Growth Inhibition in Suspension-Cultured Rice Cells under Phosphate Deprivation Is Mediated through Putrescine Accumulation

The effects of phosphate deprivation on the growth and polyamine levels of suspension-cultured rice (Oryza sativa) cells were investigated. When rice suspension cells were deprived of phosphate, cell growth was markedly inhibited. Phosphate deprivation resulted in a higher putrescine level and lower spermidine and spermine levels in rice suspension cells. The growth of rice cells cultured in the absence of phosphate did not recover as a result of spermidine and spermine addition. D-Arginine and [alpha]-methylornithine, inhibitors of putrescine biosynthesis, caused a reduced level of putrescine in rice suspension cells cultured under phosphate deprivation. The growth of rice cells cultured in the absence of phosphate was completely recovered after the addition of D-arginine but not [alpha]-methylornithine. Our results indicate that putrescine accumulation is a factor causing growth inhibition of suspension-cultured rice cells under phosphate deprivation.     

Ethylene involvement in in vitro organogenesis and plant growth of Populus tremula L.

The possible involvements of a decrease in medium pH and accumulation of ammonium in ammonium-inhibited growth of rice cells were investigated. Ammonium, applied at concentrations ranging from 20 to 50 mM, markedly inhibited cell growth and decreased medium pH. The accumulation of ammonium in rice cells was observed only when ammonium concentration was 40 mM or higher. Ammonium-inhibited growth was alleviated when medium pH was buffered with MES [2-(N-morpholino)-ethanesulfonic acid]. However, no difference in ammonium level was observed between buffered and unbuffered ammonium-fed rice cells. Succinic acid, -ketoglutaric acid, glutamic acid and glutamine were found to be effective in reversing ammonium-inhibited growth of rice cells and reducing a fall in pH in the ammonium-fed medium. Succinic acid, -ketoglutaric acid and glutamic acid decreased the level of ammonium in ammonium-fed rice cells. However, glutamine was unable to decrease the ammonium level in ammonium-fed rice cells. The current results suggest that a decrease in medium pH is a factor responsible for growth inhibition of ammonium-fed rice cells.     

湖南稻田土壤固定态铵含量的季节变化及生物有效性

以湖南省3种固定态铵含量较高的稻田土壤为供试土壤,通过盆栽试验,研究了稻田土壤固定态铵在植稻期间的动态变化及其生物有效性。结果表明,稻田土壤的固定态铵含量处于不断的变化之中,施氮肥和有机肥使土壤固定态铵含量升高,而水稻吸收氮则使土壤固定态铵含量降低,其变化趋势与土壤碱解氮含量变化相似。“新固定的”固定态铵基本对当季水稻全部有效。而“原有的”固定态铵对当季作物和后季作物部分有效,就供试土壤而言,在水稻生育期间,土壤固定态铵的释放量是潮沙泥>紫泥田>河沙泥;就不同水稻而言,早稻生育期间土壤固定态铵的释放量大于晚稻生育期间土壤固定态铵的释放量。     

Nutrient regulation of bacterial growth and production of anti-tumor metabolites in <Emphasis Type="Italic">Stigmatella</Emphasis> WXNXJ-B fermentation

The metabolites produced by Stigmatella WXNXJ-B inhibited the growth of tumor cells. The aims of this research were to evaluate the inhibition potency to different tumor cell lines and to study the effects of ammonium, phosphate and iron salts on bacterial growth and production of bioactive metabolites in Stigmatella WXNXJ-B fermentation. The results showed that the chloroform extract (CE-ME) showed the strongest growth inhibition bioactivity on mouse melanoma cell line (B16), murine colon carcinoma cell line (CT-26), human liver carcinoma cell line (HepG2) and human breast cancer cell line (MDA-MB231) in vitro and the IC₅₀ values were 9.94, 7.33, 11.34 and 11.66 μg ml⁻¹ respectively. The IC₅₀ value was above 700 μg ml⁻¹ on normal mouse spleen cells. Morphology happened changes in B16 cells treated with CE-ME. The anti-tumor metabolites were mainly produced during the stationary phase of the bacterial growth. Cell growth was stimulated at the phosphate concentration below 5 mM, but it was inhibited partly with 10 mM phosphate. The production of bioactive substances was inhibited by the phosphate. Ammonium increased the cell growth by 250% at 5 mM addition. The inhibition rate to B16 cells was increased to 89% at the concentration of 40 mM ammonium. The bacteria showed the best growth with 4 mM iron. Iron had little effect on the production at 2 mM, but bigger inhibition effect at higher iron concentration.     

