Plant Physiology

Meza-Basso, Luis; Alberdi, Miren; Raynal, Monique; Ferrero-Cadinanos, Maria-Luz; Delseny, Michel

doi: 10.1104/pp.82.3.733pmid: 16665102

Abstract Changes induced by cold treatment in young rapeseed (Brassica napus) seedlings were investigated at the molecular level. Following germination at 18°C for 48 hours, one half of the seedlings was transferred to 0°C for another 48 hour period, the other half being kept at 18°C as a control. Newly synthesized proteins were labeled for the last 6 hours of incubation with [35S]methionine. The different polypeptides were separated by two-dimensional electrophoresis in polyacrylamide gels. Newly synthesized proteins were revealed by fluorography. Protein synthesis clearly continues at 0°C and some polypeptides preferentially accumulate at this temperature. On the other hand, synthesis of several others is repressed while many are insensitive to cold treatment. Similar changes are also observed when mRNA is prepared from cold treated seedlings, translated in vitro in a reticulocyte cell free system and compared with the products of mRNA extracted from control samples. Among the genes which are repressed we identified the small subunit of ribulose 1,6-bisphosphate carboxylase. These changes are also detectable after shorter treatments. 2 Present address: Instituto de Bioquimica, Universidad Austral de Chile, U.A.C.H. - Casilla 567, Valdivia, Chile. 3 Present address: Instituto de Botanica, Universidad Austral de Chile, U.A.C.H. - Casilla 567, Valdivia, Chile. 4 Present address: Instituto de Biologia Celular 1 CS1C, 144 Velazquez, Madrid-28006, Spain. 1 Supported by the CNRS (UA 565 and A.I.P. 95 31 67) in France and by grants from Direccioń de investigacion (UACH S-84-29, RS-83-19 and FNC 1212-84) in Chile. Part of this work was carried out while Dr. Luis Meza-Basso was on leave from UACH and was a visiting Professor at the University Louis Pasteur in Strasbourg; M.L. Ferrero-Cadinanos was supported by a student fellowship from the exchange programme between the Spanish CSIC and the French CNRS and by the Réseau européen de Biologie moléculaire végétale. This content is only available as a PDF. © 1986 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)

Maki, Hisae; Yamagishi, Kenji; Sato, Takahide; Ogura, Nagao; Nakagawa, Hiroki

doi: 10.1104/pp.82.3.739pmid: 16665103

Abstract An enzyme-linked immunosorbent assay permitting the determination of nanogram quantities of nitrate reductase (NR) in cultured spinach cells has been developed and used for studies of the mechanism by which NR activity is regulated as a function of culture age. When 8-day old spinach cells were transferred to fresh medium, NR activity increased markedly in 2 days and thereafter decreased gradually until it became undetectable on the 10th day after the transfer. Determination of the amounts of NR by the immunosorbent assay indicated that the unique alteration of NR activity could be accounted for by the concomitant change in the amount of NR protein. Immunoblotting analysis of the subunit of NR also supported this result. It is concluded that the regulation of NR in spinach cells as a function of culture age is mediated by changes in the amount of the enzyme protein rather than by activation and inactivation of the preexisting proteins. 1 Supported by Grant-in-Aid for Scientific Research (No. 60116001) from the Ministry of Education, Science and Culture. This content is only available as a PDF. © 1986 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)

MacDonald, Elizabeth M. S.; Powell, Gary K.; Regier, Dean A.; Glass, N. Louise; Roberto, Franciso; Kosuge, Tsune; Morris, Roy O.

doi: 10.1104/pp.82.3.742pmid: 16665104

Abstract Cytokinin production by strains of the phytopathogenic bacterium Pseudomonas syringae pv savastanoi was measured by immunoaffinity chromatography of the culture medium on immobilized anti-cytokinin antibodies, followed by high performance liquid chromatography, radioimmunoassay and mass spectrometry. P. savastanoi strain PB213-2 secretes zeatin (80 nanograms per milliliter) and ribosylzeatin (80 nanograms per milliliter). Even higher levels of zeatin (400 nanograms per milliliter) are produced by the olive-specific strain EW1006, which also produces 180 nanograms per milliliter of the recently identified cytokinin, ribosyl-1″ -methylzeatin. The amounts secreted were approximately 1000 times greater than those secreted by Agrobacterium tumefaciens (DA Regier, RO Morris 1982 Biochem Biophys Res Commun 104: 1560-1566). Examination of cytokinin production by plasmid deletion mutants of PB213-2 and EW1006 indicated that cytokinin biosynthesis was specified, at least in part, by plasmid-borne genes. A fragment of the 105 kilobase pair plasmid from EW1006 was cloned into Escherichia coli where its expression resulted in dimethylallyl transferase activity and the secretion of zeatin. 2 Present address: Department of Biochemistry SJ-70, University of Washington, Seattle, WA 98195. 1 Supported by Grants PCM 83-03371 from the National Science Foundation and 83-CRCR-1-1249 from the Competitive Research Grants Office, Science and Education Administration, United States Department of Agriculture. F. R. and N. L. G. supported by fellowships from funds awarded to the University of California, Davis, by the McNight Foundation. Technical Paper No. 7626 from the Oregon State University Agricultural Experiment Station. This content is only available as a PDF. © 1986 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)

