Reproductive development and the evolution of perennial life history

1. Background

We study the regulatory mechanisms that control the onset of reproduction in plants, how these are regulated by environmental cues and how they are modified with life history during plant evolution.

The initiation of reproduction is strongly regulated in all organisms and is crucial in ensuring their evolutionary success. In plants, the onset of reproduction is controlled by environmental cues at several check points, and the relative importance of these check points differs among species. Floral induction, the decision to switch from forming vegetative organs such as leaves to producing flowers, represents the first check point. This step is strictly regulated in all plants and is usually triggered by seasonal cues such as day length or winter temperatures. In response to these cues, meristems, from which all above ground tissues are derived, terminate the production of vegetative organs and initiate the formation of flowers. Floral induction requires a complex reprogramming of meristem function to increase its overall size, to switch the identity of the organs it forms and to influence its determinacy, which defines the number of flowers it ultimately produces. Floral bud growth and development is a second major check point. Growth and development of floral buds can be arrested so that they do not form mature flowers until they are exposed to appropriate environmental cues. In this way floral induction can be separated from the appearance of mature flowers and reproduction. Species in which this check point has a major role can for example undergo floral induction in autumn but the floral buds then arrest until spring, when the flowers open and reproduction is initiated. To address how each of these processes is controlled, we use Arabidopsis thaliana as a model system to define regulatory processes that control floral induction by environmental cues and the switch in identity from a vegetative to an inflorescence meristem. In addition, we have developed Arabis alpina, a relative of A. thaliana, as a model system to study regulation of reproduction in the context of the perennial life-cycle. Perennials differ from annuals such as A. thaliana in living for many years, restricting the extent and duration of flowering to ensure they survive to the following year and controlling flowering more strongly at the level of floral bud arrest.

Further reading (selected reviews)

Hyun, Y., Richter, R., Coupland, G. (2017). Competence to flower: Age-controlled sensitivity to environmental cues. Plant Physiology doi: 10.1104/pp.16.01523.

Romera-Branchat, M., Andrés, F. and Coupland, G. (2014). Flowering responses to seasonal cues: what’s new? Current Opinion in Plant Biology 21, 120 – 127.

Andrés, F. and Coupland, G. (2012). The genetic basis of flowering responses to seasonal cues. Nature Reviews Genetics 13, 627-639.


2. Environmental control of floral induction and inflorescence meristem identity in Arabidopsis thaliana

We study the mechanisms controlling floral induction in A. thaliana and the associated change in meristem identity from the vegetative meristem forming leaves to the inflorescence meristem that produces flowers. We have defined a regulatory pathway in the phloem companion cells that controls abundance of the CONSTANS (CO) transcription factor in response to day length. Under long days characteristic of summer, CO protein is more stable causing its abundance to increase and therefore to promote flowering, whereas under short days the protein is unstable. Recently, we showed that phosphorylation of CO is associated with its destabilization under short days. Furthermore, we demonstrated that if plants are exposed to high temperatures under short days then the small amount of CO present can promote flowering as if the plants were in long days, demonstrating the need to balance the input from different environmental cues. When CO accumulates under long days it promotes transcription of the FLOWERING LOCUS T ( FT) gene, whose small protein product is related to lipid-binding proteins. We have shown that under these conditions, FT protein moves to the shoot apex through the phloem and triggers floral development at the meristem. The mechanisms by which FT induces floral development at the shoot apex are being studied in detail through a combination of imaging using fluorescent protein fusions, ChIPseq to identify genes to which FT is recruited and suppressor mutagenesis using sensitized mutant screens.  

Pattern of expression of SPL15 protein in the inflorescence.

SPL15 protein is fused to the VENUS fluorescent protein is expressed from the SPL15 promoter and visualized by confocal microscopy.

Interestingly, A. thaliana does eventually flower under short days, although the CO-FT pathway is inactive under these conditions. Recently, we showed that flowering under these conditions is controlled by the SQUAMOSA BINDING PROTEIN LIKE 15 (SPL15) transcription factor. Imaging of fluorescent protein fusions showed that SPL15 is expressed directly in the shoot meristem, and our data suggest that it is a point of convergence of several other pathways that were previously implicated in regulating the floral transition. SPL15 transcription is repressed by FLOWERING LOCUS C (FLC) a transcription factor involved in the response to winter temperatures (vernalization), it is also regulated at the post-transcriptional level in the meristem by microRNA156, and it is repressed post-translationally by DELLA proteins that are components of the gibberellin signalling pathway. To induce flowering, we showed that SPL15 will directly activate in the meristem genes with known functions in flowering and inflorescence development, FRUITFULL and MIRNA172b. The transcriptional activation function of SPL15 involves interaction with other transcription factors expressed in the early inflorescence meristem, notably the MADS box factor SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1. Currently, we are exploring the regulation of SPL15 in the shoot meristem, the functions of its known target genes in conferring the switch to inflorescence meristem function and are identifying a broader range of SPL15 targets. We are also interested in why SPL15 activity is important only under short days, and how the CO-FT pathway can overcome its absence under long days.

