Modifications in Arabidopsis Plants with Altered α- l-Arabinofuranosidase Activity [C][W]Ricardo A. Chávez Montes, Philippe Ranocha, Yves Martinez, Zoran Minic,1 Lise Jouanin, Mélanie Marquis, Luc Saulnier, Lynette M. Fulton,2 Christopher S. Cobbett, Frédérique Bitton, Jean-Pierre Renou, Alain Jauneau, and Deborah Goffner* UMR 5546, CNRS-Université Paul Sabatier, Surfaces Cellulaires et Signalisation chez les Végétaux, BP 42617 Auzeville, 31326 Castanet-Tolosan, France (R.A.C.M., P.R., Y.M., A.J., D.G.); Laboratoire de Biologie Cellulaire, Institut National de la Recherche Agronomique, 78026 Versailles cedex, France (Z.M., L.J.); Biopolymères Interactions Assemblages, Unité de Recherche sur les Polysaccharides leurs Organisations et Interactions, Institut National de la Recherche Agronomique, BP 71627, 44316 Nantes cedex 03, France (M.M., L.S.); Department of Genetics, University of Melbourne, Victoria 3010, Australia (L.M.F., C.S.C.); and Unité de Recherche en Génomique Végétale, INRA-CNRS, CP 5708, 91057 Evry cedex, France (F.B., J.-P.R.) Received September 28, 2007; Accepted March 13, 2008. |
Abstract Although cell wall remodeling is an essential feature of plant growth and development, the underlying molecular mechanisms are poorly understood. This work describes the characterization of Arabidopsis (Arabidopsis thaliana) plants with altered expression of ARAF1, a bifunctional α-l-arabinofuranosidase/β-d-xylosidase (At3g10740) belonging to family 51 glycosyl-hydrolases. ARAF1 was localized in several cell types in the vascular system of roots and stems, including xylem vessels and parenchyma cells surrounding the vessels, the cambium, and the phloem. araf1 T-DNA insertional mutants showed no visible phenotype, whereas transgenic plants that overexpressed ARAF1 exhibited a delay in inflorescence emergence and altered stem architecture. Although global monosaccharide analysis indicated only slight differences in cell wall composition in both mutant and overexpressing lines, immunolocalization experiments using anti-arabinan (LM6) and anti-xylan (LM10) antibodies indicated cell type-specific alterations in cell wall structure. In araf1 mutants, an increase in LM6 signal intensity was observed in the phloem, cambium, and xylem parenchyma in stems and roots, largely coinciding with ARAF1 expression sites. The ectopic overexpression of ARAF1 resulted in an increase in LM10 labeling in the secondary walls of interfascicular fibers and xylem vessels. The combined ARAF1 gene expression and immunolocalization studies suggest that arabinan-containing pectins are potential in vivo substrates of ARAF1 in Arabidopsis. |
Cell walls undergo dynamic changes during plant growth and development. Wall composition and macromolecular assembly vary greatly among taxa, species, organs, and cell types within an individual or domains of a given cell wall. These differences contribute to cell shape and, in some cases, specialized cellular function. Cell wall-related genomic approaches in different physiological contexts have revealed that many cell wall biosynthetic/modifying enzymes and structural proteins are regulated at the transcriptional level. In the case of secondary wall formation, one can correlate morphological and cytological cellular changes with the spatial and temporal regulation of wall-modifying enzymes over a developmental xylem gradient in poplar ( Populus spp.; Schrader et al., 2004) and in in vitro tracheary elements (TEs) of zinnia ( Zinnia elegans; Milioni et al., 2001; Demura et al., 2002; Pesquet et al., 2005). In a genomic approach of zinnia TEs, Pesquet et al. (2005) identified a family 51 (Carbohydrate Active enZYmes [CAZY] database, http://www.cazy.org; Coutinho and Henrissat, 1999) α- l-arabinofuranosidase (arabinofuranosidase) that was highly expressed in TE induction medium at the onset of secondary wall formation. Interestingly, although relatively little redundancy was observed in sequence data generated from the different genomic studies in zinnia, this family 51 arabinofuranosidase gene was systematically identified. At3g10740 is the closest Arabidopsis ( Arabidopsis thaliana) homolog to the arabinofuranosidase zinnia sequence identified. Although At3g10740 was originally named ASD1, we refer to it as ARAF1 from here on, because ARAf was the name attributed to the corresponding purified protein ( Minic et al., 2004). This enzyme belongs to family 51 glycosyl-hydrolases (GHs), but similar enzymatic activities are found for enzymes that belong to family 3 GHs. α- l-Arabinofuranosidases (EC 3.2.1.55) are defined as enzymes that catalyze the hydrolysis of terminal nonreducing α- l-arabinofuranoside residues. However, several enzymes in families 3 and 51 are capable of hydrolyzing both l-Ara and d-Xyl from a variety of substrates in vitro and therefore may be considered as bifunctional arabinofuranosidase/ β- d-xylosidase (xylosidase; EC 3.2.1.37) enzymes. For example, when considering barley ( Hordeum vulgare) ARA-I, Arabidopsis XYL3 and ARAF1, and alfalfa ( Medicago sativa) MsXyl1, the kcat/ Km ratio using artificial substrates such as 4-nitrophenyl- α- l-arabinofuranose (pNPA) and 4-nitrophenyl- β- d-xylopyranose are of the same order of magnitude, suggesting that they are true bifunctional enzymes ( Lee et al., 2003; Minic et al., 2004, 2006; Xiong et al., 2007). The hydrolytic activity of many of these enzymes has also been tested with natural polysaccharidic substrates. For example, barley ARA-I preferentially hydrolyzes d-Xyl rather than l-Ara residues of wheat ( Triticum aestivum) arabinoxylan (WAX; Lee et al., 2003), whereas XYL3 and ARAF1 from Arabidopsis and MsXyl1 from alfalfa hydrolyze both l-Ara and d-Xyl from WAX and l-Ara from sugar beet ( Beta vulgaris) arabinan (SBA) at approximately the same rates in vitro ( Minic et al., 2004, 2006; Xiong et al., 2007). In contrast, barley XYL hydrolyzes only d-Xyl from WAX and has a kcat/ Km ratio for 4-nitrophenyl- β- d-xylopyranose approximately 30-fold higher than for pNPA, suggesting that it is a true xylosidase ( Lee et al., 2003). The barley AXAH-I hydrolyzes l-Ara from SBA, larchwood ( Larix dahurica) arabinogalactan, and WAX, but not d-Xyl from WAX, suggesting that it is more likely true arabinofuranosidase ( Lee et al., 2001). Although biochemical data for purified radish ( Raphanus sativus) RsAraf1 are not available, its overexpression in Arabidopsis provoked an increase in total arabinofuranosidase activity with only a minor increase in total xylosidase activity, suggesting that it is also a true arabinofuranosidase ( Kotake et al., 2006). Finally, Japanese pear ( Pyrus pyrifolia) PpARF2 hydrolyzes l-Ara from SBA but does not hydrolyze l-Ara or d-Xyl from WAX, indicating that it is a pectin-specific true arabinofuranosidase ( Tateishi et al., 2005). Because enzymatic activity does not necessarily correlate with the protein primary structure, the plant gene database annotations “ α- l-arabinofuranosidase” and “ β- d-xylosidase” should be regarded with skepticism until biochemical and physiological data become available. At the molecular level, ARAF1 gene expression and that of another closely related family 51 arabinofuranosidase gene, At5g26120 ( ARAF2), have been partially characterized in Arabidopsis ( Fulton and Cobbett, 2003). Whereas ARAF2 expression was limited to the vasculature in older root tissue and in floral organs and abscission zones, ARAF1 was expressed ubiquitously throughout the plant, and especially in vascular tissues, in