COP1 interacts with the ELF4-ELF3-LUX complex for the regulation of hypocotyl elongation in Arabidopsis thaliana

Abstract

Daily rhythms are regulated by endogenous circadian clock in plants. A circadian oscillator keeps circadian time regarding the appropriate time of day (24-h) and is entrained by environmental cues such as daily and seasonal changes. The most circadian clock is showing rhythmical pattern which can regulate hypocotyl elongation in Arabidopsis thaliana. In previous study, ELF3-ELF4-LUX complex (EC complex) functions as a circadian clock and represses the expression of the bHLF factors PHYTOCHROME INTERACTING FACTOR (PIF). ELF3 (EARLY FLOWERING 3), ELF4 (EARLY FLOWERING 4) and LUX (LUX ARRHYTHMMO

PHYTOCLOCK 1) (called the evening complex) form the protein complex in the early evening. It is required for the proper expression of the transcription factors. PIF4 and PIF5 genes are belong to these types of transcription factors. EC complex inhibits the PIF4 and PIF5, leading to promoting photomorphogenesis

yet, the mechanism by which between COP1 and EC complex is still unknown. Here, we elucidate the regulation of hypocotyl length by interaction between EC complex and PIFs through COP1. To figure out EC complex can regulate hypocotyl elongation through COP1, known as a repressor of photomorphogenesis, we investigated phenotypes of hypocotyl length. Based on hypocotyl phenotypes under LD, SD, 12L/12D and DD conditions, the phenotypes of cop1-4 mutant are shorter than these of wild type (Col-0). Also, the phenotypes of elf3-1, elf4-101 and pcl1-1 (evening complex) mutants are longer than these of wild type. In other words, a loss-function of COP1 gene leads to short hypocotyl as repressors of photomorphogenesis. To identify genetic analysis between COP1 and EC complex, we constructed various mutant lines containing double (cop1-4elf3-1, cop1-4elf4-101, cop1-4pcl1-1) and triple (cop1-4elf3-1pcl1-1) mutants. When double and triple mutants were analyzed, we found that phenotypes of cop1-4 background mutant including cop1-4elf3-1, cop1-4elf4-101, cop1-4pcl1-1, cop1-4elf3-1elf4-101 were shorter than those of wild type, indicating that COP1 acts downstream of ELF3, ELF4 and LUX. To investigate relationship between EC complex and COP1, we examined COP1 mRNA level in elf3-1, elf4-101, and pcl1-1 mutants under 12L/12D (or 8h light/16h dark) condition. There was no difference expression of COP1 mRNA in evening complex mutants compared with Col-0. To investigate relationships between COP1 and PIF, we showed that the expressions of PIFs mRNA were regulated by COP1 under 12L/12D conditions. For this reason, PIFs direct-gene expressions were also induced, suggesting that COP1 functions as activator of PIFs genes. These data showed that COP1 with EC complex regulated PIFs mRNA expressions. In this study, the expression of COP1 mRNA in evening complex mutants is no difference compared with Col-0, however genetic interaction following cop1-4 mutant phenotype was clearly showed. Therefore, we suggest that between COP1 and EC complex is associate with translational regulation. To demonstrate translational relationship between EC complex and COP1, we examined yeast-two hybrid assays. We found that COP1 physically interacted with ELF3, ELF4 and LUX. Thus, we propose a model that COP1 is translationally supperesed by EC complex. Also COP1 can transcriptionally activates PIFs mRNA levels. Overall, these results provide a molecular mechanism for the hypocotyl elongation by COP1. And EC complex through PIFs pathway is mediated photomorphogenesis.