Effects of CO 2 and O 2 on Photosynthesis and Growth of Autotrophic Tobacco Callus

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...

Section: Methodssupporting

confidence: 92%

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

Xu¹,

Blair²,

Rogers³

et al. 1988

“…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).…”

mentioning

confidence: 99%

Photosynthetic Characterization of Photoautotrophic Cells Cultured in a Minimal Medium

Goldstein¹,

Widholm²

1990

Photosynthetic properties of photoautotrophic suspensions cultured in a minimal growth medium have been evaluated to determine whether changes have occurred in ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity, phosphoenol-pyruvate (PEP) carboxylase activity, chlorophyll content, or culture growth. Five photoautotrophic lines Amaranthus powellii, Datura innoxia, Glycine max, Gossypium hirsutum, and a Nicotiana tabacum-Nicotiana glutinosa fusion hybrid were grown in a medium without organic carbon other than phytohormones, and without vitamins. These photoautotrophic lines had total Rubisco activities ranging from 85 to 266 micromoles CO2 fixed per milligram chlorophyll hour-1, with percent activation of Rubisco ranging from 16 to 53%. Inclusion of protease inhibitors in the homogenization buffer did not result in higher Rubisco activity. PEP carboxylase activity for cells cultured in minimal medium was found to range from 16 to 146 micromoles CO2 per milligram chlorophyll hour-', with no higher activity in the C4 Amaranthus cells compared with PEP carboxylase activity in the C3 species assayed. Rubisco-to-PEP carboxylase ratios ranged from 2.2 to 1 up to 9.4 to 1. Chlorophyll contents increased in all but the Nicotiana cell line, and all of the photoautotrophic culture lines were capable of growth in vitamin-free medium with the exception of SB-P, which requires thiamine.

Section: Introductionmentioning

confidence: 99%

“…Consequently, few reports dealed with the photorespiratory activity of photoautotrophic cultures (13 treated with a-HPMS, an inhibitor of the glycolate oxidase in the glycolate pathway (3). In some cases, growth of photoautotrophic cultures in atmospheric air was reported (19,22).In the present work, we have used photoautotrophic Nicotiana plumbaginifolia protoplast-derived calli, grown either under photorespiratory conditions (0.05% C02) or not (2% C02) (24), to investigate the long-term effects of C02-enrichment and of AAN, an inhibitor of the glycolate pathway, on growth and photosynthesis. …”

mentioning

confidence: 99%

“…Consequently, few reports dealed with the photorespiratory activity of photoautotrophic cultures (13). However, cultures grown at high CO2 have the enzymatic capacity to carry out photorespiration at low C02: McHale et al (19) observed a Warburg-effect in Nicotiana tabacum photoautotrophic calli; an accumulation of glycolate was noticed in similar calli 'Abbreviations: Rubisco, ribulose-1,5-bisphosphate carboxylase; a-HPMS, a-hydroxy-2-pyridinemethanesulfonate; AAN, aminoacetonitrile; PMSF, phenylmethyl-sulfonylfluoride; RuBP, ribulose-1,5-bisphosphate.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Effects of CO₂-Enrichment and of Aminoacetonitrile on Growth and Photosynthesis of Photoautotrophic Calli of Nicotiana plumbaginifolia

Rey¹,

Eymery²,

Peltier³

1990

Photoautotrophic calli of Nicotiana plumbaginifolia were grown for 3 weeks under two CO2 concentrations (500 and 20,000 microliters of CO2 per liter). Calli cultured at high CO2 exhibited a two-fold higher rate of growth. At CO2 test levels, these calli were characterized by a lower net photosynthetic capacity than calli cultured at low CO2. This diminution due to CO2 adaptation could be ascribed to a 170% stimulation of dark respiration, a 40% decrease in total ribulose-1,5-bisphosphate carboxylase (Rubisco) activity, and also to a feedback inhibition of photosynthesis: high CO2 grown calli contained about 5.5-fold more sucrose and three-fold less orthophosphate (Pi) than low CO2 grown calli.Whether the decrease in Rubisco activity is related to the accumulation of sucrose and to the Pi limitation is discussed. Both calli exhibited a Warburg-effect showing the existence of active photorespiration at low CO2. In calli grown at low CO2 with 5 millimolar aminoacetonitrile (AAN), an inhibitor of the glycolate pathway, fresh weight decreased by 25% and chlorophyll content by 40%, dark respiration increased by 50% and net CO2 uptake decreased by about 60% at 340 microliters of CO2 per liter and 35% at 10,000 microliters of CO2 per liter. In these calli, glutamine and glutamate contents were half of control calli. In contrast, AAN did not provoke any noticeable effect in calli grown at high CO2. In photoautotrophic calli, the inhibition of the glycolate pathway by AAN results in severe perturbations in glutamate metabolism and in chlorophyll biosynthesis.In vitro photoautotrophic calli cultures represent a system for studying photosynthesis of higher plants (13). These cultures generally require for sustained growth high CO2 levels (more than 1%). In these conditions, photorespiration is probably not expressed during growth because of the inhibition of the oxygenase function of Rubisco' by high CO2. Consequently, few reports dealed with the photorespiratory activity of photoautotrophic cultures (13 treated with a-HPMS, an inhibitor of the glycolate oxidase in the glycolate pathway (3). In some cases, growth of photoautotrophic cultures in atmospheric air was reported (19,22).In the present work, we have used photoautotrophic Nicotiana plumbaginifolia protoplast-derived calli, grown either under photorespiratory conditions (0.05% C02) or not (2% C02) (24), to investigate the long-term effects of C02-enrichment and of AAN, an inhibitor of the glycolate pathway, on growth and photosynthesis. MATERIALS AND METHODS Culture ProcedureCulturing of photoautotrophic protoplast-derived calli was described previously (24). Calli were grown in unsealed Petri dishes on a modified medium of Bourgin et al. (4) without metabolizable sugar. Dishes were kept in Plexiglas chambers continuously illuminated with fluorescent light ( 120 IAE* m-2 s_') and flushed with either laboratory air (500 ,tL CO2 L-') or CO2-enriched air (20,000 uL CO2-L', balance air). When specified, AAN (Sigma), was added to the medium at a final concentrati...