The Dark Fixation of CO₂ by Succulent Leaves: Metabolic Changes Subsequent to Initial Fixation.

Abstract

The methods used in these longer periods of dark fixation of C1402 are the same as those described in detail in a previous communication (15). A specially constructed apparatus permits excised leaves from B. calycinum to be exposed to C1402 in total darkness. Approximately one gm of young leaves taken from the apex of the plants immediately before use was placed in the apparatus, C1402 generated from 4 to 5 mg of BaC'403 (specific activity 0.0282 mc/mg) was admitted, and the reaction terminated at the desired time by homogenization in boiling 80 % ethanol. The extract was filtered, extracted with Skellysolv A (Pentane), and the ethanolic extract concentrated to a volume of 2.0 ml under reduced pressure. Two dimensional chromatography of an aliquot (0.1 to 0.2 ml) of the concentrated extract in 80 % phenol-20 % water (w/w) in the first direction and (79) butanol-(19) acetic acid-(50) water (v/v/v) in the second, was used to separate the compounds. Radio-autographs were made by exposing "no-screen" x-ray film to the chromatogram. Identification of radioactive compounds was made by elution and subsequent cochromatography with known compounds. The activity of each compound was measured directly on the paper with an end window Geiger tube. Derivatives of the a-keto acids were made by disrupting the tissue with 30 ml of 5 N H2SO4, adding 30 ml of 5 N H2SO4 saturated with 2,4-dinitrophenyl hydrazine, and forming the-hydrazone derivatives as described by Ranson (personal communication). The mixture was allowed to stand for one hour at room temperature and then filtered. The filtrate was extracted three times with 25-ml portions of ethyl acetate. The ethyl acetate was then extracted three times with 25-ml portions of 10 % Na2CO3. The Na2CO3 solution was acidified to pH 1.0 with cold 5 N H2SO4 and the hydrazones extracted into ethyl acetate. The ethyl acetate fraction was dried over anhydrous Na2SO4 and then concentrated to 2.0 ml under reduced pressure.The hydrazones were separated by paper chromatography by the method of Isherwood and Cruickshank (9), and were eluted and identified by cochromatography with authentic derivatives of the keto-acids.

The Influence of 0.03% Carbon Dioxide on Protein Metabolism of Etiolated Avena sativa Coleoptiles

“…This hypothesis is consistent with reports of dark fixation in which malate and aspartate are the first detectable labeled products (2,15,16), and with data indicating that in maize roots the rate of fixation of CO2 into a water soluble fraction is correlated with the rate of protein synthesis (16). In addition, atmospheric CO2 levels have been shown to stimulate the incorporation of labeled leucine into an ethanol insoluble residue from carrot and tomato roots (16), and it has been reported that malate can replace CO2 in stimulating the growth of etiolated coleoptiles (1).…”

Maintenance Carbon Cycle in Crassulacean Acid Metabolism Plant Leaves

“…Nocturnal '4C02 assimilation profiles indicate that various metabolites are labeled, especially aspartate, alanine, and intermediates ofthe citric acid cycle (17,23). The percentage of the label that accumulates in these components relative to malate, however, is low.…”

Sodium Chloride Effect on Dark Fixation of CO2 by Marine & Terrestrial Plants

“…The methods used in the study of the dark fixation of CO2 in intact spinach leaves are the same as those detailed in earlier communications (8,9). Spinach leaves were obtained commercially and thoroughly washed in distilled water and blotted dry; 1 g of young leaves was placed in an apparatus which permits exposure to the C140., in total darkness.…”

“…It will be shown in this paper that the effects of sodium chloride can be reversed by adding oxidized or reduced diphosphopyridine nucleotide (DPN+, DPNH). Two possible mechanisms are advanced to explain the (9). Radioactive compounds were located by exposing the chromatogram to no-screen X-ray film.…”