HISTONE DEACETYLASE6 interacts with FLOWERING LOCUS D and regulates flowering in Arabidopsis

Histone acetylation and deacetylation play an important role in epigenetic controls of gene expression. HISTONE DEACETYLASE6 (HDA6) is a REDUCED POTASSIUM DEPENDENCY3-type histone deacetylase, and the Arabidopsis (Arabidopsis thaliana) hda6 mutant axe1-5 displayed a late-flowering phenotype. axe1-5/flc-3 double mutants flowered earlier than axe1-5 plants, indicating that the late-flowering phenotype of axe1-5 was FLOWERING LOCUS C (FLC) dependent. Bimolecular fluorescence complementation, in vitro pull-down, and coimmunoprecipitation assays revealed the protein-protein interaction between HDA6 and the histone demethylase FLD. It was found that the SWIRM domain in the amino-terminal region of FLD and the carboxyl-terminal region of HDA6 are responsible for the interaction between these two proteins. Increased levels of histone H3 acetylation and H3K4 trimethylation at FLC, MAF4, and MAF5 were found in both axe1-5 and fld-6 plants, suggesting functional interplay between histone deacetylase and demethylase in flowering control. These results support a scenario in which histone deacetylation and demethylation cross talk are mediated by physical association between HDA6 and FLD. Chromatin immunoprecipitation analysis indicated that HDA6 bound to the chromatin of several potential target genes, including FLC and MAF4. Genome-wide gene expression analysis revealed that, in addition to genes related to flowering, genes involved in gene silencing and stress response were also affected in hda6 mutants, revealing multiple functions of HDA6. Furthermore, a subset of transposons was up-regulated and displayed increased histone hyperacetylation, suggesting that HDA6 can also regulate transposons through deacetylating histone.     

The downregulation of FLOWERING LOCUS C (FLC) expression in plants with low levels of DNA methylation and by vernalization occurs by distinct mechanisms

FLOWERING LOCUS C (FLC), a repressor of flowering, is a major determinant of flowering time in Arabidopsis. FLC expression is repressed by vernalization and in plants with low levels of DNA methylation, resulting in early flowering. This repression is not associated with changes of DNA methylation within the FLC locus in either vernalized plants or plants with low levels of DNA methylation. In both cases, there is a reduction of histone H3 trimethyl-lysine 4 (K4) and acetylation of both histones H3 and H4 around the promoter-translation start of FLC. The expression of the two genes flanking FLC is also repressed in both conditions and repression is associated with decreased histone H3 acetylation. The changes in histone modifications at the FLC gene cluster, which are similar in vernalized plants and in plants with reduced DNA methylation, must arise by different mechanisms. VERNALIZATION 1, VERNALIZATION 2 and VERNALIZATION INSENSITIVE 3 modulate FLC expression in vernalized plants; these proteins play no role in the downregulation of FLC in plants with low levels of DNA methylation. Chimeric FLC::GUS transgenes respond to vernalization but these same transgenes show a position-dependent response to low levels of DNA methylation. In plants with reduced DNA methylation, expression of the five MADS AFFECTING FLOWERING (MAF) genes is repressed, suggesting that DNA methylation alters the expression of a trans-acting regulator common to FLC and members of the related MAF gene family. Our observations suggest that DNA methylation is not part of the vernalization pathway.     

The MADS domain factors AGL15 and AGL18 act redundantly as repressors of the floral transition in Arabidopsis

The developmental roles of AGL15 and AGL18, members of the AGL15-like clade of MADS domain regulatory factors, have not been defined previously. Analysis of transgenic Arabidopsis plants showed that overexpression of AGL18 produces the same phenotypic changes as overexpression of AGL15, and the two genes have partially overlapping expression patterns. Functional redundancy was confirmed through analysis of loss-of-function mutants. agl15 agl18 double mutants, but not single mutants, flower early under non-inductive conditions, indicating that AGL15 and AGL18 act in a redundant fashion as repressors of the floral transition. Further genetic analyses and expression studies were used to examine the relationship between AGL15 and AGL18 activity and other regulators of the floral transition. AGL15 and AGL18 act upstream of the floral integrator FT, and a combination of agl15 and agl18 mutations partially suppresses defects in the photoperiod pathway. agl15 agl18 mutations show an additive relationship with mutations in genes encoding other MADS domain floral repressors, and further acceleration of flowering is seen in triple and quadruple mutants under both inductive and non-inductive conditions. Thus, flowering time is determined by the additive effect of multiple MADS domain floral repressors, with important contributions from AGL15 and AGL18.     

