Many organisms, including plants, use the circadian clock to measure the duration of day and night. Daily rhythms in the plant circadian system are generated by multiple interlocked transcriptional/translational loops and also by spatial regulations such as nuclear translocation. GIGANTEA (GI), one of the key clock components in Arabidopsis, makes distinctive nuclear bodies like other nuclear-localized circadian regulators. However, little is known about the dynamics or roles of GI subnuclear localization. Here, we characterize GI subnuclear compartmentalization and identify unexpected dynamic changes under diurnal conditions. We further identify EARLY FLOWERING 4 (ELF4) as a regulator of GI nuclear distribution through a physical interaction. ELF4 sequesters GI from the nucleoplasm, where GI binds the promoter of CONSTANS (CO), to discrete nuclear bodies. We suggest that the subnuclear compartmentalization of GI by ELF4 contributes to the regulation of photoperiodic flowering.

As most organisms age, their appearance, physiology, and behaviour alters as part of a life history strategy that maximizes their fitness over their lifetime. The passage of time is measured by organisms and is used to modulate these age-related changes. Organisms have an endogenous time measurement system called the circadian clock. This endogenous clock regulates many physiological responses throughout the life history of organisms to enhance their fitness. However, little is known about the relation between ageing and the circadian clock in plants. Here, we investigate the association of leaf ageing with circadian rhythm changes to better understand the regulation of life-history strategy in Arabidopsis. The circadian periods of clock output genes were approximately 1h shorter in older leaves than younger leaves. The periods of the core clock genes were also consistently shorter in older leaves, indicating an effect of ageing on regulation of the circadian period. Shortening of the circadian period with leaf age occurred faster in plants grown under a long photoperiod compared with a short photoperiod. We screened for a regulatory gene that links ageing and the circadian clock among multiple clock gene mutants. Only mutants for the clock oscillator TOC1 did not show a shortened circadian period during leaf ageing, suggesting that TOC1 may link age to changes in the circadian clock period. Our findings suggest that age-related information is incorporated into the regulation of the circadian period and that TOC1 is necessary for this integrative process.

In plants, the circadian clock controls daily physiological cycles as well as daylength-dependent developmental processes such as photoperiodic flowering and seedling growth. Here, we report that FIONA1 (FIO1) is a genetic regulator of period length in the Arabidopsis thaliana circadian clock. FIO1 was identified by screening for a mutation in daylength-dependent flowering. The mutation designated fio1-1 also affects daylength-dependent seedling growth. fio1-1 causes lengthening of the free-running circadian period of leaf movement and the transcription of various genes, including the central oscillators CIRCADIAN CLOCK-ASSOCIATED1, LATE ELONGATED HYPOCOTYL, TIMING OF CAB EXPRESSION1, and LUX ARRHYTHMO. However, period lengthening is not dependent upon environmental light or temperature conditions, which suggests that FIO1 is not a simple input component of the circadian system. Interestingly, fio1-1 exerts a clear effect on the period length of circadian rhythm but has little effect on its amplitude and robustness. FIO1 encodes a novel nuclear protein that is highly conserved throughout the kingdoms. We propose that FIO1 regulates period length in the Arabidopsis circadian clock in a close association with the central oscillator and that the circadian period can be controlled separately from amplitude and robustness.

A gene-trapping vector carrying a GUS/Luciferase dual reporter gene was developed to establish an efficient and convenient screening system for T-DNA-based gene trapping in plants. A key feature of this gene trap scheme is to place two different types of reporters, luciferase (Luc) and β-glucuronidase (GUS), as a fusion protein within a trapped gene to probe the activity of the gene. Luc is then utilized as a non-invasive, vital and highly sensitive screening reporter to identify trapped lines, including direct screening of the trapped lines from the primary T-DNA mutant pools. GUS is utilized as a histochemical assay reporter to analyze detailed cellular expression patterns. Transgenic expression studies in Arabidopsis showed that this fusion reporter protein retains functional enzyme activity for both GUS and Luc. Using this system in Arabidopsis, we were able to identify 3,737 trapped lines from 26,900 individual T-DNA insertion lines. Sequence determination of the T-DNA insertion loci in the genome of 78 trapped lines identified GUS/Luc fusions with 27 annotated Arabidopsis genes which included a subset of transcription factors, protein kinases, regulatory proteins and metabolic enzymes. Of these, particular expression patterns of four tagged genes were further confirmed by analyzing putative promoter regions of the corresponding wild-type genes. Furthermore, the protein stability of the GUS/Luc fusion reporter was controlled by application of luciferase substrate (luciferin), overcoming the excessive stability problem of GUS that causes misrepresentation of the transcriptional activity of a promoter. These results demonstrate the utility of the GUS/Luc dual reporter system as a gene trap reporter for studying plant genome function and also as a convenient dual reporter system for study of gene expression.

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.