A genome-wide association and fine-mapping study of white rust resistance in hexaploid chrysanthemum cultivars with a wild diploid reference genome.

White rust caused by Puccinia horiana is one of the most serious diseases of chrysanthemum (Chrysanthemum × morifolium). In this study, we report the DNA markers associated with resistance against P. horiana via a simple approach using the genome of a wild diploid relative, Chrysanthemum seticuspe. First, we identified the important region of the genome in the resistant cultivar "Ariesu" via a genome-wide association study. Simplex single nucleotide polymorphism (SNP) markers mined from ddRAD-Seq were used in a biparental population originating from crosses between resistant "Ariesu" and susceptible "Yellow Queen". The C. seticuspe genome was used as a reference. For the fine mapping of P. horiana resistance locus 2 (Phr2), a comparative whole genome sequencing study was conducted. Although the genome sequences of chrysanthemum cultivars assembled via the short-read approach were fragmented, reliable genome alignments were reconstructed by mapping onto the chromosome level of the C. seticuspe pseudomolecule. Base variants were then identified by comparing the assembled genome sequences of resistant "Ariesu" and susceptible "Yellow Queen". Consequently, SNP markers that were closer to Phr2 compared with ddRAD-Seq markers were obtained. These SNP markers co-segregated with resistance in F1 progenies originating from resistant "Ariesu" and showed robust transferability for detecting Phr2-conferring resistance among chrysanthemum genetic resources. The wild C. seticuspe pseudomolecule, a de facto monoploid genome used for ddRAD-Seq analysis and assembled genome sequence comparison, demonstrated this method's utility as a model for developing DNA markers in hexaploid chrysanthemum cultivars.

A chromosome-level genome sequence of Chrysanthemum seticuspe, a model species for hexaploid cultivated chrysanthemum.

Chrysanthemums are one of the most industrially important cut flowers worldwide. However, their segmental allopolyploidy and self-incompatibility have prevented the application of genetic analysis and modern breeding strategies. We thus developed a model strain, Gojo-0 (Chrysanthemum seticuspe), which is a diploid and self-compatible pure line. Here, we present the 3.05 Gb chromosome-level reference genome sequence, which covered 97% of the C. seticuspe genome. The genome contained more than 80% interspersed repeats, of which retrotransposons accounted for 72%. We identified recent segmental duplication and retrotransposon expansion in C. seticuspe, contributing to arelatively large genome size. Furthermore, we identified a retrotransposon family, SbdRT, which was enriched in gene-dense genome regions and had experienced a very recent transposition burst. We also demonstrated that the chromosome-level genome sequence facilitates positional cloning in C. seticuspe. The genome sequence obtained here can greatly contribute as a reference for chrysanthemum in front-line breeding including genome editing.

Constitutive expression of CsGI alters critical night length for flowering by changing the photo-sensitive phase of anti-florigen induction in chrysanthemum.

Chrysanthemum is a typical short day (SD) flowering plant that requires a longer night period than a critical minimum duration to successfully flower. We identified FLOWERING LOCUS T-LIKE 3 (FTL3) and ANTI-FLORIGENIC FT/TFL1 FAMILY PROTEIN (AFT) as a florigen and antiflorigen, respectively, in a wild diploid chrysanthemum (Chrysanthemum seticuspe). Expression of the genes that produce these proteins, CsFTL3 and CsAFT, is induced in the leaves under SD or a noninductive photoperiod, respectively, and the balance between them determines the progression of floral transition and anthesis. However, how CsFTL3 and CsAFT are regulated to define the critical night length for flowering in chrysanthemum is unclear. In this study, we focused on the circadian clock-related gene GIGANTEA (GI) of C. seticuspe (CsGI) and generated transgenic C. seticuspe plants overexpressing CsGI (CsGI-OX). Under a strongly inductive SD (8 L/16D) photoperiod, floral transition occurred at almost the same time in both wild-type and CsGI-OX plants. However, under a moderately inductive (12 L/12D) photoperiod, the floral transition in CsGI-OX plants was strongly suppressed, suggesting that the critical night length for flowering was lengthened for CsGI-OX plants. Under the 12 L/12D photoperiod, CsAFT was upregulated in CsGI-OX plants. Giving a night break (NB) 10 h after dusk was the most effective time to inhibit flowering in wild-type plants, while the most effective time for NB was extended to dawn (12 and 14 h after dusk) in CsGI-OX plants. In wild-type plants, a red-light pulse delivered 8 or 10 h after dusk induced maximal CsAFT expression, but the length of the time period over which CsAFT could be induced by red light was extended until subjective dawn in CsGI-OX plants. Therefore, CsGI-OX plants required a longer dark period to maintain lower levels of CsAFT, and their critical night length for flowering was thus lengthened. These results suggested that CsGI has an important role in the control of photoperiodic flowering through shaping the gate for CsAFT induction by light in chrysanthemum.

