Genome-Wide Analysis of the NAC Gene Family and Functional Verification of the DcNAC043s in Dendrobium catenatum

doi:10.21203/rs.3.rs-2488383/v1

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Genome-Wide Analysis of the NAC Gene Family and Functional Verification of the DcNAC043s in Dendrobium catenatum

https://doi.org/10.21203/rs.3.rs-2488383/v1

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published 09 Oct, 2023

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The NAC (NAM, ATAF1/2 and CUC2) domain protein plays an important role in plant growth and development. The function of members of NAC gene family has been deeply studied in many plants. However, the evolutionary relationships and characteristics of NAC family genes in Dendrobium catenatum (D.candidum) unclear. In this study, we identified 33 NAC genes in D.catenatum, all contain NAM conservative domain. Subcellular localization predictions indicated that all the DcNAC proteins are localized to the nucleus. Phylogenetic analysis suggested that the DcNAC gene family could be divided into four groups. Then, the amino-acid composition, physicochemical properties, gene structure, motif, and promoter cis-acting elements were analyzed, the evolutionarily conservative gene DcNAC043 was found. Using qRT-PCR, DcNAC043-1 and DcNAC043-2 were proved to respond to drought stress induction in D.catenatum. At the same time, phloroglucinol staining proved that the expression of DcNAC043-1 and DcNAC043-2 in the plant were up-regulated, which could increase the lignin content in the plant. We supplemented the relevant information of NAC gene family in D.catenatum. At the same time, the gene function of DcNAC043s and its contribution to the response of Dendrobium to drought stress were verified. These results provide a comprehensive evolutionary history of NAC genes in D.catenatum, and insight into the biological functions of DcNAC043s genes in response to drought stress.

DcNAC043

drought stress

evolution

lignin

Dendrobium catenatum

Plants often suffer from a variety of natural stress, such as salt, drought, cold, pathogens, and insect pests (Shao et al., 2015). To cope with the impact of these different environmental pressures on plant growth and development, plants have evolved a series of defense mechanisms to resile from these stresses. NAC transcription factor gene family is a large plant specific gene family, as a molecular switch that can activate or inactivate gene expression, it plays an important role in plant response to these stresses (Shao et al., 2015; Qu et al., 2016).

The NAC gene family is designated, after the initials of three genes, NAM (NO APICAL MERISTEM, petunia) (Souer et al., 1996), ATAF1/2 (ARABIDOPSIS TRANSCRIPTION ACTIVATION FACTOR, Arabidopsis) and CUC2 (CUP-SHAPE COTYLEDON, Arabidopsis) (Aida et al., 1997). The five N-terminal motifs (A, B, C, D, E) of NAC transcription factor protein form a conservative domain. The length is about 160 amino acids, and the flank contains α- Helix and β- Fold structure. The motif A related to the formation of dimers and the motifs C and D that have nuclear localization signals and participate in DNA binding are highly conserved. The substructure variation of motif B and E is large, which may be closely related to the functional diversity of NAC genes (Jia et al., 2019). On the contrary, C-terminal domains are very different, with transcriptional activation or inhibition activities, and participate in the regulation of multiple networks (Olsen et al., 2005).

At present, NAC transcription factor family has been widely studied in plants species. Under the induction of different plant development stages and various environmental factors, NAC can activate specific target proteins to regulate plant growth and development and plant secondary growth (Nakashima et al., 2012). For example, in Arabidopsis, 117 NAC genes were identified (Kim et al., 2006). Overexpression of the NAC family gene VNI2, which could inhibit the normal development of xylem vessels in roots and aboveground parts by inhibiting the expression of xylem vessel development genes (Yamaguchi et al., 2010). In rice (Oryza sative), 151 NAC genes were identified (Nuruzzaman et al., 2010). Among them, after OsNAC6 overexpression in the plants, the transgenic plants showed a semi dwarfing phenotype (Sakamoto et al., 2016). In Populus trichocarpa, 163 NAC genes were identified (Hu et al., 2010). It was reported that overexpression of PopNAC122 gene will reduce plant height, cell size and cell number (Grant et al., 2010).

In addition, NAC genes has also been proved to play an important role when plants were subjected to biotic and abiotic stresses (Lee et al., 2012). For example, many NAC genes have the function of enhancing plant drought resistance. AtNAC016 could improve plant drought resistance by inhibiting the expression of AREB1, a negative regulator of ABA signaling pathway in Arabidopsis (Sakuraba et al., 2015). In rice, after OsNAC10 gene overexpression, the drought resistance of the plant was enhanced, the results of field experiments showed that the rice yield under drought and control conditions was significantly higher than that of the wild type (WT) (Jeong et al., 2010). It was reported that after overexpression of TaNAC2, the resistance of plants to drought, salt and cold stress was enhanced in Triticum aestivum (Mao et al., 2012). To sum up, NAC gene family has been identified in most plants, and the functions of some genes have been verified. Nevertheless, little is known about this gene family in Dendrobium catenatum (D. candidum).

