The CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) gene encodes a MYB-related transcription factor involved in the phytochrome induction of a light-harvesting chlorophyll a/b-protein (Lhcb) gene. Expression of the CCA1 gene is transiently induced by phytochrome and oscillates with a circadian rhythm. Constitutive expression of CCA1 protein in transgenic plants abolished the circadian rhythm of several genes with dramatically different phases. These plants also had longer hypocotyls and delayed flowering, developmental processes regulated by light and the circadian clock. Furthermore, the expression of both endogenous CCA1 and the related LHY gene was suppressed. Our results suggest that CCA1 is a part of a feedback loop that is closely associated with the circadian clock in Arabidopsis.

We have isolated the gene for a protein designated CCAl. This protein can bind t o a region of the promoter of an Arabidopsis light-harvesting chlorophyll a/b protein gene, Lhcb7*3, which is necessary for its regulation by phytochrome. The CCAl protein interacted with two imperfect repeats in the Lhcb7*3 promoter, AAA/,AATCT, a sequence that is conserved in Lhcb genes. A region near the N terminus of CCA1, which has some homology t o the repeated sequence found in the DNA binding domain of Myb proteins, is required for binding t o the Lhcb7*3 promoter. Lines of transgenic Arabidopsis plants expressing antisense RNA for CCAl showed reduced phytochrome induction of the endogenous Lhcb7*3 gene, whereas expression of another phytochrome-regulated gene, rbcS-7A, which encodes the small subunit of ribulose-1 ,Bbisphosphate carboxylase/oxygenase, was not affected. Thus, the CCAl protein acts as a specific activator of Lhcb7*3 transcription in response t o brief red illumination. The expression of CCAl RNA was itself transiently increased when etiolated seedlings were transferred t o light. We conclude that the CCAl protein is a key element in the functioning of the phytochrome signal transduction pathway leading t o increased transcription of this Lhcb gene in Arabidopsis.

Circadian rhythms have been demonstrated in organisms across the taxonomic spectrum. In view of their widespread occurrence, the adaptive significance of these rhythms is of interest. We have previously shown that constitutive expression of the CCA1 (CIRCADIAN CLOCK ASSOCIATED 1) gene in Arabidopsis plants (CCA1-ox) results in loss of circadian rhythmicity. Here, we demonstrate that these CCA1-ox plants retain the ability to respond to diurnal changes in light. Thus, transcript levels of several circadian-regulated genes, as well as CCA1 itself and the closely related LHY, oscillate robustly if CCA1-ox plants are grown under diurnal conditions. However, in contrast with wild-type plants in which transcript levels change in anticipation of the dark/light transitions, the CCA1-ox plants have lost the ability to anticipate this daily change in their environment. We have used CCA1-ox lines to examine the effects of loss of circadian regulation on the fitness of an organism. CCA1-ox plants flowered later, especially under long-day conditions, and were less viable under very short-day conditions than their wild-type counterparts. In addition, we demonstrate that two other circadian rhythm mutants, LHY-ox and elf3, have low-viability phenotypes. Our findings demonstrate the adaptive advantage of circadian rhythms in Arabidopsis.First described in plants more than 270 years ago, circadian rhythms have been found in the vast majority of eukaryotes examined and in many prokaryotes. The basic mechanisms that are responsible for circadian rhythms are being elucidated in several model species. At the core of a circadian system is a molecular oscillator that generates a period of rhythmicity of about 24 h. The oscillator is self-sustaining, but it is influenced by environmental cues such as changes in light and temperature conditions (Dunlap, 1999). The oscillator controls a plethora of biological processes including nitrogen fixation in cyanobacteria (Johnson et al., 1996), scent emission in plants ( Kolosova et al., 2001), conidiation in Neurospora crassa. (Pittendrigh et al., 1959), olfactory responses of Drosophila melanogaster (Krishnan et al., 1999), luteinizing hormone levels in birds (Follett et al., 1974), and wheel running activity in hamsters (Mesocricetus auratus; Ralph and Menaker, 1988). The fact that circadian regulation is ubiquitous across the taxonomic spectrum and that its importance was even recognized in the Hebrew Bible (Ancoli-Israel, 2001) suggests that it is important for optimizing an organism's response to its environment and enhancing its fitness.The circadian clock plays a number of different roles. One is in the regulation of photoperiodism, which is the detection and response to changes in the duration of days and nights that enables organisms to adapt to seasonal changes in their environment (Thomas, 1998). Photoperiodism controls reproduction in many organisms, including flowering time in many plants ( Thomas and Vince-Prue, 1997). This photoperiodic response ensures that plants flower duri...

Regulation of protein turnover mediated by ZEITLUPE (ZTL) constitutes an important mechanism of the circadian clock in Arabidopsis thaliana. Here, we report that FLAVIN BINDING, KELCH REPEAT, F-BOX1 (FKF1) and LOV KELCH PROTEIN2 (LKP2) play similar roles to ZTL in the circadian clock when ZTL is absent. In contrast with subtle circadian clock defects in fkf1, the clock in ztl fkf1 has a considerably longer period than in ztl. In ztl fkf1 lkp2, several clock parameters were even more severely affected than in ztl fkf1. Although LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSO-CIATED1 (CCA1) expression levels are lower in ztl than in the wild type, introducing both fkf1 and lkp2 mutations into the ztl mutant dramatically diminished LHY expression without further affecting CCA1 expression. This demonstrates different contributions of ZTL, FKF1, and LKP2 in the regulation of LHY and CCA1 expression. In addition, FKF1 and LKP2 also interacted with TIMING OF CAB EXPRESSION1 (TOC1) and PSEUDO-RESPONSE REGULATOR5 (PRR5), and both proteins were further stabilized in ztl fkf1 and ztl fkf1 lkp2 compared with in ztl. Our results indicate that ZTL, FKF1, and LKP2 together regulate TOC1 and PRR5 degradation and are major contributors to determining the period of circadian oscillation and enhancing robustness.