Genome-wide association study overcomes the genome complexity in autohexaploid chrysanthemum and tags SNP markers onto the flower color genes.

The use of DNA markers has revolutionized selection in crop breeding by linkage mapping and QTL analysis, but major problems still remain for polyploid species where marker-assisted selection lags behind the situation in diploids because of its high genome complexity. To overcome the complex genetic mode in the polyploids, we investigated the development of a strategy of genome-wide association study (GWAS) using single-dose SNPs, which simplify the segregation patterns associated polyploids, with respect to the development of DNA markers. In addition, we employed biparental populations for the GWAS, wherein the SNP allele frequency could be predicted. The research investigated whether the method could be used to effectively develop DNA markers for petal color in autohexaploid chrysanthemum (Chrysanthemum morifolium; 2n = 6x = 54). The causal gene for this trait is already-known CmCCD4a encoding a dioxygenase which cleaves carotenoids in petals. We selected 9,219 single-dose SNPs, out of total 52,489 SNPs identified by dd-RAD-Seq, showing simplex (1 × 0) and double-simplex (1 × 1) inheritance pattern according to alternative allele frequency with respect to the SNP loci in the F1 population. GWAS, using these single-dose SNPs, discovered highly reproducible SNP markers tightly linked to the causal genes. This is the first report of a straightforward GWAS-based marker developing system for use in autohexaploid species.

A pure line derived from a self-compatible Chrysanthemum seticuspe mutant as a model strain in the genus Chrysanthemum.

Asteraceae is the largest family of angiosperms, comprising approximately 24,000 species. Molecular genetic studies of Asteraceae are essential for understanding plant diversity. Chrysanthemum morifolium is the most industrially important ornamental species in Asteraceae. Most cultivars of C. morifolium are autohexaploid and self-incompatible. These properties are major obstacles to the genetic analysis and modern breeding of C. morifolium. Furthermore, high genome heterogeneity complicates molecular biological analyses. In this study, we developed a model strain in the genus Chrysanthemum. C. seticuspe is a diploid species with a similar flowering property and morphology to C. morifolium and can be subjected to Agrobacterium-mediated transformation. We isolated a natural self-compatible mutant of C. seticuspe and established a pure line through repeated selfing and selection. The resultant strain, named Gojo-0, was favorable for genetic analyses, including isolation of natural and induced mutants, and facilitated molecular biological analysis, including whole genome sequencing, owing to the simplicity and homogeneity of its genome. Interspecific hybridization with Chrysanthemum species was possible, enabling molecular genetic analysis of natural interspecific variations. The accumulation of research results and resources using Gojo-0 as a platform is expected to promote molecular genetic studies on the genus Chrysanthemum and the genetic improvement of chrysanthemum cultivars.

Chrysanthemum requires short-day repeats for anthesis: Gradual CsFTL3 induction through a feedback loop under short-day conditions.

Chrysanthemums require continuous short-days (SD) for anthesis. FTL3 (FLOWERING LOCUS T-like 3), a floral promoter expressed in chrysanthemum leaf, forms a complex with its interacting partner FDL1 to induce floral meristem identity gene AFL1. We explored the FTL3 induction mechanism during SD repeats in Chrysanthemum seticuspe. CsFTL3 expression was not immediately induced by a shift from long-day (LD) to SD, but gradually increased until the capitulum development stage under repeated SDs. Overexpression of CsFTL3 transgene increased endogenous leaf CsFTL3 induction under SD but not LD. Overexpression of CsFDL1 promoted anthesis and increased CsAFL1 and CsFTL3 expression under SD. Loss-of-function of CsFDL1 by RNAi resulted in delayed anthesis and downregulation of leaf CsAFL1 and CsFTL3, indicating the necessity of CsFDL1 for CsFTL3 induction. Overexpression of an antagonistic protein of CsFTL3 or CsFDL1 inhibited leaf CsFTL3 induction. CsFTL3 expression was positively regulated during SDs by a feedback mechanism involving the CsFTL3-CsFDL1 complex. Furthermore, flowering was accomplished by feedback with high levels of CsFTL3 induction under repeated SDs.

