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First published online December 30, 2003; 10.1104/pp.103.035519 Plant Physiology 134:296-306 (2004) © 2004 American Society of Plant Biologists MUCILAGE-MODIFIED4 Encodes a Putative Pectin Biosynthetic Enzyme Developmentally Regulated by APETALA2, TRANSPARENT TESTA GLABRA1, and GLABRA2 in the Arabidopsis Seed Coat1Botany Department, University of British Columbia, 6270 University Boulevard, Vancouver, British Columbia, Canada V6T 1Z4
The Arabidopsis seed coat epidermis undergoes a complex process of differentiation that includes the biosynthesis and secretion of large quantities of pectinaceous mucilage, cytoplasmic rearrangement, and secondary cell wall biosynthesis. Mutations in MUM4 (MUCILAGE-MODIFIED4) lead to a decrease in seed coat mucilage and incomplete cytoplasmic rearrangement. We show that MUM4 encodes a putative NDP-L-rhamnose synthase, an enzyme required for the synthesis of the pectin rhamnogalacturonan I, the major component of Arabidopsis mucilage. This result suggests that the synthesis of monosaccharide substrates is a limiting factor in the biosynthesis of pectinaceous seed coat mucilage. In addition, the reduced cytoplasmic rearrangement observed in the absence of a key enzyme in pectin biosynthesis in mum4 mutants establishes a causal link between mucilage production and cellular morphogenesis. The cellular phenotype seen in mum4 mutants is similar to that of several transcription factors (AP2 [APETALA2], TTG1 [TRANSPARENT TESTA GLABRA1], TTG2 MYB61, and GL2 [GLABRA2]). Expression studies suggest that MUM4 is developmentally regulated in the seed coat by AP2, TTG1, and GL2, whereas TTG2 and MYB61 appear to be regulating mucilage production through alternate pathway(s). Our results provide a framework for the regulation of mucilage production and secretory cell differentiation.
The mature seed coat, in addition to forming a protective layer around the embryo, can play roles in such processes as seed dispersal and germination. One adaptation believed to act in these processes is the production of mucilage in the epidermal cells of the seed coat. This specialization, known as myxospermy, is found in many families, including the Brassicaceae, Solanaceae, Linaceae, and Plantaginaceae (Grubert, 1981  ; Boesewinkel and Bouman, 1995). Seed imbibition in these plants leads to the formation of a gel capsule around the seed that is thought to aid hydration and/or dispersal. Mucilage consists of pectins, a heterogeneous group of acidic polysaccharides that form a gel-like matrix. In the primary cell wall, pectins form the matrix in which cellulose microfibrils and hemicelluloses are embedded. Key pectins include homo-GalUA (HGA) and rhamnogalacturonan I (RGI). HGA consists of an unbranched chain of  -1,4-linked GalUA residues whose carboxyl groups may be modified through methyl-esterification. RGI has a backbone of alternating -1,2-linked Rha and -1,4-linked GalUA residues, with sugar side chains attached on varying numbers of Rha residues. Both HGA and RGI are believed to be synthesized in the Golgi apparatus through the activities of glycosyltransferases using nucleotide sugars imported from the cytoplasm (for review, see Ridley et al., 2001). Chemical analysis of Arabidopsis mucilage has shown that it is composed largely of -1,2 linked Rha and -1,4 linked GalUA, suggesting that it is comprised primarily of RGI (Penfield et al., 2001). Immunofluorescence studies of mucilage have demonstrated the presence of methylesterified HGA (Willats et al., 2001); however, the authors did not test for the presence of RGI.
