FLOWERING LOCUS C Expression Research Articles

SISTER OF FCA physically associates with SKB1 to regulate flowering time in Arabidopsis thaliana

BackgroundProper flowering time is important for the growth and development of plants, and both too early and too late flowering impose strong negative influences on plant adaptation and seed yield. Thus, it is vitally important to study the mechanism underlying flowering time control in plants. In a previous study by the authors, genome-wide association analysis was used to screen the candidate gene SISTER OF FCA (SSF) that regulates FLOWERING LOCUS C (FLC), a central gene encoding a flowering suppressor in Arabidopsis thaliana.ResultsSSF physically interacts with Protein arginine methyltransferase 5 (PRMT5, SKB1). Subcellular co—localization analysis showed that SSF and SKB1 interact in the nucleus. Genetically, SSF and SKB1 exist in the same regulatory pathway that controls FLC expression. Furthermore, RNA-sequencing analysis showed that both SSF and SKB1 regulate certain common pathways.ConclusionsThis study shows that PRMT5 interacts with SSF, thus controlling FLC expression and facilitating flowering time control.

ABA INSENSITIVE 2 promotes flowering by inhibiting OST1/ABI5-dependent FLOWERING LOCUS C transcription in Arabidopsis.

The plant hormone abscisic acid (ABA) is an important regulator of plant growth and development and plays a crucial role in both biotic and abiotic stress responses. ABA modulates flowering time, but the precise molecular mechanism remains poorly understood. Here we report that ABA INSENSITIVE 2 (ABI2) is the only phosphatase from the ABA-signaling core that positively regulates the transition to flowering in Arabidopsis. Loss-of-function abi2-2 mutant shows significantly delayed flowering both under long day and short day conditions. Expression of floral repressor genes such as FLOWERING LOCUS C (FLC) and CYCLING DOF FACTOR 1 (CDF1) was significantly up-regulated in abi2-2 plants while expression of the flowering promoting genes FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) was down-regulated. Through genetic interactions we further found that ost1-3 and abi5-1 mutations are epistatic to abi2-2, as both of them individually rescued the late flowering phenotype of abi2-2. Interestingly, phosphorylation and protein stability of ABA INSENSITIVE 5 (ABI5) were enhanced in abi2-2 plants suggesting that ABI2 dephosphorylates ABI5, thereby reducing protein stability and the capacity to induce FLC expression. Our findings uncovered the unexpected role of ABI2 in promoting flowering by inhibiting ABI5-mediated FLC expression in Arabidopsis.

Arabidopsis SEC13B Interacts with Suppressor of Frigida 4 to Repress Flowering.

SECRETORY13 (SEC13) is an essential member of the coat protein complex II (COPII), which was reported to mediate vesicular-specific transport from the endoplasmic reticulum (ER) to the Golgi apparatus and plays a crucial role in early secretory pathways. In Arabidopsis, there are two homologous proteins of SEC13: SEC13A and SEC13B. SUPPRESSOR OF FRIGIDA 4 (SUF4) encodes a C2H2-type zinc finger protein that inhibits flowering by transcriptionally activating the FLOWERING LOCUS C (FLC) through the FRIGIDA (FRI) pathway in Arabidopsis. However, it remains unclear whether SEC13 proteins are involved in Arabidopsis flowering. In this study, we first identified that the sec13b mutant exhibited early flowering under both long-day and short-day conditions. Quantitative real-time PCR (qRT-PCR) analysis showed that both SEC13A and SEC13B were expressed in all the checked tissues, and transient expression assays indicated that SEC13A and SEC13B were localized not only in the ER but also in the nucleus. Then, we identified that SEC13A and SEC13B could interact with SUF4 in vitro and in vivo. Interestingly, both sec13b and suf4 single mutants flowered earlier than the wild type (Col-0), whereas the sec13b suf4 double mutant flowered even earlier than all the others. In addition, the expression of flowering inhibitor FLC was down-regulated, and the expressions of flowering activator FLOWERING LOCUS T (FT), CONSTANS (CO), and SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1) were up-regulated in sec13b, suf4, and sec13b suf4 mutants, compared with Col-0. Taken together, our results indicated that SEC13B interacted with SUF4, and they may co-regulate the same genes in flowering-regulation pathways. These results also suggested that the COPII component could function in flowering in Arabidopsis.

