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.

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.

Analysis of BnMTL, a novel metallothionein-like protein in the bast fiber crop Ramie (Boehmeria nivea).

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.

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.

Influence of rice straw biochar on growth, antioxidant capacity and copper uptake in ramie (Boehmeria nivea L.) grown as forage in aged copper-contaminated soil.

Copper (Cu) contamination in agricultural soil poses severe threats to living organisms, and possible ecofriendly solutions need to be considered for Cu immobilization, such as using biochar. A pot study was conducted to examine the effectiveness of biochar derived from rice straw (RSB) at various application rates (0, 2.5, 5 and 10% w/w) to mitigate possible risks of Cu solubility and its uptake by ramie (Boehmeria nivea L.) as forage. The plant growth parameters as well as soil chemical properties (pH, electrical conductivity and cation exchange capacity) notably improved with the increasing RSB application. Moreover, prominent reduction was observed in soil bioavailable Cu concentration by 96% with RSB application of 10% relative to control. In addition, Cu content in B. nivea roots, leaves and stems decreased by 60, 28 and 22%, respectively, for 10% RSB application. It was noted that chlorophyll content and gas exchange parameters in leaves were significantly higher at 10% RSB application than in control. Furthermore, 10% RSB resulted in a greater reduction in oxidative stress from the Cu in soil. Thus, soil amendment with RSB demonstrated positive results for Cu stabilization in aged Cu-contaminated soil, thereby reducing its accumulation and translocation in B. nivea and mitigating livestock feed security risks.

Diplosporous development in Boehmeria tricuspis: Insights from de novo transcriptome assembly and comprehensive expression profiling.

Boehmeria tricuspis includes sexually reproducing diploid and apomictic triploid individuals. Previously, we established that triploid B. tricuspis reproduces through obligate diplospory. To understand the molecular basis of apomictic development in B. tricuspis, we sequenced and compared transcriptomic profiles of the flowers of sexual and apomictic plants at four key developmental stages. A total of 283,341 unique transcripts were obtained from 1,463 million high-quality paired-end reads. In total, 18,899 unigenes were differentially expressed between the reproductive types at the four stages. By classifying the transcripts into gene ontology categories of differentially expressed genes, we showed that differential plant hormone signal transduction, cell cycle regulation, and transcription factor regulation are possibly involved in apomictic development and/or a polyploidization response in B. tricuspis. Furthermore, we suggest that specific gene families are possibly related to apomixis and might have important effects on diplosporous floral development. These results make a notable contribution to our understanding of the molecular basis of diplosporous development in B. tricuspis.

Large-scale developing of simple sequence repeat markers and probing its correlation with ramie (Boehmeria nivea L.) fiber quality.

Marker-assisted selection is an important component of the discipline of molecular breeding. Using DNA markers to assist in plant breeding, the efficiency and precision could be greatly increased. However, the scarcity number of identified DNA markers has hindered the research and the breeding process of ramie (Boehmeria nivea L.) in many aspects, especially fiber quality, one of the top-priority breeding objectives of ramie. In this study, 4230 SSR loci were identified in 3969 unigenes (6.80 % of 58,369), which were de novo assembled from the transcriptome involving different ramie fiber developmental stages. Among these SSRs, the dinucleotides (1599, 37.80 %) and trinucleotides (772, 18.25 %) were most abundant; the motifs AG/CT (1140, 26.94 %), AT/AT (407, 9.62 %) and AGA/TCT (246, 8.31 %) comprised the three most abundant repeats. A total of 2431 primer pairs were designed flanking the SSRs and 1050 of them were employed in PCR amplification for their usefulness using three ramie cultivars. The results showed that 88.10 % of these primers could generate positive PCR bands in any of the three cultivars. Further phylogenetic analysis that conducted from the PCR amplification of 52 specifically sifted SSR primers within 17 cultivars approved that the possible correlation may exist between the primers and ramie fiber quality. These developed SSR markers could be applied in downstream studies, like genetic and physical maps, quantitative trait loci mapping, genetic diversity studies and cultivar fingerprinting, and breeding processes of ramie with better fiber quality under further confirmation of the correlation with ramie fiber quality.

Growth, photosynthesis, and defense mechanism of antimony (Sb)-contaminated Boehmeria nivea L.

