Elucidating the roles of three β-glucuronosyltransferases (GLCATs) acting on arabinogalactan-proteins using a CRISPR-Cas9 multiplexing approach in Arabidopsis

Background 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 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. Results Three 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. Conclusions This 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.