Molecular Plant Advance Access originally published online on September 2, 2009
Molecular Plant 2009 2(5):1000-1014; doi:10.1093/mp/ssp062
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Arabidopsis thaliana T-DNA Mutants Implicate GAUT Genes in the Biosynthesis of Pectin and Xylan in Cell Walls and Seed Testa
a Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA
b Complex Carbohydrate Research Center, BioEnergy Science Center, 315 Riverbend Road, University of Georgia, Athens, GA 30602, USA
c Department of Plant Biology, University of Georgia, Athens, GA 30602, USA
1 To whom correspondence should be addressed at address b. E-mail dmohnen{at}ccrc.uga.edu, fax 706-542-4412, tel. 706-542-4458.
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Abstract |
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 TOP Abstract INTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Galacturonosyltransferase 1 (GAUT1) is an
1,4-D-galacturonosyltransferase that transfers galacturonic acid from uridine 5'-diphosphogalacturonic acid onto the pectic polysaccharide homogalacturonan (Sterling et al., 2006). The 25-member Arabidopsis thaliana GAUT1-related gene family encodes 15 GAUT and 10 GAUT-like (GATL) proteins with, respectively, 56–84 and 42–53% amino acid sequence similarity to GAUT1. Previous phylogenetic analyses of AtGAUTs indicated three clades: A through C. A comparative phylogenetic analysis of the Arabidopsis, poplar and rice GAUT families has sub-classified the GAUTs into seven clades: clade A-1 (GAUTs 1 to 3); A-2 (GAUT4); A-3 (GAUTs 5 and 6); A-4 (GAUT7); B-1 (GAUTs 8 and 9); B-2 (GAUTs 10 and 11); and clade C (GAUTs 12 to 15). The Arabidopsis GAUTs have a distribution comparable to the poplar orthologs, with the exception of GAUT2, which is absent in poplar. Rice, however, has no orthologs of GAUTs 2 and 12 and has multiple apparent orthologs of GAUTs 1, 4, and 7 compared with either Arabidopsis or poplar. The cell wall glycosyl residue compositions of 26 homozygous T-DNA insertion mutants for 13 of 15 Arabidopsis GAUT genes reveal significantly and reproducibly different cell walls in specific tissues of gaut mutants 6, 8, 9, 10, 11, 12, 13, and 14 from that of wild-type Arabidopsis walls. Pectin and xylan polysaccharides are affected by the loss of GAUT function, as demonstrated by the altered galacturonic acid, xylose, rhamnose, galactose, and arabinose composition of distinct gaut mutant walls. The wall glycosyl residue compositional phenotypes observed among the gaut mutants suggest that at least six different biosynthetic linkages in pectins and/or xylans are affected by the lesions in these GAUT genes. Evidence is also presented to support a role for GAUT11 in seed mucilage expansion and in seed wall and mucilage composition.
Key Words: Carbohydrate metabolism • cell walls • Arabidopsis • biosynthesis • mutant • pectin • mucilage
Received for publication May 13, 2009. Accepted for publication July 13, 2009.
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INTRODUCTION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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The plant cell wall is composed of many structural elements that interact with specificity and elasticity to accommodate cell growth, cell shape, and cell differentiation. The cell wall protects the plant from external environmental and compressive forces, wounding, pathogen attack, and internal turgor pressure (Ridley et al., 2001). Carbohydrates, proteins, and phenolics make up the bulk of the structural framework of the plant cell wall. A detailed understanding of the structure, synthesis, and interaction of these components is important to understand wall function.
The load-bearing structural network of the wall is formed largely by high-molecular-weight cellulose microfibrils in both the primary and secondary walls. Cellulose microfibrils are composed of β-(1,4)-D-glucan chains that are synthesized at the plasma membrane by macromolecular polymeric complexes. The cellulose chains associate by inter- and intra-chain hydrogen bonding to form large microfibrils that can hydrogen bond with hemicelluloses, such as xyloglucan and xylan, to make up the cellulose–hemicellulose network (Herth, 1983; Nishiyama et al., 2002). Hemicelluloses may also play a role in the organization of cellulose microfibrils, their crystallinity, as well as the ratio of cellulose I to Iβ (McCann et al., 1990; Jarvis, 2000). Proteins such as wall glycosyl hydrolyases, extensins, expansins, arabinogalactan proteins, hydroxyproline-rich glycoproteins, proline-rich proteins, wall-associated kinases, and others contribute to wall structural integrity and the regulation of wall structural components (reviewed in Cosgrove, 2000; Showalter, 2001). The pectic polysaccharides form the wall matrix by interacting in known, as well as unidentified, ways with other pectins and with hemicelluloses, wall proteins, and phenolic compounds to yield the complex functional walls of higher plants (reviewed in Ridley et al., 2001).
The hallmark of pectic polysaccharides is the presence of 4-linked -D-galacturonic acid (GalA) residues. There are three major classes of pectic polysaccharides: homogalacturonan (HG), rhamnogalacturonan-I (RG-I), and the substituted galacturonans; rhamnogalacturonan-II (RG-II), xylogalacturonan, and apiogalacturonan (Mohnen, 2008; Scheller et al., 2007). The pectic polysaccharides are covalently linked, as evidenced by the release of RG-I, RG-II, and HG oligosaccharides upon digestion of the wall with endopolygalacturonase, which cleaves the glycosidic linkages of the HG backbone (Keegstra et al., 1973). Additional evidence exists for the possible cross-linking of pectic polysaccharides to xylan (DuPont and Selvendran, 1987), xyloglucan (Popper and Fry, 2007), and phenolics (Ishii and Tobita, 1993), suggesting a high level of structural complexity in the plant cell wall matrix. HG is a polymer of -(1,4)-linked-D-GalA that is largely unbranched, but may be modified by acetyl groups at the O-2 and O-3, and methylesters at the C-6 of many GalA residues (Mort et al., 1993). The tricellular junctions, middle lamellae, and primary wall are rich in HG, which may constitute up to 69% of primary wall pectin (Ridley et al., 2001). The galacturonan of some plant species may be substituted by terminal xylose residues, or by a disaccharide (Xylp-(1,2)-Xylp
) at O3 of GalA residues in xylogalacturonan (Ovodov et al., 1971; Le Goff et al., 2001), or by 2-linked and 3-linked apiose residues in apiogalacturonan (Hart and Kindel, 1970). The substituted galacturonan RG-II represents approximately 10% of pectin (Scheller et al., 2007; Mohnen et al., 2008). An RG-II structural unit, which is thought to be embedded within a larger HG region, consists of four highly complex side chains (A–D) attached to an HG backbone of seven to nine GalA residues: a structure highly conserved across species. RG-II sidechain A is involved in the dimerization of two RG-II molecules, via a borate diester, which confers strength to the wall (reviewed in O'Neill et al., 2004). RG-I has a repeating disaccharide backbone of [-D-GalpA-1,2--L-Rhap-1,4]n. The GalA residues in the RG-I backbone may be O-acetylated as in HG. The rhamnose residues of the RG-I backbone may be branched with -(1,5)-linked arabinan, β-(1,4)-linked galactan, and various arabinogalactan structures (Carpita and Gibeaut, 1993; Mohnen et al., 2008). The regulation of RG-I sidechain structure and abundance is developmentally determined, suggesting that RG-I sidechains have important and diverse structure–function relationships in primary walls (Willats et al., 1999).
