Among 15 independent TaLEA transgenic lines, one line showed significantly reduced height in all plants, and this line was named dwf1. In addition, the mutant showed lower root biomass, fewer lateral roots, crinkly and leathery leaves, dark green leaf blades and deep red petioles, in comparison to the wild type and the other transgenic lines (Table 1). Chlorophyll content of dwf1 was higher compared to the wild type and the transgenic line 5 and 6. Dwf1 exhibited a less leaf length-width ratio. These characteristics indicated that dwf1 had multiple morphological defects (Figures 1 and 2).

TAIL-PCR method was employed to determine the T-DNA insertion sites in dwf1. We obtained an 847 bp right border and a 698 bp left border flanking sequence (Table S1). Sequence alignments of the sequences against the pROK binary vectors sequence and the NCBI poplar genome database showed that the right and the left border mapped to different chromosomes (Figure 3). The right border was located at Chr IV (10,570,141 bp), while the left border was located at Chr VIII (7,459,761 bp). The results indicated that there was more than one independent T-DNA copy.

NCBI Map Viewer was used to search for potential expressed genes adjacent to the insertions [15]. Only a putative AP2 transcription factor (GI: 224073210) was found 3 kb downstream the insertion site of the left border (Table 2). Quantitative RT-PCR analysis showed that AP2 was up-regulated in dwf1 compared with the wild type and the other transgenic lines (Figure 4).

Sequence analysis revealed that the AP2 gene encodes a protein of 369 amino acids containing two DNA-binding domains, an AP2 and a B3 DNA-binding domain (Figure 5).

The AP2/ERF superfamily is defined by the AP2/ERF domain, which consists of about 60 to 70 amino acids that are involved in DNA-binding. Members of this superfamily can be divided further into three families. The AP2 family proteins contain two repeat AP2/ERF domains, the ethylene response factor (ERF) family proteins contain a single AP2/ERF domain, while the RAV family proteins contain a B3 domain and a single AP2/ERF domain [16,17]. The AP2/ERF proteins are involved in a variety of biological processes related to plant growth and development, as well as various responses to environmental stimuli [18–20].

Recently, members of the RAV family were reported to be involved in plant responses to ethylene and brassinosteroids (BRs) [21,22]. Transgenic tobacco overexpressing the GmRAV (Glycine max RAV) gene had slower plant growth rates, reduced root elongation, delayed flowering times, reduced photosynthetic rates and reduced chlorophyll contents in leaves [23]. The RAV1 transcription factor also positively regulates leaf senescence in Arabidopsis [24].

It has been proven that RAV genes play an important role in maintaining brassinosteroid homeostasis in higher plants. Brassinosteroids (BRs) are plant steroid hormones that are perceived by the cell surface receptor kinase Brassinosteroid Insensitive1 (BRI1). In rice RAVL1 regulates the expression of the BR receptor [25]. Furthermore, RAVL1 is also required for the expression of the BR biosynthetic genes. According to the microarray analysis results, putative BRI1-associated receptor kinase 1 precursor genes (PtpAffx.35408.1.S1_at, PtpAffx.48409.1.S1_at) were up-regulated in dwf1. All of these results indicated that there might be some relationship between the overexpression of AP2 and the dwarf phenotype of dwf1. However, the functional role of AP2 in dwarf formation still needs a further confirmation.

To analyze further the molecular mechanisms responsible for the dwf1 phenotype, the mRNA profile of dwf1 and the wild type were compared using an Affymetrix whole-genome microarray chip. Compared with the wild type, 537 genes were differentially expressed, including 257 down-regulated genes and 280 up-regulated genes in the dwf1 mutant (Tables S. 2,3). To validate these microarrays data, six genes were selected at random for quantitative RT-PCR assays. Each of these six genes displayed an expression pattern consistent with the microarray analysis results (Figure 6). Therefore, further analyses focused on these 537 differentially expressed genes.

