SUPPLEMENTAL DATA Supplemental Table I. Sequence and annealing temperatures of the primers used in this work. Supplemental Figure 1. Analysis of AtBGAL10 activity. A. β-Galactosidase activity against XLLG in flowers and developing siliques. Proteins were extracted from one wild type sample (black) and two replicates of bgal10-1 (white). Error bars show standard deviations. B. β-Galactosidase activity against XLLG of 4 Pichia pastoris clones transformed with empty vector and 20 clones transformed with AtBGAL10. C. MALDI- TOF/TOF spectrum of the ion at 1247 m/z units produced by digestion of XLLG with secreted proteins from Pichia transformed with AtBGAL10, as shown in Figure 1F. Error bars show standard deviations. Only fragments resulting from breaks of glycosidic bonds have been labeled with their size and losses (Pent for pentose, Hex for hexose and H 2 O for losses that include the reducing end). Grey labels show putative products of double cleavages of XXLG. In the upper left corner the structures of XXLG and XLXG show the expected ions resulting from fragmentation between the central glucose residues. The ion at 659 m/z, diagnostic of XXLG is much more abundant than the 773 m/z ion (grey triangles), suggesting that XLXG is, at most, a minor component. It might not be present since several double and triple fragmentations can produce ions of 773 m/z units from XXLG (one is shown by dotted lines). D. Gel electrophoresis of secreted proteins from three Pichia clones transformed with empty vector and three clones transformed with AtBGAL10. Proteins extracts are the same used for activity assays in Figure 1D. The sizes of marker proteins are indicated to the left. Black arrowheads indicate putative AtBGAL10 bands. E. MALDI-TOF of a 24 h digestion of XLLG at 1.5 mM with Columbia protein extract. Digestion products are indicated by triangles and labeled with possible structures and m/z of the corresponding ion. Putative products of transglycosylation by α-xylosidase are indicated as +Xyl. F. MALDI-TOF/TOF spectrum of the ion at 1247 m/z units from E. G. MALDI-TOF/TOF spectrum of the 1277 m/z ion in the digestion of XLLG with bgal10 protein extract shown in Figure 1G. The structure of GLLG is shown with the sizes of the different products of single cleavages. A red triangle shows the expected position of the fragment resulting from a pentose cleavage. H. MALDI-TOF/TOF spectrum of the 821 m/z ion from the digestion in Figure 1G. Symbols are as in F. Supplemental Figure 2. Phylogenetic tree of AtBGAL10 and close homologs from 23 species with sequenced genomes. The clade where AtBGAL10 and its putative orthologs are located is shown in grey. Bootstrap values based on 500 replicates are shown next to each node. Gene annotations for species other than rice and Arabidopsis are taken from Phytozome V7.0 database (www.phytozome.net). Supplemental Figure 3. Xyloglucan composition. A Xyloglucan composition in stems of bgal10-1 and bgal10-2 mutants. Alcohol-insoluble cell wall residues were obtained from two samples of wild type (black), bgal10-1 (white) or bgal10-2 stems (grey). Xyloglucan was extracted with endoglucanase and peak areas from three MALDI-TOF spectra were quantified to estimate standard deviations, presented as error bars. The proportion of acetylated subunits corresponds to the area above the horizontal lines. B. Xyloglucan
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SUPPLEMENTAL DATA - Plant physiology2012/01/20 · SUPPLEMENTAL DATA Supplemental Table I. Sequence and annealing temperatures of the primers used in this work. Supplemental Figure
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SUPPLEMENTAL DATA
Supplemental Table I. Sequence and annealing temperatures of the primers used in this work.
