RNA-induced silencing complexes (RISCs) play central roles in posttranscriptional gene silencing. In plants, the mechanism of RISC assembly has remained elusive due to the lack of cell-free systems that recapitulate the process. In this report, we demonstrate that plant AGO1 protein synthesized by in vitro translation using an extract of evacuolated tobacco protoplasts incorporates synthetic small interfering RNA (siRNA) and microRNA (miRNA) duplexes to form RISCs that sequester the single-stranded siRNA guide strand and miRNA strand, respectively. The formed RISCs were able to recognize and cleave the complementary target RNAs. In this system, the siRNA duplex was incorporated into HSP90-bound AGO1, and subsequent removal of the passenger strand was triggered by ATP hydrolysis by HSP90. Removal of the siRNA passenger strand required the ribonuclease activity of AGO1, while that of the miRNA star strand did not. Based on these results, the mechanism of plant RISC formation is discussed.

FLOWERING LOCUS T (FT) genes encode proteins that function as the mobile floral signal, florigen. In this study, we characterized five FT-like genes from the model legume, Medicago (Medicago truncatula). The different FT genes showed distinct patterns of expression and responses to environmental cues. Three of the FT genes (MtFTa1, MtFTb1, and MtFTc) were able to complement the Arabidopsis (Arabidopsis thaliana) ft-1 mutant, suggesting that they are capable of functioning as florigen. MtFTa1 is the only one of the FT genes that is up-regulated by both long days (LDs) and vernalization, conditions that promote Medicago flowering, and transgenic Medicago plants overexpressing the MtFTa1 gene flowered very rapidly. The key role MtFTa1 plays in regulating flowering was demonstrated by the identification of fta1 mutants that flowered significantly later in all conditions examined. fta1 mutants do not respond to vernalization but are still responsive to LDs, indicating that the induction of flowering by prolonged cold acts solely through MtFTa1, whereas photoperiodic induction of flowering involves other genes, possibly MtFTb1, which is only expressed in leaves under LD conditions and therefore might contribute to the photoperiodic regulation of flowering. The role of the MtFTc gene is unclear, as the ftc mutants did not have any obvious flowering-time or other phenotypes. Overall, this work reveals the diversity of the regulation and function of the Medicago FT family.

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.

The MADS-domain transcription factor SHORT VEGETATIVE PHASE plays a key role as a repressor of the transition to flowering and as a regulator of early floral development in Arabidopsis thaliana (Arabidopsis). However, no flowering-time repressors have been functionally identified in the model legume Medicago truncatula (Medicago). In this study, phylogenetic analysis of two closely-related MtSVP-like sequences, MtSVP1 and MtSVP2, showed that their predicted proteins clustered together within the eudicot SVP clade. To determine if the MtSVP-like genes have a role in flowering, they were functionally characterized in Medicago and Arabidopsis. Transcripts of both MtSVP genes were abundant and broadly expressed in vegetative tissues but were detected at much lower levels in flowers in Medicago. Over-expression of the MtSVP genes in Arabidopsis resulted in delayed flowering and flowers with many abnormal phenotypes such as leafy sepals, changes to floral organ number and longer pedicels than the wild type. By contrast, in transgenic Medicago, over-expression of MtSVP1 resulted in alterations to flower development, but did not alter flowering time, suggesting that MtSVP1 may not function to repress the transition to flowering in Medicago.

The timing of the transition to flowering is carefully controlled by plants in order to optimize sexual reproduction and the ensuing production of seeds, grains, and fruits. The genetic networks that regulate floral induction are best characterized in the temperate eudicot Arabidopsis in which the florigen gene FT plays a major role in promoting the transition to flowering. Legumes are an important plant group, but less is known about the regulation of their flowering time. In the model legume Medicago truncatula (Medicago), a temperate annual plant like Arabidopsis, flowering is induced by prolonged cold (vernalization) followed by long day lengths (LD). Recent molecular-genetic experiments have revealed that a FT-like gene, MtFTa1, is a central regulator of flowering time in Medicago. Here, we characterize the three Medicago FRUITFULL (FUL) MADS transcription factors, MtFULa, MtFULb, and MtFULc using phylogenetic analyses, gene expression profiling through developmental time courses, and functional analyses in transgenic plants. MtFULa and MtFULb have similarity in sequence and expression profiles under inductive environmental conditions during both vegetative and reproductive development while MtFULc is only up regulated in the apex after flowering in LD conditions. Sustained up regulation of MtFULs requires functional MtFTa1 but their transcript levels are not affected during cold treatment. Overexpression of MtFULa and MtFULb promotes flowering in transgenic Arabidopsis plants with an additional terminal flower phenotype on some 35S:MtFULb plants. An increase in transcript levels of the MtFULs was also observed in Medicago plants overexpressing MtFTa1. Our results suggest that the MtFULs are targets of MtFTa1. Overall, this work highlights the conserved functions of FUL-like genes in promoting flowering and other roles in plant development and thus contributes to our understanding of the genetic control of the flowering process in Medicago.

Overexpression of Medicago SOC1a leads to precocious primary shoot axis elongation.