Investigation into the conservation of the regulatory pathways controlling flowering time
Flowering is a prerequisite for crop production and its timing needs to be optimized to suit local conditions. In the model plant Arabidopsis thaliana, several flowering pathways regulate FT, a highly conserved protein known as florigen. FT is expressed in the leaves under long day conditions and moves to the shoot apical meristem to induce flowering. For flowering to occur, FLC - the major repressor of FT - needs to be suppressed by the prolonged winter cold, allowing FT to be expressed in the spring once the day length is long enough. This project set out to investigate the genetic regulation of flowering time in non-Arabidopsis model plants. Unlike FT, which is a universal floral promoter, a role for FLC has only been established within the members of the Brassicaceae family. To elucidate whether FLC-like genes control flowering time in plant families other than the Brassicaceae, Nicotiana benthamiana was used as a model plant belonging to the Solanaceae (tomato) family. To distinguish FLC-like genes from the other MADS-box genes, 10 amino acid residues were identified which are only conserved between FLC-like proteins, and are not conserved in other MADS-box sequences. Based on the conserved residues, two FLC-like sequences were identified in N. benthamiana. To discover whether these could function to regulate Arabidopsis flowering time, transgenic Arabidopsis lines were produced over-expressing NbFLC-like genes. A late-flowering phenotype was observed in the transgenic plants that suggested a conserved floral repressing function for NbFLC-like genes. Although the roles of the NbFLC-like genes could not be tested endogenously, two other genes - NbFCA and NbFPA - were investigated, as their homologues in Arabidopsis repress FLC. The late-flowering phenotypes of the knock-down Nbfca and Nbfpa RNAi mutants revealed that NbFCA and NbFPA contribute to the promotion of flowering time. Based on qRT-PCR analyses, it was appeared that unlike their counterparts in Arabidopsis, these genes potentially promote flowering through the activation of NbFT, rather than through the down-regulation of NbFLC. However, RNA-seq analyses of the Nbfca and Nbfpa RNAi lines indicated that FCA regulates NbFLC-L1 through alternative splicing. While FLC-like genes were identified from a range of plant families, there do not appear to be any FLC-like genes in legumes. As FLC plays a key role in the vernalization process, the lack of its homologues in legumes suggests that the process of vernalization might have involved an alternative mechanism. Our laboratory has recently identified a novel gene from the model legume Medicago that we hypothesize may have obtained an FLC-like function. In this study the up-regulation of this gene in response to prolonged cold, and its down-regulation in the subsequent warm temperatures, was identified by qRT-PCR analyses. Its up-regulation preceded the upregulation of MtFTa1 suggesting a potential function as an upstream regulator of MtFTa1 in the vernalization process.By studying knock-out mutants, the MtFD gene was identified as a downstream gene of MtFTa1 in the promotion of flowering time. Moreover, various candidate transcription factors from other organisms were identified as potential trans-activators of MtFTa1, MtFTb1 and the FT-derived ncRNA promoters, by transient assays. Amongst them, members of the DOF family were identified as novel regulators of these genes which have not been previously reported. Overall, this study provided further evidence that the floral repressing function of FLC-like genes is potentially conserved outside the Brassicaceae family. Evidence (NbFCA and NbFPA) suggested that the function of some components of the autonomous pathway may be conserved in N. benthamiana, although they may have diverged regulatory mechanisms. In Medicago, the vernalization-response of the FT-derived ncRNA and its potential function upstream of MtFTa1 suggested an alternative mechanism, independent of FLC.
Advisor: Macknight, Richard
Degree Name: Doctor of Philosophy
Degree Discipline: Biochemistry
Publisher: University of Otago
Keywords: Flowering time; Medicago; Nicotiana benthamiana; FLC; FCA; FPA; FT; Alternative splicing; ncRNA; Transient assay
Research Type: Thesis