Identification and analysis of Chrysanthemum nankingense NAC transcription factors and an expression analysis of OsNAC7 subfamily members

Gene retrieval of C. nankingense NAC TFs

The A. thaliana NAC TF sequences were obtained from the Arabidopsis Information Resource (http://www.arabidopsis.org/). C. nankingense genomic data were downloaded from the website of the Institute of Chinese Medicine, Chinese Academy of Chinese Medical Sciences (http://www.amwayabrc.com/zh-cn/download.htm). Based on the A. thaliana NAC protein sequences, HMMER3.0 software was used to construct a multiple comparison HMM model, which was then used to search the C. nankingense genome protein data to obtain the CnNAC protein sequences. The CnNAC protein sequences were identified and screened using the SMART tool (http://smart.embl-heidelberg.de/) and Pfam (https://pfam.xfam.org/), and the predicted CnNAC TF members were obtained. The CnNAC TFs were used for subsequent analyses based on this comprehensive screening (Li et al., 2019).

Bioinformatics analysis of C. nankingense NAC TFs

The NAC TFs of C. nankingense, A. thaliana, and some of those of O. sativa were used to construct a rootless phylogenetic tree using the multiple comparison function in ClustalW (default parameters) and the neighbor-joining (NJ) method in MEGA-X software according to the existing A. thaliana and O. sativa NAC gene family classification system (Duan et al., 2019; Ooka et al., 2003; Hu et al., 2015). The rootless phylogenetic tree was used to study the phylogenetic relationships of the CnNAC TFs (Zhang et al., 2019). MEME online software (http://meme-suite.org/tools/meme) was used to predict the conserved motifs of the CnNAC TFs. The ProtParam online tool (https://web.expasy.org/protparam/) was used to analyze the physical and chemical properties of the CnNAC TF amino acid sequences and predict their subcellular locations (Li et al., 2019). The TMHMM online tool (http://www.cbs.dtu.dk/services/TMHMM-2.0/) was used to predict the protein transmembrane structure of the CnNAC TFs. The three-dimension (3D) models of the conserved NAC domains of the CnNAC proteins were predicted by Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) (Kelley et al., 2015).

Growth conditions and treatment of the plant materials

C. nankingense seeds (from Beijing Forestry University) were sown on peat to produce seedlings under growing conditions of 25 °C/23 °C (day/night), 16 h/8 h (day/night), and 2,000 Lux. The roots, stems, and 3–5 new leaves on the top of the seedlings were collected and quickly flash frozen in liquid nitrogen as experimental materials at 28, 44, and 60 d after seeding, respectively, and stored at −80 °C. Three replicates were taken for each period.

Rooted C. nankingense seedlings obtained from stem hydroponics were subjected to osmotic and salt stresses. The side shoots of C. nankingense seedlings grown for 44 d were cut out and inserted into cultivation medium containing MS medium and 0.1 mg/L IBA. The growing conditions were 25 °C/23 °C (day/night), 16 h/8 h (day/night), and 2,000 Lux. The cultivation medium was changed every 3–5 d, and rooted seedlings were obtained after 20 d. Then, the cultivation solution was changed to 20% PEG6000 or 200 mM NaCl solution for the osmotic and salt stress treatments, respectively. Samples were taken at 0, 1, 4, 8, 12, 24, and 36 h (Fan et al., 2016). The entire rooted seedling was taken as a sample and stored at −80 °C after flash-freezing in liquid nitrogen. Three replicates were taken for each period.

RNA isolation and real-time quantitative-polymerase chain reaction (RT-qPCR) analysis

Total RNA was isolated using the FastPure Plant Total RNA Isolation Kit (Code No. RC401-01; Nanjing Vazyme Biotechnical Co., Ltd., Nanjing, China). The reverse transcription of RNA was carried out using the PrimeScript™ RT reagent Kit with the gDNA Eraser (Perfect Real Time, Code No. RR047A; Takara Bio., Shiga, Japan). The primers used to analyze the expression of the OsNAC7 subfamily members were designed based on the C. nankingense genome (Table 1). RT-qPCR was carried out with on the Applied Biosystems StepONEPlus Real Time PCR System (Foster City, CA, USA), using ChamQ Universal SYBR qPCR Master Mix (Code No. Q711-03; Nanjing Vazyme Biotechnical Co., Ltd., Nanjing, China). The reaction system was 40 cycles of 95 °C for 20 s, 95 °C for 3 s, 60 °C for 30 s, 95 °C for 15 s, and 60 °C for 1 min (Wei et al., 2016), with actin7 from C. nankingense as the internal reference gene (Chen et al., 2018). The relative expression of genes was calculated by the 2^−ΔCt method.