Supplemental Figure 1. Analysis of AtBGAL10 activity. A. β-Galactosidase activity against XLLG in
flowers and developing siliques. Proteins were extracted from one wild type sample (black) and two replicates
of bgal10-1 (white). Error bars show standard deviations. B. β-Galactosidase activity against XLLG of 4
Pichia pastoris clones transformed with empty vector and 20 clones transformed with AtBGAL10. C. MALDI-
TOF/TOF spectrum of the ion at 1247 m/z units produced by digestion of XLLG with secreted proteins from
Pichia transformed with AtBGAL10, as shown in Figure 1F. Error bars show standard deviations. Only
fragments resulting from breaks of glycosidic bonds have been labeled with their size and losses (Pent for
pentose, Hex for hexose and H2O for losses that include the reducing end). Grey labels show putative products
of double cleavages of XXLG. In the upper left corner the structures of XXLG and XLXG show the expected
ions resulting from fragmentation between the central glucose residues. The ion at 659 m/z, diagnostic of
XXLG is much more abundant than the 773 m/z ion (grey triangles), suggesting that XLXG is, at most, a
minor component. It might not be present since several double and triple fragmentations can produce ions of
773 m/z units from XXLG (one is shown by dotted lines). D. Gel electrophoresis of secreted proteins from
three Pichia clones transformed with empty vector and three clones transformed with AtBGAL10. Proteins
extracts are the same used for activity assays in Figure 1D. The sizes of marker proteins are indicated to the
left. Black arrowheads indicate putative AtBGAL10 bands. E. MALDI-TOF of a 24 h digestion of XLLG at
1.5 mM with Columbia protein extract. Digestion products are indicated by triangles and labeled with possible
structures and m/z of the corresponding ion. Putative products of transglycosylation by α-xylosidase are
indicated as +Xyl. F. MALDI-TOF/TOF spectrum of the ion at 1247 m/z units from E. G. MALDI-TOF/TOF
spectrum of the 1277 m/z ion in the digestion of XLLG with bgal10 protein extract shown in Figure 1G. The
structure of GLLG is shown with the sizes of the different products of single cleavages. A red triangle shows
the expected position of the fragment resulting from a pentose cleavage. H. MALDI-TOF/TOF spectrum of
the 821 m/z ion from the digestion in Figure 1G. Symbols are as in F.
Supplemental Figure 2. Phylogenetic tree of AtBGAL10 and close homologs from 23 species with
sequenced genomes. The clade where AtBGAL10 and its putative orthologs are located is shown in grey.
Bootstrap values based on 500 replicates are shown next to each node. Gene annotations for species other than
rice and Arabidopsis are taken from Phytozome V7.0 database (www.phytozome.net).
Supplemental Figure 3. Xyloglucan composition. A Xyloglucan composition in stems of bgal10-1 and
bgal10-2 mutants. Alcohol-insoluble cell wall residues were obtained from two samples of wild type (black),
bgal10-1 (white) or bgal10-2 stems (grey). Xyloglucan was extracted with endoglucanase and peak areas from
three MALDI-TOF spectra were quantified to estimate standard deviations, presented as error bars. The
proportion of acetylated subunits corresponds to the area above the horizontal lines. B. Xyloglucan
composition in elongated siliques. MALDI-TOF spectra correspond to Columbia xyloglucan and three
independent extractions of bgal10-1 xyloglucan. Characteristic peaks of bgal10 xyloglucan are indicated in
grey. C. Xyloglucan subunits observed during short endoglucanase digestions of bgal10-1 cell wall residues.
Supplemental Figure 4. Fragmentation analysis of unusual xyloglucan subunits in bgal10-1 leaves. A.
MALDI-TOF/TOF spectrum of the 1277 m/z ion. Only fragments resulting from breaks of glycosidic bonds
have been labeled with their size and losses (Pent for pentose, Hex for hexose and H2O for losses that include
the reducing end). Grey labels show putative products of double cleavages. The structure of GLLG is shown
with the sizes of the different products of single cleavages. A red triangle shows the expected position of the
peak for a pentose cleavage. B. MALDI-TOF/TOF spectrum of the 1115 m/z ion. Symbols are as in A, except
that the red triangle shows the position of the fragment resulting from two pentose cleavages. Two possible
components are shown with their single cleavage products. C. MALDI-TOF/TOF spectrum of the 953 m/z
ion. Symbols are as in A. D. MALDI-TOF/TOF spectrum of the 821 m/z ion. Symbols are as in A. E.
MALDI-TOF/TOF spectrum of the 659 m/z ion. Symbols are as in A.
Supplemental Table I. Sequence and annealing temperatures of the primers used in this work.