Despite the increasing detail with which primary wall structure is understood, few pectin biosynthetic genes have been identified. Thus far, only a single gene, AtGAUT1, has been definitively shown to encode a protein having HG-galacturonosyltransferase (GalAT) activity (Sterling et al., 2006). AtGAUT1 is a Golgi localized type-II membrane protein that catalyzes the elongation of HG oligosaccharides (oligogalacturonides) in an 1,4-configuration, consistent with HG-GalAT function. A BLAST search (www.ncbi.nlm.nih.gov/) of the Arabidopsis genome identified 25 members of the GAUT1-related gene family. The Arabidopsis GAUT1-related gene family has 15 putative galacturonosyltransferase (GAUT) and 10 galacturonosyltransferase-like (GATL) members (Sterling et al., 2006). All the GAUT1-related genes are members of the CAZy GT8 family (Cantarel et al., 2009; www.cazy.org/).
Thus far, three mutants in three GAUT1-related family genes, irx8, parvus, and quasimodo, have been studied and the observed phenotypes suggest that these GAUT proteins, in addition to GAUT1, are likely to have GalAT activity and are involved in the synthesis of cell wall polysaccharides. The Arabidopsis IRX8 and PARVUS proteins are encoded by the GAUT12 and GATL1 genes, respectively, and have 61 and 49% amino acid similarity to GAUT1. The cell walls of irx8-1 and irx8-5 have reduced β-(1,4)-linked xylose and -(1,4)-linked GalA residues (Persson et al., 2007) and are lacking the reducing end pentasaccharide [4)-β-D-Xylp-(1,4)-β-D-Xylp-(1,3)--L-Rhap-(1,2)--D-GalpA-(1,4)-D-Xylp], purified from a sub-fraction of xylan (Peña et al., 2007). It has been suggested that the GalA in this xylan reducing-end pentasaccharide may be inserted by IRX8/GAUT12 (Peña et al., 2007). Alternatively, it has been hypothesized that IRX8/GAUT12 may synthesize a sub-fraction of HG to which xylan is attached (Persson et al., 2007). Recent studies of parvus/gatl1 have demonstrated that parvus and irx8 have a similar reduction in Xyl and β1,4-XylT activity, and, therefore, may be involved in a similar xylan biosynthetic pathway (Brown et al., 2007; Lee et al., 2007). The data provide descriptive evidence for possible functions of IRX8/GAUT12 and PARVUS/GATL1. However, biochemical evidence of enzymatic activity has not been obtained to confirm these hypotheses.
QUA1/GAUT8 has 77% amino acid similarity to GAUT1. Qua1-1 is a severely dwarfed mutant that has reduced cell adhesion in expanding leaves and callus tissue and has cell walls with 25% reduced GalA levels (Bouton et al., 2002; Leboeuf et al., 2005). Membrane preparations from qua1-1 stem tissue are reduced in both 1,4-GalAT and β1,4-XylT activities (Orfila et al., 2005), providing ambiguous results and preventing the definitive identification of QUA1/GAUT8 function. QUA1/GAUT8 has higher amino acid similarity to GAUT1 than either GAUT12 or GATL1, and QUA1/GAUT8 is hypothesized to be an 1,4-GalAT involved in HG biosynthesis (Orfila et al., 2005). However, the observed reductions in xylan and XylT activity of the qua1-1 mutant remain to be clarified.
In order to probe the function of the family of GAUT genes in wall biosynthesis, Arabidopsis T-DNA mutants corresponding to 13 GAUT genes were isolated and analyzed for changes in wall structure. Analysis of the cell wall glycosyl residue compositions of 26 gaut mutants described in this work demonstrates aberrant wall composition among mutants of GAUTs 6, 8, 9, 10, 11, 12, 13, and 14. The changes in the mol% of galacturonic acid, rhamnose, xylose, galactose, and arabinose in the walls of mutants of these eight GAUT genes, compared to wild-type (WT), have distinct patterns, suggesting that these GAUTs have at least six unique functions in pectin and/or xylan biosynthesis. Our results also confirm wall phenotypes of irx8/gaut12 mutants and lethality phenotypes associated with irx8/gaut12 and qua1/gaut8 mutants. Finally, GAUT11 is shown to be involved in the production of Arabidopsis seed testa cell wall and mucilage.
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RESULTS |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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The GAUT Family of Arabidopsis, Poplar, and Rice
The Arabidopsis GAUT1-related gene family encodes 15 GAUT and 10 GATL proteins with 56–84 and 42–53% amino acid sequence similarity, respectively, to GAUT1 (Sterling et al., 2006). Previous phylogenetic analyses of the Arabidopsis GAUT1-related gene family resulted in the designation of three GAUT clades, clades A through C, and one GATL clade (Sterling et al., 2006). The GATL clade, which consists of genes that cluster tightly and somewhat independently of the GAUT genes, was not included in the study reported here. It was previously determined that some Arabidopsis GAUT genes had conserved orthologs among species of both vascular and non-vascular plants (Sterling et al., 2006). The genomes of rice (Oryza sativa) and poplar (Populus trichocarpa) have now been sequenced and a BLAST search of Arabidopsis GAUT motifs against the poplar and rice genomes revealed GAUT1-related gene families of 21 members in poplar and 22 members in rice (Figure 1). Due to a recent genome duplication event in Populus (Tuskan et al., 2006), there are one to two apparent poplar orthologs for each Arabidopsis GAUT. A similar distribution of GAUTs in poplar and Arabidopsis is observed, except for the absence of a GAUT2 ortholog in poplar. In contrast, rice has major distinctions from Arabidopsis and poplar in the distribution of GAUT gene orthologs. Rice does not have apparent orthologs of GAUT2 or GAUT12. In addition, there are multiple apparent isoforms of GAUTs 1, 4, 7, and 9, suggesting an expansion of the role of these GAUT genes in rice.
