The phytochrome-interacting vascular plant one-zinc finger1 and VOZ2 redundantly regulate flowering in Arabidopsis

Abstract

The timing of the transition to flowering in plants is regulated by various environmental factors, including daylength and light quality. Although the red/far-red photoreceptor phytochrome B (phyB) represses flowering by indirectly regulating the expression of a key flowering regulator, FLOWERING LOCUS T (FT), the mechanism of phyB signaling for flowering is largely unknown. Here, we identified two Arabidopsis thaliana genes, VASCULAR PLANT ONE-ZINC FINGER1 (VOZ1) and VOZ2, which are highly conserved throughout land plant evolution, as phyB-interacting factors. voz1 voz2 double mutants, but neither single mutant, showed a late-flowering phenotype under long-day conditions, which indicated that VOZ1 and VOZ2 redundantly promote flowering. voz1 voz2 mutations suppressed the early-flowering phenotype of the phyB mutant, and FT expression was repressed in the voz1 voz2 mutant. Green fluorescent protein-VOZ2 signal was observed in the cytoplasm, and interaction of VOZ proteins with phyB was indicated to occur in the cytoplasm under far-red light. However, VOZ2 protein modified to localize constitutively in the nucleus promoted flowering. In addition, the stability of VOZ2 proteins in the nucleus was modulated by light quality in a phytochrome-dependent manner. We propose that partial translocation of VOZ proteins from the cytoplasm to the nucleus mediates the initial step of the phyB signal transduction pathway that regulates flowering.

Figure 1.

Flowering Phenotype of voz Mutants and Genetic Interaction between phyB and VOZs. (A) Rosette leaf number and number of days to bolting of the voz1, voz2, and double mutants grown under LD conditions (16 h white light/8 h dark). Data are the mean ± sd (n ≥ 12). Asterisks indicate a significant difference from Col at P < 10⁻⁴. (B) Rosette leaf number and number of days to bolting of the voz1, voz2, and double mutants grown under SD conditions (8 h white light/16 h dark). Data are the mean ± sd (n ≥ 12). Asterisk indicates a significant difference from Col at P < 10⁻⁴. (C) Complementation of voz1-1 voz2-1 mutant by VOZ1 and VOZ2 genomic fragments under LD conditions. Two independent lines were examined for each genomic construct (gVOZ1 and gVOZ2, respectively). Data are the mean ± sd (n ≥ 18). Asterisk indicates significant difference from Col at P < 10⁻⁴. (D) Rosette leaf number at bolting of voz1 voz2, phyB, and the triple mutants grown under LD conditions. Data are the mean ± sd (n ≥ 35). (E) Rosette leaf number at bolting of voz1 voz2, phyB, and the triple mutants grown under SD conditions. Data are the mean ± sd (n ≥ 10). (F) to (I) Plants at bolting. Plants were grown under LD conditions. Col (F) and phyB mutant (H) at day 16. voz1-1 voz2-1 mutant (G) and phyB voz1-1 voz2-1 mutant (I) at day 24. Bars = 1 cm. [See online article for color version of this figure.]

Figure 2.

Spatial Patterns of VOZ Expression. (A) and (B) Mesophyll protoplasts and vascular bundles were isolated from cotyledons and VOZ1 or VOZ2 RNA levels were determined by quantitative RT-PCR and then normalized to ACT2. Seedlings were grown under LD conditions for 10 d. ACT2 was used as a control. RNA extraction was performed three times independently. Data are the mean ± se (n = 3). (C) to (J) Representative GUS staining of ProVOZ1:GUS-VOZ1 #2 ([C], [E], [G], and [I]) and ProVOZ2:GUS-VOZ2 #1 ([D], [F], [H], and [J]) transgenic lines. LD-grown seedlings were analyzed on day 4 ([C] and [D]), day 7 ([E] and [F]), day 10 ([G] and [H]), and day 14 ([I] and [J]). Bars = 1 mm. (K) and (L) Transverse sections through cotyledon of ProVOZ1:GUS-VOZ1 #2 (K) and ProVOZ2:GUS-VOZ2 #1 (L) transgenic plants. P, phloem; X, xylem. Bars = 10 μm.

Figure 3.

Relative Expression Levels of FT, CO, and FLC in the Wild Type and voz1 voz2 Mutant. Relative expression levels of FT ([A] and [B]), CO ([C] and [D]), and FLC ([E] and [F]) were determined by quantitative RT-PCR in Col (triangles) and voz1 voz2 mutant (squares). Plants were grown for 10 d under LD conditions and harvested at the indicated times. RNA was extracted from rosette leaves ([A], [C], and [E]) or cotyledons ([B], [D], and [F]). ACT2 was used as a control. RNA extraction was performed three times independently. Data are the mean ± se (n = 3). ZT, zeitgeber time.

