Vernalization Response Research Articles

A GWAS study highlights significant associations between a series of indels in a FLOWERING LOCUS T gene promoter and flowering time in white lupin (Lupinus albus L.)

BackgroundWhite lupin (Lupinus albus L.) is a high-protein Old World grain legume with remarkable food and feed production interest. It is sown in autumn or early spring, depending on the local agroclimatic conditions. This study aimed to identify allelic variants associated with vernalization responsiveness, in order to improve our knowledge of legume flowering regulatory pathways and develop molecular selection tools for the desired phenology as required for current breeding and adaptation to the changing climate.ResultsSome 120 white lupin accessions originating from a wide range of environments of Europe, Africa, and Asia were phenotyped under field conditions in three environments with different intensities of vernalization, namely, a Mediterranean and a subcontinental climate sites of Italy under autumn sowing, and a suboceanic climate site of France under spring sowing. Two hundred sixty-two individual genotypes extracted from them were phenotyped in a greenhouse under long-day photoperiod without vernalization. Phenology data, and marker data generated by Diversity Arrays Technology sequencing (DArT-seq) and by PCR-based screening targeting published quantitative trait loci (QTLs) from linkage map and newly identified insertion/deletion polymorphisms in the promoter region of the FLOWERING LOCUS T homolog, LalbFTc1 gene (Lalb_Chr14g0364281), were subjected to a genome-wide association study (GWAS). Population structure followed differences in phenology and isolation by distance pattern. The GWAS highlighted numerous loci significantly associated with flowering time, including four LalbFTc1 gene promoter deletions: 2388 bp and 2126 bp deletions at the 5’ end, a 264 bp deletion in the middle and a 28 bp deletion at the 3’ end of the promoter. Besides LalbFTc1 deletions, this set contained DArT-seq markers that matched previously published major QTLs in chromosomes Lalb_Chr02, Lalb_Chr13 and Lalb_Chr16, and newly discovered QTLs in other chromosomes.ConclusionsThis study highlighted novel QTLs for flowering time and validated those already published, thereby providing novel evidence on the convergence of FTc1 gene functional evolution into the vernalization pathway in Old World lupin species. Moreover, this research provided the set of loci specific for extreme phenotypes (the earliest or the latest) awaiting further implementation in marker-assisted selection for spring- or winter sowing.

Distinct roles of H3K27me3 and H3K36me3 in vernalization response, maintenance, and resetting in winter wheat.

Winter plants rely on vernalization, a crucial process for adapting to cold conditions and ensuring successful reproduction. However, understanding the role of histone modifications in guiding the vernalization process in winter wheat remains limited. In this study, we investigated the transcriptome and chromatin dynamics in the shoot apex throughout the life cycle of winter wheat in the field. Two core histone modifications, H3K27me3 and H3K36me3, exhibited opposite patterns on the key vernalization gene VERNALIZATION1 (VRN1), correlating with its induction during cold exposure. Moreover, the H3K36me3 level remained high at VRN1 after cold exposure, which may maintain its active state. Mutations in FERTILIZATION-INDEPENDENT ENDOSPERM (TaFIE) and SET DOMAIN GROUP 8/EARLY FLOWERING IN SHORT DAYS (TaSDG8/TaEFS), components of the writer complex for H3K27me3 and H3K36me3, respectively, affected flowering time. Intriguingly, VRN1 lost its high expression after the cold exposure memory in the absence of H3K36me3. During embryo development, VRN1 was silenced with the removal of active histone modifications in both winter and spring wheat, with selective restoration of H3K27me3 in winter wheat. The mutant of Tafie-cr-87, a component of H3K27me3 "writer" complex, did not influence the silence of VRN1 during embryo development, but rather attenuated the cold exposure requirement of winter wheat. Integrating gene expression with H3K27me3 and H3K36me3 patterns identified potential regulators of flowering. This study unveils distinct roles of H3K27me3 and H3K36me3 in controlling vernalization response, maintenance, and resetting in winter wheat.

