LncRNAs: the art of being influential without protein.

The plant long noncoding (lnc)RNA field is on the brink of transitioning from large-scale identification of lncRNAs to their functional characterization. Due to the cross-kingdom conservation of interaction types and molecular functions, there is much to be learned from mammalian lncRNA research. Here, we discuss the different molecular processes involving lncRNAs from the regulation of chromatin to splicing. Furthermore, we discuss the lncRNA interactome, which includes proteins, other RNAs, and DNA. We explore and discuss how mammalian lncRNA functionalities could be reflected in similar pathways in plants and hypothesize that several breakthroughs in mammalian research could lead to the discovery of novel plant lncRNA molecular functions. Expanding our knowledge of the biological role of lncRNAs and their multiple applications paves the way for future agricultural applications.

A gene regulatory network critical for axillary bud dormancy directly controlled by Arabidopsis BRANCHED1.

The Arabidopsis thaliana transcription factor BRANCHED1 (BRC1) plays a pivotal role in the control of shoot branching as it integrates environmental and endogenous signals that influence axillary bud growth. Despite its remarkable activity as a growth inhibitor, the mechanisms by which BRC1 promotes bud dormancy are largely unknown. We determined the genome-wide BRC1 binding sites in vivo and combined these with transcriptomic data and gene co-expression analyses to identify bona fide BRC1 direct targets. Next, we integrated multi-omics data to infer the BRC1 gene regulatory network (GRN) and used graph theory techniques to find network motifs that control the GRN dynamics. We generated an open online tool to interrogate this network. A group of BRC1 target genes encoding transcription factors (BTFs) orchestrate this intricate transcriptional network enriched in abscisic acid-related components. Promoter::ß-GLUCURONIDASE transgenic lines confirmed that BTFs are expressed in axillary buds. Transient co-expression assays and studies in planta using mutant lines validated the role of BTFs in modulating the GRN and promoting bud dormancy. This knowledge provides access to the developmental mechanisms that regulate shoot branching and helps identify candidate genes to use as tools to adapt plant architecture and crop production to ever-changing environmental conditions.

Protein interaction mapping reveals widespread targeting of development-related host transcription factors by phytoplasma effectors.

Phytoplasmas are pathogenic bacteria that reprogram plant host development for their own benefit. Previous studies have characterized a few different phytoplasma effector proteins that destabilize specific plant transcription factors. However, these are only a small fraction of the potential effectors used by phytoplasmas; therefore, the molecular mechanisms through which phytoplasmas modulate their hosts require further investigation. To obtain further insights into the phytoplasma infection mechanisms, we generated a protein-protein interaction network between a broad set of phytoplasma effectors and a large, unbiased collection of Arabidopsis thaliana transcription factors and transcriptional regulators. We found widespread, but specific, interactions between phytoplasma effectors and host transcription factors, especially those related to host developmental processes. In particular, many unrelated effectors target specific sets of TCP transcription factors, which regulate plant development and immunity. Comparison with other host-pathogen protein interaction networks shows that phytoplasma effectors have unusual targets, indicating that phytoplasmas have evolved a unique and unusual infection strategy. This study contributes a rich and solid data source that guides further investigations of the functions of individual effectors, as demonstrated for some herein. Moreover, the dataset provides insights into the underlying molecular mechanisms of phytoplasma infection.

HD-ZIP Transcription Factors and Brassinosteroid Signaling Play a Role in Capitulum Patterning in Chrysanthemum.

Chrysanthemum is a genus in the Asteraceae family containing numerous cut flower varieties with high ornamental value. It owes its beauty to the composite flower head, which resembles a compact inflorescence. This structure is also known as a capitulum, in which many ray and disc florets are densely packed. The ray florets are localized at the rim, are male sterile, and have large colorful petals. The centrally localized disc florets develop only a small petal tube but produce fertile stamens and a functional pistil. Nowadays, varieties with more ray florets are bred because of their high ornamental value, but, unfortunately, this is at the expense of their seed setting. In this study, we confirmed that the disc:ray floret ratio is highly correlated to seed set efficiency, and therefore, we further investigated the mechanisms that underlie the regulation of the disc:ray floret ratio. To this end, a comprehensive transcriptomics analysis was performed in two acquired mutants with a higher disc:ray floret ratio. Among the differentially regulated genes, various potential brassinosteroid (BR) signaling genes and HD-ZIP class IV homeodomain transcription factors stood out. Detailed follow-up functional studies confirmed that reduced BR levels and downregulation of HD-ZIP IV gene Chrysanthemum morifolium PROTODERMAL FACTOR 2 (CmPDF2) result in an increased disc:ray floret ratio, thereby providing ways to improve seed set in decorative chrysanthemum varieties in the future.

