L. H. W. Van Der Plas

Establishment of hairy root cultures of Linum flavum producing the lignan 5-methoxypodophyllotoxin

Hairy root cultures were induced from leaf explants of Linum flavum by infection with Agrobacterium rhizogenes. The transformed nature of tissue was confirmed by the production of opines. The cultures produced 1.5 to 3.5% of the lignan 5-methoxypodophyllotoxin (5-MPT) on a dry weight basis, which was 2 to 5 times higher than the 5-MPT content in untransformed root cultures and 5 to 12 times higher than in L. flavum cell suspensions. The 5-MPT production as a function of time was up to four times higher than that in cell suspensions.

Cytochrome c dependent, antimycin-A resistant respiration in mitochondria from potato tuber (Solanum tuberosum L.). Influence of wounding and storage time on outer membrane NADH-cytochrome-c-reductase.

Cytochrome c has two stimulatory effects on respiration of mitochondria especially those from wounded potato tuber. In the first place a stimulation of succinate- and NADH-consuming, antimycin-A-sensitive respiration, which reaches a maximal value at low cytochrome c concentrations, has been found. In the second place, at higher concentrations of cytochrome c a stimulation of NADH-consuming respiration occurs, which is antimycin-A-resistant, but KCN-sensitive. This antimycin-A-resistant, NADH-consuming respiration is absent, when no cytochrome c is added to the reaction medium. It is insensitive to metal chelators, to which the antimycin-A-and KCN-resistant plant mitochondrial alternative oxidase is sensitive. By measurements of NADH-cytochrome c reductase activities a corresponding antimycin-A-resistant NADH-cytochrome c reductase has been found, which is insensitive to osmotic shock treatment. A localization of this antimycin-A-resistant electron transport with NADH as the electron donor in the outer mitochondrial membrane is likely. In the mitochondrial preparations cytochrome c might stimulate by acting as an electron-carrier between the outer membrane reductase and the inner membrane cytochrome oxidase. A big increase of the outer membrane mediated electron transport in the mitochondria has been observed after wounding of potato tuber tissue. The ability of the tissue to produce this electron transport pathway after wounding disappeared after prolonged storage of the tubers. A possible function of this electron transport pathway in fatty acid desaturation during the wound-reaction is suggested.

Potato Tuber Storage: Biochemical and Physiological Changes

Apart from the changes that accompany tuber deterioration after very long storage or after sprouting and the production of new plants, potato tubers are often considered as biochemically and physiologically invariable for a rather long time during normal storage. Yet, many changes in various biochemical and physiological processes occur, which have important consequences for the usage of the tubers as a source of food, as starting material for new plants and a new crop, and also as an object for plant physiological research.

CN-resistant respiration in potato (Solanum tuberosum L.) changes after storage of the tuber

Abstract The resistance of respiration to KCN induced in potato tuber tissue after wounding is located in the mitochondria and sensitive to metal chelators. The succinate-consuming respiration of isolated mitochondria becomes resistant after wounding, while the NADH-consuming respiration remains KCN-sensitive. The alternative oxidase most likely is located at the succinate dehydrogenase complex. From the inhibition characteristics of metal chelators it can be concluded that the alternative oxidase functions also in uninhibited respiration. Induction of alternative oxidase is low or absent in tubers before harvest and increases afterwards. While uninhibited respiration decreases during storage the relative importance of the cyanide (CN)-resistant respiration rises sharply. In the same period the formation of wound periderm cell layers decreases which might be related to a less efficient energy production (ATP) from substrate.

CN-resistant respiration in a convolvulus arvensis L. cell culture

Abstract Respiration was studied of the root tissue of Convolvulus arvensis L., callus tissue derived from these roots, cell cultures derived from this callus tissue and of mitochondria isolated from cells of cell cultures. In all cases a considerable part of respiration is CN-resistant, while addition of CN to callus tissue or cells from a cell culture even leads to a slight stimulation. This CN-resistant respiration is chelator-sensitive indicating that the alternative oxidase, known from other plant tissues, is used.

