Citrulline and DRIP-1 Protein (ArgE Homologue) in Drought Tolerance of Wild Watermelon

Drought-affected plants experience more than just desiccationof their organs due to water deficit. Plants transpire 1000times more molecules of water than of CO₂ fixed by photosynthesisin full sunlight. One effect of transpiration is to cool theleaves. Accordingly, drought brings about such multi-stressesas high temperatures, excess photoradiation and other factorsthat affect plant viability. Wild watermelon serves as a suitablemodel system to study drought responses of C₃ plants, sincethis plant survives drought by maintaining its water contentwithout any wilting of leaves or desiccation even under severedrought conditions. Under drought conditions in the presenceof strong light, wild watermelon accumulates high concentrationsof citrulline, glutamate and arginine in its leaves. The accumulationof citrulline and arginine may be related to the induction ofDRIP-1, a homologue of ArgE in Escherichia coli, where it functionsto incorporate the carbon skeleton of glutamate into the ureacycle. Immunogold electron microscopy reveals the enzyme tobe confined exclusively to the cytosol. DRIP-1 is also inducedby treating wild watermelon with 150 mM NaCl, but is not inducedfollowing treatment with 100 µM abscisic acid. The salttreatment causes the accumulation of -aminobutyrate, glutamineand alanine, in addition to a smaller amount of citrulline.Citrulline may function as a potent hydroxyl radical scavenger.     

Responses of wild watermelon to drought stress: accumulation of an ArgE homologue and citrulline in leaves during water deficits

Wild watermelon from the Botswana desert had an ability to survive under severe drought conditions by maintaining its water status (water content and water potential). In the analysis by two-dimensional electrophoresis of leaf proteins, seven spots were newly induced after watering stopped. One with the molecular mass of 40 kilodaltons of the spots was accumulated abundantly. The cDNA encoding for the protein was cloned based on its amino-terminal sequence and the amino acid sequence deduced from the determined nucleotide sequences of the cDNA exhibited homology to the enzymes belong to the ArgE/DapE/Acy1/Cpg2/YscS protein family (including acetylornithine deacetylase, carboxypeptidase and aminoacylase-1). This suggests that the protein is involved in the release of free amino acid by hydrolyzing a peptidic bond. As the drought stress progressed, citrulline became one of the major components in the total free amino acids. Eight days after withholding watering, although the lower leaves wilted significantly, the upper leaves still maintained their water status and the content of citrulline reached about 50% in the total free amino acids. The accumulation of citrulline during the drought stress in wild watermelon is an unique phenomenon in C3-plants. These results suggest that the drought tolerance of wild watermelon is related to (1) the maintenance of the water status and (2) a metabolic change to accumulate citrulline.     

Citrulline, a novel compatible solute in drought-tolerant wild watermelon leaves, is an efficient hydroxyl radical scavenger.

Drought-tolerant wild watermelon accumulates high levels of citrulline in the leaves in response to drought conditions. In this work, the hydroxyl radical-scavenging activity of citrulline was investigated in vitro. The second-order rate constant for the reaction between citrulline and hydroxyl radicals was found to be 3.9x10(9) M(-1) s(-1), demonstrating that citrulline is one of the most efficient scavengers among compatible solutes examined so far. Moreover, citrulline effectively protected DNA and an enzyme from oxidative injuries. Liquid chromatography-mass spectrometry analysis revealed that at least four major products were formed by the reaction between citrulline and hydroxyl radicals. Activities of metabolic enzymes were not inhibited by up to 600 mM citrulline, indicating that citrulline does not interfere with cellular metabolism. We reasoned, from these results, that citrulline contributes to oxidative stress tolerance under drought conditions as a novel hydroxyl radical scavenger.     

The effects of drought stress on free amino acid accumulation and protein synthesis in Brassica napus

Changes in the concentrations of free amino acids and specific organic acids were analysed during the induction of drought stress in Brassica napus . Most of the amino acids showed a characteristic linear increase with the induction of drought stress in Brassica leaves, increasing an average of 5.9-fold over control levels, followed by a reduction in concentration upon rehydration of the plants. Pyruvate concentrations doubled after 4 days of drought stress whereas 2-oxoglutarate concentrations remained relatively constant. The activities of two of the enzymes involved in amino acid biosynthesis, alanine aminotransferase (EC 2.6.1.2) and aspartate aminotransferase (EC 2.6.1.1), were also measured. Neither enzyme showed any increase in activity, except when the plants were rehydrated. This suggests that the increase in both alanine and aspartate levels results from the increase in their precursors pyruvate and glutamate and may not require increased enzyme activity. The effect of drought stress upon changes in protein synthesis was analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. We found that there was an overall decrease in protein synthesis with the induction of drought stress, followed by a resumption of synthesis upon rehydration. In addition, the synthesis of a number of specific polypeptides was found to decrease upon water loss in the leaves.     

