Plant Physiology

Scheibe, Renate

doi: 10.1104/pp.96.1.1pmid: 16668135

Abstract Assimilation of C, N, and S into organic compounds requires effective and flexible cooperation among the energy-converting, tightly coupled, thylakoid-bound processes and stromal metabolism. Fluctuations of light, temperature, and changing concentrations of the various reducible substrates pose unique regulatory problems to photoautotrophic plant cells. Covalent redox modification of enzyme proteins as mediated by the ferredoxin/thiore-doxin-system is suited to provide short-term adaptation of various enzymatic activities in the chloroplast. This mode of regulation is based on the continuous turnover of interconvertible enzyme forms, as in the systems driven by protein phosphorylation/dephosphorylation, but is particularly adapted to the unique conditions of a compartment performing oxygenic photosynthesis by depending on the simultaneous presence of reducing power and of oxygen. Individual fine control of each of the enzymes subjected to redox modification is achieved by specific metabolites acting as additional positive or negative effectors of the reductive (and/or oxidative) modification reaction. The biochemical prerequisite for such a control is the presence of regulatory (extra) sequences carrying cysteine residues which are subjected to reversible redox changes. Although no common amino acid sequence has yet been identified among the known regulatory peptides, in all cases the evolution of autotrophy should be related to the presence of extrasequences in otherwise very conserved enzyme molecules. 1 Supported by grants from the Deutsche Forschungsgemeinschaft. This content is only available as a PDF. © 1991 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)

Veierskov, Bjarke; Ferguson, Ian B.

doi: 10.1104/pp.96.1.4pmid: 16668183

Abstract Conjugation of the polypeptide ubiquitin to endogenous proteins was studied in oat (Avena sativa L.) plants, and particularly in green tissues. Conjugating activity in leaf extracts was different from that in root extracts, and in both was less than in etiolated tissue. The conjugates were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and their formation was both time- and ATP-dependent and had a pH optimum of about 8.2. The assay had a high affinity for ATP with a probable K m of less than 50 micromolar. The ubiquitin conjugating system was also shown to be present in isolated chloroplasts, and ubiquitin could be conjugated to endogenous proteins of lyzed chloroplasts in which the ATP concentrations were reduced by preincubation or desalting. SDS-PAGE analysis led to the suggestion that the large and small subunits of ribulose-1,5-bisphosphate carboxylase (RuBPCase) may be able to be ubiquitinated, and we have shown that ubiquitin can stimulate the in vitro breakdown of 125I-labeled RuBPCase. These results invite the speculation that ubiquitin may be involved in the regulation of protein turnover in green plants. 2 Present address: Institute of Plant Biology, Royal Veterinary & Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark. 1 Supported in part by grant 13-4090-M from the Danish Agricultural and Veterinary Council. This content is only available as a PDF. © 1991 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)

