The promoter of the cereal VERNALIZATION1 gene is sufficient for transcriptional induction by prolonged cold

Abstract

The VERNALIZATION1 (VRN1) gene of temperate cereals is transcriptionally activated by prolonged cold during winter (vernalization) to promote flowering. To investigate the mechanisms controlling induction of VRN1 by prolonged cold, different regions of the VRN1 gene were fused to the GREEN FLUORESCENT PROTEIN (GFP) reporter and expression of the resulting gene constructs was assayed in transgenic barley (Hordeum vulgare). A 2 kb segment of the promoter of VRN1 was sufficient for GFP expression in the leaves and shoot apex of transgenic barley plants. Fluorescence increased at the shoot apex prior to inflorescence initiation and was subsequently maintained in the developing inflorescence. The promoter was also sufficient for low-temperature induction of GFP expression. A naturally occurring insertion in the proximal promoter, which is associated with elevated VRN1 expression and early flowering in some spring wheats, did not abolish induction of VRN1 transcription by prolonged cold, however. A translational fusion of the promoter and transcribed regions of VRN1 to GFP, VRN1::GFP, was localised to nuclei of cells at the shoot apex of transgenic barley plants. The distribution of VRN1::GFP at the shoot apex was similar to the expression pattern of the VRN1 promoter-GFP reporter gene. Fluorescence from the VRN1::GFP fusion protein increased in the developing leaves after prolonged cold treatment. These observations suggest that the promoter of VRN1 is targeted by mechanisms that trigger vernalization-induced flowering in economically important temperate cereal crops.

Figure 1. Schematic representation of VRN1reporter gene constructs.

(A) A 2.0 kb fragment (SpeI-NcoI) from the VRN1 promoter was fused to the GFP reporter gene with the NOPALINE SYNTHASE (NOS) terminator sequence to generate the P_VRN1:GFP construct. (B) The P_VRN1EX1-2:GFP construct was generated by fusing the same promoter region to exon1 and exon 2 of VRN1 (EX1 and EX2), separated by a 0.35 kb segment of intron 1, followed by the GFP-NOS cassette. Scale indicates base pairs from the translational start site of MADS box open reading frame, indicated as negative relative to the coding regions of the VRN1 locus. Arrow indicates transcriptional start site, which did not vary between control or prolonged cold treatments.

Figure 2. GFP fluorescence in the leaves and shoot apices of transgenic plants with the P_VRN1:GFP construct.

(A) GFP fluorescence in the vegetative shoot apex of 12 day old transgenic plants carrying the P_VRN1:GFP construct. (B) The shoot apex at the late vegetative stage of 18 day old plants. (C,D) Shoot apices at different stages of reproductive development. (E,F) Fluorescence in the cytoplasm of cells in the developing leaves of 18 day old plants. am indicates the apical meristem, fl indicates the developing florets, gl indicates glume primordia.

Figure 3. Expression of the P_VRN1:GFP and P_VRN1EX1-2::GFP constructs in control versus cold treated seedlings.

(A) GFP transcript levels in transgenic seedlings carrying the P_VRN1:GFP construct. Expression was assayed by quantitative reverse transcriptase PCR in control seedlings, germinated in darkness at normal glasshouse temperatures (20 degrees for 5 days), compared to seedlings germinated and grown in darkness to an identical stage of development at low temperatures (4 degrees for 28 days). Expression levels were assayed in three independent transgenic lines. (B) Expression of the P_VRN1EX1-2::GFP, assayed as outlined above, in three independent transgenic lines. Expression is shown relative to ACTIN. Error bars show standard error for a minimum of 3 biological replicates. * indicates P<0.05.

Figure 4. Activity of a VRN1::GFP fusion protein in transgenic barley plants.