Epigenetic control on transcription of vernalization genes and whole-genome gene expression profile induced by vernalization in common wheat

Vernalization is necessary for winter wheat to flower. However, it is unclear whether vernalization is also required for spring wheat, which is frequently sown in fall, and what molecular mechanisms underlie the vernalization response in wheat varieties. In this study, we examined the molecular mechanisms that regulate vernalization response in winter and spring wheat varieties. For this purpose, we determined how major vernalization genes (VRN1, VRN2, and VRN3) respond to vernalization in these varieties and whether modifications to histones play a role in changes in gene expression. We also identified genes that are differentially regulated in response to vernalization in winter and spring wheat varieties. We found that in winter wheat, but not in spring wheat, VRN1 expression decreases when returned to warm temperature following vernalization. This finding may be associated with differences between spring and winter wheat in the levels of tri-methylation of lysine 27 on histone H3 (H3K27me3) and tri-methylation of lysine 4 on histone H3 (H3K4me3) at the VRN1 gene. Analysis of winter wheat transcriptomes before and after vernalization revealed that vernalization influences the expression of several genes, including those involved in leucine catabolism, cysteine biosynthesis, and flavonoid biosynthesis. These findings provide new candidates for further study on the mechanism of vernalization regulation in wheat.

Epigenetic variation in early and late flowering plants of the rubber-producing Russian dandelion Taraxacum koksaghyz provides insights into the regulation of flowering time

The Russian dandelion (Taraxacum koksaghyz) grows in temperate zones and produces large amounts of poly(cis-1,4-isoprene) in its roots, making it an attractive alternative source of natural rubber. Most T. koksaghyz plants require vernalization to trigger flower development, whereas early flowering varieties that have lost their vernalization dependence are more suitable for breeding and domestication. To provide insight into the regulation of flowering time in T. koksaghyz, we induced epigenetic variation by in vitro cultivation and applied epigenomic and transcriptomic analysis to the resulting early flowering plants and late flowering controls, allowing us to identify differences in methylation patterns and gene expression that correlated with flowering. This led to the identification of candidate genes homologous to vernalization and photoperiodism response genes in other plants, as well as epigenetic modifications that may contribute to the control of flower development. Some of the candidate genes were homologous to known floral regulators, including those that directly or indirectly regulate the major flowering control gene FT. Our atlas of genes can be used as a starting point to investigate mechanisms that control flowering time in T. koksaghyz in greater detail and to develop new breeding varieties that are more suited to domestication.

A transcriptomic time-series reveals differing trajectories during pre-floral development in the apex and leaf in winter and spring varieties of Brassica napus

Oilseed rape (Brassica napus) is an important global oil crop, with spring and winter varieties grown commercially. To understand the transcriptomic differences between these varieties, we collected transcriptomes from apex and leaf tissue from a spring variety, Westar, and a winter variety, Tapidor, before, during, and after vernalisation treatment, until the plants flowered. Large transcriptomic differences were noted in both varieties during the vernalisation treatment because of temperature and day length changes. Transcriptomic alignment revealed that the apex transcriptome reflects developmental state, whereas the leaf transcriptome is more closely aligned to the age of the plant. Similar numbers of copies of genes were expressed in both varieties during the time series, although key flowering time genes exhibited expression pattern differences. BnaFLC copies on A2 and A10 are the best candidates for the increased vernalisation requirement of Tapidor. Other BnaFLC copies show tissue-dependent reactivation of expression post-cold, with these dynamics suggesting some copies have retained or acquired a perennial nature. BnaSOC1 genes, also related to the vernalisation pathway, have expression profiles which suggest tissue subfunctionalisation. This understanding may help to breed varieties with more consistent or robust vernalisation responses, of special importance due to the milder winters resulting from climate change.

Identification of vernalization-related genes and cold memory element (CME) required for vernalization response in radish (Raphanus sativus L.).

