Ectopic expression of an orchid (Oncidium Gower Ramsey) AGL6-like gene promotes flowering by activating flowering time genes in Arabidopsis thaliana

An AP1/AGL9 group of MADS box gene, OMADS1, with extensive homology to the Arabidopsis AGAMOUS-like 6 gene (AGL6) was characterized from orchid (Oncidium Gower Ramsey). OMADS1 mRNA was detected in apical meristem and in the lip and carpel of flower. Yeast two-hybrid analysis indicated that OMADS1 is able to strongly interact with OMADS3, a TM6-like protein that was involved in flower formation and floral initiation in orchid. Transgenic Arabidopsis and tobacco ectopically expressed OMADS1 showed similar novel phenotypes by significantly reducing plant size, flowering extremely early, and losing inflorescence indeterminacy. In addition, homeotic conversion of sepals into carpel-like structures and petals into staminoid structures were also observed in flowers of 35S::OMADS1 Arabidopsis. This result indicated that OMADS1 was involved in floral formation and initiation in transgenic plants. Further analysis indicated that the expression of flowering time genes FT, SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1) and flower meristem identity genes LEAFY (LFY), APETALA1 (AP1) was significantly up-regulated in 35S::OMADS1 transgenic Arabidopsis plants. Furthermore, ectopic expression of OMADS1 rescued late-flowering phenotype in gi-1, co-3 but not for ft-1 and fwa-1 mutants. These results supported that ectopic expression of OMADS1 influenced flower transition and formation by acting as an activator for FT and SOC1 in Arabidopsis.     

Two lily SEPALLATA-like genes cause different effects on floral formation and floral transition in Arabidopsis

Two AGL2-like MADS-box genes, Lily MADS Box Gene (LMADS) 3 and LMADS4, with extensive homology of LMADS3 to the Arabidopsis SEPALLATA3 were characterized from the lily (Lilium longiflorum). Both LMADS3 and LMADS4 mRNA were detected in the inflorescence meristem, in floral buds of different developmental stages, and in all four whorls of the flower organ. LMADS4 mRNA is also expressed in vegetative leaf and in the inflorescence stem where LMADS3 expression is absent. Transgenic Arabidopsis, which ectopically expresses LMADS3, showed novel phenotypes by significantly reducing plant size, flowering extremely early, and loss of floral determinacy. By contrast, 35S::LMADS4 transgenic plants were morphologically indistinguishable from wild-type plants. The early-flowering phenotype in 35S::LMADS3 transgenic Arabidopsis plants was correlated with the up-regulation of flowering time genes FT, SUPPRESSOR OF OVEREXPRESSION OF CO 1, LUMINIDEPENDENS, and flower meristem identity genes LEAFY and APETALA1. This result was further supported by the ability of 35S::LMADS3 to rescue the late-flowering phenotype in gigantea-1 (gi-1), constans-3 (co-3), and luminidependens-1 but not for ft-1 or fwa-1 mutants. The activation of these flowering time genes is, however, indirect because their expression was unaffected in plants transformed with LMADS3 fused with rat glucocorticoid receptor in the presence of both dexamethasone and cycloheximide.     

AGL24 acts in concert with SOC1 and FUL during Arabidopsis floral transition

Arabidopsis plants flower in response to long days (LDs). Exposure of leaves to inductive day lengths activates expression of FLOWERING LOCUS T (FT) protein which moves to the shoot apical meristem (SAM) to induce developmental reprogramming. SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and FRUITFULL (FUL) are induced by FT at the apex. We previously screened the SAM for mRNAs of genes required to promote the floral transition in response to photoperiod, and conducted detailed expression and functional analyses on several putative candidates. Here, we show that expression of AGAMOUS-LIKE 24 (AGL24) is detected at the SAM under SD conditions and increases upon exposure to LDs. Mutations in AGL24 further delay flowering of a soc1 ful double mutant, suggesting that flowering is controlled by AGL24 partly independently of SOC1 and FUL.     

LEAFY controls floral meristem identity in Arabidopsis.

The first step in flower development is the generation of a floral meristem by the inflorescence meristem. We have analyzed how this process is affected by mutant alleles of the Arabidopsis gene LEAFY. We show that LEAFY interacts with another floral control gene, APETALA1, to promote the transition from inflorescence to floral meristem. We have cloned the LEAFY gene, and, consistent with the mutant phenotype, we find that LEAFY RNA is expressed strongly in young flower primordia. LEAFY expression procedes expression of the homeotic genes AGAMOUS and APETALA3, which specify organ identify within the flower. Furthermore, we demonstrate that LEAFY is the Arabidopsis homolog of the FLORICAULA gene, which controls floral meristem identity in the distantly related species Antirrhinum majus.     

