Hybrid poplar (Populus tremula L.×P. alba L., clone ‘INRA 7171-B4’), was used in this study. This clone was selected because of uniform growth and ease of transformation by Agrobacterium (^{Leplé et al., 1992}). Transgenic lines with ectopic expression of pine glutamine synthetase GS1a (^{Gallardo et al., 1999}) and non-transgenic controls were grown in sterile potting mix in 15 cm diameter pots (MetroMix 200, Scotts Co., Marysville, Ohio, USA) in a growth chamber (16 h photoperiod; 295–330 μmol m⁻² s⁻¹; 24 °C). All plants, including transgenic lines and non-transgenic controls, were clonally propagated from rooted stem cuttings. Plants were supplied with 30 ml of 1 mM ammonium and 3 mM nitrate applied to each pot twice weekly (^{Johnson et al., 1957}). Uniform experimental plants approximately 60 cm tall of high- and low-performing transgenic lines (lines 4-29 and 12-9, respectively) and non-transformed controls were randomly selected. In order to account for minor within-clone differences based on growth conditions, eight plants for each transgenic line and eight non-transgenic controls were used. All plants were numbered and their locations in the growth chamber were assigned randomly. Tobacco plants (Nicotiana tabacum L., line SR-1) were also grown in potting mix in a growth chamber under the same growth conditions described above.

For biochemical and molecular analyses, fully expanded mature leaves (leaf ranks 7–10) were taken from four plants of the two transgenic poplar lines and controls. Leaves were cut into 0.5×0.5 cm segments, weighed, frozen in liquid nitrogen, and stored at –86 °C (^{Man et al., 2005}). Each experiment was performed at least twice (in duplicate).

The content of free amino acids in leaves was determined using high performance liquid chromatography (HPLC). Leaf discs (approximately 200 mg) from GS1a transgenic and control plants were placed in Eppendorf tubes containing 800 μl of 5% (v/v) ice-cold perchloric acid. Samples were frozen at −20 °C. The frozen samples were thawed and refrozen three times, and centrifuged at 13 500 g for 10 min. Amino acids were analysed by HPLC according to the method of ^{Minocha and Long (2004)}. Four replicate determinations were run for each treatment. External standards consisted of a mix of 23 amino acids. The detectable total free amino acid pool of poplar leaf tissue consisted mainly of aspartic acid, glutamic acid, glutamine, serine, arginine, cysteine, glycine, proline, γ-aminobutyric acid (GABA), valine, methionine, isoluecine, leucine, tryptophan, phenylalanine, ornithine, lysine, and histidine. Threonine, asparagine, tyrosine, cysteine, and hydroxyproline could not be quantified using this method (^{Minocha and Long, 2004}).

Tobacco leaves were supplied with exogenous amino acids as previously described (^{Tsuno et al., 1983}). Four fully-expanded tobacco leaves from four different tobacco plants were used for each treatment. One half of each leaf was cut from the middle vein longitudinally and placed petiole down into a beaker containing an aqueous test solution of amino acids (30 mM, pH 7.0). The remaining half of each leaf was placed in a beaker containing MilliQ water (control). Immediately after being placed in the beakers, approximately 1 cm of each petiole was excised below the surface of the solution in order to maintain the intact water column of the cut leaves and to facilitate uniform uptake of the experimental solution. Experimental leaves were maintained in the growth chamber for 3 h (conditions defined above) after which they were blotted dry, cut into 0.4×0.4 cm segments, frozen in liquid nitrogen, and stored at –80 °C.

