A novel pectate lyase with high specific activity from Bacillus sp. B58-2: Gene cloning, heterologous expression and use in ramie degumming.

Pectinase plays a crucial role in ramie degumming. A gene encoding a putative pectate lyase from Bacillus sp. strain B58-2 was cloned and heterologously expressed in Escherichia coli. The amplified gene BvelPL1 encoded a mature protein of 400 amino acids. BvelPL1 shared the highest amino acid sequence identity (78.75%) with the enzymatically characterized pectate lyase Pel from Bacillus subtilis strain RCK (GenBank: AFH66771.1). The purified recombinant enzyme rBvelPL1-Ec exhibited a maximum specific activity of 2433.26 U/mg at pH 8.5 and 50 °C towards polygalacturonic acid. This specific activity was higher than that of most reported pectate lyases. Remarkably, the enzymatic activity of rBvelPL1-Ec increased by 23.28 times in the presence of 0.4 mM calcium ion. The effect of calcium ion on promoting the enzymatic activity of rBvelPL1-Ec was greater than that for all reported pectate lyases. After degumming with rBvelPL1-Ec, a weight loss of 21.27 ± 1.17% of circled ramie fibers was obtained, and the surfaces of the ramie fibers became smoother. Moreover, a weight loss of 30.47 ± 0.46% was obtained through enzymatic treated and subsequent NaOH treated circled ramie fibers. The excellent performance in degumming suggests that rBvelPL1-Ec may serve as a promising biocatalyst in the textile industry.

Integrated microRNA and whole-transcriptome sequencing reveals the involvement of small and long non-coding RNAs in the fiber growth of ramie plant.

BACKGROUND: MicroRNAs (miRNAs) and long non-coding RNAs (lncRNAs) are the two main types of non-coding RNAs that play crucial roles in plant growth and development. However, their specific roles in the fiber growth of ramie plant (Boehmeria nivea L. Gaud) remain largely unknown. METHODS: In this study, we performed miRNA and whole-transcriptome sequencing of two stem bark sections exhibiting different fiber growth stages to determine the expression profiles of miRNAs, lncRNAs, and protein-encoding genes. RESULTS: Among the identified 378 miRNAs and 6,839 lncRNAs, 88 miRNAs and 1,288 lncRNAs exhibited differential expression. Bioinformatics analysis revealed that 29 and 228 differentially expressed protein-encoding genes were targeted by differentially expressed miRNAs and lncRNAs, respectively, constituting eight putative competing endogenous RNA networks. lncR00022274 exhibited downregulated expression in barks with growing fibers. It also had an antisense overlap with the MYB gene, BntWG10016451, whose overexpression drastically increased the xylem fiber number and secondary wall thickness of fibers in the stems of transgenic Arabidopsis, suggesting the potential association of lncR00022274-BntWG10016451 expression with fiber growth. CONCLUSIONS: These findings provide insights into the roles of ncRNAs in the regulation of fiber growth in ramie, which can be used for the biotechnological improvement of its fiber yield and quality in the future.

Genome-Wide Identification and Expression Analysis of BnPP2C Gene Family in Response to Multiple Stresses in Ramie (Boehmeria nivea L.).

The protein phosphatase 2C (PP2C), a key regulator of the ABA signaling pathway, plays important roles in plant growth and development, hormone signaling, and abiotic stress response. Although the PP2C gene family has been identified in many species, systematic analysis was still relatively lacking in ramie (Boehmeria nivea L.). In the present study, we identified 63 BnPP2C genes from the ramie genome, using bioinformatics analysis, and classified them into 12 subfamilies, and this classification was consistently supported by their gene structures and conserved motifs. In addition, we observed that the functional differentiation of the BnPP2C family of genes was restricted and that fragment replication played a major role in the amplification of the BnPP2C gene family. The promoter cis-regulatory elements of BnPP2C genes were mainly involved in light response regulation, phytohormone synthesis, transport and signaling, environmental stress response and plant growth and development regulation. We identified BnPP2C genes with tissue specificity, using ramie transcriptome data from different tissues, in rhizome leaves and bast fibers. The qRT-PCR results showed that the BnPP2C1, BnPP2C26 and BnPP2C27 genes had a strong response to drought, high salt and ABA, and there were a large number of stress-responsive elements in the promoter region of BnPP2C1 and BnPP2C26. The results suggested that BnPP2C1 and BnPP2C26 could be used as the candidate genes for drought and salt tolerance in ramie. These results provide a reference for further studies on the function of the PP2C gene and advance the development of the mechanism of ramie stress response, with a view to providing candidate genes for the molecular breeding of ramie for drought and salt tolerance.

