A modern circadian clock in the common angiosperm ancestor of monocots and eudicots

The circadian clock enhances fitness through temporal organization of plant gene expression, metabolism and physiology. Two recent studies, one in BMC Evolutionary Biology, demonstrate through phylogenetic analysis of the CCA1/LHY and TOC1/PRR gene families that the common ancestor of monocots and eudicots had components sufficient to construct a circadian clock consisting of multiple interlocked feedback loops. See research article http://www.biomedcentral.com/1471-2148/10/126

'evening' loop based again on the time of peak mRNA accumulation, TOC1 represses a component, 'Y' , that includes GIGANTEA (GI) and possibly PRR5. Th is component in turn positively regulates TOC1 expression at least in part through modulation by GI of proteasomal degradation of TOC1 mediated by the F-box protein ZEITLUPE (ZTL) [4,5]. In addition, proper regulation of CCA1 and LHY requires other clock genes, including EARLY FLOWERING 4 (ELF4), which encodes a protein of unknown function, and LUX ARRHYTHMO/PHYTOCLOCK1 (LUX/PCL), which encodes a Myb domain transcription factor; these and other clock components have yet to be fully incorporated into current clock models [4,5]. Th e number of interlocked feedback loops will undoubtedly increase as the regulatory relationships among clock components are more fully described.
Th e value of model organisms such as Arabidopsis stems from the generalization of knowledge acquired in the model to all fl owering plants, and especially to those of agricultural signifi cance. Th e increasing availability of genomic sequences from multiple plants is now permitting our fi rst insights into this issue.

Phylogenetic analysis of the PRR and CCA1/LHY gene families shows that circadian clocks composed of multiple interlocked feedback loops evolved prior to the divergence of monocots and eudicots
Molecular phylogenetic analysis of the PRR genes indicates that the common ancestor of the monocots and eudicots had three PRR gene clades [2]. Since the divergence of the monocots and eudicots, the clades corresponding to PRR3/PRR7 and PRR5/PRR9 have expanded independently in both lineages as a result of genome duplications [2]. Within the eudicots, or 'true dicots' , a subset of the former broad classifi cation of dicots that includes more than half of extant plant species, two further genome duplications occurred in Arabidopsis following its divergence from papaya (Carica papaya) but, after each duplication, one of the paralogs was lost. In contrast, poplar has retained the duplicate copies of PRR5, PRR7 and PRR9, which originated in a genome duplication, termed the Salicoid duplication, that occurred in the poplar lineage after its separation from the papaya-Arabidopsis lineage. PRR3 has been completely lost from the poplar genome, although it is unclear whether this loss predated or followed the Salicoid duplication. Th e Brassica rapa genome has triploidized since its divergence form Arabidopsis approximately 14.5 million years ago, yet for no members of the B. rapa TOC1/PRR gene family have all three copies persisted, making it clear that diff erential PRR gene loss has occurred [8].
Takata et al. [9] have conducted a parallel analysis of angiosperm CCA1/LHY genes, and their observations are consistent with those obtained in their analysis of the PRR genes; the common ancestor of monocots and eudicots had one CCA1/LHY gene and there has been independent duplication of the LHY/CCA1 genes in the monocots and eudicots. Within the eudicots, there has been independent duplication in poplar and Arabidopsis.
Th e key conclusion from these studies is that the common ancestor of the monocots and eudicots had the basic components necessary for the construction of a circadian clock with multiple interlocked feedback loops prior to the separation of these groups 200 million years ago [2]. Th is makes it very likely that the Arabidopsis clock will prove a useful model for most agricultural species. It will be interesting to determine whether the more basal angiosperms, such as the Magnoliales, also share this common clock architecture.

Sub-and neo-functionalization among clock genes
One consequence of gene duplication is that it allows the two copies to subdivide the functions of the ancestral copy (functional specialization or sub-functionalization), or for one copy to acquire a new function (neo-functionalization) while the other retains the original function, thus preserving fi tness; but is there evidence for either functional specialization or acquisition of novel functions among PRR genes during evolution of the angiosperms? Th e strongest evidence comes from Arabidopsis, where clock function is best studied. TOC1 and four other PRR genes each show circadian oscillations in transcript abundance, with peak abundance occurring at intervals spanning the day starting at dawn with PRR9, followed by PRR7, PRR5, PRR3, and fi nally at dusk with TOC1 (PRR1) [4,5]. As shown in Figure 2, TOC1 is recruited to the CCA1 promoter and is a positive regulator of CCA1 expression, although the molecular details remain incompletely described [1]. PRR9, PRR7, and PRR5 are recruited to the promoters of CCA1 and LHY and negatively regulate their expression [10]. It is likely that the sequential expression of PRR9, PRR7, and PRR5 contributes to sustained repression of CCA1 and LHY expression throughout the day. Th is indicates that, while the function of these three genes is partially redundant, with normal expression of the three genes the temporal window of CCA1/LHY repression is extended. Th us, PRR9, PRR7, and PRR5 off er an example of subfunctionalization in the temporal domain. Although the function of the rice (Oryza sativa) orthologs of PRR9, PRR7, and PRR5 has not been established, there is a similar sequential pattern of expression of OsPRR73/ OsPRR37 and then OsPRR95/OsPRR59, followed by OsTOC1 (OsPRR1) [11].
In Arabidopsis, the PRR3 gene off ers an example of acquisition of a novel function. PRR9, PRR7, and PRR5 all have a similar role in negatively regulating CCA1 and LHY, suggesting that this represents the ancestral function ( Figure 2). PRR3 appears, instead, to have acquired a novel and specialized function in the vascular tissue, where PRR3 binds to TOC1 and, in doing so, blocks the interaction of TOC1 with ZTL, the F-box protein that targets TOC1 for proteasomal degradation [12]. Th us, PRR3 exhibits a restricted domain of expression and has acquired a novel function, the regulation of TOC1 stability through protein-protein interaction (Figure 2). In Arabi dop sis, loss of PRR3 function confers only a very small shortening of circadian period [13], which is consistent with the apparent loss of PRR3 in poplar, without conco mitant perturbation of clock function.
Th ere are additional suggestions of evolving function in the PRR7 lineage. In Arabidopsis, PRR7 contributes to the determination of fl owering time, although the eff ects are not large and PRR7 is not a major determinant of fl owering time among natural populations [14]. In contrast, in the monocots barley and wheat, PRR7 (Ppd-H1 and Ppd-D1, respectively) is one of the major determinants of photoperiod sensitivity and fl owering time [15,16]. Whether this represents a true acquisition of novel function in the monocots or a loss of function in the eudicots remains uncertain and will require more detailed dissection of the roles of PRR7 in the fl owering pathways of monocots and eudicots.

Future directions
Th ere remains a great deal of work to achieve a mechanistic understanding of how the circadian clock keeps time. Four of the fi ve PRR proteins are recruited to   [2,9] establish that the common ancestor of monocots and eudicots had PRR and CCA1/LHY genes and, therefore, the materials with which to construct a functional circadian clock. How has the diff erential amplifi cation of these two gene families in the angiosperm lineages allowed modulation of circadian timekeeping? How well does the outline presented in Figures 1 and 2 apply across the angiosperms and to more primitive plants? Within species, has variation among clock genes contributed to fi tness? Th ere is no shortage of questions and the increasing availability of genome sequences and tools to probe gene function in many species make this a wonderful time to study the basis of circadian timing.