Cloning, functional expression, and characterization of the raffinose oligosaccharide chain elongation enzyme, galactan:galactan galactosyltransferase, from common bugle leaves

Abstract

Galactan:galactan galactosyltransferase (GGT) is a unique enzyme of the raffinose family oligosaccharide (RFO) biosynthetic pathway. It catalyzes the chain elongation of RFOs without using galactinol (alpha-galactosyl-myoinositol) by simply transferring a terminal alpha-galactosyl residue from one RFO molecule to another one. Here, we report the cloning and functional expression of a cDNA encoding GGT from leaves of the common bugle (Ajuga reptans), a winter-hardy long-chain RFO-storing Lamiaceae. The cDNA comprises an open reading frame of 1215 bp. Expression in tobacco (Nicotiana plumbaginifolia) protoplasts resulted in a functional recombinant protein, which showed GGT activity like the previously described purified, native GGT enzyme. At the amino acid level, GGT shows high homologies (>60%) to acid plant alpha-galactosidases of the family 27 of glycosylhydrolases. It is clearly distinct from the family 36 of glycosylhydrolases, which harbor galactinol-dependent raffinose and stachyose synthases as well as alkaline alpha-galactosidases. Physiological studies on the role of GGT confirmed that GGT plays a key role in RFO chain elongation and carbon storage. When excised leaves were exposed to chilling temperatures, levels of GGT transcripts, enzyme activities, and long-chain RFO concentrations increased concomitantly. On a whole-plant level, chilling temperatures induced GGT expression mainly in the roots and fully developed leaves, both known RFO storage organs of the common bugle, indicating an adaptation of the metabolism from active growth to transient storage in the cold.

Figure 1.

Comparison of the deduced amino acid sequence of GGT (ArGGT-1) from common bugle source leaves with highly homologous plant α-Gals from C. papaya (CpAGAL; accession no. AAP04002), rice (OsAGAL; accession no. BAB12570/1UAS), P. vulgaris (PvAGAL; accession no. AAA73964), and C. arabica (CaAGAL; accession no. AAA33022). Numbers indicate codons of ArGGT-1. Underlined amino acid residues in ArGGT-1 indicate the identified peptide sequences from the purified common bugle GGT and were used to design degenerate DNA primers. The α-Gal motif is marked with asterisks. Of the rice active site, the nucleophile Asp is marked by a bold arrow and the Asp functioning as the general acid/base catalyst by a normal arrow. A secretory signal peptide of the C. arabica precursor peptide is indicated by bold italic letters; putative secretory signals of the other peptides are in italics. Amino acids identical to the ArGGT-1 sequence are boxes in black. Gaps, marked by dots, are included for a better match. The sequence reported here has been deposited in the GenBank database (ArGGT-1; accession no. AY386246). The alignment was generated using PileUp/GCG.

Figure 2.

DNA gel blot analysis of GGT in common bugle leaves. Genomic DNA of common bugle (10 μg per lane) was digested with different restriction enzymes, blotted, and hybridized with a 250-bp radiolabeled cDNA fragment from the GGT 3′ end, including 148 bp from the 3′ UTR. Lane 1, HindIII; lane 2, XhoI; lane 3, undigested; and lane 4, DNA M_r marker (kb).

Figure 3.

Unrooted phylogenetic tree containing α-galactoside hydrolase and galactosyltransferase cDNA-derived amino acid sequences from plants. Phylogenetic analysis was performed by ClustalX (http://inn-prot.weizmann.ac.il/software/ClustalX.html), and the unrooted tree was drawn with the TreeView32 software (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html). Four subgroups can be discerned. Group I, acid α-Gals and GGT, including C. arabica (Ca, AAA33022), Cyamopsis tetragonoloba (Ct, AAE69558), Glycine max (Gm, AAA73963), P. vulgaris (Pv, AAA73964), Lycopersicum esculentum (Le, AAF04591), Helianthus annuus (Ha, BAC66445), rice (Os2, BAB12570/1UAS), and common bugle (Ar1, AY386246), as well as sequences homologous to α-Gal or GGT, respectively, including Arabidopsis (At1, CAC08338; At2, NP_568193; At3, CAC08337), C. papaya (Cp, AAP04002), and rice (Os1, AC023240). Group II, alkaline α-Gals, including Cucumis melo (Cm1, AY114164; Cm2, AY114165) and L. esculentum (Le, AAN32945), as well as seed imbibition proteins (SIPs) of Arabidopsis (At1, NP_175970; At2, CAB66109), Brassica oleracea (Bo, CAA55893), Hordeum vulgare (Hv, AAA32975), Persea americana (Pa, CAB77245), and rice (Os1, CAD41092; Os2, BAC82968). Group III, raffinose synthase (RS), including sequences of Cucumis sativus (Cm, AAD02832) and pea (Ps, CAD20127), as well as sequences homologous to RS, including Arabidopsis (At, BAB11595) and rice (Os, BAB64768). Group IV, stachyose synthase (STS) and verbascose synthase (VS), including Alonsoa meridionalis (Am, CAD31704), pea (Ps1-STS_VS, CAC38094; Ps2-STS, CAD55555), Stachys affinis (Sa, CAC86963), and Vigna angularis (Va, CAB64363), as well as the sequence homologous to STS of Arabidopsis (At, AAD22659). Family 27 of glycosylhydrolase (according to the CAZY database, http://afmb.cnrs-mrs.fr/CAZY/) includes the newly characterized GGT and eukaryotic acid α-Gals (group I). Family 36 of glycosylhydrolases includes eukaryotic alkaline α-Gals, SIPs, and RS and STS genes (groups II, III, and IV), respectively. Scale bar indicates distance value of 0.1 substitutions per site.

Figure 4.

HPLC-PAD chromatograms showing enzymatic activities of transfected tobacco protoplasts. Extracted and desalted enzyme samples were incubated with 50 mm stachyose for 30, 60, and 120 min in McIlvaine buffer, pH 5.0. A, Protoplasts transfected with GGT-1, producing mainly verbascose and raffinose. The melibiose and the Fru peaks originate from intrinsic tobacco protoplast invertase activity (high on raffinose and low on stachyose as substrates). B, Empty vector control, showing intrinsic invertase activity. IS, internal standard.

Figure 5.

GGT mRNA levels, carbohydrate concentrations, and GGT enzyme activities from pools of cold-grown excised leaves of common bugle plants. A, Radiosignals of ³²P-labeled GGT hybridized on RNA gel blots and ³²P-labeled 400-bp fragment of 26S rRNA as a control of the total RNA loaded. B–E, Carbohydrate concentration and extractable GGT activity of cold-grown (8°/3°C day/night, 12 h photoperiod) excised leaves. Concentrations of carbohydrates were determined by HPLC-PAD. Enzyme activity was measured in desalted crude enzyme extracts incubated with 50 mm stachyose as the substrate. Values are means ± se of four replicates with pools of 12 leaves each. Leaf size ranged from 4 to 9 cm².

Figure 6.

GGT mRNA levels in different parts of common bugle plants grown for 6 weeks in the warm (22°C) or the cold (4°C). Radiosignals of ³²P-labeled GGT hybridized on RNA gel blots and ³²P-labeled 400-bp fragment of 26S rRNA as a control of the total RNA loaded.