Systematic Identification and Analysis of NAC Gene Family in Moso Bamboo (Phyllostachys edulis)

Background: NAC (NAM/ATAF1/2/CUC2) gene family is a large plant-specific transcription factor family, which is implicated in many functions, such as morphogenesis, the thickness formation of secondary cell walls as well as biotic and abiotic stress and more. In moso bamboo ( Phyllostachys edulis ), 94 PeNACs have been identified and three members are predicted to relate to the secondary cell wall. However, there were few studies on moso bamboo NAC genes under stress. Results: In this study, we re-identified 165 PheNACs with the latest moso bamboo genome data and divided them into 12 subfamilies using NAM domains. Gene structure and motif distribution manifested the NAC gene family was fairly conserved. Evolutionary analysis showed that the segmental duplication played a significant role in the expansion of NAC genes and the relationship between moso bamboo and Brachypodium distachyon was closest than beween moso bamboo and other four species ( Arabidopsis thaliana, Oryza sativa , Sorghum bicolor and Zea mays ). Based on the promoter analysis of the 27 NAC members in A subfamily, quantitative real-time PCR exhibited these genes reacted differently under drought, high salt, abscisic acid and methyl jasmonate treatments. Finally, we selected out four potential stress-associated genes (PheNAC001, -056, -080 and -100) and found they all localized in the tobacco nucleus and had transcriptional activity in yeast. Conclusions: These preliminary results provide valuable information for mining potential resistance NAC genes and lay theoretical basis for breeding new stress-resistant varieties in moso bamboo.

3 chromosome-level reference and alternative splicing atlas in 2018, which was helpful for the classification, evolution and functional analyses of moso bamboo genes [1,2].
Moso bamboo, like other plants, is also forced to encounter multiple adverse environmental stresses throughout the life time, such as drought, cold and high salinity etc. To adapt to these unfavorable conditions, moso bamboo has been and is constantly undergoing evolutionary mechanisms at physiology, biochemical and molecular levels [3]. Generally, when plants are subjected to external stress, they will be induced the production of pivotal enzymes and metabolic proteins, such as dehydrins and late embryogenesis abundant (LEA) proteins [4]. Meanwhile, the receptors on the cell membrane sense the stress stimulation and convert it into the signal for intracellular transmission to mediate the expression levels of some responsive genes called regulatory proteins [5]. These regulatory genes will further modulate the expression levels of downstream genes by binding to DNA fragments in the promoter region of the target genes, for instance transcription factors (TFs): AP2/ERF [6], bZIP [7,8], WRKY [9], MYB [10] and NAC [11] have been identified in rice and Arabidopsis.
For plant-specific transcription factor NAC, one NAM domain is generally located at the N-terminus of the gene and has ~ 150 amino acids [12], which can be further divided into 5 sub-domains (A-E): the ACD sub-domains are relatively conservative; the B and E sub-domains are divergent [13]. The domains from ANAC019-(1-168), NAC2/ORE1-(1-165), ATAF1-(1-165) and VND7-(1-163) can be combined with CGT [GA] or CGT core sequences in vitro [14]. Comparatively, the diverse C-terminal transcription regulation region (TRR) can activate or suppress the transcription of multiple target genes, for instance ANAC019 [14,15]. Although the C-terminal sequence is variable, simple repeat regions rich in acidic amino acids, prolines, serines, and threons are necessary for transcriptional activation [13]. Also, the area might have transmembrane motifs (TMs), which anchor to the plasma membrane and might relate to signal perception and conduction [15].
To date, researchers have paid more and more attention to NAC genes due to their numerous functions. Accumulating functional reports have showed many NAC genes in rice play key roles in biotic and abiotic stress containing SANC1/OsNAC9 [ and Botrytis cinerea [26], and it also directly regulates abscisic acid (ABA) biosynthetic gene NCED3 by binding its promoter in vivo [27]. Until now, increasing evidences have uncovered NAC genes have great functions not only in biotic and abiotic stress, but also in many other aspects such as cell division [28], the formation and maintenance of sepals and stamens (floral organs) [ predicted three members (PeNAC8, PeNAC36 and PeNAC73) were related to the secondary cell wall [40], we use the newly issued moso bamboo genome data to re-identify NAC family genes, analyze their basic characteristics, the evolutionary patterns and the expression levels under different stress treatments to quickly find key genes association with stress in this study. Therefore, these results are helpful to understand the characteristics of NAC genes and lay the foundation for revealing moso bamboo internal molecular mechanisms under stress.

