To extend our understanding of flowering time control in eudicots, we screened for mutants in the model legume Medicago truncatula (Medicago). We identified an early flowering mutant, spring1, in a T-DNA mutant screen, but spring1 was not tagged and was deemed a somaclonal mutant. We backcrossed the mutant to wild type R108. The F1 plants and the majority of F2 plants were early flowering like spring1, strongly indicating that spring1 conferred monogenic, dominant early flowering. We hypothesized that the spring1 phenotype resulted from over expression of an activator of flowering. Previously, a major QTL for flowering time in different Medicago accessions was located to an interval on chromosome 7 with six candidate flowering- time activators, including a CONSTANS gene, MtCO, and three FLOWERING LOCUS T (FT) genes. Hence we embarked upon linkage mapping using 29 markers from the MtCO/FT region on chromosome 7 on two populations developed by crossing spring1 with Jester. Spring1 mapped to an interval of ∼0.5 Mb on chromosome 7 that excluded MtCO, but contained 78 genes, including the three FT genes. Of these FT genes, only FTa1 was up-regulated in spring1 plants. We then investigated global gene expression in spring1 and R108 by microarray analysis. Overall, they had highly similar gene expression and apart from FTa1, no genes in the mapping interval were differentially expressed. Two MADS transcription factor genes, FRUITFULLb (FULb) and SUPPRESSOR OF OVER EXPRESSION OF CONSTANS1a (SOC1a), that were up-regulated in spring1, were also up-regulated in transgenic Medicago over-expressing FTa1. This suggested that their differential expression in spring1 resulted from the increased abundance of FTa1. A 6255 bp genomic FTa1 fragment, including the complete 5′ region, was sequenced, but no changes were observed indicating that the spring1 mutation is not a DNA sequence difference in the FTa1 promoter or introns.
SUMMARYMolecular-genetic control of the flowering time of temperate-climate plants is best understood in Arabidopsis and the cereals wheat and barley. However, key regulators such as FLC and cereal VRN2 are not found in legumes. Therefore, we used forward genetics to identify flowering time genes in the model legume Medicago truncatula (Medicago) which is induced to flower by vernalisation and long-day photoperiods. A screen of a Tnt1 retroelement tagging population yielded two mutants, spring2 and spring3, with a dominant early flowering phenotype. These mutants overexpress the floral activator FTa1 and two candidate downstream flowering genes SOC1a and FULb, similar to the spring1 somaclonal variant that we identified previously. We demonstrate here that an increase in the expression of FTa1, SOC1a and FULb and early flowering does not occur in all conditions in the spring mutants. It depends on long-day photoperiods but not on vernalisation. Isolation of flanking sequence tags and linkage analysis identified retroelement insertions at FTa1 that co-segregated with the early flowering phenotype in all three spring mutants. These were Tnt1 insertions in the FTa1 third intron (spring3) or the 3′ intergenic region (spring2) and an endogenous MERE1-4 retroelement in the 3′ intergenic region in spring1. Thus the spring mutants form an allelic series of gain-of-function mutations in FTa1 which confer a spring growth habit. The spring retroelement insertions at FTa1 separate long-day input from vernalisation input into FTa1 regulation, but this is not due to large-scale changes in FTa1 DNA methylation or transcript processing in the mutants.
Flowering time is an important contributor to plant productivity and yield. Plants integrate flowering signals from a range of different internal and external cues in order to flower and set seed under optimal conditions. Networks of genes controlling flowering time have been uncovered in the flowering models Arabidopsis, wheat, barley and rice. Investigations have revealed important commonalities such as FT genes that promote flowering in all of these plants, as well as regulators that are unique to some of them. FT genes also have functions beyond floral promotion, including acting as floral repressors and having a complex role in woody polycarpic plants such as vines and trees. However, much less is known overall about flowering control in other important groups of plants such as the legumes. This review discusses recent efforts to uncover flowering-time regulators using candidate gene approaches or forward screens for spring early flowering mutants in the legume Medicago truncatula. The results highlight the importance of a Medicago FT gene, FTa1, in flowering-time control. However, the mechanisms by which FTa1 is regulated by environmental signals such as long days (photoperiod) and vernalisation (winter cold) appear to differ from Arabidopsis.
BackgroundThe ability to induce flowering on demand is of significant biotechnological interest. FT protein has been recently identified as an important component of the mobile flowering hormone, florigen, whose function is conserved across the plant kingdom. We therefore focused on manipulation of both endogenous and heterologous FT genes to develop a floral induction system where flowering would be inhibited until it was induced on demand. The concept was tested in the model plant Arabidopsis thaliana (Arabidopsis).ResultsOur starting point was plants with strongly delayed flowering due to silencing of FT with an artificial microRNA directed at FT (amiR-FT) [1]. First, we showed that constitutive expression of a heterologous FT gene (FTa1), from the model legume Medicago truncatula, (Medicago) was able to rescue the amiR-FT late-flowering phenotype. In order to induce flowering in a controlled way, the FTa1 gene was then expressed under the control of an alcohol-inducible promoter in the late flowering amiR-FT plants. Upon exposure to ethanol, FTa1 was rapidly up regulated and this resulted in the synchronous induction of flowering.ConclusionsWe have thus demonstrated a controlled-inducible flowering system using a novel combination of endogenous and heterologous FT genes. The universal florigenic nature of FT suggests that this type of system should be applicable to crops of economic value where flowering control is desirable.
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