Fig 1.
Complete cDNA sequences of the AhERF-VII and AhDOF-AI transcription factor genes and the predicted proteins coded by their open reading frames (ORF).
(A) The AhERF-VII cDNA has an ORF of 1,022 bp and encodes a protein of 254 amino acids. (B) The AhDOF-AI cDNA has an ORF of 1,734 bp and encodes a protein of 337 amino acids. The sequences corresponding to the putative AP2 (in A) and Dof zinc finger (in B) DNA-binding domains are underlined in purple. Also shown, in blue and green, are sequence regions selected to design the primers used to isolate their cDNA sequences and to quantify their expression levels by RT-qPCR, respectively.
Fig 2.
The amino acid alignment of AhERF-VII and AhDOF-AI proteins with other TFs family members from selected plant species.
Amino acid residues that are conserved in at least three of the seven sequences are shaded, whereas identical amino acids are shown in black. In (A), the cyan line drawn above the sequences represents the highly conserved N-terminal MCGGAII/L motif of unknown function. A green line was drawn to represent putative nuclear localization signal (NLS), and the red line indicates the conserved DNA-binding domain (or AP2/ ERF domain). In (B) the cyan and red lines show the four regions associated with the zinc finger structure. The green line represents the cluster I conserved motif. Bv = Beta vulgaris; Cc = Cajanus cajan; Gb = Gossypium barbadense; Gh = Gossypium hirsutum; JERF = Jasmonate and Ethylene Responsive Factor; Le = Lycopersicum esculentum; Ns = Nicotiana sylvestris, and Sl = Solanum lycopersicum.
Fig 3.
Phylogenetic tree including the AhERF-VII of A. hypochondriacus together with all the Arabidopsis thaliana ERF proteins.
Also shown are the highly homologous GbERF1 and GbERF2 proteins from Gossipum barbadense, and the GhERF protein from G. hirsutum. The phylogenetic tree was constructed using the neighbor joining method with amino acid sequence data. It was drawn using the TreeView program, based on alignments obtained using MUSCLE software. The bootstrap values shown are in percent.
Fig 4.
Phylogenetic tree of the AhDOF-AI protein of A. hypochondriacus together with all Dof domain-containing proteins from soybean (Glycine max, GmDof), Arabidopsis thaliana (AtDof), and rice (Oryza sativa, OsDof).
Also shown is a highly homologous Dof protein from Beta vulgaris (BvDof). The phylogenetic tree was constructed using the neighbor joining method with amino acid sequence data. It was drawn using the TreeView program, based on alignments obtained using MUSCLE software. The 1050 bootstrap values shown are in percent.
Fig 5.
Tissue-specific- and stress-induced expression patterns of the AhERF-VII and AhDOF-AI transcription factor genes in A. hypochondricus.
Panels (A) and (B), and (C) and (D) show the changes in AhERF-VII gene and AhDOF-AI expression levels, respectively, in leaves and roots of plants exposed for different time periods (2-to-8 days) to water-deficit (WS) or salinity stress (SS) conditions. The expression levels are relative to those detected in control plants maintained in optimal conditions (Op), whose expression was set to 1.0. Panels (E and F) show the tissue-specific expression levels of the AhERF-VII and AhDof-AI genes, respectively, measured in relation to those detected in young stems (YS), whose expression levels were set at 1.0. The tissues examined were axilar meristem (AxM), intermediate and young leaves (IL and YL, respectively), panicles (P) and roots (R). Bars and error bars indicate mean values and ES, respectively (n = 6). Different letters over the bars represent statistically significant differences at P ≤ 0.05 (Tukey Kramer test). The discontinuous horizontal lines indicate the 1.5-fold threshold value above which gene expression was considered to be induced.
Fig 6.
Relative expression levels of the AhERF-VII and AhDOF-AI transcription factor genes in A. hypochondriacus produced shortly after the application of exogenous phytohormones or phytohormone-like compounds.
