Expression of LhFT1, the Flowering Inducer of Asiatic Hybrid Lily, in the Bulb Scales

Abstract

Asiatic hybrid lily leaves emerge from their bulbs in spring, after cold exposure in winter, and the plant then blooms in early summer. We identified four FLOWERING LOCUS T (FT)-like genes, LhFT1, LhFT4, LhFT6, and LhFT8, from an Asiatic hybrid lily. Floral bud differentiation initiated within bulbs before the emergence of leaves. LhFT genes were mainly expressed in bulb scales, and hardly in leaves, in which the FT-like genes of many plants are expressed in response to environmental signals. LhFT1 was expressed in bulb scales after vernalization and was correlated to flower bud initiation in two cultivars with different flowering behaviors. LhFT8 was upregulated in bulb scales after cold exposure and three alternative splicing variants with a nonsense codon were simultaneously expressed. LhFT6 was upregulated in bulb scales after flower initiation, whereas LhFT4 was expressed constantly in all organs. LhFT1 overexpression complemented the late-flowering phenotype of Arabidopsis ft-10, whereas that of LhFT8 did so partly. LhFT4 and LhFT6 overexpression could not complement. Yeast two-hybrid and in vitro analyses showed that the LhFT1 protein interacted with the LhFD protein. LhFT6 and LhFT8 proteins also interacted with LhFD, as observed in AlphaScreen assay. Based on these results, we revealed that LhFT1 acts as a floral activator during floral bud initiation in Asiatic hybrid lilies. However, the biological functions of LhFT4, LhFT6, and LhFT8 remain unclear.

Keywords: FLOWERING LOCUS T like genes; Lilium sp.; alternative splicing; cold exposure; flower initiation; geophytes; ornamental plants.

FIGURE 1

Vegetative and reproductive development stages in the Asiatic hybrid lily ‘Lollypop.’ (A) Bulbs before chilling exposure. Bar, 2 cm. (B) Anthesis. Bar, 2 cm. (C–H) Definition of the floral development stages of the SAM. Bar, 500 μm. br, bract; op, outer perianth; ip, inner perianth; st, stamen; pi, pistil. (C) Vegetative SAM. (D) Flower initiation, rounded SAM. (E) Bract formation. (F) Formation of outer and inner perianthes primordia. (G) Formation of pistil primordia. (H) Pistil development. (I) Flower bud transition after planting. Flower developmental stages are defined in “Materials and Methods.” We observed three independent SAMs each week. (J) Alteration of plant height and leaf number after planting. The blue arrow indicates leaves emerged from the soil at three weeks after planting. The red arrow indicates ‘Lollypop’ blooming at 75 days after planting.

FIGURE 2

Phylogenetic tree of FT-like protein sequences from different monocot plants. The phylogenetic tree was constructed using the neighbor-joining method. Bootstrap values from 1,000 replicates were used to assess the robustness of the tree. The scale indicates the average number of substitutions per site. The FT-like gene names and GenBank accession numbers were as follows: Allium cepa AcFT1 (AG081838.1), AcFT2 (AGZ20208.1), AcFT3 (AGZ20209.1), AcFT4 (AGZ20210.1), AcFT5 (AGZ20211.1), and AcFT6 (AGZ20212.1); Arabidopsis thaliana AtFT (NP_176726.1), AtMFT (NP_173250.1), AtTFL1 (NP_196004.1), and AtTSF (NP_193770.1); Cymbidium goeringii CymFT (ADI58462.1); Hordeum vulgare HvFT1 (AAZ38709.1), HvFT2 (ABB99414.1), HvFT3 (ABD75336.2), HvFT4 (ABD75337.1), HvFT5 (ABM26903.1), HvMFT1 (BAH24198.1), and HvTFL1 (ANO81637.1); Lilium hybrid LfFT1 (AIF76295.1), LhFT1, LhFT4, LhFT6, and LhFT8 (this study); L. longiflorum FlFT (ATU06687.1); Lilium sp. LiFTL1 (ATU06689.1), LiFTL2 (ATU06688.1), and LiFTL3 (ATU06690.1); Narcissus tazetta NtFT (AFS50164.1); Oncidium hybrid OncFT (AIG6303.1) and OncTFL1 (AIU44253.1); Oryza sativa OsHd3a (XP_01641951.1) and OsRFT1 (XP_015642519.1); Phalaenopsis aphrodite PaFT1 (AJF93423.1); Triticum aestivum TaFT (ACA25437.1); Tulipa gesneriana TgFT1 (ATU06684.1), TgFT2 (ATU006685.1), and TgFT3 (ATU006686.1); Zea mays ZCN1 (ONM42318.1), ZCN2 (NP_00110624.1), ZCN3 (NP_001106242.1), ZCN4 (NP_001106243.1), ZCN5 (NP001106244.1), ZCN6 (NP_001106245.1), ZCN7 (NP_001106246.1), ZCN8 (NP_001106247.1), ZCN9 (ABW96232.1), ZCN10 (NP_00106249.1), ZCN11 (AQK83984.1), ZCN12 (NP_001106250.1), ZCN14 (NP_001106251.1), ZCN15 (NP_001106252.1), ZCN16 (NP_001106253.1), ZCN17 (NP_001106254.1), ZCN18 (NP_001354358.1), ZCN19 (NP_001106256.1), ZCN20 (NP_001296779.1), ZCN25 (NP_001106257.1), and ZCN26 (NP_001106265.1).