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The rice and poplar genes included in this comparative phylogenetic analysis resolved the GAUT genes into seven clades. In order to preserve previous clade identity between the original three Arabidopsis clades (Sterling et al., 2006) and the more finely resolved seven clades presented here, the following clade identities are assigned. Arabidopsis GAUT clade A is subdivided into clades A-1, A-2, A-3, and A-4; GAUT clade B is subdivided into clades B-1 and B-2; and GAUT clade C remains undivided. The corresponding GAUTs in each clade are: A-1 (1 to 3); A-2 (4), A-3 (5 and 6) and A-4 (7); B-1 (8 and 9), B-2 (10 and 11) and C (12 to 15).
GAUT Gene Transcript Expression in Arabidopsis Tissues
Available transcript expression of AtGAUTs compiled from the Whole Genome Array, Massively Parallel Signature Sequence, and Genevestigator bioinformatic databases (Table 1) was used to select tissues used for the cell wall analyses reported here. In addition, total RNA from 8-week-old Arabidopsis WT inflorescence, silique, stem, and leaf tissues was used for qualitative and semi-quantitative RT–PCR using GAUT gene-specific primers. PCR products corresponding to the transcripts of 14 GAUT genes, excluding GAUT2, were detected in the WT inflorescence, leaf, stem, silique, and root tissues tested. GAUT2 may be expressed at a very low level or at different stages of development that have not yet been tested (Figure 2). Qualitative RT–PCR results partially agree with the published transcript expression data (see Table 1). In several instances, we detected GAUT transcript in tissues where it had not been previously reported. The data available from the Whole Genome Analysis (Yamada et al., 2003) did not detect GAUT5, while the Massively Parallel Signature Sequence data did not indicate detection of GAUTs 7, 10, 11, and 12 in leaf, GAUTs 1, 3, and 7 in stem, and GAUTs 1, 3, 4, 8, 9, 10, 13, and 15 in silique (Meyers et al., 2004). Overall, the data supplied by Whole Genome Analysis and Massively Parallel Signature Sequences under-reported GAUT gene transcript expression. The relative transcript expression of the GAUT genes, however, more closely agrees with that reported by Genevestigator (Zimmermann et al., 2004). Genevestigator does not list a probe for GAUT5, and therefore has no expression data for this gene, while the MPSS database reports low to moderate expression of GAUT5, in agreement with the result reported here.
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In general, RT–PCR indicated that relative transcript expression in Arabidopsis was highest for GAUTs 1, 4, 8, 9, and 12, moderate for GAUTs 3, 5, 6, 10, 14, and 15, and low for GAUTs 2, 7, 11, and 13. It should be noted that RT–PCR of GAUT7 repeatedly produced two bands, one of the expected size and a minor band of a smaller size. Whether the smaller band represents a splice variant has not been investigated. The RT–PCR data indicated that the GAUT genes were expressed at some level in all tissues tested; therefore, inflorescence, silique, leaf, and stems were used for the chemical and biochemical studies of the GAUT mutants.
Isolation of Homozygous Mutants of 13 of the 15 GAUT Genes
Twenty-six Arabidopsis homozygous T-DNA insertion seed lines in 13 distinct GAUT genes were isolated from mutagenized seed obtained from the SALK Institute (http://signal.salk.edu/cgi-bin/tdnaexpress) through the Arabidopsis Biological Resource Center (Alonso et al., 2003). Mutant seed lines were preferentially selected with the T-DNA insertion site in an exon, 5’ UTR, or intron of the GAUT gene, if such lines were available. SALK insertion seed lines of GAUT1 were not available and neither homozygous nor heterozygous mutants were recovered from the SALK insertion seed lines for GAUT4. RT–PCR of total RNA isolated from homozygous gaut mutant lines identified 10 knockout mutants and 10 knockdown mutants (Table 2).
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Growth Phenotypes of gaut Mutants
The gaut mutants plants were initially inspected visually for obvious growth phenotypes, such as dwarfing and/or organ malformation, compared to WT plants. Major abnormalities were not observed in plant growth or morphology for most gaut mutants isolated in this study, with the exception of gaut8 and gaut12. The presence of subtle growth phenotypes may require more sensitive methods than those applied here. Functional redundancy among the GAUT proteins may contribute to the lack of severe phenotypes observed among gaut mutants. Estimates put forth by Østergaard and Yanofsky (2004) predict that mutations in only approximately 10% of genes may result in detectable mutant phenotypes due to gene redundancy among large gene families in higher organisms. Thus far, two out of 13 GAUT genes (
15%) have yielded mutants with severe growth phenotypes, which is in line with the predicted outcome (Østergaard and Yanofsky, 2004). Previously analyzed qua1-1 insertion mutants (insertion in the 5'UTR) had severe dwarfing, sterility, and bumpy epidermal surfaces as a result of reduced cell adhesion (Bouton et al., 2002). Mutants allelic to qua1-1 (gaut8-2, gaut8-3, and gaut8-4) produced only heterozygous and WT progeny, suggesting an embryo-lethal phenotype. A single homozygous mutant was isolated, gaut8-1, with a predicted insertion in the 3'UTR that did not show the expected qua1-1 phenotype and was experimentally determined to have detectable GAUT8 transcript by RT–PCR, which may account for the WT-like phenotype of these plants.
The irx8-1/gaut12-1 and irx8-5/gaut12-2 mutant plants were severely dwarfed and sterile, which necessitated recovery of homozygous plants from the progeny of heterozygous parental plants, as previously reported (Persson et al., 2007). The phenotype of irx8-1/gaut12-1 and irx8-5/gaut12-2 was recognized in plants at least 4 weeks old. Such plants were small and with darkened leaves compared to WT. Surprisingly, the gaut12-5 promoter mutant (SALK_038620) did not produce homozygous progeny. In addition, gaut12-5 heterozygous mutants were dwarfed compared to WT, and more severely dwarfed compared to the irx8-1/gaut12-1 or irx8-5/gaut12-2 heterozygotes. RT–PCR of RNA from homozygous irx8-1/gaut12-1 and irx8-5/gaut12-2 plants did not yield PCR products using 5'- and 3'-end coding region-specific primers, showing that the full-length GAUT12 transcript was not produced. Because of the lethal phenotype, only heterozygous gaut12-5 was obtained and therefore was not included in our analyses of gaut homozygous mutants.
