Effects of anticoagulant from Spirodela polyrhiza in rats

A fibrinolytic protease was purified from an Oriental medicinal herb, Spirodela polyrhiza (Choi, H. S., et al., Biosci. Biotechnol. Biochem., 65, 781-786 (2001)). The protease hydrolyzed not only fibrin but also fibrinogen. The enzyme had an anticoagulant activity measured with activated partial thromboplastin time, thrombin time, and prothrombin time in rat plasma. It doubled all three at 69, 29, and 221 nM, respectively. The protein had anticoagulant activity when given intravenously and orally. The maximum delay in the activated partial thromboplastin time was at the dose of 0.52 and 4.2 mg/kg for intravenous and oral administration, respectively. This protein may be useful in clinical applications for anticoagulation.     

铜对紫背浮萍的影响

利用紫萍(Spirodela polyrhiza)作材料,在实验室条件下研究了增加铜对紫萍生长、光合作用色素含量、叶绿素a荧光的产生和植物体内铜累积的影响。结果表明,环境中增加1μmol L-1d的Cu2+时即可显著地抑制紫萍的生长(用鲜重和干重表示),随增加的铜的量加大,对生长的抑制作用增加,鲜重在8 μmol L-1Cu2+时,干重在2 μmol L-1 Cu2+以上时出现负增长;铜水平的升高可使叶绿素含量大幅下降,但对类胡萝卜素的含量影响不大;对叶绿素a荧光的产生也有影响,短时间的处理(24 h内)使Fo和Fm升高,但Fv/Fm变化很小,随着处理时间的延长和处理浓度的增加,Fo和Fm下降至很低水平,特别是Fm下降更快,以至Fv/Fm可能出现负值,表明光合作用器官已受到破坏;当铜处理水平增加时植物体内的铜含量水平也增加,表明其对铜有一定的累积作用,但因为在高铜浓度水平下植物的生物量显著下降,因而从植物修复的角度考虑,在中等或微量铜污染的情况下,才能选择适宜来源的紫萍用于进行铜污染环境的植物修复。     

Cadmium Removal by Lemna minor and Spirodela polyrhiza

The present study investigates the ability of two genus of duckweed (Lemna minor and Spirodela polyrhiza) to phytoremediate cadmium from aqueous solution. Duckweed was exposed to six different cadmium concentrations, such as, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mg/L and the experiment was continued for 22 days. Water samples were collected periodically for estimation of residual cadmium content in aqueous solution. At the end of treatment period plant samples were collected and accumulated cadmium content was measured. Cadmium toxicity was observed through relative growth factor and changes in chlorophyll content. Experimental results showed that Lemna minor and Spirodela polyrhiza were capable of removing 42–78% and 52–75% cadmium from media depending upon initial cadmium concentrations. Cadmium was removed following pseudo second order kinetic model. Maximum cadmium accumulation in Lemna minor was 4734.56 mg/kg at 2 mg/L initial cadmium concentration and 7711.00 mg/kg in Spirodela polyrhiza at 3 mg/L initial cadmium concentration at the end of treatment period. Conversely in both cases maximum bio-concentration factor obtained at lowest initial cadmium concentrations, i.e., 0.5 mg/L, were 3295.61 and 4752.00 for Lemna minor and Spirodela polyrhiza respectively. The present study revealed that both Lemna minor and Spirodela polyrhiza was potential cadmium accumulator.     

