Nucleoside 5′-Phosphoramidates Control the Phenylpropanoid Pathway in Vitis vinifera Suspension-Cultured Cells

It is known that cells contain various uncommon nucleotides such as dinucleoside polyphosphates (NpnN’s) and adenosine 5′-phosphoramidate (NH2-pA) belonging to nucleoside 5′-phosphoramidates (NH2-pNs). Their cellular levels are enzymatically controlled. Some of them are accumulated in cells under stress, and therefore, they could act as signal molecules. Our previous research carried out in Arabidopsis thaliana and grape (Vitis vinifera) showed that NpnN’s induced the expression of genes in the phenylpropanoid pathway and favored the accumulation of their products, which protect plants against stress. Moreover, we found that NH2-pA could play a signaling role in Arabidopsis seedlings. Data presented in this paper show that exogenously applied purine (NH2-pA, NH2-pG) and pyrimidine (NH2-pU, NH2-pC) nucleoside 5′-phosphoramidates can modify the expression of genes that control the biosynthesis of both stilbenes and lignin in Vitis vinifera cv. Monastrell suspension-cultured cells. We investigated the expression of genes encoding for phenylalanine ammonia-lyase (PAL1), cinnamate-4-hydroxylase (C4H1), 4-coumarate:coenzyme A ligase (4CL1), chalcone synthase (CHS1), stilbene synthase (STS1), cinnamoyl-coenzyme A:NADP oxidoreductase (CCR2), and cinnamyl alcohol dehydrogenase (CAD1). Each of the tested NH2-pNs also induced the expression of the trans-resveratrol cell membrane transporter VvABCG44 gene and caused the accumulation of trans-resveratrol and trans-piceid in grape cells as well as in the culture medium. NH2-pC, however, evoked the most effective induction of phenylpropanoid pathway genes such as PAL1, C4H1, 4CL1, and STS1. Moreover, this nucleotide also induced at short times the accumulation of N-benzoylputrescine (BenPut), one of the phenylamides that are derivatives of phenylpropanoid and polyamines. The investigated nucleotides did not change either the lignin content or the cell dry weight, nor did they affect the cell viability throughout the experiment. The results suggest that nucleoside 5′-phosphoramidates could be considered as new signaling molecules.

Characterisation of extracellular polysaccharides from suspension cultures of members of the Poaceae

Microscopic examination of suspension-cultured cells of Phleum pratense L., Panicum miliaceum L., Phalaris aquatica L. and Oryza sativa L. showed that they were comprised of numerous root primordia. Polysaccharides secreted by these suspension cultures contained glycosyl linkages consistent with the presence of high proportions of root mucilage-like polysaccharides. In contrast, suspension-cultured cells of Hordeum vulgare L. contained mostly undifferentiated cells more typical of plant cells in suspension culture. The polysaccharides secreted by H. vulgare cultures contained mostly linkages consistent with the presence of glucuronoarabinoxylan. The soluble polymers secreted by cell-suspension cultures of Phleum pratense contained 70% carbohydrate, 14% protein and 6% inorganic material. The extracellular polysaccharides were separated into four fractions by anion-exchange chromatography using a gradient of imidazole-HCl at pH 7.0. From glycosyl-linkage analyses, five polysaccharides were identified: an arabinosylated xyloglucan (comprising 20% of the total polysaccharide), a glucomannan (6%), a type-II arabinogalactan (an arabinogalactan-protein; 7%), an acidic xylan (3%), and a root-slime-like polysaccharide, which contained features of type-II arabinogalactans and glucuronomannans (65%).

