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

Abstract

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.

Figure 1.

Reproducible diel response in phosphorylation of SnRK1 reporters under equinoctial growth conditions. SnRK1-reporter lines were grown in a 12-h photoperiod (irradiance 160 µmol m⁻² s⁻¹) for 22 d as controls in three independent experiments, in which additional plants were grown in parallel and subjected to different treatments. Whole rosettes were harvested at 4-h intervals through the diel cycle. A) Phosphorylation of the NUC or GEN polypeptide. This was quantified by immunoblotting as described in Materials and Methods and in Supplemental Fig. S1. B) Selected metabolites. Data are shown for the control samples from the following experiments: (i) Control A: blue symbols, NUC and GEN lines (from Experiment 3 in Supplemental Data Set S1, also shown in Fig. 2); (ii) Control B: red symbols, NUC and GEN lines (from Experiments 4 and 5 in Supplemental Data Set S1, also shown in Figs. 3 and 4; (iii) Control C: gray symbols; NUC line only (from Supplemental Data Set S1, Experiment 2, also shown in Supplemental Fig. S3B). Data are shown as mean ± SD (n = 3–4 biological replicates, each containing 3–5 pooled rosettes). Statistical analyses are provided separately for each time series in Figs. 2 and 3 and Supplemental Data Set S1, Experiment 2, respectively.

Figure 2.

SnRK1 activity in Arabidopsis plants transferred to low irradiance for one light period. NUC and GEN lines were grown in a 12-h photoperiod at 160 µmol m⁻² s⁻¹ irradiance for 19 d. At 20 DAS, starting at dawn (ZT0), irradiance was reduced to 60 µmol m⁻² s⁻¹ for the following 12 h (treatment light period), plants were darkened at ZT12 (treatment night), and at ZT24 were re-illuminated at an irradiance of 160 µmol m⁻² s⁻¹ (recovery day), as described in Moraes et al. (2019). Samples from both genotypes and from the corresponding control treatment (plants left in the original growth light regime; corresponds to Control A in Fig. 1) were harvested at 4-h intervals from ZT0-ZT36 (with respect to dawn on the treatment day). A) Soluble sugars and starch. B) Phosphorylation of NUC (top) and GEN (bottom) SnRK1-reporter polypeptides. The dashed gray background denotes the low-light treatment day. Results are shown as mean ± SD (n = 3–4 biological replicates). Statistical analysis: letters indicate significant (P < 0.05) changes between different times for a given genotype and asterisks indicate significant (P < 0.05) differences between lines at a given time point, according to one-way ANOVA and pairwise multiple comparison post testing using the Holm–Sidak method. The original and additional data are available in Supplemental Data Set S1, Experiment 3.

Figure 3.

SnRK1 activity in Arabidopsis plants transferred to continuous low light. NUC and GEN lines were grown in a 12-h photoperiod at 160 µmol m⁻² s⁻¹ irradiance for 19 d. At 20 DAS, plants were shifted at dawn (ZT0) to continuous low light (90 µmol m⁻² s⁻¹) for 32 h, while corresponding controls were kept in the original growth conditions (12-h photoperiod, 160 µmol m⁻² s⁻¹; corresponds to Control B in Fig. 1). A) Diel changes of sugars and starch. B) Phosphorylation of NUC (top) and GEN (bottom) SnRK1-reporter polypeptides. The dashed gray background represents the night for control samples and subjective night for treated samples. Data points for control samples at ZT28 and ZT32 were not measured and the plotted values are replicated from those at ZT4 and ZT8, respectively (black circles). Results are shown as mean ± SD (n = 3 biological replicates). Statistical analysis: letters indicate significant (P < 0.05) changes between different times for a given treatment and asterisks represent significant (P < 0.05) differences between the low-light treatment and the control at a given time point according to one-way ANOVA with pairwise multiple comparison post testing using the Holm–Sidak method. The original and additional data are available in Supplemental Data Set S1, Experiment 4.

Figure 4.