Phylogenetic tree of PheNACs and OsNACs domains
After determining the specific location of the NAC protein domain using pfam website, the conserved domain sequences of 143 OsNACs and 165 PheNACs were accurately extracted from their complete 6 amino acid sequence by tbtools software [47]. Then an unrooted tree was constructed using MEGA6 program by the neighbor-joining method with 100 bootstrap values, whereas the single phylogenetic tree of PheNACs was set to 1000 bootstrap values [48]. Moreover, multiple alignments were also carried out through DNAMAN software [49].

Gene structure and conserved motifs
In order to more clearly see the specific positions of exons, introns and domains on genes, GSDS (http://gsds.cbi.pku.edu.cn/index.php) [50,51] was employed to visualize the gene structure.

Scaffold location, duplication relationship and selection pressure
First, an analytical view for 165 PheNACs were mapped in offline MapInspect. Then, we used Blastp and MCScanX to examine duplication genes [53]. The paralogous pairs were visualized using Amazing Super Circos; and orthologous pairs between moso bamboo and the other five plants were displayed using Dual Synteny Plotter (https://github.com/CJ-Chen/TBtools). Even all the non-synonymous substitution rate (Ka) and the synonymous substitution (Ks) values of homologous pairs were also measured by TBtools [47]. In genetics, the Ka/Ks ratio is an indicator of selective pressure. Ka/Ks >1 means positive selection; Ka/Ks =1 implies neutral evolution and Ka/Ks <1 represents purifying selection [54] . Using the formal T=Ks/2λ (λ=6.5×10−9), divergence time was also calculated [2] Gene ontology annotation 165 NAC protein sequences were loaded into Blast2GO program, then blasted, mapped and annotated to obtain GO terms, which were mainly divided into three categories: biological process, cellular component and molecular function [55].
RNA isolation of moso bamboo leaves were manipulated with RNAiso Plus (TaKaRa Code No.9108) according to the manufacturer's protocol. Then the cDNA was synthesized using thePrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa Code No. 6210A) as the instructions described. The reverse transcription reaction was at 30 °C for 10 min followed by 42 °C for 60 min. In addition, Gene primers were designed using Primer Premier 5.0 software, and moso bamboo CDS library was uploaded manually on the NCBI website to check the primer specificity (Additional file 8: Table S6). The intrinsic membrane protein 41 (TIP41) was used as a reference gene [57,58]. The qRT-PCR program was set to 95 °C for 30 s; 40 cycles of 95 °C for 10 s, 55 °C for 15 s, and 72 °C for 10 s.

Subcellular Localization and Transcriptional Activity
The pCAMBIAI1305 vector with GFP protein driven by 35S was inserted by CDS with stop codon removed, the fusion vectors were transferred into EH105 strains respectively (BIO-RAD, Hercules, CA, USA) for injection into Nicotiana benthamiana to observe the fusion protein location using confocal laser scanning microscop (CarlZeiss LSM710). In addition, the PheNACs were cloned and independently insert into the pGBKT7 vector (Clontech, Palo Alto, CA, United States), which was used to detect whether the genes had transcriptional activity in yeast. The positive control pGBKT7-53 + pGADT7-T, the negative control pGBKT7 and pGBKT7-PheNAC were respectively transferred to Y2HGold strains (The competent cells purchased from Shanghai Weidi biotechnology Co., Ltd.) according to the instructions. The positive strains were cultured on double deficiency medium (SD/-Trp/-Leu) and the other two groups were grown on single deficiency selection medium (SD/-Trp).

Identification and classification subfamily of moso bamboo NAC genes
To acquire NAC members in moso bamboo, 697 NAC protein sequences including 116 AtNACs, 143 OsNACs, 114 BNACs, 180 SbNACs and 144 ZmNACs were used as guidance for BLASTP in moso bamboo protein database (Additional file 3: Table S1). Correspondingly, the number of collected moso and PheNAC162 were the largest and smallest genes, accounting for 69% and 39%, respectively. The protein MWs were from 15.82 (PheNAC064) to 99.32 kDa (PheNAC008), and the pIs ranged from 4.53 (PheNAC111) to 9.89 (PheNAC006). Unexpectedly, except for PheNAC160 in the cytoplasm, the remaining 164 PheNACs were predicted to be located in the nucleus (Additional file 4: Table S2). In addition to PheNAC030 and PheNAC096, there were also 10 PheNACs with a membrane-bound motif at their own C-terminus, 8 of which were from H subfamily (Fig. S1).

