Photosynthetic electron transport in thylakoids from cotton cultivars (Gossypium sp.) differing in tolerance to prometryn.

A method to determine photosynthetic electron transport in thylakoid membranes is described for Gossypium barbadense (cv. Pima S-7) and G. hirsutum (cv. DP 5415). These cultivars differed markedly in tolerance to prometryn, a PS II inhibitor. The rates of photosynthetic electron transport obtained were 245 µmole oxygen mg(-1) chl h(1). Plant age and leaf size influenced the activity of the thylakoid preparations. Thylakoids from leaves of plants 24 to 37 d and 50-70 mm in diameter had the highest activities; thylakoids from cotyledons, fully expanded leaves and young leaves had low activity. Thylakoids from both species had similar photosynthetic activities and I50's for prometryn, atrazine and diuron. Thus, tolerance to prometryn was not due to differential binding at D1 protein.

Effect of Different Carbon Sources on the Ammonium Induction of Different Forms of NADP-Specific Glutamate Dehydrogenase in Chlorella sorokiniana Cells Cultured in the Light and Dark.

The ammonium induction of the chloroplast-localized NADP-specific glutamate dehydrogenase (NADP-GDH) was shown not to be a light-dependent process per se in Chlorella sorokiniana. In the dark without exogenous organic substrates, the cells synthesized low levels of fully active NADP-GDH, provided endogenous starch reserves had not been depleted. When cells were supplied with exogenous acetate, the rate of induction of NADP-GDH activity per milliliter of culture in the dark was equal to or slightly greater than the rate observed under photosynthetic conditions without an organic carbon source. Glucose supported only a low rate of induction of NADP-GDH activity in the dark. Both acetate and glucose inhibited induction of enzyme activity in the light. The NADP-GDH holoenzyme had at least 7 different electrophoretic forms. These forms differed in net charge and/or molecular weight. Their difference in molecular weight was due to the presence of 2 subunits with similar antigenic properties but different molecular weights (M(r) = 55,500 and 53,000; alpha-and beta-subunits, respectively). Depending upon the cultural conditions and length of the induction period, a wide variation was observed in the alpha:beta subunit ratio and in the numbers and sizes of the NADP-GDH holoenzymes.

Ploidy effects in isogenic populations of alfalfa : I. Ribulose-1,5-bisphosphate carboxylase, soluble protein, chlorophyll, and DNA in leaves.

The influence of polyploidization on ribulose-1,5-bisphosphate carboxylase (RuBPCase), buffer-soluble protein (BSP), chlorophyll (Chl), and DNA was examined in fully expanded leaves of isogenic diploid-tetraploid (DDC 2X-4X) and tetraploid-octoploid (IC 4X-8X) sets of alfalfa (Medicago sativa L.). The concentration of RuBPCase in leaf extracts was determined by rocket immunoelectrophoresis. Activities of RuBPCase, expressed per milligram protein or per milligram Chl, and leaf tissue concentrations of RuBPCase, BSP, Chl, and DNA were similar between ploidy levels of the DDC 2X-4X set. Tetraploids and octoploids were similar in RuBPCase activities, expressed per milligram protein or per milligram Chl, and in leaf tissue concentrations of RuBPCase and DNA. Octoploids were significantly lower than tetraploids in concentrations of Chl and BSP.When compared on a per leaf basis, tetraploids were 80% higher in BSP and essentially double comparable diploids in fresh weight, RuBPCase, Chl, and DNA. The observation that leaves of the DDC tetraploid population contain twice as much DNA as comparable diploids suggests that leaves of both ploidy levels contain similar numbers of cells. Leaves of the octoploid population were 33% to 80% higher than corresponding tetraploids in BSP, fresh weight, RuBPCase, Chl, and DNA. Ratios of RuBPCase to DNA and Chl to DNA were similar across ploidy levels of both isogenic sets suggesting that cellular content of Chl and RuBPCase increases proportionately with the amount of DNA per cell.

Ploidy Effects in Isogenic Populations of Alfalfa : III. Chloroplast Thylakoid-Bound Coupling Factor 1 in Protoplasts and Leaves.

