Nitro-Oxidative Stress Correlates with Se Tolerance of Astragalus Species.

At high concentrations, selenium (Se) exerts phytotoxic effects in non-tolerant plant species partly due to the induction of nitro-oxidative stress; however, these processes are not fully understood. In order to obtain a more accurate view of the involvement of nitro-oxidative processes in plant Se sensitivity, this study aims to characterize and compare Se-triggered changes in reactive oxygen (ROS) and nitrogen species (RNS) metabolism and the consequent protein tyrosine nitration as a marker of nitrosative stress in the non-accumulator Astragalus membranaceus and the Se hyperaccumulator Astragalus bisulcatus. The observed parameters (Se accumulation, microelement homeostasis, tissue-level changes in the roots, germination, biomass production, root growth and cell viability) supported that A. membranaceus is Se sensitive while the hyperaccumulator A. bisulcatus tolerates high Se doses. We first revealed that in A. membranaceus, Se sensitivity coincides with the Se-induced disturbance of superoxide metabolism, leading to its accumulation. Furthermore, Se increased the production or disturbed the metabolism of RNS (nitric oxide, peroxynitrite and S-nitrosoglutathione), consequently resulting in intensified protein tyrosine nitration in sensitive A. membranaceus. In the (hyper)tolerant and hyperaccumulator A. bisulcatus, Se-induced ROS/RNS accumulation and tyrosine nitration proved to be negligible, suggesting that this species is able to prevent Se-induced nitro-oxidative stress.

Effects of Elevated CO2 on the Swainsonine Chemotypes of Astragalus lentiginosus and Astragalus mollissimus.

Rapid changes in the Earth's atmosphere and climate associated with human activity can have significant impacts on agriculture including livestock production. CO2 concentration has risen from the industrial revolution to the current time, and is expected to continue to rise. Climatic changes alter physiological processes, growth, and development in numerous plant species, potentially changing concentrations of plant secondary compounds. These physiological changes may influence plant population density, growth, fitness, and toxin concentrations and thus influence the risk of toxic plants to grazing livestock. Locoweeds, swainsonine-containing Astragalus species, are one group of plants that may be influenced by climate change. We evaluated how two different swainsonine-containing Astragalus species responded to elevated CO2 concentrations. Measurements of biomass, crude protein, water soluble carbohydrates and swainsonine concentrations were measured in two chemotypes (positive and negative for swainsonine) of each species after growth at CO2 levels near present day and at projected future concentrations. Biomass and water soluble carbohydrate concentrations responded positively while crude protein concentrations responded negatively to elevated CO2 in the two species. Swainsonine concentrations were not strongly affected by elevated CO2 in the two species. In the different chemotypes, biomass responded negatively and crude protein concentrations responded positively in the swainsonine-positive plants compared to the swainsonine-negative plants. Ultimately, changes in CO2 and endophyte status will likely alter multiple physiological responses in toxic plants such as locoweed, but it is difficult to predict how these changes will impact plant herbivore interactions.

Small RNA and degradome sequencing reveals important microRNA function in Astragalus chrysochlorus response to selenium stimuli.

Selenium (Se), an essential element, plays important roles in human health as well as environmental sustainability. Se hyperaccumulating plants are thought as an alternative selenium resource, recently. Astragalus species are known as hyperaccumulator of Se by converting it to nonaminoacid compounds. However, Se-metabolism-related hyperaccumulation is not elucidated in plants yet. MicroRNAs (miRNAs) are key molecules in many biological and metabolic processes via targeting mRNAs, which may also play an important role in Se accumulation in plants. In this study, we identified 418 known miRNAs, belonging to 380 families, and 151 novel miRNAs induced by Se exposure in Astragalus chyrsochlorus callus. Among known miRNAs, the expression of 287 families was common in both libraries, besides 71 families were expressed only in Se-treated sample, whereas 60 conserved families were expressed in control tissue. miR1507a, miR1869 and miR2867-3p were mostly up-regulated, whereas miR1507-5p and miR8781b were significantly down-regulated by Se exposure. Computational analysis shows that the targets of miRNAs are involved in different types of biological mechanisms including 47 types of cellular component, 103 types of molecular function and 144 types of biological process. Degradome analysis shows that 1256 mRNAs were targeted by 499 miRNAs. We conclude that some known and novel miRNAs such as miR167a, miR319, miR1507a, miR4346, miR7767-3p, miR7800, miR9748 and miR-n93 target transcription factors, disease resistance proteins and some specific genes like cysteine synthase and might be related to plant hormone signal transduction, plant-pathogen interaction and sulphur metabolism pathways.

