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Article

Bnt05G007257, a Novel NAC Transcription Factor, Predicts Developmental and Synthesis Capabilities of Fiber Cells in Ramie (Boehmeria nivea L.)

by
Xuehua Bai
1,†,
Yafen Fu
1,†,
Xin Wang
1,†,
Guangyao Chen
2,
Yanzhou Wang
1,
Tongying Liu
1,
Guang Li
1 and
Siyuan Zhu
1,*
1
Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Changsha 410205, China
2
Yuan LongPing High-Tech Agriculture Co., Ltd., Changsha 410125, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2023, 13(6), 1575; https://doi.org/10.3390/agronomy13061575
Submission received: 16 April 2023 / Revised: 6 June 2023 / Accepted: 8 June 2023 / Published: 10 June 2023
(This article belongs to the Section Crop Breeding and Genetics)
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Abstract

:
NAC transcription factors are one of the largest transcription factor families in plants, and they play a key role in the growth and development of a secondary cell wall. Despite the fact that ramie is well-known for its high fiber yield, the role of NAC transcription factors in ramie secondary cell wall synthesis and fiber development remains unknown. In this study, based on our previous study, we describe the characterization, physicochemical property analysis, protein structure and function prediction, subcellular localization, and functional validation of Bnt05G007257, which encodes an NAC transcription factor from ramie, in transgenic A. thaliana. Our findings show that the open reading frame of Bnt05G007257 was 1035 bp long and encodes for a protein comprising 344 amino acids, having a relative molecular mass of 39.0945 kDa and a theoretical isoelectric point of 6.55. The secondary structure of the encoded protein mainly consisted of random coiling, with a typical conserved structural domain of NAC. The phylogenetic tree revealed that Bnt05G007257 is a homolog of the NAC transcription factor SND2, which regulates secondary wall biosynthesis in A. thaliana. Subcellular localization showed that Bnt05G007257 was tentatively predicted to be localized in the cytoplasm. Furthermore, in stem sections, the secondary wall fiber cells’ thickness in Bnt05G007257 transgenic plants was 31.50% thicker than that in wild-type plants, and the radial width was significantly increased by approximately 21.75%. This indicates that the NAC family homolog Bnt05G007257 may have the potential function of promoting fiber cell development and secondary cell wall synthesis, providing a theoretical basis for the selection of high-fiber-yielding ramie varieties in the future.

1. Introduction

Ramie (Boehmeria nivea L.), which belongs to the genus Boehmeria Jacq. in the family Urticaceae, is an ancient perennial, persistent fiber crop that is commonly known as Chinese grass [1]. The cropped area and yield in China accounts for 90% of the ramie production in the world [2]. Ramie has a variety of utilization values, as the leaves are rich in nutrients, which can be used as animal feed [3], and the roots and leaves contain many bioactive compounds, which have been shown to have medicinal value [4]. Additionally, ramie is also the second largest fiber crop in China, and its planting area and fiber yield are second only to cotton [5]. It has been used in fabrics for 4700 years due to its stronger natural fibers [6]. Ramie fiber is mainly derived from the phloem, and its fiber development is mostly divided into the following four stages: fiber cell differentiation, elongation (primary cell wall synthesis), thickening (secondary cell wall synthesis), and maturation [7]. The plant cell wall usually consists of the primary cell wall, secondary cell wall, and middle lamella [8]. Ramie phloem fiber is an isolated single-cell fiber, which is a hollow dead cell formed by secondary wall thickening after the cell stops growing. Genes related to cell elongation and cell wall synthesis play an important role in the development of ramie bast fiber.
However, there has been little progress in improving ramie fiber yield and quality. Zeng et al. [9] found that a candidate gene, Bnt03G004997, which is overexpressed in Arabidopsis thaliana (A. thaliana), enhances the number of xylem and fiber parts in transgenic A. thaliana. In the last decade, hundreds of genes potentially associated with fiber development have been identified based on homology or expression analysis, few of which have been proven to be associated with fiber development [10,11,12]. Therefore, it is crucial to investigate the development of ramie fiber and the mining of functional characteristics of critical genes in ramie fiber for cultivating high-quality ramie varieties and improving ramie yields.
NAC transcription factors are one of the largest families of transcription factors in plants and are widely distributed in A. thaliana, rice, cucumber, and wheat, playing critical roles in their growth and development. These transcription factors respond to adversity stress [13] and regulate seed and embryo development [14], cell division, stem-tip meristem formation [15], plant lateral root development [16], flower morphogenesis [17], fruit ripening [18], leaf senescence [19], and secondary wall formation [20], among others. Currently, NAC transcription factors are being identified in an increasing number of plants in the plant model system A. thaliana. The NAC transcription factor family that acts as an upstream transcription factor plays an important role in secondary cell wall synthesis [21].
Although the NAC family is known to regulate these functions in A. thaliana, the role of the NAC transcription factors in ramie secondary cell wall synthesis and fiber development remains elusive. To address this, we built on the results of previous studies [9,22] and identified one candidate gene (Bnt05G007257) in ramie transcripts that showed homology with the genes in the NAC transcription family. Therefore, in this study, we performed physicochemical property analysis, protein structure and function prediction, subcellular localization, and functional validation of the ramie NAC transcription factor Bnt05G007257 to generate valuable genes and provide a theoretical basis for breeding high-yielding ramie plants.

