RG-I contains L-arabinose in multiple linkages (see Table 5.2). Most of the arabinose is in the furanose ring form, although a terminal arabinose exists in the pyranose form in some RG-I side chains (268). AraT activity was originally identified in microsomes from mung bean (Phaseolus aureus) (388) and bean (Phaseolus vulgaris) (389) although definitive evidence that those AraT activities were involved in pectin synthesis was not demonstrated [see Ref. (205) for review]. The bean AraT activity was primarily associated with enriched Golgi, and to a lesser extent enriched endoplasmic reticulum (390).

The study of AraTs specifically involved in pectin synthesis has been problematic for several reasons. Multiple wall polysaccharides and proteoglycans contain arabinose, in­cluding pectin, hemicelluloses (e. g., glucuronoarabinoxylan), and arabinogalactan proteins. Thus, experiments aimed at studying pectin biosynthetic AraTs by incubating microsomal membranes with radiolabeled UDP-Ara have not been very successful. The nucleotide — sugar donor, UDP-Ara, was not available and had to be synthesized (391), although more recently the UDP-p-L-arabinopyranose form has become available through CarbSource (http://www. ccrc. uga. edu/~carbosource/CSS_home. html). However, while most Ara in pectin is in the furanose form, the nucleotide-sugar synthesized by the 4-epimerization of UDP-a-D-Xyl is UDP-p-L-arabinopyranose. Thus, this has been the nucleotide-sugar form most readily available for experimental use. However, there is uncertainty as to the nature of the nucleotide-sugar substrate used for pectin synthesis. Is it UDP-p-L- arabinopyranose (UDP-Arap) or UDP-p-L-arabinofuranose (UDP-Ara/)? IfitisUDP-p-L — arabinofuranose, how is this synthesized by the plant and, experimentally, what is the most facile way to produce it? Is it synthesized by enzyme-catalyzed ring contraction of UDP-l- arabinopyranose by a mutase (392)? Indeed, recently, Ishii and colleagues (393) identified a UDP-arabinopyranose mutase (UAM) from rice that catalyzes the reversible formation of UDP-Ara/ from UDP-Arap. Interestingly, UAMs are the same proteins that previously were identified as reversibly glycosylated polypeptides (RGPs), proteins that are reversibly glycosylated in the presence of UDP-Glc and several other nucleotide-sugars (394-396). The significance of the reverse glycosylation detected in vitro, in regards to the role(s) of UAM in wall synthesis, remains to be determined.

Recent efforts to investigate the AraT activity in mung bean confronted some of the above-mentioned problems. Incubation of mung bean microsomal membranes with UDP — p-L-[14C]arabinopyranose (UDP-[14C]Ara) resulted in the incorporation ofboth [14C]Ara and [14C]Xyl into elongated endogenous acceptors because of the epimerization of some of the UDP-[14C]Ara into UDP-[ 14C]Xylby a UDP-Xyl-4-epimerase present in the microsomal fraction (397). Furthermore, digestion of the radiolabeled product synthesized in the micro — somes with endo-arabinase yielded very little radiolabeled Ara or arabinose-containing small oligosaccharides, suggesting that the conditions used were not conducive for the synthesis of arabinans. Conversely, digestion of the product with arabinofuranosidase did release some [14C]Ara, indicating that an enzyme activity that could add at least a single Ara residue was present in the microsomes. A breakthrough in identifying pectin biosynthetic AraTs came when detergent-solubilized microsomal membranes and defined arabinooligosaccha — rides were used as acceptors for study of pectin biosynthetic AraTs (397). The incubation of detergent-solubilized microsomal membranes with (1—5)-a-L-arabinooligosaccharides of DP 5-8 and with UDP-p-L-[14C]arabinopyranose led to the identification of an AraT activity that could transfer a single arabinopyranose residue onto the non-reducing end of a1,5-arabinooligosaccharide acceptors. The enzyme had a pH optimum of 6.5 and was shown to reside predominantly in the Golgi by subcellular fractionation of organelles (397). The anomeric configuration and linkage of the arabinopyranose residue transferred by the mung bean AraT was subsequently shown to be (3-(1—>3) through the use of fluorescently labeled a-L-arabinooligosaccharide acceptors (271). Thus, mung bean contains an a1,5- arabinan:p-(1—3)arabinopyranose AraT (271).

