CRISPR-Cas9 multiplex genome editing of the hydroxyproline-O-galactosyltransferase gene family alters arabinogalactan-protein glycosylation and function in Arabidopsis

Abstract

Background Arabinogalactan-proteins (AGPs) are a class of hydroxyproline-rich proteins (HRGPs) that are heavily glycosylated (> 90%) with type II arabinogalactans (AGs). AGPs are implicated in various plant growth and development processes including cell expansion, somatic embryogenesis, root and stem growth, salt tolerance, hormone signaling, male and female gametophyte development, and defense. To date, eight Hyp-O-galactosyltransferases (GALT2–6, HPGT1–3) have been identified; these enzymes are responsible for adding the first sugar, galactose, onto AGPs. Due to gene redundancy among the GALTs, single or double galt genetic knockout mutants are often not sufficient to fully reveal their biological functions. Results Here, we report the successful application of CRISPR-Cas9 gene editing/multiplexing technology to generate higher-order knockout mutants of five members of the GALT gene family (GALT2–6). AGPs analysis of higher-order galt mutants (galt2 galt5, galt3 galt4 galt6, and galt2 galt3 galt4 galt5 gal6) demonstrated significantly less glycosylated AGPs in rosette leaves, stems, and siliques compared to the corresponding wild-type organs. Monosaccharide composition analysis of AGPs isolated from rosette leaves revealed significant decreases in arabinose and galactose in all the higher-order galt mutants. Phenotypic analyses revealed that mutation of two or more GALT genes was able to overcome the growth inhibitory effect of β-D-Gal-Yariv reagent, which specifically binds to β-1,3-galactan backbones on AGPs. In addition, the galt2 galt3 galt4 galt5 gal6 mutant exhibited reduced overall growth, impaired root growth, abnormal pollen, shorter siliques, and reduced seed set. Reciprocal crossing experiments demonstrated that galt2 galt3 galt4 galt5 gal6 mutants had defects in the female gametophyte which were responsible for reduced seed set. Conclusions Our CRISPR/Cas9 gene editing/multiplexing approach provides a simpler and faster way to generate higher-order mutants for functional characterization compared to conventional genetic crossing of T-DNA mutant lines. Higher-order galt mutants produced and characterized in this study provide insight into the relationship between sugar decorations and the various biological functions attributed to AGPs in plants.

Arabinogalactan Proteins in Plant Roots – An Update on Possible Functions

“…Together with the lower glycosylation status of AGPs, the mutant displays reduced seedcoat cellulose content, swollen root-tip cells, and other root growth defects, e.g., inhibition of root growth, reduction of root hair length and density (Basu et al, 2015). Shorter roots were observed also in the quintuple mutant galt2 galt3 galt4 galt5 galt6, but surprisingly this mutant formed longer root hairs compared to wild type (Zhang et al, 2021). All these observations highlight the importance of O-glycosylation in cell growth and cell wall deposition (Basu et al, 2015;Showalter and Basu, 2016).…”

Sweet Modifications Modulate Plant Development

“…All AtGALTs are expressed in the seed coat during different stages of embryogenesis. Knocked out galt2 and galt5 mutants showed reduced seed coat mucilage, while galt4 and galt6 revealed a reduced seed set, pointing towards a probable function during seed development [ 168 , 169 , 242 ].…”

“…Hyp-O-GALT is responsible for the transfer of a Gal residue onto Hyp residues of AGPs. Eight Hyp-O-GALTs (i.e., GALT2-6 and HPGT1-3) in Arabidopsis have been characterized and catalyse the transfer of Gal onto Hyp residues [ 169 ]. The single mutants did not display any aberrant leaf phenotype, with the exception of the galt6 mutation that resulted in accelerated leaf senescence in Arabidopsis .…”

“…The phenotype of galt6 resembles the altered phenotype observed in hpat1 / hpat2 [ 162 ]. The quintuple mutant galt2 / galt3 / galt4 / galt5 / galt6 had fewer and smaller rosette leaves [ 169 ]. The triple mutant hpgt1 / hpgt2 / hpgt3 displayed a dwarf phenotype with smaller rosettes which is in line with the phenotype observed for the quintuple mutant [ 166 ].…”

Functional Characterization of Hydroxyproline-O-Galactosyltransferases For Arabidopsis Arabinogalactan-Proteins synthesis

“…Whereas the hpgt1 hpgt2 hpgt3 triple mutant exhibited reductions in plant height, leaf size, seed set, silique length as well as an increase in root length. Moreover, Zhang et al (2021) utilized CRISPR/Cas9 gene editing/multiplexing approach to generate the quintuple mutant galt2 galt3 galt4 galt5 galt6 which exhibited reduced overall growth, impaired root growth, abnormal pollen, and reduced seed set with shorter siliques [35].…”