The production of mucilage in the epidermal cells of the Arabidopsis seed coat is part of a coordinated developmental process that begins with cell growth, followed by biosynthesis and polar secretion of large quantities of pectin, formation of a cytoplasmic column through cytoplasmic constriction and vacuolar contraction, and, finally, the synthesis of a secondary cell wall (columella; Beeckman et al., 2000
Several genes encoding putative transcription factors have been implicated in seed coat epidermal development because mutations in these genes result in seeds that fail to release mucilage upon hydration. Mutants in AP2 (APETALA2), in addition to their defects in floral morphogenesis, lack differentiation past the growth phase of mucilage secretory cells (Jofuku et al., 1994 The objective of this study was to characterize the role of MUM4 in the development of the Arabidopsis seed coat. Using positional cloning of MUM4, we demonstrate that MUM4 encodes a putative NDP-L-Rha synthase. Mutations in this gene lead to reduced mucilage in the seed coat and an altered columella. Expression studies show that MUM4 is developmentally regulated during seed coat differentiation such that its transcript levels are increased at the time of mucilage production. Furthermore, MUM4 appears to be a downstream target of a cascade of transcription factors that includes AP2, TTG1, and GL2. These results demonstrate the importance of mucilage production for the morphology of seed coat epidermal cells and suggest a regulatory framework for the control of seed coat epidermal differentiation.
mum4 Mutant Phenotype
Two alleles of mum4 (mum4-1 and mum4-2) were identified in a screen for mutants that lacked extruded mucilage as visualized by staining with Ruthenium red (compare Fig. 1, A with D; Western et al., 2001
Viewed with scanning electron microscopy, mature mum4 seed coats are marked not only by the absence of mucilage extrusion upon hydration but also by the absence of volcano-shaped columellae (compare Fig. 1, B with E). Light microscopy of sections of mature seeds revealed that both columellae and mucilage are present in mum4 but reduced in comparison with wild type (compare Fig. 1, C with F). No other phenotypes were observed.
To more fully characterize the mum4 seed coat epidermal cell defects, their development was studied with light microscopy (Fig. 1, G-L). Under our growth conditions, seed maturation to dry seed takes approximately 18 d and has been staged by DPA. In terms of embryo growth, 4 DPA is early to mid globular stage, 7 DPA is mid-torpedo stage, whereas 10 DPA is late upturned-U stage (this study; Western et al., 2000
The possibility of a change in mucilage quantity or composition in mum4 versus wild-type seed coats was addressed by quantifying monosaccharides using gas chromatography. Because mum4 seed coats release mucilage in the presence of ammonium oxalate, mucilage could be extracted from intact mum4 seeds for direct comparison with wild-type mucilage. Trimethylsilane (TMS) derivatives were used because this method allows detection of GalUA and neutral sugars simultaneously. The results showed that, for equal seed masses, less sugar was extracted from mum4 mucilage, suggesting that less mucilage is synthesized compared with wild-type seeds (Fig. 2). In addition, the decrease in monosaccharides in mum4 mucilage was limited primarily to two sugars, GalUA and a single neutral sugar (identified as either Rha or Fuc; Fig. 2; H0 µ1 = µ2, GalUA, T = 7.897, P < 0.01; Rha, T = 6.960, P = 0.01). Because the identity of the neutral sugar could not be completely resolved using TMS derivatives, alditol acetate derivatization, which gives a single derivative for each monosaccharide, was used to confirm that it is Rha (Fuc found only at trace levels in wild-type mucilage; data not shown). These results are consistent with the suggestion that Arabidopsis mucilage is comprised primarily of relatively unbranched RGI (Penfield et al., 2001
To further investigate the effect of mum4 mutations on mucilage production, immunofluorescence with antipectin antibodies was used. Whole-mount immunofluorescence performed on wild-type Arabidopsis seeds using the polyclonal antipectin antibody
To examine the mucilage in situ, samples were cryo-fixed/freeze substituted, resin embedded, sectioned, and then labeled by indirect immunofluorescence. Labeling was seen on thick sections of developing wild-type seed coats stained with
To identify why mum4 seeds had reduced mucilage, we cloned MUM4 by map-based cloning. MUM4 was mapped to a 144-kb region spanning the bacterial artificial chromosomes F12M16 and T3F20. To identify the exact locus, the mum4-3 T-DNA insertion allele was probed with sequences derived from these bacterial artificial chromosomes, and an insertion was identified in a region of T3F20 containing four predicted genes. PCR performed using gene-specific primers in combination with a T-DNA right border primer led to the identification of a T-DNA insertion in the predicted gene At1g53500. Next, we isolated a putative full-length cDNA by reverse transcriptase (RT)-PCR using a series of primers upstream from the predicted translation start site. Sequencing of a putative full-length At1g53500 transcript revealed that the gene consists of three exons and two introns (including one in the 5'-untranslated region [UTR]) and encodes a transcript of 2,477 bp (Fig. 4A).
