Cloning, Functional Expression, and Characterization of the Raffinose Oligosaccharide Chain Elongation Enzyme, Galactan:Galactan Galactosyltransferase, from Common Bugle Leaves^{<a href="#fn1" rid="fn1" class=" fn">1</a>}

Abstract

Raffinose family oligosaccharides (RFOs) are sucrosyl galactosides, in which the Gal residues are linked in an α-1,6 fashion to the C-6 of the Glc moiety of Suc. These water-soluble, nonreducing carbohydrates are widespread in the plant kingdom. In many cases, they are direct photosynthetic products and are used for storage, translocation, and utilization of carbon as well as for protection against different abiotic stresses such as those caused by frost, drought, and salt (for review, see Kandler and Hopf, 1984; Keller and Pharr, 1996; Avigad and Dey, 1997; Peterbauer and Richter, 2001). The best-studied and most widespread RFOs are the two short-chain RFOs, the trisaccharide raffinose (Gal₁-Suc) and the tetrasaccharide stachyose (Gal₂-Suc). Much less is known about the long-chain RFOs, which may occur with degrees of polymerization (DPs) of up to 15 (Bachmann et al., 1994). The focus of our recent research efforts has, therefore, been the study of the physiology and metabolism of the whole range of RFOs. One plant species that allows such studies is the common bugle (Ajuga reptans). This frost-hardy, perennial labiate runs on both the short-chain as well as the long-chain RFOs. It produces, translocates, and stores RFOs and might even use them as antifreeze or antisalt stress agents (Bachmann et al., 1994; Bachmann and Keller, 1995; Gilbert et al., 1997; Sprenger and Keller, 2000; Inan Haab and Keller, 2002).

While the biosynthetic pathway of raffinose and stachyose is well established and known to be galactinol (α-galactosyl-myoinositol) dependent, a novel galactinol-independent pathway responsible for the synthesis of long-chain RFOs was recently described (Bachmann et al., 1994; Inan Haab and Keller, 2002). RFO chains are elongated from raffinose and stachyose by the activity of the enzyme galactan:galactan galactosyltransferase (GGT). GGT catalyzes the direct transfer of a terminal Gal residue from one RFO molecule to another, resulting in the next higher and lower RFO oligomers, respectively. This soluble galactosyltransferase is a glycoprotein, has an acid pH optimum, and resides in the vacuole. Its activity correlates positively with the accumulation of long-chain RFOs (Braun and Keller, 2000; Inan Haab and Keller, 2002). Such a galactinol-independent mode of RFO chain elongation has only been described in two other instances so far. Coleus blumei leaves clearly showed increased GGT activities when plants were salt stressed in their root zones (Gilbert et al., 1997). Secondly, a GGT-like activity was observed in maturing pea (Pisum sativum) seeds producing verbascose (Gal₃-Suc) from stachyose alone, but it showed an atypical neutral pH optimum (Peterbauer et al., 2001). Subsequent detailed studies revealed that a multifunctional stachyose synthase, not a bona fide GGT, was responsible for RFO chain elongation in pea seeds (Peterbauer et al., 2002, 2003).

To further the understanding of the physiological importance of GGT, we have continued to study the common bugle. In this article, we report on the successful cloning of GGT, based on peptide sequence information derived from the major 48-kD band of the purified GGT preparation of the common bugle published previously (Inan Haab and Keller, 2002). We functionally expressed and characterized recombinant GGT in tobacco (Nicotiana plumbaginifolia) protoplasts and showed that GGT transcript levels and enzyme activities, as well as long-chain RFO concentrations, clearly increased in cold-treated plants. The results favor the conclusion that GGT plays a key role in long-chain RFO formation in the common bugle.

Reaction mixtures contained galactosyl donors and acceptors at concentrations of 50 mm (sugars) and approximately 50 m (water), respectively. Empty vector controls were used to correct for the endogenous activities of the tobacco protoplasts. Products were analyzed by HPLC-PAD on a Benson BC-200 column. ND, Not detected; ppl, protoplasts.

Acknowledgments

Notes

^{This work was supported by the Swiss National Foundation.}

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.036210.