The expression of these genes relative to control was measured 1, 3, 6, 12 and 24 h after the application of methyl jasmonate (MeJA, panel A), benzothiadiazole (BTH, panels B and D), and abscisic acid (ABA, panels C and E). The response was measured in treated leaves (local response, LR) and in untreated, distal leaves (systemic response, SR). Bars and error bars indicate mean values and ES, respectively (n = 6). Different letters over the bars represent statistically significant differences at P ≤ 0.05 (Tukey Kramer test). The discontinuous horizontal lines indicate the 1.5-fold threshold value above which gene expression was considered to be induced.
Fig 7.
Localization by fluorescence microscopy of the GFP-ERF and GFP-DOF fusion proteins in root cells.
A nuclear localization was observed in root cells near the root tip of transgenic Arabidopsis plants constitutively expressing the GFP-AhERF-VII (panels E and F) or the GFP-AhDOF-AI (panels G and H) fusion proteins. The results were compared with those obtained from control transgenic Arabidopsis plants constitutively expressing the 35S:: GFP-GUS (panels A and B) or the H2B:: GFP fusion proteins (panels C and D). The latter overexpress a histone 2B fused with GFP (H2B::GFP), which is a commonly employed nuclear marker.
Fig 8.
The overexpression of the AhERF-VII gene confers water-deficit stress tolerance in transgenic Arabidopsis plants.
Panel (A) shows the aspect of WT control plants and of three lines of AhERF-VII overexpressing transgenic Arabidopsis plants (EL25, EL2, and EL15) in optimal conditions (Op), after 6 days of water-deficit stress (WS), and one day after watering was resumed to allow recovery (R). In panel (B), the survival rate of the plants shown in A is presented. Panel (C) shows the water loss rate measured in detached rosette leaves taken from transgenic and WT control plants. Panels (D) and (E) show the calculated “Aspect Ratio” parameter employed to quantify stomata aperture, and the regions of interest (ROI) cropped from A. thaliana leaves, respectively, in WT and transgenic (line EL25) plants in optimal (Op) or water-deficit stress (WS) conditions. Bars and error bars indicate mean values and ES, respectively (n = 25). Asterisks (in C) and different letters over the bars (in B and D) represent statistically significant differences at P ≤ 0.05 (Tukey Kramer test). The results shown are those obtained from a representative experiment that was repeated thrice with similar results.
Fig 9.
The overexpression of the AhDOF-AI gene confers acute salt stress tolerance in transgenic Arabidopsis plants.
Panel (A) shows the aspect of WT control plants and of three lines of AhDof-AI overexpressing transgenic Arabidopsis plants (DL2, DL31, and DL4) in optimal conditions (Op), at the end of acute salt stress (SS), and 2 weeks after watering was resumed to allow recovery (R). In panel (B), the survival rate of the plants shown in (A) is presented. Bars and error bars indicate mean values and ES, respectively (n = 25). Different letters over the bars represent statistically significant differences at P ≤ 0.05 (Tukey Kramer test). The results shown are those obtained from a representative experiment that was repeated thrice with similar results.
Fig 10.
Reactive oxygen species scavenging enzyme activity in transgenic OE-AhERF-VII and OE-AhDof-AI plants.
Activity levels of three reactive oxygen species scavenging enzymes (superoxide dismutase [SOD], catalase [CAT], and glutathione reductase [GR]) and were quantified in vitro in leaf extracts of transgenic OE-AhERF-VII (line EL25) Arabidopsis plants (panels A-C) growing in optimal conditions (Op), 6 days after water-deficit stress (WS) and 1 day after normal watering was restored (R). Antioxidant enzyme activity was also determined in leaves of transgenic OE-AhDof-AI (line DL2) Arabidopsis plants (panels D-F) maintained under optimal growing conditions (Op) or at the end of the acute salt stress (SS) treatment. In each case, gray and empty bars represent transgenic and WT plants, respectively. Different letters over the bars represent statistically significant differences at P ≤ 0.05 (Tukey Kramer test). Bars and error bars indicate mean values and ES, respectively (n = 20). The results shown are those obtained from a representative experiment that was repeated thrice with similar results.
Fig 11.