Strategy to Identify Glycosyl Residue Composition Differences between gaut Mutant and WT Walls
Gas chromatography–mass spectrometry (GC–MS) has been used to detect the changes in glycosyl residue composition in cell walls arising from mutations in cell wall-related genes (Reiter et al., 1997). Analysis of wall glycosyl residue composition by GC–MS of trimethylsilyl (TMS) derivatives allows detection of acidic and neutral sugars in a single analysis (Doco et al., 2001), in contrast to composition analysis by formation of alditol acetate derivatives that detects neutral but not acidic sugars (Reiter et al., 1997). Since uronic acids make up the largest proportion of glycosyl residues in the non-cellulosic wall polysaccharides of WT Arabidopsis tissues (Figure 3), the TMS method was chosen to analyze gaut mutant walls. A statistical assessment of the TMS method showed that at least four independent TMS analyses per wall sample are necessary to detect a 15% difference between the glycosyl residue composition of different wall samples with 90% or greater statistical confidence (Supplemental Table 1). The mutant glycosyl residue composition results were normalized to the composition of WT plants grown in the same experiment, in order to minimize the variability observed in the glycosyl residue compositions of plants grown in different experiments. Thus, for example, rhamnosyl compositions would be normalized according to the following formula:
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(2) = 0.1). As an extra measure of stringency, a 15% point or greater departure from the normalized WT mean, in addition to a statistically different outcome by ANOVA, was required for declaration of a real difference from WT.
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18. Glycosyl residues are abbreviated as arabinose (Ara), rhamnose (Rha), fucose (Fuc), xylose (Xyl), galacturonic acid (GalA), mannose (Man), galactose (Gal), and glucose (Glc).Wall Glycosyl Residue Composition Is Altered in Multiple gaut Gene Mutants
TMS glycosyl residue composition analyses of walls from two or more tissues of WT and mutant lines, representing 13 GAUT genes, revealed that specific gaut mutants have unique wall composition changes, which include increases and decreases in GalA, as well as significant changes in other glycosyl residues (Table 3). The wall glycosyl residue compositions that were statistically different in the gaut mutants compared to WT are shown in bold italics in Table 3. Reproducible mutant phenotypes were identified by comparing the natural log transformed data for all mutants that had statistically different mol% GalA, Xyl, Rha, Gal, and Ara levels compared to WT in at least two mutant alleles of the same gene or in at least two tissues of the same mutant allele (Supplemental Figure 1).
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Eight gaut mutants had statistically different mol% levels of GalA, Xyl, Rha, Gal, or Ara in at least two mutant alleles of the same gene or in at least two tissues of the same mutant allele compared to WT, resulting in distinguishable patterns of glycosyl residue composition changes in the walls of gaut mutants (summarized in Table 4). The silique tissues of gaut6-1 and gaut6-3 were consistently reduced in GalA, increased in Xyl, Rha, and Fuc, and similar to WT in Gal and Ara wall composition. Viable gaut8 homozygous knockout mutants were not isolatable, and, therefore, the wall composition of qua1-1 is used to establish a phenotype grouping for gaut8 mutants. The leaves of qua1-1 that were previously analyzed (Bouton et al., 2002) were decreased in GalA and Xyl, but were not changed in Rha or other sugars. The gaut9-1 stems were reduced in wall GalA and increased in Xyl and Fuc. The gaut10-1, gaut10-2, and gaut11-1 were consistently reduced in silique GalA only. The irx8-1/gaut12-1 and irx8-5/gaut12-2 mutant stems were severely reduced in Xyl, coincident with elevated Ara, Rha, and Gal content. The gaut12-1 and gaut12-2 are analogous to irx8-1 and irx8-5, and, consequently, show similar stem glycosyl residue composition as previously reported (Brown et al., 2005; Peña et al., 2007; Persson et al., 2007). Gaut13-1, gaut14-1, and gaut14-2 had increased GalA and Gal and reduced Xyl, Rha, Ara, and Fuc, with greater mol% changes in gaut14-1 (T-DNA insertion in an exon) than gaut14-2 (T-DNA insertion in the 3’ region). There were also some changes in Fuc, Man, and Glc in walls of several gaut mutants. For example, increased Fuc was observed in gaut6-1, gaut6-2, gaut6-3, gaut9-1, gaut9-2, and gaut9-3; decreased Fuc in gaut8-1, gaut11-2, gaut14-1, and gaut14-2; increased Man in gaut5-1 and gaut5-2; increased Glc in gaut3-1, gaut3-2, and gaut6-2; and decreased Glc in mutants of gaut5-1, gaut5-2, and gaut10-2. Few significant changes were found in the walls of gauts 2, 3, 5, 7, and 15, and those that did occur were not consistent between two or more mutants or in more than one tissue of a single mutant.
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Survey of Seed Mucilage Reveals GAUT11 Involved in Mucilage Extrusion
The seeds of myxospermous species, such as Arabidopsis, extrude mucilage from the seed coat epidermal cells when hydrated to protect against desiccation and to aid in seed dispersal. The mucilage of WT and gaut mutant seeds was investigated by ruthenium red staining as a facile method to determine whether specific GAUT genes are involved in mucilage polysaccharide extrusion or synthesis. The mucilage extruded from Arabidopsis seeds is enriched in the pectic polysaccharide RG-I, which efficiently binds ruthenium red stain due to the negative charge on the GalA residues in mucilage. This method has been successfully employed to identify mucilage or testa polysaccharide biosynthesis mutants (Western et al., 2001). The seed mucilage was evaluated by observing the staining intensity of mucilage and measuring the mucilage thickness under a dissecting microscope after application of aqueous 0.05% ruthenium red to the seeds of WT and the 26 gaut mutant lines. A single mutant (gaut11-2) was identified that displayed a reproducible reduced mucilage thickness phenotype compared to WT seed mucilage thickness.
