2.1 Plant materials and growth conditions
A. sinensis calli from plant leaves were obtained as described in previous reports (Wang et al. 2016). The calli were subcultured on fresh Murashige and Skoog (MS) media comprising 1 µg/mL 6-benzyladenin (6-BA), 2 µg/mL naphthylacetic acid (NAA), 1 µg/mg kinetin (KT), and 1 µg/mL dichlorophenoxyacetic acid (2,4-D) for 15 days and then cultured on media supplemented with 100 µM ABA, 200 mM NaCl, 750 mM mannitol, or 500 µM CdCl2. For low-temperature treatment, the calli were cultured in a 4°C environment. Calli from different treatment groups and the control group were harvested at different time points (0 h, 12 h, 24 h, 36 h, 48 h). Transgenic and wild-type N. benthamiana seeds were surface sterilized for 30 s in 75% ethanol, sterilized with 2% NaClO for 10 min, rinsed with sterile water four times, and then cultivated in 1/2-strength MS media supplemented with different concentrations of ABA in a growth chamber at 25°C (14 h light/10 h dark).
2.2 RNA preparation and cDNA for AsTal1 analysis
Total RNA was isolated from the samples of A. sinensis calli using Total RNA Purification Reagent (Norgen, Cat# 17200) according to the manufacturer’s instructions. The quantity and integrity of the RNA samples were determined by a NanoDrop ND-2000 spectrophotometer and 1.2% agarose electrophoresis. Reverse-transcription reactions were performed with 1 µg of total RNA, 200 U of M-MLV transcriptase (Promega, Madison, WI, USA), dNTPs (0.2 mM each), 20 U of RNase inhibitor (Invitrogen) and 0.1 µM oligo dT (18) primer in a final volume of 30 µL. Reverse transcription was carried out at 42°C for 90 min. The AsTal1 cDNA sequence containing class-specific TAL conserved domains and nucleotide sequences was obtained from the A. sinensis callus transcriptomic dataset. To isolate this gene from A. sinensis calli, open reading frames (ORFs) were isolated via RT-PCR using specific primers (Table 1). The resulting DNA fragments were ligated into a pMD19-T vector, and three clones were sequenced in both directions. The ORFs of the cDNA sequence of AsTAL1 were predicted with the ORF finder online tool (http://www.ncbi.nlm.nih.gov/projects/gorf/). The amino acid alignments were performed by DNAMAN software, and phylogenetic trees were constructed using the neighbor-joining tree algorithm of the MEGA 6.0 program.
2.3 Subcellular localization
The ORF of AsTal1 was isolated by PCR amplification using specific primers (Table S1). The fragments were fused to the C-terminus of the green fluorescent protein (GFP) gene in a pCAMBIA1300-35S-EGFP binary vector. The binary vectors were then transformed into Agrobacterium tumefaciens strain EHA105, after which the transformed Agrobacterium were infiltrated into Nicotiana benthamiana leaves as described in previous reports. Subcellular localization was observed via laser confocal scanning microscopy.
2.4 Quantitative real-time PCR (qRT-PCR) assays
qRT-PCR was performed on a CFX ConnectTM Real-time System (Bio-Rad) using TransStart Tip Green qPCR SuperMix (Transgen) according to the manufacturer’s protocol. The A. sinensisGAPDH gene and the N. benthamiana β-actin gene were used as internal controls to normalize RNA levels. Real-time PCR was initiated with 30 s of incubation at 94°C, followed by 40 cycles of 94°C for 5 s and 60°C for 30 s. The primers used for qRT-PCR were generated by the Primer 5.0 program (Table S1), and the 2-ΔΔCT method was used to analyze the expression level of AsTal1 genes. Three biological experiments and three experimental replicates were included to analyze gene expression. The primers used for qRT-PCR are listed in Table S1.
2.5 N. benthamiana transformation
To construct AsTal1 overexpression vectors, the AsTal1 ORF was obtained by specific primers and subsequently subcloned into a pCAMBIA1300-35S binary vector to yield pCAMBIA1300-35S-AsTal1 vectors. The pCAMBIA1300-35S-AsTal1 plasmids were then transformed into wild-type N. benthamiana using the Agrobacterium-mediated leaf disc transformation method. Positive transgenic N. benthamiana plants were identified through PCR and qPCR. T3 seeds of three independent overexpression lines (OE) and wild type (WT) plants were used to analyze the function of the AsTal1 gene.
2.6 Analysis of ROS
The concentration of total protein extracted from the roots of OE and WT lines was quantified with the BCA method. The H2O2 and O2- contents and the activities of antioxidant enzymes such as peroxidase (APX), superoxide dismutase (SOD), and peroxidase (POD) were measured using detection kits (Nanjing Jianchen Bioengineering Institute, China) according to the manufacturer’s protocols.