Deciphering the Role of POLYCOMB REPRESSIVE COMPLEX1 Variants in Regulating the Acquisition of Flowering Competence in Arabidopsis

Abstract

Polycomb group (PcG) proteins play important roles in regulating developmental phase transitions in plants; however, little is known about the role of the PcG machinery in regulating the transition from juvenile to adult phase. Here, we show that Arabidopsis (Arabidopsis thaliana) B lymphoma Moloney murine leukemia virus insertion region1 homolog (BMI1) POLYCOMB REPRESSIVE COMPLEX1 (PRC1) components participate in the repression of microRNA156 (miR156). Loss of AtBMI1 function leads to the up-regulation of the primary transcript of MIR156A and MIR156C at the time the levels of miR156 should decline, resulting in an extended juvenile phase and delayed flowering. Conversely, the PRC1 component EMBRYONIC FLOWER (EMF1) participates in the regulation of SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE and MIR172 genes. Accordingly, plants impaired in EMF1 function displayed misexpression of these genes early in development, which contributes to a CONSTANS-independent up-regulation of FLOWERING LOCUS T (FT) leading to the earliest flowering phenotype described in Arabidopsis. Our findings show how the different regulatory roles of two functional PRC1 variants coordinate the acquisition of flowering competence and help to reach the threshold of FT necessary to flower. Furthermore, we show how two central regulatory mechanisms, such as PcG and microRNA, assemble to achieve a developmental outcome.

Figure 1.

FLC, MAF4, and MAF5 expression is significantly altered in atbmi1 mutants. A to G, Phenotypes of strong (A), intermediate (B and C), and weak (D) atbmi1a/b, wild-type (WT) Columbia (Col; E), emf1-2 (F), and emf2-2 (G) at 10 d after germination (DAG). Bars = 2 mm. H, Expression levels of FLC, MAF1, MAF2, MAF3, MAF4, and MAF5 in 7- and 14-d-old plants at ZT1 under LD conditions. The expression levels of these genes were also analyzed in 7-d-old FRI-Col seedlings. Quantifications were normalized to ACTIN2 (ACT2). The y axis indicates fold change compared with wild-type Col.

Figure 2.

H3K27me3 levels at MAF4, MAF5, and FLC are altered in atbmi1 mutants. A, Schematic diagram of MAF4, MAF5, and FLC genomic regions. Exons and untranslated regions are represented by black and gray boxes, respectively, while introns and other genomic regions are represented by black lines. The translation start site (ATG) and stop codon (TAA or TAG) are indicated. DNA fragments amplified in chromatin immunoprecipitation (ChIP) assays are indicated below the genomic regions. B, ChIP analysis of H3K27me3 levels at the FLC, MAF4, and MAF5 first intron region in wild-type (WT), atbmi1a/b weak, and atbmi1a/b/c seedlings at 10 DAG. ACT7 was used as a negative control. The immunoprecipitated DNAs were quantified and normalized to ACT7. Error bars indicate the sd of two biological replicates.

Figure 3.

FT expression in atbmi1 mutants is CO dependent. A, Expression levels of FT in 7- and 14-d-old plants at ZT1 under LD conditions. ACT2 was used as an internal control (samples are as in Fig. 1H). B, FT mRNA levels in the indicated seedlings over an LD cycle at 7 and 14 DAG. C, CO mRNA levels over an LD cycle at 14 DAG. FT and CO transcript levels were normalized to ACT2; error bars indicate the sd of two biological repeats. D, FLC and FT transcript levels in 7-d-old wild-type (WT) Col, atbmi1a/b weak, and FRI-Col seedlings under LD conditions at Zeitgeber time 16 (ZT16). E, Vasculature organization of 10-d-old cotyledons from wild-type Col and different atbmi1a/b phenotypes.

Figure 4.

atbmi1a/b mutants misexpress MIR156A and MIR156C. A, Flowering time of wild-type (WT) Col and atbmi1a/b weak plants (left). The time was measured by the number of rosette leaves produced from the SAM prior to flowering; 16 to 20 plants for each line were scored. Error bars indicate sd. Juvenile (J) and transition (T) leaves were differentiated from adult leaves (A) by shape (right). B, Expression levels of pri-MIR156A, pri-MIR156C, and the seed maturation genes LEAFY COTYLEDON1 (LEC1) and FUS3 in the different mutants at 7 and 14 DAG growing under LD conditions at ZT1.

Figure 5.

MIR156A and MIR156C are direct targets of AtBMI1. A, ChIP analysis of H2Aub levels at MIR156A and MIR156C TSS in wild-type (WT) and atbmi1a/b weak seedlings at 10 DAG. FUS3 was used as a positive control. B, ChIP analysis of H3K27me3 levels at MIR156A and MIR156C TSS in wild-type, atbmi1a/b weak, and atbmi1a/b/c seedlings at 10 DAG. FUS3 was used as a positive control. The immunoprecipitated DNAs were quantified and normalized to ACT7. Error bars indicate the sd of at least two biological replicates. C, Expression levels of pri-MIR156A and pri-MIR156C in the wild type, atbmi1a/b strong, and val1/2 mutants at 10 DAG. ACT2 was used as an internal control. D, ChIP analysis of H3K27me3 levels at the TSS of MIR156A and MIR156C in wild-type and val1/2 seedlings at 7 DAG. WUSCHEL (WUS) was included as a negative target of VAL and a positive control of H3K27me3 (Yang et al., 2013a). The immunoprecipitated DNAs were quantified and normalized to ACT7. Error bars indicate the sd of two biological replicates. E, Schematic representation of MIR156A/C regulation by VAL-AtBMI1-PRC1/PRC2 and FUS3. Lines with bars indicate the repression of gene expression, and the line with the arrow indicates activation.

Figure 6.

AtBMI1-PRC1- and EMF1-PRC1-mediated regulation of miR156 and miR172. Expression levels of pri-MIR156C, pri-MIR172b, SPL3, SPL9, and FT are shown for wild-type (WT) and mutant seedlings at 10 DAG. Quantifications were normalized to ACT2. Error bars represent the sd of two biological replicates.

Figure 7.

Model of the roles of AtBMI1-PRC1 and EMF1-PRC1 variants in regulating juvenile-to-adult phase transition through miR156 and miR172 repression. EMF1-PRC1 represses MIR172 and SPLs to maintain the juvenile phase. As the plant ages, the levels of miR156 decrease by AtBMI1-PRC1-mediated repression, which allows the development of adult traits and the acquisition of flowering competence. Solid purple lines with bars indicate negative regulation; solid red lines with arrows indicate positive regulation; orange lines with bars indicate EMF1-PRC1/PRC2 repression; pink lines with bars indicate AtBMI1-PRC1/PRC2 repression (the dashed pink line indicates a possible negative regulation); and the dotted black line with arrow indicates the movement of FT from leaves to the SAM. Repressed genes are indicated in light blue italic type and activated genes in dark blue italic type; proteins and miRNAs are indicated in black type. FUL, FRUITFULL.