A DUF-246 family glycosyltransferase-like gene affects male fertility and the biosynthesis of pectic arabinogalactans
© Stonebloom et al. 2016
Received: 12 January 2016
Accepted: 13 April 2016
Published: 18 April 2016
Pectins are a group of structurally complex plant cell wall polysaccharides whose biosynthesis and function remain poorly understood. The pectic polysaccharide rhamnogalacturonan-I (RG-I) has two types of arabinogalactan side chains, type-I and type-II arabinogalactans. To date few enzymes involved in the biosynthesis of pectin have been described. Here we report the identification of a highly conserved putative glycosyltransferase encoding gene, Pectic ArabinoGalactan synthesis-Related (PAGR), affecting the biosynthesis of RG-I arabinogalactans and critical for pollen tube growth.
T-DNA insertions in PAGR were identified in Arabidopsis thaliana and were found to segregate at a 1:1 ratio of heterozygotes to wild type. We were unable to isolate homozygous pagr mutants as pagr mutant alleles were not transmitted via pollen. In vitro pollen germination assays revealed reduced rates of pollen tube formation in pollen from pagr heterozygotes. To characterize a loss-of-function phenotype for PAGR, the Nicotiana benthamiana orthologs, NbPAGR-A and B, were transiently silenced using Virus Induced Gene Silencing. NbPAGR-silenced plants exhibited reduced internode and petiole expansion. Cell wall materials from NbPAGR-silenced plants had reduced galactose content compared to the control. Immunological and linkage analyses support that RG-I has reduced type-I arabinogalactan content and reduced branching of the RG-I backbone in NbPAGR-silenced plants. Arabidopsis lines overexpressing PAGR exhibit pleiotropic developmental phenotypes and the loss of apical dominance as well as an increase in RG-I type-II arabinogalactan content.
Together, results support a function for PAGR in the biosynthesis of RG-I arabinogalactans and illustrate the essential roles of these polysaccharides in vegetative and reproductive plant growth.
KeywordsArabidopsis thaliana Nicotiana benthamiana Cell wall Rhamnogalacturonan-I Pectin Pollen tube
Plant cell wall polysaccharides are mainly synthesized by glycosyltransferases, enzymes that transfer monosaccharides from an activated donor substrate, usually a nucleotide sugar, onto acceptor molecules, forming glycosidic bonds. Most glycosyltransferases exhibit strong selectivity for donor sugar and acceptor substrates . Glycosyltransferases are classified as “inverting” or “retaining” enzymes depending on whether glycosylation occurs with retention or inversion of stereochemistry at the anomeric carbon atom of the donor substrate. These enzymes have been further classified into families on the basis of amino acid sequence similarities in the Carbohydrate Active enZyme database (CAZy) . The Arabidopsis genome contains 463 genes classified into 41 distinct glycosyltransferase gene families and an additional 100 glycosyltransferase-like genes possessing insufficient similarity to characterized enzymes to be assigned to a CAZy gene family.
To date, only a few enzymes involved in the biosynthesis of pectin have been characterized. These include GAUT1, which is a homogalacturonan galacturonosyltransferase in glycosyltransferase (GT) family 8 . Another GT family 8 member, GATL5, has been shown to be involved in the synthesis of Arabidopsis seed coat mucilage, which is largely composed of RG-I, though its precise role in mucilage biosynthesis has not been established . Xylosyltransferases found in GT family 77, RhamnoGalacturonan-II XylosylTransferase-1 (RGXT1), RGXT2 and RGXT3, have been shown to xylosylate l-fucose in the A-chain of RG-II [10, 11]. ARAD1 and 2 belong to GT family 47 and are involved in the biosynthesis of arabinans on RG-I; however their catalytic activity has not been demonstrated [12, 13]. XGD1 also belongs to GT family 47 and is a xylosyltransferase that adds xylose to homogalacturonan to form xylogalacturonan . A recently characterized galactan synthase, GALS1, in GT family 92 extends pectic β-1,4-galactan . GALS1 requires at least a galactotetraose oligosaccharide as a substrate indicating that it extends but does not initiate RG-I galactan biosynthesis. Thus, additional unidentified enzymes are apparently required for the initial branching of RG-I. A recent study suggests that at least some RG-I may be produced as a proteoglycan attached to AGP-polysaccharides .