SnRK1 activity in Arabidopsis plants grown in T24 and T28 light-dark cycles. NUC and GEN lines were grown from sowing in a 14-h light/14 h dark (T28) cycle under 160 µmol m⁻² s⁻¹ irradiance for 19 d, in parallel with control plants that were grown in a 12-h light/12-h dark (T24) cycle. At 20 DAS (as defined by the T24 cycle), starting at dawn, plants were harvested at 4-h intervals throughout the light-dark cycle (open symbols). At the end of the light-dark cycle, additional plants were left in darkness and harvested for two further time points for the plants grown in a T24 cycle (i.e. at ZT28 and ZT32), and for one further time point for plants grown in a T28 cycle (i.e. at ZT32). This experiment was carried out in parallel with that shown in Fig. 3. The T24 data from ZT0 to ZT24 are identical to the control treatment in the continuous low-light treatment of Fig. 3 (Control B), with additional time points added for plants that were left in the dark from ZT24 onwards and harvested at ZT28 and ZT32. The vertical dotted line indicates the time at which control plants were transferred to continued darkness. A) Soluble sugars and starch. B) Phosphorylation of NUC (top) and GEN (bottom) SnRK1-reporter polypeptides. The white background represents light in both T cycles and the gray-shaded areas represent darkness (ZT12–ZT24 for the T24 cycle and ZT14–ZT28 for the T28 cycle) in one or both T cycles: pale gray denotes darkness in the T24 and light in the T28 cycle, mid-gray denotes darkness in both T-cycles, and dark gray represents extended night in both T cycles. Black closed symbols represent time points in extended darkness. Results are shown as mean ± SD (n = 3 biological replicates). Statistical analysis: letters indicate significant (P < 0.05) changes between different times for a given genotype and asterisks represent significant (P < 0.05) differences between T28 versus T24 at a given time point according to a one-way ANOVA with pairwise multiple comparison post testing using the Holm–Sidak method. The original and additional data are available in Supplemental Data Set S1, Experiment 5.

Figure 5.

Elevated Tre6P in the light period inhibits nuclear SnRK1 activity. Line #6.3 (NUC × iTPS) was grown in a 12-h photoperiod at 160 µmol m⁻² s⁻¹ irradiance for 20 d. At 21 DAS, plants were sprayed at ZT4 (arrow) with 2% (v/v) ethanol to induce the expression of bacterial TPS (otsA) or with water as a mock-induction control, and then harvested at ZT7, ZT9, and ZT11 for measurement of Tre6P (top panel), sucrose (middle), and the phosphorylation status of the NUC polypeptide (bottom) as a readout of nuclear SnRK1 activity in vivo. Results are shown as mean ± SD (n = 3–4 biological replicates). Statistical analysis: asterisks indicate significant (P < 0.05) differences between the ethanol-induced plants and water controls according to a one-way ANOVA with pairwise multiple comparison post testing using the Holm–Sidak method. Additional metabolites are shown in Supplemental Fig. S7. The original data are available in Supplemental Data Set S1, Experiment 7.

Figure 6.

Transiently high Tre6P at the end of the night leads to an increase in nuclear SnRK1 activity. Line #6.3 (NUC × iTPS) and #1.2 control (NUC × alcR) were grown in short-day conditions (6 h light/18 h dark) at 160 µmol m⁻² s⁻¹ irradiance for 25 d. At 26 DAS, plants were sprayed with 2% (v/v) ethanol at ZT20 (arrow) to induce expression of bacterial TPS (otsA). Rosettes were harvested at ZT20, ZT22, and ZT24 (end of normal night), and at ZT25 and ZT27 (extended darkness) for measurement of: Tre6P (top panel) and the phosphorylation of the NUC polypeptide (bottom) as a readout of nuclear SnRK1 activity in vivo. Results are shown as mean ± SD (n = 3–4 biological replicates). Statistical analysis: letters indicate significant (P < 0.05) changes between different times for a given genotype and asterisks indicate significant (P < 0.05) differences between the #6.3 (NUC × iTPS) and #1.2 control (NUC × alcR) lines according to a one-way ANOVA with pairwise multiple comparison post testing using the Holm–Sidak method. Additional metabolites are shown in Supplemental Fig. S8. The original data are available in Supplemental Data Set S1, Experiment 8.