E3 SUMO ligase SIZ1 splicing variants localize and function according to external conditions.

SIZ1 (SAP and MIZ1) is a member of the Siz/PIAS-type RING family of E3 SUMO (small ubiquitin-related modifier) ligases that play key roles in growth, development, and stress responses in plant and animal systems. Nevertheless, splicing variants of SIZ1 have not yet been characterized. Here, we identified four splicing variants of Arabidopsis (Arabidopsis thaliana) SIZ1, which encode three different protein isoforms. The SIZ1 gene encodes an 873-amino acid (aa) protein. Among the four SIZ1 splicing variants (SSVs), SSV1 and SSV4 encode identical 885 aa proteins; SSV2 encodes an 832 aa protein; and SSV3 encodes an 884 aa protein. SSV2 mainly localized to the plasma membrane, whereas SIZ1, SSV1/SSV4, and SSV3 localized to the nucleus. Interestingly, SIZ1 and all SSVs exhibited similar E3 SUMO ligase activities and preferred SUMO1 and SUMO2 for their E3 ligase activity. Transcript levels of SSV2 were substantially increased by heat treatment, while those of SSV1, SSV3, and SSV4 transcripts were unaffected by various abiotic stresses. SSV2 directly interacted with and sumoylated cyclic nucleotide-gated ion channel 6 (CNGC6), a positive thermotolerance regulator, enhancing the stability of CNGC6. Notably, transgenic siz1-2 mutants expressing SSV2 exhibited greater heat stress tolerance than wild-type plants, whereas those expressing SIZ1 were sensitive to heat stress. Furthermore, transgenic cngc6 plants overaccumulating a mutated mCNGC6 protein (K347R, a mutation at the sumoylation site) were sensitive to heat stress, similar to the cngc6 mutants, while transgenic cngc6 plants overaccumulating CNGC6 exhibited restored heat tolerance. Together, we propose that alternative splicing is an important mechanism that regulates the function of SSVs during development or under adverse conditions, including heat stress.

Arabidopsis retromer subunit AtVPS29 is involved in SLY1-mediated gibberellin signaling.

KEY MESSAGE: Retromer protein AtVPS29 upregulates the SLY1 protein and downregulates the RGA protein, positively stimulating the development of the root meristematic zone, which indicates an important role of AtVPS29 in gibberellin signaling. In plants, the large retromer complex is known to play roles in multiple development processes, including cell polarity, programmed cell death, and root hair growth in Arabidopsis. However, many of its roles in plant development remain unknown. Here, we show that Arabidopsis trimeric retromer protein AtVPS29 (vacuolar protein sorting 29) modulates gibberellin signaling. The SLEEPY1 (SLY1) protein, known as a positive regulator of gibberellic acid (GA) signaling, exhibited lower abundance in vps29-3 mutants compared to wild-type (WT) plants. Conversely, the DELLA repressor protein, targeted by the E3 ubiquitin ligase SCF (Skp, Cullin, F-box) complex and acting as a negative regulator of GA signaling, showed increased abundance in vps29-3 mutants compared to WT. The vps29-3 mutants exhibited decreased sensitivity to exogenous GA supply in contrast to WT, despite an upregulation in the expression of GA receptor genes within the vps29-3 mutants. In addition, the expression of the GA synthesis genes was downregulated in vps29-3 mutants, implying that the loss of AtVPS29 causes the downregulation of GA synthesis and signaling. Furthermore, vps29-3 mutants exhibited a reduced meristematic zone accompanied by a decreased cell number. Together, these data indicate that AtVPS29 positively regulates SLY1-mediated GA signaling and plant growth.

A mutation in the pPLA-IIα gene encoding PATATIN-RELATED PHOSPHOLIPASE a causes late flowering in Arabidopsis.

Arabidopsis PATATIN-RELATED PHOSPHOLIPASE 2A (pPLA-IIα) participates in the responses to various growth conditions. The factors affecting pPLA-IIα gene expression and pPLA-IIα protein activity for gycerolipids have been studied thoroughly, but the role of pPLA-IIα during the reproductive phase remains unclear. The effect of pPLA-IIα on flowering time was therefore investigated. ppla-iiα mutants flowered later than wild-type plants under long day conditions. Expression of the floral stimulators FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) was downregulated in ppla-iiα mutants compared with their expression in wild-type plants, but expression of the floral repressor FLOWERING LOCUS C (FLC) was upregulated. In addition, expression levels of COLDAIR, a long intronic noncoding RNA, decreased in ppla-iiα mutants. Taken together, these data indicate that pPLA-IIα acts as a positive regulator of flowering time through repression of FLC expression.

