Changes in intracellular amino acids and organic acids induced by nitrogen starvation and nitrate or ammonium resupply in the cyanobacterium Phormidium laminosum

In Phormidium laminosum cells, nitrogen starvation caused a decrease in the intracellular levels of all amino acids, except glutamate, and an increase in the total level of the analyzed organic acids. The addition of nitrate or ammonium to N-starved cells resulted in substantial increases in the pool size of most amino acids. Upon addition of ammonium the total level of organic acids diminished, whereas it increased upon addition of nitrate, after a transient decay during the first minutes. Nitrogen resupply stimulated amino acid synthesis, the effect being faster and higher when ammonium was assimilated. The data indicate that nitrate and ammonium assimilation induced an enhancement of carbon flow through the glycolytic and the tricarboxylic-acid pathways to amino acid biosynthesis, with a concurrent decrease in the carbohydrate reserves. The results suggest that the availability of carbon skeletons limited the rate of ammonium assimilation, whereas the availability of reducing equivalents limited the rate of nitrate assimilation.Abbreviations Chl chlorophyll - GOGAT ferredoxin-dependent glutamate synthase (EC 1.4.7.1) - GS glutamine synthetase (EC 6.3.1.2) This work has been supported by grants from the Spanish Ministry of Education and Science (DGICYT and PB92-0464) and the University of the Basque Country (042.310-EC203/94) M.I.T. and J.A.G. were the recipients of fellowships from the Basque Government.     

Effect of nitrogen starvation on ammonium assimilation by Chlamydomonas reinhardtii

In nitrogen-starved Chlamydomonas reinhardtii , wild type, strain 21 gr cells, consumption of nitrate, nitrite and ammonium may occur in the dark in the absence of an added carbon source. Consumption of ammonium in the dark was about 25% higher than in the light, while consumption of nitrate or nitrite in the dark was lower than in the light.
N starvation produced a linear increase with time in the intracellular level of glutamine synthetase (GS, EC 6.3.2.1) and glutamate synthase (NADH-GOGAT, EC 1.4.1.14 and ferredoxin-GOGAT, EC 1.4.7.1) activities in C. reinhardtii . The effect on GS₁ (3-fold) and NADH-GOGAT (4.5-fold) was higher than that on GS₂ (1.5-fold) and ferredoxin-GOGAT (1.5-fold).
Experiments with methylammonium, L-methionine-D, L-sulfoximine (MSX) and azaserine suggest that: 1) Ammonium itself decreases the intracellular levels of glutamine synthetase and ferredoxin-glutamate synthase activities; and 2) a metabolite resulting from ammonium assimilation by the alga may be a negative modulator of NADH-glutamate synthase activity.     

The effect of nitrogen refeeding on the carbohydrate content into nitrogen-starved cells of Platymonas striata Butcher

Studies on the mean cellular carbohydrate contents of Platymonas striata Butcher under conditions of nitrogen-starvation, and after refeeding these starved cultures with either nitrate or ammonium ions (growing under continuous illumination or with an alternating light/dark regime) have shown that nitrogen-starved cells accumulated abnormal amounts of cellular carbohydrate and that nitrogen refeeding produced a marked drop in the cellular carbohydrate. Cells grown in a light/dark regime accumulated less carbohydrates than those grown in continuous light. The mean cellular carbohydrate levels 16 h after nitrogen refeeding were still much in excess of those of cells grown with normal nutrition. It was therefore suggested that the differences in nitrogen uptakes in this period — when comparing either the uptake of cells grown in continuous light with that of cells grown in a light/dark regime; or when comparing the uptakes of cells presented with either nitrate or ammonium ions and grown in a light/dark regime —cannot be directly due to shortages of carbohydrate for the provision of carbon skeletons for nitrogen assimilation.     

