The 24-hour rhythmicity in the levels of many eukaryotic mRNAs contrasts with the long half-lives of most detected proteins. Such stable molecular species are not expected to reflect the RNA rhythms, emphasizing the need to test the circadian regulation of protein abundance and modification. Here we present circadian proteomic and comparable phosphoproteomic time-series from Arabidopsis thaliana plants, estimating that 0.4% of quantified proteins and a much larger proportion of quantified phosphosites were rhythmic under constant light conditions. Approximately half of the quantified phospho-sites were most phosphorylated at subjective dawn, when SnRK1 protein kinase was a candidate regulator.A CCA1-over-expressing line that disables the plant clock gene circuit lacked most or all circadian protein phosphorylation, indicating that the phospho-dawn is regulated by the canonical clock mechanism. The few phospho-sites that fluctuated despite CCA1-over-expression still tended to peak in abundance close to subjective dawn, indicating that their temporal regulation was less dependent on the clock gene circuit. To test the potential functional relevance of our datasets, we conduct phosphomimetic experiments using the bifunctional enzyme fructose-6-phosphate-2-kinase / phosphatase (F2KP), as an example. The clock gene circuit controls diverse protein targets in metabolism and physiology via phosphorylation, including where the bulk abundance of the cognate proteins is arrhythmic.

Twenty-four-hour, circadian rhythms control many eukaryotic mRNA levels, whereas the levels of their more stable proteins are not expected to reflect the RNA rhythms, emphasizing the need to test the circadian regulation of protein abundance and modification. Here we present circadian proteomic and phosphoproteomic time series from Arabidopsis thaliana plants under constant light conditions, estimating that just 0.4% of quantified proteins but a much larger proportion of quantified phospho-sites were rhythmic. Approximately half of the rhythmic phospho-sites were most phosphorylated at subjective dawn, a pattern we term the “phospho-dawn.” Members of the SnRK/CDPK family of protein kinases are candidate regulators. A CCA1 -overexpressing line that disables the clock gene circuit lacked most circadian protein phosphorylation. However, the few phospho-sites that fluctuated despite CCA1 -overexpression still tended to peak in abundance close to subjective dawn, suggesting that the canonical clock mechanism is necessary for most but perhaps not all protein phosphorylation rhythms. To test the potential functional relevance of our datasets, we conducted phosphomimetic experiments using the bifunctional enzyme fructose-6-phosphate-2-kinase/phosphatase (F2KP), as an example. The rhythmic phosphorylation of diverse protein targets is controlled by the clock gene circuit, implicating posttranslational mechanisms in the transmission of circadian timing information in plants.

Background and Aims ATP-dependent phosphofructokinases (PFKs) catalyse phosphorylation of the carbon-1 position of fructose-6-phosphate, to form fructose-1,6-bisphosphate. In the cytosol, this is considered a key step in channelling carbon into glycolysis. Arabidopsis thaliana has seven genes encoding PFK isoforms, two chloroplastic and five cytosolic. This study focusses on the four major cytosolic isoforms of PFK in vegetative tissues of A. thaliana. Methods We have isolated homozygous knock-out individual mutants (pfk1, pfk3, pfk6, pfk7) and two double mutants (pfk1/7 and pfk3/6) and characterized their growth and metabolic phenotypes. Key Results In contrast to single mutants and the double mutant pfk3/6 for the hypoxia-responsive isoforms, the double mutant pfk1/7 had reduced PFK activity and shows a clear visual and metabolic phenotype with reduced shoot growth, early flowering, and elevated hexose levels. This mutant also has an altered ratio of short/long aliphatic glucosinolates and an altered root-shoot distribution. Surprisingly, this mutant does not show any major changes in short-term carbon flux and in levels of hexose-phosphates. Conclusions We conclude that the two isoforms PFK1 and PFK7 are important for sugar homeostasis in leaf metabolism and apparently source/sink relations in Arabidopsis, while PFK3 and PFK6 only play a minor role under normal growth conditions.