The TCP transcription factor HvTB2 heterodimerizes with VRS5 and controls spike architecture in barley.

KEY MESSAGE: Understanding the molecular network, including protein-protein interactions, of VRS5 provide new routes towards the identification of other key regulators of plant architecture in barley. The TCP transcriptional regulator TEOSINTE BRANCHED 1 (TB1) is a key regulator of plant architecture. In barley, an important cereal crop, HvTB1 (also referred to as VULGARE SIX-ROWED spike (VRS) 5), inhibits the outgrowth of side shoots, or tillers, and grains. Despite its key role in barley development, there is limited knowledge on the molecular network that is utilized by VRS5. In this work, we performed protein-protein interaction studies of VRS5. Our analysis shows that VRS5 potentially interacts with a diverse set of proteins, including other class II TCP's, NF-Y TF, but also chromatin remodelers. Zooming in on the interaction capacity of VRS5 with other TCP TFs shows that VRS5 preferably interacts with other class II TCP TFs in the TB1 clade. Induced mutagenesis through CRISPR-Cas of one of the putative VRS5 interactors, HvTB2 (also referred to as COMPOSITUM 1 and BRANCHED AND INDETERMINATE SPIKELET 1), resulted in plants that have lost their characteristic unbranched spike architecture. More specifically, hvtb2 mutants exhibited branches arising at the main spike, suggesting that HvTB2 acts as inhibitor of branching. Our protein-protein interaction studies of VRS5 resulted in the identification of HvTB2 as putative interactor of VRS5, another key regulator of spike architecture in barley. The study presented here provides a first step to underpin the protein-protein interactome of VRS5 and to identify other, yet unknown, key regulators of barley plant architecture.

Passiflora organensis FT/TFL1 gene family and their putative roles in phase transition and floral initiation.

KEY MESSAGE: Comprehensive analysis of the FT/TFL1 gene family in Passiflora organensis results in understanding how these genes might be involved in the regulation of the typical plant architecture presented by Passiflora species. Passion fruit (Passiflora spp) is an economic tropical fruit crop, but there is hardly any knowledge available about the molecular control of phase transition and flower initiation in this species. The florigen agent FLOWERING LOCUS T (FT) interacts with the bZIP protein FLOWERING LOCUS D (FD) to induce flowering in the model species Arabidopsis thaliana. Current models based on research in rice suggest that this interaction is bridged by 14-3-3 proteins. We identified eight FT/TFL1 family members in Passiflora organensis and characterized them by analyzing their phylogeny, gene structure, expression patterns, protein interactions and putative biological roles by heterologous expression in Arabidopsis. PoFT was highest expressed during the adult vegetative phase and it is supposed to have an important role in flowering induction. In contrast, its paralogs PoTSFs were highest expressed in the reproductive phase. While ectopic expression of PoFT in transgenic Arabidopsis plants induced early flowering and inflorescence determinacy, the ectopic expression of PoTSFa caused a delay in flowering. PoTFL1-like genes were highest expressed during the juvenile phase and their ectopic expression caused delayed flowering in Arabidopsis. Our protein-protein interaction studies indicate that the flowering activation complexes in Passiflora might deviate from the hexameric complex found in the model system rice. Our results provide insights into the potential functions of FT/TFL1 gene family members during floral initiation and their implications in the special plant architecture of Passiflora species, contributing to more detailed studies on the regulation of passion fruit reproduction.

Parasitic modulation of host development by ubiquitin-independent protein degradation.

Certain obligate parasites induce complex and substantial phenotypic changes in their hosts in ways that favor their transmission to other trophic levels. However, the mechanisms underlying these changes remain largely unknown. Here we demonstrate how SAP05 protein effectors from insect-vectored plant pathogenic phytoplasmas take control of several plant developmental processes. These effectors simultaneously prolong the host lifespan and induce witches' broom-like proliferations of leaf and sterile shoots, organs colonized by phytoplasmas and vectors. SAP05 acts by mediating the concurrent degradation of SPL and GATA developmental regulators via a process that relies on hijacking the plant ubiquitin receptor RPN10 independent of substrate ubiquitination. RPN10 is highly conserved among eukaryotes, but SAP05 does not bind insect vector RPN10. A two-amino-acid substitution within plant RPN10 generates a functional variant that is resistant to SAP05 activities. Therefore, one effector protein enables obligate parasitic phytoplasmas to induce a plethora of developmental phenotypes in their hosts.