Abscisic acid is involved in phenolic compounds biosynthesis,mainly anthocyanins,in leaves of Aristotelia chilensis plants (Mol.) subjected to drought stress

Abscisic acid (ABA) regulates the physiological and biochemical mechanisms required to tolerate drought stress, which is considered as an important abiotic stress. It has been postulated that ABA might be involved in regulation of plant phenolic compounds biosynthesis, especially anthocyanins that accumulate in plants subjected to drought stress; however, the evidence for this postulate remains elusive. Therefore, we studied whether ABA is involved in phenolic compounds accumulation, especially anthocyanin biosynthesis, using drought stressed Aristotelia chilensis plants, an endemic berry in Chile. Our approach was to use fluridone, an ABA biosynthesis inhibitor, and then subsequent ABA applications to young and fully‐expanded leaves of drought stressed A. chilensis plants during 24, 48 and 72 h of the experiment. Plants were harvested and leaves were collected separately to determine the biochemical status. We observed that fluridone treatments significantly decreased ABA concentrations and total anthocyanin (TA) concentrations in stressed plants, including both young and fully‐expanded leaves. TA concentrations following fluridone treatment were reduced around fivefold, reaching control plant levels. ABA application restored ABA levels as well as TA concentrations in stressed plant at 48 h of the experiment. We also observed that TA concentrations followed the same pattern as ABA concentrations in the ABA treated plants. Quantitative real‐time PCR revealed that AcUFGT gene expression decreased in fully‐expanded leaves of stressed plants treated with fluridone, while a subsequent ABA application increased AcUFGT expression. Taken together, our results suggest that ABA is involved in the regulation of anthocyanin biosynthesis under drought stress.     

Amino acid precursor supply in the biosynthesis of the RNA polymerase inhibitor streptolydigin by Streptomyces lydicus

Biosynthesis of the hybrid polyketide-nonribosomal peptide antibiotic streptolydigin, 3-methylaspartate, is utilized as precursor of the tetramic acid moiety. The three genes from the Streptomyces lydicus streptolydigin gene cluster slgE1-slgE2-slgE3 are involved in 3-methylaspartate supply. SlgE3, a ferredoxin-dependent glutamate synthase, is responsible for the biosynthesis of glutamate from glutamine and 2-oxoglutarate. In addition to slgE3, housekeeping NADPH- and ferredoxin-dependent glutamate synthase genes have been identified in S. lydicus. The expression of slgE3 is increased up to 9-fold at the onset of streptolydigin biosynthesis and later decreases to ～2-fold over the basal level. In contrast, the expression of housekeeping glutamate synthases decreases when streptolydigin begins to be synthesized. SlgE1 and SlgE2 are the two subunits of a glutamate mutase that would convert glutamate into 3-methylaspartate. Deletion of slgE1-slgE2 led to the production of two compounds containing a lateral side chain derived from glutamate instead of 3-methylaspartate. Expression of this glutamate mutase also reaches a peak increase of up to 5.5-fold coinciding with the onset of antibiotic production. Overexpression of either slgE3 or slgE1-slgE2 in S. lydicus led to an increase in the yield of streptolydigin.     

Structural studies on the subunits of glutamate synthase from Azospirillum brasilense

The amino acid composition and the N-terminal sequences of the two dissimilar subunits of glutamate synthase from Azospirillum brasilense have been determined along with the sequences of selected CNBr peptides. Comparison of our data with those available for Escherichia coli glutamate synthase revealed an overall good homology between the enzymes from the two sources. This is more evident for the heavy subunits where the highly conserved N-terminal sequence containing Cys-1, suggests that this region may be involved in catalysis. However, it appears that the light subunits are different with respect to both their amino acid composition and their N-terminal region, suggesting that the latter may not be part of the enzyme active site. Finally, an extinction coefficient at 444 nm of 62.66 +/- 4.61 mM-1.cm-1 was determined.     

Effect of drought stress at supraoptimal temperature on polyamine concentrations in transgenic soybean with increased proline levels

The effect of drought stress at supraoptimal temperature on free proline and polyamine levels was compared in wild type and transgenic soybean (Glycine max cv. Ibis) plants having increased proline levels. Since glutamate and arginine are precursors of both proline and polyamines, it was assumed that the genetic manipulation of proline levels would also affect the polyamine levels. The proline and spermine concentrations increased, while the putrescine concentration generally decreased or did not change after the treatments in both genotypes. Following drought higher proline and lower spermine levels were detected in the transgenic plants compared to the wild type ones, which could be explained by the increased use of their common precursors for proline biosynthesis in the transgenic plants.     