To confirm that At1g53500 is MUM4, we sequenced this locus in mum4-1, mum4-2, and mum4-3. All three alleles contain mutations in the second exon. mum4-1 and mum4-2 contain point mutations leading to G to A transitions at nucleotides 595 and 886, respectively (Fig. 4A). These missense mutations should result in a conserved Asp residue being replaced with an Asn in mum4-1 and a Gly being replaced with an Arg in mum4-2 (Fig. 5). mum4-3 contains a T-DNA insertion at nucleotide 1,637 (Fig. 4A). The similar phenotype between the missense alleles and that of the mum4-3 insertional allele that lacks detectable transcript (data not shown) suggests that these changes have a significant effect on protein activity. A database search of the Salk Institute sequence-indexed Arabidopsis T-DNA insertion lines identified two new alleles of mum4, mum4-4, and mum4-5 (Salk_085051 and 038898, respectively), both of which also have insertions in the second exon (Fig. 4A). Based on the Ruthenium red assay, both of these mutants have a phenotype (data not shown) similar to mum4-1 (Fig. 1D). The wild-type At1g53500 sequence plus 2.1 kb upstream and 0.4 kb downstream sequence was cloned to give the plasmid pMUM4g. Transformation of mum4-1 plants with pMUM4g versus an empty vector control showed that the wild-type At1g53500 genomic clone could rescue the mucilage phenotype (Fig. 4, B and C). Together, these results demonstrate that At1g53500 is MUM4.
MUM4 encodes a protein of 667 amino acids (Fig. 5). Comparison with GenBank sequences suggested that MUM4 contains two domains: an N-terminal domain with similarity to bacterial dTDP-D-Glc-4,6-dehydratases (44% identical, 61% similar to RfbB [COG1088] consensus) and a C-terminal domain with some similarity to bacterial 4-reductases (20% identical, 34% similar to RfbD [COG1091] consensus) (Tatusov et al., 2000
A comparison with other Arabidopsis genes predicted from genome annotation revealed that MUM4 is a member of a small gene family of putative nucleotide sugar interconversion factors that was initially described in an in silico analysis by Reiter and Vanzin (2001
The expression pattern for MUM4 mRNA in different tissues was determined qualitatively using RT-PCR at a saturating cycle number. As suggested by expressed sequence tag data, MUM4 transcripts are apparent in all tissues tested: stems, roots, leaves, seedlings, inflorescence tips, and siliques (Fig. 6A). A similar analysis was done for RHM1 and RHM3. As with MUM4, both RHM1 and RHM3 transcripts were detected in all tissues (Fig. 6A). RHM3, however, unlike MUM4 and RHM1, appeared to have only low expression in some tissues (leaves and inflorescences) because bands for these tissues were not saturated in several trials, even at the high cycle number used for qualitative RT-PCR.
