Genome-Wide Identification of NAC Genes Associated with Bast Fiber Growth in Ramie (Boehmeria nivea L.)

Abstract

NAM, ATAF, and CUC (NAC) proteins are plant-specific transcription factors that play crucial roles in fiber growth by regulating the secondary wall thickening. In this study, a systematical investigation of NAC genes was performed in the genome of ramie, an important fiber crop, resulting in a total of 60 ramie NAC genes identified. Phylogenetic analysis of these 60 NAC members in conjunction with 111 Arabidopsis NAC proteins identified 11 subfamilies, three of which showed considerable contraction in the ramie genome. Ten ramie NAC genes were identified to encode the orthologs of Arabidopsis NAC regulators involved in the control of secondary wall biosynthesis. Of these ten genes, most showed relatively high expression in the stems, and eight displayed a differential expression between the barks from the top and middle section of the stems where fiber growth is under different stages. Furthermore, the overexpression of three of these ten NAC genes significantly promoted fiber growth in transgenic Arabidopsis. These results indicated that these ten NAC genes were associated with the fiber growth of ramie. This study provided an important basis for researching the regulatory mechanism of fiber growth.

1. Introduction

Transcription factors (TFs) are a protein type that can bind to or modulate the DNA structure in the regulatory region of genes, and then affect their transcription ability, thereby regulating gene expression in eukaryotes. In plants, at least 58 TF families containing 320,370 TFs have been identified in 165 plant species (up to April of 2022) [1]. Of these, the NAM, ATAF, and CUC (NAC) family is a plant-specific TF family, derived originally from the names of three proteins, no apical meristem (NAM), ATAF1-2, and CUC2 (cup-shaped cotyledon). The NAC family is a large family of TFs, and generally comprises hundreds of members, such as a total of 163, 151, 101, and 117 non-redundant NAC members reported in populus, rice, soybean, and the Arabidopsis genome [2,3,4]. NAC proteins comprise two domains, i.e., N-terminal DNA-binding domain and a C-terminal domain that are highly conserved and variable, respectively [5,6]. The unstable C-terminal domain of NAC proteins has been identified as a functional domain in transcriptional activation or repression [7,8,9]. The NAC transcription factors are multifunctional proteins, and widely participate in the plant growth and development, such as maintenance of the shoot apical meristem, lateral root development, flower formation, plant stress responses, embryo development, cell growth and division, seed germination, senescence, and flowering time [10,11,12,13,14,15,16,17,18,19,20,21,22].

Plant fiber is essential for humans because of its value as a raw material to produce paper, textiles, and composites. Plant fibers typically comprise specialized secondary cellular walls which mainly consist of hemicelluloses (xylan and glucomannan), cellulose, and lignin [23], and fiber growth mainly involved in the biosynthesis of secondary walls. Previous studies in Arabidopsis revealed that the biosynthesis of secondary walls was mainly controlled by a complex regulatory network in which NAC proteins are the top-level master switches [23,24]. Therefore, NAC proteins play crucial roles in regulating the biosynthesis of secondary walls and fiber growth.

Ramie, an important fiber crop in China, has been cultivated over 4700 years [25]. The identification of ramie NAC genes involved in secondary wall biosynthesis will be helpful for understanding the regulatory mechanism underlying fiber growth. A previous study identified 32 NAC genes from the ramie transcriptome [26]. However, because the genes with a transient expression in specific time or low expression level are challenging for detecting by transcriptome analysis, the currently reported number of NAC members from the ramie transcriptome was far less than in other plants [2,3,4]. Recently, a high-quality chromosome-level ramie genome has been assembled [27], which provided an opportunity to investigate, genome-wide, some important gene families in this crop. Therefore, this study performed an exploration of the NAC family in the ramie genome, and then identified the NAC genes associated with fiber growth

2. Materials and Methods

2.1. Identifying NAC Members of Ramie

Sequences of the Arabidopsis NAC protein were collected by downloading from the TAIR database (www.arabidopsis.org/index.jsp (accessed on 15 February 2022)). After removing the redundant proteins, a total of 111 Arabidopsis NAC proteins were used as reference queries to identify NAC proteins in the ramie genome using BLASTP [28]. The identified ramie proteins were subjected to conserved domain region analysis using the online CDD program in NCBI (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi (accessed on 15 February 2022)) [29] and MEME (http://meme-suite.org/tools/meme (accessed on 15 February 2022)) [30], and only the proteins with NAM domain were reserved.

