FT Homolog Research Articles

Involvement of CBF in the fine-tuning of litchi flowering time and cold and drought stresses.

Litchi (Litchi chinensis) is an economically important fruit tree in southern China and is widely cultivated in subtropical regions. However, irregular flowering attributed to inadequate floral induction leads to a seriously fluctuating bearing. Litchi floral initiation is largely determined by cold temperatures, whereas the underlying molecular mechanisms have yet to be identified. In this study, we identified four CRT/DRE BINDING FACTORS (CBF) homologs in litchi, of which LcCBF1, LcCBF2 and LcCBF3 showed a decrease in response to the floral inductive cold. A similar expression pattern was observed for the MOTHER OF FT AND TFL1 homolog (LcMFT) in litchi. Furthermore, both LcCBF2 and LcCBF3 were found to bind to the promoter of LcMFT to activate its expression, as indicated by the analysis of yeast-one-hybrid (Y1H), electrophoretic mobility shift assays (EMSA), and dual luciferase complementation assays. Ectopic overexpression of LcCBF2 and LcCBF3 in Arabidopsis caused delayed flowering and increased freezing and drought tolerance, whereas overexpression of LcMFT in Arabidopsis had no significant effect on flowering time. Taken together, we identified LcCBF2 and LcCBF3 as upstream activators of LcMFT and proposed the contribution of the cold-responsive CBF to the fine-tuning of flowering time.

Impacts of RNA Mobility Signals on Virus Induced Somatic and Germline Gene Editing.

Viral vectors are being engineered to deliver CRISPR/Cas9 components systemically in plants to induce somatic or heritable site-specific mutations. It is hypothesized that RNA mobility signals facilitate entry of viruses or single guide RNAs (sgRNAs) into the shoot apical meristem where germline mutations can occur. Our objective was to understand the impact of RNA mobility signals on virus-induced somatic and germline gene editing in Nicotiana benthamiana and Zea mays. Previously, we showed that foxtail mosaic virus (FoMV) expressing sgRNA induced somatic mutations in N. benthamiana and Z. mays expressing Cas9. Here, we fused RNA mobility signals to sgRNAs targeting the genes encoding either N. benthamiana phytoene desaturase (PDS) or Z. mays high affinity potassium transporter 1 (HKT1). Addition of Arabidopsis thaliana Flowering Locus T (AtFT) and A. thaliana tRNA-Isoleucine (AttRNAIle) did not improve FoMV-induced somatic editing, and neither were sufficient to facilitate germline mutations in N. benthamiana. Maize FT homologs, Centroradialus 16 (ZCN16) and ZCN19, as well as AttRNAIle were found to aid somatic editing in maize but did not enable sgRNAs delivered by FoMV to induce germline mutations. Additional viral guide RNA delivery systems were assessed for somatic and germline mutations in N. benthamiana with the intention of gaining a better understanding of the specificity of mobile signal-facilitated germline editing. Potato virus X (PVX), barley stripe mosaic virus (BSMV), and tobacco rattle virus (TRV) were included in this comparative study, and all three of these viruses delivering sgRNA were able to induce somatic and germline mutations. Unexpectedly, PVX, a potexvirus closely related to FoMV, expressing sgRNA alone induced biallelic edited progeny, indicating that mobility signals are dispensable in virus-induced germline editing. These results show that PVX, BSMV, and TRV expressing sgRNA all have an innate ability to induce mutations in the germline. Our results indicate that mobility signals alone may not be sufficient to enable virus-based delivery of sgRNAs using the viruses, FoMV, PVX, BSMV, and TRV into cell types that result in germline mutations.

Temporally distinct regulatory pathways coordinate thermo-responsive storage organ formation in potato.

Tuberization is an important developmental process in potatoes, but it is highly affected by environmental conditions. Temperature is a major environmental factor affecting tuberization, with high temperatures suppressing tuber development. However, the temporal aspects of thermo-responsive tuberization remain elusive. In this study, we show that FT homolog StSP6A is suppressed by temporally distinct regulatory pathways. Experiments using StSP6A-overexpressing plants show that post-transcriptional regulation plays a major role at the early stage, while transcriptional regulation is an important late-stage factor, suppressing StSP6A at high temperatures in leaves. Overexpression of StSP6A in leaves restores tuber formation but does not recover tuber yield at the late stage, possibly because of suppressed sugar transport at high temperatures. Transcriptome analyses lead to the identification of potential regulators that may be involved in thermo-responsive tuberization at different stages. Our work shows that potato has temporally distinct molecular mechanisms that finely control tuber development at high temperatures.

