bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning of Sucrose:Sucrose 1-Fructosyltransferase from Onion and Synthesis of Structurally Defined Fructan Molecules from Sucrose1
Authors: Vijn Irma , van Dijken Anja , Lüscher Marcel , Bos Antoine , Smeets Edward , Weisbeek Peter , Wiemken Andres , Smeekens Sjef
Published: July 2002

Sucrose (Suc):Suc 1-fructosyltransferase (1-SST) is the key enzyme in plant fructan biosynthesis, since it catalyzes de novo fructan synthesis from Suc. We have cloned 1-SST from onion (Allium cepa) by screening a cDNA library using acid invertase from tulip (Tulipa gesneriana) as a probe. Expression assays in tobacco (Nicotiana plumbaginifolia) protoplasts showed the formation of 1-kestose from Suc. In addition, an onion acid invertase clone was isolated from the same cDNA library. Protein extracts of tobacco protoplasts transformed with this clone showed extensive Suc-hydrolyzing activity. Conditions that induced fructan accumulation in onion leaves also induced 1-SST mRNA accumulation, whereas the acid invertase mRNA level decreased. Structurally different fructan molecules could be produced from Suc by a combined incubation of protein extract of protoplasts transformed with 1-SST and protein extract of protoplasts transformed with either the onion fructan:fructan 6G-fructosyltransferase or the barley Suc:fructan 6-fructosyltransferase.
Piperonylic Acid, a Selective, Mechanism-Based Inactivator of the trans-Cinnamate 4-Hydroxylase: A New Tool to Control the Flux of Metabolites in the Phenylpropanoid Pathway1
Authors: Schalk Michel , Cabello-Hurtado Francisco , Pierrel Marie-Agnès , Atanassova Rossitza , Saindrenan Patrick , Werck-Reichhart Danièle
Published: July 2002

Piperonylic acid (PA) is a natural molecule bearing a methylenedioxy function that closely mimics the structure of trans-cinnamic acid. The CYP73A subfamily of plant P450s catalyzes trans-cinnamic acid 4-hydroxylation, the second step of the general phenylpropanoid pathway. We show that when incubated in vitro with yeast-expressed CYP73A1, PA behaves as a potent mechanism-based and quasi-irreversible inactivator of trans-cinnamate 4-hydroxylase. Inactivation requires NADPH, is time dependent and saturable (KI = 17 μm,kinact = 0.064 min−1), and results from the formation of a stable metabolite-P450 complex absorbing at 427 nm. The formation of this complex is reversible with substrate or other strong ligands of the enzyme. In plant microsomes PA seems to selectively inactivate the CYP73A P450 subpopulation. It does not form detectable complexes with other recombinant plant P450 enzymes. In vivo PA induces a sharp decrease in 4-coumaric acid concomitant to cinnamic acid accumulation in an elicited tobacco (Nicotiana tabacum) cell suspension. It also strongly decreases the formation of scopoletin in tobacco leaves infected with tobacco mosaic virus.
Intermediates of Salicylic Acid Biosynthesis in Tobacco
Authors: Ribnicky David M. , Shulaev Vladimir , Raskin Ilya
Published: July 2002

Salicylic acid (SA) is an important component of systemic-acquired resistance in plants. It is synthesized from benzoic acid (BA) as part of the phenylpropanoid pathway. Benzaldehyde (BD), a potential intermediate of this pathway, was found in healthy and tobacco mosaic virus (TMV)-inoculated tobacco (Nicotiana tabacum L. cv Xanthi-nc) leaf tissue at 100 ng/g fresh weight concentrations as measured by gas chromatography-mass spectrometry. BD was also emitted as a volatile organic compound from tobacco tissues. Application of gaseous BD to plants enclosed in jars caused a 13-fold increase in SA concentration, induced the accumulation of the pathogenesis-related transcript PR-1, and increased the resistance of tobacco to TMV inoculation. [13C6]BD and [2H5]benzyl alcohol were converted to BA and SA. Labeling experiments using [13C1]Phe in temperature-shifted plants inoculated with the TMV showed high enrichment of cinnamic acids (72%), BA (34%), and SA (55%). The endogenous BD, however, contained nondetectable enrichment, suggesting that BD was not the intermediate between cinnamic acid and BA. These results show that BD and benzyl alcohol promote SA accumulation and expression of defense responses in tobacco, and provide insight into the early steps of SA biosynthesis.
The Role of the Alternative Oxidase in Stabilizing the in Vivo Reduction State of the Ubiquinone Pool and the Activation State of the Alternative Oxidase
Authors: Millenaar Frank F. , Benschop Joris J. , Wagner Anneke M. , Lambers Hans
Published: July 2002