Kaushal, Gur P.; Elbein, Alan D.

doi: 10.1104/pp.82.3.748pmid: 16665105

Abstract The GlcNAc-1-P-transferase was solubilized from microsomal preparations of soybean cultured cells by treatment with 1% Triton X-100. The solubilized enzyme catalyzed the formation of dolichyl pyrophosphoryl-GlcNAc when incubated with UDP-GlcNAc and dolichyl phosphate. The GlcNAc-1-P-transferase activity was stimulated by the addition of phosphatidylglycerol and phosphatidylinositol, but was inhibited by phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. The Km value for dolichyl-phosphate was 6.2 micromolar and that determined for UDP-GlcNAc was 0.42 micromolar. The pH optimum for the GlcNAc-1-P reaction was between 7.2 and 7.6; maximum activity occurred at about 10 millimolar Mg2+. The addition of unlabeled GDP-mannose or UDP-glucose considerably inhibited enzyme activity which could be restored to nearly the original value by addition of more dolichyl phosphate to the incubation mixture. On the other hand, the addition of unlabeled ADP-glucose and GDP-glucose enhanced the enzyme activity. This stimulation by these sugar nucleotides was found to be due to the protection of the substrate UDP-[3H]-GlcNAc from pyrophosphatase degradation. The GlcNAc-1-P-transferase reaction was very sensitive to tunicamycin and 50% inhibition required less than 1 microgram of antibiotic per milliliter. Amphomycin, showdomycin, and diumycin also inhibited this reaction but at higher concentrations. 1 Supported by a grant from the National Institutes of Health (AM 21800). This content is only available as a PDF. © 1986 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)

Weigel, Pierre; Weretilnyk, Elizabeth A.; Hanson, Andrew D.

doi: 10.1104/pp.82.3.753pmid: 16665106

Abstract Chenopods synthesize betaine by a two-step oxidation of choline: choline → betaine aldehyde → betaine. Both oxidation reactions are carried out by isolated spinach (Spinacia oleracea L.) chloroplasts in darkness and are promoted by light. The mechanism of betaine aldehyde oxidation was investigated with subcellular fractions from spinach leaf protoplasts. The chloroplast stromal fraction contained a specific pyridine nucleotide-dependent betaine aldehyde dehydrogenase (about 150 to 250 nanomoles per milligram chlorophyll per hour) which migrated as one isozyme on native polyacrylamide gels stained for enzyme activity. The cytosol fraction contained a minor isozyme of betaine aldehyde dehydrogenase. Leaves of pea (Pisum sativum L.), a species that lacks betaine, had no betaine aldehyde dehydrogenase isozymes. The specific activity of betaine aldehyde dehydrogenase rose three-fold in spinach plants grown at 300 millimolar NaCl; both isozymes contributed to the increase. Stimulation of betaine aldehyde oxidation in illuminated spinach chloroplasts was due to a thylakoid activity which was sensitive to catalase; this activity occurred in pea as well as spinach, and so appears to be artifactual. We conclude that in vivo, betaine aldehyde is oxidized in both darkness and light by the dehydrogenase isozymes, although some flux via a light-dependent, H2O2-mediated reaction cannot be ruled out. 2 Permanent address: Laboratoire de Biologie et Physiologie Végétales, Université de Rennes I, Rennes, France. P. W. was supported by a fellowship from the French Ministry of Foreign Affairs. 1 Funded by Department of Energy Contract DE-AC02-76ERO-1338, and by grants from CIBA-GEIGY Corporation and the Michigan Sugar Company. This content is only available as a PDF. © 1986 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)

Mayfield, Stephen P.; Nelson, Timothy; Taylor, William C.