The MADS-Domain Factors AGAMOUS-LIKE15 and AGAMOUS-LIKE18, along with SHORT VEGETATIVE PHASE and AGAMOUS-LIKE24, Are Necessary to Block Floral Gene Expression during the Vegetative Phase

Multiple factors, including the MADS-domain proteins AGAMOUS-LIKE15 (AGL15) and AGL18, contribute to the regulation of the transition from vegetative to reproductive growth. AGL15 and AGL18 were previously shown to act redundantly as floral repressors and upstream of FLOWERING LOCUS T (FT) in Arabidopsis (Arabidopsis thaliana). A series of genetic and molecular experiments, primarily focused on AGL15, was performed to more clearly define their role. agl15 agl18 mutations fail to suppress ft mutations but show additive interactions with short vegetative phase (svp) mutations in ft and suppressor of constans1 (soc1) backgrounds. Chromatin immunoprecipitation analyses with AGL15-specific antibodies indicate that AGL15 binds directly to the FT locus at sites that partially overlap those bound by SVP and FLOWERING LOCUS C. In addition, expression of AGL15 in the phloem effectively restores wild-type flowering times in agl15 agl18 mutants. When agl15 agl18 mutations are combined with agl24 svp mutations, the plants show upward curling of rosette and cauline leaves, in addition to early flowering. The change in leaf morphology is associated with elevated levels of FT and ectopic expression of SEPALLATA3 (SEP3), leading to ectopic expression of floral genes. Leaf curling is suppressed by sep3 and ft mutations and enhanced by soc1 mutations. Thus, AGL15 and AGL18, along with SVP and AGL24, are necessary to block initiation of floral programs in vegetative organs.Appropriate timing of the shift from vegetative to reproductive growth is an important determinant of plant fitness. The time at which a plant flowers is determined through integration of signals reflecting extrinsic and intrinsic conditions, such as photoperiod, the duration of cold, plant health, and age (for review, see Amasino, 2010). One of the most important pathways regulating the timing of the floral transition is the photoperiod pathway (for review, see Imaizumi and Kay, 2006). Under long-day (LD) inductive conditions in Arabidopsis (Arabidopsis thaliana), photoperiod pathway components act to promote flowering by inducing CONSTANS (CO) and downstream genes. The floral integrator FLOWERING LOCUS T (FT) is a major target of multiple flowering pathways and the photoperiod pathway in particular. It is directly activated by CO (Samach et al., 2000). Under LD conditions, the peak of CO expression is coincident with the presence of light, and CO activates FT expression in the leaf vascular system (Yanovsky and Kay, 2003). FT travels through the phloem to the shoot apex (Corbesier et al., 2007), where, together with FLOWERING LOCUS D (Abe et al., 2005; Wigge et al., 2005), it activates APETALA1 (AP1) and other floral meristem identity genes, starting the flowering process. Other flowering time pathways converge on FT and/or directly impact gene expression in the meristem. The changes in gene expression that accompany the floral transition must be rapid, robust, largely irreversible, and strictly controlled spatially. This is achieved through positive feed-forward and negative feedback loops involving multiple regulatory factors (for recent review, see Kaufmann et al., 2010).Members of the MADS-box family of regulatory factors are central players in the regulatory loops controlling the floral transition (for a recent review, see Smaczniak et al., 2012a). MADS-domain factors typically act in large multimeric complexes and are well suited for regulation that involves combinatorial action. During the floral transition, MADS-domain proteins can act either as repressors or activators. In Arabidopsis, important floral repressors include SHORT VEGETATIVE PHASE (SVP) and members of the FLOWERING LOCUS C (FLC)-like group, including FLC, FLOWERING LOCUS M (FLM)/MADS AFFECTING FLOWERING1 (MAF1), and MAF2 to MAF5. Promoters