Photoperiodic regulation of flowering

The length of day and night is equal at the Equator, but towards the poles day length increases during the summer and decreases during the winter. Plants developing at the various geographical latitudes accommodated to these conditions adjusting their life cycle, including flowering, to the cycle of seasons. Plants can be classified as short-day, long-day or day-neutral plants depending on the day length that is required to induce flowering. The short-day and long-day plants have different critical values of day length and flower if the light period is shorter (short-day plants) or longer (long-day plants) than this threshold. Long-day plants usually flower in the spring and early summer, while short-day plants at the end of summer or during the autumn. However, the photoperiod is an ambiguous signal, since each day/night ratio manifests itself twice a year. Therefore, plants either combine temperature sensing with photoperiodism (see later for vernalisation) or they record the tendency whether the night periods are shortening or lengthening during subsequent days. Moreover, the developmental state can also affect flowering (see also later). A plant can be in a juvenile non-competent state in a given season and in a mature flowering-non-competent state only in the other.

Although plants are classified according to their day-length requirement for flowering, a series of experiments showed that plants measure the length of the night (dark period) not the day (light period) (Fig. 9.4) (8). At a defined day length, the flowering response is dependent on the critical value of the dark period (Fig. 9.4). However, if the dark period is set, changing the length of the light period does not affect the flowering response (Fig. 9.4). Furthermore, disrupting the dark period by short time of irradiation (few minutes) is enough to cancel the effect of the dark period on flowering, but disrupting the light period by dark has no effect (Fig. 9.4). If the dark period is interrupted by light, the plants restart measuring its length, while the opposite does not take place.

94

Short day (long night) plant Long day (short night) plant

= critical value

day/light night/dark

Figure 9.4. Effect day- and night length on the photoperiodic regulation of flower initiation. The length of the night exceeding a critical value promotes (short day plants) or inhibits (long day plants) flowering. Interruption of the dark period by a light pulse resets the start of measurement. Interruption of the light period by dark has no effect. (Figure of A. Fehér)

Based on the observation that the flowering of short-day plants can be delayed by a short light pulse applied during the night, the researches could investigate the effect of several light parameters on flower induction (9). They observed that:

 the effect of light depends on its wavelength

 the spectrum of the effective wavelengths overlaps with the absorption spectrum of phytochromes

 if the dark-interrupting light is red, flowering is delayed, but far-red light is inefficient in this regard

 if several red and far-red pulses are applied alternately the last pulse is the decisive

 the efficiency of the light signal that interrupts the dark period and delays flowering depends on the time of application: it is most effecting around the middle of the dark period.

 if plants are grown under continuous dark, the efficiency of the light pulse cycles with an app.

24h-long period.

From these observations it was concluded that the red/far-red light sensitive photoreceptors, the phytochromes, have role in the measurement of day/night length. The periodicity in the efficiency of the dark-interrupting light suggested that the circadian clock might also be included in the regulation.

The circadian clock is an endogenous time-keeping mechanism of living organisms. It allows the synchronization of physiological and metabolic processes with the exogenous dark-light cycle, which strongly influences the biological activities of the organisms including plants (10). The circadian clock has an app. 24-hour period, and is “free-running”, what means that it maintains its rhythmicity even under constitutive environmental conditions (constant light/dark). This endogenous mechanism is

95 based on multi-component molecular oscillators. These oscillators are transcriptional/translational regulatory loops of transcriptional factors and regulatory proteins. The output is altered gene expression, app. 10% of all Arabidopsis genes exhibit circadian rhythmicity in their expression pattern.

The input is light. Although the clock is free-running, it needs light to be precisely adjusted to the changing environmental dark/light cycle as well as to maintain its robustness (the amplitude of the cycles diminishes under constant conditions).

Based on the above observations, Erwin Bünning (1936) established the model of photoperiodic regulation of flowering (9). According to this model, the plant’s sensitivity towards a flowering-inducing signal exhibits a circadian rhythmicity and it has its maximum value around sunset. During long days, but not under short days, the sensitivity maximum coincides with the presence of light and this coincidence that is required for flowering initiation in long-day plants or its inhibition in short-day plants (“Bünning hypothesis” or “coincidence model”).