Subcellular localization and functional analysis

The super1300 vector was used as the overexpression vector (kindly provided by Professor Huitang Pan of Beijing Forestry University, Beijing, China. Data S1). The vector uses super35S as the promoter. According to the open reading frame (ORF) of the CHR00069684 gene (Data S2) and the polyclonal sites of the super1300 vector, the endonucleases PstI and KpnI (NEB, Beijing, China) were selected to construct the binary expression vector according to the double enzyme method. The recombinant plasmid was transformed into Escherichia coli competent cells (DH5α), which were inoculated on solid LB medium containing 50 mg/L kanamycin; the resistance E. coli cells were validated by colony PCR. The obtained positive plaques were sent to the sequencing company (Beijing Ruibio BiotechCo.,Ltd, Amsterdam, the Netherlands) for sequencing and plasmid recovery. The super35S::GFP and super35S::CHR00069684::GFP vectors were introduced into Agrobacterium tumefaciens strain GV3101 (Weidi Biotechnology, Shanghai, China) by the freeze-thaw method and then inoculated on LB medium supplemented with 50 mg/L kanamycin, 50 mg/L gentamicin, and 50 mg/L rifampicin. The surviving plaques were validated by colony PCR, and the positive plaques were inoculated into LB liquid medium containing 50 mg/L kanamycin, 50 mg/L gentamicin, and 50 mg/L rifampicin for propagation. The transgenic tobacco (NT78) was obtained through A. tumefaciens-mediated transformation (Horsch et al., 1985), screened by 30 mg/L hygromycin, and validated by PCR identification of the target gene. CHR000696884::GFP fusion protein and GFP protein signals were observed by a laser confocal microscopy (Leica TCS SP5) in roots, stems, and leaves of the transgenic tobacco plants. The super35S::GFP vector was used as control.

Identification of the CnNAC TFs and the phylogenetic analysis

According to the A. thaliana NAC protein sequences, 153 predicted CnNAC TFs were identified from the C. nankingense genome data using HMMER3.0. Using ClustalW in MEGA, 153 C. nankingense NAC proteins, 105 A. thaliana NAC proteins, and 6 O. sativa NAC proteins were multi-sequence aligned, and a rootless phylogenetic tree was constructed using the NJ method by MEGA (Fig. 1, Data S3). According to the existing A. thaliana and O. sativa NAC gene family classification system (Duan et al., 2019; Ooka et al., 2003; Hu et al., 2015) and the topology of the rootless phylogenetic tree, the CnNAC TFs were divided into two groups of 19 subfamilies, including 17 known subfamilies and two unknown subfamilies. Group 1 contained 15 subfamilies, including OsNAC7, NAC1, NAM, ANAC077, ANAC011, OsNAC8, TIP, NAC2, ONAC022, TERN, SENU5, NAP, AtNAC3, ATAF, and unclassified 2, with a total of 111 members. Group 2 contained five subfamilies, which were ANAC063, ONAC003, ANAC007, unclassified 1, and ONAC001, with a total of 42 members. Among them, the ANAC077 subfamily occurred in both groups. The different branches of the phylogenetic tree testify to the functional diversity of this family.

According to further analysis of the CnNAC TFs (Fig. S1), 104 NAC genes were detected in the 17 known subfamilies, and 49 NAC genes were detected in the two unknown subfamilies. Three known subfamilies (ONAC022, NAC2, and OsNAC7) contained 13, 11, and 12 members respectively, with the greatest numbers of genes among all known subfamilies. However, the two unknown subfamilies, unclassified 1 and unclassified 2, contained 28 and 21 NAC genes, respectively.

Prediction of the protein structure is considered as a reliable analytical method to understand the molecular function of proteins (Kelley et al., 2015). Therefore, the 3D models of one gene from each branch of the NAC phylogenetic trees (Fig. 1), and all 60 CnNAC family members were constructed using the Phyre2 server (Fig. 2). c3ulxA (Chen et al., 2011) and d1ut7a (Ernst et al., 2004) were used as the reference model template, and the confidence level was set as 100%. The c3ulxA template is a DNA binding protein and annotated as stress-induced NAC1 according to the crystal structure of the conserved domain of rice stress responsive NAC1 in the PDB database. The d1ut7a template is annotated as a NAC domain. The 3D modeling results revealed that these CnNAC TFs possessed similar tertiary structures, implying that CnNAC TFs may evolved from the same ancestor sequence and/or under purifying selection to maintain stabilization during the long-term evolution after the initial divergence (Zhu et al., 2019).