林肯链霉菌合成林可霉素代谢调节的研究

在摇瓶条件下研究了葡萄糖、铵盐、磷酸盐对林可霉素产生菌林肯链霉菌的生长及林可霉素生物合成的影响。发酵过程中林可霉素的合成主要发生在菌体生长期,逐渐下降。使用6%的葡萄糖未发现通常所说的“葡萄糖效应”。0.2%铵盐有利于细胞生长,但0.8%NH+4对林可霉素的生物合成具有抑制作用。发酵48h后补加0.6% NH^＋_４,对林可霉素的生成没有显著影响。0.05%～0.1%磷酸盐对林可霉素合成具有较强的抑制作用。并就磷酸盐对菌体由初级代谢转向次级代谢的作用作了初步考察。     

Disturbed ammonium assimilation is associated with growth inhibition of roots in rice seedlings caused by NaCl

The effects of NaCl on changes in ammonium level and enzyme activities of ammonium assimilation in roots growth of rice (Oryza sativa L.) seedlings were investigated. NaCl was effective in inhibiting root growth and stimulated the accumulation of ammonium in roots. Accumulation of ammonium in roots preceded inhibition of root growth caused by NaCl. Both effects caused by NaCl are reversible. Exogenous ammonium chloride and methionine sulfoximine (MSO), which caused ammonium accumulation in roots, inhibited root growth of rice seedlings. NaCl decreased glutamine synthetase and glutamate synthase activities in roots, but increased glutamate dehydrogenase activity. The growth inhibition of roots by NaCl or MSO could be reversed by the addition of L-glutamic acid or L-glutamine. The current results suggest that disturbance of ammonium assimilation in roots may be involved in regulating root growth reduction caused by NaCl.Abbreviations GDH glutamate dehydrogenase - GOGAT glutamate synthase - GS glutamine synthetase - MSO methionine sulfoximine     

Inhibition of ammonium assimilation restores elongation of seminal rice roots repressed by high levels of exogenous ammonium

Elongation of seminal and lateral roots of rice seedlings was markedly inhibited by high ammonium levels in growth medium. However, high exogenous nitrate concentrations had little inhibitory effect on root growth. The objective of this study was to elucidate the relationship between inhibition of rice root growth induced by high ammonium conditions and ammonium assimilation in the seedlings. Activity of glutamine synthetase (GS) was kept at a low level in the seminal roots of the seedlings grown under high nitrate levels. In contrast, high ammonium levels significantly enhanced the GS activity in the roots, so that Gln abundantly accumulated in the shoots. These results indicate that ammonium assimilation may be activated in the seminal roots under high ammonium conditions. Application of methionine sulfoximine (MSO), an inhibitor of GS, relieved the repression of the seminal root elongation induced by high ammonium concentrations. However, the elongation of lateral roots remained inhibited even under the same condition. Furthermore, MSO drastically increased ammonium level and remarkably decreased Gln level in the shoots grown under high ammonium conditions. These results show that, for rice seedlings, an assimilatory product of ammonium, and not ammonium itself, may serve as an endogenous indicator of the nitrogen status involved in the inhibition of seminal root elongation induced by high levels of exogenous ammonium.     

Osmoregulation in Rhizobium meliloti: inhibition of growth by salts

A defined medium of low osmolarity was developed permitting growth of Rhizobium meliloti with generation times of approximately 2.8 h doubling^-1. The effects of sodium, potassium, magnesium, ammonium, chloride, sulfate, phosphate, bicarbonate and acetate ions on the growth rate of R. meliloti were determined. Sodium, potassium and ammonium ions had little effect on growth at concentrations of 100 mEq or less; magnesium ion inhibited growth severely at concentrations of 50 mEq (25 mM). Of the anions, chloride and sulfate appeared to have little effect while phosphate, bicarbonate, and acetate inhibited growth at concentrations of as little as 25 mEq. The addition of proline, glutamate, or betaine to cells growing in inhibitory concentrations of NaCl did not relieve the inhibition. When grown in the presence of inhibitory levels of NaCl, the intracellular concentration of glutamate but not of proline or gamma amino butyric acid increased 5-fold.     