Nitrate reductase inactivator from Spirodela polyrhiza (L.) Schleiden

NADH:nitrate reductase (EC 1.6.6.1) activity in the crude extract from Spirodela polyrhiza was relatively labile in vitro. Inclusion of polyvinylpolypyrrolidone into the extraction medium had only a slight effect on the stability of the enzyme, whereas addition of 3 % casein, azocasein, or other proteins to the extraction medium greatly increased the nitrate reductase (NR) activity. Various protease inhibitors were tested for their ability to prevent the loss of NR activity in vitro. Iodoacetate and para-chloromercuric benzoate, the thiol-protease inhibitors, as well as pepstatin, the aspartic-protease inhibitor had no effect on stability of the nitrate reductase. EDTA had a slight stimulatory effect, whereas 5 mM o-phenantroline, another inhibitor of the metallo-proteases increased the activity of nitrate reductase. The highest enzyme activity was found in the presence of phenylmethylsulphonyl fluoride and di-isopropyl phosphorofluoridate both being serine-protease inhibitors. The protease-like inactivator was separated from Spirodela polyrhiza by ammonium sulfate fractionation and acid treatment (pH 4.0). After centrifugation the protein of inactivator in supernatant adjusted to pH 7.5 was removed. When this fraction was examined by electrophoresis in polyacrylamide which copolymerized with edestin, the protein of the nitrate reductase inactivator remained at the cathode. Fractions containing a protein of inactivator degraded casein to products soluble in trichloroacetic acid. Inhibition of the inactivator proteolytic activity by phenylmethylsulphonyl fluoride and di-isopropyl phosphorofluoridate but not by other reagents (thiol- and metallo-protease inhibitors) suggested the involvement of a serine residue at its active site. The inactivator fraction from Spirodela polyrhiza resulted in a loss of the nitrate reductase activity in crude extracts from both cucumber and corn seedlings. A biochemical nature a protein of the nitrate reductase inactivator from S. polyrhiza is discussed.     

紫萍营养繁殖过程的解剖观察

以采自海南陵水的紫萍(Spirodela polyrhiza DW2501-4)为材料,观察紫萍的营养繁殖过程。紫萍DW2501-4培养在0.5倍Hoagland′s液体培养基中,先不断长出新的叶状体,随着营养物质的减少,长出休眠体进入休眠状态。休眠体经过4℃处理7 d后,可在含1%蔗糖的Hoagland′s固体培养基中重新萌发。为了进一步观察叶状体、类休眠体和休眠体的组织结构差异,以及休眠体萌发过程中组织结构的变化,制作了石蜡切片。观察结果显示:紫萍叶状体有数条叶脉,细胞中含少量的淀粉粒,表皮层以下有分层的气室,通常分上下两层,上层气室比下层气室小。类休眠体也有数条叶脉和少量气室,细胞中有较多淀粉粒。休眠体的细胞差异不明显,几乎没有气室,细胞内有大量的淀粉粒,部分细胞含有单宁;随着休眠体萌发,细胞中淀粉粒不断变小,同时分生组织分化出根和新的叶状体。     

Circadian Rhythmicity in the Activities of Phenylalanine Ammonia-Lyase from Lemna perpusilla and Spirodela polyrhiza

The oscillations in phenylalanine ammonia-lyase activity from Spirodela polyrhiza and phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities from Lemna perpusilla displayed a circadian rhythm under continuous light. Rhythmicity in enzymic activity could not be detected in continuous darkness since under this condition phenylalanine ammonia-lyase activity remains at a fairly constantly low level. Results from our studies of the oscillatory pattern of the respective activities of phenylalanine and tyrosine ammonia-lyase support their “inseparability.”     

铈对紫萍抗铜能力的影响

铈能减缓铜胁迫导致的紫萍(Spirodela polyrhiza L.)叶片膜透性的增加、可溶性蛋白含量和叶绿素a/b 值的下降以及超氧阴离子(O₂·)、丙二醛(MDA)的积累。与同浓度的单一铜处理相比, 加入20 mg/L 的铈后, 超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)的活性和类胡萝卜素(Car)、抗坏血酸(AsA)含量不同程度的增强或增多, 暗示铈可能通过提高紫萍活性氧清除能力以增强对铜的抗性和耐性。     

Aminoacylation of tRNA and Protein Turnover in Nitrateand Sulphate-Deficient Spirodela polyrhiza

