Properties of a small basic Peptide from pumpkin seeds

A small basic peptide with an unusual amino acid composition has been isolated from the seeds of pumpkin, Cucurbita maxima. Amino acid analysis and sequence data show the protein to be about 36 residues in length, with an approximate composition Lys₁, Arg₁₄, Asp₃, (Glu + Gln)₁₅, Gly₁, Pro₁, Trp₁. On the basis of composition, the molecular weight is approximately 5000 daltons and the nitrogen content by weight is 20.4%. Twelve amino acids are entirely lacking. The peptide is slightly toxic to mouse B-16 melanoma cells, but its in vivo function is unknown. It does not appear to be derived from cucurbitin, the pumpkin storage globulin; however, it could be a storage peptide involved in nitrogen mobilization during the early stages of germination.     

Modifications of extracellular electric and ionic gradients preceding the transition from tip growth to isodiametric expansion in the apical cell of the fern gametophyte

Fern (Onoclea sensibilis L.) gametophytes exposed to blue light are induced to undergo a morphological transition from a tip-growing filament to a planar prothallus. Extracellular measurements of electric currents and localized ion activities around the apical cell of 8 to 10 day-old gametophytes were made with a vibrating probe and ion selective electrodes. In darkness, we observed exit current densities of an average of 75 nanoamperes per square centimeter near the tip and 2 to 15 nanoamperes per square centimeter along the lateral walls of this cell. Measurements with ion selective electrodes for H⁺, K⁺, and Ca²⁺ showed that this cell was bounded by a thin layer of medium that was depleted in K⁺ and Ca²⁺ and exhibited a lower pH than the bulk solution. Both the K⁺ and Ca²⁺ depletion zones and the zone of higher acidity were particularly pronounced at the tip end of the cell; the pH at 2 micrometers from the tip was nearly 0.5 units more acid than the bulk medium at pH 6. Disruption of steady state, external gradients with media that contained lower concentrations of H⁺, K⁺, Ca²⁺, or Cl⁻ produced certain differences in the rates of restoration of particular ion zones, raising the possibility that some of the ion migrations are interdependent. Within 15 minutes after irradiation with blue light, current leaving the tip declined to levels which were indistinguishable from those leaving the lateral walls and there was a rapid lowering in the rates of tip acidification and K⁺ depletion near the tip. The rapid dissipation of both the longitudinally aligned electrical field and the tip-localized asymmetries in external cation distribution in blue light suggest that loss of electrical polarity in this tip growing cell may be an initial step in the chain of events which govern changes in cell shape.     

A Starchless Mutant of Nicotiana sylvestris Containing a Modified Plastid Phosphoglucomutase

A mutant (NS 458) of Nicotiana sylvestris (Spegazzini and Comes) unable to synthesize leaf starch was isolated in the M₂ generation following ethyl methanesulfonate mutagenesis by testing with iodine. Segregation ratios in reciprocal F₂ progenies showed that the starchless phenotype resulted from a recessive mutation in a single nuclear gene. DEAE-agarose chromatography showed that the mutant is grossly deficient in plastid phosphoglucomutase (EC 2.1.5.1) activity. The structure of the enzyme is changed, as evidenced by increased Michaelis constants and by the prolonged activation period (>40 minutes) observed when the enzyme is assayed in triethanolamine buffer rather than imidazole buffer. The activity of the wild-type enzyme with saturating glucose 6-P alone was 7% of the activity when saturating glucose 1,6-P₂ was also present. The results suggest that glucose 1,6-P₂ is both an effector and a dissociable reaction intermediate. The growth rate of mutant and wild-type plants were not significantly different in continuous light and on an 8-hour dark, 16-hour light cycle and the mutants grew normally under greenhouse conditions. The mutant supports growth during diurnal periods of darkness by vacuolar storage of sugars instead of chloroplast storage of starch. The simplification in metabolism achieved by blocking the diversion of plastid fructose-6-P to starch facilitates the induction of oscillations in CO₂ fixation.     