A consensus primer pair specific to the ASA α-subunit was designed using the multiple alignment system of ‘Vector NTI Suite, Version 6.0’ (InforMax Inc., Bethesda, MD, USA). The pair (sense primer 5′-AATGATGTTGGAAAGGTGTC-3′; antisense primer, 5′-ACCTTTGGTGCTCCACTAAC-3′) were used to detect an expected 184 bp PCR fragment of the ASA α-subunit. Primer pairs were aligned with available DNA sequences at the NCBI database using the ‘Gapped BLAST and PSI-BLAST 2.0’ (^{Altschul et al., 1997}). Results indicated that the identified primer pair is specific to plant anthranilate synthase (ASA) α-subunit. Furthermore, in order to ensure that no identical sequences specific to the ASA α-subunit exist in the transformation vector used to construct the pine GS1a transformed poplar, the primer sequences were also aligned with the transformation vector sequences using ‘PAIRWISEBLAST’. No identical or similar sequences were found.

Total RNA was isolated from poplar and tobacco leaves according to ^{Chang et al. (1993)} with modifications. After LiCl precipitation, the DNA-free kit (Ambion, Austin, TX, USA) was used to remove genomic DNA residues from total RNA according to the manufacturer's protocol.

An iScriptSelect cDNA Synthesis Kit (Bio-Rad laboratories, Hercules, CA, USA) was used in the RT reaction. Oligo (dT) primers were used to produce the entire length of cDNAs from mRNAs. The detailed RT reaction procedures were followed as recommended by the manufacturer. RT-PCR was performed using the 18S rRNA gene (Ambion, Austin, TX, USA) as an internal standard (324 bp amplicon) according to manufacturer's protocol. The resulting RT-PCR products were analysed on 1.5% (w/v) agarose gels using standard procedures.

Quantitative real-time PCR assays (q-PCR) were performed using a LightCycler 480^® instrument (Roche Diagnostics, Indianapolis, IN, USA) with polyubiquitin (UBQ11) as the reference gene (^{Brunner et al., 2004}). All reactions were carried out in a 20 μl final volume containing 8 μl of 1/15 diluted cDNA sample, 1 μl of 10 μM of each primer (0.5 μM final concentration), 10 μl 2× LightCycler^® 480 SYBR Green I MasterMix. For each q-PCR run, reactions were done in triplicate. Two independent q-PCR runs gave identical results. The conditions for q-PCR were as follows: 95 °C for 5 min followed by 45 thermal cycles of 95 °C for 10 s, 60 °C for 20 s, 72 °C for 20 s, with a ramping rate of 4.4, 2.2, and 2.2 °C s⁻¹, respectively. SYBR specific fluorescence (483–533 nm) at the end of each cycle was measured and recorded via CCD camera and software. Melting curve analyses to verify sequence specificity were run at of 95 °C for 5 s (4.4 °C s⁻¹), 70 °C for 1 min (2.2 °C s⁻¹), 95 °C (0.11 °C s⁻¹). Fluorescence change profiles of thermal dynamic dissociation of DNA were recorded in the last heating step through the continuous data acquisition process (5 acquisitions s⁻¹). The relative expression ratio of target mRNAs was calculated using the LightCycler^® 480 Software release 1.5.0 (Roche Diagnostics, Indianapolis, IN, USA). Relative standard curves were generated using 1/10, 1/20, and 1/40 dilutions of cDNA, respectively.

Total soluble proteins were extracted from poplar leaves as described previously (^{Gallardo et al., 1999}). Protein content was estimated (^{Bradford, 1976}) using bovine serum albumin as the standard. Proteins were separated by SDS-PAGE (10% v/v acrylamide) using the discontinuous buffer system of ^{Laemmli (1970)}. Equal amounts of protein (25 μg) were loaded in each lane. Gels were stained with Coomassie blue.

