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.

Purpose Name Sequence Annealing

Absence of insertion gal10-1

BGAL10-1L GCCTACTCTGTTGCTCGGTTTT 58ºC

BGAL10-1R CACCGCCTTGTCATTTTTATCA

Presence of insertion gal10-1

BGAL10-1L GCCTACTCTGTTGCTCGGTTTT 58ºC

SYN-LB CATCTGAATTTCATAACCAATCTCGA

Absence of insertion gal10-2

BGAL10-2L GCAACTCTTGGACAAGAAGCAA 58ºC

BGAL10-2R GCCATAAATCTCTCGCATTCCT

Presence of insertion gal10-2

BGAL10-2L GCAACTCTTGGACAAGAAGCAA 58ºC

SALK-LB GATTTCGGAACCACCATCAAAC

Absence of insertion Atxyl1-2

XYL1-2L TTTATGGTTCGCATCCGATGTA 58ºC

XYL1-2R CAAGAACCGTGGTCCATGTCTA

Presence of insertion Atxyl1-2

XYL1-2L TTTATGGTTCGCATCCGATGTA 58ºC

GABI-LB CCATATTGACCATCATACTCATTGC

Promoter BGAL10

PRGAL10L1 TTGGGTAATTGGGGGTGTTTTA 58ºC

PRGAL10R1 CGTCGTACTTCCCTCGAGTGTT

BGAL10 heterologous expression

BGAL10L1 GGTACCGCGAATGTATCCTACGACCAC 56ºC

BGAL10R1 GCGGCCGCCACCACCGATACTTTCCTTTTG

Supplemental Figure 1B

C

Inte

nsity

(arb

itry

units

)

Mass (m/z)

1247 peak

1247

(pre

curs

or io

n)

1115

(-P

ent)

1085

(-H

ex)

953

(-H

ex, -

Pen

t)

659

(-2

Hex

, -2

Pen

t)

983

(-2

Pen

t)

773

(-2

Hex

, -P

ent,

-H20

)

821

(-H

ex, -

2 P

ent)

935

(-H

ex, -

Pen

t, -H

20)

641

(-2

Hex

, -2

Pen

t, -H

20)

611

(-3

Hex

, -P

ent,

-H20

)

497

(-3

Hex

, - 2

Pen

t)47

9 (-

3 H

ex, -

2 P

ent,

-H20

)

Glc Glc Glc Glc

Xyl Xyl Xyl

Gal

659oo o

o611

o o

o

Glc Glc Glc Glc

Xyl Xyl Xyl

Gal

497oo o

o773

o o

o

XXLG XLXG10

67 (-

Hex

, -H

2O)

0

5

10

15

20

25

30

1 2 3 4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Act

ivity

pka

t mg

-1

Vector AtBGAL10

D

70100

130kDa M 1 2 3 1 2 3

Vector AtBGAL10

A

0

10

20

30

40

50

60

70

Act

ivity

pka

t mg

-1

Col bgal10-1

G

Mass (m/z)

Inte

nsity

(arb

itry

units

)

E

Glc Glc Glc Glc

Xyl Xyl

GalGal

659oo o

o641

o

o o

983

1115

1097

GLLG

1277

(pre

curs

or io

n)

1115

(-H

ex)

1097

(-H

ex, -

H2O

)

983

(-H

ex, -

Pen

t)

659

(-3

Hex

, -P

ent)

641

(-3

Hex

, -P

ent,

-H2O

)

821

(-2

Hex

, -P

ent)

953

(-2

Hex

)93

5 (-

2 H

ex, -

H2O

)

803

(-2

Hex

, -P

ent,

-H2O

)

689

(-2

Hex

, -2

Pen

t)

479

(-4

Hex

, - P

ent,

-H2O

)49

7 (-

4 H

ex, -

Pen

t)

114

1 (-

Pen

t)

Mass (m/z)

Inte

nsity

(arb

itry

units

)

