Flowering in darkness in Arabidopsis thaliana

A modified method for studying the initiation of flowering in darkness (dark flowering, DF) in Arabidopsis thaliana has been developed, and the DF process has been examined with the aid of late-flowering mutants. A majority of plants developed floral buds by the use of liquid-shaken cultures in darkness. The late-flowering phenotype in gi and co mutants and early-flowering phenotype in a hy2 mutant disappeared in DF. It was found that wild-type plants grown under DF conditions express light-regulated genes and develop appropriate leaf architecture, as do the light-grown plants, without the apparent differentiation of chloroplasts. The shift experiments from darkness to light revealed the critical duration of growth in darkness for the initiation of DF. These results indicate that the DF process to the initiation of flowering is a mode of development distinct from that in light in Arabidopsis .     

Flowering of Arabidopsis cop1 mutants in darkness

To elucidate the role of the COP1 gene in flowering, we analyzed flowering of cop1 mutant lines in darkness. When grown in the presence of 1% (w/v) sucrose, the cop1-6 mutant flowered in darkness, but cop1-1 and cop1-4 did not. However, cop1-1 and cop1-4 flowered in darkness when grown in the presence of 5% (w/v) sucrose. Therefore, the COP1 gene represses not only photomorphogenesis in seedlings but also flowering in darkness. Comparison of mRNAs levels of floral identity genes in cop1-6 and wild-type plants grown in darkness revealed increased mRNA levels of genes that act downstream of CO and reduced FLC mRNA level in cop1-6. Double mutants of cop1-6 and each of the late-flowering mutations cry2-1, gi-2, co-1, and ld-1 flowered in darkness. All of the double mutants except cry2-1 cop1-6 flowered later than cop1-6, demonstrating that cop1-6 is epistatic to cry2-1 for early flowering. The ld-1 cop1-6 double mutant flowered much earlier than the ld-1 mutant. The delay in flowering in the double mutants was not strongly influenced by the light conditions, whereas that of the gi-2 cop1-6 double mutant was reduced in darkness.     

Effects of sucrose supply on growth and paraquat tolerance of the late-flowering gi-3 mutant

Mutations at the GI locus in Arabidopsis are pleiotropic: gi mutants are late-flowering, tolerant to the toxicity of the herbicide paraquat, and have an increased starch content. We tested the effects of exogenous sucrose supply on the level of paraquat tolerance and on growth and development of the gi-3 mutant. Paraquat tolerance was the highest in gi-3 seedlings grown on medium containing 1% sucrose. As expected, all measured growth parameters (root length, fresh weight, anthocyanin, and sucrose content) were influenced by the sucrose dose, but in a number of assays (effect on fresh weight and developmental characteristics) the sucrose-dependent response in gi-3 was heterochronic. Additionally, the late-flowering phenotype of the gi-3 mutant was reverted to wild type after prolonged growth in darkness on sucrose-containing media.     

The effect of daylength on the transition to flowering in phytochrome-deficient, late-flowering and double mutants of Arabidopsis thaliana

The effect of daylength on flowering was investigated in the following mutants of Arabidopsis thaliana : phytochrome B deficient ( hy3=phyB ); phytochrome chromophore deficient ( hy2 ); late-flowering ( co, gi. fca and fwa ); the hy2 and hy3 , late-flowering double mutants and the hy2, hy3 , late-flowering triple mutants. The hy mutants flower with fewer rosette leaves than the Landsberg erecta wild type under both long day and short day conditions and express this effect to a different degree in all late-flowering mutant backgrounds and under both daylengths, with the exception of fca under short days. The number of cauline leaves and days to flowering is less affected by the hy genotype. The hy2, hy3 double mutants flower with even fewer rosette leaves than the hy2 and hy3 monogenic mutants, suggesting an inhibitory role for phytochrome B and other stable phytochromes on flowering. The complex interaction between phytochrome, daylength and the effect of the late-flowering genes on the various parameters that describe the transition to flowering in Arabidopsis is discussed.     