Transcriptomic analysis reveals the molecular mechanisms of Boehmeria nivea L. in response to antimonite and antimonate stresses.

Soil antimony (Sb) pollution is a global concern that threatens food security and human health. Boehmeria nivea L. (ramie) is a promising phytoremediation plant exhibiting high tolerance and enrichment capacity for Sb. To reveal the molecular mechanisms and thus enhance the ramie uptake, transport, and detoxification of Sb with practical strategies, a hydroponic experiment was conducted to compare the physiological and transcriptomic responses of ramie towards antimonite (Sb(Ⅲ)) and antimonate (Sb(Ⅴ)). Phenotypic results showed that Sb(Ⅲ) had a stronger inhibitory effect on the growth of ramie. Root Sb content under Sb(Ⅲ) was 2.43 times higher than that in Sb(Ⅴ) treatment. Based on the ribonucleic acid sequencing (RNA-Seq) technique, 3915 and 999 significant differentially expressed genes (DEGs) were identified under Sb(Ⅲ) and Sb(Ⅴ), respectively. Transcriptomic analysis revealed that ramie showed different adaptation strategies to Sb(Ⅲ) and Sb(V). Key DEGs and their involved pathways such as catalytic activity, carbohydrate metabolisms, phenylpropanoid biosynthesis, and cell wall modification were identified to perform crucial roles in Sb tolerance and detoxification. Two heavy metal-associated domain-type genes, six heavy metal-associated isoprenylated plant proteins, and nine ABC transporters showed possible roles in the transport and detoxification of Sb. The significant upregulation of NRAMP5 and three NIPs suggested their roles in the transport of Sb(V). This study is the basis for future research to identify the exact genes and biological processes that can effectively enhance Sb accumulation or improve plant tolerance to Sb, thereby promoting the phytoremediation of Sb-polluted soils.

Genome-Wide Analysis of AP2/ERF Gene Superfamily in Ramie (Boehmeria nivea L.) Revealed Their Synergistic Roles in Regulating Abiotic Stress Resistance and Ramet Development.

AP2/ERF transcription factors (TFs) are one of the largest superfamilies in plants, and play vital roles in growth and response to biotic/abiotic stresses. Although the AP2/ERF family has been extensively characterized in many species, very little is known about this family in ramie (Boehmeria nivea L.). In this study, 138 AP2/ERF TFs were identified from the ramie genome and were grouped into five subfamilies, including the AP2 (19), RAV (5), Soloist (1), ERF (77), and DREB (36). Unique motifs were found in the DREB/ERF subfamily members, implying significance to the AP2/ERF TF functions in these evolutionary branches. Segmental duplication events were found to play predominant roles in the BnAP2/ERF TF family expansion. Light-, stress-, and phytohormone-responsive elements were identified in the promoter region of BnAP2/ERF genes, with abscisic acid response elements (ABRE), methyl jasmonate response elements, and the dehydration response element (DRE) being dominant. The integrated transcriptome and quantitative real-time PCR (qPCR) revealed 12 key BnAP2/ERF genes positively responding to waterlogging. Five of the genes are also involved in ramet development, with two (BnERF-30 and BnERF-32) further showing multifunctional roles. The protein interaction prediction analysis further verified their crosstalk mechanism in coordinating waterlogging resistance and ramet development. Our study provides new insights into the presence of AP2/ERF TFs in ramie, and provides candidate AP2/ERF TFs for further studies on breeding varieties with coupling between water stress tolerance and high yield.

Identification of the Xyloglucan Endotransglycosylase/Hydrolase (XTH) Gene Family Members Expressed in Boehmeria nivea in Response to Cadmium Stress.