Halkier, Barbara Ann; Møller, Birger Lindberg

doi: 10.1104/pp.96.1.10pmid: 16668136

Abstract The biosynthesis of the tyrosine-derived cyanogenic glucoside dhurrin involves N-hydroxytyrosine, (E)- and (Z)-p-hydroxyphenylacetaldehyde oxime, p-hydroxyphenylacetonitrile, and p-hydroxymandelonitrile as intermediates and has been studied in vitro using a microsomal enzyme system obtained from etiolated sorghum (Sorghum bicolor [L.] Moench) seedlings. The biosynthesis is inhibited by carbon monoxide and the inhibition is reversed by 450 nm light demonstrating the involvement of cytochrome P-450. The combined use of two differently prepared microsomal enzyme systems and of tyrosine, p-hydroxyphenylacetaldehyde oxime, and p-hydroxyphenylacetonitrile as substrates identify two cytochrome P-450-dependent monooxygenases: the N-hydroxylase which converts tyrosine into N-hydroxytyrosine and the C-hydroxylase converting p-hydroxyphenylacetonitrile into p-hydroxymandelonitrile. The inhibitory effect of a number of putative cytochrome P-450 inhibitors confirms the involvement of cytochrome P-450. Monospecific polyclonal antibodies raised toward NADPH-cytochrome P-450-reductase isolated from sorghum inhibits the same metabolic conversions as carbon monoxide. No cytochrome P-450-dependent monooxygenase catalyzing an N-hydroxylation reaction has previously been reported in plants. The metabolism of p-hydroxyphenylacetaldehyde oxime is completely dependent on the presence of NADPH and oxygen and results in the production of p-hydroxymandelonitrile with no accumulation of the intermediate p-hydroxyphenylacetonitrile in the reaction mixture. The apparent NADPH and oxygen requirements of the oxime-metabolizing enzyme are identical to those of the succeeding C-hydroxylase converting p-hydroxyphenylacetonitrile to p-hydroxymandelonitrile. Due to the complex kinetics of the microsomal enzyme system, these requirements may not appertain to the oxime-metabolizing enzyme, which may convert p-hydroxyphenylacetaldehyde oxime to p-hydroxyacetonitrile by a simple dehydration. 1 This work was partially supported by the Danish International Development Agency (DANIDA), the Danish Government Program for Biotechnology Research, The Rockefeller Foundation, the Commission of the European Communities programme Science and Technology for Development, the Danish Agricultural and Veterinary Research Council, the Carlsberg Foundation and Stiftelsen Hofmansgave. This content is only available as a PDF. © 1991 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)

Hunt, Dale C.; Chrispeels, Maarten J.

doi: 10.1104/pp.96.1.18pmid: 16668149

Abstract A cytosolic pea (Pisum sativum) seed albumin (ALB) and a chimeric protein (PHALB) consisting of the signal peptide and first three amino acids of phytohemagglutinin (PHA) and the amino acid sequence of ALB were expressed in parallel suspension cultures of tobacco (Nicotiana tabacum) cells and their intracellular fates examined. PHALB was efficiently secreted by the cells whereas ALB remained intracellular. These experiments show that the information contained in the signal peptide of a vacuolar protein is both necessary and sufficient for efficient secretion, and define secretion as a default or bulk-flow pathway. Entry into the secretory pathway was accompanied by glycosylation and the efficient conversion of the high mannose glycans into complex glycans indicating that transported glycoproteins do not need specific recognition domains for the modifying enzymes in the Golgi. Tunicamycin depressed the accumulation of the unglycosylated polypeptide in the culture medium much less than the accumulation of other glycoproteins. We interpret this as evidence that glycans on proteins that are not normally glycosylated do not have the same function of stabilizing and protecting the polypeptide as on natural glycoproteins. 1 Supported by a grant from the National Science Foundation (Cell Biology) and a contract from the United States Department of Energy (Basic Energy Biosciences). This content is only available as a PDF. © 1991 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)

Lax, Alan R.; Vaughn, Kevin C.

doi: 10.1104/pp.96.1.26pmid: 16668162

Abstract Polyphenol oxidase (PPO) appears to be ubiquitous in higher plants but, as yet, no function has been ascribed to it. Herein, we report on the localization of PPO based upon biochemical fractionation of chloroplast membranes in Vicia faba (broad bean) into various complexes and immunocytochemical electron microscopic investigations. Sucrose density gradient fractionations of thylakoid membranes after detergent solubilization reveals that PPO protein (by reactivity with anti-PPO antibody) and activity (based upon ability to oxidize di-dihydroxyphenylalanine) are found only in fractions enriched in photosystem II (PSII). Furthermore, of the PSII particles isolated using three different protocols utilizing several plant species, all had PPO. Immunogold localization of PPO on thin sections reveals exclusive thylakoid labeling with a distribution pattern consistent with other PSII proteins (80% grana, 20% stroma). These data strongly indicate that PPO is at least peripherally associated with the PSII complex. This content is only available as a PDF. © 1991 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)

Ji, Lin; Wood, Stephen; Becana, Manuel; Klucas, Robert V.