Fire Blight Susceptibility in Lilium spp. Correlates to Sensitivity to Botrytis elliptica Secreted Cell Death Inducing Compounds.

Fire blight represents a widespread disease in Lilium spp. and is caused by the necrotrophic Ascomycete Botrytis elliptica. There are >100 Lilium species that fall into distinct phylogenetic groups and these have been used to generate the contemporary commercial genotypes. It is known among lily breeders and growers that different groups of lilies differ in susceptibility to fire blight, but the genetic basis and mechanisms of susceptibility to fire blight are unresolved. The aim of this study was to quantify differences in fire blight susceptibility between plant genotypes and differences in virulence between fungal isolates. To this end we inoculated, in four biological replicates over 2 years, a set of 12 B. elliptica isolates on a panel of 18 lily genotypes representing seven Lilium hybrid groups. A wide spectrum of variation in symptom severity was observed in different isolate-genotype combinations. There was a good correlation between the lesion diameters on leaves and flowers of the Lilium genotypes, although the flowers generally showed faster expanding lesions. It was earlier postulated that B. elliptica pathogenicity on lily is conferred by secreted proteins that induce programmed cell death in lily cells. We selected two aggressive isolates and one mild isolate and collected culture filtrate (CF) samples to compare the cell death inducing activity of their secreted compounds in lily. After leaf infiltration of the CFs, variation was observed in cell death responses between the diverse lilies. The severity of cell death responses upon infiltration of the fungal CF observed among the diverse Lilium hybrid groups correlated well to their fire blight susceptibility. These results support the hypothesis that susceptibility to fire blight in lily is mediated by their sensitivity to B. elliptica effector proteins in a quantitative manner. Cell death-inducing proteins may provide an attractive tool to predict fire blight susceptibility in lily breeding programs.

The Seed Development Factors TT2 and MYB5 Regulate Heat Stress Response in Arabidopsis.

HEAT SHOCK FACTOR A2 (HSFA2) is a regulator of multiple environmental stress responses required for stress acclimation. We analyzed HSFA2 co-regulated genes and identified 43 genes strongly co-regulated with HSFA2 during multiple stresses. Motif enrichment analysis revealed an over-representation of the site II element (SIIE) in the promoters of these genes. In a yeast 1-hybrid screen with the SIIE, we identified the closely related R2R3-MYB transcription factors TT2 and MYB5. We found overexpression of MYB5 or TT2 rendered plants heat stress tolerant. In contrast, tt2, myb5, and tt2/myb5 loss of function mutants showed heat stress hypersensitivity. Transient expression assays confirmed that MYB5 and TT2 can regulate the HSFA2 promoter together with the other members of the MBW complex, TT8 and TRANSPARENT TESTA GLABRA 1 (TTG1) and that the SIIE was involved in this regulation. Transcriptomic analysis revealed that TT2/MYB5 target promoters were enriched in SIIE. Overall, we report a new function of TT2 and MYB5 in stress resistance and a role in SIIE-mediated HSFA2 regulation.

PHOSPHATIDYLETHANOLAMINE-BINDING PROTEINS: the conductors of dual reproduction in plants with vegetative storage organs.

Geophytes, the plants that form vegetative storage organs, are characterized by a dual reproduction system, in which vegetative and sexual propagation are tightly regulated to ensure fitness in harsh climatic conditions. Recent findings highlight the role of the PEBP (PHOSPHATIDYLETHANOLAMINE-BINDING PROTEIN) gene family in geophytes as major players in the molecular cascades underlying both types of reproduction. In this review, we briefly explain the life cycle and reproduction strategies of different geophytes and what is known about the physiological aspects related to these processes. Subsequently, an in-depth overview is provided of the molecular and genetic pathways driving these processes. In the evolution of plants, the PEBP gene family has expanded, followed by neo- and subfunctionalization. Careful characterization revealed that differential expression and differential protein complex formation provide the members of this gene family with unique functions, enabling them to mediate the crosstalk between the two reproductive events in geophytes in response to environmental and endogenous cues. Taking all these studies into account, we propose to regard the PEBPs as conductors of geophyte reproductive development.

Comprehensive phenotyping reveals interactions and functions of Arabidopsis thaliana TCP genes in yield determination.