Ruthenium red staining of WT and gaut11-2 seeds (Figure 4A–4C) revealed that 68% of gaut11-2 seeds had little extruded mucilage, while the remaining gaut11-2 seeds (32%) had reduced thickness of the mucilage layer to approximately half that of WT. Samples of WT and gaut11-2 seed were tested three separate times independently, with similar results obtained in seed derived from different parental plants (Table 5). Analysis of the uronic acid content of the hot water-extracted mucilage (WEM) of gaut11-2 and WT seed indicated that WEM of WT had 59 µg uronic acid per 200 extracted seeds, while gaut11-2 mucilage had 48 µg uronic acid per 200 extracted seeds (see Table 4). The total carbohydrate extracted, as detected by a phenol sulfuric acid assay, was similar for WT and gaut11-2 WEM. This suggests that even though very little mucilage was observed by ruthenium red staining, a similar amount of carbohydrate was able to be extracted over several hours, but that the uronic acid content of that mucilage was reduced by 19%. The gaut11-2 WEM was subjected to glycosyl residue composition analysis (Figure 4) and found to have statistically significant reductions in GalA and Xyl content and increases in Man and Gal content, as determined by ANOVA (t(2) = 0.05). The glycosyl residue composition of residual gaut11-2 seed material that represents the remaining mucilage, some testa wall, and possibly some storage polysaccharide was also reduced in GalA (69%) and Gal (68%) and increased in Ara (110%), Man (128%), and Glc (138%) compared to WT.
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DISCUSSION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Newly Resolved GAUT Gene Clades in Arabidopsis, Poplar, and Rice
The relatedness of GAUT genes has been re-evaluated based on the analysis of phylogenetic relationships of Arabidopsis, poplar, and rice GAUT genes. This comparative phylogenetic analysis distinguished seven GAUT clades (Figure 1), instead of three, as previously proposed by Sterling et al. (2006). The previous Arabidopsis GAUT clade A that included AtGAUT1–GAUT7 has been subdivided into four clades; GAUT clade A-1 (AtGAUT 1 through 3), GAUT clade A-2 (AtGAUT4), clade A-3 (AtGAUT5 and AtGAUT6), and GAUT clade (AtGAUT7). The former Arabidopsis clade B has been subdivided into GAUT clade B-1 (AtGAUT8 and AtGAUT9) and GAUT clade B-2 (AtGAUT10 and AtGAUT11). The former Arabidopsis GAUT clade C has not been subdivided and contains AtGAUT12 through AtGAUT15.
GAUT2 does not appear to have a direct ortholog in either rice or poplar. It is possible that GAUT2 may not be a complete copy of a GAUT1 duplication event, based on a shorter N-terminus compared to GAUTs 1–7; however, its length is comparable to the other GAUTs. GAUT2 also does not have detectable transcript in the tissues tested and GAUT2 T-DNA insertion mutants did not have reproducible phenotypes. These data, combined with the phylogenetic analysis of GAUT2, support the hypothesis that GAUT2 may be a non-functional truncated homolog. It cannot be ruled out, however, that GAUT2 may have a very low abundance transcript and a unique function in Arabidopsis alone, although this seems unlikely based on the current data.
The Arabidopsis and poplar genomes have one (At2g38650) and two (XP_002323701, XP_002326255) copies of GAUT7, respectively, while the rice genome contains five GAUT7-like sequences. There is considerable evidence that the AtGAUT7 protein resides in a complex with AtGAUT1, a complex that has homogalacturonan 1,4-GalAT activity (M.A. Atmodjo, unpublished data). GalAT activity was detected in immunoprecipitates from HEK cells transiently transfected with GAUT1, but not in HEK cells transiently transfected with GAUT7 (Sterling et al., 2006). Based on these data, GAUT7 may be expressed in an inactive state with limited activity itself or may function as an ancillary protein necessary for GAUT1-associated GalAT activity. Whatever the role of GAUT7, its function appears to be dramatically expanded in rice. Because the role of GAUT7 in wall polysaccharide biosynthesis is currently unknown, the underlying biological reason for five copies of GAUT7 in rice remains to be determined.
Poplar and rice each have putative orthologs of GAUT9: XP_002332802 (poplar), Os06g12280 (rice), and Os02g51130 (rice). Poplar also has at least one putative ortholog of GAUT8 (XP_002301803). There is not an obvious ortholog of GAUT8 in rice, although there is one rice gene (Os02g29530) positioned between GAUT8 and GAUT9. Phylogenic analyses using additional sequenced plant genomes may clarify the relatedness of the latter gene to GAUT8 and GAUT9.
GAUT12 has two poplar orthologs but no orthologs in rice (Figure 1). GAUT12 has been linked xylan synthesis. The putative functions that have been hypothesized for GAUT12 include an 1,4-GalAT that adds GalA into a primer or cap for xylan synthesis or as a novel linkage in xylan or pectic polysaccharides (Brown et al., 2005; Peña et al., 2007; Persson et al., 2007). GAUT12 has been shown to be essential for normal growth and more specifically for the synthesis of secondary wall glucuronoxylan and/or wall HG synthesis. Rice does not have an apparent homolog of GAUT12, and appears to produce secondary wall xylan and glucuronoarabinoxylan, but not 4-O-methylglucuronoxylan (Ebringerova and Heinze, 1999). Thus, GAUT12 may have a specialized function in glucuronoxylan synthesis of dicot plants. GAUT12 transcript has been shown to be localized closely with glucuronoxylan-rich vascular tissues, suggesting that GAUT12 has a specialized role in the synthesis of secondary wall glucuronoxylan of dicot walls (Persson et al., 2007). GAUT12 has an expression profile distinct from that of other GAUT genes according to semi-quantitative RT–PCR; it is much more highly expressed in stem than in other tissues compared to other GAUT transcripts. The unique transcript expression profile, role in secondary wall 4-O-methylglucuronoxylan synthesis, and exclusivity among the dicot species suggest that GAUT12 has undergone a differentiation that has rendered it essential in dicots and nonessential in monocots.
GAUT Gene Transcripts are Expressed Ubiquitously in Arabidopsis Tissues
The transcript expression of GAUT8 and GAUT12 has been associated with vascular tissues in Arabidopsis stem (Orfila et al., 2005; Persson et al., 2007). The GAUT12 results described here agree with previous analyses of GAUT12/IRX8 gene expression by RT–PCR analysis (Persson et al., 2007) and GAUT8 RT–PCR data agree with reports of QUA1 expression (by Northern blot) in ‘Flowers II’ and ‘Rosette Leaves II’ RNA, but do not agree with the low transcript expression reported in ‘Stems II’ by Bouton and colleagues (2002). We report high relative expression of GAUT8 in stems. In situ PCR of QUA1/GAUT8 in WT stems (Orfila et al., 2005), however, did reveal prominent expression in that tissue, which is more closely aligned with our results. The detectable expression of all of the GAUT genes in all of the tissues tested correlates with a function in wall biosynthesis, as this is a process required by all plant cells. GUS reporter gene studies have shown that QUA2, a putative pectinmethyltransferase involved in pectin biosynthesis, also has ubiquitous expression (Mouille et al., 2007).