The structure and composition of pectins are altered during growth, development and in response to changing conditions. RG-I has specific roles in many plant organs and tissues, and is present in all primary plant cell walls . RG-I is likely present in the cell walls of all vascular plants and has been detected in the walls of basal land plants such as Physcomitrella patens as well as the inner cell wall of the Charophyte alga Penium magaritaceum . Homogalacturonan plays a critical role in the walls of tip growing cells such as root hairs and pollen tubes where de-methyl esterification and cross-linking of homogalacturonan at the edge of the growing tip is thought to solidify the nascent cell wall . RG-I arabinogalactans are also critical components of the pollen tube cell wall. In olive plants pectic galactan forms a ring around the aperture where pollen tubes emerge from the pollen grain . Pectic arabinans likely present as sidechains on RG-I are a critical part of the pollen cell wall .
A better understanding of the biosynthesis and processing of pectins is critical to elucidating how this enigmatic class of polymers functions in regulating properties of the plant cell wall. Many uncharacterized GT activities are required for the biosynthesis of pectic polysaccharides. Here we report the identification of a new gene affecting pollen tube growth and present evidence that it is involved in the biosynthesis of pectic arabinogalactans attached to RG-I. We have named the Arabidopsis gene, At3g26370, Pectic ArabinoGalactan synthesis-Related (PAGR).
PAGR is a highly conserved DUF-246 domain containing protein
Land plants have an expanded group of genes encoding proteins related to GT family 65, the DUF246 family of GT65-like proteins with 39 members in Arabidopsis . The mammalian GT family 65 protein POFUT1 fucosylates serine/threonine in epidermal growth factor repeats . As the genes that contain a DUF246 domain are an expanded family in plants, some of them are likely to be involved in plant-specific processes . The only previously studied plant DUF246-encoding genes are MSR1 and MSR2, which affect the production and secretion of mannans in Arabidopsis; however their specific role in mannan biosynthesis remains elusive . In examining the predicted amino acid sequences of plant DUF246-containing proteins we identified a gene, At3g26370 (PAGR), encoding a protein that is more highly conserved throughout the land plants than other DUF246 containing proteins. The Selaginella moellendorffii and Physcomitrella patens orthologs of PAGR are 76.0 % and 70.9 % identical to the Arabidopsis protein, respectively (Additional file 1: Figure S1). Other Arabidopsis DUF246 proteins have basal land plant orthologs with pairwise amino acid identities between 45 and 67.7 %. The strong conservation of PAGR throughout the land plants can be observed as the short branch length within the PAGR clade in phylogenetic analysis of the DUF246 containing proteins in Arabidopsis, Selaginella and Physcomitrella (Additional file 2: Figure S2). PAGR is predicted to encode a type-II membrane protein with an N-terminal disordered domain, a transmembrane domain and the highly conserved DUF246 domain (Fig. 1c).
PAGR is expressed ubiquitously
We examined the expression pattern of PAGR in Arabidopsis using quantitative RT-PCR of RNA prepared from various tissues (Fig. 1d). PAGR was expressed in all tissues tested with higher levels of transcript detected in reproductive tissues and roots. Transcript levels varied less than 7-fold between the tissues with the highest (flowers) and lowest (mature leaves) expression. This result is in agreement with publicly available microarray data showing expression of PAGR throughout the plant (Additional file 3: Figure S3) .
PAGR mutant alleles are not transmitted via pollen
Segregation ratio of PAGR mutant alleles. The genotype of offspring from selfed heterozygotic pagr mutants was determined by PCR. The observed segregation ratios are consistent with a 1:1 segregation as expected with non-functional pollen
p-value, χ2 test of 1:2:1 segregation
p-value, χ2 test of 0:1:1 segregation
Segregation of pagr mutant alleles in the progeny of reciprocal crosses between pagr heterozygotes and wild type
p-value, χ2 test of 1:1 segregation
Col ♀ x pagr-1/PAGR ♂
pagr-1/PAGR ♀ x Col ♂
Col ♀ x pagr-2/PAGR ♂
pagr-2/PAGR ♀ x Col ♂
Pollen tube growth analysis of pagr heterozygotes and the wild type
proportion of germinating pollen
wild type (Col-0)
PAGR is localized to the Golgi apparatus and to small punctate structures
Silencing of PAGR in Nicotiana benthamiana affects pectin biosynthesis
As PAGR was predicted to be involved in the production of plant cell wall polysaccharides, we analyzed the monosaccharide composition of cell wall material from NbPAGR-silenced and virus-infected non-silenced control plants. NbPAGR-silenced plants exhibited a significantly altered monosaccharide composition including a 33 % decrease in galactose content, a small decrease in glucuronic acid content, and increased galacturonic acid content (Fig. 4c). Sequential extraction of the cell wall material showed that less galactose and arabinose were present in pectic extracts obtained from NbPAGR-silenced cell walls with the chelating agent cyclohexane diamine tetraacetic acid (CDTA) and with sodium carbonate (Additional file 7: Figure S7). The cell wall material remaining after the extraction of pectic polysaccharides did not exhibit differences in composition between silenced and virus-infected control plants.
