GIGANTEA and EARLY FLOWERING 4 in Arabidopsis exhibit differential phase-specific genetic influences over a diurnal cycle

Abstract

The endogenous circadian clock regulates many physiological processes related to plant survival and adaptability. GIGANTEA (GI), a clock-associated protein, contributes to the maintenance of circadian period length and amplitude, and also regulates flowering time and hypocotyl growth in response to day length. Similarly, EARLY FLOWERING 4 (ELF4), another clock regulator, also contributes to these processes. However, little is known about either the genetic or molecular interactions between GI and ELF4 in Arabidopsis. In this study, we investigated the genetic interactions between GI and ELF4 in the regulation of circadian clock-controlled outputs. Our mutant analysis shows that GI is epistatic to ELF4 in flowering time determination, while ELF4 is epistatic to GI in hypocotyl growth regulation. Moreover, GI and ELF4 have a synergistic or additive effect on endogenous clock regulation. Gene expression profiling of gi, elf4, and gi elf4 mutants further established that GI and ELF4 have differentially dominant influences on circadian physiological outputs at dusk and dawn, respectively. This phasing of GI and ELF4 influences provides a potential means to achieve diversity in the regulation of circadian physiological outputs, including flowering time and hypocotyl growth.

Figure 1.

Photoperiodic Flowering Time in WT and Mutants. (A) Whole plant images. Plants were grown under LD for 4 weeks: Col (WT), elf4, gi, and gi elf4 mutants. The scale bar represents 5 cm. (B) Total numbers of leaves. Total leaves including rosette and cauline leaves were counted at the first flower bloom. White and black bars represent LD and SD, respectively. Data represent the means ± 95% confidence interval (CI) from at least 16 plants. Asterisks indicate statistically significant differences (Col versus elf4; P < 0.01). (C, E) CO mRNA expression levels under LD (C) and SD (E). (D, F) FT mRNA expression levels under LD (D) and SD (F). Total RNA was isolated from 7-day-old seedlings, and CO and FT mRNA were measured by quantitative PCR and normalized against Actin 2 (ACT). White and black bars represent day and night, respectively. Data represent the means ± standard error (SE) from biological triplicates.

Figure 2.

Seedling Growth in WT and Mutants. (A) Seedling images of Col (WT), elf4, gi, and gi elf4 seedlings. Plants grown under LD for 7 d were imaged. The scale bar represents 1 cm. (B) Relative hypocotyl lengths under LD and SD. Hypocotyl lengths were measured and normalized to the mean hypocotyl length under continuous dark (DD) conditions. Data represent the means ± 95% CI from at least 20 seedlings. (C, D) PIF4 mRNA expression levels under LD (C) and SD (D). Total RNA was isolated from 7-day-old seedlings, and PIF4 mRNA expression levels were measured by quantitative PCR and normalized to that of ACT. White and black bars represent day and night, respectively. Data represent the means ± SE from experiments performed in triplicate. Asterisks indicate statistically significant differences (Col versus elf4; P = 0.02 in LD and P = 0.12 in SD).

Figure 3.

Endogenous Clock Activities in WT and Mutants. (A) Leaf movements in LL. Tip-to-tip distances between first and second leaves were measured using the leaf movement assay (LMA) program. (B) Relative amplitude errors (RAE) analyzed by FTT-NLLS. (C, E) CCR2 promoter activities in LL (C) and DD (E). Luminescence intensities were measured every hour, and the absolute luminescence intensities were normalized to the mean intensity in each background. Data represent the means ± SE. (D, F) RAEs analyzed by FTT-NLLS. Period lengths computed from each experiment and statistics are shown in Table 1. (G, H) LHY expression levels in LD (G) and SD (H). Total RNA was isolated from 7-day-old seedlings, and LHY mRNA levels were measured by quantitative PCR and then normalized to that of ACT. White and black bars represent day and night, respectively. Data represent the means ± SE from experiments performed in triplicate.

Figure 4.

Differential Expression Patterns between Col-0 and Each of elf4, gi, and gi elf4 Mutants. (A) Gene expression patterns representing the relative dominance of gi to elf4. (B) Gene expression patterns representing the relative dominance of elf4 to gi. (C) Gene expression patterns reflecting the synergistic effects of gi and elf4 mutations. Red and green colors represent log₂-fold-changes for up- and down-regulation in gi, elf4, and gi elf4 mutants relative to WT, respectively. Patterns were numbered from one to six according to their differential expression. The number of genes in each pattern is denoted in the box. In each pattern, the genes associated with the regulations of flowering time, hypocotyl growth, and circadian rhythmicity.

Figure 5.

Biological Processes Represented by the Genes in the Six Patterns at Dawn and Dusk. (A, B) The heat maps of the enrichment scores. A high enrichment score (see ‘Methods’) in a coordinate (x-axis and y-axis) of the heat map indicates that the corresponding GOBP (y-axis) is significantly represented by the genes in the corresponding pattern (x-axis). The red color represents the enrichment score as described in the color bar. Blue letters indicate GOBPs associated with the regulations of flowering time, hypocotyl growth, and circadian rhythmicity.