De novo whole-genome assembly in Chrysanthemum seticuspe, a model species of Chrysanthemums, and its application to genetic and gene discovery analysis.

Cultivated chrysanthemum (Chrysanthemum morifolium Ramat.) is one of the most economically important ornamental crops grown worldwide. It has a complex hexaploid genome (2n = 6x = 54) and large genome size. The diploid Chrysanthemum seticuspe is often used as a model of cultivated chrysanthemum, since the two species are closely related. To expand our knowledge of the cultivated chrysanthemum, we here performed de novo whole-genome assembly in C. seticuspe using the Illumina sequencing platform. XMRS10, a C. seticuspe accession developed by five generations of self-crossing from a self-compatible strain, AEV2, was used for genome sequencing. The 2.72 Gb of assembled sequences (CSE_r1.0), consisting of 354,212 scaffolds, covered 89.0% of the 3.06 Gb C. seticuspe genome estimated by k-mer analysis. The N50 length of scaffolds was 44,741 bp. For protein-encoding genes, 71,057 annotated genes were deduced (CSE_r1.1_cds). Next, based on the assembled genome sequences, we performed linkage map construction, gene discovery and comparative analyses for C. seticuspe and cultivated chrysanthemum. The generated C. seticuspe linkage map revealed skewed regions in segregation on the AEV2 genome. In gene discovery analysis, candidate flowering-related genes were newly found in CSE_r1.1_cds. Moreover, single nucleotide polymorphism identification and annotation on the C. × morifolium genome showed that the C. seticuspe genome was applicable to genetic analysis in cultivated chrysanthemums. The genome sequences assembled herein are expected to contribute to future chrysanthemum studies. In addition, our approach demonstrated the usefulness of short-read genome assembly and the importance of choosing an appropriate next genome sequencing technology based on the purpose of the post-genome analysis.

Photoperiod-insensitive floral transition in chrysanthemum induced by constitutive expression of chimeric repressor CsLHY-SRDX.

A wide variety of physiological processes including flowering are controlled by the circadian clock in plants. In Arabidopsis, LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) constitute the central oscillator, and their gain of function and loss of function disrupt the circadian clock and affect flowering time through FLOWERING LOCUS T (FT), a gene encoding a florigen. Chrysanthemum is a typical short-day (SD) plant and responds to shortening of day length by the transition from the vegetative to reproductive phase. We identified FLOWERING LOCUS T-LIKE 3 (FTL3) and ANTI-FLORIGENIC FT/TFL1 FAMILY PROTEIN (AFT) as a florigen and antiflorigen, respectively, in a wild diploid chrysanthemum (Chrysanthemum seticuspe f. boreale). CsFTL3 and CsAFT are induced under SD or a noninductive photoperiod, respectively, and their balance determines the floral transition and anthesis. Meanwhile, the time-keeping mechanism that regulates the photoperiodic flowering in chrysanthemum is poorly understood. Here, we focused on a LHY/CCA1-like gene called CsLHY in chrysanthemum. We fused CsLHY to a gene encoding short transcriptional repressor domain (SRDX) and constitutively expressed it in chrysanthemum. Although the transcription of clock-related genes was conditionally affected, circadian rhythm was not completely disrupted in CsLHY-SRDX transgenic plants. These plants formed almost the same number of leaves before floral transition under SD and long-day conditions. Thus, CsLHY-SRDX chrysanthemum showed photoperiod-insensitive floral transition, but further development of the capitulum was arrested, and anthesis was not observed. Simultaneously with the flowering phenotype, CsFTL3 and CsAFT were downregulated in CsLHY-SRDX transgenic plants. These results suggest that CsLHY-SRDX affects CsFTL3 and CsAFT expression and causes photoperiod-insensitive floral transition without a severe defect in the circadian clock.

CsTFL1, a constitutive local repressor of flowering, modulates floral initiation by antagonising florigen complex activity in chrysanthemum.

