Plant Physiology

Youle, Richard J.; Huang, Anthony H. C.

doi: 10.1104/pp.58.6.703pmid: 16659749

Abstract Protein bodies from the storage endosperm of dry castor bean (Ricinus communis L.) were isolated by successive nonaqueous linear density gradient centrifugation. The isolated protein bodies were lysed by the addition of water, and the various structural components of the organelles were separated by sucrose gradient centrifugation. The matrix protein remained at the top of the gradient while the membrane, the crystalloids, and the globoids migrated to densities 1.15 g/cm3, 1.30 g/cm3, and > 1.46 g/cm3, respectively. The protein of the protein bodies was distributed evenly between the crystalloids and the matrix, and little protein was present in the globoids or the membrane. The proteins of the protein bodies were resolved into protein components of diverse molecular weights in sodium dodecyl sulfate-acrylamide gel electrophoresis. The protein components of the organelle matrix were distinct from those of the crystalloids. Whereas the matrix proteins had very diverse molecular weights, the crystalloid proteins were mainly composed of several proteins with molecular weights between 50,000 and 60,000 daltons. Also, the matrix proteins were soluble in water while the crystalloid proteins were insoluble in water but soluble in salt solution, thus representing albumins and globulins, respectively. Two of the matrix proteins with molecular weights approximately 120,000 and 65,000 daltons were identified as the phytohemagglutimin and the toxic protein ricin, respectively. During germination, the crystalloid proteins served as the storage protein and went through rapid degradation with smaller polypeptides formed as intermediates. In contrast, the proteins of the matrix under-went a much slower degradation during the same period and did not appear to be storage protein. 1 This work was supported by National Science Foundation Grant BMS 75-02320. 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)

Tully, Raymond E.; Beevers, Harry

doi: 10.1104/pp.58.6.710pmid: 16659750

Abstract Protein bodies in the endosperm of castor bean seeds (Ricinus communis L.) contain phytin globoids and protein crystalloids embedded in an amorphous proteinaceous matrix. The protein bodies are apparently surrounded by a single membrane. The protein bodies were isolated by grinding and centrifuging in glycerol. Such isolated protein bodies were almost identical (after cytological fixation) to those observed in situ, except that the globoids were lost. However, membrane-like structures appear to have surrounded the globoids. Histochemical analysis of the isolated protein bodies showed that carbohydrates (glycoproteins) are localized only in the matrix region. Addition of water to protein bodies in glycerol caused dissolution of the matrix, and release of the globoids and crystalloids. When the crystalloids were centrifuged on sucrose density gradients, they were recovered at an equilibrium density of 1.29 to 1.30 g/ml. The crystalloids were only slightly soluble in most aqueous buffers but were very soluble in sodium dodecyl sulfate, urea, or NaOH solutions. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and chromatography on ion exchange celluloses show that the protein bodies are composed of one major and several minor anodic proteins. The major protein, along with a few of the minor proteins, is localized in the crystalloids. The major protein (molecular weight 65,000) was converted by mercaptoethanol into subunits with molecular weights of 32,000 and 15,800. It is proposed that the protein is made up of two of the smaller subunits and one of the larger, linked by disulfide bridges. None of the crystalloid proteins appear to be glycosylated. The water-soluble matrix fraction is composed mainly of two proteins, with molecular weights of 12,500 and 10,300 on the gels. Neither is a glycoprotein, and neither can be reduced with mercaptoethanol to give subunits. The soluble fraction also contains other lesser components among which are several glycoprotein lectins. One of these is ricin D, which is the main glycoprotein in the protein bodies. 1 This work was supported by National Science Foundation Grant PCM 75-23566 to H. B., and by a National Science Foundation Graduate Fellowship to R. T. 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)

Falkner, Gernot; Horner, Franz; Werdan, Karl; Heldt, Hans W.

doi: 10.1104/pp.58.6.717pmid: 16659751

Abstract The pH in the cytoplasmic and thylakoid spaces of the blue-green alga, Anacystis nidulans, has been determined in the light and in the dark by uptake of 5,5-dimethyloxazolidine-2,4-dione and methylamine into the sucrose-impermeable 3H-H2O space, as measured by silicon layer filtering centrifugation. Illumination causes an alkalinization in the cytoplasm which is accompanied by an acidification in the thylakoid space, reflecting light-dependent proton transport across the thylakoid membrane. Under light conditions, a pH gradient of approximately 2.8 between the cytoplasmic and thylakoid spaces has been measured that can be abolished almost completely by addition of the uncoupler, 3-chlorocarbonyl cyanide phenylhydrazone. The pH in the cytoplasm is independent of the pH in the medium. 1 This work has been supported by a grant from the Österreichischer Forschungsförderungsfonds and the Deutsche Forschungsgemeinschaft. 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)

Vasconcelos, Aurea C.