Gene structure and protein motifs for PheNACs
To better understand the structural characteristics of moso bamboo NAC gene family, exon/intron structure and motif composition were examined. We counted 9 kinds of exon number (1, 2, 3, 4, 5, 6, 7, 9 and 11). Among these, the NAC genes with three exons were 120 members (72.73%) and with four exons had 16 (9.7%). PheNAC008 had the most exons (11) and PheNAC097 contained an entire exon. And each gene had only one NAM domain basically distributed on the first two exons. In subfamily H and J, some genes had longer UTR (untranslated region) (Fig. 2B). Additionally, the genes in a common cluster shared the similar gene structure and motif compositions. For example, the members in subfamily L had three exons, even their lengths were so close and the formation and order of motifs seemed to be completely identical. Surprisingly, 134 members (81.2%) of nine subfamilies shared the motif 1, 6, 4, 3, 5, 2 and 7. And the motif 1 represented the NAM domain, which was why each NAC gene had it. Moreover, the seven members in subfamily E, their motif compositions were quite consistent and they all contained motif 8 and 10. And motif 9 was discovered in three members PheNAC095, -096 and -097, suggesting they might play special functions (Fig. 2C).

Gene distribution and evolutionary pattern analysis of PheNACs
A total of 165 PheNACs were mapped onto 23 moso bamboo scaffolds and the highest frequency was  Table S3). Based on previous reports, moso bamboo had undergone two whole-genome duplication (WGD) events and the time of two events was very close (peak Ks:0.499 ~ 1.556, recent peak Ks: 0.197 ~ 0.382). Therefore, we roughly attributed 63 genes to derive from the first WGD event ( Ks values of 53 pairs were in the range of 0.499 ~ 1.556) and the remaining 82 NAC members might result from the recent WGD event [59]. Meanwhile, we noticed 42 NAC genes correspond to 3 paralogs and 32 members had 2 homologous genes in moso bamboo genome (Additional file 5: Table S3), which might prove moso bamboo genome had experienced two WGD events. Additionally, the ka/ks ratios of 150 paralogous pairs (Phe-Phe) were lower than 1 (5 paralogous pairs have no Ks value), implying a purifying selection for these PheNACs (Fig. 3C).

Microsynteny analysis of NAC genes between moso bamboo and other five grass species
To further explore the evolutionary relationships of NAC genes between moso bamboo and and other  Table S4).

Gene Ontology Annotations
To clarify the function of the 165 PheNACs, the gene ontology (GO) was performed and gained 624 GO terms which were divided into three main categories: biological process, cellular component and molecular function (Fig. 5). Both 104 items were implicated in bio-synthetic process and cellular nitrogen compound metabolic process, and 52 were commented on transcriptional regulation, DNAtemplated. Totally, the three entries occupied 81.5% in biological process. Meanwhile, we found 11 items were related to positive regulation of response to salt stress (GO: 1901002), 8 involved in positive regulation of response to water deprivation (GO: 1902584), and 4 responded to cold (GO: 0009409). Actually, the 11 salt-stress genes included the drought and cold-stress genes. In addition, it was unexpected that only one gene (PheNAC041) was interpreted to participate in the cell wall organization or bio-genesis, which was not consistent with the previous report (Shan et al., 2019).
However, 125 terms in cellular component were classified into 5 kinds, 120 of them were annotated nucleus (GO: 0005634). Two gene were noted with membrane (PheNAC074 and PheNAC125, GO: 0016020) and one was plasma membrane (PheNAC096, GO: 0061617), which signified the three genes might play an important role in membrane composition. Among total 180 notes of molecular function, 170 terms were assigned to involve binding: 150 were associated with DNA binding, regulation of transcription; 15 were related to sequence-specific DNA (GO: 0043565) and 5genes (PheNAC003, -063, -064, -080 and -100) might concern protein binding (GO: 0005515). The details were seen in Additional file 7: Table S5.