To determine if the observed changes in pectic galactose content were caused by a reduction in β-1,4-galactan, cell wall material was digested with endo-1,4-β-galactanase from Aspergillius niger and the solubilized and residual materials were subsequently analyzed. Treatment with the 1,4-β-galactanase released 68.9 % less galactose from cell walls of NbPAGR-silenced plants than from cell walls of the control plants (Fig. 4d-e), indicating that the reduction in total galactose content was related to a significant reduction in pectic galactan content and/or enzymatic accessibility. Interestingly, less arabinose was released following β-1,4 galactanase treatment from NbPAGR-silenced cell wall material as well, suggesting that type-I arabinogalactans were affected by NbPAGR- silencing in addition to β-1,4-galactan. Residual material following galactanase digestion was similar in composition in NbPAGR-silenced and control cell walls (Fig. 4e).
Overexpression of PAGR in Arabidopsis causes ectopic phenotypes
NbPAGR silenced cell walls contain rhamnogalacturonan-I deficient in arabinogalactan
The mol% monosaccharide composition of RG-I purified from NbPAGR-silenced and control N. benthamiana plants
The estimated average number of monosaccharide residues making up each RG-I molecule in NbPAGR-Silenced and control N. benthamiana plants
PAGR overexpression increases rhamnogalacturonan-I arabinan content
The mol% monosaccharide composition of RG-I purified from 35S::PAGR-YFP lines and the wild type (Col0)
35S::PAGR-YFP line 6
35S-PAGR-YFP line 9
The estimated average number of monosaccharide residues making up each RG-I molecule in 35S::PAGR-YFP and wild type (Col-0) plants
35S::PAGR-YFP line 6
35S-PAGR-YFP line 9
PAGR affects the abundance of arabinogalactan and rhamnogalacturonan-I backbone epitopes in purified rhamnogalacturonan-I
In order to better understand the changes in RG-I composition induced by altered PAGR expression, the glycan epitope composition of RG-I samples purified by size exclusion chromatography from NbPAGR-silenced and control Nicotiana benthamina plants as well as PAGR-overexpressing and wild type Arabidopsis plants were analyzed by ELISA using a comprehensive set of plant glycan-directed antibodies  (Fig. 7c, Additional file 11: Table S3). These RG-I preparations were recognized nearly exclusively by antibodies directed at pectic backbone and arabinogalactan epitopes. Weak binding by antibodies against de-esterified homogalacturonan (HG) epitopes was observed in RG-I preparations from all plant lines, as expected given the methods used to generate the RG-I. RG-I from NbPAGR-silenced plants yielded stronger signals for antibodies that recognize un-branched RG-I backbone epitopes than did RG-I from the controls. RG-I from silenced plants also exhibited altered signal strength for antibodies binding arabinogalactan epitopes. RG-I from silenced plants produced reduced signals for some antibodies belonging to the AG-2, −3, and −4 groups of antibodies that recognize distinct arabinogalactan epitopes and subtly enhanced signals for many antibodies belonging to the AG-I group. RG-I from PAGR-overexpressing Arabidopsis plants showed no changes in signals for antibodies recognizing RG-I or HG backbone epitopes. Antibodies belonging to the AG-1 group produced stronger signals for RG-I from PAGR-overexpressing plants (lines 6 and 9) as did some antibodies in groups AG-3 and −4 that target other arabinogalactan epitopes. Thus, the ELISA analyses support the conclusion that NbPAGR-silencing affects branching of the RG-I backbone and the overall arabinogalactan composition of RG-I. ELISA results for RG-I from 35S::PAGR-YFP lines also support the conclusion that the arabinogalactan substitution of RG-I is altered in these lines.
