Mobile FT mRNA contributes to the systemic florigen signalling in floral induction

Abstract

In inducing photoperiodic conditions, plants produce a signal dubbed "florigen" in leaves. Florigen moves through the phloem to the shoot apical meristem (SAM) where it induces flowering. In Arabidopsis, the FLOWERING LOCUS T (FT) protein acts as a component of this phloem-mobile signal. However whether the transportable FT mRNA also contributes to systemic florigen signalling remains to be elucidated. Using non-conventional approaches that exploit virus-induced RNA silencing and meristem exclusion of virus infection, we demonstrated that the ArabidopsisFT mRNA, independent of the FT protein, can move into the SAM. Viral ectopic expression of a non-translatable FT mRNA promoted earlier flowering in the short-day (SD) Nicotiana tabacum Maryland Mammoth tobacco in SD. These data suggest a possible role for FT mRNA in systemic floral signalling, and also demonstrate that cis-transportation of cellular mRNA into SAM and meristem exclusion of pathogenic RNAs are two mechanistically distinct processes.

Figure 1. Arabidopsis FT RNA enables virus-induced RNA silencing (VIGS) in shoot apex.

(a) A diagrammatic representation of recombinant PVX vectors. The introduced stop codon (*) that replaces the FT start codon in the FT mutant mFT to prevent translation is indicated. Gene of interest (GOI) indicates the position of individual gene insertion in the PVX genome. (b–d) Systemic VIGS of GFP expression in leaves and stems of the GFP transgenic Nicotiana benthamiana line 16c plants. Plants were mock inoculated (b), or inoculated with PVX/GFP (c), PVX/FT-GFP (d) or PVX/mFT-GFP (e). (f) VIGS in stem tips of line 16c plants mock inoculated (mock), or infected with PVX/GFP (GFP), PVX/FT-GFP (FT-GFP), or PVX/mFT-GFP (mFT-GFP). (g–h) VIGS in shoot apices and surrounding young leaves of line 16c plants mock inoculated (g) or infected with PVX/GFP (h), PVX/FT-GFP (i), or PVX/mFT-GFP (j). Photographs were taken at 21 days post-inoculation under long-wavelength UV illumination through a yellow Kodak No. 58 filter (b–f), and using a Zeiss LSM710 Laser Scanning Microscope (g–j) through transmitted light (TM) to show the outlines of the shoot apices and their surrounding tissues, or through lasers to show green (GFP) and red (Chlorophyll) fluorescence. The merged images of green and red fluorescence (GFP + Chlorophyll) are displayed (g–h). GFP-expressing tissues showed green fluorescence and tissues with silencing of GFP mRNA by VIGS appeared red due to chlorophyll fluorescence. Shoot apices and surrounding tissues are indicated by arrows. Bar = 1 mm.

Figure 2. FT RNA-mediated PVX entry into SAM.

Arabidopsis FT mRNA enables entry of PVX into the shoot apical meristem (SAM). Nicotiana benthamiana plants were infected with PVX/GFP, PVX/FT or PVX/mFT (Fig. 1a). Plant tissues were collected at 12 days post-inoculation and sections were probed with (+) or without (−) an antiserum specifically raised against the PVX coat protein (CP). Pink colour indicates the presence and distribution of recombinant viruses within infected cells and tissues. PVX/GFP (a) cannot enter SAM, but PVX/FT (b) and PVX/mFT (c) overcome SAM exclusion. Bar = 100 µm.

Figure 3. Viral ectopic expression of the Arabidopsis FT protein.

Total soluble proteins were extracted from young leaf tissues of Nicotiana benthamiana plants mock-inoculated or infected with PVX/GFP, PVX/FT or PVX/mFT at 12 days post-inoculation and analysed by western blot using antibodies specific to PVX CP (a), GFP (b) and FT (c). Coomassie blue-stained gel (d) shows equal loading of soluble protein samples. The position and sizes of the protein markers are indicated.

Figure 4. Viral transient expression of a non-translatable mFT RNA triggers early flowering.

(a) Virus-based flowering assay. MM plants were either mock inoculated (mock) or treated with PVX/mFT (mFT) and grown under SD with a 12-hr photoperiod of light at 25 °C. An arrow indicates a flower bud at the shoot tip of a mock-inoculated plant whilst a dozen flowers are blossoming in an mFT RNA expressing plant. Photographs were taken at 10 weeks post-inoculation (wpi). (b) RT-PCR detection of viral transient mFT RNA in MM plant treated with PVX/mFT (mFT), but not mock inoculation (mock). The 1-Kb DNA marker (M) (1500, 1000, 750, 500 and 400-bp from top to bottom) is included to show the expected size (684 bp) of the RT-PCR product. (c) Direct sequence of the RT-PCR product verified the viral transient mFT RNA, showing the double mutations. Mutation of the native FT start codon ATG with TAG (asterisked) and a nucleotide deletion (Δ) are indicated. The codon triplets are underlined. (d–f) Western blot detection of the Arabidopsis FT protein (d) in MM plants treated with PVX/FT (FT), but not with mock inoculation (mock) or PVX/mFT (mFT); of the PVX coat protein (e) in plants treated with PVX/mFT or PVX/FT, but not in mock-inoculated plant. Coomassie blue-stained gel indicates the equal loading of protein samples (f). The positions and sizes of the protein marker (M) are indicated. (g–i) MM plants mock-inoculated (g), or treated with PVX/mFT (h) or PVX/FT (i) were grown under a non-inducing LD 16-hr photoperiod and only plants treated with PVX/FT flowered. Photographs were taken at 10 wpi. (j–l) MM plants mock-inoculated (j), or treated with PVX/mFT (k) or PVX/FT (l) were grown in SD. Plants with PVX/FT treatment developed buds at 4 wpi and flowered at 6 wpi whilst plants mock-inoculated or inoculated with PVX/mFT were only bolting during these periods. Photographs were taken at 6 wpi.

Figure 5. Analyses of budding and flowering time and numbers of floral buds and flowers.

(a) PVX/mFT-based flowering assays in SD. Seven – nine N. tabacum MM plants were either mock-inoculated (mock) or infected with PVX/mFT (mFT) that had the capacity to express a non-translatable Arabidopsis FT mRNA, but not the FT protein. Numbers of floral buds and flowers in individual plants were counted weekly starting at 7 weeks post-inoculation (wpi) until 13 wpi. Among PVX/mFT-treated MM plants, first floral buds and flowers appeared at 7 and 10 wpi, respectively. At 11 wpi, more than 45% of floral buds converted into flowers. However, only half of mock-inoculated plants started to produce floral buds at 9 wpi and less than 18% of floral buds converted into flowers at 11 wpi. The average numbers of combining floral buds and flowers per plant [25 ± 7 (n = 7) vs 10 ± 9 (n = 9), p = 0.002; 30 ± 5 vs 18 ± 9, p = 0.005] were significantly higher (Student's t-Test) in PVX/mFT- than mock-treated plants at (before) 10 and 11 wpi, but showed no significant difference (33 ± 3 vs 24 ± 11, p = 0.094) at 13 wpi. These data suggest that viral transient expression of the non-translatable Arabidopsis FT mRNA induced earlier budding and flowering in MM plants in SD. (b) PVX/FT-based flowering assays in SD. Eight MM plants were treated with PVX/FT (FT) that was capable of expressing a wild-type FT gene. Viral transient expression of wild-type FT mFT RNA and the FT protein facilitated much earlier development of floral buds and flowers. This important baseline information indicates that the FT mRNA and its protein product had a synergetic effect on systemic florigenic signalling.