The Cell Wall Arabinose-Deficient Arabidopsis thaliana Mutant murus5 Encodes a Defective Allele of REVERSIBLY GLYCOSYLATED POLYPEPTIDE2

Abstract

Traditional marker-based mapping and next-generation sequencing was used to determine that the Arabidopsis (Arabidopsis thaliana) low cell wall arabinose mutant murus5 (mur5) encodes a defective allele of REVERSIBLY GLYCOSYLATED POLYPEPTIDE2 (RGP2). Marker analysis of 13 F2 confirmed mutant progeny from a recombinant mapping population gave a rough map position on the upper arm of chromosome 5, and deep sequencing of DNA from these 13 lines gave five candidate genes with G→A (C→T) transitions predicted to result in amino acid changes. Of these five, only insertional mutant alleles of RGP2, a gene that encodes a UDP-arabinose mutase that interconverts UDP-arabinopyranose and UDP-arabinofuranose, exhibited the low cell wall arabinose phenotype. The identities of mur5 and two SALK insertional alleles were confirmed by allelism tests and overexpression of wild-type RGP2 complementary DNA placed under the control of the 35S promoter in the three alleles. The mur5 mutation results in the conversion of cysteine-257 to tyrosine-257 within a conserved hydrophobic cluster predicted to be distal to the active site and essential for protein stability and possible heterodimerization with other isoforms of RGP.

Molecular characteristics of plant UDP-arabinopyranose mutases

“…The presence of a Rossmann fold is supported by 3D modeling, since both StRGP and AtRGP2 have been threaded on glycosyltransferase templates containing such a nucleotide-binding domain (De Pino et al. 2007; Dugard et al. 2016).…”

“…2010). Other noteworthy amino acids are Cys257 of AtRGP2, which has been found to be mutated to a tyrosine in the cell wall arabinose-deficient Arabidopsis mutant murus5 , most likely impacting AtRGP2 folding or oligomerization (Dugard et al. 2016), and a conserved arginine adjacent to the autoglycosylated residue (Arg165 in OsUAM1), which was shown to be essential for mutase activity (Konishi, Ohnishi-Kameyama et al.…”

“…Secondary structure predictions and hydrophobic cluster analyses indicate that UAMs contain both α-helices and β-sheets, and that at least class 1 UAMs could adopt a Rossmann fold commonly found in nucleotide binding proteins (De Pino et al. 2007; Dugard et al. 2016; Kuttiyatveetil and Sanders 2017).…”

“…A search for functional homologs using the ab initio model did, however, yield glycosyltransferase hits with Rossmann folds (Kuttiyatveetil and Sanders 2017). Given the similar reaction catalyzed by UAMs and UGMs, Dugard et al. (2016) also did an attempt to model AtRGP2 on an Escherichia coli UGM template, despite the low sequence identity (21%).…”

“…The discovery of UDP-Ara f transporters (UAfTs) in the Golgi membrane (Rautengarten et al. 2017), together with recent genotype/phenotype studies (Dugard et al. 2016; Willis et al.…”