Katsuhiko Sumitomo

Carotenoid Cleavage Dioxygenase (CmCCD4a) Contributes to White Color Formation in Chrysanthemum Petals

The white petals of chrysanthemum (Chrysanthemum morifolium Ramat.) are believed to contain a factor that inhibits the accumulation of carotenoids. To find this factor, we performed polymerase chain reaction-Select subtraction screening and obtained a clone expressed differentially in white and yellow petals. The deduced amino acid sequence of the protein (designated CmCCD4a) encoded by the clone was highly homologous to the sequence of carotenoid cleavage dioxygenase. All the white-flowered chrysanthemum cultivars tested showed high levels of CmCCD4a transcript in their petals, whereas most of the yellow-flowered cultivars showed extremely low levels. Expression of CmCCD4a was strictly limited to flower petals and was not detected in other organs, such as the root, stem, or leaf. White petals turned yellow after the RNAi construct of CmCCD4a was introduced. These results indicate that in white petals of chrysanthemums, carotenoids are synthesized but are subsequently degraded into colorless compounds, which results in the white color.

Analysis of Carotenoid Composition in Petals of Calendula (Calendula officinalis L.)

Nineteen carotenoids were identified in extracts of petals of orange- and yellow-flowered cultivars of calendula (Calendula officinalis L.). Ten carotenoids were unique to orange-flowered cultivars. The UV–vis absorption maxima of these ten carotenoids were at longer wavelengths than that of flavoxanthin, the main carotenoid of calendula petals, and it is clear that these carotenoids are responsible for the orange color of the petals. Six carotenoids had a cis structure at C-5 (C-5′), and it is conceivable that these (5Z)-carotenoids are enzymatically isomerized at C-5 in a pathway that diverges from the main carotenoid biosynthesis pathway. Among them, (5Z,9Z)-lycopene (1), (5Z,9Z,5′Z,9′Z)-lycopene (3), (5′Z)-γ-carotene (4), and (5′Z,9′Z)-rubixanthin (5) has never before been identified. Additionally, (5Z,9Z,5′Z)-lycopene (2) has been reported only as a synthesized compound.

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.

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.

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.

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.

Spectral sensitivity of flowering and FT-like gene expression in response to night-break light treatments in the chrysanthemum cultivar, ‘Reagan’

Summary A night-break (i.e., a short exposure to light near the middle of the dark period) inhibits flowering in chrysanthemum (Chrysanthemum morifolium Ramat.), a short-day plant. We studied the effect of night-break light quality (wavelength) on flowering and on expression of the FLOWERING LOCUS T (FT)-like gene, CmFTL3. Night-break treatments with the yellow-to-red wavelength range showed strong inhibitory effects on flowering. Further studies using monochromatic light from panels of light emitting diodes (LEDs) showed that the maximum effect on the inhibition of flowering was around 596 nm, and that levels of CmFTL3 mRNA were reduced in a pattern consistent with the observed inhibition of flowering. Wavelengths from ultraviolet-A to blue, and far-red, had no inhibitory effects on flowering. Our results also showed that the inhibitory effect of red light on flowering responses could be reduced by far-red light. These results suggest that phytochromes are involved in night-break responses, and the absorption spectrum of leaf extracts indicated that the effect of wavelength might be distorted by the screening effect of the other pigments found in green leaves. We also discuss further developments of lighting techniques for commercial production of chrysanthemum.

Significance of CmCCD4a orthologs in apetalous wild chrysanthemum species, responsible for white coloration of ray petals

Most chrysanthemum (Chrysanthemum morifolium Ramat.) flowers have a central capitulum, composed of many disc florets that is surrounded by ray petals. CmCCD4a, a gene that encodes a carotenoid cleavage dioxygenase (CCD), is expressed specifically in the ray petals of chrysanthemum cultivars, and its expression leads to white ray petals as a result of carotenoid degradation. Here, we show that wild chrysanthemums with white ray petals have CmCCD4a orthologs, whereas those with yellow ray petals lack these orthologs, as is the case in chrysanthemum cultivars. CmCCD4a orthologs also exist in some lines of Chrysanthemum pacificum and Chrysanthemum shiwogiku, even though these species lack ray petals. Interspecific hybridization between C. shiwogiku and a yellow-flowered chrysanthemum cultivar showed that the CmCCD4a orthologs from C. shiwogiku lead to the development of white ray petals. This indicates that the translation products of the CmCCD4a orthologs maintain enzymatic activity that can degrade carotenoids in chrysanthemums, irrespective of whether or not the ray petals that CmCCD4a expression actually occurred.

Memory of prolonged Winter cold inhibits flowering and increases long-day leaf number in the chrysanthemum cultivar ‘Nagano Queen’

Summary Chrysanthemum (Chrysanthemum morifolium Ramat.) is a short-day plant; however, flowering is eventually initiated under non-inductive long-day or night-break conditions. When using Summer-to-Autumn-flowering cultivars, such flowering is undesirable for stable horticultural production, particularly for Summer cropping, because of the short length of the cut flower, the formation of a crown bud, and the development of a branched spray of flowers. Under non-inductive conditions, flowering depends on the number of leaves that form below the terminal flower bud (i.e., the long-day leaf number). Long-day leaf numbers varied among cultivars in this study. ‘Nagano Queen’ showed a seasonal change in long-day leaf number, being high in Spring and decreasing in Summer. In addition, rooted cuttings under night-break conditions initiated very early flowering. Here, we show how exposure to prolonged Winter cold influenced long-day leaf number, and its inhibitory effect on flowering. Cold pre-treatments inhibited flower initiation under subsequent short-day conditions, with noticeable genetic variation among cultivars. For instance, flower initiation in ‘Nagano Queen’ was strongly inhibited in Spring, but the inhibition decreased in Summer. This result indicated that the inhibitory effect of Winter cold caused a delay in Spring flowering, which gradually declined from Spring to Summer. Exposure to prolonged Winter cold also inhibited flower initiation under long-day and night-break conditions, and increased long-day leaf number in Spring. These results indicate that this seasonal change in the inhibitory effect of Winter cold influences long-day leaf number in ‘Nagano Queen’.