Constitutive expression of <i>PeHAB1</i>confers ABA insensitivity in transgenic plants during seed germination and root growth development.

<p>(A) The emergence rate of green cotyledons from Col-0 and <i>PeHAB1</i> transgenic seeds plated on MS medium supplemented with different concentration of ABA. Approximately 100 seeds were used in each experiment. Error bars represent the standard deviation. (B-D) Col-0 and two independent <i>35S</i>::<i>PeHAB1</i> lines were germinated and grown in medium (MS) supplemented with 0.75 μM ABA. Photographs were taken after 14 days. (E-F) Root length in MS medium supplemented with 10 μM ABA and its correspondingly statistical analysis. Data means ±SE from three independent experiments (<i>n</i> = 30 seeds).</p

Phenotype tests for water loss and stomata opening under ABA treatment.

<p>(A) Water loss analysis. Time courses for the percentage of initial fresh weight were recorded from the detached leaves of wild-type controls, L2, and L6. Data are averages ±SE from three independent experiments (<i>n</i> = 10 plants). Asterisks indicate significant difference between transgenic lines and wild-type at different time points (* <i>P</i><0.05; Student’s <i>t</i>-test). (B) Stomatal photographs of <i>35S</i>::<i>PeHAB1</i> transgenic lines and wild-type plants in the presence or absence of ABA. (C) Average stomatal aperture of <i>PeHAB1</i> overexpression transgenic lines and wild type plants in response to ABA. Data are averages ±SE from three independent experiments (<i>n</i> > 20 stomata). Asterisks indicate significant difference between transgenic lines and wild-type with or without ABA (** <i>P</i><0.01; Student’s <i>t</i>-test).</p

Real-time PCR analysis of several Clade A PtPP2Cs and <i>PeHAB1</i> under ABA, NaCl, drought, and cold treatment.

<p>(A) Relative gene expression analysis of four Clade A PtPP2Cs under various abiotic stress treatments. (B) Quantitative RT-PCR was performed with <i>PeActin</i> as an internal reference and <i>PeDREB2</i> as an experimental control. The expression level in untreated leaves was assigned a value of 1. The capital characters represent 200 μM ABA (ABA), 300 mM NaCl (S), dehydration (D), and 4°C cold, respectively. Data are presented as means ± SE from three independent biological replicates.</p

A Putative PP2C-Encoding Gene Negatively Regulates ABA Signaling in <i>Populus euphratica</i>

<div><p>A PP2C homolog gene was cloned from the drought-treated cDNA library of <i>Populus euphratica</i>. Multiple sequence alignment analysis suggested that the gene is a potential ortholog of HAB1. The expression of this <i>HAB1</i> ortholog (<i>PeHAB1</i>) was markedly induced by drought and moderately induced by ABA. To characterize its function in ABA signaling, we generated transgenic <i>Arabidopsis thaliana</i> plants overexpressing this gene. Transgenic lines exhibited reduced responses to exogenous ABA and reduced tolerance to drought compared to wide-type lines. Yeast two-hybrid analyses indicated that PeHAB1 could interact with the ABA receptor PYL4 in an ABA-independent manner. Taken together; these results indicated that PeHAB1 is a new negative regulator of ABA responses in poplar.</p></div

Yeast two-hybrid assay for PeHAB1- interacting proteins.

<p>(A) Yeast two-hybrid interaction test of PeHAB1 with PePYL4. Interactions were detected by growth of yeast on selective medium and the detection of a GFP signal. (a-d) indicated negative control; yeast harbored PeHAB1 and PePYL4; Supplied interacting pairs that served as positive control; yeast harbored PePYL4 and PeHAB1 of substitution mutation W420A and yeast harbored PeHAB1 and PePYL4 of substitution mutation P124S. (B) <i>β</i>-galactosidase activity (blue) in streaks of yeast cells on a culture plate as readouts of protein-protein interactions by using the yeast two-hybrid system.</p

Sequence analysis of PeHAB1.

