Linus H. W. van der Plas

Role of oxidative damage in tulip bulb scale micropropagation

The activation of oxygen stress-related enzymes was compared in regenerating and non-regenerating tulip bulb scale explants and regenerating stalk explants. The phospholipid composition of scale explants showed an increase of linolenic acid (1-15%) and a decrease in linoleic acid (70-55%). After incubation it was comparable to that of stalk explants in which no changes were observed. In all tested systems an increase in activity of catalase, peroxidase, SOD, lipoxygenase, polyphenoloxidase and phenylalanine ammonia lyase, was observed during incubation of the explants. The reaction can be divided into two phases. The first one (observed for scale explant lipoxygenase and to a lesser extent for SOD) occurs rapidly (1-2 h) after cutting the explants and appears to be wounding related. In the second phase (observed for all enzymes), starting during the first week of incubation, wound healing and regeneration can be observed. The activation of catalase, peroxidase and phenylalanine ammonia lyase was comparable in all tested systems and appears not to be related with the differences in tissue culture performance. In the second phase, the activity of lipoxygenase, peroxidase, catalase and phenylalanine ammonia lyase decreases in regenerating explants, while in non-regenerating explants they remain high. Our conclusion from these results is that oxidative damage is not the prime cause of the low regenerability of tulip bulb scale explants.

Relation between primary and secondary metabolism in plant cell suspensions

Cell suspensions ofMorinda citrifolia are able to produce large amounts of anthraquinones (AQ) when they are cultivated on a B5-medium containing 1 mg 1-1 naphtyl acetic acid (NAA); this production is inhibited by addition of 2,4-dichloro-phenoxyacetic acid (2,4-d). Also during cultivation on 1 mg 1-1 2,4-d AQ-production is absent.It appeared that in the presence of NAA a kind of ‘AQ-production’ program is switched on: cell division rate is low as well as metabolic activity, while endogenous sugar levels are high. The same properties develop in the presence of auxins like indolyl-acetic acid and p-chloro-phenylacetic acid. With 2,4-d and related auxins (like p-chloro-phenoxyacetic acid) AQ production is absent and emphasis is laid on a developmental program characterized by high cell division rates, high metabolic activity and low endogenous sugar contents. Independent of the type of auxin applied, the cells grow as a suspension consisting of finely dispersed cells. The ‘AQ-producing differentiation program’ cannot be maintained during a consecutive series of subculturings: with increasing AQ-contents the viability of the cells and the cell division rate decrease.The possible mechanisms of regulation of AQ-production by auxins are discussed as well as the advantages of the use of theMorinda model system in the study of the relation between growth, primary and secondary metabolism.

Effect of oxygen on growth and respiration of potato tuber callus

Growth and oxygen uptake of potato callus is faster in an oxygen-enriched atmosphere (70% oxygen, v/v; “oxygen-callus”) than in air (20% oxygen, v/v; “air-callus”). Especially the non-mitochondrial, so-called ‘residual respiration’ is increased in “oxygen-callus”. The capacities of the mitochondrial respiratory pathways (cytochrome pathway, Vcyt and alternative pathway, Valt) are also higher in this callus. In both callus types only a small part of the alternative pathway capacity is used in uninhibited respiration. The lower oxygen uptake of “air-callus” at normal air oxygen pressures is partially due to diffusional impedance. Measurement of the respiratory parameters of “air-callus” in oxygen-saturated medium leads to higher values than measurement in air-saturated medium, although these values are still lower than those of “oxygen-callus”.ATP-production was calculated from the oxygen-uptake data and compared with the dry weight production of the callus to give values of 10.0 and 10.8 g dry weight produced.-mol ATP-1, for “air-callus” and “oxygen-callus” respectively. As no harmful side-effects are observed, cultivation of callus under elevated oxygen pressures may be useful, when rapid callus-growth is necessary.

