Populus is widely distributed in the middle latitudes of the world, and is one of the important industrial wood and ecological protection tree species in China (An, 2013, Modern Horticulture, (14): 69-71). Poplars have fast growth, strong adaptability and relatively simple reproduction, which have important economic and ecological values (Shao, 2012). Poplar plantation is limited by desertification and salinization, and poplar is often subjected to abiotic and biological stresses such as drought, low temperature and infection of pathogenic microorganisms in the growth process, so it is of practical value to enhance the stress resistance of poplar. At the same time, poplars have full genomic information and complete genetic engineering technology platform, so poplars are also model species for exploring the gene function of woody plants (Wang et al., 2016).

Plants rely on transcription factor (TF) to regulate defense genes, control plant secondary metabolism and respond to hormone signals, so as to cooperate with a large number of genes in response to adversity. Transcription factors can bind specifically to cis-acting elements in upstream promoters of genes to control the expression of target genes (Ao, 2017). According to the sequence specificity of DNA binding domain, transcription factors can be divided into multiple transcription factor families, among which NAC transcription factors with NAM, ATAF1,2 and CUC2 DNA-binding domains are the largest in higher plants (Kasuga et al., 1999). ATAF1 was found to be one of the archetypal NAC transcription factors in Arabidopsis thaliana (Souer et al., 1996). AtATAF1 can be induced to be up-regulate expression under high salt, drought, Botrytis cinerea and ABA (Huh et al., 2012). NCED3 is a regulatory target gene of ATAF1 and plays a key role in ABA biosynthesis. In plants overexpressed by AtATAF1, the transcriptional abundance of NCED3 and ABA level increased, indicating that AtATAF1 activates ABA response genes and regulates its biosynthesis (Jensen et al., 2013). Overexpression of AtATAF1 in Arabidopsis thaliana also increased the sensitivity of plants to ABA and salt, thus enhancing drought tolerance of transgenic plants (Liu et al., 2016). However, overexpression of AtATAF1 can increase plant susceptibility to pathogens such as Pseudomonas syringe (Cahill et al., 2018). Therefore, ATAF1 plays an important role in regulating both biological and abiotic stress in model plant Arabidopsis thaliana (Mauch-Mani and Flors, 2009).

In this study, we cloned a ATAF1 homolog (PtATAF1-1) from Poplar, and studied its bioinformation, gene expression pattern and transient expression of poplar protoplast. It is preliminarily speculated that ATAF1 gene is involved in the response to biological and abiotic stress. This study provides a preliminary theoretical reference for further exploring the function of ATAF1 gene in poplar.

1 Result and Analysis

1.1 Cloning and sequence analysis of PtATAF1-1 homolog in Populus trichocarpa

Blastp analysis of Arabidopsis AtATAF1 (AT1G01720) amino acid sequences in Populus trichocarpa database revealed 8 highly similar proteins, identified as Potri.002G081000, Potri.011G046700, Potri.011G123500, Potri.001G404100, Potri.005G180200, Potri.004G038000, Potri.011G123300, Potri.001G404400, E-Value is 1e-116, 6e-063, 2e-062, 2e-062, 6e-063, 7e-112, 3e-063, 5e-063. Identity is 70.65%, 70.55%, 69.23%, 69.14%, 68.81%, 68.86%, 68.52%, 68.21% respectively. Blastp analysis was performed using these homologous proteins in The Arabidopsis Information Resource (TAIR), it was found that Potri.005G180200 and Potri.002G081000 were most likely to be ATAF1 homologous proteins in poplars. Therefore, they were named PtATAF1-1 and PtATAF1-2, respectively. Under the cultivation conditions of Populus trichocarpa in our laboratory, only the full length of PtATAF1-1 gene sequence can be amplified from cDNA of Populus trichocarpa leaves by RT-PCR, so PtATAF1-1 is only used as the research object in the future.

Rapid amplification of cDNA ends (RACE) was used to amplify 5¢ and 3¢ ends of Populus trichocarpa PtATAF1-1. A full-length cDNA sequence of 1 242 bp, containing an 873 bp ORF encoding 291 amino acids was obtained. It also contains 5¢ UTR (97 bp) and 3¢ UTR (272 bp) (Figure 1).