Citrate, a possible precursor of astaxanthin in Phaffia rhodozyma: influence of varying levels of ammonium, phosphate and citrate in a chemically defined medium.

The influence of ammonium, phosphate and citrate on astaxanthin production by the yeast Phaffia rhodozyma was investigated. The astaxanthin content in cells and the final astaxanthin concentration increased upon reduction of ammonium from 61 mM to 12.9 mM (from 140 microg/g to 230 microg/g and 1.2 microg/ml to 2.3 microg/ml, respectively). Similarly, both the astaxanthin content and astaxanthin concentration increased by reducing phosphate from 4.8 mM to 0.65 mM (160 microg/g to 215 microg/g and 1.7 microg/ml to 2.4 microg/ml, respectively). Low concentrations of ammonium or phosphate also increased the fatty acid content in cells. By analogy with lipid synthesis in other oleaginous yeasts, an examination of the data for varying nitrogen and phosphate levels suggested that citrate could be the source of carbon for fatty acids and carotenoid synthesis. Supporting this possibility was the fact that supplementation of citrate in the medium at levels of 28 mM or higher notably increased the final pigment concentration and pigment content in cells. Increased carotenoid synthesis at low ammonium or phosphate levels, and stimulation by citrate were both paralleled by decreased protein synthesis. This suggested that restriction of protein synthesis could play an important role in carotenoid synthesis by P. rhodozyma.     

Metabolic changes during phosphate deprivation in Englena in air and in oxygen

1. Euglena cells were grown in culture media containing either 20mm-phosphate or 20mum-phosphate, with ethanol or glucose as the sole source of carbon, and gassed with either air+carbon dioxide (95:5) or oxygen+carbon dioxide (95:5) at atmospheric pressure. 2. After growth in low-phosphate medium with ethanol as substrate, the cells developed signs of oxygen toxicity, as indicated by a decreased rate of respiration, a decreased net synthesis of paramylum and a failure to resume growth on replenishment of phosphate. 3. After growth in low-phosphate medium with glucose as substrate, the signs of oxygen toxicity were less apparent. 4. During phosphate deprivation the carotenoid content of Euglena increased more than threefold. This increase was largely prevented by exposure of the cells to oxygen+carbon dioxide (95:5) during growth. Oxygenation appears to interfere with ring closure of the common carotenoid precursor. 5. Mitochondria obtained from Euglena exposed to oxygen during phosphate deprivation, i.e. when signs of oxygen toxicity were evident, had greatly decreased activities of succinate dehydrogenase, succinate-cytochrome c oxidoreductase and NADH-cytochrome c oxidoreductase, compared with mitochondria obtained from Euglena exposed to oxygen in medium containing 20mm-phosphate.     

Properties ofAzotobacter vinelandii in phosphate-limited batch cultures

Batch cultures ofA. vinelandii in ammonium phosphate-limited and N-free phosphate-limited media were compared with control cultures (N-free phosphate-sufficient media). The effects of phosphate limitation on growth were determined by viable cells counts. Under phosphate-limitation conditions, growth inhibition and decreased viability were observed. Intracellular levels of RNA, poly-3-hydroxybutyrate, phosphate and oxygen uptake were significantly affected by phosphate limitation. When phosphate-limited cultures were examined microscopically, pleomorphism was more marked than in control cultures. Also phosphate-limited cells showed an increase in resistance to UV irradiation, mechanical disruption, desiceation and the combined action of ethylenediaminetetraacetie acid and lysozyme.     

Regulation of sugar metabolism in rice (Oryza sativa L.) seedlings under arsenate toxicity and its improvement by phosphate

The effect of arsenate with or without phosphate on the growth and sugar metabolism in rice seedlings cv. MTU 1010 was studied. Arsenate was found to be more toxic for root growth than shoot growth and water content of the seedlings gradually decreased with increasing concentrations. Arsenate exposure at 20 μM and 100 μM resulted in an increase in reducing sugar content and decrease in non-reducing sugar content. There was a small increase in starch content, the activity of starch phosphorylase was increased but α-amylase activity was found to be decreased. Arsenate toxicity also affected the activities of different carbohydrate metabolizing enzymes. The activities of sucrose degrading enzymes viz., acid invertase and sucrose synthase were increased whereas, the activity of sucrose synthesizing enzyme, viz. sucrose phosphate synthase declined. The combined application of arsenate with phosphate exhibited significant alterations of all the parameters tested under the purview of arsenate treatment alone which was congenial to better growth and efficient sugar metabolism in rice seedlings. Thus, the use of phosphorus enriched fertilizers may serve to ensure the production of healthy rice plants in arsenic contaminated soils.     