2.2. Phylogenetic Analysis of NAC Proteins

Phylogenetic analysis was performed using the 111 Arabidopsis NAC proteins in company with the ramie-identified NAC proteins. Multiple sequence alignments of the protein sequences, including the highly conserved N-terminal binding domain and variable C-terminal functional domain, were carried out using the Clustal program (version 1.83) [31]. Then, an unrooted phylogenetic tree was constructed based on the Neighbor-Joining (NJ) method using the software MEGA 7.0 [32] and the bootstrap test carried out with 1000 replicates. According to the phylogenetic tree, the ramie NAC protein and its Arabidopsis ortholog with the closest evolutionary relationship was ascertained.

2.3. Expression Analysis

In our previous study, the mRNAs of the roots, leaves, and stems of two ramie varieties, Zhongsizhu 1 and tenacissima, were sequenced [27], and these data were deposited in the NCBI GEO database (accession no. GSE130587). Additionally, the transcriptome of sections of bark from the middle and top ramie stems of Zhongzhu 1 were characterized (GEO accession no. GSE158630) [27]. The clean reads from the RNA sequencing were aligned with the ramie reference (project ID in NCBI: PRJNA663427), using the software hisat2 (version: 2.2.1.0) [33], with the default parameters. Fragments per kilobase per million read (FPKM) values of the ramie genes were estimated to measure their expression level [34]. The expression difference of each gene between two different fiber development stages were estimated using the DEseq program (version: 1.18.0) [35], and the significant difference was determined according to the parameters: p < 0.05, fold-change > 2. Then, the FPKM value and expression difference of these 60 NAC genes were filtered from the whole transcriptomic investigation. A heatmap of the gene expression was visualized using an online tool (https://cloud.oebiotech.cn/task/detail/heatmap/ (accessed on 10 March 2022)).

2.4. Overexpression of Ramie NAC Genes

The full-length sequences of three ramie NAC genes were obtained by performing a high-fidelity amplification from a ramie cDNA library, using the primers of the corresponding gene. The primer sequences have been shown in Table S1. Subsequently, the amplified sequence of three genes was separately ligated into the PBI121 vector to initiate their expression from driving the CaMV 35S promoter. Then, a heat shock method was used to introduce the plasmid construct into Agrobacterium tumefaciens strain GV3101, and the resulting Agrobacterium was introduced into wild Arabidopsis using the floral dip method [36]. Transgenic Arabidopsis were planted in a greenhouse under the following conditions: temperature, 22 °C; 15 h light/9 h dark cycle. The 40-day-old transgenic plants were sectioned, stained with Safranin O-Fast Green, then the stem cells were examined via the transmission light microscopy.

2.5. Subcellular Localization

Amplification of the full-length cDNA sequence of Bnt01G000429 was carried out. Subsequently, the amplicon was fused with the enhanced green fluorescent protein (EGFP) cDNA in an in-frame way, and then was ligated to the vector PEZR (K)-LN to drive expression by the CaMV 35S promoter. The heat shock method introduced the plasmid construct into the cells of the Agrobacterium tumefaciens strain GV3101 at 37 °C, and then was transiently expressed in the epidermal cells of tobacco according to the method described by Sparkes et al. [37]. After 48 h incubation, the fluorescence signals of transfected tobacco leaves were examined via the Leica TCS SP5 spectral confocal microscope.

