Expression and Functional Analysis of the ZCN1(ZmTFL1) Gene, a TERMINAL FLOWER 1 Homologue that Regulates the Vegetative to Reproductive Transition in Maize

TERMINAL FLOWER 1 (TFL1) homologs play critical roles in regulating flowering time and/or maintaining flowering of meristems. In this study, the gene of maize TFL1 ortholog ZmTFL1 (ZCN1) was cloned from both the tropical inbred line CML288 and temperate inbred line Huangzao 4, and the function of ZmTFL1 (ZCN1) was determined during different periods of floral development. Spatial and temporal expression patterns revealed that ZCN1 was predominantly localized in shoot apical meristems that develop into flowers, and only at low levels in leaves. To further identify the role of ZCN1 in floral development of maize, the morphology of shoot apices in maize during floral development was investigated using laser scanning confocal microscopy. Moreover, the relative levels of expression of ZCN1, ZCN8, DLF1, and ZAP1 genes were determined. Over-expression of ZCN1 partially complemented the late flowering phenotype in the tfl1-14 Arabidopsis mutant. Moreover, transgenic Arabidopsis plants exhibited indeterminate inflorescence with increased shoot length and higher numbers of trichomes on leaves. In addition, expression levels of AP1 were significantly down-regulated in 35S::ZCN1 transgenic Arabidopsis plants. These results indicated that ZCN1 as well as its homolog TFL1 in Arabidopsis are involved in the regulation of floral transition in maize.     

BROTHER OF FT AND TFL1 (BFT) has TFL1‐like activity and functions redundantly with TFL1 in inflorescence meristem development in Arabidopsis

The FLOWERING LOCUS T (FT)/TERMINAL FLOWER 1 (TFL1) family is a small gene family that encodes important regulators that control flower development in Arabidopsis. Here, we investigated the biological role of the product of BROTHER OF FT AND TFL1 (BFT), a member of this family, whose function remains unknown. Comparison of the critical residues that play a role in distinguishing FT‐ or TFL1‐like activity revealed that BFT is more similar to FT. Similar to FT expression, BFT expression showed a diurnal oscillation pattern, peaking in the evening. In situ hybridization revealed BFT expression in the shoot apical meristem, young leaf and axillary inflorescence meristem. Transgenic plants over‐expressing BFT exhibited delayed flowering and severe floral defects (floral indeterminacy and compact inflorescences surrounded by serrate leaves), similar to 35S::TFL1 plants. LEAFY (LFY) and APETALA1 (AP1) expression was significantly reduced in 35S::BFT plants. BFT over‐expression failed to rescue the terminal flower phenotype of tfl1 mutants; however, it delayed both terminal flower formation in the primary inflorescence and axillary inflorescence development in the tfl1 mutant background. Consistent with this, the loss‐of‐function BFT alleles, bft‐2 and an BFT RNAi line, accelerated termination of the primary inflorescence and formation of axillary inflorescences in the tfl1 mutant background. Taken together, our results suggest that, despite similarities in the critical residues of BFT and FT, BFT possesses a TFL1‐like activity and functions redundantly with TFL1 in inflorescence meristem development, and possibly contributes to the regulation of plant architecture.     

Characterization and expression analysis of TERMINAL FLOWER1 homologs from cultivated alloteraploid cotton (Gossypium hirsutum) and its diploid progenitors

The seasonal cycle and persistence of a plant is governed by a combination of the determinate or indeterminate status of shoot and root apical meristems. A perennial plant is one in which the apical meristem of at least one of its shoot axes remains indeterminate beyond the first growth season.TERMINAL FLOWER1 (TFL1) genes play important roles in regulating flowering time, the fate of inflorescence meristem and perenniality. To investigate the role of TFL1-like genes in the determination of the apical meristems in an industrially important crop cultivated for its fibers, we isolated and characterized two TFL1 homologs (TFL1a and TFL1b) from tetraploid cultivated cotton (Gossypium hirsutum) and its diploid progenitors (Gossypium arboreum and Gossypium raimondii). All isolated genes maintain the same exon–intron organization. Their phylogenetic analysis at the amino acid level confirmed that the isolated sequences are TFL1-like genes and collocate in the TFL1 clade of the PEBP protein family. Expression analysis revealed that the genes TFL1a and TFL1b have slightly different expression patterns, suggesting different functional roles in the determination of the meristems. Additionally, promoter analysis by computational methods revealed the presence of common binding motifs in TFL1-like promoters. These are the first reported TFL1-like genes isolated from cotton, the most important crop for the textile industry.     

