Contribution of Nonautotrophic Carbon Dioxide Fixation to Protein Synthesis in Suspension Cultures of Paul's Scarlet Rose

Abstract

Bicarbonate-'4C was provided to 5-and 11-day-old suspension cultures of Paul's Scarlet rose, and the incorporation of 4C into lipid, protein, amino acids, and organic acids was determined. The rate of bicarbonate uptake was approximately the same by 5-and 11-day-old cells, but the distribution of 4C among cell constituents was markedlv different. In 5-dav-old cells a larger proportion of the "4C entered protein, whereas in 11-day-old cells there was a greater tendency for "4C to accumulate in malate.The '4C in protein was distributed among 10 amino acids each having greater than 1 % of the total 14C recovered in protein. The distribution of "4C among tricarboxylic acid cycle intermediates indicated that the aspartate family of amino acids was synthesized directly from oxaloacetate produced as a result of nonautotrophic C02 fixation. However, this was not the sole source of oxaloacetate used for the synthesis of aspartate, for in a double labeling study with bicarbonate-14C and acetate-3H it was shown that oxaloacetate was drained simultaneously from the tricarboxvlic acid cycle for this purpose.Privation studies have shown that CO2 is required for the normal growth of microbial (22), animal (6, 15), and nonphotosynthetic plant cells (16,19). In explaining this requirement, some authors have emphasized the importance of nonautotrophic CO2 fixation for replenishment of carbon skeletons diverted from the TCA2 cycle into other metabolic pathways (2,16,17,23). Other authors have proposed that the product of CO2 fixation served as a direct source of carbon for amino acid synthesis (7,8,10), and was therefore necessary for growth. In previous work we showed that suspension cultures of Paul's Scarlet rose require CO2 for nonautotrophic growth (13). The present study compared the use of bicarbonate-'4C by 5-versus 11-day-old cells and looked specifically at the relationship of nonautotrophic CO2 fixation to the TCA cycle and the synthesis of amino acids incorporated into protein. growth conditions and composition of the medium were the same as used previously (5, 14), with two exceptions; the concentration of molybdic acid in the medium was 0.01 mg/l, and potassium iodide was 0.5 mg/l. One-gram samples of cells were harvested at selected times and incubated in 25-ml Erlenmeyer flasks containing 5 ml of the medium (pH 7.5). The medium contained 4 ,uCi of NaHW4CO3 (specific radioactivity 10 /Ci/ptmole). The depletion of bicarbonate-"C from the medium was determined by removing 10 ,l of the incubation medium at various time intervals and assaying for radioactivity. The uptake of bicarbonate-4C by cells was corrected for the direct loss of "4CO2 to the atmosphere by running controls which consisted of duplicate amounts of bicarbonate-'4C in a medium containing no cells. MATERIALS AND METHODSAt the end of the incubation period the medium was filtered through Miracloth. The recovered cells were washed thoroughly with deionized water, were placed into 15 ml of hot 80% ethanol, and homogenized for 3 min on a VirTis grind...

Activities of Ribulose Bisphosphate Carboxylase and Phosphoenolpyruvate Carboxylase and 14C-Bicarbonate Fixation during in Vitro Culture of Pinus radiata Cotyledons

“…However, it has been reported that the activity of PEPC is high in exponentially growing cell cultures (4, 5, 18,19) as well as in callus cultures (21). Similar observations (10, 13) suggesting that the carbon fixed by an increased activity of PEPC might be important for a supply of carbon skeletons to biosynthetic pathways in young, exponentially growing parts of intact plants have also been made.…”

14C-metabolism during callus induction in tobacco

“…The label from bicarbonate went mainly into organic acids and amino acids and a small amount into lipids ( Figure 6). Similar results have been observed in sycamore [17] and Paul's Scarlet rose [9] cells. The incorporation of label from bicarbonate into organic and amino acids was greater in the pith-phloem explants than in the subcultured callus from day 2 onwards.…”

Quantification of organelle changes in plant suspension cultures during growth

“…Since this cell line has the tendency to grow as aggregates (Nesius & Fletcher 1975) the volume of individual cells was difficult to determine. To ensure an accurate measurement, two methods were used to determine cell volume: the classical, micrometer method (Nesius et al 1972) was applied to external cells and a new method was developed using volume of the cell mass divided by the number of cells in the aggregate as determined by nuclear DNA fluorochromes.…”

“…Each period lasts several days and is similar to one of the developmental stages characteristic of cells present in meristematic regions of intact plants. With rose cultures it has been shown that as the cells progress through the various growth periods, a number of important physiological processes, organic acid metabolism (Fletcher & Beevers 1970), amino acid synthesis (Hunt & Fletcher 1976;Nesius & Fletcher 1975), respiration (Hunt & Fletcher 1976), and protein synthesis (Hunt & Fletcher 1976;Mohanty & Fletcher 1980) change dramatically. It has also been established with various cell lines that during culture growth cells experience numerous ultrastructural changes (Fowke 1986;Sutton-Jones & Street 1968).…”