2. Results

2.1. Isolation and Characterization of Bnt05G007257

In our previous study, we examined genomic regions undergoing selection for a significant decrease in nucleotide diversity during ramie breeding, including 1128 predicted genes. The expression levels of the transcriptome [22] and proteome [9] were analyzed in stem bark tissues, including from the top and middle of the stem. Results showed that there were significant differences in the transcript or protein abundance of 156 predicted genes in the selected elimination region. Through genome annotation, a NAC gene, Bnt05G007257, with a significant difference in gene expression was found, which may belong to the NAC transcription family through genome annotation [9]. Sequence analysis showed that the full-length coding sequence of Bnt05G007257 is 1035 bp, which encodes 344 amino acid proteins. Serine and threonine were found to be the most abundant amino acids. The analyses also revealed the presence of 47 negatively charged (Asp and Glu) and 43 positively charged amino acid residues (Arg and Lys). The molecular weight (MW) and theoretical isoelectric point (pI) of the protein are 39.0945 kDa and 6.55, respectively.

2.2. Protein Physicochemical Property Analysis

We quantified the hydrophilicity/hydrophobicity of the Bnt05G007257 protein (Figure 1a), and found that the lowest and highest scores for the protein appeared in amino acids 211 and 236, which were −3.289 and 1.178, respectively. The negative score represents the extent of hydrophilicity, whereas the positive score depicts the hydrophobicity. Overall, the amino acid scores were mostly negative, indicating that the content of hydrophilic amino acids in the peptide chain was higher. The average scores of hydrophilic amino acids were greater than those of hydrophobic amino acids, implying Bnt05G007257 is a hydrophilic protein. To further characterize the structural properties of the Bnt05G007257 protein, we performed transmembrane structure analysis (Figure 1b). Our data showed that there was no transmembrane region in the whole peptide chain of Bnt05G007257, and the probability of the existence of transmembrane structure was less than 0.07142. Based on the findings, it is unlikely that Bnt05G007257 is a transmembrane protein. We also probed for the existence of any potential phosphorylation sites in the Bnt05G007257 protein, using phosphorylation site-specific online prediction tools. The prediction analysis showed many potential phosphorylation sites with all of their scores above the threshold of 0.500 (Figure 1c), indicating Bnt05G007257 has multiple phosphorylation sites.