agreement with data available in the Genevestigator database ( https://www.genevestigator.ethz.ch/; Zimmermann et al., 2004). The deduced ARAF1 protein sequence contains an N-terminal signal peptide that predicts an extracellular localization. Moreover, proteomic analyses undertaken in Arabidopsis indicated that ARAF1 is present in the extracellular fraction ( Charmont et al., 2005; Jamet et al., 2006; Minic et al., 2007). Together, these results suggest that ARAF1 acts on polysaccharides at the cell wall. Here, we have adopted a genetic approach to determine which of the l-Ara-containing (or d-Xyl-containing) cell wall components may be the physiological substrate of ARAF1 in planta. We identified T-DNA-tagged insertional mutants lacking ARAF1 activity and produced transgenic Arabidopsis plants that overexpress ARAF1. Beyond global sugar analysis, we probed for wall modifications at the cellular level by comparing immunolocalization patterns using antibodies raised against α-(1,5)-linked l-Ara (LM6) and β-(1,4)-linked d-Xyl (LM10 and LM11) residues. The data presented suggest that arabinans are potential in vivo substrates of ARAF1. |
RESULTS pARAF1 GUS Analysis Revealed New Cell-Specific Expression Sites Reverse transcription (RT)-PCR expression analysis previously indicated that ARAF1 transcript was detected in all organs examined ( Fulton and Cobbett, 2003). Data retrieved from the Genevestigator database and RT-PCR analysis carried out herein confirmed these results ( https://www.genevestigator.ethz.ch/; Zimmermann et al., 2004; data not shown). To provide a comprehensive view of ARAF1 expression, the pARAF1GUS transformants originally described by Fulton and Cobbett (2003) were used. Fulton and Cobbett (2003) originally showed that ARAF1 is preferentially expressed in the vascular tissues of different organs. Therefore, we undertook a detailed analysis, particularly in stems and roots, of the precise cell types expressing ARAF1. In 7-d-old seedlings, GUS expression was observed in the vascular cylinders of hypocotyls and roots ( Fig. 1, A and B), in emerging lateral roots ( Fig. 1B), and in the vasculature of cotyledons (data not shown). In the apical portion of the stem, GUS expression was localized in the phloem, cambial region, and cells surrounding the protoxylem and developing metaxylem ( Fig. 1C). In the basal portion of the stem, GUS expression was identical to that in the apical portion ( Fig. 1D). It is interesting that ARAF1 was not expressed in the secondary xylem ( Fig. 1D). In roots of adult plants, GUS expression was detected in the primary xylem, in patches throughout extraxylary tissues ( Fig. 1E), and in developing secondary xylem vessels close to the cambium ( Fig. 1F). In roots with xylem fibers, GUS activity was also present in the cambial region ( Fig. 1G). Finally, GUS expression was observed in the guard cells in stems ( Fig. 1H).  | Figure 1.Histochemical localization of GUS activity in pARAF1GUS transformants. In 7-d-old seedlings, GUS activity is visible in the vascular cylinder of the hypocotyl (A) and in root and emerging lateral roots (B). In the upper (C) and lower (D) parts (more ...) |
T-DNA-Tagged ARAF1 Mutants Were Identified araf1-1, a mutant line in the ecotype Wassilewskija (Ws) background, was identified in the Versailles T-DNA insertional mutant collection ( Bechtold et al., 1993). Segregation analysis on selective medium containing kanamycin and Southern-blot analysis indicated that araf1-1 contained only one T-DNA insertion (data not shown). Sequencing confirmed that the T-DNA insertion was located in the 17th exon ( Fig. 2A). No ARAF1 transcript could be detected by RT-PCR in the araf1-1 mutant using the rt-araf1-1 primers ( Fig. 2B). However, the presence of a hybrid ARAF1-T-DNA mRNA transcript in the araf1-1 mutant was detected using a gene-specific primer, hyb-araf1-1, and T-DNA right border primer, RBver ( Fig. 2A; data not shown). As this could have led to the translation of a modified, yet active, form of ARAF1, wild-type and araf1-1 stem crude protein extracts were analyzed. The arabinofuranosidase activity profiles following CM-Sepharose chromatography were obtained ( Fig. 2C). From fractions 1 to 45, wild-type stems exhibited three arabinofuranosidase peaks (peaks I, II, and III), whereas araf1-1 stems exhibited only two peaks (peaks I and III). Peak III in fractions 35 to 45 corresponded to XYL1, a previously identified and characterized bifunctional arabinofuranosdase/xylosidase ( Minic et al., 2004). Fractions 10 to 20 showed two arabinofuranosidase activity peaks for the wild-type line and one in the mutant line. Peak II, which was absent in the mutant line, corresponded to ARAF1. In addition, this elution position was similar to that of previously analyzed ARAF1 ( Minic et al., 2004). Peak I corresponded to ARAF2, encoded by the At5g26120 gene (Z. Minic and L. Jouanin, unpublished data). A second araf1 mutant, araf1-2, a mutant in the ecotype Columbia 0 (Col0) background from the GABI-Kat collection ( Rosso et al., 2003), was also identified. The available flanking sequence tag from the GABI-Kat database indicates that the T-DNA insertion in araf1-2 was located in the 11th intron. No ARAF1 transcript could be detected in this mutant using the hmz-araf1-2-F and rt-araf1-2-R primers ( Fig. 2B).  | Figure 2.Schematic illustration of the intron-exon structure of the α-l-arabinofuranosidase At3g10740 gene, RT-PCR analysis of the ARAF1 transcript in wild-type and mutant lines, and chromatographic analysis of arabinofuranosidase enzyme activities. A, (more ...) |
When grown under different conditions, including in soil or in vitro, in the greenhouse or in growth chambers, araf1-1 and araf1-2 showed no reproducible developmental phenotype. Germination was not affected by the mutation. Because ARAF1 was expressed in guard cells, stomatal function measurements according to Jones et al. (2003) were performed. No alterations in guard cell function could be detected as a result of the ARAF1 mutation (S. McQueen-Mason, unpublished data). 35SARAF1 Transgenic Lines Exhibited Developmental Stem Phenotypes To generate ARAF1-overexpressing lines, Col0 plants were transformed with a 35S ARAF1 construct. Homozygous transformant seedlings were screened by semiquantitative RT-PCR for increased ARAF1 transcript accumulation, and three independent lines, 35S ARAF1a, 35S ARAF1b, and 35S ARAF1c, were selected ( Fig. 3A). Activity assays performed on total protein extracts of adult stems using pNPA as a substrate indicated an 8-fold increase in total arabinofuranosidase activity in the 35S ARAF1a line and a 25-fold increase in the 35S ARAF1b and 35S ARAF1c lines compared with Col0 plants ( Fig. 3B). Many developmental parameters, including germination, stem elongation rate, silique development, and seed yield, were unchanged in all 35S ARAF1 lines. However, detailed observation showed that the three lines exhibited a delay in inflorescence emergence (growth stage 5.10; Boyes et al., 2001) of approximately 1 week ( Fig. 4A).  | Figure 3.Semiquantitative RT-PCR analysis and total arabinofuranosidase activity in crude stem extracts of three independent ARAF1-overexpressing lines. A, Semiquantitative RT-PCR analysis of ARAF1 transcript levels in 7-d-old seedlings of the wild type (Col0) (more ...) |
 | Figure 4.Developmental phenotypes in ARAF1-overexpressing lines. A, Number of days after imbibition required for inflorescence emergence (as defined by Boyes et al. [2001]) in Col0 and 35SARAF1 transformants. n = 20 per line. Error bars represent (more ...) |
