The Behavior of Isocitric Acid in Excised Leaves of Bryophyllum Calycinum During Culture in Alternating Light and Darkness

Abstract

In an earlier study of the diurnal variation of organic acids and starch in excised leaves of Bryophyllum calycinum (3), it was observed that the quantity of isocitric acid present did not change significantly during a threeday period of culture in water under greenhouse conditions of illumination. Because of the contrast between the behavior of this substance and that of malic and citric acids, it seemed essential to repeat the experiment under even more carefully controlled conditions and to employ improvements in the method for the determination of isocitric acid which have now become available (1). The outcome has been a confirmation of the earlier experiment. The changes in the quantity of isocitric acid in Bryophyllum leaves exposed to normal diurnal alternations of light and darkness, if significant at all, are scarcely in excess of the combined errors involved in the sampling and in the method of determination.Experimental procedure The plants were propagated from the same clone used in earlier work. They had been transplanted to sand in pots February 15, 1949, and were grown with the culture solution previously described (2). The samples of leaflets were collected July 28 by the statistical method (8), the upper three pairs of fully developed opposite leaves (five leaflets each) from 20 plants being taken to provide 10 samples each of 60 leaflets. Collection was begun at 4 A.M. (standard time) and all samples were in position in the culture troughs with the petioles in water by 5:30. Sunrise occurred at 4:43 A.M., sunset at 7:14 P.M. Zero time was taken at 5:00 A.M. when the control sample was placed in the drying oven.The troughs were set up in a greenhouse under white parachute cloth shade. During the first day, there was a slight haze in the morning and light cloud was present in the early afternoon; the temperature reached 330 C and the humidity was high. The second day was clear save for a little cloudiness near noon and the maximum temperature was 360 C. The effect of the cloth shade was to diffuse the light but the intensity was of the order of 1600 to 2000 foot-candles throughout most of both days.Samples were removed at times selected to give information on the rates of change of the organic acids and starch during the day and night periods. They were dried at 800 C, broken up and equilibrated at 240 C and 50% relative humidity before being finally ground in a Wiley mill for analysis, and were then stored in closed bottles in the air-conditioned room.

The Effect of Temperature on the Behavior of Malic Acid and Starch in Leaves of Bryophyllum calycinum Cultured in Darkness.

“…A promotion of the accumulaigure 2. For the first 8 hours, owing to tion of organic acids by culture at low temperature ion of acids, the pH dropped at the same is thus clearly evident. th temperatures.…”

“…Although the maximum does not correspond exactly in point of time with the minimum value for starch, the general behavior is exactly that to be anticipated if interconversion of these two substances were assumed to be taking place. The statistical analysis suggests that in the experiment at 240 C there is less than one chance in 50 that the apparent relationship is fortuitous and at 60 C, as well as in several other cases (8,10), far higher correlations have been found.…”

“…The details of the organic acid composition of the was maintained for another 8 hours, but samples are plotted in the lower part of the figure. little further change and a slight increase Isocitric acid is the major organic acid component of irred later.…”

“…The ratio for the interval between 8 and' it were, a centrally located substance in a complex series of metabolic reactions, some of which lead to its increase, some to its diminution. Starch would seem to play the part of a storage substance which, in darkness, rapidly begins to contribute to the reactions that synthesize malic acid.…”

The Capacity of Leaves of Bryophyllum calycinum to Recover from Prolonged Exposure to Darkness or to Light.

“…The curve for citric acid at the left of figure 2 illustrates this point un-usually well. The citric acid in the leaves cultured in darkness increased 6- (8), is masked in the present instance because of the slow and continuous loss of malic acid which supervened after the leaves had been in darkness for 18 hours.…”