2.3. Protein Structure and Function Prediction

Secondary structure analysis of Bnt05G007257 showed that it mainly consists of 19.19% alpha helices, 21.80% extended chains, 6.10% beta turn angles and 52.91% unregulated coiled regions (Figure 2a). In addition, functional domain prediction analysis showed the presence of a conserved structural domain specific to the NAM subfamily (Figure 2b). This conserved functional domain exists in the amino acid region of the Bnt05G007257 protein at positions 84–223, and is responsible for recognition. The functional domain prediction also showed that the probability of the presence of a signal peptide was less than 0.0008, implying the protein does not have any signal peptides (Figure 2c).

2.4. Evolutionary Analysis of Protein Homology

The NAC family transcription factors VND1, VND2, VND3, VND4, VND5, VND6, VND7, SND1, and SND2, as well as other genes, have been found to be the key genes regulating the formation of the secondary cell wall. We constructed a phylogenetic tree to analyze the relationship between this gene and these key genes (Figure 3b). Our previous research showed that the Bnt05G007257 gene has high homology with SND2/SND3 of A. thaliana [9], which is consistent with the findings from this study. SND2/SND3, a NAC transcription factor gene, regulates genes involved in secondary cell walls in A. thaliana fibers. Evolutionary tree analysis showed that Bnt05G007257 is closer to the SND2/SND3 genes. We hypothesized that this gene could regulate the secondary cell wall of ramie fiber; thus, we selected the Bnt05G007257 gene for further analysis.

2.5. Subcellular Localization of Bnt05G007257

To determine the subcellular localization of Bnt05G007257, a comprehensive computational prediction was performed. The computational predictions showed that Bnt05G007257 may be localized to the peroxisome, cytoplasm, nucleus, and cytoskeleton (https://wolfpsort.hgc.jp/, accessed on 3 March 2022). However, a search on SignalP-5.0 (http://www.cbs.dtu.dk/services/SignalP/, accessed on 4 March 2022) revealed that Bnt05G007257 does not carry a signal peptide (Figure 2c). To verify this contradictory prediction, the coding region of Bnt05G007257 was fused with the EGFP reporter gene under the control of the CaMV 35S promoter, and then injected into tobacco by Agrobacterium tumefaciens for transient expression and confocal microscopy. As shown in Figure 3a, the fluorescent signal of the null PCAMBIA1301-EGFP was found to be dispersed in the cytoplasm and cell membrane of the cells, while that of PCAMBIA1301-Bnt05G007257-EGFP was only seen in the cytoplasm, most likely as the predicted peroxisomal material. This result suggests that the Bnt05G007257 gene is localized in the cytoplasm.

2.6. Function Validation of Bnt05G007257

The completed pCAMBIA1301-Bnt05G007257 expression vector was transferred into Agrobacterium GV3101, and the Agrobacterium was transformed into A. thaliana plants via the flower dip method. Furthermore, As verified by direct amplification and qRT-PCR in triplicate, the target gene was transferred into A. thaliana, as shown in Figure 4c, and had significant expression in the transformed A. thaliana plants compared to wild-type plants, as shown in Figure 4d. Then, the stem sections were observed.
Apparently, the height of the Bnt05G007257 transgenic A. thaliana plants was slightly lower than that of the WT plants; cross-sectional sections from the bottoms of A. thaliana stems revealed that the stem sections of the WT and transgenic plants were different in anatomy, and at the same magnification, compared with that in the WT plants, Bnt05G007257 showed a greater increase in the number of fibers in both the bast and xylem, as well as an increase in the cell cross-sectional area (Figure 4b,c). Safranin staining was observed in the secondary xylem and phloem fibers in the stems of WT and Bnt05G007257 plants, and stronger staining of fiber cells was seen in the Bnt05G007257 stem sections compared with the WT plants. Bnt05G007257 showed a significant increase in the number of secondary wall fiber cells. The thickness and diameter of the cell wall of secondary wall fiber cells in wild-type (WT) and Bnt05G007257 transgenic A. thaliana were measured and statistically analyzed. The results showed that the thickness of the secondary wall fiber cells in Bnt05G007257 transgenic plants was significantly thicker than that in WT plants (p < 0.01), with an increase of approximately 31.50%, and the diameter of fiber cells was significantly larger than that in WT plants (p < 0.05), with an increase of approximately 21.75% (Table 1). This indicated that Bnt05G007257 may promote fiber cell development.

