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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.    Supplementary Information  The online version contains supplementary material available at 10.1186/s12870-020-02791-9.

bmc plant biology

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