Plant materials and culture
Populus simonii×P. nigra is a specific hybrid poplar widely grown in the northeast, northwest and southwest of China. The growing twigs of wild-type Populus simonii×P. nigra from one clone of the experimental forest of Northeast Forestry University were hydroponic cultured at room temperature with 16/8-hour light/dark cycles and 70% relative humidity for two months. The new roots, stems and leaves from the twigs were frozen in liquid nitrogen for RNA-Seq. And the roots, petioles, leaves, xylem and cambiums were harvested for expression pattern analysis. Three biological replicates were prepared for each tissue.
The seeds of wild-type Nicotiana tobacum were preserved in the state key laboratory of tree genetics and breeding of Northeast Forestry University. To prepare sterilized tobacco explants, the tobacco seeds were sterilized using 70% (v/v) ethanol for 30 s, followed by NaClO solution (1% NaClO, 0.05% TWEEN20) for 10 min and rinsed using sterile water for 5 times. Then the seeds were placed on 1/2 MS solid medium (pH 5.8–6.0) at 24±2°C, 16/8-hour light/dark cycles for germination. And germinated seeds were transferred into tissue culture bottles containing 1/2 MS solid medium. The aseptic seedlings at one month old were used for gene transformation .
NAC expression analysis by RNA-Seq
A total of nine samples including leaves, stems, and roots with respective three biological replicates were shipped with dry ice to GENEWIZ Company (www.genewiz.com) for RNA isolation, mRNA-purification, and RNA-Seq with Illumina Hi-seq platform. The raw sequences were cleaned using Trimmomatic v0.30 . The cleaned reads were aligned to Populus trichocarpa reference genome using STAR 2.4.2a . The mRNA abundance of each gene in each sample was quantified as FPKM (fragment per kilo bases per million reads).
The FPKM information of 289 NAC family members was drawn from RNA-Seq data (Supplemental Excel 1). The NACs with FPKM≥ 4 in at least one tissue were applied to count differentially expressed NAC genes in the three tissues. The fold change (FC) in the different tissues was standardized by Log2 FPKM ratios [35, 36]. The hierarchical clustering of the differentially expressed NAC genes in the three tissues was conducted by Heatmapper (http://www.heatmapper.ca/expression/).
The total RNA was extracted using Column Plant RNAout Kit (CAT#:71203, Tiandz, Beijing, China) and reverse-transcribed into cDNA using PrimeScript™ RT reagent Kit with gDNA Eraser (RR047A, Takara, Dalian, China). RT-qPCR experiment was performed by ABI 7500 fast real-time PCR detection system using SYBR Premix Ex Taq™Ⅱ (DRR081A, TaKaRa, Dalian, China). The relative expression level of genes was calculated by 2-△△Ct method with three biological replicates . The primer pairs of poplar NAC15 gene (NAC15–1), reference gene, and lignin- and cellulose-related genes (Supplemental Table 1) were designed based on Populus trichocarpa v3.1 in Phytozome12 (https://phytozome.jgi.doe.gov/pz/portal.html).
Phylogenetic analysis of NAC15 protein
Amino acid sequences of NACs from Populus trichocarpa and other species were derived from PlantTFDB (http://planttfdb.cbi.pku.edu.cn/). Multiple alignment of conserved NAC domain was performed by Clustal W . Phylogenetic tree of NAC proteins was constructed by Neighbor-Joining method with MEGA 6 program .
Subcellular localization of NAC15
To confirm subcellular localization of NAC15, the coding region of NAC15 gene without stop codon was cloned into pBI121 vector with specific primers (NAC15–2, Supplemental Table 1) and expressed with green fluorescent protein (GFP) under the control of CaMV35S promoter. The combined vectors 35S::NAC15-GFP and 35S::GFP as control were transferred into onion epidermal cells by particle bombardment, separately. The fluorescence signal of GFP was detected by fluorescence microscopy system (LSM 700, Zeiss, Germany).
Generation of transgenic tobacco overexpressing NAC15 gene
The 1515 bp transcript sequence of NAC15 gene was cloned into pBI121 vector under the control of CaMV35S promoter with specific primers (NAC15–3, Supplemental Table 1). The recombined vector and empty vector as control were transformed into EHA105 Agrobacterium strain by electroporation, separately. The transformed EHA105 strain was confirmed by PCR and sequencing.
The tobacco transformation was conducted as following: 1) the leaves from aseptic plants at one month old were cut into 1cm × 1cm discs and soaked in the positively transformed EHA105 liquid medium (OD 0.3- 0.5) for 10 min; 2) the leave discs were dried with sterilized filter paper and put on the 1/2 MS solid medium for co-culture in dark for two days; 3) the leave discs were transferred on the pre-cultural medium (1/2 MS solid medium containing 0.5mg/L 6-BA, 0.05 mg/L NAA and 100 mg/L Kan) until callus emerged; 4) healthy callus were transferred on the shooting medium (1/2 MS solid medium containing 0.1 mg/L 6-BA, 0.05 mg/L NAA and 100 mg/L Kan) until shoots grew; 5) the shoots were transferred into the rooting medium (1/2 MS containing 0.2 mg/L IBA and 100 mg/L Kan) until roots generated; 6) the transgenic tobacco seedlings were confirmed by PCR and RT-PCR . The specific primers for PCR (NAC15–4) and RT-PCR (NAC15–5) were list in the Supplemental Table 1.
Determination of secondary wall composition
The relative content of lignin, hemicellulose and cellulose in tobacco plants was measured at maturation stage with three biological replicates. The sample preparations, determination procedures and calculation formulas referred to the description by Sukjun et al .
The tobacco stems at growth period were used for histological analysis. The procedure was conducted as follows: 1) fixed the stems in the FAA solution (70% ethanol: glacial acetic acid: formaldehyde; 90: 5: 5, v/v) and embedded them in the frozen sectioning medium (OCT; Thermo Scientific, Waltham, MA); 2) cut the embedded stems into slices and put the slices on the slides; 3) stained the slides with phloroglucinol solution for 2 min; 4) soaked the slides in 50% (v/v) HCl; 5) put coverslips on the slides and wiped the slides with lens paper; 6) examined the slides with optical light microscope .
All the data in the study were the mean and standard error of three biological replicates. Student’s t-test was used to identify significant differences between transgenic tobacco and control plants. And the statistical significance was controlled at p<0.05.