Characterisation of extracellular polysaccharides from suspension cultures of members of the Poaceae

Microscopic examination of suspension-cultured cells of Phleum pratense L., Panicum miliaceum L., Phalaris aquatica L. and Oryza sativa L. showed that they were comprised of numerous root primordia. Polysaccharides secreted by these suspension cultures contained glycosyl linkages consistent with the presence of high proportions of root mucilage-like polysaccharides. In contrast, suspension-cultured cells of Hordeum vulgare L. contained mostly undifferentiated cells more typical of plant cells in suspension culture. The polysaccharides secreted by H. vulgare cultures contained mostly linkages consistent with the presence of glucuronoarabinoxylan. The soluble polymers secreted by cell-suspension cultures of Phleum pratense contained 70% carbohydrate, 14% protein and 6% inorganic material. The extracellular polysaccharides were separated into four fractions by anion-exchange chromatography using a gradient of imidazole-HCl at pH 7.0. From glycosyl-linkage analyses, five polysaccharides were identified: an arabinosylated xyloglucan (comprising 20% of the total polysaccharide), a glucomannan (6%), a type-II arabinogalactan (an arabinogalactan-protein; 7%), an acidic xylan (3%), and a root-slime-like polysaccharide, which contained features of type-II arabinogalactans and glucuronomannans (65%).

Structural characterisation of galactoglucomannan secreted by suspension-cultured cells of Nicotiana plumbaginifolia

Galactoglucomannan (GGM) from cultures of Nicotiana plumbaginifolia has Man:Glc:Gal:Ara:Xyl in 1.0:1.1:1.0:0.1:0.04 ratio. Linkage analysis contained 4- and 4,6-Manp, 4-Glcp, terminal Galp and 2-Galp, small amounts and terminal Arap and terminal Xyl p, and ~ 0.03 mol acetyl per mol of glucosyl residue. Treatment with α- and β-D-galactosidases showed that the majority of the side-chains were either single Galp-α-(1 → residues or the disaccharide Galp-β-(1 → 2)-Galp-α-(1 → linked to O-6 of the 4-Manp residues of the glucomannan backbone. Analysis of the oligosaccharides generated by endo-(1 → 4)-β-mannanase digestion confirmed that the GGM comprises a backbone of predominantly alternating → 4)-D-Manp-β-(1 → and → 4)-D-Glc p-β-(1 → branched at O-6 of 65% of the 4-Manp residues. The major oligosaccharide identified was D-Glcp-β-(1 → 4)-[D-Galp-β-(1 → 2)-D-Galp-α-(1 → 6)]-D-Manp-p-β-(1 → 4)-D-Glcp-β-(1 → 4)-[D-Galp-α-(1 → 6)]-D-Manp-β-(1 → (27%), and most of the other oligosaccharides produced in significant quantities were based on this structure.

Structural characterisation of galactoglucomannan secreted by suspension-cultured cells of Nicotiana plumbaginifolia

Galactoglucomannan (GGM) from cultures of Nicotiana plumbaginifolia has Man:Glc:Gal:Ara:Xyl in 1.0:1.1:1.0:0.1:0.04 ratio. Linkage analysis contained 4- and 4,6-Manp, 4-Glcp, terminal Galp and 2-Galp, small amounts and terminal Arap and terminal Xyl p, and ~ 0.03 mol acetyl per mol of glucosyl residue. Treatment with α- and β-D-galactosidases showed that the majority of the side-chains were either single Galp-α-(1 → residues or the disaccharide Galp-β-(1 → 2)-Galp-α-(1 → linked to O-6 of the 4-Manp residues of the glucomannan backbone. Analysis of the oligosaccharides generated by endo-(1 → 4)-β-mannanase digestion confirmed that the GGM comprises a backbone of predominantly alternating → 4)-D-Manp-β-(1 → and → 4)-D-Glc p-β-(1 → branched at O-6 of 65% of the 4-Manp residues. The major oligosaccharide identified was D-Glcp-β-(1 → 4)-[D-Galp-β-(1 → 2)-D-Galp-α-(1 → 6)]-D-Manp-p-β-(1 → 4)-D-Glcp-β-(1 → 4)-[D-Galp-α-(1 → 6)]-D-Manp-β-(1 → (27%), and most of the other oligosaccharides produced in significant quantities were based on this structure.