Selenium increases antimony uptake in ramie (Boehmeria nivea L.) by enhancing the physiological, antioxidative, and ionomic mechanisms.

Ramie (Boehmeria nivea L.) is a promising phytoremediation candidate due to its high tolerance and enrichment capacity for antimony (Sb). However, challenges arise as Sb accumulated mainly in roots, complicating soil extraction. Under severe Sb contamination, the growth of ramie may be inhibited. Strategies are needed to enhance Sb accumulation in ramie's aboveground parts and improve tolerance to Sb stress. Considering the beneficial effects of selenium (Se) on plant growth and enhancing resistance to abiotic stresses, this study aimed to investigate the potential use of Se in enhancing Sb uptake by ramie. We investigated the effects of Se (0.5, 1, 2, 5, or 10 µM) on ramie growth, Sb uptake and speciation, antioxidant responses, and ionomic profiling in ramie under 10 mg/L of SbIII or antimonate (SbV) stresses. Results revealed that the addition of 0.5 µM Se significantly increased shoot biomass by 75.73% under SbIII stress but showed minimal effects on shoot and root length in both SbIII and SbV treatments. Under SbIII stress, 2 µM Se significantly enhanced Sb concentrations by 48.42% in roots and 62.88% in leaves. In the case of SbV exposure, 10 µM Se increased Sb content in roots by 42.57%, and 1 µM Se led to a 91.74% increase in leaves. The speciation analysis suggested that Se promoted the oxidation of SbIII to less toxic SbV to mitigate Sb toxicity. Additionally, Se addition effectively minimized the excess reactive oxygen species produced by Sb exposure, with the lowest malondialdehyde (MDA) content at 0.5 µM Se under SbIII and 2 µM Se under SbV, by activating antioxidant enzymes including superoxide dismutase, catalase, peroxidase, and glutathione peroxidase. Ionomic analysis revealed that Se helped in maintaining the homeostasis of certain nutrient elements, including magnesium, potassium (K), calcium (Ca), iron (Fe), and copper (Cu) in the SbIII-treated roots and K and manganese (Mg) in the SbV-treated roots. The results suggest that low concentrations of Se can be employed to enhance the phytoremediation of Sb-contaminated soils using ramie.

Uptake, tolerance, and detoxification mechanisms of antimonite and antimonate in Boehmeria nivea L.

Boehmeria nivea L. (ramie) is a promising phytoremediation plant for antimony (Sb)-contaminated soils. However, the uptake, tolerance, and detoxification mechanisms of ramie to Sb, which are the basis for finding efficient phytoremediation strategies, remain unclear. In the present study, ramie was exposed to 0, 1, 10, 50, 100, and 200 mg/L of antimonite (Sb(III)) or antimonate (Sb(V)) for 14 days in hydroponic culture. The Sb concentration, speciation, subcellular distribution, and antioxidant and ionomic responses in ramie were investigated. The results illustrated that ramie was more effective in the uptake of Sb(III) than Sb(V). Most of the Sb accumulated in ramie roots, with the highest level reaching 7883.58 mg/kg. Sb(V) was the predominant species in leaves, with 80.77-96.38% and 100% in the Sb(III) and Sb(V) treatments, respectively. Immobilization of Sb on the cell wall and leaf cytosol was the primary mechanism of accumulation. Superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) contributed significantly to root defense against Sb(III), while CAT and glutathione peroxidase (GPX) were the major antioxidants in leaves. CAT and POD played crucial roles in the defense against Sb(V). B, Ca, K, Mg, and Mn in Sb(V)-treated leaves and K and Cu in Sb(III)-treated leaves may be related to the biological processes of Sb toxicity mitigation. This study is the first to investigate the ionomic responses of plants toward Sb and could provide valuable information for the phytoremediation of Sb-polluted soils.

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.

Comparative transcriptomics of stem bark reveals genes associated with bast fiber development in Boehmeria nivea L. gaud (ramie).

BACKGROUND: Boehmeria nivea L. Gaud (Ramie) produces one of the longest natural fibers in nature. The bark of ramie mainly comprises of the phloem tissue of stem and is the raw material for fiber. Therefore, identifying the molecular regulation of phloem development is important for understanding of bast fiber biosynthesis and improvement of fiber quality in ramie. RESULTS: In this study, we collected top bud (TB), bark from internode elongating region (ER) and bark from internode fully elongated region (FER) from the ramie variety Zhongzhu No. 1. Histological study indicated that these samples contain phloem tissues at different developmental and maturation stages, with a higher degree of maturation of phloem tissue in FER. RNA sequencing (RNA-seq) was performed and de novo transcriptome was assembled. Unigenes and differentially expressed genes (DEGs) in these three samples were identified. The analysis of DEGs by using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed clear differences in gene expression between ER and FER. Some unigenes involved in secondary cell wall biosynthesis were up-regulated in both ER and FER, while unigenes for some cell wall components or cell wall modifications showed differential expression between ER and FER. In addition, the ethylene respond factors (ERFs) in the ethylene signaling pathway were up-regulated in FER, and ent-kaurenoic acid oxidase (KAO) and GA 20-oxidase (GA20ox) for gibberellins biosynthesis were up-regulated while GA 2-oxidase (GA2ox) for gibberellin inactivation was down-regulated in FER. CONCLUSIONS: Both morphological study and gene expression analysis supported a burst of phloem and vascular developmental processes during the fiber maturation in the ramie stem, and ethylene and gibberellin are likely to be involved in this process. Our findings provide novel insights into the phloem development and fiber maturation in ramie, which could be useful for fiber improvement in ramie and other fiber crops.

