Supplementary Material

Mucilage secretory cells, complex regulation of a key extracellular matrix

Edith Francoz1,2* Philippe Ranocha1,2*, Vincent Burlat1,2 and Christophe Dunand1,2

1Université de Toulouse, UPS, UMR 5546, Laboratoire de Recherche en Sciences Végétales, BP 42617, F-31326 Castanet-Tolosan, France.

2CNRS, UMR 5546, BP 42617, F-31326 Castanet-Tolosan, France.

*These authors contributed equally to this work.

Corresponding authors: Burlat, V. () and Dunand, C. ()

Table S1: Mucilage secretory cell (MSC) tool boxa
AGI Code / Annotation / Role in seed physiology / Mutant seed phenotype / Refs
1. Transcriptional pathways
At1g09540 / MYB DOMAIN PROTEIN 61 (MYB61) / Regulation of mucilage synthesis / Lack of mucilage deposition and extrusion / [S1-S3]
At1g52880 / NAC-REGULATED SEED MORPHOLOGY 2 (NARS2) / NAM / Regulation of embryogenesis and seed coat synthesis / Embryogenesis defects and no mucilage extrusion (in nars1 nars2 double mutant) / [S4]
At1g56650 / MYB DOMAIN PROTEIN 75 (MYB75) / Regulation of seed coat wall synthesis / Distorted cell shape; thicker radial walls / [S5]
At1g62990 / KNOTTED-LIKE HOMEOBOX OF ARABIDOPSIS THALIANA 7 (KNAT7) / Regulation of seed coat cell wall synthesis and mucilage adherence / Reduced mucilage release; thicker MSC radial cell walls / [S3,S5]
At1g63650 / ENHANCER OF GLABRA 3 (EGL3) / Regulation of mucilage synthesis / Reduced mucilage and columellae (in egl3 tt8 double mutant) / [S6,S7]
At1g76880 / DE1 BINDING FACTOR 1 (DF1) / Unknown / Disturbed coat structure; impaired pectin synthesis and mucilage release / [S8,S9]
At1g79840 / GLABRA2 (GL2) / Regulation of mucilage synthesis / No mucilage release; Higher seed oil content / [S10,S11]
At2g32700 / MUCILAGE-MODIFIED 1 (MUM1) / LEUNIG_HOMOLOG (LUH) / Regulation of mucilage and cell wall synthesis / No mucilage extrusion; slightly irregular columellae / [S10, S12,S13]
At2g37260 / TRANSPARENT TESTA GLABRA 2 (TTG2) / DR. STRANGELOVE 1 (DSL1) / Regulation of mucilage synthesis / Reduced mucilage and columellae / [S14]
At3g13540 / MYB DOMAIN PROTEIN 5 (MYB5) / Regulation of mucilage synthesis / Reduced mucilage extrusion in myb5 simple mutant; no mucilage extrusion in myb5 tt2 double mutant / [S7,S15]
At3g15510 / NAC-REGULATED SEED MORPHOLOGY 1 (NARS1) / NAC2 / Regulation of embryogenesis and seed coat synthesis / Embryogenesis defects and no mucilage extrusion (in nars1 nars2 double mutant) / [S4]
At4g09820 / TRANSPARENT TESTA 8 (TT8) / Regulation of mucilage synthesis / Reduced mucilage and columellae (in egl3 tt8 double mutant) / [S6,S7,S16]
At4g21030 / DNA-BINDING WITH ONE FINGER 4.2 (DOF4.2) / Unknown / Collapsed seed epidermal cells, reduced mucilage content, altered mucilage monosaccharide composition in DOF4.2 over-expressing line / [S17]
At4g32551 / LEUNIG (LUG) / Regulation of mucilage synthesis or maturation / Slightly reduced mucilage extrusion / [S13,S18]
At4g36920 / APETALA 2 (AP2) / Regulation of oi1/oi2 differentiation / No mucilage and columellae / [S10]
At5g24520 / TRANSPARENT TESTA GLABRA 1 (TTG1) / Regulation of mucilage synthesis / Reduced mucilage and columellae / [S7,S10,S15]
