Table2_Identification of Dof transcription factors in Dendrobium huoshanense and expression pattern under abiotic stresses.XLSX

Introduction: DNA-binding with one finger (Dof) transcription factors (TFs) are a unique family of TFs found in higher plants that regulate plant responses to light, hormones, and abiotic stresses. The specific involvement of Dof genes in the response to environmental stresses remains unknown in D. huoshanense.Methods: A total of 22 Dof family genes were identified from the D. huoshanense genome.Results: Chromosome location analysis showed that DhDof genes were distributed on 12 chromosomes, with the largest number of Dof genes located on chromosome 8. The phylogenetic tree revealed that DhDofs could be categorized into 11 distinct subgroups. In addition to the common groups, DhDof4, DhDof5, DhDof17, and the AtDof1.4 ortholog were clustered into the B3 subgroup. Group E was a newly identified branch, among which DhDof6, DhDof7, DhDof8, and DhDof9 were in an independent branch. The conserved motifs and gene structure revealed the differences in motif number and composition of DhDofs. The dof domain near the N-terminus was highly conserved and contained a C2-C2-type zinc finger structure linked with four cysteines. Microsynteny and interspecies collinearity revealed gene duplication events and phylogenetic tree among DhDofs. Large-scale gene duplication had not occurred among the DhDofs genes and only in one pair of genes on chromosome 13. Synteny blocks were found more often between D. huoshanense and its relatives and less often between Oryza sativa and Arabidopsis thaliana. Selection pressure analysis indicated that DhDof genes were subject to purifying selection. Expression profiles and correlation analyses revealed that the Dof gene under hormone treatments showed several different expression patterns. DhDof20 and DhDof21 had the highest expression levels and were co-expressed under MeJA induction. The cis-acting element analysis revealed that each DhDof had several regulatory elements involved in plant growth as well as abiotic stresses. qRT-PCR analysis demonstrated that DhDof2 was the main ABA-responsive gene and DhDof7 was the main cold stress-related gene. IAA suppressed the expression of some Dof candidates, and SA inhibited most of the candidate genes.Discussion: Our results may provide new insights for the further investigation of the Dof genes and the screening of the core stress-resistance genes.</p

Table1_Identification of Dof transcription factors in Dendrobium huoshanense and expression pattern under abiotic stresses.XLSX

Introduction: DNA-binding with one finger (Dof) transcription factors (TFs) are a unique family of TFs found in higher plants that regulate plant responses to light, hormones, and abiotic stresses. The specific involvement of Dof genes in the response to environmental stresses remains unknown in D. huoshanense.Methods: A total of 22 Dof family genes were identified from the D. huoshanense genome.Results: Chromosome location analysis showed that DhDof genes were distributed on 12 chromosomes, with the largest number of Dof genes located on chromosome 8. The phylogenetic tree revealed that DhDofs could be categorized into 11 distinct subgroups. In addition to the common groups, DhDof4, DhDof5, DhDof17, and the AtDof1.4 ortholog were clustered into the B3 subgroup. Group E was a newly identified branch, among which DhDof6, DhDof7, DhDof8, and DhDof9 were in an independent branch. The conserved motifs and gene structure revealed the differences in motif number and composition of DhDofs. The dof domain near the N-terminus was highly conserved and contained a C2-C2-type zinc finger structure linked with four cysteines. Microsynteny and interspecies collinearity revealed gene duplication events and phylogenetic tree among DhDofs. Large-scale gene duplication had not occurred among the DhDofs genes and only in one pair of genes on chromosome 13. Synteny blocks were found more often between D. huoshanense and its relatives and less often between Oryza sativa and Arabidopsis thaliana. Selection pressure analysis indicated that DhDof genes were subject to purifying selection. Expression profiles and correlation analyses revealed that the Dof gene under hormone treatments showed several different expression patterns. DhDof20 and DhDof21 had the highest expression levels and were co-expressed under MeJA induction. The cis-acting element analysis revealed that each DhDof had several regulatory elements involved in plant growth as well as abiotic stresses. qRT-PCR analysis demonstrated that DhDof2 was the main ABA-responsive gene and DhDof7 was the main cold stress-related gene. IAA suppressed the expression of some Dof candidates, and SA inhibited most of the candidate genes.Discussion: Our results may provide new insights for the further investigation of the Dof genes and the screening of the core stress-resistance genes.</p

<i>In vivo</i> airway reactivity of Pak1<i>^−/−</i> mice was lower than that of WT mice to aerosolized (A) and intra-venous (B) acetylcholine.