Arabidopsis plants have a nonuniform number of secondary stems (defined as stems emerging from the main stem). Under our standard growth conditions, the majority of individuals in a wild-type population had n secondary stems. The value for n varied among experiments (between 3 and 5). 35SARAF1 populations exhibited an altered distribution in the number of secondary stems, with the majority of plants having n − 1 secondary stems ( Fig. 4C). A representative experiment is shown in Figure 4B. The majority of wild-type Col0 plants (63%) had an average of three secondary stems, with fewer than 20% of the individuals having either two or four secondary stems. In contrast, nearly 80% of the 35SARAF1a and 35SARAF1b individuals had only two secondary stems, and plants with four secondary stems were absent in 35SARAF1a and 35SARAF1b populations. Modification of ARAF1 Activity in Planta Revealed Subtle Changes in Global Monosaccharide Composition As a first step in assessing the role of ARAF1 in dictating cell wall polysaccharide composition, we determined the repercussions of the absence or overexpression of ARAF1 on the monosaccharide content of alcohol-insoluble residue (AIR) from the wild type, the araf1-1 mutant, and 35SARAF1 transformants ( Table I). Dry seeds, roots, and the upper and lower portions of the stem were all examined because of the ubiquitous expression of ARAF1. In general, l-Ara content was high in seeds (16 mol %) compared with the other organs studied (1–5 mol %). Minor yet significant differences in the amount of some sugars were observed. For example, in dry seeds, l-Ara content was higher in the araf1-1 mutant and lower in 35SARAF1 transformants. In roots and the basal portion of the stem, d-Xyl content was significantly higher in 35SARAF1 lines than in Col0. However, no differences in d-Xyl content were observed for the araf1-1 mutant. Uronic acid content was also significantly lower in the 35SARAF1 lines compared with Col0 in the basal portion of the stem.  | Table I. Monosaccharide composition of AIR from wild-type, mutant, and overexpressing lines |
Immunolocalization Studies Point to Cell Type-Specific Modifications of Cell Wall Polysaccharides in Plants with Modified ARAF1 Activity Because changes in ARAF1 gene expression had only a limited impact on the global monosaccharide composition of the cell wall, we undertook a more detailed analysis of the cell wall by immunolocalization using antibodies raised against specific polysaccharidic wall structures. These studies provide clues to identify in vivo enzyme substrates at the cellular level, even those too subtle to detect by global analyses. Based on in vitro biochemical data for ARAF1, we first compared LM6 epitope distribution in wild-type, mutant, and overexpressing lines. LM6 recognizes arabinans found in pectin and some proteins ( Willats et al., 1998; Lee et al., 2005; Harholt et al., 2006). The observations described below for araf1-1 were confirmed with the araf1-2 mutant (data not shown). The distribution of LM6 epitopes was first investigated in the basal portions of the stem. In the wild type, relatively intense labeling was found in the epidermal cell walls, with very weak labeling in the cortical parenchyma and the pith ( Fig. 5, A and G). LM6 epitopes were more abundant in vascular bundles with an active intrafascicular cambium ( Fig. 5B) than without ( Fig. 5A). Within the vascular bundles, labeling was observed in the cambial region, phloem, and xylem parenchyma cells in close contact with xylem vessels ( Fig. 5B). No signal was observed in cells with secondary walls including xylem vessels ( Fig. 5I) and interfascicular fibers (data not shown).  | Figure 5.Indirect immunofluorescence micrographs of resin-embedded Ws (A, B, C, G, and I) and araf1-1 (D, E, F, H, and J) stem (A, B, D, and E) and root (C and F) sections labeled with LM6 monoclonal antibody. LM6 signal and autofluorescence of tissues at 488 (more ...) |
In the araf1-1 mutant, no qualitative differences in the cell types that were labeled with LM6 were observed. However, an increase in labeling intensity was observed in all cell types ( Fig. 5, D versus A and E versus B). In vascular bundles, labeling was intense in the walls of parenchyma cells in close contact with vessels ( Fig. 5, J versus I). Labeling was also more intense in the cortical parenchyma ( Fig. 5, H versus G) and the pith (data not shown). In the apical portion of the wild-type stems, LM6 labeling was uniformly very low, with the exception of the epidermis, suggesting that α-(1,5)-arabinans are present in very low amounts at this stage of stem development. No differences in LM6 signal intensity were observed in the araf1-1 mutant in this tissue (data not shown). In wild-type roots, LM6 labeling was detected in the phloem, cambial region, and parenchyma cells in close association with xylem vessels ( Fig. 5C). The epidermal and cortical parenchyma cell walls showed barely detectable LM6 labeling. No labeling was detected in xylem vessel walls. Again, in the araf1-1 mutant root, although no qualitative differences in LM6 labeling were detected, signal intensity was higher in all cell types ( Fig. 5, F versus C). As a complementary means to assess the link between ARAF1 and LM6 epitopes, resin-embedded sections of wild-type stems were incubated with ARAF1 enzyme or a commercial arabinofuranosidase from Aspergillus niger. This resulted in a nearly complete loss of LM6 signal ( Fig. 6). These results, together with the mutation-induced increase of LM6 labeling, suggest that LM6 epitopes are likely to be in vivo ARAF1 substrates. Unexpectedly, however, we were unable to detect significant variations in LM6 signal localization or intensity compared with the wild type in any tissues in the three independent 35SARAF1 lines (data not shown).  | Figure 6.In-section arabinofuranosidase activity assays and subsequent LM6 labeling. LR White resin-embedded sections were treated with buffer (A), partially purified ARAF1 (B), or a commercial arabinofuranosidase from A. niger (C) and subsequently labeled with (more ...) |
As LM6 has been shown to bind pectic arabinan but also some proteins, presumably arabinogalactan proteins ( Lee et al., 2005; Harholt et al., 2006), the observed differences in LM6 labeling could be due to a difference in the amount of pectic arabinan, protein arabinan, or both. To determine whether the amount of protein arabinan is altered in mutant or ARAF1-overexpressing lines, ELISAs using the LM6 antibody were performed on total protein extracts from the lower part of stems of wild-type, araf1 mutant, and 35SARAF1 lines. No significant differences in the quantity of LM6 epitopes could be detected ( Fig. 7), suggesting that the absence or overexpression of ARAF1 does not alter the structure of the arabinan moiety of proteins. These data, together with immunolocalization, argue in favor of the arabinan component of pectins rather than proteins as the potential in vivo substrate of ARAF1.  | Figure 7.ELISA quantification of LM6-reactive proteins in crude stem extracts. A, Ws (black squares) and araf1-1 (white squares). B, Col0 (black squares), 35SARAF1b (white circles), and 35SARAF1c (white triangles). It should be noted that the (more ...) |