Glycosidic linkage analysis of rhamnogalacturonan-I fractions
Linkage analysis of RG-I from NbPAGR-silenced N. benthamiana plants and PAGR-overexpressing Arabidopsis lines
35S::PAGR-YFP line 6
35S-PAGR-YFP line 9
1.1 ± 0.4
1.0 ± 0.2
1.9 ± 0.5
1.4 ± 0.2
2.3 ± 0.6
6.2 ± 0.5
8.9 ± 0.1*
8.5 ± 1.0
7.1 ± 1.8
9.6 ± 1.1
0.7 ± 0.1
0.7 ± 0.1
1.3 ± 0.1
1.3 ± 0.1
1.2 ± 0.1
4.9 ± 0.4
6.7 ± 0.2
6.3 ± 0.3
6.0 ± 0.7
5.8 ± 0.5
8.4 ± 0.5
9.2 ± 0.7
16.5 ± 0.7
16.5 ± 0.6
16.0 ± 0.7
7.9 ± 0.1
8.8 ± 0.4
7.4 ± 1.2
7.6 ± 1.1
6.8 ± 0.4
4.4 ± 0.1
4.5 ± 0.3
3.5 ± 0.1
4.6 ± 0.2*
4.7 ± 0.2*
1.2 ± 0.1
1.4 ± 0.1*
6.4 ± 0.2
6.4 ± 0.5
5.9 ± 0.5
47.9 ± 0.4
42.0 ± 1.6*
19.4 ± 0.4
16.0 ± 0.6*
14.7 ± 0.6*
6.6 ± 0.4
6.1 ± 0.5
5.5 ± 0.5
2.5 ± 0.1
3.0 ± 0.2*
4.6 ± 0.3
5.6 ± 0.1*
6.3 ± 0.1*
1.7 ± 0.2
1.4 ± 0.1
8.9 ± 0.5
9.3 ± 1.3
8.0 ± 1.0
0.7 ± 0.1
0.6 ± 0.1
1.6 ± 0.1
1.6 ± 0.2
1.8 ± 0.1
1.5 ± 0.1
1.3 ± 0.1
10.6 ± 0.2
10.4 ± 0.4
6.8 ± 0.3
10.7 ± 0.2*
11.7 ± 1.1*
Results reported here indicate that PAGR positively affects the biosynthesis of type-II arabinogalactans when overexpressed in Arabidopsis while silencing of the N. benthamiana ortholog, NbPAGR, decreases substitution of RG-I with type-I arabinogalactans and reduces branching of the RG-I backbone. Proposing a simple hypothesis for the glycosyltransferase activity of PAGR to explain these discordant results is challenging. Many distinct arabinogalactan sidechain structures have been detected as branches on the RG-I backbone . The pattern of these substitutions on the RG-I backbone has yet to be described.
In the biosynthesis of glucuronoxylan and xyloglucan, glycosyltransferases display strong acceptor substrate specificity. In the biosynthesis of glucuronoxylan, GUX1 shows a strong preference for the addition of glucuronic acid to evenly spaced xylan residues at intervals of around 8 residues while GUX2 adds glucuronic acid at intervals of between 5 and 7 residues with no preference for even or odd spacing . The activity of these two glucuronosyltransferases creates distinct xylan domains within the same molecule with distinct substitution patterns. In the biosynthesis of xyloglucan, XLT2 and MUR3 specifically add galactose to distinct xylosyl residues in each xyloglucan subunit [43, 44]. We speculate that by altering the substitution of the RG-I backbone, PAGR may affect recognition of the RG-I backbone by enzymes synthesizing type-I and type-II arabiongalactans. Alternatively, PAGR might primarily affect substitution of the most abundant RG-I arabinogalactan sidechain. We observed that in N. benthamiana, where silencing of NbPAGR primarily affects substitution of RG-I with type-I arabinogalactans, these are the most abundant sidechains. In Arabidopsis RG-I, type-II arabinogalactans are significantly more abundant.