春化作用相关基因FLC的研究进展

拟南芥春化作用相关基因FLOWERING LOCUS C（FLC）属于MADS盒基因,它编码的蛋白转录因子对开花具抑制作用。春化作用通过负调控FLC的转录及蛋白表达水平,促进拟南芥的某些晚花生态型和晚花突变体开花。主要介绍了FLC基因在春化途径中的关键作用,及其春化作用通过FLC基因与其它开花途径相联系等内容。     

The fission yeast mitotic activator cdc25 and sucrose induce early flowering synergistically in the day-neutral Nicotiana tabacum cv. Samsun

Here, the tobacco (Nicotiana tabacum) day-neutral (DN) cv. Samsun transformed with the Schizosaccharomyces pombe mitotic activator gene Spcdc25 was used to study the onset of flowering. Wild type (WT) and cdc25 plants were grown from seeds in vitro until they were 20 cm high. Apical and basal nodes were then subcultured repeatedly and the regenerated plants were used to document time to flowering and the number of leaves formed before flowering. Three sucrose treatments (3, 5 or 7% (weight/volume)) were used and measurements of leaf endogenous soluble carbohydrates were performed. In the 3% treatment, cdc25 plants flowered but WT plants did not. The higher sucrose treatments enabled WT flowering; two-thirds of the plants flowered at 5%, while all plants flowered at 7% sucrose. However, in all treatments, cdc25 plants exhibited significantly earlier flowering and fewer leaves compared with wild type. Remarkably, a typical acropetal flowering gradient in WT plants did not occur in cdc25 plants. In cdc25 leaves, there were significantly higher amounts of endogenous sugars with a higher proportion of sucrose compared with WT. Our data demonstrate that Spcdc25 expression and sucrose act synergistically to induce precocious flowering.     

Late-flowering genes interact with early-flowering genes to regulate flowering time in Arabidopsis thaliana.

To investigate the genetic mechanisms regulating the transition from the vegetative to reproductive growth in Arabidopsis, double mutants between three different early-flowering mutants, early flowering 1-1, 2-1, 3-1, (elf 1-1, 2-1, 3-1) and five different late-flowering mutants, gi-1, ft-1, fwa-1, ld-1, and fca-9, were constructed and phenotypes analyzed. Double mutants in all combinations displayed the late-flowering phenotypes which resembled their respective late-flowering parents in both flowering time and the number of vegetative leaves produced. The results indicate that five late-flowering mutants are epistatic to all three early-flowering mutants tested here. This epistatic relationship suggests that ELF1, ELF2, and ELF3 genes function upstream of these five late-flowering genes no matter if they are functioning in autonomous or photoperiod pathways. These three early-flowering genes may negatively modify the activity of most late-flowering genes to influence the time of the vegetative-to-reproductive transition in Arabidopsis.     

高羊茅FaCONSTANS基因的表达及功能分析

植物由营养生长向生殖生长转变过程中光周期调控起着重要的作用。CONSTANS (CO) 是光周期途径中的特有基因,为探讨高羊茅FaCONSTANS (FaCO) 基因响应日照长短从而启动植物开花的机理,利用实时荧光定量qRT-PCR技术分析在长日照、短日照、持续光照、持续黑暗条件下FaCO基因的表达水平。构建过表达载体p1300-FaCO,利用农杆菌介导法遗传转化拟南芥,构建沉默载体p1300-FaCO-RNAi遗传转化高羊茅。结果表明,FaCO基因的表达受光周期调控,与生物钟控制的昼夜节律相关。在长日照条件下FaCO基因促进拟南芥开花,且恢复拟南芥突变体开花表型。RNAi沉默FaCO基因的高羊茅转基因植株晚花或者一直处于营养生长阶段。本研究初步探究高羊茅FaCO基因对开花过程的调控,这将有助于更进一步了解该基因的生物学功能。     

Epistatic natural allelic variation reveals a function of AGAMOUS-LIKE6 in axillary bud formation in Arabidopsis

