An improved method for the highly specific detection of transcription start sites.

Precise detection of the transcriptional start site (TSS) is a key for characterizing transcriptional regulation of genes and for annotation of newly sequenced genomes. Here, we describe the development of an improved method, designated 'TSS-seq2.' This method is an iterative improvement of TSS-seq, a previously published enzymatic cap-structure conversion method to detect TSSs in base sequences. By modifying the original procedure, including by introducing split ligation at the key cap-selection step, the yield and the accuracy of the reaction has been substantially improved. For example, TSS-seq2 can be conducted using as little as 5 ng of total RNA with an overall accuracy of 96%; this yield a less-biased and more precise detection of TSS. We then applied TSS-seq2 for TSS analysis of four plant species that had not yet been analyzed by any previous TSS method.

A transcriptional program underlying the circannual rhythms of gonadal development in medaka.

To cope with seasonal environmental changes, organisms have evolved approximately 1-y endogenous circannual clocks. These circannual clocks regulate various physiological properties and behaviors such as reproduction, hibernation, migration, and molting, thus providing organisms with adaptive advantages. Although several hypotheses have been proposed, the genes that regulate circannual rhythms and the underlying mechanisms controlling long-term circannual clocks remain unknown in any organism. Here, we show a transcriptional program underlying the circannual clock in medaka fish (Oryzias latipes). We monitored the seasonal reproductive rhythms of medaka kept under natural outdoor conditions for 2 y. Linear regression analysis suggested that seasonal changes in reproductive activity were predominantly determined by an endogenous program. Medaka hypothalamic and pituitary transcriptomes were obtained monthly over 2 y and daily on all equinoxes and solstices. Analysis identified 3,341 seasonally oscillating genes and 1,381 daily oscillating genes. We then examined the existence of circannual rhythms in medaka via maintaining them under constant photoperiodic conditions. Medaka exhibited approximately 6-mo free-running circannual rhythms under constant conditions, and monthly transcriptomes under constant conditions identified 518 circannual genes. Gene ontology analysis of circannual genes highlighted the enrichment of genes related to cell proliferation and differentiation. Altogether, our findings support the "histogenesis hypothesis" that postulates the involvement of tissue remodeling in circannual time-keeping.

Seasonal pigment fluctuation in diploid and polyploid Arabidopsis revealed by machine learning-based phenotyping method PlantServation.

Long-term field monitoring of leaf pigment content is informative for understanding plant responses to environments distinct from regulated chambers but is impractical by conventional destructive measurements. We developed PlantServation, a method incorporating robust image-acquisition hardware and deep learning-based software that extracts leaf color by detecting plant individuals automatically. As a case study, we applied PlantServation to examine environmental and genotypic effects on the pigment anthocyanin content estimated from leaf color. We processed >4 million images of small individuals of four Arabidopsis species in the field, where the plant shape, color, and background vary over months. Past radiation, coldness, and precipitation significantly affected the anthocyanin content. The synthetic allopolyploid A. kamchatica recapitulated the fluctuations of natural polyploids by integrating diploid responses. The data support a long-standing hypothesis stating that allopolyploids can inherit and combine the traits of progenitors. PlantServation facilitates the study of plant responses to complex environments termed "in natura".

The evolution of the hypotetraploid Catolobus pendulus genome - the poorly known sister species of Capsella.

The establishment of Arabidopsis as the most important plant model has also brought other crucifer species into the spotlight of comparative research. While the genus Capsella has become a prominent crucifer model system, its closest relative has been overlooked. The unispecific genus Catolobus is native to temperate Eurasian woodlands, from eastern Europe to the Russian Far East. Here, we analyzed chromosome number, genome structure, intraspecific genetic variation, and habitat suitability of Catolobus pendulus throughout its range. Unexpectedly, all analyzed populations were hypotetraploid (2n = 30, ~330 Mb). Comparative cytogenomic analysis revealed that the Catolobus genome arose by a whole-genome duplication in a diploid genome resembling Ancestral Crucifer Karyotype (ACK, n = 8). In contrast to the much younger Capsella allotetraploid genomes, the presumably autotetraploid Catolobus genome (2n = 32) arose early after the Catolobus/Capsella divergence. Since its origin, the tetraploid Catolobus genome has undergone chromosomal rediploidization, including a reduction in chromosome number from 2n = 32 to 2n = 30. Diploidization occurred through end-to-end chromosome fusion and other chromosomal rearrangements affecting a total of six of 16 ancestral chromosomes. The hypotetraploid Catolobus cytotype expanded toward its present range, accompanied by some longitudinal genetic differentiation. The sister relationship between Catolobus and Capsella allows comparative studies of tetraploid genomes of contrasting ages and different degrees of genome diploidization.