E3 SUMO ligase AtSIZ1 regulates the cruciferin content of Arabidopsis seeds.

Arabidopsis thaliana E3 SUMO ligase SIZ1 (AtSIZ1) controls vegetative growth and development, including responses to nutrient deficiency and environmental stresses. Here, we analyzed the effect of AtSIZ1 and its E3 SUMO ligase activity on the amount of seed proteins. Proteomic analysis showed that the level of three major nutrient reservoir proteins, CRUCIFERIN1 (CRU1), CRU2, and CRU3, was reduced in the siz1-2 mutant compared with the wild type. However, quantitative real-time PCR (qRT-PCR) analysis showed that transcript levels of CRU1, CRU2, and CRU3 genes were significantly higher in the siz1-2 mutant than in the wild type. Yeast two-hybrid analysis revealed direct interaction of AtSIZ1 with CRU1, CRU2, and CRU3. The sumoylation assay revealed that CRU2, and CRU3 proteins were modified with a small ubiquitin-related modifier (SUMO) by the E3 SUMO ligase activity of AtSIZ1. Additionally, high-performance liquid chromatography (HPLC) analysis showed that the amino acid content was slightly higher in siz1-2 mutant seeds than in wild type seeds. Taken together, our data indicate that AtSIZ1 plays an important role in the accumulation and stability of seed storage proteins through its E3 ligase activity.

Biosynthesis of DDMP saponins in soybean is regulated by a distinct UDP-glycosyltransferase.

2,3-Dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP) saponins are one of the major saponin groups that are widely distributed in legumes such as pea, barrel medic, chickpea, and soybean. The steps involved in DDMP saponin biosynthesis remain uncharacterized at the molecular level. We isolated two recessive mutants that lack DDMP saponins from an ethyl methanesulfonate-induced mutant population of soybean cultivar Pungsannamul. Segregation analysis showed that the production of DDMP saponins is controlled by a single locus, named Sg-9. The locus was physically mapped to a 130-kb region on chromosome 16. Nucleotide sequence analysis of candidate genes in the region revealed that each mutant has a single-nucleotide polymorphism in the Glyma.16G033700 encoding a UDP-glycosyltransferase UGT73B4. Enzyme assays and mass spectrum-coupled chromatographic analysis reveal that the Sg-9 protein has glycosyltransferase activity, converting sapogenins and group B saponins to glycosylated products, and that mutant proteins had only partial activities. The tissue-specific expression profile of Sg-9 matches the accumulation pattern of DDMP saponins. This is the first report on a new gene and its function in the biosynthesis of DDMP saponins. Our findings indicate that Sg-9 encodes a putative DDMP transferase that plays a critical role in the biosynthesis of DDMP saponins.

Nitrate Reductases Are Relocalized to the Nucleus by AtSIZ1 and Their Levels Are Negatively Regulated by COP1 and Ammonium.

Nitrate reductases (NRs) catalyze the first step in the reduction of nitrate to ammonium. NR activity is regulated by sumoylation through the E3 ligase activity of AtSIZ1. However, it is not clear how NRs interact with AtSIZ1 in the cell, or how nitrogen sources affect NR levels and their cellular localization. Here, we show that the subcellular localization of NRs is modulated by the E3 SUMO (Small ubiquitin-related modifier) ligase AtSIZ1 and that NR protein levels are regulated by nitrogen sources. Transient expression analysis of GFP fusion proteins in onion epidermal cells showed that the NRs NIA1 and NIA2 localize to the cytoplasmic membrane, and that AtSIZ1 localizes to the nucleoplasm, including nuclear bodies, when expressed separately, whereas NRs and AtSIZ1 localize to the nucleus when co-expressed. Nitrate did not affect the subcellular localization of the NRs, but it caused AtSIZ1 to move from the nucleus to the cytoplasm. NRs were not detected in ammonium-treated cells, whereas the localization of AtSIZ1 was not altered by ammonium treatment. NR protein levels increased in response to nitrate but decreased in response to ammonium. In addition, NR protein levels increased in response to a 26S proteasome inhibitor and in cop1-4 and DN-COP1-overexpressing transgenic plants. NR protein degradation occurred later in cop1-4 than in the wild-type, although the NR proteins did not interact with COP1. Therefore, AtSIZ1 controls nuclear localization of NR proteins, and ammonium negatively regulates their levels. The function and stability of NR proteins might be post-translationally modulated by ubiquitination.

