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Apurinic/apyrimidinic endonuclease 1 (APE1) exonuclease plays a vital proofreading role in the base excision repair pathway by removing 3’end group, including oxidatively damaged DNA bases, chain terminating nucleotide analog drugs, terminal blocking groups and mismatched bases. Herein, a simple and real time fluorometric method was developed for detecting APE1 exonuclease activity using molecular beacon as substrate. Experimental results demonstrated that the detection limit of this method was 0.01 U/μL for APE1, and the Km value was (0.27 ± 0.10) μmol/L. Moreover, the method was used to screen antibiotics and the results showed that streptomycin sulfate and gentamycin sulfate had a significant inhibitory effect on APE1 exonuclease activity in a concentration-dependent manner. Meanwhile, the assay was used for investigating the inhibitory effects of heavy metal ions on APE1 exonuclease activity. Finally, the method was further used to monitor the effect of heavy metal ions treatment of the APE1 exonuclease activity of ramie, and the results indicated that APE1 exonuclease activity was inhibited in a concentration-dependent manner after treatment with Pb2+ and Cd2+, which was consistent with heavy metal ions assay in vitro. In summary, this method provided an alternative tool for the biochemical analysis for APE1 exonuclease activity, which was hopeful for the assessment for heavy metal ions adsorption according to the enzymatic activity change in phytoremediation.

Background The bast fiber crop ramie can be used as high-quality forage resources, especially in tropical or subtropical region where there is lack of high-quality protein feed. Hongxuan No.1 (HX_1) is a unique ramie variety with a light reddish brown leaf color, which is obviously different from elite cultivar, Zhongzhu No.1 (ZZ_1, green leaf). While, the regulatory mechanism of color difference or secondary metaboliates synthesis between these two varieties have not been studied. Results In this study, phenotypic, transcriptomic and metabolomic analysis of HX_1 and ZZ_1 were conducted to elucidate the mechanism of leaf color formation. Chromaticity value and pigment content measuring showed that anthocyanin was the main metabolites imparting the different leaf color phenotype between the two varieties. Based on LC/MS, at least 14 anthocyanins were identified in leaves of HX_1 and ZZ_1, and the HX_1 showed the higher relative content of malvidin-, pelargonidin-,and cyanidin-based anthocyanins. Transcriptome and metabolome co-analysis revealed that the up-regulated expression of flavonoids synthesis gene was positively correlated with total anthocyanins accumulation in ramie leaf, and the differentfially expression of “blue gene” (F3’5’H) and the “red gene” (F3’H) in leaves bring out HX_1 metabolic flow more input into the cyanidin branch. Furthermore, the enrichment of glycosylated modification pathway (UGT and AT) and the expression of flavonoid 3-O-glucosyl transferase (UFGT), anthocyanidin reductase (ANR), in leaves were significantly influenced the diversity of anthocyanins between HX_1 and ZZ_1. Conclusions Phenotypic, transcriptomic and metabolomic analysis of HX_1 and ZZ_1 indicated that the expression levels of genes related to anthocyanin metabolism contribute to the color formation of ramie variety. Anthocyanins are important plant secandary metabilates with many physiological functions, the results of this study will deepened our understanding of ramie leaf color formation, and provided basis for molecular breeding of functional forage ramie.

Background: The characterization of gene families especially MYB being the largest transcription factor (TFs) families in plants is a crucial step for functional studies. The completion of ramie genome sequencing provides a great opportunity to investigate the organization and evolutionary traits of ramie MYB genes at the genome-wide level. Results: A total of 105 BnGR2R3-MYB genes were identified in ramie and divided into 35 distinct subfamilies according to phylogeny divergence and sequences similarity. These genes were unevenly distributed among 14 chromosomes. Collinearity analysis showed that the segmental and tandem duplication events is the dominant form of the gene family expansion, and duplications prominent in distal telomeric regions. 88 BnGR2R3-MYB genes showed syntenic relationship with those in Apocynum venetum, followed by Cannabis sativa (58), Arabidopsis (53), maize (8) and rice (4). 103 of the 105 BnGMYB proteins were predicted nuclear subcellular, the remaining two were in either chloroplast or cytoplasm. The combination of transcriptome data and phylogenetic tree allows us to propose some powerful MYB candidates that might be involved in the regulation of secondary wall-associated cellulose synthases (BnGMYB14) secondary cell wall thickening (BnGMYB66/67) and flavonoids synthesis (BnGMYB60). The MYBs subgroups involve in regulating anthocyanin were different from arabidopsis and tomato pointing that BnGMYB in other groups play a role in regulating anthocyanin synthesis. qPCR results revealed 8 MYB TFs candidate genes for cadmium stress in ramie. There was an increased in synthesis of procyanidins under the cadmium stress, which suggest a new regulatory pathway in response to the stress. The predicted network identifies the interface between flavonoid metabolic pathways and adversity stress, and found evidence for the involvement of flavonoid synthetic pathways in the stress regulation.Conclusions: This work provides a basic understanding of BnGR2R3-MYB gene family characteristics and provides valuable information that may contribute in improving the tolerance of ramie to cadmium stress and fiber quality.

