Analysis of flowering pathway integrators in Arabidopsis

Flowering is regulated by an integrated network of several genetic pathways in Arabidopsis. The key genes integrating multiple flowering pathways are FT, SOC1 and LFY. To elucidate the interactions among these integrators, genetic analyses were performed. FT and SOC1 share the common upstream regulators CO, a key component in the long day pathway, and FLC, a flowering repressor integrating autonomous and vernalization pathways. However, the soc1 mutation further delayed the flowering time of long day pathway mutants including ft, demonstrating that SOC1 acts partially independently of FT. Although soc1 did not show an obvious defect in flower meristem determination on its own, it dramatically increased the number of coflorescences in a lfy mutant, which is indicative of a defect in floral initiation. Therefore, double mutant analysis shows that the three integrators have both overlapping and independent functions in the determination of flowering time and floral initiation. The expression analysis showed that FT regulates SOC1 expression, and SOC1 regulates LFY expression, but not vice versa, which is consistent with the fact that FT and LFY have the least overlapping functions among the three integrators. The triple mutation ft soc1 lfy did not block flowering completely under long days, indicating the presence of other integrators. Finally, vernalization accelerated flowering of flc ft soc1 and ft soc1 lfy triple mutants, which shows that the vernalization pathway also has targets other than FLC, FT, SOC1 and LFY. Our genetic analysis reveals the intricate nature of genetic networks for flowering.     

Mutagenesis of plants overexpressing CONSTANS demonstrates novel interactions among Arabidopsis flowering-time genes

CONSTANS (CO) promotes flowering of Arabidopsis in response to long photoperiods. Transgenic plants carrying CO fused with the cauliflower mosaic virus 35S promoter (35S::CO) flowered earlier than did the wild type and were almost completely insensitive to length of day. Genes required for CO to promote flowering were identified by screening for mutations that suppress the effect of 35S::CO. Four mutations were identified that partially suppressed the early-flowering phenotype caused by 35S::CO. One of these mutations, suppressor of overexpression of CO 1 (soc1), defines a new locus, demonstrating that the mutagenesis approach is effective in identifying novel flowering-time mutations. The other three suppressor mutations are allelic with previously described mutations that cause late flowering. Two of them are alleles of ft, indicating that FT is required for CO to promote early flowering and most likely acts after CO in the hierarchy of flowering-time genes. The fourth suppressor mutation is an allele of fwa, and fwa soc1 35S::CO plants flowered at approximately the same time as co mutants, suggesting that a combination of fwa and soc1 abolishes the promotion of flowering by CO. Besides delaying flowering, fwa acted synergistically with 35S::CO to repress floral development after bolting. The latter phenotype was not shown by any of the progenitors and was most probably caused by a reduction in the function of LEAFY. These genetic interactions suggest models for how CO, FWA, FT, and SOC1 interact during the transition to flowering.     

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.     

Flowering and expression of flowering-related genes under long-day conditions with light-emitting diodes

The effects of light quality on flowering time were investigated in Gypsophila paniculata, which is a long-day cut flower, and with Arabidopsis under long-day conditions with light-emitting diodes (LEDs). Gypsophila paniculata plants were grown under natural daylight and flowering was controlled by long-day treatment with a weak LED light of a single color in the night. Flowering was promoted not by blue light, but by far-red light in G. paniculata, while flowering was promoted by both light colors in Arabidopsis. FT homologs of G. paniculata GpFT1 and GpFT2 were differentially expressed under long-day conditions with white light, suggesting that they play roles in flowering at different stages of reproductive development. GpFTs and FT gene expression was not induced by far-red light in G. paniculata or Arabidopsis. Instead, the expression of the SOC1 homolog of G. paniculata GpSOC1 and SOC1 was induced by far-red light in G. paniculata and Arabidopsis. Flowering was promoted by induction of FT and SOC1 expression with blue light in Arabidopsis, whereas GpFTs and GpSOC1 expression was low with blue light induction in G. paniculata. The relationship between flowering and the expression of FT and SOC1 in Arabidopsis was confirmed with ft and soc1 mutants. These results suggest that long-day conditions with far-red light promote flowering through SOC1 and its homologs, while the conditions with blue light do not promote flowering in G. paniculata, because of low expression of GpFTs and GpSOC1 in contrast to that 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.     