A possible function for the alternative (nonphosphorylating) pathway is to stabilize the reduction state of the ubiquinone pool (Qr/Qt), thereby avoiding an increase in free radical production. If the Qr/Qt were stabilized by the alternative pathway, then Qr/Qt should be less stable when the alternative pathway is blocked. Qr/Qtincreased when we exposed roots of Poa annua (L.) to increasing concentrations of KCN (an inhibitor of the cytochrome pathway). However, when salicylhydroxamic acid, an inhibitor of the alternative pathway, was added at the same time, Qr/Qt increased significantly more. Therefore, we conclude that the alternative pathway stabilizes Qr/Qt. Salicylhydroxamic acid increasingly inhibited respiration with increasing concentrations of KCN. In the experiments described here the alternative oxidase protein was invariably in its reduced (high-activity) state. Therefore, changes in the reduction state of the alternative oxidase cannot account for an increase in activity of the alternative pathway upon titration with KCN. The pyruvate concentration in intact roots increased only after the alternative pathway was blocked or the cytochrome pathway was severely inhibited. The significance of the pyruvate concentration and Qr/Qt on the activity of the alternative pathway in intact roots is discussed.
The Glycosylphosphatidylinositol-Anchored Phosphatase fromSpirodela oligorrhiza Is a Purple Acid Phosphatase
Authors: Nakazato Hiroshi , Okamoto Takashi , Nishikoori Miwa , Washio Kenji , Morita Naoki , Haraguchi Kensaku , Thompson Guy A. , Okuyama Hidetoshi
Published: July 2002

We recently presented clear evidence that the major low-phosphate-inducible phosphatase of the duckweedSpirodela oligorrhiza is a glycosylphosphatidylinositol (GPI)-anchored protein, and, to our knowledge, is the first described from higher plants (N. Morita, H. Nakazato, H. Okuyama, Y. Kim, G.A. Thompson, Jr. [1996] Biochim Biophys Acta 1290: 53–62). In this report the purified 57-kD phosphatase is shown to be a purple metalloenzyme containing Fe and Mn atoms and having an absorption maximum at 556 nm. The phosphatase activity was only slightly inhibited by tartrate, as expected for a purple acid phosphatase (PAP). Furthermore, the protein cross-reacted with an anti-Arabidopsis PAP antibody on immunoblots. The N-terminal amino acid sequence of the phosphatase was very similar to those of Arabidopsis, red kidney bean (Phaseolus vulgaris), and soybean (Glycine max) PAP. Extracts of S. oligorrhiza plants incubated with the GPI-specific precursor [3H]ethanolamine were treated with antibodies raised against the purified S. oligorrhizaphosphatase. Radioactivity from the resulting immunoprecipitates was specifically associated with a 57-kD band on sodium dodecyl sulfate-polyacrylamide gels. These results, together with previous findings, strongly indicate that the GPI-anchored phosphatase ofS. oligorrhiza is a PAP.
Export of Carbon from Chloroplasts at Night
Authors: Schleucher Jürgen , Vanderveer Peter J. , Sharkey Thomas D.
Published: July 2002