doi: 10.1104/pp.82.3.760pmid: 16665107

Abstract Maize seedlings, treated with the herbicide norflurazon to produce a deficiency in carotenoid pigments, were grown in low-fluence-rate light. Under these conditions, which induced chlorophyll biosynthesis while minimizing photooxidation, carotenoid-deficient seedlings showed identical patterns of chloroplast protein accumulation compared with normal seedlings. Carotenoid pigments thus play no direct role in regulating the accumulation of chloroplast proteins. When shifted to high-fluence-rate light, chlorophyll was rapidly photooxidized in carotenoid-deficient seedlings. Chloroplast proteins showed varying degrees of sensitivity to photooxidation. The P-700 apoprotein of photosystem I was rapidly degraded. Most stromal and thylakoid proteins either decreased progressively in photooxidative conditions or appeared to be unaffected. The relative quantity of the light-harvesting chlorophyll a/b-binding protein of photosystem II increased significantly in the first few hours of high-fluence-rate light. It then appeared to be only minimally affected 18 hours after complete photooxidation of chlorophyll. 2 Present address: Department de Biologie Moleculaire, Sciences II, Universite de Geneve, 30, quai Ernest-Ansermet, CH-1211 Geneve 4, Switzerland. 3 Present address: Department of Biology, Yale University, P.O. Box 6666, New Haven, CT 06511. 1 Supported by grants from the Competitive Research Grants Office of the United States Department of Agriculture to W. T. This content is only available as a PDF. © 1986 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)

Augé, Robert M.; Schekel, Kurt A.; Wample, Robert L.

doi: 10.1104/pp.82.3.765pmid: 16665108

Abstract Osmotic adjustment in Rosa hybrida L. cv Samantha was characterized by the pressure-volume approach in drought-acclimated and unacclimated plants brought to the same level of drought strain, as assayed by stomatal closure. Plants were colonized by either of the vesicular-arbuscular mycorrhizal fungi Glomus deserticola Trappe, Bloss and Menge or G. intraradices Schenck and Smith, or were nonmycorrhizal. Both the acclimation and the mycorrhizal treatments decreased the osmotic potential (Ψπ) of leaves at full turgor and at the turgor loss point, with a corresponding increase in pressure potential at full turgor. Mycorrhizae enabled plants to maintain leaf turgor and conductance at greater tissue water deficits, and lower leaf and soil water potentials, when compared with nonmycorrhizal plants. As indicated by the Ψπ at the turgor loss point, the active Ψπ depression which attended mycorrhizal colonization alone was 0.4 to 0.6 megapascals, and mycorrhizal colonization and acclimation in concert 0.6 to 0.9 megapascals, relative to unacclimated controls without mycorrhizae. Colonization levels and sporulation were higher in plants subjected to acclimation. In unacclimated hosts, leaf water potential, water saturation deficit, and soil water potential at a particular level of drought strain were affected most by G. intraradices. G. deserticola had the greater effect after drought preconditioning. 1 Scientific paper No. 7485, College of Agriculture and Home Economics Research Center, Washington State University. This content is only available as a PDF. © 1986 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)

Terzaghi, William B.

doi: 10.1104/pp.82.3.771pmid: 16665109

Abstract The development of a system for modifying the membrane fatty acid composition of cultured soybean cells (Glycine max [L.] Merr.) is described. Tween-fatty acid esters carrying specific fatty acids were synthesized and added to the medium of suspension cultures. Cells transferred large quantities of exogenous fatty acids from Tweens to all acylated membrane lipids; up to 50% of membrane fatty acids were exogenously derived. C15 to C20 saturated fatty acids and C16, C18, and C20 unsaturated fatty acids with either cis or trans double bonds were incorporated into lipids. Cells elongated saturated fatty acids of C16 or less, and unsaturated fatty acids with cis double bonds were further desaturated. No other types of modifications were observed. Growth ceased in cells treated with excessive concentrations of Tween-fatty acid esters, but frequently not for several days. Cessation of cell growth was correlated with changes in membrane fatty acid composition resulting from incorporation of large amounts of exogenous fatty acids into membrane lipids, although cells tolerated large variations in fatty acid composition. Maximum tolerable Tween concentrations varied widely according to the fatty acid supplied. Potential uses of this system and implications of the observed modifications on the pathway of incorporation are discussed. 2 Supported by National Science Foundation predoctoral fellowship SPE 835-0132 and by a University of Utah graduate research fellowship. Present address: Carnegie Institution of Washington, Department of Plant Biology, Stanford CA 94305. 1 Supported by National Institute of Environmental Health Sciences grant 01498 to Dr. Karl G. Lark. This research was conducted in partial fulfillment of the doctoral degree requirement of the Department of Biology, University of Utah. This content is only available as a PDF. © 1986 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)