Chrysanthemums require repeated cycles of short-day (SD) photoperiod for successful anthesis, but their vegetative state is strictly maintained under long-day (LD) or night-break (NB) conditions. We have previously demonstrated that photoperiodic flowering of a wild diploid chrysanthemum (Chrysanthemum seticuspe f. boreale) is controlled by a pair of systemic floral regulators, florigen (CsFTL3) and anti-florigen (CsAFT), produced in the leaves. Here, we report the functional characterisation of a local floral regulator, CsTFL1, a chrysanthemum orthologue of TERMINAL FLOWER 1 gene in Arabidopsis. Constitutive expression of CsTFL1 in C. seticuspe (CsTFL1-ox) resulted in extremely late flowering under SD and prevented up-regulation of floral meristem identity genes in shoot tips and leaves. Bimolecular fluorescence complementation assay showed that both CsTFL1 and CsFTL3 interacted with CsFDL1, a bZIP transcription factor FD homologue, in the nucleus. The transient gene expression assay indicated that CsTFL1 suppresses flowering by directly antagonising the flower inductive activity of the CsFTL3-CsFDL1 complex. Our results suggest that strict maintenance of vegetative state under non-inductive photoperiod is achieved by the coordinated action of both the systemic floral inhibitor and local floral inhibitor CsTFL1, which is constitutively expressed in shoot tips.

Environmental responses of the FT/TFL1 gene family and their involvement in flower induction in Fragaria × ananassa.

Flowering time control is important for fruit production in Fragaria × ananassa. The flowering inhibition pathway has been extensively elucidated in the woodland strawberry, Fragaria vesca, whereas the factors involved in its promotion remain unclear. In this study, we investigated the environmental responses of F. × ananassa FT and TFL1-like genes, which are considered key floral promoters and repressors in many plants, respectively. A putative floral promoter, FaFT3, was up-regulated in the shoot tip under short-day and/or low growth temperature, in accordance with the result that these treatments promoted flowering. FaFT3 mRNA accumulated before induction of a floral meristem identity gene, FaAP1. FaFT2, a counterpart of FvFT2, expressed in the flower bud of F. vesca, was not induced in the shoot tip differentiating sepal or stamen, suggesting that this gene works at a later stage than stamen formation. In F. vesca, FvFT1 transmits the long-day signal perceived in the leaves to the shoot tip, and induces the potent floral inhibitor FvTFL1. FaFT1 was expressed in the leaves under long-day conditions in F. × ananassa. Expression of FaTFL1 was higher in the shoot tip under long-day than short-day conditions. Independent of day-length, FaTFL1 expression was higher under high temperature than low temperature conditions. These results suggest that FaFT3 induction by short-day or low temperature stimuli is a key step for flowering initiation. As in F. vesca, F. × ananassa floral inhibition pathways depend on FaTFL1 regulation by day-length via FaFT1, and by temperature.

The gated induction system of a systemic floral inhibitor, antiflorigen, determines obligate short-day flowering in chrysanthemums.

Photoperiodic floral induction has had a significant impact on the agricultural and horticultural industries. Changes in day length are perceived in leaves, which synthesize systemic flowering inducers (florigens) and inhibitors (antiflorigens) that determine floral initiation at the shoot apex. Recently, FLOWERING LOCUS T (FT) was found to be a florigen; however, the identity of the corresponding antiflorigen remains to be elucidated. Here, we report the identification of an antiflorigen gene, Anti-florigenic FT/TFL1 family protein (AFT), from a wild chrysanthemum (Chrysanthemum seticuspe) whose expression is mainly induced in leaves under noninductive conditions. Gain- and loss-of-function analyses demonstrated that CsAFT acts systemically to inhibit flowering and plays a predominant role in the obligate photoperiodic response. A transient gene expression assay indicated that CsAFT inhibits flowering by directly antagonizing the flower-inductive activity of CsFTL3, a C. seticuspe ortholog of FT, through interaction with CsFDL1, a basic leucine zipper (bZIP) transcription factor FD homolog of Arabidopsis. Induction of CsAFT was triggered by the coincidence of phytochrome signals with the photosensitive phase set by the dusk signal; flowering occurred only when night length exceeded the photosensitive phase for CsAFT induction. Thus, the gated antiflorigen production system, a phytochrome-mediated response to light, determines obligate photoperiodic flowering response in chrysanthemums, which enables their year-round commercial production by artificial lighting.

