Abstract
BackgroundArabinogalactan-proteins (AGPs) are one of the most complex protein families in the plant kingdom and are present in the cell walls of all land plants. AGPs are implicated in diverse biological processes such as plant growth, development, reproduction, and stress responses. AGPs are extensively glycosylated by the addition of type II arabinogalactan (AG) polysaccharides to hydroxyproline residues in their protein cores. Glucuronic acid (GlcA) is the only negatively charged sugar added to AGPs and the functions of GlcA residues on AGPs remain to be elucidated.ResultsThree members of the CAZy GT14 family (GLCAT14A-At5g39990, GLCAT14B-At5g15050, and GLCAT14C-At2g37585), which are responsible for transferring glucuronic acid (GlcA) to AGPs, were functionally characterized using a CRISPR/Cas9 gene editing approach in Arabidopsis. RNA seq and qRT-PCR data showed all three GLCAT genes were broadly expressed in different plant tissues, with GLCAT14A and GLCAT14B showing particularly high expression in the micropylar endosperm. Biochemical analysis of the AGPs from knock-out mutants of various glcat single, double, and triple mutants revealed that double and triple mutants generally had small increases of Ara and Gal and concomitant reductions of GlcA, particularly in the glcat14a glcat14b and glcat14a glcat14b glcat14c mutants. Moreover, AGPs isolated from all the glcat mutants displayed significant reductions in calcium binding compared to WT. Further phenotypic analyses found that the glcat14a glcat14b and glcat14a glcat14b glcat14c mutants exhibited significant delays in seed germination, reductions in root hair length, reductions in trichome branching, and accumulation of defective pollen grains. Additionally, both glcat14b glcat14c and glcat14a glcat14b glcat14c displayed significantly shorter siliques and reduced seed set. Finally, all higher-order mutants exhibited significant reductions in adherent seed coat mucilage.ConclusionsThis research provides genetic evidence that GLCAT14A-C function in the transfer of GlcA to AGPs, which in turn play a role in a variety of biochemical and physiological phenotypes including calcium binding by AGPs, seed germination, root hair growth, trichome branching, pollen development, silique development, seed set, and adherent seed coat mucilage accumulation.
Highlights
Arabinogalactan-proteins (AGPs) are one of the most complex protein families in the plant kingdom and are present in the cell walls of all land plants
This research provides genetic evidence that GLCAT14A-C function in the transfer of glucuronic acid (GlcA) to Arabinogalactan protein (AGP), which in turn play a role in a variety of biochemical and physiological phenotypes including calcium binding by AGPs, seed germination, root hair growth, trichome branching, pollen development, silique development, seed set, and adherent seed coat mucilage accumulation
GLCAT14A, GLCAT14B, and GLCAT14C belong to the Carbohydrate Active Enzyme (CAZy) GT14 family This study focused on functional characterization of GLCAT14A (At5g39990), GLCAT14B (At5g15050), and GLCAT14C (At2g37585) found in the CAZy GT14 family, which includes 11 proteins in Arabidopsis
Summary
Arabinogalactan-proteins (AGPs) are one of the most complex protein families in the plant kingdom and are present in the cell walls of all land plants. AGPs are extensively glycosylated by the addition of type II arabinogalactan (AG) polysaccharides to hydroxyproline residues in their protein cores. Arabinogalactan-proteins (AGPs) are one of the most complex protein families in plants due to the diversity of the core proteins and the heterogeneity of the glycan chains attached to these protein cores. AGPs are Olinked glycoproteins characterized by the presence of type II (β-1,3 and β-1,6) AGs attached to hydroxyproline (Hyp) residues present in the protein core. The Hyp residues in AGP core proteins are modified by the addition of β-1,3-galactose sugar backbones, which are further modified by the addition of multiple, branching β1,6 galactose side chains, which are further modified by the addition of arabinose (Ara) residues and less extensively by other sugars, such as rhamnose (Rha), fucose (Fuc), xylose (Xyl) and glucuronic acid (GlcA) [3, 4]
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