The influence of polyploidization on chloroplast coupling factor 1 (CF1) was examined in leaves and leaf protoplasts from isogenic diploid-tetraploid (DDC 2X-4X) and tetraploid-octoploid (IC 4X-8X) sets of alfalfa (Medicago sativa L.). Alfalfa CF1 was purified to homogeneity and was found to contain five subunits with molecular weights of 57,900, 54,300, 38,700, 23,100, and 15,200. Rocket immunoelectrophoresis with anti-spinach CF1 gamma immunoglobulins was used to quantify CF1 from protoplasts and leaves. In the DDC 2X-4X set, fresh weight per cell and cellular content of CF1, chlorophyll (Chl), and DNA doubled with ploidy. Ratios of CF1 to Chl of leaf protoplasts and leaves were similar between diploids and tetraploids. In the IC 4X-8X set, octoploid protoplasts were 90% higher in Chl than were comparable tetraploids, whereas octoploids were 50 to 60% higher than tetraploids in fresh weight per cell and cellular content of CF1 and DNA. Concentrations of CF1 and Chl in leaves and ratios of CF1 to DNA in protoplasts were similar across ploidy levels of both isogenic sets. Therefore, cellular content of CF1 increases proportionately with the amount of DNA per cell or gene dosage.

Ploidy Effects in Isogenic Populations of Alfalfa : II. Photosynthesis, Chloroplast Number, Ribulose-1,5-Bisphosphate Carboxylase, Chlorophyll, and DNA in Protoplasts.

Photosynthetically-active protoplasts isolated from isogenic sets of diploid-tetraploid and tetraploid-octoploid alfalfa (Medicago sativa L.) leaves were used to investigate the consequences of polyploidization on several aspects related to photosynthesis at the cellular level. Protoplasts from the tetraploid population contained twice the amount of DNA, ribulose-1,5-bisphosphate carboxylase (RuBPCase), chlorophyll (Chl), and chloroplasts per cell compared to protoplasts from the diploid population. Although protoplasts from the octoploid population contained nearly twice the number of chloroplasts and amount of Chl per cell as tetraploid protoplasts, the amount of DNA and RuBPCase per octoploid cell was only 50% higher than in protoplasts from the tetraploid population. The rate of CO(2)-dependent O(2) evolution in protoplasts nearly doubled with an increase in ploidy from the diploid to tetraploid level, but increased only 67% with an increase in ploidy from the tetraploid to octoploid level. Whereas leaves and protoplasts had similar increases in RuBPCase, DNA, and Chl with increase in ploidy level, it was concluded that increased cell volume rather than increased cell number per leaf is responsible for the increase in leaf size with ploidy.

A simple and sensitive DNA assay for plant extracts.

A sensitive fluorimetric method was developed for the quantitative determination of DNA in plant (Zea mays L. and Medicago sativa L.) extracts. This method takes advantage of the specific increase in fluorescence intensity of the complex of DNA and the dye 4',6'-diamidino-2-phenylindole (DAPI). Recovery of DNA and dissociation of histones from DNA were maximized by the addition of 2.0 molar NaCl to the homogenates. Treatment of the homogenate with chloroform to remove pigments and proteins decreased the quenching of fluorescence of the DAPI-DNA complex. The fluorescence intensity of RNA with DAPI was less than 2% of that produced by an equivalent weight of DNA. Comparisons were made between this fluorimetric DNA method and the commonly used diphenylamine assay for DNA. The diphenylamine DNA assay was more timeconsuming, less sensitive, and consistently resulted in lower estimates of DNA concentrations than did the fluorimetric DNA assay.

Light requirement for induction and continuous accumulation of an ammonium-inducible NADP-specific glutamate dehydrogenase in chlorella.

