Contreras_Planesas_whole_thesis.pdf (3.89 MB)
Exploring flowering and photoperiod response mechanisms in legumes
thesisposted on 2023-05-28, 12:23 authored by Contreras Planesas, B
Flowering defines the reproductive stage of a plant, and its timing is regulated by environmental factors to align reproduction with favourable conditions to maximise the success of a plant. Daylength (photoperiod) and temperature are the two primary factors that signal seasonal changes and drive plant responses. The genetic pathways through which these factors act to influence flowering time has been extensively studied in model plants like Arabidopsis, and underlying molecular mechanisms have been substantially elucidated. The molecular knowledge regarding flowering time control is helping to describe and predict the adaptation of plants and crops, and is particularly useful in an agricultural context. The molecular pathway conferring photoperiod-responsiveness of flowering was first described in the model Arabidopsis and involves a central role for the hormone-like florigen protein FT that integrates environmental information and signals from leaf to shoot apex. Under LD conditions, FT gene expression is highly induced by CO, a direct FT regulator and key element of the pathway, that integrates input from light and from the circadian clock to determine photoperiod sensitivity. While many other genes participate in the regulation of flowering, research across diverse angiosperm crop and model species plants seem to support the conserved nature of this central mechanism, in which light perceived by photoreceptors is translated to photoperiod-specific CO expression, which in turn activates FT, providing the ultimate inductive signal for flowering to occur. However, other evidence suggests that this conservation may not be universal. In legumes, the FT gene family is expanded, but several genes show strong photoperiod-dependent expression and promotive effects on flowering, similar to Arabidopsis FT. In contrast, CO-like genes are present, but have not been shown to have any clear or substantial role in regulation of flowering, at least in the temperate LD legumes. An increasing number of other legume flowering gene homologs have also been examined, but this is ongoing, and many potential components still remain to be characterised. Also, the finding that the role of CO may not be conserved in legumes raises questions about how photoperiod-specific FT expression occurs. This thesis explores this question by characterising several loci implicated in photoperiod sensitive flowering in other systems. In the case of soybean, a SD legume, the integrating role of CO may instead be played by E1, a legume specific gene encoding a transcription factor that directly represses FT gene expression under LD. E1 is also the most important locus explaining latitudinal variation for flowering time across different cultivars. Chapter 3 of this thesis examines E1 phylogeny, compares E1 protein structures and investigates the potential role of E1 genes in two temperate LD legumes, pea and Medicago, through the characterization of mutants obtained by reverse genetics. Protein-level control is an important feature of the photoperiod pathway, and is critical for the precise timing of the activity of some elements in the pathway, most notably CO. In Arabidopsis, COP1 is the main E3 ubiquitin ligase involved in the regulation of CO protein abundance. COP1 has been shown to participate in diverse light-mediated processes and it is particularly well characterised for its role in photomorphogenesis. Its participation in flowering regulation includes a direct interaction with CO but also with other components such as photoreceptors, GIGANTEA (GI) and ELF3 thereby also modulating transcriptional regulation of CO. In pea, the LIP1 gene has been characterized as the COP1 ortholog, and Chapter 4 examines its potential role in flowering time control and developmental rate, including how it might interact genetically with key elements of the pea flowering network such as LATE1 (PsGI), HR (PsELF3a) and PHYA through the characterisation of the double and triple mutants with lip1. In addition, the predicted protein structure of LIP1 was compared to COP1 to identify any possible functionally-significant differences. Another important component in the Arabidopsis photoperiod pathway is FKF1, which is able to perceive blue light and participates in the transcriptional and post-transcriptional regulation of CO. Its function is associated with the formation of protein complexes controlling protein degradation. One such complex, formed between FKF1 and GI, is able to regulate members of the CDF transcription factor family, which are repressors of CO transcription. This complex serves as one important point in the integration of light and circadian clock regulation for the response to photoperiod in Arabidopsis. Chapter 5 investigates whether FKF1 might participate in flowering time control in pea and Medicago, through isolation of mutants and examination of potential effects on flowering and photomorphogenesis. A phylogenetic study of the legume FKF1 family is included together with an examination of the possible physical interaction of FKF1 and LATE1 using a yeast two-hybrid assay. Results from the thesis suggest that some regulatory factors of the pathway are conserved and participate in flowering control, but others have minimal regulatory roles. E1 in pea and Medicago has a small promoting effect on flowering, opposite to that observed in the SD legume, soybean. Thus, although potentially retaining a role in flowering time regulation, it is not the sole point of integration for the photoperiod response. LIP1 also has a small effect on flowering time due in part to a slow developmental rate (plastochron). Genetic interactions with important flowering components like LATE1 and HR suggest it is unlikely to have a role in the photoperiod response similar to Arabidopsis COP1. Finally, FKF1 protein is able to interact with LATE1 in pea but its flowering effect in both pea and Medicago seems to be limited, with a minimal late-flowering phenotype in some photoperiod conditions. Overall, this research makes a significant contribution to the current understanding of photoperiod regulation of flowering time in temperate long-day legumes.
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