Following electrophoresis, proteins were electrotransferred on to nitrocellulose membranes (0.45 μm pore size, Whatman PROTRAN BA 85; Dassel, Germany) using the Bio-Rad wet blotting system at 4 °C with constant current (40 mA) overnight. After transfer, the membranes were incubated with polyclonal primary rabbit antibodies prepared against Arabidopsis ASA1 (anthranilate synthase α-subunit 1, 595 amino acids, approximately 66 kDa) (^{Niyogi and Fink, 1992}; ^{Bernasconi et al., 1994}; ^{Rutherford et al., 1998}) (1:10 000 dilution) (antibodies were generously provided by Professor Jack Widholm, University of Illinois). The Vectastain ABC-AmP kit (Vector Laboratories, Burlingame, CA, USA) with biotinylated secondary antibody (alkaline phosphatase conjugated, goat-anti-rabbit IgG) was used for immunodetection according to the manufacturer's protocols. Scanned images of bands were quantified with ImageJ 1.42q (National Institute of Health, Bethesda, MD, USA) according to established protocols (^{Tarlton and Knight, 1996}; ^{Vierck et al., 2001}; ^{Mousli et al., 2003}). Means of 4–8 measurements of digital counts of luminosity of each band were used to calculate relative band densities for comparisons of the different treatments.

Free IAA contents were measured using GS-MS according to the procedure of Muller (^{Mueller and Weiler, 2000}; ^{Pollmann et al., 2009}). Final values are reported as means of 12 measurements from triplicate feeding treatments.

The t test for all treatments was carried out using SigmaPlot 11.0 (Systat Inc., San Jose, CA). In addition, each pair of amino acid feeding treatments was compared with each other. Significance was accepted at P ≤0.05 and P ≤0.01.

Transgenic poplar plants expressing the pine cytosolic GS1a exhibited significant increases in growth over non-transgenic controls during 4 weeks growth (Fig. 1). Increases were shown for relative total leaf area (Fig. 1A) and relative plant height (Fig. 1B). Transgenics achieved maximum increases in relative height growth and relative leaf area growth after 3 weeks. Furthermore, after 4 weeks, GS activitiy (means of 4–6 plants) in leaves of transgenic poplar were significantly enhanced (93.1 μmol g⁻¹ FW h⁻¹) compared with the amounts in leaves of non-transgenic controls (54.2 μmol g⁻¹ FW h⁻¹) (Fig. 2). These results are consistent with previously published results (^{Gallardo et al., 1999}; ^{Fu et al., 2003}).

In order to assess the consequences of GS overexpression on nitrogen homeostasis, glutamine and total free amino acid contents in leaves of transgenic and control poplars were measured (Table 1). Free glutamine contents in GS transgenic leaves were significantly higher than contents in non-transgenic controls (1381.4 versus 418.78 nmol g⁻¹ FW; 229.9%). Furthermore, glutamate, tryptophan, and GABA were also significantly enhanced in the GS1a transgenic poplars. Although aspartate contents in transgenics were higher, the differences were not significant. Contents of total free amino acid were significantly enhanced in GS poplar leaves (approximately 20% higher than for non-transgenic controls) (Table 1).

To confirm the results obtained with GS1a transgenic poplar, a leaf feeding technique was used to supply tobacco leaves with exogenous glutamine, as previously described by ^{Tsuno et al. (1983)}. The contents of free amino acids, including glutamine, glutamate, phenylalanine, tryptophan, aspartate, γ-amino butyric acid (GABA), and arginine in tobacco leaves supplied with 30 mM glutamine and 2 mM chorismate are reported in Table 2. Free glutamine in tobacco leaves supplied with 30 mM glutamine was significantly enhanced over the paired control supplied with water alone (97.6 mmol g⁻¹ FW versus 0.922 mmol g⁻¹ FW; P <0.01). Glutamine contents in tobacco leaves supplied with 30 mM L-glutamine and 2 mM chorismate were also increased over the contents in leaves supplied with water alone (56.9 mmol g⁻¹ FW versus 0.98 mmol g⁻¹ FW; P <0.01). In tobacco leaves supplied with 2 mM chorismate alone, free glutamine showed low values with little difference from the paired control provided with water alone (0.99 mmol g⁻¹ FW versus 1.16 mmol g⁻¹ FW). Furthermore, glutamate, aspartate, and GABA were elevated in leaves supplied with glutamine, either with or without chorismate. Phenylalanine was enhanced in leaves supplied with chorismate, either in the presence of or absence of glutamine (Table 2).