Diffusible factor(s) controlling density inhibition of 3T3 cell growth: a new approach

In 3T3 Swiss mouse fibroblasts, incorporation of phosphate into cells and phosphorylation of small organic compounds were increased by shaking dense cultures. This response was not obtained with SV40 transformed Swiss 3T3 cells (SV-3T3). It appeared likely that these results could be accounted for by an inhibitor released from 3T3 cells but not from SV-3T3 cells. Our new method of co-incubation of sparse and dense cultures allowed us to demonstrate inhibition of growth and phosphate metabolism in sparse 3T3 cultures which were shaken in the presence of dense cultures. The inhibition was much less when the cultures were co-cultivated but not shaken. The inhibition of phosphate incorporation in acid-soluble and acid-insoluble fractions of sparse cultures was observed as early as 20 minutes of co-incubation in the presence of dense cultures, so this inhibition is not the result of depletion of growth factors in the medium. Our experiments suggest that an inhibitor(s) was released from dense cultures of 3T3 cells.     

Enzyme changes during Bacillus subtilis sporulation caused by deprivation of guanine nucleotides

When sporulation is initiated by nutrient limitation, e.g., at the end of growth, certain biochemical processes occur in sequence. To determine which of these processes occur, even when the cells sporulate in the presence of a rapidly metabolizable carbon source, we induced sporulation of Bacillus subtilis by deprivation of guanine nucleotides, in a synthetic medium containing excess glucose, ammonium ions, and phosphate. The deprivation was produced either by decoyinine addition to a standard strain or by guanosin limitation of a guanine auxotroph. At 1 h after the onset of this deprivation, an extensive turnover of proteins began whose appearance was chloramphenicol sensitive. At least one enzyme (aspartate transcarbamylase) lost 70% of its activity within 15 min, indicating its rapid destruction. Whereas the magnitude of the above two changes was similar to that observed during sporulation at the end of growth in nutrient sporulation medium, protease (intracellular and extracellular) increased to less than one-tenth of the specific activity in nutrient sporulation medium, and alkaline phosphatase increased to less than one-half. However, glucose dehydrogenase, an enzyme made only in forespores, increased to the same specific activity under both conditions, presumably because the forespore compartment is protected from media (e.g., glucose) influences by the double membrane (two bilayers with opposite polarity).     

COMPARATIVE pH DEPENDENT METAL INHIBITION OF NUTRIENT UPTAKE BY SCENEDESMUS QUADRICAUDA(CHLOROPHYCEAE)1,2

Cadmium and copper inhibition of nutrient uptake by the green alga Scenedesmus quadricauda is highly pH dependent in an inorganic medium; both metals are less toxic at low pH. The alga was grown in chemostats with both N and P approaching limiting levels; it was then possible to study metal toxicity to the nitrate, ammonium, and phosphate uptake systems of algae in an identical physiological state. When the logarithm of the Cd concentration causing 25% inhibition of nitrate, ammonium, and phosphate uptake was regressed against pH almost perfect linear relationships were obtained. This was also true at the 50% inhibition level, except for a smaller than predicted increase in Cd toxicity to ammonium uptake at pH 8, which may be due to the beginning of Cd precipitation at this pH. Cu²⁺ toxicity was linearly related to pH for ammonium and phosphate uptake and although, its toxicity for nitrate uptake also increased with pH, the increase was not perfectly linear. The toxicity of total Cu showed no linear relationship to pH. Cd²⁺ and Cu²⁺ toxicity increased by up to four orders of magnitude from pH 5 to 8. Competition between free metal and hydrogen ions for uptake sites on the cell surface is suggested as a mechanism increasing the toxicity of free metal, ions as the hydrogen ion content decreases (i.e. at higher pH).     