<p>(A) Sequence alignment of the deduced amino acid sequence of PeHAB1 with four Clade A <i>Arabidopsis</i> PP2C members including AtABI1, AtABI2, AtHAB1, AtHAB2, and one <i>Populus trichocarpa</i> gene Potri.018G060300. Identical amino acid residues are highlighted in black, while conserved amino acid residues are indicated by colored shading. Asterisk codes indicated the amino acid residues involved in interaction with PYLs. Trp420 was marked with a small triangle. (B) Phylogenetic tree of PeHAB1, 27 representative PP2C members from <i>Arabidopsis</i>, and 45 PP2C candidates from <i>P</i>. <i>trichocarpa</i>. The ABA-related Clade A PP2Cs were indicated by the grey shade. The tree was constructed by Neighbor–Joining method based on multiple alignments of full-length amino acid sequences with bootstrap values of 1000 replicates.</p

Image_5_Populus trichocarpa PtNF-YA9, A Multifunctional Transcription Factor, Regulates Seed Germination, Abiotic Stress, Plant Growth and Development in Arabidopsis.JPEG

<p>NF-YAs play important roles in abiotic stress. However, their characteristics and functions in abiotic stress of poplar, a model woody plant, have not been fully investigated. Here, the biological functions of PtNF-YA9 (Potri.011G101000), an NF-YA gene from Populus trichocarpa, were first fully investigated. PtNF-YA9 is located in the nucleus. The expression of PtNF-YA9 was reduced by mannitol, NaCl, and abscisic acid (ABA). The GUS staining of ProNF-YA9::GUS transgenic lines was also reduced by mannitol treatments. In the PtNF-YA9-overexpressed Arabidopsis (OxPtNA9), OxPtNA9 lines exhibited sensitivity to simulated drought, ABA, and salinity stress during germination stage, and growth arrest emerged at post-germination stage. These phenomena might involve the ABA signaling pathway via the regulation of ABI3, ABI4, and ABI5. At vegetative stages, OxPtNA9 lines decreased in water loss via promoting stomatal closure and displayed high instantaneous water-use efficiency (WUE) of the leaf to exhibit enhanced drought tolerance. Furthermore, OxPtNA9 lines exhibited long primary root in the half-strength Murashige–Skoog agar medium supplemented with NaCl and conferred strong tolerance in the soil under salt stress. Additionally, PtNF-YA9 exhibited dwarf phenotype, short hypocotyl, small leaf area and biomass, delayed flowering, and increased chlorophyll content. Above all, our research proposes a model in which PtNF-YA9 not only plays a key role in reducing plant growth but also can play a primary role in the mechanism of an acclimatization strategy in response to adverse environmental conditions.</p

Image_3_Populus trichocarpa PtNF-YA9, A Multifunctional Transcription Factor, Regulates Seed Germination, Abiotic Stress, Plant Growth and Development in Arabidopsis.JPEG

<p>NF-YAs play important roles in abiotic stress. However, their characteristics and functions in abiotic stress of poplar, a model woody plant, have not been fully investigated. Here, the biological functions of PtNF-YA9 (Potri.011G101000), an NF-YA gene from Populus trichocarpa, were first fully investigated. PtNF-YA9 is located in the nucleus. The expression of PtNF-YA9 was reduced by mannitol, NaCl, and abscisic acid (ABA). The GUS staining of ProNF-YA9::GUS transgenic lines was also reduced by mannitol treatments. In the PtNF-YA9-overexpressed Arabidopsis (OxPtNA9), OxPtNA9 lines exhibited sensitivity to simulated drought, ABA, and salinity stress during germination stage, and growth arrest emerged at post-germination stage. These phenomena might involve the ABA signaling pathway via the regulation of ABI3, ABI4, and ABI5. At vegetative stages, OxPtNA9 lines decreased in water loss via promoting stomatal closure and displayed high instantaneous water-use efficiency (WUE) of the leaf to exhibit enhanced drought tolerance. Furthermore, OxPtNA9 lines exhibited long primary root in the half-strength Murashige–Skoog agar medium supplemented with NaCl and conferred strong tolerance in the soil under salt stress. Additionally, PtNF-YA9 exhibited dwarf phenotype, short hypocotyl, small leaf area and biomass, delayed flowering, and increased chlorophyll content. Above all, our research proposes a model in which PtNF-YA9 not only plays a key role in reducing plant growth but also can play a primary role in the mechanism of an acclimatization strategy in response to adverse environmental conditions.</p