Flowering retardation by high temperature in chrysanthemums: involvement of FLOWERING LOCUS T-like 3 gene repression.

Flowering time of the short-day plant Chrysanthemum morifolium is largely dependent upon daylength, but it is also distinctly influenced by other environmental factors. Flowering is delayed by summer heat. Here, the underlying basis for this phenomenon was investigated. Heat-induced flowering retardation occurred similarly in C. morifolium and C. seticuspe, a wild-type diploid chrysanthemum. In both plants, this flowering retardation occurred mainly because of inhibition of capitulum development. Concurrently, expression of flowering-related genes in the shoot tip was delayed under high temperature conditions. In chrysanthemums, FLOWERING LOCUS T-like 3 (FTL3) has been identified as a floral inducer produced in the leaves after short-day stimuli and transported to the shoot tip. In C. seticuspe, heat-induced flowering retardation was accompanied by a reduction in FTL3 expression in the leaves. Two C. morifolium cultivars with flowering times that are differently affected by growth temperature were also examined. High temperature-induced FTL3 repression was observed in the leaves of both cultivars, although the degree of repression was greater in the heat-sensitive cultivar than in the heat-tolerant cultivar. When a scion of the heat-sensitive cultivar was grafted onto the stock of the heat-tolerant cultivar, flowering in the shoot tip was less sensitive to heat. Conversely, a scion of the heat-tolerant cultivar grafted onto the heat-sensitive cultivar showed increased heat sensitivity. Thus, several lines of evidence suggest that the reduction of FTL3 signalling from the leaves to the shoot tip at high temperatures is involved in flowering retardation in chrysanthemums.

Day light quality affects the night-break response in the short-day plant chrysanthemum, suggesting differential phytochrome-mediated regulation of flowering.

Chrysanthemum (Chrysanthemum morifolium) is a short-day plant, which flowers when the night length is longer than a critical minimum. Flowering is effectively inhibited when the required long-night phase is interrupted by a short period of exposure to red light (night break; NB). The reversal of this inhibition by subsequent exposure to far-red (FR) light indicates the involvement of phytochromes in the flowering response. Here, we elucidated the role of light quality in photoperiodic regulation of chrysanthemum flowering, by applying a range of different conditions. Flowering was consistently observed under short days with white light (W-SD), SD with monochromatic red light (R-SD), or SD with monochromatic blue light (B-SD). For W-SD, NB with monochromatic red light (NB-R) was most effective in inhibiting flowering, while NB with monochromatic blue light (NB-B) and NB with far-red light (NB-FR) caused little inhibition. In contrast, for B-SD, flowering was strongly inhibited by NB-B and NB-FR. However, when B-SD was supplemented with monochromatic red light (B+R-SD), no inhibition by NB-B and NB-FR was observed. Furthermore, the inhibitory effect of NB-B following B-SD was partially reversed by subsequent exposure to a FR light pulse. The conditions B-SD/NB-B (no flowering) and B+R-SD/NB-B (flowering) similarly affected the expression of circadian clock-related genes. However, only the former combination suppressed expression of the chrysanthemum orthologue of FLOWERING LOCUS T (CmFTL3). Our results suggest the involvement of at least 2 distinct phytochrome responses in the flowering response of chrysanthemum. Furthermore, it appears that the light quality supplied during the daily photoperiod affects the light quality required for effective NB.

CsFTL3, a chrysanthemum FLOWERING LOCUS T-like gene, is a key regulator of photoperiodic flowering in chrysanthemums.

Chrysanthemum is a typical short-day (SD) plant that responds to shortening daylength during the transition from the vegetative to the reproductive phase. FLOWERING LOCUS T (FT)/Heading date 3a (Hd3a) plays a pivotal role in the induction of phase transition and is proposed to encode a florigen. Three FT-like genes were isolated from Chrysanthemum seticuspe (Maxim.) Hand.-Mazz. f. boreale (Makino) H. Ohashi & Yonek, a wild diploid chrysanthemum: CsFTL1, CsFTL2, and CsFTL3. The organ-specific expression patterns of the three genes were similar: they were all expressed mainly in the leaves. However, their response to daylength differed in that under SD (floral-inductive) conditions, the expression of CsFTL1 and CsFTL2 was down-regulated, whereas that of CsFTL3 was up-regulated. CsFTL3 had the potential to induce early flowering since its overexpression in chrysanthemum could induce flowering under non-inductive conditions. CsFTL3-dependent graft-transmissible signals partially substituted for SD stimuli in chrysanthemum. The CsFTL3 expression levels in the two C. seticuspe accessions that differed in their critical daylengths for flowering closely coincided with the flowering response. The CsFTL3 expression levels in the leaves were higher under floral-inductive photoperiods than under non-inductive conditions in both the accessions, with the induction of floral integrator and/or floral meristem identity genes occurring in the shoot apexes. Taken together, these results indicate that the gene product of CsFTL3 is a key regulator of photoperiodic flowering in chrysanthemums.

