Circadian rhythms inKalanchoë: the pathway of(14)CO 2 fixation during prolonged light.

(14)CO2 was applied repeatedly at 3- to 6-h intervals toKalanchoë daigremontiana leaves during continuous light of differing irradiances. The circadian rhythm in net CO2 uptake in gasexchange measurements and its disappearance at high irradiances was confirmed by oscillating rates of(14)CO2 incorporation. At 10-30 W m(-2) a markedly circadian oscillation in the(14)CO2-uptake rate was measured; with increasing energy fluence rate the oscillation levelled off at a constant high uptake rate. The labelling patterns obtained during the 10 min of(14)CO2 fixation indicated that the rhythm of CO2 exchange is the consequence of a rhythmic behaviour in the C4 pathway of CO2 fixation. During the mininum of(14)CO2 uptake no C4 products were labelled; however, substantial amounts of label were transferred to C4 products during the peaks of(14)CO2 uptake. Metabolism of C3 and C4 products was also studied in pulsechase experiments at different points of the circadian cycle. In bright light (100 W m(-2)), when the(14)CO2 uptake was constantly high, the transfer of label into C4 products (malic acid) was high in spite of the fact that the malate pool is known to be reduced to a permanently low level under these conditions. This led us to the conclusion that it is not the capacity of the phosphoenolpyruvatecarboxylase-mediated CO2 fixation but rather the storage of malic acid in the vacuole that is disturbed under bright-light conditions when the circadian oscillation levelled off.

Circadian rhythms in crassulacean acid metabolism: phase relationships between gas exchange, leaf water relations and malate metabolism in Kalanchoë daigremontiana.

Gas exchange, leaf water relations, malate content and phosphoenolpyruvate (PEP) carboxylase activity in crude extracts were examined for circadian rhythmicity in the crassulacean acid metabolism plant Kalanchoë daigremontiana. At low irradiance (20 W m(-2)) the rhythm in CO2 uptake continued for several days with a period length of approx. 22 h, whereas the transpiration rhythm was no longer apparent after 24 h. This shows that the CO2 rhythm in continuous light (LL) is not under stomatal control. Circadian oscillations in malate content were detectable for up to 72 h in LL but were of much reduced amplitude. This was reflected in the changes in leaf water relations, which quickly damped after transfer to LL. The activity of PEP carboxylase assayed immediately after extraction showed a rhythmicity for at least 18 h, but after 36 h, values from different plants were scattered. We suggest that the CO2-uptake rhythm is primarily the result of endogenous changes in the activity of PEP carboxylase, which competes to varying degrees with ribulose-1,5-bisphosphate carboxylase for CO2.

Circadian rhythms in Kalanchoë: effects of irradiance and temperature on gas exchange and carbon metabolism.

Gas exchange in K. blossfeldiana shows a circadian rhythm in net CO2 uptake and transpiration when measured under low and medium irradiances. The period length varies between 21.4 h at 60 W m(-2) and 24.0 h at 10 W m(-2). In bright light (≧80 W m(-2)) or darkness there are no rhythms. High leaf temperatures result in a fast dampening of the CO2-uptake rhythm at moderate irradiances, but low leaf temperatures can not overcome the dampening in bright light. The rhythm in CO2 uptake is accompanied by a less pronounced and more rapidly damped rhythm in transpiration and by oscillations in malate levels with the amplitude being highly reduced. The oscillations in starch content, usually observed to oscillate inversely to the acidification in light-dark cycles, disappear after the first cycle in continuous light. The balance between starch and malate levels depends in continuous light on the irradiance applied. Leaves show high malate and low starch content at low irradiance and high starch and low malate in bright light. During the first 12 h in continuous light replacing the usual dark period, malate synthesis decreases with the increasing irradiance. Up to 50 W m(-2) starch content decreases; at higher irradiances it increases above the values usually measured at the end of the light period of the 12:12 h light-dark cycle.

Changes of the chlorophyll fluorescence induction kinetics of C3 and CAM plants during day/night cycles.

The induction kinetics of the 680 nm chlorophyll fluorescence were measured on attached leaves of Kalanchoë daigremontiana R. Hamet et Perr. (CAM plant), Sedum telephium L. and Sedum spectabile Bor. (C3 plant in spring, CAM plant in summer) and Raphanus sativus L. (C3 plant) at three different times during a 12/12 h day/night cycle. During the fluorescence transient the fluorescence intensity at the O, P and T-level (fO, fmax, fst,) was different for the plant species tested; this may be due to their different leaf structure, pigment composition and organization of their photosystems. The kinetics of the fluorescence induction depended on the time of preillumination or dark adaptation during the light/dark cycle but not on the type of primary CO2 fixation mechanism (C3 and CAM). For dark adapted leaves measured either at the end of the dark phase or after dark adaptation of plants taken from the light phase a higher P-level fluorescence, a higher variable fluorescence (P-O) and a larger complementary area were found than for leaves of plants taken directly from the light phase. This indicates the presence of largely oxidized photosystem 2 acceptor pools during darkness. During the light phase the fluorescence decline after the P-level was faster than during the dark phase; from this we conclude that the light adaptation of the photosynthetic apparatus (state 1→state 2 transition, Δ pH) during the induction period proceeded faster in plants taken from the light phase than in plants taken from the dark phase.

