Abstract
Following pollination, the epidermal cells of the Arabidopsis (Arabidopsis thaliana) ovule undergo a complex differentiation process that includes the synthesis and polar secretion of pectinaceous mucilage followed by the production of a secondary cell wall. Wetting of mature seeds leads to the rapid bursting of these mucilage secretory cells to release a hydrophilic gel that surrounds the seed and is believed to aid in seed hydration and germination. A novel mutant is identified where mucilage release is both patchy and slow and whose seeds display delayed germination. While developmental analysis of mutant seeds reveals no change in mucilage secretory cell morphology, changes in monosaccharide quantities are detected, suggesting the mucilage release defect results from altered mucilage composition. Plasmid rescue and cloning of the mutant locus revealed a T-DNA insertion in AtBXL1, which encodes a putative bifunctional beta-d-xylosidase/alpha-l-arabinofuranosidase that has been implicated as a beta-d-xylosidase acting during vascular development. Chemical and immunological analyses of mucilage extracted from bxl1 mutant seeds and antibody staining of developing seed coats reveal an increase in (1-->5)-linked arabinans, suggesting that BXL1 is acting as an alpha-l-arabinofuranosidase in the seed coat. This implication is supported by the ability to rescue mucilage release through treatment of bxl1 seeds with exogenous alpha-l-arabinofuranosidases. Together, these results suggest that trimming of rhamnogalacturonan I arabinan side chains is required for correct mucilage release and reveal a new role for BXL1 as an alpha-l-arabinofuranosidase acting in seed coat development.
Highlights
Following pollination, the epidermal cells of the Arabidopsis (Arabidopsis thaliana) ovule undergo a complex differentiation process that includes the synthesis and polar secretion of pectinaceous mucilage followed by the production of a secondary cell wall
A screen for mutants affected in mucilage extrusion led to the identification of the MUCILAGE-MODIFIED genes (MUM1 to MUM5; Western et al, 2001). mum4 mutants make a reduced amount of mucilage, mum3 and mum5 appear to be affected in mucilage composition, while mum1 and mum2 mutants are defective in mucilage release upon seed hydration
MUM4 encodes a UDP-LRha synthase (RHAMNOSE SYNTHASE2 [RHM2]), an enzyme required for the synthesis of rhamnogalacturonan I (RG I), the primary pectin found in Arabidopsis seed mucilage (Usadel et al, 2004; Western et al, 2004; Oka et al, 2007)
Summary
To identify genes required for the synthesis and extrusion of seed coat mucilage, pools of T-DNA insertion lines (Feldmann, 1991) were screened for the presence of mucilage when hydrated by staining with the pectin dye ruthenium red. The remaining seeds were analyzed, but no significant differences in monosaccharide levels were observed (Table II) These results suggest that there is a change in mucilage composition that may be responsible for the slow and patchy mucilage release observed in the patchy mutants. Patchy mutants transformed with PTYg were found to have wild-type mucilage (9/10 independent transformants), while pGREEN0229-transformed plants retained the patchy mucilage phenotype (11/11) (Fig. 5, A and B; Supplemental Table S1) These results are consistent with the hypothesis that an insertion in AtBXL1 is responsible for the seed coat phenotype. To confirm changes existed in the levels of (1/5)linked arabinans in bxl versus wild-type mucilage, immunoblots with extracted mucilage were performed using the arabinan-specific antibody LM6 (Willats et al, 1998, 2001b; Supplemental Fig. S2). Treatment with all three arabinofuranosidases led to rescue of the patchy mucilage
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