Characterization of FLC, SOC1 and FT homologs in Eustoma grandiflorum: effects of vernalization and post-vernalization conditions on flowering and gene expression.

A rosette plant of Eustoma grandiflorum requires vernalization (exposure to a period of cold temperature) and long-day conditions to promote flowering, while prolonged cold or cool temperatures in post-vernalization periods delay flowering. This study aimed to investigate the effect of growth conditions on flowering regulation in Eustoma. In Arabidopsis, vernalization suppresses a floral repressor gene, FLOWERING LOCUS C (FLC) and upregulates floral promoter genes, such as SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and FLOWERING LOCUS T (FT). We identified and characterized the Eustoma homologs of these genes. In contrast to Arabidopsis FLC, Eustoma grandiflorum FLC-like (EgFLCL) expression was upregulated by cold temperature and downregulated by subsequent warm temperature exposure. The expression of Eustoma grandiflorum SOC1-like (EgSOC1L) and FT-like (EgFTL) genes was not significantly induced during vernalization, but their transcripts increased during a warm post-vernalization period in the long days. Vernalized plants grown under cool post-vernalization temperatures exhibited higher EgFLCL expression, lower EgSOC1L and EgFTL expression and flowered later than those grown under warm temperatures. Overexpression of EgFLCL cDNA repressed flowering in transgenic Arabidopsis, whereas overexpression of EgSOC1L or EgFTL cDNA promoted flowering. Our results suggest that flowering regulation by vernalization in Eustoma differs from the paradigm developed for Arabidopsis. EgFLCL is regulated by temperature and may be involved in floral repression during cold and cool seasons. Warm- and long-day conditions following vernalization are required to induce two putative floral promoters, EgSOC1L and EgFTL, effectively.

Involvement of the ethylene response pathway in dormancy induction in chrysanthemum.

Temperature plays a significant role in the annual cycling between growth and dormancy of the herbaceous perennial chrysanthemum (Chrysanthemum morifolium Ramat.). After exposure to high summer temperatures, cool temperature triggers dormancy. The cessation of flowering and rosette formation by the cessation of elongation are characteristic of dormant plants, and can be stimulated by exogenous ethylene. Thus, the ethylene response pathway may be involved in temperature-induced dormancy of chrysanthemum. Transgenic chrysanthemums expressing a mutated ethylene receptor gene were used to assess this involvement. The transgenic lines showed reduced ethylene sensitivity: ethylene causes leaf yellowing in wild-type chrysanthemums, but leaves remained green in the transgenic lines. Extension growth and flowering of wild-type and transgenic lines varied between temperatures: at 20 degrees C, the transgenic lines showed the same stem elongation and flowering as the wild type; at cooler temperatures, the wild type formed rosettes with an inability to flower and entered dormancy, but some transgenic lines continued to elongate and flower. This supports the involvement of the ethylene response pathway in the temperature-induced dormancy of chrysanthemum. At the highest dosage of ethephon, an ethylene-releasing agent, wild-type plants formed rosettes with an inability to flower and became dormant, but one transgenic line did not. This confirms that dormancy is induced via the ethylene response pathway.

The nature of floral signals in Arabidopsis. II. Roles for FLOWERING LOCUS T (FT) and gibberellin.

