Model for the Photoperiod Pathway

Flowering in Arabidopsis occurs much earlier in long days than in short days. Mutations that delayed flowering in long days but had little, if any, effect under short days defined genes in the long-day photoperiod pathway (Redei, 1962; Koornneef et al., 1991) . Genes involved in this photoperiod pathway include GI, CO, FD, FE, FHA, FT and FWA (Koornneef et al., 1991; figure 1.1).

The expression of GI has a circadian rhythm and mutants of the gene have altered expression of CO. GI encodes for a nuclear protein (Huq et al., 2000) . The gi mutant has low CO mRNA levels and is late flowering (Fowler et al., 1999; Park et al., 1999) . When CO is overexpressed in a gi mutant background it is able to overcome the late flowering phenotype, and is therefore downstream of GI (Suarez-Lopez et al., 2001) .

Photoperiod control

CO plays a central role in photoperiodic flowering control in Arabidopsis. (Putterill et al.,1995) . The protein contains two major regions required for its function that were identified by sequencing mutant alleles and identifying regions of homology with other proteins (Robson et al., 2001) . The CCT domain, which is approximately 60 aa long, was named because of its similarity to CO-like proteins and TIMING OF CHLOROPHYLL A/B BINDING PROTEIN 1 (TOC1) (Strayer et al., 2000; Ledger et al., 2001) . The second domain contains two zinc fingers related to B-box zinc fingers of animal proteins (Putterill et al., 1995; Robson et al., 2001) . By analogy with their function in animal proteins such as the transcription factor XNF7 and ribonucleoprotein PwA33 (Borden, 1998) , these are probably involved in protein-protein interactions (Robson et al., 2001).

The role of Constans in the photoperiod response

CO mRNA is expressed at low levels in Arabidopsis, however in situ hybridisation performed on 8 day-old seedlings showed that CO is expressed in the shoot apical meristem and young leaves (Simon et al., 1996) , whilst in older plants it is expressed in the inflorescence and young floral buds (Coupland, 1997) . A fusion of the CO promoter to a GUS reporter gene was generated to further analyse the spatial expression (Putterill et al., 1995; An et al., 2004) . CO:GUS was expressed in the vascular tissue of the hypocotyl, the cotyledons and the leaves, and was also present in the shoot apex. Cross sections demonstrated that CO:GUS was expressed predominantly in the phloem (An et al., 2004).

Flowering time genes

The quantity of CONSTANS expression has a rate-limiting effect upon flowering time, with greater amounts of the protein leading to earlier flowering (Samach et al., 2000) . Overexpression of CO from the 35S promoter causes an early-flowering phenotype and increased expression of the flowering-time genes FT and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1). As the addition of dexamethasone and cycloheximide results in a rapid increase of FT and SOC1 mRNA levels in transgenic plants that carry the inducible 35S::CO:GR construct, it is thought that both genes are direct targets of CO (Samach et al., 2000) . Overexpression of CO in ft or soc1 mutants causes a partial suppression of the early flowering phenotype. However 35S::CO ft soc1 plants still flower earlier than wild-type plants, suggesting that CO is able to regulate the expression of additional flowering-time genes.

Overexpression of FT and SOC1 results in extreme early flowering and complements the co mutation, confirming that they act downstream of CO (Kardailsky et al., 1999; Kobayashi et al., 1999; Borner et al., 2000; Lee et al., 2000; Onouchi et al., 2000; Samach et al., 2000; Hepworth et al., 2002) . It follows that the main route of activation in the long-day pathway is from GI to CO to FT and SOC1. A class of genes that form part of the photoperiod pathway are also components of the circadian clock (Figure 1.1), these include CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), LATE ELONGATED HYPOCOTYL (LHY), EARLY FLOWERING 3 (ELF3), TOC1 and GI. Single mutants of any of these genes cause early flowering. (Schaffer et al., 1998; Wang and Tobin, 1998; Fowler et al., 1999; Park et al., 1999; McWatters et al., 2000; Strayer et al., 2000; Hicks et al., 2001) . Photoreceptors mediate input signals to the circadian clock entraining it to oscillate with a 24Hr period. Although the photoreceptors have a strong influence upon the circadian clock, more important is the coordination of clock rhythms and light, which has a dramatic impact upon flowering time. (Roden et al., 2002; Yanovsky and Kay, 2002, 2003) . The external coincidence model proposes that there is a light sensitive phase in which flowering is promoted if a plant is exposed to light during a certain phase of the circadian clock. Arabidopsis plants are able to distinguish between photoperiod lengths, by integration of the circadian clock at the CONSTANS level.