Floral transition is accelerated by exposure to long-term cold like winter in plants, which is called as vernalization. Acceleration of floral transition by vernalization is observed in a diversity of biennial and perennial plants including Brassicaceae family plants. Scientific efforts to understand molecular mechanism underlying vernalization-mediated floral transition have been intensively focused in model plant Arabidopsis thaliana. To get a better understanding on floral transition by vernalization in radish (Raphanus sativus L.), we investigated transcriptomic changes taking place during vernalization in radish. Thousands of genes were differentially regulated along time course of vernalization compared to non-vernalization (NV) sample. Twelve major clusters of DEGs were identified based on distinctive expression profiles during vernalization. Radish FLC homologs were shown to exert an inhibition of floral transition when transformed into Arabidopsis plants. In addition, DNA region containing RY motifs located within a Raphanus sativus FLC homolog, RsFLC1 was found to be required for repression of RsFLC1 by vernalization. Transgenic plants harboring disrupted RY motifs were impaired in the enrichment of H3K27me3 on RsFLC1 chromatin, thus resulting in the delayed flowering in Arabidopsis. Taken together, we report transcriptomic profiles of radish during vernalization and demonstrate the requirement of RY motif for vernalization-mediated repression of RsFLC homologs in radish (Raphanus sativus L.).

Allelic diversity of the Vrn genes and the control of growth habit and earliness in wheat.

Wheat is one of three main food crops around the world, which has the largest distribution area due to its adaptation to the different environments. This review considers polymorphisms and allelic variation of the vernalization response genes Vrn controlling the major adaptation traits in wheats (the genus Triticum L.): growth habit (spring vs. winter) and length of vegetative period (earliness). The review summarizes available information on the allelic diversity of the Vrn genes and discusses molecular-level relationships between Vrn polymorphisms and their effect on growth habit (spring vs. winter) and earliness (length vegetative period in spring plants) in di-, tetra- and hexaploid wheat species. A unique attempt has been made to relate information on mutations (polymorphisms) in dominant Vrn alleles to the values of the commercially most important trait "length of plant vegetative period (earliness)". The effects of mutations (polymorphisms) in the recessive vrn genes on vernalization requirement in winter wheats are considered, and this trait was formalized. The evolution of the winter/spring growth habit in the genus Triticum species is discussed. A scheme of phylogenetic interactions between Vrn alleles was constructed on the basis of these polymorphisms; the paper considers the possibilities to enhance the diversity of polymorphisms for the dominant Vrn genes and their alleles using wheat related species and rarely used alleles and discusses the prospects of breeding for improved earliness for concrete agroecological zones.

Modeling the Flowering Activation Motif during Vernalization in Legumes: A Case Study of M. trancatula.

In many plant species, flowering is promoted by the cold treatment or vernalization. The mechanism of vernalization-induced flowering has been extensively studied in Arabidopsis but remains largely unknown in legumes. The orthologs of the FLC gene, a major regulator of vernalization response in Arabidopsis, are absent or non-functional in the vernalization-sensitive legume species. Nevertheless, the legume integrator genes FT and SOC1 are involved in the transition of the vernalization signal to meristem identity genes, including PIM (AP1 ortholog). However, the regulatory contribution of these genes to PIM activation in legumes remains elusive. Here, we presented the theoretical and data-driven analyses of a feed-forward regulatory motif that includes a vernalization-responsive FT gene and several SOC1 genes, which independently activate PIM and thereby mediate floral transition. Our theoretical model showed that the multiple regulatory branches in this regulatory motif facilitated the elimination of no-sense signals and amplified useful signals from the upstream regulator. We further developed and analyzed four data-driven models of PIM activation in Medicago trancatula in vernalized and non-vernalized conditions in wild-type and fta1-1 mutants. The model with FTa1 providing both direct activation and indirect activation via three intermediate activators, SOC1a, SOC1b, and SOC1c, resulted in the most relevant PIM dynamics. In this model, the difference between regulatory inputs of SOC1 genes was nonessential. As a result, in the M. trancatula model, the cumulative action of SOC1a, SOC1b, and SOC1c was favored. Overall, in this study, we first presented the in silico analysis of vernalization-induced flowering in legumes. The considered vernalization network motif can be supplemented with additional regulatory branches as new experimental data become available.