GW2 Functions as an E3 Ubiquitin Ligase for Rice Expansin-Like 1.

Seed size is one of the most important traits determining the yield of cereal crops. Many studies have been performed to uncover the mechanism of seed development. However, much remains to be understood, especially at the molecular level, although several genes involved in seed size have been identified. Here, we show that rice Grain Width 2 (GW2), a RING-type E3 ubiquitin ligase, can control seed development by catalyzing the ubiquitination of expansin-like 1 (EXPLA1), a cell wall-loosening protein that increases cell growth. Microscopic examination revealed that a GW2 mutant had a chalky endosperm due to the presence of loosely packed, spherical starch granules, although the grain shape was normal. Yeast two-hybrid and in vitro pull-down assays showed a strong interaction between GW2 and EXPLA1. In vitro ubiquitination analysis demonstrated that EXPLA1 was ubiquitinated by GW2 at lysine 279 (K279). GW2 and EXPLA1 colocalized to the nucleus when expressed simultaneously. These results suggest that GW2 negatively regulates seed size by targeting EXPLA1 for degradation through its E3 ubiquitin ligase activity.

COP1 regulates plant growth and development in response to light at the post-translational level.

Photoreceptors perceive different wavelengths of light and transduce light signals downstream via a range of proteins. COP1, an E3 ubiquitin ligase, regulates light signaling by mediating the ubiquitination and subsequent proteasomal degradation of photoreceptors such as phytochromes and cryptochromes, as well as various development-related proteins including other light-responsive proteins. COP1 is itself regulated by direct interactions with several signaling molecules that modulate its activity. The control of photomorphogenesis by COP1 is also regulated by its localization to the cytoplasm in response to light. COP1 thus acts as a tightly regulated switch that determines whether development is skotomorphogenic or photomorphogenic. In this review, we discuss the effects of COP1 on the abundance and activity of various development-related proteins, including photoreceptors, and summarize the regulatory mechanisms that influence COP1 activity and stability in plants.

Post-translational modifications of FLOWERING LOCUS C modulate its activity.

Flowering Locus C (FLC) is a key floral repressor that precisely controls flowering time. The role of FLC has been extensively studied at the transcriptional level using molecular biological and epigenetic approaches. However, how FLC functions and how its stability is controlled at the post-translational level are only beginning to be understood. Recent studies show that various post-translational modifications (PTMs) control the stability and activity of FLC. In this review, we focus on three types of PTMs that regulate FLC function: phosphorylation, ubiquitination, and sumoylation. This report should serve as a model to guide post-translational studies of other important floral regulators.

The E3 SUMO ligase AtSIZ1 functions in seed germination in Arabidopsis.

Seed germination is an important stage in the lifecycle of a plant because it determines subsequent vegetative growth and reproduction. Here, we show that the E3 SUMO ligase AtSIZ1 regulates seed dormancy and germination. The germination rates of the siz1 mutants were less than 50%, even after a short period of ripening. However, their germination rates increased to wild-type levels after cold stratification or long periods of ripening. In addition, exogenous gibberellin (GA) application improved the germination rates of the siz1 mutants to the wild-type level. In transgenic plants, suppression of AtSIZ1 caused rapid post-translational decay of SLEEPY1 (SLY1), a positive regulator of GA signaling, during germination, and inducible AtSIZ1 overexpression led to increased SLY1 levels. In addition, overexpressing wild-type SLY1 in transgenic sly1 mutants increased their germination ratios to wild-type levels, whereas the germination ratio of transgenic sly1 mutants overexpressing mSLY1 was similar to that of sly1. The germination ratios of siz1 mutant seeds in immature developing siliques were much lower than those of the wild-type. Moreover, SLY1 and DELAY OF GERMINATION 1 (DOG1) transcript levels were reduced in the siz1 mutants, whereas the transcript levels of DELLA and ABSCISIC ACID INSENSITIVE 3 (ABI3) were higher than those of the wild-type. Taken together, these results indicate that the reduced germination of the siz1 mutants results from impaired GA signaling due to low SLY1 levels and activity, as well as hyperdormancy due to high levels of expression of dormancy-related genes including DOG1.

