Equilibration of Label in Malate during Dark Fixation of CO₂ in Kalanchoë fedtschenkoi

Abstract

In vitro studies of dark 14CO2 fixation with isolated cell aggregates of Kalanchocefedtschenkoi showed that malate synthesized after 20 Bradbeer et al. (3) to occur via two CO2 fixation steps: first, RuDP2 carboxylase was thought to fix CO2 generating two molecules of 3-PGA, which subsequently were converted to PEP; second, this PEP should be subject to a second carboxylation step yielding OAA, finally giving rise to malate. This hypothesis was based upon the finding that label in carbons 4 and 1 in malate was distributed at a constant ratio of 2:1 after short and long periods of dark CO2 fixation. Recently this contention gained some support from the observation of labeled 3-PGA after dark CO2 fixation of isolated cells of Sedum telephium (2). Reinvestigating the early reports of Bradbeer et al. (3) and Jolchine (6), Sutton and Osmond (10) reported that malate was labeled exclusively in the C-4 position after a short time of CO2 dark fixation in leaves of four CAM species, whereas after longer fixation times label also appeared in the C-1 position. These investigators attributed the formerly observed 2:1 ratio to an erroneous method of malate degradation. Additional insight into this matter was provided by in vitro studies of Kluge et al. (7). Upon feeding PEP to tissue slices of two CAM plants during 14CO2 fixation in the dark, the incorporation of 14C was stimulated, but the ratio of label in C-4 to C-1 remained about 7:3, similar to that of the control. This observation was a first indication that the labeling pattern in malate might not be a consequence of carboxylation reactions, but of reactions occurring after carboxylation. This paper presents additional evidence that carboxylation of RuDP is not involved during synthesis of malate in the dark and that label appearing in C-1 of malate is presumably due to the action of fumarase. MATERIALS AND METHODSPlant Material. Mature plants of Kalanchoe fedtschenkoi were kept in an environmental control chamber at a rhythm of 9 hr light (40,000 lux fluorescent tubes plus red light) at 28 C and 40% relative humidity and corresponding 15 hr dark at 17 C and 78% relative humidity. Diurnal changes of acidity in medium size leaves were about 100 ueq/g fresh weight.Selection of Incubation Medium. At the end of the light period, pieces of 2 cm2 were cut out of mature leaves and the epidermis of the lower side was carefully peeled off. These pieces of tissue were floated on water upside down and allowed to take up 14CO2 in the dark for 10 min. After rinsing with distilled H20 for 5 min, the tissue was incubated at 25 C in 5 ml of various solutions, which were renewed each hour. Solution one was distilled H20, solution two contained 50 mm HEPES, pH 7.5, 0.1 mm Ca(NO3)2, 1 mM MgCl2, and 1 mm K2HPO4; solutions three to six contained buffer as solution two plus 0.4, 0.6, 0.8, and 1 M sorbitol, respectively.After 5 hr the leaf tissues were rinsed with distilled H2O and extracted in boiling ethanol. The leakage of radioactivity from the tissues was determined and com...

Effect of Varying CO2 Partial Pressure on Photosynthesis and on Carbon Isotope Composition of Carbon-4 of Malate from the Crassulacean Acid Metabolism Plant Kalanchoë daigremontiana Hamet et Perr.

“…The isotopic composition of carbon4 of malic acid isolated from K. daigremontiana in the morning reflects not only the isotopic composition of carbon4 of newly synthesized malic acid but also reflects the isotopic composition of the small pool of malic acid which remained at the end of the previous day. In addition, carbons I and 4 of malic acid are to some extent randomized by the action of fumarase following carboxylation (1,6,10). Provided that fumarase randomization is incomplete, it is possible to use the isotopic compositions of carbons I and 4 to calculate the isotopic composition prior to randomization.…”

“…Note that E3 contains the fractionations due to C02-HCO3 interchange and PEP carboxylase. The equation can thus be restated as: k3/k2 = (0.9933 -k12/k 3)/(k12/k 13 _ 1.0044) (6) Calculation of Intemal CO2 Concentration, C02(i) C02(i) was derived from gas exchange measurements by using the relationship: (l) where Fis the proportion of total malate extracted which is present at the beginning of the night and I-F is the proportion which is formed during the night. An equation analogous to equation 1 can be applied to the combined isotopic composition of carbons I and 4.…”

Postillumination CO2 fixation by wheat leaves

“…It is concluded that in the dark 70 to 80% of the carbon is fixed by RuBPCase (sum of the radioactivity percentages found in C3 metabolites plus 30% of the labelling of malate and aspartate) is the same. These figures may be overestimated because a randomization between C1 and C4 of malate occurs by mitochondrial fumarase [3]. Thus phosphoenolpyruvate labelling indicates that a fraction of the radioactivity in C1 of malate came from PGA carbons.…”

Crassulacean Acid Metabolism (CAM): Einige stoffwechselphysiologische und ökologische Aspekte

“…KLUGE und TING 1978 (vgl. KLUGE und TING 1978, COCKBURN et al 1979 (BÖCHER und KLUGE 1977, 1978 1976, KLUGE und TING 1978, OSMOND 1978 (vgl. KLUGE 1976, KLUGE und TING 1978.…”