Mapping QTL for vernalization requirement identified adaptive divergence of the candidate gene Flowering Locus C in polyploid Camelina sativa.

Vernalization requirement is an integral component of flowering in winter-type plants. The availability of winter ecotypes among Camelina species facilitated the mapping of quantitative trait loci (QTL) for vernalization requirement in Camelina sativa. An inter and intraspecific crossing scheme between related Camelina species, where one spring and two different sources of winter-type habit were used, resulted in the development of two segregating populations. Linkage maps generated with sequence-based markers identified three QTLs associated with vernalization requirement in C. sativa; two from the interspecific (chromosomes 13 and 20) and one from the intraspecific cross (chromosome 8). Notably, the three loci were mapped to different homologous regions of the hexaploid C. sativa genome. All three QTLs were found in proximity to Flowering Locus C (FLC), variants of which have been reported to affect the vernalization requirement in plants. Temporal transcriptome analysis for winter-type Camelina alyssum demonstrated reduction in expression of FLC on chromosomes 13 and 20 during cold treatment, which would trigger flowering, since FLC would be expected to suppress floral initiation. FLC on chromosome 8 also showed reduced expression in the C. sativa ssp. pilosa winter parent upon cold treatment, but was expressed at very high levels across all time points in the spring-type C. sativa. The chromosome 8 copy carried a deletion in the spring-type line, which could impact its functionality. Contrary to previous reports, all three FLC loci can contribute to controlling the vernalization response in C. sativa and provide opportunities for manipulating this requirement in the crop.

Long-Term In Situ Conservation Drove Microevolution of Solina d'Abruzzo Wheat on Adaptive, Agronomic and Qualitative Traits.

Solina is an example of a bread wheat landrace that has been conserved in situ for centuries in Central Italy. A core collection of Solina lines sampled in areas at different altitudes and climatic conditions was obtained and genotyped. A clustering analysis based on a wide SNP dataset generated from DArTseq analysis outlined the existence of two main groups, which, after Fst analysis, showed polymorphism in genes associated with vernalization and photoperiod response. Starting from the hypothesis that the different pedoclimatic environments in which Solina lines were conserved may have shaped the population, some phenotypic characteristics were studied in the Solina core collection. Growth habit, low-temperature resistance, allelic variations at major loci involved in vernalization response, and sensitivity to photoperiod were evaluated, together with seed morphologies, grain colour, and hardness. The two Solina groups showed different responses to low temperatures and to photoperiod-specific allelic variations as well as the different morphology and technological characteristics of the grain. In conclusion, the long-term in situ conservation of Solina in environments sited at different altitudes has had an impact on the evolution of this landrace which, despite its high genetic diversity, remains clearly identifiable and distinct so as to be included in conservation varieties.

Vernalization-triggered expression of the antisense transcript COOLAIR is mediated by CBF genes

To synchronize flowering time with spring, many plants undergo vernalization, a floral-promotion process triggered by exposure to long-term winter cold. In Arabidopsis thaliana, this is achieved through cold-mediated epigenetic silencing of the floral repressor, FLOWERING LOCUS C (FLC). COOLAIR, a cold-induced antisense RNA transcribed from the FLC locus, has been proposed to facilitate FLC silencing. Here, we show that C-repeat (CRT)/dehydration-responsive elements (DREs) at the 3'-end of FLC and CRT/DRE-binding factors (CBFs) are required for cold-mediated expression of COOLAIR. CBFs bind to CRT/DREs at the 3'-end of FLC, both in vitro and in vivo, and CBF levels increase gradually during vernalization. Cold-induced COOLAIR expression is severely impaired in cbfs mutants in which all CBF genes are knocked-out. Conversely, CBF-overexpressing plants show increased COOLAIR levels even at warm temperatures. We show that COOLAIR is induced by CBFs during early stages of vernalization but COOLAIR levels decrease in later phases as FLC chromatin transitions to an inactive state to which CBFs can no longer bind. We also demonstrate that cbfs and FLCΔCOOLAIR mutants exhibit a normal vernalization response despite their inability to activate COOLAIR expression during cold, revealing that COOLAIR is not required for the vernalization process.

