Combinatorial signal integration by APETALA2/Ethylene Response Factor (ERF)-transcription factors and the involvement of AP2-2 in starvation response

Abstract

Transcription factors of the APETALA 2/Ethylene Response Factor (AP2/ERF)- family have been implicated in diverse processes during development, stress acclimation and retrograde signaling. Fifty-three leaf-expressed AP2/ERFs were screened for their transcriptional response to abscisic acid (ABA), 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), methylviologen (MV), sucrose and high or low light, respectively, and revealed high reactivity to these effectors. Six of them (AP2-2, ARF14, CEJ1, ERF8, ERF11, RAP2.5) were selected for combinatorial response analysis to ABA, DCMU and high light. Additive, synergistic and antagonistic effects demonstrated that these transcription factors are components of multiple signaling pathways. AP2-2 (At1g79700) was subjected to an in depth study. AP2-2 transcripts were high under conditions linked to limited carbohydrate availability and stress and down-regulated in extended light phase, high light or in the presence of sugar. ap2-2 knock out plants had unchanged metabolite profiles and transcript levels of co-expressed genes in extended darkness. However, ap2-2 revealed more efficient germination and faster early growth under high sugar, osmotic or salinity stress, but the difference was abolished in the absence of sugar or during subsequent growth. It is suggested that AP2-2 is involved in mediating starvation-related and hormonal signals.

Keywords: Arabidopsis thaliana; abscisic acid; apetala2/ethylene response factor; germination; photosynthesis; retrograde signaling; transcription factor.

Figure 1

Transcript levels of marker genes in response to effector treatments. Leaf discs floated in 0.1 mM CaCl₂ solution supplemented with 30 μM abscisic acid (ABA), 10 μM 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 1 μM MV and 30 mM sucrose, respectively, or without additions as control for 4 h. Marker transcripts responsive to sugar (β-amylase), ABA (RD29) or ROS (H₂O₂) (PKRP) were quantified and expressed as log fold change relative to control. Strong and significant >2.5 fold up regulation was seen for each selected marker. Data are means ± SE of three biological experiments (sqRT-PCR), p-value < 0.05, Student t test, normalized to the control with actin2.

Figure 2

Transcript profiling of selected APETALA 2/Ethylene Response Factor Transcription Factors (AP2/ERF-TFs). Transcript levels of 53 AP2-ERF-TFs were analyzed by sqRT-PCR for their response to different effector treatments (30 μM ABA, 10 μM DCMU, 1 μM MV, 30 mM sucrose, high light (HL) and low light (LL) for 4 h using leaf discs floating on 0.1 mM CaCl₂). The results served as a first screen to select interesting candidates for investigation of combinatorial regulation. Data were obtained from two independent biological experiments and are presented as log₂-fold change (green color means down-regulation, red color up-regulation, respectively) following normalization to actin2. Data of selected TFs represent data from n = 6 experiments for DCMU and ABA.

Figure 3

DCMU and ABA concentration dependencies of ΦPSII and expression levels of AP2-2. Chlorophyll a fluorescence analysis of leaf discs was used to quantify the quantum yield of photosystem II. In identically treated discs, AP2-2-transcript levels were quantified by sqRT-PCR. Data were taken from three experiments with six leaf discs in each experiment, ±SD.

Figure 4

Transcript regulation of six AP2/ERF-TFs in response to combinatorial effector application. Leaf discs were treated with 30 μM ABA (A), 10 μM DCMU (D) and high light (H), respectively, and each combination. (A) AP2-2; (B) RAP2.5; (C) ERF11; (D) CEJ1; (E) ERF8, and (F) ARF14. The qRT-PCR data were obtained from n = 3 experiments with technical replicates and are presented as means ± SD. Identical letters indicate groups of same significance level (p < 0.05, Student t test).

Figure 5

(A) Response of AP2-2 to single effector application. AP2-2 transcript level increased upon treatments which induce or indicate metabolic demand like extended darkness (5 h over normal), 30 μM ABA or 10 μM DCMU. In contrast, no reaction or even down regulation occurred under conditions encompassing metabolic and energy abundance, i.e., high light, continuous light, heat treatment or sucrose (30 mM). The qRT-PCR data are means of n = 3 ± SE. Identical letters indicate same significance groups (p < 0.1, Student t test); (B) Up-regulation of AP2-2-transcript in low light or darkness with and without sugar supplementation. AP2-2-transcript was quantified in 13 days old seedlings by qRT-PCR at low light (8 μmol quanta·m⁻²·s⁻¹) or at the end of the subsequent dark period. Seedlings were grown at 80 μmol quanta·m⁻²·s⁻¹ in 0.25% sucrose/MS medium for 11 days and transferred to either MS medium with 0% or 2% sucrose. Samples were taken 2 days after transfer to the new conditions. Data are means of n = 4 from two independent experiments, ±SE.

Figure 6

Regulation of transcripts identified as co-regulated with AP2-2 in wild type and ap2-2 knock out plants. Bioinformatic analysis of publicly available transcriptome data generated a gene list of co-regulated transcripts (Table S.2). Among the top 13 genes in the list were β-xylosidase 1 (BXL1), a protein kinase (AKINβ), dark induced raffinose synthase family protein (DIN10), a glutamine-dependent asparagine synthase (ASN1, also called DIN6), and a mitochondrial substrate carrier family protein (MSCP). Their transcript abundance was determined by qRT-PCR with n = 3, presented as log 2 ± SE. Differences were not seen in the response to extended darkness.

Figure 7

Glucose-6-phosphate (G6P) and ATP levels in WT and ap2-2 KO after extended dark phase in wild type and ap2-2 knock out plants. In parallel to transcript determinations (Figure 6) levels of ATP and G6P were quantified at 5, 10 and 24 h of extended darkness. Data are means of n = 3 experiments with 3 measurements each, ±SD.

Figure 8

Root elongation of ap2-2 knock out plants versus wild type under different treatments. (A) Root length of 2 days old ap2-2 knock out or wild type seedlings under various treatments. Seeds were germinated in 0.25% sucrose/MS medium as control. Media were supplemented with 200 mM mannitol to induce osmotic stress or 200 mM NaCl in 0.25% sucrose to establish salt stress. Germination without sugar supplementation was also tested. The data were obtained from 60 seedlings and are presented as means ± SE; (B) Root development in 2% sucrose-containing MS medium. The data were obtained from 40 seedlings and are presented as means ± SE; (C) Root development in 200 mM NaCl. The data were obtained from 60 seedlings and are presented as means ± SE; (D) Comparison of 2 days old seedlings of wild type and ap2-2 KO grown without sugars, 0.25% sucrose or 200 mM mannitol plus 0.25% sucrose.