To evaluate the potential functions of genes with significant transcriptional changes between the dwf1 mutant and the wild type, gene ontology (GO) categories were assigned to 443 genes of the 537 genes based on the plant GO slim provided by blast2GO [26]. The categorization of differentially expressed genes according to their cellular component, molecular function and biological process is shown in Figure 7. According to cellular component, the analysis revealed a high percentage of genes corresponding to the nucleus, plastid, plasma membrane, cell wall, extracellular region, mitochondrion and vacuole. Based on molecular function, the most represented GO terms were hydrolase activity, protein binding, nucleotide binding, kinase activity and transcription factor activity. Differentially expressed genes related to more than 20 biological processes, including responses to stress, transcription, protein modification processes, lipid and carbohydrate metabolism, transport, catabolic processes, responses to abiotic and biotic stimuli, responses to endogenous stimuli and cellular component organization.

KEGG pathway analysis was used to identify the biological pathways of the genes that were significantly differentially expressed. More than 60 different metabolic pathways were involved; some were consistent with biological processes revealed already by GO analysis. The most represented pathways included pathways involved with the biosyntheses of secondary metabolites, flavonoids, phenylpropanoids, starch, sucrose, terpenoids, steroids, plant hormones and alkaloids derived from the shikimate pathway, and the metabolism of galactose and glycerolipids. Many of these metabolic pathways are related to plant hormones, which are responsible for controlling plant growth and development.

Many plant hormone related genes showed differential expression patterns in dwf1. These genes included plant hormone receptors, such as F-box, LRR receptor-like and G-proteins, ubiquitin, cytochrome P450 genes, and plant hormone response genes (Table 3).

In plants, nearly every aspect of the plant’s life is regulated by plant hormones. Central to comprehending hormonal control of plant growth and development is the understanding of how the hormones are perceived. In the past few years, some plant hormone receptors have been identified. F-box proteins such as TIR1 [27] and AFBs are receptors of auxin. Auxin plays a vital role in regulating plant growth and development [28,29]. Jasmonic acid shares the same receptors with auxin [30]. Leucine-rich repeat receptor-like kinases, BRI1, are the receptors of Brassinosteroids [14]. In this study, three potential F-box family proteins PtpAffx.200885.1.S1_x_at, PtpAffx.45694.1.S1_at and PtpAffx.125908.1.A1_at showed differential expression levels (Figure 8).

Plant cytochromes P450 (CYPs) are involved in a wide range of biosynthetic reactions, leading to various fatty acid conjugates, plant hormones, defensive compounds, or medically important drugs. Terpenoids, which represent the largest class of characterized natural plant compounds, are often substrates for plant CYPs. In this study more than 10 cytochrome P450s showed different expression patterns.

The ubiquitin-proteasome system (UPS) is involved in nearly every aspect of plant growth and development. Ubiquitin is attached to target proteins through the action of three enzymes known as E1, E2, and E3. Ubiquitination is a recurring theme in plant signal transduction. E3 ubiquitin ligases in particularly participate in hormone perception [31–34]. In this study 5 ubiquitins and 2 ubiquitins ligases showed different expression patterns. All of these might affect the perception and the transduction of plant hormones, and plants’ growth will be abnormal if they are sensitive or insensitive to plant hormones.

Transcription factors regulate gene expression at the level of transcription via the recognition of promoter elements. In this study 27 differentially regulated genes encoded putative transcription factors, including 23 up-regulated and 4 down-regulated genes (Table 4). More than half of the transcription factors were from WRKY, AP2, and ERF families, while the others belonged to various families, including heat shock transcription factors and myb families. WRKY transcription factors are one of the largest families of transcriptional regulators in plants and form integral parts of signaling webs that modulate many plant processes [35–38].

Using the microarrays method we obtained differently expressed genes in dwf1 compared with wild type plants. However, misregulation could be due to the LEA transgene on the T-DNA. We performed a Digital Gene Expression (DGE) to analysis the gene expression patterns in transgenic line 5 (XL-5), 6 (XL-6) with normal phenotype, and dwf1. Specifically, we analyzed the expression patterns of the plant hormone related genes and transcription factors in transgenic line 5, 6, and dwf1. According to the digital gene expression (DGE) results (Table 5), nearly all of these differentially expressed genes, especially plant hormone related, between dwf1 and WT did not change expression level in XL-5 and XL-6 as compared to the wild type. The results indicated that dwf1 showed a unique gene expression profile compared to wild type and the other transgenic lines.