Genetic interactions reveal the antagonistic roles of FT/TSF and TFL1 in the determination of inflorescence meristem identity in Arabidopsis

During the transition to the reproductive phase, the shoot apical meristem switches from the developmental program that generates vegetative organs to instead produce flowers. In this study, we examined the genetic interactions of FLOWERING LOCUS T (FT)/TWIN SISTER OF FT (TSF) and TERMINAL FLOWER 1 (TFL1) in the determination of inflorescence meristem identity in Arabidopsis thaliana. The ft‐10 tsf‐1 mutants produced a compact inflorescence surrounded by serrated leaves (hyper‐vegetative shoot) at the early bolting stage, as did plants overexpressing TFL1. Plants overexpressing FT or TSF (or both FT and TFL1) generated a terminal flower, as did tfl1‐20 mutants. The terminal flower formed in tfl1‐20 mutants converted to a hyper‐vegetative shoot in ft‐10 tsf‐1 mutants. Grafting ft‐10 tsf‐1 or ft‐10 tsf‐1 tfl1‐20 mutant scions to 35S::FT rootstock plants produced a normal inflorescence and a terminal flower in the scion plants, respectively, although both scions showed similar early flowering. Misexpression of FT in the vasculature and in the shoot apex in wild‐type plants generated a normal inflorescence and a terminal flower, respectively. By contrast, in ft‐10 tsf‐1 mutants the vasculature‐specific misexpression of FT converted the hyper‐vegetative shoot to a normal inflorescence, and in the ft‐10 tsf‐1 tfl1‐20 mutants converted the shoot to a terminal flower. TFL1 levels did not affect the inflorescence morphology caused by FT/TSF overexpression at the early bolting stage. Taking these results together, we proposed that FT/TSF and TFL1 play antagonistic roles in the determination of inflorescence meristem identity, and that FT/TSF are more important than TFL1 in this process.     

Early evolution of the MFT-like gene family in plants

Angiosperm genes sharing a conserved phosphatidylethanolamine-binding (PEPB) domain have been shown to be involved in the control of shoot meristem identity and flowering time. The family is divided into three subfamilies, FT-like, TFL1-like and MFT-like. This study is focused on the evolution of the MFT-like clade, suggested to be ancestral to the two other clades. We report that the bryophyte Physcomitrella patens and the lycopod Selaginella moellendorfii contain four and two MFT-like genes respectively. Neither species have any FT or TFL1-like genes. Furthermore, we have identified a new subclade of MFT-like genes in Angiosperms. Quantitative expression analysis of MFT-like genes in Physcomitrella patens reveals that the expression patterns are circadian and reaches maximum in gametangia and sporophytes. Our data suggest that the occurrence FT and TFL1-like genes, is associated with the evolution of seed plants. Expression data for Physcomitrella MFT-like genes implicates an involvement in the development of reproductive tissues in the moss.     

The alleles at the E1 locus impact the expression pattern of two soybean FT-like genes shown to induce flowering in Arabidopsis

A small gene family of phosphatidyl ethanolamine-binding proteins (PEBP) has been shown to function as key regulators in flowering; in Arabidopsis thaliana the FT protein promotes flowering whilst the closely related TFL1 protein represses flowering. Control of flowering time in soybean [Glycine max (L.) Merrill] is important for geographic adaptation and maximizing yield. Soybean breeders have identified a series of loci, the E-genes, that control photoperiod-mediated flowering time, yet how these loci control flowering is poorly understood. The objectives of this study were to evaluate the expression of GmFT-like genes in the E1 near-isogenic line (NIL) background. Of the 20 closely related PEBP proteins in the soybean genome, ten are similar to the Arabidopsis FT protein. Expression analysis of these ten GmFT-like genes confirmed that only two are detectable in the conditions tested. Further analysis of these two genes in the E1 NILs grown under short-day (SD) and long-day (LD) conditions showed a diurnal expression and tissue specificity expression commensurate with soybean flowering time under SD and LD conditions, suggesting that these were good candidates for flowering induction in soybean. Arabidopsis ft mutant lines flowered early when transformed with the two soybean genes, suggesting that the soybean genes can complement the Arabidopsis FT function. Flowering time in E1 NILs is consistent with the differential expression of the two GmFT-like genes under SD and LD conditions, suggesting that the E1 locus, at least in part, impacts time to flowering through the regulation of soybean FT expression.     