Identification and characterization of small RNAs from the phloem of Brassica napus

Systemic signalling is indispensable for the coordination of diverse physiological processes during development, defence and nutrient allocation. Indirect evidence suggests that plant small RNAs (smRNAs) could be involved in long-distance information transfer via the vasculature of the plant. Analyses of the smRNA complements of vascular exudates from oilseed rape ( Brassica napus ) showed that xylem sap is devoid of RNA, whereas phloem sap contained a large number of smRNAs. In addition to 32 annotated microRNAs (miRNAs) from 18 different families that could be identified and approved, a set of unknown smRNAs, predominantly of 21 and 24 nucleotides in length, was obtained, and selected candidates were found to be highly abundant in phloem sap. Moreover, we could demonstrate that the levels of three miRNAs known to respond to nutrient deprivation in non-vascular tissue, miR395 (sulphate), miR398 (copper) and miR399 (phosphate), were increased in phloem sap during the growth of plants under the respective starvation conditions. Interestingly, only mature miRNA molecules were found to be stress responsive, demonstrating that single-stranded sense miRNAs are most likely to represent the physiologically relevant molecules. The strong responses in the phloem suggest a role of miRNAs in systemic information transfer via this long-distance transport system.     

Effect of L-methionine-DL-sulphoximine, a specific inhibitor of glutamine synthetase, on ammonium and nitrate metabolism in the unicellular alga Cyanidium caldarium

In the unicellular alga Cyanidium caldarium nitrate utilization is strongly inhibited by ammonium and it is resumed when ammonium has been depleted. In the presence of L-methionine-DL-sulphoximine (MSX), which prevents ammonium assimilation through a specific irreversible inhibition of glutamine synthetase, nitrate reduction is no longer inhibited by ammonium, and most of the ammonium derived from nitrate reduction is excreted into the external medium. However, in the presence of MSX, nitrate reduction to ammonium proceeds at a reduced rate (45 to 70% of the control); this is particularly marked at low nitrate concentration. It is hypothesized that either MSX or accumulating ammonium bring about decrease in the rate of nitrate entry into the cell.     

Detection of mRNAs correlated with proliferation of cells in suspension cultures of Catharanthus roseus

Gene expression correlated with proliferation was investigated in Catharanthus roseus (L.) G. Don. cells. Polyadenylated RNAs were isolated from cells in proliferative states or in nonproliferative states and the variations in the population and levels of mRNA were analyzed by in vitro translation followed by separation of the corresponding polypeptides by two-dimensional gel electrophoresis. Levels of two mRNAs increased in the actively cycling cells, while they were hardly detected in cells in stationary phase or in cells arrested by starvation of phosphate, sucrose or nitrogen. The molecular masses of the translated products of these two mRNAs were 53 and 60 kDa. No mRNAs were specifically synthesized in common among the various cultures of cells whose growth was arrested by nutrient starvation.     

草地贪夜蛾幼虫耐饥力及饥饿对其生长发育和繁殖的影响

本文旨在明确草地贪夜蛾Spodoptera frugiperda(J.E.Smith)幼虫耐饥力及饥饿处理对其生长发育、繁殖力的影响.选取初孵幼虫(幼虫孵化1 h内)、2、4、6、8和10日龄的幼虫进行饥饿处理,测定存活率和存活时间分析其耐饥力;进一步选取8日龄幼虫分别饥饿1、2、3和4d后再复食,分别统计和分析饥饿胁...     

The influence of starvation on mortality,development, and protein content in parasitized and unparasitized Tribolium castaneum

The effects of starvation on mortality, development, and protein content in Nosema whitei-infected and uninfected Tribolium castaneum were investigated. T. castaneum larvae, starved for 4, 6, 8, and 10 days postinfection, showed an increase in larval mortality. Pupal mortality also increased, producing a decrease in adult emergence. Starvation of larvae for 6 days or more delayed development and the average time to adult emergence increased. These effects tended to be more marked in Nosema-infected larvae. No consistent pattern of weight changes was observed in either the uninfected or infected pupae and adults. Infected T. castaneum larvae showed a significant decrease in protein compared to controls. Starvation apparently does not aggravate this condition nor does it have any significant effects on the total hemolymph protein content of uninfected and infected larvae.     