Selenium hyperaccumulation by Astragalus (Fabaceae) does not inhibit root nodule symbiosis.

PREMISE OF STUDY: A survey of the root-nodule symbiosis in Astragalus and its interaction with selenium (Se) has not been conducted before. Such studies can provide insight into how edaphic conditions modify symbiotic interactions and influence partner coevolution. In this paper plant-organ Se concentration ([Se]) was investigated to assess potential Se exposure to endophytes. • METHODS: Selenium distribution and molecular speciation of root nodules from Se-hyperaccumulators Astragalus bisulcatus, A. praelongus, and A. racemosus was determined by Se K-edge x-ray absorption spectroscopy. A series of greenhouse experiments were conducted to characterize the response of root-nodule symbiosis in Se-hyperaccumulators and nonhyperaccumulators. • KEY RESULTS: Nodules in three Se-hyperaccumulators (Astragalus crotalariae, A. praelongus, and A. preussii) are reported for the first time. Leaves, flowers, and fruits from Se-hyperaccumulators were routinely above the hyperaccumulator threshold (1,000 µg Se g(-1) DW), but root samples rarely contained that amount, and nodules never exceeded 110 µg Se g(-1) DW. Nodules from A. bisulcatus, A. praelongus, and A. racemosus had Se throughout, with a majority stored in C-Se-C form. Finally, an evaluation of nodulation in Se-hyperaccumulators and nonhyperaccumulators indicated that there was no nodulation inhibition because of plant Se tolerance. Rather, we found that in Se-hyperaccumulators higher levels of Se treatment (up to 100 µM Se) corresponded with higher nodule counts, indicating a potential role for dinitrogen fixation in Se-hyperaccumulation. The effect was not found in nonhyperaccumulators. • CONCLUSIONS: As the evolution of Se hyperaccumulation in Astragalus developed, root-nodule symbiosis may have played an integral role.

The effects of excess copper on antioxidative enzymes, lipid peroxidation, proline, chlorophyll, and concentration of Mn, Fe, and Cu in Astragalus neo-mobayenii.

To probe the physiological and biochemical tolerance mechanisms in Astragalus neo-mobayenii Maassoumi, an endemic plant around the Cu-rich areas from the North West of Iran, the effect of different copper concentrations at toxic levels on this plant was investigated. Copper was applied in the form of copper sulfate (CuSO4·5H2O) in four levels (0, 50, 100, and 150 µM). We observed no visible symptoms of Cu toxicity in this plant species. During the exposure of plants to excess copper, the antioxidant defense system helped the plant to protect itself from the damage. With increasing copper concentration, superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities increased in leaves and roots (P < 0.001) compared with that of the control group. The chlorophyll amount gradually declined with increasing Cu concentrations. However, reduction in the 50 µM level showed insignificant changes. Enhanced accumulation of proline content in the leaves was determined, as well as an increase of MDA content (oxidative damage biomarker) (P < 0.001). The results indicated that Cu contents in leaves and roots enhanced with increasing levels of Cu application. The Fe and Mn contents in both shoots and roots significantly decreased with increasing Cu concentration. Finally, the mechanisms of copper toxicity and copper tolerance in this plant were briefly discussed.

Production of cycloartane triterpene glycosides in shoot cultures of Astragalus sieberi DC.

Shoot tips of the germinated seeds of Astragalus sieberi DC. were cultured on MS-medium supplemented with 0.1 mg L(-1) each of naphthalene acetic acid (NAA) and benzyl adenine (BA) for establishment of shoot cultures. The effect of different concentrations of growth regulators on saponin content was studied and optimised. Saponin content was monitored by HPLC analysis. The most appropriate growth regulator with which to produce the highest content of saponins in shoot cultures was 0.5 mg L(-1) each of NAA and BA. The isolated cycloastragenol-3-O-glucoside was identified by spectral analysis and compared with an authentic sample.

