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Article

Identification of Stress Responsive NAC Genes in Casuarina equisetifolia L. and Its Expression Analysis under Abiotic Stresses

by
Xiaomei Li
1,†,
Nan Li
2,†,
Dandan Wen
1,
Jianfeng Yu
1,
Jiadu Hong
1,
Mengjie Wu
1,
Longjun Cheng
1,* and
Shuai Meng
1,*
1
State Key Laboratory of Subtropical Silviculture, Zhejiang Agriculture and Forestry University, Hangzhou 311300, China
2
Zhejiang Forest Resources Monitoring Centre, Hangzhou 310020, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2024, 14(3), 535; https://doi.org/10.3390/agronomy14030535
Submission received: 27 January 2024 / Revised: 24 February 2024 / Accepted: 2 March 2024 / Published: 5 March 2024
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Abstract

:
NAC (NAM, ATAF and CUC)-like transcription factors, a class of plant-specific transcription factors, play a pivotal role in plant growth, development, metabolism, and stress response. Notably, a specific subclass of NAC family, known as SNAC (stress-responsive NAC), is particularly involved in the plant’s response to abiotic stress. As a very useful tree, Casuarina equisetifolia L. also has excellent stress resistance properties. To explore gene resources of C. equisetifolia which are associated with stress resistance and the molecular mechanisms that it employed is very helpful to its molecular-assisted breeding. In this study, 10 CeSNAC transcription factors were identified by constructing the phylogenetic tree of 94 CeNACs from the genome of C. equisetifolia L. together with 79 SNAC in different plant species. Phylogenetic tree analysis revealed that these 10 CeSNAC genes are classified into the ATAF (Arabidopsis transcription activation factor), NAP (NAC-like, activated by AP3/P1), and AtNAC3 subfamilies of the NAC family, all featuring the typical NAM (no apical meristem) domain, with the exception of CeSNAC7. In addition, all NAC transcription factors, except CeSNAC9, were localized in the nucleus. Examination of the CeSNAC promoter unveiled the presence of stress response elements such as a STRE (stress responsive element), an MBS (MYB binding site), an ABRE (abscisic acid responsive element) and a LTR (low temperature responsive element). Under various stress treatments, the majority of CeSNAC expressions exhibited induction in response to low temperature, drought, and high salt treatments, as well as ABA (abscisic acid) treatment. However, CeSNAC6, CeSNAC7, and CeSNAC9 were found to be inhibited specifically by drought treatment. Additionally, only CeSNAC3 and CeNAC9 expression was hindered while the rest of the CeSNAC expression were induced by MeJA (methyl jasmonate) treatment. These findings shed light on the relationship between different CeSNAC genes and their responses to abiotic stress conditions, providing valuable insights for further research into CeSNAC functions and aiding the development of stress-resistant varieties in C. equisetifolia.