Characterization of Chrysanthemum ClSOC1-1 and ClSOC1-2, homologous genes of SOC1

The MADS-box gene SOC1/TM3 (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1/ Tomato MADS-box gene 3) is a main integrator in the Arabidopsis flowering pathway; its structure and function are highly conserved in many plant species. SOC1-like genes have been isolated in chrysanthemum, one of the most well-known ornamental plants, but it has not been well characterized thus far. We isolated and characterized ClSOC1-1 and ClSOC1-2, two putative orthologs of Arabidopsis SOC1, from the wild diploid chrysanthemum, Chrysanthemum lavandulifolium, to investigate the regulatory mechanisms of flowering time control in chrysanthemum. Expression analysis indicated that ClSOC1-1 and ClSOC1-2 were expressed in all examined organs/tissues (leaves, shoot apices, petioles, stems and roots) with different expression levels, and with high expression in the shoot apices and leaves during the early stage of floral transition. The expression levels of ClSOC1-1 and ClSOC1-2 in the shoot apices increased at different developmental stages with the highest expression levels after 7 days of short-day treatment. Overexpression of ClSOC1-1 and ClSOC1-2 in wild-type Arabidopsis resulted in early flowering, which was coupled with the upregulation of one of the flowering promoter genes LEAFY. Our results suggested that the ClSOC1-1 and ClSOC1-2 genes play an evolutionarily conserved role in promoting flowering in Chrysanthemum lavandulifolium and could serve as a vital target for the genetic manipulation of flowering time in the chrysanthemum.     

CONSTANS delays Arabidopsis flowering under short days

Long days (LD) promote flowering of Arabidopsis thaliana compared with short days (SD) by activating the photoperiodic pathway. Here we show that growth under very‐SD (3 h) or darkness (on sucrose) also accelerates flowering on a biological scale, indicating that SD actively repress flowering compared with very‐SD. CONSTANS (CO) repressed flowering under SD, and the early flowering of co under SD required FLOWERING LOCUS T (FT). FT was expressed at a basal level in the leaves under SD, but these levels were not enhanced in co. This indicates that the action of CO in A. thaliana is not the mirror image of the action of its homologue in rice. In the apex, CO enhanced the expression of TERMINAL FLOWER 1 (TFL1) around the time when FT expression is important to promote flowering. Under SD, the tfl1 mutation was epistatic to co and in turn ft was epistatic to tfl1. These observations are consistent with the long‐standing but not demonstrated model where CO can inhibit FT induction of flowering by affecting TFL1 expression.     

Regulation of the <Emphasis Type="Italic">ALBINO3</Emphasis>-mediated transition to flowering in <Emphasis Type="Italic">Arabidopsis</Emphasis> depends on the expression of <Emphasis Type="Italic">CO</Emphasis> and <Emphasis Type="Italic">GA1</Emphasis>

ALBINO3, a homologue of PPF1 in Arabidopsis, encodes a chloroplast protein, and is essential for chloroplast differentiation. In the present study, ALBINO3(−) transgenic plants exhibited a significant decrease in both the number of rosette leaves at bolting and the days before bolting, suggesting the important roles of ALBINO3 in regulating flowering during non-inductive short-day photoperiods. ALBINO3 mRNA was apparently accumulated in shoot apical meristem and floral meristems around the shoot apical meristem in wild-type plants. ALBINO3 might be predominantly involved in inducing the floral repression pathway by activating the expression of TFL1, and by suppressing the expression of LFY, respectively, in the shoot apical meristem. Moreover, the function of ALBINO3 in regulating flowering transition depended on the expression of CO and GA1, because ALBINO3 might function in the downstream integration of the photoperiod-dependent and the photoperiod-independent pathways. These results suggest that ALBINO3 may have an important integrative function in the flowering process in Arabidopsis.     

