Phytochrome B in the mesophyll delays flowering by suppressing FLOWERING LOCUS T expression in Arabidopsis vascular bundles

Abstract

Light is one of the most important environmental factors that determine the timing of a plant's transition from the vegetative to reproductive, or flowering, phase. Not only daylength but also the spectrum of light greatly affect flowering. The shade of nearby vegetation reduces the ratio of red to far-red light and can trigger shade avoidance responses, including stem elongation and the acceleration of flowering. Phytochrome B (phyB) acts as a photoreceptor for this response. Physiological studies have suggested that leaves can perceive and respond to shade. However, little is known about the mechanisms involved in the processing of light signals within leaves. In this study, we used an enhancer-trap system to establish Arabidopsis thaliana transgenic lines that express phyB-green fluorescent protein (GFP) fusion protein in tissue-specific manners. The analysis of these lines demonstrated that phyB-GFP in mesophyll cells affected flowering, whereas phyB-GFP in vascular bundles did not. Furthermore, mesophyll phyB-GFP suppressed the expression of a key flowering regulator, FLOWERING LOCUS T, in the vascular bundles of cotyledons. Hence, a novel intertissue signaling from mesophyll to vascular bundles is revealed as a critical step for the regulation of flowering by phyB.

Figure 1.

PBT and Bpro Constructions and Physiological Phenotypes of Representative PBT Lines. (A) Constructs used in this study. m35S, CaMV 35S minimal promoter; PHYB, full-length PHYB coding sequence; GFP, sGFP coding sequence; nosT, nopaline synthase terminator; PHYB promoter, PHYB authentic promoter (Goosey et al., 1997). (B) Flowering time of PBT lines and PBG10 under continuous white light (cW) (50 μmol m⁻² s⁻¹). Mean ± se (n = 15 to 20). (C) Flowering time of PBT lines and PBG10 under long-day (LD) conditions (16 h white light/8 h dark; 65 μmol m⁻² s⁻¹). Mean ± se (n = 10 to 30). (D) Flowering time of PBT lines and PBG10 under short-day (sd) conditions (8 h white light/16 h dark; 110 μmol m⁻² s⁻¹). Mean ± se (n = 15 to 30). (E) Hypocotyl lengths of PBT lines and PBG10 under continuous white light (6 μmol m⁻² s⁻¹). Seedlings were grown for 5 d. Mean ± se (n = 20 to 30). (F) Cotyledon area of PBT lines and PBG10 under continuous white light (35 μmol m⁻² s⁻¹). Seedlings were grown for 5 d. Mean ± se (n = 20 to 30).

Figure 2.

Confocal Microscopic Detection of phyB-GFP Fluorescence in the Cotyledon. PhyB-GFP was detected in nuclei (Yamaguchi et al., 1999). Seedlings were grown for 5 d under continuous white light (50 μmol m⁻² s⁻¹). Green fluorescence from GFP and red fluorescence from chlorophyll were overlaid electronically. A differential interference contrast image was overlaid as well for epidermis. Arrows and arrowheads indicate phyB-GFP fluorescence in mesophyll and vascular bundle cells, respectively. (A) to (H), (I) to (P), and (Q) to (X) show phyB-GFP fluorescence in mesophyll, vascular bundle, and epidermis, respectively. PhyB-GFP fluorescence in PBT48 ([A], [I], and [Q]), PBT56 ([B], [J], and [R]), PBT6 ([C], [K], and [S]), PBT239 ([D], [L], and [T]), PBT133 ([E], [M], and [U]), PBT390 ([F], [N], and [V]), PBG10 ([G], [O], and [W]), and Bpro7 ([H], [P], and [X]). Bars = 50 μm.

Figure 3.

