Metabolite Control Overrides Circadian Regulation of Phosphoenolpyruvate Carboxylase Kinase and CO(2) Fixation in Crassulacean Acid Metabolism

Abstract

Phosphoenolpyruvate carboxylase (PEPc) catalyzes the primary fixation of CO(2) in Crassulacean acid metabolism plants. Flux through the enzyme is regulated by reversible phosphorylation. PEPc kinase is controlled by changes in the level of its translatable mRNA in response to a circadian rhythm. The physiological significance of changes in the levels of PEPc-kinase-translatable mRNA and the involvement of metabolites in control of the kinase was investigated by subjecting Kalanchoë daigremontiana leaves to anaerobic conditions at night to modulate the magnitude of malate accumulation, or to a rise in temperature at night to increase the efflux of malate from vacuole to cytosol. Changes in CO(2) fixation and PEPc kinase activity reflected those in kinase mRNA. The highest rates of CO(2) fixation and levels of kinase mRNA were observed in leaves subjected to anaerobic treatment for the first half of the night and then transferred to ambient air. In leaves subjected to anaerobic treatment overnight and transferred to ambient air at the start of the day, PEPc-kinase-translatable mRNA and activity, the phosphorylation state of PEPc, and fixation of atmospheric CO(2) were significantly higher than those for control leaves for the first 3 h of the light period. A nighttime temperature increase from 19 degrees C to 27 degrees C led to a rapid reduction in kinase mRNA and activity; however, this was not observed in leaves in which malate accumulation had been prevented by anaerobic treatment. These data are consistent with the hypothesis that a high concentration of malate reduces both kinase mRNA and the accumulation of the kinase itself.

Figure 1

Rates of net CO₂ uptake and malate content in leaves exposed to anaerobic conditions for part or all of the dark period. A, Leaves were enclosed in an atmosphere of N₂ for the first half (half N₂) or entire duration (full N₂) of the 13-h dark period before transfer to ambient air. Rates of net CO₂ assimilation were measured. Control leaves were exposed to the ambient atmosphere in the growth chamber. Each gas exchange curve is representative of three replicate runs with se <10%. B, Malate content was measured in leaves subjected to the above treatments with each point being the mean of three replicates with se <10%. ●, Control leaves; ○, full-N₂ leaves; ▪, half-N₂ leaves. The solid bar on the x axis represents the period of darkness.

Figure 2

PEPc kinase activity and levels of translatable PEPc kinase mRNA under ambient and anaerobic conditions at night. Leaves were enclosed in an atmosphere of N₂ overnight to prevent malate accumulation or maintained in ambient air. Samples for PEPc kinase assays (A) and RNA isolation and measurement of PEPc kinase translatable mRNA (B) were taken simultaneously from the same leaves at intervals over the 13-h dark period. Shown are autoradiographs of the ³²P-labeled PEPc bands following SDS-PAGE. The doublet of PEPc bands is caused by the presence in a ratio of about 10:1 of two related subunits in K. fedtschenkoi PEPc, both of which are phosphorylated by PEPc kinase (Carter et al., 1991). The relative intensity of the PEPc bands, shown below each track, was determined by phosphor imaging. The total incorporation of [³⁵S]Met into in vitro translation products using RNA isolated from control and N₂-treated leaves was similar (data not shown). The results are from duplicate experiments

Figure 3

Apparent K_i of PEPc for l-malate, PEPc kinase activity, and translatable kinase mRNA under ambient and anaerobic conditions at night. Control leaves (●) were kept in ambient air throughout. Full-N₂ leaves (○) were enclosed in an atmosphere of N₂ overnight to prevent malate accumulation. Half-N₂ leaves (▪) were enclosed in an atmosphere of N₂ to prevent malate accumulation for the first half of the dark period before transfer to ambient air. Samples for PEPc and PEPc kinase assays and RNA isolation were taken simultaneously from the same leaves at intervals over the dark period. Kinase activity and translatable mRNA values are expressed as percentages of the maximum reached during the 13-h dark period. The results are from duplicate experiments.

Figure 4

Changes in the apparent K_i for l-malate, PEPc kinase activity, and translatable kinase mRNA at the start of the photoperiod after a night in ambient or anaerobic conditions. Leaves that had been maintained in an atmosphere of N₂ overnight to prevent malate accumulation were transferred to ambient air at the start of the photoperiod (○). Control leaves were maintained in ambient air (●). Samples for PEPc and PEPc kinase assays and RNA isolation were taken simultaneously from the same leaves at intervals over the light period. Kinase activity and translatable mRNA values are expressed as percentages of the maximum reached during the photoperiod. The results are from duplicate experiments.

Figure 5

Modulation of net CO₂ assimilation rates and malate accumulation by a temperature increase at night. Control leaves (●) were exposed to ambient air. Half-N₂ leaves (○) were enclosed in an atmosphere of N₂ for the first half of the dark period to prevent malate accumulation. Leaves were subjected to an 8°C rise in temperature from 2:30 to 3 am. The half-N₂ leaves were subsequently exposed to ambient air at 27°C for the duration of the dark period. A, Rates of net CO₂ uptake by leaves under the two treatments with each gas exchange curve representative of three replicate runs with se <10%. B, Malate content was measured in leaves subjected to the above treatments, with each point the mean of three replicates with se <10%. The solid bar on the x axis represents the period of darkness.

Figure 6

Modulation of the apparent K_i for l-malate, PEPc kinase activity, and translatable kinase mRNA by an increase in temperature at night. Control leaves (●) were exposed to ambient air. Half-N₂ leaves (○) were enclosed in an atmosphere of N₂ for the first half of the dark period to prevent malate accumulation. All leaves were subjected to an 8°C rise in temperature from 2:30 to 3 am. The half-N₂ leaves were subsequently exposed to ambient air at 27°C for the duration of the dark period. Samples for PEPc and PEPc kinase assays and RNA isolation were taken simultaneously from the same leaves at intervals over the dark period. Kinase activity and translatable mRNA values are expressed as percentages of the maximum reached in leaves during a normal dark period at 19°C. The results are from duplicate experiments.