At5g35550 / TRANSPARENT TESTA 2 (TT2) / Regulation of mucilage synthesis / No mucilage extrusion (in myb5 tt2 double mutant) / [S7]
At5g40330 / MYB DOMAIN PROTEIN 23 (MYB23) / Regulation of mucilage synthesis. Control of GL2 expression level / Reduced mucilage and columellae / [S15,S19]
At5g42630 / ABERRANT TESTA SHAPE (ATS) / KANADI 4 (KAN4) / Regulation of integument formation during ovule ontogenesis / Slightly reduced dormancy; abnormal testa development; reduced mucilage extrusion / [S20,S21]
At5g65410 / ARABIDOPSIS THALIANA HOMEOBOX PROTEIN 25 (ATHB25) / ZINC FINGER HOMEODOMAIN 2 (ZFHD2) / Regulation of GA biosynthesis / Increased seed longevity in ATHB25 over-expressing lines / [S22]
At5g66730 / ENHYDROUS (ENY) / INDETERMINATE DOMAIN 1 (IDD1) / Regulation of seed germination in response to light and hormones / Reduced mucilage extrusion in ENY over-expressing line / [S23]
2.1. Mucilage synthesis
At1g02720 / GALACTURONOSYLTRANSFERASE-LIKE 5 (GATL5) / Biosynthesis of mucilage pectins / Reduced mucilage extrusion; defective epidermal cell wall adhesion / [S24]
At1g18580 / GALACTURONOSYLTRANSFERASE 11 (GAUT11) / Biosynthesis of mucilage pectins / Reduced mucilage extrusion / [S25]
At1g53500 / MUCILAGE-MODIFIED 4 (MUM4) / RHAMNOSE BIOSYNTHESIS 2 (RHM2) / Biosynthesis of mucilage pectins (RGI) / No mucilage release; Higher seed oil content / [S10,S11, S26-28]
At5g22740 / CELLULOSE SYNTHASE-LIKE A 2 (CSLA2) / Biosynthesis of mucilage glucomannan / More compact adherent mucilage; Altered spatial organization of mucilage pectin and cellulose / [S29]
2.2. Mucilage secretion
At1g09330 / ECHIDNA (ECH) / With YIP, required for the secretion of cell wall polysaccharides (hemicelluloses, pectins) and mucilage / Reduced mucilage extrusion; intracellular accumulation of pectin in seed coat, impaired cell wall polysaccharides trafficking / [S30,S31]
At1g63930 / FROM THE CZECH 'ROH' MEANING 'CORNER' (ROH1) / Regulation of pectin secretion; interacts with EXO70A1 / Reduced mucilage release (in a gain of function mutant) / [S32]
At2g18840 / YPT/RAB GTPASE INTERACTING PROTEIN 4A (YIP4A) / With ECH, required for the secretion of cell wall polysaccharides (hemicelluloses, pectins) and mucilage / Reduced mucilage extrusion; intracellular accumulation of pectin in seed coat (in yip4a yip4b double mutant) / [S30]
At2g35630 / MICROTUBULE ORGANIZATION 1 (MOR1) / Cortical microtubules-mediated vesicle guiding during mucilage production (and cell wall maturation?) / Altered mucilage release (in mutant seeds matured at 29°C) / [S33]
At3g10380 / SUBUNIT OF EXOCYST COMPLEX 8 (SEC8) / Pectin secretion / Reduced mucilage release; lower pectin content; almost no columellae / [S32]
At4g30260 / YPT/RAB GTPASE INTERACTING PROTEIN 4B (YIP4B) / With ECH, required for the secretion of cell wall polysaccharides (hemicelluloses, pectins) and mucilage / Reduced mucilage extrusion; intracellular accumulation of pectin in seed coat (in yip4a yip4b double mutant) / [S30]
At5g03540 / EXOCYST SUBUNIT EXO70 FAMILY PROTEIN A1 (EXO70A1) / Pectin secretion / Reduced mucilage release; lower pectin content; flattened seed epidermal cells / [S32]
2.3. Mucilage modification
