Control of Flowering Time (George Coupland)
Plant development is often synchronised to the changing seasons. Flowering, tuberisation and onset of bud dormancy all occur at appropriate times of the year. Changing daylength is one of the environmental signals used by plants to trigger these developmental processes, as was first shown in the 1920s. The ability to monitor and respond to daylength, called photoperiodism, is now known to be widespread throughout the plant and animal Kingdoms. This response is an important character in adaptation of both wild species and crop plants to life at different latitudes. My research group is focused on understanding the molecular mechanisms used by plants to detect daylength and to use this information to trigger flowering. We are also interested in how this process interacts with responses to other environmental signals such as temperature, and how the mechanisms underlying daylength responses are modified in other species to generate responses distinct to those shown by Arabidopsis.
So far, most of our work has focused on the isolation and characterization of genes that regulate flowering of the model species Arabidopsis thaliana. Flowering of Arabidopsis occurs rapidly under long days of 16 hours light, but is dramatically delayed under short days of 10 hours light. We and others have described genetic and molecular interactions between a set of genes that defines a pathway that promotes flowering in response to long days (reviewed in Mouradov et al, 2002). We are now extending our understanding of this long-day pathway by identifying more genes that act within it, seeking to explain the molecular interactions between the gene products at the level of biochemistry and describing in which plant tissues the different components of the pathway act to promote flowering. We are also beginning to investigate whether the long-day pathway occurs in plant species unrelated to Arabidopsis that flower in response to short days, and if so how its activity is modified to create these distinct responses.
CONSTANS - a light sensitive timer
The most extensively studied of the Arabidopsis flowering time genes that confer a daylength response is called CONSTANS (CO). Inactivation of the gene causes late flowering, while its overexpression induces early flowering (Putterill et al, 1995; Simon et al, 1996; Onouchi et al, 2000).
The CO protein is located in the nucleus and is involved in the regulation of the transcription of other genes that regulate flowering time (Robson et al, 2001). For example, CO directly activates the transcription of the flowering time gene FT, which encodes a regulator of phosphorylation (Samach et al, 2000). Although the level of FT transcription is extremely sensitive to CO levels and is induced rapidly in response to initiating CO expression, CO is not predicted to bind directly to the FT promoter but probably acts within a protein complex that includes a DNA binding protein that enables attachment of the complex to the FT promoter.
In wild-type plants, CO activates FT expression under long but not short days, so how does daylength regulate the activity of CO? Progress in answering this question came from the demonstration that CO is expressed only at certain times of day (Suarez-Lopez et al, 2001). These times correspond to the interval when plants are exposed to light in long days, but under short days are in darkness. Therefore, activation of CO protein function by light may explain how its activity is restricted to long days. We obtained further support for this model by studying the environmental conditions under which CO will activate FT transcription. Transgenic plants that express CO mRNA at high level from a viral promoter also express very high levels of FT mRNA, as long as the plants are exposed to light, but when these plants are shifted to darkness, FT transcription declines rapidly. This suggests that exposure to light is required to activate CO protein function.
Our studies on CO explain in broad terms how diurnal rhythms in transcription and post-transcriptional regulation of protein function by light could trigger flowering in response to daylength. We are working intensively to identify the signal transduction pathway that triggers CO activity in response to light, and to understand the effect of exposure to light on CO protein.
Generating diurnal patterns in CO mRNA abundance
The importance of the diurnal patterns in CO transcription for the control of flowering in response to daylength, led us to study the mechanisms by which these patterns are generated. Other flowering-time genes that act within the daylength pathway are required for these diurnal patterns in CO expression. For example, the gigantea (gi) and lhy-1 mutations both cause late flowering, and these mutations reduce the abundance of the CO mRNA at times when it reaches peak levels in wild- type plants (Suarez-Lopez et al, 2001). That these reductions in CO mRNA are the basis of the late flowering phenotype was further suggested by showing that overexpression of CO in these mutant backgrounds corrected the late flowering phenotype associated with the mutations. We have studied the functions of the LHY and GI genes in some detail (Schaffer et al, 1997; Fowler et al, 1999). LHY encodes a MYB transcription factor that, along with the related protein CCA1, controls diurnal rhythms in a wide range of Arabidopsis genes, particularly those, like CO, that peak late in the day (Mizoguchi et al, 2002). When plants are removed from daily cycles of light and dark into continuous light then LHY and CCA1 are required to maintain the rhythms in all circadian clock regulated genes tested. We continue to study the roles of LHY and CCA1 in generating diurnal rhythms, and particularly their function in regulating the expression of genes such as GI and CO that control flowering time.