Distinct responses to autumn and spring temperatures by the key flowering-time regulator FLOWERING LOCUS C.

Despite the similarity in temperature regimes between late autumn and early spring, plants exhibit distinct developmental responses that result in distinct morphologies, that is, overwintering and reproductive forms. In Arabidopsis, the control of autumn-spring distinction involves the transcriptional regulation of the floral repressor FLOWERING LOCUS C (FLC). The memory of winter cold is registered as epigenetic silencing of FLC. Recent studies on A. thaliana FLC revealed detailed and additional mechanisms of silencing in response to autumn and winter cold. Studies on perennial Arabidopsis FLC revealed that its expression responds to spring warmth and is robustly upregulated, ignoring cold. These new studies provide mechanistic insights into the distinct regulation of FLC under autumn and spring temperature regimes.

Effects of fruit dimorphism on genetic structure and gene flow in the coastal shrub Scaevola taccada.

BACKGROUND AND AIMS: Plant propagules often possess specialized morphologies that facilitate dispersal across specific landscapes. In the fruit dimorphism of a coastal shrub, Scaevola taccada, individual plants produce either cork-morph or pulp-morph fruits. The former is buoyant and common on sandy beaches, whereas the latter does not float, is bird-dispersed, and is common on elevated sites such as slopes on sea cliffs and behind rocky shores. We hypothesized that beach populations bridge the heterogeneous landscapes by serving as a source of both fruit types, while dispersal is biased for the pulp morph on elevated sites within the islands and for the cork morph between beaches of different islands. Based on this hypothesis, we predicted that populations in elevated sites would diverge genetically over time due to isolation by distance, whereas beach populations would maintain high genetic similarity via current gene flow. METHODS: The genetic structure and gene flow in S. taccada were evaluated by investigating genome-wide single nucleotide polymorphisms in plants from 17 sampling sites on six islands (belonging to the Ryukyu, Daito and Ogasawara Islands) in Japan. KEY RESULTS: Geographical isolation was detected among the three distant island groups. Analyses within the Ryukyu Islands suggested that sandy beach populations were characterized by genetic admixture, whereas populations in elevated sites were relatively isolated between the islands. Pairwise FST values between islands were lowest between sandy beaches, intermediate between sandy beaches and elevated sites, and highest between elevated sites. CONCLUSIONS: Dispersal across the ocean by cork morphs is sufficiently frequent to prevent genetic divergence between beaches of different islands. Stronger genetic isolation of elevated sites between islands suggests that bird dispersal by pulp morphs is restricted mainly within islands. These contrasting patterns of gene flow realized by fruit dimorphism provide evidence that fruit characteristics can strongly mediate genetic structure.

Circadian-period variation underlies the local adaptation of photoperiodism in the short-day plant Lemna aequinoctialis.

Phenotypic variation is the basis for trait adaptation via evolutionary selection. However, the driving forces behind quantitative trait variations remain unclear owing to their complexity at the molecular level. This study focused on the natural variation of the free-running period (FRP) of the circadian clock because FRP is a determining factor of the phase phenotype of clock-dependent physiology. Lemna aequinoctialis in Japan is a paddy field duckweed that exhibits a latitudinal cline of critical day length (CDL) for short-day flowering. We collected 72 strains of L. aequinoctialis and found a significant correlation between FRPs and locally adaptive CDLs, confirming that variation in the FRP-dependent phase phenotype underlies photoperiodic adaptation. Diel transcriptome analysis revealed that the induction timing of an FT gene is key to connecting the clock phase to photoperiodism at the molecular level. This study highlights the importance of FRP as a variation resource for evolutionary adaptation.

Combination of genetic analysis and ancient literature survey reveals the divergence of traditional Brassica rapa varieties from Kyoto, Japan.