of flowering include such MADS-domain factors as SUPPRESSOR OF CONSTANS1 (SOC1) and AGAMOUS-LIKE24 (AGL24). Together with non-MADS-box proteins FT and TWIN SISTER OF FT, SOC1 and AGL24 function as floral integrators. These operate downstream of the flowering time pathways but upstream of the meristem identity regulators such as LEAFY (LFY) and the MADS-domain factor AP1.The MADS-domain factors AGL15 and AGL18 also contribute to regulation of the floral transition in Arabidopsis. While single mutants have no phenotype, agl15 agl18 double mutants flower earlier than the wild type (Adamczyk et al., 2007). Therefore, AGL15 and AGL18 appear to act in a redundant fashion in seedlings, and like SVP, FLC, and MAF1 to MAF5, they act as floral repressors. The contributions of AGL15 and AGL18 are most apparent in the absence of strong photoperiodic induction: the agl15 agl18 double mutant combination partially suppresses the delay in flowering observed in co mutants, as well as the flowering delay associated with growth under short-day (SD) noninductive conditions. The earlier flowering in agl15 agl18 mutants under these conditions is associated with up-regulation of FT, and both AGL15 and AGL18 are expressed in the vascular system and shoot apex of young seedlings (Adamczyk et al., 2007), raising the possibility that AGL15 and AGL18 act directly on FT in leaves, as well as other targets in the meristem.AGL15, and to a lesser extent AGL18, have been further implicated in the networks that control flowering through molecular studies. Zheng et al. (2009) performed a chromatin immunoprecipitation (ChIP) analysis using AGL15-specific antibodies, tissue derived from embryo cultures, and a tiling array. Floral repressors (SVP and FLC), floral integrators (FT and SOC1), and a microRNA targeting AP2-like factors (miR172a) were identified as possible AGL15 targets (Zheng et al., 2009), suggesting that AGL15 may contribute to regulation through multiple avenues during the floral transition. AGL15 itself is directly bound and activated by AP2, which is both an A-class floral identity gene and a floral repressor (Yant et al., 2010). AGL15 is down-regulated in ap2 mutants, which are early flowering, while AGL18 is the nearest locus to multiple AP2-bound sites (Yant et al., 2010). Both AGL15 and AGL18 were identified as SOC1 targets through ChIP analyses (Immink et al., 2009; Tao et al., 2012). In yeast (Saccharomyces cerevisiae) two-hybrid assays, AGL15 interacts with a number of other MADS-domain proteins (de Folter et al., 2005), and in a one-hybrid study based on the SOC1 promoter, AGL15-SVP, AGL15-AGL24, and AGL15-SOC1 heterodimers were shown to bind to regions containing CArG boxes (Immink et al., 2012). AGL18 may act redundantly to AGL15 in these contexts. However, AGL18 either does not interact or only interacts weakly with other proteins in yeast two-hybrid assays (de Folter et al., 2005; Hill et al., 2008; Causier et al., 2012). It remains to be determined whether this truly reflects weaker or nonredundant in planta interactions or a technical problem in the artificial yeast system.Guided by the knowledge gained about AGL15 targets and interactions from molecular studies, we asked the following question: what is the functional significance of these molecular relationships in the context of the floral transition? We performed a series of genetic experiments combining agl15 agl18 mutations and mutations in interacting factors such as SVP, AGL24, and SOC1, as well as targets such as FT and SOC1. We also performed further molecular experiments focused on AGL15, for which a variety of tools are available. Among other things, we show that AGL15 and AGL18, along with AGL24 and SVP, play a role in blocking expression of the floral MADS-domain factor SEPALLATA3 (SEP3) during the vegetative phase. In the absence of these four factors, reproductive programs are initiated early, and floral genes are expressed in the youngest rosette leaf and cauline leaves.     