In the Arabidopsis multiparent recombinant inbred line mapping population, a limited number of plants were detected that lacked axillary buds in most of the axils of the cauline (stem) leaves, but formed such buds in almost all rosette axils. Genetic analysis showed that polymorphisms in at least three loci together constitute this phenotype, which only occurs in late-flowering plants. Early flowering is epistatic to two of these loci, called REDUCED SHOOT BRANCHING1 (RSB1) and RSB2, which themselves do not affect flowering time. Map-based cloning and confirmation by transformation with genes from the region where RSB1 was identified by fine-mapping showed that a specific allele of AGAMOUS-Like6 from accession C24 conferred reduced branching in the cauline leaves. Site-directed mutagenesis in the Columbia allele revealed the causal amino acid substitution, which behaved as dominant negative, as was concluded from a loss-of-function mutation that showed the same phenotype in the late-flowering genetic background. This causal allele occurs at a frequency of 15% in the resequenced Arabidopsis thaliana accessions and correlated with reduced stem branching only in late-flowering accessions. The data show the importance of natural variation and epistatic interactions in revealing gene function.     

FLOWERING LOCUS C encodes a novel MADS domain protein that acts as a repressor of flowering.

Winter-annual ecotypes of Arabidopsis are relatively late flowering, unless the flowering of these ecotypes is promoted by exposure to cold (vernalization). This vernalization-suppressible, late-flowering phenotype results from the presence of dominant, late-flowering alleles at two loci, FRIGIDA (FRI) and FLOWERING LOCUS C (FLC). In this study, we report that flc null mutations result in early flowering, demonstrating that the role of active FLC alleles is to repress flowering. FLC was isolated by positional cloning and found to encode a novel MADS domain protein. The levels of FLC mRNA are regulated positively by FRI and negatively by LUMINIDEPENDENS. FLC is also negatively regulated by vernalization. Overexpression of FLC from a heterologous promoter is sufficient to delay flowering in the absence of an active FRI allele. We propose that the level of FLC activity acts through a rheostat-like mechanism to control flowering time in Arabidopsis and that modulation of FLC expression is a component of the vernalization response.     

Monogenic Recessive Mutations Causing Both Late Floral Initiation and Excess Starch Accumulation in Arabidopsis

A recessive Arabidopsis mutation, carbohydrate accumulation mutant1 (cam1), which maps to position 22.8 on chromosome 3, was identified by screening leaves of ethyl methanesulfonate-mutagenized M2 plants stained with iodine for altered starch content. Increased starch content in leaves of the cam1 mutant was observed at the onset of flowering. This mutant also had a delayed floral initiation phenotype with more rosette leaves than the parental line. In addition, activities of several enzymes associated with starch metabolism were altered in the cam1 mutant. The late-flowering mutant gigantea (gi) also manifested an elevated starch level in leaves. However, not all late-flowering mutants had increased leaf starch content. Double mutants cam1 adg1 (for ADP-glucose pyrophosphorylase), cam1 pgm (for phosphoglucomutase), and gi pgm had no observable starch in leaves but showed the late-flowering phenotype, demonstrating that the elevated starch content is not the cause of late floral initiation. The pleiotropic effects of cam1 and gi suggest that they may play regulatory roles in starch metabolism and floral initiation. These data suggest that starch accumulation and floral initiation may share a common regulatory pathway.     

Effect of Light Quality and Vernalization on Late-Flowering Mutants of Arabidopsis thaliana

We have analyzed the response to vernalization and light quality of six classes of late-flowering mutants (fb, fca, fe, fg, ft, and fy) previously isolated following mutagenesis of the early Landsberg race of Arabidopsis thaliana (L.) Heynh. When grown in continuous fluorescent illumination, four mutants (fca, fe, ft, and fy) and the Landsberg wild type exhibited a reduction in both flowering time and leaf number following 6 weeks of vernalization. A significant decrease in flowering time was also observed for all the mutants and the wild type when constant fluorescent illumination was supplemented with irradiation enriched in the red and far red regions of the spectrum. In the most extreme case, the late-flowering phenotype of the fca mutant was completely suppressed by vernalization, suggesting that this mutation has a direct effect on flowering. The fe and fy mutants also showed a more pronounced response than wild type to both vernalization and incandescent supplementation. The ft mutant showed a similar response to that of the wild type. The fb and fg mutants were substantially less sensitive to these treatments. These results are interpreted in the context of a multifactorial pathway for induction of flowering, in which the various mutations affect different steps of the pathway.     

