Arasuke Nishi

Isolation and antimicrobial activity of the phytoalexin 6-methoxymellein from cultured carrot cells

Abstract 6-Methoxymellein was identified as a common phytoalexin produced by carrot roots irrespective of the species of challenging fungi. It was also shown that cultured carrot cells produced 6-methoxymellein when the culture was inoculated with fungi. This compound has a broad antimicrobial spectrum and inhibits the growth of several fungi, yeasts and bacteria.

Production and metabolism of 6-methoxymellein in cultured carrot cells

Abstract The carrot phytoalexin, 6-methoxymellein, accumulated in carrot suspension culture when it was incubated with a partial hydrolysate of carrot cells obtained by pectinase or trypsin treatment. 6-Methoxymellein also accumulated when these enzymes were added to the culture. In both cases, phytoalexin content decreased rapidly after maximum accumulation was attained, suggesting that the cultured carrot cells metabolized the phytoalexin. Cultured carrot cells incubated with the phytoalexin formed three different metabolites: among them, two which accumulated in the medium, were identified as the β-glucoside of 6-methoxymellein and 6-hydroxymellein. 6-Methoxymellein was found to be toxic to the host plant and induced a lag period in the growth of cultured carrot cells at low concentration. When it was added at concentrations higher than 0·5 m m , it caused a rapid decrease in viable cell number.

Invertase in cultured Daucus carota cells

Abstract Invertase activity of cultured carrot cells was distributed between cell wall and supernatant fractions of the cell homogenate. The enzyme associated with the cell wall fraction was solubilized by alkaline NaCl solution and the proportions found in the cell wall and soluble fractions depended on the concentration of NaCl. Formation of protoplasts by the action of cellulase and pectinase was accompanied by release of 50–60% of the invertase activity from the cells.

Isolation of variant carrot cell lines with altered pigmentation

Abstract Cultured carrot cells were treated with a known mutagenic compound, N -methyl- N ′-nitro- N -nitroso-guanidine, and plated on a nutrient agar medium. Four variant cell lines whose pigmentation properties differed from stock calluses have been isolated. The contents of major carotenoid components, β-carotene and lycopene, of these cells were determined and compared with those of parent strains.

Biosynthesis of Cell-Wall Polysaccharides in Cultured Carrot Cells

Biosynthesis of the cell wall in carrot cells (Daucus carota L.) cultured in a synthetic liquid medium was studied by measuring the incorporation of radioactive glucose and myo-inositol (MI). When the cells were fed with [14C]glucose in the presence of 0.01% MI, the label soon appeared in the neutral sugars in the cell wall but little radioactivity was found in the uronic-acid residues even after a prolonged incubation. On the other hand, radioactivity derived from [3H]MI was found to be distributed among uronic acids and pentoses but not in the hexose residues in the wall. The data indicate that MI is an important intermediate for the synthesis of acidic sugars in the wall of cultured carrot cells.

Formation of phenolic acid in carrot cells in suspension cultures.

Abstract The formation of phenolic acid in carrot cells grown in suspension culture was examined in relation to cell growth and the concentration of 2,4-dichlorophenoxyacetic acid (2,4-D). Cells multiplied and grew through logarithmic, linear and stationary phases. At all these phases, caffeic, ferulic and p -hydroxybenzoic acids were detected. Biosynthesis of these acids was higher in the early logarithmic phase than at any other phase and rapidly declined in the stationary phase. By lowering the concentration of 2,4-D, these organic acids, and the activity of phenylalanine ammonia-lyase were markedly increased.

Pectic polysaccharides in carrot cells growing in suspension culture.

Pectic polysaccharides in the cell wall of suspension-cultured carrot cells (Daucus carota L.) were fractionated into high- and low-molecular-weight components by molecular-sieve chromatography with a Sepharose 4B column. During the phase of cell-wall expansion, the relative content of low-molecular-weight polymers rapidly increased. Electrophoretic analyses of these fractions showed that the high-molecular-weight components were largely composed of neutral and weakly acidic polymers while the low-molecular-weight fraction contained, in addition to neutral polymers, strongly acidic polyuronides in which the content of neutral sugars was very small. The accumulation of a large amount of the strongly acidic polyuronides occurred in a late stage of cell-wall growth, concomitant with a marked decrease in the high-molecular-weight components.

Effect of auxin on the metabolism of mevalonic acid in suspension-cultured carrot cells

Abstract The rate of incorporation of [ 14 C]mevalonate into carotenoid and steroid fractions in suspension-cultured carrot cells decreased markedly after 2,4-dichlorophenoxyacetic acid was removed from the medium. In parallel to this change, the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase in a microsomal fraction was reduced to ca 33% of the control value, while that of a particulate fraction showed no significant change. The activities of mevalonate activating enzymes remained unchanged after auxin deprivation.

Changes in non-cellulosic cell-wall polysaccharides during the growth of carrot cells in suspension cultures

The cell-wall composition of carrot (Daucus carota L.) cells has been studied during their growth in suspension culture. Pectic and hemicellulosic polymers were fractionated according to molecular size by a Sepharose 4B column. Polyuronides in the pectic fraction were resolved into high- and low-molecular-weight components. The low-molecular-weight polyuronides were relatively free of neutral sugars and showed a marked increase during the growth of the cell wall. Hemicellulosic polysaccharides were of disperse molecular size. As cell expansion proceeded, the contents of glucose and xylose in the high-molecular-weight region increased while those in the low-molecular-weight fraction decreased. Removal of auxin from the medium apparently caused degradation of high-molecular-weight polymers in both the pectic and hemicellulosic fractions.

Increases in enzyme levels during the formation of phenolic acids in carrot cell cultures

Abstract The levels of glutamic acid dehydrogenase (GDH), phenylalanine ammonia-lyase (PAL), cinnamic acid 4-hydroxylase (CAH) and O -methyltransferase (OMT) were measured during the formation of phenolic acids in carrot cells in suspension culture. Caffeic, ferulic and p -hydroxybenzoic acids were always present as the culture proceded. Total content of these acids increased at the early logarithmic and linear phases. GDH showed high activity at the early logarithmic and stationary phases. PAL activity was much enhanced at the linear and stationary phases. CAH activity was found in actively growing cells, especially at the early and late logarithmic phases OMT behaved similarly to PAL. The increases in GDH and CAH might be responsible for the rapid synthesis of phenolic acid at the early logarithmic phase. The increase in phenolic acid at the linear phase would certainly be due to enhancements of both PAL and OMT. On the other hand, the accumulation of vanillic acid was observed in cells which were transferred and cultured on an agar medium, but not in cells in suspension culture. This accumulation is related to increases in OMT levels and also to changes in the degree of β-oxidation.