Introduction

Herbaceous peony (Paeonia lactiflora Pall.) is a perennial herbaceous plant of the Paeoniaceae family. It is a traditional flower in China and has a cultivation history of nearly 4,000 years, which was watched in the palace in the Xia Dynasty (Lu et al. 2017). According to historical records, tree peony and herbaceous peony are both known as "King of the flowers" and "Premier of the flowers"; both of them mean wealth. But compared with tree peony, the quality of herbaceous peony cut flowers is better. In recent years, herbaceous peony has been quite popular in the fresh cut flower market because of its high ornamental value including erect flower stem, elegant flower postures and various colors (Tang et al. 2018). In the production of herbaceous peony cut flowers, the upright strength of the flower stem is an important indicator of the quality of cut flowers. Existing studies have shown that the stem strength of plants is closely related to factors such as plant height, stem thickness, flower weight and flower stem (Xia et al. 2018). At present, there are few reports about the molecular regulation mechanism of the stem strength of herbaceous peony. Therefore, studying the molecular mechanism of the stem strength of herbaceous peony and enhancing the stem strength have great significance for the growth and development of herbaceous peony and the quality of cut flowers.

NAC transcription factor family is one of the largest transcription factor families found in the plant genome (Kang et al. 2012). According to genome-wide analysis, at least 151 and 117 NAC family transcription factors were found in Oryza sative and Arabidopsis thaliana (Nuruzzaman et al. 2010). Populus trichocarpa contains at least 163 members (Hu et al. 2010), and at least 152 members have been found in Glycine max and Nicotiana tabacum (Rushton et al. 2008; Le et al. 2011). It is reported that 48 and 45 members of NAC family transcription factors were found in the EST database of Hordeum vulgare and Citrus limonia, respectively (Christiansen et al. 2011; de Oliveira et al. 2011). As a large family, NAC transcription factors have a variety of biological functions, and are mainly involved in plant growth and development, stress resistance, disease resistance regulation, secondary growth regulation, and hormone signal transduction (Wang and Zhang 2018). Existing studies have shown that multiple NAC genes can promote the formation of secondary cell walls (SCW) in fiber cells, such as NST1/2/3 (NAC secondary walls thicken promoting factor) and VND6/7 (vascular-related NAC domain) (Kubo et al. 2005), while other NAC genes inhibit the SCW formation in fiber cells. For example, over-expression of AtNAC012 in Arabidopsis could slightly thicken the cell walls of xylem ducts, but strongly inhibit the production of fiber cells’ SCW (Ko et al. 2007).

Although NAC family transcription factors have been cloned in many plants, so far, no study has been reported on NAC transcription factor in herbaceous peony. In view of this, the aim of this study was to clone PlNAC2 in herbaceous peony, perform sequence comparison of PlNAC2 protein with other proteins and construct phylogenetic tree. Also, we predicted protein property, secondary structure and tertiary structure of PlNAC2, and analyzed its spatio-temporal expression patterns.

Materials and Methods

Preparation of RNA and cDNA synthesis

The stems of the herbaceous peony cultivar ‘Xixia Yingxue’ from S1 (Stage 1: flower-bud stage) to S3 (Stage 3: full-flowering stage) and the roots, stems, leaves and flowers of ‘Hongyan Zhenghui’ were used as the materials. Total RNA of herbaceous peony samples were extracted by the CTAB method. The reverse transcription was performed by using 5×M-MLV Buffer (TAKARA, Dalian, China) reverse transcription kit, and the cDNA was synthesized according to the instructions.

Cloning of PlNAC2 in herbaceous peony

Based on PlNAC2 sequence obtained from the transcriptome data, a pair of primers was designed and selected for final PCR amplification (Table 1). The total volume of the RT-PCR reaction system was 25 μL, containing 12.5 μL High enzyme, 2 μL cDNA, 1 μL for each of the forward primers and reverse primers, and 8.5 μL ddH₂O. The reaction program was pre-denaturation at 98°C for 2 min. PCR was performed: 40 cycles at 98°C for 10 s, 50°C for 15 s and 72°C for 30 s by using a thermal cycler. The Agarose Gel DNA Recovery Kit (Tiangen, Beijing, China) was used to purify and recover the PCR products and then ligated it with the pMD18-T (Tiangen, Beijing, China) vector. Finally, the identified positive recombinants were sent to Qingke Biotechnology Co., Ltd for sequencing.