Rice homolog of Sin3-associated polypeptide 30, OsSFL1, mediates histone deacetylation to regulate flowering time during short days.

Rice homolog of Sin3-associated polypeptide 30, OsSFL1, mediates histone deacetylation to regulate flowering time during short days.

Source-Sink Regulation Is Mediated by Interaction of an FT Homolog with a SWEET Protein in Potato

Potato plants form tuberous storage organs on underground modified stems called stolons. Tubers are rich in starch, proteins, and other important nutrients, making potato one of the most important staple food crops. The timing of tuber development in wild potato is regulated by day length through a mechanism that is closely related to floral transition [1, 2]. Tuberization is also known to be regulated by the availability of assimilates, in particular sucrose, the transported form of sugar, required for starch synthesis. During the onset of tuber development, the mode of sucrose unloading switches from apoplastic to symplastic [3]. Here, we show that this switch maybe mediated by the interaction between the tuberization-specific FT homolog StSP6A and the sucrose efflux transporter StSWEET11 [4]. The binding of StSP6A to StSWEET11 blocked the leakage of sucrose to the apoplast, and is therefore likely to promote symplastic sucrose transport. The direct physical interaction between StSWEET11 and StSP6A proteins represents a link between the sugar and photoperiodic pathways for the regulation of potato tuber formation. Our data suggest that a previously undiscovered function for the FT family of proteins extends their role as mobile signals to mediators of source-sink partitioning, opening the possibility for modifying source-sink interactions.

Non-vernalization Flowering and Seed Set of Cabbage Induced by Grafting Onto Radish Rootstocks.

Cabbage (Brassica oleracea var. capitata) requires a long-term low-temperature exposure for floral induction, causing a delay in the breeding cycle. The objective of this study is to develop a method to induce flowering in cabbage without low-temperature treatment, using a grafting method. We conducted grafting experiments using two flower-induced Chinese kale cultivars (B. oleracea var. alboglabra) and seven radish cultivars/accessions as rootstocks and investigated the flowering response of grafted cabbage scions without low-temperature treatment. “Watanabe-seiko No.1” cabbage, when grafted onto the two Chinese kale cultivars, did not formed flower buds. Flowering was successfully induced in “Watanabe-seiko No.1” by grafting onto three out of the seven tested radish cultivars, and in “Kinkei No.201” and “Red cabbage” by grafting onto one tested radish cultivar. In “Watanabe-seiko No.1,” the earliest flower bud appearance was observed at 29 days after grafting. Seeds were also obtained from the three cabbage cultivars that flowered by grafting. Gene expression analysis of “Watanabe-seiko No.1” cabbage scions which formed flower buds by grafting, revealed high expression of the homolog of the floral integrator, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (BoSOC1), at the time of flower bud appearance. However, in the same leaf samples, we observed low expression of two homologs of florigen, FLOWERING LOCUS T (BoFT.C2 and BoFT.C6). In addition, two homologs of the floral repressor FLOWERING LOCUS C (BoFLC3 and BoFLC4), which are known to be down-regulated before flower bud differentiation in the vernalization pathway, were highly expressed, indicating that grafting onto radish induces cabbage flowering independently of the vernalization pathway. The expression level of the radish FT homolog (RsFT) in “Rat’s tail-G2,” which had highly induced flowering in the grafted cabbage scion, was higher than in the other radish cultivars. However, although “Rat’s tail-CH” effectively induced flowering in the cabbage scion, the expression of RsFT was low in this cultivar. In this study, floral induction of non-vernalized cabbage cannot be explained by the expression levels of RsFT in rootstock plants, alone. The flowering of non-vernalized cabbage would be induced by transmissible agents from rootstocks and not by the expression of cabbage FT, BoFT, from the scion itself.

Isolation and functional characterization of a novel FLOWERING LOCUS T homolog (NtFT5) in Nicotiana tabacum