<p>Resistance in response to increasing concentrations of aerosolized acetylcholine (ACh) for wild-type (WT; N = 8) and Pak1<i>^−/−</i> mice (N = 8) (A); values are means ± SEM. There was no difference in resistance at baseline. When analyzed by repeated measures ANOVA, resistance increased with increasing ACh dose (p<0.0001), Pak1<i>^−/−</i> mice had a significantly smaller slope of the dose response curve (p<0.03), and a significantly smaller increase in resistance compared to WT mice (p<0.03). Post-hoc analysis demonstrated Pak 1<i>^−/−</i> compared to WT mice had significantly smaller resistances at all ACh concentrations ≥7.5 mg/ml (p<0.05). Resistance in response to increasing concentrations of intravenous acetylcholine (ACh) for wild-type (WT; N = 4) and Pak1<i>^−/−</i> mice (N = 4) (B); values are means ± SEM. There was no difference in resistance at baseline. When analyzed by repeated measures ANOVA, resistance for Pak1<i>^−/−</i> mice increased less with increasing ACh dose (p<0.0001) compared to WT mice. Post-hoc analysis demonstrated Pak1<i>^−/−</i> compared to WT mice had significantly lower resistances at ACh concentrations ≥0.42 mg (p<0.05).</p

Aerosolized IPA3 inhibited airway contractility <i>in vivo</i> and suppressed Pak activation.

<p>When assessed by repeated ANOVA, resistance increased with increasing ACh dose (p<0.0001), and IPA3 dissolved in 1% DMSO (N = 3) and aerosolized 1-hour prior to bronchial challenge of WT mice significantly reduced the slope of the increase in resistance (p<0.0001), as well as the magnitude of the increase in resistance compared to control vehicle (1%DMSO; N = 5) (p<0.001) (A). Post-hoc analysis indicated that IPA3 treatment resulted in lower resistances at MCh doses ≥33 mg/ml (p<0.05). Tracheal smooth muscle isolated from WT mice treated <i>in vivo</i> with IPA3 demonstrated significantly lower Pak activation as assessed by Pak T423 phosphorylation following stimulation with ACh compared to airway smooth muscle isolated from WT mice (B). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042601#s2" target="_blank">Results</a> represent 2 samples of tracheal smooth muscle from each group. Each sample consisted of pooled tracheal muscle tissues from 3 separate mice with the same treatment.</p

Comparison of the Lung Parenchyma (N = 3–5 in each group) for WT (black) and Pak1^−/− (grey) mice.

<p>There were no significant differences for lung volumes at 30 cmH₂O (p>0.37) (A); pressure volume curves normalized to volume at 30 cmH₂O (B); or Alveolar Mean Linear Intercepts (MLI) (p>0.40) (C).</p

Pak1, Pak2 and Pak3 isoforms were detected in WT murine tracheal smooth muscle by immunoblot.

<p>No Pak1 was detected in extracts of isolated tracheal smooth muscle (A) or whole tracheas (B) from <i>Pak1</i>^−/− mice. Immunoblots of tracheal smooth muscle were obtained from tracheal smooth muscle extracts pooled from 3 mice of each type. Whole trachea extracts were each from a single mouse.</p

Tracheas isolated from <i>Pak1</i>^−/− mice showed reduced contractility to ACh in vitro.

<p>Isometric force generation (% of maximal force to ACh stimulation in WT mice) was significantly lower in tracheas isolated from <i>Pak1</i>^−/− (grey squares) mice compared to WT (black diamonds) mice (N = 10 or 11 in each group, p<0.01).</p

Comparison of Lung Histology for WT and <i>Pak1</i>^−/− mice.

<p>Airway wall area (A), airway smooth muscle area (B), and airway epithelium area (C) were not significantly different between the WT (black squares) and <i>Pak1</i>^−/− (grey circles) mice when assessed by ANOVA adjusting for airway perimeter (p>0.15). (N = 5 mice in each group).</p