A role for multiple circadian clock genes in the response to signals that break seed dormancy in Arabidopsis

Abstract

Plant seeds can sense diverse environmental signals and integrate the information to regulate developmental responses, such as dormancy and germination. The circadian clock confers a growth advantage on plants and uses environmental information for entrainment. Here, we show that normal circadian clock gene function is essential for the response to dormancy-breaking signals in seeds. We show that mutations in the clock genes LATE ELONGATED HYPOCOTYL, CIRCADIAN CLOCK ASSOCIATED1 (CCA1), and GIGANTEA (GI) cause germination defects in response to low temperature, alternating temperatures, and dry after-ripening. We demonstrate that the transcriptional clock is arrested in an evening-like state in dry seeds but rapidly entrains to light/dark cycles in ambient temperatures upon imbibition. Consistent with a role for clock genes in seed dormancy control, CCA1 expression is transcriptionally induced in response to dry after-ripening and that after-ripening affects the amplitude of subsequent transcriptional clock gene oscillations. Control of abscisic acid- and gibberellin-related gene expression in seeds requires normal circadian function, and GI and TIMING OF CAB EXPRESSION1 regulate the response to ABA and GA in seeds. We conclude that circadian clock genes play a key role in the integration of environmental signaling controlling dormancy release in plants.

Figure 1.

The Germination Behavior of lhy cca1 and gi-11 Mutants. (A) The germination of freshly harvested seed before and after cold stratification at 22°C. (B) The germination behavior of seed after-ripened for 3 months at the indicated temperatures in degrees Celsius. 27-17 indicates that 27°C was given during the light period and 17°C during the dark period. (C) The germination of freshly harvested wild-type, LHY-, and CCA1-overexpressing seed at the indicated temperatures. 27-17 indicates that 27°C was given during the light period and 17°C during the dark period. (D) The germination of wild-type and gi mutant seeds in white light with and without the indicated dormancy breaking treatments at 22°C. Con, control. Values given are means ± se for five to eight independent seed batches. Asterisks indicate germination significantly different from the wild type (P < 0.01).

Figure 2.

A Role for the Clock-Associated Genes ZTL and LUX in Seed Dormancy and Germination Control. (A) The germination response of freshly harvested wild type (Col or C24) ztl-3, lux-2, lux-5, and toc1 seed to imbibition at different ambient temperatures. (B) The response of freshly harvested wild-type and ztl-3 seeds to cold stratification treatment. Data points in (A) and (B) represent mean and se of data from five to eight replicate seed batches for each genotype.

Figure 3.

The Transcript Levels of Circadian Clock-Associated Genes in Dormant and After-Ripened Seeds The expression of circadian clock–associated genes in imbibed freshly harvested dormant Col-0 seeds at 22°C in 12-h light/dark cycles (D22) and in seeds in which dormancy has been or is being broken by dry after-ripening (ND22) or by imbibition and maintenance at constant 12°C (ND12), a temperature at which primary dormancy is broken. Expression levels were determined by real-time RT-PCR. Data points represent mean ± sd using two biological replicates.

Figure 4.

Seed Germination in Response to Paclobutrazol and Norfluorazon. The germination of Ws, cca1, lhy, gi-11, and double and triple mutant seeds (as indicated) under control conditions (water agarose; Con) or in response to applied GA (100 μM), NOR (50 μM), or both (GA+NOR) at 22°C. Data points represent the mean and se of data from five to eight independent seed batches. Asterisks indicate germination significantly different from the wild type (P < 0.01).

Figure 5.

The Expression of Selected Genes Central to Hormone Metabolism Associated with Germination Control in Dormant and After-Ripened Seeds. The expression of ABA- and GA-related genes in freshly harvested imbibed Col-0 seeds at 22°C (D22), in dry after-ripened seeds at 22°C (ND22), and in freshly harvested seeds maintained at 12°C (ND12), a temperature at which primary dormancy is broken. Data points represent mean ± sd of data from two biological replicates.

Figure 6.

The Expression of ABA- and GA-Related Genes in 3-Month after-Ripened lhy cca1 and gi-11 Seeds during the first 48 h of Imbibition. (A) Visual illustration of the germination of Ws, lhy cca1, and gi-11 seeds after 48 h of imbibition at 22°C. (B) The expression of GA3OX1, NCED6, ABI3, and CYP707A2 in Ws, lhy cca1, and gi-11. Data points represent means and sd of data from two biological replicates.

Figure 7.

The Transcriptional Induction of CCA1 Expression by after-Ripening Is Independent of the Light Signal. After-ripened Landsberg erecta wild-type seeds were imbibed for 9 h in the light or dark, and the expression of CCA1 and of the after-ripening and light-induced CYP707A2 gene was compared with the expression in freshly harvested seeds. Closed circles, freshly harvested seeds in white light; closed triangles, 3-month after-ripened seed maintained in darkness; open circles, 3-month after-ripened seeds in white light. This treatment only resulted in germination at high frequency. Data points represent the mean and sd of data from two biological replicates.

Figure 8.

The Germination of Cold-Stratified Wild-Type and Clock Mutant Seeds in Control Conditions or in Response to Applied ABA and PAC. Bars represent the mean and se of five replicate seed batches for each genotype. Numbers in brackets indicate the concentration of ABA or PAC in micromoles. TMG refers to the TOC1 minigene (Más et al., 2003), the TOC1 cDNA expressed under its own promoter. WA, water agarose.