A second mungbean arabinopyranosetransferase activity was identified (273) that could transfer an individual arabinopyranosyl residue from UDP-p-L-[14C]arabinopyranose onto the non-reducing end of fluorescently labeled 1,4-linked p-D-galactooligosaccharides of DP 3-7, with significantly better activity manifested with 1,4-linked p-D — galactooligosaccharides of DP 5 or greater. The p-1,4-galactan:AraT activity transferred the Ara residue in an a configuration on the O-4 position of the galactooligosaccharides, identi­fying the AraT as a p-1,4-galactan:a1,4AraT. The enzyme had a pH optimum of 6.0-6.5 and apparent Km(s) for UDP-p-L-[14C]arabinopyranose and fluorescently labeled galactohep — tasaccharide of 330 pM and 45 pM, respectively. Interestingly, the enzyme would not use fluorescently labeled galactooligosaccharides of DP 6-10 that had a single a-L-Arap residue at the non-reducing end as acceptors for the previously described p -1,4-galactan: p -1,4-GalT (386), indicating that the enzyme cannot use mono-arabinosylated galactooligosaccharides as acceptors. The authors propose that the presence of the a-L-arabinopyranosyl residue on the p-1,4-galactan oligosaccharides prevents further galactosylation of the galactooligosac­charides (273).

Recently, a gene encoding a putative arabinan:a-1,5-arabinosyltransferase (ARAD1; At2g35100) has been identified in Arabidopsis (270) through analysis of the CAZy GT47 family glycosyltransferase gene At2g35100 (138) (http://afmb. cnrs-mrs. fr/CAZY/) and phe­notypic biochemical and immunochemical analyses of the corresponding Arabidopsis T — DNA insert mutant ARABINAN DEFICIENT 1. ARAD1 encodes a protein with a predicted molecular mass of 52.8 kDa and a single transmembrane helix region near the N-terminus.

Although homozygous knockout mutants of ARAD1 show no visible growth differences from wild type, isolated walls from mutant leaves and stems had 25 and 54%, respectively, reduced levels of Ara compared to wild type walls (270). Transformation of the mutant plant with the ARAD1 gene complemented the mutant phenotype (i. e. restored the amount of Ara in the wall to wild type levels), thus providing evidence that ARAD1 affects wall arabinose levels. Immunocytochemical analysis of leaf, inflorescence stem, and stem revealed a reduc­tion in immunolabeling with the anti-a-1,5-arabinan antibody LM6. A lack of difference between the labeling of protein extracts from wild type and mutant with LM6 suggested that the mutant was affected in the synthesis of a-1,5-arabinans, but not in glycoprotein synthesis (270). This observation was confirmed by comparison of RG-I isolated from wild type and ARAD1 walls. Mutant RG-I had a 68% reduction in Ara content, which linkage data showed was predominantly due to a reduction in 5-linked Ara/ and also in 2,5 f — linked Ara and 2,3,5-linked Araf. These results strongly suggest that ARAD1 is a putative RG-I arabinan:a-1,5-arabinosyltransferase. Confirmation of this activity will require expression of enzymatically active enzyme expressing a-1,5-arabinosyltransferase activity.

Recently, a novel approach to identify genes involved in cell wall synthesis has been taken

(398) and offers promise in leading to the positional cloningfor a gene that affects the number of arabinan side chains in RG-I. The method takes advantage of the natural variation that occurs in cell wall synthesis in natural plant populations and of the availability of Arabidopsis recombinant inbred line (RIL) populations which facilitate the identification and cloning of quantitative trait loci (QTLs). Through the use of multiple techniques to analyze cell walls of an RIL population from a cross between Arabidopsis Bay-0 and Shahdara, including global monosaccharide composition and Fourier-transform infrared (FTIR) microspectroscopy, a major QTL was identified that accounted for 51% of the heritable variation observed for the arabinose-rhamnose ratio in the cell walls, a difference that appeared to be due to variation in the amount of RG-linked arabinan. Whether this strategy will lead to the identification of RG-I biosynthetic AraTs remains to be shown.

Two Arabidopsis putative arabinosyltransferases, designated reduced residual arabinose — 1 and -2 (RRA1; At1g75120 and RRA2; At1g75110) were recently identified by Egelund et al.

(399) based on a 20% reduction in the arabinose content in pectin — and xyloglucan-depleted cell wall fractions from meristematic tissue of rra1 and rra2 mutant plants. However, whether these genes, which are classified in CAZy family GT77, encode functional arabinosyltrans­ferases, and if so, whether they function in pectin, arabinoxylan, wall structural protein, or other syntheses, remains to be determined.