Integrated miRNA-mRNA analysis reveals the roles of miRNAs in the replanting benefit of Achyranthes bidentata roots.

The yield and quality of the medicinal plant Achyranthes bidentata can be increased when it is replanted into a field cultivated previously with the same crop, however, fundamental aspects of its biology (so-called "replanting benefit") still remain to be elucidated. miRNAs are sRNA molecules involved in the post-transcriptional regulation of gene expression in plant biological processes. Here, 267 conserved and 36 novel miRNAs were identified in A. bidentata roots. We compared the miRNA content of the roots (R1) from first-year planting with that of the roots (R2) of second-year replanting, and screened 21 differentially expressed (DE) miRNAs. Based on in silico functional analysis, integrated miRNA-mRNA datasets allowed the identification of 10 miRNA-target family modules, which might participate in the benefit. The expression profiles of the miRNA-target modules were potentially correlated with the presence of the replanting benefit. The indication was that the miRNA-responsive continuous monoculture could reprogram miRNA-mRNA expression patterns, which possibly promote the root growth and development, enhance its transport activity and strengthen its tolerance to various stresses, thereby improving A. bidentata productivity as observed in the replanting benefit. Our study provides basic data for further research on the molecular mechanisms of the benefit in A. bidentata.

NAA and 6-BA promote accumulation of oleanolic acid by JA regulation in Achyranthes bidentata Bl.

Application of plant growth regulators has become one of the most important means of improving yield and quality of medicinal plants. To understand the molecular basis of phytohormone-regulated oleanolic acid metabolism, RNA-seq was used to analyze global gene expression in Achyranthes bidentata treated with 2.0 mg/L 1-naphthaleneacetic acid (NAA) and 1.0 mg/L 6-benzyladenine (6-BA). Compared with untreated controls, the expression levels of 20,896 genes were significantly altered with phytohormone treatment. We found that 13071 (62.5%) unigenes were up-regulated, and a lot of differentially expressed genes involved in hormone or terpenoid biosynthesis, or transcription factors were significantly up-regulated. These results suggest that oleanolic acid biosynthesis induced by NAA and 6-BA occurs due to the expression of key genes involved in jasmonic acid signal transduction. This study is the first to analyze the production and hormonal regulation of medicinal A. bidentata metabolites at the molecular level. The results herein contribute to a better understanding of the regulation of oleanane-type triterpenoid saponins accumulation and define strategies to improve the yield of these useful metabolites.

The root transcriptome of Achyranthes bidentata and the identification of the genes involved in the replanting benefit.

KEY MESSAGE: The transcriptome profiling in replanting roots revealed that expression pattern changes of key genes promoted important metabolism pathways, antioxidant and pathogen defense systems, adjusted phytohormone signaling and inhibited lignin biosynthesis. The yield of the medicinal plant Achyranthes bidentata could be significantly increased when replanted into a field cultivated previously for the same crop, but the biological basis of this so-called "replanting benefit" is unknown. Here, the RNA-seq technique was used to identify candidate genes responsible for the benefit. The analysis of RNA-seq libraries prepared from mRNA extracted from the roots of first year planting (normal growth, NG) and second year replanting (consecutive monoculture, CM) yielded about 40.22 GB sequencing data. After de novo assembly, 87,256 unigenes were generated with an average length of 1060 bp. Among these unigenes, 55,604 were annotated with public databases, and 52,346 encoding sequences and 2881 transcription factors were identified. A contrast between the NG and CM libraries resulted in a set of 3899 differentially transcribed genes (DTGs). The DTGs related to the replanting benefit and their expression profiles were further analyzed by bioinformatics and qRT-PCR approaches. The major differences between the NG and CM transcriptomes included genes encoding products involved in glycolysis/gluconeogenesis, glutathione metabolism and antioxidant defense, in aspects of the plant/pathogen interaction, phytohormone signaling and phenylpropanoid biosynthesis. The indication was that replanting material enjoyed a stronger level of defense systems, a balance regulation of hormone signals and a suppression of lignin formation, thereby promoting root growth and development. The study provides considerable significant insights for a better understanding of the molecular mechanism of the replanting benefit and suggests their possible application in developing methods to reinforce the effects in medicinal plants.

