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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 and seed coat structure of controls and mum4 enhancer lines resulting from mutations in MUM2 and MYB61. (A) Seeds pretreated in EDTA with shaking and stained with ruthenium red. Note the thick layer of mucilage surrounding wild-type Col-2 seeds and the very thin layer around mum4-1 seeds. (B) Scanning electron microscopy of dry seeds. Note the prominent hexagonal cell walls and central volcano-shaped columella in the centre of Col-2 cells. (C) Toluidine blue-stained resin sections of 13 DPA seeds. Wild-type mucilage cells have burst open in the aqueous fixative, leaving tall, blue-stained, volcano-shaped columellae with some cell wall material attached to the centre of the columella. mum4-1 cells do not burst, and contain small pockets of purple-stained mucilage in the upper apical corners, subtended by a blue dome of secondary cell wall. ttg1-1 cells, similar to mum4-1, do not burst, however, the mucilage pockets are smaller and the secondary cell wall is thinner in appearance. Scale bars: (A) 500 μm, (B) 50 μm, (C) 10 μm.
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fig1: Mucilage release and seed coat structure of controls and mum4 enhancer lines resulting from mutations in MUM2 and MYB61. (A) Seeds pretreated in EDTA with shaking and stained with ruthenium red. Note the thick layer of mucilage surrounding wild-type Col-2 seeds and the very thin layer around mum4-1 seeds. (B) Scanning electron microscopy of dry seeds. Note the prominent hexagonal cell walls and central volcano-shaped columella in the centre of Col-2 cells. (C) Toluidine blue-stained resin sections of 13 DPA seeds. Wild-type mucilage cells have burst open in the aqueous fixative, leaving tall, blue-stained, volcano-shaped columellae with some cell wall material attached to the centre of the columella. mum4-1 cells do not burst, and contain small pockets of purple-stained mucilage in the upper apical corners, subtended by a blue dome of secondary cell wall. ttg1-1 cells, similar to mum4-1, do not burst, however, the mucilage pockets are smaller and the secondary cell wall is thinner in appearance. Scale bars: (A) 500 μm, (B) 50 μm, (C) 10 μm.

Mentions: When Arabidopsis seeds are hydrated, the seed coat mucilage swells rapidly, leading to the bursting of the primary cell wall and the release of mucilage to surround the seed in a gel-like capsule (Fig. 1A) (Western et al., 2000; Windsor et al., 2000). mum4 mutants make a significantly reduced amount of mucilage (Western et al., 2001, 2004) that remains within the cells when seeds are hydrated. Addition of a heavy metal chelator such as EDTA or EGTA, however, leads to the release of mum4 mucilage. This is probably due to the withdrawl of Ca2+ ions from the cell wall pectins, leading to weakening of the cell wall and/or permitting increased swelling of the mucilage present. mum4 seeds shaken in EDTA prior to ruthenium red staining reveal a thin layer of stained mucilage around the seeds, consistent with their reduced mucilage production (Fig. 1A) (Western et al., 2004). By contrast, mutants for TTG1, which acts upstream of both the GL2 and TTG2 pathways of mucilage production, make very little mucilage and show no obvious mucilage release when EDTA-treated (Fig. 1A). The moderate level of mucilage release found for mum4 mutants, as well as the ability to differentiate mum4 mutants from mutants with further reduced mucilage, allowed us to perform a genetic screen for phenotypic enhancers of mum4. mum4-1 seeds were mutagenized with ethyl methanesulphonate and seeds from individual M2 plants (M3 lines) were collected and screened for reduced levels of mucilage compared with mum4-1 as observed with ruthenium red staining after EDTA pretreatment. Over 5000 M3 lines derived from ten parental M1 batches (1000–1500 M1 parents) were screened, leading to the identification of nine mum4-1 enhancers [named mum enhancers (men)] that have no visible mucilage release when treated with EDTA (Figs 