Since ancient times, humans have bred several plants that we rely on today. However, little is known about the divergence of most of these plants. In the present study, we investigated the divergence of Mibuna (Brassica rapa L. subsp. nipposinica L. H. Bailey), a traditional leafy vegetable in Kyoto (Japan), by combining genetic analysis and a survey of ancient literature. Mibuna is considered to have been bred 200 years ago from Mizuna, another traditional leafy vegetable in Kyoto. Mibuna has simple spatulate leaves, whereas Mizuna has characteristic serrated leaves. The quantitative trait loci (QTL) and gene expression analyses suggested that the downregulation of BrTCP15 expression contributed to the change in the leaf shape from serrated to simple spatulate. Interestingly, the SNP analysis indicated that the genomic region containing the BrTCP15 locus was transferred to Mibuna by introgression. Furthermore, we conducted a survey of ancient literature to reveal the divergence of Mibuna and found that hybridization between Mizuna and a simple-leaved turnip might have occurred in the past. Indeed, the genomic analysis of multiple turnip cultivars showed that one of the cultivars, Murasakihime, has almost the same sequence in the BrTCP15 region as Mibuna. These results suggest that the hybridization between Mizuna and turnip has resulted in the establishment of Mibuna.

The Flowering Season-Meter at FLOWERING LOCUS C Across Life Histories in Crucifers.

Many plant species overwinter before they flower. Transition to flowering is aligned to the seasonal transition as a response to the prolonged cold in winter by a process called vernalization. Multiple well-documented vernalization properties in crucifer species with diverse life histories are derived from environmental regulation of a central inhibitor of the flowering gene, Flowering Locus C (FLC). Episode(s) of flowering are prevented during high FLC expression and enabled during low FLC expression. FLC repression outlasts the winter to coincide with spring; this heterochronic aspect is termed "winter memory." In the annual Arabidopsis thaliana, winter memory has long been associated with the highly conserved histone modifiers Polycomb and Trithorax, which have antagonistic roles in transcription. However, there are experimental limitations in determining how dynamic, heterogenous histone modifications within the FLC locus generate the final transcriptional output. Recent theoretical considerations on cell-to-cell variability in gene expression and histone modifications generating bistable states brought support to the hypothesis of chromatin-encoded memory, as with other experimental systems in eukaryotes. Furthermore, these advances unify multiple properties of vernalization, not only the winter memory. Similarly, in the perennial Arabidopsis halleri ssp. gemmifera, recent integration of molecular with mathematical and ecological approaches unifies FLC chromatin features with the all-year-round memory of seasonal temperature. We develop the concept of FLC season-meter to combine existing information from the contrasting annual/perennial and experimental/theoretical sectors into a transitional framework. We highlight simplicity, high conservation, and discrete differences across extreme life histories in crucifers.

Altitudinal differentiation in the leaf wax-mediated flowering bud protection against frost in a perennial Arabidopsis.

An altitudinal gradient of leaf water repellency is often observed between and within species. In a previous study of Arabidopsis halleri, cauline leaves (stem leaves that wrap flowering buds) showed higher water repellency in exposed semi-alpine plants than in understory low-elevation plants. Here, we examined altitudinal variations in the cuticular wax content of the leaf surface and experimentally evaluated the role of high water repellency of cauline leaves. Leaf cuticular wax was analysed using comprehensive two-dimensional gas chromatography (GC)-mass spectrometry and a GC-flame ionisation detector. Young flowering buds wrapped by cauline leaves were exposed to freezing temperatures with or without water, and frost damage to the flowering buds was compared between plants from semi-alpine and low-elevation habitats. Higher amounts of C29, C31, and C33 alkanes were observed in the cauline leaves of semi-alpine plants than in those of low-elevation plants. In the freezing experiment, water application increased damage to the flowering buds of low-elevation plants, and the extent of damage to the flowering buds was lower in semi-alpine plants than in low-elevation plants when water was applied to the plant surface. Genetic variations in the amounts of alkanes on the leaf surface depending on the altitude occurred specifically in cauline leaves. Our results indicate that the water repellency of cauline leaves presumably minimises frost damage to flowering buds at high altitudes.

Phylogeographic and demographic modeling analyses of the multiple origins of the rheophytic goldenrod Solidago yokusaiana Makino.