Transcriptomic Analysis of Flowering Time Genes in Cultivated Chickpea and Wild Cicer.

Chickpea (Cicer arietinum L.) is a major grain legume and a good source of plant-based protein. However, comprehensive knowledge of flowering time control in Cicer is lacking. In this study, we acquire high-throughput transcriptome sequencing data and analyze changes in gene expression during floral transition in the early flowering cultivar ICCV 96029, later flowering C. arietinum accessions, and two wild species, C. reticulatum and C. echinospermum. We identify Cicer orthologs of A. thaliana flowering time genes and analyze differential expression of 278 genes between four species/accessions, three tissue types, and two conditions. Our results show that the differences in gene expression between ICCV 96029 and other cultivated chickpea accessions are vernalization-dependent. In addition, we highlight the role of FTa3, an ortholog of FLOWERING LOCUS T in Arabidopsis, in the vernalization response of cultivated chickpea. A common set of differentially expressed genes was found for all comparisons between wild species and cultivars. The direction of expression change for different copies of the FT-INTERACTING PROTEIN 1 gene was variable in different comparisons, which suggests complex mechanisms of FT protein transport. Our study makes a contribution to the understanding of flowering time control in Cicer, and can provide genetic strategies to further improve this important agronomic trait.

Common evolutionary trajectory of short life-cycle in Brassicaceae ruderal weeds

Weed species are detrimental to crop yield. An understanding of how weeds originate and adapt to field environments is needed for successful crop management and reduction of herbicide use. Although early flowering is one of the weed trait syndromes that enable ruderal weeds to overcome frequent disturbances, the underlying genetic basis is poorly understood. Here, we establish Cardamine occulta as a model to study weed ruderality. By genome assembly and QTL mapping, we identify impairment of the vernalization response regulator gene FLC and a subsequent dominant mutation in the blue-light receptor gene CRY2 as genetic drivers for the establishment of short life cycle in ruderal weeds. Population genomics study further suggests that the mutations in these two genes enable individuals to overcome human disturbances through early deposition of seeds into the soil seed bank and quickly dominate local populations, thereby facilitating their spread in East China. Notably, functionally equivalent dominant mutations in CRY2 are shared by another weed species, Rorippa palustris, suggesting a common evolutionary trajectory of early flowering in ruderal weeds in Brassicaceae.

Contemplation on wheat vernalization.

Vernalization is a period of low non-freezing temperatures, which provides the competence to flower. This mechanism ensures that plants sown before winter develop reproductive organs in more favourable conditions during spring. Such an evolutionary mechanism has evolved in both monocot and eudicot plants. Studies in monocots, represented by temperate cereals like wheat and barley, have identified and proposed the VERNALIZATION1 (VRN1) gene as a key player in the vernalization response. VRN1 belongs to MADS-box transcription factors and is expressed in the leaves and the apical meristem, where it subsequently promotes flowering. Despite substantial research advancement in the last two decades, there are still gaps in our understanding of the vernalization mechanism. Here we summarise the present knowledge of wheat vernalization. We discuss VRN1 allelic variation, review vernalization models, talk VRN1 copy number variation and devernalization phenomenon. Finally, we suggest possible future directions of the vernalization research in wheat.

Wild emmer wheat, the progenitor of modern bread wheat, exhibits great diversity in the VERNALIZATION1 gene.

Wild emmer wheat is an excellent reservoir of genetic variability that can be utilized to improve cultivated wheat to address the challenges of the expanding world population and climate change. Bearing this in mind, we have collected a panel of 263 wild emmer wheat (WEW) genotypes across the Fertile Crescent. The genotypes were grown in different locations and phenotyped for heading date. Genome-wide association mapping (GWAS) was carried out, and 16 SNPs were associated with the heading date. As the flowering time is controlled by photoperiod and vernalization, we sequenced the VRN1 gene, the most important of the vernalization response genes, to discover new alleles. Unlike most earlier attempts, which characterized known VRN1 alleles according to a partial promoter or intron sequences, we obtained full-length sequences of VRN-A1 and VRN-B1 genes in a panel of 95 wild emmer wheat from the Fertile Crescent and uncovered a significant sequence variation. Phylogenetic analysis of VRN-A1 and VRN-B1 haplotypes revealed their evolutionary relationships and geographic distribution in the Fertile Crescent region. The newly described alleles represent an attractive resource for durum and bread wheat improvement programs.

