Identification and Functional Characterization of FLOWERING LOCUS T in Platycodon grandiflorus

Abstract

Platycodon grandiflorus roots have been used as a foodstuff and traditional medicine for thousands of years in East Asia. In order to increase the root development of P. grandiflorus, cultivators removed the inflorescences, suggesting the possible negative effect of flowering on root development. This indicates that the genetic improvement of P. grandiflorus by late flowering is a potential approach to increase productivity. However, nothing is known about key genes integrating multiple flowering pathways in P. grandiflorus. In order to fill this gap, we identified potential homologs of the FLOWERING LOCUS T (FT) gene in P. grandiflorus. The alignment with other FT members and phylogenetic analysis revealed that the P. grandiflorus FT (PlgFT) protein contains highly conserved functional domains and belongs to the FT-like clade. The expression analysis revealed spatial variations in the transcription of PlgFT in different organs. In addition, the expression level of PlgFT was increased by high temperature but not by photoperiodic light input signals, presumably due to lacking the CONSTANS binding motif in its promoter region. Furthermore, PlgFT induced early flowering upon its overexpression in P. grandiflorus, suggesting the functional role of PlgFT in flowering. Taken together, we functionally characterized PlgFT as a master regulator of P. grandiflorus flowering under inductive high temperature, which will serve as an important target gene for improving the root productivity.

Keywords: CONSTANS; FLOWERING LOCUS T; Platycodon grandiflorus; temperature.

Figure 1

Sequence alignment of PlgFT protein and other reported FT proteins. Red boxes indicate the conserved motifs and segments, including DPDxP, GXHR, segment A, and LYN. Blue boxes indicate key amino acids for flowering activator.

Figure 2

Subcellular localization and yeast two-hybrid assay for P. grandiflorus FT. (A) Subcellular localization of PlgFT in Nicotiana benthamiana leaves. PlgFT recombinant proteins transiently expressed in N. benthamiana leaves through agro-infiltration. Scale bar = 20 μm. (B) Analysis of protein–protein interaction between PlgFT and Arabidopsis FD (AtAD) using yeast two-hybrid assay. Yeast cells (AH109) were grown on SD agar medium lacking either Trp and Leu (−LT) or Trp, Leu, and His (−LTH).

Figure 3

Expression pattern of PlgFT in different organs (A) and flower development stages (B). Expression levels of PlgFT were normalized to actin. Data represent the means ± SD of three independent experiments. Different letters correspond to means that are statistically different (p < 0.05). The scale bars represent 10 cm (A) and 1 cm (B).

Figure 4

Diurnal expression pattern of PlgFT under different photoperiodic conditions. The expression levels of PlgFT were measured every 4 h throughout the diurnal cycle in LD and SD conditions. Expression levels of PlgFT were normalized to actin. Values represent three independent replicates ± SD. Uppercase denotes significance (p < 0.05) between the expression levels under LD, whereas lowercase denotes significance (p < 0.05) between the expression levels under SD.

Figure 5

Regulation of PlgFT expression by GA₃ (A) and high-temperature conditions (B). Two-month-old plants were treated with GA₃ or high temperature (30 °C). The expression levels of PlgFT were normalized to actin. Values represent three independent replicates ± SD. Uppercase denotes significance (p < 0.05) between the expression levels after treatment of GA3 or high temperature (30 °C), whereas lowercase denotes significance (p < 0.05) between the expression levels after treatment of mock or normal temperature (22 °C).

Figure 6

Ectopic expression of PlgFT in P. grandiflorus induced early flowering. (A) The expression of PlgFT-GFP in the selected transgenic plants was confirmed by RT-PCR. Phenotypes of TC and PlgFT-GFP overexpression plants (PlgFT-OX) grown in the in vitro condition (B) and soil (C). Scale bar = 1 cm (B) or 10 cm (C).