Phylogenetic tree including the AhERF-VII of <i>A</i>. <i>hypochondriacus</i> together with all the <i>Arabidopsis thaliana</i> ERF proteins.

<p>Also shown are the highly homologous GbERF1 and GbERF2 proteins from <i>Gossipum barbadense</i>, and the GhERF protein from <i>G</i>. <i>hirsutum</i>. 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.</p

Overexpression of Grain Amaranth (<i>Amaranthus hypochondriacus</i>) AhERF or AhDOF Transcription Factors in <i>Arabidopsis thaliana</i> Increases Water Deficit- and Salt-Stress Tolerance, Respectively, via Contrasting Stress-Amelioration Mechanisms

<div><p>Two grain amaranth transcription factor (TF) genes were overexpressed in Arabidopsis plants. The first, coding for a group VII ethylene response factor TF (i.e., AhERF-VII) conferred tolerance to water-deficit stress (WS) in transgenic Arabidopsis without affecting vegetative or reproductive growth. A significantly lower water-loss rate in detached leaves coupled to a reduced stomatal opening in leaves of plants subjected to WS was associated with this trait. WS tolerance was also associated with an increased antioxidant enzyme activity and the accumulation of putative stress-related secondary metabolites. However, microarray and GO data did not indicate an obvious correlation between WS tolerance, stomatal closure, and abscisic acid (ABA)-related signaling. This scenario suggested that stomatal closure during WS in these plants involved ABA-independent mechanisms, possibly involving reactive oxygen species (ROS). WS tolerance may have also involved other protective processes, such as those employed for methyl glyoxal detoxification. The second, coding for a class A and cluster I DNA binding with one finger TF (i.e., AhDof-AI) provided salt-stress (SS) tolerance with no evident fitness penalties. The lack of an obvious development-related phenotype contrasted with microarray and GO data showing an enrichment of categories and genes related to developmental processes, particularly flowering. SS tolerance also correlated with increased superoxide dismutase activity but not with augmented stomatal closure. Additionally, microarray and GO data indicated that, contrary to AhERF-VII, SS tolerance conferred by AhDof-AI in Arabidopsis involved ABA-dependent and ABA-independent stress amelioration mechanisms.</p></div

The overexpression of the <i>AhDOF-AI</i> gene confers acute salt stress tolerance in transgenic <i>Arabidopsis</i> plants.

<p>Panel (A) shows the aspect of WT control plants and of three lines of <i>AhDof-AI</i> 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 <i>P</i> ≤ 0.05 (Tukey Kramer test). The results shown are those obtained from a representative experiment that was repeated thrice with similar results.</p

Reactive oxygen species scavenging enzyme activity in transgenic OE-<i>AhERF-VII</i> and OE-<i>AhDof-AI</i> plants.

<p>Activity levels of three reactive oxygen species scavenging enzymes (superoxide dismutase [SOD], catalase [CAT], and glutathione reductase [GR]) and were quantified <i>in vitro</i> in leaf extracts of transgenic OE-<i>AhERF-VII</i> (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-<i>AhDof-AI</i> (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 <i>P</i> ≤ 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.</p

Localization by fluorescence microscopy of the GFP-ERF and GFP-DOF fusion proteins in root cells.

<p>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.</p

Phylogenetic tree of the AhDOF-AI protein of <i>A</i>. <i>hypochondriacus</i> together with all Dof domain-containing proteins from soybean (<i>Glycine max</i>, GmDof), <i>Arabidopsis thaliana</i> (AtDof), and rice (<i>Oryza sativa</i>, OsDof).

<p>Also shown is a highly homologous Dof protein from <i>Beta vulgaris</i> (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.</p

Highly expressed genes detected in both OE-<i>AhERF-VII</i> and OE-<i>AhDOF-AI</i> Arabidopsis transgenic plants under optimal, stress and recovery conditions.

<p>Highly expressed genes detected in both OE-<i>AhERF-VII</i> and OE-<i>AhDOF-AI</i> Arabidopsis transgenic plants under optimal, stress and recovery conditions.</p

Complete cDNA sequences of the AhERF-VII and AhDOF-AI transcription factor genes and the predicted proteins coded by their open reading frames (ORF).

<p>(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.</p

The overexpression of <i>AhDof-AI</i> in Arabidopsis modifies its metabolic pattern in optimal growing conditions and under acute salt stress conditions.

<p>Metabolic heat map obtained from acidified methanol extracts obtained from leaves collected from wild type (WT) and OE-<i>AhDof-AI Arabidopsis</i> 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.</p

Proline content in transgenic OE-<i>AhERF-VII</i> and OE-<i>AhDof-AI</i> plants.

<p>Proline accumulation levels were quantified <i>in vitro</i> in leaf extracts of transgenic <i>OE-AhERF-VII</i> (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 <i>OE-AhDof-AI</i> (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 <i>P</i> ≤ 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.</p