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.

UHPLC-MS/MS Quantification Combined with Chemometrics for Comparative Analysis of Different Batches of Raw, Wine-Processed, and Salt-Processed Radix Achyranthis Bidentatae.

An accurate and reliable method using ultra-high performance liquid chromatography combined with triple quadrupole tandem mass spectrometry (UHPLC-MS/MS) was established for simultaneous quantification of five major bioactive analytes in raw, wine-processed, and salt-processed Radix Achyranthis bidentatae (RAB). The results showed that this method exhibited desirable sensitivity, precision, stability, and repeatability. The overall intra-day and inter-day variations (RSD) were in the range of 1.57-2.46 and 1.51-3.00%, respectively. The overall recoveries were 98.58-101.48% with a relative standard deviation (RSD) of 0.01-1.86%. In addition, the developed approach was applied to 21 batches of raw, wine-processed, and salt-processed samples of RAB. Hierarchical clustering analysis (HCA), principal component analysis (PCA), heat map, and boxplot analysis were performed to evaluate the quality of raw, wine-processed, and salt-processed RAB collected from different regions. The chemometrics combined with the quantitative analysis based on UHPLC-MS/MS results indicated that the content of five analytes increased significantly in processed RAB compared to raw RAB.

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.

Deuterium labeling for investigating de novo synthesis of terpene volatiles in Achyranthes bidentata.

PURPOSE OF WORK: Plants synthesize and accumulate secondary metabolites as defensive volatiles against diverse stresses. We aim to unravel the jasmonate-inducible volatile de novo synthetic metabolites in plants using a deuterium-labeling technique. Jasmonic acid and its methyl ester (MeJA) are well-documented for inducing defensive volatiles. Here, we have developed an efficient deuterium oxide (D2O)-based labeling approach to determine the extent of de novo synthetic metabolites in a model plant A. bidentata bidentata. The labeling approach was demonstrated on quantitative profiling of terpene volatile organic compounds (VOCs) elicited by airborne MeJA in Achyranthes plants. We show, for the first time that airborne MeJA-elicited terpene VOCs are predominantly and differentially de novo synthesized except for a homoterpene, (3E)-4,8-dimethyl-1,3,7-nonatriene, which is weakly and least labelled with deuterium. D2O is therefore an efficient labeling source for investigating de novo synthetic metabolites of terpene VOCs in planta.

Interplant communication: airborne methyl jasmonate is essentially converted into JA and JA-Ile activating jasmonate signaling pathway and VOCs emission.

Methyl jasmonate (MeJA) was identified as an airborne signal involved in mediating interplant defense response communications over a decade ago. However, how MeJA activates plant defense systems and what becomes of the compound after it has done so has, thus far, remained unknown. To investigate this, Achyranthes bidentata plants were exposed to deuterated methyl jasmonate (d(2)MeJA) followed by absolute quantification of metabolic products of d(2)MeJA, and emissions of volatile organic compound (VOC) as defensive markers. We found that d(2)MeJA was metabolized mainly into deuterated jasmonic acid (d(2)JA) and jasmonoyl isoleucine (d(2)JA-Ile), and to a much lesser extent, deuterated jasmonoyl leucine (d(2)JA-Leu). Increases in d(2)JA-Ile/Leu and also endogenous JA-Ile/Leu were tightly co-related with, and significantly influenced the pattern and amount of, VOC emissions. The amount of accumulated d(2)JA-IIe was 13.1-fold higher than d(2)JA-Leu, whereas the amounts of JA-IIe and JA-Leu accumulated were almost identical. This study demonstrates that exogenous MeJA activates defensive systems (such as VOC emissions) in receiver plants by essentially converting itself into JA and JA-IIe and initiating a signal transduction leading to VOC emissions and induction of endogenous JA-IIe and JA-Leu, which in turn cause further amplification of VOC emissions.

[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.

Estrogenic and Pregnancy Interceptory Effects of Achyranthes Aspera Linn. Root

Achyranthes aspera Linn. (Amaranthaceae) is an abundant indigenous herb in India. It is traditionally being used as an abortifacient. Four successive solvent extracts of the root were screened for antifertility activity in female albino rats. The chloroform and ethanol extracts exhibited 100anti-implantation activity when given orally at 200 mg/kg body weight. Both the extracts at the dose of 200 mg/kg body weight also exhibited estrogenic activity. Histological studies of the uterus were carried out to confirm this estrogenic activity