Bioinformatics analyses

Software for bioinformatics analysis were listed in Table 2. BLAST was used to identify the homology of nucleic acid and protein sequences. ORF Finder was used to find open reading frames and translate it into protein sequences. Based on the sequence alignment of PlNAC2 with other homologous sequences, a phylogenetic tree was constructed by using MEGA 5.0. ProtParam was used to predict the physical and chemical properties of PlNAC2 protein. Signal P 5.0 Server was used to predict signal peptide. TMHMM 2.0 Server was used to analyze the transmembrane domain of PlNAC2 protein. The online software SOPMA was used to infer the secondary structure of PlNAC2 protein. The online software ProtScale was used to predict the hydrophobicity/hydrophilicity of the amino acid sequence of PlNAC2 protein. SWISS-MODEL workspace was used to simulate the tertiary structure of PlNAC2 protein.

Quantitative real-time PCR

Based on PlNAC2 sequence obtained from the transcriptome data, a pair of primers was designed to analyze the distribution of PlNAC2 during the stem growth of ‘Xixia Yingxue’ and the distribution of PlNAC2 in the roots, stems, flowers and leaves of ‘Hongyan Zhenghui’ (Table 1). The PCR mixture contained 1 μL cDNA, 10 μM forward and reverse primers 0.5 μL each, 4.3 μL ddH₂O and 6.3 μL SYBR Master Mix (TAKARA, Dalian, China). The reaction program was pre-denaturation at 95°C for 30 s, 40 cycles included 95°C denaturation for 5 s, 50°C annealing for 30 s, and 72°C extension for 30 s. The comparative CT method calculated the relative quantification of PlNAC2 expression level. The cDNA samples to be tested were set up in triplicate, and the data analysis was performed by using 2^–ΔΔCt method.

Results

Cloning and sequencing analysis of PlNAC2 in herbaceous peony

Table 1: Primers used for PlNAC2 isolation and expression analysis

Table 2: Software used for bioinformatics analysis

The full-length sequence of PlNAC2 was obtained by RT-PCR. The PCR product was visualized by agarose gel electrophoresis (Fig. 1), which showed that the size of product was 1,109 bp. Analysis of the sequencing results showed that the open reading frames of PlNAC2 was 939 bp in length and encoded 313 amino acids (Fig. 2). The NCBI website was used to perform a BLAST alignment on the amino acid sequence of PlNAC2. The comparison results showed that the amino acid sequence of the conserved domain of PlNAC2 had high homology with other NAC transcription factors. The structural function domain of the encoded amino acids was analyzed by using DNAMAN software. Compared with Quercus lobata, Suaeda liaotungensis, Durio zibethinus, Chenopodium quinoa, Herrania umbratica, Camellia sinensis, Theobroma cacao, Vitis quinquangularis and A. thaliana, the N-terminal of PlNAC2 protein had a complete NAM (no apical meristem) characteristic domain and this sequence had a typical NAM conserved domain consisting of 155 amino acids between amino acids 6–161. This domain contained 5 sub-domains (A–E), while the C-terminal sequence had a transcriptional regulation domain with high diversity (Fig. 3). It could be inferred that PlNAC2 belonged to the NAC family. Moreover, the sequence contained two nuclear localization signal sequences, which led to speculate that PlNAC2 protein was located in the nucleus (Fig. 2). PlNAC2 protein was longer than Q. lobata (293 aa), S. liaotungensis (302 aa), D. zibethinus (298 aa), C. quinoa (304 aa), H. umbratica (303 aa), C. sinensis (301 aa), T. cacao (296 aa), V. quinquangularis (294 aa) and A. thaliana (289 aa).

Fig. 1: Amplification of PlNAC2

M: 2 kb DNA Marker; 1: PlNAC2 cDNA result

MEGA 5.0 software was used to construct a phylogenetic tree of PlNAC2 protein and other protein sequences including QlNAC2 (XP_030949572.1), SlNAC2 (AGZ15313.1), DzNAC2 (XP_022740811.1), CqNAC2 (XP_021772603.1), HuNAC2 (XP_021274745.1), CsNAC2 (XP_028092031.1), TcNAC2 (XP_007048529.2), VqNAC2 (ALM02085.1) and AtNAC2 (NP_171677.1) (Fig. 4). Among them, PlNAC2 had a high homology with QlNAC2, which indicated that PlNAC2 and QlNAC2 were relatively closely related in evolution.

