Circadian rhythms in the activity of a plant protein kinase.

Abstract

Bryophyllum fedtschenkoi is a Crassulacean acid metabolism plant whose phosphoenolpyruvate carboxylase is regulated by reversible phosphorylation in response to a circadian rhythm. A partially purified protein kinase phosphorylated phosphoenolpyruvate carboxylase in vitro with a stoichiometry approaching one per subunit and caused a concomitant 5‐ to 10‐fold decrease in the sensitivity of the carboxylase to inhibition by malate. The sites phosphorylated in vitro were identical to those phosphorylated in intact tissue. The activity of the protein kinase was controlled in a circadian fashion. During normal diurnal cycles, kinase activity appeared between 4 and 5 h after the onset of darkness and disappeared 2‐‐‐‐3 h before the end of darkness. Kinase activity displayed circadian oscillations in constant environmental conditions. The activity of protein phosphatase 2A, which dephosphorylates phosphoenolpyruvate carboxylase, did not oscillate. Treatment of detached leaves with the protein synthesis inhibitors puromycin and cycloheximide blocked the nocturnal appearance of the protein kinase activity, maintained phosphoenolypyruvate carboxylase in the dephosphorylated state and blocked the circadian rhythms of CO2 output that is observed in constant darkness and CO2‐free air. The simplest explanation of the data is that there is a circadian rhythm in the synthesis of phosphoenolpyruvate carboxylase kinase.

Phosphoenolpyruvate Carboxylase Kinase in Tobacco Leaves Is Activated by Light in a Similar but Not Identical Way as in Maize

“…To overcome this experimental limitation, chromatography on BDA was used to enrich for the tobacco leaf kinase. Previous experiments by Wang and Chollet (1993a) demonstrated that the tobacco enzyme, like C, (Bakrim et al, 1992;Wang and Chollet, 1993b) and CAM (Carter et al, 1991;Li and Chollet, 1994) PEPC-kinases, binds tightly to this "affinity" matrix and is eluted with buffered 0.5 M NaCl. Figure 1 depicts typical chromatographic elution profiles of tobacco PEPC-PK activity from a small column of BDA following extraction from illuminated or darkened leaves, ammonium sulfate concentration, and desalting.…”

“…therein). Finally, a protein phosphatase type 2A inhibitor such as microcystin-LR or okadaic acid should be included during enzyme extraction to minimize in vitro dephosphorylation of phosAs has been observed in C, and CAM leaves (Echevarria et al, 1990;Carter et al, 1991;Jiao et al, 1991a;Bakrim et al, 1992;Jiao and Chollet, 1992;Li and Chollet, 1994), the regulatory phosphorylation of PEPC in C, leaves from tobacco appears to be largely influenced by the activity state of PEPC-PK. However, a simultaneous assessment of the activity of the native PEPC-phosphatase heteromeric protein is necessary before a final conclusion can be drawn.…”

“…Cytosolic levels of Ca2+, pH, and certain photosynthetic metabolites also have been suggested as signaling elements in the light activation of C, PEPC-PK in situ Duff et al, 1996;Giglioli-Guivarc'h et al, 1996). In CAM leaves, however, PEPC-kinase activity is up-/ down-regulated through an endogenous circadian rhythm rather than by a light signal (Carter et al, 1991;Nimmo, 1993;Li and Chollet, 1994). As a result, the CAM leaf kinase exhibits a higher activity during the night than during the day.…”

Conservation and Divergence of Circadian Clock Operation in a Stress-Inducible Crassulacean Acid Metabolism Species Reveals Clock Compensation against Stress

“…PEPc is activated at night by a phosphorylation event that is driven by a circadian clock. Circadian oscillations in the phosphorylation state of PEPc are due to circadian control of the expression and activity of PEPc kinase (PPCK; Carter et al, 1991;Hartwell et al, 1996Hartwell et al, , 1999Hartwell et al, , 2002. Phosphorylation reduces the sensitivity of PEPc to feedback inhibition by malate, and this makes phospho-PEPc more active in vivo at night, allowing malate to accumulate in the vacuole.…”

Multiple cDNAs of phosphoenolpyruvate car☐ylase in the C4 dicotFlaveria trinervia

“…The structural motif Lys/Arg-X-X-Ser of this phosphorylation site i~ also present at the corresponding position of the F. trinervia enzyme. Thus, this element is shared by the C4 and CAM isoforms but is lacking in the C:isoenzyme [3,18].…”