The ammonium-inducible NADP-specific glutamate dehydrogenase of Chlorella sorokiniana was shown to require light for both its induction by ammonia in uninduced cells, and its continuous accumulation in fully induced cells. Addition of ammonia to uninduced cells in the light resulted in a 35-minute induction lag followed by linear and coincident increases in enzyme activity and antigen. Enzyme activity was not induced in the dark; however, transfer of these cells to the light resulted in an immediate increase in enzyme activity and antigen. The absence of an induction lag suggested that mRNA sequences and/or an enzyme precursor with different antigenic properties than the active holoenzyme accumulated in cells in the dark in ammonium medium. When fully induced cells were transferred to the dark, the activity of the enzyme quickly ceased to accumulate. In contrast to the NADP-specific isozyme, the cells also contain a constitutive NAD-specific isozyme which was shown to accumulate in cells in the dark in either ammonium or nitrate medium.

Evidence for messenger ribonucleic acid of an ammonium-inducible glutamate dehydrogenase and synthesis, covalent modification, and degradation of enzyme subunits in uninduced Chlorella sorokiniana cells.

The cells of Chlorella sorokiniana cultured in nitrate medium contain no detectable catalytic activity of an ammonium-inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH). However, several lines of experimental evidence indicated that the NADP-GDH messenger ribonucleic acid was present at high levels and was being translated in uninduced cells. First, binding studies with 125I-labeled anti-NADP-GDH immunoglobulin G and total polysomes isolated from uninduced and induced cells showed that NADP-GDH subunits were being synthesized on polysomes from both types of cells. Second, when polyadenylic acid-containing ribonucleic acid was extracted from polysomes from uninduced and induced cells and placed into a messenger ribonucleic acid-dependent in vitro translation system, NADP-GDH subunits were synthesized from the ribonucleic acid from both sources. Third, when ammonia was added to uninduced cells, NADP-GDH antigen accumulated without an apparent induction lag. Fourth, by use of a specific immunoprecipitation procedure coupled to pulse-chase studies with [35S]sulfate, it was shown that the NADP-GDH subunits are rapidly synthesized, covalently modified, and then degraded in uninduced cells.

Thiamin Phosphorylation by Thiamin Pyrophosphotransferase during Seed Germination.

Thiamin pyrophosphotransferase activity was present in seedling extracts from several monocot and dicot species of agronomic as well as noncultivated plants. Changes in thiamin pyrophosphotransferase activity and thiamin pyrophosphate content were followed for 6 days in soybean (Merr.) seedlings. Maximum enzyme activity occurred 48 to 96 hours from imbibition. Thiamin pyrophosphate content peaked sharply at 36 hours and was preceded by increased thiamin pyrophosphotransferase activity. Addition of pyrithiamin, an inhibitor of in vitro thiamin pyrophosphotransferase activity, to the imbibition medium at various times inhibited subsequent fresh weight gains of soybean seedlings. These results indicated that, although not among the earliest phosphorylation events after initiation of water imbibition by soybean seeds, a substantial increase in thiamin pyrophosphate content did precede the onset of rapid seedling growth and development. Since both enzyme activity and thiamin appear to be available in unimbibed soybean seeds, ATP or other nucleoside triphosphate concentration may represent an important factor in modulating thiamin phosphorylation during early seedling development.

Isolation and Characterization of Thiamin Pyrophosphotransferase from Glycine max Seedlings.

Thiamin:ATP pyrophosphotransferase (EC2.7.6.2) activity from soybean (Merr.) seedlings grown for 48 hours was determined by measuring the rate of [2-(14)C]thiamin incorporation into thiamin pyrophosphate. With partially purified (11-fold) enzyme, optimal activity occurred between pH 7.1 and 7.3, depending on the buffer system that was used. Assays were routinely conducted at a final pH of 8.1 in order to minimize interference from competing reactions. Enzyme activity required the presence of a divalent cation, and a number of nucleoside triphosphates proved to be active as pyrophosphate donors. Apparent K(m) values of 18.3 millimolar and 4.64 micromolar were obtained for Mg.ATP and thiamin, respectively. Among the compounds tested, pyrithiamin and thiamin pyrophosphate were most effective in inhibiting thiamin pyrophosphotransferase activity. Based on Sephadex G-100 gel filtration, soybean thiamin pyrophosphotransferase has a molecular weight of 49,000.