The Wall Compositions of Multiple gaut Mutants are Altered Compared to WT
Analysis of the walls of gaut mutants using the TMS method (Doco et al., 2001) allowed the GalA content of the walls to be quantified. An accurate quantification of wall GalA content is important when attempting to identify mutants of putative pectin biosynthesis genes, because GalA is a major component of the pectic polysaccharides (Ridley et al., 2001). Mutants of GAUTs 6, 9, 10, and 11 had statistically significant reductions in GalA content in more than one mutant sampling. Two other gaut mutants, gaut13 and gaut14, had statistically significant increased wall GalA content. The wall compositional phenotypes of the gaut mutants are discussed below.
The wall glycosyl residue composition phenotype of gaut6 provides compelling evidence that GAUT6 is a putative pectin biosynthetic GalAT. GAUT6 has 64% amino acid similarity to GAUT1 and gaut6 has reduced wall GalA that coincides with higher levels of Xyl and Rha wall compositions. It is possible that the increased Xyl and Rha content signifies the compensatory reinforcement of the wall by xylans and an apparent enrichment of RG-I in proportion to reduced HG polymers. Further work is necessary to test this hypothesis; however, preliminary results are in agreement with this hypothesis (Caffall, Ph.D. thesis, 2008).
GAUTs 8, 9, 10 and 11 have been placed in two separate sub-clades (B-1 and B-2). However, all mutants in the two B clades show marked reductions in wall GalA content. Qua1-1 mutant plants have walls with both reduced GalA and Xyl, and microsomal membrane protein preparations from qua1-1 stems had reduced GalAT and xylan synthase activity compared to WT (Orfila et al., 2005; Brown et al., 2007). The QUA1 cumulative experimental evidence argues in favor of a putative pectin biosynthetic GalAT, based on the significant reduction in homogalacturonan and the strong defect in cell adhesion (Bouton et al., 2002; Leboeuf et al., 2005). Deficiencies in cell adhesion have been associated with changes in pectin synthesis (Iwai et al., 2002) and pectin localization (Shevell et al., 2000). In addition, the transcript expression of a pair of Golgi-localized putative pectinmethyltranserfases is strongly correlated with QUA1/GAUT8 expression, as well as with the expression of GAUT9 and GAUT1 (Mouille et al., 2007). The gaut9, gaut10, and gaut11 mutant plants did not have any obvious physical growth or cell adhesion defects, but the wall compositional phenotypes of these gaut plants, and the high amino acid similarity with QUA1/GAUT8, suggest that these GAUTs are putative pectin biosynthetic GalATs. The mutant alleles of GAUT9, GAUT10, and GAUT11 have reduced wall GalA content but were not decreased in Xyl, which has been observed in some mutants thought to be involved in xylan synthesis (Brown et al., 2007; Lee et al., 2007; Peña et al., 2007; Persson et al., 2007). Based on the evidence, a role for the genes in GAUT clades A as well as a role for the genes in clade B and C in pectin biosynthesis is proposed.
In contrast to QUA1/GAUT8, IRX8/GAUT12 is believed to function in glucuronoxylan synthesis essential for secondary wall function. The irx8-1/gaut12-1 and irx8-5/gaut12-2 mutant plants have reduced Xyl content with increases in the GalA content in stem and silique walls, consistent with previous reports and consistent with the proposed function of IRX8/GAUT12 in the synthesis of an oligosaccharide essential for xylan synthesis. Mutants of IRX8/GAUT12 and other putative xylan biosynthetic genes, IRX7, IRX8, IRX9, IRX14, and PARVUS, have similar wall compositional phenotypes (Peña et al., 2007; Persson et al., 2007). IRX8/GAUT12 may play a specialized role, among the GAUTs, in secondary wall synthesis and vascularization in dicot species (Brown et al., 2007). Xylans are abundant in stem and silique tissues, where the Xyl compositional phenotype is observed; however, reductions in Xyl are not observed in inflorescence where IRX8/GAUT12 is also expressed. In inflorescences, irx8/gaut12 mutants show a reduction in GalA to 82% that of WT. Thus, the changes brought about by the lesion in GAUT12 additionally impact the pectin component of the wall. The underlying causes for the reduced GalA content in the inflorescence may be of significance to understand how pectin and xylan synthesis are regulated and connected.
The walls of gaut13 and gaut14 have increased GalA and Gal content and reduced Xyl and Rha content compared to WT. It seems unlikely that a mutant showing an increased wall GalA phenotype is involved in the synthesis of HG. However, reduced Rha, primarily a component of RG-I, may lead to walls enriched in HG, driving up GalA content. A Gal containing wall component is increased in the walls of gaut13 and gaut14 (and also gaut12). Pectic galactans have been associated with wall strengthening (McCartney et al., 2000) and are also increased in irx8/gaut12 walls (Persson et al., 2007). A galactan in gaut13 and gaut14 may be up-regulated in response to wall weakening in a similar manner. GAUT13 and GAUT14 are very closely related to GAUT12, which would also suggest that the Xyl containing polysaccharide that is reduced in mutants of these genes is also a xylan and that GAUT13 and GAUT14 share overlapping function with GAUT12. Based on the strong transcript expression of GAUT12, most notably in the stem tissues of 8-week-old Arabidopsis plants, it is conceivable that gaut13 or gaut14, which have WT-like growth phenotypes, may be partially rescued by existing GAUT12 expression, if function is shared between GAUT12, GAUT13, and GAUT14, thus resulting in mild or undetectable growth phenotypes.