Because the only obvious phenotype in mum4 plants is the defect in the seed coat epidermis, we looked at the expression of MUM4 during seed coat differentiation (Fig. 6B). RNA was extracted from siliques at three stages of development: (a) before mucilage synthesis (4 DPA; see Fig. 1G), (b) midmucilage synthesis (7 DPA; Fig. 1H), and (c) after the completion of mucilage synthesis (10 DPA; Fig. 1I), and subjected to northern analysis. This experiment showed that although MUM4 is expressed throughout seed coat differentiation, transcript levels are highest at the time of mucilage synthesis (7 DPA; Fig. 6B). To test if the higher levels of MUM4 detected at 7 DPA may be largely restricted to the seed coat, ap2-7 mutants were used as a control in gel blots (Fig. 6B). ap2 was chosen because the outer two layers of the seed coat of ap2 mutant seeds fail to differentiate, whereas all other cell types of the seed (embryo, endosperm, and seed coat endothelium) appear normal (Western et al., 2001
Mutations in AP2, TTG1, TTG2, GL2, and MYB61, which encode putative transcription factors, lead to seeds with reduced mucilage in the seed coat. Furthermore, developmental analyses have shown that the cellular phenotypes for ttg1, gl2, and myb61 mucilage secretory cells are almost identical to those of mum4 mutants, whereas the ap2 mutant cellular phenotype is more severe (Penfield et al., 2001
The ap2 mutant phenotype differs from that of ttg1, ttg2, gl2, and myb61 in that the differentiation of the mucilage secretory cells is halted before mucilage and columella production in ap2 whereas the others resemble mum4, having low levels of mucilage and flattened columellae. Thus, phenotypically, AP2 appears to be working upstream of the other genes. To determine whether AP2 acts by controlling the transcription of the other genes, semiquantitative RT-PCR was used to determine the levels of TTG1, TTG2, GL2, and MYB61 in ap2-7 mutants versus wild type at 4 and 7 DPA. The results showed a significant decrease in transcript levels for GL2 at both 4 and 7 DPA (Fig. 7B) and TTG2 at 4 DPA (TTG2 levels at 7 DPA too low to differentiate between wild type and ap2; Fig. 7B). This result suggests that AP2 is required for maximal levels of both GL2 and TTG2 expression during seed coat differentiation.
TTG1 has been shown to be required for maximal expression of both TTG2 and GL2 in leaves (Johnson et al., 2002
The development of the mucilage secretory cells of the Arabidopsis seed coat is a complex process involving the biosynthesis and secretion of a large quantity of pectinaceous mucilage, intracellular cytoplasmic rearrangement, and secondary cell wall production. We have shown that the putative NDP-L-Rha synthase encoded by MUM4 is required for wild-type levels of mucilage biosynthesis and columella formation. Expression of MUM4 is up-regulated during seed coat differentiation and is regulated by several transcription factors required for seed coat epidermal cell differentiation. These data validate the utility of the mucilage secretory cells as a model system for polysaccharide biosynthesis, establish a regulatory framework for seed coat epidermal differentiation, and provide a causal link between pectin biosynthesis and cell morphogenesis.
Our data support the hypothesis that MUM4 encodes an enzyme involved in RGI biosynthesis. First, analysis of mum4 mucilage using antibodies and gas chromatography have demonstrated a significant reduction in RGI and its composite backbone monosaccharides Rha and GalUA in comparison with wild-type seeds. Second, cloning of MUM4 revealed a putative protein containing similarity to bacterial nucleotide sugar interconversion enzymes, suggesting that it, too, is required for the production of activated sugars. The N-terminal portion of MUM4 is most similar to bacterial dTDP-D-Glc 4,6-dehydratases, the first of three enzymes required for the conversion of dTDP-D-Glc to dTDP-L-Rha (Tonetti et al., 1998
Gram-negative bacteria such as Escherichia coli encode three separate enzymes (4,6-dehydratase, 3,5-epimerase, and 4-reductase) to convert dTDP-D-Glc to dTDP-L-Rha (Tonetti et al., 1998
MUM4 is a member of a small gene family consisting of three genes (RHM1, MUM4/RHM2, and RHM3; Reiter and Vanzin, 2001
MUM4 transcript increases in differentiating siliques at the time of mucilage production. Two lines of evidence suggest that this up-regulation occurs in the seed coat epidermis to support mucilage biosynthesis. First, the only obvious phenotypic defect in mum4 plants occurs in the seed coat epidermis. Second, MUM4 expression is severely attenuated in siliques of ap2 mutants that fail to differentiate the outer two layers of the seed coat. Such a specific up-regulation of a putative NDP-L-Rha synthase may be required to provide extra Rha for the production of the large quantity of RGI required for mucilage synthesis. If so, the amount of this enzyme must be the limiting factor in Rha biosynthesis and the amount of Rha a limiting factor in RGI biosynthesis.