ProtParam was used to analyze the amino acid sequence encoded by PlNAC2, and we found that its molecular formula was C₁₆₂₁H₂₄₆₀N₄₂₆O₄₇₁S₁₆, the number of amino acid residues was 313; the relative molecular mass was 35964.90 Da, the theoretical isoelectric point was 8.89; and there were 34 amino acid residues (Asp + Glu) were negatively charged, and 40 amino acid residues (Arg + Lys) were positively charged. The instability coefficient was 45.51, which suggested that it was an unstable protein (< 40, protein is stable), and the aliphatic coefficient was 56.39. The total hydrophilic coefficient was -0.783.

SignalP 5.0 Server was used to analyze the signal peptide of PlNAC2 protein. As shown in Fig. 5 that no signal peptide existed in PlNAC2 protein. And TMHMM 2.0 Server was used to analyze the transmembrane domain of PlNAC2 protein. As shown in Fig. 6, there was no obvious transmembrane domain in PlNAC2 protein. The online software SOPMA was used to infer the secondary structure of PlNAC2 protein. The results showed that this protein was mainly random coil (66.13%), and contained 19.17% alpha helix, 2.56% beta turn and 12.14% extended strand and other structures (Fig. 7). The online software ProtScale was used to predict the hydrophobicity/hydrophilicity of the amino acid sequence of PlNAC2 protein. The results showed that the proportion of hydrophilic amino acids in the protein was greater than proportion of hydrophobic amino acids. It could be inferred that this protein was hydrophilic (Fig. 8).

The SWISS-MODEL workspace was used to simulate the tertiary structure of PlNAC2 protein. The prediction result showed that the similarity between PlNAC2 protein sequence and the template sequence was 0.54, and the identity was 70.73% (Fig. 9). It could be inferred that the protein was a NAC protein, which was consistent with the target gene.

Tissue distribution of PlNAC2 in herbaceous peony

Fig. 2: Nucleotide and amino acid sequence of PlNAC2

Putative nuclear localization signal are shown by underline

Fig. 3: Alignments of NAC domain between PlNAC2 and other NAC proteins

Conserved sub-domains A to E are shown by underline in the NAC domain

qRT-PCR (quantitative real-time PCR) was used to detect the distribution of PlNAC2 during the growth and development of herbaceous peony and different tissues. And the results showed that PlNAC2 existed in the whole process of stem growth and development in ‘Xixia Yingxue’. The expression of PlNAC2 gradually decreased with its development. Among different tissues in ‘Hongyan Zhenghui’, PlNAC2 was highly expressed in stems, followed by leaves and roots, and was the lowest in flowers (Fig. 10).

Discussion

Fig. 4: Phylogenetic tree of PlNAC2 and some other NAC proteins

Fig. 5: Prediction of signal peptide of PlNAC2 protein

Fig. 6: Prediction of the transmembrane domain of PlNAC2 protein

This study evaluated PlNAC2 and its distribution in the tissues of herbaceous peony to determine whether PlNAC2 plays a role in the formation of SCW. Based on the transcriptome data (Xia 2019), the full-length sequence of PlNAC2 was obtained by RT-PCR. It proved that PlNAC2 product was 1,109 bp in size and encoded 313 amino acids. The relative molecular mass was 35964.90 Da and the theoretical isoelectric point was 8.89. It was an unstable hydrophilic protein without signal peptide and transmembrane domain.

Fig. 7: Prediction of the secondary structure of protein of PlNAC2 protein

Blue: Alpha helix; Red: Extended strand; Green: Beta turn; Purple: Random coil

Fig. 8: Hydrophilicity/hydrophobicity analysis of PlNAC2 protein

Fig. 9: Prediction of the tertiary structure of protein of PlNAC2 protein

Fig. 10: The expression patterns of PlNAC2

S1 (Stage 1) = flower-bud stage, S2 (Stage 2) = pigmented stage, S3 (Stage 3) = full-flowering stage. The values represented mean ± SE, and different letters marked significant differences (P < 0.05)

NCBI website was used for analysis and multiple sequence alignment. The results showed that the amino acid sequence of the conserved domain of PlNAC2 had high homology with other NAC transcription factors. Among them, PlNAC2 had the highest homology with QlNAC2. In addition, the N-terminal of PlNAC2 protein had a complete NAM characteristic domain which meant that PlNAC2 belonged to the NAC family. PlNAC2 protein contained two nuclear localization signal sequences. It was initially determined that this gene was located in nucleus.