doi: 10.1104/pp.58.6.719pmid: 16659752

Abstract Intact Euglena gracilis chloroplasts, which had been purified on gradients of silica sol, incorporated [35S]methionine or [3H]leucine into soluble and membrane-bound products, using light as the only source of energy. The chloroplasts were osmotically shocked, fractionated on discontinuous gradients of sucrose, and the products of protein synthesis of the different fractions characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The soluble fraction resolved into three zones of radioactivity, the major one corresponding to the large subunit or ribulose diphosphate carboxylase. The thylakoid membrane fraction contained nine labeled polypeptides, the two most prominent in the region of 31 and 42 kilodaltons. The envelope fraction contained a major radioactive peak of about 48 kilodaltons and four other minor peaks. The patterns of protein synthesis by isolated Euglena chloroplasts are broadly similar to those observed with chloroplasts of spinach and pea. 1 This work was supported in part by grants from the United States Public Health Service (No. HD-05602) and the Charles and Johanna Busch Memorial Fund to C. A. Price, and by a grant from the Rutgers Research Council to A. C. Vasconcelos. 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)

Strand, Larry L.; Rechtoris, Carol; Mussell, Harry

doi: 10.1104/pp.58.6.722pmid: 16659753

Abstract Purified polygalacturonases from two fungi released proteins from wall fractions prepared from three plant species. Peroxidase activity was associated with the proteins released from the cell walls, and several of the protein fractions released contained hydroxyproline. Cellulase, purified free of pectic enzyme activity, was ineffective in releasing cell wall proteins. Specific inhibition of endopolygalacturonase activity prevented release of the proteins. 1 Work supported in part by United States Department of Agriculture, CSRS Grant No. 316-15-42. 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)

Neyra, Carlos A.; Hageman, Richard H.

doi: 10.1104/pp.58.6.726pmid: 16659754

Abstract The observation that exposure of the leaf canopy to increasing concentrations of CO2 (100-400 μl/l) decreases the influx of nitrate to the leaf blades, but not to the roots or stalks (largely leaf sheaths), was reconfirmed using 15NO3−. Decreases in leaf nitrate supply were associated with decreases in induction of nitrate reductase, thus supporting the view that the influx of nitrate to a tissue is a major factor in regulation of the level of nitrate reductase. The whole plant 15N distribution data show that the CO2 effects were due to decreased influx of nitrate into the leaf blade rather than CO2-enhanced nitrate reduction. The decreases in nitrate accumulation by the leaf blade with increases in CO2 concentration were only partially accounted for by differences in transpiration. Because the initial malate concentration of root tissue (detopped plants) had no subsequent effect on nitrate uptake, it seems unlikely that high levels of malate induced by CO2 were responsible for the exclusion of nitrate from the leaf blades. Time course changes in nitrate and malate concentrations in root tissue (detopped plants) during nitrate uptake showed that oxidation of extra malate does not stimulate nitrate uptake and that malate is not specifically required as an energy source at the ion carrier level. The observation that nitrate and malate concentrations in corn leaf blades were negatively correlated was reconfirmed with 25 additional corn genotypes. However, using the same tissue, a higher correlation was obtained between malate plus aconitate and nitrate, suggesting that organic acids other than malate could be involved. The proposal that reduction of nitrate in the leaf is stoichiometrically related to malate production is a valid explanation of the relationship only if malate oxidation does not provide NADH for nitrate reduction. However, addition of malate and NAD to crude extracts (in vitro assay) or malate to leaf blade sections (in vivo assay) caused nitrate reduction. Because of these observations and the known intracellular location of NAD-malate dehydrogenase and nitrate reductase, we believe that malate oxidation is one of the major sources of NADH for nitrate reduction in corn leaf blades in situ. 2 Present address: Embrapa R. J., Km 47, Via Campo Grande, Rio de Janeiro, Brazil. 1 This work was supported by Hatch funds and a Frasch Foundation grant. C. A. N. gratefully acknowledges the assistance of a fellowship grant from MUCIA-Universidad Agraria La Molina, Peru. 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)

Nicholas, Joseph C.; Harper, James E.; Hageman, Richard H.

doi: 10.1104/pp.58.6.731pmid: 16659755

Abstract The optimum in vivo nitrate reductase (NR) assay medium for soybean (Glycine max [L.] Merr.) leaves was 50 mm KNO3, 1% (v/v) 1- propanol, and 100 mm potassium phosphate buffer (pH 7.5). Loss of in vivo NR activity from leaves of soybeans exposed to dark was fastest at 40 C and slowest at 20 C. However, by the end of a 16-hr dark period, even those plants exposed to the lowest (20 C) temperature had lost 95% of the initial activity. Upon re-exposure to light, following a 16 hr-30 C dark period, in vivo NR activity increased rapidly to maximum levels after 4 hr light. The rate of increase was proportional to light intensity (6, 16, and 45 klux) and independent of temperature (20, 30, and 40 C). Studies with field-grown soybeans indicated that mighttime temperature (16-27 C) had no effect on the subsequent in vivo NR activity in sunlight at ambient temperature. There was a marked decrease in in vivo NR activity in late afternoon with the field-grown plants. This decrease continued throughout the night with elevated temperature (27 C) while NR activity increased when a cooler (16 C) night temperature was imposed. The changes in in vivo NR activity in response to light and dark treatments were quite rapid and thought to be related to energy limitations as well as enzyme level. 1 Cooperative investigation of the North Central Region, Agricultural Research Service, U. S. Department of Agriculture and the Illinois Agricultural Experiment Station. Research was supported in part by the National Soybean Crop Improvement Council. 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)