At2g47670 / PECTIN METHYLESTERASE INHIBITOR 6 (PMEI6) / Regulation of pectin methylesterase activity on soluble (and adherent) mucilage / Delayed mucilage release; absence of methylesterified homogalacturonans / [S34]
At3g09820 / ADENOSINE KINASE 1 (ADK1) / Involved in pectin methylation / Less methylesterified pectins / [S35]
At4g28370 / transmembrane E3 ubiquitin
ligase FLYING SAUCER 1 (FLY1) / Regulation of degree of methylesterification in pectins / Reduced mucilage extrusion; increased mucilage adherence / [S36,S37]
At5g49360 / BETA-XYLOSIDASE 1 (BXL1) / Modification of mucilage pectic arabinan / Patchy and slow mucilage release; delayed germination / [S38]
At5g63800 / MUCILAGE-MODIFIED 2 (MUM2) / BETA-GALACTOSIDASE 6 (BGAL6) / Maturation of mucilage pectins (RGI) / No mucilage extrusion; slightly irregular columellae / [S10,S37,S39-S41]
2.4. Mucilage stabilization
At2g35620 / LEUCINE-RICH REPEAT RECEPTOR KINASE ( FEI2) / With SOS5, influences the biosynthesis of mucilage cellulose by MUM3/CESA5 / Reduced adherent mucilage layer; altered spatial organization of mucilage pectin and cellulose / [S42]
At3g29810 / COBRA-LIKE 2 (COBL2) / influences the biosynthesis of mucilage cellulose by MUM3/CESA5; also controls cellulose microfibrils production in the radial primary wall and the columella / reduction in the rays of cellulose present in
mucilage; increased solubility of the pectic component of the mucilage / [S43]
At3g46550 / SALT OVERLY SENSITIVE 5 (SOS5) / With FEI2, influences the biosynthesis of mucilage cellulose by MUM3/CESA5 / Reduced adherent mucilage layer; slightly smaller columellae; altered spatial organization of mucilage pectin and cellulose / [S37,S42]
At5g09870 / MUCILAGE-MODIFIED 3 (MUM3) / CELLULOSE SYNTHASE 5 (CESA5) / Biosynthesis of mucilage cellulose; also functions redundantly with CESA2 and CESA9 for primary wall and columella synthesis / Reduced adherent mucilage layer; altered spatial organization of mucilage pectin and cellulose / [S10,S37,S42,S44,S45]
2.5. Mucilage synthesis, secretion or modification
Unknown / MUCILAGE-MODIFIED 5 (MUM5) / De-esterification of mucilage pectins (RGI) / Altered mucilage staining / [S10,S44]
Unknown / MUM ENHANCER 1 (MEN1) / Unknown / Reduced mucilage content (in a mum4 background) / [S46]
Unknown / MUM ENHANCER 2 (MEN2) / Unknown / Reduced mucilage release (in a mum4 background) / [S46]
Unknown / MUM ENHANCER 4 (MEN4) / Unknown / Reduced mucilage content (in a mum4 background) / [S46]
Unknown / MUM ENHANCER 5 (MEN5) / Unknown / Reduced mucilage content (in a mum4 background) / [S46]
Unknown / MUM ENHANCER 6 (MEN6) / Unknown / Reduced mucilage release (in a mum4 background) / [S46]
3. Cell wall synthesis and modification
At2g21770 / CELLULOSE SYNTHASE A9 (CESA9) / Cell wall synthesis / Altered seed coat cell shape; lower cellulose content / [S45,S47]
At3g50990 / CLASS III PEROXIDASE 36 (PRX36) / Regulation of the outer cell wall degradation/loosening during embryogenesis / No mucilage release; normal mucilage composition / [S48]
At4g39350 / CELLULOSE SYNTHASE A2 (CESA2) / Cell wall synthesis / Altered seed coat cell shape; lower cellulose content / [S45]