Our data indicate that MUM4 transcription is decreased in ttg1 and gl2 mutants but is essentially wild type in ttg2 and myb61 mutants (Fig. 7A). This result suggests a regulatory framework for mucilage secretory cell differentiation in which there are at least two pathways controlling mucilage biosynthesis in the seed coat. In one of these pathways, it appears that TTG1 and GL2 control the transcription of MUM4 (Fig. 8). In other epidermal systems, such as trichomes and root hairs, TTG1 has been shown to interact with a tissue-specific MYB protein through the bHLH protein GLABRA3 (Payne et al., 2000
Correct regulation of MUM4 transcription at the time of mucilage production does not require TTG2 or MYB61 (Fig. 7A). However, TTG1 is required to activate TTG2 in the seed coat (Fig. 7C) and in other tissues (Johnson et al., 2002
AP2 encodes a putative transcription factor that is required for the differentiation of the outer two layers of the seed coat (Western et al., 2001
The mum4 phenotype is characterized by both a reduction in the amount of mucilage produced and by a flattened columella in the seed coat epidermis. The flattened appearance of the columella in mum4 seeds appears to be the result of incomplete cytoplasmic rearrangement and vacuole constriction, leaving a dome of cytoplasm over which the secondary cell wall is laid. Although the defect in mucilage production is readily explained by the putative function of MUM4 as an NDP-L-Rha synthase, the connection with the columella shape change is less obvious. At least two hypotheses can explain the dependence of cell morphogenesis on mucilage synthesis. The formation of the apoplastic space and cytoplasmic column may be driven by the pressure of accumulating mucilage in the extracytoplasmic space and/or the formation of an osmotic gradient between the cell and the hydrophilic pectin may lead to the loss of turgor and size reduction of the vacuole. Such passive processes may contribute to cell morphogenesis but seem too simple to account for the apparent complexity of the shape of the cytoplasmic column. An alternate but not mutually exclusive explanation is that the synthesis of mucilage may represent or lead to the production of an oligosaccharide signal that stimulates the cell to undergo morphogenesis (Dumville and Fry, 2000
Plant Material and Growth Conditions
Lines of Arabidopsis used were mum4-1, mum4-2 (Col-2 ecotype; Western et al., 2001
mum4-3 was isolated by screening T-DNA mutagenized lines (Feldmann and Marks, 1987
Flowers were staged as by Western et al. (2001
Developing seeds for bright-field microscopy were prepared as described by Western et al. (2000 For immunofluorescence, developing seeds were placed in copper hats in a 0.2 M Suc solution and high-pressure frozen using a Balzers HPM 101 (Balzers Instruments, Balzers, Liechtenstein). The samples were freeze substituted over 5 d in 0.25% (v/v) glutaraldehyde/8% (v/v) dimethoxypropane in acetone using a dry ice/acetone bath at -80°C, then gradually warmed to -20°C, 4°C, and then 20°C (3 h at each temperature) before embedding in LR White resin. Sectioning and microscopy were performed as for bright field.
Immersion immunofluorescence was performed according to the procedure of Willats et al. (2001 For immunofluorescence, 0.5-µm sections were incubated in 50 mM EGTA for 1 h, blocked with 5% (w/v) NFDM for 20 min, then incubated for 1 h with primary antibodies at 1:5 and 1:10 (v/v) dilutions in 5% (w/v) NFDM, and for 1 h in secondary antibodies diluted at 1:100 (v/v). Rinses were performed before and after each incubation with 1x Tris-buffered saline/0.1% (v/v) Tween 20. Primary antibodies, secondary antibodies, pre-immune controls, and mounting were done as described for immersion immunofluorescence.