The FLX gene of Arabidopsis is required for FRI-dependent activation of FLC expression

The Arabidopsis FLOWERING LOCUS C (FLC) gene encodes a MADS box protein that acts as a dose-dependent repressor of flowering. Mutants and ecotypes with elevated expression of FLC are late flowering and vernalization responsive. In this study we describe an early flowering mutant in the C24 ecotype, flc expressor (flx), that has reduced expression of FLC. FLX encodes a protein of unknown function with putative leucine zipper domains. FLX is required for FRIGIDA (FRI)-mediated activation of FLC but not for activation of FLC in autonomous pathway mutants. FLX is also required for expression of the FLC paralogs MADS AFFECTING FLOWERING 1 (MAF1) and MAF2.     

PIE1, an ISWI family gene,is required for FLC activation and floral repression in Arabidopsis

Proper control of the floral transition is critical for reproductive success in flowering plants. In Arabidopsis, FLOWERING LOCUS C (FLC) is a floral repressor upon which multiple floral regulatory pathways converge. Mutations in PHOTOPERIOD-INDEPENDENT EARLY FLOWERING1 (PIE1) suppress the FLC-mediated delay of flowering as a result of the presence of FRIGIDA or of mutations in autonomous pathway genes. PIE1 is required for high levels of FLC expression in the shoot apex, but it is not required for FLC expression in roots. PIE1 is similar to ATP-dependent, chromatin-remodeling proteins of the ISWI and SWI2/SNF2 family. The role of PIE1 as an activator of FLC is consistent with the general role of ISWI and SWI2/SNF2 family genes as activators of gene expression. The pie1 mutation also causes early flowering in noninductive photoperiods independently of FLC; thus, PIE1 appears to be involved in multiple flowering pathways. PIE1 also plays a role in petal development, as revealed by the suppression of petal defects of the curly leaf mutant by the pie1 mutation.     

RFI2, a RING-domain zinc finger protein, negatively regulates CONSTANS expression and photoperiodic flowering

The red and far-red light-absorbing phytochromes interact with the circadian clock, a central oscillator that sustains a 24-h period, to measure accurately seasonal changes in day-length and regulate the expression of several key flowering genes. The interactions and subsequent signalling steps upstream of the flowering genes such as CONSTANS (CO) and FLOWERING LOCUS T (FT) remain largely unknown. We report here that a photomorphogenic mutant, red and far-red insensitive 2-1 ( rfi2-1), flowered early particularly under long days. The rfi2-1 mutation also enhanced the expression of CO and FT under day/night cycles or constant light. Both co-2 and gigantea-2 (gi-2) were epistatic to rfi2-1 in their flowering responses. The gi-2 mutation was also epistatic to the rfi2-1 mutation in the expression of CO and hypocotyl elongation. However, the rfi2-1 mutation did not affect the expression of GI, a gene that mediates between the circadian clock and the expression of CO. Like many other flowering genes, the expression of RFI2 oscillated under day/night cycles and was rhythmic under constant light. The amplitude of the rhythmic expression of RFI2 was significantly reduced in phyB-9 or lhy-20 plants, and was also affected by the gi-2 mutation. As previously reported, the gi-2 mutation affects the period length and amplitude of CCA1 and LHY expression, and GI may act through a feedback loop to maintain a proper circadian function. We propose a regulatory step in which RFI2 represses the expression of CO, whereas GI may maintain the proper expression of RFI2 through its positive action on the circadian clock. The regulatory step serves to tune the circadian outputs that control the expression of CO and photoperiodic flowering.     

The microRNA156-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE3 module regulates ambient temperature-responsive flowering via FLOWERING LOCUS T in Arabidopsis

