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

Abstract

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 knockout 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.

Calcium Binding by Arabinogalactan Polysaccharides Is Important for Normal Plant Development

“…Mutants in the AGP Hyp O-galactosyltransferases, which initiate the AG glycans, also show reduced stem length (Ogawa-Ohnishi and Matsubayashi, 2015). The recent observation that knockout glcat14a/b, glcat14b/ c, and glcat14a/b/c mutants have shorter inflorescences (Zhang et al, 2020) supports our finding that AGP glucuronidation is important for plant development. The triple glcat14a/b/e mutant showed very limited growth and was unable to produce progeny.…”

“…glcat14 mutants glcat14a/ b and glcat14a/b/d had reduced trichome branching. The reduction of trichome branching in glcat14a/b mutants was recently reported in another study (Zhang et al, 2020). The reduction of trichome branching together with the crooked trichome, seedlings, and characteristic etiolated hypocotyl phenotype of the glcat14a/b/e triple mutants suggest that glucuronidation of AGs is essential for cell shape formation and expansion.…”

“…However, unfertilized embryo sac7 and tube growth defective12, both mutants in AT3G03690, a member of the GT14 family, were identified in two independent screening assays for defects in fertilization and pollen tube elongation (Pagnussat et al, 2005;Boavida et al, 2009). Deficiencies in pollen germination and development have recently been reported in glcat14abc compound mutants (Zhang et al, 2020). AT3G03690 is likely to be an AG GlcAT, and so further studies on the effect of these mutations on pollen growth may provide additional evidence for the biological function of the AGP-Ca 21 interaction at the pollen cell surface.…”

On the Potential Function of Type II Arabinogalactan O-Glycosylation in Regulating the Fate of Plant Secretory Proteins

“…A very productive area of AGP-related research is floral development and sexual reproduction (Pereira et al, 2016a;Su et al, 2020). Some classical AGPs and AG-peptides are required for normal pollen tube growth (Levitin et al, 2008;Pereira et al, 2016b) and in the light of the Ca 2+ -capacitor hypothesis it would be interesting to test if the pollen tube phenotype previously observed in triple mutants lacking some of the type II AG specific glucuronyl transferases (Zhang et al, 2020) is modulated by external Ca 2+ levels. However, sometimes the glycan does not seem to be directly involved in the function of an AGP.…”

“…However, as was previously noted, arabinose in type II AG is α-linked and the precise biochemical role of RAY1 in AGP biosynthesis remains to be clarified ( Showalter and Basu, 2016a ). By contrast, several type II AG specific β-1-6-glucuronosyl transferases are included in family GT14—called GlcAT14-A, -B, and -C. The glcat14 single and multiple mutants in these three loci showed a variety of developmental defects as well a reduction of AGP-bound calcium ( Knoch et al., 2013 ; Dilokpimol and Geshi, 2014 ; Zhang et al., 2020 ). Finally, two members of family GT34 called FUT4 and FUT6 redundantly acted in the α-1-2 fucosylation of type II AG ( Liang et al., 2013 ; Tryfona et al., 2014 ).…”