When Brassica nigra leaf petiole suspension cells were subjected to 7 days of inorganic phosphate (Pi) starvation the extractable activity of: (a) pyrophosphate:fructose 6-phosphate 1-phosphotransferase, nonphosphorylating NADP-glyceraldehyde 3-phosphate dehydrogenase, phosphoenolpyruvate phosphatase, and phosphoenolpyruvate carboxylase increased at least fivefold, (b) phosphorylating NAD-glyceraldehyde 3-phosphate dehydrogenase decreased about sixfold, and (c) ATP:fructose 6-phosphate 1-phosphotransferase, 3-phosphoglycerate kinase, pyruvate kinase, or NAD malic enzyme was not altered. Pi deprivation also resulted in significant reductions in extractable levels of Pi, ATP, ADP, fructose 2,6-bisphosphate, and soluble protein, but caused a sixfold elevation in free amino acid concentrations. No change in inorganic pyrophosphate concentration was observed following Pi starvation. It is hypothesized that pyrophosphate:fructose 6-phosphate 1-phosphotransferase, nonphosphorylating NADP-glyceraldehyde 3-phosphate dehydrogenase, and phosphoenolpyruvate phosphatase bypass nucleotide phosphate or Pi-dependent glycolytic reactions during sustained periods of Pi depletion.

Phosphoenolpyruvate phosphatase from Brassica nigra leaf petiole suspension cells has been purified 1700-fold to apparent homogeneity and a final specific activity of 380 micromole pyruvate produced per minute per milligram protein. Purification steps included: ammonium sulfate fractionation, S-Sepharose, chelating Sepharose, concanavalin A Sepharose, and Superose 12 chromatography. The native protein was monomeric with a molecular mass of 56 kilodaltons as estimated by analytical gel filtration. The enzyme displayed a broad pH optimum of about pH 5.6 and was relatively heat stable. Western blots of microgram quantities of the final preparation showed no cross-reactivity when probed with rabbit polyclonal antibodies prepared against either castor bean endosperm cytosolic pyruvate kinase, or sorghum leaf phosphoenolpyruvate carboxylase. The final preparation exhibited a broad substrate selectivity, showing high activity toward p-nitrophenyl phosphate, adenosine diphosphate, adenosine triphosphate, gluconate 6-phosphate, and phosphoenolpyruvate, and moderate activity toward several other organic phosphates. Phosphoenolpyruvate phosphatase possessed at least a fivefold and sixfold greater affinity and specificity constant, respectively, for phosphoenolpyruvate (apparent Michaelis constant = 50 micromolar) than for any other nonartificial substrate. The enzyme was activated 1.7-fold by 4 millimolar magnesium, but was strongly inhibited by molybdate, fluoride, zinc, copper, iron, and lead ions, as well as by orthophosphate, ascorbate, glutamate, aspartate, and various organic phosphate compounds. It is postulated that phosphoenolpyruvate phosphatase functions to bypass the adenosine diphosphate dependent pyruvate kinase reaction during extended periods of orthophosphate starvation.

Induction of phosphatase activity is an important component of the plant cell response to phosphate deficiency. Suspension cell cultures of Brassica nigra contain two major inducible acid phosphatase (APase) isozymes; vacuolar phosphoenolpyruvate (PEP) APase and cell wall nonspecific APase. Polyclonal antibodies raised against purified PEPAPase cropsreacted specifically with both isozymes. Furthermore, anti-(PEP-AJPase) IgG *To whom reprint requests should be addressed. 9538The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Barley plants were grown in nutrient solutions, which were maintained at either 0 (‐P) or 15 μM orthophosphate (+P). After 11 days phosphate influx into the intact roots of the ‐P plants began to increase by comparison with +P plants. During this period differences became apparent between the treatments in absolute growth rates, as well as in the root:shoot ratios. Phosphate influx in the ‐P plants continued to increase as a function of time, to a maximum value of 2.4 μmol (g fresh wt)‐1h‐1 at 16 days after germination. This rate was 6 times higher than influx values for +P plants of the same age. During the period of enhanced uptake phosphate was strongly correlated (r2= 0.77) with root organic phosphate concentration. – The enhancement of inorganic phosphate influx into intact roots of ‐P plants was rapidly reduced by the provision of 15 μM orthophosphate. Typically, within 4 h of exposure to this concentration of phosphate, influx values fell from 1.80 ± 0.20 to 0.75 ± 0.03 μmol (g fresh wt)‐1 h‐1, while inorganic phosphate concentrations of the roots increased from 0.12 to 1.15 μmol (g fresh wt)‐1 during the same period. Hill plots of the influx data obtained during this period, treating root inorganic phosphate as an inhibitor of influx, gave Hill coefficients close to 2. The rapidity of the reduction of influx associated with increased root inorganic phosphate together with the Hill plot data provide evidence for an allosteric inhibition of influx by internal inorganic phosphate.