Image_6_Populus trichocarpa PtNF-YA9, A Multifunctional Transcription Factor, Regulates Seed Germination, Abiotic Stress, Plant Growth and Development in Arabidopsis.JPEG

<p>NF-YAs play important roles in abiotic stress. However, their characteristics and functions in abiotic stress of poplar, a model woody plant, have not been fully investigated. Here, the biological functions of PtNF-YA9 (Potri.011G101000), an NF-YA gene from Populus trichocarpa, were first fully investigated. PtNF-YA9 is located in the nucleus. The expression of PtNF-YA9 was reduced by mannitol, NaCl, and abscisic acid (ABA). The GUS staining of ProNF-YA9::GUS transgenic lines was also reduced by mannitol treatments. In the PtNF-YA9-overexpressed Arabidopsis (OxPtNA9), OxPtNA9 lines exhibited sensitivity to simulated drought, ABA, and salinity stress during germination stage, and growth arrest emerged at post-germination stage. These phenomena might involve the ABA signaling pathway via the regulation of ABI3, ABI4, and ABI5. At vegetative stages, OxPtNA9 lines decreased in water loss via promoting stomatal closure and displayed high instantaneous water-use efficiency (WUE) of the leaf to exhibit enhanced drought tolerance. Furthermore, OxPtNA9 lines exhibited long primary root in the half-strength Murashige–Skoog agar medium supplemented with NaCl and conferred strong tolerance in the soil under salt stress. Additionally, PtNF-YA9 exhibited dwarf phenotype, short hypocotyl, small leaf area and biomass, delayed flowering, and increased chlorophyll content. Above all, our research proposes a model in which PtNF-YA9 not only plays a key role in reducing plant growth but also can play a primary role in the mechanism of an acclimatization strategy in response to adverse environmental conditions.</p

Image_1_Populus trichocarpa PtNF-YA9, A Multifunctional Transcription Factor, Regulates Seed Germination, Abiotic Stress, Plant Growth and Development in Arabidopsis.TIF

<p>NF-YAs play important roles in abiotic stress. However, their characteristics and functions in abiotic stress of poplar, a model woody plant, have not been fully investigated. Here, the biological functions of PtNF-YA9 (Potri.011G101000), an NF-YA gene from Populus trichocarpa, were first fully investigated. PtNF-YA9 is located in the nucleus. The expression of PtNF-YA9 was reduced by mannitol, NaCl, and abscisic acid (ABA). The GUS staining of ProNF-YA9::GUS transgenic lines was also reduced by mannitol treatments. In the PtNF-YA9-overexpressed Arabidopsis (OxPtNA9), OxPtNA9 lines exhibited sensitivity to simulated drought, ABA, and salinity stress during germination stage, and growth arrest emerged at post-germination stage. These phenomena might involve the ABA signaling pathway via the regulation of ABI3, ABI4, and ABI5. At vegetative stages, OxPtNA9 lines decreased in water loss via promoting stomatal closure and displayed high instantaneous water-use efficiency (WUE) of the leaf to exhibit enhanced drought tolerance. Furthermore, OxPtNA9 lines exhibited long primary root in the half-strength Murashige–Skoog agar medium supplemented with NaCl and conferred strong tolerance in the soil under salt stress. Additionally, PtNF-YA9 exhibited dwarf phenotype, short hypocotyl, small leaf area and biomass, delayed flowering, and increased chlorophyll content. Above all, our research proposes a model in which PtNF-YA9 not only plays a key role in reducing plant growth but also can play a primary role in the mechanism of an acclimatization strategy in response to adverse environmental conditions.</p