D. candidum is the third largest genus of Orchidaceae and contains approximately 1,450 species, with high ornamental and medicinal value in China (Zhang et al., 2016). NAC gene plays an important role in plant response to drought stress, which is crucial for plant growth and development (Hu et al., 2006; Zheng et al., 2009; Chen et al., 2018; Negi et al., 2018). However, few studies have focused on identifying NAC genes in D. candidum. We also examined that DcNAC043-1, DcNAC043-2 and DcMYB46 (the gene encoding DcNAC043 interacting protein) expression under drought stress. Simultaneously, the lignin content in the plant was detected under the condition of up regulation of DcNAC043s expression, to reveal the relationship) between DcNAC043s and drought stress responses. Our research provides insight into the evolution of NAC genes in plants and lays the foundation for elucidating the roles of NACs in regulating drought stress in D. candidum.

2.1 Plant Materials and Treatments

The materials used in this study were collected by Ming-jin Huang, associate professor of Agricultural College/Dendrobium Institute of Guizhou University, in Yandang Mountain, Zhejiang Province, China, and then planted in Dendrobium Institute of Guizhou University (26°26′34.510″N/106°53′59.955″E). It was identified as Dendrobium candidum by the Department of Agriculture and Rural Affairs of Guizhou Province, named as Jinhu No. 1, and identified as "Qianren 20210012" (Non endangered species). To investigate the expression patterns of DcNAC043 and DcMYB46 genes under drought stress, the Dendrobium sown in nutritious soil in a growth chamber, with a 16 h light (25°C)/8 h dark (20°C) photoperiod, 60% humidity, and an 800 µmol·m^− 2·s^− 1 light intensity. The photosynthetic photon flux density (PPFD) was measured using a quantum radiometer/photometer (LI 185 B; LI-COR Biosciences, Lincoln, NE, United States). One-years-old plants were transferred into new Nutrient soil for treatment. Dendrobium was treated with natural drought, three biological replicates of leaves were harvested after 0, 3, 6 and 9 day (d) of treatment (Fig. 1A a-d). All samples were immediately frozen in liquid nitrogen and stored at -80 ℃. All the samples were prepared for RNA extraction and quantitative real-time PCR (qRT-PCR) detection (Liu et al., 2021). The remaining dendrobium materials used in the experiment continue to grow in the Dendrobium Institute.

2.2 Identification of NAC Family Genes in D. catenatum

D. candidum Genomic data (BioProject ID PRJNA453230) were obtained from NCBI (http://www.ncbi.nlm.nih.gov/) publicly availabl (Liu et al., 2021). Pfam website (http://pfam.xfam.org/) was used to download the hidden Markov model (HMM) profile of the NAC domain (PF02365) (Mistry et al., 2021). To identify NAC proteins in these species, 117 NAC proteins sequences from Arabidopsis were used as queries in a reciprocal Basic Local Alignment Search Tool Protein (BLASTP) analysis (Liu et al., 2018). Multiple sequence alignment of the NAC proteins was performed in ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2) using the default parameters (E value < 1e-5). According to the aligned sequences, a new HMM in HMMER (HMMER 3.1, http://hmmer.org/) was constructedand and used as the query (E value < 0.01) to search against the D. candidum genome sequence data. NAC gene candidate was based on the gene encoding NAC domain protein (Shen et al., 2019).

2.3 Phylogenetic and Protein Property Analysis of the NAC Family in D. catenatum

Multiple sequence alignments of NAC coding sequences between D. candidum and A. thaliana sequences mentioned above were conducted using ClustalW2 with default parameters (http://www.genome.jp/toolsbin/clustalw) (Liu et al., 2018). After screening the sequences that were closely related to Arabidopsis, based on these sequences (NAC031, NAC033, NAC043, NAC009, NAC018, NAC002 and NAC029), the related sequences of species that were closely related to D. candidum were obtained from NCBI (http://www.ncbi.nlm.nih.gov/) (Table S1), and then ClustalW2 Performs was usd multiple sequence alignments, with default parameters. The phylogenetic trees were generated using the Molecular Evolutionary Genetics Analysis (MEGA) 7 program (Tokyo Metropolitan University, Tokyo, Japan) (Kumar et al., 2016), with the NJ method, the p-distance + G substitution model, 1000 bootstrap replications, and conserved sequences with a coverage of 80% (Liu et al., 2018). Using FigTree v1.4.0 (http://tree.bio.ed.ac.uk/software/figtree/) to realize the phylogenetic trees visualization.

The physicochemical properties of each DcNAC protein were predicted using the ExPASy online program (http://web.expasy.org/translate/) (Duvaud et al., 2021). The transmembrane transport peptides were predicted using Tmpred (http://www.ch.embnet.org/software/TMPRED_form.html), and signal peptides were predicted using SignalP4.1 (http://www.cbs.dtu.dk/services/SignalP/) (Cruz et al., 2017). he subcellular localization of proteins were predicted using Plant-mPLoc online program (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/) (Li et al., 2020). The coding sequence alignments were imported into KaKs_calculator3.0 (Chinese Academy of Sciences, Beijing, China) to calculate the synonymous mutation rate (Ks) and non-synonymous mutation rate (Ka) using the NG method (Zhang et al., 2021). STRING datebase (https://string-db.org/) was used to display the protein-protein networks of NAC proteins (Szklarczyk et al., 2021). Gene Structure Display Server (GSDS 2.0, http://gsds.cbi.pku.edu.cn) was used to display the exon/intron structures of DcNAC genes.

