Physiological approaches to the study of flowering-time control
Mature leaves perceive photoperiod, yet floral evocation occurs in the shoot apex at the SAM. Therefore, long-distance signalling by floral stimuli must occur (Bernier et al., 1993).
The first demonstration of the floral stimulus was by Knott (1934) , who demonstrated that exposing only spinach leaves to photoperiodic cycles of light and dark would initiate flowering, whereas exposing only the apex to these conditions would not. He therefore concluded that an unknown factor must be transported from the leaves to the shoot apex, where it promotes floral development.
In 1937, Mikhail Chailakhyan suggested that the floral stimulus was a flowering hormone, which he named florigen (Chailakhyan, 1937; Aksenova, 2002) . This hypothesis was extended by the proposal that an anti-florigen molecule exists and has an inhibitory effect upon flowering. This substance was proposed to be expressed in non-induced leaves (Lang et al., 1977).
Grafting experiments designed to study the photoperiod response have been performed in numerous species. In these experiments single leaves or the lower shoot and root (stock) of a plant are exposed to one photoperiod and grafted to the shoot apex or upper shoot (scion) of a second plant. The effect of the leaves or stock on flowering of the scion can then be measured. This approach has been used in Tobacco, Perilla, pea and more recently Arabidopsis (Zeevaart, 1962; Lang, 1965; Murfet, 1992; Weller et al., 1997; Turnbull et al., 2002; An et al., 2004) .
Grafting Perilla and Citrus
Classical grafting experiments using Perilla crispa or Xanthium demonstrated exposure of a leaf or the stock to inductive photoperiods is required for the production of the floral stimulus. In these experiments grafting of a single Perilla leaf induced by exposure to short days onto a non-induced long-day grown Perilla shoot resulted in the axiliary shoots of the non-induced plant producing flowers (Zeevaart, 1962) . The same leaf was sequentially grafted onto seven non-induced stems and induced flowering in all of them indicating that a single leaf is highly effective in floral induction. Furthermore, grafting citrus experiments showed that floral signals could be transmitted between different plant species. Although for this to be effective, the plant species must be taxonomically closely related such as grafting citrus trees (Zeevaart, 1976) . Continued production of the floral stimulus seems to be required, at least in some species. If induced leaves were removed from Impatiens balsamina plants then floral shoots reverted to a vegetative identity (Tooke et al., 1998).
Florigen
Three main models were proposed to describe the nature of the floral transmissible stimulus. The florigen/anti-florigen model proposes that the promotive and inhibitor stimuli are single universal growth regulators. In contrast, the nutrient diversion model proposes that the balance of source/sink relationships are modified during flowering to enable the apex to receive increased assimilates. Finally, the multifactorial control model suggests that many different nutrients and phytohormones are required to comprise the floral stimulus (Bernier et al., 1981; Sachs and Hackett, 1983; Lang, 1984).
Many micro and macromolecules that are involved in flowering move readily from the leaf to the shoot apex through the symplastic pathway connections of the phloem (Imlau et al., 1999; Oparka and Cruz, 2000; Ruiz-Medrano et al., 2001) . These molecules include sucrose, cytokinins, and gibberellins, all of which appear to have a role in the promotion of flowering of mustard (Wilson et al., 1992; Blazquez et al., 1998; Bonhomme et al., 2000) . Sucrose and cytokinin concentrations increase at the apex during floral evocation (Corbesier et al., 1996). Interestingly, there is a decrease in the size exclusion limit of plasmodesmata at the shoot apex on floral induction (Gisel et al., 1999; Gisel et al., 2002) . This alteration in the aperture of plasmodesmata may restrict trafficking in a way that enhances the impact of the floral stimulus. (Crawford and Zambryski, 1999; Gisel et al., 2002).
Sucrose at the shoot apex
Sucrose is the metabolite that has been shown to reach the SAM earliest after floral induction. Although there is a large accumulation of sucrose at the shoot apex during floral induction, there is no immediate effect on mitotic activity. The source of the sucrose is not increased photosynthesis, but is mobilisation of carbohydrates stored in the leaves and stem (Bodson and Outlaw, 1985; Bernier et al., 1993; Corbesier et al., 1998) . Following the surge in sucrose, cytokinin levels increase in the vascular tissues; the source of the cytokinins is likely to be the roots, where it is released following stimulation by sucrose (Bernier et al., 1993; Lejeune, 1994) . The released cytokinins are mainly zeatin riboside and isopentenyladenine riboside. Bernier and colleagues (1993) inhibited the transient root to shoot flux of cytokinins in white mustard (Sinapis alba ) during a long-day and demonstrated this abolished the flowering response.
