Periodically taken photographs reveal the effect of pollinator insects on seed set in lotus flowers.

Understanding of pollination systems is an important topic for evolutionary ecology, food production, and biodiversity conservation. However, it is difficult to grasp the whole picture of an individual system, because the activity of pollinators fluctuates depending on the flowering period and time of day. In order to reveal effective pollinator taxa and timing of visitation to the reproductive success of plants under the complex biological interactions and fluctuating abiotic factors, we developed an automatic system to take photographs at 5-s intervals to get near-complete flower visitation by pollinators during the entire flowering period of selected flowers of Nelumbo nucifera and track the reproductive success of the same flowers until fruiting. Bee visits during the early morning hours of 05:00-07:59 on the second day of flowering under optimal temperatures with no rainfall or strong winds contributed strongly to seed set, with possible indirect negative effects by predators of the pollinators. Our results indicate the availability of periodic and consecutive photography system in clarifying the plant-pollinator interaction and its consequence to reproductive success of the plant. Further development is required to build a monitoring system to collect higher-resolution time-lapse images and automatically identify visiting insect species in the natural environment.

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.

Physcomitrella STEMIN transcription factor induces stem cell formation with epigenetic reprogramming.

Epigenetic modifications, including histone modifications, stabilize cell-specific gene expression programmes to maintain cell identities in both metazoans and land plants1-3. Notwithstanding the existence of these stable cell states, in land plants, stem cells are formed from differentiated cells during post-embryonic development and regeneration4-6, indicating that land plants have an intrinsic ability to regulate epigenetic memory to initiate a new gene regulatory network. However, it is less well understood how epigenetic modifications are locally regulated to influence the specific genes necessary for cellular changes without affecting other genes in a genome. In this study, we found that ectopic induction of the AP2/ERF transcription factor STEMIN1 in leaf cells of the moss Physcomitrella patens decreases a repressive chromatin mark, histone H3 lysine 27 trimethylation (H3K27me3), on its direct target genes before cell division, resulting in the conversion of leaf cells to chloronema apical stem cells. STEMIN1 and its homologues positively regulate the formation of secondary chloronema apical stem cells from chloronema cells during development. Our results suggest that STEMIN1 functions within an intrinsic mechanism underlying local H3K27me3 reprogramming to initiate stem cell formation.

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.

Florigen and anti-florigen: flowering regulation in horticultural crops.

Flowering time regulation has significant effects on the agricultural and horticultural industries. Plants respond to changing environments and produce appropriate floral inducers (florigens) or inhibitors (anti-florigens) that determine flowering time. Recent studies have demonstrated that members of two homologous proteins, FLOWERING LOCUS T (FT) and TERMINAL FLOWER 1 (TFL1), act as florigen and anti-florigen, respectively. Studies in diverse plant species have revealed universal but diverse roles of the FT/TFL1 gene family in many developmental processes. Recent studies in several crop species have revealed that modification of flowering responses, either due to mutations in the florigen/anti-florigen gene itself, or by modulation of the regulatory pathway, is crucial for crop domestication. The FT/TFL1 gene family could be an important potential breeding target in many crop species.

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.

In vivo imaging of cortical interneurons migrating in the intermediate/subventricular zones.

We developed an imaging system that enables migrating cortical interneurons (CIs) through the lower intermediate zone/subventricular zone (IZ/SVZ) in mouse embryos. CIs were labeled by in utero electroporation performed at embryonic day (E) 11.5 and were observed, through the skull of living embryos, detached from the dam with the umbilical cord remain attached. To identify imaged cell locations, we used GAD67-GFP mice and GFP fluorescence was photo-bleached after the recording. We found that CIs in the IZ/SVZ migrated medially straight toward the midline on the tangential plane, while those in the marginal zone migrated in all directions.

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.

Constitutive expression of the GIGANTEA ortholog affects circadian rhythms and suppresses one-shot induction of flowering in Pharbitis nil, a typical short-day plant.

GIGANTEA (GI) is a key regulator of flowering time, which is closely related to the circadian clock function in Arabidopsis. Mutations in the GI gene cause photoperiod-insensitive flowering and altered circadian rhythms. We isolated the GI ortholog PnGI from Pharbitis (Ipomoea) nil, an absolute short-day (SD) plant. PnGI mRNA expression showed diurnal rhythms that peaked at dusk under SD and long-day (LD) conditions, and also showed robust circadian rhythms under continuous dark (DD) and continuous light (LL) conditions. Short irradiation with red light during the flower-inductive dark period did not change PnGI expression levels, suggesting that such a night break does not abolish flowering by affecting the expression of PnGI. In Pharbitis, although a single dusk signal is sufficient to induce expression of the ortholog of FLOWERING LOCUS T (PnFT1), PnGI mRNA expression was not reset by single lights-off signals. Constitutive expression of PnGI (PnGI-OX) in transgenic plants altered period length in leaf-movement rhythms under LL and affected circadian rhythms of PnFT mRNA expression under DD. PnGI-OX plants formed fewer flower buds than the wild type when one-shot darkness was given. In PnGI-OX plants, expression of PnFT1 was down-regulated, suggesting that PnGI functions as a suppressor of flowering, possibly in part through down-regulation of PnFT1.