1. Introduction

To adapt to adverse environments, plants have evolved intricate mechanisms spanning from physiological to molecular levels. Transcription factors (TFs), including AP2/ERF (APETALA2/ethylene responsive factor), MYB (v-Myb myeloblastosis viral oncogene homolog), NAC (NAM, ATAF, and CUC), WRKY, Bhlh (basic helix-loop-helix), zinc finger proteins, and ZIP (basic leucine zipper), play a pivotal role in plant stress responses at the molecular level [1,2]. The NAC transcription factors, a distinctive class among plant TFs, are assumed to be involved in regulating abiotic stress responses like low temperature, drought, and salinity [3,4]. NACTFs are characterized by the NAC domain, sharing a high sequence similarity with proteins encoded by the NAM gene in Petunia hybrida and the ATAF1 (Arabidopsis transcription activation factor1), ATAF2 (Arabidopsis transcription activation factor2), and CUC2 (cup-shaped cotyledon 2) genes in Arabidopsis thaliana [5,6,7]. The NAC domain, typically located at the N-terminal, consists of approximately 150 amino acid residues divided into five subdomains: A, B, C, D, and E, with the D and E parts serving as DNA-binding domains. The transcriptional activation region (TAR) is variable and located at the C-terminal of the protein [8].
NAC TFs are present in a wide range of plant species, contributing to growth, development, stress response, hormone signal transduction, and secondary metabolism [9,10,11]. A large number of NAC TFs have been proven to play vital roles in facilitating plant adaptation and survival to challenge an adverse environment [12,13]. In Arabidopsis, ANAC019 and ANAC055 intricately regulate the ABA (abscisic acid) signaling pathway under drought stress, thereby enabling the plant to adapt to drought stress [14,15]. SlNAC4 was proved to be involved in drought and salt stress response possibly through the JA (jasmonate) pathway [16]. ANAC072 regulates the ion balance and ROS scavenging pathways under salt stress, enhancing the plant’s tolerance to saline environments [17,18]. In maize, ZmNAC55 regulates the ABA signaling pathway and water utilization under drought stress, increasing the resistance of plants to drought [19]. Overexpression of ZmNAC111 significantly improves drought tolerance in the seedling stage by upregulating ABA-dependent stress-responsive genes [20]. OsNAC10 is strongly induced by drought, high salinity, and ABA. When it was overexpressed specifically in the roots of rice, tolerance to drought, high salinity and the low temperature of transgenic plants all were increased at the reproductive stage [21], while overexpression of OsNAC17 could enhance drought tolerance through increased lignin accumulation in plants [21,22]. Notably, stress-responsive NAC TFs form a distinct group known as SNAC (stress-responsive NAC) with a close evolutionary relationship [23,24,25,26,27]. Casuarina equisetifolia L. is a very useful tree which is distributed in tropical and subtropical regions. Because of its superior biological characteristics, such as fast growth, wind and salt resistance, and nitrogen fixation, it is widely planted for wood production, paper pulp, shelterbelts along the coastal area, and ecological restoration in the region of high salinity, drought and low nutrient soil [28,29]. However, it is sensitive to cold stress [30]. Leveraging Casuarina’s excellent stress resistance properties, it serves as an ideal model to study the molecular mechanisms of abiotic stress responses in plants. With the accomplishment of the C. equisetifolia genome sequence, research focused on understanding the molecular mechanisms of salinity and cold responses has commenced [29,30,31,32]. Nevertheless, the molecular mechanisms underlying its response to abiotic stresses remain insufficiently understood. To further explore the stress resistance gene resource and better understand their mechanism is very meaningful for the breeding of C. equisetifolia.
In this study, ten CeSNACs were identified from the C. equisetifolia genome through phylogenetic analysis of CeNAC and SNAC proteins from other species. The characteristics of CeNAC genes and the protein structure were investigated. Additionally, the expression patterns of CeSNACs under different abiotic stress conditions such as ABA, MeJA, low temperature, drought, and high salt were also analyzed with RT-qPCR. These results lay the foundation for further exploration into the molecular mechanisms regulating stress responses in C. equisetifolia and offering potential implications for the development of stress-tolerant varieties.

2. Materials and Methods

2.1. Identification of CeSNAC Genes

The Hidden Markov Model (PF02365) file containing conserved domains of NAC TFs was downloaded from the Pfam database (http://pfam.xfam.org/, accessed on 9 March 2023). HMMER (ver3.3.1) software was used to screen for NAC family members in C. equisetifolia with the protein database file downloaded from https://www.ncbi.nlm.nih.gov/datasets/genome/GCA_028551475.1/ (accessed on 26 January 2024); the expect value (e-value) was set as 1 × 10−20. The resulting sequences were employed to construct a new, specific NAC HMM for C. equisetifolia using the hmmerbuild program in HMMER software. A subsequent search of the protein database was conducted with an e-value of 0.01. Duplicate genes were then removed to identify the NAC family members in C. equisetifolia. To find SNAC members within the CeNAC family, SNAC proteins from other species were gathered by reviewing the relevant literature. A total of 74 SNAC proteins, along with all CeNAC protein sequences, were used to construct a phylogenetic tree using MEGA7.0 with the maximum likelihood method (bootstrapping repeated 1000 times). CeNAC members clustering with SNACs from other species were classified as CeSNACs. For the characteristics of CeSNAC proteins, the molecular weight and isoelectric point were analyzed using the online software ExPASy (https://web.expasy.org/compute_pi/, accessed on 12 May 2023). Additionally, the subcellular localization of CeSNACs proteins was predicted using the PSORT program (https://psort.hgc.jp/, accessed on 12 May 2023).

2.2. Analysis Structural Characterization of CeSNAC Genes and Coding Proteins

CeSNACs protein sequences were submitted to the MEME online website (https://meme-suite.org/meme/tools/meme, accessed on 13 January 2024) to search for motifs in the CeSNAC protein sequences and the motifs with e-values greater than 0.01 were discarded. Additionally, the CD-Search program (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 19 May 2023) was employed to search conserved domains within CeSNAC proteins. Additionally, the gene structure of CeSNACs was analyzed by GSDS2.0 online software (http://gsds.gao-lab.org/, accessed on 19 May 2023). Tbtools (V1.09) software was used to create figure files for these results [33].