The phosphatidyl ethanolamine-binding proteins (PEBPs) function primarily in regulating flowering in plants. Here, we report a genome-wide identification of PEBPs and functional characterization of a novel FLOWERING LOCUS T homolog (NtFT5) in tobacco, Nicotiana tabacum. Seven new PEBPs were identified by a genome-wide analysis from N. tabacum. Expression profile showed that NtFT5 was mainly expressed in flowers. Overexpression of NtFT5 conferred an early flowering phenotype. By optimizing rooting medium, heritable short life-cycle tobacco lines were obtained by overexpression of NtFT5. Several orthologs of flowering genes downstream of FT gene were up-regulated in the NtFT5-overexpression transgenic plant lines. The NtFT5-overexpressing tobaccos formed fewer flowers and seeds per capsule compared with wild type. The seed-to-seed life cycle of NtFT5 overexpressing tobacco lines was about 2.5 months. Gene identification was effectively undertaken in the short life-cycle tobacco line by a second transformation via a gusA reporter gene and transient expression of Ros1 via PVX (Potato Virus X)system. Our findings indicate that NtFT5 is a novel FT homolog that has potential to induce flowering, which will improve our understanding of the mechanism underlying flowering control in N. tabacum. In addition, the results show that the generated heritable short life-cycle transgenic tobacco line is an effective and stable host system to accelerate gene function study, which promises to provide a better tobacco research model for plants.

Abscisic acid affects expression of citrus FT homologs upon floral induction by low temperature in Satsuma mandarin (Citrus unshiu Marc.).

After a long juvenile period, citrus trees undergo seasonal flowering cycles. Under natural conditions, citrus flowering is regulated mainly by low ambient temperatures around 15-20 °C and water deficit stress. Recent studies have revealed that fluctuations in the expression of citrus homologs of FLOWERING LOCUS T (FT, encoding a flowering integrator) are correlated with their presumed role as flower-promoting signals. Previous ectopic expression analyses have demonstrated the flower-promoting function of citrus FT homologs. In this study, we examined whether abscisic acid (ABA) affects the expression of FT homologs and the flowering induced by low ambient temperatures. Application of exogenous ABA to potted Satsuma mandarin (Citrus unshiu Marc.) trees resulted in transient accumulation of citrus FT homolog transcripts. The promoter of one citrus FT homolog, CiFT3, was active in transgenic A. thaliana (Arabidopsis thaliana) and responded to exogenous and endogenous ABA. CiFT3 is preferentially expressed in shoots, and its expression was affected by flower-inductive treatments. Endogenous ABA accumulated in mandarin shoots during the floral induction period at 15 °C and under field conditions. The accumulation of ABA was correlated with the accumulation of FT homolog transcripts and flowering intensity. It was consistent with changes in the expression of genes related to ABA metabolism. The abundance of carotenoid precursors that serve as substrates for ABA biosynthesis decreased in leaves during the accumulation of ABA. Our data indicate that ABA and carotenoid precursors in leaves influence the flowering of mandarin trees induced by low temperature.

Functional Analysis of the FT Homolog from Eustoma grandiflorum Reveals Its Role in Regulating A and C Functional MADS Box Genes to Control Floral Transition and Flower Formation

High temperatures cause rosetting and the continued vegetative growth of Eustoma grandiflorum. Eustoma requires a period of cold treatment to promote flowering. Ortholog of flowering time gene FT (EgFT) was isolated and characterized from E. grandiflorum. The ectopic expression of EgFT significantly promotes flowering in transgenic E. grandiflorum even when grown at high (28–30 °C) temperature conditions without any cold treatment. In addition, a severe defect in stamen and carpel formation in which they are converted into sepal/petal-like structures were observed in transgenic E. grandiflorum that strongly expressed EgFT. This homeotic conversion was correlated with the upregulation of A (EgAP1/EgFUL) and E (EgSEP1/3) and downregulation of C (EgAG) functional MADS box genes in transgenic E. grandiflorum. In wild-type E. grandiflorum, EgFT mRNA was detected in leaves and was expressed higher in young than in mature flower buds. In flowers, EgFT mRNA was strongly expressed in sepals, moderately expressed in petal, and was almost undetectable in stamen and carpel. This expression pattern was very similar to that found for EgAP1 and completely inverse to that found for EgAG. These results indicated that EgFT is able to promote flowering and regulate sepal/petal formation by activating the A/E genes and suppressing the C gene in E. grandiflorum. 35S::EgFT also caused early flowering in transgenic Arabidopsis. However, the flower organ formation and the expression of the Arabidopsis AG gene were not affected. Thus, the ability for EgFT to regulate EgAG expression may represent a distinct mechanism in E. grandiflorum for the regulation of flower organ formation.