Xyloglucan endotransglycosylase/hydrolase (XTH) genes play an important role in plant resistance to abiotic stress. However, systematic studies of the response of Boehmeria nivea (ramie) XTH genes (BnXTHs) to cadmium (Cd) stress are lacking. We sought to identify the BnXTH-family genes in ramie through bioinformatics analyses and to investigate their responses to Cd stress. We identified 19 members of the BnXTH gene family from the ramie genome, referred to as BnXTH1-19, among which BnXTH18 and BnXTH19 were located on no chromosomes and the remaining genes were unevenly distributed across 11 chromosomes. The 19 members were divided into four groups, Groups I/II/IIIA/IIIB, according to their phylogenetic relationships, and these groups were supported by analyses of intron-exon structure and conserved motif composition. A highly conserved catalytic site (HDEIDFEFLG) was observed in all BnXTH proteins. Additionally, three gene pairs (BnXTH6-BnXTH16, BnXTH8-BnXTH9, and BnXTH17-BnXTH18) were obtained with a fragment and tandem-repeat event analysis of the ramie genome. An analysis of cisregulatory elements revealed that BnXTH expression might be regulated by multiple hormones and abiotic and biotic stress responses. In particular, 17 cisregulatory elements related to abiotic and biotic stress responses and 11 cisregulatory elements related to hormone responses were identified. We also found that most BnXTH genes responded to Cd stress, and BnXTH1, BnXTH3, BnXTH6, and BnXTH15 were most likely to contribute to the Cd tolerance of ramie, as evidenced by the substantial increases in expression under Cd treatment. Heterologous expression of BnXTH1, BnXTH6, and BnXTH15 significantly enhanced the Cd tolerance of transgenic yeast cells. These results suggest that the BnXTH gene family is involved in Cd stress responses, laying a theoretical foundation for functional studies of BnXTH genes and the innovative breeding of Cd-tolerant ramie.

Probing the transcriptome of Boehmeria nivea reveals candidate genes associated with the biosynthesis of chlorogenic acid.

Boehmeria nivea (L.) Gaudich is used in traditional Chinese medicine. Chlorogenic acids are major medically active components of Boehmeria nivea, which can be used clinically to treat hyperglycemia, pneumonia, and cancer. To identify the genes involved in chlorogenic acid biosynthesis, we analyzed transcriptome data from leaf, root, and stem tissues of Boehmeria nivea using the Illumina Hi-Seq 4000 platform. A total of 146,790 unigenes were obtained from Boehmeria nivea, of which 106,786 were annotated in public databases. In analyses of the KEGG (Kyoto Encyclopedia of Genes and Genome) database, 484 unigenes that encode the five key enzymes involved in chlorogenic acid biosynthesis were identified, and shikimate O-hydroxycinnamoyl transferase was spatially simulated. Some of these key enzyme unigenes expression levels were verified by RT-qPCR (real-time quantitative Polymerase Chain Reaction). Furthermore, multiple genes encoding plant resistance proteins or transcription factors were identified and analyzed. Differentially expressed genes were identified by performing pairwise comparison of genes between tissues. This study increases the number of public transcript datasets of this species and identifies candidate genes related to the biosynthesis of chlorogenic acid, laying a foundation for the further exploration of this pathway in Boehmeria nivea.

Resequencing of 301 ramie accessions identifies genetic loci and breeding selection for fibre yield traits.

Ramie is an important fibre-producing crop in China; however, the genetic basis of its agronomic traits remains poorly understood. We produced a comprehensive map of genomic variation in ramie based on resequencing of 301 landraces and cultivars. Genetic analysis produced 129 signals significantly associated with six fibre yield-related traits, and several genes were identified as candidate genes for respective traits. Furthermore, we found that natural variations in the promoter region of Bnt14G019616 were associated with extremely low fibre abundance, providing the first evidence for the role of pectin methylesterase in fibre growth of plants. Additionally, nucleotide diversity analysis revealed that breeding selection has been markedly focussed on chromosome 9 in which ~ 39.6% sequence underwent selection, where one gibberellin-signalling-repressed DELLA gene showed distinct selection signatures in the cultivars. This study provides insights into the genetic architecture and breeding history of fibre yield traits in ramie. Moreover, the identification of fibre yield-related genetic loci and large-scale genomic variation represent valuable resources for genomics-assisted breeding of this crop.