Induction of flowering by Gibberellins
Gibberellins are a large group of tetracyclic diterpenoid carboxylic acids, which have an ent-gibberellane skeleton. They induce flowering in many plant species. The application of exogenous gibberellins to Arabidopsis or the dual-day length plant Bryophyllum (which flowers only after a sequence of short days followed by long days) grown under short days initiates flowering (Lang, 1965; Zeevaart, 1985) . This effect also occurs when gibberellins are provided to some short-day plants grown under long-day photoperiods (Taiz and Zeiger, 2002) . Application of exogenous gibberellic acid can also promote flowering in non-vernalised Arabidopsis plants and is able to overcome the age related autonomous floral evocation required by some plants (Wilson et al., 1992; Taiz and Zeiger, 2002) . This is in agreement with the observation that exogenous application of inhibitors of gibberellin biosynthesis to Arabidopsis delays flowering (Chandler and Dean, 1994).
Work on Spinacia oleracia gave new insights into the regulation of gibberellins by photoperiod. This long-day plant contains low levels of gibberellins when grown in short-day photoperiods, and grows vegetatively (Taiz and Zeiger, 2002) . However when spinach is grown under long days there is a marked increase in the levels of gibberellins made by the 13-hydroxylated pathway (GA 53 – GA 44 – GA 19 - GA 20 – GA 1). This results in a five-fold increase of GA 1 and leads to stem elongation (Zeevaart et al., 1993) . Similar increases in GA biosynthesis in response to photoperiod were described in Arabidopsis (Olszewski et al., 2002).
The effect of the plant growth hormone gibberellin on flowering
The effects of gibberellins on flowering can differ between species. They promote flowering in herbaceous plants and some conifers but inhibit flowering in many woody species, such as apple, citrus and pear (Luckwill, 1980) . The role of the plant growth hormone gibberellin in flowering is therefore complex. Although their role in long-distance signalling or as part of the floral stimulus has not been demonstrated, they are commonly found in the vascular tissue (Xu et al., 1997) . However, as enzymes involved in gibberellin biosynthesis are found throughout the plant, it is possible that translocation of gibberellins may not be a prerequisite for a role in the initiation of flowering (Colasanti and Sundaresan, 2000).
Experiments on pea (Pisum sativa) have provided insight into how mutations can affect the translocation of signals from the shoot to the shoot apex (Howell, 1998) . Murfet and colleagues (1985) determined the site of action of mutations that affect photoperiodic control of flowering by grafting mutant scions onto wild-type or mutant stocks. This allowed tissues in which particular genes function to regulate flowering to be determined. If the shoot apex were mutant for VEGETATIVE (VEG1) gene this totally abolished flowering, even if the stock were veg1 mutant. VEG1 is not involved in the photoperiodic response, but is likely to be a general initiator of flowering, rather than to be involved in the perception of a floral stimulus (Murfet, 1985) . Three genes STERILE NODES (SN), DAY NEUTRAL (DNE) and PHOTOPERIOD RESPONSE (PPD) are required in the leaves and cotyledons and are photoperiod responsive. Mutations in any of the three genes results in flowering under non-inductive short-day photoperiods. These genes therefore appear to control a graft-transmissible inhibitor of flowering (Murfet and Reid, 1993; Weller et al., 1997) . The GIGAS gene, which is required in the leaf, promotes flowering. The gigas mutant flowers late when grown under long-day conditions, and therefore GIGAS is thought to promote the synthesis or export of a floral stimulus that is produced in the leaf and can cross graft junctions to initiate flowering at the apex (Weller et al., 1997) .
Commercial maize varieties are daylength insensitive, flowering with the same number of leaves regardless of photoperiod. Mutations in INDETERMINATE 1 (ID1) delay flowering and affect tassel and ear morphology. Expression of ID1 mRNA is restricted to the leaves, and is absent from the SAM (Colasanti et al., 1998) . Maize does not have a vascular cambium and so is not suitable for grafting experiments. Therefore genetic chimeras were analysed to test whether ID1 gene function in the leaves was sufficient to promote flowering. Chimeric id1 plants that contained sectors of ID1 flowered earlier than mutant id1 plants. This suggests that ID1 is involved in producing a transmissible signal from young leaves that promotes flowering at the apex.
Although Arabidopsis has been an effective model species in which to study genetic flowering-time networks, the involvement of transmissible signals in flowering has only been studied recently. An and colleagues (2004) as well as Ayre and Turgeon (2004) showed that the flowering-time gene CONSTANS (CO) is active in the phloem. A Y-grafted junction could form phloem connections across which radiolabelled sucrose was transferred. Grafting of induced donor shoots to plants grown in non-inductive short-day conditions demonstrated that a floral stimulus was transferred from the long-day grown shoot to induce flowering in the short-day grown plant. Finally, a wild-type donor shoot was grafted to a co-2 mutant and this resulted in accelerated flowering of the mutant. This suggested that CO or downstream targets of CO act in the phloem in response to long days to regulate a signal that induces flowering (An et al., 2004; Ayre and Turgeon, 2004).