Differential effects of nitrogen and sulfur deprivation on growth and biodiesel feedstock production of Chlamydomonas reinhardtii

Biodiesel production from microalgae is a promising approach for energy production; however, high cost of its process limits the use of microalgal biodiesel. Increasing the levels of triacylglycerol (TAG) levels, which is used as a biodiesel feedstock, in microalgae has been achieved mainly by nitrogen starvation. In this study, we compared effects of sulfur (S) and nitrogen (N) starvation on TAG accumulation and related parameters in wild-type Chlamydomonas reinhardtii CC-124 mt(-) and CC-125 mt(+) strains. Cell division was interrupted, protein and chlorophyll levels rapidly declined while cell volume, total neutral lipid, carotenoid, and carbohydrate content increased in response to nutrient starvation. Cytosolic lipid droplets in microalgae under nutrient starvation were monitored by three-dimensional confocal laser imaging of live cells. Infrared spectroscopy results showed that relative TAG, oligosaccharide and polysaccharide levels increased rapidly in response to nutrient starvation, especially S starvation. Both strains exhibited similar levels of regulation responses under mineral deficiency, however, the degree of their responses were significantly different, which emphasizes the importance of mating type on the physiological response of algae. Neutral lipid, TAG, and carbohydrate levels reached their peak values following 4 days of N or S starvation. Therefore, 4 days of N or S starvation provides an excellent way of increasing TAG content. Although increase in these parameters was followed by a subsequent decline in N-starved strains after 4 days, this decline was not observed in S-starved ones, which shows that S starvation is a better way of increasing TAG production of C. reinhardtii than N starvation.     

一个血清抑制基因的克隆

比较血清培养细胞和血清饥饿细胞的基因表达差异,获得了一段血清饥饿细胞中特异表达的cDNA序列,以此序列出发,通过搜索表达序列标签(EST),拼接出完整的基因序列,通过PCR分段克隆获得全长cDNA序列.该基因全长5 429 bp,编码框预测有791个氨基酸残基.GenBank搜索,该基因与已有的细胞周期调控基因没有同源性.所以,该基因是一个新的与细胞周期有关的基因（GenBank接受号：AY050169）.由于该基因最初发现在无血清培养条件下表达,故叫血清抑制基因（serum inhibit gene,Si-1基因）.     

Possible role of adenylates in the engagement of the cyanideresistant pathway in nutrient-starved Catharanthus roseus Cells

In Cathuranthus roseus (L.) G. Don cells the cyanide-resistant pathway is engaged after phosphate or nitrogen starvation. Re-addition of these nutrients disengaged it again. Re-addition of phosphate leads to a transient disengagement which becomes only permanent after a second addition of phosphate. Disengagement after re-addition of nitrogen is slow: it takes 9 days before the activity has disappeared. In this system the mechanism of engagement of the cyanide-resistant pathway was studied. Addition of phosphate to phosphate-starved cells induced cell division within 24 h. The disengagement of the cyanide-resistant pathway was probably only an indirect effect of phosphate because the cellular P, content, which increased rapidly after addition, was low again before the cyanide-resistant pathway was disengaged. A better correlation was observed between high ADP and adenylate content of the cells and disengagement of the cyanide-resistant pathway. In addition it appeared that the engagement of the cyanide-resistant pathway was not the result of a limited carrier capacity of the cytochrome pathway. It is tentatively concluded that the engagement of the cyanide-resistant pathway in phosphate-starved cells was the result of a limited adenylate content. After nitrogen addition to N-starved cells, it took 5 days until the first growth occurred. Before the cyanide-resistant pathway was disengaged, its activity increased with the increased respiration rate which preceded growth. Within 72 h a higher ADP content was observed, which was still high after 10 days. The stimulation of the cytochrome pathway by uncoupler was small and more or less the same with and without added nitrogen, as long as the cyanide-resistant pathway was engaged. After disengagement the stimulation by uncoupler was significantly larger. It is suggested that the engagement during N-starvation was the result of a limited carrier capacity of the cytochrome pathway. Stimulation of the metabolism by re-addition of phosphate, nitrogen or sucrose resulted in a rapid increase in the levels of uracil nucleotides and uridine diphosphoglucose (UDPG) which are involved in sucrose metabolism.     