Regulating role of abscisic acid on cadmium enrichment in ramie (Boehmeria nivea L.).

Abscisic acid (ABA) is known as an important hormone regulating plant stress resistance, such as salt, drought and heavy metal resistance. However, the relationship between ABA and cadmium (Cd) enrichment in ramie (Boehmeria nivea L.) is still unclear to date. This study aimed to reveal the effect of ABA on Cd enrichment in ramie, and we received the following results: (1) Under Cd treatment, the Cd uptake of ramie increased with the increase of Cd concentration, but the chlorophyll content decreased. Under Cd treatment, the ABA content was highest in roots of ramie, followed by that in old leaves, and lowest in new leaves. Long-time treatment of high Cd concentration reduced the ability of endogenous ABA biosynthesis. (2) Spraying ABA on ramie plants (SORP) and adding ABA directly to the culture solution (ADCS) with low concentration can promote the growth of ramie and increase the amount of Cd uptake, and the effect of SORP is better. (3) The molecular reason for the decrease of chlorophyll content due to Cd stress, may be resulted from the down-regulated expression of the chlorophyll synthesis genes (BnPAO and BnNYC1) and the up-regulated expression of the chlorophyll degradation genes (BnCHLH, BnCHLG, BnHAP3A and BnPPR1). The elevated ABA content in ramie plants may due to the up-regulated expression of the ABA synthesis related genes (BnABA1, BnNCED3, and BnNCED5) and the genes (BnABCG40, BnNFXL2, BnPYL9, BnGCR2, BnGTG1, BnBGLU1, BnUTG1, BnVHAG1 and BnABI5) that encoding ABA transport and response proteins, which was consistent with the enhance the Cd uptake in ramie. Our study revealed the relationship between ABA and Cd uptake in ramie, which provided a reference for improving the enrichment of Cd in ramie.

Phosphoproteome analysis reveals an extensive phosphorylation of proteins associated with bast fiber growth in ramie.

BACKGROUND: Phosphorylation modification, one of the most common post-translational modifications of proteins, widely participates in the regulation of plant growth and development. Fibers extracted from the stem bark of ramie are important natural textile fibers; however, the role of phosphorylation modification in the growth of ramie fibers is largely unknown. RESULTS: Here, we report a phosphoproteome analysis for the barks from the top and middle section of ramie stems, in which the fiber grows at different stages. A total of 10,320 phosphorylation sites from 9,170 unique phosphopeptides that were assigned to 3,506 proteins was identified, and 458 differentially phosphorylated sites from 323 proteins were detected in the fiber developmental barks. Twelve differentially phosphorylated proteins were the homologs of Arabidopsis fiber growth-related proteins. We further focused on the function of the differentially phosphorylated KNOX protein whole_GLEAN_10029667, and found that this protein dramatically repressed the fiber formation in Arabidopsis. Additionally, using a yeast two-hybridization assay, we identified a kinase and a phosphatase that interact with whole_GLEAN_10029667, indicating that they potentially target this KNOX protein to regulate its phosphorylation level. CONCLUSION: The finding of this study provided insights into the involvement of phosphorylation modification in ramie fiber growth, and our functional characterization of whole_GLEAN_10029667 provide the first evidence to indicate the involvement of phosphorylation modification in the regulation of KNOX protein function in plants.

Genomic analyses provide comprehensive insights into the domestication of bast fiber crop ramie (Boehmeria nivea).

Ramie (Boehmeria nivea) is an economically important natural fiber-producing crop that has been cultivated for thousands of years in China; however, the evolution of this crop remains largely unknown. Here, we report a ramie domestication analysis based on genome assembly and resequencing of cultivated and wild accessions. Two chromosome-level genomes representing wild and cultivated ramie were assembled de novo. Numerous structural variations between two assemblies, together with the genetic variations from population resequencing, constituted a comprehensive genomic variation map for ramie. Domestication analysis identified 71 high-confidence selective sweeps comprising 320 predicted genes, and 29 genes from sweeps were associated with fiber growth in the expression. In addition, we identified seven genetic loci associated with the fiber yield trait in the segregated population derived from the crossing of two assembled accessions, and two of which showed an overlap with the selective sweeps. These findings indicated that bast fiber traits were focused on during the domestication history of ramie. This study sheds light on the domestication of ramie and provides a valuable resource for biological and breeding studies of this important crop.

