Induction of Arabidopsis tryptophan pathway enzymes and camalexin by amino acid starvation, oxidative stress, and an abiotic elicitor.

The tryptophan (Trp) biosynthetic pathway leads to the production of many secondary metabolites with diverse functions, and its regulation is predicted to respond to the needs for both protein synthesis and secondary metabolism. We have tested the response of the Trp pathway enzymes and three other amino acid biosynthetic enzymes to starvation for aromatic amino acids, branched-chain amino acids, or methionine. The Trp pathway enzymes and cytosolic glutamine synthetase were induced under all of the amino acid starvation test conditions, whereas methionine synthase and acetolactate synthase were not. The mRNAs for two stress-inducible enzymes unrelated to amino acid biosynthesis and accumulation of the indolic phytoalexin camalexin were also induced by amino acid starvation. These results suggest that regulation of the Trp pathway enzymes under amino acid deprivation conditions is largely a stress response to allow for increased biosynthesis of secondary metabolites. Consistent with this hypothesis, treatments with the oxidative stress-inducing herbicide acifluorfen and the abiotic elicitor alpha-amino butyric acid induced responses similar to those induced by the amino acid starvation treatments. The role of salicylic acid in herbicide-mediated Trp and camalexin induction was investigated.     

The effect of amino acid starvation on nucleoside uptake and RNA synthesis in Tetrahymena

The uptake of nucleosides and the synthesis of RNA in Tetrahymena thermophila were examined following amino acid starvation. Omission of leucine, phenylalanine, or arginine from the medium resulted in a rapid decrease in the incorporation of [3H]uridine into the acid-soluble pool and acid-insoluble material (RNA). Amino acid starvation inhibited the uptake of all ribo- and deoxyribonucleosides tested but did not affect the uptake of amino acids or glucose. In addition, under the conditions used, the omission of an amino acid did not result in a large decrease in amino acid incorporation into total protein. Treatment of cells with cycloheximide or emetine gave results similar to the effects of amino acid starvation, but in these experiments the inhibition of protein synthesis was essentially complete. Nucleotide pool sizes were also measured following amino acid starvation. ATP and UTP levels were essentially unchanged, but the dTTP pool size was decreased by 40%. The decrease in RNA synthesis in vivo in the absence of an essential amino acid was reflected in the endogenous RNA synthetic activity of isolated nuclei. However, when solubilized RNA polymerase activity was measured with calf thymus DNA as template, no significant difference was observed between control and amino acid-starved cells.     

Decreased purine synthesis during amino acid starvation of human lymphoblasts

Normal human lymphoblasts starved for each of several essential, but not essential, amino acids had decreased DNA and RNA synthesis but no change in free intracellular purine nucleotides. The rates of purine nucleotide synthesis via the de novo and salvage pathways were measured by incorporating [14C]formate and [14C]hypoxanthine labels, respectively, into lymphoblasts starved for an amino acid or treated with a protein synthesis inhibitor. After 3 h of starvation, purine synthesis via the de novo pathway decreased 90% and via the salvage pathway decreased 60%. Cycloheximide and puromycin each reduced de novo synthesis by 96% and salvage synthesis by 72%. The decrease in purine synthesis de novo after removal of the amino acid was of first order kinetics and was fully and rapidly reversed by reconstitution with the amino acid. The synthesis of alpha-N-formylglycinamide ribonucleotide declined 97% after amino acid starvation; the synthesis of purines from 5-aminoimidazole-4-carboxamide riboside decreased 41%. The synthesis of guanylates decreased more than the synthesis of adenylates during amino acid starvation.     

Delayed-relaxed response explained by hyperactivation of RelE

Escherichia coli encodes two rel loci, both of which contribute to the control of synthesis of macromolecules during amino acid starvation. The product of relA (ppGpp synthetase I) is responsible for the synthesis of guanosine tetraphosphate, ppGpp, the signal molecule that exerts stringent control of stable RNA synthesis. The second rel locus, relBE, was identified by mutations in relB that confer a so-called 'delayed-relaxed response' characterized by continued RNA synthesis after a lag period of approximately 10 min after the onset of amino acid starvation. We show here that the delayed-relaxed response is a consequence of hyperactivation of RelE. As in wild-type cells, [ppGpp] increased sharply in relB101 relE cells after the onset of starvation, but returned rapidly to the prestarvation level. RelE is a global inhibitor of translation that is neutralized by RelB by direct protein-protein interaction. Lon protease activates RelE during amino acid starvation by degradation of RelB. We found that mutations in relB that conferred the delayed-relaxed phenotype destabilized RelB. Such mutations confer severe RelE-dependent inhibition of translation during amino acid starvation, indicating hyperactivation of RelE. Hyperactivation of RelE during amino acid starvation was shown directly by measurement of RelE-mediated cleavage of tmRNA. The RelE-mediated shutdown of translation terminated amino acid consumption and explains the rapid restoration of the ppGpp level observed in relB mutant cells. Restoration of the prestarvation level of ppGpp, in turn, allows for the resumption of stable RNA synthesis seen during the delayed-relaxed response.     

