Insight into missing genetic links between two evening-expressed pseudo-response regulator genes TOC1 and PRR5 in the circadian clock-controlled circuitry in Arabidopsis thaliana

In Arabidopsis thaliana, many circadian clock-associated genes have been identified. Among them, the evening-expressed TOC1 (TIMING OF CAB EXPRESSION 1) gene plays a role by forming a transcriptional feedback core loop together with the morning-expressed CCA1 (CIRCADIAN CLOCK-ASSOCIATED 1) gene and its homologous LHY (LATE ELONGATED HYPOCOTYL) gene. TOC1 encodes a member of the PSEUDO-RESPONSE REGULATOR (PRR) family, including PRR9, PRR7, PRR5, PRR3,and PRR1/TOC1. The PRR genes other than TOC1 (or PRR1) also appear to be crucial for certain circadian-associated events. To clarify missing genetic linkages amongst these PRR genes, here we constructed a toc1 prr5 double knockdown mutant. In free-running circadian rhythms, the resulting toc1-2 prr5-11 mutant plants showed an extremely short period and reduced amplitude phenotype, which was more severe than that of the toc1-2 single mutant plant, suggesting a non-linear genetic interaction between TOC1 and PRR5. Surprisingly, the hallmark early flowering phenotype of toc1-2 in the short-day conditions had been converted to a markedly late flowering phenotype in the long-day conditions, when combined with the prr5-11 allele, which itself showed a subtle flowering phenotype. This unexpected genetic result (i.e. phenotypic sign conversion) suggested that the TOC1 and PRR5 genes are coordinately implicated in a non-linear and closed genetic circuitry. In the toc1-2 prr5-11 double mutant, the diurnal expression profile of CDF1 (CYCLING DOF FACTOR 1) was markedly de-repressed in the evening in the long-day conditions. These and other results of this study led us to propose the novel view that TOC1 might play bipartite roles in the control of flowering time within a closed circuitry; the one is a GI (GIGANTEA)-dependent negative role through CCA1/LHY, and the other is a CDF1-dependent positive role through cooperating closely with PRR5.