FLZ13 interacts with FLC and ABI5 to negatively regulate flowering time in Arabidopsis.

The transition from vegetative to reproductive growth, known as flowering, is a critical developmental process in flowering plants to ensure reproductive success. This process is strictly controlled by various internal and external cues; however, the underlying molecular regulatory mechanisms need to be further characterized. Here, we report a plant-specific protein, FCS-LIKE ZINC FINGER PROTEIN 13 (FLZ13), which functions as a hitherto unknown negative modulator of flowering time in Arabidopsis thaliana. Biochemical analysis showed that FLZ13 directly interacts with FLOWERING LOCUS C (FLC), a major flowering repressor, and that FLZ13 largely depends on FLC to repress the transcription of two core flowering integrators: FLOWERING LOCUS T and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1. In addition, FLZ13 works together with ABSCISIC ACID INSENSITIVE 5 to activate FLC expression to delay flowering. Taken together, our findings suggest that FLZ13 is an important component of the gene regulatory network for flowering time control in plants.

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.

The nuclear pore Y-complex functions as a platform for transcriptional regulation of FLOWERING LOCUS C in Arabidopsis.

The nuclear pore complex (NPC) has multiple functions beyond the nucleo-cytoplasmic transport of large molecules. Subnuclear compartmentalization of chromatin is critical for gene expression in animals and yeast. However, the mechanism by which the NPC regulates gene expression is poorly understood in plants. Here we report that the Y-complex (Nup107-160 complex, a subcomplex of the NPC) self-maintains its nucleoporin homeostasis and modulates FLOWERING LOCUS C (FLC) transcription via changing histone modifications at this locus. We show that Y-complex nucleoporins are intimately associated with FLC chromatin through their interactions with histone H2A at the nuclear membrane. Fluorescence in situ hybridization assays revealed that Nup96, a Y-complex nucleoporin, enhances FLC positioning at the nuclear periphery. Nup96 interacted with HISTONE DEACETYLASE 6 (HDA6), a key repressor of FLC expression via histone modification, at the nuclear membrane to attenuate HDA6-catalyzed deposition at the FLC locus and change histone modifications. Moreover, we demonstrate that Y-complex nucleoporins interact with RNA polymerase II to increase its occupancy at the FLC locus, facilitating transcription. Collectively, our findings identify an attractive mechanism for the Y-complex in regulating FLC expression via tethering the locus at the nuclear periphery and altering its histone modification.

Causal role of a promoter polymorphism in natural variation of the Arabidopsis floral repressor gene FLC

Noncoding polymorphism frequently associates with phenotypic variation, but causation and mechanism are rarely established. Noncoding single-nucleotide polymorphisms (SNPs) characterize the major haplotypes of the Arabidopsis thaliana floral repressor gene FLOWERING LOCUS C (FLC). This noncoding polymorphism generates a range of FLC expression levels, determining the requirement for and the response to winter cold. The major adaptive determinant of these FLC haplotypes was shown to be the autumnal levels of FLC expression. Here, we investigate how noncoding SNPs influence FLC transcriptional output. We identify an upstream transcription start site (uTSS) cluster at FLC, whose usage is increased by an A variant at the promoter SNP-230. This variant is present in relatively few Arabidopsis accessions, with the majority containing G at this site. We demonstrate a causal role for the A variant at -230 in reduced FLC transcriptional output. The G variant upregulates FLC expression redundantly with the major transcriptional activator FRIGIDA (FRI). We demonstrate an additive interaction of SNP-230 with an intronic SNP+259, which also differentially influences uTSS usage. Combinatorial interactions between noncoding SNPs and transcriptional activators thus generate quantitative variation in FLC transcription that has facilitated the adaptation of Arabidopsis accessions to distinct climates.

MED8 regulates floral transition in Arabidopsis by interacting with FPA.

Success in plant reproduction is highly dependent on the correct timing of the floral transition, which is tightly regulated by the flowering pathways. In the model plant Arabidopsis thaliana, the central flowering repressor FLOWERING LOCUS C (FLC) is precisely regulated by multiple flowering time regulators in the vernalization pathway and autonomous pathway, including FPA. Here we report that Arabidopsis MEDIATOR SUBUNIT 8 (MED8) promotes floral transition in Arabidopsis by recruiting FPA to the FLC locus to repress FLC expression. Loss of MED8 function leads to a significant late-flowering phenotype due to increased FLC expression. We further show that MED8 directly interacts with FPA in the nucleus and recruits FPA to the FLC locus. Moreover, MED8 is indispensable for FPA's function in controlling flowering time and regulating FLC expression. Our study thus reveals a flowering mechanism by which the Mediator subunit MED8 represses FLC expression by facilitating the binding of FPA to the FLC locus to ensure appropriate timing of flowering for reproductive success.