Vernalisation and photoperiod responses of diverse wheat genotypes

Context Wheat (Triticum aestivum L.) adaptation is highly dependent on crop lifecycle duration, particularly the time at which flowering occurs in a specific environment. Frost, low solar radiation, heat and drought can significantly reduce yield if a crop flowers too early or late. Wheat genotypes have different lifecycle durations determined by plant responses to temperature (thermal time accumulation and vernalisation) and photoperiod. These responses are largely controlled by five phenology genes (two PPD1 and three VRN1 genes). Advances in crop phenology modelling suggest that flowering time under field conditions could be accurately predicted with parameters derived from photoperiod and vernalisation responses obtained in controlled environments. Aims This study quantified photoperiod and vernalisation responses of 69 Australian wheat genotypes selected for diversity at the PPD1 and VRN1 loci. Methods Spring and winter genotypes were grown in four controlled environments at a constant temperature of 22°C with photoperiod (17 or 8 h) and vernalisation (0 or 8 weeks) treatments as factors. Key results Thermal time from coleoptile emergence to flowering in spring genotypes was typically decreased more by long photoperiod than by vernalisation; the opposite was true for winter genotypes. Spring genotypes that were sensitive to vernalisation contained a sensitive allele at the Vrn-A1 locus. Conclusions There is large diversity in phenological responses of wheat genotypes to photoperiod and vernalisation, including among those with matching multi-locus genotype. Implications Data from this study will be used to parameterise and test a wheat phenology model in a future study.

Flowering responses of serradella (Ornithopus spp.) and subterranean clover (Trifolium subterraneum L.) to vernalisation and photoperiod and their role in maturity type determination and flowering date stability

Context Serradellas (Ornithopus spp.) are promising alternative annual legumes to subterranean clover (Trifolium subterraneum L.), for permanent, temperate pastures. However, many cultivars exhibit unstable flowering dates across years. This is a risk for seed production and persistence. Aim This study assessed how vernalisation and photoperiod cues determine maturity type and flowering date stability among serradella cultivars. Methods First flower appearance was recorded for early and late maturing cultivars of yellow serradella (Ornithopus compressus L.), French serradella (Ornithopus sativus Brot.) and subterranean clover after exposure to six vernalisation treatments (0, 1, 3, 5, 7 or 9 weeks at 5°C) with subsequent growth under four photoperiods (8, 12, 16 or 20 h). Key results ‘Intrinsic earliness’ differed by only zero to three nodes for cultivars within species, indicating that maturity type was determined primarily by a cultivar’s responses to vernalisation and photoperiod. An interaction between these responses was observed, with a precipitous decline in the requirement for vernalisation when photoperiods exceeded 12 h. Many cultivars also displayed a persistent component to their vernalisation response, whereby long photoperiods (20 h) did not completely negate the response to vernalisation. Conclusions Later maturity was associated particularly with need for long exposure to the vernalisation treatment to minimise the duration from sowing to first flower appearance. Stable flowering is more likely when a cultivar has components of its vernalisation requirement that are not satisfied before autumn ends to prevent premature flowering, and a photoperiod response in spring that overrides any unmet vernalisation requirement. Implications Persistence by serradella cultivars requires selection for suitable responsiveness to vernalisation and photoperiod.

