GIGANTEA regulates phytochrome A-mediated photomorphogenesis independently of its role in the circadian clock

Abstract

GIGANTEA (GI) is a nuclear protein involved in the promotion of flowering by long days, in light input to the circadian clock, and in seedling photomorphogenesis under continuous red light but not far-red light (FR). Here, we report that in Arabidopsis (Arabidopsis thaliana) different alleles of gi have defects in the hypocotyl-growth and cotyledon-unfolding responses to hourly pulses of FR, a treatment perceived by phytochrome A (phyA). This phenotype is rescued by overexpression of GI. The very-low-fluence response of seed germination was also reduced in gi. Since the circadian clock modulates many light responses, we investigated whether these gi phenotypes were due to alterations in the circadian system or light signaling per se. In experiments where FR pulses were given to dark-incubated seeds or seedlings at different times of the day, gi showed reduced seed germination, cotyledon unfolding, and activity of a luciferase reporter fused to the promoter of a chlorophyll a/b-binding protein gene; however, rhythmic sensitivity was normal in these plants. We conclude that while GI does not affect the high-irradiance responses of phyA, it does affect phyA-mediated very-low-fluence responses via mechanisms that do not obviously involve its circadian functions.

Figure 1.

The effects of hourly pulses of FR or hourly pulses of R on cotyledon unfolding and hypocotyl-growth inhibition are reduced in different gi mutant alleles compared to the wild type and rescued by ectopic expression of GI. One-day-old seedlings were transferred to the indicated light treatments for 3 d before measurements. The phyA phyB mutant is included for comparative purposes. Data are means and se of at least five replicate boxes. Factorial ANOVA with GI versus gi and accession as main factors yielded significant effects of the gi mutations at P < 0.0001 for each response (cotyledon unfolding or hypocotyl growth) and light condition (R or FR). In the case of Ler, only gi-5 was included in the analysis to maintain the balance of one wild type and one mutant per accession.

Figure 2.

The VLFR of seed germination is reduced in gi compared to the wild type. Chilled seeds were exposed to a pulse of either long-wavelength FR or R and returned to darkness for 3 d before counting germinated seeds. Data are means and se of two (left) or three (right) replicate boxes. Factorial ANOVA yielded significant (P < 0.0001) interactions between light condition and genotype in both cases. Bonferroni tests indicate significant differences between gi-4 and wild type in darkness (P < 0.05) and in response to a FR pulse (P < 0.001), between gi-5 and wild type only in response to a FR pulse (P < 0.001), and between gi-2 and wild type in response to a FR (P < 0.001) or R (P < 0.05) pulse.

Figure 3.

The circadian clock gates VLFR of seed germination but gi does not affect the phase of rhythmic sensitivity. The seeds were sown at 7 pm, incubated 104 h in darkness at 5°C, and transferred to 22°C (always in darkness) for the time indicated in abscissas, before a 5-min pulse of FR. The number of germinated seeds was counted 4 d later. White and gray boxes represent subjective day and night, respectively. Data are means and se of 12 replicate boxes. Factorial ANOVA of the data shown in the top panel indicates that the effect of GI versus gi is significant at P < 0.0001; the effect of time is significant at P < 0.0001 and the interaction is significant at P < 0.04. Seeds of the wild type or of the gi mutant exposed to a pulse or R at time = 0 germinated more than 90%.

Figure 4.

gi affects the sensitivity of cotyledon unfolding in response to light, but not circadian gating of this response. The seeds were sown at 7 pm, incubated 61 h in darkness at 5°C, and given 11 h R to induce germination. Seed imbibition synchronizes biological rhythms to the beginning of the night phase (Zhong et al, 1998), and this coincided with the time when the R treatment to induce germination was terminated. Time 0 in abscissas is the beginning of the R treatment. White and gray boxes represent subjective day and night, respectively. The seedlings were exposed to five pulses of FR (3 min) starting at the time indicated in abscissas. The distance between cotyledons was measured 2 d later. Data are means ± se of six replicate boxes. Factorial ANOVA indicates that the effect of genotype is significant at P < 0.0001; the effect of time is significant at P < 0.0001 and the interaction is not significant.

Figure 5.

The response of CAB2∷LUC activity to a pulse of FR is affected in the gi-201 mutant. The seeds were sown at 7 pm, incubated 61 h in darkness at 5°C, and given 11 h R to induce germination. Time 8 h in abscissas is subjective dawn of day 5 after the R treatment to induce germination. White and gray boxes represent subjective day and night, respectively. The seedlings were exposed to a pulse of FR (3 min) at the time indicated in abscissas. Bioluminescence values integrated for 6 h after the pulse are expressed relative to the levels in darkness. Data are means and se of 50 seedlings. Factorial ANOVA yielded significant effects of time (P < 0.0001), genotype (P < 0.0001), and interaction (P < 0.001).

Figure 6.

GI affects the hypocotyl gravitropic response in the phyA mutant background but not in the presence of phyA. One-day-old seedlings grown on vertical agar were transferred to the indicated light treatments for 3 d before measurements of the angle between the hypocotyl and the vertical axis. Data are means and se of 12 replicate boxes. Factorial ANOVA (GI/gi and PHYA/phyA as main factors) was conducted for each light and dark condition (the phyB mutant was not included in the analysis). In darkness, the effect of GI versus gi was significant at P < 0.05. Under hourly FR, the effect of PHYA versus phyA was significant at P < 0.0001. Under hourly R, the interaction was significant at P < 0.04 because the effect of gi versus GI was significant only in the phyA background.