Cultures of Spirodela polyrhiza were maintained in completeHoagland's medium at 25°C in continuous light. Nitrate-and sulphate-deficient plants were cultured in media containing1/20 Hoagland's nitrate and 1/200 Hoagland's sulphate respectively.After 10 days of growth the plants were examined for total aminoacyltRNA levels. Turnover of leucyl-tRNA and rates of protein synthesiswere assessed by pulse feeding [³H]leucine. Control and nutrient-deficientplants had similar levels of tRNA-associated amino acids. Howeverthe amounts of tRNA, expressed on a fresh weight basis, weresignificantly lower in nitrate- and sulphate-deficient plants.Although the specific radioactivities of leucyltRNA were highestin deficient cultures the rate of turnover of this pool wasless than non-deficient control or nitrate- and sulphate-supplementedplants. Calculation of the average rate constants for proteinsynthesis and degradation showed that nitrate deficiency, althoughnot affecting rates of synthesis, supported rates of proteindegradation that were higher than control cultures. ¹ Present address: The Biological Laboratories, Harvard University,16 Divinity Avenue, Cambridge, Massachusetts 02138, U.S.A. (Received March 7, 1983; Accepted August 16, 1983)     

Physiological and biochemical responses induced by lead stress in Spirodela polyrhiza

The effects of increasing lead concentration on the activities of superoxide dismutase (SOD), peroxides (POD) and catalase (CAT), levels of ascorbate (AsA), reduced glutathione (GSH), Pb accumulation and its influence on nutrient elements, polyamines (PAs) content, as well as activities of polyamine oxidase (PAO) and ornithine decarboxylase (ODC), were investigated in Spirodela polyrhiza. POD and CAT activities increased progressively followed by a decline, while SOD activity gradually fell. The effect of Pb application on AsA content was similar to that seen for POD and CAT activities. GSH content initially rose but then declined. A significant enhancement in Pb accumulation was observed, except in the 25?μM Pb treatments. Nutrient elements were also affected. Moreover, Pb stress induced a considerable decrease in total spermidine (Spd), while the levels of total putrescine (Put) and spermine (Spm) initially increased at 25?μM Pb but then declined. Free and perchloric acid soluble conjugated (PS-conjugated) PAs contents changed in a similar way to total PAs. In addition, Pb stress induced a continuous accumulation of perchloric acid insoluble bound (PIS-bound) Spm and an initial accumulation of PIS-bound Put and Spd. The ratio of free (Spd?+?Spm)/Put significantly declined whereas the ratio of total (Spd?+?Spm)/Put rose at low Pb concentrations (25 and 50?μM). PAO activity rose gradually with an increase in Pb concentration, reaching peak values at 100?μM, while ODC activity first increased at 25?μM Pb and then declined. The results indicated that the tolerance of S. polyrhiza to Pb stress was enhanced by activating the antioxidant system, preventing the entry of the Pb ion and altering the content of polyamines.     

Subcellular localization, purification, and some catalitic properties of aspartate aminotransferase from Spirodela polyrhiza

Intracellular distribution of aspartate aminotransferase (AAT) in Spirodela polyrhiza (Lemnaceae), strain SJ, has been studied by differential centrifugation. The bulk of the enzyme (73% of total cellular content) was localized in the cytoplasm and 24% activity was localized in chloroplasts. Purified cytoplasmic and chloroplastic isozymes differed by their affinity for substrates. The reaction balance was shifted towards direct and reverse transamination in the cytoplasm and chloroplast, respectively. Competitive inhibition of AAT by excessive substrates and enzyme affinity modulation by certain intermediates of the tricarboxylic acid cycle (isocitrate, succinate, and citrate) were observed. Possible involvement of AAT isozymes in the coordination of carbon and nitrogen metabolism through the regulation of 2-oxoglutarate synthesis and utilization in different cellular compartments is discussed.     

