40S Ribosomal protein S6 kinase integrates daylength perception and growth regulation in Arabidopsis thaliana

Abstract

Plant growth occurs via the interconnection of cell growth and proliferation in each organ following specific developmental and environmental cues. Therefore, different photoperiods result in distinct growth patterns due to the integration of light and circadian perception with specific Carbon (C) partitioning strategies. In addition, the TARGET OF RAPAMYCIN (TOR) kinase pathway is an ancestral signaling pathway that integrates nutrient information with translational control and growth regulation. Recent findings in Arabidopsis (Arabidopsis thaliana) have shown a mutual connection between the TOR pathway and the circadian clock. However, the mechanistical network underlying this interaction is mostly unknown. Here, we show that the conserved TOR target, the 40S ribosomal protein S6 kinase (S6K) is under circadian and photoperiod regulation both at the transcriptional and post-translational level. Total S6K (S6K1 and S6K2) and TOR-dependent phosphorylated-S6K protein levels were higher during the light period and decreased at dusk especially under short day conditions. Using chemical and genetic approaches, we found that the diel pattern of S6K accumulation results from 26S proteasome-dependent degradation and is altered in mutants lacking the circadian F-box protein ZEITLUPE (ZTL), further strengthening our hypothesis that S6K could incorporate metabolic signals via TOR, which are also under circadian regulation. Moreover, under short days when C/energy levels are limiting, changes in S6K1 protein levels affected starch, sucrose and glucose accumulation and consequently impacted root and rosette growth responses. In summary, we propose that S6K1 constitutes a missing molecular link where day-length perception, nutrient availability and TOR pathway activity converge to coordinate growth responses with environmental conditions.

Figure 1.

S6K1 and S6K2 expression and protein levels are regulated by photoperiod and the circadian clock. S6K1 and S6K2 transcript levels were detected by RT-qPCR in 2-week-old wild type seedlings grown under short day (SD, A), long day (LD, A) and continuous light conditions (LL, B). Results were analyzed in technical triplicates and normalized to Actin 2 in two independent biological replicates in (A), in (B) the results of one representative biological replicate are shown. Error bars refer to ± standard deviation values. S6K total protein levels determined in 10 day-old seedlings grown under either short day (SD) (C) or long day (LD) conditions (E). In parallel, the levels of phosphorylated S6K protein (S6K-P) were detected in the same samples, in SD (D) and in LD (F). Loading control corresponds to Coomassie Brilliant Blue staining of the same western membranes used either for anti-S6K or anti-S6K-P detection. Arrows point to S6K or S6K-phosphorylated bands (S6K-P), star indicates an unspecific band. Underneath each western blot is shown the average signal intensity of either the S6K or S6K-P signals after normalization with loading control in two independent biological replicates. Error bars represent ± standard error values. MW represents protein molecular weight marker bands in KDa. Time (h) refers to hours after lights on. Dark gray rectangles correspond to the dark period under LD, whereas lighter gray rectangles correspond to the dark period under SD. Dotted rectangles refer to the subjective night period.

Figure 2.

S6K total protein levels are regulated by the 26S proteasome. A) S6K total protein levels present in 10 day-old seedlings grown under SDs conditions, either mock-treated (DMSO—Dimethyl sulfoxide; left panels) or incubated with the 26S proteasome inhibitor (MG132; right panels). Seedlings were incubated at ZT3 for the times described (h). The upper panels show total S6K levels, whereas the lower panels reflect the actin levels. Asterisk (*) refers to an unspecific band. MW refers to protein molecular weight marker bands shown in KDa. B) Plotted averages of S6K specific band in two independent biological duplicate experiments under the exact same conditions as described in (A). Error bars represent ± standard error values. Details for quantification of results are provided in the Materials and methods section. Statistically significant differences (* indicates P < 0.05) were calculated using Student's t test for each time point analysed.

Figure 3.

The circadian F-box protein ZTL modulates S6K protein levels and half-life. Total S6K protein accumulates at higher levels and for a longer period of time in ztl-3 mutant plants than in wild type (WT, Col-0) seedlings grown under SD conditions. A) Total S6K and actin protein levels in WT plants (upper panels) and ztl-3 mutants (lower panels) were detected by western blot analysis using anti-S6K and anti-actin specific antibodies. Arrows point to S6K and actin bands, respectively. Star indicates an unspecific band. B) Average S6K signal intensity after normalizing with the corresponding actin signal in three independent biological replicates. C) Similarly, phosphorylated S6K is present for a longer period of time in the ztl-3 mutants (lower panels) when compared with WT (upper panels) seedlings. Loading control was determined by actin levels in the same blots. Arrows point to S6K-P and actin bands, respectively. Star indicates an unspecific band. D) Average signal intensity for S6K-P after normalizing with actin loading control in two independent biological replicates. E) Upper panels: SD grown 10 day-old WT and ztl-3 seedlings were incubated at ZT3 with the protein synthesis inhibitor cycloheximide (CHX) and maintained under white light for the indicated hours (h). The s6k1.1 mutant was used as negative control. Shown is one representative experiment out of two biological replicates. Arrows point to S6K and actin bands, respectively. Star indicates an unspecific band. Lower panel: average S6K signal intensity after normalizing with the loading control in two independent biological replicates. F) Upper panels: SD grown 10 day-old WT and ztl-3 seedlings were incubated at ZT3 with cycloheximide (CHX) and maintained in the dark for the indicated hours (h). Shown one representative experiment out of two biological replicates. Arrows indicate S6K or actin levels, asterisk points to unspecific band. Lower panel: Quantification of the average S6K signal after normalization with the loading control (actin) in two independent biological replicates. Gray rectangle represents the incubation in the dark. Error bars correspond to ± standard error values. MW represents protein molecular weight marker bands shown in KDa. Time refers to hours (h) after lights on; white and gray rectangles indicate the light and dark periods, respectively. Statistically significant differences (* P < 0.05, ***P < 0.001) were calculated using Student's t test for each time point analyzed.