3. Discussion

NAC transcription factors are a class of regulatory proteins, and their regulation is linked to a series of physiological and biochemical responses in plants. NAC genes not only regulate basic plant metabolism and the adversity stress response, but also play a key role in secondary plant growth and secondary cell wall development. The NAC transcription factor family within many species is attracting increasing interest, as a large number of genes from the family are being identified in a variety of plants, including A. thaliala [14], Oryza sativar [23], and Solanum lycopersicum [24] with 171, 115, and 104 genes, respectively. The plant NAC transcription factor family includes three types of genes: NAM, ATAF, and CUC2 (NAC is named after the initial letters of these three genes) [25]. These genes encode homologous proteins, with highly conserved NAC structural domains at the N-terminus and variable transcriptional regulation domains at the C-terminus [26]. This conserved NAC domain consists of five subdomains (A-E), with the C-terminus consisting mainly of acid-rich amino acid groups responsible for the function of the transcriptional regulatory domain, which is mainly involved in transcriptional activation. The main hydrophilic regions of abiotic stress-related NAC proteins are located in the A, C, and D subdomains, being mostly in the nucleus and individually in the cytoplasm or mitochondria [27,28]. The full-length cDNA clone of Bnt05G007257 was successfully obtained from ramie leaves using the transcriptomics method [9], which has an open reading frame of 1035 bp and encodes 344 amino acids. Additionally, it is highly hydrophilic and has no obvious membrane-spanning domains. The encoded protein contains a structural domain typical of NAC transcription factors, which is located in the amino acid region at positions 84–223, and is subcellularly localized in the cytoplasm, meaning that it is tentatively presumed to be a NAC transcription factor. A signal peptide usually refers to the amino acid sequence at the N-terminal of a newly synthesized polypeptide chain that guides the transmembrane transfer (localization) of a protein. In this study, the transmembrane structure of Bnt05G007257 was analyzed, and the results showed that there was no transmembrane region in the whole peptide chain of Bnt05G007257; thus, it was speculated that Bnt05G007257 is not a transmembrane protein. The subcellular localization results showed that Bnt05G007257 was located in the cytoplasm, further confirming that Bnt05G007257 is not a transmembrane protein.
The NAC-mediated transcriptional regulation of secondary wall biosynthesis is a conserved mechanism in vascular plants [29], with a more conserved structure and biological function across species. MtNST1 in Tribulus terrestris is a homolog of NST1-3, which if mutated in alfalfa fiber cells, results in the loss of this secondary cell wall [30]. OsSWN1, a homolog of NST3/SND1, also regulates secondary wall synthesis in rice, and the ectopic expression of OsSWN1 in A. thaliana causes ectopic secondary wall deposition in the chloroplast [31]. Thus, gene homology analysis is clearly an important reference in the speculation of function and identification of NAC genes associated with secondary wall thickening. The phylogenetic tree analysis indicated that Bnt05G007257 is a homolog of the NAC transcription factor SND2, which is responsible for the regulation of secondary wall biosynthesis in A. thaliana and is more closely related to SND3. Our findings suggest that the Bnt05G007257-encoded protein shares structural similarity with NAC transcription factors [32] and may promote ramie fiber cell development and secondary cell wall synthesis.
Some NAC family transcription factors have been identified as structural domain protein 1 (SND1), NAC secondary wall-thickening promoter 1 (NST1) and vascular-associated NAC structural domain (VND), which directly regulate the expression of genes related to secondary cell wall formation [33]. SND2 has been reported to be an indigenous target site of SND1, which increases secondary wall thickness in fiber cells when it is overexpressed, and carries out the opposite when its expression is repressed [34,35,36]. It has been shown that SND2 plays a role in poplar secondary growth as well as in secondary cell wall development; overexpression of SND2 in Eucalyptus stems increases the fiber cross-sectional cell area and promotes fiber cell development [37]. Simultaneously, in A. thaliana, SND2 is known to activate the promoters of genes related to the secondary cell wall biosynthesis of cellulose, xylem, and lignin [38,39]. In this study, the sequence of the Bnt05G007257 gene had a very high homology to SND2, and the overexpression of Bnt05G007257 in A. thaliana led to an extremely significant increase in the number of xylem and bast fiber cells in transgenic plants; these results are basically consistent with our previous research results. Additionally, this is similar to the phenotypic results of the candidate gene Bnt03G004997, selected by Zeng et al. [9], that was overexpressed in A. thaliana and had greater numbers of xylem and fiber cells, which demonstrated the function of the Bnt05G007257 gene in promoting fiber cell development and secondary cell wall synthesis in A. thaliana. Taken together, these results predicted that the Bnt05G007257 gene positively regulates both lignin and cellulose synthesis in A. thaliana, and we hypothesized that the Bnt05G007257 gene may also be a positive regulator of ramie xylem and fiber cell development. To summarize, the theoretical predictions and transgenic functional verification in A. thaliana of Bnt05G007257 are consistent with the properties of NAC transcription factors. Further experiments are needed to verify the exact underlying mechanisms.
The overexpression of SND2/3 and its homologous gene SND4/5 fuses with the activation domain of viral protein VP16, resulting in ectopic secondary wall deposition in cells that are normally parenchyma cells. SND2/3/4/5 regulates the secondary wall NAC binding elements (SNBEs) with the secondary wall NAC master switch by binding and activating secondary wall NAC binding elements (SNBEs) and switches (SWNs), which are expressed in identical downstream target genes [40]. Bnt05G007257 is a homologous gene of NAC transcription factor SND2 in A. thaliana, which regulates secondary wall biosynthesis and is closely related to SND3. It is of obvious significance to explore the function of Bnt05G007257 in regulating the expression of downstream genes of SNBEs and SWNs.

