Arabidopsis GT34 family contains five xyloglucan α‐1,6‐xylosyltransferases

Abstract

Summary The Arabidopsis genome includes seven family 34 glycosyltransferase (GT34) encoding genes. XXT1 and XXT2 have previously been shown to encode XyG α‐1,6‐xylosyltransferases, while knockout mutants of a third, XXT5, exhibit decreased XyG content, suggesting a similar activity. Here, we extend the study to the rest of the Arabidopsis GT34 genes in terms of biochemical activity and their roles in XyG biosynthesis. The enzyme activity of XXTs was investigated using recombinant protein expressed in E. coli. XyG analysis of single and double T‐DNA insertion knockouts, together with overexpression of GT34s in selected mutant lines, provided detailed function of each gene. We reveal the activity of the third member of the GT34 gene family (XXT4) that exhibits xylosyltransferase activity. Double mutants for either xxt2 or xxt5 had a large impact on XyG content, structure and size distribution. Overexpression of the remaining member, XXT3, was able to restore XyG epitopes in xxt2, xxt5 and xxt2 xxt5 double knockouts, suggesting that it also encodes a protein with XXT activity. Our work demonstrates that five of the seven Arabidopsis GT34 genes encode XXT enzymes.

Enzymatic Activity of Xyloglucan Xylosyltransferase 5

“…The xxt5 mutant had a 50% reduction in XyG content as well as a distinct root hair phenotype. In addition, XXT1 and XXT2 have been confirmed to be catalytically active in vitro as a-1,6-xylosyltransferases (Cavalier and Keegstra, 2006), while the catalytic activity of XXT5 has not been shown despite several attempts (Faik et al, 2002;Zabotina et al, 2008;Vuttipongchaikij et al, 2012). Therefore, it was suggested that XXT5 might lack enzymatic activity and, most likely, perform other functions.…”

“…5A). Vuttipongchaikij et al (2012) reported the only attempt to produce recombinant XXTs in E. coli cells, expressing XXTs without transmembrane domains in BL21 cells, and demonstrated enzymatic activity for XXT1, XXT2, and XXT4. However, protein expression levels in that study were low, as indicated by faint bands on SDS-PAGE and by reported yields of 40 to 90 mg of total protein per 1 L of cell culture.…”

“…S1B). A previous study reported the expression of truncated XXTs with an N-terminal glutathione S-transferase tag in E. coli BL21 cells (Vuttipongchaikij et al, 2012). Here, we tested different E. coli strains and various N-terminal tags to determine the optimal conditions for expressing XXTs (Supplemental Fig.…”

“…All of the enzymes that decorate the glucan backbone of XyG are type II transmembrane proteins containing a short N terminus localized in the cytosol, a transmembrane domain, a stem region, and a large catalytic domain localized in the Golgi lumen. Xylosylation of the glucan backbone is catalyzed by at least three glycosyltransferases, Xyloglucan Xylosyltransferase 1 (XXT1), XXT2, and XXT5, which are members of the GT34 family (Faik et al, 2002;Cavalier and Keegstra, 2006;Cavalier et al, 2008;Zabotina et al, 2008Zabotina et al, , 2012Vuttipongchaikij et al, 2012) in the Carbohydrate Active Enzyme database (Campbell et al, 1997;Coutinho et al, 2003). Analysis of a phylogenetic tree of GT34 glycosyltransferases revealed three major clades (Mansoori et al, 2015).…”

“…The generation of knockout mutants in Arabidopsis showed that these enzymes, along with the putative xylosyltransferase XXT5, function to synthesize XyG Zabotina et al, 2008Zabotina et al, , 2012. Another xylosyltransferase, XXT4, was later demonstrated to be catalytically active in vitro, although xxt4 mutants had no detectable decrease in XyG content (Vuttipongchaikij et al, 2012). The xxt1 xxt2 double mutant, xxt1 xxt2 xxt5 triple mutant, and xxt5 single mutant all show severe root hair phenotypes, displaying root hairs that were short and thick, formed bubble-like extrusions near the tips, and were lower in abundance compared with wild-type roots Zabotina et al, 2008Zabotina et al, , 2012.…”