Transcriptional regulation of the ethylene response factor LeERF2 in the expression of ethylene biosynthesis genes controls ethylene production in tomato and tobacco

Abstract

Fine-tuning of ethylene production plays an important role in developmental processes and in plant responses to stress, but very little is known about the regulation of ethylene response factor (ERF) proteins in ethylene biosynthesis genes and ethylene production. Identifying cis-acting elements and transcription factors that play a role in this process, therefore, is important. Previously, a tomato (Solanum lycopersicum [f. sp. Lycopersicon esculentum]) ERF protein, LeERF2, an allele of TERF2, was reported to confer ethylene triple response on plants. This paper reports the transcriptional modulation of LeERF2/TERF2 in ethylene biosynthesis in tomato and tobacco (Nicotiana tabacum). Using overexpressing and antisense LeERF2/TERF2 transgenic tomato, we found that LeERF2/TERF2 is an important regulator in the expression of ethylene biosynthesis genes and the production of ethylene. Expression analysis revealed that LeERF2/TERF2 is ethylene inducible, and ethylene production stimulated by ethylene was suppressed in antisense LeERF2/TERF2 transgenic tomato, indicating LeERF2/TERF2 to be a positive regulator in the feedback loop of ethylene induction. Further research showed that LeERF2/TERF2 conservatively modulates ethylene biosynthesis in tobacco and that such regulation in tobacco is associated with the elongation of the hypocotyl and insensitivity to abscisic acid and glucose during germination and seedling development. The effects on ethylene synthesis were similar to those of another ERF protein, TERF1, because TERF1 and LeERF2/TERF2 have overlapping roles in the transcriptional regulation of ethylene biosynthesis in tobacco. Biochemical analysis showed that LeERF2/TERF2 interacted with GCC box in the promoter of NtACS3 and with dehydration-responsive element in the promoter of LeACO3, resulting in transcriptional activation of the genes for ethylene biosynthesis in tomato and tobacco, which is a novel regulatory function of ERF proteins in plant ethylene biosynthesis.

Figure 1.

Expression of LeERF2/TERF2 is inducible by ethylene. Each lane was loaded with 20 μg of total RNA from 4-week-old tomato plants grown under ethylene gas for the time indicated. RNA was analyzed by RNA gel blotting and a gene-specific probe from the 3′ flanking sequences of TERF2. RNAs were used as the loading control.

Figure 2.

TERF2-OEm increases while TERF2-RI decreases the production of ethylene by regulating the expression of ethylene biosynthesis genes. A, Expression of LeERF2/TERF2 in transgenic tomato. After reverse transcription, the synthesized cDNA was used as a template for RT-PCR amplifications. The mRNA levels were detected with ethidium bromide staining. B, Production of ethylene in wild-type (WTM), TERF2-OEm, and TERF2-RI plants was measured in three independent assays with three plant samples each. The data show ethylene production by wild-type, TERF2-OEm, and TERF2-RI plants, expressed in terms of the increase over the control (sealed vials without any plant). FW, Fresh weight. C, Expression of LeACS and LeACO in the wild type, TERF2-OEm, and TERF2-RI by Q-PCR. The expression level of each gene in the wild type was standardized to 100, referring to the internal control of Actin. The assay was repeated three times. Error bars represent se.

Figure 3.

TERF2-OE decreases the sensitivity to ABA and Glc during tobacco seed germination. A, TERF2-OE decreases the sensitivity of tobacco to Glc during seed germination. B, TERF2-OE decreases the sensitivity of tobacco to ABA during seed germination. Data are from germinating seeds on the day 4 after culturing on half-strength MS with different concentrations of ABA or Glc. Fifty seeds were used for each independent triple assay. Error bars represent sd. WT, Wild type.

Figure 4.

TERF1-OE or TERF2-OE increases the production of ethylene by regulating the expression of ethylene biosynthesis genes in tobacco. A, Production of ethylene in wild-type, TERF1-OE, and TERF2-OE plants was measured in three independent assays with three plant samples each. FW, Fresh weight. B, Expression of NtACS and NtACO in wild-type, TERF1-OE, and TERF2-OE plants by Q-PCR. The expression level of each gene in the wild type was standardized as 100 with reference to the internal control of Actin. The assay was repeated three times. Error bars represent se. WT, Wild type.

Figure 5.

LeERF2/TERF2 identifies GCC box of the NtACS3 promoter in in vivo transient expression assays. A, Interaction of LeERF2/TERF2 or TERF1 with the promoter of NtACS3 in vivo. P1, P2, P3, and dGCC indicate that the GUS reporter gene was driven by 1,239-, 508-, and 248-bp sequences and the removal of GCC box of the NtACS3 promoter, respectively. B, GCC box of the NtACS3 promoter is essential for interaction with LeERF2/TERF2. 4×GCC box and 4×mGCC box indicate that four repeated GCC box or mutated GCC box sequences of the NtACS3 promoter, respectively, were inserted upstream of the minimal promoter of 35S in pBI121. Mini is the reporter containing the basic promoter TATA box upstream of the GUS reporter gene. TERF2 indicates the constitutive expression of full-length LeERF2/TERF2. None, as the negative control of the effector, indicates the empty vector plasmid. Agrobacterium containing the reporter vector and effector vector was simultaneously infiltrated into tobacco leaves. Relative GUS activity is the ratio of GUS activity in 35S:TERF2 to that in the empty vector. The assay was repeated three times. Error bars indicate se.

Figure 6.

LeERF2/TERF2 specifically interacts with GCC box of the NtACS3 promoter in vitro and in vivo. A, LeERF2/TERF2 binds to GCC box of the NtACS3 promoter and DRE/CRT of the LeACO3 promoter in electrophoretic mobility shift assays. GCC and mGCC indicate the native and mutant GCC box of the NtACS3 promoter, and DRE/CRT and mDRE/CRT indicate the native and mutant DRE/CRT of the LeACO3 promoter, respectively. B, LeERF2/TERF2 interacts with GCC box of the NtACS3 promoter in chromatin immunoprecipitation (ChIP) assays. Tobacco plants transiently overexpressing 35S:GFP-His or 35S:TERF2-His were subjected to ChIP with anti-His antibodies. PCR was performed using primers corresponding to sequences located at −508 and −223 bp in the NtACS3 promoter. As controls for DNA input into the ChIP assays, aliquots of sonicated chromatin were analyzed by PCR using the primers of Actin. Tobacco plants expressing 35S:GFP-His were used as the negative control.

Figure 7.

Site mutation of LeERF2/TERF2 ERF-binding domain alters the specific binding to the promoter of NtACS3. A, Identification of essential sites of LeERF2/TERF2 for interaction with the promoter of NtACS3 using the yeast one-hybrid assay. The reporter was a pLacZ vector containing the lacZ reporter gene driven by four repetitions of GCC box of the NtACS3 promoter. Effectors TERF2, dERF, W84A, V85A, and W86A indicate the plasmids pB42AD containing native and LeERF2/TERF2 mutant in which the ERF domain had been deleted and mutations of W84A, V85A, and W86A, respectively. B, Transient assays in tobacco leaves to confirm the interaction of essential amino acids of LeERF2/TERF2 with the promoter of NtACS3. The reporter was the pCAMBIA 1303 vector, in which the full-length promoter of NtACS3 substituted for the 35S to drive the expression of the GUS reporter gene. Effectors TERF2, dERF, W84A, V85A, and W86A indicate the native and LeERF2/TERF2 ERF mutants in which the ERF domain had been deleted and mutations of W84A, V85A, and W86A, respectively. CaMV, Cauliflower mosaic virus; NOS, nopaline synthase.