Plant materials, growth conditions, and treatments
T. androssowii seedlings were grown in pots containing a mixture of turf peat and sand (2:1 v/v). Thoroughly watered 2-month-old seedlings were each exposed to the following treatments: 0.4 M NaCl, 20% (w/v) PEG6000, 0.3 M NaHCO3, 150 μM CdCl2, and 150 μM ABA for 0, 6, 12, 24, 48, and 72 h, respectively. Following these treatments, leaves, stems and roots of seedlings from each sample (sample size of 10 seedlings) were harvested at the indicated times after initiation of each treatment and pooled for real-time RT-PCR analyses.
Seedlings of Arabidopsis were grown into pots filled with perlite/soil mixture in a growth chamber under the controlled conditions (16 h light: 8 h dark; 70-75 % relative humidity; 22°C). Three week-old seedlings were each exposed to the following treatments: 20% (w/v) PEG6000, or 100 μM ABA for 0, 3, 6 and 12 h, respectively. After these treatments, the leaves and roots of seedlings were respectively harvested and pooled (sample size of 10 seedlings) for real-time RT-PCR analyses.
Cloning and expression analysis of TaeIF5A1
A TaeIF5A1 (AY587771) gene was cloned from T. androssowii cDNA library
. The sequence alignments of the eIF5A proteins from different species, including plants, yeast, mammalian and other eukaryotes was conducted using CLUSTALX1.81, and a phylogenetic tree was constructed using the Neighbor-Joining method provided by the computer program MEGA5. The promoter of TaeIF5A1 was PCR-amplified from genomic DNA of T. androssowii using the Genome Walking Kit (TaKaRa, China). To analyze the activity of promoter of TaeIF5A1, the 35S promoter in pCAMBIA1301 was replaced with the TaeIF5A1 promoter (1,486 bp in length) to drive the β-glucuronidase (GUS) gene (Figure
3A). The TaeIF5A1 promoter::GUS construct was transferred into Arabidopsis plants by floral dip method. The T3 seedlings were employed for spatial expression analysis of TaeIF5A1 using GUS staining.
Real-time RT-PCR was performed in Opticon 2 System (Bio-Rad, Hercules, CA) with α-tubulin
β-tubulin and β-actin genes as internal references. Primers used for RT-PCR are listed in Additional file
3: Table S1. The amplification was performed using the following cycling parameters: 94°C for 30 s followed by 45 cycles at 94°C for 12 s, 60°C for 30 s, 72°C for 40 s and 82°C for 1 s for plate reading. A melting curve was generated for each sample at the end of each run to assess the purity of the amplified products. Each reaction was conducted in triplicate to ensure reproducibility of results. Expression levels were calculated from the cycle threshold according to the delta delta Ct method
Subcellular localization of the TaeIF5A1 protein
The TaeIF5A1 coding region without the termination codon was ligated in frame to N-terminal of the green fluorescent protein (GFP) to generate the TaeIF5A1::GFP fusion gene. A CaMV 35S promoter was employed to drive TaeIF5A1::GFP, and the GFP gene under the control of the CaMV 35S promoter (35S::GFP) was used as a control. The constructs were introduced into the onion epidermis cells by particle bombardment (Bio-Rad). The transformed cells were analyzed using confocal laser scanning microscopy LSM410 (Zeiss, Jena, Germany).
Identification of the upstream regulator of TaeIF5A1
One W-box motif (“CTGACT”)
 was found to exist in the promoter of TaeIF5A1 (Additional file
1). To study which gene can recognize this W-box and regulate the expression of TaeIF5A1, the three tandem copies of promoter sequence fragment (“AGGCTGACT”) containing W-box motif sequence were cloned into a pHIS2 vector (construct R3, Figure
5A), and were screened with Tamarix cDNA library for a one-hybrid assay (Clontech, Palo Alto, CA, USA). To investigate the interactions between the W-box and positive clones, we mutated the W-box core motif “TGAC”
[31, 32] with “TGGC”, “TAAC” or “TTTT” (constructs R4, R5, R6, Figure
5A) , and the interactions between the mutant W-box sequences and the positive clones were performed using yeast one hybrid. To further confirm the upstream regulator of TaeIF5A1, a 461 bp fragment of TaeIF5A1 promoter (from −456 to −916) containing the W-box motif (construct R1, Figure
5A ), and a 165 bp fragment of TaeIF5A1 promoter (from −591 to −755) containing the W-box motif and a 165 bp fragment of TaeIF5A1 promoter (from −591 to −755) containing the mutated W-box core motif “TTTT” (construct R2, mR2, Figure
5A), were cloned into pHIS2, respectively. The interactions between putative upstream regulators and the promoter fragments containing W-box or mutated W-box were performed using a yeast one-hybrid assay. In the above experiments, the p53HIS2 plasmid (pHIS2 contains three copies of p53 DNA element) was used as a negative control vector. All primers are shown in Additional file
3: Table S2.
