Plant Physiology

Wainwright, Irene M.; Ting, Irwin P.

doi: 10.1104/pp.58.4.447pmid: 16659695

Abstract The properties of the microbody malate dehydrogenase (EC 1.1.1.37) (MDH) isozyme from cotyledons of Cucumus sativus L. were compared during development. It is concluded that the isozyme remains unaltered, despite the transition from glyoxysomal to peroxisomal function that occurs during greening of the cotyledons. This conclusion is based on electrophoretic behavior, chromatographic elution from DEAE-cellulose, molecular weight, kinetic behavior, and immunological identity. In most cases, the distinct properties of the other MDH isozymes in the tissue during development provide additional support for an unchanging microbody isozyme. A method for assaying specifically the microbody isozyme was developed; a diluted preparation was assayed spectrophotometrically before and after complete immunological precipitation. The turnover of the microbody MDH isozyme was investigated by a radioactive labeling study. There is incorporation into both glyoxysomal and peroxisomal MDH. Degradation rates do not correspond with either decline of glyoxysomal activity or the continuation of peroxisomal activity. Apparently, the microbody MDH isozyme is continually turned over throughout cotyledon development. 2 Recipient of Phi Beta Kappa grant. 1 This work was supported in part by National Science Foundation Grant BMS74-11842. This content is only available as a PDF. © 1976 American Society of Plant Biologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Guilfoyle, Tom J.

doi: 10.1104/pp.58.4.453pmid: 16659696

Abstract DNA-dependent RNA polymerases were solubilized from nuclei of cauliflower inflorescences and purified by agarose A-1.5m, DEAE-cellulose, DEAE-Sephadex, and phosphocellulose chromatography and sucrose density gradient centrifugation. RNA polymerases I + III were separated from II by DEAE-cellulose chromatography. Subsequent chromatography on DEAE-Sephadex resolved RNA polymerase I from III. RNA polymerases I and II were further purified to high specific activity by phosphocellulose chromatography and sucrose density gradient centrifugation. RNA polymerase I was refractory to α-amanitin at 2 mg/ml. RNA polymerase II was 50% inhibited at 0.05 μg/ml, and RNA polymerase III was 50% inhibited at 1 to 2 mg/ml of α-amanitin. The enzymes were characterized with respect to divalent cation optima, ionic strength optima, and abilities to transcribe cauliflower, synthetic, and cauliflower mosaic virus DNA templates. 1 This research was supported by Public Health Service Grant CA-11624 from the National Cancer Institute to J. L. Key and a postdoctoral fellowship from the National Cancer Institute to T. J. G. This content is only available as a PDF. © 1976 American Society of Plant Biologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Howard, Russell J.; Wright, Simon W.; Grant, Bruce R.

doi: 10.1104/pp.58.4.459pmid: 16659697

Abstract The properties of the soluble β-glucans formed during photosynthesis of the green siphonous alga Caulerpa simpliciuscula are described. There are two components in the soluble β-glucan fraction. One has an apparent degree of polymerization of 37 glucose units and the other of 270 glucose units. The β-glucan with the lower apparent molecular weight accounts for most of the mass in the β-glucan fraction and is similar in properties to soluble laminarins reported in other algal and fungal species. The β-glucan with the high apparent molecular weight contains most of the radioactivity accumulated in the β-glucan fraction during short periods of photosynthesis. 1 This work was supported in part by the Australian Research Grants Committee, and by a C.S.I.R.O. postgraduate award to R. J. H. This content is only available as a PDF. © 1976 American Society of Plant Biologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Shaw, Roy; Varns, Jerry L.; Miller, Karon A.; Talley, Eugene A.

