8.3.2 The processes of floral induction and initiation
Following the discovery of photoperiod-regulated flowering, there soon followed evidence of leaves as photoperiod sensors, of a timekeeper involving endogenous circadian rhythms, of transmissible florigenic signals and of a resulting cascade of developmental changes at the apex.
Although sometimes used loosely, it has long been clear that the term ‘flowering’ embraces an amazing series of signalling systems and developmental transitions. Photoperiodic induction refers to photoreceptor-driven, leaf-speciﬁc processes. Flower initiation at the apex is now divided into floral evocation and floral differentiation; evocation describes the early processes occurring at the apex before irreversible commitment and differentiation of flower primordia. Although the term ‘florigen’ was coined initially, there may be multiple transmitted florigenic stimuli so ‘floral stimuli’ or ‘florigens’ are more appropriate.
(a) Photoperiod and leaf photoresponse
Sensing of photoperiod requires photoreceptor pigments and a responsive organ. Elegant experiments involving selective light exposure of different parts of the plant conﬁrmed that the leaf blade is the photoresponsive site. Defoliated plants show little or no photoperiodic response and direct illumination of the shoot tip is mostly ineffective. A leaf, once photoperiodically treated, may be permanently changed. Leaves of the SDP Perilla, for example, exhibit a remarkable permanently induced state to the extent that a single leaf is capable of causing flowering when grafted in sequence to six vegetative receptor plants over a period of 14 weeks (Figure 8.26).
There are at least three plant pigments that could regulate photoperiodic flowering responses: chlorophyll via photosynthesis, phytochrome and the blue light receptor (see Section 8.4). Photosynthetic input will enhance flowering as shown earlier for the LDP Lolium (Figure 8.21). Measurements of shoot apex sugars show that increased photosynthetic sucrose supply to the shoot apex may be important, but on its own it is insufﬁcient. The primary requirement is instead for activation of phytochrome (see Section 8.4). For example, Lolium can flower in response to a single long day extended with non-photosynthetic light. Far-red-rich wavelengths from tungsten lamps are more effective than red-rich wavelengths from fluorescent lamps (Table 8.9), and this is typical for LDPs. For another LDP, Arabidopsis, involvement of phytochrome in flowering is revealed by a brief (10 min) end-of-day exposure to pure far-red (FR) light which promotes flowering with classic R/FR photoreversibility (Figure 8.27). What in perhaps surprising, considering the range of phytochrome mutants in Arabidopsis, is that none of the mutants presently known for phytochrome A or B (see Section 8.4) delays flowering (Figure 8.27).
Phytochrome’s role in flowering in SDPs relates to increases in the duration of the dark period (Figure 8.28). Light in the middle of the long inductive dark period (a ‘night break’) inhibits flowering of SDPs — they experience a ‘pseudo’ long day. Conversely, night breaks may promote flowering of LDPs. For SDPs, the night-break duration may be amazingly brief (1–300s) and the response often shows R/FR photoreversibility (Vince-Prue 1975; see also Section 8.4). Other evidence from action spectra emphasises the importance of red wavelengths of light for SDPs in contrast to the response to far-red for LDPs.
(b) Photoperiodic timekeeping
Accurate measurement of daylength for control of flowering requires a ‘photo’ response via a photoreceptor, and a measure of ‘period’ generally involving a circadian, rhythmic, timer. Circadian, meaning ‘about a day’, refers to the natural period of these rhythms often being not exactly 24h. In the absence of external stimuli, most rhythms manifest as free-running circadian cycles. However, the timing of dawn and/or dusk entrain the rhythm to synchronise with exact 24h cycles and hence provide an accurate daily clock used by both SDPs and LDPs. The currently favoured explanation of photoperiodic timekeeping involves rhythmic biochemical processes.