Effects of Iron Chelators,Iron Salts,and Iron Oxide Nanoparticles on the Proliferation and the Iron Content of Oligodendroglial OLN-93 Cells

The oligodendroglial cell line OLN-93 was used as model system to investigate the consequences of iron deprivation or iron excess on cell proliferation. Presence of ferric or ferrous iron chelators inhibited the proliferation of OLN-93 cells in a time and concentration dependent manner, while the application of a molar excess of ferric ammonium citrate (FAC) prevented the inhibition of proliferation by the chelator deferoxamine. Proliferation of OLN-93 cells was not affected by incubation with 300 μM iron that was applied in the form of FAC, FeCl₂, ferrous ammonium sulfate or iron oxide nanoparticles, although the cells efficiently accumulated iron during exposure to each of these iron sources. The highest specific iron content was observed for cells that were exposed to the nanoparticles. These data demonstrate that the proliferation of OLN-93 cells depends strongly on the availability of iron and that these cells efficiently accumulate iron from various extracellular iron sources.     

磷酸氢二铵对糙皮侧耳菌丝生长及基质降解酶活性的影响

氮源种类和浓度均会对糙皮侧耳Pleurotus ostreatus菌丝长势、生长速率以及代谢产生影响,而氮匮乏与氮过量积累都会对糙皮侧耳的生长产生不利影响。为了研究氮源对糙皮侧耳菌丝生长和基质降解酶活性的影响,本研究选取磷酸氢二铵[(NH4)2HPO4]作为唯一氮源,通过在培养基中添加不同浓度的磷酸氢二铵检测糙皮侧耳菌丝生长速率和木质纤维素降解酶的活性,筛选出糙皮侧耳生长的最适磷酸氢二铵浓度。结果表明,当磷酸氢二铵的添加浓度区间为10–20 mmol/L时菌丝长势最好。以缺氮时的酶活作为对照,当磷酸氢二铵的添加浓度为10 mmol/L时,羧甲基纤维素酶的活力最强,显著高于对照组(P<0.05);当添加浓度为5 mmol/L时,滤纸纤维素酶的活力最强,显著高于对照组(P<0.05),而10 mmol/L浓度下的酶活力比5 mmol/L时稍有下降,但二者之间无显著性差异(P>0.05);当添加浓度为40 mmol/L时,木聚糖酶的活力最强,显著高于对照组(P<0.05);当添加浓度为10mmol/L时,漆酶的活力最强,显著高于对照组(P<0.05)。基质降解酶...     

Initiation of Yeast Sporulation by Partial Carbon, Nitrogen, or Phosphate Deprivation

In this paper we show that partial deprivation of a carbon source, a nitrogen source, or phosphate in the presence of all other nutrients needed for growth initiates meiosis and sporulation of Saccharomyces cerevisiae homothallic strain Y55. For carbon deprivation experiments, cells were grown in synthetic medium (pH 5.5) containing an excess of one carbon source and then transferred to the same medium containing different concentrations of the same carbon source. In the case of transfer to different acetate concentrations, the log optical density at 600 nm increased at the previous rate until the cells had used up all of the acetate, whereupon the cells entered a stationary phase and did not sporulate. The same was observed with ethanol. In contrast, at different concentrations of dihydroxy-acetone or pyruvate, cells grew at different rates and sporulated optimally at intermediate concentrations (50 to 75 mM). The response to galactose was similar but reflected the presence of a low-affinity galactose transport system and the induction of a high-affinity galactose transport system. Cells could also sporulate when a glucose medium ran out of glucose, apparently because they initiated sporulation during the subsequent lag period and then used the produced ethanol as a carbon source. For phosphate deprivation experiments, cells growing with excess ethanol or pyruvate and phosphate were transferred to the same medium containing limiting amounts of phosphate. First, they used up the intracellular phosphate reserves for rapid growth, and then they sporulated optimally when an intermediate concentration (30 μM) of phosphate had been added to the medium. For nitrogen deprivation experiments, cells grown with excess acetate, ethanol, or pyruvate and NH₄⁺ were transferred to the same medium from which all nitrogen had been removed. These cells sporulated well in acetate medium but poorly in ethanol and pyruvate media. However, the sporulation frequency in the latter media could be increased greatly by adding intermediate concentrations (1 mM) of the slowly metabolizable amino acids glycine, histidine, or phenylalanine. If one assumes that the sporulation response to partial deprivation of carbon-, nitrogen-, or phosphorus-containing compounds reflects control by a single metabolite, the intracellular concentration of this metabolite may decide at the START position (G1 phase) of the cell cycle whether a/α cells enter mitosis or meiosis.