In vitro callus induction and plantlet regeneration of Achyranthes aspera L., a high value medicinal plant.

OBJECTIVE: To study callus induction from different explants (internode, leaf, root) and in vitro plantlets propagation from medicinally important plant Achyranthes aspera L. METHODS: Sterilized explants were prepared by using 0.1% HgCl2 and 0.5% Bavistin and callus was obtained when cultured onto Murashige Skoog's (MS) medium by using different concentrations and combination of 2,4-D, NAA, BAP, IAA, IBA with 3% sucrose and 0.8% agar. Induced callus was immediately transferred to MS medium containing at different concentrations of phytohormones for shootlets and rootlets induction respectively. RESULTS: Sterilization treatment of 0.1% HgCl2 for 2-3 min and Bavistin 0.5% for 10-12 min showed the highest percentage of asepsis and survival rate. Maximum induction of callus was obtained from a combination of 2.0 mg/L 2,4-D and 0.5 mg/L NAA from leaf. Highest shootlets number (4.83±0.17) and length (3.8±0.16) cm were observed on full strength MS medium when fortified with BAP 4.0 mg/L and KIN 0.5 mg/L. Concerted efforts of BAP 2.0 mg/L and NAA 0.5 mg/L on full strength MS medium showed highest leaf number (6.77±0.94). In vitro raised shoots were allowed to root on different strengths of MS medium fortified with IAA and IBA at different concentrations. Experimentally, 3.0 mg/L IBA was enabled to induce maximum rootlets number (10.0±9.82) on full strength MS medium. Afterwards, regenerated shoots with well developed roots were successfully subjected to hardening process and were acclimatized. The survived plantlets showed 66.67% survival frequency without any morphological abnormality. CONCLUSIONS: The results demonstrated that different explants were good source of callus induction, morphology analysis as well as indirect plantlets regeneration.

Assessment of mineral phosphate-solubilizing properties and molecular characterization of zinc-tolerant bacteria.

Plant growth-promoting bacteria with the ability to tolerate heavy metals have importance both in sustainable agriculture and phytoremediation. The present study reports on the isolation and characterization of mineral phosphate-solubilizing (MPS) bacteria associated with the Achyranthes aspera L. plant (prickly chaff, flower plant). Out of 35 bacterial isolates, 6 isolates, namely RS7, RP23, EPR1, RS5, RP11 and RP19, with high MPS activity were selected and subjected to the assessment of MPS activity under various stress conditions, viz. ZnSO(4) (0.30-1.5 M), NaCl and temperature. MPS activity by the selected isolates was observed at concentrations of as high as >1.2 M ZnSO(4). Significant improvement in plant growth was observed on bacterization of seeds (pearl millet) with all of the six selected isolates. Plant growth was measured in terms of root length, shoot length, fresh weight and % increase in root biomass. The molecular diversity among the phosphate-solubilizing bacteria was studied employing enterobacterial repetitive intergenic sequence-PCR (ERIC-PCR). Representative strains from each ERIC type were identified, on the basis of a partial sequence of the 16S rRNA gene, as members of the genera Pseudomonas, Citrobacter, Acinetobacter, Serratia, and Enterobacter. Among all the isolates, RP19 was the best in terms of phosphate-solubizing activity and its response to various stresses. The ability of RP19 and other isolates to exhibit MPS activity at high ZnSO(4) concentrations suggests their potential as efficient biofertilizer for growing plants in metal (ZnSO(4))-contaminated soil.

In vitro clonal propagation of Achyranthes aspera L. and Achyranthes bidentata Blume using nodal explants.