doi: 10.1104/pp.58.4.464pmid: 16659698

Abstract Callus was initiated from explants of tubers of the Norchip cultivar of Solanum tuberosum L. and grown on medium with a single carbon source and without addition of coconut milk, protein hydrolysate, or amino acid. Callus samples were harvested at intervals and compared to mature tubers for which there was good biochemical knowledge. The amino acid spectrum, the glycoalkaloid content, and the properties of the isolated invertase and sucrose synthetase were similar in callus and in tuber. Significantly the level of sucrose synthetase varied with the age of the developing callus just as it did with the age of the developing tuber. Of greater significance, levels of reducing sugars and invertase varied with the age of developing callus and also with time and temperature of storage after the callus has ceased growth. Similar changes occur in intact tubers. Callus and tuber biochemistry differed in the amount of deposited starch and in the absence of potato invertase inhibitor. 2 Present address: The International Potato Center, Apartado 5969, Lima, Peru. 1 Agricultural Experiment Station, University of Minnesota Scientific Journal Series No. 9308. 3 A laboratory cooperatively operated by North Central Region, Agricultural Research Service, United States Department of Agriculture; the Minnesota Agricultural Experiment Station; the North Dakota Agricultural Experiment Station; and the Red River Valley Potato Growers Association. This content is only available as a PDF. © 1976 American Society of Plant Biologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Pirie, Andrew; Mullins, Michael G.

doi: 10.1104/pp.58.4.468pmid: 16659699

Abstract Sucrose (0.04 to 0.12 m) induces accumulation of both total phenolics and anthocyanin in leaf discs of grapevine (Vitis vinifera L.) incubated in intermittent light. Abscisic acid (20 μM) and 2-chloroethyl phosphonic acid (60 μm) act synergistically with the sucrose to enhance its induction of both total phenolics and anthocyanin. The magnitude of this interaction depends on leaf age. Nitrate (30 mm) inhibits sucrose induction of phenolics and anthocyanin. Levels of total phenolics and anthocyanin changed independently. 2 T. L. Pawlett Postgraduate Scholar. 1 This work was supported in part by a grant from the Australian Wine Board. This content is only available as a PDF. © 1976 American Society of Plant Biologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Hasson, Edna P.; West, Charles A.

doi: 10.1104/pp.58.4.473pmid: 16659700

Abstract The rates of oxidation of ent-kaur-16-ene to ent-kaur-16-en-19-ol, ent-kaur-16-en-19-al, ent-kaur-16-en-19-oic acid, and ent-kaur-16-en-7α-ol-19-oic acid are maximal in microsomes prepared from the endosperm of immature Marah macrocarpus seeds in which the cotyledons are approximately one-half the overall length of the seed. The supernatant fraction remaining from the preparation of the microsomes contains factors which stimulate the rates of oxidation catalyzed by the microsomes. Added TPNH is more effective than added DPNH in meeting the requirement for reduced pyridine nucleotide. A mixture of DPNH, ATP, and TPN+ is much more effective than DPNH alone. Experiments with 2,4-dinitrophenol as a selective inhibitor indicate that the ATP-stimulated synthesis of TPNH which occurs in these microsomes in the presence of this mixture of coenzymes provide TPNH for use in the mixed function oxidations. Relatively low concentrations of DPNH and TPNH together are much more effective than either alone at equivalent concentration. This is consistent with the involvement of two pathways of electron transfer associated with the mixed function oxidations, one of which preferentially utilizes TPNH and the other favoring DPNH. FAD added to microsomes at an optimal concentration of about 10 μm in the presence of TPNH stimulates the rate of the oxidations; higher concentrations are inhibitory. FMN by itself does not produce this stimulation. However, FMN and FAD added together at low concentrations (0.5 μm each) have approximately the same effectiveness as FAD alone at 10 μm. This suggests a role for both flavin nucleotides in the normal electron transfer pathways associated with these oxidations. Some of the stimulatory properties of the supernatant fraction may be accounted for by its content of reduced pyridine nucleotides, FAD, and FMN; the concentrations of FAD and FMN were determined to be 1.1 μm and 0.4 μm, respectively. However, the effects of the supernatant fraction are not completely explained by its content of these coenzymes since other experiments indicate the presence of a heat-labile, nondialyzable stimulatory factor(s) in the supernatant fraction in addition to heat-stable, dialyzable fractors. 1 This work was supported in part by National Institutes of Health Grant GM 07065 from the National Institute of General Medical Sciences. This content is only available as a PDF. © 1976 American Society of Plant Biologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Hasson, Edna P.; West, Charles A.