2.3. Analysis of Cis-Acting Elements of CeSNAC Gene Promoters

A total of 2000 base pairs upstream of ATG (the start codon) of CeSNAC genes were retrieved from genome sequence files to act as promoter sequences. The PlantCare platform (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 19 May 2023) was used to predict cis-acting elements on these promoters. Stress-related cis-elements of CeSNAC promoters were analyzed with Excel software (2022).

2.4. Subcellular Localization of CeSNAC Proteins

The coding sequences of CeSNACs were amplified using cDNA templates reverse-transcribed from mRNA extracted from leaves of 3-month-old seedlings. Then, they were infused with GFP driven by 35S promoter to construct the vector with pCambia1300. The recombinant plasmid was transformed into GV3101 (Agrobacterium tumefaciens) and transiently transformed into tobacco plants. GFP checking was conducted with confocal laser scanning microscopy on Zeiss LSM810 systems (Jena, Germany).

2.5. Plant Material and Stress Treatments

One-month-old clones of C. equisetifolia, which were planted in 0.5 L pots (one plant for each pot), were transferred to the growth chamber (snijder M1000, Tilburg, The Netherlands) to continue growing until 3 months, under a temperature of 26/22 °C for day/night, a 16/8 h photoperiod, 150 μmol·m−2·s−1 light intensity and 65% relative humidity. The stems, leaves, and roots tissue of the seedlings were harvested to extract RNA to perform analysis of tissue-specific expression of CeSNACs.
For low temperature, drought, and salinity, ABA and MeJA treatments with 3-month-old seedlings were carried out as follows.
High-salt treatment: The seedlings were irrigated with a concentration of 300 mmol·L−1 NaCl solution with 200 mL for each seedling, while the control group was treated with the same volume of water. To keep the salt concentration of the treatment, salt solution was used every 24 h for a total of 7 days. Then, the RNA of leaves was harvested to extract the total RNA.
Drought treatment: The seedlings in the drought treatment group were deprived of water for a duration of 7 days, and the seedlings in the control group were given a regular watering (200 mL per pot each day). After the treatment was completed, the total RNA of leaves was extracted to perform CeSNAC genes expression analysis.
Low-temperature treatment: For the cold treatment group, the seedlings were exposed to 4 °C in the growth chamber. The seedlings were placed in the chamber in the time order of 0, 24, 36, 42, and 46 h, respectively, to serve as treatments for 2, 6, 12, 24, and 48 h. The control group was kept in normal conditions in the growth chamber. When the treatment was over, the RNA of leaves for the treatment and control groups was extracted.
ABA and MeJA treatment: ABA and MeJA solutions with a concentration of 100 μmol·L−1 were sprayed to the seedings evenly; each seedling in the treatment group was sprayed 20 mL every 12 h. For the control group, an equal amount of water was used to spray. The treatment lasted for 24 h and leaves were harvested to extract RNA when it was over.
All experiments were carried out in the growth chamber. There are five clone seedlings for each treatment and control and every treatment involved three biological replicates.

2.6. RNA Extraction and qRT-PCR Analysis of CeSNAC Genes

The stems, leaves, and roots tissue of 3-month seedlings which grew under normal conditions and the leaves of the same old seedlings from the stresses treatment and control group were also harvested. The tissues collected were used to extract the RNA with TRizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s recommendations. The extracted RNA was treated with DNase I (Thermo Fisher Scientific, Wilmington, MA, USA) to remove genome DNA contamination. NanoDrop™ One (Thermo Fisher Scientific, Waltham, MA, USA) was used to determine the concentration and purity of the RNA; then, reverse transcription of it was performed with an M-MULV reverse transcriptase kit (Thermo Fisher Scientific, Wilmington, MA, USA) to transcribe into cDNA.
Primers of CeNACs and the CeEF1α, the internal reference gene, were designed using primer premie 5 software (Table S1). The qRT-PCR amplifications were performed on the PCR cycler BIO-RAD CFX96 (Biorad, Hercules, CA, USA). The cycling program began with an initial denaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s, annealing at 60 °C for 20 s, and an extension at 72 °C for 20 s. The CeActin gene was used as an internal control to normalize the data and the relative expression levels of genes of interest were calculated using the 2−ΔΔCT method.

2.7. Statistical Analysis

All experiments were conducted with three replicates for each trial. The analysis of the final data was carried out using SPSS (ver 18.0, Chicago, IL, USA) software through single factor ANOVA. Statistically significant results are denoted by asterisks. The error bar represents the standard deviation of the three replicates.