The overall biosynthetic process by which pectic polysaccharides are made continues to be debated [1, 16]. Two main models for the pectin biosynthetic process have been proposed; the consecutive GT model and the domain synthesis model. In the consecutive GT model GTs sequentially add sugars from nucleotide-sugars onto a growing pectin polysaccharide as they move through the Golgi apparatus. In the domain synthesis model oligo- or polysaccharide primers are synthesized from nucleotide sugars or lipid-linked sugars and elongated into pectic glycan domains. These pectic domains are then transferred onto a growing pectin molecule. Our data could also support PAGR being involved in a biosynthetic step prior to the transfer of glycosides or oligosaccharides onto a nascent RG-I and thus affect the production of more than one type of RG-I sidechain. This possible function for PAGR is similar to the hypothesized roles of Mannan Synthase-Related (MSR)-1 and −2 . The MSRs are DUF246 domain containing, Golgi-localized GT-like proteins involved in mannan biosynthesis (see Additional file 2: Figure S2). In msr1 msr2 double mutants mannosyl levels are reduced by approximately 50 % and mannan synthase activity is reduced. Wang et al., (2012) hypothesized that MSR proteins may function in the production of oligosaccharide primers for the synthesis of mannans or in stabilization of the Mannan Synthase.
That PAGR appears to be necessary for pollen tube growth but not pollen development suggests that PAGR has a role in the production of polysaccharides present in the pollen tube cell wall. Experiments with transgenic expression of enzymes capable of digesting pectic polysaccharides in potatoes have shown that these polysaccharides are essential for pollen viability . A collapsed pollen phenotype and decreased male fertility were observed in potato lines expressing enzymes digesting the RG-I backbone or pectic arabinans. The presence of conserved PAGR orthologs in Physcomitrella and Selaginella support that its role has been conserved throughout the evolution of land plants. The involvement of PAGR in production of a critical polysaccharide structure, such as an early bond in RG-I arabinogalactan sidechains, could drive such a high level of conservation.
The morphological phenotypes induced by overexpression and silencing of PAGR and NbPAGR provide evidence supporting a role for pectic arabinogalactans in regulating the extensibility of plant cell walls. Pleiotropic morphological phenotypes are manifested only in specific organs of PAGR overexpressors, which suggests that critical properties of RG-I are substantially altered by PAGR overexpression only in specific tissues. The elasticity of cell walls in the shoot apical meristem is thought to play a key role in regulating organ formation . Pectin methylesterification affects the elasticity of cell walls and is highly regulated in the shoot apical meristem, where regions of de-methyl-esterification underlie new lateral organ primordia . Alteration of pectin methylesterification in the shoot apical meristem also affects the elasticity of cell walls and the production of new organs . The altered phyllotaxy and fasciation of 35S::PAGR-YFP plants may be attributable to altered extensibility of cell walls in the shoot apical meristem. In NbPAGR-silenced plants decreased internode expansion and shortened roots are likely due to decreased cell wall extensibility. If PAGR has a role in the biosynthesis of RG-I arabinogalactan sidechains it is possible that both the biochemical and morphological phenotypes of PAGR overexpression may depend upon the degree of RG-I substitution normally present in a particular tissue. For example, in the young PAGR-overexpressing seedlings analyzed for RG-I monosaccharide composition, we observed a significant increase in RG-I arabinan content, while the similar linkage analysis of RG-I from rosette leaves did not show alterations in arabinan in PAGR-overexpressors.
Together, the results presented here support that PAGR functions in the biosynthesis of RG-I arabinogalactans and illustrates the essential roles of these polysaccharides in vegetative and reproductive plant growth. More research is needed to understand the detailed structure of RG-I, particularly with respect to the pattern and nature of branching, and the biosynthetic process by which RG-I is produced in plants. Such research will better enable efforts to identify the specific biochemical role of PAGR in the biosynthesis of RG-I arabinogalactan sidechains.
