Expression Pattern and Functional Characterization of PISTILLATA Ortholog Associated With the Formation of Petaloid Sepals in Double-Flower Eriobotrya japonica (Rosaceae)

Abstract

Double-flower Eriobotrya japonica, of which one phenotype is homeotic transformation of sepals into petals, is a new germplasm for revealing the molecular mechanisms underlying the floral organ transformation. Herein, we analyzed the sequence, expression pattern and functional characterization of EjPI, which encoded a B-class floral homeotic protein referred to as PISTILLATA ortholog, from genetically cognate single-flower and double-flower E. japonica. Phylogenetic analysis suggested that the EjPI gene was assigned to the rosids PI/GLO lineage. Analysis of protein sequence alignments showed that EjPI has typical domains of M, I, K, and C, and includes a distinctive PI motif at the C-terminal region. Compared with asterids PI/GLO lineage, the K1 and K3 subdomains of EjPI both contain a single amino acid difference. Subcellular localization of EjPI was determined to be in the nucleus. Expression pattern analysis revealed that EjPI expressed not only in petals, filament, and anther in single-flower E. japonica, but also in petaloid sepals in double-flower E. japonica. Meanwhile, there were high correlation between EjPI transcript level and petaloid area within a sepal. Furthermore, 35S::EjPI transgenic wild-type Arabidopsis caused the homeotic transformation of the first whorl sepals into petaloid sepals. Ectopic expression of EjPI in transgenic pi-1 mutant Arabidopsis rescued normal petals and stamens. These results suggest expression pattern of EjPI is associated with the formation of petaloid sepal. Our study provides the potential application of EjPI for biotechnical engineering to create petaloid sepals or regulate floral organ identity in angiosperms.

Keywords: Eriobotrya japonica; MADS-box gene; PISTILLATA; double-flower; ectopic expression; expression pattern.

Figure 1

Comparative morphological observation in the single-flower and double-flower of E. japonica. (A) Single-flower E. japonica; (B) Double-flower E. japonica, showing homeotic conversional petaloid sepals from sepals in the first whorl (blue arrows) and petals in the second whorl (red arrows). (C) Petaloid sepals (blue arrow) in double-flower E. japonica. (D) Sepal. (E) Petaloid sepal. (F) Comparison of petaloid sepal and petal. Sep, sepal; Pe-se, petaloid sepals; Pet, petal.

Figure 2

Phylogenetic analysis of PI/GLO-like MADS-box proteins. The EjPI protein sequence is blasted with twenty-six B-class proteins from other angiosperms, with two A-class proteins, four C-class proteins and four E-class proteins as out group. Black arrows show that the gene lineages are obtained through gene duplication. EjPI protein is marked. PI, PISTILLATA.

Figure 3

Sequence comparisons of EjPI and the other PI/GLO orthologous proteins. First underlined region represents the MADS domain. Second underlined region represents the K domain. The PI-motif is boxed. Dots indicate the amino acid residues identical to EjPI. Dashes are introduced into the sequences to improve the alignment. The K domain contains K1, K2, and K3 subdomains with (abcdefg)n heptad repeats (Yang et al., 2003), which are also underlined. Meanwhile, Asp-92/Glu-92 and Asn-149 in K1 and K3 subdomains in rosids PI/GLO lineage are boxed.

Figure 4

Subcellular localization of EjPI. GFP, GFP fluorescence; 4,6-diamidino-2-phenylindole (DAPI) staining shows nuclear localization; BF, bright-field; Merged, merged image of GFP and DAPI. GFP, green fluorescent protein.

Figure 5

Spatial expression of EjPI in single-flower and double-flower E. japonica by semi-quantitative RT-PCR. (A) Spatial expression of EjPI in double-flower E. japonica. (B) Spatial expression of EjPI in single-flower E. japonica. Sep, sepals; Pe-se, petaloid sepals; Pet, petals; Sta, stamens; Car, carpels; Fi, filaments; An, anthers.

Figure 6

Relative expression levels of EjPI in petaloid sepals in double-flower E. japonica by qRT-PCR. (A) Different types of petaloid sepals. (B) Petaloid area within one sepal. (C) Relative expression levels of EjPI in petaloid sepals. Sep, sepals; Pe-se, petaloid sepals; Pet, petals. Error bars indicate the standard deviation of three biological replicates. Different letters indicate significant differences (P < 0.05).

Figure 7

Comparison of the phenotypes of the wild-type and 35S::EjPI transgenic wild-type lines. (A) Flower of the wild-type Arabidopsis, showing tightly closed sepals and stamens (line 1^#). (B) Inflorescence of the transgenic wild-type Arabidopsis with the pBI121 vector only (negative control, line 2^#). (C) Flower of the transgenic wild-type Arabidopsis with the pBI121 vector only, showing no phenotypic alteration (line 3^#). (D) Inflorescence of 35S::EjPI, showing completely separating petaloid sepals (red arrows) and opening stamens (white arrows) (line 4^#). (E) Flower of 35S::EjPI transgenic wild-type Arabidopsis, showing green/white petaloid sepals in the first whorl (red arrows) (line 5^#). (F) Flower of 35S::EjPI transgenic wild-type Arabidopsis, showing completely separating petaloid sepals (red arrows) and opening stamens (white arrows) (line 6^#). (G) Cell shapes of adaxial surface in a wild-type Arabidopsis sepals, showing irregular cell margin; (H) Cell shapes of adaxial surface in petals in wild-type Arabidopsis; (I) Cell shapes of adaxial surface in petaloid sepals in 35S::EjPI transgenic wild-type lines, showing a petaloid margins (white arrow). Bars = 500 µm in (A), (B), (C), (D), (E), and (F) and Bars = 100 µm in (G), (H), and (I).

Figure 8

PCR analysis of 35S::EjPI transgenic homozygous pi-1 mutant lines. Lane M: DL2000 DNA marker. Lane 1-27: the PCR with DNA of 35S::EjPI transgenic homozygous pi-1 Arabidopsis as templates. Lane pi-1: the PCR with DNA of pi-1 line containing the pBI121 vector only as a template. Lane P: the PCR with plasmid containing EjPI full-length CDS as a template.

Figure 9

Phenotypic comparison of the homozygous pi-1 mutant and 35S::EjPI transgenic homozygous pi-1 Arabidopsis. (A) Flower of a homozygous pi-1 mutant Arabidopsis (line 1^#). (B) Flower of the transgenic homozygous pi-1 mutant with the pBI121 vector only (negative control) (line 2^#). (C) The flower of 35S::EjPI transgenic homozygous pi-1 Arabidopsis, showing shortened petals (red arrows) and runtish stamens (white arrows) (line 3^#). (D) Flower of 35S::EjPI transgenic homozygous pi-1 Arabidopsis, showing normal petals (red arrows) and completely rescuing stamens (white arrows) (line 4^#). Bars = 500 µm.