Signals produced in leaves are transported to the shoot apex where they cause flowering. Protein of the gene FLOWERING LOCUS T (FT) is probably a long day (LD) signal in Arabidopsis. In the companion paper, rapid LD increases in FT expression associated with flowering driven photosynthetically in red light were documented. In a far red (FR)-rich LD, along with FT there was a potential role for gibberellin (GA). Here, with the GA biosynthesis dwarf mutant ga1-3, GA(4)-treated plants flowered after 26 d in short days (SD) but untreated plants were still vegetative after 6 months. Not only was FT expression low in SD but applied GA bypassed some of the block to flowering in ft-1. On transfer to LD, ga1-3 only flowered when treated simultaneously with GA, and FT expression increased rapidly (<19.5 h) and dramatically (15-fold). In contrast, in the wild type in LD there was little requirement for GA for FT increase and flowering so its endogenous GA content was near to saturating. Despite this permissive role for endogenous GA in Columbia, RNA interference (RNAi) silencing of the GA biosynthesis gene, GA 20-OXIDASE2, revealed an additional, direct role for GA in LD. Flowering took twice as long after silencing the LD-regulated gene, GA 20-OXIDASE2. Such independent LD input by FT and GA reflects their non-sympatric expression (FT in the leaf blade and GA 20-OXIDASE2 in the petiole). Overall, FT acts as the main LD floral signal in Columbia and GA acts on flowering both via and independently of FT.

The nature of floral signals in Arabidopsis. I. Photosynthesis and a far-red photoresponse independently regulate flowering by increasing expression of FLOWERING LOCUS T (FT).

Arabidopsis flowers in long day (LD) in response to signals transported from the photoinduced leaf to the shoot apex. These LD signals may include protein of the gene FLOWERING LOCUS T (FT) while in short day (SD) with its slower flowering, signalling may involve sucrose and gibberellin. Here, it is shown that after 5 weeks growth in SD, a single LD up-regulated leaf blade expression of FT and CONSTANS (CO) within 4-8 h, and flowers were visible within 2-3 weeks. Plants kept in SDs were still vegetative 7 weeks later. This LD response was blocked in ft-1 and a co mutant. Exposure to different LD light intensities and spectral qualities showed that two LD photoresponses are important for up-regulation of FT and for flowering. Phytochrome is effective at a low intensity from far-red (FR)-rich incandescent lamps. Independently, photosynthesis is active in an LD at a high intensity from red (R)-rich fluorescent lamps. The photosynthetic role of a single high light LD is demonstrated here by the blocking of the flowering and FT increase on removal of atmospheric CO(2) or by decreasing the LD light intensity by 10-fold. These conditions also reduced leaf blade sucrose content and photosynthetic gene expression. An SD light integral matching that in a single LD was not effective for flowering, although there was reasonable FT-independent flowering after 12 SD at high light. While a single photosynthetic LD strongly amplified FT expression, the ability to respond to the LD required an additional but unidentified photoresponse. The implications of these findings for studies with mutants and for flowering in natural conditions are discussed.

The involvement of gibberellin 20-oxidase genes in phytochrome-regulated petiole elongation of Arabidopsis.

Long day (LD) exposure of rosette plants causes rapid stem/petiole elongation, a more vertical growth habit, and flowering; all changes are suggestive of a role for the gibberellin (GA) plant growth regulators. For Arabidopsis (Arabidopsis thaliana) L. (Heynh), we show that enhancement of petiole elongation by a far-red (FR)-rich LD is mimicked by a brief (10 min) end-of-day (EOD) FR exposure in short day (SD). The EOD response shows red (R)/FR photoreversibility and is not affected in a phytochrome (PHY) A mutant so it is mediated by PHYB and related PHYs. FR photoconversion of PHYB to an inactive form activates a signaling pathway, leading to increased GA biosynthesis. Of 10 GA biosynthetic genes, expression of the 20-oxidase, AtGA20ox2, responded most to FR (up to a 40-fold increase within 3 h). AtGA20ox1 also responded but to a lesser extent. Stimulation of petiole elongation by EOD FR is reduced in a transgenic AtGA20ox2 hairpin gene silencing line. By contrast, it was only in SD that a T-DNA insertional mutant of AtGA20ox1 (ga5-3) showed reduced response. Circadian entrainment to a daytime pattern provides an explanation for the SD expression of AtGA20ox1. Conversely, the strong EOD/LD FR responses of AtGA20ox2 may reflect its independence of circadian regulation. While FR acting via PHYB increases expression of AtGA20ox2, other GA biosynthetic genes are known to respond to R rather than FR light and/or to other PHYs. Thus, there must be different signal transduction pathways, one at least showing a positive response to active PHYB and another showing a negative response.