3. Results

3.1. Genome-Wide Identification of Ramie NAC Genes

BLAST analysis using Arabidopsis NAC regulators as inquiries identified 375 NAC candidates in the ramie genome. After filtering the NAC proteins without a conserved NAM domain, a total of 60 NAC ramie genes was identified (Table S2). Of them, 30 genes showed identical coding sequences with previously reported NAC genes that were identified from the ramie transcriptome [26]. Two previously reported NAC genes, BnNAC12 and BnNAC23, were found to be the same gene, and both were transcribed from Bnt03G004378; their slight difference of transcript sequence should result from alternative splicing. Phylogenetic analysis for the NAC members of Arabidopsis and ramie revealed that these NAC proteins could be classified into 11 groups (Figure 1). The number of NAC members in these 11 subfamilies varied from 1 to 18. Three groups (IV, V, and VI) comprised only one or two ramie members, indicating a contraction of these three subfamilies during the evolution of the ramie genome. Stress-responsive NAC subfamilies of ramie had been proposed [26], and its members fell into the group of VIII, indicating that the VIII group was a potential stress-responsive subfamily. Forty-one ramie NAC members showed high conservation in the protein sequence with Arabidopsis members (Table S2). There were 13 Arabidopsis NAC genes involved in the regulation of secondary wall-biosynthesis, and they distributed in three phylogenetic branches (Figure 1). In these three phylogenetic branches, there were 13 ramie NAC proteins, 10 of which were the orthologs involved in the regulation of Arabidopsis secondary wall biosynthesis (Table S2).

3.2. Expression Analysis of Ramie NAC Genes

The expression of ramie NAC genes was investigated in the stems, leaves, and roots of two ramie variates, the cultivated Zhongsizhu 1 (ZSZ1) and wild Boehmeria nivea var. tenacissima (tenacissima). The result revealed five NAC genes (Bnt04G006656, Bnt05G007366, Bnt05G008828, Bnt13G018465, and BntUnG020221) without any expression in the investigated tissues of two varieties (Figures S1 and S2). Additionally, four genes (Bnt03G004611, Bnt07G011376, Bnt08G012167, and Bnt09G014314) were only expressed in the leaves and/or roots of ZSZ1, but not in any investigated tissues of tenacissima. Among these expressed NAC genes, 14, 3, and 24 displayed remarkably higher expression in the stems, leaves, and roots than in the other investigated tissues in ZSZ1, respectively (Figure S1); whereas 15, 10, and 10 showed specifically higher expression in the stems, leaves, and roots than in the other two tissues in tenacissima, respectively (Figure S2). Two genes, Bnt08G013056 and Bnt09G014278, showed extremely high expression in the roots, with the fragments per kilobase per million read (FPKM) value of >300 (Figure 2a). Both Bnt08G013056 and Bnt09G014278 fell into the VIII group, a potential stress-responsive subfamily; Bnt08G013056 and Bnt09G014278 encodes an ortholog of stress-responsive ANAC072 and AtNAP [7,38], respectively. Furthermore, previous studies indicated that drought stress could cause an up-regulated expression for Bnt09G014278 (namely BnNAC07) [26]. Taken together, this evidence supported that these two roots of highly expressed NAC genes were involved in the regulation of the stress response.

We further focused on the expression of NAC genes encoding the ortholog of the Arabidopsis secondary wall biosynthetic NAC regulator. Of ten NAC orthologous genes, nine showed relatively high expression in the stems, except Bnt01G000429 that mainly expressed in the roots, in ZSZ1 (Figure 2b). In wild tenacissima, six of these ten NAC genes displayed high expression in stems (Figure 2c). Three genes, Bnt03G004997, Bnt06G010588, and Bnt03G004081, showed relatively high expression level in the stems of ZSZ1, but in tenacissima, they mainly expressed in the leaves, indicating a difference in the expression pattern of them. Furthermore, the expression level of these 10 NAC genes were compared between the barks from the top and middle section of the stems where fiber growth is under different stages [39], and revealed that the transcript abundance of eight genes had a more than two-fold difference (Figure 2d), indicating the potential roles of these NAC regulators in the fiber growth of ramie.