Integrating analog and digital modes of gene expression at Arabidopsis FLC.

Quantitative gene regulation at the cell population level can be achieved by two fundamentally different modes of regulation at individual gene copies. A 'digital' mode involves binary ON/OFF expression states, with population-level variation arising from the proportion of gene copies in each state, while an 'analog' mode involves graded expression levels at each gene copy. At the Arabidopsis floral repressor FLOWERING LOCUS C (FLC), 'digital' Polycomb silencing is known to facilitate quantitative epigenetic memory in response to cold. However, whether FLC regulation before cold involves analog or digital modes is unknown. Using quantitative fluorescent imaging of FLC mRNA and protein, together with mathematical modeling, we find that FLC expression before cold is regulated by both analog and digital modes. We observe a temporal separation between the two modes, with analog preceding digital. The analog mode can maintain intermediate expression levels at individual FLC gene copies, before subsequent digital silencing, consistent with the copies switching OFF stochastically and heritably without cold. This switch leads to a slow reduction in FLC expression at the cell population level. These data present a new paradigm for gradual repression, elucidating how analog transcriptional and digital epigenetic memory pathways can be integrated.

The P-body component DECAPPING5 and the floral repressor SISTER OF FCA regulate FLOWERING LOCUS C transcription in Arabidopsis.

Flowering is the transition from vegetative to reproductive growth and is critical for plant adaptation and reproduction. FLOWERING LOCUS C (FLC) plays a central role in flowering time control, and dissecting its regulation mechanism provides essential information for crop improvement. Here, we report that DECAPPING5 (DCP5), a component of processing bodies (P-bodies), regulates FLC transcription and flowering time in Arabidopsis (Arabidopsis thaliana). DCP5 and its interacting partner SISTER OF FCA (SSF) undergo liquid-liquid phase separation (LLPS) that is mediated by their prion-like domains (PrDs). Enhancing or attenuating the LLPS of both proteins using transgenic methods greatly affects their ability to regulate FLC and flowering time. DCP5 regulates FLC transcription by modulating RNA polymerase II enrichment at the FLC locus. DCP5 requires SSF for FLC regulation, and loss of SSF or its PrD disrupts DCP5 function. Our results reveal that DCP5 interacts with SSF, and the nuclear DCP5-SSF complex regulates FLC expression at the transcriptional level.

Non-vernalization requirement for flowering in Brassica rapa conferred by a dominant allele of FLOWERING LOCUS T

We identified and characterized a dominant FT allele for flowering without vernalization in Brassica rapa, while demonstrating its potential for deployment in breeding to accelerate flowering in various Brassicaceae crops. Controlling the timing of flowering is key to improving yield and quality of several agricultural crops including the Brassicas. Many Brassicaceae crops possess a conserved flowering mechanism in which FLOWERING LOCUS C (FLC) represses the transcription of flowering activators such as FLOWERING LOCUS T (FT) during vernalization. Here, we employed genetic analysis based on next-generation sequencing to identify a dominant FT allele, BraA.FT.2-C, for flowering in the absence of vernalization in the Brassica rapa cultivar 'CHOY SUM EX CHINA 3'. BraA.FT.2-C harbors two large insertions upstream of its coding region and is expressed without vernalization, despite FLC expression. We show that BraA.FT.2-C offers an opportunity to introduce flowering without vernalization requirement into winter-type brassica crops, including B. napus, which have many functional FLC paralogs. Furthermore, we demonstrated the feasibility of using B. rapa harboring BraA.FT.2-C as rootstock for grafting to induce flowering in radish (Raphanus sativus), which requires vernalization for flowering. We believe that the ability of BraA.FT.2-C to overcome repression by FLC can have significant applications in brassica crops breeding to increase yields by accelerating or delaying flowering.

Two Arabidopsis Splicing Factors, U2AF65a and U2AF65b, Differentially Control Flowering Time by Modulating the Expression or Alternative Splicing of a Subset of FLC Upstream Regulators.