Ramie (Boehmeria nivea) is a perennial herb that is highly tolerant of heavy metals. In the present study, we cloned a novel metallothionein-like gene from ramie; this gene, termed BnMTL, encodes a putative 46 amino acid protein with a molecular mass of 4.38 kDa. Analysis using quantitative RT-PCR revealed that cadmium (Cd2+ ) treatment results in elevated expression of BnMTL in the roots. We heterologously overexpressed BnMTL in Escherichia coli cells to examine its binding to Cd2+ and its possible role in homeostasis. Recombinant E. coli cells expressing BnMTL exhibited a high tolerance of Cd2+ stress up to a concentration of 1 mm, and the observed accumulation of Cd2+ was almost eight-fold higher than the control. These results demonstrate that BnMTL (i) is highly expressed in the root following exposure to Cd2+ and (ii) encodes a typical metallothionein-like protein with high cadmium-binding activity.

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.

Additional file 4 Fig. S2: MA map and Volcano map. (A) MA map of differential expression; (B) Volcano map of differential expression.

Additional file 6 Fig. S3: Top20 enrichment analysis of KEGG. (A) HX1-vs-ZZ1 (Up); (B) HX1-vs-ZZ1 (Down).

Additional file 7 Fig. S4: LC-MS data analysis. (A) PCA; (B) PLS-DA; (C) OPLS-DA analysis; (D) 200 times response sequencing test of OPLS-DA model; (E) volcano plot.

Additional file 3 Fig. S1: Analysis of mRNA expression level. A is distribution of mRNA expression in 8 samples; B is Box plot.

Background: The occurrence of adventitious roots is necessary for the survival of cuttings. In this study, comparative transcriptome analysis between two ramie (Boehmeria nivea L.) varieties with different adventitious root (AR) patterns, was performed by RNA-Seq under water culture (treatment, T) and non-water culture conditions (control, CK) to reveal the regulatory mechanism of rooting. Results: Characterization of the cuttings of two ramie cultivars, Zhongzhu No 2 (Z2) and Huazhu No 4 (H4), indicated that Z2 had high adventitious rooting rate but H4 had low rooting rate. Twelve cDNA libraries of the two varieties were constructed, and a total of 26723 genes were identified. In non-water culture condition, the number of the distinctive genes in H4 was 2.7 times of that in Z2, while in water culture condition, the number of the distinctive genes in Z2 was nearly 2 times of that in H4. A total of 4411 and 5195 DEGs were identified in the comparison of H4CK vs. H4T and Z2CK vs. Z2T, respectively. After water culture, more DEGs were up-regulated in Z2, but more DEGs were down-regulated in H4. Gene Ontology (GO) Gene functional analysis of the DEGs indicated that polysaccharide metabolic process, carbohydrate metabolic process, cellular carbohydrate metabolic process, cell wall macromolecule metabolic process, and photosystem GO terms were distinctive significantly enriched in H4. Simultaneously, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that photosynthesis, photosynthesis-antenna proteins and starch and sucrose metabolism pathways were identified in H4. Moreover, KEGG analysis showed that jasmonic acid (JA) can interact with ethylene to regulate the occurrence and number of AR in Z2.Conclusions:This study reveals the transcriptomic divergence of two ramie varieties with high and low adventitious rooting rates, and provides insights into the molecular regulatory mechanism of adventitious root formation in ramie.