2.4 Motif Identification and Cis-Acting Elements Analysis

Conserved motifs in the proteins were identified using the Expectation Maximization for Motif Elucidation program (MEME v4.12.0, http://meme-suite.org) with the following parameter settings: The maximum number of motifs was 15, and the optimum width was set from 6 to 200. Only motifs with an e-value of 1e-10 were retained for further analysis. The results were visualized using TBtools. PlantCARE software (http://bioinformatics.psb.ugent.be/webtools/plantcare) was used to predict the cis-acting elements within 2000 bp upstream of all DcNAC genes.

2.5 Expression Analysis of the DcNAC043 and DcMYB46 by qRT-PCR

Total RNA was extracted from the Dendrobium leaf using an RNA Extraction Kit (Tiangen, Beijing, China), and cDNA was synthesized from 1 µg of total RNA using M-MLV transcriptase (TaKaRa Biotechnology, Dalian, China) according to the manufacturer’s instructions. Total RNA was extracted from the Dendrobium leaf using an RNA Extraction Kit (Tiangen, Beijing, China), and cDNA was synthesized from 1 µg of total RNA using M-MLV transcriptase (TaKaRa Biotechnology, Dalian, China) according to the manufacturer’s instructions. And cDNA was synthesized using the PrimeScript™ 1st Strand cDNA Synthesis Kit (Takara, Japan) according to the manufacturer’s instructions. Reverse transcription quantitative PCR (RT-qPCR) was performed on a Bio-Rad CFX96 Real Time System (USA) according to a previously described method (Ge et al., 2018).

Gene-specific primer sequences for the DcNAC043 and DcMYB46 were designed using Geneious Prime (Version.2022.2) (Zhang et al., 2021) (Table S2). Each reaction was carried out in biological triplicate in a reaction volume of 20 µL containing 1.6 µL of gene-specific primers (1.0 µM), 2.0 µL of cDNA, 10 µL of SYBR green, 0.2 µL of ROX Reference Dye II, and 6 µL of sterile distilled water. The PCR program was as follows: 95℃ for 3 min; 45 cycles of 10 s at 95℃ and 30 s at 60℃; and then melt curve 65℃ to 95℃, increment 0.5℃ for 5 s (Liu et al., 2018). Melting curves were generated to estimate the specificity of these reactions. Relative expression levels were calculated using the 2^−∆∆Ct method, with ACT2 used as internal controls (Wu et al., 2015).

2.6 Observation on Lignin in Cell Wall of Stem in D. catenatum

Stems of Dendrobium after 0, 3, 6 and 9 d of natural drought stress were sliced into thin sections. Thin sections were stained for lignin with a HCl-phloroglucinol solution [2% (w/v) phloroglucinol (dissolved in 95% (v/v) ethanol): 2M HCl = 1:1, fresh preparation] and observed under a Zeiss Axioscope A1 microscope (Leica) with a × 0.5 optical adapter.

2.7 Statistical Analysis

All experiments were assessed using at least three independent repeats, and all data are represented as the mean ± SD. The statistical analysis was performed using a Student’s t-test, and the significance was indicated using asterisks: *(p-value < 0.05) or **(p-value < 0.01).

1. Identification of NAC Proteins in D. candidum and other plants

Using the 117 AtNAC protein sequences in Arabidopsis as queries, 33 NAC proteins were identified from D. candidum. All DcNAC proteins share a conserved domains: The NAM domain. Relationships based on the phylogenetic tree, the nomenclature used for DcNAC protein was based on the corresponding AtNAC orthologs (Table 1). Based on the phylogenetic tree, eight relatively conservative NAC members were selected for subsequent analysis (Fig. 1A). Using the nine DcNAC protein sequences in D. candidum as queries, other 12 NAC043, 1 NAC033, 13 NAC031, 13 NAC029, 5 NAC018, 10 NAC009 and 9 NAC002 proteins were identified from 37 crops (P. equestris. and others) (Table 1).