Because in vitro assays suggested that ARAF1 can hydrolyze l-Ara and d-Xyl present in arabinoxylans ( Minic et al., 2006), we tested the effect of modified ARAF1 expression on the distribution and amounts of xylan using LM10 and LM11 antibodies ( McCartney et al., 2005). The general belief is that LM10 recognizes low-substituted arabinoxylans whereas LM11 recognizes both low- and higher-substituted arabinoxylans ( Carafa et al., 2005; Bauer et al., 2006; Zhou et al., 2006). In Arabidopsis stems, LM10 and LM11 signals are found in cells with secondary walls ( http://www.bmb.leeds.ac.uk/staff/jpk/gal5.htm; Bauer et al., 2006; Zhou et al., 2006). In agreement with these results, we detected LM10 label in xylary and interfascicular fibers and in xylem vessels in the basal portion of wild-type stems ( Fig. 8A). One to two layers of parenchyma cells in the pith and in the cortex adjacent to interfascicular fibers were also labeled ( Fig. 8A, dashed arrows). In ARAF1-overexpressing lines, although the distribution of the LM10 signal was not modified, the intensity was enhanced significantly ( Fig. 8, B versus A). The difference in signal intensity was greater in 35SARAF1b and 35SARAF1c than in the 35SARAF1a line (data not shown). This correlated with the more enhanced arabinofuranosidase activity measured in these lines ( Fig. 3B). No differences in LM10 labeling were observed in the araf1-1 mutant (data not shown). This is perhaps not surprising because ARAF1 is not normally expressed in LM10-containing cell types.  | Figure 8.Indirect immunofluorescence micrographs of resin-embedded Col0 and 35SARAF1b stem (A and B) and root (C and D) sections labeled with the LM10 monoclonal antibody. LM10 signal and autofluorescence of tissues at 488 nm are indicated by the red (more ...) |
In wild-type roots, LM10 epitopes were localized exclusively in cells within the stele ( Fig. 8C). In the outermost portion of the xylem, vessels and fibers were labeled, whereas in the inner part of the xylem, the signal was restricted to the vessels. In ARAF1-overexpressing roots, as is the case in stems, LM10 signal intensity was increased significantly, although no change in distribution was observed ( Fig. 8D). Again, LM10 labeling was unchanged in the araf1-1 mutant (data not shown). Using the LM11 antibody, similar increases in signal intensity were observed for all lines and tissues examined (data not shown). The increase of LM10 and LM11 labeling in 35SARAF1 transformants suggested that ARAF1 is able to alter xylan composition. To determine the effect of arabinofuranosidase activity on LM10 and LM11 binding, resin-embedded sections of the lower part of the stem from wild-type plants were treated with ARAF1 or a commercial A. niger arabinofuranosidase and subsequently labeled with LM10 or LM11 antibodies. The commercial arabinofuranosidase from A. niger is reported to hydrolyze l-Ara, but not d-Xyl, from wheat arabinoxylan. Arabinofuranosidase treatment of sections did not alter LM10 or LM11 signal intensity (data not shown), suggesting that, at least at this particular stage of stem development, neither of the arabinofuranosidases altered the LM10 or LM11 epitope structures. Alternatively, the increase in LM10 and LM11 signal in the ARAF1 overexpressors may be due to an increase in xylan content, as indicated by monosaccharide analysis or structural secondary wall modifications leading to increased antibody accessibility to xylan. Transcriptomic Analysis of araf1-1 and 35SARAF1bPairwise transcriptomic comparisons of the basal portion of stems were performed on Ws versus araf1-1 and Col0 versus 35SARAF1b using the complete Arabidopsis transcriptome microarray (CATMA; Crowe et al., 2003). The CATMA contains 24,576 sequences corresponding to known and predicted genes. The araf1-1 mutation had only a very minor impact on global gene expression, in keeping with the subtle cell-specific wall modifications (Supplemental Table S1). Among the 54 differentially expressed genes, six that are classified in the CAZY database were up-regulated 2- to 3-fold: a putative β-amylase (At5g18670), an endoxyloglucan transferase (AtXth4; At2g06850), a putative β-galactosidase (At5g56870), a putative α-galactosidase (At5g20250), and two putative trehalose-P synthases, TPS2 (At1g70290) and TPS11 (At2g18700). Four genes participating in hormone metabolism or hormone response were also found to be differentially expressed in araf1-1. None of the genes associated with global l-Ara metabolism, including l-arabinosyl-transferases and Ara-kinases, exhibited differential expression as a result of the araf1 mutation. In contrast to araf1-1, the overexpression of ARAF1 resulted in far-reaching effects on global gene expression, with 1,784 differentially expressed genes in 35SARAF1b (Supplemental Table S2). Fifty of them are classified in the CAZY database. Among them are genes with a putative role in l-Ara metabolism, including the other arabinofuranosidase family 51 member, ARAF2 (At5g26120), which was up-regulated, and a putative endo-arabinanase (At5g67540), which was down-regulated ( Table II). No GH family 3 members or putative β-1,4-xylanases were differentially expressed. The putative functions of the CAZY genes that were differentially regulated in 35SARAF1b were highly variable; they included putative pectin methyl-esterases, putative family 1 pectate-lyases, several β-glucosidases, and several glucosyl-transferases. We also found three down-regulated trehalose-P synthases, TPS1 (At1g78580), TPS7 (At1g06410), and TPS11 (At2g18700; Table II). TPS1 has been shown to participate in the regulation of carbon metabolism and the transition from the vegetative to the reproductive stage in Arabidopsis ( Schluepmann et al., 2003; van Dijken et al., 2004). Many genes involved in secondary wall metabolism were also differentially expressed ( Table II). They included genes encoding enzymes of the shikimate (a putative chorismate synthase, a putative dehydroquinate dehydratase, and a hydroxy-cinnamoyltransferase) and phenylpropanoid (among them were two Phe ammonia-lyases, including PAL1, cinnamoyl-CoA reductase 1 and 2, a putative 4-coumaroyl-CoA synthase, and a cinnamic acid 4-hydroxylase) pathways. Furthermore, Bglu45 (At1g61810), a monolignol β-glucosidase, was also down-regulated. These results suggest that the alteration of polysaccharide metabolism leads to the regulation of the lignin biosynthetic pathway. However, UV autofluorescence and phloroglucinol staining of the lower part of the stem fresh sections did not reveal any alterations in lignin distribution in any of the 35SARAF1 lines (data not shown). Finally, a large number of genes participating in hormone metabolism, in particular auxin, abscisic acid, and ethylene, and genes involved in abiotic stimuli response and plant-microorganism interactions, were also among the differentially expressed genes in 35SARAF1b. These are presumably indirect consequences of increased ARAF1 activity on global gene expression and suggest that the modification of cell wall structure may have important consequences in plant development and responses to the environment. | Table II.List of selected CAZY and lignin-related genes differentially expressed in 35SARAF1b stems |