Arabidopsis thaliana Heyn. (L) accession Columbia-0 seeds were obtained from Lehle Seeds (Round Rock, Texas). Arabidopsis plants were grown under a 10-h photoperiod 22 °C with 90 μmol m−2 s−1 illumination during the day period. After 4 weeks, plants were transferred to a 16-h light photoperiod to induce flowering. Nicotiana benthamiana seeds were kindly provided by the Dinesh-Kumar Lab (Univerisity of California, Davis). N. benthamiana plants were grown under 16-h photoperiod at 25 °C, 60 % humidity with 200 μmol m−2s−1 illumination during the day period. Arabidopsis T-DNA lines CS836448 (pagr-1) and SALK_064738C (pagr-2) were acquired from the Arabidopsis Biological Resource Center (ABRC, Ohio State University). Genotyping of T-DNA lines was performed by PCR using genomic primers Cs836448F 5’-TCTTCCAGAGATAGAGCAGATGGCTG-3’ and Cs836448R 5’-TGCGCTTCTGCAAGGCGAGC-3’ for pagr-1 and S_64738F 5’-TGGCGTCACTGGGTGCTCCT-3’ and S_64738R 5’-TCAGCCATCTGCTCTATCTCTGGAAG-3’ for pagr-2. T-DNAs were detected using the forward genomic primers and the appropriate left-border T-DNA primers pDAP101-Lb1 5’-GCCTTTTCAGAAATGGATAAATAGCCTTGCTTCC-3’ for pagr-1 and LB1 5’-TGGTTCACGTAGTGGGCCATCG-3’ for pagr-2. In vitro pollen germination assays were performed as described . Differential staining of aborted pollen grains was performed as described  and imaged using a Leica DMB4000B microscope.
Virus Induced Gene Silencing (VIGS)
NbPAGR-A (Niben101Scf07590g07020) and NbPAGR-B (Niben101Scf35628g00005) were identified in the Sol Genomics Network Database  as reciprocal best BLAST hits for PAGR in the N. benthamiana genome. Alignment and phylogenetic analysis of the NbPAGR predicted amino acid sequences with the Arabidopsis DUF246 family proteins showed NbPAGR-A and -B to be the close homologs of Arabidopsis PAGR (Additional file 2: Figure S2). The NbPAGR-A sequence used to induce VIGS was amplified from total N. benthamiana cDNA using primers 5’-TTATCTAGACGATGACGATTACCGTGGCCGT-3’ and 5’-TTATCTAGAGCTGGTTTAGACCACCCTCAGCG-3’. Subsequently the fragment was cloned into the Xba1 site in pYL156  i.e. pTRV2, to generate pYL156-NbPAGR. As a non-silencing control plasmid, pYL156 with a fragment of the GUS gene was used (pYC1) . pYC1 and pYL156-NbPAGR were independently transformed into Agrobacterium tumefaciens strain GV3101. Virus-induced gene silencing was induced in 2–3 week old N. benthamiana plants according to standard protocols . Tissue was collected 14 days post infection for all analyses. Sequence alignments were performed in Geneious 4.6.5 (Biomatters, New Zealand) Phylogenetic analyses were performed using http://www.phylogeny.fr .
PAGR Overexpression and localization
The coding sequence of At3g26370 (PAGR) was amplified from total Arabidopsis cDNA using Phusion High-Fidelity DNA polymerase (New England Biolabs) and the primers 5’-CACCATGGCAGAGTTACGGCACTCGAGCTCTCTC-3’ and 5’-TCCAGCTTTACACATGCATGGAGTGAGAGG-3’. The PCR product was cloned into pENTR-D-Topo (Invitrogen). A Gateway LR recombination reaction was performed according to the manufacturer’s protocol (Invitrogen) to transfer the coding sequence of PAGR into pGWB41  to produce 35S::PAGR-YFP for production of transgenic Arabidopsis plants. This construct was transformed into A. tumefaciens strain GV3101 and Arabidopsis plants of the Columbia-0 ecotype were transformed via the floral dip method . For total cell wall and RG-I monosaccharide composition analysis of 35S::PAGR-YFP lines, T3 seedlings were grown in liquid culture at 22 °C for 14 days. For localization studies, the coding sequence of PAGR was recombined into pGWB44 to product 35S::PAGR-CFP. PAGR-CFP was transiently co-expressed with α-mannosidase-1  in 4-week-old N. benthamiana leaves following described procedures  except that 100 mM 2-(N-morpholino)ethanesulfonic acid, 100 mM MgCl2, 10 μM acetosyringone was used as the infiltration medium. Expression in N. benthamiana epidermal cells was imaged using a Zeiss 710 confocal laser-scanning microscope (Carl Zeiss).
