Characteristics of Five New Photoautotrophic Suspension Cultures Including Two Amaranthus Species and a Cotton Strain Growing on Ambient CO₂ Levels

Abstract

Suspension cultures of cotton (Gossypium hirsutum), Amaranthus cruentus, A. powellii, Datura innoxia, and a Nicotiana tabacum-N. glutinosa fusion hybrid were adapted to grow photoautotrophically under continuous light. The cotton strain grew with an atmosphere of ambient CO2 (about 0.06 to 0.07% in the culture room) while the other strains required elevated CO2 levels (5%). Photoautotrophy was indicated by the requirement for CO2 and for light for growth. The strains grew with doubling times near 14 days and had from 50 to 600 micrograms of chlorophyll per gram of fresh weight. The cells grew in small to moderate sized clumps with cell sizes from 40 to 70 micrometers (diameter). Like most photoautotrophic cultures described so far the ribulose 1,5-bisphosphate carboxylase (RuBPcase) activity levels were well below those of mature leaves. The phosphoenolpyruvate carboxylase levels were not elevated in the C4 Amaranthus species. The cells showed high dark respiration rates and had lower net CO2 fixation under high 02 conditions. Dark CO2 fixation rates ranged from near 10 to 30% of that in light.Fluorescence emission spectra measurements show that the cell antenna pigments systems of the four strains examined are similar to that of chloroplasts of green plants. The cotton strain which was capable of growth under ambient CO2 conditions showed the unique properties of a high RuBPcase activation level in ambient CO2 and a stable ability to show net CO2 fixation in 21% 02 conditions. The first higher plant tissue culture described as being photoautotrophic, i.e. growing with CO2 and light as the sole carbon and energy source, respectively, was that of Bergmann (3) lower RuBPcase2 activity which often leads to RuBPcase to PEPcase activity ratios near one. These differing characteristics are not really surprising since photoautotrophic cultured cells are growing and dividing unlike a mature leaf. Thus, the cultured cells should be compared to developing leaves which do have high respiration rates and a lower ratio of RuBPcase to PEPcase activity (1,14). Probably because of these characteristics all photoautotrophic cultures described thus far require elevated CO2 levels, usually 1 to 5%, for growth and have CO2 compensation concentrations higher than that ofmature leaves (reviewed in 11, 20).To date, no species with the C4 photosynthesis pathway has been grown photoautotrophically. Photoheterotrophic, green cultures of the C4 species, Gisekia pharnaceoides (25), Portulaca oleracea (12, 13) and Froelichia gracilis (15) have been initiated and used in photosynthesis studies, but these cultures were grown with 2 to 2.5% sucrose in the culture medium.Photoautotrophic cultures can be used for many purposes including the selection of mutants resistant to photosynthetic herbicides, for screening for herbicidal activity, and for herbicide mechanism ofaction studies. The photoautotrophic cultures may be valuable for producing desired compounds if chloroplasts are involved in the biosynthesis. Studies of chloroplast dev...

Photosynthetic Characterization of Photoautotrophic Cells Cultured in a Minimal Medium

“…The cotton and soybean photoautotrophic lines have more rapid doubling times than the other lines, averaging 6 to 7 d for COT-62, 7 d for COT-P, and 8 to 9 d for doubling ofboth SB 1-P and SB-P. A longer doubling time of 12 d was found for DAT-P, NTG-P, and APO-P. The cells cultured with MIN medium do not differ in doubling time from those cultured on KTP°or KN°me-dium (22,26).…”

“…The (26) for these same culture lines and 14 d for COT-P by Blair et al (3). The cotton and soybean photoautotrophic lines have more rapid doubling times than the other lines, averaging 6 to 7 d for COT-62, 7 d for COT-P, and 8 to 9 d for doubling ofboth SB 1-P and SB-P. A longer doubling time of 12 d was found for DAT-P, NTG-P, and APO-P.…”

“…var 'Corsoy,' described previously by Horn et al (6); SB 1-P, G. max PI 437833, described by Rogers and Widholm (22); COT-P, Gossypium hirsutum cv 'Stoneville 825,' described by Blair et al (3); and COT-62, a G. hirsutum line selected from COT-P for high Chl. The other lines, DAT-P, Datura innoxia; NTG-P, a Nicotiana tabacum-Nicotiana glutinosa hybrid; and APO-P, Amaranthus powellii, were initiated as described by Xu et al (26) (2.75 MCi/,Mmol), and 0.5 mM RuBP. Initial activity reactions were started with 50 ML of cell extract and quenched after 30 s with 0.2 mL 6 N acetic acid.…”

“…The first photoautotrophic culture line established in our laboratory was Glycine max var 'Corsoy' in 1982 (6). 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 in Photoautotrophic Cell Suspension Cultures Grown at Low and High CO2

“…In the previous studies with photosyn- proposed that the low levels of RuBP carboxylase present were analogous to the low levels observed in developing leaves (13,32). The reason for the difference in RuBP carboxylase levels in the different cell lines is unknown, but it is evident that all four of the cell suspensions studied in detail here are much more like cells in mature leaves.…”