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MUM ENHANCERS are important for seed coat mucilage production and mucilage secretory cell differentiation in Arabidopsis thaliana.

Arsovski AA, Villota MM, Rowland O, Subramaniam R, Western TL - J. Exp. Bot. (2009)

Bottom Line: A number of genes have been identified affecting mucilage secretory cell differentiation, including MUCILAGE-MODIFIED4 (MUM4). mum4 mutants produce a reduced amount of mucilage and cloning of MUM4 revealed that it encodes a UDP-L-rhamnose synthase that is developmentally up-regulated to provide rhamnose for mucilage pectin synthesis.Eight mum enhancers (men) have been identified, two of which result from defects in known mucilage secretory cell genes (MUM2 and MYB61).Conversely, mutations in MEN2 and MEN6 appear to affect mucilage release rather than quantity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, McGill University, Montreal, QC, Canada H3A 1B1.

ABSTRACT
Pollination triggers not only embryo development but also the differentiation of the ovule integuments to form a specialized seed coat. The mucilage secretory cells of the Arabidopsis thaliana seed coat undergo a complex differentiation process in which cell growth is followed by the synthesis and secretion of pectinaceous mucilage. A number of genes have been identified affecting mucilage secretory cell differentiation, including MUCILAGE-MODIFIED4 (MUM4). mum4 mutants produce a reduced amount of mucilage and cloning of MUM4 revealed that it encodes a UDP-L-rhamnose synthase that is developmentally up-regulated to provide rhamnose for mucilage pectin synthesis. To identify additional genes acting in mucilage synthesis and secretion, a screen for enhancers of the mum4 phenotype was performed. Eight mum enhancers (men) have been identified, two of which result from defects in known mucilage secretory cell genes (MUM2 and MYB61). Our results show that, in a mum4 background, mutations in MEN1, MEN4, and MEN5 lead to further reductions in mucilage compared to mum4 single mutants, suggesting that they are involved in mucilage synthesis or secretion. Conversely, mutations in MEN2 and MEN6 appear to affect mucilage release rather than quantity. With the exception of men4, whose single mutant exhibits reduced mucilage, none of these genes have a single mutant phenotype, suggesting that they would not have been identified outside the compromised mum4 background.

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Mucilage release of men4-1 versus wild-type seeds in ruthenium red with and without shaking. (A, B) Col-2 wild-type seeds. (A) Seeds put directly into ruthenium red without shaking are surrounded by an outer, diffuse layer of mucilage and an inner, dense layer of mucilage, while those shaken in dye lose the soluble outer layer (B). (C, D) men4-1 seeds lack mucilage release when treated directly with ruthenium red, with or without agitation. Scale bar: 500 μm. (This figure is available in colour at JXB online.)
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fig3: Mucilage release of men4-1 versus wild-type seeds in ruthenium red with and without shaking. (A, B) Col-2 wild-type seeds. (A) Seeds put directly into ruthenium red without shaking are surrounded by an outer, diffuse layer of mucilage and an inner, dense layer of mucilage, while those shaken in dye lose the soluble outer layer (B). (C, D) men4-1 seeds lack mucilage release when treated directly with ruthenium red, with or without agitation. Scale bar: 500 μm. (This figure is available in colour at JXB online.)