Dry-mounted seeds were prepared and observed as by Western et al. (2001
Mucilage was extracted from samples of approximately 5 mg of intact seeds by shaking in 0.2% (w/v) ammonium oxalate for 2 h at 30°C. No significant difference in mass was observed between Col-2 and mum4-1 seed (four lots of 100 counted seed weighed in triplicate; Col-2 = 2.0 ± 0.3 mg, mum4-1 = 2.2 ± 0.1 mg SD). Ten microliters of internal standard (5 mg mL-1 myo-inositol) was added before precipitation with 5 volumes of absolute ethanol and drying under nitrogen gas. Derivatization, gas chromatography, and identification of trimethylsilyl ethers were performed as described previously (Western et al., 2000 For alditol acetates, samples were hydrolyzed in trifluoroacetic acid for 2 h at 100°C, dried under nitrogen gas, then reduced for 90 min at 40°C in 245 µL of 2.6 M ammonia and 700 µL of 2% (w/v) sodium borohydride in dimethylsulfoxide. After addition of 175 µL of acetic acid, the samples were acetylated for 10 min at room temperature with 175 µL of 1-methylimidazole and 2.8 mL of acetic anhydride. Water (5.6 mL) was added, and the alditol acetates were extracted with 1.05 mL of dichloromethane (DCM). The recovered phase was dried and resuspended in 200 µL of DCM, then re-extracted with 1 mL of water. This recovered DCM phase was used for analysis. Samples were run on a gas chromatograph (model 5890A, Hewlett-Packard, Mississauga, ON, Canada) on a HP-23 glass capillary column (30-m x 0.25-mm i.d.) with helium as the carrier gas. The temperature program was 2 min at 180°C, increase 10°C min-1 up to 200°C, 5 min at 200°C, increase 10°C min-1 to 250°C, and hold at 250°C for 10 min. Monosaccharides were identified through comparison with the retention times obtained with individual sugar standards and a composite standard consisting of Rib, Fuc, Man, Gal, Glc, Ara, Rha, Xyl, GlcUA, and GalUA.
A mapping population of 519 plants derived from a cross between mum4-1 and wild-type Ler was used for progressive fine mapping using simple sequence length polymorphism and cleaved-amplified polymorphic sequence markers generated from sequence information provided by the Arabidopsis Genome Initiative (2000
To confirm that the gene was MUM4, mum4-1 and mum4-2 were sequenced. Sequencing of the Salk Institute sequence-indexed Arabidopsis T-DNA insertion lines 085051 and 038898 revealed two further alleles, named mum4-4 and mum4-5, respectively. Molecular complementation of MUM4 was carried out using a 5.2-kb SalI/BstZ17I fragment of T3F20, including At1g53500 plus 2.1 kb upstream and 0.4 kb downstream sequence, into pGREEN0229 (Hellens et al., 2000
The 5'-UTR of MUM4 was located using RT-PCR with primers located up to 900 bp upstream from the predicted ATG in combination with an internal primer. RNA was isolated from developing siliques according to the protocol of Downing et al. (1992
RNA was isolated as described above. For RNA gel blots, 10 µg of total RNA was separated on formaldehyde gels, blotted to Hybond-XL nylon membranes (Amersham Biosciences Corp., Baie d'Urfé, QC, Canada), and hybridized according to the manufacturer's instructions. A 32P-labeled MUM4 probe corresponding to the degenerate region between the two predicted enzymatic domains was synthesized using PCR (MUM4p11 5'-ggaagactttctgatggatctagt-3'/MUM4p12 5'-gatcaaaaacttcaacgaagc-3'). 