Establishment of a vernalization requirement in Brachypodium distachyon requires REPRESSOR OF VERNALIZATION1

Abstract

A requirement for vernalization, the process by which prolonged cold exposure provides competence to flower, is an important adaptation to temperate climates that ensures flowering does not occur before the onset of winter. In temperate grasses, vernalization results in the up-regulation of VERNALIZATION1 (VRN1) to establish competence to flower; however, little is known about the mechanism underlying repression of VRN1 in the fall season, which is necessary to establish a vernalization requirement. Here, we report that a plant-specific gene containing a bromo-adjacent homology and transcriptional elongation factor S-II domain, which we named REPRESSOR OF VERNALIZATION1 (RVR1), represses VRN1 before vernalization in Brachypodium distachyon That RVR1 is upstream of VRN1 is supported by the observations that VRN1 is precociously elevated in an rvr1 mutant, resulting in rapid flowering without cold exposure, and the rapid-flowering rvr1 phenotype is dependent on VRN1 The precocious VRN1 expression in rvr1 is associated with reduced levels of the repressive chromatin modification H3K27me3 at VRN1, which is similar to the reduced VRN1 H3K27me3 in vernalized plants. Furthermore, the transcriptome of vernalized wild-type plants overlaps with that of nonvernalized rvr1 plants, indicating loss of rvr1 is similar to the vernalized state at a molecular level. However, loss of rvr1 results in more differentially expressed genes than does vernalization, indicating that RVR1 may be involved in processes other than vernalization despite a lack of any obvious pleiotropy in the rvr1 mutant. This study provides an example of a role for this class of plant-specific genes.

Keywords: Brachypodium; RVR1; VRN1; flowering; vernalization.

Fig. 1.

A gene with a BAH and TFIIS domain (RVR1) represses flowering time. (A) Flowering times of Bd21-3 wild-type, rvr1-1, rvr1-2, UBI:VRN1, and UBI:FT1 (VRN1 and FT1 cDNA under control of the maize ubiquitin promoter) grown in 20, 16, 15, 14, 12, 10, and 8 h of light either without cold exposure (NV) and with cold exposure (V) for 4 wk (4wkV). Bars represent the average of 12 plants ± standard deviation. The experiment was repeated with similar results. Arrows above bars indicate that none of the plants flowered at the end of the experiment (150 d). (B) Representative image of Bd21-3 wild-type, the two alleles of rvr1, UBI:VRN1, and UBI:FT1 grown in 16-h of light for 90 d after germination without vernalization (NV). (Scale bar, 5 cm.) (C) Domain structure of the 675-aa-long RVR1 protein showing the location and corresponding amino acid changes of the ethyl methane sulfonate-induced mutations of the two mutant alleles rvr1-1 and rvr1-2. Domain structure of RVR1 includes a putative nuclear localization signal (NLS), BAH, and TFIIS domains. (D) Image of representative T1 plants grown in a 16-h photoperiod. Bd21-3, rvr1-1, and segregating nontransgenic sibling plants are controls for the effect of overexpression of the wild-type RVR1 cDNA in the rvr1-1 background (UBI:RVR1::rvr1-1). (Scale bar, 5 cm.)

Fig. S1.

Mapping frequencies of the five chromosomes in Brachypodium showing a skew toward the Bd21-3 genotype around 40 mb on chromosome 2: (A) rvr1-1, (B) rvr1-2. HA, Hawaiian; LG, linkage group.

Fig. S2.

(A) RVR1 gene expression in Bd21-3 and rvr1-1 grown in 1× 6-h days NV. Newly emerged third leaf was harvested; data relative to Bd21-3. (B) Days to heading in Bd21-3, rvr1-1, and 10 transgenic (rescue) lines overexpressing the RVR1 cDNA from Bd21-3 in the rvr1-1 background grown in 16-h NV. (C and D) Subset of the transgenic lines in A grown in 20-h days NV and V. In B–D, values represent the average of six plants ± standard deviation. *P < 0.01.

Fig. S3.

Maximum-likelihood inference of the phylogenetic relationships among RVR1 genes based on a nucleotide alignment of the conserved BAH domain. Maximum-likelihood bootstrap support values (Right) and Bayesian posterior probabilities (Left) are indicated at each branch. Scale bar indicates substitutions per site. Focal species are labeled in boldface font.

Fig. S4.

Mutant characterization of the RVR1 ortholog in Arabidopsis thaliana Atrvr1 (At4g11560). (A) Gene diagram of RVR1 from Arabidopsis with the location of the two T-DNAs analyzed, Atrvr1-1 (Salk_017758) and Atrvr1-2 (Sail_1246_E10). (B) Semiquantitative PCR of AtRVR1 in frigida (fri, wild-type; Columbia ecotype) and the two T-DNA alleles confirming the presence of the given T-DNA. UBQ10 used as a loading control. (C and D) Flowering time of the T-DNAs as reported via leaf counts in LD and SD NV. (E) Flowering times after a vernalization time course of Atrvr1-2 introgressed within the FRI background grown in 16-h LD. Imbibed seeds were vernalized at 5 °C for either 10, 20, or 40 d.

Fig. 2.