Confocal Microscopic Detection of phyB-GFP Fluorescence in Shoot Apex, Hypocotyls, and Roots. Seedlings were grown for 5 d under continuous white light (50 μmol m⁻² s⁻¹). Green fluorescence from GFP, red fluorescence from chlorophyll, and a differential interference contrast image were overlaid electronically. (A) to (H), (I) to (P), and (Q) to (X) show phyB-GFP fluorescence in shoot apex, hypocotyls, and roots, respectively. PhyB-GFP fluorescence in PBT48 ([A], [I], and [Q]), PBT56 ([B], [J], and [R]), PBT6 ([C], [K], and [S]), PBT239 ([D], [L], and [T]), PBT133 ([E], [M], and [U]), PBT390 ([F], [N], and [V]), PBG10 ([G], [O], and [W]), and Bpro7 ([H], [P], and [X]). Bars = 50 μm.

Figure 4.

Quantitative Analysis of the phyB-GFP Fluorescence in Epidermal Cells. PhyB-GFP fluorescence in epidermis was observed by confocal microscopy with different photomultiplier settings. The gain was set to 650 (top), 620 (middle), or 590 (bottom). Numbers ±se below indicate relative intensity of the phyB-GFP fluorescence in epidermal cells. The gain was set to 650, and the fluorescence intensity within the nuclear region was integrated for each nucleus. n = 10 to 14. Bar = 20 μm.

Figure 5.

Immunoblot Detection of phyB-GFP and Endogenous phyB in Cotyledons, the Shoot Apex, the Hypocotyls, and the Roots. Proteins were extracted from 5-d-old seedlings grown under continuous white light (50 μmol m⁻² s⁻¹) and subjected to immunoblotting analysis with anti-Arabidopsis phyB antibody, mBA2. Closed and open arrowheads indicate positions of phyB-GFP and endogenous phyB, respectively. Asterisks indicate bands that are presumed to be a degradation product of phyB-GFP. MW, molecular weight (k). Each lane contained 20 μg of total proteins.

Figure 6.

Flowering Time of the Wild Type, phyB, ft, and phyB ft. Plants were grown under continuous white light (50 μmol m⁻² s⁻¹). Mean ± se (n = 20).

Figure 7.

FT Expression in PBT Lines. Seedlings were grown under continuous white light (50 μmol m⁻² s⁻¹). TUB2/TUB3 was used as a control. a.u., arbitrary unit. (A) FT expression in the seedlings on days 2 through 7. The samples were analyzed by relative quantification using real-time PCR. RNA extraction was performed three times independently. Mean ± se (n = 3). (B) FT expression in different parts of the seedlings on day 5. Seedlings were separated into three parts (cotyledon, shoot apex, and the remainder) and analyzed by relative quantification using real-time PCR. RNA extraction was performed four times independently. Mean ± se (n = 4).

Figure 8.

FT and PHYB-GFP Expression in Mesophyll and Vascular Bundles. Mesophyll protoplasts and vascular bundles were isolated from cotyledons. Seedlings were grown for 5 d under continuous white light (50 μmol m⁻² s⁻¹). TUB2/TUB3 was used as a control. a.u., arbitrary unit. (A) Mesophyll protoplasts isolated from the cotyledons. Bar = 100 μm. (B) Vascular bundles isolated from the cotyledons. Bar = 1 mm. (C) Expression of RbcS (a mesophyll maker) and Sultr (a vascular bundle marker) in the mesophyll and vascular bundle samples. Total RNA was extracted from ∼10⁴ protoplasts or from vascular bundles prepared from 20 cotyledons and subjected to the analysis. The samples were analyzed by relative quantification using real-time PCR. RNA extraction was performed three times independently. Mean ± se (n = 3). (D) Expression of PHYB-GFP in the mesophyll protoplasts and vascular bundles. The samples were analyzed by relative quantification using real-time PCR. RNA extraction was performed three times independently. Mean ± se (n = 3). (E) Expression of FT in the mesophyll protoplasts and vascular bundles. The samples were analyzed by relative quantification using real-time PCR. RNA extraction was performed three times independently. Mean ± se (n = 3).