Water and N are the most important factors affecting ramie (Boehmeria nivea (L.) Gaudich) growth. In this study, de novo transcriptome assembly and Tandem Mass Tags (TMT) based quantitative proteome analysis of ramie under nitrogen and water co-limitation conditions were performed, and exposed to treatments, including drought and N-deficit (WdNd), proper water but N-deficit (WNd), proper N but drought (WdN), and proper N and water (CK), respectively. A total of 64,848 unigenes (41.92% of total unigenes) were annotated in at least one database, including NCBI non-redundant protein sequences (Nr), Swiss-Prot, Protein family (Pfam), Gene Ontology (GO) and KEGG Orthology (KO), and 4268 protein groups were identified. Most significant changes in transcript levels happened under water-limited conditions, but most significant changes in protein level happened under water-limited conditions only with proper N. Poor correlation between differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) was observed in ramie responding to the treatments. DEG/DEP regulation patterns related to major metabolic processes responding to water and N deficiency were analyzed, including photosynthesis, ethylene responding, glycolysis, and nitrogen metabolism. Moreover, 41 DEGs and 61 DEPs involved in regulating adaptation of ramie under water and N stresses were provided in the study, including DEGs/DEPs related to UDP&mdash, glucuronosyhransferase (UGT), ATP synthase, and carbonate dehydratase. The strong dependency of N-response of ramie on water conditions at the gene and protein levels was highlighted. Advices for simultaneously improving water and N efficiency in ramie were also provided, especially in breeding N efficient varieties with drought resistance. This study provided extensive new information on the transcriptome, proteome, their correlation, and diversification in ramie responding to water and N co-limitation.

Flavonoids contained in ramie (Boehmeria nivea) root are important resources for officinal use. The plant seedling can grow optimally and develop an easy-harvesting adventitious root system in a hydroponic system. This research was carried out to unravel the regulation mechanism of flavonoid biosynthesis under hydroponic culturing condition, which hitherto remained unknown. Root samples of the ramie plants grown in soil and hydroponic conditions at different developmental stages were collected and pooled for transcriptome sequencing, and the expression levels of flavonoid biosynthesis-related genes and total flavonoid content were quantified. A total of 43,541 unigenes were finally annotated and the expression of 9 key unigenes were confirmed by qRT-PCR. The flavonoid biosynthesis-related gene BnF3H was significantly up-regulated in the roots of hydroponic ramie, and higher total flavonoids content in the hydroponic ramie roots was also observed. The large dataset of transcripts and unigenes provided abundant genetic information for identifying flavonoid biosynthesis genes in ramie roots. Our results suggest flavonoid biosynthesis in ramie roots is positively affected in aquatic environment, and hydroponic culturing could have a potential for enhancing total flavonoids content in ramie roots.

In this paper, we present the transcriptome profiles of the A. venetum L. by RNA-Seq approach. A total of 6.57 Gb raw data were obtained, and 52,983 unigenes with an average length of 1009 bp and N50 of 1632 bp were annotated with the 7 databases. The unigenes annotated to KEGG database were divided into 21 categories from 6 main groups. Among these, 4952 (22.21%) unigenes were clustered to “Global and overview maps”, and 1834 (8.23%) unigenes were clustered to “Carbohydrate metabolism”. In addition, 6340 unigenes containing 7579 SSRs were identified and the mononucleotide, dinucleotide, trinucleotide motifs were the most common motif type (95.59%), accounting for 39.62%, 36.02%, and 19.95%, respectively.

Apocynum plants, especially A. venetum and A. hendersonii, are rich in flavonoids. In the present study, a whole genome survey of the two species was initially carried out to optimize the flavonoid biosynthesis-correlated gene mining. Then, the metabolome and transcriptome analyses were combined to elucidate the flavonoid biosynthesis pathways. Both species have small genome sizes of 232.80 Mb (A. venetum) and 233.74 Mb (A. hendersonii) and showed similar metabolite profiles with flavonols being the main differentiated flavonoids between the two specie. Positive correlation of gene expression levels (flavonone-3 hydroxylase, anthocyanidin reductase, and flavonoid 3-O-glucosyltransferase) and total flavonoid content were observed. The contents of quercitrin, hyperoside, and total anthocyanin in A. venetum were found to be much higher than in A. hendersonii, and such was thought to be the reason for the morphological difference in color of A. venetum and A. hendersonii. This study provides valuable genomic and metabolome information for understanding of A. venetum and A. hendersonii, and lays a foundation for elucidating Apocynum genus plant flavonoid biosynthesis.