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DISCUSSION Pectic Arabinans Are Potential in Vivo ARAF1 Substrates in Arabidopsis In this study, we have determined the sites of ARAF1 gene expression at the cellular level and, in combination with genetic and immunochemical approaches, have identified pectic arabinans as potential in vivo substrates of ARAF1. ARAF1 is expressed in vascular tissues, including the cambium, phloem, and xylem parenchyma cells, and is largely overlapping with LM6 labeling in wild-type plants. In-section assays in which wild-type stem sections were treated with ARAF1 enzyme resulted in the nearly complete disappearance of LM6 signal. In addition, in ARAF1-deficient plants, an increase in LM6 labeling was observed, suggesting that the absence of ARAF1 causes the accumulation of LM6 epitopes. As indicated by ELISA using crude protein extracts, this increase was not due to an increase in LM6 epitope-containing glycoproteins. Furthermore, Harholt et al. (2006) have shown that an important proportion of the LM6 signal in the stem can be attributed to the arabinan component of rhamnogalacturonan-I. Finally, biochemical data indicate that pectic arabinan is a substrate of ARAF1 in vitro ( Minic et al., 2004, 2006). Together, these results suggest that pectic arabinans are potential substrates of ARAF1 in vivo. Unexpectedly, 35SARAF1 lines showed no reduction in LM6 signal intensity. One explanation could be that, in muro, ARAF1 alone cannot fully hydrolyze arabinans and that the wild-type ARAF1 enzyme is already hydrolyzing LM6 epitope-containing wall components at a maximal rate. This scenario is not likely because wild-type stem sections treated with native Arabidopsis ARAF1 resulted in the complete disappearance of LM6 signal, suggesting that ARAF1 can indeed completely hydrolyze arabinans when present in sufficient quantities. Another more plausible explanation is that fine-tuning mechanisms are controlling arabinan dynamics at the cell wall. The existence of such mechanisms is apparent when considering a wild-type plant. LM6 signal is readily detected in cells, despite the presence of ARAF1, which hydrolyzes arabinans in these same cell types. An equilibrium between active arabinofuranosidases and arabinan-containing substrates has already been described in Arabidopsis microcalli cultures ( Leboeuf et al., 2004). It is possible that an increase in ARAF1 activity in the overexpressing lines might accelerate the rate of overall arabinan cycling; that is, not just hydrolysis but also reincorporation into wall polysaccharides, without having an impact on their final content and/or structure. l-[ 3H]Ara or [ 14C]Ara feeding experiments in proso millet ( Panicum miliaceum), maize ( Zea mays; Gibeaut and Carpita, 1991), and Arabidopsis ( Dolezal and Cobbett, 1991; Burget et al., 2003) showed that l-Ara is actively cycled between a monosaccharide and a polysaccharide state, suggesting that arabinans are undergoing constant remodeling. A final argument for fine-tuning mechanisms in cell wall arabinan metabolism is that biosynthetic and hydrolytic enzymes are at least partially colocalized in the same cell types. For example, ARAD1, a putative pectin arabinosyltransferase, is expressed in certain ARAF1-expressing cell types in stems and roots ( Harholt et al., 2006). Interestingly, the overexpression of ARAD1 in a wild-type background did not lead to an increase in l-Ara content in the cell wall, suggesting that arabinan biosynthesis is not regulated at the transcriptional level of glycosyltransferase genes. A detailed analysis of the possible interactions between arabinan hydrolytic and biosynthetic pathways would help in understanding what determines cell wall l-Ara content. Localized Arabinan Alterations Do Not Lead to a Visible Phenotype When considering the restricted cell-specific ARAF1 gene expression, it is perhaps not surprising that ARAF1-deficient plants did not exhibit major differences in cell wall global monosaccharide composition, particularly l-Ara, under normal growth conditions. The cell-specific localization of ARAF1 may also explain the limited number of genes that were differentially expressed in the araf1-1 mutant background. Two mutants with reduced l-Ara content, mur4 and arad1, have been characterized previously. arad1 is affected in an arabinosyltransferase gene and is characterized by a 70% reduction in the l-Ara content of the rhamnogalacturonan-I fraction ( Harholt et al., 2006). mur4 is a mutant of a UDP- d-Xyl4-epimerase and has a 50% to 75% reduction in l-Ara content that affects not only pectic arabinan but also other l-Ara-containing molecules such as rhamnogalacturonan-II and arabinogalactan proteins ( Burget and Reiter, 1999; Burget et al., 2003). Similar to araf1, neither of these mutants exhibits an apparent phenotype. Together, these results indicate that a large plasticity is tolerated in the amount of l-Ara present not only in arabinans but also in a wider range of l-Ara-containing molecules. Other structural features of pectins besides arabinan composition appear to be more critical for normal plant development. For example, the qua1 mutant, which is defective in a family 8 putative glycosyltransferase, exhibited a reduction of 25% in homogalacturonan, resulting in a wide range of phenotypic defects, including dwarfing and a loss of cell adhesion ( Bouton et al., 2002; Leboeuf et al., 2004). The mur1 mur4 double mutant has an extreme dwarfed phenotype that has been attributed to defects in rhamnogalacturonan-II structure ( Burget et al., 2003). Although arabinan has been shown to play an important role in guard cell function in isolated epidermal strips treated with exogenous fungal endo-arabinanse ( Jones et al., 2003), it is unclear whether endogenous arabinan-remodeling enzymes actually participate in this process. Guard cell function was not affected in either araf1 (S. McQueen-Mason, personal communication) or arad1 mutants, although in the latter case it is unknown whether ARAD1 is expressed in guard cells ( Harholt et al., 2006). It would be of interest to determine whether the overexpression of an endogenous arabinanase with a major impact on LM6 labeling would affect stomatal function. Another important factor that may explain a lack of visible phenotype in araf1 mutants is the presence of other arabinofuranosidase activities with overlapping spatiotemporal expression that may compensate for ARAF1 activity. In Arabidopsis stems, there are at least two other enzymes that fit these criteria: XYL1 (encoded by At5g49360) and ARAF2 (encoded by At5g26120). Although XYL1 is a GH family 3 member, its biochemical characteristics are almost identical to those of ARAF1 ( Minic et al., 2004). Genevestigator database mining indicated nearly identical organ expression between the two genes ( https://www.genevestigator.ethz.ch/). Furthermore, the analysis of pXYL1GUS plants showed that XYL1 is expressed in the fibers, protoxylem, metaxylem, and intrafascicular cambium but not in the phloem ( Goujon et al., 2003). ARAF2 is a family 51 GH with 78% identity to ARAF1 and has been shown to hydrolyze pNPA (Z. Minic and L. Jouanin, unpublished data). Analysis of pARAF2GUS plants indicated that ARAF2 is expressed in the cambium and phloem but not in the xylem (R.A. Chávez Montes and D. Goffner, unpublished data). The partially overlapping expression patterns of these enzymes suggest that functional redundancy is plausible in some cell types. That said, the araf2 single mutant exhibited no apparent phenotype, and more importantly, the phenotype of araf1 araf2 double mutants was not additive compared with that of the single araf1-1 mutant (data not shown), suggesting that compensation by ARAF2 in the araf1 mutant background was unlikely. Compensation by other family 3 or even family 43 GHs, however, cannot be excluded because there is no information currently available on the expression or substrate specificity