Members of the Arabidopsis thaliana TCP transcription factor (TF) family affect plant growth and development. We systematically quantified the effect of mutagenizing single or multiple TCP TFs and how altered vegetative growth or branching influences final seed yield. We monitored rosette growth over time and branching patterns and seed yield characteristics at the end of the lifecycle. Subsequently, an approach was developed to disentangle vegetative growth and to determine possible effects on seed yield. Analysis of growth parameters showed all investigated tcp mutants to be affected in certain growth aspects compared with wild-type plants, highlighting the importance of TCP TFs in plant development. Furthermore, we found evidence that all class II TCPs are involved in axillary branch outgrowth, either as inhibitors (BRANCHED-like genes) or enhancers (JAW- and TCP5-like genes). Comprehensive phenotyping of plants mutant for single or multiple TCP TFs reveals that the proposed opposite functions of class I and class II TCPs in plant growth needs revision and shows complex interactions between closely related TCP genes instead of full genetic redundancy. In various instances, the alterations in vegetative growth or in branching patterns result into negative trade-off effects on seed yield that were missed in previous studies, showing the importance of comprehensive and quantitative phenotyping.

Improved inference of intermolecular contacts through protein-protein interaction prediction using coevolutionary analysis.

MOTIVATION: Predicting residue-residue contacts between interacting proteins is an important problem in bioinformatics. The growing wealth of sequence data can be used to infer these contacts through correlated mutation analysis on multiple sequence alignments of interacting homologs of the proteins of interest. This requires correct identification of pairs of interacting proteins for many species, in order to avoid introducing noise (i.e. non-interacting sequences) in the analysis that will decrease predictive performance. RESULTS: We have designed Ouroboros, a novel algorithm to reduce such noise in intermolecular contact prediction. Our method iterates between weighting proteins according to how likely they are to interact based on the correlated mutations signal, and predicting correlated mutations based on the weighted sequence alignment. We show that this approach accurately discriminates between protein interaction versus non-interaction and simultaneously improves the prediction of intermolecular contact residues compared to a naive application of correlated mutation analysis. This requires no training labels concerning interactions or contacts. Furthermore, the method relaxes the assumption of one-to-one interaction of previous approaches, allowing for the study of many-to-many interactions. AVAILABILITY AND IMPLEMENTATION: Source code and test data are available at www.bif.wur.nl/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Arabidopsis thaliana ambient temperature responsive lncRNAs.

BACKGROUND: Long non-coding RNAs (lncRNAs) have emerged as new class of regulatory molecules in animals where they regulate gene expression at transcriptional and post-transcriptional level. Recent studies also identified lncRNAs in plant genomes, revealing a new level of transcriptional complexity in plants. Thousands of lncRNAs have been predicted in the Arabidopsis thaliana genome, but only a few have been studied in depth. RESULTS: Here we report the identification of Arabidopsis lncRNAs that are expressed during the vegetative stage of development in either the shoot apical meristem or in leaves. We found that hundreds of lncRNAs are expressed in these tissues, of which 50 show differential expression upon an increase in ambient temperature. One of these lncRNAs, FLINC, is down-regulated at higher ambient temperature and affects ambient temperature-mediated flowering in Arabidopsis. CONCLUSION: A number of ambient temperature responsive lncRNAs were identified with potential roles in the regulation of temperature-dependent developmental changes, such as the transition from the vegetative to the reproductive (flowering) phase. The challenge for the future is to characterize the biological function and molecular mode of action of the large number of ambient temperature-regulated lncRNAs that have been identified in this study.

Novel functions of the Arabidopsis transcription factor TCP5 in petal development and ethylene biosynthesis.

The flowers of most dicotyledons have petals that, together with the sepals, initially protect the reproductive organs. Later during development petals are required to open the flower and to attract pollinators. This diverse set of functions demands tight temporal and spatial regulation of petal development. We studied the functioning of the Arabidopsis thaliana TCP5-like transcription factors (TFs) in petals. Overexpression of TCP5 in petal epidermal cells results in smaller petals, whereas tcp5 tcp13 tcp17 triple knockout lines have wider petals with an increased surface area. Comprehensive expression studies revealed effects of TCP5-like TFs on the expression of genes related to the cell cycle, growth regulation and organ growth. Additionally, the ethylene biosynthesis genes 1-amino-cyclopropane-1-carboxylate (ACC) synthase 2 (ACS2) and ACC oxidase 2 (ACO2) and several ETHYLENE RESPONSE FACTORS (ERFs) are found to be differentially expressed in TCP5 mutant and overexpression lines. Chromatin immunoprecipitation-quantitative PCR showed direct binding of TCP5 to the ACS2 locus in vivo. Ethylene is known to influence cell elongation, and the petal phenotype of the tcp5 tcp13 tcp17 mutant could be complemented by treatment of the plants with an ethylene pathway inhibitor. Taken together, this reveals a novel role for TCP5-like TFs in the regulation of ethylene-mediated petal development and growth.