doi: 10.1104/pp.96.1.32pmid: 16668174

Abstract A ferric leghemoglobin reductase from the cytosol of soybean (Glycine max) root nodules was purified to homogeneity and partially characterized. The enzyme is a flavoprotein with flavin adenine dinucleotide as the prosthetic group and consists of two identical subunits, each having a molecular mass of 54 kilodaltons. The pure enzyme shows a high activity for ferric leghemoglobin reduction with NADH as the reductant in the absence of any exogenous mediators. The enzyme also exhibits NADH-dependent 2,6-dichloroindophenol reductase activity. A sequence of the first 50 N-terminal amino acids of the purified protein was obtained. Comparisons with known protein sequences have shown that the sequence of the ferric leghemoglobin reductase is highly related to those of the flavin-nucleotide disulfide oxido-reductases, especially dihydrolipoamide dehydrogenase of the pyruvate dehydrogenase complex. 2 Permanent address: Laboratorio de Fijación de Nitrógeno, E.E. Aula Dei, Apdo. 202, 50080 Zaragoza, Spain. 1 Journal Series No. 9404, Agricultural Research Division, University of Nebraska. This content is only available as a PDF. © 1991 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)

Lewinsohn, Efraim; Gijzen, Mark; Savage, Thomas J.; Croteau, Rodney

doi: 10.1104/pp.96.1.38pmid: 16668182

Abstract Cell-free extracts from Pinus ponderosa Lawson (ponderosa pine) and Pinus sylvestris L. (Scotch pine) wood exhibited high levels of monoterpene synthase (cyclase) activity, whereas bark extracts of these species contained no detectable activity, and they inhibited cyclase activity when added to extracts from wood, unless polyvinylpyrrolidone was included in the preparation. The molecular mass of the polyvinylpyrrolidone added was of little consequence; however, polyvinylpolypyrrolidone (a cross-linked insoluble form of the polymer) was ineffective in protecting enzyme activity. Based on these observations, methods were developed for the efficient extraction and assay of monoterpene cyclase activity from conifer stem (wood and bark) tissue. The level of monoterpene cyclase activity for a given conifer species was shown to correlate closely with the monoterpene content of the oleoresin and with the degree of anatomical complexity of the specialized resin-secreting structures. Cyclase activity and monoterpene content were lowest in the stems of species containing only isolated resin cells, such as western red cedar (Thuja plicata D. Don). Increasing levels of cyclase activity and oleoresin monoterpenes were observed in advancing from species with multicellular resin blisters (true firs [Abies]) to those with organized resin passages, such as western larch (Larix occidentalis Nutt.), Colorado blue spruce (Picea pungens Engelm.) and Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco). The highest levels of cyclase activity and oleoresin monoterpenes were noted in Pinus species that contain the most highly developed resin duct systems. The relationship between biosynthetic capacity, as measured by cyclase activity, monoterpene content, and the degree of organization of the secretory structures for a given species, may reflect the total number of specialized resin-producing cells per unit mass of stem tissue. 2 McKnight Foundation Postdoctoral Fellow. 3 McKnight Foundation Graduate Fellow. 1 Research supported in part by U.S. Department of Agriculture grant 88-37253-3942, Project 0268 from the Washington State University Agricultural Research Center, and by a McKnight Foundation Grant. This content is only available as a PDF. © 1991 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)

