An AC-Rich Bean Element Serves as an Ethylene-Responsive Element in Arabidopsis

Abstract

Ethylene-responsive elements (EREs), such as the GCC box, are critical for ethylene-regulated transcription in plants. Our previous work identified a 19-bp AC-rich element (ACE) in the promoter of bean (Phaseolus vulgaris) metal response element-binding transcription factor 1 (PvMTF-1). Ethylene response factor 15 (PvERF15) directly binds ACE to enhance PvMTF-1 expression. As a novel ERF-binding element, ACE exhibits a significant difference from the GCC box. Here, we demonstrated that ACE serves as an ERE in Arabidopsis. It conferred the minimal promoter to respond to the ethylene stress and inhibition of ethylene. Moreover, the cis-acting element ACE could specifically bind the nuclear proteins in vitro. We further revealed that the first 9-bp sequence of ACE (ACE_core) is importantly required by the binding of nuclear proteins. In addition, PvERF15 and PvMTF-1 were strongly induced by ethylene in bean seedlings. Since PvERF15 activates PvMTF-1 via ACE, ACE is involved in ethylene-induced PvMTF-1 expression. Taken together, our findings provide genetic and biochemical evidence for a new ERE.

Keywords: AC-rich element; Arabidopsis; ethylene response factor; ethylene-responsive element.

Figure 1

GUS transient assays for ACE as an ERE in the etiolated Arabidopsis seedlings. (A) The Arabidopsis seeds were grown in 1/2 glutathione S-transferase (MS) without (control) and with 10 μM ACC (ACC) or 50 μM AgNO₃ (AgNO₃) in the dark for two weeks and then subjected to hypocotyl or root length analysis. Data are means ± standard error (SE) (n = 100). The significance was assessed using a one-sided Student’s t-test (* p ≤ 0.05). (B,C) GUS transient assays in the etiolated Arabidopsis seedlings. Schematic diagram of constructs used in the experiments (B). The etiolated Arabidopsis seedlings were co-transformed with reporter and internal control constructs. The transformed etiolated Arabidopsis seedlings were grown on the solution medium without (control) or with 10 μM ACC (ACC) or 50 μM AgNO₃ (AgNO₃) for 2 days. The expression of GUS or GFP was determined by immunoblot using an anti-GUS antibody and anti-GFP antibody from the same protein samples. (C) Relative GUS activity was measured as the relative band intensity of GUS to GFP (the control was set as 1). The data represent an average from two independent experiments. Means ± SE are shown. The significance was assessed by a one-sided Students t-test (* p ≤ 0.05).

Figure 2

ACE specifically binds to the nuclear proteins in vitro. (A) Western blot verified the successful isolation of nuclear proteins using an anti-histone H3 (nuclei marker) antibody and anti-glucose pyrophosphorylase (UGPase) (cytoplasm marker) antibody. (B) EMSA was performed using biotin-labeled 2 × ACE probes with the affinity-purified GST-PvERF15 (positive control), GST mock (negative control), and nuclear proteins from the etiolated Arabidopsis seedlings. The bound complex is indicated by the arrows. The experiments were performed two times showing similar results.

Figure 3

Competitive EMSA identifies the required sequences of ACE for nuclear protein binding. (A) ACE (2×) was used as the probe in the EMSA with the nuclear proteins, and the mutant variants (red cases) were used as competitors (50-fold molar). (B) Gel shifts with the ACE_core probe (2×) showing specific binding to the same and/or similar nuclear proteins as ACE. Mutations introduced into the ACE_core are shown in red. A repeat experiment confirmed the results (Figure S1).

Figure 4

ACC regulates the mRNA levels of both PvMTF-1 and PvERF15 in bean seedlings. (A) Bean seeds were germinated and grown on the MS without (control) or with 500 μM ACC for 10 days in the dark. ACC induced a typical ethylene-responsive phenotype, indicating the effects of ethylene stress. Bar = 1 cm. (B) The etiolated bean seedlings were subjected to quantitative reverse transcription qRT-PCR analysis. ACC-induced expression level of PvERF15 or PvMTF-1 was expressed as a ratio relative to control, which was set to a value of 1. Data shown are averages of three independent qRT-PCR experiments. Error bars represent SEs. Significance between experimental values was assessed using one-sided Student’s t test (*, p ≤ 0.05, and **, p ≤ 0.01).