Figure 4.

Modulation of S6K1 levels in ztl-3 mutants affects plant growth responses. A) Upper panel: representative photos of genotypes used to generate ztl-3 s6k1.1 double mutants. Seedlings were grown in vitro for 10 days under SD conditions. Lower panel: Boxplot analysis of corresponding root length of Col-0 (green, n = 84) seedlings, ztl-3 (orange, n = 38), s6k1.1 (dark blue, n = 40) mutants, and ztl-3 s6k1.1 cross lines #1.1.1 (light blue; n = 16) and #1.1.5 (lilac, n = 19) in three independent biological replicates. Results shown for Col-0 and ztl-3 are the same as in (C). However, different representative photos were selected in each upper panel to provide a better overview of these two genotypes. B) Boxplot analysis of rosette area of 21-days old SD-grown Col-0 (n = 176), ztl-3 (n = 124), s6k1.1 (n = 64), and ztl-3 s6k1.1 lines #1.1.1 (n = 84) and #1.1.5 (n = 88) in two independent biological replicates. Colors represent same genotypes as described in (A). C) Upper panel: representative photos of genotypes used to generate ztl-3; S6K1p::S6K1g-CFP lines. Seedlings were grown in vitro for 10 days under SD conditions. Lower panel: Boxplot analysis of corresponding root length of Col-0 (green, n = 84) seedlings, ztl-3 (orange, n = 38) mutants, S6K1p::S6K1g-CFP#11.6 lines (dark red, n = 35) and ztl-3; S6K1p::S6K1g-CFP cross lines #2.1.3 (beige; n = 15) and #3.11.2 (light red, n = 21) in three independent biological replicates. D) Boxplot analysis of total rosette areas from 21 day-old SD-grown Col-0 (n = 176), ztl-3 mutants (n = 124), S6K1p::S6K1g-CFP#11.6 seedlings (n = 67), and crossed ztl-3; S6K1p::S6K1g-CFP lines #2.1.3 (n = 65) and #3.11.2 (n = 66) from three independent biological replicates. Scale bar represents 1 cm in all images of panels (A) and (C). Boxplots show the median (black line), and box limits extend from the 25th to the 75th percentiles, whiskers represent the 10th and 90th percentiles, whereas outliers are shown as individual black dots. Different letters correspond to statistically significant differences (P < 0.05) determined by One-Way ANOVA followed by Tukey's test (GraphPad Prism).

Figure 5.

Regulation of S6K1 levels under SD conditions is required for efficient use of resources and adequate growth responses. A, B) Variable S6K1 levels affect diel accumulation of sugars. Top to bottom: accumulation of Starch, Sucrose, Glucose, and Fructose in 30-day old soil-grown seedlings under SD conditions. Pools of three rosettes were analyzed in five biological replicates in Col-0 and s6k1.1 mutants (A) and in three biological replicates in Col-0 and S6K1p::S6K1g-CFP#11.6 overexpression line (B). The results are the mean ± SD. Statistically significant differences are indicated by asterisks (Student's t-test): *P < 0.05, **P < 0.01, and ***P < 0.001. FW refers to fresh weight of analyzed plants (g). C) Current working model describing overall impact of circadian (clock drawing) and photoperiod (sun drawing) regulation of S6K1 and consequently, TOR pathway activity. S6K1 protein levels start to increase at dawn and will be maximal between ZT3-6 when TOR-dependent phosphorylated S6K also accumulates. At dusk, ZTL will mediate S6K1 proteasome-dependent degradation. S6K1 protein levels will then be minimal during the long dark period. This regulatory network will allow the coordination between C/energy use and TOR pathway activity (depicted as translational capacity) to ensure that plant growth responses are perfectly matched with the available resources especially during the long night. White cubes show available C for growth and its pattern of accumulation is depicted by the yellow line, green rosettes represent Arabidopsis, and gray circles connected to the black lines refer to ribosomes associated to mRNAs (to depict translational capacity). White and gray rectangles indicate the light and dark periods, respectively.