Differential Proteomic Analysis to Identify Proteins Associated with Apomeiosis in Boehmeria tricuspis (Hance) Makino Using an iTRAQ-Based Strategy.

Numerous candidate genes related to apomixis have been identified through transcriptomics; however, the molecular mechanism underlying apomixis remains unclear. Elucidation of the underlying mechanisms is essential to expand its application in crop breeding. Therefore, here, we employed the isobaric tags for a relative and absolute quantification labeling technology to investigate the protein expression in Boehmeria tricuspis generated through different reproductive modes at the functional megaspore stage. We identified 40 differential abundance proteins associated with apomeiosis, most of which were involved in "response to stress". Functional analysis suggested that lower levels of reactive oxygen species (ROS) play a role in inducing the development of apomeiosis. Proteins related to ROS regulation, cell wall modifications, and stability under heat stress play a crucial role in the development of diplosporic apomeiosis. Our results give evidence to the insight that stress can induce a switch from apomixis to sexuality by ROS content, and an increased composition of stress tolerance as well as secondary metabolites can buffer ROS effects. Precise coordination of these proteins involved in inter-related regulatory control mechanisms may act together in the transition from the sexual to apomixis development.

A MYB transcription factor, BnMYB2, cloned from ramie (Boehmeria nivea) is involved in cadmium tolerance and accumulation.

MYB-related transcription factors play important roles in plant development and response to various environmental stresses. In the present study, a novel MYB gene, designated as BnMYB2 (GenBank accession number: MF741319.1), was isolated from Boehmeria nivea using rapid amplification of cDNA ends (RACE) and RT-PCR on a sequence fragment from a ramie transcriptome. BnMYB2 has a 945 bp open reading frame encoding a 314 amino acid protein that contains a DNA-binding domain and shares high sequence identity with MYB proteins from other plant species. The BnMYB2 promoter contains several putative cis-acting elements involved in stress or phytohormone responses. A translational fusion of BnMYB2 with enhanced green fluorescent protein (eGFP) showed nuclear and cytosolic subcellular localization. Real-time PCR results indicated that BnMYB2 expression was induced by Cadmium (Cd) stress. Overexpression of BnMYB2 in Arabidopsis thaliana resulted in a significant increase of Cd tolerance and accumulation. Thus, BnMYB2 positively regulated Cd tolerance and accumulation in Arabidopsis, and could be used to enhance the efficiency of Cd removal with plants.

Identification and characterization of microRNAs in phloem and xylem from ramie (Boehmeria nivea).

Ramie (Boehmeria nivea) is a widely cropped species in southern China due to its high economic value of natural fiber for industry. Development of phloem and xylem is key evidence for generating fiber. However, the MicroRNA (miRNA) profiles of phloem and xylem in ramie have not been reported yet. miRNA belong to a small RNA family which has been recognized as an important regulator for various biological processes. In the present study, we aimed to identify differently expressed miRNAs between phloem and xylem in adult ramie. The results showed that 137 and 122 unique conserved miRNAs were identified from phloem and xylem libraries, respectively. Meanwhile, 4 novel miRNAs were identified from ramie by miRDeep2. Of these miRNAs, 77 conserved miRNAs in ramie were differentially expressed. Among the differentially expressed miRNAs, 44 miRNAs and 33 miRNAs were up-regulated and down-regulated in phloem compared to that in xylem, respectively. The functions of differentially expressed miRNAs were associated with regulating the development and differentiation of phloem and xylem. The present study provides a glance of miRNA profiles for further understanding of miRNA role in ramie development.

Selection of Reference Genes for qPCR Analyses of Gene Expression in Ramie Leaves and Roots across Eleven Abiotic/Biotic Treatments.

