The Arabidopsis thaliana CBF cold response pathway plays a central role in cold acclimation. It is characterized by rapid cold induction of genes encoding the CBF1-3 transcription factors, followed by expression of the CBF gene regulon, which imparts freezing tolerance. Our goal was to further the understanding of the cis-acting elements and trans-acting factors involved in expression of CBF2. We identified seven conserved DNA motifs (CM), CM1 to 7, that are present in the promoters of CBF2 and another rapidly cold-induced gene encoding a transcription factor, ZAT12. The results presented indicate that in the CBF2 promoter, CM4 and CM6 have negative regulatory activity and that CM2 has both negative and positive activity. A Myc binding site in the CBF2 promoter was also found to have positive regulatory effects. Moreover, our results indicate that members of the calmodulin binding transcription activator (CAMTA) family of transcription factors bind to the CM2 motif, that CAMTA3 is a positive regulator of CBF2 expression, and that double camta1 camta3 mutant plants are impaired in freezing tolerance. These results establish a role for CAMTA proteins in cold acclimation and provide a possible point of integrating low-temperature calcium and calmodulin signaling with cold-regulated gene expression.

The first described feedback loop of the Arabidopsis circadian clock is based on reciprocal regulation between TIMING OF CAB EXPRESSION 1 (TOC1) and CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1)/LATE ELONGATED HYPOCOTYL (LHY). CCA1 and LHY are Myb transcription factors that bind directly to the TOC1 promoter to negatively regulate its expression. Conversely, the activity of TOC1 has remained less well characterized. Genetic data support that TOC1 is necessary for the reactivation of CCA1/LHY, but there is little description of its biochemical function. Here we show that TOC1 occupies specific genomic regions in the CCA1 and LHY promoters. Purified TOC1 binds directly to DNA through its CCT domain, which is similar to known DNA-binding domains. Chemical induction and transient overexpression of TOC1 in Arabidopsis seedlings cause repression of CCA1/LHY expression, demonstrating that TOC1 can repress direct targets, and mutation or deletion of the CCT domain prevents this repression showing that DNAbinding is necessary for TOC1 action. Furthermore, we use the Gal4/UAS system in Arabidopsis to show that TOC1 acts as a general transcriptional repressor, and that repression activity is in the pseudoreceiver domain of the protein. To identify the genes regulated by TOC1 on a genomic scale, we couple TOC1 chemical induction with microarray analysis and identify previously unexplored potential TOC1 targets and output pathways. Taken together, these results define a biochemical action for the core clock protein TOC1 and refine our perspective on how plant clocks function.ost organisms that experience day/night cycles have a circadian clock that phases cellular processes and behavior to specific times of day while also anticipating daily diurnal changes to confer a fitness advantage (1). The basic molecular architecture of most clocks consists of negative-feedback loops where positive and negative components control each other's expression to generate oscillations with an approximate 24-h period (1).At the core of the Arabidopsis clock, CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) (2) and LATE ELONGATED HY-POCOTYL (LHY) (3) are morning-expressed Myb transcription factors that directly bind the evening element (4, 5) in the promoters of evening-expressed genes and act as transcriptional repressors. CCA1 and LHY have redundant functions (6, 7), are often coexpressed, and make up the negative arm of the first described feedback loop in Arabidopsis by binding the promoter of TIMING OF CAB EXPRESSION 1 (TOC1) (4). Genetically, TOC1 is the positive component of this feedback loop because CCA1/LHY morning reactivation is dependent on TOC1 (4), yet TOC1 overexpression also results in lower CCA1/LHY expression (8, 9), confusing our understanding of TOC1's role in the core clock feedback loop. TOC1 is an evening-expressed protein (10) that is part of a five-member family called the PSEUDO-RESPONSE REGULATORS (PRRs) that are expressed in succession from the morning to the night in the order PRR9, PRR7, PRR5, PRR3, and then TOC1 (11). Sequence similari...