We investigated the transcriptomic changes in the shoot apices during floral transition in Arabidopsis mutants of two closely related splicing factors: AtU2AF65a (atu2af65a) and AtU2AF65b (atu2af65b). The atu2af65a mutants exhibited delayed flowering, while the atu2af65b mutants showed accelerated flowering. The underlying gene regulatory mechanism of these phenotypes was unclear. We performed RNA-seq analysis using shoot apices instead of whole seedlings and found that the atu2af65a mutants had more differentially expressed genes than the atu2af65b mutants when they were compared to wild type. The only flowering time gene that was significantly up- or down-regulated by more than two-fold in the mutants were FLOWERING LOCUS C (FLC), a major floral repressor. We also examined the expression and alternative splicing (AS) patterns of several FLC upstream regulators, such as COOLAIR, EDM2, FRIGIDA, and PP2A-b'ɤ, and found that those of COOLAIR, EDM2, and PP2A-b'ɤ were altered in the mutants. Furthermore, we demonstrated that AtU2AF65a and AtU2AF65b genes partially influenced FLC expression by analyzing these mutants in the flc-3 mutant background. Our findings indicate that AtU2AF65a and AtU2AF65b splicing factors modulate FLC expression by affecting the expression or AS patterns of a subset of FLC upstream regulators in the shoot apex, leading to different flowering phenotypes.

FLK is an mRNA m6A reader that regulates floral transition by modulating the stability and splicing of FLC in Arabidopsis

N6-methyladenosine (m6A), which is added, removed, and interpreted by m6A writers, erasers, and readers, respectively, is the most abundant modification in eukaryotic mRNAs. The m6A marks play a pivotal role in the regulation of floral transition in plants. FLOWERING LOCUS K (FLK), an RNA-binding protein harboring K-homology (KH) motifs, is known to regulate floral transition by repressing the levels of a key floral repressor FLOWERING LOCUS C (FLC) in Arabidopsis. However, the molecular mechanism underlying FLK-mediated FLC regulation remains unclear. In this study, we identified FLK as a novel mRNA m6A reader protein that directly binds the m6A site in the 3ʹ-untranslated region of FLC transcripts to repressing FLC levels by reducing its stability and splicing. Importantly, FLK binding of FLC transcripts was abolished in vir-1, an m6A writer mutant, and the late-flowering phenotype of the flk mutant could not be rescued by genetic complementation using the mutant FLKm gene, in which the m6A reader encoding function was eliminated, indicating that FLK binds and regulates FLC expression in an m6A-dependent manner. Collectively, our study has addressed a long-standing question of how FLK regulates FLC transcript levels and established a molecular link between the FLK-mediated recognition of m6A modifications on FLC transcripts and floral transition in Arabidopsis.

Arabidopsis SRPKII family proteins regulate flowering via phosphorylation of SR proteins and effects on gene expression and alternative splicing.

Alternative splicing of pre-mRNAs is crucial for plant growth and development. Serine/arginine-rich (SR) proteins are a conserved family of RNA binding proteins that are critical for both constitutive and alternative splicing. However, how phosphorylation of SR proteins regulates gene transcription and alternative splicing during plant development is poorly understood. We found that the Arabidopsis thaliana L. SR protein-specific kinase II family proteins (SRPKIIs) play an important role in plant development, including flowering. SRPKIIs regulate the phosphorylation status of a subset of specific SR proteins, including SR45 and SC35, which subsequently mediates their subcellular localization. A phospho-dead SR45 mutant inhibits the assembly of the ASAP (apoptosis-and splicing-associated protein) complex and thereby upregulates the expression of FLC (FLOWERING LOCUS C) via epigenetic modification. The splicing efficiency of FLC introns was significantly increased in the shoot apex of the srpkii mutant. Transcriptomic analysis revealed that SRPKIIs regulate the alternative splicing of approximately four hundred genes, which largely overlap with those regulated by SR45 and SC35-SCL family proteins. In summary, we found that Arabidopsis SRPKIIs specifically affect the phosphorylation status of a subset SR proteins and regulate the expression and alternative splicing of FLC to control flowering time.

Genome-wide association study identifies a novel BMI1A QTL allele that confers FLC expression diversity in Arabidopsis thaliana.