Hexose export from chloroplasts at night has been inferred in previous studies of mutant and transgenic plants. We have tested whether hexose export is the normal route of carbon export from chloroplasts at night. We used nuclear magnetic resonance to distinguish glucose (Glc) made from hexose export and Glc made from triose export. Glc synthesized in vitro from fructose-6-phosphate in the presence of deuterium-labeled water had deuterium incorporated at C-2, whereas synthesis from triose phosphates caused C-2 through C-5 to become deuterated. In both tomato (Lycopersicon esculentum L.) and bean (Phaseolus vulgaris L.), Glc from sucrose made at night in the presence of deuterium-enriched water was deuterated only in the C-2 position, indicating that >75% of carbon is exported as hexoses at night. In darkness the phosphate in the cytosol was 28 mm, whereas that in the chloroplasts was 5 mm, but hexose phosphates were 10-fold higher in the cytosol than in the chloroplasts. Therefore, hexose phosphates would not move out of chloroplasts without the input of energy. We conclude that most carbon leaves chloroplasts at night as Glc, maltose, or higher maltodextrins under normal conditions.
The Wheat Peptidyl Prolylcis-trans-Isomerase FKBP77 Is Heat Induced and Developmentally Regulated1
Authors: Kurek Isaac , Aviezer Keren , Erel Noa , Herman Eliot , Breiman Adina
Published: July 2002

We isolated a cDNA encoding a 568-amino acid, heat-stress-induced peptidyl prolyl isomerase belonging to the FK506-binding-protein (FKBP) family. The open reading frame encodes for a peptidyl prolyl isomerase that possesses three FKBP-12-like domains, a putative tetratricopeptide motif, and a calmodulin-binding domain. Specific antibodies showed that the open reading frame encodes a heat-induced 77-kD protein, the wheat FKBP77 (wFKBP77), which exhibits 84% identity with the wFKBP73 and 42% identity with the human FKBP59. Because of the high similarity in sequence to wFKBP73, wFKBP77 was designated as the heat-induced isoform. The wFKBP77 mRNA steady-state level was 14-fold higher at 37°C than at 25°C. The wFKBP77 transcript abundance was the highest in mature embryos that had imbibed and 2-d-old green shoots exposed to 37°C, and decreased to 6% in 6-d-old green shoots. The transcript level returned to the level detected at 25°C after recovery of the embryos for 90 min at 25°C. We compared wFKBP73 and wFKBP77 with the heat-shock proteins having cognate and heat-stress-induced counterparts.
Antisense Expression of the CK2 α-Subunit Gene in Arabidopsis. Effects on Light-Regulated Gene Expression and Plant Growth1
Authors: Lee Yew , Lloyd Alan M. , Roux Stanley J.
Published: July 2002

The protein kinase CK2 (formerlycasein kinase II) is thought to be involved in light-regulated gene expression in plants because of its ability to phosphorylate transcription factors that bind to the promoter regions of light-regulated genes in vitro. To address this possibility in vivo and to learn more about the potential physiological roles of CK2 in plants, we transformed Arabidopsis with an antisense construct of the CK2 α-subunit gene and investigated both morphological and molecular phenotypes. Antisense transformants had a smaller adult leaf size and showed increased expression ofchs in darkness and of cab andrbcS after red-light treatment. The latter molecular phenotype implied that CK2 might serve as one of several negative and quantitative effectors in light-regulated gene expression. The possible mechanism of CK2 action and its involvement in the phytochrome signal transduction pathway are discussed.
Characterization ofMdMADS2, a Member of theSQUAMOSASubfamily of Genes, in Apple1
Authors: Sung Soon-Kee , Yu Gyung-Hee , An Gynheung
Published: July 2002