Model for activation of transcription by SPL15.
SPL15 acts in a protein complex with the MADS box transcription factor SOC1 to activate transcription of target genes.

FLC is a key repressor of flowering that blocks floral induction until plants are exposed to winter temperatures that repress FLC transcription. FLC directly binds to and represses transcription of several key genes involved in floral induction, as described above for SPL15. We have performed a range of genome-wide approaches to identify FLC direct targets and by cross referencing these with targets of other floral regulators have defined a set of putative key regulators of flowering. Currently, we are analysing these systematically by reverse genetics, imaging and transcriptome analyses to widen our knowledge of the transcriptional network that controls floral induction at the shoot meristem.

By these and related approaches we aim to define the regulatory functions controlling floral induction from environmental perception in the leaf vasculature, via systemic signalling through the phloem sieve elements to the meristematic transition that occurs at the shoot apex. Finally, once the meristem has initiated the production of flowers, this developmental state is stable and maintained independently of environmental cues. We aim to understand how the regulatory networks expressed in the shoot meristem stabilize the identity of the inflorescence meristem.


Further reading (selected recent papers)

Richter, R., Kinoshita, A., Vincent, C., Martinez-Gallegos, R., Gao, H., van Driel, AD., Hyun, Y., Mateos, JL., Coupland, G. (2019). Floral regulators FLC and SOC1 directly regulate expression of the B3-type transcription factor TARGET OF FLC AND SVP 1 at the Arabidopsis shoot apex via antagonistic chromatin modifications. PLoS genetics 15 (4), e1008065,

Hayama, R., Sarid-Krebs, L., Richter, R., Fernández, V., Jang, S. and Coupland, G. (2017). PSEUDO RESPONSE REGULATORs stabilize CONSTANS protein to promote flowering in response to day length. EMBO Journal 36(7): 904-918, doi:10.15252/embj.201693907.

Hyun, Y., Richter, R., Vincent, C., Martinez-Gallegos, R., Porri, A. and Coupland, G. (2016). Multi-layered Regulation of SPL15 and Cooperation with SOC1 Integrate Endogenous Flowering Pathways at the Arabidopsis Shoot Meristem. Developmental Cell 37, 254-266

Fernández, V., Takahashi, Y, Le Gourrierec, J. and Coupland, G. (2016). Photoperiodic and thermosensory pathways interact through CONSTANS to promote flowering at high temperature under short days. The Plant Journal 86 59.

De Montaigu, A., Giakountis, A., Rubin, M., Tóth, R., Cremer, F., Sokolova, V., Porri, A., Reymond, M., Weinig, C. and Coupland, G. (2015). Natural diversity in daily rhythms of gene expression contributes to phenotypic variation. Proceedings of the National Academy of Sciences 112, 905-910.


3. Arabis as a model system to study life-history evolution and flowering patterns in perennials

Perennial Arabis alpina growing in a natural population in the French Alps.

Annual and perennial plants show many differences in their patterns of reproduction. Annuals flower once in their life cycle and then transfer all resources into seeds leading to death of the plant. By contrast perennials live for many years, flower each year and intersperse periods of vegetative growth and flowering. Therefore, the balance between vegetative and reproductive development differs fundamentally between annuals and perennials. We are interested in the mechanisms that control the flowering patterns of perennials, and particularly those processes not found in annuals. Perennials must terminate flowering and restore vegetative growth, they induce flowering multiple times in their life cycle and they balance vegetative growth and reproduction creating a trade-off between the number of progeny produced and survival to flower the following year. In addition to flowering patterns, other related life-history traits differ between annuals and perennials. For example, perennials restrict senescence to particular regions of the plant after flowering whereas annual plants undergo whole plant senescence after flowering. Perennials also store nutrients at the end of the growing season in vegetative tissues and these are reused the following year, whereas annuals transfer all nutrients to seeds. We have developed Arabis alpina as a perennial model system to study these processes. This species is a member of the same family as A. thaliana allowing comparison of regulatory mechanisms, and is closely related to annual sister species A. montbretiana and A. iberica. To facilitate our studies, we have sequenced the genome of A. alpina and are completing the genomes of its annual relatives. In addition, we have intercrossed annual A. montbretiana and perennial A. alpina and in the following generations are developing introgression lines to identify genes that diverge in function during evolution and confer life-history traits.

