The molecular mechanism of vernalization in Arabidopsis and cereals: role of Flowering Locus C and its homologs

Winter varieties of plants can flower only after exposure to prolonged cold. This phenomenon is known as vernalization and has been widely studied in the model plant Arabidopsis thaliana as well as in monocots. Through the repression of floral activator genes, vernalization prevents flowering in winter. In Arabidopsis, FLOWERING LOCUS C or FLC is the key repressor during vernalization, while in monocots vernalization is regulated through VRN1, VRN2 and VRN3 (or FLOWERING LOCUS T). Interestingly, VRN genes are not homologous to FLC but FLC homologs are found to have a significant role in vernalization response in cereals. The presence of FLC homologs in monocots opens new dimensions to understand, compare and retrace the evolution of vernalization pathways between monocots and dicots. In this review, we discuss the molecular mechanism of vernalization-induced flowering along with epigenetic regulations in Arabidopsis and temperate cereals. A better understanding of cold-induced flowering will be helpful in crop breeding strategies to modify the vernalization requirement of economically important temperate cereals.     

A root chicory MADS box sequence and the Arabidopsis flowering repressor FLC share common features that suggest conserved function in vernalization and de‐vernalization responses

Root chicory (Cichorium intybus var. sativum) is a biennial crop, but is harvested to obtain root inulin at the end of the first growing season before flowering. However, cold temperatures may vernalize seeds or plantlets, leading to incidental early flowering, and hence understanding the molecular basis of vernalization is important. A MADS box sequence was isolated by RT‐PCR and named FLC‐LIKE1 (CiFL1) because of its phylogenetic positioning within the same clade as the floral repressor Arabidopsis FLOWERING LOCUS C (AtFLC). Moreover, over‐expression of CiFL1 in Arabidopsis caused late flowering and prevented up‐regulation of the AtFLC target FLOWERING LOCUS T by photoperiod, suggesting functional conservation between root chicory and Arabidopsis. Like AtFLC in Arabidopsis, CiFL1 was repressed during vernalization of seeds or plantlets of chicory, but repression of CiFL1 was unstable when the post‐vernalization temperature was favorable to flowering and when it de‐vernalized the plants. This instability of CiFL1 repression may be linked to the bienniality of root chicory compared with the annual lifecycle of Arabidopsis. However, re‐activation of AtFLC was also observed in Arabidopsis when a high temperature treatment was used straight after seed vernalization, eliminating the promotive effect of cold on flowering. Cold‐induced down‐regulation of a MADS box floral repressor and its re‐activation by high temperature thus appear to be conserved features of the vernalization and de‐vernalization responses in distant species.     

Potent induction of Arabidopsis thaliana flowering by elevated growth temperature

The transition to flowering is an important event in the plant life cycle and is modulated by several environmental factors including photoperiod, light quality, vernalization, and growth temperature, as well as biotic and abiotic stresses. In contrast to light and vernalization, little is known about the pathways that mediate the responses to other environmental variables. A mild increase in growth temperature, from 23 °C to 27 °C, is equally efficient in inducing flowering of Arabidopsis plants grown in 8-h short days as is transfer to 16-h long days. There is extensive natural variation in this response, and we identify strains with contrasting thermal reaction norms. Exploiting this natural variation, we show that FLOWERING LOCUS C potently suppresses thermal induction, and that the closely related floral repressor FLOWERING LOCUS M is a major-effect quantitative trait locus modulating thermosensitivity. Thermal induction does not require the photoperiod effector CONSTANS, acts upstream of the floral integrator FLOWERING LOCUS T, and depends on the hormone gibberellin. Analysis of mutants defective in salicylic acid biosynthesis suggests that thermal induction is independent of previously identified stress-signaling pathways. Microarray analyses confirm that the genomic responses to floral induction by photoperiod and temperature differ. Furthermore, we report that gene products that participate in RNA splicing are specifically affected by thermal induction. Above a critical threshold, even small changes in temperature can act as cues for the induction of flowering. This response has a genetic basis that is distinct from the known genetic pathways of floral transition, and appears to correlate with changes in RNA processing.     