Amylase and trypsin activities during Artemia development on artificial axenic media; effect of starvation and specific deletions

The amylase and trypsin activities of Artemia reared on synthetic axenic media were studied during development and compared with earlier results obtained with phytoplankton-fed Artemia. The enzymatic activity responses are also analysed with regard to the Provasoli growth index, protein growth and survival during starvation experiments or during ones involving specific deletions (starch or albumin). These experiments demonstrated a repressive regulatory mechanism for amylase and trypsin under substrate-saturating conditions. Differences in response of amylase and trypsin to starch and albumin deletions are discussed in terms of the existence of internal carbohydrate and protein pools in crustaceans. The ability of Artemia to shift from a high metabolism with exponential growth to a low metabolism without any growth but permitting survival is observed. The related enzyme responses are discussed. The importance of interactions between nutritional conditions, metabolic requirements, and the regulation of digestive enzymes is stressed. The response times and implications for the field studies on Zooplankton ecology are discussed.     

Plant cells selected for resistance to phosphate starvation show enhanced P use efficiency

Summary In many organisms, phosphate starvation induces multigene systems that act to increase the availability and uptake of exogenous phosphates. Tissue-cultured tomato cells were plated onto solid media containing starvation levels of phosphate. While most cells died, we identified isolated clumps of callus capable of near-normal rates of growth. Starvation-resistant cells were used to start suspension cultures that were kept under phosphate starvation conditions. A selected cell line showed constitutively enhanced secretion of acid phosphatase and greatly increased rates of phosphate uptake. These pleiotropic effects suggest modification of a regulatory apparatus that controls coordinated changes in the expression of a multigene system. The somaclonal variant cell line grew normally under phosphate-sufficient conditions, but did significantly better than unselected cells under phosphate-limited conditions. In vitro selection may be a useful system for developing phosphate ultraefficient crop plants.     

Disruption of the phosphate-starvation response of oilseed rape suspension cells by the fungicide phosphonate

The influence of the anti-fungal agent phosphonate (Phi) on the response of oilseed rape (Brassica napus L. cv. Jet Neuf ) cell suspensions to inorganic phosphate (Pi) starvation was examined. Subculture of the cells for 7 d in the absence of Pi increased acid phosphatase (APase; EC 3.1.3.2) and pyrophosphate (PPi)-dependent phosphofructokinase (PFP; EC 2.7.1.90) activities by 4.5- and 2.8-fold, respectively, and led to a 19-fold increase in V _max and a 14-fold decrease in K _m (Pi) for Pi uptake. Addition of 2 mM Phi to the nutrient media caused dramatic reductions in the growth and Pi content of the Pi-starved, but not Pi-sufficient cells, and largely abolished the Pi-starvation-dependent induction of PFP, APase, and the high-affinity plasmalemma Pi translocator. Immunoblotting indicated the cells contain three APase isoforms that are synthesized de novo following Pi stress, and that Phi treatment represses this process. Phosphonate treatment of Pi-starved cells significantly altered the relative extent of in-vivo ³²P-labelling of polypeptides having M_rs of 66, 55, 45 and 40 kDa. However, Phi had no effect on the total adenylate pool of Pi-starved cells which was about 32% lower than that of Pi-sufficient cells by day 7. Soluble protein levels, and activities of pyruvate kinase (EC 2.7.1.40) and ATP-dependent phosphofructokinase (EC 2.7.1.11) were unaffected by Pi starvation and/or Phi treatment. The effects of Phi on the growth, and APase and PFP activities of Pi-starved B. napus seedlings were similar to those observed in the suspension cells. The results are consistent with the hypothesis that a primary site of Phi action in higher plants is at the level of the signal transduction chain by which plants perceive and respond to Pi stress at the molecular level. Received: 30 December 1996 / Accepted: 19 February 1997