Analysis of the CnNAC TF conserved motifs

The online tool MEME-Motif Suite was used to analyze the motifs of the 153 predicted CnNAC TFs (Fig. 3) and the positions of the 15 motifs on different genes were determined (Fig. 4). As a result (Fig. S2), five genes (CHR00045488-RA, CHR00007510-RA, CHR00036356-RA, CHR00079376-RA, and CHR00089260-RA) containing 11 motifs, respectively, constituted the most abundant motifs. These five genes all belonged to the unknown unclassified 1 subfamily. Nine genes (CHR00007511-RA, CHR00035414-RA, CHR00039887-RA, CHR00039888-RA, CHR00055754-RA, CHR00055752-RA, CHR00088453-RA, CHR00024060-RA, and CHR00058504-RA) containing 10 motifs, respectively, were also in the unknown subfamilies (six in unclassified 1 and three in unclassified 2). Sixty-two genes, which contained the highest number of 7 motifs, were distributed in 12 known subfamilies and two unknown subfamilies.

According to the comparative analysis of the motifs in different subfamilies (Fig. 4), the included motif bases were in motifs 1–7 in the known subfamilies of Group 1, but different subfamilies contained different numbers of motifs and different conserved motifs. For example, motifs 4, 6, 3, and 7 were in the NAM subfamily and motifs 1, 4, 2, 5, and 6 were in the OsNAC7 subfamily. However, some members in the unknown subfamily unclassified 2 had more motifs, such as motifs 10, 13, and 14, than in the other subfamilies. Each member in the known subfamilies of Group 2 contained far fewer motifs than those in the known subfamilies of Group 1. Members in the known subfamilies of Group 2 contained motifs 1, 3, 4, 5, 6, and 7, and lacked motif 2 compared to those in the known subfamilies of Group 1. However, the CnNAC TFs in unclassified 1 of Group 2 had the highest number of motifs, most of which were motifs 7–11.

Subcellular localization, protein transmembrane analysis, and physicochemical property analysis of CnNAC TFs

According to the subcellular localization and transmembrane analysis results (Table S1), 106 of the 153 CnNAC TFs were located in the nucleus and 47 were located outside the cell. Of the 111 members in Group 1, 106 were nuclear and 15 were extracellular, of which the 21 members within the unclassified 2 subfamily were all nuclear. Only 10 of the 42 members of Group 2 were located in the nucleus, the other 32 were located outside the cell, and 23 of the 28 members of the unclassified 1 subfamily of Group 2 were located outside the cell. However, the protein transmembrane analysis of the 153 NAC TFs revealed that only one member of Group 1 was expressed inside the membrane and eight members were expressed transmembrane, while the remaining 102 members were all expressed outside the membrane. There were no transmembrane-expressed Group 2 members, while two members were expressed inside the membrane and 40 members were expressed outside the membrane. The three genes expressed inside the membrane were all located in the nucleus, and the genes expressed transmembrane and outside the membrane all had members located in the nucleus and extracellularly, respectively.

The physicochemical properties of the CnNAC TFs were analyzed (Table S2). The lengths of the CnNAC TF amino acids ranged from 68 to 693 (mean length = 307.99). The molecular weight range of the CnNAC TFs was 7,787.81–80,985.3 Da with an average molecular weight of 35,195.1 Da. The theoretical isoelectric point (pI) range of the CnNAC TFs was 4.43–10.2, and the average pI was 7.09. The grand average of hydropathy was −0.72896, while the lowest value was −1.37, and the highest value was −0.275. The aliphatic amino acid index was 44.15–94.84, with a mean value of 64.58.

Analysis of the OsNAC7 subfamily gene expression levels during different C. nankingense growth stages

The OsNAC7 subfamily included 12 genes in A. thaliana, including SND1, NST1, URP7, BRn1/2, and VND1/2/3/4/5/6/7, the functions of which are all related to regulation of the formation of secondary cell walls in plant stems, roots, and anthers. Here, we analyzed the expression levels of the OsNAC7 subfamily genes in the roots, stems, and leaves of C. nankingense during different growth stages (Figs. 5 and 6, Table S3).