Mentions: Backcrosses to mum4-1 plants revealed, in each case, that the seed phenotype was the result of a recessive mutation to a single locus (Table 1). Complementation tests were also performed between the nine men mum4-1 lines. Only one pair of mutants did not complement each other, revealing the identification of eight mutant loci. To determine if any of the men mum4-1 lines represented known mucilage mutant loci beyond MUM4, several assays were performed. First, no changes in seed shape were observed as in ap2 and ats mutants (Léon-Kloosterziel et al., 1994). Second, their identity as new alleles of GL2, TTG1, and TTG2 was tested through an examination of seed coat colour and trichome presence (Koornneef, 1981; Rerie et al., 1994; Johnson et al., 2002). All men mum4-1 lines had trichomes, and none had obviously yellow seeds, suggesting that they are different genes. Third, to eliminate tt mutants that were not obviously yellow, tetrazolium red staining was used to detect the increased permeability to solutes found for most tt mutants, including ttg1, tt2, and tt8 that are known to affect mucilage production (Debeaujon et al., 2000). One line that appeared to be wild-type seed colour (named men3-1 mum4-1) showed significant staining with tetrazolium red (data not shown). However, closer examination of men3-1 mum4-1 seeds revealed that they were slightly paler than wild-type seeds. The seed colour phenotype was found to segregate away from the mucilage phenotype, suggesting a background mutation in a tt or related gene that is unlikely to significantly affect mucilage release (data not shown). Fourth, complementation tests were performed with myb61, another reduced mucilage mutant (Penfield et al., 2001). One line was found not to complement myb61-1 and sequencing confirmed that it is a new allele of MYB61, which has been named myb61-6 (G to A transition leading to the conversion of Trp at position 252 to a stop codon). Fifth, the remaining men mum4-1 lines were backcrossed to wild-type Columbia-2 (Col-2) plants to determine if there was a mutant phenotype in the absence of mum4-1, as all mucilage mutants identified to date other than egl3, tt2, and tt8 have detectable mucilage release phenotypes in a wild-type background. Only two mutant lines had detectable single mutant phenotypes where no mucilage was released when shaken in ruthenium red stain without EDTA pretreatment (Fig. 3; data not shown). Since they were already shown not to be allelic to known reduced mucilage mutants, both of these lines were backcrossed to the mucilage release mutants mum1-1, mum2-1, and patchy (Western et al., 2001; AA Arsovski, TL Western, unpublished results). One line was found to complement all three mutants, while the other only complemented mum1-1 and patchy. While sequencing did not reveal an obvious mutation in the coding sequence of MUM2 for the non-complementing line, regulatory region or intron-related mutations cannot be ruled out. This line did, however, map to the MUM2 region, which, in combination with multiple complementation tests using both the double and isolated single mutants, and similar results between the single mutant and mum2 in all further assays (data not shown), suggest that this is, indeed, a new allele of MUM2 that has been named mum2-13. The other line represents a different gene that has been named MEN4. EDTA pretreatment of men4 single mutants revealed the release of a reduced amount of mucilage compared to wild-type seeds (compare Fig. 1A with Fig. 2A).


MUM ENHANCERS are important for seed coat mucilage production and mucilage secretory cell differentiation in Arabidopsis thaliana.

Arsovski AA, Villota MM, Rowland O, Subramaniam R, Western TL - J. Exp. Bot. (2009)

Mucilage release of men4-1 versus wild-type seeds in ruthenium red with and without shaking. (A, B) Col-2 wild-type seeds. (A) Seeds put directly into ruthenium red without shaking are surrounded by an outer, diffuse layer of mucilage and an inner, dense layer of mucilage, while those shaken in dye lose the soluble outer layer (B). (C, D) men4-1 seeds lack mucilage release when treated directly with ruthenium red, with or without agitation. Scale bar: 500 μm. (This figure is available in colour at JXB online.)
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2692007&req=5