The <Emphasis Type="Italic">FT/TFL1</Emphasis> gene family in grapevine

The FT/TFL1 gene family encodes proteins with similarity to phosphatidylethanolamine binding proteins which function as flowering promoters and repressors. We show here that the FT/TFL1 gene family in Vitis vinifera is composed of at least five genes. Sequence comparisons with homologous genes identified in other dicot species group them in three major clades, the FT, MFT and TFL1 subfamilies, the latter including three of the Vitis sequences. Gene expression patterns are in agreement with a role of VvFT and VvMFT as flowering promoters; while VvTFL1A, VvTFL1B and VvTFL1C could be associated with vegetative development and maintenance of meristem indetermination. Overexpression of VvFT in transgenic Arabidopsis plants generates early flowering phenotypes similar to those produced by FT supporting a role for this gene in flowering promotion. Overexpression of VvTFL1A does not affect flowering time but the determination of flower meristems, strongly altering inflorescence structure, which is consistent with the biological roles assigned to similar genes in other species.     

EARLY IN SHORT DAYS 7 (ESD7) encodes the catalytic subunit of DNA polymerase epsilon and is required for flowering repression through a mechanism involving epigenetic gene silencing

We have characterized a mutation affecting the Arabidopsis EARLY IN SHORT DAYS 7 (ESD7) gene encoding the catalytic subunit of DNA polymerase epsilon (ε), AtPOL2a. The esd7‐1 mutation causes early flowering independently of photoperiod, shortened inflorescence internodes and altered leaf and root development. esd7‐1 is a hypomorphic allele whereas knockout alleles displayed an embryo‐lethal phenotype. The esd7 early flowering phenotype requires functional FT and SOC1 proteins and might also be related to the misregulation of AG and AG‐like gene expression found in esd7. Genes involved in the modulation of chromatin structural dynamics, such as LHP1/TFL2 and EBS, which negatively regulate FT expression, were found to interact genetically with ESD7. In fact a molecular interaction between the carboxy terminus of ESD7 and TFL2 was demonstrated in vitro. Besides, fas2 mutations suppressed the esd7 early flowering phenotype and ICU2 was found to interact with ESD7. Discrete regions of the chromatin of FT and AG loci were enriched in activating epigenetic marks in the esd7‐1 mutant. We concluded that ESD7 might be participating in processes involved in chromatin‐mediated cellular memory.     

T-DNA integration into genomic DNA of rice following Agrobacterium inoculation of isolated shoot apices

This paper establishes that the isolated shoot meristem of monocotyledons can be infected and transformed using Agrobacterium. Since this explant from nearly any cereal cultivar can rapidly regenerate into a plant, using this explant effectively eliminates the genotype regeneration restrictions to cereal crop transformation allowing direct transformation of elite germplasm. Shoot apices of Oryza sativa L. Tropical Japonica, cv. Maybelle were explants used for cocultivation, and gene transfer was accomplished using Agrobacterium containing plasmids for the bar gene expression driven by the CaMV 35S promoter or by the rice actin 1 promoter. Experiments to determine the survival rates of isolated shoot apices on media containing the herbicide, glufosinate-ammonium (PPT), established that no shoot apices survived on 0.5 or 1.0 mg/l PPT. After shoot apices were cocultivated with Agrobacterium, 2.8% (overall 20 out of 721 shoot apices) survived on 0.5 mg/l PPT. Results demonstrated that the use of the actin 1 promoter-based expression vector and an extra-wounding treatment of the meristematic cells appeared to be most effective in promoting transformation. Integration, expression and transmission of the transferred foreign genes in primary, R1 and R2 generation plants were confirmed by molecular analyses and herbicide application tests. A germination test of R2 progeny from one of the transgenic plants (R1) established a phenotype segregation ratio showing a non-Mendelian inheritance pattern. Inactivation of the transferred foreign gene in R2 progeny appeared to result from transgene methylation.     