GAUT11 Effects Mucilage Extrusion
The composition and linkage analysis of gaut11-2 mucilage suggests a minor reduction in RG-I-like extractable polysaccharides. The gaut11-2 mutant has reduced mucilage expansion and reduced GalA content of extracted mucilage and testa, suggesting a role in the synthesis of mucilage polysaccharides. The gaut11-2 mutant has reduced GalA in silique walls, while gaut11-1 has reduced GalA in inflorescence, silique, and leaf walls. The gaut11-1 seeds, however, did not appear to have inhibited mucilage expansion. The predicted insertion site location of the T-DNA insertion present in gaut11-2 is in the 3'UTR, a location that may alter the targeting or regulation of GAUT11 expression rather than knocking out function (Lai, 2002) and account for the difference in phenotype between gaut11-1 and gaut11-2. The visible phenotype of gaut11-1 is similar in character to the mucilage modified (mum) mutants (Western et al., 2001, 2004). Three types of mum mutants have been described: mutants of pectin modification (mum2 and mum1), mutants affecting cytoplasmic rearrangement (transparent testa glabra-1; ttg1, glabra-2; gl2), and mutants of mucilage biosynthesis (mum3, mum5, and mum4) (Western et al., 2001). Preliminary data suggest a role for GAUT11 in wall modification or biosynthesis based on the reduction in GalA in the extractable mucilage and based on the observation that the majority of the polysaccharides may be extracted over time, but are inefficiently released from the seed epidermal cells. It is known that unbranched RG-I, or reductions in intact RG-I, may lead to increased Ca2+ cross-linking of HG in the wall (Jones et al., 2003), and thus inhibit expansion and release of mucilage by hydration. Additionally, accumulation of less RG-I in the epidermal cells of the seed coat may prevent extrusion of the mucilage by reducing the internal pressure that is required to break through the epidermal cell wall necessary to release mucilage (Western et al., 2000).
Lethality of gaut Mutants: Something Lost, Something Gained
GAUT1 is an HG-GalAT. GAUT1 was the most abundant glycosyltransferase isolated from Arabidopsis suspension culture microsomal membrane fractions (Sterling et al., 2006). In addition, GAUT1 and GAUT4 are expressed highly in the tissues of 8-week-old plants according to semi-quantitative RT–PCR and to the GENEVESTIGATOR and MPSS databases (Figure 2 and Table 1) (Meyers et al., 2004; Zimmermann et al., 2004). Proteins that share high amino acid similarity often have a similar function and it is likely that GAUT4 (83% amino acid similarity to GAUT1) also has a function in synthesizing HG in the walls of Arabidopsis similar to that of GAUT1. The lack of recoverable mutants for GAUT1 and GAUT4 may speak to the importance of these genes in plant growth and development. Indeed, a gaut1 SAIL mutant yielded only heterozygous and WT progeny; homozygotes were not obtained (M.A. Atmodjo and I. Petrascu, 2008, unpublished data). More vigorous attempts to isolate and characterize GAUT1 and GAUT4 and their respective mutants will undoubtedly aid in the clarification of their roles in pectin and wall biosynthesis. A degree of lethality has also been demonstrated in gaut8 and gaut12 mutants, both in this report and elsewhere (Bouton et al., 2002; Persson et al., 2007). Qua1-1, irx8-1, and irx8-5 mutants are severely dwarfed and semi-sterile (Brown et al., 2005; Orfila et al., 2005).
Conclusion
The data presented establish the foundation for multiple hypotheses regarding GAUT gene function. The rigorous testing of these hypotheses is expected to lead to the identification of additional genes involved in specific pectin and wall biosynthetic pathways. The wall compositional phenotypes support the proposition that (1) GAUT proteins play a role in wall biosynthesis, (2) GAUTs 6, 9, 10, and 11, which have the highest amino acid similarity to GAUT1, have putative functions in pectin biosynthesis, and (3) GAUTs 13 and 14 are likely to have putative functions in xylan biosynthesis like GAUT12, or in pectin RG-I biosynthesis. The mutant wall composition phenotypes presented here are not sufficient to prove GAUT function, but serve to support hypotheses regarding GAUT function. The data demonstrate that mutants corresponding to more than half of the gaut mutants have significantly altered wall polysaccharides and strongly support a role for the family in pectin and/or xylan synthesis and function. Potential gene redundancy could explain the lack of wall phenotypic changes in some of the gaut mutants, and the generation of double mutants might uncover phenotypes masked by such potential redundancy.
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Sequence Alignment of GAUT Family Proteins and Phylogenetic Analysis
Protein sequences were identified by BLAST search of Arabidopsis thaliana (www.Arabidopsis.org/index.jsp), Oryza sativa (www.tigr.org/tdb/e2k1/osa1/), and Populus trichocarpa (http://genome.jgi-psf.org/Poptr1_1/Poptr1_1.home.html) genomes, using AtGAUT1 as the search probe. The GAUT protein sequences were aligned using ClustalX (Thompson et al., 1997) and suggested protein alignment parameters (Hall, 2004). Phylogenetic Bayesian analysis was carried out employing MrBayes (Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck, 2003). Full-length protein sequences were used in the analysis for all proteins except Os09g36180, whose C-terminal 404 amino acid extension was excluded.
Plant Materials and Growth Conditions
Arabidopsis thaliana var. Columbia S6000 T-DNA insertion mutant seeds were obtained from the Arabidopsis Biological Resource Center (www.biosci.ohio-state.edu/pcmb/Facilities/abrc/abrchome.htm). Arabidopsis WT and gaut mutant seeds were sown on pre-moistened soil and grown to maturity under 60% constant relative humidity with a 14/10 light/dark cycle (14 h (19°C; 150 µEi m–2 s–1)/10 h (15°C)). The plants were fertilized (Peters 20/20/20 with micronutrients) once a week or as needed. WT and T-DNA seeds were sown in ‘growth sets’ of 20 plants. Walls were harvested from multiple 8-week-old WT and PCR-genotyped mutant plants and pooled, respectively, together for wall glycosyl residue composition analysis. The following tissues were harvested for the wall analyses: the apical inflorescence excluding the young siliques; the young fully expanded leaves approximately 3 cm long; green siliques; and the top 8 cm of actively growing stem minus the inflorescence and siliques.
DNA Extraction and Mutant Genotyping
Fresh, flash-frozen leaf tissue (100–200 mg) was ground with a mortar and pestle and suspended in 0.5 ml extraction buffer (100 mM Tris-HCl pH 8.0, 100 mM EDTA pH 8.0, 250 mM NaCl, 100 µg ml–1 proteinase K and 1% (w/v) n-lauroylsarcosine) and extracted with an equal volume of phenol:chloroform:isoamyl alcohol (49:50:1, v/v). RNA was degraded by addition of 2 µl of DNase-free RNase A (10 mg ml–1) for 20 min at 37°C. The DNA was precipitated twice with 70% (v/v) ethanol and suspended in a final volume of 50 µl. Primers used for mutant genotyping were designed by ISECT tools (http://signal.salk.edu/isects.html). The genotype of mutant plants was determined based on the ability of the LB primers to anneal and produce T-DNA-specific PCR products when combined with the appropriate GAUT gene-specific primer. Gene-specific primer pairs were similarly used to determine the presence of intact GAUT genes (see Supplemental Table 2).