4. Conclusions

Plant fibers and fiber cell secondary walls play an important role in the growth and development of plants, which indicates practical and industrial applications. In this study, we reported the cloning of the gene Bnt05G007257 obtained from ramie leaves, which encodes 344 amino acids. It contains a structural domain typical of the NAC transcription factor located in the amino acid region at positions 84–223 and was judged to be a gene homologous to SND2. SND2, a key regulator of secondary wall growth in the A. thaliana family, was cloned in ramie and its function was initially identified in the model plant A. thaliana. This study not only provides a novel framework for investigating the mechanisms underlying fiber cell development and secondary wall biosynthesis in ramie, but it also lays the foundation for the genetic improvement of fiber yield traits in breeding.

5. Materials and Methods

5.1. Plant Material Collection

The ramie cultivar (Zhongzhu 1) was obtained from the Ramie Germplasm Resource Nursery of the Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Changsha, China (28°12′35.91″ N, 112°41′51.07″ E). Ramie fresh stem tissue material was harvested in June 2021. Colombian wild-type A. thaliana (WT) was used as the model plant material for transgene functional validation. All experimental procedures were approved by the Professional Committee of Plant Protection of the Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences.

5.2. RNA Extraction and cDNA Synthesis

The sequences of the candidate genes are listed in Supplementary Material S1. Specific primers were designed using the software Primer Premier 5.0, and Changsha Dyke Biologicals was commissioned to synthesize the primers. The details of the primers are provided in Table S1. RNA was extracted from the fresh stem tissue material of ramie using a plant RNA extraction kit (TsingKe Biotechnology, Beijing, China), and cDNA was obtained by reverse transcription of the extracted RNA using the reverse transcription kit Prime Script TM IV 1st strand cDNA Synthesis Mix (NEB Biotechnology, Beijing, China).

