Role of Chitinase and Chitin Oligosaccharides in Lignification Response of Cultured Carrot Cells Treated with Mycelial Walls

Chitinase activity was induced in cultured carrot cells by incubationwith mycelial walls of a fungus, Chaetomium globosum. Both intra-and extracellular chitinases were resolved into four componentsby gel filtration chromatography. The extracellular enzymesliberated soluble oligosaccharides of different sizes from insolublechitin, suggesting that these carrot chitinases are endo-hydrolases.The solubilized chitinase digests obtained from insoluble mycelialwalls of C. globosum and chitin were fractionated by gel filtrationchromatography, and the elicitor activity of each fraction forthe accumulation of phenolic acids in cultured carrot cellswas determined. In both solubilized fragments of fungal wallsand of chitin, elicitor-active oligosaccharides were distributedin many fractions, however, potent activity for inducing phenolicacid synthesis was observed in the high molecular weight fractions. (Received October 5, 1987; Accepted February 12, 1988)     

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

The rate of incorporation of [¹⁴C]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.     

Colchicine-binding activity of carrot tubulin at different culture age

Tubulin contents in the extract from cultured carrot cells at different growth phases were investigated by measuring colchicine-binding activity. The addition of vinblastine and dithiothreitol to the reaction mixture appreciably improved the stability of both free and colchicine-bound tubulins. Colchicine-binding activity in the cell extract obtained from stationary phase was more labile than that from log phase though the extract showed higher affinity to colchicine. After purification, however, tubulin from the cells at different growth phases showed the same affinity and its colchicine-binding activity was much more stable than in crude extract. The colchicine-binding activity in the crude extract was corrected for the decay during measurement and apparent difference in the affinity so that the activity in the cells containing different kind and amount of interefering substances could be compared. The corrected amount of colchicine that binds to the 100,000×g extract was 46 pmol/10⁵ cells at log phase. It decreased with the progression of culture age from linear to stationary phase. Combining the data with the morphological observation, it was suggested that the log phase cells contained larger free tubulin pool than the linear or stationary phase cells.     

Phytoalexin production in cultured carrot cells treated with pectinolytic enzymes

6-Methoxymellein, a phytoalexin of carrot, was produced in cultured cells upon addition of partial hydrolysates of carrot cells obtained by treatment with purified endo-polygalacturonase or endo-pectin lyase. Direct addition of these enzymes to the cell culture also stimulated the accumulation of this 6-methoxymellein. When the hydrolysates obtained by these enzymes were subsequently treated within pectin esterase, the activity for the elicitation of 6-methoxymellein production decreased appreciably. These results suggest that pectinolytic enzymes release elicitor-active cell wall fragments from carrot cells and that a certain degree of esterification of the galacturonosyl moiety in these pectic polysaccharides is required for elicitor activity.     

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.Abbreviation MW molecular weight     

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

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.     

Changes in membrane glycoproteins during fruit-body formation inPhysarum polycephalum

Sporangia formation ofPhysarum polycephalum was induced by starvation and illumination, and the morphogenic process during the differentiation was studied by scanning electron microscopy. Plasma membranes were prepared from these differentiating plasmodia and the membrane proteins were analyzed by polyacrylamide gel electrophoresis. Many glycoproteins appeared during the fruit-body formation. Of these a protein of apparent molecular mass of 66 kD was prominent in sporangia forming stage which showed a high affinity to RCA lectin. Inhibition of the glycosylation and processing of these glycoproteins resulted in the prevention of fruit-body formation suggesting that the synthesis of these membrane components is a prerequisite process for the sporangia formation in the slime mold.     

Involvement of plasma membrane-located calmodulin in the response decay of cyclic nucleotide-gated cation channel of cultured carrot cells

Increase in cytoplasmic cyclic AMP concentration stimulates Ca²⁺ influx through the cyclic AMP-gated cation channel in the plasma membrane of cultured carrot cells. However, the Ca²⁺ current terminated after a few minutes even in the presence of high concentrations of cyclic AMP indicating that hydrolysis of the nucleotide is not responsible for stop of the Ca²⁺ influx. Cyclic AMP evoked discharge of Ca²⁺ from inside-out sealed vesicles of carrot plasma membrane, and it was strongly inhibited when the suspension of the vesicles was supplemented with 1 μM of free Ca²⁺, while Ca²⁺ lower than 0.1 μM did not affect the Ca²⁺-release. The Ca²⁺ flux across plasma membrane was restored from this Ca²⁺-induced inhibition by the addition of calmodulin inhibitors or anti-calmodulin. These results suggest that Ca²⁺ influx initiated by the increase in intracellular cAMP in cultured carrot cells is terminated when the cytosolic Ca²⁺ concentration reaches the excitatory level in the cells, and calmodulin located in the plasma membrane plays an important role in the response decay of the cyclic nucleotide-gated Ca²⁺ channel.     

Factors Affecting Phytoalexin Production in Cultured Carrot Cells

The production of 6-methoxymellein, a phytoalexin, in culturedcarrot cells under various growth conditions was studied usingtwo induction methods; by adding partial hydrolysates obtainedby treating the cells with pectinase or trypsin, or by directlyadding these enzymes to growing cells to cause the release ofcellular components as endogenous elicitor. 6-Methoxymellein production depended greatly on the cell cultureage. Maximal production was found in cells at the early stationaryphase, while actively dividing cells had only negligible amounts.Release of elicitor from the cells by pectinase or trypsin wasalso influenced by the culture stage. Effective elicitor wasobtained only from cells in the late logarithmic and early stationaryphases. 6-Methoxymellein production required the presence of 2,4-D.IAA could not substitute for 2,4-D, though partial hydrolysatesprepared from these cells grown with IAA or without auxin showedsignificant elicitor activity. On the other hand, the productionwas inhibited by actinomycin D or cycloheximide, suggestingthat de novo syntheses of RNA and protein are required for thephytoalexin production. (Received November 17, 1984; Accepted March 2, 1985)     

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.Abbreviation MI myo-inositol