Role of SVP in the control of flowering time by ambient temperature in Arabidopsis

Abstract

Plants must perceive and rapidly respond to changes in ambient temperature for their successful reproduction. Here we demonstrate that Arabidopsis SHORT VEGETATIVE PHASE (SVP) plays an important role in the response of plants to ambient temperature changes. The loss of SVP function elicited insensitivity to ambient temperature changes. SVP mediates the temperature-dependent functions of FCA and FVE within the thermosensory pathway. SVP controls flowering time by negatively regulating the expression of a floral integrator, FLOWERING LOCUS T (FT), via direct binding to the CArG motifs in the FT sequence. We propose that this is one of the molecular mechanisms that modulate flowering time under fluctuating temperature conditions.

Figure 1.

Role of SVP in the temperature-dependent control of flowering in Arabidopsis. (A) Flowering time of a group of flowering time mutants at 23°C and 16°C under long-day conditions. The numbers listed above the genotypes denote the ratios of flowering time at 16°C and 23°C (16°C/23°C). Error bars indicate the standard deviation. The inset shows wild-type Columbia (Col) plants and svp-32 plants grown at 23°C and 16°C. (B) Effects of low temperature on SVP expression in wild-type plants (Col). SVP, FLC, and FT expression levels were measured by real-time PCR in the leaf of 10-d-old seedlings grown at the indicated temperatures. The average of three technical replicates is shown. FLC and FT were used as control genes, the expressions of which are thermo-regulated (Blázquez et al. 2003). Tubulin was used as an internal control. (C) Histochemical analysis of 10-d-old seedlings of SVP∷GUS and FT∷GUS plants grown at 23°C and 16°C. Bars, 500 μm. (D) Nuclear localization of SVP-GFP fusion protein in onion epidermal cells incubated at 16°C and 23°C. The nucleus is indicated by an arrow. DAPI (4′-6-Diamidino-2-phenylindole) was used for nuclear staining. Bars, 10 μm.

Figure 2.

Genetic interaction of SVP with FCA, FVE, and FLC. (A) Flowering time of the svp-32 fca-9, and svp-32 fve-3 double mutants at 23°C and 16°C under long-day conditions. The numbers listed below denote the ratios of flowering time (16°C/23°C). (B) Effects of fca and fve mutations on SVP expression in 10-d-old seedlings. (C) SVP expression in 10-d-old seedlings of loss- and gain-of-function alleles of FLC. The FRI locus originates from the Sf2 ecotype, and flc is flc-3 (Michaels and Amasino 1999). 35S∷FLC plants were used rather than an allele harboring functional FRI and FLC. (D) FLC expression in 10-d-old seedlings of the loss- and gain-of-function mutants of SVP. (E) Flowering time of mutants harboring various combinations of svp, fri, and flc mutations at 23°C and 16°C. SVP fri FLC and svp fri FLC indicate wild-type Columbia plants and svp-32 plants, respectively. The numbers listed below denote the ratios of flowering time (16°C/23°C).

Figure 3.

Role of SVP as an FT repressor. (A) Time-course expression of FT in wild-type (Col) and svp-32 plants at 23°C and 16°C. FT expression level was monitored in 6-, 8-, 10-, 12-, and 14-d-old seedlings. (B) pFT∷GUS expression patterns in 10-d-old seedlings of wild-type (Col) and svp-32 plants at 23°C. Bars, 500 μm. (C) Flowering time of svp-32 ft-10, svp-32 35S∷FT, and ft-10 soc1-2 double mutants at 23°C and 16°C. The numbers listed below denote the flowering time ratios (16°C/23°C).

Figure 4.

Binding of SVP protein to the vCArG III in the FT promoter. (A) A ChIP assay using protoplasts transfected with SVP-HA and FLC-HA constructs. The location of six vCArG motifs (vCArG I to vCArG VI) identified in a 1.8-kb FT promoter and the different fragments analyzed by PCR are represented. A CArG motif to which FLC is known to bind within the first intron of FT (Helliwell et al. 2006; Searle et al. 2006) is designated as CArG VII. A fourfold dilution series of the input DNA was used as a semiquantitative standard. Relative enrichment indicates the amplified signal value normalized against that of input DNA. The value of enrichment in vCArG I/II was set to 1 for SVP-HA and FLC-HA. Similar results were obtained from five independent experiments. (Input) Total input chromatin DNA; (HA) DNA selected using HA antibodies; (Myc) DNA selected using Myc antibodies. (B) The effects of SVP-HA protein on the FT promoter activities. A schematic representation of the reporters and the effectors used in this assay is shown. vCArG motifs are shaded in gray and mutations introduced in vCArG motifs are indicated in lowercase. m3FT∷LUC indicates the FT∷LUC construct harboring a mutated vCArG III. Luciferase activities were normalized by GUS activities. This experiment was repeated five times, with similar results.