In-vivo control of valine and leucine synthesis in the duckweed Spirodela polyrhiza

Duckweed colonies were grown on 1 l of nutrient solution supplied with 10 M l-[¹⁴C]leucine or with 25 M l-[¹⁴C]valine. Under these conditions the exogenously supplied amino acid did not inhibit growth, but caused in the plants a moderately increased pool of that amino acid, which remained essentially constant during the culture period. The effect of the increased pool of valine or leucine on the biosynthesis of these amino acids was determined from isotope dilution in the protein-bound valine and-or leucine. An increase in the leucine pool from 1.1 to 5.0 nmol mg^–1 dry weight resulted in a 21% reduction of metabolite flow through the common part of the valine-leucine biosynthetic pathway; leucine synthesis was reduced by 35%, but valine synthesis by only 5% and isoleucine synthesis was apparently unaffected. An increase in the valine pool from 3.2 to 6.6 nmol mg^–1 dry weight reduced the metabolite flow through the valine-leucine pathway by 48%, valine synthesis by 70%, and leucine synthesis from pyruvate by 29%, which was compensated by leucine synthesis from exogenous valine, whereas the synthesis of isoleucine was not changed. It is concluded that the biosynthesis of valine and leucine is mainly controlled by feedback inhibition of acetohydroxyacid synthetase. In vivo, the feedback inhibition can be exerted in such a way that synthesis of acetolactate (the precursor of valine and leucine) is appreciably reduced, whereas synthesis of acetohydroxybutyrate (the isoleucine precursor) is not inhibited.     

Phytochrome-induced transmissible signal elicits germination response in turions of Spirodela polyrhiza

The role of cell competence, including the spatiotemporal aspect of phytochrome-induced long-distance signal transmission, was investigated in turions of Spirodela polyrhiza (L.) Schleiden. Irradiation of the dorsal surface of the turions triggered a significant germination response, while identical treatment of the ventral surface was less effective. Red-light (R) microbeam irradiation of a subregion (ca. 1 μm²) of the dorsal surface could induce the germination response. Therefore, photoactivation of phytochrome in a single cell or few cells is sufficient to trigger the photomorphogenetic response. The ultimate response occurs at the proximal end of the turion by way of growth and emergence of the frond primordia about 1.3 mm away from the microbeam-irradiated distal cell(s). This photoinduction was reversible by a pulse of far-red light (FR) given less than 24 h after R microbeam irradiation. Microsurgical separation of distal (irradiated) and proximal (primordium-bearing) halves of the turions following microbeam irradiation further revealed that the light-induced transmissible signal can be intercepted and that it required more than 48 h to traverse one half distance of the turions. Based on the kinetics of the signal transmission, the possible involvement of light scattering, light piping, or transfer of electrophysiological signals can be excluded. Taken together, the results indicate that a transmissible signal is generated by the irradiated cell(s) and propagated across to the non-irradiated cells, leading to induction of the photomorphogenetic response.     

Phytochrome Control of Turion Formation in Spirodela polyrhiza L. Schleiden

Turion yield in Spirodela polyrhiza, strain SJ, is increasedby increasing the daily light period. This effect is more pronouncedin autotrophic than in mixotrophic conditions. Night-break irradiation(15 mins) increased turion yield by 150 % under the conditionsof an 8-h daily light period. Besides the effect of night-breakirradiation, end-of-day far-red irradiation decreased turionyield with increasing photoperiod, whereas end-of-day red irradiationwas without any effect. This demonstrates the promoting effectof the P_fr form of phytochrome on formation of light-grown turions. Formation of dark-grown turions was increased by about 240%by a single red light pulse and was reversed by an immediatelyapplied far-red light pulse. Consequently, under heterotrophicconditions phytochrome modulates the turion formation process. Spirodela polyrhiza L. Schleiden, duckweed, Lemnaceae, photomorphogenesis, phytochrome, turion     

Effect of low temperature on eutrophicated waterbody restoration by Spirodela polyrhiza

Spirodela polyrhiza exposed to low temperature was investigated to study the possible application for recovering eutrophicated waterbody at 10-12 degrees C. The cell growth and enzymatic resistance to oxidative stress were compared with that grown at optimum temperature (26-28 degrees C). The frond number, fresh weight, soluble protein, chlorophyll, carrot pigment, root length, peroxidase (POD), catalase (CAT), superoxide dismutase (SOD) and malondialdehyde (MDA) were measured. The results demonstrated that the cell growth, the synthesis and absorption ability decreased and the protective enzymes increased. S. polyrhiza encountered seriously oxidative damage at such low temperature condition, and it was not suitable for recovering the eutrophicated natural waterbody at low temperature of 10-12 degrees C.     

