Novel thrombolytic protease from edible and medicinal plant Aster yomena (Kitam.) Honda with anticoagulant activity: purification and partial characterization.

A thrombolytic protease named kitamase possessing anticoagulant property was purified from edible and medicinal plant Aster yomena (Kitam.) Honda. Kitamase showed a molecular weight of 50 kDa by SDS-PAGE and displayed a strong fibrin zymogram lysis band corresponding to the similar molecular mass. The enzyme was active at high temperatures (50°C). The fibrinolytic activity of kitamase was strongly inhibited by EDTA, EGTA, TPCK and PMSF, inhibited by Zn(2+). The Km and Vmax values for substrate S-2251 were determined as 4.31 mM and 23.81 mM/mg respectively. It dissolved fibrin clot directly and specifically cleaved the α, Aα and γ-γ chains of fibrin and fibrinogen. In addition, kitamase delayed the coagulation time and increased activated partial thromboplastin time and prothrombin time. Kitamase exerted a significant protective effect against collagen and epinephrine induced pulmonary thromboembolism in mice. These results suggest that kitamase may have the property of metallo-protease like enzyme, novel fibrino(geno)lytic enzyme and a potential to be a therapeutic agent for thrombosis.

Molecular characterization of an oxidosqualene cyclase that yields shionone, a unique tetracyclic triterpene ketone of Aster tataricus.

Shionone is the major triterpenoid component of Aster tataricus possessing a unique all six-membered tetracyclic skeleton and 3-oxo-4-monomethyl structure. To clarify its biosynthetic process, an oxidosqualene cyclase cDNA was isolated from A. tataricus, and the function of the enzyme was determined in lanosterol synthase-deficient yeast. The cyclase yielded ca. 90% shionone and small amounts of ß-amyrin, friedelin, dammara-20,24-dienol, and 4-epishionone and was designated as a shionone synthase (SHS). Transcripts of SHS were detected in A. tataricus organs, confirming its involvement in shionone biosynthesis. SHS was shown to have evolved in the Asteraceae from ß-amyrin synthase lineages and acquired characteristic species- and product-specificities.

The role of Aster amellus Linn. in the degradation of a sulfonated azo dye Remazol Red: a phytoremediation strategy.

Phytoremediation is a novel and promising approach for the treatment of pollutants. This study did explore the potential of Aster amellus Linn. to decolorize a sulfonated azo dye Remazol Red (RR), a mixture of dyes and a textile effluent. Induction in the activities of lignin peroxidase, tyrosinase, veratryl alcohol oxidase and riboflavin reductase was observed during RR decolorization, suggesting their involvement in the metabolism of RR. UV-Visible absorption spectrum, HPLC and FTIR analysis confirmed the degradation of RR. Four metabolites after the degradation of the dye were identified as 2-[(3-diazenylphenyl) sulfonyl] ethanesulfonate, 4-amino-5-hydroxynaphthalene-2,7-disulfonate, naphthalene-2-sulfonate and 3-(1,3,5-triazin-2-ylamino)benzenesulfonate by using GC/MS. Textile effluent and mixture of dyes showed 47% and 62% decrease respectively in American Dye Manufacturers Institute value. BOD of textile effluent and mixture of dyes were reduced by 75% and 48% respectively, COD of industrial effluent and mixture of dyes was reduced by 60% and 75% and TOC was reduced by 54% and 69% respectively after the treatment by A. amellus for 60 h; this indicated that the plant can be used for cleaning textile effluents. Toxicity study revealed the phytotransformation of RR into non-toxic products.

Elevated atmospheric CO2 concentration enhances salinity tolerance in Aster tripolium L.