Quantitative real-time PCR (qPCR) is commonly used for deciphering gene functions. For effective qPCR analyses, suitable reference genes are needed for normalization. The objective of this study is to identify the appropriate reference gene(s) for qPCR analyses of the leaves and roots of ramie (Boehmeria nivea L.), an important natural fiber crop. To accomplish this goal, we investigated the expression patterns of eight common plant qPCR reference genes in ramie leaves and roots under five abiotic stresses, five hormonal treatments, and one biotic stress. The relative expression stabilities of the eight genes were evaluated using four common but different approaches: geNorm, NormFinder, BestKeeper, and RefFinder. Across the 11 tested conditions, ACT1 was the most stably expressed among the eight genes while GAPDH displayed the biggest variation. Overall, while variations in the suggested reference genes were found for different tissue x treatment combinations, our analyses revealed that together, genes ACT1, CYP2, and UBQ can provide robust references for gene expression studies of ramie leaves under most conditions, while genes EF-1α, TUB, and ACT1 can be used for similar studies of ramie roots. Our results should help future functional studies of the genes in ramie genome across tissues and environmental conditions.

Linkage mapping of quantitative trait loci for fiber yield and its related traits in the population derived from cultivated ramie and wild B. nivea var. tenacissima.

Ramie is an important natural fiber crop, and the fiber yield and its related traits are the most valuable traits in ramie production. However, the genetic basis for these traits is still poorly understood, which has dramatically hindered the breeding of high yield in this fiber crop. Herein, a high-density genetic map with 6,433 markers spanning 2476.5 cM was constructed using a population derived from two parents, cultivated ramie Zhongsizhu 1 (ZSZ1) and its wild progenitor B. nivea var. tenacissima (BNT). The fiber yield (FY) and its four related traits-stem diameter (SD) and length (SL), stem bark weight (BW) and thickness (BT)-were performed for quantitative trait locus (QTL) analysis, resulting in a total of 47 QTLs identified. Forty QTLs were mapped into 12 genomic regions, thus forming 12 QTL clusters. Among 47 QTLs, there were 14 QTLs whose wild allele from BNT was beneficial. Interestingly, all QTLs in Cluster 10 displayed overdominance, indicating that the region of this cluster was likely heterotic loci. In addition, four fiber yield-related genes underwent positive selection were found either to fall into the FY-related QTL regions or to be near to the identified QTLs. The dissection of FY and FY-related traits not only improved our understanding to the genetic basis of these traits, but also provided new insights into the domestication of FY in ramie. The identification of many QTLs and the discovery of beneficial alleles from wild species provided a basis for the improvement of yield traits in ramie breeding.

Integration of Quantitative Trait Loci Mapping and Expression Profiling Analysis to Identify Genes Potentially Involved in Ramie Fiber Lignin Biosynthesis.

Ramie fibers, one of the most important natural fibers in China, are mainly composed of lignin, cellulose, and hemicellulose. As the high lignin content in the fibers results in a prickly texture, the lignin content is deemed to be an important trait of the fiber quality. In this study, the genetic basis of the fiber lignin content was evaluated, resulting in the identification of five quantitative trait loci (QTLs). Three genes, whole_GLEAN_10021050, whole_GLEAN_10026962, and whole_GLEAN_10009464 that were identified on the QTL regions of qLC7, qLC10, and qLC13, respectively, were found to be homologs of the Arabidopsis lignin biosynthetic genes. Moreover, all three genes displayed differential expression in the barks located in the top and middle parts of the stem, where lignin was not being synthesized and where it was being biosynthesized, respectively. Sequence comparison found that these three genes had wide variations in their coding sequences (CDSs) and putative promoter regions between the two parents, especially the MYB gene whole_GLEAN_10021050, whose protein had insertions/deletions of five amino acids and substitutions of two amino acids in the conserved domain. This evidence indicates that these three genes are potentially involved in lignin biosynthesis in ramie fibers. The QTLs identified from this study provide a basis for the improvement of lignin content and fiber quality in ramie breeding. The characterization of the three candidate genes here will be helpful for the future clarification of their functions in ramie.

A genomic resource derived from the integration of genome sequences, expressed transcripts and genetic markers in ramie.