OBJECTIVE: To develop the reproducible in vitro propagation protocols for the medicinally important plants viz., Achyranthes aspera (A. aspera) L. and Achyranthes bidentata (A. bidentata) Blume using nodal segments as explants. METHODS: Young shoots of A. aspera and A. bidentata were harvested and washed with running tap water and treated with 0.1% bavistin and rinsed twice with distilled water. Then the explants were surface sterilized with 0.1% (w/v) HgCl2 solutions for 1 min. After rinsing with sterile distilled water for 3-4 times, nodal segments were cut into smaller segments (1 cm) and used as the explants. The explants were placed horizontally as well as vertically on solid basal Murashige and Skoog (MS) medium supplemented with 3% sucrose, 0.6% (w/v) agar (Hi-Media, Mumbai) and different concentration and combination of 6-benzyl amino purine (BAP), kinetin (Kin), naphthalene acetic acid (NAA) and indole acetic acid (IAA) for direct regeneration. RESULTS: Adventitious proliferation was obtained from A. aspera and A. bidentata nodal segments inoculated on MS basal medium with 3% sucrose and augmented with BAP and Kin with varied frequency. MS medium augmented with 3.0 mg/L of BAP showed the highest percentage (93.60±0.71) of shootlets formation for A. aspera and (94.70±0.53) percentages for A. bidentata. Maximum number of shoots/explants (10.60±0.36) for A. aspera and (9.50±0.56) for A. bidentata was observed in MS medium fortified with 5.0 mg/L of BAP. For A. aspera, maximum mean length (5.50±0.34) of shootlets was obtained in MS medium augmented with 3.0 mg/L of Kin and for A. bidentata (5.40±0.61) was observed in the very same concentration. The highest percentage, maximum number of rootlets/shootlet and mean length of rootlets were observed in 1/2 MS medium supplemented with 1.0 mg/L of IBA. Seventy percentages of plants were successfully established in polycups. Sixty eight percentages of plants were well established in the green house condition. Sixty five percentages of plants were established in the field. CONCLUSIONS: The results have shown that use of nodal buds is an alternative reproducible and dependable method for clonal propagation of A. aspera and A. bidentata. The high rate of direct shoot-root multiplication and their high rate of post-hardening survival indicate that this protocol can be easily adopted for commercial large scale cultivation.

[Determination of metal elements in Achyranthis bidentatae radix from various habitats].

OBJECTIVE: To establish an atomic absorption spectrometry method for determination of the contents of metal elements in Achyranthis Bidentatae Radix and analyze 21 batches of samples from different areas. METHODS: Fe, Mn, Ca, Mg, K, Zn and Cu were detected by atomic absorption spectrometry with hydrogen flame detector, Pb, As and Cd were detected by graphite furnace atomic absorption, Hg was detected by cold atomic absorption. RESULTS: The heavy metal contents met the requirement of Chinese Pharmacopoeia. The contents of K, Mg, Cu and Mn in the samples of geo-authentic areas were higher,while the contents of Fe, Zn, Hg and Pb in the samples of non-authentic areas were higher. CONCLUSION: This method is sample, accurate, repeatable and could be used to evaluate the quality of Achyranthis Bidentatae Radix.

[Effect of phosphorus on copper tolerance in Achyranthes bidentata].

OBJECTIVE: To study the effect of phosphorus on copper tolerance in Achyranthes bidentata. METHOD: A PVC pipe experiment was conducted to study the interactive effects of phosphorus (P) and copper (Cu), on growth, elemental accumulation and chemical constituents of A. bidentata. Two levels of elemental P were applied at 0 (P0) and 100 ( P100) mg x kg(-1) soil with 5 levels of Cu at 0 (Cu0), 100 (Cu100), 200 (Cu200), 200 (Cu400), 200 (Cu600) mg x kg(-1) soil, respectively. RESULT AND CONCLUSION: The biomass production between different Cu treatments, phosphorus treatment showed significant differences. The biomass reached the maximum value as the concentration of Cu and P was 100 mg x kg(-1). Low concentration of Cu improved the growth of A. bidentata. The growth was blocked as Cu concentration reached 200 mg x kg(-1) in soil, however the contents of oleanolic acid and ecdysterone in roots of A. bidentata had not influenced by Cu. P could improved the copper tolerance in A. bidentata and increased root yield. The Cu concentration in soil of the cultivation bases must be below 200 mg x kg(-1) in order to produce good quality of medicinal material.