During the past decades, molecular genetic studies confirmed the basic hypothesis of this model. In Arabidopsis, a mutation was identified which made the flowering time of this plant independent of the photoperiod. Arabidopsis thaliana is a facultative long-day plant species: it has an early flowering phenotype under long days, and a late flowering phenotype under short days. The mutant exhibited late flowering under both conditions and the corresponding gene was named CONSTANS (CO) (11).

Expression of the CO gene exhibits circadian rhythmicity with an expression maximum at evening around sunset (12). Therefore, this gene coding for a transcription factor, display the characteristics of the “sensitivity factor” hypothesized by Bünning. But how CO as a transcription factor regulates flowering?

The decision to flower takes place in the shoot meristem (see before). However, CO is expressed in the leaf primarily in the cells around the phloem. It means that the signal that evokes flowering is perceived in the leaf and gets transferred to the shoot apex. If in the co mutant background, CO was overexpressed under the control of a meristem specific promoter it could not complement the late flowering phenotype. If the same experiment was repeated using a promoter working in phloem companion cells, the complementation worked. These experiments proved the CO has its function in the leaf and there should be a mobile signal that transfers the information generated by CO from the leaf to the shoot meristem (12). Scientists has long supposed the existence of a mobile molecule that accumulates in leaves and induce flowering in the shoot apex and called this hypothetical hormone as

“florigen”, after Mikahail Chailakhyan (1937). Early experiments showed that from a plant that was induced to flower, the flowering signal could be transferred by grafting a single induced leaf to a none-induced plant.

Molecular genetic investigations revealed that the target of the CO transcription factor is the FLOWERING LOCUS T (FT) gene, one of the flowering integration proteins (see above) (12). Since CO is expressed in leaves, FT is also formed in leaves. However, scientists showed that if the FT protein is marked with a green-fluorescent protein (GFP) and expressed in the phloem companion cells of leaves, the fluorescent FT protein soon accumulates in the shoot meristem. FT in the meristem interacts with the FD transcription factor and initiates the expression of the flower meristem identity genes AP1 and LFY (via SOC1) (Fig. 9.5). Based on this, the FLOWERING LOCUS T (FT) protein is the long-searched

“florigen”; the mobile factor formed in leaves but triggering the flowering response in the soot meristem.

96 Figure 9.5. The model of photoperiod-dependent signalling between the leaf and the shoot meristem. CONSTANS (CO) switches on the expression of the FLOWERING LOCUS T (FT) gene under long-day photoperiod in the leaf.

FT is transferred in the phloem to the shoot meristem where it associates with the FLOWERING D (FD) transcription factor to trigger the expression of the flower meristem identity factors APETALA 1 (AP1) and LEAFY (LFY). Induction of LFY expression is indirect via the expression of the flowering integrator SUPPRESSOR OF CONSTANS 1 (SOC1) transcription factor. The synergistic AP1 and LFY are antagonistic with TFL1 to establish the inflorescence meristem (see Fig. 9.2). (Figure of A. Fehér)

But why the FT gene is switched on by CO only under long days? Why the activity of the CO gene during the dark period of long nights is not sufficient to evoke the flowering response? The answer is in the stability of the CO protein that is regulated by light (Fig. 9.6) (9). During short days, the maximum of CO expression is in the dark. Although CO proteins are produced, those are rapidly degraded in the dark and cannot switch on efficient transcription of FT (Fig. 9.6) (12). Under long days, however, CO protein is already produced in the evening when light is still present. Light prevents the degradation of CO that switches on FT gene expression and thus FT protein accumulates at a sufficient level to get transported to the shoot meristem and trigger the flowering response (Fig. 9.6) (12). Light exerts its effect on the CO protein via the PhyA and Cry1 photoreceptors at the evening and PhyB in the morning.

Among the short-day plants, the flowering regulation of rice (Oryza sativa) has been studied in details (13). The rice genome also codes for CO- and FT-like proteins called HEADING DATE 1 (Hd1) and HEADING DATE 3a (Hd3a), respectively. However, the mechanism of regulation differs between Arabidopsis and rice. In rice the CO homologue protein Hd1 is converted by light from an inducer to a repressor. Thus, Hd1 activates Hd3a (FT homologue) expression during the dark evenings under short days but inhibits it at long days due to the presence of light in the evening period when the Hd1 gene is transcribed.