3. Results

3.1. Identification of CeSNAC Genes

By constructing a specific Hidden Markov model file for C. equisetifolia NAC transcription factors and conducting the second search in the protein database of C. equisetifolia, a total of 94 non-redundant NAC transcription factor genes were identified. To find SNAC genes from the CeNAC family, 79 SNAC genes in other plant species were collected from the literature and their protein sequences were downloaded from NCBI, such as 18 SNAC genes of from A. thaliana, 24 from Oryza sativa, 4 from Lycopersicon esculentum, 3 from Triticum aestivum, and 3 from Populus. CeNAC proteins together with the 94 CeNAC proteins were used to generate a phylogenetic tree by MEGA. The results showed that 10 CeNAC TFs were clustered into three subfamilies including ATAF, NAP, and AtNAC3, which were considered as a stress-responsive NAC protein subfamily together with OsNAC3. The E subdomain within the NAC domain of these four subfamilies exhibits a significant conservation and is widely recognized as a characteristic of SNAC transcription factors [34]. However, there are no CeNAC members in the OsNAC3 subfamily, mainly distributed in monocotyledons (Figure 1). For the 10 CeSNACs, there are 5 members in the NAP subfamily, 3 in the AtNAC3 family, and 2 in the ATAF subfamily.
The CeSNAC genes, named CeSNAC1~10, have a gene length ranging from 823 to 2852 bp. The open reading frames vary from 528 to 1056 bp and the number of amino acid residues ranges from a minimum of 175 to a maximum of 348. In terms of protein molecular weights, they range from 20.02 to 39.38 kDa. The isoelectric points span from 4.66 to 9.02. According to the subcellular localization predictions made by the PSORT programmer, it was found that seven CeSNAC proteins are nuclear-localized proteins, whereas CeSNAC8 possibly expressed in the cytoplasm, and proteins of CeSNAC4 and CesNAC7 were predicted to function in the chloroplasts (Table 1).

3.2. Characteristics of CeSNAC Gene and Its Encoding Proteins

To have a more comprehensive understanding of the gene and encoded protein of CeSNACs, a conserved domain search (CDs) tool on NCBI was employed to analyze the CeSNAC proteins sequence, and NAC domains (NAMs) were found in all CeSNAC proteins (Figure 2B).
Exon/intron structure analysis showed that there are two introns in most of the CeSNACs, while CeSNAC1, CeSNAC5, and CeSNAC7, which are from the NAP and AtNAC3 subfamily, respectively, have only one intron (Figure 2C). To further detail the NAM domain in different CeSNACs, motifs of the CeSNAC proteins sequence were scanned by MEME tools. Finally, five motifs were found and motifs 1 to 5 correspond to the five subdomains of A, B, C, D, and E in the NAC domain [35,36] (Figure 2A,D). All the NAC domains of CeSNACs contain the five subdomains. However, CeSNAC7, a member of the NAP subfamily, has a changed subdomain order, compared to other NAC domains of CeSNACs. CeSNAC members in different subfamilies have a certain degree of divergence in the number of introns and the composition of the NAC domain, suggesting that the evolution of SNAC in C. equisetifolia may be relatively independent among subfamilies.

3.3. Analysis of Protein Sequences and Prediction of the Structure for CeSNACs

To conduct a more in-depth analysis of the seven candidate CeSNACs, a comparison of the amino acid sequences of candidate CeSNACs from each of the two subfamilies individually was conducted. Notably, a substantial sequence difference was observed between the two subfamilies. Within the ATAF subfamily, the sequences of CeSNAC1 and CeSNAC2 showed a higher degree of similarity. Conversely, in the NAP subfamily, the sequences of CeSNAC3 and CeSNAC4 were more similar (Figure 3A). The SOPMA website was used to predict the secondary structure, and it was found that the 10 CeSNACs proteins have four different structures: an alpha helix, an extended strand, a beta turn, and a random coil secondary structure (Figure 3B). The proportion of random coils in 10 proteins was found to be over 50%. CeSNAC4 exhibited the highest percentage at 69.25%, while CeSNAC7 had the lowest percentage at 50.40%. In contrast, beta turn was the least prevalent among the four structures, accounting for less than 5.70% overall and only 2.30% in CeSNAC6 (Figure 3B). CeSNAC7 displayed a higher proportion of alpha helices compared to other CeSNACs, whereas the alpha helices proportion in the remaining CeSNACs remained relatively stable, fluctuating between 15% and 20% across the other six SNACs. The percentage of extended strands remained relatively constant across the seven CeSNACs, with values ranging from 13% to 16% (Figure 3B). This suggests that random coils play a significant role in the structure of these proteins. The tertiary structure of NAC proteins was predicted by the SWISS-MODEL, and the findings indicated that the structure of ATAF and NAP family proteins was relatively conserved (Figure 3C). This suggests that these proteins may have similar functions and play important roles in biological processes.