In the current study, the active flavonoids from Apocynum venetum and Apocynum hendersonii leaf were efficiently characterized using UPLC-ESI-MS/MS, and yielding the highest content of 15.35 mg/g (A. venetum) and 13.28 mg/g (A. hendersonii) respectively. The antioxidant assay in vitro showed that the isolated flavonoid ingredient groups exhibited free radical scavenging activities to DPPH, ABTS and linoleic acid. The antimicrobial assay revealed the isolated flavonoid ingredient from both A. venetum and A. hendorsonii have exerted anti-MRSA and anti-P. aeruginosa effect through disrupting cell integrity and declining ATP. In vivo assay demonstrated that these flavonoid ingredients effectively accelerated MRSA-infected and P. aeruginosa-infected Balb/c mice wound healing. In summary, these results showed that the characterized flavonoid ingredients exhibited great potential as natural antioxidant and antimicrobial agents, and shed light into future potential applications of Apocynum spp.

Nitrogen-use efficiency (NUE) has significant impacts on plant growth and development. NUE in plants differs substantially in physiological resilience to nitrogen stress, however, the molecular mechanisms underlying enhanced resilience of high-NUE plants to nitrogen deficiency remains unclear. We compared transcriptome-wide gene expression between high-NUE and low-NUE ramie (Boehmeria nivea (L.) Gaudich) genotypes under nitrogen (N)-deficient and normal conditions to identify the transcriptomic expression patterns that contribute to ramie resilience to nitrogen deficiency. Two ramie genotypes with contrasting NUE were used in the study, including T29 (NUE = 46.01%) and T13 (NUE = 15.81%). Our results showed that high-NUE genotypes had higher gene expression under the control condition across 94 genes, including frontloaded genes such as GDSL esterase and lipase, gibberellin, UDP-glycosyltransferase, and omega-6 fatty acid desaturase. Seventeen stress-tolerance genes showed lower expression levels and varied little in response to N-deficiency stress in high-NUE genotypes. In contrast, 170 genes were upregulated under N deficiency in high-NUE genotypes but downregulated in low-NUE genotypes compared with the controls. Furthermore, we identified the potential key genes that enable ramie to maintain physiological resilience under N-deficiency stress, and categorized these genes into three groups based on the transcriptome and their expression patterns. The transcriptomic and clustering analysis of these nitrogen-utilization-related genes could provide insight to better understand the mechanism of linking among the three gene classes that enhance resilience in high-NUE ramie genotypes.

Background MicroRNAs (miRNAs) regulate numerous crucial abiotic stress processes in plants. However, information is limited on their involvement in cadmium (Cd) stress response and tolerance mechanisms in plants, including ramie (Boehmeria nivea L.) that produces a number of economic valuable as an important natural fibre crop and an ideal crop for Cd pollution remediation. Results Four small RNA libraries of Cd-stressed and non-stressed leaves and roots of ramie were constructed. Using small RNA-sequencing, 73 novel miRNAs were identified. Genome-wide expression analysis revealed that a set of miRNAs was differentially regulated in response to Cd stress. In silico target prediction identified 426 potential miRNA targets that include several uptake or transport factors for heavy metal ions. The reliability of small RNA sequencing and the relationship between the expression levels of miRNAs and their target genes were confirmed by quantitative PCR (q-PCR). We showed that the expression patterns of miRNAs obtained by q-PCR were consistent with those obtained from small RNA sequencing. Moreover, we demonstrated that the expression of six randomly selected target genes was inversely related to that of their corresponding miRNAs, indicating that the miRNAs regulate Cd stress response in ramie. Conclusions This study enriches the number of Cd-responsive miRNAs and lays a foundation for the elucidation of the miRNA-mediated regulatory mechanism in ramie during Cd stress. Electronic supplementary material The online version of this article (10.1186/s12870-018-1561-5) contains supplementary material, which is available to authorized users.