ABSTRACT
The Ajuga reptans L. galactan:galactan galactosyltransferase (ArGGT) is a vacuolar enzyme that synthesizes long-chain raffinose family oligosaccharides (RFOs), the major storage carbohydrates of this plant. ArGGT is structurally and functionally related to acid plant alpha-galactosidases (alpha-Gals) of the glycosylhydrolase family 27, present in the apoplast or the vacuole. Sequence comparison of acid alpha-Gals with ArGGT revealed that they all contain an N-terminal signal sequence and a highly similar core sequence. Additionally, ArGGT and some acid alpha-Gals contain C-terminal extensions with low sequence similarities to each other. Here, we show that the C-terminal pentapeptide, SLQMS, is a non-sequence-specific vacuolar sorting determinant. Analogously, we demonstrate that the C-terminal extensions of selected acid alpha-Gals from Arabidopsis, barley, and rice, are also non-sequence-specific vacuolar sorting determinants, suggesting the presence of at least one vacuolar form of acid alpha-Gal in every plant species.
Subject(s)
Galactosyltransferases/chemistry , Galactosyltransferases/metabolism , Protein Sorting Signals , Vacuoles/metabolism , Ajuga/enzymology , Amino Acid Sequence , Molecular Sequence Data , Protein Structure, Tertiary/physiology , Protein Transport , Sequence Homology, Amino AcidABSTRACT
The concentration of cytochrome P450 and ecdysone 20-monooxygenase activity in plants and callus cell culture of the bugleweed Ajuga reptans L. were determined. The maximal ecdysone 20-monooxygenase activity of cytochrome P450 was found in vegetative rosettes of intact plants. During the stage of flowering, the ecdysone 20-monooxygenase activity of cytochrome P450 in plant leaves was higher than in other organs. It was demonstrated that the content of ecdysteroids in callus cell culture is higher than in the intact plant with concurrent retention of a high ecdysone-20-monooxygenase activity.
Subject(s)
Ajuga/enzymology , Aryl Hydrocarbon Hydroxylases/metabolism , Cytochrome P-450 Enzyme System/metabolism , Steroid Hydroxylases/metabolism , Aryl Hydrocarbon Hydroxylases/chemistry , Cells, Cultured , Flowering Tops/enzymology , Plant Leaves/enzymology , Seasons , Steroid Hydroxylases/chemistryABSTRACT
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.
Subject(s)
Ajuga/enzymology , Galactosyltransferases/genetics , Plant Leaves/enzymology , Raffinose/metabolism , Ajuga/chemistry , Ajuga/genetics , Amino Acid Sequence , Cloning, Molecular , Cold Temperature , DNA, Complementary/chemistry , DNA, Complementary/genetics , Galactosyltransferases/metabolism , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Plant , Molecular Sequence Data , Phylogeny , Plant Leaves/chemistry , Plant Leaves/genetics , Plant Proteins/genetics , Plant Proteins/metabolism , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Nicotiana/enzymology , Nicotiana/geneticsABSTRACT
Three new withanolides, bracteosin A (= (22R)-5beta,6beta : 22,26-diepoxy-4beta,28-dihydroxy-3beta-methoxyergost-24-ene-1,26-dione; 1), bracteosin B (= (22R)-5beta,6beta : 22,26-diepoxy-4beta,28-dihydroxy-3beta-methoxy-1,26-dioxoergost-24-en-19-oic acid; 2), and bracteosin C (= (22R)-22,26-epoxy-4beta,6beta,27-trihydroxy-3beta-methoxyergost-24-ene-1,26-dione; 3), have been isolated from the whole plants of Ajuga bracteosa. Their structures were deduced by spectral analysis, including 1D- and 2D-NMR techniques. In addition, dihydroclerodin-1, clerodinin A, lupulin A, and dihydroajugapitin have also been isolated for the first time from this species. Compounds 1-3 exhibited evident inhibitory potential against cholinesterase enzymes in a concentration-dependent fashion.