Level of glutathione is regulated by ATP-dependent ligation of glutamate and cysteine through photosynthesis in Arabidopsis thaliana: mechanism of strong interaction of light intensity with flowering

Glutathione (GSH) is associated with flowering in Arabidopsis thaliana, but how GSH biosynthesis is regulated to control the transition to flowering remains to be elucidated. Since the key reaction of GSH synthesis is catalyzed by gamma-glutamylcysteine synthetase (gamma-ECS) and all the gamma-ECS cDNAs examined contained extra sequences for plastid targeting, we investigated the relationships among GSH levels, photosynthesis and flowering. The GSH level in Arabidopsis increased with the light intensity. The ch1 mutants defective in a light-harvesting antenna in photosystem II showed reduced GSH levels with accumulation of the GSH precursor cysteine, and introduction of the gamma-ECS gene GSH1 under the control of the cauliflower mosaic virus 35S promoter (35S-GSH1) into the ch1 mutant altered the GSH level in response to the gamma-ECS mRNA level. These indicate that photosynthesis limits the gamma-ECS reaction to regulate GSH biosynthesis. Like the glutathione-biosynthesis-defective cad2-1 mutant, the ch1 mutants flowered late under weak-light conditions, and this late-flowering phenotype was rescued by supplementation of GSH. Introduction of the 35S-GSH1 construct into the ch1 mutant altered flowering in response to the gamma-ECS mRNA and GSH levels. These findings indicate that flowering in A. thaliana is regulated by the gamma-ECS reaction of GSH synthesis that is coupled with photosynthesis.     

Differential expression of genes important for adaptation in Capsella bursa-pastoris (Brassicaceae)

Understanding the genetic basis of natural variation is of primary interest for evolutionary studies of adaptation. In Capsella bursa-pastoris, a close relative of Arabidopsis (Arabidopsis thaliana), variation in flowering time is correlated with latitude, suggestive of an adaptation to photoperiod. To identify pathways regulating natural flowering time variation in C. bursa-pastoris, we have studied gene expression differences between two pairs of early- and late-flowering C. bursa-pastoris accessions and compared their response to vernalization. Using Arabidopsis microarrays, we found a large number of significant differences in gene expression between flowering ecotypes. The key flowering time gene FLOWERING LOCUS C (FLC) was not differentially expressed prior to vernalization. This result is in contrast to those in Arabidopsis, where most natural flowering time variation acts through FLC. However, the gibberellin and photoperiodic flowering pathways were significantly enriched for gene expression differences between early- and late-flowering C. bursa-pastoris. Gibberellin biosynthesis genes were down-regulated in late-flowering accessions, whereas circadian core genes in the photoperiodic pathway were differentially expressed between early- and late-flowering accessions. Detailed time-series experiments clearly demonstrated that the diurnal rhythm of CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) and TIMING OF CAB EXPRESSION1 (TOC1) expression differed between flowering ecotypes, both under constant light and long-day conditions. Differential expression of flowering time genes was biologically validated in an independent pair of flowering ecotypes, suggesting a shared genetic basis or parallel evolution of similar regulatory differences. We conclude that genes involved in regulation of the circadian clock, such as CCA1 and TOC1, are strong candidates for the evolution of adaptive flowering time variation in C. bursa-pastoris.     

HISTONE DEACETYLASE6 interacts with FLOWERING LOCUS D and regulates flowering in Arabidopsis