When grown under long days there is a broad peak of CO expression starting approximately 12 hours after dawn, this peak remains high until close to dawn. The expression pattern is different in short-day growth conditions. There is a minor peak in expression after 12 hours, which rapidly disappears, followed by a larger peak in expression 20 hours after dawn (Suarez-Lopez et al., 2001; Yanovsky and Kay, 2002) . The clock regulates CO expression so that it is expressed in the dark period in short-days, yet expressed in the light period in long-day conditions (Suarez-Lopez et al., 2001; Yanovsky and Kay, 2003; Valverde et al., 2004) .

Perception of Light

Many of the genes that show a response to day length in Arabidopsis encode proteins that are involved in the perception of light. These include the photoreceptors PhyA, PhyB and CRY2 (Johnson et al., 1994; Guo et al., 1998; Guo et al., 1999; El-Din El-Assal et al., 2001; Valverde et al., 2004) .

Late flowering mutants

cry2 (fha) mutants were first identified as late-flowering mutants in long-day/white light photoperiods (Koornneef et al., 1991; Guo et al., 1999) . PhyB is an inhibitor of floral initiation; phyB loss of function mutants are early flowering (Parks and Quail, 1993) . When grown in red light conditions PhyB acts to repress the function of CO. As co phyB double mutants are not early flowering it would seem that CO is required for the early-flowering phenotype of phyB mutants (Putterill et al., 1995) . cry2 mediates a blue light–dependent inhibition of phyB function (Guo et al., 1998; Lin et al., 1998) . Interestingly CO mRNA levels are similar in wild-type and cry2 backgrounds but FT mRNA levels are greatly reduced in these backgrounds. This suggests that cry2 may regulate CO post transcriptionally (Suarez-Lopez et al., 2001; Yanovsky and Kay, 2002) .

The regulation of CO protein stability by light also plays a major role in daylength perception. When plants are exposed to either blue or far-red light, which are known to induce flowering, an accumulation of CO protein is observed in the nucleus. However, when plants are grown in either non-inductive red-light or total darkness, then there is no accumulation of CO protein in the nucleus, probably due to degradation of the protein by ubiquitination (Valverde et al., 2004) . The photoreceptors CRY1, CRY2 and PhyA promote flowering and stabilise CO protein, and in the cry1 cry2 double mutant there is a reduction in the morning and evening peaks of CO protein abundance. Conversely PhyB both delays flowering and enhances the degradation of CO protein. There is no degradation of CO protein in the early flowering phyB mutant (Valverde et al., 2004) . Therefore, the stability of CO protein is strongly influenced by light. It is rapidly degraded in the dark, and therefore only plants growing in long-day photoperiods have stable CO protein (Valverde et al., 2004) .

Epigenetics - FWA

Another gene that affects the photoperiod pathway is FWA. fwa mutants are gain of function epigenetic mutants, which delay flowering in long-day photoperiods (Koornneef et al., 1991; Soppe et al., 2000) . The mutation has a similar effect to that of ft mutations, suggesting that they impair a related step in flowering control (Soppe et al., 2000) . In wild-type plants, FWA is only expressed in the developing seed suggesting that it is not a flowering time gene per se, but has a role in endosperm development (Kinoshita et al., 2004) , and that the mutation delays flowering by mainly ectopic expression of FWA. The C-terminal of FWA has been shown to interact with FT in vitro (Ikeda., 2004). FWA may be able to repress the interaction between FT and FLOWERING LOCUS D (FD), which is proposed to be required for flowering and is described in section 1.7.2 (Ikeda et al., 2004) .

Repressors of flowering

Two repressors of thephotoperiod flowering-time pathway are FLM and SHORT VEGETATIVE PHASE (SVP) (Hartmann et al., 2000; Ratcliffe et al., 2001; Scortecci et al., 2001) . It is thought that these two genes act in the same pathway and that the photoperiod alleviates FLM repression (Scortecci et al., 2003) . Although FLM is closely related to FLC it is not vernalisation responsive (Ratcliffe et al., 2001) . flm mutants are able to suppress the late flowering phenotype of co and gi mutants in a dose-dependent manner, strongly suggesting that FLM acts downstream of these two genes (Scortecci et al., 2003).

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