5.3. Biological Information Analysis and Phylogenetic Tree Analysis

Physicochemical property analysis of the protein was performed with the ProtPraram tool on the ExPASy server 54 (https://web.expasy.org/protparam/, accessed on 6 March 2022). Hydrophilicity/hydrophobicity analysis was carried out using Expasy software 2 (https://web.expasy.org/protscale/, accessed on 6 March 2022). The transmembrane structural domain was determined using Expasy’s TMHMM-2.0 online software (http://www.cbs.dtu.dk/services/TMHMM/, accessed on 6 March 2022). Protein phosphorylation sites were predicted using the software Netphos-3.1 (http://www.cbs.dtu.dk/services/NetPhos/, accessed on 6 March 2022). The protein secondary structure was determined using SOPMA (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html, accessed on 7 March 2022). Conserved domains were analyzed through NCBI (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 7 March 2022). SignalP-5.0 (http://www.cbs.dtu.dk/services/SignalP/, accessed on 8 March 2022) was used to predict the presence of signal peptides. Subcellular localization prediction was performed by WoLF PSORT (https://wolfpsort.hgc.jp/, accessed on 8 March 2022).
To identify the homologs of Bnt05G007257 from 13 NAC genes known to be functionally related to A. thaliana fiber growth, sequence alignment was performed using the Clustal program [41], and a phylogenetic tree was constructed using the MEGA program (version 5.0, National Institutes of Health, Bethesda, MD, USA). The neighbor-joining method was utilized along with a 1000-repeat bootstrap test.

5.4. Gene Cloning and Overexpression Vector Construction

The cDNA was subjected to PCR amplification using ApexHF HS DNA polymerase (Accurate Biotechnology Co., Ltd, Hunan, China). PCR products were ligated with a 5 min TA/Blunt-Zero Cloning Kit (Vazyme Biotech Co., Ltd, Nanjing, China), and subsequently were transformed into Escherichia coli DH5α. The PCR products were checked using 1% agarose gel electrophoresis (Figure 5a), which showed that the band was clearly visible and the length of the gene sequence was consistent with that of the target gene. The positive clone was sequenced by Tsingke China (Shanghai, China), and the results demonstrated that we successfully ligated the target gene to the T vector (Figure 5b,c). The PCAMBIA1301-Bnt05G007257-EGFP vector was constructed according to the homologous recombination strategy described in the user manual of the Clon Express II one-step cloning kit (Vazyme Biotech, Nanjing, China), and then transformed into Agrobacterium GV3101 using the freeze–thaw method. Next, Agrobacterium GV3101 was transformed into A. thaliana plants using the flower dip method. and transgenic plants were obtained. The primers used for this part of the experiment are listed in Table S1.

5.5. Subcellular Localization of Bnt05G007257

Subcellular localization assays were performed with 6-week-old tobacco (Nicotiana tabacum L.) leaves. The coding region of Bnt05G007257 without a stop codon was cloned into the PCAMBIA1301 vector using the EGFP sequence and the cauliflower mosaic virus (CaMV) 35S promoter. The Agrobacterium tumefaciens strain GV3101, which carries the fusion construct (PCAMBIA1301- Bnt05G007257-EGFP) and the control vector (PCAMBIA1301-EGFP), was injected into the leaves of tobacco plants using a 2 mL needle-free syringe. Then, the EGFP green fluorescence signal was observed by using the Zeiss LSM Image Browser Version 4.2.0.121 (Carl Zeiss MicroImaging GmbH, Germany), and all transient expression assays were repeated at least three times.

