Joel A. Kreps

Orchestrated Transcription of Key Pathways in Arabidopsis by the Circadian Clock

Like most organisms, plants have endogenous biological clocks that coordinate internal events with the external environment. We used high-density oligonucleotide microarrays to examine gene expression in Arabidopsis and found that 6% of the more than 8000 genes on the array exhibited circadian changes in steady-state messenger RNA levels. Clusters of circadian-regulated genes were found in pathways involved in plant responses to light and other key metabolic pathways. Computational analysis of cycling genes allowed the identification of a highly conserved promoter motif that we found to be required for circadian control of gene expression. Our study presents a comprehensive view of the temporal compartmentalization of physiological pathways by the circadian clock in a eukaryote.

Transcriptome Changes for Arabidopsis in Response to Salt, Osmotic, and Cold Stress

To identify genes of potential importance to cold, salt, and drought tolerance, global expression profiling was performed on Arabidopsis plants subjected to stress treatments of 4°C, 100 mm NaCl, or 200 mm mannitol, respectively. RNA samples were collected separately from leaves and roots after 3- and 27-h stress treatments. Profiling was conducted with a GeneChip microarray with probe sets for approximately 8,100 genes. Combined results from all three stresses identified 2,409 genes with a greater than 2-fold change over control. This suggests that about 30% of the transcriptome is sensitive to regulation by common stress conditions. The majority of changes were stimulus specific. At the 3-h time point, less than 5% (118 genes) of the changes were observed as shared by all three stress responses. By 27 h, the number of shared responses was reduced more than 10-fold (< 0.5%), consistent with a progression toward more stimulus-specific responses. Roots and leaves displayed very different changes. For example, less than 14% of the cold-specific changes were shared between root and leaves at both 3 and 27 h. The gene with the largest induction under all three stress treatments was At5g52310 (LTI/COR78), with induction levels in roots greater than 250-fold for cold, 40-fold for mannitol, and 57-fold for NaCl. A stress response was observed for 306 (68%) of the known circadian controlled genes, supporting the hypothesis that an important function of the circadian clock is to “anticipate” predictable stresses such as cold nights. Although these results identify hundreds of potentially important transcriptome changes, the biochemical functions of many stress-regulated genes remain unknown.

Genes Encoding Glycine-Rich Arabidopsis thaliana Proteins with RNA-Binding Motifs Are Influenced by Cold Treatment and an Endogenous Circadian Rhythm

We have characterized the expression of two members of a class of Arabidopsis thaliana glycine-rich, putative RNA-binding proteins that we denote Ccr1 and Ccr2. Southern blot analysis indicates that Ccr1 and Ccr2 are members of a small gene family. Both Ccr1 and Ccr2 mRNA levels were influenced by a circadian rhythm that has an unusual phase for plants, with maximal accumulation at 6:00 PM and minimal accumulation at 10:00 AM. The level of CCR1 protein, however, remained relatively constant throughout the cycle. The transcript accumulation patterns of the Ccr1 and Ccr2 genes differed considerably from conditions that affect the expression of similar genes from maize, sorghum, and carrot. Levels of Ccr1 and Ccr2 mRNAs were unchanged in wounded plants, increased at least 4-fold in cold-stressed plants, and decreased 2- to 3-fold in abscisic acid-treated plants. Ccr1 transcript levels decreased in response to drought, whereas Ccr2 transcript levels increased under the same conditions. Based on the presence of additional Ccr transcripts in dark-grown plants, we propose that Ccr transcripts may be subjected to a light- or dark-mediated regulation.

GIGANTEA Acts in Blue Light Signaling and Has Biochemically Separable Roles in Circadian Clock and Flowering Time Regulation