Table 1. List of NAC genes identified in D. candidum and other plants

Gene Name in D. candidum	Domain (bp)	Protein Length (aa)	Protein Molecular Weight (kDa)	Protein Isoelectric Point (pI)	Protein Subcellular Localization	Transmembrane Helix and Signal Peptide
DcNAC002	NAM	262	30176.03	5.32	Nucleus	No
AtNAC002	NAM	289	32922.33	6.15	Nucleus	No
CsaNAC002	NAM	287	32827.1	6.33	Nucleus	No
DnNAC002	NAM	302	33680.36	8.83	Nucleus	No
EgNAC002	NAM	270	31123.62	8.76	Nucleus	No
NtNAC002	NAM	298	34247.9	6.56	Nucleus	No
RcNAC002	NAM	291	33061.55	7.59	Nucleus	No
SpNAC002	NAM	275	31772.16	8.75	Nucleus	No
SsNAC002	NAM	296	33730.2	6.4	Nucleus	No
ZjNAC002	NAM	298	33873.48	8.09	Nucleus	No
DcNAC009-1	NAM	398	45003.12	6.55	Nucleus	No
DcNAC009-2	NAM	377	42976.89	8.05	Nucleus	No
DnNAC009	NAM	398	45003.12	6.55	Nucleus	No
AoNAC009	NAM	391	44450.15	6.84	Nucleus	No
AtNAC009	NAM	425	48507.11	6.06	Nucleus	No
HbNAC009	NAM	395	44217.91	6.83	Nucleus	No
PdNAC009	NAM	405	45628.6	7.45	Nucleus	No
PeNAC009-1	NAM	398	45070.01	6.67	Nucleus	No
PeNAC009-2	NAM	394	44964.97	6.35	Nucleus	No
QlNAC009	NAM	398	44760.62	6.13	Nucleus	No
VvNAC009	NAM	401	44780.9	7.57	Nucleus	No
ZjNAC009	NAM	401	45306.99	6.67	Nucleus	No
Gene Name in D. candidum	Domain (bp)	Protein Length (aa)	Protein Molecular Weight (kDa)	Protein Isoelectric Point (pI)	Protein Subcellular Localization	Transmembrane Helix and Signal Peptide
DcNAC018	NAM	338	36977.52	8.62	Nucleus	No
AtNAC018	NAM	320	35442.72	8.52	Nucleus	No
BnNAC018	NAM	285	32254.72	6.91	Nucleus	No
DkNAC018	NAM	296	33394.87	5.78	Nucleus	No
MnNAC018	NAM	391	42990.96	7.25	Nucleus	No
TuNAC018	NAM	220	24481.67	7.01	Nucleus	No
DcNAC029	NAM	262	30176.03	5.32	Nucleus	No
AtNAC029	NAM	268	31425.7	6.38	Nucleus	No
CcNAC029	NAM	286	33067.03	6.46	Nucleus	No
CsNAC029	NAM	286	33075.1	7.69	Nucleus	No
MnNAC029	NAM	293	33463.01	7.6	Nucleus	No
PeNAC029	NAM	283	32461.73	9.06	Nucleus	No
PjNAC029	NAM	264	30560.28	8.82	Nucleus	No
PtNAC029	NAM	283	32597.9	8.77	Nucleus	No
QsNAC029	NAM	285	32980.53	8.69	Nucleus	No
RcNAC029	NAM	281	32920.41	8.69	Nucleus	No
TcNAC029	NAM	284	32767.04	8.46	Nucleus	No
TgNAC029	NAM	254	28729.7	7.69	Nucleus	No
VvNAC029	NAM	282	32475.8	7.7	Nucleus	No
ZjNAC029	NAM	284	32816.02	8.54	Nucleus	No
DcNAC031	NAM	331	37091.00	6.27	Nucleus	No
AtNAC031	NAM	334	38017.62	6.01	Nucleus	No
PeNAC031	NAM	330	37201.87	6.49	Nucleus	No
AcNAC031	NAM	323	36632.40	5.72	Nucleus	No
Gene Name in D. candidum	Domain (bp)	Protein Length (aa)	Protein Molecular Weight (kDa)	Protein Isoelectric Point (pI)	Protein Subcellular Localization	Transmembrane Helix and Signal Peptide
EgNAC031	NAM	310	34929.50	5.42	Nucleus	No
MaNAC031	NAM	317	35960.62	5.32	Nucleus	No
CnNAC031	NAM	310	34870.56	5.34	Nucleus	No
PdNAC031	NAM	309	34759.41	5.49	Nucleus	No
DnNAC031	NAM	331	37091.00	6.27	Nucleus	No
ClNAC031	NAM	291	33379.80	5.85	Nucleus	No
DcaNAC031	NAM	284	31983.24	5.71	Nucleus	No
JrNAC031	NAM	373	41646.96	5.48	Nucleus	No
PtNAC031	NAM	444	49926.63	5.74	Nucleus	No
ZmNAC031	NAM	338	37020.28	6.46	Nucleus	No
DcNAC033	NAM	319	36482.09	7.15	Nucleus	No
AtNAC033	NAM	371	42381.79	6.71	Nucleus	No
AsNAC043	NAM	449	49215.13	7.29	Nucleus	No
DcNAC043-1	NAM	377	41966.82	6.6	Nucleus	No
DcNAC043-2	NAM	355	39597.09	6.43	Nucleus	No
AtNAC043	NAM	365	40780.67	6.27	Nucleus	No
CnNAC043	NAM	388	43369.1	6.36	Nucleus	No
EgNAC043	NAM	387	43458.33	6.43	Nucleus	No
MeNAC043	NAM	393	44919.23	6.52	Nucleus	No
OsNAC043	NAM	400	42429.1	6.43	Nucleus	No
PaNAC043	NAM	396	44870.75	5.77	Nucleus	No
PdNAC043	NAM	387	43302.08	6.39	Nucleus	No
PeNAC043	NAM	386	43226.09	6.43	Nucleus	No
Gene Name in D. candidum	Domain (bp)	Protein Length (aa)	Protein Molecular Weight (kDa)	Protein Isoelectric Point (pI)	Protein Subcellular Localization	Transmembrane Helix and Signal Peptide
PtNAC043	NAM	382	43782.81	6.02	Nucleus	No
QsNAC043	NAM	392	44599.59	5.92	Nucleus	No
TcNAC043	NAM	388	43843.62	6.24	Nucleus	No
VvNAC043	NAM	390	44509.41	6.24	Nucleus	No