Blastp comparison was performed on Populus trichocarpa PtATAF1-1 in NCBI non-redundant database, and ATAF1 sequences of other 6 representative plants, namely Populus euphratica (XP_011027492.1), Salix Brachista (KAB5568578.1), Manihot esculenta (XP_021604672.1), Hevea brasiliensis (XP_021649171.1) Jatropha curcas (AEI25534.1) and Arabidopsis thaliana (NP_171677.1) were selected for multiple sequence comparison. The comparison results showed that the conserved sequence of NAM (PF02365) was found in the Pfam database, and the amino acid sequences of PtATAF1-1 and other ATAF1 were found to be highly conserved in the NAM domain, indicating that PtATAF1-1 belonged to the NAC family (Figure 2).

1.2 Evolutionary analysis of PtATAF1-1 protein

In order to investigate the evolutionary relationship of PtATAF1-1 transcription factor, the amino acid sequences of ATAF1 homologous proteins in 16 species such as Populus trichocarpa, Hevea brasiliensis, Jatropha curcas and Salix brachista were used to construct phylogenetic trees. The phylogenetic trees were constructed by MEGA X software. And it turns out, ATAF1 of Populus trichocarpa and Salix brachista in Salicaceae, and Ricinus communis, Hevea brasiliensis, and Jatropha curca in Euphorbiaceae constituted a highly supported clade (94%), which was consistent with the close relationship between Salicaceae and Euphorbiaceae. However, phylogenetic analysis with Vitis vinifera in Vitaceae, Rhamnella rubrinervis in Rhamnaceae, and Boehmeria nivea in Urticaceae did not receive more than 50% support (Figure 3).

1.3 Physical and chemical properties and protein structure analysis of PtATAF1-1

According to the analysis of PtATAF1-1 gene sequence by ProtParam, the theoretical molecular weight of PtATAF1-1 protein was 33.27 kD, the isoelectric point (pI) was 6.40, and the instability coefficient was 52.88. Among the 291 amino acid residues encoded, the proportion of proline was the largest (8.6%), followed by lysine (7.9%), leucine (7.2%) and alanine (6.9%). The hydrophilicity/hydrophobicity of PtATAF1-1 protein was predicted by ProtScale online analysis website, and the result showed that the average hydrophilicity coefficient was -0.49, suggesting that the protein was a basic hydrophilic protein (Figure 4A). The secondary structure of PtATAF1-1 was predicted by SOPMA, an online tool of PRABI-GERLAND. The results showed that the secondary structure of PtATAF1-1 had a large amount of random curl, 18.56% α-helix, 11.34% elongation and 2.06% β-rotation (Figure 4B). Using Protein Homology/analogY Recognition Engine V 2.0, a 3d model of PtATAF1-1 Protein was established online and its tertiary structure was predicted (Figure 4C), indicating that the protein was prone to homodimer formation.

1.4 Subcellular localization analysis of PtATAF1-1 protein

The PtATAF1-1-GFP fusion vector was constructed and transient expression of PtATAF1-1 in the protoplast of Populus 895 of Nanjing Forestry University was achieved by PEG mediated method. The localization of PtATAF1-1 in the protoplast was observed by fluorescence microscope with GFP as the reporter gene. The results showed that in the case of single-transformed GFP, green fluorescence existed in the cell membrane and nucleus, while the green fluorescence occurred in the PtATAF1-1-GFP fusion protein only appeared in the nucleus, indicating that PtATAF1-1 protein was a nuclear localization protein, in line with the characteristics of its transcription factor (Figure 5).

1.5 Determination of PtATAF1-1 gene expression under ABA and Flg22 treatment

The conserved regions of bacterial flagellin N-terminal Flg22 (2 μmol/L) and ABA (0.01 mmol/L) were used to treat tissue culture seedlings of Populus trichocarpa. Real-time quantitative PCR was used to detect the expression of PtATAF1-1 gene. The results showed that after treatment with ABA hormone stress, the expression level of PtATAF1-1 gene decreased at 1 h, but increased at 12 h and 24 h, and reached the peak at 24 h. Compared with the control group at 12 h and 24 h, the expression level of PtATAF1-1 gene in ABA treatment group increased. The results showed that the expression level of PtATAF1-1 gene responded to ABA treatment (Figure 6A). After Flg22 treatment, the expression level of PtATAF1-1 increased at 1 h and 12 h but decreased at 24 h. The expression level of PtATAF1-1 was higher at 1 h, 12 h and 24 h than that of the control group, indicating that the expression level of PtATAF1-1 responded to Flg22 treatment (Figure 6B). The blank control group was treated with water.