SUMMARYExposure of Arabidopsis thaliana plants to low non-freezing temperatures results in an increase in freezing tolerance that involves action of the C-repeat binding factor (CBF) regulatory pathway. CBF1, CBF2 and CBF3, which are rapidly induced in response to low temperature, encode closely related AP2/ERF DNA-binding proteins that recognize the C-repeat (CRT)/dehydration-responsive element (DRE) DNA regulatory element present in the promoters of CBF-regulated genes. The CBF transcription factors alter the expression of more than 100 genes, known as the CBF regulon, which contribute to an increase in freezing tolerance. In this study, we investigated the extent to which cold induction of the CBF regulon is regulated by transcription factors other than CBF1, CBF2 and CBF3, and whether freezing tolerance is dependent on a functional CBF-CRT/DRE regulatory module. To address these issues we generated transgenic lines that constitutively overexpressed a truncated version of CBF2 that had dominant negative effects on the function of the CBF-CRT/DRE regulatory module, and 11 transcription factors encoded by genes that were rapidly cold-induced in parallel with the 'first-wave' CBF genes, and determined the effects that overexpressing these proteins had on global gene expression and freezing tolerance. Our results indicate that cold regulation of the CBF regulon involves extensive co-regulation by other first-wave transcription factors; that the low-temperature regulatory network beyond the CBF pathway is complex and highly interconnected; and that the increase in freezing tolerance that occurs with cold acclimation is only partially dependent on the CBF-CRT/DRE regulatory module.

Organisms have evolved endogenous biological clocks as internal timekeepers to coordinate metabolic processes with the external environment. Here, we seek to understand the mechanism of synchrony between the oscillator and products of metabolism known as Reactive Oxygen Species (ROS) in Arabidopsis thaliana. ROS-responsive genes exhibit a time-of-day-specific phase of expression under diurnal and circadian conditions, implying a role of the circadian clock in transcriptional regulation of these genes. Hydrogen peroxide production and scavenging also display time-ofday phases. Mutations in the core-clock regulator, CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), affect the transcriptional regulation of ROSresponsive genes, ROS homeostasis, and tolerance to oxidative stress. Mis-expression of EARLY FLOWERING 3, LUX ARRHYTHMO, and TIMING OF CAB EXPRESSION 1 affect ROS production and transcription, indicating a global effect of the clock on the ROS network. We propose CCA1 as a master regulator of ROS homeostasis through association with the Evening Element in promoters of ROS genes in vivo to coordinate time-dependent responses to oxidative stress. We also find that ROS functions as an input signal that affects the transcriptional output of the clock, revealing an important link between ROS signaling and circadian output. Temporal coordination of ROS signaling by CCA1 and the reciprocal control of circadian output by ROS reveal a mechanistic link that allows plants to master oxidative stress responses.redox homeostasis | transcriptional coordination

The circadian clock in Arabidopsis exerts a critical role in timing multiple biological processes and stress responses through the regulation of up to 80% of the transcriptome. As a key component of the clock, the Myb-like transcription factor CIRCADIAN CLOCK ASSOCIATED1 (CCA1) is able to initiate and set the phase of clockcontrolled rhythms and has been shown to regulate gene expression by binding directly to the evening element (EE) motif found in target gene promoters. However, the precise molecular mechanisms underlying clock regulation of the rhythmic transcriptome, specifically how clock components connect to clock output pathways, is poorly understood. In this study, using ChIP followed by deep sequencing of CCA1 in constant light (LL) and diel (LD) conditions, more than 1,000 genomic regions occupied by CCA1 were identified. CCA1 targets are enriched for a myriad of biological processes and stress responses, providing direct links to clock-controlled pathways and suggesting that CCA1 plays an important role in regulating a large subset of the rhythmic transcriptome. Although many of these target genes are evening expressed and contain the EE motif, a significant subset is morning phased and enriched for previously unrecognized motifs associated with CCA1 function. Furthermore, this work revealed several CCA1 targets that do not cycle in either LL or LD conditions. Together, our results emphasize an expanded role for the clock in regulating a diverse category of genes and key pathways in Arabidopsis and provide a comprehensive resource for future functional studies.genome-wide | circadian clock | clock-controlled outputs | transcriptional regulation I n anticipation of the daily changes in light and temperature, the plant's internal circadian clock regulates a large portion of the transcriptome, nearly 80% in rice, poplar, and Arabidopsis (1, 2). The clock provides temporal coordination of multiple biological functions to ensure optimal efficiency. For example, before dawn, photosynthetic transcripts accumulate in anticipation of the expected sunlight. Even when shifted to constant light (LL) and temperature, in the absence of day/night cues, transcripts associated with photosynthesis are still up-regulated before the subjective day (1-4). Not only are light-and energyrelated activities restricted to particular times, but other aspects of plant growth, including water use, hormone activity, UVB response, and low-temperature response, show this gating effect (5-9). This temporal partitioning of biological responses ultimately provides an optimal integration of the plant's organismal functions with the environment.The circadian clock in plants involves a posttranscriptional component and a well-studied transcriptional-translation feedback regulation between the Myb-like transcription factors CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) and a member of the PSEUDO RESPONSE REGULATOR (PRR) family, TIMING OF CAB2 EXPRESSION 1 (TOC1) (10-16). Together these three components contribute t...