XXLGXLXG1247

XXLG + Xyl1379

XXXGXLG +Xyl

1085

XLGGXXG

953

XXG791

GXGLG659 GLG

821

GXLGGLXGLLG1115

F 1247 peak

1115

124765

9

773

1085

1067

953

821

479

Mass (m/z)

Inte

nsity

(arb

itry

units

)

H

Glc Glc Glc

Xyl

Gal

o o

o

o659

641527

GLG

659

(-H

ex)

641

(-H

ex, -

H2O

)

689

(-P

ent) 82

1 (p

recu

rsor

ion)

527

(-H

ex, -

Pen

t)

479

(-2

Hex

, -H

2O)

497

(-2

Hex

)

Supplemental Figure 2

AtBGAL10(At5g63810) Al-951190

ppa001345m POPTR 0007s04740

Rc-30193.t000019 Medtr3g142650 Glyma06g16420

Glyma04g38590 orange1.1g046146m

clementine0.9 002762m orange1.1g004525m clementine0.9 003301m

TmBGAL1 Eucgr.I00063 Eucgr.I00108

Cucsa.228210 mgv1a001532m

AcoGoldSmith v1.000910m Vv-GSVIVT01022339001

Os01g65460 Bradi2g56610

Si000291m Zm-GRMZM2G038281

Sb03g041450 orange1.1g003095m clementine0.9 002277m

Rc-28076.t000019 cassava4.1 001639m Cp-evm.model.supercontig 216.21 AcoGoldSmith v1.000899m

Vv-GSVIVT01025170001 Medtr2g110570

AtBGAL8(At2g28470) Al-481656 mgv1a001263m

mgv1a001293m Os03g15020

Bradi1g67760 Si034234m Zm-GRMZM2G127123

Sb01g040750 Os03g06940

AcoGoldSmith v1.000905m AtBGAL3(At4g36360)

Cp-evm.TU.supercontig 6.189 AtBGAL5(At1g45130)

Zm-GRMZM2G081583 Sb10g022620

Os06g37560 Os02g12730

Si000317m Os01g39830 Bradi2g41830

Rc-28694.t000018 cassava4.1 001652m POPTR 0003s03060

Cp-evm.model.supercontig 25.57 AtBGAL1(At3g13750) Al-929130

Medtr2g121320 Glyma08g20650 Cucsa.229010 Eucgr.C00898

Vv-GSVIVT01015203001 Cucsa.397370

AtBGAL12(At4g26140) AtBGAL2(At3g52840) AtBGAL4(At5g56870)

ppa001415m ppa001412m

POPTR 0006s14680 cassava4.1 021365m

Pp1s10 144V6 Pp1s189 71V6

AtBGAL9(At2g32810) Os12g24170

Pp1s24 113V6 Sm-404798

Sm-269483 Sm-413650

AtBGAL7(At5g20710) AtBGAL15(At1g31740)

Os10g19960 Os10g18400

Os01g34920 AtBGAL6(At5g63800)

AtBGAL16(At1g77410) Os05g35360

Os08g43570 Os09g36810

AtBGAL14(At4g38590) AtBGAL11(At4g35010)

AtBGAL13(At2g16730) AtBGAL17(At1g72990)

Os05g46200100

100

100

100

100

100

100

100

100

100

100

96

100

99

100

93100

95

79

88

100

100

100

65

99

100

86

100

100

61

65

79

65100

85

100

31

12

5

7

16

3

5

98

99

94

100

100

94

63

99

3063

4533

37

56

4041

98

60

91

3322

36

33

27

5499

30

44

4748

99

99

9369

63

38

61100

22

96

51

93

73

6848

37

30

27

100

0,1

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

Rel

ativ

e ab

unda

nce

Ion species

XXXG(1085)

XXLGXLXG(1247)

XXFG(1393)

XLFG(1555)

XLLG(1409)

GLLG(1277)

GLXGGXLGLLG

(1115)