of these enzymes. However, transcriptomic comparisons between araf1-1 and wild-type plants suggest that compensation mechanisms are not regulated at the transcriptional level because none of the above-mentioned GHs exhibited increased expression in mutant lines. In any case, it would be of interest to characterize other combinatorial mutants to assess the effects of different arabinan modifications on plant development and to determine to what extent these enzymes may work in a coordinated manner in remodeling pectin structure. Ectopic Expression of ARAF1 Leads to Secondary Wall Alterations and a Developmental Phenotype The increase in LM10 and LM11 labeling intensity in the secondary walls of 35SARAF1 lines is intriguing. Although there are several possible explanations, none of them provides information regarding the in vivo substrate of ARAF1, because these wall modifications occurred in cell types in which ARAF1 is not normally expressed. One possibility is that the ectopic overexpression of ARAF1 could lead to the excess hydrolysis of arabinosyl residues of arabinoxylans in secondary walls. This would result in a decrease in the degree of substitution of arabinoxylans and would thereby generate more low and nonsubstituted xylans (epitopes of LM10 and LM11) and/or enhance antibody accessibility to the epitopes. In keeping with this hypothesis, it has been shown that ARAF1 hydrolyzes l-Ara from arabinoxylans in vitro ( Minic et al., 2004, 2006). However, if this were true, one would expect an increase in LM10 and LM11 labeling in ARAF1-treated sections, and this was not the case. Moreover, recent mass spectrometry analysis has shown that xylans present in mature Arabidopsis stems are glucuronoxylans rather than arabinoxylans (L. Jouanin, unpublished data). Interestingly, the overexpressing lines exhibit an increase in d-Xyl content in roots and stems, and perhaps this in itself is sufficient to explain an increase in LM10 and LM11 signal in these organs. In this case, altered secondary wall structure is most likely an indirect consequence of ARAF1 overexpression. Comparative transcriptomic analysis of 35SARAF1 lines indicates that ARAF1 overexpression has far-reaching consequences on global gene expression, some of which could be associated with changes in secondary cell wall structure. For example, several key genes involved in monolignol biosynthesis are down-regulated in 35SARAF1b stems. This could result in an altered lignin network that, in turn, could lead to enhanced accessibility of LM10 and LM11 epitopes. However, UV autofluorescence and phloroglucinol staining of 35SARAF1a, - b, and - c stems did not reveal any major alterations in lignin distribution in these lines. A detailed analysis of the modified pathways in the 35SARAF1 lines will be necessary to determine the causal relationship between ARAF1 overexpression and secondary cell wall alterations. A developmental phenotype was observed when ARAF1 was ectopically expressed in Arabidopsis. In 35SARAF1 lines, a delay in inflorescence emergence and fewer secondary stems were observed. Similarly, potato ( Solanum tuberosum) plants overexpressing an A. niger endo-arabinanase exhibited altered stem architecture, flowering defects, and even starch accumulation in the stem ( Skjøt et al., 2002; Borkhardt et al., 2005). Together, these results suggest a close relationship between arabinans, carbon status, and overall plant development. Stem emergence is the result of a vegetative-to-reproductive stage transition and is controlled by hormone ( Koornneef et al., 1998) and metabolic (mostly carbon status) levels. In particular, trehalose has been shown to be important for this transition ( van Dijken et al., 2004). Interestingly, in the 35SARAF1b line, three trehalose-P synthases, TPS1 (At1g78580), TPS7 (At1g06410), and TPS11 (At2g18700), were all down-regulated. Moreover, many hormone-related genes were also differentially expressed. The observed delay in inflorescence emergence in the 35SARAF1 lines may be the result of these transcriptional modifications. The Role of ARAF1 Is Likely To Be Cell Type-Dependent Although expression of the zinnia and hybrid aspen ( Populus tremula × Populus tremuloides) ARAF1 homologs has been shown to be correlated with xylem differentiation ( Demura et al., 2002; Aspeborg et al., 2005; Pesquet et al., 2005), the expression of ARAF1 in cell types with distinct cellular functions suggests a cell type-specific role for ARAF1. Besides vascular tissues such as phloem, intrafascicular cambium, and xylem, ARAF1 is also expressed in guard cells, root tips, trichomes, newly emerging leaves, floral abscission zones ( Fulton and Cobbett, 2003), and developing embryos ( Gomez et al., 2006). Interestingly, coexpression analysis of ARAF1 and ARAF2 in plant organs, tissues, and mutants using the Expression Angler from the University of Toronto ( http://bbc.botany.utoronto.ca/ntools/cgi-bin/ntools_expression_angler.cgi) on NASCArrays indicated that among genes that had the highest degree of coexpression with ARAF1 were four cell wall-related genes: UGE1 (At1g12780), a UDP- d-Glu/UDP- d-Gal4 epimerase, a Xyl epimerase (At5g57655), XYL1 (At5g49360), and FRA8 (At2g28110), whereas for ARAF2, no easily identifiable cell wall-related genes exhibited high levels of coexpression. The integration of gene expression data at the cellular level, enzymatic characterization, and detailed cell wall composition analyses of plants with modified family 3 and 51 arabinofuranosidase activities should enable us to identify their in vivo substrates and understand their roles in plant physiology. |
MATERIALS AND METHODS Plant Materials and Growth Conditions Arabidopsis (Arabidopsis thaliana) ecotypes Ws and Col0 were used in this work. Two insertional mutants for the At3g10740 gene, araf1-1 and araf1-2, were obtained. araf1-1, a mutant in the Ws background, was obtained from the T-DNA insertion line FLAG_091G07 of the Versailles collection. araf1-2, a mutant in the Col0 background, was obtained from the T-DNA insertion line GABI_204A04 of the GABI-Kat collection. T3 homozygous mutant lines were identified by PCR. For araf1-1, gene-specific primers hmz-araf1-1-F (5′-TCAGGTGGAACCCTGATGCA-3′) and hmz-araf1-1-R (5′-GGAATGTGGCGGGAGAACAA-3′) and the insert-specific primer LBver (5′-CGGCTATTGGTAATAGGACACTGG-3′) were used. The wild-type form of the gene was detected with the hmz-araf1-1 primers, and T-DNA insertion was detected with the hmz-araf1-1-R and LBver primers. For araf1-2, gene-specific primers hmz-araf1-2-F (5′-CCAGTATCATGCCCTTTGTT-3′) and hmz-araf1-2-R (5′-CAAAGCCTGGGAAGAGAAAT-3′) and the insert-specific primer LB-o8409 (5′-ATATTGACCATCATACTCATTGC-3′) were used. The wild-type form of the gene was detected with the hmz-araf1-2 primers, and T-DNA insertion was detected with the hmz-araf1-2-R and LB-o8409 primers. Wild-type and mutant plants were grown in soil in growth chambers at 22°C (day) and 20°C (night) with 16 h of light and 70% humidity. Adult plants were 6 to 7 weeks old with a 40- to 45-cm-high main stem. Stem emergence (i.e. appearance of the first flower buds) is defined as growth stage 5.10 as described by Boyes et al. (2001). Tissue was frozen in liquid nitrogen, ground to a fine powder, and stored at −80°C until use. For some experiments (AIR composition, immunohistochemistry, and ELISA), the main stem was harvested and divided into lower and upper parts. The lower and upper parts of the stem corresponded to the bottom and top 10 cm of the main