doi: 10.1104/pp.58.4.479pmid: 16659701

Abstract Cytochrome P-450 and cytochrome b 5 at levels of approximately 0.10 and 0.60 nanomole per milligram of microsomal protein were detected by spectral measurements in microsomes prepared from endosperm tissue of immature Marah macrocarpus seeds. TPNH-cytochrome c reductase, DPNH-cytochrome c reductase, andDPNH-cytochrome b 5 reductase activities were also present in these microsomes at levels of approximately 0.060, 0.22, and 0.52 unit per milligram of microsomal protein, respectively. (One unit of reductase is the amount of enzyme catalyzing the reduction of 1 micromole of electron acceptor per minute.) Treatments of microsomes with steapsin or trypsin were not effective in solubilizing any of these electron transport components in detectable form. However, treatment of a microsomal suspension in 25% glycerol with 1% sodium deoxycholate led to the release of about 60% of the protein and each of the above hemoproteins and electron transfer activities to the fraction which was not pelleted after centrifugation for 2 hours at 105,000g. Some ent-kaur-16-ene oxidase activity could be detected in the solubilized fraction after removal of the detergent. Cytochrome b 5 and DPNH-cytochrome b 5 reductase activity were largely separated from one another and from an overlapping mixture of TPNH-cytochrome c reductase and DPNH-cytochrome c reductase when the sodium deoxycholate-solubilized fraction was chromatographed on a DEAE-cellulose column. No cytochrome P-450 or cytochrome P-420 was detected in the column fractions and no ent-kaur-16-ene oxidase activity was detected when the column fractions were tested singly or in combination. The possible participation of these components in the mixed function oxidation of ent-kaur-16-ene and a number of its oxidized derivatives catalyzed by these microsomes is discussed in relation to the model which has been developed to explain the function of analogous components in mixed function oxidase reactions in mammalian liver microsomes. 1 This work was supported in part by National Institutes of Health Grant GM 07065 from the National Institute of General Medical Sciences. This content is only available as a PDF. © 1976 American Society of Plant Biologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Kitamoto, Yutaka; Gruen, Hans E.

doi: 10.1104/pp.58.4.485pmid: 16659702

Abstract Flammulina velutipes (Curt. ex Fr.) Sing. was grown on potato-glucose solution freed of most starch. Glucose uptake and dry weight changes in the colony indicated that the large fruitbodies derived their substrates partly from glucose remaining in the medium and partly from cellular constituents stored in the mycelium and small fruitbodies. Changes in the amounts of low molecular weight carbohydrates, glycogen, and four cell wall polysaccharide fractions were followed in the mycelium and fruitbodies. Trehalose, arabitol, and smaller amounts of mannitol were the main stored low molecular weight carbohydrates. A large net loss of these compounds occurred in the mycelium and small fruitbodies after their growth ceased. The carbohydrates accumulated in the large fruitbodies, but were also partly metabolized in the colony. Reducing sugars were minor components, and included about 30 to 50% glucose and a small undetermined quantity of fructose. Glycogen was the main storage carbohydrate in the mycelium, and was also stored in the small fruitbodies. It was broken down in both structures during growth of the large fruitbodies which accumulated only small amounts. During the same period, almost 45% of the maximum amount of cell wall polysaccharides were degraded in the small fruitbodies, but not in the mycelium. By feeding 14C-glucose in replacement medium, incorporation of radioactivity into carbohydrates was followed in the colony during fruit-body development. Total incorporation was highest in trehalose, next highest in glycogen, and the rest was found in polyols and cell wall polysaccharides except for a few per cent which remained in endogenous glucose. In the large fruitbodies, specific radioactivity in glucose was much lower than in trehalose and mannitol. The labeling patterns in the mycelium and large fruitbodies suggested that trehalose, mannitol, and possibly arabitol were translocated into the stipes and pilei. 2 Present address: Department of Agricultural Chemistry, Faculty of Agriculture, Tottori University, 1-1 Koyama, Tottori 680, Japan. 1 This work was supported by Grant A-2371 from the National Research Council of Canada. This content is only available as a PDF. © 1976 American Society of Plant Biologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Zeiger, Eduardo; Hepler, Peter K.