Figure 4.

Subcellular Localization of GFP-VOZ2 Fusion Protein. Confocal images of GFP ([A], [D], [G], and [J]), chloroplast autofluorescence (Chl) ([B], [E], [H], and [K]), and merged fluorescence (Merge) ([C], [F], [I], and [L]) from epidermal cells ([A] to [C]), vascular bundle cells ([D] to [F]), palisade mesophyll cells ([G] to [I]), and spongy mesophyll cells ([J] to [L]) in leaves of Pro35S:GFP-VOZ2/voz1 voz2 plants grown under LDs for 7 d. Bars = 20 μm.

Figure 5.

Subcellular Localization Analysis of Functional VOZ2. (A) Diagrams of GFP-VOZ2 constructs with NLS or NES. (B) GFP-VOZ2 protein levels in the seedlings on day 10. Total soluble proteins were subjected to protein immunoblot analysis with anti-GFP and anti-VOZ2 antibodies. Coomassie blue staining of ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (RBCL) is shown as a loading control. (C) Rosette leaf number at bolting of NLS and NES lines grown under LD conditions. Data are the mean ± sd (n ≥ 27). (D) Protein gel blot of cytosolic and nuclear fractions of Col and the Pro35S:GFP-VOZ2/voz1 voz2 line grown under continuous white light for 10 d was probed with anti-VOZ2, anti-UGPase, and anti-histone H3 antibodies. Asterisk represents nonspecific detection. C, cytosolic fraction; N, nuclear fraction; N (×5), fivefold concentrated nuclear fraction; T, total fraction.

Figure 6.

Interaction between phyB and VOZ1/VOZ2 in Vivo. (A1) to (F6) BiFC analysis of phyB and VOZ1/VOZ2. Confocal images of YFP ([A1] to [F1], [A3] to [F3], and [A5] to [F5]) and bright-field (BF) images ([A2] to [F2], [A4] to [F4], and [A6] to [F6]) from epidermal cells of N. benthamiana infected with Agrobacterium harboring the constructs described below under dark ([A1] to [F1] and [A2] to [F2]), red ([A3] to [F3] and [A4] to [F4]), and far-red ([A5] to [F5] and [A6] to [F6]) conditions. Bars = 10 μm. BiFC analysis of the interaction between VOZ1 and phyB ([A1] to [A6]) and VOZ2 and phyB ([B1] to [B6]). VOZ1 and VOZ2 were fused to nYFP, and phyB was fused to cYFP to generate nYFP-VOZ and phyB-cYFP, respectively. A vector containing only nYFP or cYFP was used as a negative control ([C1] to [C6], [D1] to [D6], and [E1] to [E6]). phyB subcellular localization was observed with the Pro35S:PHYB-YFP construct ([F1] to [F6]). Arrows indicate nuclear speckles. (G) and (H) Co-IP of GFP-VOZ2 or GFP from Pro35S:GFP-VOZ2 and Pro35S:GFP plant extracts, respectively, using anti-GFP antibody-tagged microbeads. Plants were grown under continuous white light for 9 d and treated with either red (R) or far-red (FR) light for 8 h. B, bound fraction; T, total fraction; U, unbound fraction. [See online article for color version of this figure.]

Figure 7.

Degradation and Phosphorylation of VOZ2 Protein. Protein immunoblotting with anti-VOZ2 antibodies. Coomassie blue staining of ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (RBCL) is shown as a loading control. Asterisks represent nonspecific detection. (A) to (C) Protein levels of VOZ2, GFP-VOZ2-NLS, and GFP-VOZ2-NES in Col (A), Pro35S:GFP-VOZ2-NLS/voz1 voz2 line #7 (B), and Pro35S:GFP-VOZ2-NES/voz1 voz2 line #8 (C), respectively. Plants were grown under continuous white light for 10 d and treated with either white (W), red (R), far-red (FR) light, or darkness (D) for 24 h. Each lane contained 60 μg (A), 100 μg (B), or 50 μg (C) of total proteins. (D) Seedlings grown under continuous white light for 10 d were pretreated with (+) or without (−) 50 μM MG132 for 3 h and transferred to far-red light for 12 h. ×, a control treated with only far-red light. Each lane contained 100 μg of total proteins. (E) Proteins were extracted from 10-d-old seedlings under continuous white light and incubated with (+) or without (−) λ-PPase. A control sample before λ-PPase treatment is indicated by the letter B. Each lane contained 100 μg of total proteins. (F) VOZ2 protein levels in Ler and hy1 hy2 mutant. Plants were grown under continuous white light for 10 d and treated with either red or far-red light for 24 h. Each lane contained 65 μg of total proteins.