Understanding adaptation mechanisms is important in evolutionary biology. Parallel adaptation provides good opportunities to investigate adaptive evolution. To confirm parallel adaptation, it is effective to examine whether the phenotypic similarity has one or multiple origins and to use demographic modeling to consider the gene flow between ecotypes. Solidago yokusaiana is a rheophyte endemic to the Japanese Archipelago that diverged from Solidago virgaurea. This study examined the parallel origins of S. yokusaiana by distinguishing between multiple and single origins and subsequent gene flow. The haplotypes of noncoding chloroplast DNA and genotypes at 14 nuclear simple sequence repeat (nSSR) loci and single-nucleotide polymorphisms (SNPs) revealed by double-digest restriction-associated DNA sequencing (ddRADseq) were used for phylogeographic analysis; the SNPs were also used to model population demographics. Some chloroplast haplotypes were common to S. yokusaiana and its ancestor S. virgaurea. Also, the population genetic structures revealed by nSSR and SNPs did not correspond to the taxonomic species. The demographic modeling supported the multiple origins of S. yokusaiana in at least four districts and rejected a single origin with ongoing gene flow between the two species, implying that S. yokusaiana independently and repeatedly adapted to frequently flooding riversides.

Fine-scale empirical data on niche divergence and homeolog expression patterns in an allopolyploid and its diploid progenitor species.

Polyploidization is pervasive in plants, but little is known about the niche divergence of wild allopolyploids (species that harbor polyploid genomes originating from different diploid species) relative to their diploid progenitor species and the gene expression patterns that may underlie such ecological divergence. We conducted a fine-scale empirical study on habitat and gene expression of an allopolyploid and its diploid progenitors. We quantified soil properties and light availability of habitats of an allotetraploid Cardamine flexuosa and its diploid progenitors Cardamine amara and Cardamine hirsuta in two seasons. We analyzed expression patterns of genes and homeologs (homeologous gene copies in allopolyploids) using RNA sequencing. We detected niche divergence between the allopolyploid and its diploid progenitors along water availability gradient at a fine scale: the diploids in opposite extremes and the allopolyploid in a broader range between diploids, with limited overlap with diploids at both ends. Most of the genes whose homeolog expression ratio changed among habitats in C. flexuosa varied spatially and temporally. These findings provide empirical evidence for niche divergence between an allopolyploid and its diploid progenitor species at a fine scale and suggest that divergent expression patterns of homeologs in an allopolyploid may underlie its persistence in diverse habitats.

Duration of cold exposure defines the rate of reactivation of a perennial FLC orthologue via H3K27me3 accumulation.

Vernalisation is the process in which long-term cold exposure makes plants competent to flower. In vernalisation of Arabidopsis thaliana, a floral repressor, AtFLC, undergoes epigenetic silencing. Although the silencing of AtFLC is maintained under warm conditions after a sufficient duration of cold, FLC orthologues are reactivated under the same conditions in perennial plants, such as A. halleri. In contrast to the abundant knowledge on cold requirements in AtFLC silencing, it has remained unknown how cold duration affects the reactivation of perennial FLC. Here, we analysed the dynamics of A. halleri FLC (AhgFLC) mRNA, H3K4me3, and H3K27me3 over 8 weeks and 14 weeks cold followed by warm conditions. We showed that the minimum levels of AhgFLC mRNA and H3K4me3 were similar between 8 and 14 weeks vernalisation; however, the maximum level of H3K27me3 was higher after 14 weeks than after 8 weeks vernalisation. Combined with mathematical modelling, we showed that H3K27me3 prevents a rapid increase in AhgFLC expression in response to warm temperatures after vernalisation, which controls AhgFT expression and the initiation of flowering. Thus, the duration of cold defines the rate of AhgFLC reactivation via the buffering function of H3K27me3 against temperature increase.

Altered stomatal patterning accompanies a trichome dimorphism in a natural population of Arabidopsis.

Trichomes are large epidermal cells on the surface of leaves that are thought to deter herbivores, yet the presence of trichomes can also negatively impact plant growth and reproduction. Stomatal guard cells and trichomes have shared developmental origins, and experimental manipulation of trichome formation can lead to changes in stomatal density. The influence of trichome formation upon stomatal development in natural populations of plants is currently unknown. Here, we show that a natural population of Arabidopsis halleri that includes hairy (trichome-bearing) and glabrous (no trichomes) morphs has differences in stomatal density that are associated with this trichome dimorphism. We found that glabrous morphs had significantly greater stomatal density and stomatal index than hairy morphs. One interpretation is that this arises from a trade-off between the proportions of cells that have trichome and guard cell fates during leaf development. The differences in stomatal density between the two morphs might have impacts upon environmental adaptation, in addition to herbivory deterrence caused by trichome development.