doi: 10.1104/pp.58.4.492pmid: 16659703

Abstract Guard cell protoplasts (GCP) from young cotyledons of onion and tobacco were isolated in culture microchambers where optimal isolating and culture conditions could be determined in situ. The digestion course was quantified by following under polarized light the loss of.retardation of the birefringent cellulose of the guard cells. The assay showed that driselase has a 5-fold higher cellulytic activity than cellulysin. Driselase is, however, harmful to the GCP. Calcofluor staining was less adequate for establishing digestion courses because it increases sharply after exposing guard cells to cellulysin. Osmotic conditions were crucial for GCP survival. Onion guard cells fragment in the presence of strong plasmolyticum (>0.45 m) indicating cytoplasmic connections between neighboring guard cells and/or cytoplasmic attachments to the wall. Tobacco guard cells plasmolyzed with 0.7 m mannitol revealed several areas of strong attachment to the wall which resulted in severe damage to the cells. Healthy tobacco GCP are obtained by an initial digestion with 4% (w/v) cellulysin in 0.23 m mannitol for 2 to 3 hours followed by an increase in the osmoticum to 0.7 m to stabilize the forming protoplasts. Onion GCP were obtained by digesting paradermal slices with 4% (w/v) cellulysin in 0.23 m mannitol. Protoplasts can be osmotically released by replacing the enzyme solution with 0.23 m mannitol at early stages of digestion. They are also available after prolonged digestion (6-12 hours). Paradermal slices also yield mesophyll and epidermal cell protoplasts but they can be selectively washed away if a pure preparation of GCP is desired. Onion GCP have been kept alive in a simple culture solution for up to 10 days. 1 This work was supported by Grant BMS-74-15245 from the National Science Foundation. This content is only available as a PDF. © 1976 American Society of Plant Biologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Shaner, Dale L.; Boyer, John S.

doi: 10.1104/pp.58.4.499pmid: 16659704

Abstract The roles that leaf nitrate content and nitrate flux play in regulating the levels of nitrate reductase activity (NRA) were investigated in 8- to 14-day old maize (Zea mays L.) plants containing high nitrate levels while other environmental and endogenous factors were constant. The nitrate flux of intact plants was measured from the product of the transpiration rate and the concentration of nitrate in the xylem. NRA decreased when the seedlings were deprived of nitrate. The nitrate flux and the leaf nitrate content also decreased. When nitrate was resupplied to the roots, all three parameters increased. Attempts to alter the nitrate flux by varying transpiration rates were unsuccessful due to a relatively constant rate of delivery of nitrate to the xylem as transpiration rates fell. However, cooling the roots resulted in a decrease in the nitrate flux. Plants with a lower nitrate flux rapidly lost NRA, although the leaf nitrate content was initially unaffected. If the roots remained cool for a long enough time, the leaf nitrate content eventually decreased. Rewarming the roots increased the nitrate flux, leaf nitrate content, and NRA to control levels. When the nitrate flux in excised shoots was varied in three separate ways, decreasing the nitrate flux to the leaves resulted in a rapid decrease in NRA, although leaf nitrate contents were unchanged. These experiments show that the nitrate flux to the leaves from the roots plays a much larger regulatory role than the leaf nitrate content in controlling the level of NRA in intact plants. 2 Present address: Department of Plant Sciences, University of California-Riverside, Riverside, Calif. 92502. 1 This work was supported by University of Illinois fellowships to D. L. S. and National Science Foundation Grant GB 41314. This content is only available as a PDF. © 1976 American Society of Plant Biologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)