Isolation of Cell Walls
Cell wall samples were harvested from selected tissues of multiple 8-week-old plants from WT and mutant lines (n = 4). The plant tissues for cell wall extraction were weighed (100–200 mg), flash frozen in liquid N2 and ground to a fine powder. The tissues were consecutively extracted with 2 ml of 80% (v/v) ethanol, 100% ethanol, chloroform:methanol (1:1, v/v), and 100% acetone. Centrifugation in a table-top centrifuge at 6000 g for 10 min was used to pellet the sample between all extractions. The remaining pellet was immediately treated with -amylase (Sigma, porcine Type-I) in 100 mM ammonium formate pH 6.0. The resulting pellet was washed three times with sterile water, twice with acetone, and dried in a rotary speed-vac overnight at 40°C and weighed.
Mucilage Extraction
Mucilage was extracted from 200 Arabidopsis seeds incubated with sterile water at 60°C over the course of 6 h as follows. Each hour during the 6-h period, the seeds were centrifuged and the supernatant was transferred to a sterile tube. The combined supernatants were lyophilized and re-suspended in 600 µl of sterile water. Phenol-sulfuric (Dubois et al., 1956) and m-hydroxybiphenyl (Blumenkrantz and Asboe-Hansen, 1973) assays, to quantify total sugars and uronic acids, respectively, were carried out using 100 µl of the mucilage extracts. Duplicate 200 µl aliquots of the mucilage extract were used for glycosyl residue composition analyses. To analyze the seed coat material remaining after extraction, the water-extracted seeds were aliquoted in water to glass tubes and 20 µg of inositol was added. The seeds were lyophilized to dryness and used for glycosyl residue composition analyses.
TMS GC–MS Glycosyl Residue Composition
The cell walls were aliquoted (1–3 mg) as acetone suspensions to individual tubes and allowed to air dry. Inositol (20 µg) was added to each tube and the samples were lyophilized and analyzed for glycosyl residue composition by combined gas chromatography–mass spectrometry (GC–MS) of the per-O-trimethylsilyl (TMS) derivatives of the monosaccharide methyl glycosides produced from the sample by acid methanolysis basically as described by York et al. (1985). The dry samples were hydrolyzed for 18 h at 80°C in 1 M methanolic-HCl. The samples were cooled and evaporated under a stream of dry air and further dried two additional times with anhydrous methanol. The walls were derivatized with 200 µl of TriSil Reagent (Pierce-Endogen, Rockford, IL, USA) and heated to 80°C for 20 min. The cooled samples were evaporated under a stream of dry air, re-suspended in 3 ml of hexane, and filtered through packed glass wool. The dried samples were re-suspended in 150 µl of hexane and 1 µl of sample was injected onto an HP 5890 gas chromatograph interfaced to a 5970 MSD using a Supelco DB1 fused silica capillary column.
Statistical Analyses
The variance ratio test ( = 0.05) was used to compare the variances of standards and samples. ANOVA analyses, standard deviation, variance, t, and the mean of sample were calculated using SAS 9.1.3 software (© 2002–2004 produced by the SAS Institute Inc., Cary, NC, USA). Significant differences between WT and mutant compositions were determined with t(2) = 0.1 (90% confidence), but was set to 0.05 (95% confidence) for all other analyses. The appropriate sample size was predicted using equation 7.7, p. 105 of Biostatistical Analysis, 4th edn (Zar, 1999) (Supplemental Table 1).
RNA Extraction and RT–PCR
Total RNA was extracted from 0.5 g of stem, inflorescence, silique, and leaf tissue from 8-week-old plants. The tissues were homogenized in 10 ml of Homogenization Buffer (2% (w/v) SDS in 50 mM Tris-HCl pH 7.8 and 40% water-saturated phenol) and shaken for 15 min at 25°C. Tissue samples were centrifuged for 10 min at 8000 g and 4°C, and the supernatant removed to a clean tube. The samples were extracted two times with phenol:chloroform:isoamyl alcohol (25:24:01, v/v) and the aqueous phases were pooled. RNA was precipitated overnight with 0.1 vol. of 3 M sodium acetate and 2.5 vol. of cold ethanol. The samples were DNase-treated with RQ1 RNase-Free DNase (Promega, Madison, WI, USA) according to the manufacturer's instructions.
RT–PCR products were generated using primer sequences unique to each of the 15 GAUT genes (see Supplemental Table 2). Each GAUT gene primer set was designed to span at least one intron such that unique PCR products were produced from RNA for each GAUT gene. Control RT reactions were carried out alongside GAUT-specific reactions, utilizing primers designed to the small ribosomal protein L23 alpha, wherein the primers do not produce a product in genomic DNA (Volkov et al., 2003). Qualitative RT–PCR was carried out using 5 µg of total RNA in a 20-µl RT first-strand synthesis reaction that contained oligo(dT) primers. The RT first-strand reaction (2 µl) was added to a PCR reaction mix containing the respective GAUT gene-specific primers and amplified for 30 cycles. Semi-quantitative RT–PCR was done using 2 µg of total RNA in a 20-µl RT first-strand synthesis reaction containing oligo(dT) primers. An aliquot (1.5 µl) of the RT first-strand reaction was amplified through 26 cycles of PCR using GAUT gene-specific primers. The PCR parameters were: Step 1: 95°C for 5 min; Step 2: 95°C for 0.5 min; Step 3: 55°C for 0.5 min; Step 4: 72°C for 1.5 min; Step 5: Return to step 2 (29 or 25) times; Step 6: 72°C for 2 min; and Step 7: 4°C forever.
Mutant transcript levels were assessed as follows: knockouts (KO) were defined as mutants with RT–PCR reactions that yielded no detectable PCR product using gene-specific primers. Knockdown (KD) mutants were those that yielded a PCR product with significantly decreased intensity compared to the WT.
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Supplementary Data are available at Molecular Plant Online.
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FUNDING |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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This project was supported in part by NSF MCB awards 0313509 and 0646109, NRI, CSREES, USDA Awards 2003–35318–15377 and 2006–35318–17301, and DOE DE-FG02-93-ER20097.
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Acknowledgements |
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Great appreciation is given to the CCRC analytical services and to Mu Yun Gao for her expertise in TMS glycosyl residue compositional analyses. Thanks are also expressed to Dennis Warrenfeltz and Stefan Eberhard for GC–MS and growth chamber maintenance, respectively. Appreciation is also extended to Z. Caffall, family, and friends for their support. No conflict of interest declared.
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