1A, 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 and seed coat structure of controls and mum4 enhancer lines resulting from mutations in MUM2 and MYB61. (A) Seeds pretreated in EDTA with shaking and stained with ruthenium red. Note the thick layer of mucilage surrounding wild-type Col-2 seeds and the very thin layer around mum4-1 seeds. (B) Scanning electron microscopy of dry seeds. Note the prominent hexagonal cell walls and central volcano-shaped columella in the centre of Col-2 cells. (C) Toluidine blue-stained resin sections of 13 DPA seeds. Wild-type mucilage cells have burst open in the aqueous fixative, leaving tall, blue-stained, volcano-shaped columellae with some cell wall material attached to the centre of the columella. mum4-1 cells do not burst, and contain small pockets of purple-stained mucilage in the upper apical corners, subtended by a blue dome of secondary cell wall. ttg1-1 cells, similar to mum4-1, do not burst, however, the mucilage pockets are smaller and the secondary cell wall is thinner in appearance. Scale bars: (A) 500 μm, (B) 50 μm, (C) 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig1: Mucilage release and seed coat structure of controls and mum4 enhancer lines resulting from mutations in MUM2 and MYB61. (A) Seeds pretreated in EDTA with shaking and stained with ruthenium red. Note the thick layer of mucilage surrounding wild-type Col-2 seeds and the very thin layer around mum4-1 seeds. (B) Scanning electron microscopy of dry seeds. Note the prominent hexagonal cell walls and central volcano-shaped columella in the centre of Col-2 cells. (C) Toluidine blue-stained resin sections of 13 DPA seeds. Wild-type mucilage cells have burst open in the aqueous fixative, leaving tall, blue-stained, volcano-shaped columellae with some cell wall material attached to the centre of the columella. mum4-1 cells do not burst, and contain small pockets of purple-stained mucilage in the upper apical corners, subtended by a blue dome of secondary cell wall. ttg1-1 cells, similar to mum4-1, do not burst, however, the mucilage pockets are smaller and the secondary cell wall is thinner in appearance. Scale bars: (A) 500 μm, (B) 50 μm, (C) 10 μm.
Mentions: When Arabidopsis seeds are hydrated, the seed coat mucilage swells rapidly, leading to the bursting of the primary cell wall and the release of mucilage to surround the seed in a gel-like capsule (Fig. 1A) (Western et al., 2000; Windsor et al., 2000). mum4 mutants make a significantly reduced amount of mucilage (Western et al., 2001, 2004) that remains within the cells when seeds are hydrated. Addition of a heavy metal chelator such as EDTA or EGTA, however, leads to the release of mum4 mucilage. This is probably due to the withdrawl of Ca2+ ions from the cell wall pectins, leading to weakening of the cell wall and/or permitting increased swelling of the mucilage present. mum4 seeds shaken in EDTA prior to ruthenium red staining reveal a thin layer of stained mucilage around the seeds, consistent with their reduced mucilage production (Fig. 1A) (Western et al., 2004). By contrast, mutants for TTG1, which acts upstream of both the GL2 and TTG2 pathways of mucilage production, make very little mucilage and show no obvious mucilage release when EDTA-treated (Fig. 1A). The moderate level of mucilage release found for mum4 mutants, as well as the ability to differentiate mum4 mutants from mutants with further reduced mucilage, allowed us to perform a genetic screen for phenotypic enhancers of mum4. mum4-1 seeds were mutagenized with ethyl methanesulphonate and seeds from individual M2 plants (M3 lines) were collected and screened for reduced levels of mucilage compared with mum4-1 as observed with ruthenium red staining after EDTA pretreatment. Over 5000 M3 lines derived from ten parental M1 batches (1000–1500 M1 parents) were screened, leading to the identification of nine mum4-1 enhancers [named mum enhancers (men)] that have no visible mucilage release when treated with EDTA (Figs 1A, 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