Putative rhamnogalacturonan-II glycosyltransferase identified through callus gene editing bypasses embryo lethality

• Published in: Plant physiology (Bethesda) 2024-05, Vol.195 (4)
• Main Authors: Zhang, Yuan, Sharma, Deepak, Liang, Yan, Downs, Nick, Dolman, Fleur, Thorne, Kristen, Black, Ian M., Pereira, Jose Henrique, Adams, Paul, Scheller, Henrik V., O’Neill, Malcolm, Urbanowicz, Breeanna, Mortimer, Jenny C.
• Format: Article
• Language: English
• Subjects: BASIC BIOLOGICAL SCIENCES; pectin; plant cell wall; Rhamnogalacturonan II (RG-II)
• ISSN: 0032-0889

Abstract Rhamnogalacturonan II (RG-II) is a structurally complex and conserved domain of the pectin present in the primary cell walls of vascular plants. Borate cross-linking of RG-II is required for plants to grow and develop normally. Mutations that alter RG-II structure also affect cross-linking and are lethal or severely impair growth. Thus, few genes involved in RG-II synthesis have been identified. Here, we developed a method to generate viable loss-of-function Arabidopsis (Arabidopsis thaliana) mutants in callus tissue via CRISPR/Cas9-mediated gene editing. We combined this with a candidate gene approach to characterize the male gametophyte defective 2 (MGP2) gene that encodes a putative family GT29 glycosyltransferase. Plants homozygous for this mutation do not survive. We showed that in the callus mutant cell walls, RG-II does not cross-link normally because it lacks 3-deoxy-d-manno-octulosonic acid (Kdo) and thus cannot form the α-L-Rhap-(1→5)-α-d-kdop-(1→sidechain). We suggest that MGP2 encodes an inverting RG-II CMP-β-Kdo transferase (RCKT1). Our discovery provides further insight into the role of sidechains in RG-II dimerization. Our method also provides a viable strategy for further identifying proteins involved in the biosynthesis of RG-II.