(A) Schematic representation of a VRN1::GFP fusion construct that fuses the entire VRN1 gene, minus most of the first intron, to the GFP reporter gene followed by the 3′ UTR and terminator sequence of the VRN1 gene. Vertical bars represent exons, dotted line represents the missing segment from intron 1, relative to the wildtype version of VRN1 from vernalization responsive barleys (B) Low signal from the VRN1::GFP fusion in the vegetative shoot apical meristem (m) and developing leaf (l) of 7 day old seedlings. (C) Nuclear localisation of the VRN1::GFP fusion protein in the meristem of a reproductive shoot apex. (D–H) Time course of shoot apex development showing vegetative shoot apices (D,E), a shoot apex at the transition to reproductive development (F), indicated by double ridges (dr), and reproductive shoot apices (G,H). am indicates the apical meristem, fl indicates the developing florets, gl indicates glume primordia. Images B–H were taken on identical settings to allow direct comparison of fluorescence levels. (note: image C is a section from image H, 2 fold expanded).

Figure 5. Activity of a VRN1::GFP fusion protein in the developing leaves of transgenic barley plants.

(A) Low signal from the VRN1::GFP fusion in the developing leaves of plants grown at normal glasshouse temperatures. (B) Nuclear localisation of the VRN1::GFP fusion protein in cells within the developing leaves of seedlings germinated and grown at low temperatures to an identical stage of development. Images were taken with identical settings to allow comparison of fluorescence levels. Similar results were seen in two independent transgenic lines. Some background signals, “green lines” caused by reflection, are present in both images.

Figure 6. Potential regulatory motifs at the promoter of the VRN1 locus.

(A) Schematic representation of potential transcription factor binding sites at the promoter of VRN1. ERE indicates potential ethylene response element (GCCGCC), CRT/DRE: C-repeat transcription factor core binding site (CCGAC), MYC: MYC transcription factor binding site (CANNTG), B-ZIP: B-ZIP transcription factor binding site (ACGT). VRN Box: indicates the position of the putative vernalization regulatory motif suggested by Pidal et al. . uORF denotes the position of the small upstream open reading frame. Scale indicates bp from transcriptional start site, which is indicated by an arrow. (B) Alignment of the predicted amino acid sequences of the upstream open reading frame from the 5′ untranslated regions of VRN1 genes from temperate cereals (wheat, rye, barley, oats), temperate grasses (Lolium, Festuca, Brachypodium) and VRN1 orthologues from warm climate cereals (rice and maize). Scale indicates amino acid residues from initiation codon.

Figure 7. Transcript levels for the VRN1 gene (A genome) of wheat near-isogenic lines with different VRN1 genotypes.

Transcript levels of the A genome copy of VRN1 were assayed in hexaploid bread wheat (Triticum aestivum) near-isogenic lines that vary for VRN1 genotype. Expression was assayed by quantitative reverse transcriptase PCR, using A genome specific primers, in seedlings germinated in darkness at 20° for 5 days (control) or 4° for 6 weeks (cold). The wheat lines used carry different combinations of wildtype VRN1 alleles on each of the three genomes (a, b or d) or alleles with high basal activity (A1a, A-lang., B or D). The A1a allele (line W11) has a promoter insertion in the VRN box of the VRN1 gene on the A genome, but a full-length first intron . The A-lang. allele (line W15), first identified in the Langdon cultivar of the tetraploid wheat Triticum durum , has a 7.2 kb deletion within the first intron, but has the wildtype promoter sequence. Similarly, the B and D alleles have deletions within the first intron of the VRN1 genes on the B and D genomes respectively . Expression is shown relative to ACTIN, error bars show standard error for four biological replicates, *** indicates P<0.001.

Figure 8. A model for transcriptional regulation of VRN1.

Prior to winter, chromatin at the VRN1 locus is maintained in an inactive state by histone modifications deposited by a plant Polycomb Repressor Complex 2 (PRC2), resulting in low transcript levels. When plants are exposed to prolonged cold (snowflake) the promoter of VRN1 becomes more active, leading to increased transcription and higher steady state transcript levels. This triggers a change in the state of chromatin at the VRN1 locus, with a shift towards an active state.