Circadian clocks are widespread in nature. In higher plants, they confer a selective advantage, providing information regarding not only time of day but also time of year. Forward genetic screens in Arabidopsis (Arabidopsis thaliana) have led to the identification of many clock components, but the functions of most of these genes remain obscure. To identify both new constituents of the circadian clock and new alleles of known clock-associated genes, we performed a mutant screen. Using a clock-regulated luciferase reporter, we isolated new alleles of ZEITLUPE, LATE ELONGATED HYPOCOTYL, and GIGANTEA (GI). GI has previously been reported to function in red light signaling, central clock function, and flowering time regulation. Characterization of this and other GI alleles has helped us to further define GI function in the circadian system. We found that GI acts in photomorphogenic and circadian blue light signaling pathways and is differentially required for clock function in constant red versus blue light. Gene expression and epistasis analyses show that TIMING OF CHLOROPHYLL A/B BINDING PROTEIN1 (TOC1) expression is not solely dependent upon GI and that GI expression is only indirectly affected by TOC1, suggesting that GI acts both in series with and in parallel to TOC1 within the central circadian oscillator. Finally, we found that the GI-dependent promotion of CONSTANS expression and flowering is intact in a gi mutant with altered circadian regulation. Thus GI function in the regulation of a clock output can be biochemically separated from its role within the circadian clock.

A Putative CCAAT-Binding Transcription Factor Is a Regulator of Flowering Timing in Arabidopsis

Flowering at the appropriate time of year is essential for successful reproduction in plants. We found that HAP3b in Arabidopsis (Arabidopsis thaliana), a putative CCAAT-binding transcription factor gene, is involved in controlling flowering time. Overexpression of HAP3b promotes early flowering while hap3b, a null mutant of HAP3b, is delayed in flowering under a long-day photoperiod. Under short-day conditions, however, hap3b did not show a delayed flowering compared to wild type based on the leaf number, suggesting that HAP3b may normally be involved in the photoperiod-regulated flowering pathway. Mutant hap3b plants showed earlier flowering upon gibberellic acid or vernalization treatment, which means that HAP3b is not involved in flowering promoted by gibberellin or vernalization. Further transcript profiling and gene expression analysis suggests that HAP3b can promote flowering by enhancing expression of key flowering time genes such as FLOWERING LOCUS T and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1. Our results provide strong evidence supporting a role of HAP3b in regulating flowering in plants grown under long-day conditions.

Coordination of Plant Metabolism and Development by the Circadian Clock.

Many plant cellular activities occur with a daily rhythmicity. In some cases, the rhythmicity of these cellular activities is maintained in plants growing under constant environmental conditions, such as continuous light (LL) or darkness (DD) and constant temperature. Because rhythms can persist in the absence of externa1 time cues (known as free-running conditions), they must be driven by an internal oscillator. This oscillator, which is known as the circadian clock, gen- erates circadian rhythms. For the circadian clock to regulate rhythms such that they occur at the correct time of day throughout the year, it must be able to perceive the seasonal changes in day length. This adjustment of the clock is known as entrainment, and the environmental cues that are perceived are called Zeitgebers, from the German word meaning “time giver.” Thus, the circadian clock can be con- sidered to be an internal processor of temporal inputs from the environment (such as light and temperature). Output from the processor regulates the timing of metabolic and developmental events within the plant. Although the molecular basis of the circadian clock in plants is not known, work from other systems has estab- lished a transcriptional negative feedback loop as a para- digm. In Drosophila, the clock components encoded by the period (per) and timeless

Fluorescent Differential Display Identifies Circadian Clock-Regulated Genes in Arabidopsis thaliana

Circadian rhythms in gene expression were first observed in plants more than 13 years ago, but the underlying mechanism controlling rhythmic gene expression is still not understood. The isolation of novel circadian clock-controlled genes (ccgs) is likely to provide new tools for studying circadian rhythms. Fluorescent differential display (FDD) was used to screen Arabidopsis thaliana mRNAs for cycling transcripts. Seventy PCR primer pairs were screened, and 17 different cycling bands were observed out of an estimated 10,500 bands screened. The identities of 10 bands were determined, and the rhythmic gene expression was confirmed using northern blot analysis. The 10 cycling bands represent 7 different genes, 6 of which are present in the databases and 1 that does not match anything in current databases. The rhythmic expression of the 7 genes is composed of four distinct phases of clock regulation. The results demonstrate that FDD can be used to isolate ccgs. The genes identified in this screen range from known A. thaliana ccgs, as well as genes shown to be clock controlled in other plant species, to a novel gene that may encode a pioneer protein. Further study of these ccgs is likely to increase our understanding of circadian-regulated gene expression.