The number of amino acid residues in the DcNAC proteins ranged from 220 (TuNAC018) to 449 (AsNAC043), with an average of 336. The relative molecular weights (MWs) ranged from 24.48 (TuNAC018) to 49.93 kDa (PtNAC031). The isoelectric points (pIs) were predicted to range from 5.32 (DcaNAC002) to 9.06 (PeNAC029), and 28 members had pI values > 7 and others had pI values < 7. No transmembrane helix or signal peptide was found in any NAC protein (Table 1). All the NAC proteins were predicted to localize in the nucleus, which were consistent with the general nuclear localization of TFs.

2. Phylogenetic Analysis and Classification of the NAC Proteins in D. candidum and other plants

To clarify the evolutionary relationships between NAC proteins from Arabidopsis and D. candidum, we aligned the sequences of 33 NAC proteins using AtNACs. Based on affinities presented by the evolutionary tree, and we removed groups with only AtNAC or DcNAC, NAC proteins were divided into four groups (Fig. 2A). Although different copies of the same species were still more closely related in evolutionary relationship than the same copies of different species, we can still find the closely related copies of Arabidopsis and D. candidum in these groups. Including the NAC31 clade (class II), the NAC033 and NAC043 clade (class III), and the NAC009, NAC029, NAC018 and NAC002 clade (class IV).

To further investigate the evolutionary relationships of NAC proteins in plants, we constructed a larger neighbor-joining (NJ) tree based on 8 NAC protein sequences identified from D. candidum, 63 NAC proteins from other 37 species (Phalaenopsis equestris et al.) were found and constructed a phylogenetic tree together. According to kinship, they could be divided into 4 classes (Fig. 2A). Class I and III were the two most conservative groups, class I contains only NAC031 clade and class III contains only NAC009 clade. Class II contains NAC033 and NAC043 two clade, probably because NAC033 had only two copies of AtNAC033 and DcNAC033. Class IV as the largest group, including NAC002, NAC018 and NAC029, with a total of 30 members (Fig. 2A). Interestingly, both DcNAC043, DcNAC031 and DcNAC009 appeared to be more closely related to Phalaenopsis equestris, probably because they were both orchids.

3. Gene Structure and Conserved Motif Analyses

To compare the exon/intron structures of AtNACs and DcNACs genes, the coding sequences with their corresponding genomic sequences were aligned. 74 genes (43 AtNACs and 31 DcNACs) were mapped using the GSDS 2.0 software package. The results showed that all AtNACs used for analysis contained no introns, while almost all DcNACs contained at least one intron (except DcNAC002, DcNAC033, DcNAC070 and DcNAC074) (Fig. 1B). Among DcNACs, there were 6 copies of NAC100 and 3 copies of NAC098 in class Ⅰ, except for NAC100-1 and NAC098-1, the rest of the copies contain an intron and 2 CDS regions. Similarly, two copies of DcNAC43 in class Ⅲ, two copies of DcNAC009 in class Ⅳ, and three copies of DcNAC068 in class Ⅳ have similar gene structures (2 introns, 3 CDS regions; 1 intron, 2 CDS regions; 2 introns, 2 CDS regions, respectively) (Fig. 1B). It suggested that the gene structures of different copies of these genes were relatively conserved in the evolutionary process, and their functions may also be similar. In contrast, there seemed to be no regularity in the gene structure between different copies of other DcNAC genes, maybe there was a big difference in functionality.

To further understand the structural diversity of DcNAC proteins, we identified 15 putative motifs (motif 1–15) in the proteins using the MEME/MAST program (Fig. 2B, C). As shown in Fig. 2, all NAC protein sequences involved in the analysis contained motif 1, motif 2, motif 3, motif 4 and motif 5. However, each subgroup had its own unique motif, for example, in class Ⅰ, the specific motif 14 of individual members, the specific motif 9 and motif 12 of class Ⅱ, the specific motif 7, 11, 13 of class Ⅲ, and the specific motif 6 of group Ⅳ. The finding of similar gene structures and conserved motifs within the same subfamily further supports the accuracy of the phylogenetic tree. The other side of the shield, the motif differences between different subfamilies also indicate functional diversity of the NAC gene family in D. candidum.