A MADS-box gene,MdMADS2, was isolated from the apple (Malus × domestica Borkh.) var Fuji and its developmental expression pattern was studied during flower development. MdMADS2 shares a high degree of amino acid sequence identity with the SQUAMOSA subfamily of genes. RNA blot analysis showed that MdMADS2 is transcribed through all stages of flower development, and its transcription was seen in the four floral organs. RNA in situ hybridization revealed that the MdMADS2 mRNA is expressed both in the inflorescence meristem and in the floral meristem. The MdMADS2transcript was detected at all stages of flower development. Protein localization analysis showed that MdMADS2 protein was excluded from the stamen and carpel primordia, in which a considerableMdMADS2 mRNA signal was detected. This indicates that posttanscriptional regulation may be involved in theMdMADS2-mediated control of flower development. Transgenic tobacco expressing the MdMADS2 gene from the cauliflower mosaic virus 35S promoter showed early flowering and shorter bolts, but did not show any homeotic changes in the floral organs. These results suggest that MdMADS2 plays an important role during early stages of flower development.
Oligogalacturonic Acid and Chitosan Reduce Stomatal Aperture by Inducing the Evolution of Reactive Oxygen Species from Guard Cells of Tomato and Commelina communis
Authors: Lee Sumin , Choi Hyunjung , Suh SuJeoung , Doo In-Suk , Oh Ki-Young , Jeong Choi Eun , Schroeder Taylor Ann T. , Low Philip S. , Lee Youngsook
Published: July 2002

Stomatal opening provides access to inner leaf tissues for many plant pathogens, so narrowing stomatal apertures may be advantageous for plant defense. We investigated how guard cells respond to elicitors that can be generated from cell walls of plants or pathogens during pathogen infection. The effect of oligogalacturonic acid (OGA), a degradation product of the plant cell wall, and chitosan (β-1,4-linked glucosamine), a component of the fungal cell wall, on stomatal movements were examined in leaf epidermis of tomato (Lycopersicon esculentum L.) andCommelina communis L. These elicitors reduced the size of the stomatal aperture. OGA not only inhibited light-induced stomatal opening, but also accelerated stomatal closing in both species; chitosan inhibited light-induced stomatal opening in tomato epidermis. The effects of OGA and chitosan were suppressed when EGTA, catalase, or ascorbic acid was present in the medium, suggesting that Ca2+ and H2O2 mediate the elicitor-induced decrease of stomatal apertures. We show that the H2O2 that is involved in this process is produced by guard cells in response to elicitors. Our results suggest that guard cells infected by pathogens may close their stomata via a pathway involving H2O2 production, thus interfering with the continuous invasion of pathogens through the stomatal pores.
The Enzymatic Activity of Fungal Xylanase Is Not Necessary for Its Elicitor Activity
Authors: Enkerli Jürg , Felix Georg , Boller Thomas
Published: July 2002

Fungal xylanases fromTrichoderma spp. are potent elicitors of defense responses in various plants. To determine whether enzymatic activity is necessary for elicitor activity, we used site-directed mutagenesis to reduce the catalytic activity of xylanase II from Trichoderma reesei. For this, the glutamic acid residue at position 210, which is part of the active center in this family of enzymes, was changed to either aspartic acid (E210D) or serine (E210S). Wild-type and mutated forms of xylanase II were expressed in yeast cells and purified to homogeneity. Compared with the wild-type form of xylanase II, E210D had >100-fold and E210S 1,000-fold lower enzymatic activity. In contrast, these mutated forms showed no comparable drop in elicitor activity. They fully stimulated medium alkalinization and ethylene biosynthesis in suspension-cultured tomato (Lycopersicon esculentum) cells, as well as hypersensitive necrosis in leaves of tomato and tobacco (Nicotiana tabacum) plants. These results provide direct evidence that enzyme activity is not necessary for elicitor activity of fungal xylanase.
A Transglutaminase Immunologically Related to Tissue Transglutaminase Catalyzes Cross-Linking of Cell Wall Proteins inChlamydomonas reinhardtii
Authors: Waffenschmidt Sabine , Kusch Thomas , Woessner Jeffrey Paul
Published: July 2002