Lewinsohn, Efraim; Gijzen, Mark; Croteau, Rodney

doi: 10.1104/pp.96.1.44pmid: 16668184

Abstract Levels of monoterpene cyclase activity were determined in extracts from wounded and unwounded saplings of 10 conifer species to assess whether oleoresin biosynthesis is induced by stem wounding. Species of Abies and Picea, with low to moderate levels of constitutive monoterpene cyclase activity, exhibited a five- to 15-fold increase in cyclase activity 7 days after wounding relative to unwounded controls. In contrast, species of genera such as Pinus, with high levels of constitutive cyclase activity, did not significantly respond to wounding by alteration in the level of cyclase activity. The highest fold increase in monoterpene cyclase activity was consistently observed in Abies grandis, and the time-course of induction of activity following stem wounding in this species demonstrated a threefold increase at 2 days relative to unwounded controls, rising to a maximum increase in the response at 9 days (greater than 10-fold) followed by an apparent decline. The wound response was localized, and both bark (phloem) and wood (xylem) tissues displayed increased cyclase activity at the wound site. The magnitude of the increase in cyclase activity was dependent on the severity of the wound. 2 McKnight Foundation Postdoctoral Fellow. 1 Research supported in part by U.S. Department of Agriculture Grant 88-37253-3942, Project 0268 from the Washington State University Agricultural Research Center, and by a McKnight Foundation Grant. This content is only available as a PDF. © 1991 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)

Chitlaru, Edith; Pick, Uri

doi: 10.1104/pp.96.1.50pmid: 16668185

Abstract Changes in phosphometabolites, following osmotic shock, were analyzed by two-dimensional thin layer chromatography, in extracts of the halotolerant alga Dunaliella salina in order to clarify the regulation of glycerol synthesis from starch. The experiments were carried out in wild-type and in osmotically defective mutant cells. It is demonstrated that hyperosmotic shock induces a decrease in fructose 6-phosphate and an increase in fructose-1,6-bisphosphate indicating the activation of phosphofructokinase. Two mutants, which are specifically defective in their response to hyperosmotic shock, accumulate glucose 6-phosphate or phosphogluconate following shock, and have remarkably reduced activities of glucose-6-phosphate dehydrogenase and of phosphogluconate dehydrogenase, respectively. These results indicate that the pentose-phosphate oxidative pathway has a major role in glycerol synthesis. Hyperosmotic shock leads to a transient accumulation of phosphorylcholine and to a decrease of inositolbisphosphate in D. salina extracts. Accumulation of phosphorylcholine is not detected in osmotically defective mutants. Hypoosmotic shock induces an increase in inositolbisphosphate but not in phosphorylcholine. These results are consistent with previous indications for differential activations of phospholipases by hyper or hypoosmotic shock in Dunaliella. Based on these results we suggest that (a) phosphofructokinase is an important checkpoint enzyme in the regulation of glycerol production, and (b) that the pentose-phosphate pathway has a major role in keeping oxidation-reduction balance during glycerol synthesis. The possible role of lipid breakdown products as second messengers in regulating glycerol production in Dunaliella is discussed. This content is only available as a PDF. © 1991 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)

Singh, Anuradha; Ai, Yunjun; Kao, Teh-hui

doi: 10.1104/pp.96.1.61pmid: 16668186

Abstract Three S-allele-associated proteins (S-proteins) of Petunia inflata, a species with gametophytic self-incompatibility, were previously found to share sequence similarity with two fungal ribonucleases, RNase T2 and RNase Rh. In this study, the S-proteins from P. inflata plants of S1S2 and S2S3 genotypes were purified to homogeneity by gel filtration and cation-exchange chromatography, and their enzymatic properties were characterized. The three S-proteins (S1, S2, and S3), with pairwise sequence identity ranging from 73.1 to 80.5%, were similar in most of the enzymatic properties characterized. The ribonuclease activity had a pH optimum of 7.0 and a temperature optimum of 50°C. Diethylpyrocarbonate at 1 millimolar almost completely abolished the ribonuclease activity; cupric sulfate and zinc sulfate at 1 millimolar reduced the ribonuclease activity of the three S-proteins by 50 to 75%. EDTA and RNasin had no inhibitory effect. All three S-proteins hydrolyzed polycytidylic acid preferentially, but varied in their nucleolytic activity toward polyadenylic acid and polyuridylic acid. 1 This work was supported by National Science Foundation Grant DCB-8904008, U.S. Department of Agriculture Competitive Grant 90-37261-5560, Inter-College Competitive Grant (Project No. 2997) from the Pennsylvania Agricultural Experiment Station, and Biomedical Research Support Grant from the National Institutes of Health to T.-h. K. This content is only available as a PDF. © 1991 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)