In addition, phytochrome is clearly involved (Figure 8.27), but may not act as an instantaneous on/off switch with respect to the light/dark cycle. Phytochrome is rapidly activated in light but on return to darkness there can be a slow (~ 0.5 to 4h) delay in disappearance of active phytochrome (the Pfr form) as it is degraded or decays back to the inactive Pr form. The consequence may be an offset between when it is actually dark and when the plant perceives it is dark. In the 1950s, Borthwick and Hendricks proposed that this natural offset, acting like an hourglass, accounted for photoperiodic time measurement in flowering (Hendricks 1960). Nowadays, the hourglass theory is often dismissed, especially as it would be limited to measuring dark periods only up to 4h. How-ever, it does provide a rational explanation of flowering of SDPs exposed to an extended long dark period and may well be a necessary component of photoperiodic timekeeping but perhaps not the limiting factor. There may also be an essential stabilisation period after Pfr decay during which other forms of timing may occur.
Although daily light/dark cycles set the phase and entrain 24h rhythms, this does not explain photoperiodic control of flowering. For example, there are distinct phase settings of leaf movement rhythms for the SDP Pharbitis nil when in long or short days, but flowering is stimulated only by short days. In 1936, Bünning deduced that there is a second, additional, light response allowing or preventing expression of the rhythm (see Bünning 1960; Lumsden 1991). The phase of the rhythm imposes or determines sensitivity of flowering to this second light input. The consequence is that, depending on daylength, light may or may not be synchronised with the dark-requiring part of the rhythm (Figure 8.29) and so flowering is either prevented or allowed.
Other rhythms have been revealed at the genetic and molecular levels. For example, Arabidopsis plants transformed with a luciferase gene (see Chapter 10) for bioluminescence coupled to the promoter sequence for a clock-regulated plant gene gave a simple, visually assayed, indicator rhythm which was then used to screen for period length mutants (Millar et al. 1995c). None of the mutants influenced flowering response, so it appears that there may be several independent clocks operating.
(c) Floral stimuli and inhibitors
The diverse environmental influences on flowering make it unlikely that plants possess a simple, unique regulatory signalling system. At least for photoperiod responses, grafting experiments indicate the presence both of transmissible promoters and inhibitors. However, isolation of florigenic chemicals from induced plants (Table 8.10) remains at a preliminary stage. We are still uncertain whether the floral stimulus (or inhibitor) is a single compound, a complex of compounds, whether it is photoperiod class speciﬁc, species speciﬁc or more universal.
Grafting experiments have conﬁrmed that leaves produce photoperiodic stimuli that are transmitted to the shoot apex, as discussed earlier for Perilla (Figure 8.26). For several long-day and short-day species, pre-induced, grafted leaves or leafy shoots cause flowering of vegetative recipient plants held in non-inductive conditions (see Lang 1965 and Bernier et al. 1981). Intriguingly, grafted leaves from day-neutral species may even be effective donors to LDPs or SDPs held in non-inductive photoperiods. In a few cases, such as Sedum spectabile (LDP) and Kalanchoe blossfeldiana (SDP), interspecies grafts have also been successful. This tells us that, despite photo-periodic differences, there may be common stimuli or common perception by the apex of different stimuli.
Many unsuccessful, frustrating attempts to extract and identify flowering stimuli have led florigens sometimes to be called hypothetical, non-existent or the holy grail of plant physiology. In addition to the tobacco extract example in Table 8.10, some positive results have also been reported for the SDP Pharbitis nil (Ishioka et al. 1991). In both studies, there was activity only in extracts from induced plants. Importantly, there was no activity for extracts of non-induced long-day leaves or their phloem exudates. We predict from experiments measuring speed of transmission that the signal moves in the phloem but no florigen has been chemically identiﬁed. The identity of inhibitory compounds is a further mystery. The main evidence for floral inhibitors comes again from grafting studies, for example in day-neutral tobacco. When grafted with an LDP tobacco, Nicotiana sylvestris (Figure 8.30), the day-neutral line flowers late if the graft partner is in non-inductive conditions; we deduce that it is producing an inhibitor that can pass across the graft union. The converse experiment with the long-day partner in inductive days led to early flowering of the day-neutral plant, so there is also a transmitted promoter (Figure 8.30). However, Maryland Mammoth, a short-day tobacco, lacks the graft-transmissible inhibitor, indicating how difﬁcult it is to unravel the complexities of signalling.