Morpho-physiological traits, antioxidant capacity and phytoextraction of copper by ramie (Boehmeria nivea L.) grown as fodder in copper-contaminated soil.

Ramie (Boehmeria nivea L.), the oldest fiber crop in China, can also be grown as fodder crop because of its huge biomass production. Moreover, it has the potential to colonize heavy metal-contaminated soils which showed the possibilities of phytoremediation using B. nivea. Therefore, the present study was conducted to investigate the potential of B. nivea for phytoextraction of copper (Cu)-contaminated soil. Moreover, the impact of different concentrations of Cu on growth and antioxidant enzymatic activity by B. nivea were also studied. For this purpose, a pot experiment was conducted to examine the growth, antioxidative response, and localization (distribution) of Cu in B. nivea plant under different Cu concentrations (0, 50, 100, 200, 300, and 400 mg kg-1 soil). Results revealed that B. nivea tolerated up to 100 mg kg-1 Cu concentration without a significant decrease in biomass, but further increase in Cu concentration from 200 to 400 mg kg-1 exhibited a significant reduction in chlorophyll content, fresh and dry biomass, plant height, and number of leaves. It was further observed that B. nivea accumulated more Cu in roots (26 to 53 mg kg-1), followed by the leaves (23 to 28 mg kg-1) and stems (14 to 21 mg kg-1), while the values for both bioaccumulation factor (BAF) and translocation factor (TF) at all treatments were less than 1. Moreover, activities of antioxidative enzymes (superoxide dismutase and peroxidase) were initially increased with the exposure of 50, 100, and 200 mg kg-1 Cu, but decreased by further increasing the Cu concentration to 300 and 400 mg kg-1 indicating the oxidative stress which is manifested by high malondialdehyde (MDA) and proline contents also. Thus, based on results, it can be concluded that B. nivea accumulated relatively low Cu contents in aboveground parts and could be grown as fodder crop for phytoremediation of Cu-contaminated sites.

The miRNAome of ramie (Boehmeria nivea L.): identification, expression, and potential roles of novel microRNAs in regulation of cadmium stress response.

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.

Genome-wide association study discovered favorable single nucleotide polymorphisms and candidate genes associated with ramet number in ramie (Boehmeria nivea L.).

BACKGROUND: Ramie (Boehmeria nivea L.) is one of the most important natural fiber crops and an important forage grass in south China. Ramet number, which is a quantitative trait controlled by multigenes, is one of the most important agronomic traits in plants because the ramet number per plant is a key component of grain yield and biomass. However, the genetic variation and genetic architecture of ramie ramet number are rarely known. RESULTS: A genome-wide association study was performed using a panel of 112 core germplasms and 108,888 single nucleotide polymorphisms (SNPs) detected using specific-locus amplified fragment sequencing technology. Trait-SNP association analysis detected 44 significant SNPs that were associated with ramet number at P < 0.01. The favorable SNP Marker20170-64 emerged at least twice in the three detected stages and was validated to be associated with the ramie ramet number using genomic DNA polymerase chain reaction with an F1 hybrid progeny population. Comparative genome analysis predicted nine candidate genes for ramet number based on Marker20170-64. Real-time quantitative polymerase chain reaction analysis indicated that six of the genes were specific to upregulation in the ramie variety with high ramet number. These results suggest that these genes could be considered as ramet number-associated candidates in ramie. CONCLUSIONS: The identified loci or genes may be promising targets for genetic engineering and selection for modulating the ramet number in ramie. Our work improves understanding of the genetics of ramet number in ramie core germplasms and provides tools for marker-assisted selection for improvement of agricultural traits.

Ramie (Boehmeria nivea)'s uranium bioconcentration and tolerance attributes.