Acceleration of flowering by overexpression of MFT (MOTHER OF FT AND TFL1)

MFT (MOTHER OF FT AND TFL1) is a member of a gene family that includes two important regulators, FT (FLOWERING LOCUS T) and TFL1 (TERMINAL FLOWER 1), in determination of flowering time in Arabidopsis. Although the functions of FT and TFL1 are assigned in the family, the roles of other members are largely unknown. Especially the sequence of MFT is homologous to both FT and TFL1, which act as a floral promoter and an inhibitor, respectively, making it difficult to predict the role of MFT. We performed genetic analyses of MFT to understand its role in floral development. Constitutive expression of MFT led to slightly early flowering under long days. However, a T-DNA insertion allele of MFT did not show obvious phenotype. Further genetic analyses with the loss-of-function alleles of FT, TFL1, and ATC (Arabidopsis Thaliana CENTRORADIALIS homologue) showed that a decrease of MFT activity did not enhance the phenotypes of the single mutants. Taken together, we suggest that MFT functions as a floral inducer and that it may act redundantly in determination of flowering time in Arabidopsis.     

CRYPTIC PRECOCIOUS/MED12 is a novel flowering regulator with multiple target steps in Arabidopsis

The proper timing of flowering is of crucial importance for reproductive success of plants. Regulation of flowering is orchestrated by inputs from both environmental and endogenous signals such as daylength, light quality, temperature and hormones, and key flowering regulators construct several parallel and interactive genetic pathways. This integrative regulatory network has been proposed to create robustness as well as plasticity of the regulation. Although knowledge of key genes and their regulation has been accumulated, there still remains much to learn about how they are organized into an integrative regulatory network. Here, we have analyzed the CRYPTIC PRECOCIOUS (CRP) gene for the Arabidopsis counterpart of the MED12 subunit of the Mediator. A novel dominant mutant, crp-1D, which causes up-regulation of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), FRUITFULL (FUL) and APETALA1 (AP1) expression in a FLOWERING LOCUS T (FT)-dependent manner, was identified in an enhancer screen of the early-flowering phenotype of 35S::FT. Genetic and molecular analysis of both crp-1D and crp loss-of-function alleles showed that MED12/CRP is required not only for proper regulation of SOC1, FUL and AP1, but also for up-regulation of FT, TWIN SISTER OF FT (TSF) and FD, and down-regulation of FLOWERING LOCUS C (FLC). These observations suggest that MED12/CRP is a novel flowering regulator with multiple regulatory target steps both upstream and downstream of the key flowering regulators including FT florigen. Our work, taken together with recent studies of other Mediator subunit genes, supports an emerging view that the Mediator plays multiple roles in the regulation of flowering.     

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.     

Functional conservation and divergence of FVE genes that control flowering time and cold response in rice and Arabidopsis

Recent molecular and genetic studies in rice, a short-day plant, have elucidated both conservation and divergence of photoperiod pathway genes and their regulators. However, the biological roles of rice genes that act within the autonomous pathway are still largely unknown. In order to better understand the function of the autonomous pathway genes in rice, we conducted molecular genetic analyses of OsFVE, a rice gene homologous to Arabidopsis FVE. OsFVE was found to be ubiquitously expressed in vegetative and reproductive organs. Overexpression of OsFVE could rescue the flowering time phenotype of the Arabidopsis fve mutants by up-regulating expression of the SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1) and down-regulating FLOWERING LOCUS C (FLC) expression. These results suggest that there may be a conserved function between OsFVE and FVE in the control of flowering time. However, OsFVE overexpression in the fve mutants did not rescue the flowering time phenotype in in relation to the response to intermittent cold treatment.     