Ramie (Boehmeria nivea L.) is the oldest cash fiber crop in China and is widely grown in antimony (Sb) mining areas. To evaluate the extent of Sb resistance and tolerance, the growth, tolerance index (TI), Sb content in plant parts and in Hoagland solution, bioaccumulation factor (BF), photosynthesis, and physiological changes in Sb-contaminated B. nivea (20, 40, 80, and 200 mg L(-1) Sb) grown hydroponically were investigated. The Sb tolerance and resistance of ramie were clearly revealed by growth inhibition, a TI between 13 and 99 %, non-significant changes in the maximum quantum efficiency of photosystem (F v /F m ), energy-harvesting efficiency (photosystem II (PSII)) and single-photon avalanche diode (SPAD) value, a significant increase in Sb in plant parts, BF >1, and an increase in catalase (CAT) and malondialdehyde (MDA) at 200 mg L(-1) Sb. Under increasing Sb stress, nearly the same non-significant decline in the maximum quantum efficiency of photosystem (F v /F m ), energy-harvesting efficiency (PSII), relative quantum yield of photosystem II (φPSII), and photochemical quenching (qP), except for F v /F m at 20 mg L(-1) Sb, were recorded. SPAD values for chlorophyll under Sb stress showed an increasing trend, except for a slight decrease, i.e., <2 %, than the control SPAD value at 200 mg L(-1) Sb. With a continuous increase in MDA, superoxide dismutase (SOD), peroxidase (POD), and CAT activities were suppressed under Sb addition up to 40 mg L(-1) Sb and the addition of Sb enhanced enzyme production at 80 and 200 mg L(-1) Sb. A continuous decrease in SOD, POD, and CAT up to 40 mg L(-1) Sb and enhancements at ≥80 mg L(-1), along with the continuous enhancement of MDA activity and inhibited biomass production, clearly reveal the roles of these enzymes in detoxifying Sb stress and the defense mechanism of ramie at 80 mg L(-1) Sb. Thus, B. nivea constitutes a promising candidate for Sb phytoremediation at mining sites.

Variability of cadmium, lead, and zinc tolerance and accumulation among and between germplasms of the fiber crop Boehmeria nivea with different root-types.

Crop germplasms substantially vary in their tolerance for and accumulation of heavy metals, and assessment of this variability plays a significant role in selecting species to use in phytoremediation projects. Here, we examined germplasm-variations in cadmium (Cd), lead (Pb), and zinc (Zn) tolerance and accumulation in ramie (Boehmeria nivea), a fiber crop native to China, which has received little attention. In an 8-week greenhouse test, fourteen germplasms of ramie, among and within deep, middle, and shallow rooted-types, were compared for growth and metal accumulation traits. Results showed that both tolerance and accumulation traits varied across germplasms and rooted-types. The deep rooted-type germplasms produced more biomass and had higher tolerance to metals than the two others. In addition, considerable variations in metal accumulation were observed among plant organs (root, stem, and leaf), rooted-types, germplasms, and metal supply. However, the observed variations in metal tolerance and accumulation among both germplasms and rooted-types were not significant in most cases. In addition to supporting the idea of a certain degree of constitutional metal tolerance for ramie, our results also contribute to deep-rooted germplasms of ramie as a good candidate, rather than middle-/shallow- ones as a least-bad option, for the remediation of multi metal-contaminated soils.

Effect of exogenous nitric oxide on antioxidative system and S-nitrosylation in leaves of Boehmeria nivea (L.) Gaud under cadmium stress.

Cadmium (Cd)-induced growth inhibition is one of the primary factors limiting phytoremediation effect of Boehmeria nivea (L.) Gaud in contaminated soil. Sodium nitroprusside (SNP), a donor of nitric oxide (NO), has been evidenced to alleviate Cd toxicity in many plants. However, as an important mechanism of NO in orchestrating cellular functions, S-nitrosylation is still poorly understood in its relation with Cd tolerance of plants. In this study, higher exogenous NO levels were found to coincide with higher S-nitrosylation level expressed as content of S-nitrosothiols (SNO). The addition of low concentration (100 µM) SNP increased the SNO content, and it simultaneously induced an alleviating effect against Cd toxicity by enhancing the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), and glutathione reductase (GR) and reduced the accumulation of H2O2 as compared with Cd alone. Application of S-nitrosoglutathione reductase (GSNOR) inhibitors dodecanoic acid (DA) in 100 µM SNP group brought in an extra elevation in S-nitrosylation level and further reinforced the effect of SNP. While the additions of 400 µM SNP and 400 µM SNP + 50 µM DA further elevated the S-nitrosylation level, it markedly weakened the alleviating effect against Cd toxicity as compared with the addition of 100 µM SNP. This phenomenon could be owing to excess consumption of glutathione (GSH) to form SNO under high S-nitrosylation level. Therefore, the present study indicates that S-nitrosylation is involved in the ameliorating effect of SNP against Cd toxicity. This involvement exhibited a concentration-dependent property.