Role of Tulipa gesneriana TEOSINTE BRANCHED1 (TgTB1) in the control of axillary bud outgrowth in bulbs.

KEY MESSAGE: Tulip vegetative reproduction. Tulips reproduce asexually by the outgrowth of their axillary meristems located in the axil of each bulb scale. The number of axillary meristems in one bulb is low, and not all of them grow out during the yearly growth cycle of the bulb. Since the degree of axillary bud outgrowth in tulip determines the success of their vegetative propagation, this study aimed at understanding the mechanism controlling the differential axillary bud activity. We used a combined physiological and "bottom-up" molecular approach to shed light on this process and found that first two inner located buds do not seem to experience dormancy during the growth cycle, while mid-located buds enter dormancy by the end of the growing season. Dormancy was assessed by weight increase and TgTB1 expression levels, a conserved TCP transcription factor and well-known master integrator of environmental and endogenous signals influencing axillary meristem outgrowth in plants. We showed that TgTB1 expression in tulip bulbs can be modulated by sucrose, cytokinin and strigolactone, just as it has been reported for other species. However, the limited growth of mid-located buds, even when their TgTB1 expression is downregulated, points at other factors, probably physical, inhibiting their growth. We conclude that the time of axillary bud initiation determines the degree of dormancy and the sink strength of the bud. Thus, development, apical dominance, sink strength, hormonal cross-talk, expression of TgTB1 and other possibly physical but unidentified players, all converge to determine the growth capacity of tulip axillary buds.

Tulipa gesneriana and Lilium longiflorum PEBP Genes and Their Putative Roles in Flowering Time Control.

Floral induction in Tulipa gesneriana and Lilium longiflorum is triggered by contrasting temperature conditions, high and low temperature, respectively. In Arabidopsis, the floral integrator FLOWERING LOCUS T (FT), a member of the PEBP (phosphatidyl ethanolamine-binding protein) gene family, is a key player in flowering time control. In this study, one PEBP gene was identified and characterized in lily (LlFT) and three PEBP genes were isolated from tulip (TgFT1, TgFT2 and TgFT3). Overexpression of these genes in Arabidopsis thaliana resulted in an early flowering phenotype for LlFT and TgFT2, but a late flowering phenotype for TgFT1 and TgFT3. Overexpression of LlFT in L. longiflorum also resulted in an early flowering phenotype, confirming its proposed role as a flowering time-controlling gene. The tulip PEBP genes TgFT2 and TgFT3 have a similar expression pattern in tulip, but show opposite effects on the timing of flowering in Arabidopsis. Therefore, the difference between these two proteins was further investigated by interchanging amino acids thought to be important for the FT function. This resulted in the conversion of phenotypes in Arabidopsis upon overexpressing the substituted TgFT2 and TgFT3 genes, revealing the importance of these interchanged amino acid residues. Based on all obtained results, we hypothesize that LlFT is involved in creating meristem competence to flowering-related cues in lily, and TgFT2 is considered to act as a florigen involved in the floral induction in tulip. The function of TgFT3 remains unclear, but, based on our observations and phylogenetic analysis, we propose a bulb-specific function for this gene.

A cautionary note on the use of chromosome conformation capture in plants.

BACKGROUND: The chromosome conformation capture (3C) technique is a method to study chromatin interactions at specific genomic loci. Initially established for yeast the 3C technique has been adapted to plants in recent years in order to study chromatin interactions and their role in transcriptional gene regulation. As the plant scientific community continues to implement this technology, a discussion on critical controls, validations steps and interpretation of 3C data is essential to fully benefit from 3C in plants. RESULTS: Here we assess the reliability and robustness of the 3C technique for the detection of chromatin interactions in Arabidopsis. As a case study, we applied this methodology to the genomic locus of a floral integrator gene SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1), and demonstrate the need of several controls and standard validation steps to allow a meaningful interpretation of 3C data. The intricacies of this promising but challenging technique are discussed in depth. CONCLUSIONS: The 3C technique offers an interesting opportunity to study chromatin interactions at a resolution infeasible by microscopy. However, for interpretation of 3C interaction data and identification of true interactions, 3C technology demands a stringent experimental setup and extreme caution.