At5g67360 / SUBTILISIN-LIKE SERINE PROTEASE 1.7 (SBT1.7) / ARABIDOPSIS AG12 (ARA12) / Regulation of cell wall (and mucilage?) pectin methylesterases during seed coat maturation / No mucilage release / [S49]
4. Hormone synthesis and perception
At1g80330 / GIBBERELLIN 3-OXIDASE 4 (GA3OX4) / Regulation of a-amylase activity during seed maturation / Reduced mucilage; abnormal columellae / [S50]
At3g05120 / GA INSENSITIVE DWARF1A (GID1A) / Gibberellin perception / Altered seed coat cell shape (in a gid1a gid1b double mutant) / [S51]
At3g63010 / GA INSENSITIVE DWARF1B (GID1B) / Gibberellin perception / Altered seed coat cell shape (in a gid1a gid1b double mutant) / [S51]
At4g02780 / GA REQUIRING 1 (GA1) / Gibberellin biosynthesis / Altered seed coat cell shape / [S51]
At5g67030 / ABA DEFICIENT 1 (ABA1) / Hormonal control of mucilage biosynthesis / Reduced mucilage release / [S52]
5. Other genes affecting mucilage synthesis or release
At2g27600 / SUPPRESSOR OF K+ TRANSPORT GROWTH DEFECT1 (SKD1) / Unknown / Transparent testa and no mucilage release (in a dominant-negative SKD1 line) / [S53]
At5g15180 / CLASS III PEROXIDASE 56 (PRX56) / ? / Altered mucilage release / [S54]
At5g23940 / DEFECTIVE IN CUTICULAR RIDGES (DCR) / Surface cutin synthesis / No mucilage release / [S55,S56]
At5g63840 / RADIAL SWELLING 3 (RSW3) / Part of the ER-based quality control of mucilage synthesis enzymes? / No mucilage release (at 30°C) / [S57]
Unknown / PRAIRIE (PRA) / Unknown / Reduced mucilage and columellae / [S58]
Name / Annotation / Role in seed physiology / Mutant seed phenotype / Refs
Ecotypes (naturally occurring mutants)
Dja / PECTIN METHYLESTERASE INHIBITOR 6 (PMEI6) / Regulation of pectin methylesterase activity on soluble (and adherent) mucilage / Delayed mucilage release; absence of methylesterified homogalacturonans / [S34]
Sha / MUCILAGE-MODIFIED 2 (MUM2) / BETA-GALACTOSIDASE 6 (BGAL6) / Maturation of mucilage pectins (RGI) / No mucilage extrusion; slightly irregular columellae / [S41,S59]
Had-3 / MUCILAGE-MODIFIED 2 (MUM2) / BETA-GALACTOSIDASE 6 (BGAL6) / Maturation of mucilage pectins (RGI) / No mucilage extrusion; slightly irregular columellae / [S60]
Lom-3 / MUCILAGE-MODIFIED 2 (MUM2) / BETA-GALACTOSIDASE 6 (BGAL6) / Maturation of mucilage pectins (RGI) / No mucilage extrusion; slightly irregular columellae / [S60]
Neo / MUCILAGE-MODIFIED 2 (MUM2) / BETA-GALACTOSIDASE 6 (BGAL6) / Maturation of mucilage pectins (RGI) / No mucilage extrusion; slightly irregular columellae / [S60]
Nfro-1 / MUCILAGE-MODIFIED 2 (MUM2) / BETA-GALACTOSIDASE 6 (BGAL6) / Maturation of mucilage pectins (RGI) / No mucilage extrusion; slightly irregular columellae / [S60]
Sk-1 / CLASS III PEROXIDASE 36 (PRX36) / Regulation of the outer cell wall degradation/loosening during embryogenesis / No mucilage release; normal mucilage composition / [S60]
Sku / MUCILAGE-MODIFIED 2 (MUM2) / BETA-GALACTOSIDASE 6 (BGAL6) / Maturation of mucilage pectins (RGI) / No mucilage extrusion; slightly irregular columellae / [S60]
Sus / MUCILAGE-MODIFIED 2 (MUM2) / BETA-GALACTOSIDASE 6 (BGAL6) / Maturation of mucilage pectins (RGI) / No mucilage extrusion; slightly irregular columellae / [S60]