[Effect of medium components on callus induction and contents of polysaccharides in calli from Achyranthes bidentata].

OBJECTIVE: To study the effect of medium components on the callus induction and the contents of polysaccharides in calli from Achyranthes bidentata. METHOD: Leaves and stems were selected as explants. The effects of six kinds of factors including basal culture medium, carbon source, 2,4-D, 6-BA, TDZ, CH on the callus induction and the contents of ABPS in calli on the high growth point were studied by orthogonal design method. The data were analyzed with range analysis and variance analysis. RESULT: To leaf, the optimal medium of callus induction was B5 with 2 mg x L(-1) 2,4-D, 0.5 mg x L(-1) 6-BA, 30 g x L(-1) glucose and 1 g x L(-1) CH; to stem, the optimal one was B5 with2 mg x L(-1) 2,4-D, 1 mg x L(-1) 6-BA, 30 g x L(-1) glucose and 0.5 g x L(-1) CH. In order to obtain higher contents ABPS, to leaf calli, the optimal medium was LS with 1 mg x L(-1) 6-BA, 1 mg x L(-1) TDZ and 30 g x L(-1) sucrose; to stem calli, the optimal one was LS with 1 mg L(-1) 2,4-D, 0.5 mg x L(-1) 6-BA, 1 mg x L(-1) TDZ and 30 g x L(-1) glucose. CONCLUSION: The optimal media of callus induction were established with stems and leaves of A. bidentata as explants and with a view to an industrial production of polysaccharides by tissue and cell culture.

[Structural development of root and their relationship to accumulation of triterpenoid saponins in Achyranthes bidentata Bl].

A study concerning the relationship of dynamic accumulation of triterpenoid saponins and anatomical characteristics of Achyranthes bidentata Bl roots was undertaken by anatomical, histochemical and phytochemical method respectively. Results revealed that the primary and secondary structures of the root resembled those of usual dicots. The continual thickening growth of root principally resulted from the differentiation and development of the tertiary structure. The first ring of supernumerary cambium originated from the parenchyma and vascular ray cells of secondary phloem and each of the followed rings initiated in the outmost foundamental parenchyma cells which were derived from the immediate preceding ring. In the supernumerary cambiums, there had not distinction between the fusiform initial and the ray initial. Its cells present stratifide arranged from a longitudinal section through root. Regular and concentric rings of tertiary vascular bundles who differentiate centrifugally were enclosed by the connective parenchyma. The number of the rings continually increase with the development of the root itself. Triterpenoid saponins accumulated mainly in pericycle, primary phloem and parenchyma between primary phloem and xylem in the primary structure of root but came into existence in cells of secondary phloem and phelloderm with secondary structure development of root, and as well as in supernumerary cambium and phloem of tertiary vascular bundle after the tertiary structure maturated gradually in the roots. The investigation provides indications that the tertiary structure were not only main parts in the roots of Achyranthes bidentata Bl, but also important storage region of triterpenoid saponins in its growth and development. In addition, the analysis of using the HPLC showed that dynamic increasing trend oleanolic acid was as "S" curve during the roots growth and development and up to the highest content of triterpenoid saponins after plants grew 120 days. Meanwhile, the number of the rings of tertiary vascular bundles, length and diameter of the roots were the same as the triterpenoid saponins increasing trend. It should be optimal season for harvest.

[Study on the dynamic growth rhythm of Achyranthes bidentata under different densities].

OBJECTIVE: The experiment was conducted to study the dynamic growth rhythm of Achyranthes bidentata under different densities. METHOD: The plant samples were collected to measure the growth rate of each organ. RESULT: Under different densities, the growing dynamic rhythm of A. bidentata were similar. The growth of main root exhibited a trend of "slow-fast-slow" by stages. The increase of dry root weight was fastest during the period of 30-40 days before harvest. The dry-matter increasing rate of whole plant was fastest in the later period of branching stage. The differences of root yields among the plants growing under different densities were significant. CONCLUSION: For high yield and good quality, the density of planting of A. bidentata should be considered.