The authors sampled and analyzed 15 species of dominant wild plants in Huanan uranium tailings pond in China, whose tailings' uranium contents were 3.21-120.52 µg/g. Among the 15 species of wild plants, ramie (Boehmeria nivea) had the strongest uranium bioconcentration and transfer capacities. In order to study the uranium bioconcentration and tolerance attributes of ramie in detail, and provide a reference for the screening remediation plants to phytoremedy on a large scale in uranium tailings pond, a ramie cultivar Xiangzhu No. 7 pot experiment was carried out. We found that both wild ramie and Xiangzhu No. 7 could bioconcentrate uranium, but there were two differences. One was wild ramie's shoots bioconcentrated uranium up to 20 µg/g (which can be regarded as the critical content value of the shoot of uranium hyperaccumulator) even the soil uranium content was as low as 5.874 µg/g while Xiangzhu No. 7's shoots could reach 20 µg/g only when the uranium treatment concentrations were 275 µg/g or more; the other was that all the transfer factors of 3 wild samples were >1, and the transfer factors of 27 out of 28 pot experiment samples were <1. Probably wild ramie was a uranium hyperaccumulator. Xiangzhu No. 7 satisfied the needs of uranium hyperaccumulator on accumulation capability, tolerance capability, bioconcentration factor, but not transfer capability, so Xiangzhu No. 7 was not a uranium hyperaccumulator. We analyzed the possible reasons why there were differences in the uranium bioconcentration and transfer attributes between wild ramie and Xiangzhu No. 7., and proposed the direction for further research. In our opinion, both the plants which bioconcentrate contaminants in the shoots and roots can act as phytoextractors. Although Xiangzhu No. 7's biomass and accumulation of uranium were concentrated on the roots, the roots were small in volume and easy to harvest. And Xiangzhu No. 7's cultivating skills and protection measures had been developed very well. Xiangzhu No. 7's whole bioconcentration factors and the roots' bioconcentration factors, which were 1.200-1.834 and 1.460-2.341, respectively, increased with the increases of uranium contents of pot soil when the soil's uranium contents are 25-175 µg/g, so it can act as a potential phytoextractor when Huanan uranium tailings pond is phytoremediated.

Effects of exogenous calcium and spermidine on cadmium stress moderation and metal accumulation in Boehmeria nivea (L.) Gaudich.

Cadmium (Cd) is a detrimental metal in the environment and it is easily taken up by plants, thus entering the food chain and posing a severe threat to human health. Phytoremediation being low cost, highly stable, and environmentally friendly has been considered as a promising green technology for Cd remediation. The addition of exogenous substances to the culture media has been recognized as an efficient strategy to improve plant phytoremediation capability. Pot trials were conducted to investigate the combined effects of exogenous calcium (Ca) and spermidine (Spd) on Cd-induced toxicity in Boehmeria nivea (L.) Gaudich. (ramie). Results showed that the application of 5-mM exogenous Ca significantly alleviated Cd toxicity in ramie by reducing Cd accumulation, depressing H2O2 and malondialdehyde contents, increasing plants dry weights and chlorophyll concentrations, as well as altering the activities of total superoxide dismutase and guaiacol peroxidase. Furthermore, as a non-Cd hyperaccumulator plant, ramie hyperaccumulated Cd and suffered more severe toxic effects of Cd by the treatment of 1 mM Ca/Cd. The aggravated Cd toxicity could be compensated by the addition of exogenous Spd via the promotion of plant growth and the reduction of the oxidative stress. Overall, the combination effects of 1 mM Ca and Spd appeared to be more superior compared to other treatments in the plants under Cd stress with a higher Cd accumulation ability and the evaluated Cd stress tolerance.

Effects of selenium and silicon on enhancing antioxidative capacity in ramie (Boehmeria nivea (L.) Gaud.) under cadmium stress.

Hydroponic experiments were performed to investigate the ameliorating effects and mitigation mechanisms of selenium and silicon on Cd toxicity in Boehmeria nivea (L.) Gaud. Metal accumulation, chlorophyll content, activities of antioxidant enzymes, and antioxidant contents in ramie were evaluated. The results revealed that cadmium was mainly accumulated in the roots of plants rather than in the aerial parts. Additionally, under 5 mg L(-1) Cd stress, both Se (1 µmol L(-1)) and Si (1 mmol L(-1)) treatments decreased the Cd concentrations in plants. Besides, the treatments also inhibited the translocation ability of Cd from roots to the aboveground parts, which might be related to the decline of generation of reactive oxygen species (ROS). The application of Se and/or Si ameliorated Cd toxicity via stimulating the activities of antioxidant enzymes such as superoxide dismutase (SOD), guaiacol peroxidase (POD), and ascorbate peroxidase (APX), which resulted in the significant decrease of the contents of malondialdialdehyde (MDA) and hydrogen peroxide (H2O2) in ramie leaves. In addition, the content of nonenzymatic antioxidant such as glutathione (GSH) was increased significantly through the addition of selenite and silicate. Also, ascorbate (AsA) and vitamin E played a crucial role in scavenging excess ROS within plants. On the whole, appropriate doses of Se and Si were found to benefit plant growth and enhance the ability of ramie to alleviate Cd-induced stress. Moerover, the effects of combination of Se and Si appeared to be more superior compared to addition separately in response to Cd stress.