Total protein was extracted from 1 week old Arabidopsis seedlings by grinding in 100 mM Tris pH 7.5, 1 mM EDTA, 1 % (v/v) Triton X-100, 10 % (v/v) glycerol, 1 mM phenylmethylsulfonyl fluoride. Cellular debris was pelleted by centrifugation at 16,000 x g for 15 min at 4 °C. Extracted protein was quantified by Bradford Assay . For electrophoresis, 40 μg of protein was separated by SDS-PAGE, electrotransferred onto a PVDF membrane and incubated with AttB2 site ‘universal’ primary antibody and anti-rabbit secondary antibody as described by , except that 5 % w/v BSA (Sigma) in TBS-T was used as a blocking agent and membranes were incubated with universal antibody at a 1:3000 dilution.
RNA was extracted from Arabidopsis and N. benthamiana tissues using the RNEasy plant mini kit (Qiagen). For cloning purposes RNA was extracted from N. benthamiana tissues using Trizol Reagent (Invitrogen). cDNA was synthesized with Superscript-III reverse transcriptase (Invitrogen). Quantitative RT-PCR was performed on a StepOne-Plus Real-Time PCR system (Applied BioSystems) using Syber-Select Real-Time PCR reagents (Invitrogen). PAGR was detected using primer At3g26370 q1F 5’-GAGGTCGTCGCAGATCTTCAGGTTCATGT-3’ and At3g26370 q1R 5’-GGCTCCCACTGTTCTTCTTCATCAGGCTT-3’. MONENSIN SENSITIVITY1, At2g28390, a gene with exceptional transcript-level stability , was analyzed as a reference gene using primers 5’-AACTCTATGCAGCATTTGATCCACT-3’ and 5’-TGATTGCATATCTTTATCGCCATC-3’. Data were analyzed using the comparative Ct method. For quantitative real-time RT-PCR analysis of NbPAGR silencing, data were analyzed using the geometric mean of three reference genes as the common reference as described . For NbPAGR-A and –B, primers NbPAGR-AqF 5’-CTACGCCACTCAAGCTCGATCGGAAA-3’, NbPAGR-AqR 5’-GCCACGGTAATCGTCATCGTCATCGTCAA-3’, NbPAGR-BqF 5’-CTACGCCACTCAAGCTCGATCGGAAG-3’ and NbPAGR-BqR 5’-GCCACGGTAATCGTCATCGTCATCGTCAT-3’ were used. Elongation Factor 1A, Actin-2 and Ubiquitin 3 were used as reference genes using Elongation Factor 1A primers NbEF1qF 5’-AGGGTCCAACCCTCCTTGAGGC-3’ and NbEF1qR 5’-GCCCCTTTGGCTGGGTCGTC-3’; Actin-2 primers ACT2F 5’-TTGAGACTTTTAATACCCCAGC-3’ and ACT2R 5’-AACATGTAACCACGCTCGGTAA-3’ and Ubiquitin-3 primers UBQ3F 5’-GCCGATTACAACATCCAGAAGG-3’ and UBQ3R 5’-TGAAGTACAGCGAGCTTAACC-3’.
Cell wall isolation and monosaccharide composition analysis
Alcohol-insoluble residue (AIR) was prepared as described by . Lyophilized AIR was hydrolyzed in 2 M trifluoroacetic acid at 120 °C for 1 h and analyzed by high-performance anion exchange chromatography (HPAEC) as described by . Glucose was not determined for samples of total cell wall material due to the presence of residual starch. Sequential extraction of AIR was performed essentially as described  with the exception that samples from PAGR-overexpressors were not extracted with 1 M KOH prior to the extraction with 4 M KOH.
Cell wall preparations from VIGS plants were further analyzed by digestion with endo-β-1,4-galactanase from Aspergillus niger purified to a single band on a silver-stained gel (Megazyme, product code E-EGALN). AIR (2 mg) was dissolved in 0.1 mL of 1 M KOH and adjusted to pH 4.7 with 2 mL of 100 mM acetic acid. Galactanase was added and samples were incubated at 40 °C for 1 h at 40 °C. After incubation, cold ethanol with 10 mM EDTA was added to a final concentration of 70 % (v/v) and the sample was centrifuged for 5 min at 14,000 x g at 4 °C. The supernatant and pellet were separated, hydrolyzed and analyzed by HPAEC as described above.