Flowering time regulation produces much fruit

Many of the molecular details regarding the promotion of flowering in response to prolonged exposure to cold temperatures (vernalization) and daylength have recently been elucidated in Arabidopsis. The daylength and vernalization pathway converge in the regulation of floral promoters referred to as floral integrators. In the meristem, vernalization promotes flowering through the epigenetic repression of the floral repressor FLOWERING LOCUS C. This allows for the induction of floral integrators by CONSTANS under inductive long days. In the vasculature of leaves, CONSTANS protein is produced only in long days where it acts to promote the expression of FLOWERING LOCUS T (FT). FT protein is then translocated to the meristem where it acts to promote floral induction. Thus a detailed molecular framework for the regulation of flowering time has now been established in Arabidopsis.     

miR824-Regulated AGAMOUS-LIKE16 Contributes to Flowering Time Repression in Arabidopsis

The timing of flowering is pivotal for maximizing reproductive success under fluctuating environmental conditions. Flowering time is tightly controlled by complex genetic networks that integrate endogenous and exogenous cues, such as light, temperature, photoperiod, and hormones. Here, we show that AGAMOUS-LIKE16 (AGL16) and its negative regulator microRNA824 (miR824) control flowering time in Arabidopsis thaliana. Knockout of AGL16 effectively accelerates flowering in nonvernalized Col-FRI, in which the floral inhibitor FLOWERING LOCUS C (FLC) is strongly expressed, but shows no effect if plants are vernalized or grown in short days. Alteration of AGL16 expression levels by manipulating miR824 abundance influences the timing of flowering quantitatively, depending on the expression level and number of functional FLC alleles. The effect of AGL16 is fully dependent on the presence of FLOWERING LOCUS T (FT). Further experiments show that AGL16 can interact directly with SHORT VEGETATIVE PHASE and indirectly with FLC, two proteins that form a complex to repress expression of FT. Our data reveal that miR824 and AGL16 modulate the extent of flowering time repression in a long-day photoperiod.     

FLC: A key regulator of flowering time in Arabidopsis

The timing of floral transition has significant consequences for reproductive success in plants. The molecular genetic dissection of flowering time control in Arabidopsis identified an integrated network of pathways that quantitatively control this developmental switch. A central player in this process is the FLOWERING LOCUS C gene (FLC), which blocks flowering by inhibiting the genes required to switch the meristem from vegetative to floral development. Three systems (the FRIGIDA gene, vernalization, and the autonomous pathway) all influence the state of FLC. Last years many new genes have been identified that regulate FLC expression, and most of them are involved in the modification of FLC chromatin. This review focuses on recent insights in FLC regulation.     

The E3 Ubiquitin Ligase HOS1 Regulates Arabidopsis Flowering by Mediating CONSTANS Degradation Under Cold Stress

The timing of flowering is coordinated by a web of gene regulatory networks that integrates developmental and environmental cues in plants. Light and temperature are two major environmental determinants that regulate flowering time. Although prolonged treatment with low nonfreezing temperatures accelerates flowering by stable repression of FLOWERING LOCUS C (FLC), repeated brief cold treatments delay flowering. Here, we report that intermittent cold treatments trigger the degradation of CONSTANS (CO), a central activator of photoperiodic flowering; daily treatments caused suppression of the floral integrator FLOWERING LOCUS T (FT) and delayed flowering. Cold-induced CO degradation is mediated via a ubiquitin/proteasome pathway that involves the E3 ubiquitin ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 1 (HOS1). HOS1-mediated CO degradation occurs independently of the well established cold response pathways. It is also independent of the light signaling repressor CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) E3 ligase and light wavelengths. CO has been shown to play a key role in photoperiodic flowering. Here, we demonstrated that CO served as a molecular hub, integrating photoperiodic and cold stress signals into the flowering genetic pathways. We propose that the HOS1-CO module contributes to the fine-tuning of photoperiodic flowering under short term temperature fluctuations, which often occur during local weather disturbances.     