Decreased glutamate, glutamine and citrulline concentrations in plasma and muscle in endotoxemia cannot be reversed by glutamate or glutamine supplementation: a primary intestinal defect?

Endotoxemia affects intestinal physiology. A decrease of circulating citrulline concentration is considered as a reflection of the intestinal function. Citrulline can be produced in enterocytes notably from glutamate and glutamine. The aim of this work was to determine if glutamate, glutamine and citrulline concentrations in blood, intestine and muscle are decreased by endotoxemia, and if supplementation with glutamate or glutamine can restore normal concentrations. We induced endotoxemia in rats by an intraperitoneal injection of 0.3?mg?kg^?1 lipopolysaccharide (LPS). This led to a rapid anorexia, negative nitrogen balance and a transient increase of the circulating level of IL-6 and TNF-α. When compared with the values measured in pair fed (PF) animals, almost all circulating amino acids (AA) including citrulline decreased, suggesting a decrease of intestinal function. However, at D2 after LPS injection, most circulating AA concentrations were closed to the values recorded in the PF group. At that time, among AA, only glutamate, glutamine and citrulline were decreased in gastrocnemius muscle without change in intestinal mucosa. A supplementation with 4% monosodium glutamate (MSG) or an isomolar amount of glutamine failed to restore glutamate, glutamine and citrulline concentrations in plasma and muscle. However, MSG supplementation led to an accumulation of glutamate in the intestinal mucosa. In conclusion, endotoxemia rapidly but transiently decreased the circulating concentrations of almost all AA and more durably of glutamate, glutamine and citrulline in muscle. Supplementation with glutamate or glutamine failed to restore glutamate, glutamine and citrulline concentrations in plasma and muscles. The implication of a loss of the intestinal capacity for AA absorption and/or metabolism in endotoxemia (as judged from decreased citrulline plasma concentration) for explaining such results are discussed.     

Glutamate:glyoxylate aminotransferase modulates amino acid content during photorespiration

In photorespiration, peroxisomal glutamate:glyoxylate aminotransferase (GGAT) catalyzes the reaction of glutamate and glyoxylate to produce 2-oxoglutarate and glycine. Previous studies demonstrated that alanine aminotransferase-like protein functions as a photorespiratory GGAT. Photorespiratory transamination to glyoxylate, which is mediated by GGAT and serine glyoxylate aminotransferase (SGAT), is believed to play an important role in the biosynthesis and metabolism of major amino acids. To better understand its role in the regulation of amino acid levels, we produced 42 GGAT1 overexpression lines that express different levels of GGAT1 mRNA. The levels of free serine, glycine, and citrulline increased markedly in GGAT1 overexpression lines compared with levels in the wild type, and levels of these amino acids were strongly correlated with levels of GGAT1 mRNA and GGAT activity in the leaves. This accumulation began soon after exposure to light and was repressed under high levels of CO(2). Light and nutrient conditions both affected the amino acid profiles; supplementation with NH(4)NO(3) increased the levels of some amino acids compared with the controls. The results suggest that the photorespiratory aminotransferase reactions catalyzed by GGAT and SGAT are both important regulators of amino acid content.     

Yeast argininosuccinate synthetase. Purification; structural and kinetic properties.

Yeast argininosuccinate synthetase has been purified to homogeneity. The enzyme was found to have a molecular weight of 228,000 as determined by gel sieving. It is composed of identical subunits of Mr 49,000 as shown by gel electrophoresis. The quaternary structure as determined by cross-linking of the subunits with glutaraldehyde, followed by gel electrophoresis with dodecylsulfate, is tetrameric. The saturation functions by citrulline and aspartate are hyperbolic; with MgATP as the variable substrate a sigmoid character, dependent on the concentration of citrulline, aspartate, argininosuccinate and arginine, was observed. The positive cooperativity is reduced by increasing concentrations of citrulline and aspartate; it is increased by argininosuccinate and arginine. Kinetic analysis provided evidence for a random addition of substrates. Initial velocity studies as well as product and dead-end inhibition studies comply with a rapid-equilibrium random model, except for the interconversion of the central quaternary complexes; the different kinetic constants have been established on the basis. Yeast argininosuccinate synthetase has a double metabolic function: anabolic in the biosynthesis of arginine, catabolic as the first enzyme of citrulline utilization as nitrogen source. The kinetic properties of the enzyme point to a physiologically well-adjusted activity for both roles and to an economic and efficient utilization of ATP.     

