In vivo protein kinase activity of SnRK1 fluctuates in Arabidopsis rosettes during light-dark cycles

Sucrose-nonfermenting 1 (SNF1)-related kinase 1 (SnRK1) is a central hub in carbon and energy signaling in plants, and is orthologous with SNF1 in yeast and the AMP-activated protein kinase (AMPK) in animals. Previous studies of SnRK1 relied on in vitro activity assays or monitoring of putative marker gene expression. Neither approach gives unambiguous information about in vivo SnRK1 activity. We have monitored in vivo SnRK1 activity using Arabidopsis (Arabidopsis thaliana) reporter lines that express a chimeric polypeptide with an SNF1/SnRK1/AMPK-specific phosphorylation site. We investigated responses during an equinoctial diel cycle and after perturbing this cycle. As expected, in vivo SnRK1 activity rose toward the end of the night and rose even further when the night was extended. Unexpectedly, although sugars rose after dawn, SnRK1 activity did not decline until about 12 h into the light period. The sucrose signal metabolite, trehalose 6-phosphate (Tre6P), has been shown to inhibit SnRK1 in vitro. We introduced the SnRK1 reporter into lines that harbored an inducible trehalose-6-phosphate synthase construct. Elevated Tre6P decreased in vivo SnRK1 activity in the light period, but not at the end of the night. Reporter polypeptide phosphorylation was sometimes negatively correlated with Tre6P, but a stronger and more widespread negative correlation was observed with glucose-6-phosphate. We propose that SnRK1 operates within a network that controls carbon utilization and maintains diel sugar homeostasis, that SnRK1 activity is regulated in a context-dependent manner by Tre6P, probably interacting with further inputs including hexose phosphates and the circadian clock, and that SnRK1 signaling is modulated by factors that act downstream of SnRK1.

Automated summary

Sucrose-nonfermenting 1 (SNF1)-related kinase 1 (SnRK1) is a central hub in carbon and energy signaling in plants, with orthologues in yeast (SNF1) and animals (AMP-activated protein kinase, AMPK). Previous studies focused on in vitro assays or marker gene expression, providing limited information about in vivo SnRK1 activity. This study used Arabidopsis reporter lines to monitor in vivo SnRK1 activity and found that it increased towards the end of the night and further when the night was extended. Surprisingly, SnRK1 activity did not decline until about 12 hours into the light period, despite the rise in sugars after dawn. The metabolite trehalose 6-phosphate (Tre6P), which inhibits SnRK1 in vitro, was introduced into the plants, and it was found that elevated Tre6P decreased SnRK1 activity during the light period. These findings suggest that SnRK1 operates within a network that controls carbon utilization and diel sugar homeostasis, and its activity is regulated by Tre6P in a context-dependent manner.