3.4. Cis-Elements Analysis of CeSNAC Promoter

To analyze cis-elements on the promoter of genes can provide more information about the gene possible roles that they are involved, because cis-elements are the key sequences bound by special factors to regulate gene spatiotemporal expression [37]. 2.0 kb base pairs upstream of the start codon (ATG) in the CeSNACs gene sequence, regarded as the promoter, were to be analyzed by PlantCare. As shown in the figure, stress responsive cis-elements were widely distributed on the CeSNAC promoters, such as the STRE (stress responsive element), the DRE (dehydration-responsive element), the LTR (low-temperature responsive element) and the MBS (MYB binding site involved in drought inducibility). STRE is distributed on all CeSNAC promoters except CeSNAC7. CeSNAC1, 4, and 5 each have six STREs, while the numbers are from one to three for other CeSNAC promoters. There are one to three DRE elements on the promoters of CeSNAC, other than CeSNAC7 and CeSNAC10. And 5 and the LTR element are present on the promoters of CeSNAC1 and CeSNAC8, while CeSNAC2, 4, and 5 have one each. MBS is an element which is related with drought stress. There is no MBS element on the promoter of ATAF, AtNAC3 and NAP subfamilies, each of which has one member; they are CeSNAC1, CeSNAC7, and CeSNAC8. Furthermore, elements responsive to both stress and hormones were identified, such as the MYB and MYC elements that responded to both ABA and drought, which were also widely distributed in the CeSNAC promoters. In addition to stress-related response elements, hormonal signaling may also be involved in the response of the EgrSNAC gene to adversity stress. A total of 20, 18, and 15 abscisic acid response elements (ABREs) were found in CeSNAC8, CeSNAC2 and CeSNAC1, respectively. The salicylic acid and auxin responsive element as-1 and the gibberellin responsive element P-box were also distributed in the promoters of most CeSNAC genes. The MeJA responsive elements CGTCA-motif and TGACG-motif were also present on all the CeSNAC promoters (Figure 4).

3.5. Subcellular Localization of CeSNACs

To verify the subcellular location of CeSNACs, we cloned their entire coding sequence and used a tobacco epidermal cell system to conduct subcellular localization experiments. The outcomes of the subcellular positioning revealed that, except for CeSNAC9 which was located in the cytoplasm and nucleus, all other CeSNACs were only localized in the nucleus (Figure 5). Discrepancies in the localization patterns of CeSNAC4, CeSNAC7, and CeSNAC9 compared to the predictions in Table 1 suggest a level of conservation in the functions of these transcription factors.

3.6. Tissue Specific Expression Analysis of CeSNAC

To investigate the expression of 10 CeSNAC genes across various tissues of C. equisetifolia, we measured gene expression levels in leaves, stems, and roots. Within the ATAF subfamily, it was observed that the expression levels of CeSNAC1 and CeSNAC2 were predominantly present in the stems and leaves of the C. equisetifolia, particularly in the leaves. Simultaneously, the expression levels of CeSNAC8, CeSNAC9, and CeSNAC10 in the roots were also significantly lower than those in the stems and leaves. As for the NAP subfamily, it was noted that the levels of CeSNAC3, CeSNAC4, CeSNAC5, CeSNAC6, and CeSNAC7 expression in the roots were significantly higher when compared to the levels in the stems and leaves (Figure 6). These results indicate that the role of distinct CeSNACs in responding to stress might vary depending on the tissue.