5.6. Molecular Validation of Transgenic Plants

Direct PCR was performed on A. thaliana transgenic plants using the T5 direct PCR kit (TsingKe Biotech Co., Ltd, Beijing, China), which was followed by positive identification of transgenic A. thaliana. RNA was extracted from A. thaliana transgenic lines and untransformed plants using a plant RNA extraction kit (Accurate Biotechnology Co., Ltd., Hunan, China). qRT-PCR was conducted using M-MLV reverse transcriptase and GoTaq SYBR Green Master Mix (Accurate Biotechnology Co., Ltd., Hunan, China). Primers used for quantitative PCRs are listed in Table S2. Relative gene expression values were calculated using the 2−ΔΔCT method [42]. The A. thaliana actin gene was used as an internal control, and the sequences of primers were as follows: Actin-F—5′-AATTACCCGATGGGCA-3′; Actin-R—5′-TCATACTCGGCCTTGGA-3″.
The lower stem segments of T2 transgenic A. thaliana plants were selected and cut into 2 mm segments. The segments were washed with 1× phosphate-buffered saline, fixed with 4 % (w/v) paraformaldehyde in 1× phosphate-buffered saline at 4 °C, and then embedded in paraffin (Sigma-Aldrich) for sectioning. Sections were stained with safranin O and fast green. Images were taken with a Dmi8 microscope (Leica) and analyzed by LAS X software 3.1.0 (LAS-AF, HCS Basic Module, Leica microsystems, Wetzlar, Germany). For statistical analysis between the two groups of WT and Bnt05G007257 transgenic plants, 8–10 sections of the bottom part of the A. thaliana stem were observed separately. The thickness and radial direction of fiber cell walls were measured by ImageJ software (ImageJ software v1.6.0, NIH, Bethesda, MA, USA), with at least 30 replicates per group.

5.7. Plant Guidelines Statement

Ramie and A. thaliana plants were used in this study. The ramie cultivar (Zhongzhu 1) was obtained from Ramie Germplasm Resource Nursery (the Institute of Bast Fiber Crops, Chinese Academic of Agricultural Sciences, Changsha, China), and Colombian wild-type A. thaliana was kindly provided by Dr. Siyuan Zhu (the Institute of Bast Fiber Crops, Chinese Academic of Agricultural Sciences, Changsha, China). The collection of plant materials and the experimental research carried out on these plants complied with institutional, national, and international guidelines.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy13061575/s1. Supplementary material S1 (Gene sequences and amino acid sequences); Supplementary material S2 (Table S1. Primer dates in the manuscript).

Author Contributions

S.Z. and Y.W. designed the experiments. X.B., T.L. and G.L. provided the experimental methods. G.C. performed the research. X.B., Y.F. and X.W. analyzed the data, wrote the manuscript, and reviewed the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by grants from the National Natural Science Foundation of China (31571618 and 31771734) and the Agricultural Science and Technology Innovation Program (ASTIP-IBFC), and was supported by the China Agriculture Research System of MOF and MARA (CARS-16-E12).