Resequencing of introgression lines carrying segments of the annual A. montbretiana genome in perennial A. alpina.

Two segments (blue arcs) of the A. montbretiana genome (black outer ring) are present in the

A. alpina genome (green outer ring) and were delimited by Illumina resequencing.

In flowering regulation of A. alpina, we have focused on perennial specific traits, in particular limitation of the duration of flowering and restoration of vegetative growth and the age at which plants become sensitive to environmental cues to flower. We have shown that the FLC orthologue PERPETUAL FLOWERING 1 is differentially regulated in perennial Brassicaceae compared to annuals and has a key role in terminating a reproductive episode in perennials. In addition, the age at which plants become sensitive to environmental cues to flower, so called competence to flower, is critical in perennials because it allows the plant to acquire sufficient biomass to survive flowering. This trait is strongly evident in A. alpina, because the plant must reach 3-5 weeks old before it will respond to vernalization to flower. We showed that miR156 and its target mRNAs encoding SPL transcription factors are required to confer the time at which perennial A. alpina becomes sensitive to winter temperatures to induce flowering. We are now using CRISPR-cas9 to target specific SPL genes in the perennial background to understand better how this process is controlled. In addition, we are studying variation in flowering responses among natural accessions of A. alpina distributed across its European range to determine how important variation in reproductive characters is to adaptation to different environments. Interestingly, in some of these populations flowering and reproduction appear to be mainly controlled by regulation of bud dormancy rather than floral induction.

Using these approaches, we aim to understand which genes vary during divergence of annual and perennial life-history, and particularly how evolution of life history impacts upon the regulatory network controlling initiation of reproduction. In addition, we aim to compare the natural-genetic variation for flowering found in natural perennial populations with the well-described variation found in annuals such as A. thaliana.

Further reading (selected recent papers)

Hyun, Y., Vincent, C., Tilmes, V., Bergonzi, S., Kiefer, C., Richter, R., Martinez-Gallegos, R., Severing, E., Coupland, G. (2019) A regulatory circuit conferring varied flowering response to cold in annual and perennial plants. Science 363, 6425, 409-412, doi: 10.1126/science.aau8197.

Kiefer, C., Severing, E., Karl, R., Bergonzi, S., Koch, M., Tresch, A., & Coupland, G. (2017). Divergence of annual and perennial species in the Brassicaceae and the contribution of cis-acting variation at FLC orthologues. Molecular Ecology 26:3437-3457, doi:10.1111/mec.14084.

Mateos, J. L., Tilmes, V., Madrigal, P., Severing, E., Richter, R., Rijkenberg, C. W. M., Krajewski, P., & Coupland, G. (2017). Divergence of regulatory networks governed by the orthologous transcription factors FLC and PEP1 in Brassicaceae species. Proceedings of the National Academy of Sciences of the United States of America, 114, E11037-E11046. doi:10.1073/pnas.1618075114.

Willing, E.-M., Rawat, V., Mandáková, T., Maumus, F., Velikkakam James, G., Nordström, K.J.V., Becker, C., Warthmann, N., Chica, C., Szarzynska, B., Zytnicki, M., Albani, M.C., Kiefer, C., Bergonzi, S., Castaings, L., Mateos, J.L., Berns, M.C., Bujdoso, N., Piofczyk, T., de Lorenzo, L., Barrero-Sicilia. C., Mateos, I., Piednoël, M., Hagmann, J., Chen-Min-Taó, R., Iglesias-Fernández, R., Schuster, S.C., Alonso-Blanco, C., Roudier, F., Carbonero, P., Paz-Ares, J., Davis, S.J., Pecinka, A., Quesneville, H., Colot, V., Lysak, M.A., Weigel, D., Coupland, G.* and Schneeberger, K.*(2015). Genome expansion of Arabis alpina linked with retrotransposition and reduced symmetric DNA methylation. Nature Plants 1, 14023.

Toräng, P., Wunder, J., Ramón Obeso, J., Herzog, M., Coupland, G. and Agren, J. (2015). Large-scale adaptive differentiation in the alpine perennial herb Arabis alpina. New Phytologist 206, 459-470.


4. More on our work

The above is a summary of some aspects of our most recent work. A full list of publications is available on Google Scholar

Most of our publications are available as PDFs for download on ResearchGate at the link

If you would like more information on our work or are interested in joining us as an Intern, Masters student, PhD student or Post doc, please contact by Email.

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