The addition of primary amines to the growth medium of the unicellular green alga Chlamydomonas reinhardtii disrupts cell wall assembly in both vegetative and zygotic cells. Primary amines are competitive inhibitors of the protein-cross-linking activity of transglutaminases. Two independent assays for transglutaminase confirmed a burst of extracellular activity during the early stages of cell wall formation in both vegetative cells and zygotes. When non-inhibiting levels of a radioactive primary amine (14C-putrescine) were added to the growth medium, both cell types were labeled in a reaction catalyzed by extracellular transglutaminase. The radioactive label was found specifically in the cell wall proteins of both cell types, and acid hydrolysis of the labeled material released unmodified 14C-putrescine. Western blots of the proteins secreted at the times of maximal transglutaminase activity in both cell types revealed a single highly cross-reactive 72-kD band when screened with antibodies to guinea pig tissue transglutaminase. Furthermore, the proteins immunoprecipitated by this antiserum in vivo exhibited transglutaminase activity. We propose that this transglutaminase is responsible for an early cell wall protein cross-linking event that temporally precedes the oxidative cross-linking mediated by extracellular peroxidases.
Testing Models of Fatty Acid Transfer and Lipid Synthesis in Spinach Leaf Using in Vivo Oxygen-18 Labeling
Authors: Pollard Mike , Ohlrogge John
Published: July 2002

Oxygen-18 labeling has been applied to the study of plant lipid biosynthesis for the first time. [13C2  18O2]Acetate was incubated with spinach (Spinacia oleracea) leaves and the 18O content in fatty acid methyl esters isolated from different lipid classes measured by gas chromatography-mass spectometry. Fatty acids isolated from lipids synthesized within the plastid, such as monogalactosyldiacylglycerol, show an 18O content consistent with the exogenous acetate undergoing a single activation step and with the direct utilization of acyl-acyl carrier protein by the acyl transferases of the chloroplast. In contrast, fatty acids isolated from lipids assembled in the cytosol, such as phosphatidylcholine, show a 50% reduction in the 18O content. This is indicative of export of the fatty acyl groups from the plastid via a free carboxylate anion, and is consistent with the acyl-acyl carrier protein thioesterase:acyl-coenzyme A (CoA) synthetase mediated export mechanism. If this were not the case and the acyl group was transferred directly from acyl-acyl carrier protein to an acyl acceptor on the cytosolic side, there would be either complete retention of 18O or, less likely, complete loss of18O, but not a 50% loss of 18O. Thus, existing models for fatty acid transfer from the plastid and for spatially separate synthesis of “prokaryotic” and “eukaryotic” lipids have both been confirmed.
Soil Compaction. A Role for Ethylene in Regulating Leaf Expansion and Shoot Growth in Tomato?
Authors: Hussain Ahmed , Black Colin Ramsay , Taylor Ian Blackwood , Roberts Jeremy Alan
Published: July 2002

The role of ethylene in regulating growth in tomato (Lycopersicon esculentum Mill.) during compaction stress was examined using wild-type (cv Ailsa Craig) and transgenic (ACO1AS) genotypes; the latter has a reduced capacity to produce ethylene. Ethephon or silver ions were applied to increase ethylene production or block its action. Shoot growth in both genotypes was comparable in uncompacted (1.1 g cm−3) and uniformly compacted soil (1.5 g cm−3). However, a 1.1/1.5-g cm−3 split-pot treatment invoked marked genotypic differences: growth was reduced in cv Ailsa Craig but was comparable to uncompacted control plants in ACO1AS. As xylem sap abscisic acid levels were similar, abscisic acid was not responsible for inhibiting growth in cv Ailsa Craig. These genotypic differences in growth were accompanied by increased ethylene evolution in cv Ailsa Craig, suggesting that the ability of ACO1AS to maintain growth in the split-pot treatment reflected its lower ethylene levels, a view supported by the observation that excising the roots in the compacted compartment reduced ethylene evolution and restored shoot growth in cv Ailsa Craig. Treatment with silver restored shoot growth in cv Ailsa Craig, whereas treatment with ethephon reduced growth in ACO1AS. Thus, ethylene apparently has a key role in determining growth when tomato plants encounter differential soil compaction.
Wound-Induced Expression of the FAD7Gene Is Mediated by Different Regulatory Domains of Its Promoter in Leaves/Stems and Roots
Authors: Nishiuchi Takumi , Kodama Hiroaki , Yanagisawa Shuichi , Iba Koh
Published: July 2002