Transcript profiling reveals auxin and cytokinin signaling pathways and transcription regulation during in vitro organogenesis of Ramie (Boehmeria nivea L. Gaud).

In vitro organogenesis, one of the most common pathways leading to in vitro plant regeneration, is widely used in biotechnology and the fundamental study of plant biology. Although previous studies have constructed a complex regulatory network model for Arabidopsis in vitro organogenesis, no related study has been reported in ramie. To generate more complete observations of transcriptome content and dynamics during ramie in vitro organogenesis, we constructed a reference transcriptome library and ten digital gene expression (DGE) libraries for illumina sequencing. Approximately 111.34 million clean reads were obtained for transcriptome and the DGE libraries generated between 13.5 and 18.8 million clean reads. De novo assembly produced 43,222 unigenes and a total of 5,760 differentially expressed genes (DEGs) were filtered. Searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database, 26 auxin related and 11 cytokinin related DEGs were selected for qRT-PCR validation of two ramie cultivars, which had high (Huazhu No. 5) or extremely low (Dazhuhuangbaima) shoot regeneration abilities. The results revealed differing regulation patterns of auxin and cytokinin in different genotypes. Here we report the first genome-wide gene expression profiling of in vitro organogenesis in ramie and provide an overview of transcription and phytohormone regulation during the process. Furthermore, the auxin and cytokinin related genes have distinct expression patterns in two ramie cultivars with high or extremely low shoot regeneration ability, which has given us a better understanding of the in vitro organogenesis mechanism. This result will provide a foundation for future phytohormone research and lead to improvements of the ramie regeneration system.

Identification of 32 full-length NAC transcription factors in ramie (Boehmeria nivea L. Gaud) and characterization of the expression pattern of these genes.

NAM, ATAF, and CUC (NAC) genes are plant-specific transcription factors (TFs) that play key roles in plant growth, development, and stress tolerance. To date, none of the ramie NAC (BnNAC) genes had been identified, even though ramie is one of the most important natural fiber crops. In order to mine the BnNAC TFs and identify their potential function, the search for BnNAC genes against two pools of unigenes de novo assembled from the RNA-seq in our two previous studies was performed, and a total of 32 full-length BnNAC genes were identified in this study. Forty-seven function-known NAC proteins published in other species, in concert with these 32 BnNAC proteins were subjected to phylogenetic analysis, and the result showed that all the 79 NAC proteins can be divided into eight groups (NAC-I-VIII). Among the 32 BnNAC genes, 24, 2, and 1 gene showed higher expression in stem xylem, leaf, and flower, respectively. Furthermore, the expression of 14, 11 and 4 BnNAC genes was regulated by drought, cadmium stress, and infection by root lesion nematode, respectively. Interestingly, there were five BnNAC TFs which showed high homology with the NAC TFs of other species involved in regulating the secondary wall synthesis, and their expressions were not regulated by drought and cadmium stress. These results suggested that the BnNAC family might have a functional diversity. The identification of these 32 full-length BnNAC genes and the characterization of their expression pattern provide a basis for future clarification of their functions in ramie growth and development.

[Cloning and expression of atp6 and atp9 genes from ramie (Boehmeria nivea (L.) Gaud.) and their relationship with cytoplasmic male sterility].

The atp6 and apt9 gene fragments associated with cytoplasmic male sterility (CMS) were cloned from the mitochondrial DNA of a ramie (Boehmeria nivea (L.) Gaud.) cytoplasmic male sterile line and its maintainer and restorer lines using PCR and degenerated primer strategy. The primers were designed according to the reserved sequences in the encoding region of mitochondrial genes atp6 and atp9 of some dicotyledons from GenBank. These fragments did not have complete encoding region but showed the homology of 94% and 85% with atp6 and atp9 genes from the referred dicotyledons in GenBank. The complete atp6 and atp9 genes including the complete open reading frames were cloned by means of amplifying the 3' and 5'end unknown sequences of these gene fragments using DNA Walking method. The atp6 gene showed no difference among ramie male sterile line, maintainer and restorer lines at mtDNA sequence, transcription and translation control and protein level. However, compared to the maintainer and restorer lines, the atp9 gene of the male sterile line was different and deletion in several bases at the 3' end of the encoding region. An abnormally high expression of atp9 gene in the male sterile line at the budding stage and full-bloom stage was analyzed by RT-PCR analysis. These results indicated that the variation in DNA sequence and/or abnormality in expression of atp9 gene in the male sterile line maybe closely related to ramie CMS.