Elaine M. Tobin

Constitutive Expression of the CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) Gene Disrupts Circadian Rhythms and Suppresses Its Own Expression

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.

A Myb-related transcription factor is involved in the phytochrome regulation of an Arabidopsis Lhcb gene.

We have isolated the gene for a protein designated CCA1. This protein can bind to a region of the promoter of an Arabidopsis light-harvesting chlorophyll a/b protein gene, Lhcb1*3, which is necessary for its regulation by phytochrome. The CCA1 protein interacted with two imperfect repeats in the Lhcb1*3 promoter, AAA/cAATCT, a sequence that is conserved in Lhcb genes. A region near the N terminus of CCA1, which has some homology to the repeated sequence found in the DNA binding domain of Myb proteins, is required for binding to the Lhcb1*3 promoter. Lines of transgenic Arabidopsis plants expressing antisense RNA for CCA1 showed reduced phytochrome induction of the endogenous Lhcb1*3 gene, whereas expression of another phytochrome-regulated gene, rbcS-1A, which encodes the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, was not affected. Thus, the CCA1 protein acts as a specific activator of Lhcb1*3 transcription in response to brief red illumination. The expression of CCA1 RNA was itself transiently increased when etiolated seedlings were transferred to light. We conclude that the CCA1 protein is a key element in the functioning of the phytochrome signal transduction pathway leading to increased transcription of this Lhcb gene in Arabidopsis.

Circadian Rhythms Confer a Higher Level of Fitness to Arabidopsis Plants

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 theCCA1 (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.

F-box proteins FKF1 and LKP2 act in concert with ZEITLUPE to control Arabidopsis clock progression.

In Arabidopsis thaliana, the F-box protein ZTL affects the period length of the circadian clock by regulating the stability of the core clock proteins TOC1 and PRR5. This study shows that, together with ZTL, the ZTL homologs FKF1 and LKP2 are also involved in the same protein stability regulation to determine the pace and robustness of the plant circadian clock. 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 ASSOCIATED1 (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.

All in good time: the Arabidopsis circadian clock.

Biological time-keeping mechanisms have fascinated researchers since the movement of leaves with a daily rhythm was first described >270 years ago. The circadian clock confers a approximately 24-hour rhythm on a range of processes including leaf movements and the expression of some genes. Molecular mechanisms and components underlying clock function have been described in recent years for several animal and prokaryotic organisms, and those of plants are beginning to be characterized. The emerging model of the Arabidopsis clock has mechanistic parallels with the clocks of other model organisms, which consist of positive and negative feedback loops, but the molecular components appear to be unique to plants.

Photochrome-mediated regulation of messenger RNAs for the small subunit of ribulose 1, 5-bisphosphate carboxylase and the light-harvesting chlorophyll a/b-protein in Lemna gibba

Brief red illumination (10 min/8 hr) of Lemna gibba L. G-3 growing heterotrophically in the dark increases the growth of the plants and results in a substantial increase in the levels of mRNA for two major chloroplast polypeptides. These two nuclear-coded polypeptides are the light-harvesting chlorophyll a/b-protein, an intrinsic thylakoid membrane protein, and the small subunit of the stromal enzyme ribulose 1, 5-bisphosphate carboxylase [RuP2; 3-phospho-D-glycerate carboxylyase (dimerizing), E.C.4.1.1.39]. The effect of 10 min red illumination on the dark growth of the plants is reversed by immediate far-red illumination, but the effect on the mRNA levels is not. However, this latter response can be reversed by far-red light if the time between the beginnings of the red and far-red illumination is reduced to one minute. Thus phytochrome is the photoreceptor mediating both responses, and the effect on amounts of the translatable mRNAs has a remarkably short escape time.As expected from the high level of its mRNA in the plants grown in the dark with intermittent red illumination, the small subunit of ribulose 1, 5-bisphosphate carboxylase is synthesized in these plants and accumulates without further illumination. However, despite the relatively high levels of mRNA for the chl a/b-protein in the dark grown plants, this protein does not appear to be synthesized and inserted into the thylakoid membranes until the plants are transfered into white light. Thus, the normal synthesis of this protein must require light for some post-transcriptional process.

The interaction of light and abscisic acid in the regulation of plant gene expression.