Changes of the chlorophyll fluorescence induction kinetics of C3 and CAM plants during day/night cycles.

The induction kinetics of the 680 nm chlorophyll fluorescence were measured on attached leaves of Kalanchoë daigremontiana R. Hamet et Perr. (CAM plant), Sedum telephium L. and Sedum spectabile Bor. (C3 plant in spring, CAM plant in summer) and Raphanus sativus L. (C3 plant) at three different times during a 12/12h day/night cycle. During the fluorescence transient the fluorescence intensity at the O, P and T-level (fO, fmax, fst,) was different for the plant species tested; this may be due to their different leaf structure, pigment composition and organization of their photosystems. The kinetics of the fluorescence induction depended on the time of preillumination or dark adaptation during the light/dark cycle but not on the type of primary CO2 fixation mechanism (C3 and CAM). For dark adapted leaves measured either at the end of the dark phase or after dark adaptation of plants taken from the light phase a higher P-level fluorescence, a higher variable fluorescence (P-O) and a larger complementary area were found than for leaves of plants taken directly from the light phase. This indicates the presence of largely oxidized photosystem 2 acceptor pools during darkness. During the light phase the fluorescence decline after the P-level was faster than during the dark phase; from this we conclude that the light adaptation of the photosynthetic apparatus (state 1→ state 2 transition, Δ pH) during the induction period proceeded faster in plants taken from the light phase than in plants taken from the dark phase.

Crassulacean acid metabolism (CAM) in Kalanchoë daigremontiana: Temperature response of phosphoenolpyruvate (PEP)-carboxylase in relation to allosteric effectors.

Net CO2 dark fixation of Kalanchoë daigremontiana varies with night temperature. We found an optimum of fixation at about 15° C; with increasing night temperature fixation decreased. We studied the temperature dependence of the activity of phosphoenolpyruvate (PEP)-carboxylase, the key enzyme for CO2 dark fixation. We varied the pH, the substrate concentration (PEP), and the L-malate and glucose-6-phosphate (G-6-P) concentration in the assay. Generally, lowering the pH and reducing the amount of substrate resulted in an increase in activation by G-6-P and in an increase in malate inhibition of the enzyme. Furthermore, malate inhibition and G-6-P activation increased with increasing temperature. Activity measurements between 10° C and 45°C at a given concentration of the effectors revealed that the temperature optimum and maximum activities at that optimum varied with the effector applied. Under the influence of 5 mol m(-3) L-malate the temperature optimum and maximum activity dropped drastically, especially when the substrate level was low (at 0.5 mol m(-3) PEP from 32° C to 20° C). G-6-P raised the temperature optimum and maximum activity when the substrate level was low. If both malate and G-6-P were present, intermediate values were measured. We suggest that changes in metabolite levels in K. daigremontiana leaves can alter the temperature features of PEP-carboxylase so that the observed in vivo CO2 dark fixation can be explained on the basis of PEP-carboxylase activity.

Inhibition of the circadian rhythm of CO2 metabolism in Bryophyllum leaves by cycloheximide and dinitrophenol.

The circadian rhythm of CO2 output in darkened leaves of Bryophyllum fedtschenkoi R. Hamet and Perrier can be inhibited by cycloheximide (≧10(-6) mol) and 2,4-dinitrophenol (≧10(-5) mol) applied via the transpiration stream. After having been suppressed by 10(-6) M cycloheximide, the rhythm can be reinitiated with a 12-h exposure to light. Experiments using (14)CO2 show that cycloheximide abolishes the rhythm by inhibiting the dark fixation of CO2. Cycloheximide inhibits malate accumulation and acidification of the leaves, but does not affect the amount of the CO2-fixing enzyme phosphoenol-pyruvate carboxylase (PEP-C, EC 4.1.1.31) which can be extracted from the leaves during the 45 h of the experiment. Cycloheximide has no direct effect on the activity of the enzyme as measured in the assay. PEP-C from desalted leaf extracts was inhibited by L-malate (Ki=0.4 mmol). The most likely explanation for the inhibitory effect of cycloheximide and dinitrophenol is that they cause changes in tonoplast properties which result in a redistribution of malate from the vacuole to the cytoplasm. An increase in malate concentration in the cytoplasm will lead to inhibition of PEP-carboxylase, and hence the suppression of the rhythm of CO2 output.