Identification, Functional Study, and Promoter Analysis of HbMFT1, a Homolog of MFT from Rubber Tree (Hevea brasiliensis)

Abstract

A homolog of MOTHER OF FT AND TFL1 (MFT) was isolated from Hevea brasiliensis and its biological function was investigated. Protein multiple sequence alignment and phylogenetic analysis revealed that HbMFT1 conserved critical amino acid residues to distinguish MFT, FLOWERING LOCUS T (FT) and TERMINAL FLOWER1 (TFL1)-like proteins and showed a closer genetic relationship to the MFT-like group. The accumulation of HbMFT1 was generally detected in various tissues except pericarps, with the highest expression in embryos and relatively higher expression in roots and stems of seedlings, flowering inflorescences, and male and female flowers. HbMFT1 putative promoter analysis showed that tissue-specific, environmental change responsive and hormone-signaling responsive elements were generally present. HbMFT1 was strongly induced under a short-day condition at 28 °C, with the highest expression after the onset of a day. Overexpression of HbMFT1 inhibited seed germination, seedling growth, and flowering in transgenic Arabidopsis. The qRT-PCR further confirmed that APETALA1 (AP1) and FRUITFULL (FUL) were drastically down-regulated in 35S::HbMFT1 plants. A histochemical β-glucuronidase (GUS) assay showed that HbMFT1::GUS activity was mainly detected in stamens and mature seeds coinciding with its original expression and notably induced in rosette leaves and seedlings of transgenic Arabidopsis by exogenous abscisic acid (ABA) due to the presence of ABA cis-elements in HbMFT1 promoter. These results suggested that HbMFT1 was mainly involved in maintenance of seed maturation and stamen development, but negatively controlled germination, growth and development of seedlings and flowering. In addition, the HbMFT1 promoter can be utilized in controlling transgene expression in stamens and seeds of rubber tree or other plant species.

Keywords: Arabidopsis; Flowering; Germination; MFT homolog; Phosphatidyl ethanolamine-binding protein (PEBP) family; Rubber tree.

Figure 1

(A) Genomic organization of HbMFT1. Yellow boxes represent exons. Lines represent introns; (B) Protein multiple alignment between deduced amino acid sequence of HbMFT1 in rubber tree and phosphatidyl ethanolamine-binding protein (PEBP) family of other species. Sequence alignment was carried out using DNAMAN 6.0 software (http://www.lynnon.com/). Three triangles refer to the intron positions. I, D-P-D-x-P motif. II, G-x-H-R motif. III, the region is essential for FT/TFL1-like activity in exon IV. An asterisk indicates amino acids that are related to antagonistic functions between TFL1 and FT protein. Different colors refer to the different homology levels of aligned amino acid residues among MFT homologs. Darkblue represents 100% identity. Hotpink represents more than 75% identity. Turquoise represents more than 50% identity. The aforementioned proteins and their accession numbers: Arabidopsis (AtMFT, NP_173250.1; AtFT, NP_176726.1; AtTSF, NP_193770.1; AtBFT, NP_201010.1; AtTFL1, NP_196004.1; ATC, NP_180324.1), Jatropha curcas (JcCEN, NP_001295672.1; JcMFT1, KC874668; JcMFT2, KF944352), Malus domestica (MdCEN, NP_001280770.1), Vitis vinifera (VvMFT, NP_001267935.1).

Figure 2

Phylogenetic analysis of the members in PEBP family. The tree was constructed using the Neighbor-Joining (N-J) method for members of the PEBP family in Hevea brasiliensis (HbMFT1), Jatropha curcas (JcCEN1, NP_001295672.1; JcMFT1, KC874668; JcMFT2, KF944352), Arabidopsis thaliana (TFL1, NP_196004.1; TSF, NP_193770.1; FT, NP_176726.1; MFT, NP_173250.1; BFT, NP_201010.1; ATC, NP_180324.1), Triticum aestivum (TaMFT, BAK78908.1), Populus trichocarpa (PtMFT, XP_002321507.1), Glycine max (GmMFT, ACA24491.1), Picea abies (PaMFT1, AEH59565.1), Pinus sylvestris (PsMFT, AIJ02001.1), Gossypium barbadense (GbMFT1, AGJ98454.1), Gossypium arboreum (GaMFT1, KHG10593.1), Sinapis alba (SaFT, ACM69283.1), Brassica napus (BnFT, ACY03404.1), Brassica oleracea (BoFT, ACH86033.1), Eutrema japonicum (EjFT, ADV18466.1), Boechera stricta (BsFT, AIU56794.1), Cardamine hirsuta (ChFT, AKC05615.1), Zea mays (ZmTFL1, ABI98712.1; ZCN3, ABX11005.1), Medicago truncatula (MtTFL1, XP_013443336.1), Vitis vinifera (VvMFT, NP_001267935.1), Malus domestica (MdCEN, NP_001280770.1). All of the protein sequences were downloaded from the NCBI according to their accession numbers. Abbreviations: ATC, ARABIDOPSIS THALIANA CENTRORADIALIS; BFT, BROTHER OF FT AND TFL1; FT, FLOWERING LOCUS T; MFT, MOTHER OF FT AND TFL1; TFL1, TERMINAL FLOWER1; TSF, TWIN SISTER OF FT; CEN, CENTRORADIALIS.