TheFAD7 gene is expressed preferentially in the chlorophyllous tissues of unwounded plants. Wounding activates the expression of the FAD7 gene not only in chlorophyllous tissues, but also in nonchlorophyllous tissues of stems and roots. Our previous study suggested that wound-responsive transcriptional activation by the FAD7 promoter in leaves/stems and roots is brought about by a jasmonic acid (JA)-independent and JA-dependent signaling pathway, respectively. In this paper, we show that a specific region (from −259 to −198) in the FAD7promoter is required for wound-activated expression of this gene in leaves and stems, while another region (from −521 to −363) is necessary not only for wound-activated but also for JA-responsive expression of this gene in roots. Thus, different regulatory regions of the FAD7 promoter mediate distinct wound-induced expression of this gene in leaves/stems and roots. Gel mobility shift assays revealed the wound-inducible DNA-binding activity to the −242/−223 region in both stem and leaf nuclear extracts. In fact, deletion of this region abolished wound response of theFAD7 promoter, suggesting the in vivo role of this site. Furthermore, we detected root nuclear factors interacting with the region from −433 to −363 of this promoter. Wounding and methyl jasmonate treatments induced differently these DNA-binding activities. These results suggest that different regulatory mechanisms mediate the wound-induced expression of the FAD7 gene in aerial and subterranean organs.
Amino Acid Transporters Are Localized to Transfer Cells of Developing Pea Seeds
Authors: Tegeder Mechthild , Offler Christina E. , Frommer Wolf B. , Patrick John W.
Published: July 2002

To determine the nature and cellular localization of amino acid transport in pea seeds, two cDNA clones belonging to the AAP family of H+/amino acid co-transporters (PsAAP1 and PsAAP2) were isolated from a cotyledon cDNA library of pea (Pisum sativum L.). Functional expression in the yeast amino acid uptake mutants 22Δ6AAL and 22Δ8AA showed that PsAAP1 mediates transport of neutral, acidic, and basic amino acids. RNA-blot analyses showed that PsAAP1 is expressed in seeds and vegetative organs, including amino acid sinks and sources, whereasPsAAP2 could not be detected. For developing seeds, transcripts of PsAAP1 were detected in coats and cotyledons, with seed coats giving a weak signal. In cotyledons, expression was highest in epidermal-transfer-cell-enriched tissue. RNA in situ hybridization analysis showed that PsAAP1 was predominantly present in epidermal transfer cells forming the outer surface of cotyledons, which abuts the seed coats. Overall, our observations suggest that this transporter, which is localized in transfer cells of cotyledons, might play a role in the uptake of the full spectrum of amino acids released from seed coats.
Post-Transcriptional Regulation Prevents Accumulation of Glutathione Reductase Protein and Activity in the Bundle Sheath Cells of Maize
Authors: Pastori Gabriela M. , Mullineaux Philip M. , Foyer Christine H.
Published: July 2002

Glutathione reductase (GR; EC1.6.4.2) activity was assayed in bundle sheath and mesophyll cells of maize (Zea mays L. var H99) from plants grown at 20°C, 18°C, and 15°C. The purity of each fraction was determined by measuring the associated activity of the compartment-specific marker enzymes, Rubisco and phosphoenolpyruvate carboxylase, respectively. GR activity and the abundance of GR protein and mRNA increased in plants grown at 15°C and 18°C compared with those grown at 20°C. In all cases GR activity was found only in mesophyll fractions of the leaves, with no GR activity being detectable in bundle sheath extracts. Immunogold labeling with GR-specific antibodies showed that the GR protein was exclusively localized in the mesophyll cells of leaves at all growth temperatures, whereas GR transcripts (as determined by in situ hybridization techniques) were observed in both cell types. These results indicate that post-transcriptional regulation prevents GR accumulation in the bundle sheath cells of maize leaves. The resulting limitation on the capacity for regeneration of reduced glutathione in this compartment may contribute to the extreme chilling sensitivity of maize leaves.
Thermal Effect of CO2 on Apoplastic Ice in Rye and Oat during Freezing
Authors: Livingston David P. , Olien C. Robert , Premakumar Ramaswamy
Published: July 2002