Enzymes of the pentose phosphate pathway in callus-forming potato tuber discs grown at various temperatures

Abstract In callus-forming potato tuber discs growing at a low culture temperature (8°C) the activities of glucose-6-phosphate dehydrogenase, EC 1.1.1.49 (G6PDH) and 6-phosphogluconate dehydrogenase, EC 1.1.1.44 (6PGDH) were twice as high as during growth at a high culture temperature (28°C). After a transfer from 8°C to 28°C and vice versa an adaptation of 6PGDH activity to the new culture temperature took place. Phosphofructokinase, EC 2.7.1.11 (PFK) and alcohol dehydrogenase, EC 1.1.1.1 (ADH) activities tended to be lower during growth at a low culture temperature (ratio 6PGDH/PFK 3:1 in 8°C callus and 1:1 in 28°C callus). C 1 C 6 ratios were independent of culture temperature, suggesting that although the in vitro capacity of the pentose phosphate pathway (PPP) is higher at low culture temperatures, the relative in vivo PPP activity is not influenced by the culture temperature. However, products of the PPP probably will re-enter glycolysis, thereby also releasing C6. It is speculated that at a low culture temperature this bypass of part of glycolysis has a special function to avoid the cold-sensitive PFK.

Cell division versus secondary metabolite production in Morinda citrifolia cell suspensions

Summary Morinda citrifolia cell suspension cultures are capable of accumulating high levels of anthraquinones, but appear to display an inverse relationship between the rate of cell division and anthraquinone production: low auxin concentrations lead to a high anthraquinone production and a low growth rate while at high auxin concentrations the reverse was observed. NAA and 2,4-D both showed this effect although there was a difference with respect to the optimal concentration, 2,4-D being active at approximately a hundred-fold lower concentration than NAA. We studied the inverse relation between cell division and anthraquinone accumulation in two ways. First, batch cultures of Morinda citrifolia were treated with hydroxyurea, which almost completely inhibited cell division. The 2,4-D cultures still did not accumulate anthraquinones. Second, we compared continuous cultures grown in the presence of NAA or 2,4-D at the same cell division rates. Only the NAA culture accumulated anthraquinones. The overall high sugar content of the cells under the various cultivation conditions indicated that sufficient energy and carbon substrates were always present. The possible direct effect of auxins on the induction and activity of the pathways leading to anthraquinone production is discussed.

Growth and Respiratory Characteristics of Batch and Continuous Cell Suspension Cultures Derived from Fertile and Male Sterile Petunia hybrida

Summary Growth rate of Petunia hybrida cells, cultivated in batch suspension was the same for cells derived from fertile (RMF) and male sterile (RMS) Petunia hybrida cv. Rosy Morn. The oxygen uptake by the RMS cells was twice that of the RMF cells. Analysis of the oxygen uptake data showed, that the capacity of both the mitochondrial cytochrome and alternative pathway was larger in the RMS cells; the non-mitochondrial residual respiration was about the same for both cell types (at least in the logarithmic and early stationary growth phase). The engagement of the alternative pathway in uninhibited respiration generally was low. When the dry weight production per mole of respiratory ATP was calculated, the RMS-cells had a much lower «growth efficiency« than the RMF cells, the latter forming about twice as much dry matter per mole of ATP. When these Petunia cells are cultivated in a Kurz airlift fermentor, at various glucose concentrations and at various dilution rates, the conversion of glucose in cellular dry weight appeared to be less efficient in RMS-cells (0.2 g dry weight produced per g of glucose) than in RMF-cells (0.4–0.5 g dry weight per g of glucose). Also for these continuously cultured cells, the respiration of the RMS-cells was higher and the dry weight production per mole of respiratory ATP was lower than for the RMF-cells, again indicating a more «efficient» growth for these RMF-cells. However, the absolute value of these parameters showed big differences for cells grown in batch and in continuous culture. The possible relation between the inefficient growth and respiration of the RMS-cells and the fact that the plants from which these cells were derived are cytoplasmic male sterile is discussed.