Promoter analysis of SNAC subfamily
To explore the potential functions of NAC genes, the promoter region of 27 members gathered subfamily A was investigated in the first 2000bp of the translation initiation site (ATG). Promoter elements were mainly divided into three categories: plant growth and development, abiotic and biotic stress and phytohormone responses.
First and foremost, light responsive elements Sp1, Box 4 and G-Box total 268 accounted for 82.46%, of which 10 or more were detected in 13 members (PheNAC052, -127, -90, -003, -63, -032, -045, -080,   -100, -018, -103, -94 and -130). And the promoter elements of endosperm expression (GCN4_motif) and meristem expression (CAT-box) were also observed among 6 genes and 16 members, respectively. For O2-site (zein metabolism) and RY-element (seed-specific regulation), their numbers were 15 and 12, separately. In the second category, both ARE and GC-motif were associated with anaerobic induction. The former was identified within 24 PheNACs with a total of 51, while the latter had 24 out of 15 members. The low-temperature responsiveness elements (LTR) appeared 34 times among 27 PheNACs with 6 for PheNAC016, and TC-rich repeats occupied 7. And MBS, the MYB binding 12 site involved in drought-inducibility was also identified in 15 members (Fig. 6A). The abscisic acid responsiveness element ABRE ranked the first in the third category and PheNAC080 held the maximum number (22), followed by 94 responsive to MeJA cis-acting elements (CGTCA-motif). And other hormone-related elements including salicylic acid element, gibberellin response elements (Pbox, TATC-box and GARE-motif) and auxin-responsive elements (AuxRR-core, TGA-box and TGAelement) were also noticed no more than 3. In addition, three kinds of cis-acting elements (ABRE, CGTCA-motif and MBS) were shown the specific location in the promoter area. We could find 70% ABRE elements were scattered within the first 1000bp, and some were even concentrated in the first 500bp, for instance PheNAC003, -032, -045, -80 and -100 (Fig. 6B).

The expression levels of SNAC genes under different treatments
Based on the analysis of cis-acting elements and the published related articles, 27 NAC genes were selected for qRT-PCR at five time points under the treatments of drought and high salt, ABA and MeJA.
After 20% PEG6000 treatment, the expression of 15 genes were obviously increased (Fig. 7A), of which 8 had peaked over 10 times than the control group (0h), even PheNAC016 was up-regulated over 40-fold at 24h. Interestingly, almost these up-regulated genes reached their maximum at 12h.

Subcellular Localization and Transactivation Activity of Four NAC Genes
It is reported that a large proportion of NAC proteins are located in nucleus [60,61], and the EuLoc website predicted the 164 PheNACs were in the nucleus. Based on GO annotations, cis-acting elements and expression patterns analysis, PheNAC001, -056, -080 and -100 were picked for next subcellular localization and transcriptional activity analysis. The CDS without stop codon of four PheNACs independently fused with GFP constructs and the GFP control driven by CaMV 35S promoter were expressed in Nicotiana benthamiana leaves. The result showed the GFP signal was distributed throughout the cell with the 35S::GFP proteins, and PheNAC001, -056, 080 and -100 proteins were clearly localized in the nucleus, as predicted by the website (Fig. 9).
To investigate the transcriptional activity of the four PheNACs, pGBKT7-PheNACs, the positive control plasmids containing pGBKT7-53 and pGADT7-T, and the negative control plasmid pGBKT7 were independently transformed into the Y2HGold yeast strain. All of these transformants could grow and displayed visible white colonies on the SD/-Trp medium. In the SD/-Ade/-His/-Trp/X-α-Gal medium, the yeast cells with PheNACs and the positive control grew well and turned blue. In contrast, the negative control did not grow on this medium (Fig. 10). Thence, the four NAC fusion constructs could activate the transcription of the His3 and LacZ reporter genes, demonstrating they had self-transcription activity in yeast strains.