Specification of reproductive meristems requires the combined function of SHOOT MERISTEMLESS and floral integrators FLOWERING LOCUS T and FD during Arabidopsis inflorescence development

In Arabidopsis floral meristems are specified on the periphery of the inflorescence meristem by the combined activities of the FLOWERING LOCUS T (FT)-FD complex and the flower meristem identity gene LEAFY. The floral specification activity of FT is dependent upon two related BELL1-like homeobox (BLH) genes PENNYWISE (PNY) and POUND-FOOLISH (PNF) which are required for floral evocation. PNY and PNF interact with a subset of KNOTTED1-LIKE homeobox proteins including SHOOT MERISTEMLESS (STM). Genetic analyses show that these BLH proteins function with STM to specify flowers and internodes during inflorescence development. In this study, experimental evidence demonstrates that the specification of flower and coflorescence meristems requires the combined activities of FT-FD and STM. FT and FD also regulate meristem maintenance during inflorescence development. In plants with reduced STM function, ectopic FT and FD promote the formation of axillary meristems during inflorescence development. Lastly, gene expression studies indicate that STM functions with FT-FD and AGAMOUS-LIKE 24 (AGL24)-SUPPRESSOR OF OVEREXPRESSION OF CONTANS1 (SOC1) complexes to up-regulate flower meristem identity genes during inflorescence development.     

A Quantitative and Dynamic Model of the Arabidopsis Flowering Time Gene Regulatory Network

Various environmental signals integrate into a network of floral regulatory genes leading to the final decision on when to flower. Although a wealth of qualitative knowledge is available on how flowering time genes regulate each other, only a few studies incorporated this knowledge into predictive models. Such models are invaluable as they enable to investigate how various types of inputs are combined to give a quantitative readout. To investigate the effect of gene expression disturbances on flowering time, we developed a dynamic model for the regulation of flowering time in Arabidopsis thaliana. Model parameters were estimated based on expression time-courses for relevant genes, and a consistent set of flowering times for plants of various genetic backgrounds. Validation was performed by predicting changes in expression level in mutant backgrounds and comparing these predictions with independent expression data, and by comparison of predicted and experimental flowering times for several double mutants. Remarkably, the model predicts that a disturbance in a particular gene has not necessarily the largest impact on directly connected genes. For example, the model predicts that SUPPRESSOR OF OVEREXPRESSION OF CONSTANS (SOC1) mutation has a larger impact on APETALA1 (AP1), which is not directly regulated by SOC1, compared to its effect on LEAFY (LFY) which is under direct control of SOC1. This was confirmed by expression data. Another model prediction involves the importance of cooperativity in the regulation of APETALA1 (AP1) by LFY, a prediction supported by experimental evidence. Concluding, our model for flowering time gene regulation enables to address how different quantitative inputs are combined into one quantitative output, flowering time.     

Wheat SOC1 functions independently of WAP1/VRN1, an integrator of vernalization and photoperiod flowering promotion pathways

Heading time in bread wheat ( Triticum aestivum L.) is determined by three characters – vernalization requirement, photoperiodic sensitivity and narrow-sense earliness (earliness per se) – which are involved in the phase transition from vegetative to reproductive growth. The wheat APETALA1 ( AP1 )-like MADS-box gene, wheat AP1 ( WAP1 , identical with VRN1 ), has been identified as an integrator of vernalization and photoperiod flowering promotion pathways. A MADS-box gene, SUPPRESSOR OF OVEREXPRESSION OF CO 1 ( SOC1 ) is an integrator of flowering pathways in Arabidopsis . In this study, we isolated a wheat ortholog of SOC1 , wheat SOC1 ( WSOC1 ), and investigated its relationship to WAP1 in the flowering pathway. WSOC1 is expressed in young spikes but preferentially expressed in leaves. Expression starts before the phase transition and is maintained during the reproductive growth phase. Overexpression of WSOC1 in transgenic Arabidopsis plants caused early flowering under short-day conditions, suggesting that WSOC1 functions as a flowering activator in Arabidopsis . WSOC1 expression is affected neither by vernalization nor photoperiod, whereas it is induced by gibberellin at the seedling stage. Furthermore, WSOC1 is expressed in transgenic wheat plants in which WAP1 expression is cosuppressed. These findings indicate that WSOC1 acts in a pathway different from the WAP1 -related vernalization and photoperiod pathways.     

Conservation of floral homeotic gene function between Arabidopsis and antirrhinum.