The flowering time of plants is affected by modest changes in ambient temperature. However, little is known about the regulation of ambient temperature-responsive flowering by small RNAs. In this study, we show that the microRNA156 (miR156)-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE3 (SPL3) module directly regulates FLOWERING LOCUS T (FT) expression in the leaf to control ambient temperature-responsive flowering. Overexpression of miR156 led to more delayed flowering at a lower ambient temperature (16°C), which was associated with down-regulation of FT and FRUITFULL expression. Among miR156 target genes, SPL3 mRNA levels were mainly reduced, probably because miR156-mediated cleavage of SPL3 mRNA was higher at 16°C. Overexpression of miR156-resistant SPL3 [SPL3(-)] caused early flowering, regardless of the ambient temperature, which was associated with up-regulation of FT and FRUITFULL expression. Reduction of miR156 activity by target mimicry led to a phenotype similar to that of SUC2::rSPL3 plants. FT up-regulation was observed after dexamethasone treatment in GVG-rSPL3 plants. Misexpression and artificial microRNA-mediated suppression of FT in the leaf dramatically altered the ambient temperature-responsive flowering of plants overexpressing miR156 and SPL3(-). Chromatin immunoprecipitation assay showed that the SPL3 protein directly binds to GTAC motifs within the FT promoter. Lesions in TERMINAL FLOWER1, SHORT VEGETATIVE PHASE, and EARLY FLOWERING3 did not alter the expression of miR156 and SPL3. Taken together, our data suggest that the interaction between the miR156-SPL3 module and FT is part of the regulatory mechanism controlling flowering time in response to ambient temperature.     

Chromatin regulation of flowering

The transition to flowering is a major developmental switch in the life cycle of plants. In Arabidopsis (Arabidopsis thaliana), chromatin mechanisms play critical roles in flowering-time regulation through the expression control of key flowering-regulatory genes. Various conserved chromatin modifiers, plant-specific factors, and long noncoding RNAs are involved in chromatin regulation of FLOWERING LOCUS C (FLC, a potent floral repressor). The well-studied FLC regulation has provided a paradigm for chromatin-based control of other developmental genes. In addition, chromatin modification plays an important role in the regulation of FLOWERING LOCUS T (FT, encoding florigen), which is widely conserved in angiosperm species. The chromatin mechanisms underlying FT regulation in Arabidopsis are likely involved in the regulation of FT relatives and, therefore, flowering-time control in other plants.     

AGL24 acts in concert with SOC1 and FUL during Arabidopsis floral transition

Arabidopsis plants flower in response to long days (LDs). Exposure of leaves to inductive day lengths activates expression of FLOWERING LOCUS T (FT) protein which moves to the shoot apical meristem (SAM) to induce developmental reprogramming. SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and FRUITFULL (FUL) are induced by FT at the apex. We previously screened the SAM for mRNAs of genes required to promote the floral transition in response to photoperiod, and conducted detailed expression and functional analyses on several putative candidates. Here, we show that expression of AGAMOUS-LIKE 24 (AGL24) is detected at the SAM under SD conditions and increases upon exposure to LDs. Mutations in AGL24 further delay flowering of a soc1 ful double mutant, suggesting that flowering is controlled by AGL24 partly independently of SOC1 and FUL.     

The nuclear actin-related protein ARP6 is a pleiotropic developmental regulator required for the maintenance of FLOWERING LOCUS C expression and repression of flowering in Arabidopsis

Actin-related proteins (ARPs) are found in the nuclei of all eukaryotic cells, but their functions are generally understood only in the context of their presence in various yeast and animal chromatin-modifying complexes. Arabidopsis thaliana ARP6 is a clear homolog of other eukaryotic ARP6s, including Saccharomyces cerevisiae ARP6, which was identified as a component of the SWR1 chromatin remodeling complex. We examined the subcellular localization, expression patterns, and loss-of-function phenotypes for this protein and found that Arabidopsis ARP6 is localized to the nucleus during interphase but dispersed away from the chromosomes during cell division. ARP6 expression was observed in all vegetative tissues as well as in a subset of reproductive tissues. Null mutations in ARP6 caused numerous defects, including altered development of the leaf, inflorescence, and flower as well as reduced female fertility and early flowering in both long- and short-day photoperiods. The early flowering of arp6 mutants was associated with reduced expression of the central floral repressor gene FLOWERING LOCUS C (FLC) as well as MADS AFFECTING FLOWERING 4 (MAF4) and MAF5. In addition, arp6 mutations suppress the FLC-mediated late flowering of a FRIGIDA-expressing line, indicating that ARP6 is required for the activation of FLC expression to levels that inhibit flowering. These results indicate that ARP6 acts in the nucleus to regulate plant development, and we propose that it does so through modulation of chromatin structure and the control of gene expression.