In vivo structural modification of type II arabinogalactans with fungal endo-ß-1, 6-galactanase in Arabidopsis.

Arabinogalactan-proteins (AGPs) are mysterious extracellular glycoproteins in plants. Although AGPs are highly conserved, their molecular functions remain obscure. The physiological importance of AGPs has been extensively demonstrated with ß-Yariv reagent, which specifically binds to AGPs and upon introduction into cells, causes various deleterious effects including growth inhibition and programmed cell death. However, structural features of AGPs that determine their functions have not been identified with ß-Yariv reagent. It is known that AGPs are decorated with large type II arabinogalactans (AGs), which are necessary for their functions. Type II AGs consist of a ß-1,3-galactan main chain and ß-1,6-galactan side chains with auxiliary sugar residues such as L-arabinose and 4-O-methyl-glucuronic acid. While most side chains are short, long side chains such as ß-1,6-galactohexaose (ß-1,6-Gal6) also exist in type II AGs. To gain insight into the structures important for AGP functions, in vivo structural modification of ß-1,6-galactan side chains was performed in Arabidopsis. We generated transgenic Arabidopsis plants expressing a fungal endo-ß-1,6-galactanase, Tv6GAL, that degrades long side chains specifically under the control of dexamethasone (Dex). Two of 6 transgenic lines obtained showed more than 40 times activity of endo-ß-1,6-galactanase when treated with Dex. Structural analysis indicated that long side chains such as ß-1,6-Gal5 and ß-1,6-Gal6 were significantly reduced compared to wild-type plants. Tv6GAL induction caused retarded growth of seedlings, which had a reduced amount of cellulose in cell walls. These results suggest that long ß-1,6-galactan side chains are necessary for normal cellulose synthesis and/or deposition as their defect affects cell growth in plants.

Expression of a fungal exo-ß-1,3-galactanase in Arabidopsis reveals a role of type II arabinogalactans in the regulation of cell shape.

Arabinogalactan-proteins (AGPs) are a family of plant extracellular proteoglycans implicated in many physiological events. AGP is decorated with type II arabinogalactans (AGs) consisting of a ß-1,3-galactan backbone and ß-1,6-galactan side chains, to which other sugars are attached. Based on the fact that a type II AG-specific inhibitor, ß-Yariv reagent, perturbs growth and development, it has been proposed that type II AGs participate in the regulation of cell shape and tissue organization. However, the mechanisms by which type II AGs participate have not yet been established. Here, we describe a novel system that causes specific degradation of type II AGs in Arabidopsis, by which a gene encoding a fungal exo-ß-1,3-galactanase that specifically hydrolyzes ß-1,3-galactan backbones of type II AGs is expressed under the control of a dexamethasone-inducible promoter. Dexamethasone treatment increased the galactanase activity, leading to a decrease in Yariv reagent-reactive AGPs in transgenic Arabidopsis. We detected the typical oligosaccharides released from type II AGs by Il3GAL in the soluble fraction, demonstrating that Il3GAL acted on type II AG in the transgenic plants. Additionally, this resulted in severe tissue disorganization in the hypocotyl and cotyledons, suggesting that the degradation of type II AGs affected the regulation of cell shape.

Structural features conserved in subclass of type II arabinogalactan.

Arabinogalactan-proteins (AGPs) are extracellular proteoglycans, which are presumed to participate in the regulation of cell shape, thus contributing to the excellent mechanical properties of plants. AGPs consist of a hydroxyproline-rich core-protein and large arabinogalactan (AG) sugar chains, called type II AGs. These AGs have a ß-1,3-galactan backbone and ß-1,6-galactan side chains, to which other sugars are attached. The structure of type II AG differs depending on source plant, tissue, and age. Type II AGs obtained from woody plants in large quantity as represented by gum arabic and larch AG, here designated gum arabic-subclass, have a ß-1,3;1,6-galactan structure in which the ß-1,3-galactan backbone is highly substituted with short ß-1,6-galactan side chains. On the other hand, it is unclear whether type II AGs found as the glycan part of AGPs from herbaceous plants, here designated AGP-subclass, also have conserved ß-1,3:1,6-galactan structural features. In the present study we explore similarities of type II AG structures in the AGP-subclass. Type II AGs in fractions obtained from spinach, broccoli, bok choy, komatsuna, and cucumber were hydrolyzed into galactose and ß-1,6-galactooligosaccharides by specific enzymes. Based on the proportion of these sugars, the substitution ratio of the ß-1,3-galactan backbone was calculated as 46-58% in the five vegetables, which is consistently lower than what is seen in gum arabic and larch AG. Although most side chains were short, long chains such as ß-1,6-galactohexaose chains were also observed in these vegetables. The results suggest a conserved ß-1,3;1,6-galactan structure in the AGP-subclass that distinguishes it from the gum arabic-subclass.

Yariv reactivity of type II arabinogalactan from larch wood.

Larch arabinogalactan (AG) is classified as a plant type II AG. Its basic structure is constituted by a ß-1,3-galactan main chain with ß-1,6-galactan side chains. But its properties are distinct from other type II AGs. Whereas most type II AGs are found as glycan moieties of arabinogalactan-protein (AGP), larch AG lacks a protein moiety. Larch AG itself is also unlike other type II AGs as it lacks Yariv reactivity, the capability of AG to form insoluble precipitate with ß-Yariv reagents, 1,3,5-tri-(p-glycosyloxyphenylazo)-2,4,6-trihydroxybenzene with ß-glucosyl or ß-galactosyl residues at the termini. For the present study, we prepared ß-galactan I, II, and III from larch AG by performing single, double, and triple Smith degradation, which breaks ß-1,6-galactan side chains, and examined Yariv reactivity of the products. Methylation analysis revealed that ß-galactans II and III had lost more than 90% of their ß-1,6-galactan branches. In the radial gel diffusion assay, ß-galactans II and III showed Yariv reactivity, indicating the presence of a Yariv-reactive structure in larch AG, although native larch AG does not have Yariv reactivity. The Yariv reactivity of the ß-galactans was completely lost after treatment with endo-ß-1,3-galactanase. These results confirm that ß-1,3-galactan is necessary for Yariv reactivity of type II AG. The present results also suggest that high substitution of ß-1,3-galactan with ß-1,6-galactan side chains affects Yariv reactivity in larch AG.