As a results, all the 12 members of the C. nankingense OsNAC7 subfamily played a role in roots, stems, and leaves. The expression levels of most of the OsNAC7 genes were generally low in the roots and leaves but high in the stems. However, differences in the expression levels were observed during different root, stem, and leaf developmental stages. Ten out of the 12 members in the OsNAC7 subfamily were expressed at higher levels in the stems at 44 d, including CHR00068684, CHR00042500, CHR00009966, CHR00043953, CHR00073261, CHR00032377, CHR00026420, CHR00003673, CHR00027311, and CHR00043175. CHR00048081 was expressed at high levels in the stems at 60 d. CHR00020838 was expressed at high levels in the stems during the whole growth process, which were much higher than those in roots and leaves. Five out of the 12 members in the OsNAC7 subfamily were significantly related to root growth. Among the 12 members. CHR00048081-RA was expressed at a higher level at 28 d, CHR00020838-RA and CHR00043175-RA had higher levels at 44 d, and CHR00009966-RA and CHR00073261-RA had higher levels at 44 d and 60 d. Five out of the 12 genes were expressed at higher levels in leaves at 60 d, such as CHR00043953-RA, CHR00032377-RA, CHR00026420-RA, CHR00003673-RA, and CHR00043175-RA.

Expression analysis of the C. nankingense OsNAC7 subfamily genes under the osmotic and salt stress treatments

All A. thaliana OsNAC7 subfamily genes are associated with secondary metabolism (Yamaguchi et al., 2008). However, among the O. sativa OsNAC7 subfamily genes, ONAC106 inhibits leaf senescence and also increases salt damage and tillering angle (Fang et al., 2008; Sakuraba et al., 2015); the expression levels of ONAC052, ONAC056, and ONAC084 are downregulated under high salt and drought conditions (Sun et al., 2015). Here, we analyzed the expression levels of the C. nankingense OsNAC7 subfamily genes under osmotic and salt stresses (Fig. 7, Fig. 8, Table S3).

The results of gene expression under osmotic stress (Fig. 7) revealed that all of the 12 genes in the OsNAC7 subfamily were regulated by osmotic stress, but their expression patterns were not consistent. Among them, the expression levels of CHR00003673-RA, CHR00027311-RA, and CHR00043175-RA continued to increase after treatment, indicating that these three genes may play a key role improving the resistance to osmotic stress in C. nankingense. The CHR00026420-RA expression fluctuated but was always higher than that of the control group, indicating that this gene is expressed during osmotic stress. CHR00069684-RA showed an obvious response to osmotic stress. The expression of CHR00009966-RA and CHR00048081-RA was inhibited at the initial stage of stress, and then gradually returned to normal expression levels. The response of CHR00042500-RA and CHR00020838-RA was not obvious, and CHR00043953-RA and CHR00073261-RA were severely inhibited by osmotic stress.

The gene expression results under salt stress (Fig. 8) revealed that all of the 12 genes in the OsNAC7 subfamily responded to salt stress, and were initially upregulated and then downregulated or were first downregulated and then upregulated; all decreased to the expression level of those of the control group or below by 36 h. The expression levels of CHR00069684-RA, CHR00009966-RA, CHR00048081-RA, CHR00043953-RA, CHR00026420-RA, CHR00003673-RA, CHR00027311-RA, and CHR00043175-RA increased first at different time points and then decreased. The significantly upregulated expression levels of these eight genes indicate that they may be important in the salt stress response. CHR00042500-RA, CHR00020838-RA, CHR00073261-RA, and CHR00032377-RA were slightly inhibited by salt stress, although their initial responses to salt stress were not obvious.

Subcellular localization and functional analysis

In this study, CHR00069684 that was closely related to the NST protein was used to construct the super35S::CHR00069684::GFP vector, which was then transformed into large leaf tobacco for subcellular localization observation and functional validation. GFP signals were observed in the nucleus and cell membrane of leaves, stems, and roots of super35S::GFP transformed tobacco plants (Fig. 9). No fluorescence signal was detected in the super35S::CHR00069684::GFP transformed tobacco leaves (Fig. S3), whereas there were strong GFP signals in the cell membrane of their roots and stems (Figs. 9E and 9F). The reason why the 35S promoter-guided CHR00069684 expression showed tissue specificity need to be further studied in the future.

The super35S::CHR00069684::GFP transformed and control tobacco plants were treated with 200 mM NaCl, 200 mM ABA, or a low temperature of 4 °C (Fig. 10). The results showed that the transgenic tobacco plants were more resistant to salt and low temperature and more sensitive to ABA than the control plants. Moreover, the differences in the growth and development of tobacco plants were observed (Fig. 11); the flowering time of the transgenic tobacco was 10 d earlier than that of the control plants.