3.7. Expression Patterns of CeSNAC Genes under Abiotic Stress

NAC transcription factors play a pivotal role in orchestrating plant responses to a wide array of abiotic stresses. To gauge the responsiveness of potential CeSNACs to such stresses, RT-qPCR was employed to quantify the relative expression levels of the CeSNACs under conditions of drought, salinity, low temperature, abscisic acid (ABA), and methyl Jasmonate (MeJA).
Under drought treatment, the majority of CeSNAC genes exhibited induced expression, with the exception of CeSNAC7. The expression of CeSNAC7 was markedly inhibited at 72 and 96 h after drought treatment, registering only 0.093 and 0.05 times that of the control, respectively. In contrast, both CeSNAC3 and CeSNAC4 reached their peak expression at 72 h after drought treatment, showcasing 51.1- and 37.8-fold increases compared to the control. Transcripts of CeSNAC6, 9, and 10 were initially repressed at 24 h but induced at 48 h after drought treatment before subsequently declining. CeSNAC1 and CeSNAC2 were strongly inhibited at 24 h after drought treatment; however, their expression began to rise at 48 h after treatment (Figure 7A).
When exposed to salt stress, all CeSNAC transcripts increased after 7 days of treatment compared to the control, with CeSNAC4 expression showing a remarkable 72.03-fold increase. However, the expression patterns of different CeSNACs varied during the 72 h of drought treatment. CeSNAC1 and CeSNAC6 exhibited similar expression patterns, being consistently inhibited throughout the treatment period. The expression of CeSNAC2 and CeSNAC7 showed a trend of repression, followed by induction and subsequent repression. CeSNAC3 and CeSNAC10 were mostly inhibited except for the 72 h and 7 days treatments, while CeSNAC8 and CeSNAC9 had the lowest expression levels after 12 and 24 h of treatment, respectively. Additionally, CeSNAC4 and CeSNAC5 increased their expression levels under salinity treatment, except for the 48 h treatment (Figure 7B).
Under low temperature, CeSNAC1 and CeSNAC2, belonging to the ATAF subfamilies, were initially repressed after 2 h of treatment but were strongly induced afterward. CeSNAC3 expression increased by 134.4- and 153.7-fold compared to the control at 24 and 48 h of treatment. CeSNAC4 and CeSNAC6 were only inhibited at 2 h of treatment. The induction levels of CeSNAC5 and CeSNAC8 continued to increase with the duration of treatment, while the expression of CeSNAC7 and CeSNAC9 was suppressed under cold treatment. In addition, CeSNAC10 only increased its expression at 48 h of treatment (Figure 7C).
In response to ABA stress, the expression levels of CeSNAC1, CeSNAC4, and CeSNAC6 were prominently induced, while the expression of other CeSNACs was significantly inhibited (Figure 7D). MeJA treatment led to the activation of NAC gene expression, with varying degrees of upregulated expression at different treatment times. Notably, the expression levels of CeSNAC1, CeSNAC2, CeSNAC4, CeSNAC5, CeSNAC6, and CeSNAC9 peaked 24 h after the induction treatment while CeSNAC3 and CeSNAC10 were inhibited under MeJA treatment (Figure 7E). These results offer insights into their potential roles in plant stress responses and adaptation.