LGGXG(659)

XXG(791)

XLGLXG

GXXG(953)

GLG(821)

A

B C

1550 1800m/z

Inte

nsity

XLLGG1571

XLLGG-Ac1613

XLFGG-Ac1759

XLFG-Ac1597

XLFG1555

600 1600

659 821 953 1115 12771157 1319

bgal10-1

bgal10-1

bgal10-1

Columbia

Inte

nsity

m/z

Supplemental Figure 3

Mass (m/z)

Inte

nsity

(arb

itry

units

)In

tens

ity (a

rbitr

y un

its)

Mass (m/z)

B

A

1115

(pre

curs

or io

n)

983

(-P

ent)

953

(-H

ex)

821

(-H

ex, -

Pen

t)

689

(-1

Hex

, -2

Pen

t)

659

(-2

Hex

, -P

ent)

803

(-H

ex, -

Pen

t, -H

20)

641

(-2

Hex

, - P

ent,

-H20

)

497

(-3

Hex

, - P

ent)

479

(-3

Hex

, - P

ent,

-H20

)

935

(-H

ex, -

H2O

)

Glc Glc Glc Glc

Xyl Xyl

GalGal

659oo o

o641

o

o o

983

1115

1097

GLLG

Glc Glc Glc Glc

Xyl Xyl

Gal

497oo o

o641

o

o

821

953

935983

GLXG

Glc Glc Glc

Xyl Xyl

Gal

659o o

o479

o

o

821

953

935

Galo

LLG

1277

(pre

curs

or io

n)

1115

(-H

ex)

1097

(-H

ex, -

H2O

)

983

(-H

ex, -

Pen

t)

659

(-3

Hex

, -P

ent)

641

(-3

Hex

, -P

ent,

-H2O

)

821

(-2

Hex

, -P

ent)

953

(-2

Hex

)93

5 (-

2 H

ex, -

H2O

)

803

(-2

Hex

, -P

ent,

-H2O

)

689

(-2

Hex

, -2

Pen

t)

479

(-4

Hex

, - P

ent,

-H2O

)49

7 (-

4 H

ex, -

Pen

t)

114

1 (-

Pen

t)

851

(-2

Pen

t)

773

(-2

Hex

, -H

20)

791

(-2

Hex

)

Supplemental Figure 4

Mass (m/z)

Inte

nsity

(arb

itry

units

)

D

C

Glc Glc Glc

Xyl

Gal

o o

o

o659

641527

GLG

659

(-H

ex)

641

(-H

ex, -

H2O

)

689

(-P

ent)

821

(pre

curs

or io

n)

527

(-H

ex, -

Pen

t)

479

(-2

Hex

, -H

2O)

497

(-2

Hex

)

821

(-P

ent)

953

(pre

curs

or io

n)

791

(-H

ex)

659

(-H

ex, -

Pen

t)

773(

-Hex

, -H

2O)

479

(-2

Hex

, -P

ent,

-H2O

) 49

7 (-

2 H

ex, -

Pen

t)

641

(-H

ex, -

Pen

t, -H

2O)

689

(-2

Pen

t)

611

(-2

Hex

, -H

2O)

689

(-H

ex, -

2 P

ent)

XLG

Glc Glc Glc

Xyl

Gal

o o

o

o791

773659

Xylo821

LXG

Glc Glc Glc

Xyl

Gal

o o

o

o791

773821

Xylo659

479

497

Glc Glc Glc

Xyl

o o

o773

Xylo821

Glc o791

479

497

GXXG

Inte

nsity

(arb

itry

units

)

Mass (m/z)

E

659

(pre

curs

or io

n)

497

(-H

ex)

365

(-H

ex, -

Pen

t)

479

(-H

ex, -

H2O

)

LG

Glc Glc

Xyl

Gal

o

o

o497

479365

527

(-P

ent)

317

(-2

Hex

, -H

2O)

335

(-2

Hex

)