Mechanisms underlying vernalization-mediated VIN3 induction in Arabidopsis

VERNALIZATION INSENSITIVE 3 (VIN3) is required for vernalization-mediated repression of FLOWERING LOCUS C (FLC) in Arabidopsis. The induction of VIN3 by long-term exposure to cold is one of earliest events in vernalization response. However, molecular mechanisms underlying for the VIN3 induction are poorly understood. Recently, we reported that the constitutive repression of VIN3 in the absence of the cold exposure is due to multiple repressive chromatin modifying components, including a transposable element (TE)-derived sequence, LIKE-HETEROCHROMATIN PROTEIN 1 (LHP1) and POLYCOMB REPRESSION COMPLEX 2 (PRC2). In addition, the maximum level of VIN3 induction requires EARLY FLOWERING 7 (ELF7) and EARLY FLOWERING IN SHORDAYS (EFS), which are components of activating chromatin modifying complexes. Furthermore, dynamic changes in histone modifications at VIN3 chromatin are observed during the course of vernalization. Thus, mechanisms underlying the induction of VIN3 include changes at the level of chromatin.Key words: vernalization, flowering, chromatinVernalization can be defined as “the acquisition or acceleration of the ability to flower by a chilling treatment.”¹ To maximize floral reproductive capability, plants in temperate climates have evolved a vernalization requirement to prevent flowering before the winter season and to ensure flowering in the spring. Vernalization response provides plants with competence to flower, rather than to induce flowering itself, through changes that remain stable even after cold exposure. This process is an epigenetic switch, whereby molecular changes remain stable throughout subsequent mitotic divisions despite the absence of initiating stimulus, cold exposure.^2–4 Vernalization response can be thought of as being comprised of two phases. The first is a cold perception system that measures the cumulative time of exposure to cold. The second phase is essentially the output of cold perception system: when a sufficient duration of cold has been perceived, a series of changes of gene expression ensue, ultimately leading to the epigenetic repression of FLC.     

Brahma is required for proper expression of the floral repressor FLC in Arabidopsis

Background

BRAHMA (BRM) is a member of a family of ATPases of the SWI/SNF chromatin remodeling complexes from Arabidopsis. BRM has been previously shown to be crucial for vegetative and reproductive development.

Methodology/Principal Findings

Here we carry out a detailed analysis of the flowering phenotype of brm mutant plants which reveals that, in addition to repressing the flowering promoting genes CONSTANS (CO), FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1), BRM also represses expression of the general flowering repressor FLOWERING LOCUS C (FLC). Thus, in brm mutant plants FLC expression is elevated, and FLC chromatin exhibits increased levels of histone H3 lysine 4 tri-methylation and decreased levels of H3 lysine 27 tri-methylation, indicating that BRM imposes a repressive chromatin configuration at the FLC locus. However, brm mutants display a normal vernalization response, indicating that BRM is not involved in vernalization-mediated FLC repression. Analysis of double mutants suggests that BRM is partially redundant with the autonomous pathway. Analysis of genetic interactions between BRM and the histone H2A.Z deposition machinery demonstrates that brm mutations overcome a requirement of H2A.Z for FLC activation suggesting that in the absence of BRM, a constitutively open chromatin conformation renders H2A.Z dispensable.

Conclusions/Significance

BRM is critical for phase transition in Arabidopsis. Thus, BRM represses expression of the flowering promoting genes CO, FT and SOC1 and of the flowering repressor FLC. Our results indicate that BRM controls expression of FLC by creating a repressive chromatin configuration of the locus.     

Different regulatory regions are required for the vernalization-induced repression of FLOWERING LOCUS C and for the epigenetic maintenance of repression

Vernalization, the promotion of flowering by a prolonged period of low temperature, results in repression of the floral repressor FLOWERING LOCUS C (FLC) and in early flowering. This repression bears the hallmark of an epigenetic event: the low expression state is maintained over many cell division cycles, but expression is derepressed in progeny. We show that the two stages of the response of FLC to vernalization, the repression of FLC and the maintenance of the repression during growth at normal temperatures after vernalization, are mediated through different regions of the FLC gene. Both promoter and intragenic regions are required for the responses. We also identify a 75-bp region in the FLC promoter that, in addition to intragenic sequences, is required for expression in nonvernalized plants.     

Coordination of the Vernalization Response through a VIN3 and FLC Gene Family Regulatory Network in Arabidopsis

Vernalization is an environmentally induced epigenetic switch in which winter cold triggers epigenetic silencing of floral repressors and thus provides competence to flower in spring. Vernalization triggers the recruitment of chromatin-modifying complexes to a clade of flowering repressors that are epigenetically silenced via chromatin modifications. In Arabidopsis thaliana, VERNALIZATION INSENSITIVE3 (VIN3) and its related plant homeodomain finger proteins act together with Polycomb Repressive Complex 2 to increase repressive histone marks at floral repressor loci, including FLOWERING LOCUS C (FLC) and its related genes, by vernalization. Here, we show that VIN3 family of proteins nonredundantly functions to repress different subsets of the FLC gene family during the course of vernalization. Each VIN3 family protein binds to modified histone peptides in vitro and directly associates with specific sets of FLC gene family chromatins in vivo to mediate epigenetic silencing. In addition, members of the FLC gene family are also differentially regulated during the course of vernalization to mediate proper vernalization response. Our results show that these two gene families cooperated during the course of evolution to ensure proper vernalization response through epigenetic changes.