Photosynthesis in C 4 Plant Tissue Cultures

The relative transport of photosynthetic and dark carboxylation products in photoheterotrophic cells of Arachis hypogaea L. var. TMV-3 at varied phases of growth were determined. Despite the presence of an equally competent photosynthetic apparatus as determined from "CO2 incorporation rates in the dark and light, pulse-chase experiments revealed little or no change in the radioactivity of the C3 intermediates but rapid disappearance of label from the dark carbon assimilates (malate and other tricarboxylic acid cycle intermediates) with a simultaneous increase in the aminoacid pool in early log-phase (10 days old) cells. However, significant flow of carbon through the photosynthetic intermediates resulting in the accumulation of sugars occurred in the late log-phase (34 days old) cells. Limitation of exogenous sugar in the nutrient milieu and depletion of reserve carbohydrates stored in starch of the chloroplasts of the cells were considered as the decisive factors in promoting transport of C3 cycle intermediates through the reductive pentose phosphate pathway in photoheterotrophic cells. The observed drain of radioactivity even from the small amounts of tricarboxylic acid cycle intermediates synthesized during photosynthesis into glutamate indicated that the transport of carbon through the nonautotrophic pathway is not controlled by these factors.The rates and early products of carbon assimilation have been determined for photoheterotrophic tissue and cell cultures of carrot (1 1), tobacco (3, 18, 21), Ruta graveolens (7), Chenopodium rubrum (14), Portulaca oleracea (15), Kalanchoe crenata (17), and Chamaecereus sylvestril (22). However, the relative changes in the transport of the early formed photosynthetic carbon through the reductive pentose phosphate pathway using pulse-chase experiments have not been precisely monitored in these cultures. In the only system analyzed thus far to this effect, little decrease or increase in photosynthetic products was reported (15). This was attributed to the reduced cellular transport and absence of translocation supposedly present in the cultured cells. Yet another important contributing factor was the supply of carbohydrate stored as starch in the chloroplasts of the cultured cells. With a reduced demand for photosynthates due to the availability of sugar in the nutrient medium and plenty of reserve carbohydrate stored in starch, a reduced flow of carbon in the cultured cells was anticipated (15).In a previous paper, we reported the establishment of highly chlorophyllous cell suspension culture from the callus proliferation of leaf explants ofArachis hypogaea L. var. TMV-3 (23). The cells of this culture possessed well-differentiated photochemical appa- ratus and were particularly devoid of stored starch in the chloroplasts during the late log-phase growth, a situation favoring the transition to photoautotrophy. We now report on the nature of the early products of photosynthetic carbon assimilation and their interconversion in such cells. MATERIALS AND METHODS Pla...

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confidence: 96%

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confidence: 99%

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Carbon Assimilation in Photoheterotrophic Cells of Peanut (Arachis hypogaea L.) Grown in Still Nutrient Medium

Seeni

Gnanam²

1982

“…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.…”

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confidence: 99%

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

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...

“…TMV-3 could be well compared with the values of Chl reported previously in callus and cell cultures. Even the highly chlorophyllous cultures reported thus far, contained only 120 to 180 ,ug Chl on a g fresh weight basis (13,20). Another desirable feature is the highly dispersed status of these cells in the culture.…”

Section: Resultsmentioning

confidence: 99%

Growth of Photoheterotrophic Cells of Peanut (Arachis hypogaea L.) in Still Nutrient Medium

Seeni

Gnanam²

1982

Cell suspension cultures were established from the callus proliferation of leaf explants of 10-to 12-day-old seedings of the peanut (Arachis hypogaea L. var. TMV-3). The cels could be cultivated in both agitated and stil media, the latter promoting more of chlorophyll (ChM) Seeds dusted with 0.1% (w/w) Agrosan-D were germinated in garden soil in earthen pots. The growth conditions were the same as described earlier (21). Ten-to 12-d-old seedlings served as the source of explants used for callus initiation.Callus Initiation. Young but fully expanded leaves (second or third from above) were excised from the plants; leaflets were dissected out and thoroughly washed in tap water. Surface sterilization of the leaflets consisted of rapid immersion in 70%o ethanol followed by submersion during 10 min in 5% (v/v) NaOCl and three rinses in sterile distilled H20. Leaf discs of 0.5 cm diameter were punched from the lamina avoiding the midrib and major veins, washed once in sterile distilled H20, and transferred to nutrient media in 100-ml Erlenmeyer flasks. The composition of the culture medium was essentially that of Murashige and Skoog (21) supplemented with the following additives: NAA, 2.5 mg/l; BA, 1 mg/l; and D-glucose, 3.5% (w/v). The culture conditions are described elsewhere (21). Highly chlorophyllous clones of cells were selectively isolated from the calli proliferated upon the leaf explants and transferred to fresh agar media. The additives of the subculture medium, however, were: NAA, 0.6 mg/l; BA, 0.2 mg/ 1; and D-glucose, 2.5%. The stock callus cultures grown in the latter medium for two to three passages of 3 weeks each were used for the initiation of cell suspension cultures.Suspension Cultures. Stock suspension cultures were initiated by disaggregating the cells of the friable calli, scooping the cell masses out, and reinoculating into liquid medium (125 ml) in 500-ml Erlenmeyer flasks. The growth medium of the suspension culture was identical to that used for subculturing the calli except that the agar was omitted. The culture flasks were sealed with two layers of heavy duty aluminum foil and itcubated under still conditions or mounted on a gyrotory shaker at 60 rpm. The cultures were maintained at 28 + 1 C and illuminated at an irradiance of 300 ILE m-2 s-' by a bank of white fluorescent tubes (Philips TL 40/33) for a 12-h photoperiod. The irradiance was measured using a UDT-model 40X Opto-Meter. The cells grew rapidly both in still and agitated media. However, growth tended to be in clumps, particularly in the former.