Rhamnogalacturonan-I was isolated essentially as described by . Briefly, 15 mg of AIR was digested overnight with 3U of pectin methyl-esterase (Novoshape Pure PME, Novozymes) and 20U of endopolygalacturonanase M2 (Megazyme, product code E-PGALUSP) at 37 °C in 1 ml of 50 mM ammonium oxalate (pH 5.0). Following digestion, insoluble material was removed by centrifugation followed by filtration through a 0.2 μm spin filter. Oligosaccharides and the digestion buffer were removed by washing of the solubilized polysaccharides on a 10 kDa Molecular Weight Cutoff spin filter (Amicon) with sterile water. Samples were eluted from the spin concentrators in water and separated by size-exclusion chromatography in 50 mM ammonium formate (pH5.0) on a Superdex 200 10/300GL column (GE Healthcare Bio-Sciences, http://www.gelifesciences.com/) at a flow rate of 0.5 ml/min. Elution of polysaccharides from the column was monitored with a Shodex RI-101 refractive index detector (Shodex, http://www.shodex.com). Fractions were collected manually, lyophilized, hydrolyzed and analyzed by HPAEC as described above. Estimates of the MW of RG-I were made with reference to the retention times of Dextran MW standards (Sigma-Aldrich). The relative mass percentage of each monosaccharide in the RG-I fractions was determined by first calculating the mass ratio of each monosaccharide by dividing the product of the mol% and the molar mass of each monosaccharide by the sum of the products of the molar mass and mol% of each monosaccharide. We then multiplied the monosaccharide mass ratios by the estimated molecular weight of the RG-I fraction.
ELISA screening of purified rhamnogalacturonan-I preparations
Purified RG-I samples were dissolved in water and were coated onto ELISA plates [384 well clear flat bottom polystyrene high bind microplate (product no. 3700), Corning Life Sciences] on an equal weight (gravimetric) per well basis (0.5 μg/well). The samples were then subjected to ELISA screening with a comprehensive suite of cell wall glycan-directed monoclonal antibodies essentially as described earlier [40, 61]. The ELISA screening assays were done using an Robotic System (Thermo Scientific) comprising an Orbitor RS Robotic Arm (Thermo Scientific) accessing the following components: Carousel plate storage/incubation (Thermo Scientific), EON Plate Reader (Biotek), EL406 ELISA Plate Washer (Biotek), Multiflo Dispenser (Biotek) and Precision XS Fluid Dispenser (Biotek). The whole system is operated by the laboratory automation software, Momentum 3.2.7 (Thermo Scientific). Water was used as the blanks and these background values were subtracted from the sample ELISA responses. The ELISA assays were done in technical duplicates and data represent the average of the replicates. Cell wall glycan-directed antibodies were were obtained from laboratory stocks (CCRC, JIM and MAC series) at the Complex Carbohydrate Research Center (available through CarboSource Services; http://www.carbosource.net) or through BioSupplies (Australia) (BG1, LAMP).
Rhamnogalacturonan-I linkage analysis
Glycosidic linkage analysis of RG-I was performed by GC/MS analysis of their partially methylated alditol acetates . Purified RG-I samples were per-O-methylated using liquid NaOH in dimethyl sulfoxide and further derivatized to their corresponding partially methylated alditol acetates by trifluoroacetic acid hydrolysis, reduction and per-O-acetylation. The derivatives were separated using a gas chromatograph (Agilent 7890A, Agilent Technologies, www.agilent.com) equipped with a Supelco SP2380 column (Sigma-Aldrich) and a mass spectrometer (Agilent 5975C) using a temperature gradient as described . Eluted compounds were identified based on their retention time compared to standards and their ion fragmentation patterns.
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Availability of data and materials
The data sets supporting the results of this article are included within the article and its additional files. Nucleotide sequences and biological materials including Arabidopsis seeds, plasmids and bacterial strains created through this work are available at (https://registry.jbei.org). Data supporting phylogenetic analyses presented in this study are available at (http://purl.org/phylo/treebase/phylows/study/TB2:S19106).
This work was supported by the U. S. Department of Energy, Office of Science, Office of Biological and Environmental Research, through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the U. S. Department of Energy. Part of the work was supported by the Danish Strategic Research Council (Set4Future 11–116795). The generation of the CCRC series of plant cell wall glycan-directed monoclonal antibodies used for ELISA screening in this work was supported by the United States National Science Foundation Plant Genome Program (DBI-0421683 and IOS-0923992).
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