The effect of Cd on mycorrhizal development and enzyme activity of Glomus mosseae and Glomus intraradices in Astragalus sinicus L.

The influence of cadmium (Cd) on the root colonization of Astragalus by two arbuscular mycorrhizal fungi (AMF) was investigated. Astragalus sinicus L. grown in the soil in the presence of four levels of Cd was inoculated individually with Glomus mosseae, Glomus intraradices, or by a mixed inoculum of these two fungi. Histochemical vital stains were used to measure the efficiency and function of the mycorrhizal symbiosis after 7weeks. Nested PCR with ALP-active stained roots was applied to monitor the efficiency AMF in mixed community at the same time. The addition of Cd slightly decreased plant biomass and mycorrhizal colonization, and depressed the activities of alkaline phosphatase (ALP) and succinate dehydrogenase (SDH), especially in G. intraradices. Mycorrhizal colonization significantly improved plant growth regardless of the level of addition of Cd. The plants inoculated with G. mosseae or mixed inoculum immobilized more Cd in the roots, and partitioned less Cd to the shoots at the highest Cd addition level. Root colonization efficiency of G. intraradices was improved in the mixed community and G. mosseae was the dominant colonizer in this community. Based on the higher Cd tolerance of this strain of G. mosseae, it would be preferred for bioremediation in Cd contaminated soil.

Defensive and secondary metabolism in Astragalus chrysochlorus cell cultures, in response to yeast extract stressor.

In this study it was aimed to investigate inducibility of secondary metabolism in Astragalus chrysochlorus by yeast extract which is known to cause the synthesis of defensive phenolic metabolites. Cell suspension cultures of Astragalus chrysochlorus responded to elicitor treatment (10 gl(-1) yeast extract) by increasing phenylalanine ammonia lyase (PAL E.C. 4.3.1.5) activity the key enzyme of phenylpropanoid pathway and accumulation of phenolic compounds. Yeast extract was added on 13th day in the cultures when cells were at early stage of logarithmic phase. The highest PAL activity (2.71 U microg protein(-1) min(-1)) was measured at 36 hr after addition of yeast extract and increasing of total phenolics accompanied with 221 microg g(-1) value as fresh weight (FW). Total phenolic content reached maximum value, 343.86 microg g(-1) FW, at 48 hr while control's value was 162 microg g(-1) FW. Maximum PAL activity and total phenolic content were 2.88 and 2.12 times higher than the controls of A. chrysochlorus cells, respectively. Our results indicate that there is a positive correlation between induced PAL activity and accumulation of total phenolics. It is considered that early defense response given to environmental stressors such as biotic and abiotic factors by upregulation of phenolic branch of secondary metabolism occurs in A. chrysochlorus with addition of yeast extract.

[Culture-filtrate producing condition and biological activity of Fusarium solani].

OBJECTIVE: To study the culture-filtrate producing condition of Fusarium Solani isolated from Astragalus root and explore the mechanism Astragalus root rot disease caused by, in order to find theoretical support for screening resistant germ plasma via mycotoxin. METHOD: The method of germinating seeds in petri dish with filter paper and inhibition method for embryo growth were used to study the biological activity and the specialty of cultural filtrate of 10 F. solani isolates. RESULT: The toxin produced by F. solani had strong inhibition effect in the different nutrient media, at different temperatures and under different light conditions. With extension of culturing time, embryo inhibition rate went up gradually with the strongest inhibition at the 12th day and the inhibition ratio between 92.0% -52.0%. The toxin produced at 5 degrees C to 35 degrees C inhibited embryo germination of Astragalus differently with the strongest at 25 degrees C, and next to it at 20,30 degrees C. The impact of light on bioactive substances of the toxin was not statistically distinctive, but the 24-hour darkness was benefit to toxin production. PSC had a stronger inhibition rate than the other nutrient media, next to it was PDB. After autoclaving, the toxin still kept toxic to embryo of Astragalus, which indicated that the toxin was tolerant to high temperatures. CONCLUSION: The toxin produced by F. solani at different growing condition had strong biological activity, was tolerant to high temperature. The best condition for F. solani to produce toxin was that it was cultured in PSC liquid medium, in dark, at 25 degrees C for 12 d. The toxin produced by isolate HQM40 was non-host specific toxin.