Participation of the CN-resistant Alternative Oxydase Pathway in the Respiration of White and Green Soybean Cells During Growth in Batch Suspension Culture

Summary In suspension cultures of white and green soybean cells ( Glycine max L.) the capacity of the cytochrome pathway (V cyt ) and of the alternative pathway (V alt ) together with the contribution of the alternative pathway (p · V alt ) to total respiration have been determined during growth in batch culture. The capacity of the alternative pathway (V alt ) and of the cytochrome (V cyt ) is comparable in white and green cells during growth. During the first four days of growth the contribution of the alternative pathway (p · V alt ) in green cells is twice as much as that in white cells. This difference disappears at the end of the logarithmic growth phase. The reason why p · V alt in green cells is higher might be an enlarged demand for precursors in biosynthetic processes because of the presence of developing chloroplasts in green cells. When KCN is added in the absence of metal chelator, respiration of whole cells, but not of the isolated mitochondria, is stimulated. It is suggested that the enhancement of respiration by KCN is caused by stimulated glycolytic glucose catabolism. The affinity constants (1/Ki) for BHAM in presence of 0.1 µmol KCN in white and green cells during growth is found to be positively correlated to the capacity of the alternative pathway.

Evidence of medium-chain-length polyhydroxyoctanoate accumulation in transgenic potato lines expressing the Pseudomonas oleovorans Pha-C1 polymerase in the cytoplasm

The phaC1 gene from Pseudomonas oleovorans, coding for the Pha-C1 polymerase, was introduced into the potato genome. Transgenic callus and plant lines which transcribed and translated the transgene were selected and cell suspension cultures from the wild type and transgenic lines were established. The substrate for the Pha-C1 polymerase, 3-(R)-hydroxyoctanoate, was provided to the growth medium. In the transgenic lines, but not in the wild type or in transgenic cell suspension cultures without Pha-C1 expression, evidence of medium-chain-length polyhydroxyalkanoate accumulation ranging from 0.02 to 9.7 mg of polymer per gram of dry weight was observed after feeding in the growth medium the substrate.

Influence of temperature, ethylene and cyanide on the occurrence of alternative respiration in mitochondria from iris bulbs

Abstract Mitochondria, isolated from iris ( Iris hollandica cv. Ideal) bulbs that have been treated for early flowering with high temperatures (14 days at 35°C followed by 3 days at 40°C) or with ethylene (10–500 ppm), show an induction of alternative respiratory capacity and a rise in state III respiration. Mitochondria from untreated bulbs (stored at 30°C) do not have an alternative pathway capacity and state III respiration is low. Induction of the alternative respiration by ethylene is maximal after 24 h, while induction by high temperature (> 36°C) is much slower. In the temperature range from 36–40°C, the extent of the induced alternative respiratory capacity increases with higher temperatures. A temperature of 42°C is lethal within 5 days. Bulbs stored at 30°C and 35°C before 40°C treatment reach the same values for alternative respiratory capacity. A treatment of the bulbs with 2.2 mM HCN (30°C) leads to an induction of alternative respiration concomitant with a decrease in state III respiration, after a lag time of 2–3 days. A treatment of 5 days with 2.2 mM HCN or longer is lethal.

Accumulation of anthraquinones in Morinda citrifolia cell suspensions

Cell suspensions of Morinda citrifolia were cultivated in a B5-medium containing 4% sucrose as the sole carbon source and 1 mg l-1 naphthyl acetic acid (NAA) or 1 mg l-1 2,4-dichloro-phenoxyacetic acid (2,4-D). Both auxins were able to support growth but only in the presence of NAA anthraquinone production was observed. 2,4-D inhibited the production in NAA cultures. Anthraquinone synthesis took place in the growth and the stationary phase and amounts of 0.2-0.4 mmol (about 100–200 mg) g-1 dry weight could be reached.