4. Responsive Elements in DcNAC Promoters

To further investigate the potential regulatory mechanisms of DcNAC cduring the abiotic stress response, a sequence 2 kb upstream from the translation initiation site of the DcNAC gene was submitted to PlantCARE to detect cis-elements. All genes involved in the analysis of promoter elements (DcNAC008-5, DcNAC009-1, DcNAC009-2, DcNAC018, DcNAC029, DcNAC031, DcNAC033, DcNAC043-1, DcNAC043-2, DcNAC068-1, DnNAC068-2, DcNAC068-3, DcNAC070, DcNAC074, DcNAC078, DcNAC094, DcNAC098-1, DcNAC098-3, DcNAC100-2, DcNAC100-3, DcNAC100-4, DcNAC100-5, DcNAC100-6) contain CAAT-box and TATA-box elements, which proved that all the genes can be transcribed normally and thus participate in plant growth and development.

Abiotic stress response elements and phytohormone response elements were analyzed. DcNAC009-1 and DcNAC043-2 contained 20 and 18 response elements (Hormones and Abiotic Stresses), respectively, which were the two genes with the highest element content (Fig. 3). The promoters of the two genes, DcNAC009-2 and DcNAC078, had the least stress response elements, and both had only five. Among them, MYB and MYC elements (response to drought and ABA induction) were the most numerous (49 and 66, respectively), and TCA-element (response to salicylic acid induction) was the least numerous (only 4). Among the promoters of 23 genes, drought stress response elements appeared most frequently, accounting for 48%.

5. Ka/Ks Ratio Calculation

The genomic information obtained from National Center for Biotechnology Information (NCBI) does not include the chromosomal location of the genes. Therefore, chromosomal location and collinearity analysis were not performed in this study. To explore the selective pressure on DcNAC genes, the non-synonymous/synonymous mutation ratio (Ka/Ks) was calculated for the genes involved in the evolutionary analysis, and values of Ka/Ks > 1, = 1 and < 1 indicate positive selection, neutral selection and purifying selection, respectively (Nekrutenko et al., 2002).

After removing gene pairs with no numerical results, the remaining genes were counted. As shown in Table 2, the Ka/Ks ratio for all DcNAC genes were < 1, ranging from 0.0807 (AtNAC087-2/DcNAC100-6) to 0.4293 (AtNAC003/DnNAC100-4), indicating the genes were negative selection during evolution and functionally conserved, which reduced the rate of change in aa profile. A comparation of the Ka/Ks ratio of DcNAC genes among the class Ⅰ, Ⅱ, Ⅲ, Ⅳ showed that the average Ka/Ks ratio was higher in the class Ⅰ (0.2040) than in the class Ⅲ, Ⅱ and Ⅳ (0.1534, 0.1376, and 0.1327, respectively), suggesting that DcNAC genes in the class Ⅰ experienced higher selection pressure during the evolutionary history of D. candidum. However, the overall results showed that most DcNAC genes were slowly evolving in D. candidum.

Table 2

Non-synonymous and synonymous nucleotide substitution rates between *AtNACs* and the corresponding orthologs in *D. candidum*
Class	Gene ID in A. thaliana	Gene ID in D. candidum	Model	Ka	Ks	Ka/Ks	Average Ka/Ks
class Ⅰ	AtNAC087-1	DcNAC100-2	NG	0.468434197	2.557865571	0.183134799	0.20402197901
	AtNAC087-1	DcNAC100-5	NG	0.436294941	2.023831831	0.215578654
	AtNAC087-1	DcNAC100-6	NG	0.394337503	4.491721063	0.087792073
	AtNAC087-1	DcNAC098-3	NG	0.572624176	2.308115946	0.248091599
	AtNAC087-2	DcNAC100-2	NG	0.464403439	2.499483998	0.185799725
	AtNAC087-2	DcNAC100-5	NG	0.428894814	2.108583374	0.203404247
	AtNAC087-2	DcNAC100-6	NG	0.387037536	4.795193065	0.08071365
	AtNAC087-2	DcNAC098-3	NG	0.568073857	2.26892774	0.250371066
	AtNAC046	DcNAC100-1	NG	0.506388723	2.282130264	0.221892997
	AtNAC046	DcNAC100-3	NG	0.567082575	2.03160241	0.279130686
	AtNAC046	DcNAC100-5	NG	0.440093004	2.716369271	0.162015161
	AtNAC046	DcNAC098-3	NG	0.554766803	3.187560964	0.174041158
	AtNAC003	DcNAC100-3	NG	0.850019745	2.591716023	0.327975649
	AtNAC003	DcNAC100-4	NG	0.917949133	2.137835393	0.429382513
	AtNAC003	DcNAC098-1	NG	0.884168001	2.748308448	0.321713526
	AtNAC100	DcNAC100-2	NG	0.537904375	3.457938057	0.155556394
	AtNAC100	DcNAC100-5	NG	0.429689375	3.364907829	0.12769722
	AtNAC100	DcNAC100-6	NG	0.454125313	3.459733101	0.131260216
	AtNAC080-1	DcNAC098-3	NG	0.59653315	2.761880885	0.215988008
	AtNAC080-2	DcNAC100-2	NG	0.505064036	4.724742008	0.106897696
	AtNAC080-2	DcNAC098-3	NG	0.585621162	3.326929401	0.176024523
Class	Gene ID in A. thaliana	Gene ID in D. candidum	Model	Ka	Ks	Ka/Ks	Average Ka/Ks
class Ⅱ	AtNAC031	DcNAC031	NG	0.431280805	3.134691632	0.137583168	0.137583168
class Ⅲ	AtNAC070	DcNAC070	NG	0.402300976	2.483945488	0.161960469	0.153369762
class Ⅲ	AtNAC043	DcNAC043-2	NG	0.441099307	3.04670664	0.144779055	0.153369762
class Ⅳ	AtNAC009	DcNAC009-1	NG	0.437391197	3.986581962	0.109715842	0.132727745
	AtNAC009	DcNAC009-2	NG	0.457590621	2.630631012	0.173947095
	AtNAC094	DcNAC009-2	NG	0.373855525	4.065702878	0.091953479
	AtNAC034	DcNAC094	NG	0.467782298	2.701768753	0.17313928
	AtNAC029	DcNAC029	NG	0.395328956	3.792687035	0.104234531
	AtNAC102	DcNAC002	NG	0.280971227	2.615130735	0.107440604
	AtNAC081	DcNAC068-1	NG	0.460062342	3.772828441	0.121940965
	AtNAC002	DcNAC002	NG	0.223847243	1.734422845	0.129061517
	AtNAC099	DcNAC008-1	NG	0.426090186	2.466525263	0.172749167
	AtNAC099	DcNAC008-2	NG	0.40002781	3.169558772	0.126209305
	AtNAC099	DcNAC008-3	NG	0.446691438	3.164196424	0.141170578
	AtNAC099	DcNAC008-5	NG	0.446691438	3.164196424	0.141170578