A F₁ hybrid of Populus simonii × Populus nigra was generated in this study. The transgenic poplar lines were produced as described previously [39]. For co-cultivation, Agrobacterium tumefaciens strain was incubated in liquid LB medium at 28 °C with constant shaking (250 rpm) until the culture reached an optical density of about 0.6 at 600 nm. The A. tumefaciens culture was then diluted with one volume of liquid MH medium. Leaves of Populus simonii × Populus nigra excised from plantlets cultured in vitro were cut into discs and cultured in the dark for 2–3 days on MH medium containing 0.5 mg/L 6-benzylaminopurine (6-BA), 0.05 mg/L 1-naphthaleneacetic acid (NAA), and 0.8% (w/v) agar. The leaf discs were then dipped in the diluted Agrobacterium culture for about 2–5 min and cultured on co-cultivation medium. After co-cultivation in the dark for 2 days, the leaf discs were transferred to MH medium containing 0.5 mg/L 6-BA, 0.05 mg/L NAA, 200 mg/L Cefotaxime, 50 mg/L kanamycin and 0.8% (w/v) agar and cultured in the light for 7 days. The regenerated shoots were individually removed from the callus and transferred to solid MH medium containing 0.05 mg/L 6-BA, 0.01 mg/L NAA, 50 mg/L kanamycin, and 0.8% (w/v) agar. Regenerated shoots were transferred to rooting medium (MH containing 0.2 mg/L indolebutyric acid, sucrose 15 g/L, and 50 mg/L kanamycin).

The seedlings of wild type and dwf1 were transferred to a greenhouse and planted in a mixture of turfy peat and sand (2:1 v/v) with 75% relative humidity and a constant temperature of 24 °C. Young leaves were harvested from three independent biological replicates with five plants per replicate. Leaves were immersed immediately in liquid nitrogen and stored at −70 °C for subsequent DNA and RNA extractions.

Total RNA was prepared using the sodium dodecyl sulfate (SDS) method [40]. RNA integrity was examined by electrophoresis on a 1% formaldehyde denaturing gel. The samples with bright bands corresponding to ribosomal 28S and 18S RNA (with a ratio of intensity of ca. 1.5 to 2.5:1) were used for microarray analyses and qRT-PCR. First strand cDNA was generated from 1 μg RNA using PrimeScript^® RT reagent kit (Perfect Real Time; TaKaRa, Japan) with oligo-dT primers and according to the manufacturer’s protocol.

Quantitative real-time PCR (Q-PCR) was conducted using SYBR Green dye (SYBR^® Green Realtime PCR Master Mix-Plus-, Code No. QPK-212). PCR was conducted according to the manual. Parallel reactions using actin (GI:224116599) and tublin (GI:224054577) genes were performed to normalize the amount of template cDNA. The protocol of real-time PCR was as follows: initiation with a 2 min denaturation at 94 °C, followed by 45 cycles of amplification with 10 s of denaturation at 94 °C, 15 s of annealing at 60 °C, 30 s of extension at 72 °C and reading the plate for fluorescence data collection at 75 °C. A melting curve was performed from 75 to 95 °C to check the specificity of the amplified product. Primer sequences for the real-time PCR assay are listed in Table S4. Three PCR replicates were performed for each RNA sample.