4. Discussion

The SNAC group, as one of six major NAC groups, has been established in an ancient moss lineage [38]. A total of 18 NAC subfamilies were identified according to the phylogenetic tree of NAC proteins in Oryza sativa and A. thaliana, in which NAP, AtNAC3, ATAF, and OsNAC3 were classified into the SNAC group [34,39]. Their members have been proved to be involved in regulating responses to cold, drought, salinity, heat, and other stress responses [40,41,42].
There are varied numbers of SNAC in different plant species. For example, 18 BdSNACs in Brachypodium distachyon (L.) P. Beauv, 14 MsSNACs in Medicago sativa, and 17 MdSNACs in Malus domestic were identified [25,43,44]. In this study, 94 CeNAC proteins were identified in C. equisetifolia. All of them together with 79 SNAC proteins from other plants were used to construct a phylogenetic tree. Finally, 10 CeNACs and 66 SNAC proteins were found to cluster into 3 SNAC subfamilies including NAP, AtNAC3 and ATAF. No CeSNAC was grouped into the OsNAC3 subfamily, suggesting that the evolution of this subfamily is separate from eudicots.
All CeSNAC proteins have classic NAC domains except for CeSNAC7. In the structure of NAC proteins, subdomains A, C, and D were tightly conserved which are involved in DNA binding, while the subdomains B and E were divergent, affecting the roles of NAC proteins in plants [34]. The NAC domains of CeSNACs exhibit conserved A, C, and D subdomains, indicating their potential as transcription factors in plants. And the subcellular localization of CeSNACs showed that the proteins they encoded predominantly expressed in the nucleus, consistent with their function as transcription factors.
The distribution of cis-elements on gene promoters determines the spatiotemporal expression patterns of genes, providing valuable insights into gene function [45]. Analysis of cis elements on CeSNACs promoters revealed the presence of stress-responsive elements, including an STRE (stress responsive element), a DRE (dehydration responsive element), an LTR (low temperature responsive element), and an MBS (low temperature responsive element), suggesting the involvement of abiotic stresses in the regulation of CeSNACs. Moreover, ABRE (abscisic acid responsive element), as-1 (auxin and salicylic acid responsive element 1), CGTCA-motif and TGACG-motif which are ABA, MeJA (methyl jasmonate)-responsive elements were also found within the CeSNACs promoters, indicating that phytohormones were involved in the stress responses of CeSNACs. Similar cis elements were also found in SNACs promoters from other plant species [19,44,46]. The expression pattern of CeSNAC under low temperature, drought, salinity, ABA, and MeJA further supported their involvement in stress responses.
Close homologous genes tend to exhibit similar expression patterns due to their evolutionary proximity. For example, FtNAC6 and FtNAC7, the two closest homologous genes from Fagopyur tataricum, demonstrated nearly identical expression patterns when seedlings were exposed to low temperature, salinity, drought, and MeJA [26]. Similarly, ATAF2 and its closest homolog ANAC102 were both induced under drought, salt, and cold conditions [47]. CeSNAC1 and CeSNAC2, both belonging to the ATAF subfamily, displayed a close relationship in tertiary structure and exhibited nearly identical expression patterns under drought, cold, and salinity treatments. CeSNAC3-6, belonging to the NAP subfamily, were primarily induced under drought, cold, and salinity conditions. Additionally, CeSNAC1 and CeSNAC2 both have high transcripts in leaves; and CeSNAC3-7 expressed strongly in roots compared in stems and leaves while CeSNAC9 and CeSNAC10 expressed specially in stems. On the contrary, SNAC members from the same subfamily displayed varying responses to stresses, with CeSNAC1 being induced while CeSNAC2 was repressed by ABA. Additionally, CeSNAC9 exhibited a decrease in transcripts with the duration of the cold treatment, while the expression of CeSNAC10 was inhibited firstly then induced finally during the same process. These findings indicate the complex responsiveness of CeSNACs to different stress conditions, emphasizing the need to explore their functions to enhance the stress resistance of C. equisetifolia.
In summary, the ten CeSNAC proteins were identified from the NAC family of C. equisetifolia. Characteristics of their structure, protein sequence, and cis elements were analyzed. Furthermore, the expression pattern of CeSNACs under different stress treatments were also investigated, indicating that they play roles in different stress responses.

5. Conclusions

Our results identified 10 CeSNAC genes from the genome of C. equisetifolia and demonstrated their crucial roles in abiotic stress responses. These findings provided valuable insights into understanding the stress responses of C. equisetifolia and lay the foundation for further research on the roles and mechanisms of the CeSNAC genes in the stress resistance of C.equisetifolia.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy14030535/s1, Table S1: Primers of CeSNACs used in RT-qPCR.

Author Contributions

S.M. and L.C. planned and designed the research. X.L., D.W., N.L., J.Y., J.H. and M.W. performed experiments and analyzed data, etc. X.L., S.M. and L.C. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Zhejiang Provincial Natural Science Foundation of China (Grant No. LY20C160002).

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest

The authors declare no conflicts of interest.

