Flowering of strict photoperiodic <Emphasis Type="Italic">Nicotiana</Emphasis> varieties in non-inductive conditions by transgenic approaches

The genus Nicotiana contains species and varieties that respond differently to photoperiod for flowering time control as day-neutral, short-day and long-day plants. In classical photoperiodism studies, these varieties have been widely used to analyse the physiological nature for floral induction by day length. Since key regulators for flowering time control by day length have been identified in Arabidopsis thaliana by molecular genetic studies, it was intriguing to analyse how closely related plants in the Nicotiana genus with opposite photoperiodic requirements respond to certain flowering time regulators. SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and FRUITFULL (FUL) are two MADS box genes that are involved in the regulation of flowering time in Arabidopsis. SOC1 is a central flowering time pathway integrator, whereas the exact role of FUL for floral induction has not been established yet. The putative Nicotiana orthologs of SOC1 and FUL, NtSOC1 and NtFUL, were studied in day-neutral tobacco Nicotiana tabacum cv Hicks, in short-day tobacco N. tabacum cv Hicks Maryland Mammoth (MM) and long-day N. sylvestris plants. Both genes were similarly expressed under short- and long-day conditions in day-neutral and short-day tobaccos, but showed a different expression pattern in N. sylvestris. Overexpression of NtSOC1 and NtFUL caused flowering either in strict short-day (NtSOC1) or long-day (NtFUL) Nicotiana varieties under non-inductive photoperiods, indicating that these genes might be limiting for floral induction under non-inductive conditions in different Nicotiana varieties.     

利用RNA干扰技术获得晚抽薹开花转基因大白菜

LEAFY基因在植物的成花转变中起重要作用。在拟南芥中,降低LEAFY基因的表达使开花延迟,并且增加分枝。利用RNAi技术降低大白菜LEAFY基因表达,获得晚抽薹开花的转基因大白菜。开花时转基因大白菜的株高明显低于对照,而叶片数、叶面积和侧枝数则高于对照;转基因植株的LEAFY基因表达明显降低,导致大白菜抽薹开花延迟。因此可以通过降低LEAFY基因表达来延迟需低温春化作用长日植物开花。也为利用生物技术获得晚抽薹转基因大白菜提供了理论依据。     

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.     

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.     

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.     

Integration of photoperiod and cold temperature signals into flowering genetic pathways in Arabidopsis

Appropriate timing of flowering is critical for propagation and reproductive success in plants. Therefore, flowering time is coordinately regulated by endogenous developmental programs and external signals, such as changes in photoperiod and temperature. Flowering is delayed by a transient shift to cold temperatures that frequently occurs during early spring in the temperate zones. It is known that the delayed flowering by short-term cold stress is mediated primarily by the floral repressor FLOWERING LOCUS C (FLC). However, how the FLC-mediated cold signals are integrated into flowering genetic pathways is not fully understood. We have recently reported that the INDUCER OF CBF EXPRESSION 1 (ICE1), which is a master regulator of cold responses, FLC, and the floral integrator SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) constitute an elaborated feedforward-feedback loop that integrates photoperiod and cold temperature signals to regulate seasonal flowering in Arabidopsis. Cold temperatures promote the binding of ICE1 to FLC promoter to induce its expression, resulting in delayed flowering. However, under floral inductive conditions, SOC1 induces flowering by blocking the ICE1 activity. We propose that the ICE1-FLC-SOC1 signaling network fine-tunes the timing of photoperiodic flowering during changing seasons.     

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.     

CONSTANS activates SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 through FLOWERING LOCUS T to promote flowering in Arabidopsis

CONSTANS (CO) regulates flowering time by positively regulating expression of two floral integrators, FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1), in Arabidopsis (Arabidopsis thaliana). FT and SOC1 have been proposed to act in parallel pathways downstream of CO based on genetic analysis using weak ft alleles, since ft soc1 double mutants showed an additive effect in suppressing the early flowering of CO overexpressor plants. However, this genetic analysis was inconsistent with the sequential induction pattern of FT and SOC1 found in inducible CO overexpressor plants. Hence, to identify genetic interactions of CO, FT, and SOC1, we carried out genetic and expression analyses with a newly isolated T-DNA allele of FT, ft-10. We found that ft-10 almost completely suppressed the early flowering phenotype of CO overexpressor plants, whereas soc1-2 partially suppressed the phenotype, suggesting that FT is the major output of CO. Expression of SOC1 was altered in gain- or loss-of-function mutants of FT, whereas expression of FT remained unchanged in gain- or loss-of-function mutants of SOC1, suggesting that FT positively regulates SOC1 to promote flowering. In addition, inactivation of FT caused down-regulation of SOC1 even in plants overexpressing CO, indicating that FT is required for SOC1 induction by CO. Taken together, these data suggest that CO activates SOC1 through FT to promote flowering in Arabidopsis.     