Meristematic tissues from rye (Secale cereale) and oat (Avena sativa) were studied in an isothermal calorimeter at −3°C. When the frozen tissue was placed in the calorimeter, the pressure increased within 4 d to 25 and 9 kPa above ambient pressure in the sample vessels containing crowns of rye and oat, respectively. Concurrently, the thermal output went down to −194 μW in rye over the 4-d period; this negative thermal activity could be accounted for by ice melting in the plants. When the pressure was released, the output from the calorimeter went from −194 to 229 μW within 1 h, suggesting that water had frozen in the plants. We propose that CO2 from respiration had dissolved in the water in the plants and caused melting of ice (heat absorption) due to the colligative properties of solutions. When the pressure was released, the CO2 came out of solution and the water froze (heat evolution). These thermal observations were duplicated in a simplified, non-biological system using a glycol/water mixture that was partially frozen at −3°C.
Disaccharide-Mediated Regulation of Sucrose:Fructan-6-Fructosyltransferase, a Key Enzyme of Fructan Synthesis in Barley Leaves
Authors: Müller Joachim , Aeschbacher Roger A. , Sprenger Norbert , Boller Thomas , Wiemken Andres
Published: July 2002

Previous work has indicated that sugar sensing may be important in the regulation of fructan biosynthesis in grasses. We used primary leaves of barley (Hordeum vulgare cv Baraka) to study the mechanisms involved. Excised leaf blades were supplied in the dark with various carbohydrates. Fructan pool sizes and two key enzymes of fructan biosynthesis, sucrose (Suc):Suc-1-fructosyltransferase (1-SST; EC 2.4.1.99) and Suc:fructan-6-fructosyltransferase (6-SFT; EC2.4.1.10) were analyzed. Upon supply of Suc, fructan pool sizes increased markedly. Within 24 h, 1-SST activity was stimulated by a factor of three and 6-SFT-activity by a factor of more than 20, compared with control leaves supplemented with mannitol (Mit). At the same time, the level of mRNA encoding 6-SFT increased conspicuously. These effects were increased in the presence of the invertase inhibitor 2,5-dideoxy-2,5-imino-d-mannitol. Compared with equimolar solutions of Suc, glucose (Glu) and fructose stimulated 6-SFT activity to a lesser extent. Remarkably, trehalose (Tre; Glc-α-1 and 1-α-Glc) had stimulatory effects on 6-SFT activity and, to a somewhat lesser extent, on 6-SFT mRNA, even in the presence of validoxylamine A, a potent trehalase inhibitor. Tre by itself, however, in the presence or absence of validoxylamine A, did not stimulate fructan accumulation. Monosaccharides phosphorylated by hexokinase but not or weakly metabolized, such as mannose (Man) or 2-deoxy-Glc, had no stimulatory effects on fructan synthesis. When fructose or Man were supplied together with Tre, fructan and starch biosynthesis were strongly stimulated. Concomitantly, phospho-Man isomerase (EC 5.3.1.8) activity was detected. These results indicate that the regulation of fructan synthesis in barley leaves occurs independently of hexokinase and is probably based on the sensing of Suc, and also that the structurally related disaccharide Tre can replace Suc as a regulatory compound.
A Dual Function α-Dioxygenase-Peroxidase and NAD+Oxidoreductase Active Enzyme from Germinating Pea Rationalizing α-Oxidation of Fatty Acids in Plants,
Authors: Saffert Alexander , Hartmann-Schreier Jenny , Schön Astrid , Schreier Peter
Published: July 2002