Sequence features
As a plant-specific large transcription factor family, 165 NAC members were identified and divided into 12 subfamilies based on the report [42]. First, we found the NAM domain with average length 128bp in pfam was shorter as described 150bp in other articles [62]. Hence, we manually aligned the sequences outside the NAM domain and found ~50bp following by NAM domain was a certain degree of conservatism. For one subfamily, their NAM domain sequences were quite conserved, whereas great differences existed between different subfamilies (Additional file 2: Fig. S2). As transcription factor, the function of NAC genes is mainly dependent on the NAM domain which can combine the promoter region specific cis-acting element of different downstream genes or interacts with other proteins. Generally, NAC genes mainly bind to the NACRS with core sequences CACG or CATGTG sequence [16,63]. As increasing reports, more NAC genes could combine with different DNA fragments. Arabidopsis transcriptional repressor CBNAC could bind to CBNACBS with a GCTT core sequence but not the CACG sequence [31]. SNAC1 could also recognize an ABA responsive element (ABRE) from OsbZIP23 promoter to monitor the expression level of several ABA signaling genes [64].
Thence, we speculated NAC genes could bind different DNA sequences to participate in different pathways due to distinct NAM domains. On the other hand, NAC gene function need to be controlled by upstream genes. MdHY5 interacted with the G-box from MdNAC52 promoter to affect the MdNAC52 transcription and Anthocyanin and proanthocyanidin (PA) synthesis [65]; AdNAC6 and AdNAC7, the targets for miR164, were both significantly up-regulated by exogenous ethylene in various fruit [66].
For the 165 NAC promoter regions, which contains various cis-acting elements such as Sp1, GT1motif, G-Box and Box 4 involved in light responsive elements, abscisic acid responsiveness element (ABRE) and the MeJA-responsiveness regulatory elements (TGACG/CGTCA-motif), MYB binding site involved in drought-inducibility (MBS) and low-temperature responsiveness elements (LTR) and many more, suggesting they might perform different functions at different stages (statistics but not shown).
In addition, the majority of NAC genes in moso bamboo and other species contained two introns, indicating that the family was structurally conservative (Fig. 2) [43]. Strikingly, the members of subfamily F, PheNAC096/097and PheNAC095/096 were two pairs of paralogous genes, but the three members had different numbers of introns: PheNAC095-(1), PheNAC096-(8), PheNAC097-(0), indicating PheNAC095 and PheNAC097 might lose introns during evolution process. The changes in gene structure might cause gene function change. In comparison with PheNAC066, PheNAC054 and PheNAC057 of subfamily G had one less intron. And PheNAC066 lacked motif 6 and contained two GO terms, whereas PheNAC054 and PheNAC057 were short of motif 4 but had 4 GO terms, which might explain that their function had changed (Fig. 2B, C, Additional file 7: Table S5).

Evolutionary relationships
In moso bamboo genome, the number for 165 NAC genes is a little more than 118 in Brachypodium distachyon, 151 in rice, 145 in sorghum and 152 in maize, which may be because moso bamboo genome (1.91Gb) contains more protein-coding genes (51074) than these monocotyledonous plants [1]. Certainly, NAC gene family has also been widely introduced in many dicotyledonous plants such as Arabidopsis thaliana (116) [41], potato (110) [67], soybean (152) [68], Populus trichocarpa (163) [69] and Asian pears (185) [70] etc. This different numbers might be not only correlated with the size of plant genomes but also related to gene duplication. The genomes of Arabidopsis thaliana (125Mb, 25498 protein coding genes) [71], potato (844Mb, 39031) [72], poplar (385Mb, 45555) [73], Asian pears (527Mb, 59552) [74] and soybean (1.025 Gb, 52051) [75] are smaller than that of moso bamboo and the total number of protein-coding genes varies widely. On one hand, potato has experienced two rounds of whole-genome triplication events (WGT) [72]; Arabidopsis thaliana and soybean have undergone three genome duplication events, including two doublings and one triple [76]; in Populus, apart from a triploid event, there is a single "salicoid" duplication event (Pduplication) [73]; Asian pears is similar to poplar, with one doubling and one triple [59]. Moso bamboo undergoes only two whole-genome doubling events, finally leading to an unbalanced gene number in different species. On the other hand, Asian pears, like moso bamboo, segmental duplication seemed to make a greater contribution in NAC gene expansion (Fig. 3A and B). We also identified 18 OsNAC genes involved in segmental duplication and 36 members associated with tandem duplication [42].

And in Brachypodium distachyon, both tandem duplicated and segment duplicated genes reached 22
BNACs [43]. In potato, 20, 27, 10 and 46 StNACs were discovered to be segmental, tandem, proximal and dispersed duplicated, respectively [67]. Therefore, different patterns of gene duplication contributed differently to NAC family gene amplification in diverse species.

As for Ks values of NAC orthologous pairs between moso bamboo and Brachypodium distachyon,
Oryza sativa, Sorghum bicolor and Zea mays (Phe-Bd, Phe-Os and Phe-Sb, Phe-Zm), the distribution situations were very similar to that of the entire genome (Fig. 4B, Supplementary Figure 11 Table   S4), meaning the closer relationships with moso bamboo were in turn Brachypodium distachyon > Oryza sativa > Sorghum bicolor > Zea mays. This is exactly consistent with the conclusions previously reported [2]. Additionally, in terms of Ka/Ks ratios, the values of 32 paralogous pairs (Phe-Phe) containing 43 PheNACs (Fig. 3C), 20 pairs (Phe-Bd), 26 pairs (Phe-Os), 26 pairs (Phe-Sb) and 34 orthologous pairs (Phe-Zm) were between 0.5 and 1 (Fig. 4C), signifying these genes were moving forward the direction of positive selection to adapt to the environment [77].

