PLANT PHYSIOLOGY observed to take place as the culture period was prolonged. The data refer particularly to the synthesis and decomposition of starch, and the correlated reciprocal changes in malic and citric acids.During the culture period, the total protein of the leaf slowly diminished in amount. It is tentatively suggested that the changes in the rates at which the leaves are able to recover from the stressed condition are correlated with the loss of protein and specifically with the gradual, although only partial, de- It has long been realized that green leaves have the ability to fix CO2 in the dark to form organic acids with the concomitant loss of carbohydrate stores. A group of plants known as the succulents is characterized by a particularly active dark fixation of CO2. In the case of the succulents, the organic acids synthesized in the dark are transformed to carbohydrates during a subsequent light period (4).
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.
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