Circadian oscillation of gibberellin signaling in Arabidopsis

Abstract

Circadian clocks are endogenous timekeeping mechanisms that allow organisms to anticipate rhythmic, daily environmental changes. Temporal coordination of transcription results in a set of gene expression patterns with peak levels occurring at precise times of the day. An intriguing question is how a single clock can generate different oscillatory rhythms, and it has been proposed that hormone signaling might act in plants as a relay mechanism to modulate the amplitude and the phase of output rhythms. Here we show that the circadian clock gates gibberellin (GA) signaling through transcriptional regulation of the GA receptors, resulting in higher stability of DELLA proteins during daytime and higher GA sensitivity at night. Oscillation of GA signaling appears to be particularly critical for rhythmic growth, given that constitutive expression of the GA receptor expands the daily growth period in seedlings, and complete loss of DELLA function causes continuous, arrhythmic hypocotyl growth. Moreover, transcriptomic analysis of a pentuple della KO mutant indicates that the GA pathway mediates the rhythmic expression of many clock-regulated genes related to biotic and abiotic stress responses and cell wall modification. Thus, gating of GA sensitivity by the circadian clock represents an additional layer of regulation that might provide extra robustness to the diurnal growth rhythm and constitute a regulatory module that coordinates the circadian clock with additional endogenous and environmental signals.

Fig. 1.

The circadian clock controls the diurnal oscillation of DELLA proteins in the cell expansion zone of hypocotyls. Expression of GID1a, GID1b, and GID1c in 5-d-old Ler WT seedlings (A), in toc1-1 (F), and in lhy (K) mutants grown under short-day photocycles (8 h light/16 h dark). Values are expressed relative to PP2a expression. In B–E, G–J, and L–O, seedlings carrying the 35S::TAP-GAI or RGA::GFP-RGA constructs were grown for 5 d under short-day photocycles (8 h light/16 h dark). DELLA protein levels in the Ler WT (B) and in the toc1-1 (G) and lhy (J) mutants were determined by Western blot analysis. TAP-GAI and GFP-RGA proteins were detected with commercial antibodies against the myc tag and GFP, respectively. DELLA levels were normalized against levels of DET3, which was used as loading control. Data are average of three independent experiments and plotted as mean ± SEM. Protein level at ZT0 was set to 1 and used as reference for all other time points. White and gray areas represent day and night, respectively. Fluorescence of GFP-RGA oscillates in the upper part of hypocotyls of Ler WT (C–E) and toc1-1 mutant seedlings (H–J), but not in the lhy mutant (M–O). Fluorescence was detected by confocal microscopy. Images are representative of three independent biological repeats including 12 to 15 seedlings per time point and per genotype.

Fig. 2.

Blocking GA signaling at night affects hypocotyl growth. Seedlings of the HS::gai-1D line were grown under short-day photocycles (8 h light/16 h dark) and received heat treatments of 33 °C for 10 min at ZT5 or ZT17 (Materials and Methods). In A, shaded areas mark the period of the day during which gai-1D accumulates. (B) Expression of gai-1D (bars) and its target gene GA20ox2 (circles, scale on right) after heat treatments at ZT5 (white symbols) and ZT17 (dark symbols). (C) Hypocotyl length of Col-0 WT and HS::gai-1D seedlings that did not receive heat treatments (gray bars) or that received treatments at ZT5 (white bars) or ZT17 (black bars). The experiment was repeated three times with similar results. Data represent the mean ± SD (n ≥ 15 seedlings), and asterisks indicate P < 0.0001.

Fig. 3.

GA application at night releases the growth restrain imposed by DELLAs. RGA::GFP-RGA seedlings grown under short-day photocycles (8 h light/16 h dark) in the presence of 0.2 μM PAC were treated with 1 μM GA₄ at ZT0 or ZT12 or untreated (Materials and Methods). (A) Scheme of DELLA accumulation after GA₄ treatments, deduced from the GFP-RGA fluorescence of seedlings grown under the same conditions (B). Confocal images taken at the time of GA₄ treatment (ZT0 and ZT12) and 1 h (ZT1 and ZT13) and 10 h later (ZT10 and ZT22) show that the maximum period with low DELLA levels spans less than 10 h. Images are representative of three independent biological repeats including eight to 10 seedlings per time point. (C) Hypocotyl length of WT (Ler) seedlings grown in the presence of 0.2 μM PAC that did not receive any additional treatment (mock) or that were treated with 1 μM GA₄ at ZT0 (day) or ZT12 (night). The WT seedlings contain the RGA::GFP-RGA transgene. Data represent mean ± SD (n ≥ 15 seedlings). Asterisks indicate P < 0.001.

Fig. 4.

GA activity regulates diurnal rhythms of hypocotyl elongation. Col 0 and 35S::GID1a seedlings (A) and Ler and quintuple della mutant seedlings (B) were grown under short-day photocycles (8 h light/16 h dark) for 3 d before they were imaged under the same conditions for three additional days. Blue and red symbols and lines denote the WT and mutant/transgenic seedlings, respectively. Seedlings’ growth rates were measured as described in Materials and Methods. White and gray areas represent day and night, respectively. Data represent mean ± SD (n ≥ 10 seedlings). a.u., arbitrary units.

Fig. 5.

DELLAs mediate circadian regulation of transcription. (A) Scatter plot of genes differentially regulated at ZT21 versus ZT9 in WT Ler and della mutants. Genes showing statistically significant (FDR < 0.1) differential expression between Ler and della are displayed for each time point in blue and red. (B) Enrichment of gene ontology categories among genes regulated by DELLAs at ZT9 (P < 0.0001 in all cases). (C) Venn diagram showing overlap between genes regulated by DELLAs at ZT9; genes directly bound by HY5, as detected by ChIP-chip experiments in light-grown seedlings (30); and genes regulated by PIF transcription factors, as genes differentially expressed in the quadruple pif1 pif3 pif4 pif5 mutant in darkness and light, with respect to WT (31).