Interactions between the branched-chain amino acids in the growth of Spirodela polyrhiza

The joint action of L-valine and L-isoleucine, L-leucine and L-isoleucine, and L-valine and L-leucine on the growth of Spirodela polyrhiza was established. The effect of one branched-chain amino acid on growth inhibition by another one was compared with the non-specific antagonisms which glycine and L-alanine exert on growth inhibition by singly supplied branched-chain amino acids. In this way specific and non-specific interactions could be distinguished. It appeared that: (1) L-isoleucine was a specific antagonist of L-valine; (2) L-leucine was a specific antagonist of L-isoleucine; (3) L-valine and L-leucine were synergistic growth inhibitors. Further, it was found that: (4) growth inhibition by L-leucine was specifically antagonized by simultaneously supplied L-valine and L-isoleucine; (5) an excess of L-isoleucine strongly inhibited the conversion of exogenous valine into leucine; (6) accumulation of valine was typical of isoleucine-induced growth inhibition. The results are consistent with the view that growth inhibition by L-valine and L-leucine is due to the blocking of acetohydroxy acid synthetase, the first common enzyme in the valine-isoleucine biosynthetic pathway. Growth inhibition by L-isoleucine, however, seems to result from inhibition of leucine synthesis at a step after 2-oxoisovaleric acid. Some aspects of the regulation of branched-chain amino acid biosynthesis in higher plants are discussed.     

Multiple Effects of Chromate on Spirodela polyrhiza: Electron Microscopy and Biochemical Investigations

Abstract: Growth rates of S. polyrhiza were reduced by chromate concentrations higher than 50 μM. Analysis of plant cells by transmission electron microscopy revealed the accumulation of starch grains in the chloroplasts following the application of chromate at low concentrations or for short periods (100 μM for 2 days or 500 μM for 1 day). Increasing the chromate concentration (1000 μM for 1 day) or extending the period (100 μM for 4 days) resulted in the disappearance of most of the starch grains and the extensive formation of plastoglobuli. These results were confirmed by chemical analysis of the starch content. It has been suggested that this transient accumulation of starch was caused, first, by inhibition of the export of carbohydrates out of the plastids, and then by inhibition of photosynthesis. Chromate decreased the chlorophyll content and the chlorophyll a/b ratio. The quantitative analysis of the chlorophyll protein complexes showed that the photosystem II (core complex as well as connecting antenna) was more sensitive to chromate treatment than photosystem I and the peripheral light-harvesting complex of photosystem II. This explains the previous results on time-resolved chlorophyll a fluorescence (Appenroth et al. Environ. Pollut. [2001] 115, 49 - 64). Photosynthesis is clearly an important target of chromate toxicity. Electron microscopic analysis showed damage to several membrane systems, such as that of thylakoids, chloroplast envelope, plasmalemma and, at higher concentrations, that of tonoplast and mitochondria. Thus, the membrane system is another target of chromate toxicity.     

Light-induced degradation of storage starch in turions of Spirodela polyrhiza depends on nitrate

Light induces both the germination of turions of the duckweed Spirodela polyrhiza and the degradation of the reserve starch stored in the turions. The germination photoresponse requires nitrate, and we show here that nitrate is also needed for the light-induced degradation of the turion starch. Ammonium cannot substitute for nitrate in this regard, and nitrate thus acts specifically as signal to promote starch degradation in the turions. Irradiation with continuous red light leads to starch degradation via auto-phosphorylation of starch-associated glucan, water dikinase (GWD), phosphorylation of the turion starch and enhanced binding of alpha-amylase to starch granules. The present study shows that all of these processes require the presence of nitrate, and that nitrate exerts its effect on starch degradation at a point between the absorption of light by phytochrome and the auto-phosphorylation of the GWD. Nitrate acts to coordinate carbon and nitrogen metabolism in germinating turions: starch will only be broken down when sufficient nitrogen is present to ensure appropriate utilization of the released carbohydrate. These data constitute the first report of control over the initiation of reserve starch degradation by nitrate.