fig3: Mucilage release of men4-1 versus wild-type seeds in ruthenium red with and without shaking. (A, B) Col-2 wild-type seeds. (A) Seeds put directly into ruthenium red without shaking are surrounded by an outer, diffuse layer of mucilage and an inner, dense layer of mucilage, while those shaken in dye lose the soluble outer layer (B). (C, D) men4-1 seeds lack mucilage release when treated directly with ruthenium red, with or without agitation. Scale bar: 500 μm. (This figure is available in colour at JXB online.)
Mentions: Backcrosses to mum4-1 plants revealed, in each case, that the seed phenotype was the result of a recessive mutation to a single locus (Table 1). Complementation tests were also performed between the nine men mum4-1 lines. Only one pair of mutants did not complement each other, revealing the identification of eight mutant loci. To determine if any of the men mum4-1 lines represented known mucilage mutant loci beyond MUM4, several assays were performed. First, no changes in seed shape were observed as in ap2 and ats mutants (Léon-Kloosterziel et al., 1994). Second, their identity as new alleles of GL2, TTG1, and TTG2 was tested through an examination of seed coat colour and trichome presence (Koornneef, 1981; Rerie et al., 1994; Johnson et al., 2002). All men mum4-1 lines had trichomes, and none had obviously yellow seeds, suggesting that they are different genes. Third, to eliminate tt mutants that were not obviously yellow, tetrazolium red staining was used to detect the increased permeability to solutes found for most tt mutants, including ttg1, tt2, and tt8 that are known to affect mucilage production (Debeaujon et al., 2000). One line that appeared to be wild-type seed colour (named men3-1 mum4-1) showed significant staining with tetrazolium red (data not shown). However, closer examination of men3-1 mum4-1 seeds revealed that they were slightly paler than wild-type seeds. The seed colour phenotype was found to segregate away from the mucilage phenotype, suggesting a background mutation in a tt or related gene that is unlikely to significantly affect mucilage release (data not shown). Fourth, complementation tests were performed with myb61, another reduced mucilage mutant (Penfield et al., 2001). One line was found not to complement myb61-1 and sequencing confirmed that it is a new allele of MYB61, which has been named myb61-6 (G to A transition leading to the conversion of Trp at position 252 to a stop codon). Fifth, the remaining men mum4-1 lines were backcrossed to wild-type Columbia-2 (Col-2) plants to determine if there was a mutant phenotype in the absence of mum4-1, as all mucilage mutants identified to date other than egl3, tt2, and tt8 have detectable mucilage release phenotypes in a wild-type background. Only two mutant lines had detectable single mutant phenotypes where no mucilage was released when shaken in ruthenium red stain without EDTA pretreatment (Fig. 3; data not shown). Since they were already shown not to be allelic to known reduced mucilage mutants, both of these lines were backcrossed to the mucilage release mutants mum1-1, mum2-1, and patchy (Western et al., 2001; AA Arsovski, TL Western, unpublished results). One line was found to complement all three mutants, while the other only complemented mum1-1 and patchy. While sequencing did not reveal an obvious mutation in the coding sequence of MUM2 for the non-complementing line, regulatory region or intron-related mutations cannot be ruled out. This line did, however, map to the MUM2 region, which, in combination with multiple complementation tests using both the double and isolated single mutants, and similar results between the single mutant and mum2 in all further assays (data not shown), suggest that this is, indeed, a new allele of MUM2 that has been named mum2-13. The other line represents a different gene that has been named MEN4. EDTA pretreatment of men4 single mutants revealed the release of a reduced amount of mucilage compared to wild-type seeds (compare Fig. 1A with Fig. 2A).

Bottom Line: A number of genes have been identified affecting mucilage secretory cell differentiation, including MUCILAGE-MODIFIED4 (MUM4). mum4 mutants produce a reduced amount of mucilage and cloning of MUM4 revealed that it encodes a UDP-L-rhamnose synthase that is developmentally up-regulated to provide rhamnose for mucilage pectin synthesis.Eight mum enhancers (men) have been identified, two of which result from defects in known mucilage secretory cell genes (MUM2 and MYB61).Conversely, mutations in MEN2 and MEN6 appear to affect mucilage release rather than quantity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, McGill University, Montreal, QC, Canada H3A 1B1.

ABSTRACT
Pollination triggers not only embryo development but also the differentiation of the ovule integuments to form a specialized seed coat. The mucilage secretory cells of the Arabidopsis thaliana seed coat undergo a complex differentiation process in which cell growth is followed by the synthesis and secretion of pectinaceous mucilage. A number of genes have been identified affecting mucilage secretory cell differentiation, including MUCILAGE-MODIFIED4 (MUM4). mum4 mutants produce a reduced amount of mucilage and cloning of MUM4 revealed that it encodes a UDP-L-rhamnose synthase that is developmentally up-regulated to provide rhamnose for mucilage pectin synthesis. To identify additional genes acting in mucilage synthesis and secretion, a screen for enhancers of the mum4 phenotype was performed. Eight mum enhancers (men) have been identified, two of which result from defects in known mucilage secretory cell genes (MUM2 and MYB61). Our results show that, in a mum4 background, mutations in MEN1, MEN4, and MEN5 lead to further reductions in mucilage compared to mum4 single mutants, suggesting that they are involved in mucilage synthesis or secretion. Conversely, mutations in MEN2 and MEN6 appear to affect mucilage release rather than quantity. With the exception of men4, whose single mutant exhibits reduced mucilage, none of these genes have a single mutant phenotype, suggesting that they would not have been identified outside the compromised mum4 background.

Show MeSH
Related in: MedlinePlus