References

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Figure 1. Phylogenetic tree of the NAC gene family in C. equisetifolia and other species. The purple circle represents members of the NAP, AtNAC3, ATAF, and OsNAC3 subgroups of the SNAC; the red represents members of the SNAC proteins of Casuarina equisetifolia.
Figure 1. Phylogenetic tree of the NAC gene family in C. equisetifolia and other species. The purple circle represents members of the NAP, AtNAC3, ATAF, and OsNAC3 subgroups of the SNAC; the red represents members of the SNAC proteins of Casuarina equisetifolia.
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Figure 2. Analysis of the intron-exon structure and conserved domains of the NAC protein in different gene subfamilies. (A) Motifs analysis in CeSNAC proteins. (B) Analysis of NAC domain (NAM) in CeSNAC protein sequences. (C) Gene structure analysis of CeSNACs. (D) Characteristics of motifs in CeSNACs.
Figure 2. Analysis of the intron-exon structure and conserved domains of the NAC protein in different gene subfamilies. (A) Motifs analysis in CeSNAC proteins. (B) Analysis of NAC domain (NAM) in CeSNAC protein sequences. (C) Gene structure analysis of CeSNACs. (D) Characteristics of motifs in CeSNACs.
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Figure 3. Amino acid sequence alignment and structure analysis of 7 CeNACs proteins. (A) Amino acid sequence alignment analysis. (B) Analysis of the secondary structure of amino acids. (C) Protein tertiary structure analysis.
Figure 3. Amino acid sequence alignment and structure analysis of 7 CeNACs proteins. (A) Amino acid sequence alignment analysis. (B) Analysis of the secondary structure of amino acids. (C) Protein tertiary structure analysis.
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Figure 4. Analysis of the cis-elements of the CeSNAC promoters. The color represents the number of cis-acting elements, ranging from green through orange to red. Lighter green indicates fewer cis-acting elements. Darker red indicates greater quantities of cis-elements.
Figure 4. Analysis of the cis-elements of the CeSNAC promoters. The color represents the number of cis-acting elements, ranging from green through orange to red. Lighter green indicates fewer cis-acting elements. Darker red indicates greater quantities of cis-elements.
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Figure 5. Subcellular localization of CeSNACs in tobacco leaves. The 35S:GFP fusion protein of CeSNACs (in green) and H2B-mCherry (a nucleus localization marker in red) was co-expressed in tobacco leaves. Scale bar = 5 μm.
Figure 5. Subcellular localization of CeSNACs in tobacco leaves. The 35S:GFP fusion protein of CeSNACs (in green) and H2B-mCherry (a nucleus localization marker in red) was co-expressed in tobacco leaves. Scale bar = 5 μm.
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Figure 6. The relative expression of CeSNACs in different tissues. R: roots; S: stems; L: leaves. The data represent the mean ± SD from three biological replicates. ***, p < 0.001; **, p < 0.005; *, p < 0.01.
Figure 6. The relative expression of CeSNACs in different tissues. R: roots; S: stems; L: leaves. The data represent the mean ± SD from three biological replicates. ***, p < 0.001; **, p < 0.005; *, p < 0.01.
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Figure 7. The expression patterns of NAC genes under abiotic stress in Casuarina equisetifolia. (A) The expression of NAC genes in drought. (B) The expression levels of NAC genes in high salt. (C) The expression levels of NAC genes in low temperature. (D) The expression levels of NAC genes in ABA. (E) The expression levels of NAC genes under MeJA stress.
Figure 7. The expression patterns of NAC genes under abiotic stress in Casuarina equisetifolia. (A) The expression of NAC genes in drought. (B) The expression levels of NAC genes in high salt. (C) The expression levels of NAC genes in low temperature. (D) The expression levels of NAC genes in ABA. (E) The expression levels of NAC genes under MeJA stress.
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Table 1. Characterization of the sequences of CeSNAC family genes and coding proteins.
Table 1. Characterization of the sequences of CeSNAC family genes and coding proteins.
IDNameIsoelectric PointMolecular WeightNumber of Amino AcidsOpen Reading FrameGene LengthPrediction of Subcellular Localization
CCG028838CeSNAC18.5327,236.82407232852Nucleus
CCG003077CeSNAC26.0133,865.352928791914Nucleus
CCG005697CeSNAC37.0632,323.592818461471Nucleus
CCG006102CeSNAC48.9439,389.6335110561976Chloroplast
CCG006104CeSNAC54.8129,050.222607831478Nucleus
CCG022581CeSNAC69.0238,519.4134810471258Nucleus
CCG022771CeSNAC74.6613,979.68125378522Chloroplast
CCG014948CeSNAC87.7039,159.8734610411752Nucleus
CCG004745CeSNAC94.77 20,024.75175528823Cytoplasm
CCG004029CeSNAC104.9121,137.61865611651Nucleus
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Li, X.; Li, N.; Wen, D.; Yu, J.; Hong, J.; Wu, M.; Cheng, L.; Meng, S. Identification of Stress Responsive NAC Genes in Casuarina equisetifolia L. and Its Expression Analysis under Abiotic Stresses. Agronomy 2024, 14, 535. https://doi.org/10.3390/agronomy14030535

AMA Style

Li X, Li N, Wen D, Yu J, Hong J, Wu M, Cheng L, Meng S. Identification of Stress Responsive NAC Genes in Casuarina equisetifolia L. and Its Expression Analysis under Abiotic Stresses. Agronomy. 2024; 14(3):535. https://doi.org/10.3390/agronomy14030535

Chicago/Turabian Style

Li, Xiaomei, Nan Li, Dandan Wen, Jianfeng Yu, Jiadu Hong, Mengjie Wu, Longjun Cheng, and Shuai Meng. 2024. "Identification of Stress Responsive NAC Genes in Casuarina equisetifolia L. and Its Expression Analysis under Abiotic Stresses" Agronomy 14, no. 3: 535. https://doi.org/10.3390/agronomy14030535

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