Enhanced efficiency of cadmium removal by Boehmeria nivea (L.) Gaud. in the presence of exogenous citric and oxalic acids.

Boehmeria nivea (L.) Gaud. is a potential candidate for the remediation of Cd contaminated sites. The present investigation aims to explore Cd tolerance threshold and to quickly identify the role of exogenous organic acids in Cd uptake and abiotic metal stress damage. Elevated Cd levels (0-10mg/L) resulted in an obvious rise in Cd accumulation, ranging from 268.0 to 374.4 in root and 25.2 to 41.2mg/kg dry weight in shoot, respectively. Citric acid at 1.5 mmol/L significantly facilitated Cd uptake by 26.7% in root and by 1-fold in shoot, respectively. Cd translocation efficiency from root to shoot was improved by a maximum of 66.4% under 3 mmol/L of oxalic acid. Citric acid exhibited more prominent mitigating effect than oxalic acid due to its stronger ligand affinity for chelating with metal and avoiding the toxicity injury of free Cd ions more efficiently. The present work provides a potential strategy for efficient Cd remediation with B. nivea.

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.

Comparative proteome analysis of the response of ramie under N, P and K deficiency.

Ramie is an important natural fiber. There has been little research on the molecular mechanisms of ramie related to the absorption, utilization and metabolism of nitrogen (N), phosphorus (P) and potassium (K). One approach to reveal the mechanisms of N, P and K (NPK) utilization and metabolism in ramie is comparative proteome analysis. The differentially expressed proteins in the leaves of ramie were analyzed by proteome analysis after 6 days of N- and K-deficient treatments and 3 days of P-deficient treatment using MALDI-TOF/TOF mass spectrometry and 32, 27 and 51 differential proteins were obtained, respectively. These proteins were involved in photosynthesis, protein destination and storage, energy metabolism, primary metabolism, disease/defense, signal transduction, cell structure, transcription, secondary metabolism and protein synthesis. Ramie responded to NPK stress by enhancing secondary metabolism and reducing photosynthesis and energy metabolism to increase endurance. Specifically, ramie adapted to NPK deficiency by increasing signal transduction pathways, enhancing the connection between glycolysis and photosynthesis, promoting the intracellular flow of carbon and N; promoting the synthesis cysteine and related hormones and upregulating actin protein to promote growth of the root system. The experimental results provide important information for further study on the high-efficiency NPK utilization mechanism of ramie.

miRNAs expression profile in bast of ramie elongation phase and cell wall thickening and end wall dissolving phase.

MicroRNAs (miRNAs) are 20-24 nucleotide long non-coding RNAs that play critical regulatory roles during plant development, organ morphogenesis, and cell fate determination and differentiation. In this study, miRNA microarray chips were used to explore the expression profile of ramie miRNAs between the bast of fiber elongation phase and those of cell wall thickening and end wall dissolving phase. There are 150 and 148 credible miRNAs in the bast of fiber elongation phase and cell wall thickening and end wall dissolving phase, respectively. These miRNAs distributed in 27 species and mainly concentrated in nine species. Analysis showed that 51 miRNAs were differentially expressed: 27 up-regulated (miR166, miR172, miR396, miR482, miR894 and miR2911 families) and 24 down-regulated (miR156, miR159, miR164, miR319 and miR1450 families) in the bast of fiber elongation phase compared with the bast of cell wall thickening and end wall dissolving phase. To further confirm our results, we examined the expression of three miRNAs (zma-miR172b*, pvu-miR482 and vvi-172a) by quantitative real-time reverse transcriptase-PCR. Our results will provide a molecular basis for future research miRNA function on ramie genetics and breeding.

De novo assembly and characterization of transcriptome using Illumina paired-end sequencing and identification of CesA gene in ramie (Boehmeria nivea L. Gaud).

