Identification and expression analysis of ERF transcription factor genes in petunia during flower senescence and in response to hormone treatments

Abstract

Ethylene-responsive element-binding factor (ERF) genes constitute one of the largest transcription factor gene families in plants. In Arabidopsis and rice, only a few ERF genes have been characterized so far. Flower senescence is associated with increased ethylene production in many flowers. However, the characterization of ERF genes in flower senescence has not been reported. In this study, 13 ERF cDNAs were cloned from petunia. Based on the sequence characterization, these PhERFs could be classified into four of the 12 known ERF families. Their predicted amino acid sequences exhibited similarities to ERFs from other plant species. Expression analyses of PhERF mRNAs were performed in corollas and gynoecia of petunia flower. The 13 PhERF genes displayed differential expression patterns and levels during natural flower senescence. Exogenous ethylene accelerates the transcription of the various PhERF genes, and silver thiosulphate (STS) decreased the transcription of several PhERF genes in corollas and gynoecia. PhERF genes of group VII showed a strong association with the rise in ethylene production in both petals and gynoecia, and might be associated particularly with flower senescence in petunia. The effect of sugar, methyl jasmonate, and the plant hormones abscisic acid, salicylic acid, and 6-benzyladenine in regulating the different PhERF transcripts was investigated. Functional nuclear localization signal analyses of two PhERF proteins (PhERF2 and PhERF3) were carried out using fluorescence microscopy. These results supported a role for petunia PhERF genes in transcriptional regulation of petunia flower senescence processes.

Fig. 1.

Schematic analysis of PhERFs with ERF domains and EAR repressor domains. (A) Comparison of ERF domains by deduced amino acids sequences. Conserved residues are shaded in black. Grey shading indicates similar residues in 10 out of 14 of the sequences. (B) Location of ERF domains (black bars) and EAR repressor domains (grey bars).

Fig. 2.

Phylogenetic tree of ERFs. Thirteen petunia PhERFs (black circles) were aligned with the Arabidopsis ERF family, Solanum lycopersicum [SlERF2 (GenBank accession number AAO34704), SlERF3 (AAO34705), SlERF4 (AAO34706), SlERF5 (AY559315.1), Pti4 (AAC50047), JERF1 (AAK95687)]; Nicotiana tabacum [NtERF1 (UniProtKB/Swiss-Prot accession no. Q40476), NtERF2 (Q40479), NtERF3 (Q40477), NtERF4 (Q40478)]; Capsicum annuum [CaPF1 (GenBank accession no. AAP72289), CaERELP1 (AAS20427)], Malusx domestica [MdERF1 (GenBank accession no. BAF43419), MdERF2 (BAF43420)]; Prunus salicina [PsERF1a (GenBank accession no. FJ026009), PsERF1b (FJ026008), PsERF2a (FJ026007]), PsERF2b (FJ026006), PsERF3a (FJ026005), PsERF3b (FJ026004), PsERF12 (FJ026003)]; Cucumis melo [CmERELP (GenBank accession no. BAD01556)]; and Dianthus caryophyllus [DcERF1 (GenBank accession no. AB517647)]. The amino acid sequences of Arabidopsis ERFs were obtained from the Arabidopsis Information Resource or the National Center for Biotechnology Information database. The amino acid sequences were analysed with Vector NTI (version 9.0.0; Invitrogen), and the phylogenetic tree was constructed with MEGA (version 3.1) using a bootstrap test of phylogeny with minimum evolution test and default parameters.

Fig. 3.

Natural senescence of unpollinated wild-type (WT) Petunia hybrida ‘Carpet White’ flowers and changes in ethylene production of corollas and gynoecia. (A) Natural senescence of unpollinated WT P. hybrida ‘Carpet White’ flowers. (B) Changes in ethylene production of corollas. (C) Changes in ethylene production of gynoecia. (This figure is available in colour at JXB online.)

Fig. 4.

Expression profile of PhERF1-10, PhERF12, and PhERF13 during natural flower senescence, and effects of exogenous ethylene on the expression of these genes by quantitative PCR in petunia corollas. The mRNA level in corollas was measured by quantitative PCR. Relative expression levels are shown as fold change values (1=time 0). Data are means ±SD (n–3).

Fig. 5.

Expression profile of PhERF1-10, PhERF12, and PhERF13 during natural flower senescence and effects of exogenous ethylene on the expression of these genes by quantitative PCR in petunia gynoecia. The mRNA level in gynoecia was measured by quantitative PCR. Relative expression levels are shown as fold change values (1=time 0). Data are means ±SD (n=3).

Fig. 6.

Effects of STS on the expression of PhERF1-10, PhERF12, and PhERF13 by quantitative PCR in corollas and gynoecia. (A) Effects of STS on the expression of PhERF1-10, PhERF12, and PhERF13 in corollas. (B) Effects of STS on the expression of PhERF1-10, PhERF 12, and PhERF 13 in gynoecia. The mRNA level was measured by quantitative PCR. Relative expression levels are shown as fold change values (1=time 0). Data are means ±SD (n=3).

Fig. 7.

Effects of ABA, IAA, SA, MeJA, BA, and sugar on the expression of PhERF1-10, PhERF12, and PhERF13 by quantitative PCR in corollas of petunia. The mRNA level in corollas was measured by quantitative PCR. Relative expression levels are shown as fold change values (1=time 0). Data are means ±SD (n=3).

Fig. 8.

Subcellular localization of PhERF2 and PhERF3 proteins. (A) Nuclear localization of GFP–PhERF2 fusion protein in onion epidermal cells. (B) Nuclear localization of GFP–PhERF3 fusion protein in onion epidermal cells. (C) Onion epidermal cells transformed with a translational construct of GFP as a positive control showed localization throughout the cell, with the strongest signals in the cytoplasm and nucleus. GFP fluorescence (left panel) and differential contrast imagery (right panel) were compared. The positions of nuclei are indicated by arrows. The scale bars indicate 100 μm.