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Little is known about plant circadian oscillators, in spite of how important they are to sessile plants, which require accurate timekeepers that enable the plants to respond to their environment. Previously, we identified a circadian clock-associated (CCA1) gene that encodes an Mybrelated protein that is associated with phytochrome control and circadian regulation in plants. To understand the role CCA1 plays in phytochrome and circadian regulation, we have isolated an Arabidopsis line with a T DNA insertion that results in the loss of CCA1 RNA, of CCA1 protein, and of an Lhcb-promoter binding activity. This mutation affects the circadian expression of all four clock-controlled genes that we examined. The results show that, despite their similarity, CCA1 and LHY are only partially redundant. The lack of CCA1 also affects the phytochrome regulation of gene expression, suggesting that CCA1 has an additional role in a signal transduction pathway from light, possibly acting at the point of integration between phytochrome and the clock. Our results indicate that CCA1 is an important clock-associated protein involved in circadian regulation of gene expression.Organisms have internal clocks to regulate physiological and cellular processes. The timekeepers, or oscillators, controlling these clocks are proposed to be negative autoregulatory feedback loops in which one or more gene products feed back and repress their own expression (1). The oscillator can be reset by input pathways from environmental cues, such as light and temperature, and, in turn, can regulate overt rhythmicity. Although much has been learned about circadian clocks in other organisms, little is known about the molecular basis of plant circadian oscillators. Plant homologs have not been identified yet for any of the known circadian genes from other organisms, including frq from Neurospora, per and tim from Drosophila, mper from mice, and the kai genes from the cyanobacterium Synechococcus (1, 2). An Arabidopsis mutant, toc1, that affects the period of clock-controlled processes has been isolated; however, the corresponding gene has not been cloned, and its role in the oscillator is not known (3).Recently, two Arabidopsis Myb-related genes, CCA1 (circadian clock-associated) and LHY, have been shown to have an important function in the circadian control of a number of plant processes (4, 5). CCA1 and LHY are 60% similar at the amino acid level, with regions of identity extending throughout the protein. Constitutive expression of either gene affects multiple processes controlled by circadian rhythms, including oscillations of circadian clock-controlled genes (4, 5). Furthermore, both CCA1 and LHY RNAs showed circadian oscillations after a period of entrainment and showed feedback inhibition of their own synthesis. Constitutive expression of CCA1 represses the expression of the LHY gene (4). CCA1 also is regulated by the plant photoreceptor phytochrome (4).To understand how CCA1 might function in the circadian control of plant processes and in the s...

The circadian clock-associated 1 (CCA1) gene encodes a Myb-related transcription factor that has been shown to be involved in the phytochrome regulation of Lhcb1*3 gene expression and in the function of the circadian oscillator in Arabidopsis thaliana. By using a yeast interaction screen to identify proteins that interact with CCA1, we have isolated a cDNA clone encoding a regulatory (␤) subunit of the protein kinase CK2 and have designated it as CKB3. CKB3 is the only reported example of a third ␤-subunit of CK2 found in any organism. CKB3 interacts specifically with CCA1 both in a yeast two-hybrid system and in an in vitro interaction assay. Other subunits of CK2 also show an interaction with CCA1 in vitro. CK2 ␤-subunits stimulate binding of CCA1 to the CCA1 binding site on the Lhcb1*3 gene promoter, and recombinant CK2 is able to phosphorylate CCA1 in vitro. Furthermore, Arabidopsis plant extracts contain a CK2-like activity that affects the formation of a DNA-protein complex containing CCA1. These results suggest that CK2 can modulate CCA1 activity both by direct interaction and by phosphorylation of the CCA1 protein and that CK2 may play a role in the function of CCA1 in vivo.

The genome of Arabidopsis thaliana is exceedingly small, in part because it lacks the large middle repetitive DNA component characteristic of other plants. In this paper we have characterized a member of the low copy DNA component: the gene family for the light-harvesting chlorophyll a/b-protein. This gene family is unusual in that it contains far fewer members than the 7-16 coding sequences for this protein found in other plants. We used cross-hybridization with a Lemna gene encoding a light-harvesting chlorophyll a/b-protein to isolate 3 genes from Arabidopsis, all of which are clustered on an 11-kb genomic clone. Southern blot analysis suggests that there is a fourth related gene in Arabidopsis. Sequence analysis of the three genes demonstrates that within the translated region the nucleic acid sequence homology is 96%, the deduced amino acid sequence of the mature proteins is identical for the three genes, and two of the genes have a high degree of sequence homology in both their 5' and 3' immediate flanking regions. The genes have regulatory sequences typical of eukaryotic genes upstream of the translation start sites. However, not all of these genes are equally expressed in plants grown under normal light-dark conditions.