E3 SUMO ligase AtSIZ1 positively regulates SLY1-mediated GA signalling and plant development.

Gibberellins affect various plant development processes including germination, cell division and elongation, and flowering. A large number of studies have been carried out to address the molecular mechanisms that mediate gibberellin signalling effects on plant growth. However, such studies have been limited to DELLA protein degradation; the regulatory mechanisms controlling how the stability and function of SLEEPY1 (SLY1), a protein that interacts with target DELLA proteins as components of the Skp, Cullin, F-box (SCF)(SLY1) complex, are modulated at the post-translational level have not been addressed. In the present study, we show that the E3 SUMO (small ubiquitin-related modifier) ligase AtSIZ1 regulates gibberellic acid signalling in Arabidopsis species by sumoylating SLY1. SLY1 was less abundant in siz1-2 mutants than in wild-type plants, but the DELLA protein repressor of ga1-3 (RGA) was more abundant in siz1-2 mutants than in wild-type plants. SLY1 also accumulated to a high level in the SUMO protease mutant esd4. Transgenic sly1-13 mutants over-expressing SLY1 were phenotypically similar to wild-type plants; however, sly1-13 plants over-expressing a mutated mSLY1 protein (K122R, a mutation at the sumoylation site) retained the mutant dwarfing phenotype. Over-expression of SLY1 in sly1-13 mutants resulted in a return of RGA levels to wild-type levels, but RGA accumulated to high levels in mutants over-expressing mSLY1. RGA was clearly detected in Arabidopsis co-expressing AtSIZ1 and mSLY1, but not in plants co-expressing AtSIZ1 and SLY1. In addition, sumoylated SLY1 interacted with RGA and SLY1 sumoylation was significantly increased by GA. Taken together, our results indicate that, in Arabidopsis, AtSIZ1 positively controls GA signalling through SLY1 sumoylation.

FLC-mediated flowering repression is positively regulated by sumoylation.

Flowering locus C (FLC), a floral repressor, is a critical factor for the transition from the vegetative to the reproductive phase. Here, the mechanisms regulating the activity and stability of the FLC protein were investigated. Bimolecular fluorescence complementation and in vitro pull-down analyses showed that FLC interacts with the E3 small ubiquitin-like modifier (SUMO) ligase AtSIZ1, suggesting that AtSIZ1 is an E3 SUMO ligase for FLC. In vitro sumoylation assays showed that FLC is modified by SUMO in the presence of SUMO-activating enzyme E1 and conjugating enzyme E2, but its sumoylation is inhibited by AtSIZ1. In transgenic plants, inducible AtSIZ1 overexpression led to an increase in the concentration of FLC and delayed the post-translational decay of FLC, indicating that AtSIZ1 stabilizes FLC through direct binding. Also, the flowering time in mutant FLC (K154R, a mutation of the sumoylation site)-overexpressing plants was comparable with that in the wild type, whereas flowering was considerably delayed in FLC-overexpressing plants, supporting the notion that sumoylation is an important mechanism for FLC function. The data indicate that the sumoylation of FLC is critical for its role in the control of flowering time and that AtSIZ1 positively regulates FLC-mediated floral suppression.

Arabidopsis COP1 guides stomatal response in guard cells through pH regulation.

Plants rely on precise regulation of their stomatal pores to effectively carry out photosynthesis while managing water status. The Arabidopsis CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), a critical light signaling repressor, is known to repress stomatal opening, but the exact cellular mechanisms remain unknown. Here, we show that COP1 regulates stomatal movement by controlling the pH levels in guard cells. cop1-4 mutants have larger stomatal apertures and disrupted pH dynamics within guard cells, characterized by increased vacuolar and cytosolic pH and reduced apoplastic pH, leading to abnormal stomatal responses. The altered pH profiles are attributed to the increased plasma membrane (PM) H+-ATPase activity of cop1-4 mutants. Moreover, cop1-4 mutants resist to growth defect caused by alkali stress posed on roots. Overall, our study highlights the crucial role of COP1 in maintaining pH homeostasis of guard cells by regulating PM H+-ATPase activity, and demonstrates how proton movement affects stomatal movement and plant growth.

Genetic Control of Tolerance to Drought Stress in Wild Soybean (Glycine soja) at the Vegetative and the Germination Stages.