Analysis of RT-PCR products on agarose gels showed that the ASA α-subunit transcripts in GS1a transgenic poplar was enhanced (Fig. 3B) when compared with both the non-transgenic control (Fig. 3A) and the low-performing transgenic line 4-18 (Fig. 3C). The ASA α-subunit transcript in the high-performing line 4-29 was approximately 60% greater than in the non-transgenic control, as determined by image analysis (Image J software, NIH).

In the related paired leaf experiment, tobacco leaves provided with 30 mM glutamine showed an enhancement of ASA α-subunit transcriptss when compared with its corresponding paired water control (Fig. 4, lanes A and B). Tobacco leaves were also provided with 30 mM glutamine plus 2 mM chorismate, both substrates for ASA. Assessment of the ASA α-subunit transcript on gels (Fig. 4, lane D) showed the same trend as that observed with 30 mM glutamine alone. Supplying leaves with 2 mM chorismate alone showed no clear effect on the ASA α-subunit transcript compared with the corresponding control (Fig. 4, lane C).

Quantitative RT-PCR (q-PCR) analysis of ASA α-subunit transcripts in leaves of GS1a transgenic poplar are presented in Fig. 5. Transcripts are presented as relative content as a percentage increase versus the non-transgenic control. For the high-performing GS transgenic line (4-29), the ASA α-subunit transcript exceeded the non-transgenic control by approximately 118% (P <0.01). The ASA α-subunit transcript was also analysed in a second GS1a transgenic line (12-9) characterized by improved growth over the non-transgenic control, but with growth less than that of line 4-29 (Fu et al., 2001). The ASA α-subunit transcript in leaves of this second transgenic line was also enhanced over the non-transgenic control (38%; P <0.01) (Fig. 5).

q-PCR analysis was also used to assess the ASA α-subunit transcript in the corresponding tobacco experiment (Fig. 6). In tobacco leaves fed with 30 mM glutamine, the ASA α-subunit transcript was approximately 175% (P <0.01) greater than the paired control fed with water alone. Tobacco leaves provided with 30 mM glutamine and 2 mM chorismate also showed significant increases in the ASA α-subunit transcript (approximately 195%; P <0.01) over the paired control. Feeding tobacco leaves with 2 mM chorismate alone showed only a slight increase in the ASA α-subunit transcript versus the paired control (not significant).

The ASA α-subunit polypeptide (66 kDa) was detected in immunoblots of protein extracts of both poplar and tobacco leaves (Fig. 7A, B). Quantitative estimates of the ASA α-subunit polypeptide in immunoblots (ImageJ Software) showed that the ASA α-subunit was enhanced in both leaves of the GS1a transgenic poplar (line 4-29) (approximately 120% versus the non-transgenic control) and in tobacco leaves fed with 30 mM glutamine (approximately 90% higher than in the paired controls).

Quantitative determinations of free IAA in leaves of GS1a transgenic poplar and in leaves of tobacco fed with 30 mM glutamine are presented in Table 3. IAA in the high- performing GS1a transgenic poplar (line 4-29) averaged 32.15% greater than in the non-transgenic control (116.3 versus 88.0 pmol g⁻¹ FW; P <0.05). IAA in the moderately performing poplar GS1a transgenic line (12-9) was not statistically different from the non-transgenic control (91.33 versus 88.0 pmol g⁻¹ FW). In the related tobacco leaf experiment, leaves provided with 30 mM glutamine showed increased IAA contents compared with the control (140.08 versus 112.92 pmol g⁻¹ FW), however, this increase was not shown to be significant. IAA in tobacco leaves supplied with 30 mM glutamine and 2 mM chorismate was significantly greater than in the paired control (109.83 versus 77.67; 41.4%; P <0.05). Interestingly, IAA contents of tobacco leaves supplied with 2 mM chorismate alone were also significantly enhanced over the paired control (143.58 versus 97.0; 48.0%; P <0.001).