Over-expression of BcFLC1 from non-heading Chinese cabbage enhances cold tolerance in Arabidopsis

A gene (named BcFLC1) homologous to the AtFLC gene, which encodes a floral repressor, was isolated from the nonheading Chinese cabbage (Brassica campestris L. ssp. chinensis) cv. NJ074. The gene showed high similarity to AtFLC. For studying the gene function, we designed to introduce the BcFLC1 gene into Arabidopsis thaliana. The results showed that BcFLC1 had effects on flowering time similar to AtFLC. We also found that Arabidopsis cold-tolerance was enhanced by BcFLC1 overexpression. Under low temperature stress, the BcFLC1 transgenic plants exhibited stronger growth than wild-type plants. The elevated cold tolerance of the BcFLC1 over-expressing plants was also confirmed by the changes of electrolyte leakage and malonyldialdehyde and proline content.     

Regulation of flowering time by Arabidopsis MSI1

The transition to flowering is tightly controlled by endogenous programs and environmental signals. We found that MSI1 is a novel flowering-time gene in Arabidopsis. Both partially complemented msi1 mutants and MSI1 antisense plants were late flowering, whereas ectopic expression of MSI1 accelerated flowering. Physiological experiments revealed that MSI1 is similar to genes from the autonomous promotion of flowering pathway. Expression of most known flowering-time genes did not depend on MSI1, but the induction of SOC1 was delayed in partially complemented msi1 mutants. Delayed activation of SOC1 is often caused by increased expression of the floral repressor FLC. However, MSI1 function is independent of FLC. MSI1 is needed to establish epigenetic H3K4 di-methylation and H3K9 acetylation marks in SOC1 chromatin. The presence of these modifications correlates with the high levels of SOC1 expression that induce flowering in Arabidopsis. Together, the control of flowering time depends on epigenetic mechanisms for the correct expression of not only the floral repressor FLC, but also the floral activator SOC1.     

Cytokinin promotes flowering of Arabidopsis via transcriptional activation of the FT paralogue TSF

Cytokinins are involved in many aspects of plant growth and development, and physiological evidence also indicates that they have a role in floral transition. In order to integrate these phytohormones into the current knowledge of genetically defined molecular pathways to flowering, we performed exogenous treatments of adult wild type and mutant Arabidopsis plants, and analysed the expression of candidate genes. We used a hydroponic system that enables synchronous growth and flowering of Arabidopsis, and allows the precise application of chemicals to the roots for defined periods of time. We show that the application of N⁶‐benzylaminopurine (BAP) promotes flowering of plants grown in non‐inductive short days. The response to cytokinin treatment does not require FLOWERING LOCUS T (FT), but activates its paralogue TWIN SISTER OF FT (TSF), as well as FD, which encodes a partner protein of TSF, and the downstream gene SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1). Treatment of selected mutants confirmed that TSF and SOC1 are necessary for the flowering response to BAP, whereas the activation cascade might partially act independently of FD. These experiments provide a mechanistic basis for the role of cytokinins in flowering, and demonstrate that the redundant genes FT and TSF are differently regulated by distinct floral‐inducing signals.     

Effects of sugar on vegetative development and floral transition in Arabidopsis

Although sugar has been suggested to promote floral transition in many plant species, growth on high concentrations (5% [w/v]) of sucrose (Suc) significantly delayed flowering time, causing an increase in the number of leaves at the time of flowering in Arabidopsis. The effect of high concentrations of Suc seemed to be metabolic rather than osmotic. The delay of floral transition was due to extension of the late vegetative phase, which resulted in a delayed activation of LFY expression. In addition, growth on low concentrations (1% [w/v]) of Suc slightly inhibited flowering in wild-type plants. This delay resulted from effects on the early vegetative phase. This inhibition was more pronounced in tfl1, an early flowering mutant, than in the wild type. Although 1% (w/v) Suc was reported to promote floral transition of late-flowering mutants such as co, fca, and gi, floral transition in these mutants was delayed by a further increase in Suc concentration. These results suggest that sugar may affect floral transition by activating or inhibiting genes that act to control floral transition, depending on the concentration of sugars, the genetic background of the plants, and when the sugar is introduced. Growth on 1% (w/v) Suc did not restore the reduced expression levels of FT and SOC1/AGL20 in co or fca mutants. Rather, expression of FT and SOC1/AGL20 was repressed by 1% (w/v) Suc in wild-type background. The possible effects of sugar on gene expression to promote floral transition are discussed.