a60 genes/proteins with mucilage phenotype in the corresponding mutants were retrieved from exhaustive literature search and classified among 5 categories. 54 of the genes/proteins have an attributed Arabidopsis Genome Initiative (AGI) number and six have not yet been identified. Genes with underlined and double underlined AGI numbers are part of two potential chromosome clusters, respectively. Ecotypes behaving as naturally occurring mutants with mucilage release phenotype are also listed and the corresponding identified causal mutated genes are indicated.

Supplementary references

S1 Penfield, S., et al. (2001) MYB61 is required for mucilage deposition and extrusion in the Arabidopsis seed coat. Plant Cell 13, 2777-2791

S2 Newman, L.J., et al. (2004) Involvement of the R2R3-MYB, AtMYB61, in the ectopic lignification and dark-photomorphogenic components of the det3 mutant phenotype. Plant J 37, 239-250

S3 Romano, J.M., et al. (2012) AtMYB61, an R2R3-MYB transcription factor, functions as a pleiotropic regulator via a small gene network. New Phytol 195, 774-786

S4 Kunieda, T., et al. (2008) NAC Family Proteins NARS1/NAC2 and NARS2/NAM in the Outer Integument Regulate Embryogenesis in Arabidopsis. Plant Cell 20, 2631-2642

S5 Bhargava, A., et al. (2013) The interacting MYB75 and KNAT7 transcription factors modulate secondary cell wall deposition both in stems and seed coat in Arabidopsis. Planta 237, 1199-1211

S6 Zhang, F., et al. (2003) A network of redundant bHLH proteins functions in all TTG1-dependent pathways of Arabidopsis. Development 130, 4859-4869

S7 Gonzalez, A., et al. (2009) TTG1 complex MYBs, MYB5 and TT2, control outer seed coat differentiation. Dev Biol 325, 412-421

S8 Kaplan-Levy, R.N., et al. (2012) The trihelix family of transcription factors--light, stress and development. Trends Plant Sci 17, 163-171

S9 Vasilevski, A., et al. (2012) LASSO modeling of the Arabidopsis thaliana seed/seedling transcriptome: a model case for detection of novel mucilage and pectin metabolism genes. Mol Biosyst 8, 2566-2574

S10 Western, T.L., et al. (2001) Isolation and characterization of mutants defective in seed coat mucilage secretory cell development in arabidopsis. Plant Physiology 127, 998-1011

S11 Shi, L., et al. (2012) Arabidopsis glabra2 mutant seeds deficient in mucilage biosynthesis produce more oil. Plant Journal 69, 37-46

S12 Huang, J., et al. (2011) The Arabidopsis Transcription Factor LUH/MUM1 Is Required for Extrusion of Seed Coat Mucilage. Plant Physiology 156, 491-502

S13 Walker, M., et al. (2011) The Transcriptional Regulator LEUNIG_HOMOLOG Regulates Mucilage Release from the Arabidopsis Testa. Plant Physiology 156, 46-60

S14 Johnson, C.S., et al. (2002) TRANSPARENT TESTA GLABRA2, a trichome and seed coat development gene of Arabidopsis, encodes a WRKY transcription factor. Plant Cell 14, 1359-1375