A plant-based chemical genomics screen for the identification of flowering inducers.

BACKGROUND: Floral timing is a carefully regulated process, in which the plant determines the optimal moment to switch from the vegetative to reproductive phase. While there are numerous genes known that control flowering time, little information is available on chemical compounds that are able to influence this process. We aimed to discover novel compounds that are able to induce flowering in the model plant Arabidopsis. For this purpose we developed a plant-based screening platform that can be used in a chemical genomics study. RESULTS: Here we describe the set-up of the screening platform and various issues and pitfalls that need to be addressed in order to perform a chemical genomics screening on Arabidopsis plantlets. We describe the choice for a molecular marker, in combination with a sensitive reporter that's active in plants and is sufficiently sensitive for detection. In this particular screen, the firefly Luciferase marker was used, fused to the regulatory sequences of the floral meristem identity gene APETALA1 (AP1), which is an early marker for flowering. Using this screening platform almost 9000 compounds were screened, in triplicate, in 96-well plates at a concentration of 25 µM. One of the identified potential flowering inducing compounds was studied in more detail and named Flowering1 (F1). F1 turned out to be an analogue of the plant hormone Salicylic acid (SA) and appeared to be more potent than SA in the induction of flowering. The effect could be confirmed by watering Arabidopsis plants with SA or F1, in which F1 gave a significant reduction in time to flowering in comparison to SA treatment or the control. CONCLUSIONS: In this study a chemical genomics screening platform was developed to discover compounds that can induce flowering in Arabidopsis. This platform was used successfully, to identify a compound that can speed-up flowering in Arabidopsis.

Splicing-related genes are alternatively spliced upon changes in ambient temperatures in plants.

Plants adjust their development and architecture to small variations in ambient temperature. In a time in which temperatures are rising world-wide, the mechanism by which plants are able to sense temperature fluctuations and adapt to it, is becoming of special interest. By performing RNA-sequencing on two Arabidopsis accession and one Brassica species exposed to temperature alterations, we showed that alternative splicing is an important mechanism in ambient temperature sensing and adaptation. We found that amongst the differentially alternatively spliced genes, splicing related genes are enriched, suggesting that the splicing machinery itself is targeted for alternative splicing when temperature changes. Moreover, we showed that many different components of the splicing machinery are targeted for ambient temperature regulated alternative splicing. Mutant analysis of a splicing related gene that was differentially spliced in two of the genotypes showed an altered flowering time response to different temperatures. We propose a two-step mechanism where temperature directly influences alternative splicing of the splicing machinery genes, followed by a second step where the altered splicing machinery affects splicing of downstream genes involved in the adaptation to altered temperatures.

Molecular Regulation of Temperature-Dependent Floral Induction in Tulipa gesneriana.

The vegetative-to-reproductive phase change in tulip (Tulipa gesneriana) is promoted by increasing temperatures during spring. The warm winters of recent years interfere with this process and are calling for new adapted cultivars. A better understanding of the underlying molecular mechanisms would be of help, but unlike the model plant Arabidopsis (Arabidopsis thaliana), very little is known about floral induction in tulip. To shed light on the gene regulatory network controlling flowering in tulip, RNA sequencing was performed on meristem-enriched tissue collected under two contrasting temperature conditions, low and high. The start of reproductive development correlated with rounding of the shoot apical meristem and induction of TGSQA expression, a tulip gene with a high similarity to Arabidopsis APETALA1 Gene Ontology enrichment analysis of differentially expressed genes showed the overrepresentation of genes potentially involved in floral induction, bulb maturation, and dormancy establishment. Expression analysis revealed that TERMINAL FLOWER1 (TgTFL1) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1-like1 (TgSOC1-like1) might be repressors, whereas TgSOC1-like2 likely is an activator, of flowering. Subsequently, the flowering time-associated expression of eight potential flowering time genes was confirmed in three tulip cultivars grown in the field. Additionally, heterologous functional analyses in Arabidopsis resulted in flowering time phenotypes in line with TgTFL1 being a floral repressor and TgSOC1-like2 being a floral activator in tulip. Taken together, we have shown that long before morphological changes occur in the shoot apical meristem, the expression of floral repressors in tulip is suppressed by increased ambient temperatures, leading either directly or indirectly to the activation of potential flowering activators shortly before the commencement of the phase change.