RVR1 represses VRN1 expression and the rvr1-1 mutant phenotype is dependent upon VRN1. (A) qRT-PCR data from samples of the third-leaf or meristem-enriched apex (shoot/meristem) of NV Bd21-3 and rvr1-1 grown in a 16-h photoperiod to the fourth-leaf stage. VRN1 expression (Upper) and FT1 expression (Lower). Bars represent the average of three biological replicates ± standard deviation. (B) qRT-PCR of VRN1 expression (Upper) and FT1 expression (Lower). Third leaf harvested at the third-leaf stage of Bd21-3 and rvr1-1 grown in 8-h SD or 16-h LD. V plants were exposed to 4 wk of cold as an imbibed seed and the NV plants were planted at the end of the vernalization treatment. (C) qRT-PCR of VRN1 expression of leaf three or meristem-enriched shoot apex from Bd21-3 and rvr1-1 plants either NV or from plants during cold exposure. (D) Representative image of Bd21-3 wild-type, rvr1-1, and rvr1-1/amiVRN1 grown in 16-h days NV. Image taken 65 d postgermination. (Scale bar, 5 cm.) (E) Days to heading of rvr1-1, Bd21-3, three independent T1 amiVRN1/rvr1-1 transgenic plants, and nontransgenic sibling plants segregated grown in 16-h days NV. Arrows above bars indicate that none of the plants flowered at the end of the experiment. (F) qRT-PCR of VRN1 expression in amiVRN1/rvr1-1 confirming the knock-down of VRN1 expression. Asterisks above bars indicate statistically significant contrasts between rvr1 and Bd21-3 NV (*P < 0.01).

Fig. S5.

(A–F) qRT-PCR data of leaf three or shoot/meristem from fourth-leaf stage Bd21-3 and rvr1-1 plants grown NV in a 16-h photoperiod. Bars represent the average of three biological replicates ± standard deviation. Expression is relative to Bd21-3. *P < 0.01.

Fig. S6.

Third leaf harvested at the third-leaf stage of Bd21-3 and rvr1-1 grown in 8-h SD or 16-h LD. V plants were treated with 4 wk of cold as an imbibed seed and the NV were planted at end of vernalization treatment. Expression of (A) FUL2, (B) FT2, (C) VRN2, (D) OS2. In A and B expression was relative to Bd21-3 NV. In C and D expression was normalized to UBC18. *P < 0.01.

Fig. S7.

(A) RVR1 gene expression in leaves of Bd21-3 grown in a variety of different conditions. All plants are the third-leaf stage and the newly formed third leaf was harvested. NV, 4 wk V (4V), NV+11 d after cold developmental control for 3V+11 (NV+11), 3 wk V+11 d after cold (3V+11). Expression normalized to UBC18 as done in Ream et al. (29). (B) RVR1 expression in the third leaf of different B. distachyon accessions grown in 20-h days NV. In A and B, values represent the average of three biological replicates ± standard deviation.

Fig. S8.

(A–C) RVR1 expression in the third leaf grown NV in 16-h days. RVR1 expression in (A) UBI:FT, (B) UBI:VRN1, (C) UBI:VRN2. (D and E) VRN1 expression (D) and RVR1 expression (E) in the third leaf of plants grown NV in 20-h days.

Fig. 3.

The effect of vernalization on the histone modification, H3K27me3 at BdVRN1 in Bd21-3 and rvr1-1 seedlings. (A) Diagram of the 5′ end of BdVRN1 showing the regions analyzed by ChIP, followed by qRT-PCR. (B–E) Relative abundance of H3K27me3 at BdVRN1 in NV and V seedlings from Bd21-3 and rvr1-1. Data represent the mean ± SEM from three biological replicate experiments. An asterisk (*) indicates significant differences compared with 21-3 NV (*P < 0.05). Primer sequences are provided in Table S1.

Fig. 4.

Effects of rvr1-1 mutation and vernalization on the leaf transcriptome. (A) Overlap between the transcriptomic changes observed in rvr1-1 seedling and Bd21-3 wild-type plants postvernalization (FC > 2; P < 0.001). (Upper) Number of genes up-regulated; (Lower) number of genes down-regulated. (B) Comparison of the FC observed in the nonvernalized rvr1-1 mutant and in vernalized Bd21-3 seedlings. Red dotted lines indicate fold-change thresholds used for the identification of differentially expressed genes (DEG).

Fig. S9.

Analysis of RNA-seq data. (A) Principal component analysis computed using the 500 genes showing the highest mean expression levels across studied conditions (three replicates per condition). (B) Heatmap showing the clustering of RNA-seq replicates in the different experimental conditions. The heatmap was computed using all of the detected genes. (C and D) MA plots highlighting the differentially genes for (C) the rvr1-1 mutant vs. NV wild-type plants and (D) V vs. NV wild-type plants. (E) Log₂ of the FC in V vs. NV wild-type plants. Genes are categorized according to their down- or up-regulation in the rvr1-1 vs. NV wild-type plants analysis. Green dots indicate genes differentially regulated in both experiments and triangles show genes out of the scale. ***P < 0.001. (F) qRT-PCR verification of the RNA-seq data. Data were normalized using a geometric mean of three reference genes: SamDC, ACT2, and UBC18. The relative expression of the wild-type NV sample was set to one. DEG, differentially expressed genes; VERN, vernalization.