NAC genes response to stresses
The NAC functions have been well studied in rice [41,42]. Based on the N-J phylogenetic tree constructed using the domains from 165 PheNACs and 143 OsNACs, NAC genes that clustered together are predicted to have similar potential functions (Fig. 1). In SNAC group, there are six OsNACs with known functions: SNAC1 (LOC_Os03g60080.1), SNAC2/OsNAC6 (LOC_Os01g66120.1), OsNAC3 (LOC_Os07g12340.1), OsNAC5 (LOC_Os11g08210.1) and OsNAC10 (LOC_Os12g03040.1), which have been reported to be associated with biotic and abiotic stress, especially for OsNAC6 (LOC_Os01g66120.1). The gene aggregated with PheNAC080 and PheNAC100 and was induced by cold, high salt, drought and ABA [78]. Over-expressing OsNAC6 in rice exhibited increased tolerance to dehydration and high-salt stresses, as well as blast disease [79]. And the transgenic rice enhanced plant drought tolerance by affecting its direct targets NICOTIANAMINE SYNTHASE to promote the accumulation of the metal chelator NA [80]. Therefore, we speculated that PheNAC080 and PheNAC100 might also have similar functions and regarded them as the study focus. However, their performances were always the opposite under drought and salt treatments. The transcription level of PheNAC080 was suppressed under drought, but PheNAC100 was high expression at the observed time points (Fig. 7A). Under salt treatment, PheNAC080 showed high expression at 12 and 24h yet PheNAC100 presented low expression (Fig. 7B). Thus, their specific functions still need further experiments to explain. In addition, plant hormone ABA and Jasmonic acid (JA) are key regulators of stress signaling networks [81]. When plants suffer from drought or high salt stress, ABA will be generated to control stomatal closure by the SnRK/PP2C signaling pathway and endogenous jasmonic acid (JA) levels generally increase [81][82][83]. In this study, after ABA-treated, PheNAC080 and PheNAC100 were both low expression, although the former held the most ABRE elements (22) and the latter had 13 ones in their own promoter region. Meanwhile, we also found PheNAC001 was strongly induced by exogenous ABA and it had 8 ABRE elements (Fig 6 & 8A). The most noticeable is the varied performance of PheNAC100, whose expression was down-regulated first, then suddenly induced up-regulated nearly 20 times, then down and up again. PheNAC056 has the most CGTCAmotif elements (9), whereas its expression level was slightly increased but not significant under MeJA.
And OsNAC10 were annotated as the homologous gene of PheNAC001 and PheNAC056, whose overexpression improves rice drought tolerance and grain yield (Jeong et al., 2010). And a grapevine gene VaNAC17 was strongly induced exogenous ABA and methyl jasmonate (MeJA), whose overexpression in Arabidopsis plants enhanced drought tolerance by regulating endogenous JA biosynthesis and ROS scavenging [84]. Thus, these genes are also predicted to have similar functions with great possibility. Finally, we also proved that these four genes are located in the nucleus of tobacco cell and have transcriptional activity in yeast, which could explain the general properties as transcription factors.

Conclusion
In this study, we carefully re-identified the NAC members in moso bamboo using the latest genome data. After comprehensive investigation and analysis of these NAC genes, qRT-PCR assays showed the expression level of four genes (PheNAC001, -056, -080 and − 100) were induced to extent degree under different stresses, and they were all localized in the nucleus and both had transcriptional activity. To clarify the biological functions and related pathways of these four genes, more experiments need to perform. Thus, this study provides a useful reference for the functional research of NAC family genes.

Additional Files
Additional file 1: Fig S1. The specific location of membrane-bound motif protein. Additional file 3: Table S1. NAC gene ID name of five species (Arabidopsis thaliana, Brachypodium distachyon, Rice, Sorghum bicolor and maize).

Ethics approval and consent to participate
The seeds of moso bamboo were collected from Guilin in Guang Xi Province, China. And the seeds were provided and identified by the Guilin Forestry Bureau. In addition, all the materials of moso bamboo used and analyzed were available for non-commercial purpose. This article did not contain any studies with human participants or animals performed by any of the authors.

Consent for publication
Not applicable.

Supplementary Files
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