FLOWERING LOCUS T regulates stomatal opening

Stomatal pores surrounded by a pair of guard cells in the plant epidermis control gas exchange for photosynthesis in response to light, CO(2), and phytohormone abscisic acid. Phototropins (phot1 and phot2) are plant blue-light receptor kinases and mediate stomatal opening via activation of the plasma membrane H(+)-ATPase. However, the signaling mechanism from phototropins to the H(+)-ATPase has yet to be determined. Here, we show that FLOWERING LOCUS T (FT) is expressed in guard cells and regulates stomatal opening. We isolated an scs (suppressor of closed-stomata phenotype in phot1 phot2) 1-1 mutant of Arabidopsis thaliana and showed that scs1-1 carries a novel null early flowering 3 (elf3) allele in a phot1 phot2 background. scs1-1 (elf3 phot1 phot2 triple mutant) had an open-stomata phenotype with high H(+)-ATPase activity and showed increased levels of FT mRNA in guard cells. Transgenic plants overexpressing FT in guard cells showed open stomata, whereas a loss-of-function FT allele, ft-1, exhibited closed stomata and failed to activate the H(+)-ATPase in response to blue light. Our results define a new cell-autonomous role for FT and demonstrate that the flowering time genes ELF3 and FT are involved in the regulation of H(+)-ATPase by blue light in guard cells.     

Integration of flowering signals in winter-annual Arabidopsis

Photoperiod is the primary environmental factor affecting flowering time in rapid-cycling accessions of Arabidopsis (Arabidopsis thaliana). Winter-annual Arabidopsis, in contrast, have both a photoperiod and a vernalization requirement for rapid flowering. In winter annuals, high levels of the floral inhibitor FLC (FLOWERING LOCUS C) suppress flowering prior to vernalization. FLC acts to delay flowering, in part, by suppressing expression of the floral promoter SOC1 (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1). Vernalization leads to a permanent epigenetic suppression of FLC. To investigate how winter-annual accessions integrate signals from the photoperiod and vernalization pathways, we have examined activation-tagged alleles of FT and the FT homolog, TSF (TWIN SISTER OF FT), in a winter-annual background. Activation of FT or TSF strongly suppresses the FLC-mediated late-flowering phenotype of winter annuals; however, FT and TSF overexpression does not affect FLC mRNA levels. Rather, FT and TSF bypass the block to flowering created by FLC by activating SOC1 expression. We have also found that FLC acts as a dosage-dependent inhibitor of FT expression. Thus, the integration of flowering signals from the photoperiod and vernalization pathways occurs, at least in part, through the regulation of FT, TSF, and SOC1.     

The Fragaria vesca Homolog of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 Represses Flowering and Promotes Vegetative Growth

In the annual long-day plant Arabidopsis thaliana, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) integrates endogenous and environmental signals to promote flowering. We analyzed the function and regulation of the SOC1 homolog (Fragaria vesca [Fv] SOC1) in the perennial short-day plant woodland strawberry (Fragaria vesca). We found that Fv SOC1 overexpression represses flower initiation under inductive short days, whereas its silencing causes continuous flowering in both short days and noninductive long days, similar to mutants in the floral repressor Fv TERMINAL FLOWER1 (Fv TFL1). Molecular analysis of these transgenic lines revealed that Fv SOC1 activates Fv TFL1 in the shoot apex, leading to the repression of flowering in strawberry. In parallel, Fv SOC1 regulates the differentiation of axillary buds to runners or axillary leaf rosettes, probably through the activation of gibberellin biosynthetic genes. We also demonstrated that Fv SOC1 is regulated by photoperiod and Fv FLOWERING LOCUS T1, suggesting that it plays a central role in the photoperiodic control of both generative and vegetative growth in strawberry. In conclusion, we propose that Fv SOC1 is a signaling hub that regulates yearly cycles of vegetative and generative development through separate genetic pathways.