Mutual regulation of Arabidopsis thaliana ethylene-responsive element binding protein and a plant floral homeotic gene, APETALA2

Abstract

Background and aims: It has previously been shown that Arabidopsis thaliana ethylene-responsive element binding protein (AtEBP) contributed to resistance to abiotic stresses. Interestingly, it has also been reported that expression of ethylene-responsive factor (ERF) genes including AtEBP were regulated by the activity of APETALA2 (AP2), a floral homeotic factor. AP2 is known to regulate expression of several floral-specific homeotic genes such as AGAMOUS. The aim of this study was to clarify the relationship between AP2 and AtEBP in gene expression.

Methods: Northern blot analysis was performed on ap2 mutants, ethylene-related Arabidopsis mutants and transgenic Arabidopsis plants over-expressing AtEBP, and a T-DNA insertional mutant of AtEBP. Phenotypic analysis of these plants was performed.

Key results: Expression levels of ERF genes such as AtEBP and AtERF1 were increased in ap2 mutants. Over-expression of AtEBP caused upregulation of AP2 expression in leaves. AP2 expression was suppressed by the null-function of ethylene-insensitive2 (EIN2), although AP2 expression was not affected by ethylene treatment. Loss of AtEBP function slightly reduced the average number of stamens.

Conclusions: AP2 and AtEBP are mutually regulated in terms of gene expression. AP2 expression was affected by EIN2 but was not regulated by ethylene treatment.

Fig. 1.

Analysis of mRNA accumulation of AtEBP, AP2 and AtERF1 genes in WT and ap2-5. (A) Northern blot analysis. Total RNAs (10 µg) were isolated from flowers (F), stems (S) and rosette leaves (L) of 40-d-old plants. Ethidium bromide staining indicates rRNAs. (B) Relative levels of AtEBP, AP2 and AtERF1 expression. Each expression level was normalized to the rRNA bands, and the value for stems in WT was assigned as 1.

Fig. 2.

AP2 and AtERF1 mRNA accumulations in transgenic Arabidopsis lines over-expressing AtEBP. Total RNAs (10 µg) were isolated from leaves of 35-d-old plants. Ethidium bromide staining indicates rRNAs.

Fig. 3.

Comparison of mRNA level of AP2 and AtERF1 in ethylene-related mutants. Total RNAs (10 µg) were isolated from leaves of 30-d-old plants. Ethidium bromide staining indicates rRNAs.

Fig. 4.

Effects of ethylene on mRNA levels of AtEBP, ERF1 and AP2 in WT and ein3-1 after ethylene treatment. Total RNAs (10 µg) were isolated from leaves of 30-d-old plants. The plants were sampled 0, 1, 6 and 12 h after spraying with 5 mm ethephone. Ethidium bromide staining indicates rRNAs.

Fig. 5.

The T-DNA insertional mutant of AtEBP. (A) Schematic diagram of the genomic AtEBP (At3g16770). White areas indicate the exon, black shading indicates the nuclear-located signal, and grey shading indicates the AP2/EREBP domain. LB indicates the light border of the T-DNA insertion. (B) Northern blot analysis of AtEBP knockout mutant plants. Total RNAs (10 µg) obtained from 30-d-old plants were loaded. The coding region of AtEBP was used as a probe. A gel stained with ethidium bromide is shown as a control.

Fig. 6.

Floral phenotype of the AtEBP knockout mutant. (A) Flower of WT, (B) vector control plant, (C, D) AtEBP over-expressing plants, and (E, F) AtEBP knockout plants. Each arrow indicates a stamen. Scale bars = 1 mm.

Fig. 7.

Schematic diagram illustrating the relationship between AP2 and AtEBP. The translation of AP2 mRNA is suppressed by micro RNAs (Chen, 2004) and AP2 protein down-regulates AP2 and AtEBP expression (Okamuro et al., 1997; this study), as indicated in grey. In the current study, it was demonstrated that AP2 expression was regulated through AtEBP and EIN2, and that AtEBP may contribute to floral development, as indicated by in black.