Histone acetylation and deacetylation play an important role in epigenetic controls of gene expression. HISTONE DEACETYLASE6 (HDA6) is a REDUCED POTASSIUM DEPENDENCY3-type histone deacetylase, and the Arabidopsis (Arabidopsis thaliana) hda6 mutant axe1-5 displayed a late-flowering phenotype. axe1-5/flc-3 double mutants flowered earlier than axe1-5 plants, indicating that the late-flowering phenotype of axe1-5 was FLOWERING LOCUS C (FLC) dependent. Bimolecular fluorescence complementation, in vitro pull-down, and coimmunoprecipitation assays revealed the protein-protein interaction between HDA6 and the histone demethylase FLD. It was found that the SWIRM domain in the amino-terminal region of FLD and the carboxyl-terminal region of HDA6 are responsible for the interaction between these two proteins. Increased levels of histone H3 acetylation and H3K4 trimethylation at FLC, MAF4, and MAF5 were found in both axe1-5 and fld-6 plants, suggesting functional interplay between histone deacetylase and demethylase in flowering control. These results support a scenario in which histone deacetylation and demethylation cross talk are mediated by physical association between HDA6 and FLD. Chromatin immunoprecipitation analysis indicated that HDA6 bound to the chromatin of several potential target genes, including FLC and MAF4. Genome-wide gene expression analysis revealed that, in addition to genes related to flowering, genes involved in gene silencing and stress response were also affected in hda6 mutants, revealing multiple functions of HDA6. Furthermore, a subset of transposons was up-regulated and displayed increased histone hyperacetylation, suggesting that HDA6 can also regulate transposons through deacetylating histone.     

The Medicago FLOWERING LOCUS T homolog, MtFTa1, is a key regulator of flowering time

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.     

Light Quality and Vernalization Interact in Controlling Late Flowering in Arabidopsis Ecotypes and Mutants

The late flowering ecotypes of Arabidopsis thaliana L. (Heyn.)Eifel, Pitztal and Innsbruck responded to 10 d vernalization(cold treatment) by flowering earlier with less with less thanhalf the number of leaves of non-induced plants. The vernalizationresponse was cumulative: increased numbers of days of vernalizationinduced earlier flowering up to an apparent saturation in responseafter 30 to 40 d. The ratio of red:far-red (R:FR) light alsoaffected non-vernalized time-to-flower. When grown under fluorescentplus incandescent lamps (R:FR = 1·0), time-to-flowerwas approximately half that required by plants grown under fluorescentlamps (R:FR = 5·8) at the same photon flux density andphotoperiod. Leaf production rate was unaffected by either vernalizationor light quality changes and time-to-flower and leaf numberwere highly correlated (r² = 0·973). The late flowering mutants of Landsberg erecta were grown underlighting which displayed a gradient of R:FR. Some mutants likeco, flowered at the same time in all R:FR treatment, while otherlike fca took nearly twice as long to flower, with double thenumber of leaves at R:FR ratio of 5·8 compared with theR:FR = 1 treatment. The ranking of the response from least tomost responsive was co, fe, gi, WT, fd, fwa, ft, fha, fpa, fy,fve and fca. Vernalization of these Landsberg mutants always resulted inearlier flowering, although only fca, fve, fy and fpa were significantlymore sensitive to thermoinduction than the wild type parent.There was a high correlation (r² = 0·89 between the responseto thermoinduction and to R:FR ratio. Vernalization of fca for24 d largely eliminated the R:FR time-to-flower response. Vernalizationand photoinduction similarly affect late flowering and can substitutefor each another.Copyright 1993, 1999 Academic Press Light quality, vernalization, flowering, Arabidopsis thaliana, phytochrome, thermoinduction, photoperiod, photoinduction, growth conditions, photon flux density, daylength, spectral quality, far-red light     

The FLF MADS box gene: a repressor of flowering in Arabidopsis regulated by vernalization and methylation

A MADS box gene, FLF (for FLOWERING LOCUS F ), isolated from a late-flowering, T-DNA-tagged Arabidopsis mutant, is a semidominant gene encoding a repressor of flowering. The FLF gene appears to integrate the vernalization-dependent and autonomous flowering pathways because its expression is regulated by genes in both pathways. The level of FLF mRNA is downregulated by vernalization and by a decrease in genomic DNA methylation, which is consistent with our previous suggestion that vernalization acts to induce flowering through changes in gene activity that are mediated through a reduction in DNA methylation. The flf-1 mutant requires a greater than normal amount of an exogenous gibberellin (GA3) to decrease flowering time compared with the wild type or with vernalization-responsive late-flowering mutants, suggesting that the FLF gene product may block the promotion of flowering by GAs. FLF maps to a region on chromosome 5 near the FLOWERING LOCUS C gene, which is a semidominant repressor of flowering in late-flowering ecotypes of Arabidopsis.