Genomic DNA was isolated from the leaves of the dwf1 and wild type plants using a modified CTAB method as described by Porebski [41]. The T-DNA insertion site in the dwf1 mutant was determined by TAIL-PCR [2]. The primers GSPL1, GSPL2 and GSPL3 (Table S5) were used for the cloning of the flanking sequence of the left border, while the primers GSPR1, GSPR2 and GSPR3 (Table S4) were used for the right border. The arbitrary degenerate primer was provided by the Genome Walking kit (Takara Genome Walking Kit, code: D316, Japan). TAIL-PCR amplification was performed according to Sissions’ procedure [42]. After three rounds of PCR reactions, the products were separated by electrophoresis on 1.5% agarose gels. The resulting bands were purified using the Aqua-SPIN Gel Extraction Ace kit (Watson Biotechnologies, China), cloned using the pMD18-T cloning kit (TaKaRa, Japan), and sequenced (Invitrogen, China). The NCBI popular genome database was used to analyze the flanking sequences.

Three sets of biological replicates were collected independently and a total of six gene chips were analyzed. For the microarray analysis experiment, an aliquot of 2 μg of total RNA was used to synthesize double-stranded cDNA. Then, biotin-tagged cRNA was produced using the MessageAmpTMII aRNA Amplification kit. The biotin-tagged cRNA was fragmented into strands of 35 to 200 bases in length according to the Affymetrix protocol. The fragmented cRNA was hybridized to Affymetrix poplar genome AFF-900728 that contains 56,055 transcripts. Hybridization was performed at 45 °C with rotation for 16 h (Affymetrix GeneChip Hybridization Oven 640). The GeneChip arrays were washed and then stained (streptavidin phycoerythrin) on an Affymetrix Fluidics Station 450, followed by scanning on a GeneChip Scanner 3000. The hybridization data were analyzed using GeneChip Operating software (GCOS 1.4). The scanned images were assessed first by visual inspection, and then analyzed to generate raw data files (saved as CEL files) using the default settings of GCOS 1.4. A global scaling procedure was performed to normalize the different arrays using the dChip software.

Statistical analyses were performed to identify differently expressed genes between dwf1 and wild type using SAM (significance analysis of microarrays) method described previously [43]. In this study we used the fold change ≥ 2 or ≤ 0.5 and q-value ≤ 0.05 as the threshold to judge the significance of gene expression.

DGE is a method that generates absolute gene expression measurements. DGE libraries were prepared using Illumina Gene Expression Sample Preparation Kit according to the manufacturer’s instructions. Briefly, 6 μg total RNA was used for mRNA capture with magnetic Oligo(dT) beads. The first and second cDNA strands were synthesized using Oligo(dT) primers. The 5′ ends of tags were generated by Endonuclease NlaIII, which recognizes and cuts off the CATG sites. The fragments apart from the 3′ cDNA fragments connected to Oligo(dT) beads were washed away and the Illumina adaptor 1 was ligated to the sticky 5′ end of the digested bead-bound cDNA fragments. The junction of Illumina adaptor 1 and CATG site is the recognition site of MmeI, which is a type of endonuclease with separate recognition sites and digestion sites. It cuts at 17bp downstream of the CATG site, producing tags with adaptor 1. After removing 3′ fragments with magnetic beads precipitation, Illumina adaptor 2 was ligated to the 3′ ends of tags, acquiring tags with different adaptors of both ends to form a tag library. After 15 cycles of linear PCR amplification, 105 bp fragments were purified by 6% TBE PAGE gel electrophoresis. After denaturation, the single-chain molecules were fixed onto the Illumina Sequencing Chip (flowcell). Each molecule grows into a single-molecule cluster sequencing template through in situ amplification. Then four types of nucleotides labeled by four colors were added, and sequencing using the method of sequencing by synthesis (SBS) were performed. Raw sequence reads were filtered by the Illumina pipeline. Low-quality sequences such the 3′ adaptor sequence, Tags which were too long or too short, Tags with unknown sequences and Tags with only 1 copy were removed. The remaining high quality sequences (Clean Tags) were mapped to Poplar reference sequences [44]. Clean Tags mapped to poplar genome sequences from multiple genes were filed. The gene expression was normalized to transcripts per million clean tags (TPM).

A commercial chlorophyll meter SPAD-502 was used to estimate the leaves chlorophyll content. 6 independent plants were selected for each transgenic line and WT.