Drought stress, which is becoming more prevalent due to climate change, is a significant abiotic factor that adversely impacts crop production and yield stability. Cultivated soybean (Glycine max), a versatile crop for humans and animals, exhibits sensitivity to drought, resulting in reduced growth and development under drought conditions. However, few genetic studies have assessed wild soybean's (Glycine soja) response to drought stress. In this work, we conducted a genome-wide association study (GWAS) and analysis of wild soybean accessions to identify loci responsible for drought tolerance at the vegetative (n = 187) and the germination stages (n = 135) using the available resequencing data. The GWAS analysis of the leaf wilting score (LWS) identified eight single-nucleotide polymorphisms (SNPs) on chromosomes 10, 11, and 19. Of these, wild soybeans with both SNPs on chromosomes 10 (adenine) and 11 (thymine) produced lower LWS, indicating that these SNPs have an important role in the genetic effect on LWS for drought tolerance at the vegetative stage. At the germination stage, nine SNPs associated with five phenotypic measurements were identified on chromosomes 6, 9, 10, 13, 16, and 17, and the genomic regions identified at the germination stage were different from those identified for the LWS, supporting our previous finding that there may not be a robust correlation between the genes influencing phenotypes at the germination and vegetative stages. This research will benefit marker-assisted breeding programs aimed at enhancing drought tolerance in soybeans.

Arabidopsis PHYTOCHROME INTERACTING FACTOR proteins promote phytochrome B polyubiquitination by COP1 E3 ligase in the nucleus.

Many plant photoresponses from germination to shade avoidance are mediated by phytochrome B (phyB). In darkness, phyB exists as the inactive Pr in the cytosol but upon red (R) light treatment, the active Pfr translocates into nuclei to initiate signaling. Degradation of phyB Pfr likely regulates signal termination, but the mechanism is not understood. Here, we show that phyB is stable in darkness, but in R, a fraction of phyB translocates into nuclei and becomes degraded by 26S proteasomes. Nuclear phyB degradation is mediated by COP1 E3 ligase, which preferentially interacts with the PhyB N-terminal region (PhyB-N). PhyB-N polyubiquitination by CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) in vitro can be enhanced by different PHYTOCHROME INTERACTING FACTOR (PIF) proteins that promote COP1/PhyB interaction. Consistent with these results, nuclear phyB accumulates to higher levels in pif single and double mutants and in cop1-4. Our results identify COP1 as an E3 ligase for phyB and other stable phytochromes and uncover the mechanism by which PIFs negatively regulate phyB levels.

Seed size: a priority trait in cereal crops.

Crop production and productivity must be increased to provide a balanced diet for the global population. The entire genome sequences of crop species allow the elucidation of genes that regulate important traits related to the final crop seed yield, which frequently depends mainly on seed size. Seed size is a major factor that controls seed quantity and it is strongly affected by various biotic, abiotic and genetic factors. Epigenetic marks in the genome and phytohormones are also important factors affecting seed growth and development. Several genes are known to be involved in the control of seed size, but their interaction and functional characterization have yet to be resolved. In this review, we discuss the different factors that govern seed size in cereal crops and Arabidopsis.

Teosinte Branched 1 modulates tillering in rice plants.

Tillering is an important trait of cereal crops that optimizes plant architecture for maximum yield. Teosinte Branched 1 (TB1) is a negative regulator of lateral branching and an inducer of female inflorescence formation in Zea mays (maize). Recent studies indicate that TB1 homologs in Oryza sativa (rice), Sorghum bicolor and Arabidopsis thaliana act downstream of the auxin and MORE AUXILIARY GROWTH (MAX) pathways. However, the molecular mechanism by which rice produces tillers remains unknown. In this study, transgenic rice plants were produced that overexpress the maize TB1 (mTB1) or rice TB1 (OsTB1) genes and silence the OsTB1 gene through RNAi-mediated knockdown. Because lateral branching in rice is affected by the environmental conditions, the phenotypes of transgenic plants were observed in both the greenhouse and the paddy field. Compared to wild-type plants, the number of tillers and panicles was reduced and increased in overexpressed and RNAi-mediated knockdown OsTB1 rice plants, respectively, under both environmental conditions. However, the effect was small for plants grown in paddy fields. These results demonstrate that both mTB1 and OsTB1 moderately regulate the tiller development in rice.