For further verification of these interactions, the three tandem copies of the W-box and the 165 bp promoter fragments containing W-box motif or mutated core motif “TTTT” were fused with 35S CaMV −46 minimal promoter and respectively cloned into pCAMBIA1301 to replaced with its 35S promoter for driving the GUS gene (constructs containing three tandem copies of the W-box named as pCAM-W-box, containing promoter fragment with W-box and mutated W-box named as pCAM-W165 and pCAM-mW165). The effector vectors were constructed by cloning the full ORF of TaRVA or TaWRKY into pROKII under the control of 35S promoter (named as pROKII-TaRVA and pROKII-TaWRKY) (Figure
5 Da). All primers are shown in Additional file
3: Table S3. Both the reporter and effector vectors were co-transformed into tobacco leaves using the particle bombardment. GUS staining assay was performed as described by Jefferson
, and GUS activity was determined according to the method of Jefferson
To investigate the expression patterns of the upstream regulators of TaeIF5A1, real-time PCR was performed to determine the expression of TaeIF5A1 and the upstream regulators in Tamarix under ABA and osmotic stress conditions. For investigation of the expression of the homologs of TaeIF5A1, TaRAV and TaWRKY in Arabidopsis in response to ABA and osmotic stimulus, BLASTX research on Tair (
http://www.arabidopsis.org/Blast/) was performed, we identified the homologs of TaeIF5A1, TaRAV and TaWRKY in Arabidopsis are AT1G13950, AT1G68840 and AT1G13960, respectively. An actin gene (AT3G18780) was used as internal reference to normalize the amount of total RNA present in each reaction. The primers used are listed in Additional file
3: Table S1, and the real-time PCR conditions were the same as above.
Expression of TaeIF5A1 in S. cerevisiae and stress-tolerance assays
The full ORF of TaeIF5A1 was cloned into pYES2 vector (Invitrogen), and was introduced into S. cerevisiae strain, INVSc1 (MATa, his3-1, leu2, trp1-289, ura3-52. His-, Leu-, Trp-, and Ura-). To determine the expression peak of TaeIF5A1 in yeast, yeast transformants harboring the TaeIF5A1 were cultivated in induction medium (SC-U medium containing 2% galactose) at 30°C for 0, 12, 24, 36, 48 and 60 h, and harvested for RNA gel blot analysis.
For stress tolerance assays, clones harboring TaeIF5A1 and empty pYES2 (control) were cultured into SC-U medium containing 2% glucose at 30°C with overnight shaking, adjusted to OD600 of 0.4 in induction medium, and incubated at 30°C for 36 h (RNA gel blot result showed that peak level of exogenous gene induced at this time). After incubation, cell densities were adjusted to equal and incubated in different concentrations of NaCl, KCl, LiCl or sorbitol, then they were incubated at 30°C with overnight shaking. The growth rates were evaluated by measuring the OD600 for liquid medium in each sample.
To analyze protein content, yeast transformants harboring Peroxiredoxin gene (TaPrx1, GenBank number: JQ082512) from Tamarix were used as control (it can remove the protein synthesis differences between the yeast transformants harboring TaeIF5A1 and empty pYES2; since transformants harboring empty pYES2 failed in producing an exogenous gene-eIF5A compared with that harboring TaeIF5A1). The yeast transformants harboring TaeIF5A1
TaPrx1 and empty pYES2 were cultured in induction medium at 30°C for 0, 12, 24, 36 and 48 h, adjusted to equal quantity, and harvested for soluble protein content analysis. The experiment was repeated at least three times. The protein extraction followed the procedure described by Kushnirov
 and protein content analyses were performed following the Bradford method
Construction of plant expression vector and poplar transformation
The TaeIF5A1 was cloned into pROKII (Additional file
2A), in which TaeIF5A1 under the control of CaMV 35S promoter, and transferred into poplar plants (Populus davidiana Dode × P. bollena Lauche) using the Agrobacterium-mediated transformation. Kanamycin-resistant lines were detected by DNA gel blot and RNA gel blot. DNA probe for RNA and DNA gel blot were prepared by PCR amplification of the coding region of the TaeIF5A1 using digoxigenin (DIG) - PCR labeling mix (Roche). Total DNA (30 μg) from samples was digested with BamH I and Sac I and separated by electrophoresis on a 0.8% agarose gel. The DNA was denatured with NaOH and then transferred to Hybond N+ membranes (Amersham). Hybridization and detection was performed following the manual instruction (DIG High Prime DNA Labeling and Detection Starter Kit II; Roche). To detect the expression of exogenous TaeIF5A1, total RNA (20 μg) was fractionated on formaldehyde agarose gels, blotted on Hybond N+ membranes and fixed by UV cross-linking (254 nm, 8 min). Hybridization and detection were conducted following the manufacturer’s instructions (Dig Northern starter kit, Roche).