stem, respectively. Stem tissue was devoid of any cauline leaves, siliques, or flowers. RT-PCR One milliliter of Extract-All (Eurobio) was added to approximately 100 mg of ground frozen tissue. RNA was isolated according to the Extract-All protocol, then incubated with 5 units of RQ1 RNase-free DNase I (Promega) for 1 h at 37°C and again treated with Extract-All to remove DNase. RNA was quantified spectrophotometrically at 260 nm using a Biophotometer (Eppendorf) and verified by gel electrophoresis. cDNA was generated with 2 μg of RNA using ImPromII reverse transcriptase (Promega). PCR was carried out using the GoTaq Flexi (Promega) protocol with 0.25 μg of cDNA. For the araf1-1 mutant, the absence of ARAF1 transcript was determined using primers rt-araf1-1-F (5′-TGGAACCCTGATGCAATAGT-3′) and rt-araf1-1-R (5′-TCATATCCTCCTCTGCCAAC-3′), resulting in amplicons of 382 bp for cDNA and 539 bp for genomic DNA. Primers hmz-araf1-1 were not used because hmz-araf1-1-F contains part of an intron sequence. Hybrid ARAF1-T-DNA transcript was detected using primers hyb-araf1-1 (5′-TCCAAAACCAGCCGTGACTT-3′) and RBver (5′-CTGATACCAGACGTTGCCCGCATAA-3′) with an annealing temperature of 60°C, resulting in amplicons of approximately 2.3 kb for cDNA and 3.8 kb for genomic DNA. For araf1-2, transcript absence was determined using primers hmz-araf1-2-F and rt-araf1-2-R (5′-GCCATTGTTTTTCCAAGAGATA-3′) with an annealing temperature of 60°C, resulting in amplicons of 689 bp for cDNA and 1,184 bp for genomic DNA. pARAF1GUS Plant Analysis pARAF1GUS plants have been described previously ( Fulton and Cobbett, 2003). GUS staining was performed at 37°C in GUS staining solution (50 m m sodium phosphate buffer, pH 7.0, 0.1% Triton X-100, 1 m m potassium ferricyanide, 1 m m potassium ferrocyanide, and 1 m m X-glucuronoside) with 10 min of vacuum infiltration. After staining, the plant tissue was cleared for at least 1 h with ethanol:acetic acid (1:1, v/v) at room temperature, and tissues were kept at 4°C in 70% ethanol until observation. Transformation of Plants with a 35SARAF1 Overexpression Construct The ARAF1 region from start to stop codon was amplified by RT-PCR from total RNA using gene-specific primers araf1-[+1] (5′-ATGGATATGGAGTCTTGGAAGTTGCTCAGAAG-3′) and araf1-[−1] (5′-TCACACAGTGGTAGTTTTCTGATGGGAAGAAG-3′). The resulting 2,037-bp fragment was cloned into the pGEM-T Easy vector (Promega), resulting in the pGEMT-araf1 vector. This vector was digested with EcoRI, and the resulting 2,057-bp fragment was cloned into the EcoRI cloning site of a vector derived from vector pGreen0029 (pGreen), pGreen0029-35SCAMΩ (a kind gift from Julie Cullimore, Institut National de la Recherche Agronomique, Toulouse, France), which contains two tandem 35S cauliflower mosaic virus promoters, the nptII kanamycin resistance gene, and a cauliflower mosaic virus terminator. Insert sequence and orientation were verified by sequencing. Col0 plants were transformed by Agrobacterium tumefaciens C58-mediated transformation ( Clough and Bent, 1998). T1 transformant seeds were grown on selective medium containing kanamycin (100 mg mL −1), and resistant seedlings were transferred to soil and left to self-pollinate. T2 seeds from each plant were harvested and grown on selective medium containing kanamycin, and resistant seedlings from plates with a segregation ratio of 3:1, indicative of a single T-DNA insertion, were transferred to soil and left to self-pollinate. T3 seeds from each plant were harvested, and a sample was grown on selective medium containing kanamycin. Three seed lots with 100% resistance, indicating homozygous transformants, were selected based on the level of ARAF1 RNA overexpression as assessed by semiquantitative RT-PCR using primers rt-araf1-1. These lines were named 35SARAF1a through 35SARAF1c and used for subsequent analysis. α-l-Arabinofuranosidase Activity Assay Total arabinofuranosidase activity in overexpressing lines was determined in microtiter plates as follows. A crude extract from stem tissue was prepared as described by Minic et al. (2004), and total protein was determined using the bicinchoninic acid method (Sigma). The reaction mixture contained 2 m m pNPA (Fluka), 0.1 m acetate buffer (pH 5.0), and 3.2 μg of protein in a total volume of 0.2 mL. The reaction was carried out at room temperature and stopped at 20-min intervals by the addition of 50 μL of 0.4 m sodium bicarbonate to the assay mixture. The concentration of the product 4-nitrophenol was determined spectrophotometrically at 405 nm, and its amount was estimated from a calibration curve. Specific activity was expressed as the amount of protein required to release 1 nmol min −1 4-nitrophenol. Cell Wall Preparation and Composition Analysis AIRs from the upper and lower parts of the stem and roots were prepared as described by Harholt et al. (2006) with slight modifications. Briefly, ground tissue was boiled in 96% ethanol for 30 min. The supernatant was removed after centrifugation for 5 min at 10,000 g. The pellet was washed eight times with 70% ethanol with subsequent centrifugation. The pellet was then washed twice with 96% ethanol, twice with 100% acetone, and dried in an oven at 40°C overnight. Dry seed AIR was prepared as follows. One hundred to 200 mg of dry seeds from plants grown under the same environmental conditions were frozen in liquid nitrogen and ground to a fine powder. The tissue was boiled in a mixture of chloroform:methanol (1:1, v/v) for 30 min. The supernatant was removed after centrifugation at 10,000 g for 5 min. The pellet was washed twice with acetone, twice with ethyl ether, and left to evaporate overnight. The monosaccharide composition of AIRs was determined as their alditol acetates after acid hydrolysis. Samples were prehydrolyzed with 13 m H 2SO 4 for 30 min at 25°C, then H 2SO 4 concentration was adjusted to 1 m with water and samples were hydrolyzed for 2 h at 100°C as described previously ( Saulnier et al., 1995). Uronic acids were assayed on the supernatant of acid hydrolysis by the automated m-hydroxydiphenyl method ( Thibault, 1979) using GalUA as a standard. Analyses were performed in duplicate. One-way ANOVA and mean comparison tests using Fisher's lsd (95% lsd) were used to compare the sugar composition (molar ratio) of the AIR between the wild type, mutants, and transformants. Immunohistochemistry Samples of Arabidopsis tissues were fixed in 2.5% (v/v) glutaraldehyde in 50 mm cacodylate buffer (pH 7.0). They were dehydrated in a successive ethanol series (20%, 40%, 60%, 80%, 95%, and 100%) and embedded in LR White resin (Electron Microscopy Sciences; 33%, 50%, 66%, and 100% in ethanol). Thin sections (1 μm) were placed on Teflon-coated slides (Electron Microscopy Sciences), blocked in phosphate-buffered saline, 2% Tween, and 1% bovine serum albumin for 2 h (PBST-BSA), and labeled overnight (12 h) at 4°C with primary antibody diluted in PBST-BSA. Sections were washed with PBST and incubated at room temperature for 2 h with a secondary antibody diluted in PBST-BSA. Slides were then washed with deionized water and dried under a stream of dry air. Primary antibodies and dilutions were as follows: LM6 (1:1, v/v), LM10 and LM11 (1:10, v/v; Plant Probes for both). The secondary antibody was a goat anti-rat IgG coupled to the fluorescent dye Alexa Fluor 633 (Molecular Probes) and was used at a 1:1,000 (v/v) dilution. Observations were carried out using a Leica DM RXA2 microscope with a TCS SP2 scanning confocal system. Acquisition settings, including