2 Discussion

ATAF1 is a transcription factor belonging to the plant NAC family, which exists widely in the Plantae. It has been found that ATAF1 and ATAF2 genes in Arabidopsis thaliana are attacked and induced by pathogens (Zheng et al., 2009). Arabidopsis ATAF1, a NAC transcription factor, is a negative regulator of the defense response of necrotizing fungi and bacterial pathogens. When ATAF1 is silenced, the resistance of plants is enhanced and participates in the regulation of many abiotic stress and necrotrophic pathogen infection responses (Wang et al., 2009; Wu et al., 2009).

In this study, PtATAF1-1 gene was homologous cloned from Populus trichocarpa, and functional domain analysis of the protein encoded by PtATAF1-1 gene showed that it had a conserved NAM domain, and phylogenetic tree analysis results proved that the pTATAF1-1 protein did belong to the NAC family. The phylogenetic analysis of Arabidopsis thaliana and Populus trichoderma does not fully conform to the genetic relationship of the species, and it may be that the evolution of protein sequence is affected by their unique living environment, different growth and development pattern and life cycle, so there may be differences in protein function. The physical and chemical properties and protein structure of PTATAF1-1 were analyzed by bioinformatics, which provided a reference for understanding the function of plant ATAF1 gene.

Previous studies on subcellular localization of ATAF1 protein showed that ATAFl-GFP fusion protein was verified by transient expression of Arabidopsis ATAF1 gene in onion epidermal cells by gene bombardment (Lu, 2006, China Agricultural University, pp.55-56). Cucumber NAC transcription factor CsATAF1 was obtained by homologous cloning method, and subcellular localization results showed that CsATAF1 protein was nuclear localization (Wang, 2018). In this study, subcellular localization analysis of 895 Poplar of Nanjing Forestry University showed that pTATAF1-1 protein was also located in the nucleus, which was the same as previous results and also in line with the functional requirements of transcription factors.

Studies have shown that NCED3 is the regulatory target gene of ATAF1. In plants overexpressed by ATAF1, the transcription abundance of NCED3 increases and plays a regulatory role in the biosynthesis of ABA (Jensen et al., 2013). In this study, real-time quantitative PCR was used to analyze the expression pattern of PtATAF1-1. PtATAF1-1 gene was responsive to both ABA and Flg22 treatments, but the effects were different. After ABA treatment, the expression level of PtATAF1-1 gene decreased first and then increased, and was higher than that of control group at 12 h and 24 h. After treatment with Flg22, the expression level of PtATAF1-1 gene in Populus trichocarpa increased first and then decreased, which were all higher than the control group. It is concluded that PtATAF1-1 gene may be involved in response to ABA signal and bacterial infection, and has certain functions in these biological processes.

At present, the in-depth study on the function of PtATAF1-1 gene in Populus trichocarpa is not clear. The above results preliminarily analyzed the structure, subcellular localization and expression level of PtATAF1-1, providing direct genetic evidence for the role of PtATAF1-1 as a plant stress regulator.

3 Materials and Methods

3.1 Plant materials and main reagents

The tissue culture seedlings of Populus trichocarpa and poplar 895 were subcultured in our laboratory and grew in an incubator with temperature of 22°C/18°C (day/night), light of 16 h/8 h (light/dark) and 50% environmental humidity. Top10 susceptible Escherichia coli samples were purchased from Tiangen Biotech, Beijing, China. pMD19-T clone vector was purchased from Dalian TaKaRa Company. RNA extraction kit, PCR product recovery kit, DNA gel recovery kit, plasmid small extraction kit, protoplast separation and transformation related reagents and kits, plasmid small extraction medium quantity kits were purchased from Tiangen Biotech, Beijing, China. The reverse transcription kit was purchased from Promega, Madison, WI, USA. The real-time quantitative kit was purchased from Roche Applied Science, Indianapolis, IN, USA. The expression vector p2GWF7.0 was stored in our laboratory.

3.2 Cloning of PTATAF1-1 gene and amplification of ORF core sequence

Poplar leaves were taken for RNA extraction and fully ground with liquid nitrogen, according to the instructions of Plant RNA extraction Kit RNAprep Pure Plant Kit (Tiangen). Amplify 3¢-RACE and 5¢-RACE segments using the RACE Kit. Follow the SMARTer® RACE 5¢/3¢ Kit (TaKaRa) instructions. After gel cutting and recycling, 3¢-RACE and 5¢-RACE products were connected to pMD19-T vector (TaKaRa), and the reaction procedure of cloned vector connection was 16℃ for 1 h. 2.5 μL of ligand products were used for transformation of Top10 E. coli. Positive clones were selected for PCR detection and sequenced to obtain 3¢-RACE and 5¢-RACE product sequences. Further splicing was performed to obtain the full-length sequence of the gene and analyze its ORF region. The full-length coding sequence (ORF) of pTATAF1-1 was cloned from the cDNA previously obtained by KOD-Plus (Toyobo, Japan), and a tail was added for another round of PCR extension reaction. PCR product recovery kit was used to recover the amplified fragment and connect it with the pCR8^TM/GW/TOPO^TM entry vector, and then connect it with the expression vector p2GWF7.0 by LR reaction of GATEWAY technology.

