Dataset Information

Effective extraction of Arabidopsis adherent seed mucilage by ultrasonic treatment.

ABSTRACT: The Arabidopsis seed coat is composed of two layers of mucilage, a water-soluble non-adherent outer layer and an adherent inner layer. The non-adherent mucilage can easily be extracted by gentle shaking. However, adherent mucilage is extremely difficult to dissociate from the seed coat. Despite various treatments to extract the adherent mucilage, including EDTA, ammonium oxalate, dilute alkali or acid washes, most of it remains on the seed coat. Here, we show for the first time the extraction of almost all of the adherent mucilage from the Arabidopsis seed coat. Our results demonstrate that ultrasonic treatment was able to extract the adherent mucilage effectively within 20?seconds. Adherent mucilage, like non-adherent mucilage, is mainly composed of rhamnogalacturonan I (RG I). The crystalline cellulose content in adherent mucilage was measured as 3.7?mg?g^-1 of dry seed. Compared with non-adherent mucilage, the adherent mucilage exhibits relatively stable levels of sugar under various environmental conditions. In all cases, adherent mucilage showed higher levels of sugar than non-adherent mucilage. The cell wall remnant could associate with the adherent mucilage, which could prevent the extraction of the adherent mucilage. Our results show that ultrasonic treatment is an effective method for the quick extraction of Arabidopsis adherent mucilage with little effort.

SUBMITTER: Zhao X

PROVIDER: S-EPMC5238429 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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Effective extraction of Arabidopsis adherent seed mucilage by ultrasonic treatment.

Zhao Xianhai X Qiao Lijun L Wu Ai-Min AM

Scientific reports 20170116

The Arabidopsis seed coat is composed of two layers of mucilage, a water-soluble non-adherent outer layer and an adherent inner layer. The non-adherent mucilage can easily be extracted by gentle shaking. However, adherent mucilage is extremely difficult to dissociate from the seed coat. Despite various treatments to extract the adherent mucilage, including EDTA, ammonium oxalate, dilute alkali or acid washes, most of it remains on the seed coat. Here, we show for the first time the extraction of ...[more]

PMID: 28091592

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Project description:Hydrated Arabidopsis thaliana seeds are coated by a gelatinous layer called mucilage, which is mainly composed of cell wall polysaccharides. Since mucilage is rich in pectin, its architecture can be visualized with the ruthenium red (RR) dye. We screened the seeds of around 280 Arabidopsis natural accessions for variation in mucilage structure, and identified a large number of novel variants that differed from the Col-0 wild-type. Most of the accessions released smaller RR-stained capsules compared to the Col-0 reference. By biochemically characterizing the phenotypes of 25 of these accessions in greater detail, we discovered that distinct changes in polysaccharide structure resulted in gelatinous coatings with a deceptively similar appearance. Monosaccharide composition analysis of total mucilage extracts revealed a remarkable variation (from 50 to 200% of Col-0 levels) in the content of galactose and mannose, which are important subunits of heteromannan. In addition, most of the natural variants had altered Pontamine Fast Scarlet 4B staining of cellulose and significantly reduced birefringence of crystalline structures. This indicates that the production or organization of cellulose may be affected by the presence of different amounts of hemicellulose. Although, the accessions described in this study were primarily collected from Western Europe, they form five different phenotypic classes based on the combined results of our experiments. This suggests that polymorphisms at multiple loci are likely responsible for the observed mucilage structure. The transcription of MUCILAGE-RELATED10 (MUCI10), which encodes a key enzyme for galactoglucomannan synthesis, was severely reduced in multiple variants that phenocopied the muci10-1 insertion mutant. Although, we could not pinpoint any causal polymorphisms in this gene, constitutive expression of fluorescently-tagged MUCI10 proteins complemented the mucilage defects of a muci10-like accession. This leads us to hypothesize that some accessions might disrupt a transcriptional regulator of MUCI10. Therefore, this collection of publicly-available variants should provide insight into plant cell wall organization and facilitate the discovery of genes that regulate polysaccharide biosynthesis.

Project description:The plant cell wall is held together by the interactions between four major components: cellulose, pectin, hemicellulose, and proteins. Mucilage is a powerful model system to study the interactions between these components as it is formed of polysaccharides that are deposited in the apoplast of seed coat epidermal cells during seed development. When seeds are hydrated, these polysaccharides expand rapidly out of the apoplastic pocket, and form an adherent halo of mucilage around the seed. In Arabidopsis, mutations in multiple genes have similar loss of mucilage adherence phenotypes including CELLULOSE SYNTHASE 5 (CESA5)/MUCILAGE-MODIFIED 3 (MUM3), MUM5/MUCI21, SALT-OVERLY SENSITIVE 5 (SOS5), and FEI2. Here, we examine the interactions between these factors to better understand how they participate to control mucilage adherence. Double mutant phenotypes indicated that MUM5 and CESA5 function in a common mechanism that adheres pectin to the seed through the biosynthesis of cellulose and xylan, whereas SOS5 and FEI2, encoding a fasciclin-like arabinogalactan protein or a receptor-like kinase, respectively, function through an independent pathway. Cytological analyses of mucilage indicates that heteromannans are associated with cellulose, and not in the pathway involving SOS5 or FEI2. A SOS5 fluorescent protein fusion (SOS5-mCITRINE) was localized throughout the mucilage pocket, consistent with a structural role in pectin adhesion. The relationship between SOS5 and FEI2 mediated mucilage adherence was examined in more detail and while sos5 and fei2 mutants show similar phenotypes, key differences in the macromolecular characteristics and amounts of mucilage polymers were observed. FEI2 thus appears to have additional, as well as overlapping functions, with SOS5. Given that FEI2 is required for SOS5 function, we propose that FEI2 serves to localize SOS5 at the plasma membrane where it establishes interactions with mucilage polysaccharides, notably pectins, required for mucilage adherence prior to SOS5 being released into the apoplast.

Dataset Information

Effective extraction of Arabidopsis adherent seed mucilage by ultrasonic treatment.

Publications

Effective extraction of Arabidopsis adherent seed mucilage by ultrasonic treatment.

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