COP1 mutation causes low leaf temperature under various abiotic stresses in Arabidopsis thaliana.

Stomata are microscopic pores on epidermal cells of leaves and stems that regulate water loss and gas exchange between the plant and its environment. Constitutive photomorphogenic 1 (COP1) is an E3 ubiquitin ligase that is involved in plant growth and development and multiple abiotic stress responses by regulating the stability of various target proteins. However, little is known about how COP1 controls stomatal aperture and leaf temperature under various environmental conditions. Here, we show that COP1 participates in leaf temperature and stomatal closure regulation under normal and stress conditions in Arabidopsis. Leaf temperature of cop1 mutants was relatively lower than that of wild type (WT) under drought, salt, and heat stress and after abscisic acid (ABA), CaCl2, and H2O2 treatments. However, leaf temperature was generally higher in both WT and cop1 mutants after abiotic stress and chemical treatment than that of untreated WT and cop1 mutants. Stomatal aperture was wider in cop1 mutants than that in WT under all conditions tested, although the extent of stomatal closure varied between WT and cop1 mutants. Under dark conditions, leaf temperature was also lower in cop1 mutants than that in WT. Expression of the genes encoding ABA receptors, ABA biosynthesis proteins, positive regulators of stomatal closure and heat tolerance, and ABA-responsive proteins was lower in cop1 mutants that that in WT. In addition, expression of respiration-related genes was lower in cop1 mutants that that in WT. Taken together, the data provide evidence that mutations in COP1 lead to wider stomatal aperture and higher leaf temperature under normal and stress conditions, indicating that leaf temperature is highly correlated with stomatal aperture.

COP1 controls salt stress tolerance by modulating sucrose content.

The E3 ubiquitin ligase Constitutive Photomorphogenic 1 (COP1) plays evolutionarily conserved and divergent roles. In plants, COP1 regulates a large number of developmental processes including photomorphogenesis, seedling emergence, and gravitropism. Nevertheless, its function in abiotic stress tolerance remains largely unknown. Here, we demonstrate the role of COP1 in salt stress tolerance in Arabidopsis thaliana. In soil, cop1-4 and cop1-6 mutants were more tolerant to high salinity than wild-type (WT) plants during vegetative growth. However, in high salt-containing Murashige and Skoog (MS) medium, cop1-4 and cop1-6 seedlings exhibited significantly impaired growth compared with WT plants. Notably, cop1-4 and cop1-6 seedlings recovered their growth to the WT level upon exogenous sucrose treatment even under high salinity conditions. Compared with WT plants, the sucrose content of cop1-4 mutants was much higher at the vegetative growth stage but similar at the seedling stage. Upon exogenous sucrose supply, root elongation was significantly stimulated in cop1-4 seedlings but only slightly stimulated in WT plants. Thus, no significant difference was observed in root length between the two genotypes. Altogether, our data indicate that cop1 mutants are more tolerant to salt stress than WT plants, and the salt tolerance of cop1 mutants is correlated with their sucrose content.

Intronic long noncoding RNA, RICE FLOWERING ASSOCIATED (RIFLA), regulates OsMADS56-mediated flowering in rice.

Long noncoding RNAs (lncRNAs) are known to play important roles in several plant processes such as flowering, organ development and stress response. However, studies exploring the diversity and complexity of lncRNAs and their mechanism of action in plants are far fewer that those in animals. Here, we show that an intronic lncRNA in rice (Oryza sativa L.), RICE FLOWERING ASSOCIATED (RIFLA), is required for the inhibition of OsMADS56 expression. RIFLA is produced from the first intron of the OsMADS56 gene. Overexpression of RIFLA in rice repressed OsMADS56 expression but activated the expression of flowering inducers Hd3a and RFT1. Additionally, RIFLA-overexpressing transgenic rice plants flowered earlier than the wild type. Under normal conditions, the transcript level of the rice enhancer of zeste gene OsiEZ1, a homolog of Arabidopsis histone H3K27-specific methyltransferase genes SWINGER (SWN) and CURLY LEAF (CLF), was as low as that of RIFLA, whereas the transcript level of OsMADS56 was relatively high. In the osiez1 mutant, OsMADS56 expression was upregulated, whereas RIFLA expression was downregulated. Additionally, RIFLA formed a complex with OsiEZ1. Together, these results suggest that the floral repressor activity of OsMADS56 is epigenetically regulated by RIFLA and OsiEZ1.