Identification and understanding of the genetic basis of natural variations in plants are essential for comprehending their phenotypic adaptation. Here, we report a genome-wide association study (GWAS) of FLOWERING LOCUS C (FLC) expression in 727 Arabidopsis accessions. We identified B LYMPHOMA MOLONEY MURINE LEUKEMIA VIRUS INSERTION REGION 1 HOMOLOG 1A (BMI1A) as a causal gene for one of the FLC expression quantitative trait loci (QTLs). Loss of function in BMI1A increases FLC expression and delays flowering time at 16 °C significantly compared with the wild type (Col-0). BMI1A activity is required for histone H3 lysine 27 trimethylation (H3K27me3) accumulation at the FLC, MADS AFFECTING FLOWERING 4 (MAF4), and MAF5 loci at low ambient temperature. We further uncovered two BMI1A haplotypes associated with the natural variation in FLC expression and flowering time at 16 °C, and demonstrated that polymorphisms in the BMI1A promoter region are the main contributor. Different BMI1A haplotypes are strongly associated with geographical distribution, and the low ambient temperature-sensitive BMI1A variants are associated with a lower mean temperature of the driest quarter of their collection sites compared with the temperature-non-responsive variants, indicating that the natural variations in BMI1A have adaptive functions in FLC expression and flowering time regulation. Therefore, our results provide new insights into the natural variations in FLC expression and flowering time diversity in plants.

Epigenetic stress memory: A new approach to study cold and heat stress responses in plants.

Understanding plant stress memory under extreme temperatures such as cold and heat could contribute to plant development. Plants employ different types of stress memories, such as somatic, intergenerational and transgenerational, regulated by epigenetic changes such as DNA and histone modifications and microRNAs (miRNA), playing a key role in gene regulation from early development to maturity. In most cases, cold and heat stresses result in short-term epigenetic modifications that can return to baseline modification levels after stress cessation. Nevertheless, some of the modifications may be stable and passed on as stress memory, potentially allowing them to be inherited across generations, whereas some of the modifications are reactivated during sexual reproduction or embryogenesis. Several stress-related genes are involved in stress memory inheritance by turning on and off transcription profiles and epigenetic changes. Vernalization is the best example of somatic stress memory. Changes in the chromatin structure of the Flowering Locus C (FLC) gene, a MADS-box transcription factor (TF), maintain cold stress memory during mitosis. FLC expression suppresses flowering at high levels during winter; and during vernalization, B3 TFs, cold memory cis-acting element and polycomb repressive complex 1 and 2 (PRC1 and 2) silence FLC activation. In contrast, the repression of SQUAMOSA promoter-binding protein-like (SPL) TF and the activation of Heat Shock TF (HSFA2) are required for heat stress memory. However, it is still unclear how stress memory is inherited by offspring, and the integrated view of the regulatory mechanisms of stress memory and mitotic and meiotic heritable changes in plants is still scarce. Thus, in this review, we focus on the epigenetic regulation of stress memory and discuss the application of new technologies in developing epigenetic modifications to improve stress memory.

Proline Affects Flowering Time in Arabidopsis by Modulating FLC Expression: A Clue of Epigenetic Regulation?

The recent finding that proline-induced root elongation is mediated by reactive oxygen species (ROS) prompted us to re-evaluate other developmental processes modulated by proline, such as flowering time. By controlling the cellular redox status and the ROS distribution, proline could potentially affect the expression of transcriptional factors subjected to epigenetic regulation, such as FLOWERING LOCUS C (FLC). Accordingly, we investigated the effect of proline on flowering time in more detail by analyzing the relative expression of the main flowering time genes in p5cs1 p5cs2/P5CS2 proline-deficient mutants and found a significant upregulation of FLC expression. Moreover, proline-deficient mutants exhibited an adult vegetative phase shorter than wild-type samples, with a trichome distribution reminiscent of plants with high FLC expression. In addition, the vernalization-induced downregulation of FLC abolished the flowering delay of p5cs1 p5cs2/P5CS2, and mutants homozygous for p5cs1 and flc-7 and heterozygous for P5CS2 flowered as early as the flc-7 parental mutant, indicating that FLC acts downstream of P5CS1/P5CS2 and is necessary for proline-modulated flowering. The overall data indicate that the effects of proline on flowering time are mediated by FLC.

