Photosynthetic Carbon Metabolism in Photoautotrophic Cell Suspension Cultures Grown at Low and High CO₂

Abstract

MATERIALS AND METHODSPhotosynthetic carbon metabolism was characterized in four photoautotrophic cell suspension cultures. There was no apparent difference between two soybean (Glycine max) and one cotton (Gossypium hirsutum) cell line which required 5% CO2 for growth, and a unique cotton cell line that grows at ambient CO2 (660 microliters per liter). Photosynthetic characteristics in all four lines were more like C3 mesophyll leaf cells than the cell suspension cultures previously studied. The pattem of 14C-labeling reflected the high ratio of ribulosebisphosphate carboxylase to phosphoenolpyruvate carboxylase activity and showed that CO2 fixation occurred primarily by the C3 pathway. Photorespiration occurred at 330 microliters per liter CO2, 21% 02 as indicated by the synthesis of high levels of 14C-labeled glycine and seine in a pulse-chase experiment and by oxygen inhibition of CO2 fixation. Short-term CO2 fixation in the presence and absence of carbonic anhydrase showed C02, not HC03-, to be the main source of inorganic carbon taken up by the low C02-requiring cotton cells. The cells did not have a C02-concentrating mechanism as indicated by silicone oil centrifugation experiments. Carbonic anhydrase was absent in the low C02-requiring cotton cells, present in the high C02-requiring soybean cell lines, and absent in other high CO2 cell lines examined. Thus, the presence of carbonic anhydrase is not an essential requirement for photoautotrophy in cell suspension cultures which grow at either high or low CO2 Photoautotrophic Cell Suspension CulturesSoybean (Glycine max) cells and high C02-requiring cotton (Gossypium hirsutum) cells were grown at 5% C02 in a modified MS medium2 which contained thiamine and hormones as the only organic compounds (22). Cells were subcultured every 2 weeks. Low C02-requiring cotton cells were grown at ambient CO2, which was 660 ,L/L, in a modified MS medium (3,10), and were subcultured every 4 weeks. Cells were used in experiments 1 to 2 weeks after transfer, and were regularly found to be 90 to 95% viable as determined by phenosafranine staining (30). Prior to use, the cells were washed three times with the appropriate buffer (usually 30 mm Mops, pH 7.0). Cells stored for 4 h in buffer at 11 sE/m2 *s in a test tube or Petri dish showed less than a 10% decrease in the rate of '4C02-fixation. CO2 FixationCells (10-20 ,ug

Photosynthetic Characterization of Photoautotrophic Cells Cultured in a Minimal Medium

“…The activation of Rubisco for growth chamber grown spinach leaves was approximately 86%, and greenhouse grown soybean leaves were reported to be 95% activated (3,21,25). However, recent studies with the COT-P and SB 1-P cells demonstrated that Rubisco could be activated to higher levels of 93 to 94% with a 5-min preillumination prior to assaying for Rubisco activity (20).…”

“…Since then, the list of species cultured as photoautotrophic lines in our laboratory include a second G. max genotype, Datura innoxia, Gossypium hirsutum, a N. tabacum-Nicotiana glutinosa fusion hybrid, and two Amaranthus species, Amaranthus powellii and Amaranthus cruentus (3,22,26). There are reports of sustained growth of chlorophyllous photoautotrophic cells at ambient CO2 levels (18)(19)(20)26) and tobacco cells at ambient CO2 levels for up to one and a half years.…”

“…Photosynthetic carbon metabolism and activity of enzymes involved in CO2 assimilation under varying physiological conditions have been analyzed with photoautotrophic and photomixotrophic culture systems (7,13,15,20,24). Recently it has been shown that the photoautotrophic soybean culture SB-P exhibits phytochrome-mediated responses in the lightdependent expression of genes encoding Chl a/b binding protein (10).…”

Synthesis and Turnover of Cell-Wall Polysaccharides and Starch in Photosynthetic Soybean Suspension Cultures

“…The Glycine max (soybean) SB-P and SB-M cell lines (Horn et al, 1983) were used in the studies presented here. These cell lines consist of finely divided suspensions with relatively high chlorophyll and Rubisco levels and low PEP carboxylase activity, making them similar to leaves in these characteristics (Roeske et al, 1989;Goldstein and Widholm, 1990) and unlike many photoautotrophic cultures that have low chlorophyll and Rubisco and high PEP carboxylase (reviewed by Widholm, 1992). There are many other photoautotrophic cultures available that could also be used in such studies (Widholm, 1992).…”

Light Stress and Oxidative Cell Damage in Photoautotrophic Cell Suspension of Euphorbia characias L

“…A photoautotrophic cell suspension was chosen because it presents the advantages of simplified system (Widholm, 1992) and it is preferred to thylakoids in which photosynthetic carbon reduction and photorespiratory carbon oxidation cycle activities are absent, to chloroplasts because their photosynthetic activity may be unstable for long-term studies, and to protoplasts, which are already stressed because of enzymatic digestion (Schnab and Zimmermann, 1989;Criqui et al, 1992). The photoautotrophic cell suspension is also a proliferating and autonomous system that seems well adapted for long-term studies and that may exhibit photosynthetic characteristics similar to those of leaf mesophyll cells (Roeske et al, 1989;Takeda et al, 1989;Chagvardieff et al, 1990).…”