Terzaghi, William B.

doi: 10.1104/pp.82.3.780pmid: 16665110

Abstract Uptake of Tween-fatty acid esters and incorporation of the fatty acids into lipids by soybean (Glycine max [L.] Merr.) suspension cultures was investigated, together with subsequent turnover of the incorporated fatty acids and associated changes in endogenous fatty acid synthesis. Tween uptake was saturable, and fatty acids were rapidly transferred from Tweens to all acylated lipids. Patterns of incorporation into glycerolipids were similar in cells treated with Tweens carrying [1-14C]-fatty acids and in cells treated with [1-14C]acetate, indicating that exogenous fatty acids were used for glycerolipid synthesis essentially as if they had been made by the cell. In Tween-treated cells neutral lipids (which include Tweens) initially accounted for the majority of lipid radioactivity. Radioactivity was then rapidly transferred to glycerolipids. A transient pool of free fatty acids accounting for up to 10% of lipid radioactivity was observed. This was consistent with the hypothesis that fatty acids are transferred from Tweens to lipids by deacylation of the Tweens, creating a pool of free fatty acids which are then used for lipid synthesis. Sterols were only slightly labeled in cells treated with Tweens, but accounted for nearly 50% of lipid radioactivity in cells treated with acetate. This suggested very little degradation and reutilization of the radioactive fatty acids in cells treated with Tweens. In cells treated with either [1-14C]acetate or Tween-[1-14C]-18:1, 70% of the initial fatty acid radioactivity remained in fatty acids after a 100 hour chase. By contrast, fatty acids not normally present disappeared more rapidly, suggesting differential treatment of such fatty acids compared with those normally present. Cells which had incorporated large amounts of exogenous fatty acids altered fatty acid synthesis in three distinct ways: (a) amounts of [1-14C]acetate incorporated into fatty acids were reduced; (b) cells incorporating exogenous unsaturated fatty acids increased the proportion of [1-14C]acetate partitioned into saturated fatty acids, while the converse was true of cells which had incorporated exogenous saturated fatty acids; (c) desaturation of 18:1 to 18:2 and 18:3 was reduced in cells which had incorporated unsaturated fatty acids. These results suggest that Tween-fatty acid esters will be useful for supplying fatty acids to cells for a variety of studies related to fatty acid or membrane metabolism. 2 Supported by National Science Foundation predoctoral fellowship SPE 835-0132 and by a University of Utah graduate research fellowship. Present address: Carnegie Institution of Washington, Department of Plant Biology, Stanford CA 94305. 1 Supported by National Institute of Environmental Health Sciences grant 01498 to Dr. Karl G. Lark. This research was conducted in partial fulfillment of the doctoral degree requirement of the Department of Biology, University of Utah. This content is only available as a PDF. © 1986 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)

Moore, Patricia J.; Darvill, Alan G.; Albersheim, Peter; Staehelin, L. Andrew

doi: 10.1104/pp.82.3.787pmid: 16665111

Abstract Plant cell walls serve several functions: they impart rigidity to the plant, provide a physical and chemical barrier between the cell and its environment, and regulate the size and shape of each cell. Chemical studies have provided information on the biochemical composition of the plant cell walls as well as detailed knowledge of individual cell wall molecules. In contrast, very little is known about the distribution of specific cell wall components around individual cells and throughout tissues. To address this problem, we have produced polyclonal antibodies against two cell wall matrix components; rhamnogalacturonan I (RG-I), a pectic polysaccharide, and xyloglucan (XG), a hemicellulose. By using the antibiodies as specific markers we have been able to localize these polymers on thin sections of suspension-cultured sycamore cells (Acer pseudoplatanus). Our results reveal that each molecule has a unique distribution. XG is localized throughout the entire wall and middle lamella. RG-I is restricted to the middle lamella and is especially evident in the junctions between cells. These observations indicate that plant cell walls may have more distinct chemical (and functional?) domains than previously envisaged. 2 Present address: Complex Carbohydrate Research Center, Richard B. Russel Agricultural Research Center, Athens, GA 30613. 1 Supported by National Institutes of Health-GM 18639 to L. A. S., Department of Energy number DE-FG09-85-ER13426 to P. A., and National Science Foundation DMBB85-45798 to A.G.D. This content is only available as a PDF. © 1986 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)