Interactions between photoperiod and temperature
Seasonal responses are often mediated by temperature as well as photoperiod. For example, many plants that grow at high latitude or high altitude will not flower until they have been exposed to low temperatures for several weeks. This requirement for extended exposure to cold is called vernalization and ensures that plants flower in the spring, when optimal conditions for seed set prevail, and not in autumn when seed maturation would not occur prior to the onset of extreme winter conditions. Arabidopsis varieties that require vernalization will flower early if vernalized and very late if they are not vernalized. This late flowering in the absence of vernalization occurs even in long photoperiods, and therefore overrides the promotion of flowering caused by long days. We have become interested in the mechanism by which the promotion of flowering caused by CO and other long-day pathway genes is suppressed in varieties that require vernalization. Other groups have characterized in detail the genes that are present in strains requiring vernalization, and shown that a MADS box transcription factor called FLC is expressed at high levels in these strains and represses flowering. However, on vernalization FLC expression falls and the plant flowers early. We have shown that FLC suppresses the ability of CO to activate its target genes such as FT (Samach et al, 2000). FLC does not appear to influence CO transcription, and will suppresses the capacity of 35S::CO to activate FT and another target gene, SOC1. We have analysed the promoters of SOC1 and FT and shown that FLC will bind directly to the SOC1 promoter to suppress its transcription, and that this effect cannot be overcome by high level expression of CO. We are using a combination of genetic and biochemical approaches to study the mechanism by which FLC suppresses CO-mediated activation of genes such as FT and SOC1. We are also interested to determine whether this antagonism between FLC, acting in the vernalization pathway, and CO acting in the photoperiod pathway, is conserved in other plant species.
Diversity in daylength responses
Different plant species show diverse responses to daylength. For example, although flowering of Arabidopsis and crop plants such as wheat is promoted by exposure to long days, flowering in species such as rice and maize show the reverse response being promoted by short days and inhibited by long days. Does the CO gene also control flowering in these short day plants? A Japanese research group recently demonstrated the importance of CO like genes in short-day plants by showing that differences in the control of flowering of rice varieties could be explained by alterations in the structure of the rice orthologue of the CO gene (Hd1), which in rice promotes flowering in response to short days. We wish to explain how diverse responses to daylength in different plant species are generated. We have shown that CO from the short-day dicotyledonous plant Pharbitis nil will activate FT transcription in Arabidopsis. We wish to compare the protein complexes in which CO acts in Arabidopsis and Pharbitis and how these are involved in regulating FT transcription as an approach to explaining the basis of the differences underlying long and short day responses.
A role for SUMOylation in flowering time control
We have recently become interested in the role of the ubiquitin-like molecule SUMO (small ubiquitin related modifier) in plants, and particularly in the control of flowering. Previously we identified a gamma-ray induced mutation that caused an extreme early flowering phenotype under short days. We cloned the gene by map- based cloning, and showed that it is a plant homologue of ULP1, a cysteine protease that is specific for the ubiquitin-like protein SUMO (also called sentrin). ULP1 both cleaves the carboxy-terminal end of SUMO to generate the mature form, and deconjugates SUMO from target proteins. We have shown that the Arabidopsis protein will cleave SUMO precursor in vitro, and that in vivo the levels of SUMO are severely depleted in the mutant. We are introducing transgenes designed to alter SUMO metabolism in wild-type and mutant plants, with the aim of identifying SUMO-conjugated proteins in Arabidopsis that are involved in the regulation of flowering.