Our study aimed at investigating the influence of elevated atmospheric CO(2) concentration on the salinity tolerance of the cash crop halophyte Aster tripolium L., thereby focussing on protein expression and enzyme activities. The plants were grown in hydroponics using a nutrient solution with or without addition of NaCl (75% seawater salinity), under ambient (380 ppm) and elevated (520 ppm) CO(2). Under ambient CO(2) concentration enhanced expressions and activities of the antioxidant enzymes superoxide dismutase, ascorbate peroxidase, and glutathione-S-transferase in the salt-treatments were recorded as a reaction to oxidative stress. Elevated CO(2) led to significantly higher enzyme expressions and activities in the salt-treatments, so that reactive oxygen species could be detoxified more effectively. Furthermore, the expression of a protective heat shock protein (class 20) increased under salinity and was even further enhanced under elevated CO(2) concentration. Additional energy had to be provided for the mechanisms mentioned above, which was indicated by the increased expression of a beta ATPase subunit and higher v-, p- and f-ATPase activities under salinity. The higher ATPase expression and activities also enable a more efficient ion transport and compartmentation for the maintenance of ion homeostasis. We conclude that elevated CO(2) concentration is able to improve the survival of A. tripolium under salinity because more energy is provided for the synthesis and enhanced activity of enzymes and proteins which enable a more efficient ROS detoxification and ion compartmentation/transport.

Enhanced triterpene saponin biosynthesis and root nodulation in transgenic barrel medic (Medicago truncatula Gaertn.) expressing a novel beta-amyrin synthase (AsOXA1) gene.

Triterpene saponins are a group of bioactive compounds abundant in the genus Medicago, and have been studied extensively for their biological and pharmacological properties. In this article, we evaluated the effects of the ectopic expression of AsOXA1 cDNA from Aster sedifolius on the production of triterpene saponins in barrel medic (Medicago truncatula Gaertn.). AsOXA1 cDNA encodes beta-amyrin synthase, a key enzyme involved in triterpene saponin biosynthesis. One of the four transgenic lines expressing AsOXA1 accumulated significantly larger amounts of some triterpenic compounds in leaf and root than did control plants. In particular, the leaf exhibited significantly higher levels of bayogenin, medicagenic acid and zanhic acid. The amounts of medicagenic acid and zanhic acid, which represent the core of the M. truncatula leaf saponins, were 1.7 and 2.1 times higher, respectively, than the amounts extracted from the control line. In root, the production of bayogenin, hederagenin, soyasapogenol E and 2beta-hydroxyoleanolic acid was increased significantly. The increase in the total amounts of triterpenic compounds observed in the leaves of transgenic lines correlated with the AsOXA1 expression level. Interestingly, the plants expressing AsOXA1 showed, under different growth conditions, improved nodulation when compared with the control line. Nodulation enhancement was also accompanied by a significant change in the soyasapogenol B content. Our results indicate that the ectopic expression of AsOXA1 in barrel medic leads to a greater accumulation of triterpene saponins and enhanced root nodulation.

Herbicide resistance in Aster squamatus conferred by a less sensitive form of acetolactate synthase.

A biotype of Aster squamatus (Sprengel) Hieronymus with suspected resistance to the ALS-inhibiting herbicide imazapyr was detected in a chicken farm in the province of Seville, Spain, which had been treated once a year with imazapyr for 10 years. Resistance to imazapyr in this biotype was studied using dose-response experiments, absorption and translocation assays, metabolism studies and ALS activity assays. The rate of imazapyr required to inhibit A squamatus growth by 50% (ED50) was 15 times higher for the R (resistant) than for the S (susceptible) biotype. Cross-resistance existed for the ALS-inhibitors imazamox, imazethapyr, amidosulfuron, nicosulfuron, rimsulfuron, triasulfuron and tribenuron, but not for bensulfuron. Control of A squamatus using alternative herbicides was poor with clopyralid, intermediate with quinclorac, amitrole and MCPA, and excellent with 2,4-D, glufosinate and glyphosate. Absorption of [14C]imazapyr increased over time for both the R and S biotypes, and translocation from the treated leaf to shoots and roots was similar in both biotypes, with most of the radioactivity remaining in the treated leaf. No metabolites of imazapyr were detected in either biotype. Sensitivity of the ALS enzyme (target site) to imazapyr was lower for the R biotype (I50(R) = 4.28 x I50(S)). The mechanism of imazapyr resistance in this R biotype appears to be an altered ALS conferring decreased sensitivity to imazapyr at the whole-plant level.