Aging induces gradual yet massive cell death in higher organisms, including annual plants. Even so, the underlying regulatory mechanisms are barely known, despite the long-standing interest in this topic. Here, we demonstrate that ORE1, which is a NAC (NAM, ATAF, and CUC) transcription factor, positively regulates aging-induced cell death in Arabidopsis leaves. ORE1 expression is up-regulated concurrently with leaf aging by EIN2 but is negatively regulated by miR164. miR164 expression gradually decreases with aging through negative regulation by EIN2, which leads to the elaborate up-regulation of ORE1 expression. However, EIN2 still contributes to aging-induced cell death in the absence of ORE1. The trifurcate feed-forward pathway involving ORE1, miR164, and EIN2 provides a highly robust regulation to ensure that aging induces cell death in Arabidopsis leaves.

The circadian clock is essential for coordinating the proper phasing of many important cellular processes. Robust cycling of key clock elements is required to maintain strong circadian oscillations of these clock-controlled outputs. Rhythmic expression of the Arabidopsis thaliana F-box protein ZEITLUPE (ZTL) is necessary to sustain a normal circadian period by controlling the proteasome-dependent degradation of a central clock protein, TIMING OF CAB EXPRESSION 1 (TOC1). ZTL messenger RNA is constitutively expressed, but ZTL protein levels oscillate with a threefold change in amplitude through an unknown mechanism. Here we show that GIGANTEA (GI) is essential to establish and sustain oscillations of ZTL by a direct protein-protein interaction. GI, a large plant-specific protein with a previously undefined molecular role, stabilizes ZTL in vivo. Furthermore, the ZTL-GI interaction is strongly and specifically enhanced by blue light, through the amino-terminal flavin-binding LIGHT, OXYGEN OR VOLTAGE (LOV) domain of ZTL. Mutations within this domain greatly diminish ZTL-GI interactions, leading to strongly reduced ZTL levels. Notably, a C82A mutation in the LOV domain, implicated in the flavin-dependent photochemistry, eliminates blue-light-enhanced binding of GI to ZTL. These data establish ZTL as a blue-light photoreceptor, which facilitates its own stability through a blue-light-enhanced GI interaction. Because the regulation of GI transcription is clock-controlled, consequent GI protein cycling confers a post-translational rhythm on ZTL protein. This mechanism of establishing and sustaining robust oscillations of ZTL results in the high-amplitude TOC1 rhythms necessary for proper clock function.

Circadian clocks are ubiquitous molecular time-keeping mechanisms that coordinate physiology and metabolism and provide an adaptive advantage to higher plants. The central oscillator of the plant clock is composed of interlocked feedback loops that involve multiple repressive factors acting throughout the circadian cycle. PSEUDO RESPONSE REGULATORS (PRRs) comprise a five-member family that is essential to the function of the central oscillator. PRR5, PRR7, and PRR9 can bind the promoters of the core clock genes CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) to restrict their expression to near dawn, but the mechanism has been unclear. Here we report that members of the plant Groucho/Tup1 corepressor family, TOPLESS/TOPLESS-RELATED (TPL/TPR), interact with these three PRR proteins at the CCA1 and LHY promoters to repress transcription and alter circadian period. This activity is diminished in the presence of the inhibitor trichostatin A, indicating the requirement of histone deacetylase for full TPL activity. Additionally, a complex of PRR9, TPL, and histone deacetylase 6, can form in vivo, implicating this tripartite association as a central repressor of circadian gene expression. Our findings show that the TPL/TPR corepressor family are components of the central circadian oscillator mechanism and reinforces the role of this family as central to multiple signaling pathways in higher plants.T he circadian clock system consists of multiple interlocked feedback loops that generally contain activating and repressive arms within the loops to sustain robust 24-h oscillations (1-3). Many of the best-characterized elements in the plant circadian system are transcriptional repressors that act during the subjective morning to allow evening expression of their targets, or are expressed during the subjective evening to keep expression of morning genes down at night (4, 5). One well-studied loop of reciprocal repression involves the inhibition of early-day expression of the evening gene TIMING OF CAB EXPRESSION 1 (TOC1; PRR1) by the morning-expressed myb transcription factors CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) (6). TOC1 is the founding member of five closely related PSEUDO RESPONSE REGULATORS (PRRs: PRR9, PRR7, PRR5, PRR3) and binds DNA through a conserved CCT domain at the carboxy terminus, repressing evening expression of both CCA1 and LHY (7, 8). The mechanism of CCA1/LHY-mediated repression of TOC1 requires the corepressor DE-ETIOLATED1 (DET1) to interact with CCA1 and LHY at the TOC1promoter, likely in the context of a larger COP10-DET1-DDB1(CDD) complex (9). TOC1 is also regulated by the related myb-transcription factor, REV8, which binds the TOC1 promoter and likely acts as a positive activator (10, 11). In contrast, the partners and mechanism of lateevening TOC1-mediated repression of CCA1/LHY are unknown. However, a second evening-phased repressor complex, EARLY FLOWERING 3 (ELF3)-EARLY FLOWERING4 (ELF4)-LUX ARRHYTHMO (LUX) has been identified as acting to ...