The suppression of defective translation by ppGpp and its role in the stringent response.

Amino acid starvation is shown to decrease the fidelity of translation in E. coli. When proteins are analyzed by two-dimensional gel electrophoresis, missense errors are detected as an unusual heterogeneity in their isoelectric points, while premature termination of protein synthesis can be recognized by a decreased relative rate of synthesis of higher molecular weight proteins and by the the accumulation of a complex group of new small polypeptides. The types of translational errors observed are amino acid-specific. For example, starvation of a rel- strain for histidine produces severe isoelectric point heterogeneity with little evidence of premature termination, while starvation for leucine has little effect on the isoelectric points, but produces a drastic decrease in the average molecular weight of the newly synthesized protein. These differences suggest codon-specific errors in reading the genetic code. In these rel- cells, the effect of amino acid starvation on the rates of synthesis of complete individual proteins is both protein- and amino acid-specific. For example, ribosomal protein L7/12, which lacks histidine, is made at a higher level during histidine starvation than during isoleucine or leucine starvation. This suggests that in rel- cells, the modulation of gene expression caused by the lack of a particular amino acid is, at least in part, a function of the abundance of that amino acid in particular proteins-that is, the response of rel- cells to starvation is consistent with the theory that the inhibition of protein synthesis and the accompanying increase in error frequency both result from low levels of the correct substrate. In marked contrast, virtually no starvation-induced translational errors are detected in a rel+ strain, and the response is not amino acid-specific. Varoius data strongly imply that in this rel+ strain, essentially all the changes caused by starvation are due to the accumulation of ppGpp, which independently reduces protein synthesis, thereby suppressing all the direct effects of amino acid limitation seen in rel- strains (where ppGpp does not accumulate upon starvation). A model is presented which describes how ppGpp might suppress the direct effects of starvation and avoid the loss of translational fidelity. In addition, the direct and specific effects of ppGpp on gene expression are examined independently of amino acid starvation.     

Nitrogen and carbon costs of soybean and lupin root systems during phosphate starvation

Phosphate (P) starvation is one of the most limiting nutrients to N₂ fixation in legumes. Soybeans and lupins present different climatic origins, nodule morphologies and metabolic complexities, which may have various adaptive responses to short-term P starvation. Lupins and soybeans were cultivated hydroponically for 3 weeks. Short-term P starvation was induced for 14 days by switching the P-supply to 2 μM P. During P starvation, the lupins showed a lower decline in nodular P concentrations and maintained their biological N₂ fixation (BNF), in contrast to the soybeans. The lupins also maintained their photosynthetic rates and the nodular construction and growth respiration costs under P starvation, whilst soybeans showed a decrease in photosynthetic rates and an increase in nodular construction and growth respiration costs under P starvation. There was a also a shift towards more organic acid synthesis, relative to amino acid synthesis in lupin nodules than soybean nodules under P starvation. The lupins had higher amino acid concentrations in their nodules, whilst the soybean nodules maintained their ureide levels at the expense of a decline in amino acids. These results indicate that lupins may to be better adapted to maintaining BNF during short-term P starvation than the soybeans.     

Autophagy is required for maintenance of amino acid levels and protein synthesis under nitrogen starvation

Autophagy is a transport system of cytoplasmic components to the lysosome/vacuole for degradation well conserved in eukaryotes. Autophagy is strongly induced by nutrient starvation. Several specific proteins, including amino acid synthesis enzymes and vacuolar enzymes, are increased during nitrogen starvation in wild-type cells but not in autophagy-defective delta atg7 cells despite similar mRNA levels. We further examined deficiencies in these cells. Bulk protein synthesis was substantially reduced in delta atg7 cells under nitrogen starvation compared with wild-type cells. The total intracellular amino acid pool was reduced in delta atg7 cells, and the levels of several amino acids fell below critical values. In contrast, wild-type cells maintained amino acid levels compatible with life. Autophagy-defective cells fail to maintain physiologic amino acid levels, and their inability to synthesize new proteins may explain most phenotypes associated with autophagy mutants at least partly.     

Amino acid starvation in Escherichia coli K-12: characteristics of the translation process.

Some characteristics of the translation process during amino acid starvation in Escherichia coli have been examined. Once starvation has been established, premature termination of polypeptides is negligible and complete proteins are formed. There is some preference for the synthesis of shorter proteins. The number of ribosomes involved in protein synthesis appears to decline to about half during amino acid-starvation. The assembly time of proteins during amino acid starvation is increased to only about fourfold, though protein synthesis maintained by turnover is reduced to 10%. To explain these observations, a model has been proposed for the course of events that possibly take place from the onset of starvation.     