6. Protein-Protein networks Analysis of DcNAC043s

The previous analysis found that NAC043 gene was relatively conservative in the evolutionary process. Therefore, based on D candidum, with Arabidopsis and Populus protein database were refered, the protein interacting with DcNAC043s were predicted by STRING software. As the Fig. 4 showed that the two copies of DcNAC043 were consistent with Arabidopsis, and both predicted to interact with DcMYB46 (Zhong et al., 2007). Except Cellulose synthase A catalytic subunit 8 (AtCESA8), Homeobox protein knotted-1-like 3 (DcHOS66) and DcNAC031, the proteins appearing in the interaction network belonged to R2R3-MYB family. These proteins have been reported to participate in the growth and development of plant secondary cell wall (Fang et al., 2020; Im et al., 2021; Ye et al., 2021). Interestingly, although the proteins interacting with PtNAC043 didn’t belong to the R2R3-MYB family, they have also been reported to directly or indirectly regulate the growth and development of plant secondary cell wall (Xing et al., 2008; Min et al., 2020).

7. DcNAC043 responds to drought stress and regulates lignin synthesis

Previous studies showed that NAC043 responded to drought stress in ryegrass (Cheng et al., 2022). Based on the previous analysis, NAC043 had proved that its function was conservative. Therefore, we treated seedling of D. candidum under drought stress, and detected the expression of DcNAC043s during drought stress by qRT-PCR. The results showed that the change of DcNAC043s expression in leaves was related to the drought degree suffered by plants (Fig. 5B). With the increase of drought stress time, the expression of DcNAC043-1 and DcNAC043-1 in leaves also increased significantly (P_DcNAC043−1 = 0.024, 0.017, 0.009 and P_DcNAC043−2 = 0.03, 0.021, 0.017) (Fig. 5B). Consistent with the change trend of DcNAC043s, the expression of DcMYB46 also increased with the increase of drought time, which again proved that DcMYB46 was involved in regulating drought stress.

In Citrus grandis, CgNAC043 was found to regulate lignin synthesis, thereby affecting the development of secondary cell wall (Li et al., 2022). Therefore, we used the lignin specific stay HCl - phloroglucinol to dye it for analysis. We observed that the longer the drought stress time was, the more serious the lignification of the plant was, which indicates that the more lignin was accumulated (Fig. 5A). It was indirectly proved that DcNAC043s also participates in the synthesis of lignin in Dendrobium.

NAC-TF has been found and classified in many plants, but there is no detailed evidence about the NAC-TFs family of Dendrobium. In this study, although the published full genome information of Dendrobium showed that there were 75 annotated NAC genes, which was similar to the number of NAC genes identified in grapes (74) and sugarcane (88) (Wang et al., 2013; Manimekalai et al., 2017). However, based on BLASTP comparison with 117 AtNAC proteins, 33 DcNAC proteins were finally obtained in this study. It was far less than potato (110) (Singh et al., 2013), soybean (101) (Shang et al., 2016), cacao tree (102) (Shen et al., 2019), rice (151) (Nuruzzaman et al., 2010) and poplar (163) (Hu et al., 2010). The difference in the number of NAC members among these species may be caused by genome wide polyploidy events during the evolution and differentiation of each species.