3.3 Bioinformatics analysis of PtATAF1-1 gene

The RBH method was used to select the Populus trichocarpa database for Blastp in phytozome (https://phytozome.jgi.doe.gov), and the genes with high homology were found by sequence comparative analysis. Then, the genes with the highest homology were found by reverse alignment in the TAIR (https://www.arabidopsis.org/Blast/index.jsp), and the PtATATF1 gene to be cloned was determined by bidirectional alignment. NCBI Blastp was used to retrieve protein similarity, and ATAF1 amino acid sequences of other plants were downloaded. Clustalx2.1 multi-sequence alignment software was used to perform multiple alignment of amino acid sequences and predict their conserved domains. The PF sequence number of NAM was found using Pfam 32.0 (http://pfam.xfam.org/), and the domain of NAM was predicted using the Smart online tool (http://smart.embl.de/). Phylogenetic tree of PtATAF1-1 homologous protein was constructed by MEGA X. The number of Bootstrap cycles using the adjacency method is 1000. ProtParam online tools (https://web.expasy.org/protparam/) preliminary forecast of molecular weight, isoelectric point, unstable factor. The online site (http://life.nthu.edu.tw/~b861625/protscale.html) ProtScale predicts protein hydrophilicity/hydrophobicity. Secondary structure prediction of PtATAF1-1 protein was performed by SOPMA website (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html). Online tools Phyre2.0 (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) was used to predict PtATAF1-1 protein tertiary structure.

3.4 Subcellular localization of PtATAF1-1 protein

Referring to the preparation method of poplar protoplasm system (Tan et al., 2013), the protoplasts of tissue culture seedlings of Poplar 895 with good growth at about 6 weeks were extracted by enzymatic hydrolysis. p2GWF7.0-PtATAF1 and positive control plasmid were transformed into protoplasts respectively by PEG mediated method (positive control was empty vector p2GWF7.0 only carrying GFP reporter gene). The localization of PtATAF1 in protoplasts was observed by fluorescence microscope (Axioscope.A1, Carl Zeiss) using GFP as reporter gene.

3.5 qRT-PCR analysis of PtATAF1-1 gene

Treatment with ABA hormone and Flg22, ABA 0.01 mmol/L, bacterial flagellin short peptide Flg22 2 μmol/L, adding 0.01% Silwet-77 sprayed on leaves, and then harvested leaves at 0 h, 1 h, 12 h, 24 h after treatment. They were immediately frozen in liquid nitrogen and stored at -70℃. The blank control group was treated with H₂O. After growing for 4 weeks, tissue culture seedlings of Populus trichocarpa were selected and RNA was extracted from leaves of Populus trichocarpa, and cDNA was synthesized using ordinary reverse transcription kit. The qRT-PCR amplification reaction was carried out using ABI7500 fluorescence quantitative PCR instrument. Using EF1α gene as internal reference, the upstream and downstream primers used in qRT-PCR reaction were PtATAF1-1-qF and PtATAF1-1-qR respectively (Table 1). The PCR program was set as 50℃ for 2 min, 95℃ for 10 min, 95℃ for 15 s, and 60℃ for 1 min, in total, 40 cycles. According to the method of references (Schmittgen and Livak, 2008), the relative quantitative method was used to calculate the relative expression level of the target gene according to the 2^-ΔΔCT method, and 3 biological replicates were set. IBM SPSS 20.0 was used for one-way anova and multiple comparison of the data. Origin 2017 software was used to draw charts and analyze the significance of differences.

Authors’ contributions

PJ is the experimental designer and executor of this study. She has completed data analysis and written the first draft of the paper. WHR participated in experimental design and analysis of experimental results; CQ is the architect and principal of the project, directing experimental design, data analysis, paper writing and revision. All authors read and approved the final manuscript.

Acknowledgments

The study was supported by the National Natural Science Foundation of China (31570639).

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