BACKGROUND: The redundancy of genomic resources, including transcript and molecular markers, and their uncertain position in the genome have dramatically hindered the study of traits in ramie, an important natural fiber crop. RESULTS: We obtained a high-quality transcriptome consisting of 30,591 non-redundant transcripts using single-molecule long-read sequencing and proposed it as a universal ramie transcriptome. Additionally, 55,882 single nucleotide polymorphisms (SNPs) were identified and a high-density genetic map was developed. Based on this genetic map, 181.7 Mb ramie genome sequences were assembled into 14 chromosomes. For the convenient use of these resources, 29,286 (~ 95.7%) of the transcripts and all 55,882 SNPs, along with 1827 previously reported sequence repeat markers (SSRs), were mapped into the ramie genome, and 22,343 (~ 73.0%) transcripts, 50,154 (~ 89.7%) SNPs, and 1466 (~ 80.3%) SSRs were assigned to a specific location in the corresponding chromosome. CONCLUSION: This is the first study to characterize the ramie transcriptome by long-read sequencing, and the substantial number of transcripts of significant length obtained will accelerate our understanding of ramie growth and development. This integration of genome sequences, expressed transcripts, and genetic markers will provide an extremely useful resource for genetic, molecular, and breeding studies of ramie.

Correlation Analysis of Lignin Accumulation and Expression of Key Genes Involved in Lignin Biosynthesis of Ramie (Boehmeria nivea).

The phloem of the stem of ramie (Boehmeria nivea) is an important source of natural fiber for the textile industry. However, the lignin content in the phloem affects the quality of ramie phloem fiber. In this study, the lignin content and related key gene expression levels were analyzed in the phloem and xylem at different developmental periods. The results showed that the relative expression levels of lignin synthesis-related key genes in the xylem and phloem of the stem gradually decreased from the fast-growing period to the late maturation period, but the corresponding lignin content increased significantly. However, the relative expression levels of a few genes were the highest during the maturation period. During all three periods, the lignin content in ramie stems was positively correlated with the expression of genes, including PAL, C4H and 4CL1 in the phenylpropanoid pathway, F5H and CCoAOMT in the lignin-specific synthetic pathway, and CAD in the downstream pathway of lignin synthesis, but the lignin content was negatively correlated with the expression of genes including 4CL3 in the phenylpropanoid pathway and UDP-GT in the shunt pathway of lignin monomer synthesis. The ramie 4CL3 recombinant protein prefers cinnamic acid as a substrate during catalysis, and it negatively regulates lignin synthesis. It is speculated that ramie 4CL3 is mainly involved in the synthesis of ramie flavonoid compounds, and that 4CL1 is mainly involved in lignin synthesis.

Isolation and functional characterization of SUCROSE SYNTHASE 1 and SUCROSE TRANSPORTER 2 promoters from ramie (Boehmeria nivea L. Gaudich).

Sucrose synthase and sucrose transporter are involved in sucrose metabolism and partitioning of photosynthetic products, respectively. In this study, we cloned SUCROSE SYNTHASE 1 and SUCROSE TRANSPORTER 2 genes from ramie. Real-time quantitative PCR revealed that BnSUS1 and BnSUT2 were widely expressed in the analyzed tissues. Subsequently, the two promoters of BnSUS1 and BnSUT2 were isolated and truncated. The two promoters and their truncated fragments were fused GUS to transform into Arabidopsis. GUS staining showed that BnSUS1pro-1690 and BnSUS1pro-1420 had vascular specificity in cotyledons and mature leaves while BnSUT2pro-2239, BnSUT2pro-1681, BnSUT2pro-1199 and BnSUT2pro-618 had a constitutive function in seedlings and mature organs. Notably, the activity of BnSUT2pro-2239 and its fragments (except that of BnSUT2pro-231) are strongly induced by mechanical wounding. Moreover, BnSUS1pro-1051 and BnSUS1pro-485 are sensitive to CuSO4 treatment while BnSUT2pro-2239 and BnSUT2pro-1681 are sensitive to PEG and ABA treatments, respectively. Our findings will provide the foundation for deciphering the functions of BnSUS1 and BnSUT2, and also expand the promoter library to provide more options for plant genetic engineering.