3.3. Functional Analysis of Three Ramie NAC Genes

It is known that NAC genes are pivotal regulators for promoting the fiber growth and thickening of the secondary walls in plants [23,24]. However, a down-regulated expression of five NAC genes was observed in the barks from the section of the middle stems where fiber is under growing. To validate the role of these NAC genes in fiber growth, three of these five genes, Bnt01G000429, Bnt03G004081, and Bnt14G020028, were used to carry out an overexpression analysis. The result indicated that the overexpression of three NAC genes significantly increased the xylem fiber number in transgenic Arabidopsis (Figure 3a–d), especially Bnt14G020028. Furthermore, their overexpression led to a distinct thickening in the secondary walls of the fibers in transgenic Arabidopsis (Figure 3e–h). These results indicated that these three genes could promote the fiber growth and secondary wall thickening. One NAC gene, Bnt01G000429, was randomly selected for an analysis of subcellular localization, which revealed that the protein only expressed in the nucleus (Figure 4). This result suggested that Bnt01G000429 comprised a nuclear-located sequence, which verified that Bnt01G000429 is a transcription factor. Taken together, these results supported that these NAC genes positively regulate the fiber growth and thickening of secondary walls, which is accordance with their Arabidopsis orthologs.

4. Discussion

Plant fibers are sclerenchymatous cells that typically comprise thickened secondary walls. In Arabidopsis, secondary wall biosynthesis was controlled by an NAC-MYB-based regulatory network in which at least 13 NAC TFs were involved [23]. VASCULAR-RELATED NAC-DOMAIN1-7 (VND1-7) were the first seven NAC proteins to be identified as master regulators of secondary wall biosynthesis in xylem vessel cell differentiation [40]. NAC SECONDARY WALL THICKENING PROMOTING FACTOR1 (NST1) and NST2, members of a sister group to the VNDs, regulate secondary wall-biosynthesis in anther cells [41]. Subsequent work revealed that NST1 and SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN1 (SND1) are the two master switches in promoting fiber cell differentiation and the thickening of secondary walls in Arabidopsis [41,42]. In addition, SND2 and SND3 were identified as another two secondary wall-associated NACs required for normal secondary wall biosynthesis [43]. Unlike the NAC proteins above that promote the biosynthesis of the secondary wall, the NAC regulator, XND1, represses secondary wall biosynthesis [44]. Notably, in poplar, a similar NAC-MYB-based regulatory network of secondary wall-biosynthesis was identified for wood formation, and numerous NAC regulators that are the orthologs of Arabidopsis secondary wall biosynthetic NAC proteins were involved in this network [45]. These previous studies indicated a conserved function between Arabidopsis secondary wall biosynthetic NAC proteins and their orthologs of other species. In this study, a total of 10 orthologs of Arabidopsis secondary wall biosynthetic NAC regulators have been identified; of these, 9 genes showed a relatively high expression in stems of ZSZ1 (except Bnt01G00429), and 8 genes displayed a differential expression between two barks whose fibers are under different growth stages. It is probable that these 10 ramie orthologs of Arabidopsis NAC regulators involved in the secondary wall biosynthesis have a role in the fiber growth.

It is puzzling that only three of ten NAC genes displayed up-regulated expression in the barks where fiber is under-growing, and the expression of the other seven genes was either down-regulation or slight up-regulation. Bnt08G012573 displayed a slightly up-regulated expression in the barks where fiber is under growing, and its overexpression caused a significant increase in the fiber number of transgenic Arabidopsis [27]. Of the five genes with downregulated expression in fiber-growing barks, three have been achieved for overexpression analysis in this study, and one (Bnt03G004997) was used to carry out a functional analysis by Zeng et al. [46], verifying their function in promoting fiber growth. In other words, although these seven NAC genes were either down-regulated or slightly up-regulated in the fiber-growing barks, five of them were authentically involved in the regulation of fiber growth. The difference in the expression pattern of ramie NAC genes indicated a division of labor for them in the regulation of fiber growth, and this proposal provided a base for comprehensively understanding the roles of NAC genes in fiber growth in future studies. Taken together, based on the evidence from the orthologous analysis, expression pattern, and overexpression, this study identified a total of 10 NAC genes associated with fiber growth in the ramie genome, which provided an important basis for researching the regulatory mechanism of fiber growth.