Data Availability Statement

The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. (a) Hydrophilic/hydrophobic analysis of Bnt05G007257. (b) The probability distribution of each amino acid residue of Bnt05G007257 protein in the medial, lateral, and transmembrane helical regions. (c) Prediction of phosphorylation sites of Bnt05G007257.
Figure 1. (a) Hydrophilic/hydrophobic analysis of Bnt05G007257. (b) The probability distribution of each amino acid residue of Bnt05G007257 protein in the medial, lateral, and transmembrane helical regions. (c) Prediction of phosphorylation sites of Bnt05G007257.
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Figure 2. (a) Prediction of secondary structure of Bnt05G007257. (b) Prediction of conservative domain Bnt05G007257. (c) Bnt05G007257 signal peptide prediction.
Figure 2. (a) Prediction of secondary structure of Bnt05G007257. (b) Prediction of conservative domain Bnt05G007257. (c) Bnt05G007257 signal peptide prediction.
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Figure 3. (a) Subcellular localization of the Bnt05G007257 protein in Nicotiana tabacum L. epidermal cells. Chlorophyll is the chloroplast autofluorescence channel; Bright is the brightfield channel; EGFP is the green fluorescent protein channel; Merge is the merged image of the three channels of Chlorophyll, Bright, and EGFP. (b) The evolution analysis of Bnt05G007257 and fiber growth-associated NAC transcription factor in A. thaliana.
Figure 3. (a) Subcellular localization of the Bnt05G007257 protein in Nicotiana tabacum L. epidermal cells. Chlorophyll is the chloroplast autofluorescence channel; Bright is the brightfield channel; EGFP is the green fluorescent protein channel; Merge is the merged image of the three channels of Chlorophyll, Bright, and EGFP. (b) The evolution analysis of Bnt05G007257 and fiber growth-associated NAC transcription factor in A. thaliana.
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Figure 4. (a) Phenotypes of wild-type and transgenic A. thaliana. (b) Microscopic examination of the transected stems of wild-type A. thaliana (left) and Bnt05G007257-overexpressing (right) plants. The red arrow indicates fiber cells, and the scale bar is 75 μm. (c) The PCR validation of Bnt05G007257 in transgenic A. thaliana. (d) The relative expression of Bnt05G007257 in transgenic and WT A. thaliana. Bars represent the mean ± standard error, ** indicates the significant differences (Student’s t-test, ** p < 0.01).
Figure 4. (a) Phenotypes of wild-type and transgenic A. thaliana. (b) Microscopic examination of the transected stems of wild-type A. thaliana (left) and Bnt05G007257-overexpressing (right) plants. The red arrow indicates fiber cells, and the scale bar is 75 μm. (c) The PCR validation of Bnt05G007257 in transgenic A. thaliana. (d) The relative expression of Bnt05G007257 in transgenic and WT A. thaliana. Bars represent the mean ± standard error, ** indicates the significant differences (Student’s t-test, ** p < 0.01).
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Figure 5. (a) Represents gel electrophoresis of Bnt05G007257 gene. (b) Represents the successfully connected T vector and PCAMBIA1301-Bnt05G007257-EGFP recombinant sequencing alignment. (c) Represents PCR identification of Agrobacterium-positive colonies. The red circle indicates a genetic mutation.
Figure 5. (a) Represents gel electrophoresis of Bnt05G007257 gene. (b) Represents the successfully connected T vector and PCAMBIA1301-Bnt05G007257-EGFP recombinant sequencing alignment. (c) Represents PCR identification of Agrobacterium-positive colonies. The red circle indicates a genetic mutation.
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Table
Table 1. Statistics of fiber cell data for WT and Bnt05G007257 transgenic plants.
Table 1. Statistics of fiber cell data for WT and Bnt05G007257 transgenic plants.
SamplesRadial Width of Fiber Cells (μm)Fiber Cell Wall Thickness (μm)
WT9.93 ± 3.35 *1.27 ± 0.35 **
35S:Bnt05G00725712.09 ± 1.96 *1.67 ± 0.40 **
Note: significant difference (* p < 0.05, ** p < 0.01).
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Bai, X.; Fu, Y.; Wang, X.; Chen, G.; Wang, Y.; Liu, T.; Li, G.; Zhu, S. Bnt05G007257, a Novel NAC Transcription Factor, Predicts Developmental and Synthesis Capabilities of Fiber Cells in Ramie (Boehmeria nivea L.). Agronomy 2023, 13, 1575. https://doi.org/10.3390/agronomy13061575

AMA Style

Bai X, Fu Y, Wang X, Chen G, Wang Y, Liu T, Li G, Zhu S. Bnt05G007257, a Novel NAC Transcription Factor, Predicts Developmental and Synthesis Capabilities of Fiber Cells in Ramie (Boehmeria nivea L.). Agronomy. 2023; 13(6):1575. https://doi.org/10.3390/agronomy13061575

Chicago/Turabian Style

Bai, Xuehua, Yafen Fu, Xin Wang, Guangyao Chen, Yanzhou Wang, Tongying Liu, Guang Li, and Siyuan Zhu. 2023. "Bnt05G007257, a Novel NAC Transcription Factor, Predicts Developmental and Synthesis Capabilities of Fiber Cells in Ramie (Boehmeria nivea L.)" Agronomy 13, no. 6: 1575. https://doi.org/10.3390/agronomy13061575

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