BACKGROUND: Ramie fiber, extracted from vegetative organ stem bast, is one of the most important natural fibers. Understanding the molecular mechanisms of the vegetative growth of the ramie and the formation and development of bast fiber is essential for improving the yield and quality of the ramie fiber. However, only 418 expressed tag sequences (ESTs) of ramie deposited in public databases are far from sufficient to understand the molecular mechanisms. Thus, high-throughput transcriptome sequencing is essential to generate enormous ramie transcript sequences for the purpose of gene discovery, especially genes such as the cellulose synthase (CesA) gene. RESULTS: Using Illumina paired-end sequencing, about 53 million sequencing reads were generated. De novo assembly yielded 43,990 unigenes with an average length of 824 bp. By sequence similarity searching for known proteins, a total of 34,192 (77.7%) genes were annotated for their function. Out of these annotated unigenes, 16,050 and 13,042 unigenes were assigned to gene ontology and clusters of orthologous group, respectively. Searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG) indicated that 19,846 unigenes were mapped to 126 KEGG pathways, and 565 genes were assigned to http://starch and sucrose metabolic pathway which was related with cellulose biosynthesis. Additionally, 51 CesA genes involved in cellulose biosynthesis were identified. Analysis of tissue-specific expression pattern of the 51 CesA genes revealed that there were 36 genes with a relatively high expression levels in the stem bark, which suggests that they are most likely responsible for the biosynthesis of bast fiber. CONCLUSION: To the best of our knowledge, this study is the first to characterize the ramie transcriptome and the substantial amount of transcripts obtained will accelerate the understanding of the ramie vegetative growth and development mechanism. Moreover, discovery of the 36 CesA genes with relatively high expression levels in the stem bark will present an opportunity to understand the ramie bast fiber formation and development mechanisms.

Constitutional tolerance to heavy metals of a fiber crop, ramie (Boehmeria nivea), and its potential usage.

It is observed that ramie (Boehmeria nivea), an economic fiber crop, can establish and colonize metal-contaminated sites in China. Metal tolerance and accumulation by ramie originating from 13 metal-contaminated and 4 "clean" sites in China were compared under field and hydroponic conditions. All selected populations and germplasms displayed good growth performance under diverse metal-contaminated habitats; while growth responses, metal accumulation and tolerance were similar among the 8 populations and 2 germplasms when exposed to solutions containing elevated As, Cd, Pb, or Zn in the laboratory. These revealed that ramie possesses a certain degree of constitutional metal tolerance. To our knowledge, this is the first report of constitutional metal tolerance possessed by a fiber crop. Ramie can be considered as a good candidate for both fiber production and phytoremediation of sites contaminated by multi-metals, as it accumulates relative low metal concentrations, but possesses both high biomass and high economic value.

Transgenic ramie [Boehmeria nivea (L.) Gaud.]: factors affecting the efficiency of Agrobacterium tumefaciens-mediated transformation and regeneration.

In the present study, an efficient Agrobacterium-mediated gene transformation system was developed for ramie [Boehmeria nivea (L.) Gaud.] based on the examinations of several factors affecting plant transformation efficiency. The effects of Agrobacterium cell density, acetosyringone, co-cultivation temperature, co-cultivation duration, co-cultivation photoperiod and pH on stable transformation were evaluated. Agrobacterium at a concentration of OD = 0.5-0.8 improved the efficiency of transformation. Concentration of acetosyringone at 50 mg/L during co-cultivation significantly increased transformation efficiency. Co-cultivation at 20 degrees C, in comparison to 15, 25 and 28 degrees C, consistently resulted in higher transformation frequencies. A relatively short co-cultivation duration (3 days) was optimal for ramie transformation. Co-cultivation medium at pH 5.9 and co-cultivation in darkness both improved the transformation efficiencies of ramie. An overall scheme for producing transgenic ramie is presented, through which an average transformation rate from 10.5 to 24.7% in five ramie varieties was obtained. Stable expression and integration of the transgenes were confirmed by histochemical GUS assay, kanamycin painting assay, PCR and Southern blotting. This optimized transformation system should be employed for efficient Agrobacterium-mediated transformation of ramie.

[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.

Difference of toxicity and accumulation of methylated and inorganic arsenic in arsenic-hyperaccumulating and -hypertolerant plants.

The arsenic (As) hyperaccumulators, Pteris vittata and Pteris cretica and an As-tolerant plant Boehmeria nivea, were selected to compare the toxicity, uptake, and transportation of inorganic arsenate (As(V)) and its methylated counterpart dimethylarsinic acid (DMA). The XANES method was used to elucidate the effect of As species transformation on As toxicity and accumulation characteristics. Significantly higher toxicity and lower accumulation of DMAthan inorganic As(V) was shown in the As hyperaccumulators and the As-tolerant plant. Reduction of As(V) was commonly found in the plants. Arsenic complexation with thiols, which have less mobility in plants and usually occur in As-tolerant plants, was also found in rhizoids of P. cretica. Plants with greater ability to form As-thiolate have lower ability for upward transport of As. Demethylation of DMA occurred in the three plants. The DMA component decreased from the rhizoids to the fronds in both hyperaccumulators, while this tendency is reverse in B. nivea.