The course of adenine nucleotide contents in white and green soybean cells during growth in batch suspension culture

Abstract Soybean ( Glycine max L. Merr.) cells grown in suspension culture under continuous illumination in the presence of glucose show chloroplast biogenesis (green cells), which is absent in cells cultured in the dark (white cells). In both white and green cells the total amount of adenine nucleotides and the amount of ATP sharply increased during the lag phase, followed by a continuous decrease during subsequent growth. The ADP/ATP ratio and the energy charge in both kinds of cells were equal from the 2nd through the 5th day of culture. From the 6th day in green cells the ADP/ATP ratio increased and the energy charge decreased, while in white cells both parameters remained relatively constant, leading to significant differences between white and green cells. The failure of green cells to maintain the equilibrium between energy-requiring and -generating processes might be related to the energy demands of the continued development of the internal membrane system of the chloroplasts beyond the 5th day of culture.

Valinomycin : a very effective inhibitor of the cyanide-insensitive alternative pathway in plant mitochondria

The effect of valinomycin on the respiration of potato tuber callus mitochondria ( Solanum tuberosum L. cv. Bintje) was investigated. The rate of electron transfer via the cytochrome pathway can be stimulated by abolishing membrane potential with valinomycin. However, valinomycin was found to inhibit CN-resistant respiration in potato tuber callus mitochondria. The inhibition is independent of the substrate used (external NADH, succinate and malate). The inhibition was enhanced by increasing the K + concentration in the medium from 0 to 125 mM and was also observed when valinomycin was added in the presence of carbonyl cyanide 4-trifluoromethoxyphenylhydrazone (FCCP). In the presence of 125 mM K + 1 μM valinomycin inhibited electron flow, mediated by the alternative pathway, for approx. 65% and 5 μM valinomycin for approx. 85%. In mitochondria prepared from sweet potato ( Ipomea batatas ) valinomycin also inhibited CN-resistant respiration.

Respiration rate and redox state of nad during wound-induced differentiation of potato tuber tissue (Solanum tuberosum L.)

Abstract To study the involvement of NAD(H) in the activation of respiration after wounding of potato tubers, redox ratios of free cytoplasmic NAD have been determined via the pyruvate/lactate and the oxaloacetate/malate couple. These ratios change after wounding of intact tubers parallel to those of the far more oxidized total extractable NAD(H) pool. In wounded tuber tissue a positive correlation is observed between the redox state of NAD and the respiration rate. Temperature acclimatization of the tubers before wounding delays the establishment of that correlation. It is concluded that the redox ratio of free NAD may play an active role in the control of the respiration rate in wounded tuber tissue.

A cell suspension ofLinum flavum (L.) in phosphate limited continuous culture

A cell suspension ofLinum flavum was grown in phosphate limited continuous culture at two different growth rates. Energy metabolism (respiration), coniferin and lignin production and overall biomass composition were analysed, in order to establish the relations between growth, maintenance and secondary metabolism. The ATP turnover rate was higher in the faster growing cultures, corresponding with a higher energy requirement. The coniferin production was not directly correlated with the growth rate, indicating the possibility of high production at high growth rates. Steady states grown under identical conditions showed different characteristics that may have evolved during pre-culture time.

Induction of alcohol dehydrogenase in explants of potato tuber (Solanum tuberosum L.)

During callus formation a huge increase in alcoholdehydrogenase activity was observed in potato tuber tissue discs. Callus formation was no prerequisite for this increase; slicing and subsequent incubation of potato tuber tissue discs always led to an increase in alcohol dehydrogenase activity, which was dependent on cytoplasmic protein synthesis. A three-fold increase was observed during incubation in moist air (periderm formation) and during incubation on nutrient agar without carbon source. A six- to eight-fold increase occurred during incubation on nutrient agar with sucrose, ribose or pyruvate as carbon source. The extra increase in alcohol dehydrogenase activity did not occur in the presence of equimolar amounts of mannitol, sorbitol, succinate or ethanol. The extent of the activity increase was not directly correlated with the presence of a carbon source suitable for maintaining growth.