Physiological analysis of transgenic and nontransgenic poplar
The wild-type and the transgenic plants exhibiting similar height (about 1 cm in length) were grown on 1/2MS medium supplemented with 0.6% NaCl (16 h light: 8 h dark, 25°C in tube). The phenotypes of plantlets were photographed, and the heights of plantlets were measured after 20 d of growth.
For growth comparison of plants in soil, plantlets from WT and transgenic plants with similar height (about 70 cm in height) were employed. The height and basal diameter of each sample (sample size of 10) were measured before stress as baseline values. The plantlets were treated with 0.8% NaCl solution for 30 d then watered normally. Following 90 days of growth, the height and basal diameter (final values) were measured, and the relative growth rates of growth in height or basal diameter were calculated.
For physiological analysis, plantlets (60–100 cm in height) from WT and transgenic plants grown in soil were watered with 0.8% (w/v) NaCl solution for 0, 1, 4 and 7 d, and leaves were harvested for analyses. For measurement of concentration of soluble protein, a standard curve for protein level with known concentrations of bovine serum albumin (0–100 mg, at 20 mg intervals) was generated. Phosphate buffer (1.5 mL, 0.01 M, pH 7.0) was added with sample leaf powder (0.1 g), extracted for 3 min, and centrifuged. One mL of supernatant was added with 2 mL of coomassie brilliant blue G250 regent, and the light absorbance was determined at 595 nm. Water was used instead of supernatant as control, and the protein concentrations were calculated using the standard curve. For POD activity measurement, each sample powder (0.05-0.1 g) was incubated with 1.5 ml of 0.01 M phosphate buffer (pH 7.2) at 4°C for 30 min. After centrifugation, 20 μL of supernatant was diluted to 500 μL with water, then added with 0.5 mL of 0.8% H2O2 , 0.5 mL of 0.1 M phosphate buffer, 0.5 mL of 0.1 M Guaiacol buffer, and incubated at 30°C for 8 min. Light absorbance (ΔA470) of the reaction solution was measured at 470 nm. Water was used instead of H2O2 as a control. POD activity (A
) was calculated as follows: A
V) / WTv × 100. Where V: total enzyme volume, v: the volume of enzyme used in reaction, W: the material weight, T: reaction time (min). For SOD activity assay, phosphate buffer (1.5 mL) was added with the leaf powder and incubated at 4°C for 30 min. After centrifugation, 30 mL of the supernatant was diluted to 500 mL with water and added with 1.5 mL of reaction buffer (0.013 M Met, 6.3 × 10-6 M NBT, 6.5 × 10-6 M riboflavin, 1 × 10-4 M EDTA, 0.05 M phosphate buffer, pH 7.8), and incubated at 30°C for 10 min under 6000 LX. The solution was measured at 560 nm. SOD activity was calculated as ASOD[Ug-1 min-1 (FW)] = (ΔA560 × N) / (50% WT); where ΔA560 is the decrease absorbance at 560 nm (%), N: the dilution folds, W: the weight and T: the reaction time (min). Electrolyte leakage was determined according to Wang et al.. Soluble protein contents were measured following Bradford method
. A chlorophyll analyzer (Konica Minolta, Japan) was used to determinate relative chlorophyll content (RCC) in plants stressed for 1–14 d. Each sample contained at least ten plantlets and each experiment was performed in triplicate to ensure the accuracy of analyses.
For other abiotic stress tolerance tests, the plantlets with similar size were grown on 1/2MS medium supplied with 300 μM of CuSO4, CdCl2, 1 mM of ZnCl2 and 200 mM of sorbitol. Plantlets growing in normal 1/2MS medium were used as the control. After 16 d of stress, the height between WT and transgenic lines plants were compared.
Data analyses were carried out using SPSS 16.0 (SPSSInc, Chicago, III, USA) software. For all the analyses, the significance level was set at P < 0.05. Sample variability is given as the standard deviation (S.D.) of the mean.