25S rRNA bands from the ethidium bromide-stained RNA gel were used as loading controls. RNA isolation and RT of DNase-treated RNA was performed as described above. Gene-specific primers surrounding an intron were designed for each gene, with the exception of TTG1, which lacks an intron. Primers were as follows: MUM4, p1-ttcgtgtaaatgtcgcaggt/p2 5'-gagaagctcgtctagtacggtc-3'; RHM1, p1 5'-gagactatccgtgccaatgta-3'/p2 5'-taataactcgtccaacacagtc-3'; RHM3, p1 5'-ccgagtcaacgttgctgga-3'/p2 5'-ggtaagagttcatctagtatggtc-3'; GAPC, p1 5'-tcagactcgagaaagctgctac-3'/p2 5'-gatcaagtcgaccacacgg-3'; MYB61, p3 5'-tggggagacattcttgctg-3'/p4 5'-gatgggcttgtgtgtgtttg-3'; GL2, p1 5'-aacggtcactccaaggtcac-3'/p2 5'-agaaacccgcatgtcttgtc-3'; TTG2, p1 5'-gccatcttgtcctcttccac-3'/p2 5'-ccttgcgatactcctgcttc-3'; AP2, p1 5'-cagggaatcctactactccacaag-3'/p2 5'-atctgattgtgatgatgaggagag-3'; and TTG1, p1 5'-catcctccggtcacagaatc-3'/p2 5'-tttcggctctacatcgttcc-3'. Loading for all RT-PCRs was determined by standardizing against GAPC test PCRs (at 22-23 cycles) for each RT reaction. Saturating, qualitative RT-PCR for MUM4, RHM1, and RHM3 was done with 30 cycles. Semiquantitative RT-PCR (for AP2, TTG1, TTG2, GL2, and MYB61) was performed a minimum of four times for each set of primers using two to three different cycle numbers (from 25-30 cycles) to confirm results.
The GenBank accession numbers for genes mentioned in this article are AY328518 (MUM4 full-length cDNA), AY042833 (RHM1), and AY078958 (RHM3). mum4-3 has been submitted to ABRC under the seed stock number CS6382.
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We thank Ms. Yeen Ting Hwang (University of British Columbia, Vancouver, BC Canada) and Mr. Adrian Wladichuk (University of British Columbia, Vancouver, BC Canada) for technical assistance, Dr. Michael Bevan (John Innes Centre, Norwich, UK) for supplying myb61-1 seed, Dr. Mark Smith (University of British Columbia, Vancouver, BC Canada) and Ms. Michelle Fawcett (Carnegie Institution, Stanford University, CA) for help with chemical analysis of mucilage, Drs. Andrew Staehelin (University of Colorado, Boulder) and Paul Knox (University of Leeds, Leeds, UK) for gifts of antibodies, and the Salk Institute Genomic Analysis Laboratory (La Jolla, CA, USA) for providing the sequence-indexed Arabidopsis T-DNA insertion mutants. We also thank Drs. Markus Pauly (Max-Planck Institute for Molecular Plant Physiology, Golm, Germany) and Björn Usadel (Max-Planck Institute for Molecular Plant Physiology, Glom, Germany) for communicating unpublished data and delaying publication of their results on RHM2. We thank members of the Ljerka Haughn (University of British Columbia, Vancouver, BC Canada) and George Kunst (University of British Columbia, Vancouver, BC Canada) labs for comments on the manuscript. Received October 30, 2003; returned for revision November 12, 2003; accepted November 20, 2003.
1 This work was supported by the Natural Science and Engineering Research Council of Canada (Discovery Grants to G.W.H. and A.L.S. and a Cell Wall Multi-Network Grant to G.W.H.). 
2 Present address: Biology Department, McGill University, 1205 Docteur Penfield Avenue, Montreal, QC, Canada H3A 1B1.
3 Present address: Ottawa Regional Cancer Centre, Centre for Cancer Therapeutics, Third Floor, 503 Smyth Road, Ottawa, ON, Canada K1H 1C4. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.035519. * Corresponding author; email haughn{at}interchange.ubc.ca; fax 604-822-6089.
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ABSTRACT
RESULTS
, Location of the three T-DNA insertion alleles (mum4-3, mum4-4, and mum4-5). B, Complementation of mum4-1 with pMUM4g. Mucilage capsule is observed around seed stained with Ruthenium red. C, mum4-1 seed transformed with empty vector pGREEN0229. No mucilage release is observed. Scale bars = 100 µm in B and C.