A GmRAV Ortholog Is Involved in Photoperiod and Sucrose Control of Flowering Time in Soybean

Photoperiod and sucrose levels play a key role in the control of flowering. GmRAV reflected a diurnal rhythm with the highest expression at 4 h after the beginning of a dark period in soybean leaves, and was highly up-regulated under short-day (SD) conditions, despite of not following a diurnal pattern under long-day (LD) conditions. GmRAV-i (GmRAV-inhibition) transgenic soybean exhibited early flowering phenotype. Two of the FT Arabidopsis homologs, GmFT2a and GmFT5a, were highly expressed in the leaves of soybeans with inhibition (-i) of GmRAV under SD conditions. Moreover, the transcript levels of the two FT homologs in GmRAV-i soybeans were more sensitive to SD conditions than LD conditions compared to the WT plant. GmRAV-i soybeans and Arabidopsis rav mutants showed more sensitive hypocotyl elongation responses when compared with wild-type seedlings, and GmRAV-ox overevpressed in tobacco revealed no sensitive changes in hypocotyl length. These indicated that GmRAV was a novel negative regulator of SD-mediated flowering and hypocotyl elongation. Although sucrose has been suggested to promote flowering induction in many plant species, high concentration of sucrose (4% [w/v]) applied into media defer flowering time in Arabidopsis wild-type and rav mutant. This delayed flowering stage might be caused by reduction of LEAFY expression. Furthermore, Arabidopsis rav mutants and GmRAV-i soybean plants were less sensitive to sucrose by the inhibition assays of hypocotyls and roots growth. In contrast, transgenic GmRAV overexpressing (-ox) tobacco plants displayed more sensitivity to sucrose. In conclusion, GmRAV was inferred to have a fundamental function in photoperiod, darkness, and sucrose signaling responses to regulate plant development and flowering induction.

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 Coordinately Regulated Homologs of FLOWERING LOCUS T Are Involved in the Control of Photoperiodic Flowering in Soybean

FLOWERING LOCUS T (FT) is a key flowering integrator in Arabidopsis (Arabidopsis thaliana), with homologs that encode florigens in many plant species regardless of the type of photoperiodic response. We identified 10 FT homologs, which were arranged as five pairs of linked genes in different homoeologous chromosomal regions, in soybean (Glycine max), a paleopolyploid species. Two of the FT homologs, GmFT2a and GmFT5a, were highly up-regulated under short-day (SD) conditions (inductive for flowering in soybean) and had diurnal expression patterns with the highest expression 4 h after dawn. Under long-day (LD) conditions, expression of GmFT2a and GmFT5a was down-regulated and did not follow a diurnal pattern. Flowering took much longer to initiate under LD than under SD, and only the GmFT5a transcript accumulated late in development under LD. Ectopic expression analysis in Arabidopsis confirmed that both GmFT2a and GmFT5a had the same function as Arabidopsis FT, but the effect of GmFT5a was more prominent. A double-mutant soybean line for two PHYTOCHROME A (PHYA) genes expressed high levels of GmFT2a and GmFT5a under LD, and it flowered slightly earlier under LD than the wild type grown under SD. The expression levels of GmFT2a and GmFT5a were regulated by the PHYA-mediated photoperiodic regulation system, and the GmFT5a expression was also regulated by a photoperiod-independent system in LD. Taken together, our results suggest that GmFT2a and GmFT5a coordinately control flowering and enable the adaptation of soybean to a wide range of photoperiodic environments.

Ectopic Expression of an FT Homolog from Citrus Confers an Early Flowering Phenotype on Trifoliate Orange (Poncirus trifoliata L. Raf.)

Citrus FT (CiFT) cDNA, which promoted the transition from the vegetative to the reproductive phase in Arabidopsis thaliana, when constitutively expressed was introduced into trifoliate orange (Poncirus trifoliata L. Raf.). The transgenic plants in which CiFT was expressed constitutively showed early flowering, fruiting, and characteristic morphological changes. They started to flower as early as 12 weeks after transfer to a greenhouse, whereas wild-type plants usually have a long juvenile period of several years. Most of the transgenic flowers developed on leafy inflorescences, apparently in place of thorns; however, wild-type adult trifoliate orange usually develops solitary flowers in the axils of leaves. All of the transgenic lines accumulated CiFT mRNA in their shoots, but there were variations in the accumulation level. The transgenic lines showed variation in phenotypes, such as time to first flowering and tree shape. In F(1) progeny obtained by crossing 'Kiyomi' tangor (C. unshiu x sinensis) with the pollen of one transgenic line, extremely early flowering immediately after germination was observed. The transgene segregated in F(1) progeny in a Mendelian fashion, with complete co-segregation of the transgene and the early flowering phenotype. These results showed that constitutive expression of CiFT can reduce the generation time in trifoliate orange.

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.