SeeDev-binary@ldeleger:SeeDev-binary-14701918-4 JSONTXT

Our data support the hypothesis that MUM4 encodes an enzyme involved in RGI biosynthesis. First, analysis of mum4 mucilage using antibodies and gas chromatography have demonstrated a significant reduction in RGI and its composite backbone monosaccharides Rha and GalUA in comparison with wild-type seeds. Second, cloning of MUM4 revealed a putative protein containing similarity to bacterial nucleotide sugar interconversion enzymes, suggesting that it, too, is required for the production of activated sugars. The N-terminal portion of MUM4 is most similar to bacterial dTDP-d-Glc 4,6-dehydratases, the first of three enzymes required for the conversion of dTDP-d-Glc to dTDP-l-Rha (Tonetti et al., 1998). These data favor a role for MUM4 in the synthesis of NDP-l-Rha, a key step in the production of RGI. A reverse genetics approach taken by another group has come to a similar conclusion regarding the putative enzymatic function of MUM4 (also designated RHM2 [RHAMNOSE BIOSYNTHESIS 2]; B. Usadel et al., 2004). However, another role in RGI biosynthesis cannot be ruled out without evidence of enzymatic activity in the conversion of NDP-d-Glc to NDP-l-Rha. Gram-negative bacteria such as Escherichia coli encode three separate enzymes (4,6-dehydratase, 3,5-epimerase, and 4-reductase) to convert dTDP-d-Glc to dTDP-l-Rha (Tonetti et al., 1998). The putative MUM4 protein not only contains an N-terminal domain with similarity to the bacterial dTDP-d-Glc 4,6-dehydratases but also a C-terminal domain with some similarity to 4-reductases (this study; Reiter and Vanzin, 2001). There is a precedent in Arabidopsis for a bifunctional 3,5 epimerase, 4-reductase in the synthesis of GDP-l-Fuc from GDP-d-Man (GER1, GER2) (Bonin and Reiter, 2000). Therefore, based on sequence analysis, it is attractive to postulate a single, multifunctional protein acting in the conversion of NDP-d-Glc to NDP-l-Rha in Arabidopsis (Reiter and Vanzin, 2001). MUM4 is a member of a small gene family consisting of three genes (RHM1, MUM4/RHM2, and RHM3; Reiter and Vanzin, 2001) that exhibit high identity at both nucleotide and amino acid levels. The ubiquitous expression of RHM1 and RHM3 can be postulated to provide a mechanism for the formation of the normal primary cell wall in mum4 mutants and the residual mucilage present in mum4 seed coats. Redundancy in genes coding for putative NDP-l-Rha synthases is unsurprising because of the importance of the Rha-containing pectins RGI and RGII in primary cell walls. In fact, the presence of multiple genes coding enzymes involved in nucleotide sugar interconversions is common in plants (Reiter and Vanzin, 2001).