Several homeotic genes controlling floral development have been isolated in both Antirrhinum and Arabidopsis. Based on the similarities in sequence and in the phenotypes elicited by mutations in some of these genes, it has been proposed that the regulatory hierarchy controlling floral development is comparable in these two species. We have performed a direct experimental test of this hypothesis by introducing a chimeric Antirrhinum Deficiens (DefA)/Arabidopsis APETALA3 (AP3) gene, under the control of the Arabidopsis AP3 promoter, into Arabidopsis. We demonstrated that this transgene is sufficient to partially complement severe mutations at the AP3 locus. In combination with a weak ap3 mutation, this transgene is capable of completely rescuing the mutant phenotype to a fully functional wild-type flower. These observations indicate that despite differences in DNA sequence and expression, DefA coding sequences can compensate for the loss of AP3 gene function. We discuss the implications of these results for the evolution of homeotic gene function in flowering plants.     

Cloning and characterization of SOC1 homologs in barley (Hordeum vulgare) and their expression during seed development and in response to vernalization

A number of genes are involved in the vernalization pathway, such as VRN1, VRN2 and VRN3/FT1, whose function has been studied in barley and wheat. However, the function of the flowering and vernalization integrator SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) has not been well studied in Triticeae, and particularly in barley. Herein, we cloned and characterized two barley SOC1-like homologs, HvSOC1-like1 and HvSOC1-like2. Primary sequence analysis of the predicted HvSOC1-like1 and HvSOC1-like2 proteins showed that they are members of the type II MADS-box protein family. Phylogenetic analysis placed the predicted proteins with other SOC1 and SOC1-like proteins from different species neighboring those from other cereal plant species. Primary and secondary structures of the predicted proteins are conserved to each other and more distant to the recently identified barley ODDSOC1 proteins. Genomic organization of HvSOC1-like1 is very similar to the Arabidopsis and Brachypodium SOC1 genes and localized in highly syntenic chromosomal regions. Regulatory cis-acting elements detected in the HvSOC1-like1 promoter include the CArG-box, implicated in the regulation of SOC1 expression in Arabidopsis. Both HvSOC1-like1 and HvSOCI-like2 are expressed in vegetative and reproductive tissues and at different stages of seed development. Both are upregulated in a particular seed developmental stage suggesting their possible implication in seed development. Furthermore, HvSOC1-like1 was induced in two winter barley cultivars after vernalization treatment pointing to its probable involvement in the vernalization process. The study of the SOC1 genes reported here opens the way for a better understanding of both the vernalization process and seed development and germination in this important cereal crop.     

Specification of Arabidopsis floral meristem identity by repression of flowering time genes

Flowering plants produce floral meristems in response to intrinsic and extrinsic flowering inductive signals. In Arabidopsis, the floral meristem identity genes LEAFY (LFY) and APETALA1 (AP1) are activated to play a pivotal role in specifying floral meristems during floral transition. We show here that the emerging floral meristems require AP1 to partly specify their floral identities by directly repressing a group of flowering time genes, including SHORT VEGETATIVE PHASE (SVP), AGAMOUS-LIKE 24 (AGL24) and SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1). In wild-type plants, these flowering time genes are normally downregulated in emerging floral meristems. In the absence of AP1, these genes are ectopically expressed, transforming floral meristems into shoot meristems. By post-translational activation of an AP1-GR fusion protein and chromatin immunoprecipitation assays, we further demonstrate the repression of these flowering time genes by induced AP1 activity and in vivo AP1 binding to the cis-regulatory regions of these genes. These findings indicate that once AP1 is activated during the floral transition, it acts partly as a master repressor in floral meristems by directly suppressing the expression of flowering time genes, thus preventing the continuation of the shoot developmental program.     

Poppy APETALA1/FRUITFULL orthologs control flowering time, branching, perianth identity, and fruit development

Several MADS box gene lineages involved in flower development have undergone duplications that correlate with the diversification of large groups of flowering plants. In the APETALA1 gene lineage, a major duplication coincides with the origin of the core eudicots, resulting in the euFUL and the euAP1 clades. Arabidopsis FRUITFULL (FUL) and APETALA1 (AP1) function redundantly in specifying floral meristem identity but function independently in sepal and petal identity (AP1) and in proper fruit development and determinacy (FUL). Many of these functions are largely conserved in other core eudicot euAP1 and euFUL genes, but notably, the role of APETALA1 as an "A-function" (sepal and petal identity) gene is thought to be Brassicaceae specific. Understanding how functional divergence of the core eudicot duplicates occurred requires a careful examination of the function of preduplication (FUL-like) genes. Using virus-induced gene silencing, we show that FUL-like genes in opium poppy (Papaver somniferum) and California poppy (Eschscholzia californica) function in axillary meristem growth and in floral meristem and sepal identity and that they also play a key role in fruit development. Interestingly, in opium poppy, these genes also control flowering time and petal identity, suggesting that AP1/FUL homologs might have been independently recruited in petal identity. Because the FUL-like gene functional repertoire encompasses all roles previously described for the core eudicot euAP1 and euFUL genes, we postulate subfunctionalization as the functional outcome after the major AP1/FUL gene lineage duplication event.     