A developmentally regulated hydroxyproline-rich glycoprotein in maize pericarp cell walls

We have studied the accumulation of peptidyl hydroxyproline in the pericarp of developing maize (Zea mays L., Golden cross Bantam sweet corn) kernels. Although this hydroxyproline accumulates throughout development, it is most soluble and its content per milligram dry weight greatest at midmaturation stages of development. Salt-soluble proteins containing this hydroxyproline from isolated cell walls of developing kernels were fractionated on a CsCl density gradient and on a Chromatofocusing column, resulting in the purification of an hydroxyproline-rich glycoprotein, PC-1. PC-1 is a basic protein of approximately 65 to 70 kilodaltons in molecular weight with an isoelectric point of at least 10.2 and a density of 1.38 to 1.39 in CsCl. Amino acid composition data indicate that it is rich in hydroxyproline, threonine, proline, lysine, and glycine. Its relation to dicot extensin is discussed.     

Ammonia Production and Assimilation in Glutamate Synthase Mutants of Arabidopsis thaliana

Ammonia production and assimilation¹ were examined in photorespiratory mutants of Arabidopsis thaliana L. lacking ferredoxin-dependent glutamate synthase (Fd-GluS) activity. Although photosynthesis was rapidly inhibited in these mutants in normal air, NH₄⁺ continued to accumulate. The accumulation of NH₄⁺ was also seen after an initial lag of 30 minutes in 2% O₂, 350 microliters per liter of CO₂ and after 90 minutes in 2% O₂, 900 microliters per liter of CO₂. The accumulation of NH₄⁺ in normal air and low O₂ was also associated with an increase in the total pool of amino acid-N and glutamine, and a decrease in the pools of glutamate, aspartate, alanine, and serine. Upon return to dark conditions, or to 21% O₂, 1% CO₂ in the light, the NH₄⁺ which had accumulated in the leaves was reassimilated into amino acids. The addition of methionine sulfoximine (MSO) resulted in higher accumulations of NH₄⁺ in glutamate synthase mutants and prevented the reassimilation of NH₄⁺ upon return to the dark. The addition of MSO also resulted in the accumulation of NH₄⁺ in glutamate synthase mutants in the light and in 21% O₂, 1% CO₂. These results indicate that glutamine synthetase is essential for the reassimilation of photorespiratory NH₄⁺ and for primary N assimilation in the leaves and strongly suggest that glutamate dehydrogenase plays only a minimal role in the assimilation of ammonia. Levels of NADH-dependent glutamate synthase (NADH-GluS) appear to be sufficient to account for the assimilation of NH₄⁺ by a GS/NADH-GluS cycle.     

Comparison of Triazine-Resistant and -Susceptible Biotypes of Senecio vulgaris and Their F(1) Hybrids

The relationship of triazine resistance to decreased plant productivity was investigated in Senecio vulgaris L. F₁ reciprocal hybrids were developed from pure-breeding susceptible (S) and resistant (R) lines. The four biotypes (S, S × R, R, R × S) were compared in terms of atrazine response, electron transport, carbon fixation, and biomass production. Atrazine response, carbon fixation rate, and PSII and whole-chain electron transport rates of hybrids were nearly identical to those of their respective maternal parents. Significant differences occurred between the two susceptible (S, S × R) and two resistant (R, R × S) biotypes in atrazine response (I₅₀), carbon fixation rate, and PSII and whole-chain electron transport rates; PSI rates were identical in all four biotypes. Coupled and uncoupled, whole-chain electron transport rates of thylakoids of the two susceptible biotypes were approximately 50% greater than those of the two resistant biotypes at photon flux densities greater than 215 micromoles per square meter per second. Carbon exchange rates of the two susceptible biotypes were 23% greater than those of the two resistant biotypes. Hybrid biotypes (S × R, R × S) were not identical to their maternal parents in biomass production. The S, S × R, and R × S plants all achieved greater biomass than R plants. These results suggest that while the resistance mutation influences thylakoid performance, reduced productivity of triazine-resistant plants cannot be ascribed solely to decreases in electron transport or carbon assimilation rates brought about by the altered binding protein. Since the F₁ hybrids differed from their maternal parents only in nuclear genes, it appears that the detrimental effects of the triazine resistance mutation on plant growth may be attenuated by interactions of the plastid and nuclear genomes.     