Regulation of Chromosome Replication in Bacillus subtilis: Effects of Amino Acid Starvation in Strain W23

Amino acid starvation allows limited synthesis of deoxyribonucleic acid (DNA) in Bacillus subtilis strain W23. DNA synthesis increased by about 30% after leucine starvation and by about 60% after histidine starvation. Genetic analysis on the DNA synthesized after amino acid starvation showed that all genetic markers examined have replicated, regardless of which amino acid was starved for. Initially, all markers replicated equally, but upon further replication, the thr cysB and the argA to lys regions replicated ahead of their neighboring, proximal regions. This could indicate that preferred stopping sites exist in these regions or additional sites from which replication can originate reside there. The results suggest that chromosome replication continues from those sites where it had stopped during amino acid starvation.     

Processing of precursor ribosomal RNA and the presence of a modified ribosome assembly scheme in Escherichia coli relaxed strain

An electrophoretic system capable of separating 25 S, 23 S, 17.5 S and 16 S ribosomal RNA (rRNA) species was used to study the synthesis and fate of rRNA during amino acid starvation and resupplementation of E. coli relaxed strain KL99. This E. coli relAl strain responded to an amino acid starvation by increasing the rate of synthesis of 25 S and 17.5 S precursor rRNA. When the limiting amino acid was resupplemented, a previously observed 40-fold increase in the cellular guanosine 5'-diphosphate, 3'-diphosphate content [Mol. Gen. Genet. (1983) 192, 5-9] appeared to cause a reduction in new rRNA synthesis. Following amino acid resupplementation, the precursor 25 S and 17.5 S rRNA accumulated during the amino acid starvation were conserved and processed to 23 S and 16 S rRNA species, respectively. This suggests that a modified ribosome assembly scheme involving stable precursor rRNA exists in relAl bacteria during periods of amino acid limitation and resupplementation.     

Starvation and the metabolism of hepatocytes isolated from the American eel,Anguilla rostrata LeSeur

Summary Gluconeogenic, lipogenic, glycogenic and oxidative rates were estimated from¹⁴C-lactate,¹⁴C-alanine and¹⁴C-aspartate using a hepatocyte preparation isolated from starved immature American eels,Anguilla rostrata. Lactate gluconeogenesis increased significantly during starvation at 5 and 15°C. Alanine gluconeogenesis generally decreased during starvation. At the 2nd month of the starvation at 5 and 15°C, and the 8th month of starvation at 15°C, however, alanine gluconeogenesis was significantly higher than in the fed control. These increases in alanine gluconeogenesis occurred during a period of high glucose demand. Aspartate gluconeogenesis was quantitatively minor when compared to the other two substrates. Glycerol synthesis and esterification from the three substrates increased until the 5th month at 5 and 15°C followed by a gradual decline thereafter. Significant increases in glycogen synthesis occurred between the 3rd and the 5th months at 15°C, but rates were small compared to glucose synthesis. Rates of substrate oxidation appeared sufficient to provide adequate ATP to sustain gluconeogenesis in both the fed and starved eel hepatocyte. Glucagon stimulated lactate gluconeogenesis, but not amino acid gluconeogenesis in late starved eel hepatyocytes. Major changes in metabolite concentrations that occurred during starvation were increases in plasma glucose and amino acids; a significant liver glycogen depletion at the 2nd month followed by a return to control values at the third month; and, a significant protein depletion in white skeletal muscle at the 3rd month. These data suggest that lactate glucogeogenesis, but not amino acid gluconeogenesis or glycogenolysis, is the major source of tissue carbohydrates during eel starvation.This work was supported from operating grants to TWM from the National Research Council of Canada (A6944)     

Responses to multiple-nutrient starvation in marine Vibrio sp. strain CCUG 15956.

The response of marine Vibrio sp. strain S14 (CCUG 15956) to long-term (48-h) multiple-nutrient starvation (i.e., starvation for glucose, amino acids, ammonium, and phosphate simultaneously) can be described as a three-phase process. The first phase, defined as the stringent control phase, encompasses an accumulation of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and decreases in RNA and protein synthesis during the first 40 min. In the second phase, there is a temporary increase in the rates of RNA and protein synthesis between 1 and 3 h paralleling a decrease in the ppGpp pool. The third phase includes gradual decline in macromolecular synthesis after 3 h. Using two-dimensional gel electrophoresis of pulse-labeled proteins, a total of 66 proteins were identified as starvation inducible (Sti), temporally expressed throughout the three phases of starvation. The inhibition of protein synthesis during the first phase of starvation partly disrupted the subsequent temporally ordered synthesis of starvation proteins and prevented the expression of some late starvation proteins. It was also found that the early temporal class of starvation proteins, which included the majority of the Sti proteins, was the most essential for long-term survival. Vibrio sp. strain S14 cultures prestarved (1 h) for glucose, amino acids, ammonium, or phosphate as well as cultures exposed (1 h) to CdCl2 exhibited enhanced survival during the subsequent multiple-nutrient starvation in the presence of chloramphenicol or rifampin, while heat or the addition of cyclic AMP or nalidixic acid prior to starvation had no effect. It was demonstrated that amino acid starvation and CdCl2 exposure, which induced the stringent response, were the most effective in conferring enhanced survival. A few Sti proteins were common to all starvation conditions. In addition, the total number of proteins induced by multiple-nutrient starvation significantly exceeded the sum of those induced by starvation for each of the individual nutrients.     