Physiological and transcriptome analyses to infer regulatory networks in flowering transition of<i> Rosa rugosa</i>

<italic>Rosa rugosa</italic> is a famous Chinese traditional flowering species with high economic value. Flowering transition is an important process in plant growth and development. Although the flowering transition process has made great progress in some plants such as model plants, flowering transition in <italic>R. rugosa</italic> has not been rigorously characterized to establish a mechanism. In this study, the changes of buds during flowering transition in <italic>R. rugosa</italic> ‘Duoji Huangmei’ were analyzed through transcriptomic sequencing combined with morphological and physiological index determinations. Results showed that with the morphology changes of buds, both sugar and starch contents showed a similar up-down pattern while phytohormones contents displayed various trends, which implied that sugar, starch and phytohormones might play diverse roles during flowering transition in <italic>R. rugosa</italic>. Moreover, a total of 4363 differentially expressed genes (DEGs) were identified at three developmental stages. Among them, 74 DEGs were involved in metabolism, transport, and signal transduction of sugar, starch, and phytohormones, as well as photoperiod and vernalization response. We proposed that these DEGs were not regulated independently but interacted with each other to construct a gene-gene network to regulate flowering transition of <italic>R. rugosa</italic>, and the regulatory network from vegetative growth stage (S1) to flowering transition stage (S2) was more complicated. These results further enriched the study of flowering transition in Rosa and lay an important foundation for breeding new varieties with desired floral traits.

Independent recruitment of FRUITFULL-like transcription factors in the convergent origins of vernalization-responsive grass flowering

Flowering in response to low temperatures (vernalization) has evolved multiple times independently across angiosperms as an adaptation to match reproductive development with the short growing season of temperate habitats. Despite the context of a generally conserved flowering time network, evidence suggests that the genes underlying vernalization responsiveness are distinct across major plant clades. Whether different or similar mechanisms underlie vernalization-induced flowering at narrower (e.g., family-level) phylogenetic scales is not well understood. To test the hypothesis that vernalization responsiveness has evolved convergently in temperate species of the grass family (Poaceae), we carried out flowering time experiments with and without vernalization in several representative species from different subfamilies. We then determined the likelihood that vernalization responsiveness evolved through parallel mechanisms by quantifying the response of Pooideae vernalization pathway FRUITFULL (FUL)-like genes to extended periods of cold. Our results demonstrate that vernalization-induced flowering has evolved multiple times independently in at least five grass subfamilies, and that different combinations of FUL-like genes have been recruited to this pathway on several occasions.

Intron-retained alternatively spliced VIN3-LIKE 2 gene from Chimonanthus praecox promotes flowering in transgenic Arabidopsis

Vernalization is a process of acquiring or accelerating the flowering ability by prolonged cold exposure. VERNALIZATION INSENSITIVE3 (VIN3) is induced by chilling and is extremely important for the vernalization response in Arabidopsis thaliana. However, the issue of the induction of the VIN3-LIKE genes in wintersweet (Chimonanthus praecox) has been largely neglected. In the present study, we explored the molecular regulation of the PHD type finger protein-encoding gene CpVIL2 in relation to the growth and development of wintersweet in Arabidopsis. In wintersweet, quantitative real-time PCR (qRT-PCR) analysis showed that the relative expression of CpVIL2-As2i (intron-retained alternatively spliced in the second intron) was extremely higher in the pistils than in the other tissues. And the relative CpVIL2-As2i expression in flower buds (FBs) treated at 8 °C was higher than that of FBs in December, 2016 under natural conditions, which was not detected in non-flowering FBs at 16 °C. In Arabidopsis, the expression patterns of the CpVIL2-As2i gene were detected at first in CpVIL2-As2i pro::GUS (β-glucuronidase) lines, with predominantly higher expression in flowers and inflorescence. Meanwhile, the hormone-induced expression profiles of the CpVIL2-As2i promoter were confirmed using exogenous induction by abscisic acid (ABA) and indole acetic acid (IAA) phytohormones, where the GUS enzyme activity obviously decreased compared with that of control. In comparison with Arabidopsis/Col-0, early flowering was detected in ectopic 35S::CpVIL2-As2i lines. Overall, these results demonstrated the function of the CpVIL2-As2i gene, at the same time, provided us with new insights into the molecular mechanisms of early flowering and complex regulatory networks of vernalization in wintersweet.