laser power, photomultiplier gain, field of view (X,Y dimension), Z-step, and pixel size of the image, were strictly identical to ensure reliable comparisons between plant material (i.e. the wild type versus transformants or mutants). For each comparison, all sample preparation and observations were performed on the same day. For each experiment, three plants per line (the wild type, transformant, or mutant) and two sections per plant were observed. Three independent experiments for each comparison were performed. In-Section ARAF Activity Assays Resin-embedded lower parts of the stem sections (1 μm) were placed on Teflon-coated slides and blocked for 2 h at room temperature with PBST-BSA. Sections were washed with 200 mm acetate-Triton buffer (pH 4.0; 0.015% Triton X-100). Forty microliters of acetate-Triton buffer containing 120 microunits of enzyme were placed on the sections, and the reaction was carried out for 5 d at 23°C. Every 24 h, the enzyme mixture was replaced with a fresh preparation. Sections were then washed with PBST and immunohistochemistry was carried out as indicated above, starting with the addition of the primary antibody. The enzymes used were an α-l-arabinofuranosidase from Aspergillus niger (Megazyme) and an Arabidopsis ARAF1 preparation obtained as described below. One unit is defined as the amount of enzyme required to hydrolyze 1 μmol min−1 pNPA in acetate buffer (200 mm, pH 4.0). Based on its molecular mass and pI, the A. niger enzyme belongs to the GH 51 CAZY family (Megazyme technical service). ELISAs Total protein extracts were prepared according to Harholt et al. (2006), and protein concentration was determined using the bicinchoninic acid method. Two micrograms of protein extract was then diluted 1:500 (v/v) in PBS to obtain a linear response throughout the protein concentration range used. Ninety-six-well microtiter plates were coated overnight at room temperature with increasing amounts of diluted protein extract. Unbound protein was washed with PBS, and plates were blocked with PBS, 0.1% (v/v) Tween 20, and 1% (w/v) BSA (PBST-BSA). LM6 antibody (50 μL) diluted 1:200 (v/v) in PBST-BSA was added and left for 2 h at room temperature. Unbound antibodies were washed, and 50 μL of alkaline phosphatase-linked anti-rat IgG antibody (Sigma) diluted 1:2,000 (v/v) in PBST-BSA was added and left for 1 h at room temperature. Unbound antibodies were washed, 100 μL of the pNPP Liquid Substrate System for ELISA (Sigma) was added, and the A405 was measured with an ELISA plate reader (Eflab). Chromatographic Separation of α-l-Arabinofuranosidase Activities ARAF1 enzyme activity in araf1-1 mutant, wild-type, and araf1-1 stems were analyzed. Crude protein extracts were separated by cation-exchange chromatography and tested for α- l-arabinofuranosidase activity as described by Minic et al. (2004). Briefly, 2 g of stem tissue was ground in 25 m m BisTris buffer (pH 7.0) containing 200 m m CaCl 2, 10% (v/v) glycerol, 4 μm sodium cacodylate, and Protease inhibitor cocktail (1:200, v/v; Sigma). Two milliliters (1.0 mg) of soluble protein extract was equilibrated in 25 m m acetate buffer (pH 5.0) containing 2% (v/v) glycerol and 0.015% (v/v) Triton X-100 and loaded on a CM-Sepharose (Sigma) cation-exchange column (1.5 cm × 4 cm). Proteins were eluted in the same buffer with a 0 to 0.4 m NaCl discontinuous gradient in steps of 2.5 mL per 0.025 m NaCl. One-milliliter fractions were collected, and 50 μL was assayed for α- l-arabinofuranosidase activity using pNPA as substrate, as described by Minic et al. (2004). For in-section activity assays, ARAF1 enzyme from 35SARAF1b stems was obtained by lectin chromatography followed by cation-exchange chromatography. Crude protein extract was bound in-batch to 1 mL of concanavalin A resin (Sigma) equilibrated in 20 m m Tris buffer (pH 7.4) containing 0.5 m NaCl. Resin was washed with 10 volumes of the same buffer and transferred to an empty 1-mL column. Protein was eluted in a single step with 2 mL of the same buffer containing 0.2 m methyl- α- d-glucopyranose. Eluted proteins were equilibrated and separated by cation-exchange chromatography as described above. The first arabinofuranosidase activity peak eluted from the cation-exchange column contained only one arabinofuranosidase activity, ARAF1. These fractions were pooled and stored at 4°C until use. The lectin chromatography step is necessary to remove the ARAF2 enzyme (Z. Minic and L. Jouanin, unpublished data). Transcriptome Analysis The microarray analysis was carried out using the CATMA ( Crowe et al., 2003; Hilson et al., 2004), containing 24,576 gene-specific tags from Arabidopsis ( Thareau et al., 2003). The spotting of the gene-specific tag amplicons on array slides and the array analysis process have been described by Lurin et al. (2004). RNA was extracted and pooled from the lower part of the stems of two different cultures of Ws, araf1-1, Col0, and 35SARAF1b plants grown under the same exact environmental conditions. For each comparison, one technical replication with fluorochrome reversal was performed for each pool of RNA. RNA integrity, cDNA synthesis, hybridization, and array scanning were performed as described by Vergnolle et al. (2005). Statistical analysis was based on two dye swaps per comparison (Ws versus araf1-1 and Col0 versus 35SARAF1b). For each array, the raw data comprised the logarithm of median feature pixel intensity at wavelengths 635 nm (red) and 532 nm (green). No background was subtracted. In the following description, log ratio refers to the differential expression between the wild type and mutant or overexpressor. An array-by-array normalization was performed to remove systematic biases. First, we excluded spots that were considered to show badly formed features by the experimenter. Then, we performed a global intensity-dependent normalization using the LOESS procedure ( Yang et al., 2002) to correct the dye bias. Finally, on each block of the array, the log ratio median was subtracted from each value of the log ratio of the block to correct any print tip effect on each block. To determine differentially expressed genes, we performed a paired t test on the log ratios, assuming that the variance of the log ratios is the same for all genes. Spots displaying extremes of variance (too small or too large) were excluded. The raw P values were adjusted by the Bonferroni method, which controls the family-wise error rate. We considered as differentially expressed the genes with a Bonferroni P < 5%, as described by Lurin et al. (2004). Sequence data from this article were deposited in the GEO database according to MIAME standards under accession numbers GSE7420 and GSE7421. Supplemental Data The following materials are available in the online version of this article. - Supplemental Table S1. Genes differentially expressed in the lower part of araf1-1 stems.
- Supplemental Table S2. Genes differentially expressed in the lower part of 35SARAF1b stems.
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Acknowledgments We thank Amandine Freydier for all her help during mutant screening; the John Innes Centre and Julie Cullimore for their kind gift of the pGreen plasmid; Jean-Louis Luc and Patricia Panegos for their invaluable help with Arabidopsis cultures; and Simon McQueen-Mason for stomatal function experiments. A special acknowledgment goes to Fabienne Guillon for her insightful discussions on anti-xylan antibody affinity. |
Notes |
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