(d) Hormonal involvement
One reason for considering a role for plant hormones in the regulation of flowering is the frequent reports that their application dramatically alters flowering. However, cor-elations with altered endogenous hormone levels are not always evident, for example in the case of ABA content during floral induction in Lolium. By contrast, gibberellin application can cause flowering particularly of rosette plants. It may replace a need for vernalisation or long days in control of bolting and flowering (Lang 1965) and, as we will see later, endogenous gibberellin content may also increase following environmental changes that lead to flowering.
Some commercial uses of hormones have followed. For example, ethylene synchronises flowering and fruiting of bromeliads and is used worldwide for pineapple production. Conversely, inhibition of flowering of sugar cane by ethylene is practised in Hawaii where yield is greater if flowers do not develop (Moore and Osgood 1986).
With some ornamental species such as Spathiphyllum, most commercial growers use gibberellin because one application halves the time to flowering from six to three months. This early flowering is probably not related to juvenility, which is sometimes extended by applied gibberellin as in ivy (Hedera sp.) and shortened in Eucalyptus nitens when gibberellin levels are lowered. After treatment with paclobutrazol, which blocks gibberellin biosynthesis, grafted seedlings flower massively and three to ﬁve years earlier than normal (see earlier comment on juvenility and Moncur and Hasan 1994). Yet we ﬁnd there are no generalisations. For conifers, high gibberellin level may overcome juvenility and applied gibberellins, in combination with harsh cultural conditions, allow flowering at one to two years rather than after 10 to 20 years (see Pharis and King 1985). For some non-rosette species, long days and/or vernalisation can lead to rapid increases in gibberellin content (Metzger 1995) and inhibition of gibberellin biosynthesis may also block or delay flowering, which further suggests a link between gibberellins and normal reproductive responses. In species with no juvenile phase, gibberellins may replace the need for long days or vernalisation. For example, in the LDP Arabidopsis, a dwarf mutant (ga1-3) which is blocked in gib-berellin biosynthesis, flowers later than its wild type. In short days, some of these mutant plants may never flower unless treated with gibberellin (Table 8.11). On the other hand, vernalisation fails to stimulate flowering. Evidence against a role for gibberellins comes from the normal flowering of dwarf genotypes of many species (e.g. pea, corn, wheat, rice) which are blocked in gibberellin biosynthesis or in capacity to respond to gibberellin (Reid and Howell 1995).
Gibberellins can instead be inhibitory, especially for some perennials, including Fuchsia, Bougainvillea, mango and citrus, and also for species such as strawberry. Other gibberellins are known which can stimulate flowering without affecting growth. A more extreme response is seen from some novel synthetic gibberellins which can even act as growth retardants while still retaining ability to promote flowering (Evans et al. 1994a, b).
Complex relationships also exist between cytokinins and flowering. In the LDP Sinapis, endogenous cytokinin levels increase up to three-fold in long days. Applied cytokinin, however, induces only a partial flowering response (Bernier et al. 1993). There can also be indirect effects as found in Pharbitis nil where cytokinins can alter assimilate distribution to give either inhibition or promotion of flowering (Ogawa and King 1979).
We know much less about genetic and molecular events around the time of floral induction. Beginning with a late flowering mutant in Arabidopsis, a gene, Constans, has been identiﬁed whose expression is upregulated by long days (Putterill et al. 1995) and which may be one step in the sequence to florigens. Manipulation of phytochrome genes influencing flowering has also provided information on photoperiodic processes in leaves. In the future, we can expect to ﬁnd links to timekeeping genes which influence endo-genous rhythms. Analogous genes have been isolated from other organisms including Neurospora and Drosophila.