Analysis of phase of LUCIFERASE expression reveals novel circadian quantitative trait loci in Arabidopsis

Abstract

In response to exogenous rhythms of light and temperature, most organisms exhibit endogenous circadian rhythms (i.e. cycles of behavior and gene expression with a periodicity of approximately 24 h). One of the defining characteristics of the circadian clock is its ability to synchronize (entrain) to an environmental rhythm. Entrainment is arguably the most salient feature of the clock in evolutionary terms. Previous quantitative trait studies of circadian characteristics in Arabidopsis (Arabidopsis thaliana) considered leaf movement under constant (free-running) conditions. This study, however, addressed the important circadian parameter of phase, which reflects the entrained relationship between the clock and the external cycle. Here it is shown that, when exposed to the same photoperiod, Arabidopsis accessions differ dramatically in phase. Variation in the timing of circadian LUCIFERASE expression was used to map loci affecting the entrained phase of the clock in a recombinant population derived from two geographically distant accessions, Landsberg erecta and Cape Verde Islands. Four quantitative trait loci (QTL) were found with major effects on circadian phase. A QTL on chromosome 5 contained SIGNALING IN RED LIGHT REDUCED 1 and PSEUDORESPONSE REGULATOR 3, both genes known to affect the circadian clock. Previously unknown polymorphisms were found in both genes, making them candidates for the effect on phase. Fine mapping of two other QTL highlighted genomic regions not previously identified in any circadian screens, indicating their effects are likely due to genes not hitherto considered part of the circadian system.

Figure 1.

Phase in Cvi × Ler RILs and parental lines. A, Phase in response to different photoperiods. Means ± 95% confidence interval. B, Distribution of phases in RIL population after three photoperiods; parental mean phase ± 95% confidence interval.

Figure 2.

Positions (top graph) and effects (bottom graph) of phase QTL. A, ALL1, chromosome 1: Cvi alleles give early phase in LD 3:21. B, GUF5a, chromosome 5: Cvi alleles give late phase in LD 3:21. C, ALL2, chromosome 2: Cvi alleles give early phase in LD 12:12. D, GUF5b, chromosome 5: Cvi alleles give late phase in LD 12:12. Genotyping markers shown on horizontal axis. Markers asterisked where cofactors fitted. Black bar, 95% confidence interval for a QTL. Genome-wide 95% confidence levels: LD 3:21 LOD score ≥ 2.65; LD 12:12 LOD score ≥ 2.55.

Figure 3.

Confirmation of phase QTL effects (relative to Ler) and position of markers used to map breakpoints of NILs. Top, x axis depicts NIL and parental genotypes. Double-headed arrows show QTL interval predicted from NIL phase data. A, ALL1 QTL in LD 3:21: only NIL 42 shows early phase. B, GUF5a QTL in LD 3:21: all NILs except 45a show late phase. C, GUF5b QTL in LD 12:12: all NILs show late phase. Means ± 95% confidence interval. Black, Cvi introgression; white, Ler background.

Figure 4.

A, Real-time RT-PCR transcript levels of PRR3 cDNA, normalized to β-TUBULIN4 level and ZT 0 in Ler and Cvi ecotypes. Primers span first exon boundary. LD 12:12 ZT 0 to 24, then constant darkness. White bar, Day; black bar, night; gray bar, subjective night. The mean of two repeats is plotted for each ecotype; error bars show 95% confidence interval. PRR3 levels were normalized against those of β-TUBULIN4 for each time point before being normalized to ZT 0. B, Nucleotide sequence and predicted translation of PRR3 first and second exon boundary (/) from Ler and Cvi; numbering relative to coding region start site.

Figure 5.

Predicted amino acid sequence of SRR1 from Col (At5g59560), Ler, and Cvi ecotypes. Polymorphic residues (bold) are indicated by asterisk; amino acids conserved across multiple species (Staiger et al., 2003) are highlighted in gray.