Edinburgh Explorer ELF4 is required for oscillatory properties of the circadian clock

Circadian clocks are required to coordinate metabolism and physiology with daily changes in the environment. Such clocks have several distinctive features, including a free-running rhythm of approximately 24 h and the ability to entrain to both light or temperature cycles (zeitgebers). We have previously characterized the EARLY FLOWERING4 ( ELF4 ) locus of Arabidopsis ( Arabidopsis thaliana ) as being important for robust rhythms. Here, it is shown that ELF4 is necessary for at least two core clock functions: entrainment to an environmental cycle and rhythm sustainability under constant conditions. We show that elf4 demonstrates clock input defects in light responsiveness and in circadian gating. Rhythmicity in elf4 could be driven by an environmental cycle, but an increased sensitivity to light means the circadian system of elf4 plants does not entrain normally. Expression of putative core clock genes and outputs were characterized in various ELF4 backgrounds to establish the molecular network of action. ELF4 was found to be intimately associated with the CIRCADIAN CLOCK - ASSOCIATED1 ( CCA1 )/ LONG ELONGATED HYPOCOTYL ( LHY )- TIMING OF CAB EXPRESSION1 ( TOC1 ) feedback loop because, under free run, ELF4 is required to regulate the expression of CCA1 and TOC1 and, further, elf4 is locked in the evening phase of this feedback loop. ELF4 , therefore, can be considered a component of the central CCA1 / LHY - TOC1 feedback loop in the plant circadian clock. Hin dIII- Eco RI fragment. The ELF4 coding region was ampliﬁed against genomic DNA by PCR with the primers ELF435S-L, 5 # -AAAAGATCTCC-GGTCCAACTAAGAAGAAACAAT-3 # and ELF435S-R, 5 # -AAAAGATCTC- GACTTTGACGAAAATCAAAAAG-3 # and this fragment was subcloned between the 35S promoter and the terminator as a Bam HI fragment. This constructwas used togenerate multiple homozygouslines that overexpressed ELF4 ( ELF4 - ox ). All tested lines behaved similarly in all assays. Wild-type Ws lines harboring the CAB2 : LUC 1 , CCA1 : LUC , or CCR2 : LUC transgene were crossed into both the elf4 - 1 mutant and the ELF4 - ox line termed ELF4 - ox - 11 ; in each case, double homozygous lines were identiﬁed in the F 2 generation and bulked. We report experiments using the F 3 generation of these lines. Gating experiments utilized the CAB2 : LUC transgenic line (i.e. the 2CA/C insertion present in accession C24; Millar et al., 1995) introgressed into Ws and elf4 - 1 . ELF4 : LUC wasconstructed bysubcloning approximately a1.5-kb fragment from the bacterial artiﬁcial chromosome clone T28M21 as a Xba I- Nco I frag- ment into pZIP221B. This fragment is between 1.7 kb and 260 bp upstream of the translational start site. The LUC 1 gene from pLUC 1 (Promega) was sub- cloned into this resultant vector as a Nco I- Xba I fragment (pELF4-incomplete: LUC 1 ). A fragment was PCR ampliﬁed using an arbitrary upstream primer and the primer 5 # -AACCATGGTCTCGCCGTTCCTCTTCATAA-3 # . This PCR productwas digested with Nco I andthe fragment intersected into the Nco Isite of pELF4-incomplete:LUC 1 , resulting in the completed transcriptional fusion pELF4P:LUC 1 . Similarly, an upstream primer