Tamotsu Hisamatsu

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.

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.

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

Significance Photoperiodic floral initiation is thought to be regulated by a systemic flowering inducer (florigen) and inhibitor (antiflorigen) produced in the leaves. Here, we show the discovery of an antiflorigen (CsAFT) from chrysanthemum, which is produced in the leaves under a noninductive photoperiod to systemically inhibit flowering. This antiflorigen production system prevents precocious flowering and enables the year-round supply of marketable flowers by manipulation of day length. 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.

Role of ethylene in senescence of cut Eustoma flowers

Abstract Flower senescence of Eustoma grandiflorum was investigated in relation to ethylene production and sensitivity to ethylene. Ethylene production of flowers increased with flower senescence. Ethylene was mainly produced in the pistil, in particular in the style, and production in the petal increased with flower senescence. Flowers were not sensitive to ethylene at anthesis, but became more sensitive with increasing senescence. Treatment with silver thiosulphate (STS), an ethylene action inhibitor, extended flower longevity. STS inhibited ethylene production from the whole flower, particularly that from the petal. Thus, ethylene is considered to be involved in the flower senescence of Eustoma.

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.

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.

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 °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.

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.

End-of-day far-red treatment enhances responsiveness to gibberellins and promotes stem extension in chrysanthemum

SUMMARY A brief end-of-day (EOD) far-red (FR) exposure promotes extension growth in plants. This change suggests a role for gibberellins (GA). For chrysanthemum (Chrysanthemum morifolium Ramat.), we show that EOD-FR exposure enhances shoot extension and is mediated, at least in part, by increased responsiveness (sensitivity) to GA. In addition, our analysis showed how light, especially EOD-FR, influenced shoot extension. The effect of EOD-FR on promoting extension growth was maintained, not only during the dark period, but also in the subsequent light period. Furthermore, our observations suggest that dark reversion of certain phytochromes is involved in the determination of flowering time. We also discuss the applicability of EOD-FR treatment to reduce the overall duration of cultivation, while maintaining a sufficient stem height during cut chrysanthemum production.

Roles of CmFL, CmAFL1, and CmSOC1 in the transition from vegetative to reproductive growth in Chrysanthemum morifolium Ramat.

Summary The transition from vegetative to reproductive growth is marked by the termination of vegetative development and adoption of a floral identity by the shoot apical meristem. This process is called the floral transition. To elucidate the molecular determinants involved in this process in chrysanthemum (Chrysanthemum morifolium Ramat.), we isolated the following three floral identity and/or integrator genes from plants: CmFL, a homologue of FLORICAULA (FLO)/LEAFY (LFY); CmAFL1, a member of the APETALA1 (AP1)/FRUITFULL (FUL) sub-family; and CmSOC1, a member of the SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1) sub-family. Up-regulated expression of CmFL in Arabidopsis resulted in an early-flowering phenotype. Our data show significant up-regulation of expression of all three of the above-mentioned genes and a strong correlation between the floral transition and gene expression under three different photoperiodic conditions. This study strongly suggests that CmFL, CmAFL1, and CmSOC1 are involved in the transition from vegetative to reproductive growth, and are activated co-ordinately after the floral transition in early-developing inflorescences in chrysanthemum.