Interestingly, analysis of phylogenetic tree, gene structure, conservative motif and Ka/Ks value showed that NAC was unevenly distributed in Arabidopsis and Dendrobium, indicating that NAC gene family existed before the differentiation of the two species. It was different from the ancient and inactive evolutionary nature of NAC gene family in other species (Hu et al., 2010; Nuruzzaman et al., 2010; Wang et al., 2013; Singh et al., 2013; Shang et al., 2016; Singh et al., 2016; Manimekalai et al., 2017; Shen et al., 2019). It may be that Dendrobium and Arabidopsis were exposed to different environments during evolution, so gene differentiation occurred, and the number of NAC genes in their subfamilies was also different.

However, among all DcNAC genes, DcNAC009 and DcNAC043, unlike other DcNAC genes, are relatively conservative. Therefore, to further prove the evolutionary conservation of NAC009 and NAC043, we analyzed the gene structures of NAC009 in 10 different species and NAC043 in 15 different species, respectively. The results showed that NAC009 and NAC043 in all species (except Arabidopsis) have similar gene structures; even closely related species have very similar gene lengths (Fig. 6). Meanwhile, motif analysis of phylogenetic tree and gene structure based on multiple species shows that NAC043 genes were relatively conservative. All the NAC043 proteins contained not only motif 1, motif 2, motif 3, motif 4 and motif 5 that all the NAC proteins had, but also had their own unique motif 9 (SSITADTKTQMFHSSEGALDQILQYMGRSCKZESEAISNS) and motif 12 (MSISVNGQSQV) (Fig. 2B,C). Therefore, we infered that genes in this subfamily shared a common parent and may have similar functions.

In previous studies, NAC043 was shown to Ryegrass can respond to drought stress (Cheng et al., 2022). Based on this, we selected mature Dendrobium plants for drought stress treatment, and extracted plant leaves for qRT-PCR detection. The results showed that the expression of DcNAC043-1 and DcNAC043-2 in plants were up-regulated after drought stress induction, and the up-regulated trend was related to the duration of drought stress. The staining results of HCl-phloglucinol not only proved that DcNAC043s in Dendrobium responded to drought stress by regulating lignin content, but also further proved that DcNAC043s, like CgNAC043 gene in other species, had the function of regulating plant secondary cell wall growth (Li et al., 2022). Moreover, they were similar to the protein interaction network of AtNAC043 and CgNAC043. In the network, DcNAC043-1 and DcNAC043-2 also act as the master switch that activates the expression of many lignin biosynthetic genes (Zhong et al., 2006; Zhong et al., 2007; McCarthy et al., 2009; Zhou et al., 2009; Hussey et al., 2011; Li et al., 2022) (Fig. 4). It was proved that DcNAC043s were involved in the synthesis of lignin again. However, whether DcNAC043-1 and DcNAC043-2 have functional redundancy needs further experimental verification.

Our research shows that there were significant differences in the evolution of NAC genes between Dendrobium and the model plant Arabidopsis. Therefore, the functions of many members of the DcNAC family need to refer to other species for further research. But DcNAC043, which had relatively conservative function, has proved its contribution to the drought stress of Dendrobium through our verification.

In summary, we believe that DcNAC043 plays a very important role in regulating the lignin synthesis of Dendrobium. At the same time, DcNAC043, like most other NAC genes, responds positively to drought stress in plants.

Acknowledgments

We thank Ming-jin Huang for providing the Dendrobium candidum. And grateful to Hong Yi from the Instrument and Equipment Sharing Platform of Large Scale Instruments & Equipments of Guizhou University, China.

Author contributions

Mingsheng Zhang and Miao Liu: Conceptualization, Methodology and Funding acquisition. Yong-quan Li, Liu Tang, Yu-hang Tian: Analysis. Yu-jin Zhang, Li-yang Shangguan, Ye Hang: Validation. Miao Liu and Tian Tian: Writing-Original draft preparation. Ming-jin Huang and Ming-sheng Zhang: Discussion, Writing-Reviewing and Editing. All authors read and approved the final manuscript.

Funding

This work was supported by the Major Special Project of Science and Technology Program in Guizhou (2017-5411-06); the Science and Technology Support Project of Guizhou (2018-2797); the Guizhou University Cultivation Project (702029201101); Guizhou Science and Technology Plan Project ([2020]4Y071).

Availability of data and materials

The datasets analyzed during the current study could be queried and obtained in the National Biotechnology Information Center repository (NCBI https://www.ncbi.nlm.nih.gov/genome/?term=). The gene BioProject numbers and relevant references of 38 species including D. candidum are presented in Table S1.

Ethics approval

All experimental studies on plants were complied with relevant institutional, national, and international guidelines and legislation.

Consent to participate

Not applicable

Consent for publication

Yes

Competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Genome-Wide Analysis of the NAC Gene Family and Functional Verification of the DcNAC043s in Dendrobium catenatum

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Abstract

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INTRODUCTION

MATERIALS AND METHODS

2.1 Plant Materials and Treatments

2.4 Motif Identification and Cis-Acting Elements Analysis

2.7 Statistical Analysis

RESULTS

3. Gene Structure and Conserved Motif Analyses

5. Ka/Ks Ratio Calculation

6. Protein-Protein networks Analysis of DcNAC043s

DISCUSSION

Declarations

References

Supplementary Files

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