Flowering and determinacy in Arabidopsis

Meristems provide new cells to produce organs throughout the life of a plant, and their continuous activity depends on regulatory genes that balance the proliferation of meristem cells with their recruitment to organogenesis. During flower development, this balance is shifted towards organogenesis, causing the meristem to terminate after producing a genetically determined number of organs. In Arabidopsis, WUSCHEL (WUS) specifies the self-renewing cells at the core of the shoot meristems and is a key target in the control of meristem stability. The development of a determinate floral meristem is initiated by APETALA1/CAULIFLOWER (AP1/CAL) and LEAFY (LFY). The latter activates AGAMOUS (AG), partly in co-operation with WUS. AG then directs the development of the innermost floral organs and at the same time antagonizes WUS to terminate the meristem, although the mechanism of WUS repression remains unknown. All these genes participate in a series of regulatory feedback loops that maintain stable expression patterns or promote sharp developmental transitions. Although the regulators of meristem maintenance and determinacy in Arabidopsis are widely conserved, their interactions may vary in other species.     

Functional analysis of three lily (Lilium longiflorum) APETALA1-like MADS box genes in regulating floral transition and formation

Three cDNAs showing a high degree of homology to the SQUA subfamily of MADS box genes were isolated and characterized from the lily (Lilium longiflorum). Lily MADS Box Gene 5 (LMADS5) showed high sequence identity to oil palm (Elaeis guineensis) SQUAMOSA3 (EgSQUA3). LMADS6 is closely related to LMADS5 whereas LMADS7 is more related to DOMADS2, an orchid (Dendrobium) gene in the SQUA subfamily. The expression pattern for these three genes was similar and their RNAs were detected in vegetative stem and inflorescence meristem. LMADS5 and 6 were highly expressed in vegetative leaves and carpel, whereas LMADS7 expression was absent. Ectopic expression of LMADS5, 6 or 7 in transgenic Arabidopsis plants showed novel phenotypes by flowering early and producing terminal flowers. Homeotic conversions of sepals to carpelloid structures and of petal to stamen-like structures were also observed in 35S::LMADS5, 6 or 7 flowers. Ectopic expression of LMADS6 or LMADS7 was able to complement the ap1 flower defect in transgenic Arabidopsis ap1 mutant plants. These results strongly indicated that the function of these three lily genes was involved in flower formation as well as in floral induction. Furthermore, the ability of lily LMADS6 and 7 to complement the Arabidopsis ap1 mutant provided further evidence to show that the conserved motifs (paleoAP1 or euAP1) in the C-terminus of the SQUA/AP1 subfamily of MADS box genes is not strictly necessary for their function.     

A TERMINAL FLOWER1-like gene from perennial ryegrass involved in floral transition and axillary meristem identity

Control of flowering and the regulation of plant architecture have been thoroughly investigated in a number of well-studied dicot plants such as Arabidopsis, Antirrhinum, and tobacco. However, in many important monocot seed crops, molecular information on plant reproduction is still limited. To investigate the regulation of meristem identity and the control of floral transition in perennial ryegrass (Lolium perenne) we isolated a ryegrass TERMINAL FLOWER1-like gene, LpTFL1, and characterized it for its function in ryegrass flower development. Perennial ryegrass requires a cold treatment of at least 12 weeks to induce flowering. During this period a decrease in LpTFL1 message was detected in the ryegrass apex. However, upon subsequent induction with elevated temperatures and long-day photoperiods, LpTFL1 message levels increased and reached a maximum when the ryegrass apex has formed visible spikelets. Arabidopsis plants overexpressing LpTFL1 were significantly delayed in flowering and exhibited dramatic changes in architecture such as extensive lateral branching, increased growth of all vegetative organs, and a highly increased trichome production. Furthermore, overexpression of LpTFL1 was able to complement the phenotype of the severe tfl1-14 mutant of Arabidopsis. Analysis of the LpTFL1 promoter fused to the UidA gene in Arabidopsis revealed that the promoter is active in axillary meristems, but not the apical meristem. Therefore, we suggest that LpTFL1 is a repressor of flowering and a controller of axillary meristem identity in ryegrass.