Polyamines and regulation of ornithine biosynthesis in Escherichia coli.

The growth rate of several polyamine-deficient mutants of Escherichia coli was very low in minimal medium and increased markedly upon the addition of putrescine, spermidine, arginine, citrulline, or argininosuccinic acid. The endogenous content of polyamines was not significantly altered by the supplementation of polyamine-starved cultures with arginine or its precursors. In contrast, these compounds as well as putrescine or spermidine caused a 40-fold reduction in intracellular ornithine levels when added to polyamine-depleted bacteria. In vivo experiments with radioactive glutamic acid as a precursor and in vitro assays of the related enzymes showed that the decrease in ornithine levels was due to the inhibition of its biosynthesis rather than to an increase in its conversion to citrulline or delta 1-pyrroline-5-carboxylic acid and proline. High endogenous concentrations of ornithine were toxic for the E. coli strains tested. The described results indicate that the stimulatory effect of putrescine and spermidine on the growth of certain polyamine-starved bacteria may be partially due to the control of ornithine biosynthesis by polyamines.     

Programmed proteome response for drought avoidance/tolerance in the root of a C(3) xerophyte (wild watermelon) under water deficits

Water availability is a critical determinant for the growth and ecological distribution of terrestrial plants. Although some xerophytes are unique regarding their highly developed root architecture and the successful adaptation to arid environments, virtually nothing is known about the molecular mechanisms underlying this adaptation. Here, we report physiological and molecular responses of wild watermelon (Citrullus lanatus sp.), which exhibits extraordinarily high drought resistance. At the early stage of drought stress, root development of wild watermelon was significantly enhanced compared with that of the irrigated plants, indicating the activation of a drought avoidance mechanism for absorbing water from deep soil layers. Consistent with this observation, comparative proteome analysis revealed that many proteins induced in the early stage of drought stress are involved in root morphogenesis and carbon/nitrogen metabolism, which may contribute to the drought avoidance via the enhancement of root growth. On the other hand, lignin synthesis-related proteins and molecular chaperones, which may function in the enhancement of physical desiccation tolerance and maintenance of protein integrity, respectively, were induced mostly at the later stage of drought stress. Our findings suggest that this xerophyte switches survival strategies from drought avoidance to drought tolerance during the progression of drought stress, by regulating its root proteome in a temporally programmed manner. This study provides new insights into the complex molecular networks within plant roots involved in the adaptation to adverse environments.     

Responses of the photosynthetic electron transport system to excess light energy caused by water deficit in wild watermelon

In plants, drought stress coupled with high levels of illumination causes not only dehydration of tissues, but also oxidative damage resulting from excess absorbed light energy. In this study, we analyzed the regulation of electron transport under drought/high-light stress conditions in wild watermelon, a xerophyte that shows strong resistance to this type of stress. Under drought/high-light conditions that completely suppressed CO(2) fixation, the linear electron flow was diminished between photosystem (PS) II and PS I, there was no photoinhibitory damage to PS II and PS I and no decrease in the abundance of the two PSs. Proteome analyses revealed changes in the abundance of protein spots representing the Rieske-type iron-sulfur protein (ISP) and I and K subunits of NAD(P)H dehydrogenase in response to drought stress. Two-dimensional electrophoresis and immunoblot analyses revealed new ISP protein spots with more acidic isoelectric points in plants under drought stress. Our findings suggest that the modified ISPs depress the linear electron transport activity under stress conditions to protect PS I from photoinhibition. The qualitative changes in photosynthetic proteins may switch the photosynthetic electron transport from normal photosynthesis mode to stress-tolerance mode.     

Why does Escherichia coli have two primary pathways for synthesis of glutamate?

Escherichia coli has two primary pathways for glutamate synthetase-glutamate synthase pathway is known to be essential for synthesis at low ammonium concentrations and for regulation of the glutamine pool, but the necessity for glutamate dehydrogenase (GDH) has been uncertain. The results of competition experiments between the wild type and a GDH-deficient mutant during nutrient-limited growth and of direct enzyme measurements suggest that GDH is used in glutamate synthesis when the cell is limited for energy (and carbon) but ammonium and phosphate are present in excess, while the glutamine synthetase-glutamate synthase pathway is used when the cell is not under energy limitation. The use of alternative routes for glutamate synthesis implies that the energy cost of biosynthesis may be less when energy is limited than when energy is unlimited.