Seasonal plasticity and diel stability of H3K27me3 in natural fluctuating environments.

Diel and seasonal oscillations are two major environmental changes in nature. While organisms cope with the former by the well-characterized mechanism of the circadian clock1,2, there is limited information on the molecular mechanisms underlying long-term responses to the latter3-5. Histone H3 lysine 27 trimethylation (H3K27me3), a repressive histone modification, imparts stability and plasticity to gene regulation during developmental transitions6-9. Here we studied the seasonal and diel dynamics of H3K27me3 at the genome-wide level in a natural population of perennial Arabidopsis halleri and compared these dynamics with those of histone H3 lysine 4 trimethylation (H3K4me3), an active histone modification. Chromatin immunoprecipitation sequencing revealed that H3K27me3 exhibits seasonal plasticity and diel stability. Furthermore, we found that the seasonal H3K27me3 oscillation is delayed in phase relative to the H3K4me3 oscillation, particularly for genes associated with environmental memory. Our findings suggest that H3K27me3 monitors past transcriptional activity to create long-term gene expression trends during organismal responses over weeks in natural fluctuating environments.

Characterization of rhizome transcriptome and identification of a rhizomatous ER body in the clonal plant Cardamine leucantha.

The rhizome is a plant organ that develops from a shoot apical meristem but penetrates into belowground environments. To characterize the gene expression profile of rhizomes, we compared the rhizome transcriptome with those of the leaves, shoots and roots of a rhizomatous Brassicaceae plant, Cardamine leucantha. Overall, rhizome transcriptomes were characterized by the absence of genes that show rhizome-specific expression and expression profiles intermediate between those of shoots and roots. Our results suggest that both endogenous developmental factors and external environmental factors are important for controlling the rhizome transcriptome. Genes that showed relatively high expression in the rhizome compared to shoots and roots included those related to belowground defense, control of reactive oxygen species and cell elongation under dark conditions. A comparison of transcriptomes further allowed us to identify the presence of an ER body, a defense-related belowground organelle, in epidermal cells of the C. leucantha rhizome, which is the first report of ER bodies in rhizome tissue.

Complete genome sequence of a novel partitivirus from a wild brassicaceous plant, Arabidopsis halleri.

Two contigs with high similarity to partitivirus sequences were identified by de novo assembly of sequences obtained by RNA-Seq from a wild brassicaceous plant, Arabidopsis halleri subsp. gemmifera. Here, we report the complete genome sequence of a putative novel partitivirus. Excluding the poly-A tail, it consists of two RNA genome segments of 1912 and 1769 bp, which are predicted to encode a 585-amino-acid-long putative RNA-dependent RNA polymerase (RdRp) and a 487-amino-acid-long putative capsid protein (CP), respectively. Phylogenetically, this virus belongs to the genus Alphapartitivirus and is most closely related to Raphanus sativus partitivirus 1 from radish. We propose the name "Arabidopsis halleri partitivirus 1" (AhPV1) for this novel virus.

Repressive chromatin modification underpins the long-term expression trend of a perennial flowering gene in nature.

Natural environments require organisms to possess robust mechanisms allowing responses to seasonal trends. In Arabidopsis halleri, the flowering regulator AhgFLC shows upregulation and downregulation phases along with long-term past temperature, but the underlying machinery remains elusive. Here, we investigate the seasonal dynamics of histone modifications, H3K27me3 and H3K4me3, at AhgFLC in a natural population. Our advanced modelling and transplant experiments reveal that H3K27me3-mediated chromatin regulation at AhgFLC provides two essential properties. One is the ability to respond to the long-term temperature trends via bidirectional interactions between H3K27me3 and H3K4me3; the other is the ratchet-like character of the AhgFLC system, i.e. reversible in the entire perennial life cycle but irreversible during the upregulation phase. Furthermore, we show that the long-term temperature trends are locally indexed at AhgFLC in the form of histone modifications. Our study provides a more comprehensive understanding of H3K27me3 function at AhgFLC in a complex natural environment.