Proline content in transgenic OE-AhERF-VII and OE-AhDof-AI plants.
Proline accumulation levels were quantified in vitro in leaf extracts of transgenic OE-AhERF-VII (line EL25) Arabidopsis plants (panel A) growing in optimal conditions (Op), 6 days after water-deficit stress (WS) and 1 day after normal watering was restored (R). Proline levels were also determined in leaves of transgenic OE-AhDof-AI (line DL2) Arabidopsis plants (panel B) maintained under optimal growing conditions (Op) or at the end of the acute salt stress (SS) treatment. In each case, gray and empty bars represent transgenic and WT plants, respectively. Different letters over the bars represent statistically significant differences at P ≤ 0.05 (Tukey Kramer test). Bars and error bars indicate mean values and ES, respectively (n = 20). The results shown are those obtained from a representative experiment that was repeated thrice with similar results.
Table 1.
Highly expressed genes detected in both OE-AhERF-VII and OE-AhDOF-AI Arabidopsis transgenic plants under optimal, stress and recovery conditions.
Fig 12.
The overexpression of AhERF-VII in Arabidopsis modifies its metabolic pattern in optimal growing conditions and in both water-deficit stress and recovery conditions.
Metabolic heat map obtained from acidified methanol extracts obtained from leaves collected from wild type (WT) and OE-AhERF-VII Arabidopsis plants (line EL25) grown in optimal conditions (Op), or subjected to water stress for 6 d (WS), or allowed to recover from stress, 1 d after normal watering was reestablished (R). The 50 most abundant ionizable metabolites were selected to obtain the metabolic heat-map within a 80–1300 m/z range. The results shown are those obtained from a representative experiment that was repeated thrice with similar results.
Fig 13.
The overexpression of AhDof-AI in Arabidopsis modifies its metabolic pattern in optimal growing conditions and under acute salt stress conditions.
Metabolic heat map obtained from acidified methanol extracts obtained from leaves collected from wild type (WT) and OE-AhDof-AI Arabidopsis plants (line DL2) grown in optimal conditions (Op), or subjected to acute salt stress for 3 d (SS). The 50 most abundant ionizable metabolites were selected to obtain the metabolic heat-map within a 80–1300 m/z range. The results shown are those obtained from a representative experiment that was repeated thrice with similar results.
Fig 14.
Differential accumulation of water stress-related metabolites in WT and OE-AhERF-VII Arabidopsis plants in well-watered conditions and in both water-deficit stress and recovery conditions.
The box-plots show the levels (represented as different intensities of the respective m/z ion peaks) of putative (represented as an asterisk) Arabidopsis stress-related secondary metabolites that differentially accumulated in leaves of wild type (WT; empty bars) and transgenic OE-AhERF-VII (line EL25; light gray bars) plants. The plants were maintained in optimal conditions (Op), subjected to water-deficit stress for 6 d (WS), or in recovery from stress, 1 d after normal watering was reestablished (R). Each box plot graphically represents groups of numerical data (in this case 10 scans per biological replicate, i.e., n = 30), through their quartiles. The lines extending vertically from the boxes indicate variability outside the upper and lower quartiles, whereas outliers, or atypical values, are plotted as individual points. The upper and lower limits of the box represent 75% and 25% of the data, respectively, while the horizontal bar is the median, representing 50% of the data. Different letters over the bars represent statistically significant differences at P ≤ 0.05 (Tukey Kramer test).
Fig 15.
Differential accumulation of water stress-related metabolites in WT and OE-AhDof-AI Arabidopsis plants in well-watered conditions and in both water-deficit stress and recovery conditions.
The box-plots show the levels (represented as different intensities of the respective m/z ion peaks) of putative (represented as an asterisk) Arabidopsis stress-related secondary metabolites that differentially accumulated in leaves of wild type (WT; empty bars) and transgenic OE-AhDof-AI (line DL2; light gray bars) plants. The plants were maintained in optimal conditions (Op), or subjected to acute salt stress for 3 d (SS). Different letters over the bars represent statistically significant differences at P ≤ 0.05 (Tukey Kramer test).