Extended dark treatments of light-grown plants of both Lemna gibba and Arabidopsis thaliana resulted in substantial increases in abscisic acid (ABA) concentrations. The concentration of ABA could be negatively regulated by phytochrome action in Lemna. As has been noted in other species, ABA treatment reduced Lemna rbcS and Lhcb RNA levels, which are positively regulated by phytochrome in many species. In view of these observations, the possibility that phytochrome effects on gene expression may be mediated primarily by changes in ABA was tested using a transient assay in intact plants. The phytochrome responsiveness of the Lemna Lhcb2*1 promoter was still apparent in the presence of exogenous ABA. Additionally, when 2-bp mutations were introduced into this promoter so that phytochrome responsiveness was lost, a response to exogenous ABA was still present. We conclude that phytochrome- and ABA-response elements are separable in the Lhcb2*1 promoter. We tested whether the effects of ABA on RNA abundance could be inhibited by treatment with gibberellin and found no evidence for such an inhibition. We have also found that the ABA-responsive Em promoter of wheat can be negatively regulated by phytochrome action. It is likely that this regulation is mediated at least in part by phytochrome-induced changes in ABA levels. Our results demonstrate that it is essential to take into account that dark treatments and the phytochrome system can affect ABA levels when interpreting studies of light-regulated genes.

Benzyladenine modulation of the expression of two genes for nuclear-encoded chloroplast proteins in Lemna gibba: Apparent post-transcriptional regulation

Cytokinins and phytochrome have both been reported to promote chloroplast development, and possible interactions between the two have been suggested. We have examined the effects of red light (R) and a cytokinin, benzyladenine (N6-benzylaminopurine; BA), on the levels of four mRNAs coding for chloroplast proteins in Lemna gibba L. The amounts of hybridizable RNA coding for both the major chlorophyll a/b-binding protein and for the small subunit of ribulose-1,5-bisphosphate (RuBP) carboxylase decrease to a low level when white-light-grown L. gibba plants are placed in the dark. We have previously shown that a subsequent R treatment causes a several-fold increase in the levels of these two messages, and this increase is phytochrome-mediated. We have now found that addition of submicromolar concentrations of BA to plants kept in total darkness also results in an increase in levels of these two mRNAs. Furthermore, BA treatment magnifies the extent of the response to R treatment. However, the levels of mRNAs encoding the large subunit of RuBP carboxylase and the 32-kDa herbicide-binding protein, which are both chloroplastsynthesized messages, are not significantly altered by either R or BA treatment during the same time period. The relative amount of β-actin mRNA, a nuclear-encoded message for a cytoplasmic protein, is also not altered either by R or BA treatment. Thus, BA treatment does not simply alter the proportion of mRNA to total RNA. This conclusion is also supported by the observation that levels of mRNA hybridizing to a sequence abundant in dark-treated plants are not altered by BA treatment. The amplification by BA of the R-induced increase in the level of chlorophyll a/b-binding protein mRNA, consistently seen in total RNA, is not observed in RNA isolated from nuclei from plants receiving the same treatments. We therefore suggest that cytokinin is regulating expression of this message at a post-transcriptional level, possibly by affecting the stability of the RNA.

Circadian Rhythms of Ethylene Emission in Arabidopsis

Ethylene controls multiple physiological processes in plants, including cell elongation. Consequently, ethylene synthesis is regulated by internal and external signals. We show that a light-entrained circadian clock regulates ethylene release from unstressed, wild-type Arabidopsis (Arabidopsis thaliana) seedlings, with a peak in the mid-subjective day. The circadian clock drives the expression of multiple ACC SYNTHASE genes, resulting in peak RNA levels at the phase of maximal ethylene synthesis. Ethylene production levels are tightly correlated with ACC SYNTHASE 8 steady-state transcript levels. The expression of this gene is controlled by light, by the circadian clock, and by negative feedback regulation through ethylene signaling. In addition, ethylene production is controlled by the TIMING OF CAB EXPRESSION 1 and CIRCADIAN CLOCK ASSOCIATED 1 genes, which are critical for all circadian rhythms yet tested in Arabidopsis. Mutation of ethylene signaling pathways did not alter the phase or period of circadian rhythms. Mutants with altered ethylene production or signaling also retained normal rhythmicity of leaf movement. We conclude that circadian rhythms of ethylene production are not critical for rhythmic growth.

Cytokinin modulation of LHCP mRNA levels: the involvement of post-transcriptional regulation

When white-light-grown Lemna gibba plants are placed in the dark, the levels of mRNAs for two nuclear-encoded chloroplast proteins, the small subunit of ribulose-1,5-bisphosphate carboxylase (SSU) and the major chlorophyll a/b-binding protein of light-harvesting complex II (LHCP), decline to a small fraction of their previous level. We have reported [4] that red light (R), acting through phytochrome, and benzyladenine (BA), a synthetic cytokinin, independently stimulate accumulation of both mRNAs in the dark. Here, we have analyzed the products of transcription in isolated nuclei to determine if cytokinins act primarily through stimulation of transcription or if post-transcriptional processes are involved. We find that BA pretreatment may slightly stimulate transcription of LHCP RNA either with or without a red-light treatment. However, the effects of BA on the LHCP RNA accumulation were much greater than on transcription. Two naturally occurring cytokinins are also effective in increasing the mRNA abundance. We therefore conclude that, in Lemna, post-transcriptional processes are important in regulation of the LHCP RNA by cytokinins.