FIGURE 3

Expression patterns of LhFT and flowering-related genes in ‘Lollypop’ bulbs during chilling and planting. (A) Semiquantitative RT-PCR analysis of LhFT, LhFD, LhMADS5, and LhACT genes using scales (A) and SAMs (B) of ‘Lollypop’ blubs. Scale and SAM samples were collected from bulbs exposed to chilling for 0, 4, 8, 12, and 16 weeks, and from bulbs grown for 1, 2, and 3 weeks after chilling for 16 weeks. The gene names and number of cycles are indicated to the left of the panel. The amplified fragment lengths are indicated to the right of the panels. The 516-bp fragment of LhFT6 corresponds to the normal transcript, whereas the 700-bp fragment corresponds to the alternative transcript. The 540-bp fragment of LhFT8 corresponds to normal transcript, whereas the 630-bp, 725-bp, and 820-bp fragments correspond to alternative transcripts.

FIGURE 4

Structure of the genome and mRNA variants of LhFT6 (A) and LhFT8 (B). The boxes and lines indicate exons and intron, respectively. Filled and empty boxes indicate coding and non-coding regions, respectively. The numbers within the boxes correspond to the number of exons, whereas the numbers outside the boxes indicate the length (in base pairs). We were not able to obtain the complete sequences of the second intron of LhFT8 because it was too long to allow amplification and sequence analysis. Black and green arrows indicate primer position in RT-PCR and RT-qPCR analyses.

FIGURE 5

Temporal expression patterns of LhFT and flowering-related genes in different organs of ‘Lollypop.’ The quantitative gene expression analysis of LhFT1, LhFT4, LhFT6, LhFT8, LhFD, and LhMADS5 in SAM, scales, leaf samples of Asiatic hybrid ‘Lollypop’ was performed. Bulb scales samples were collected from bulbs under different development phase, including non-vernalization (NV) and vernalization (= 0 day after planting), 26, and 34 days after planting. Leaf samples were collected from 26, 34, 40, and 47 days after planting. SAM samples were collected from non-vernalized bulbs and vernalized bulb (0 day) and from shoots at 26, 34, 40, and 47 days after planting. Blue and orange fill boxes indicated chilling exposure period for 4 months and floral initiation period, respectively. The expression levels of LhFT and flowering-related genes were investigated by RT-qPCR and normalized using that of LhACT. Values are expressed as the mean ± SE (n = 6). Temporal expression pattern per each organ is shown in Supplementary Figure 5.

FIGURE 6

Flowering time phenotypes of the lines overexpressing LhFT genes in the Arabidopsis ft-10 mutant. (A) LhFT1-overexpressing transgenic plants. (B) LhFT8-overexpressing transgenic plants. (C) LhFT4-overexpressing transgenic plants. (D) LhFT6-overexpressing transgenic plants. All transgenic plants are T₂ homozygous specimens. Col-1 represents the wild-type. GFP indicates the vector control transgenic plants in the Arabidopsis ft-10 background. RT-PCR analysis was performed to investigate the expression levels of transgenes and AtACT2 (internal standard). The number of leaves in transgenic lines overexpressing each LhFT gene was investigated in 10 individuals of two or three independent lines during bolting. Values are expressed as the mean ± SE (n = 10). The different letters placed above columns are significantly different according to the Tukey–Kramer test (P < 0.05).

FIGURE 7

Protein–protein interactions between the LhFT proteins and LhFD. (A) The LhFT1, LhFT4, LhFT6, LhFT8, and LhFD proteins were fused to the GAL4 DNA-binding domain (BK) or GAL4 activation domain (AD). pGBKT7 and pGADT7 were used as the negative controls, for bait and prey, respectively. Yeast cells were grown on double-selection medium (−Leu, and −Trp; left) and quadruple-dropout medium (−Leu, −Trp, −His, and −Ade) supplemented with 15 mM 3-AT (right) at 30°C for 3 days. (B) in vitro protein–protein interaction assay by AlphaScreen. AGIA-tagged LhFTs were incubated with biotinylated LhFD. The interaction intensity between LhFTs and LhFD was analyzed by AlphaScreen Biotinylated dihydrofolate reductase (DHFR), and E. coli was used as negative control. Data are mean ± SD. of three independent experiments (n = 3). **indicates the significant difference with student’s t-test (P < 0.01). (C) AlphaScreen assay for Arabidopsis FT–FD interaction. AGIA-tagged AtFD was incubated with biotinylated AtFT. Data are represented as mean ± SD of three independent experiments (n = 3).