The N-terminal domain of the NAC family transcription factors is highly conserved and generally consists of about 160 amino acid residues, including 5 sub-conserved structural regions (A–E) where A, C and D are highly conserved domains, while B and E are not highly conserved (Chen et al. 2015). PlNAC2 had a typical NAM conserved domain consisting of 155 amino acids between the N-terminal amino acids 6–161, and this domain also contained 5 sub-domains (A–E). In addition, the C-terminal regulation region of NAC protein had a high degree of variability and specificity, conferring a variety of roles in transcription activation regulation (Wang and Dane 2013).

The NAC family transcription factors are plant-specific transcription regulation factors. Multiple NAC genes play a key role in the biosynthesis of SCW in fiber cells. VND (vascular-related NAC domain), SND (secondary wall-associated NAC domain) and NST (NAC secondary walls thicken promoting factor) are three important NAC members known to participate in the regulation of SCW synthesis (Zhou et al. 2014). In addition, some NAC transcription factors (such as SND1, VND6 and VND7) that regulate transcription at higher network levels can be used as transcription switch factors to regulate the SCW synthesis of fiber cells and ductal cells in downstream transcriptional networks (Mitsuda et al. 2005; Huang and Li 2016). CpSND1 isolated from Crataegus pinnatifida was closely homologous to AtSND1. The CpSND1 up-regulated SCW biosynthesis genes and induced SCW formation in over-expressing Arabidopsis (Chen et al. 2018). In addition, some NAC transcription factors play a role in inhibiting the SCW formation. When XND1 (xylem NAC domain) was up-regulated in fiber cells of Arabidopsis, the SCW formation was inhibited by regulating the activity of NST1. At the same time, over-expression of XND1 might lead to short plants and loss of xylem ducts (Zhao et al. 2008; Zhang et al. 2020). GhNAC1 in cotton had high homology with NSTI and NST3. It was specifically expressed in cotton fibers and gradually increased during the thickening period of SCW, while GhFSN5 (fiber secondary wall-related NAC domain) played a negative regulatory role and inhibited SCW formation (Li 2011; Sun et al. 2020).

We used qRT-PCR to detect the spatio-temporal expression pattern of PlNAC2 in herbaceous peony, and the results showed that PlNAC2 existed in the entire process of stem growth. The expression level of PlNAC2 was the highest in stems, followed by leaves and roots, and the lowest expression level was in flowers, and its expression level in stems decreased with the growth of plants, and its relative expression level was 10.66 at S1, while in S2 and S3 were 5.73 and 5.31, respectively. This result was similar to the expression patterns of XND1 and AtNAC012. Thus PlNAC2 played a down-regulating function and inhibited the SCW formation in herbaceous peony (Ko et al. 2007; Zhao et al. 2008).

Conclusion

PlNAC2 was successfully cloned and its expression patterns were studied. PlNAC2 had 1,109 nucleotides and encoded 313 amino acids. It existed throughout the growth and development of the stem in herbaceous peony, and its expression gradually decreased with the growth of the stem. Therefore, PlNAC2 acted as a negative regulator in the SCW formation. The results improve our understanding of how NAC family transcription factors regulate SCW formation. Whether PlNAC2 participates in stress resistance and other functions needs further research. Cloning of PlNAC2, analysis of the properties and expression patterns of PlNAC2 protein in this study will help further research and application about PlNAC2.

Acknowledgements

This work was supported by the Natural Science Foundation of China (31772341, 31972448), the Fifth Phase of "Project 333" Science Funding Program of Jiangsu Province (BRA2019084), the Young Talent Support Project of Jiangsu Provincial Association for Science and Technology.

Author Contributions

Jun Tao and Daqiu Zhao designed the experiments, Yuting Luan and Yuhan Tang performed the experiment, Xin Wang processed the data, Yuting Luan wrote and revised the paper.

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