Figure 3

Expression analysis of HbMFT1 in rubber tree. (A) Tissue-specific expression analysis of HbMFT1. EM, RS, SS, AS, BS, CS, PS, MS, embryos and roots, stems, shoot apices, bronze, color change, pale-green and mature leaves of three-month old seedlings; PE, MF, FF, pericarps, open male and female flowers; I1, I2, I3, I4 and I5, five different developmental-stage inflorescences; (B–E) expression of HbMFT1 in roots, stems, mature leaves and shoot apices of rubber trees at different ages (three months, two years and ten years). RS, SS, MS and AS, roots, stems, mature leaves and shoot apices of the three-month-old seedlings; RT, ST, MT and AT, roots, stems, mature leaves and shoot apices of the two-year-old trees; RR, SR, MR and AR, roots, stems, mature leaves and shoot apices of the ten-year-old trees (adult trees). HbRH2b, HbRH8 and HbYLS8 were used as reference genes for qRT-PCR analysis. Values were means ± SE from three independent biological replicates.

Figure 4

Expression changes of HbMFT1 in response to different photoperiods and temperatures. (A) Expression profiles of HbMFT1 in long-day (LD) and short-day conditions (SD); (B) Expression changes of HbMFT1 at different temperatures. Results were from three independent biological replicates.

Figure 5

HbMFT1::GUS (β-glucuronidase) activity analysis in transgenic Arabidopsis plants. (A) Schematic of T-DNA structure of pCAMBIA3301 recombinant construct. Various tissues of adult transgenic plant harboring HbMFT1::GUS fusion, including root (B); rosette leaf (C); stem with cauline leaves and axillary meristems (D); flowering inflorescence (E); mature silique (F); wild-type mature silique (G); wild-type seedling at 12 h after germination (HAG) (H); seedling at 12 HAG (I); seedling at 1 day after germination (DAG) (J); 3 DAG (K); 5 DAG (L); 7 DAG (M). bar = 2 mm for (B–G), bar = 0.2 mm for (H–J), bar = 1 mm for (K–M). Co: cotyledon, Ra: radicle, Hy: hypocotyl, S.C.: seed coat.

Figure 6

Effect of ABA on HbMFT1::GUS activity in transgenic Arabidopsis. (A,C) The phenotypes of seedlings and rosette leaves of transgenic Arabidopsis treated with 10 μM ABA for 3, 6, 12 and 24 h, respectively; (B,D) The phenotypes of seedlings and rosette leaves of transgenic Arabidopsis treated with different concentrations of ABA (10, 50, 100, 200 and 300 μM) for 24 h. Both of the transgenic Arabidopsis plant lines with HbMFT1::GUS fusions were sampled independently twice. Bar = 1 mm for (A,B); bar = 3 mm for (C,D).

Figure 6

Effect of ABA on HbMFT1::GUS activity in transgenic Arabidopsis. (A,C) The phenotypes of seedlings and rosette leaves of transgenic Arabidopsis treated with 10 μM ABA for 3, 6, 12 and 24 h, respectively; (B,D) The phenotypes of seedlings and rosette leaves of transgenic Arabidopsis treated with different concentrations of ABA (10, 50, 100, 200 and 300 μM) for 24 h. Both of the transgenic Arabidopsis plant lines with HbMFT1::GUS fusions were sampled independently twice. Bar = 1 mm for (A,B); bar = 3 mm for (C,D).

Figure 7

Seed germination comparison between 35S::HbMFT1 transgenic Arabidopsis and wild-type (wt). (A) Southern blot analysis of transgene integration of HbMFT1; (B) Expression analysis of HbMFT1 in lines transformed with 35S::HbMFT1. Values were means ± SE from three independent biological replicates; (C) Time course of the germination rate after transformation with 35S::HbMFT1; (D) Seed germination observation at different developmental periods. Results came from six independent biological replicates. Bar indicates 0.2 mm.

Figure 8

The effect of over-expressing HbMFT1 on aerial parts and roots growth. (A,B) Root growth observation for wt and 35S::HbMFT1 transgenic lines; (C) Comparison of root length between wt and 35S::HbMFT1 transgenic lines; (D) Comparison of rosette leaves between wt and 35S::HbMFT1 transgenic lines. Significant difference tests were carried out using the Student’s t-test between wt and 35S::HbMFT1 transgenic lines. The levels of significance: * indicates 0.01 < p < 0.05; ** refers to p < 0.01.

Figure 9

Effect of over-expressing HbMFT1 on flowering. (A) Flowering phenotypes of wt and 35S::HbMFT1 transgenic lines; (B) Comparison of flowering time and number of cauline and rosette leaves at flowering between wt and 35S::HbMFT1 transgenic lines; (C–G) Expression analysis of genes related with flowering in wt and 35S::HbMFT1 transgenic lines. Total RNA was isolated from seedlings growing at 25 days after germination, which was the transition phase of Arabidopsis bolting. Values were means ± SE from three independent biological replicates. The levels of significance: ** refers to p < 0.01. Abbreviations: SOC1, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1; LFY, LEAFY; FUL, FRUITFULL; AP1, APETALA1; FT, FLOWERING LOCUS T.

Figure 9

Effect of over-expressing HbMFT1 on flowering. (A) Flowering phenotypes of wt and 35S::HbMFT1 transgenic lines; (B) Comparison of flowering time and number of cauline and rosette leaves at flowering between wt and 35S::HbMFT1 transgenic lines; (C–G) Expression analysis of genes related with flowering in wt and 35S::HbMFT1 transgenic lines. Total RNA was isolated from seedlings growing at 25 days after germination, which was the transition phase of Arabidopsis bolting. Values were means ± SE from three independent biological replicates. The levels of significance: ** refers to p < 0.01. Abbreviations: SOC1, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1; LFY, LEAFY; FUL, FRUITFULL; AP1, APETALA1; FT, FLOWERING LOCUS T.