The symbiosis phenotype and expression patterns of five nodule-specific genes of Astragalus sinicus under ammonium and salt stress conditions.

In previous works, we isolated 14 nodule-specific or nodule-enhanced genes from Astragalus sinicus by suppressive subtractive hybridization. In this study, we have further identified the expression patterns of five nodule-specific genes of A. sinicus under salt and ammonium stress. Transcription levels of genes tested were quantified by quantitative fluorescence real-time RT-PCR. Results showed that: (1) About 80 mM NaCl and all stress treatments containing (NH(4))(2)SO(4) significantly inhibited nitrogen-fixing capacity of inoculated plants. About 40 mM NaCl showed relative lighter inhibition. (2) Compare with positive control at normal conditions, the expressions of all genes were significantly reduced by all ammonium stress. (3) Under salt stress without exogenous nitrogen, transcription levels of AsIIA255 and AsE246 were significantly increased after treatment for 3 days. But expressions of AsG2411, AsIIC2512, and AsB2510 were suppressed by 80 mM NaCl and not significantly affected by 40 mM NaCl. (4) Under salt stress with exogenous nitrogen, expressions of AsG2411, AsIIC2512, AsB2510, and AsIIA255 were significantly suppressed. While, the transcription level of AsE246 under 80 mM NaCl containing 1 mM (NH(4))(2)SO(4) was still higher than that of positive control. The correlation of the expression profiles of three cysteine cluster protein (CCP) genes (AsG2411, AsIIC2512, AsIIA255) and one lipid transfer protein (LTP) gene (AsE246) with the nitrogen-fixing capacities of nodules in each treatments may explain the molecular mechanisms of their supposed functions in symbiosis and nitrogen-fixing process. Our results also implied that AsIIA255 and AsE246 might play a role in the response of A. sinicus to salt stress to facilitate the nitrogen-fixation process.

[Protoplast culture and plant regeneration of the methionine resistant variant of Astragalus cicer L].

A protoplast-to-plant system for the methionine resistant variant of Astragalus cicer L. has been developed. The friable calli induced from stem segments of variant plants were used as materials for protoplast isolation through enzyme digestion. The effects of different media and plating densities on protoplast divisions and plant regeneration were studied. Sustained cell divisions and colony formation from the protoplasts of the methionine resistant cell line of Astragalus cicer L. were obtained by a DPD medium containing 2.0 mg/L 2,4- dichlorophenoxyacetic acid (2,4-D), 0.2 mg/L 6 -benzylaminopurine(6-BA), 0.3 mol/L mannitol, 200 mg/L casein hydrolysate and 2% (W/V) sucrose at a plating density of 2x10(5) /ml. The division frequency was 38.3%. At the same time, different dividing types of protoplasts were found. Organogenesis and shoot formation from the protoplast-derived calli were induced on MS medium supplemented with 0.5 mg/L NAA, 10 mg/L KT and 2% (W/V) sucrose. The protoplast-derived calli still expressed resistance to methionine. The protoplast to plant regeneration protocol developed in this study might provide the foundation for the resistant cell line as a parent for somatic hybridization.

[Change of superficial shape and cell microstructure of milk-vetch (Astragalus L.) root under simulated Cu pollution in a red soil].

This study carried out experiments to investigate changes of shape, inter-structures and cell microstructures of milk-vetch root under simulated Cu stress at non- or contaminated levels in a red soil using observation and bio-microscopic technique. It resulted that when Cu concentration ranged from 0 to 40 mg.kg-1 soil, the milk-vetch grew well and it had a whole root which worked normally. When Cu concentration reached 50 mg.kg-1 soil, the growth of milk-vetch began to get influence with decline of biomass, the taproot crooked and was less branched, root became short and hazel and had fewer shorter hairs, tubby appeared, epidermis began to shrink and cell wall cockled slightly and unevenly, the boundary between plasmalemma and organelle blurred as well. When treatment concentration reaching to 200 mg.kg-1 soil, milk-vetch roots became rotted and black, the cell wall broke and cytoplasm shrank so severely that plasmolysis happened and the plant died. So the critical Cu concentration in experimented soil was 50 mg.kg-1 soil, and the resistance of milk-vetch root to Cu contamination buildup with the growth of aboveground part, and cell wall was the main part to Cu tolerance.