Nicholas, Joseph C.; Harper, James E.; Hageman, Richard H.

doi: 10.1104/pp.58.6.736pmid: 16659756

Abstract Growth chamber studies with soybeans (Glycine max [L.] Merr.) were designed to determine the relative limitations of NO3−, NADH, and nitrate reductase (NR) per se on nitrate metabolism as affected by light and temperature. Three NR enzyme assays (+NO3−in vivo, −NO3−in vivo, and in vitro) were compared. NR activity decreased with all assays when plants were exposed to dark. Addition of NO3− to the in vivo NR assay medium increased activity (over that of the −NO3−in vivo assay) at all sampling periods of a normal day-night sequence (14 hr-30 C day; 10 hr-20 C night), indicating that NO3− was rate-limiting. The stimulation of in vivo NR activity by NO3− was not seen in plants exposed to extended dark periods at elevated temperatures (16 hr-30 C), indicating that under those conditions, NO3− was not the limiting factor. Under the latter condition, in vitro NR activity was appreciable (19 μmol NO2− [g fresh weight, hr]−1) suggesting that enzyme level per se was not the limiting factor and that reductant energy might be limiting. The addition of NADH to the in vivo NR assay medium did not stimulate NR activity, although it was not established that NADH entered the tissue. The addition of glucose, fructose 1,6-diphosphate, pyruvate, citrate, succinate, or malate to the in vivo assay medium significantly increased measurable NR activity of leaf tissue from plants pretreated to extended dark periods at elevated temperature. Glucose additions were most effective, usually stimulating increases 2- to 3-fold greater than the other metabolites. Increased NR activities from the various additives were attributed to production of NADH. The loss of in vivo NR activity in soybeans during darkness appeared to be due to the combination of a net loss of enzyme per se and energy depletion. The subsequent light stimulation of NR activity was likely due to increased availability of reductant energy as well as a net synthesis of the NR enzyme. 1 Cooperative investigation of the North Central Region, Agricultural Research Service, U. S. Department of Agriculture and the Department of Agronomy, Illinois Agricultural Experiment Station. Research was supported in part by the National Soybean Crop Improvement Council. 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)

Wedding, Randolph T.; Black, M. Kay; Pap, Dennis

doi: 10.1104/pp.58.6.740pmid: 16659757

Abstract Over a range of concentrations from less than 0.1 mm to more than 70 mm, sweet potato root mitochondria display a bimodal substrate saturation isotherm for malate. The high affinity portion of the isotherm has an apparent Km for malate of 0.85 mm and fits a rectangular hyperbolic function. The low affinity portion of the isotherm is sigmoid in character and gives an apparent S 0.5 of 40.6 mm and a Hill number of 3.7. Extracts of sweet potato mitochondria contain both malate dehydrogenase and NAD malic enzyme. The malate dehydrogenase, assayed in the forward direction at pH 7.2, shows typical Michaelis-Menten kinetics with a Km for malate of 0.38 mm. The NAD malic enzyme shows pronounced sigmoidicity in response to malate with a Hill number of 3.5 and an S 0.5 of 41.6 mm. On the basis of the normal kinetics, the Km, and the fact that oxaloacetate production from malate by mitochondria appears most active at low malate concentrations, the high affinity portion of the malate isotherm with mitochondria is attributed to malate dehydrogenase. The low affinity portion of the malate isotherm with mitochondria is thought, on the basis of the similarity of S 0.5 values, the Hill numbers, and the greater production of pyruvate from malate at high malate concentrations, to represent the activity of the NAD malic enzyme. 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)

Frasch, Wayne; Grunwald, Claus

doi: 10.1104/pp.58.6.744pmid: 16659758

Abstract In tobacco seedlings (Nicotiana tabacum L.), glucose from supplied uridine diphosphate-[U-14C]glucose was first incorporated into steryl glycosides and later into acylated steryl glycosides. However, when [14C]cholesterol was used as substrate, the acylated steryl glycosides became labeled earlier than the steryl glycosides. With [14C]cholesteryl glucoside as substrate, most of the radioactive label was recovered as free sterol, and the acylated steryl glycosides were not readily labeled; however, palmitoyl [14C]cholesteryl glucoside was rapidly converted to steryl glycoside. In feeding experiments with free sterol, an unknown, highly radioactive steroid component was isolated. Incorporation of radioactivity into the unknown occurred before the acylated steryl glycosides were labeled. It is postulated that two pathways exist for the biosynthesis of acylated steryl glycoside: one through steryl glycosides, and the other through an unidentified steroid component. It is the latter pathway which appears to be dominant in the in vivo tobacco system. 2 This work is part of M.S. thesis. 3 Present address: Botany and Plant Pathology, Illinois Natural History Survey, Urbana, Illinois 61801. 1 This paper (No. 76-3-95) is part of a project of the Kentucky Agricultural Experiment Station and is published with approval of the Director. 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)