Localization of Nitrogen-Assimilating Enzymes in the Chloroplast of Chlamydomonas reinhardtii

The specific activities of nitrate reductase, nitrite reductase, glutamine synthetase, glutamate synthase, and glutamate dehydrogenase were determined in intact protoplasts and intact chloroplasts from Chlamydomonas reinhardtii. After correction for contamination, the data were used to calculate the portion of each enzyme in the algal chloroplast. The chloroplast of C. reinhardtii contained all enzyme activities for nitrogen assimilation, except nitrate reductase, which could not be detected in this organelle. Glutamate synthase (NADH- and ferredoxin-dependent) and glutamate dehydrogenase were located exclusively in the chloroplast, while for nitrite reductase and glutamine synthetase an extraplastidic activity of about 20 and 60%, respectively, was measured. Cells grown on ammonium, instead of nitrate as nitrogen source, had a higher total cellular activity of the NADH-dependent glutamate synthase (+95%) and glutamate dehydrogenase (+33%) but less activity of glutamine synthetase (−10%). No activity of nitrate reductase could be detected in ammonium-grown cells. The distribution of nitrogen-assimilating enzymes among the chloroplast and the rest of the cell did not differ significantly between nitrate-grown and ammonium-grown cells. Only the plastidic portion of the glutamine synthetase increased to about 80% in cells grown on ammonium (compared to about 40% in cells grown on nitrate).     

Indole-3-acetic Acid oxidation and crocin bleaching by horseradish peroxidase

During indoleacetic acid (IAA) oxidation by horseradish peroxidase the water soluble model polyene, crocin, is bleached. IAA-oxidation and crocin bleaching are stimulated at acidic pH as well as by the monophenol p-hydroxyacetophenone. IAA oxidation and crocin bleaching are neither influenced by catalase or superoxide dismutase nor by different OH-radical scavengers, whereas both ascorbate and propylgallate are inhibitory.     

A Mutant of Nicotiana sylvestris Lacking Serine:Glyoxylate Aminotransferase: Substrate Specificity of the Enzyme and Fate of [2-C]Glycolate in Plants with Genetically Altered Enzyme Levels

The photorespiratory mutant of Nicotiana sylvestris, NS 349, lacking serine:glyoxylate aminotransferase (SGAT) grows in 1% CO₂ but not in normal air (NA McHale, EA Havir, I Zelitch 1988 Theor Appl Genet. In press). Alanine:hydroxypyruvate and asparagine:hydroxypyruvate aminotransferase activities were also lacking in the mutant, and plants heterozygous with respect to SGAT which grow in normal air had 50% of the activities present in homozygous plants. Therefore, all these activities are associated with the same enzyme. On feeding [2-¹⁴C]glycolate to leaf discs in the light, NS 349 showed reduced incorporation of radioactivity into the neutral and organic acid fractions and increased incorporation into the amino acid fraction, principally into serine. The effect of reducing SGAT by 50% in heterozygous plants produced little change in the metabolism of [2-¹⁴C]glycolate, showing there is a large excess of this enzyme in wild-type plants.     

Lipid peroxidation is a consequence of elicitor activity

Elicitor-active preparations from the fungal pathogen of bean Colletotrichum lindemuthianum stimulated the accumulation of products characteristic of lipid peroxidation in treated bean tissues. Bean suspension cells treated with crude and purified elicitors accumulated `lipofuscin-like pigment' (LEP) and malondialdehyde. The accumulation of LFP after about 6 h of treatment coincided with the onset of visible browning and production of the bean phytoalexins kievitone, phaseollin, and phaseollinisoflavan. The induction of phytoalexins and accumulation of LFP were also triggered by treatments with generators of activated oxygen species, xanthine:xanthine oxidase and Fe:ethylenediaminedi-o-hydroxyphenylacetic acid. These data suggest that generation of active oxygen species may be involved in lipid peroxidation triggered by elicitors.