In vivo single-molecule analysis reveals COOLAIR RNA structural diversity

Cellular RNAs are heterogeneous with respect to their alternative processing and secondary structures, but the functional importance of this complexity is still poorly understood. A set of alternatively processed antisense non-coding transcripts, which are collectively called COOLAIR, are generated at the Arabidopsis floral-repressor locus FLOWERING LOCUS C (FLC)1. Different isoforms of COOLAIR influence FLC transcriptional output in warm and cold conditions2–7. Here, to further investigate the function of COOLAIR, we developed an RNA structure-profiling method to determine the in vivo structure of single RNA molecules rather than the RNA population average. This revealed that individual isoforms of the COOLAIR transcript adopt multiple structures with different conformational dynamics. The major distally polyadenylated COOLAIR isoform in warm conditions adopts three predominant structural conformations, the proportions and conformations of which change after cold exposure. An alternatively spliced, strongly cold-upregulated distal COOLAIR isoform6 shows high structural diversity, in contrast to proximally polyadenylated COOLAIR. A hyper-variable COOLAIR structural element was identified that was complementary to the FLC transcription start site. Mutations altering the structure of this region changed FLC expression and flowering time, consistent with an important regulatory role of the COOLAIR structure in FLC transcription. Our work demonstrates that isoforms of non-coding RNA transcripts adopt multiple distinct and functionally relevant structural conformations, which change in abundance and shape in response to external conditions.

WRKY63 transcriptional activation of COOLAIR and COLDAIR regulates vernalization-induced flowering.

Arabidopsis (Arabidopsis thaliana) FLOWERING LOCUS C (FLC) acts as a key flowering regulator by repressing the expression of the floral integrator FLOWERING LOCUS T (FT). Prolonged exposure to cold (vernalization) induces flowering by reducing FLC expression. The long noncoding RNAs (lncRNAs) COOLAIR and COLDAIR, which are transcribed from the 3' end and the first intron of FLC, respectively, are important for FLC repression under vernalization. However, the molecular mechanism of how COOLAIR and COLDAIR are transcriptionally activated remains elusive. In this study, we found that the group-III WRKY transcription factor WRKY63 can directly activate FLC. wrky63 mutant plants display an early flowering phenotype and are insensitive to vernalization. Interestingly, we found that WRKY63 can activate the expression of COOLAIR and COLDAIR by binding to their promoters.WRKY63 therefore acts as a dual regulator that activates FLC directly under non-vernalization conditions but represses FLC indirectly during vernalization through inducing COOLAIR and COLDAIR. Furthermore, genome-wide occupancy profile analyses indicated that the binding of WRKY63 to vernalization-induced genes increases after vernalization. In addition, WRKY63 binding is associated with decreased levels of the repressive marker Histone H3 Lysine 27 trimethylation (H3K27me3). Collectively, our results indicate that WRKY63 is an important flowering regulator involved in vernalization-induced transcriptional regulation.

Mutation of an Essential 60S Ribosome Assembly Factor MIDASIN 1 Induces Early Flowering in Arabidopsis.

Ribosome biogenesis is tightly associated with plant growth and reproduction. Mutations in genes encoding ribosomal proteins (RPs) or ribosome biogenesis factors (RBFs) generally result in retarded growth and delayed flowering. However, the early-flowering phenotype resulting from the ribosome biogenesis defect is rarely reported. We previously identified that the AAA-ATPase MIDASIN 1 (MDN1) functions as a 60S RBF in Arabidopsis. Here, we found that its weak mutant mdn1-1 is early-flowering. Transcriptomic analysis showed that the expression of FLOWERING LOCUS C (FLC) is down-regulated, while that of some autonomous pathway genes and ABSCISIC ACID-INSENSITIVE 5 (ABI5) is up-regulated in mdn1-1. Phenotypic analysis revealed that the flowering time of mdn1-1 is severely delayed by increasing FLC expression, suggesting that the early flowering in mdn1-1 is likely associated with the downregulation of FLC. We also found that the photoperiod pathway downstream of CONSTANTS (CO) and FLOWERING LOCUS T (FT) might contribute to the early flowering in mdn1-1. Intriguingly, the abi5-4 allele completely blocks the early flowering in mdn1-1. Collectively, our results indicate that the ribosome biogenesis defect elicited by the mutation of MDN1 leads to early flowering by affecting multiple flowering regulation pathways.