Computer simulation of ribosome editing

A stochastic model of protein synthesis was modified by including the process of dissociating peptidyl-tRNA from ribosomes. To simulate ribosome editing, the probability of dissociation was assumed to be high if the peptidyl-tRNA was erroneous; that is, if it resulted from transfer of a peptide to an aminoacyl-tRNA that was inappropriate relative to the mRNA codon. The effects of amino acid starvation on protein synthesis were simulated both by increasing the probability of such erring at and by reducing the conditional probability of elongation at “hungry” codons, those whose correct amino acid was in short supply. These probabilities were varied systematically to simulate tryptophan limitation during synthesis of coat protein from bacteriophage MS2.Significant reduction, during starvation, in the synthesis of complete coat protein required large reductions in the probability of elongation at hungry codons but only small increases in the probability of erring. Enhanced dissociation of peptidyl-tRNA during starvation, followed rapidly by dissociation of ribosomes from mRNA, led to reductions in mean polysome size, a result that had been interpreted by others as due to some effect of starvation on the initiation of protein synthesis.Results from experiments by Goldman (1982) on the cell-free synthesis of MS2 coat protein during tryptophan starvation could be mimicked in detail by the computer simulations. A simple competition between correct and erroneous amino acids was sufficient to explain the tryptophan dependence of complete coat protein and internal peptide syntheses. Values for the Michaelis constants were derived from the computer simulations.     

Changes in cell dimensions during amino acid starvation of Escherichia coli.

Electron microscopic analysis was used to study cells of Escherichia coli B and K-12 during and after amino acid starvation. The results confirmed our previous conclusion that cell division and initiation of DNA replication occur at a smaller cell volume after amino acid starvation. Although during short starvation periods, the number of constricting cells decreased due to residual division, it appears that during prolonged starvation, cells of E. coli B and K-12 were capable of initiating new constrictions. During amino acid starvation, cell diameter decreased significantly. The decrease was reversed only after two generation times after the resumption of protein synthesis and was larger in magnitude than that previously observed before division (F. J. Trueba and C. L. Woldringh, J. Bacteriol. 142:869-878, 1980). This decrease in cell diameter correlates with synchronization of cell division which has been shown to occur after amino acid starvation.     

Stringent control during carbon starvation of marine Vibrio sp. strain S14: molecular cloning, nucleotide sequence, and deletion of the relA gene.

In order to evaluate the role of the stringent response in starvation adaptations of the marine Vibrio sp. strain S14, we have cloned the relA gene and generated relaxed mutants of this organism. The Vibrio relA gene was selected from a chromosomal DNA library by complementation of an Escherichia coli delta relA strain. The nucleotide sequence contains a 743-codon open reading frame that encodes a polypeptide that is identical in length and highly homologous to the E. coli RelA protein. The amino acid sequences are 64% identical, and they share some completely conserved regions. A delta relA::kan allele was generated by replacing 53% of the open reading frame with a kanamycin resistance gene. The Vibrio relA mutants displayed a relaxed control of RNA synthesis and failed to accumulate ppGpp during amino acid limitation. During carbon and energy starvation, a relA-dependent burst of ppGpp synthesis concomitant with carbon source depletion and growth arrest was observed. Also, in the absence of the relA gene, there was an accumulation of ppGpp during carbon starvation, but this was slower and smaller than that which occurred in the stringent strains, and it was preceded by a marked decrease in the [ATP]/[ADP] ratio. In both the wild-type and the relaxed strains, carbon source depletion caused an immediate decrease in the size of the GTP pool and a block of net RNA accumulation. The relA mutation did not affect long-term survival or the development of resistance against heat, ethanol, and oxidative stress during carbon starvation of Vibrio sp. strain S14.     

Membrane-bound deoxyribonucleic acid from Escherichia coli: effects of replication, protein synthesis, and ribonucleic acid synthesis.

The experiments presented in this paper suggest that the shift observed in sedimentation of deoxyribonucleic acid from cells of Escherichia coli subjected to amino acid starvation is related to inhibition of ribonucleic acid synthesis rather than to its release from the membrane at the termination of replication.