No abstract

Three mutant strains of Arabidopsis thaliana var Columbia were selected for their ability to germinate in elevated concentrations of NaCI. They were not more tolerant than wild type at subsequent development stages. Wild-type strains could not germinate at concentrations >125 mM NaCI. l w o of the mutant strains, RS17 and RS20, could withstand up to 225 mM, whereas RS19 was resistant to 175 mM. l h e RS mutants could also germinate under even lower osmotic potentials imposed by high concentrations of exogenous mannitol (550 mM), whereas the effects of elevated levels of KCI, KzS04, and LiCl were similar among the mutants and wild type. Therefore, the mutants are primarily osmotolerant, but they also possess a degree of ionic tolerance for sodium. Sodium and potassium contents of seeds exposed to high salinities indicated that the NaCI-tolerant mutants absorbed more of these respective cations during imbibition. These higher interna1 concentrations of potassium and sodium could contribute to the osmotic adjustment of the germinating seeds to the low osmotic potential of the externa1 medium. Genetic analysis of F, and Fz progeny of outcrosses suggest that the salt-tolerant mutations are recessive and that they define three complementation groups.A better understanding of the underlying mechanisms involved in the plant response to salinity is essential to confront this agronomic problem. It is known that the detrimental effects of salinity (mostly but not exclusively attributable to NaCl) occur because of (a) osmotic stress, (b) interruption of metabolic activities by ionic excesses and imbalances, and (c) interference of salt ions on the uptake of essential macro-and micronutrients (Pasternak, 1987). These adverse effects are manifested in the inhibition of germination, reduction of growth, and disturbance of development (Levitt, 1980).Plants vary greatly in their tolerances to salt. Halophytes can complete their life cycles under saline conditions (Flowers et al., 1986), but glycophytes, although generally more sensitive to saline stress, range widely in their tolerances between species and even among varieties (Greenway and Munns, 1980; Flowers and Yao, 1987). The fact that pertinent mechanisms, as a consequence, must involve many gene products emphasizes the importance of genetic analysis. Descriptions of single genes that contribute to salt tolerance are few, but they include those responsible for C1-exclusion in certain varieties of soybean (Abel, 1969) Studies of germination performance have indicated that the major effects of a saline environment on germination are the prevention of imbibition and ionic toxicity (Torres-Schumann et al., 1989). The present study characterizes mutants of Arabidopsis thaliana that are able to germinate on high NaCl concentrations. MATERIALS AND METHODS Plant MaterialSeeds of Arabidopsis thaliana var Colombia were mutagenized for 16 h in 0.2% ethyl methyl sulfonate under ambient laboratory conditions (Haughn and Somerville, 1986). Seeds were then washed thoroughly with distill...

Suspension cells of Brassica nigra responded to Pi deprivation by increasing their potential for Pi influx and by raising the active levels of intracellular, cell surface, and secreted acid phosphatases. These responses, however, were temporally distinct. Phosphate influx capacity increased 15-fold in parallel to a 10-fold decrease in endogenous Pi during 7 days of culture in basal growth medium. In contrast, intracellular and cell surface phosphatase activities changed only after alterations in cellular phosphorus status had been in place for a number of days. Even in nutrient sufficient cells the secretion of phosphatase remained relatively high as did the acfivities of the other phosphatases. The cell surface acid phosphatase had a Km of approximately 10 times that of the influx process and molybdate was a much stronger inhibitor of this phosphatase activity. From these results it appears that Pi absorption and the production or activation of phosphatases are regulated in a distinct manner. In addition, Pi uptake into Brassica nigra cells does not appear to directly involve the cell surface phosphatase under Pi-deficient conditions.

The biotransformation of Hg(II) by cyanobacteria was investigated under aerobic and pH-controlled culture conditions. Mercury was supplied as HgCl 2 in amounts emulating those found under heavily impacted environmental conditions where bioremediation would be appropriate. The analytical procedures used to measure mercury within the culture solution, including that in the cyanobacterial cells, used reduction under both acid and alkaline conditions in the presence of SnCl 2 . Acid reduction detected free Hg(II) ions and its complexes, whereas alkaline reduction revealed that meta-cinnabar (␤-HgS) constituted the major biotransformed and cellularly associated mercury pool. This was true for all investigated species of cyanobacteria: Limnothrix planctonica (Lemm.), Synechococcus leopoldiensis (Racib.) Komarek, and Phormidium limnetica (Lemm.). From the outset of mercury exposure, there was rapid synthesis of ␤-HgS and Hg(0); however, the production rate for the latter decreased quickly. Inhibitory studies using dimethylfumarate and iodoacetamide to modify intra-and extracellular thiols, respectively, revealed that the former thiol pool was required for the conversion of Hg(II) into ␤-HgS. In addition, increasing the temperature enhanced the amount of ␤-HgS produced, with a concomitant decrease in Hg(0) volatilization. These findings suggest that in the environment, cyanobacteria at the air-water interface could act to convert substantial amounts of Hg(II) into ␤-HgS. Furthermore, the efficiency of conversion into ␤-HgS by cyanobacteria may lead to the development of applications in the bioremediation of mercury.Mercury in the form of divalent ions constitutes the bulk of that in soils, where it is bound to organic compounds, to clay, and as sulfides (31). Although industrialization in the beginning of the last century is the cause of most of the mercury contamination found in the environment today, rainfall continues to carry mercury [Hg(II)] into aquatic and terrestrial systems worldwide (16,44). Despite a comprehensive knowledge of the mercury cycle and the aquatic chemistry of its constituents (12, 43, 52), several microbial taxa have not been characterized with respect to their roles in the biotransformation of this heavy metal.Several mercury biotransformation mechanisms have been described previously (4,12,39,41), and of these, prokaryotic methylation and reduction to Hg(0) may play only limited roles in the biotransformation of Hg(II) in aquatic environments (15,19). Furthermore, the reduction reaction simply refracts meteorologically precipitated Hg(II) back into the atmosphere. As such, it follows that other processes must make significant contributions to the biogeochemical cycle of Hg even though relative quantitative data are scarce (31). Insight into this area requires an understanding of the major biotransformation processes leading to mercury retention in ecosystems.The lack of quantitative, mechanistic data behind cellular accumulation versus that for volatilization of Hg is evident even in the eukar...