Populus CEN/TFL1 regulates first onset of flowering,axillary meristem identity and dormancy release in Populus

Members of the CENTRORADIALIS (CEN)/TERMINAL FLOWER 1 (TFL1) subfamily control shoot meristem identity, and loss‐of‐function mutations in both monopodial and sympodial herbaceous plants result in dramatic changes in plant architecture. We studied the degree of conservation between herbaceous and woody perennial plants in shoot system regulation by overexpression and RNA interference (RNAi)‐mediated suppression of poplar orthologs of CEN, and the related gene MOTHER OF FT AND TFL 1 (MFT). Field study of transgenic poplars (Populus spp.) for over 6 years showed that downregulation of PopCEN1 and its close paralog, PopCEN2, accelerated the onset of mature tree characteristics, including age of first flowering, number of inflorescences and proportion of short shoots. Surprisingly, terminal vegetative meristems remained indeterminate in PopCEN1‐RNAi trees, suggesting the possibility that florigen signals are transported to axillary mersitems rather than the shoot apex. However, the axillary inflorescences (catkins) of PopCEN1‐RNAi trees contained fewer flowers than did wild‐type catkins, suggesting a possible role in maintaining the indeterminacy of the inflorescence apex. Expression of PopCEN1 was significantly correlated with delayed spring bud flush in multiple years, and in controlled environment experiments, 35S::PopCEN1 and RNAi transgenics required different chilling times to release dormancy. Considered together, these results indicate that PopCEN1/PopCEN2 help to integrate shoot developmental transitions that recur during each seasonal cycle with the age‐related changes that occur over years of growth.     

Flowering Response in Relation to C and N Contents of Pharbitis nil Plants Cultured in Nitrogen-Poor Media

Flowering of seedlings of Pharbitis nil, strains Violet andTendan, cultured in modified White's medium, was promoted bymedium dilution, the critical dark period being shortened byabout 15 min. Dilution of the N source alone was enough to causethe medium-dilution effect. Dilution of the culture medium duringthe day before and on the day of exposure to the dark-period(a total of two days) caused the maximum dilution effect. TheC and N contents of the cotyledons and of the shoot apices changedrapidly in response to medium dilution. In 1/2-strength White'smedium with 1/1,000 strength NO₃^– which was most effectivefor flower promotion, the C-N ratio was highest. In 1/2-strengthmodified White's medium, in which flowering was lowest withthe longest critical dark period, the C-N ratio was lowest.Thus, there is a close relation between flowering response andthe C-N ratio in cotyledons or shoot apices of Pharbitis nil. (Received September 14, 1984; Accepted January 26, 1985)     

The gentian orthologs of the FT/TFL1 gene family control floral initiation in Gentiana

Gentians are herbaceous perennials blooming in summer through autumn. Although they are popular ornamental flowers in Japan, the regulation of their timing of flowering has not been studied. We identified and characterized gentian orthologs of the Arabidopsis FT/TFL1 gene family to elucidate the mechanisms of flowering initiation. We isolated three gentian orthologs of FT and TFL1, denoted GtFT1, GtFT2 and GtTFL1. Since up-regulation of GtFT1 and GtFT2 as well as down-regulation of GtTFL1 promoted floral initiation in gentian plantlets, these genes affected floral initiation in a similar way to Arabidopsis FT and TFL1. The expression levels of GtFT1 and GtFT2 in leaves of late-flowering gentian increased prior to floral initiation, whereas GtTFL1 was highly expressed in shoot apical meristem at the vegetative stage and decreased drastically just before flowering initiation. Comparison of gene expression patterns showed that GtFT1 expression increased earlier in early-flowering than in late-flowering gentian, whereas the timing of the increase in GtFT2 expression was similar in early- and late-flowering plants. The GtTFL1 expression in early-flowering gentian was extremely low throughout the vegetative and reproductive stages. These results indicated that either the up-regulation of GtFT1 or the down-regulation of GtTFL1 may determine flowering time. Furthermore, we found that early-flowering but not late-flowering gentians have a 320 bp insertion in the promoter region of GtTFL1. Thus, the negligible expression of GtTFL1 in early-flowering lines may be due to this insertion, resulting in a shortened vegetative stage.