Two members of the DUF579 family are responsible for arabinogalactan methylation in Arabidopsis

Abstract All members of the DUF579 family characterized so far have been described to affect the integrity of the hemicellulosic cell wall component xylan: GXMs are glucuronoxylan methyltransferases catalyzing 4‐O–methylation of glucuronic acid on xylan; IRX15 and IRX15L, although their enzymatic activity is unknown, are required for xylan biosynthesis and/or xylan deposition. Here we show that the DUF579 family members, AGM1 and AGM2, are required for 4‐O–methylation of glucuronic acid of a different plant cell wall component, the highly glycosylated arabinogalactan proteins (AGPs).

branched polysaccharide structures, attached to the hydroxyproline residues of many plant cell wall polypeptides. Proteins attached to AGs are defined as arabinogalactan proteins, and these proteins form a diverse class of cell surface proteoglycans found in most plant species throughout the plant kingdom (Showalter, 2001).
In addition, glucuronylation can also take place at the O6 of the β-1,3-galactan backbone (Tryfona et al., 2012). The AGP glycosylation occurs in the Golgi apparatus by the concerted action of different glycosyltransferases (GTs), and it has been proposed that at least ten GT activities are required for type II AG biosynthesis (Knoch, Dilokpimol, & Geshi, 2014). To date several glycosyltransferases have been described to function in AG synthesis. Among these activities, three members of the GT14 family have been reported to have β-1,6-glucuronyltransferase activity (GlcAT14A, GlcAT14B, GlcAT14C). The biological role of GlcA and MeGlcA modifications on AGPs is unclear. However, a mutant in GlcAT14A was reported to have a seedling growth phenotype (Knoch et al., 2014). While GlcA on AGs is frequently 4-O-methylated (Tryfona et al., 2012), to date an AGP O-methyltransferase has not been identified.  (Lee et al., 2012;Li et al., 2013;Urbanowicz et al., 2012)  Os02G0 6380 A tr 0 0 0 9 9 G 0 0 0 1 0 Zm 05 G 13 72 0  Clade III DUF579 family member, is also a Golgi localized protein (Nikolovski et al., 2012). To confirm the proteomics data, and investigate its sub-cellular localization more closely, we transiently expressed AGM1 fused to GFP in tobacco leaves. AGM1-GFP co-localized with the Golgi marker ST-RFP, confirming the localization in the Golgi apparatus ( Figure 1b). Additional small punctate signals were also identified, as previously noted for IRX15 proteins (Brown et al., 2011).   Figure S1) and analyzed the xylan structure of agm1, agm2 and agm1 agm2 double mutants, using Xyn11A xylanase fin- In our growth conditions agm1 agm2 did not display any obvious growth phenotypes, showing that AG methylation is not essential for viability. It has been also reported that some AGP mutants show a hypocotyl length phenotype, but we did not detect differences in | 3 hypocotyl length in etiolated plants (Supporting information Figure S5).
While we do not observe obvious fertility differences in these mutants, it has recently been shown that AG provides a signaling molecule in pollen tube guidance and the methyl group of GlcA in the AG is critical for the effectiveness of the signal (Mizukami et al., 2016).
There are other potential roles for the methyl group on GlcA. For example, GlcA on AGs can chelate calcium ions (Lamport & Varnai, 2013). 4-O-methylation of GlcA would change the calcium binding affinity, thus modulating the calcium release response to pH. Moreover, the addition of the methyl group to GlcA prevents the addition of 4-linked sugars, such as rhamnose, and will also prevent extension of 4-linked side chains to the GlcA of AG as seen in APAP1 (Tan et al., 2013). Therefore, modulating the AG structure through the activity of AGMs may provide a means of adapting the AG structure to diverse physiological processes.

CONF LICT OF I NTEREST
The authors declare no conflict of interest associated with the work described in this manuscript.

S U P P O R T I N G I N F O R M A T I O N
Additional supporting information, including materials and methods, may be found online in the Supporting Information section at the end of the article.