Abstract
BackgroundArabinogalactan-proteins (AGPs) are heavily glycosylated with type II arabinogalactan (AG) polysaccharides attached to hydroxyproline residues in their protein backbone. Type II AGs are necessary for plant growth and critically important for the establishment of normal cellular functions. Despite the importance of type II AGs in plant development, our understanding of the underlying role of these glycans/sugar residues in mucilage formation and seed coat epidermal cell development is poorly understood and far from complete. One such sugar residue is the glucuronic acid residues of AGPs that are transferred onto AGP glycans by the action of β-glucuronosyltransferase genes/enzymes.ResultsHere, we have characterized two β-glucuronosyltransferase genes, GLCAT14A and GLCAT14C, that are involved in the transfer of β-glucuronic acid (GlcA) to type II AGs. Using a reverse genetics approach, we observed that glcat14a-1 mutants displayed subtle alterations in mucilage pectin homogalacturonan (HG) compared to wild type (WT), while glcat14a-1glcat14c-1 mutants displayed much more severe mucilage phenotypes, including loss of adherent mucilage and significant alterations in cellulose ray formation and seed coat morphology. Monosaccharide composition analysis showed significant alterations in the sugar amounts of glcat14a-1glcat14c-1 mutants relative to WT in the adherent and non-adherent seed mucilage. Also, a reduction in total mucilage content was observed in glcat14a-1glcat14c-1 mutants relative to WT. In addition, glcat14a-1glcat14c-1 mutants showed defects in pectin formation, calcium content and the degree of pectin methyl-esterification (DM) as well as reductions in crystalline cellulose content and seed size.ConclusionsThese results raise important questions regarding cell wall polymer interactions and organization during mucilage formation. We propose that the enzymatic activities of GLCAT14A and GLCAT14C play partially redundant roles and are required for the organization of the mucilage matrix and seed size in Arabidopsis thaliana. This work brings us a step closer towards identifying potential gene targets for engineering plant cell walls for industrial applications.
Highlights
Arabinogalactan-proteins (AGPs) are heavily glycosylated with type II arabinogalactan (AG) polysaccharides attached to hydroxyproline residues in their protein backbone
Given the expression of GLCAT14A and GLCAT14C across seed developmental stages (Fig. 2a), we examined their expression at the linear cotyledon stage (8 DAP) in wild type (WT), glcat14a-1, glcat14c-1 and glcat14a-1glcat14c-1 mutants, and observed a significant reduction in gene expression of GLCAT14A and GLCAT14C in both the single and double mutants (Fig. 2c)
The glcat14a‐1 and glcat14a‐1glcat14c‐1 mutants have distinct seed coat mucilage phenotypes in response to different chemical extractants WT and mutant seeds were hydrated in distilled water and Na2CO3 and stained with ruthenium red (RR), a red dye which preferentially binds to unesterified pectin [21]
Summary
Arabinogalactan-proteins (AGPs) are heavily glycosylated with type II arabinogalactan (AG) polysaccharides attached to hydroxyproline residues in their protein backbone. Despite the importance of type II AGs in plant development, our understanding of the underlying role of these glycans/sugar residues in mucilage formation and seed coat epidermal cell development is poorly understood and far from complete. One such sugar residue is the glucuronic acid residues of AGPs that are transferred onto AGP glycans by the action of β-glucuronosyltransferase genes/enzymes. The SCE is an excellent model system for understanding the genetic basis of cell wall biosynthesis, secretion, assembly and modification [3, 4] because large amounts of cell wall polysaccharides can be extracted with ease and analyzed in a short timeframe. Genetic mutants that lack functional enzymes required for mucilage biosynthesis and extrusion have been identified and characterized, but many others await functional investigation
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