An enzyme with fatty acid α-oxidation activity (49 nkat mg−1; substrate: lauric acid) was purified from germinating pea (Pisum sativum) by a five-step procedure to apparent homogeneity. The purified protein was found to be a 230-kD oligomer with two dominant subunits, i.e. a 50-kD subunit with NAD+ oxidoreductase activity and a 70-kD subunit, homolog to a pathogen-induced oxygenase, which in turn shows significant homology to animal cyclooxygenase. On-line liquid chromatography-electrospray ionization-tandem mass spectrometry revealed rapid α-oxidation of palmitic acid incubated at 0°C with the purified α-oxidation enzyme, leading to (R)-2-hydroperoxypalmitic acid as the major product together with (R)-2-hydroxypalmitic acid, 1-pentadecanal, and pentadecanoic acid. Inherent peroxidase activity of the 70-kD fraction decreased the amount of the (R)-2-hydroperoxy product rapidly and increased the level of (R)-2-hydroxypalmitic acid. Incubations at room temperature accelerated the decline toward the chain-shortened aldehyde. With the identification of the dual functionα-dioxygenase-peroxidase (70-kD unit) and the related NAD+ oxidoreductase (50-kD unit) we provided novel data to rationalize all steps of the classical scheme ofα-oxidation in plants.
Molecular Characterization and Subcellular Localization of Protoporphyrinogen Oxidase in Spinach Chloroplasts
Authors: Che Fang-Sik , Watanabe Naohide , Iwano Megumi , Inokuchi Hachiro , Takayama Seiji , Yoshida Shigeo , Isogai Akira
Published: July 2002

Protoporphyrinogen oxidase (Protox) is the last common enzyme in the biosynthesis of chlorophylls and heme. In plants, there are two isoenzymes of Protox, one located in plastids and other in the mitochondria. We cloned the cDNA of spinach (Spinacia oleracea) plastidal Protox and purified plastidal Protox protein from spinach chloroplasts. Sequence analysis of the cDNA indicated that the plastid Protox of spinach is composed of 562 amino acids containing the glycine-rich motif GxGxxG previously proposed to be a dinucleotide binding site of many flavin-containing proteins. The cDNA of plastidal Protox complemented a Protox mutation inEscherichia coli. N-terminal sequence analysis of the purified enzyme revealed that the plastidal Protox precursor is processed at the N-terminal site of serine-49. The predicted transit peptide (methionine-1 to cysteine-48) was sufficient for the transport of precursors into the plastid because green fluorescent protein fused with the predicted transit peptide was transported to the chloroplast. Immunocytochemical analysis using electron microscopy showed that plastidal Protox is preferentially associated with the stromal side of the thylakoid membrane, and a small portion of the enzyme is located on the stromal side of the chloroplast inner envelope membrane.
Sugar Uptake and Transport in Rice Embryo. Expression of Companion Cell-Specific Sucrose Transporter (OsSUT1) Induced by Sugar and Light
Authors: Matsukura Chi-aki , Saitoh Toshikazu , Hirose Toshiro , Ohsugi Ryu , Perata Pierdomenico , Yamaguchi Junji
Published: July 2002

We investigated sugar uptake and transport in rice (Oryza sativa) embryo during grain germination. Endogenous sugar levels, accumulation of starch granules, and gene expression of a rice sucrose transporter (OsSUT1) were examined using rice embryos germinated with or without exogenous sugar supply. Starch granules remarkably accumulated in the cells around vascular bundles as a consequence of the sugar taken up by the embryos, indicating that the taken-up sugars are transiently converted into starch. In situ detection for OsSUT1 mRNA indicated its localization in the phloem companion cells. Furthermore, northern-blot and in situ hybridization analyses showed that OsSUT1 expression is not detectable in embryos subjected to sugar starvation conditions, whereas its expression is enhanced by an increased endogenous sugar level. Overall results indicate that the expression of companion cell-specific sucrose transporter, OsSUT1 is regulated by the endogenous sugar status as well as light exposure.
Recombinant Brassinosteroid Insensitive 1 Receptor-Like Kinase Autophosphorylates on Serine and Threonine Residues and Phosphorylates a Conserved Peptide Motif in Vitro
Authors: Oh Man-Ho , Ray William K. , Huber Steven C. , Asara John M. , Gage Douglas A. , Clouse Steven D.
Published: July 2002

BRASSINOSTEROID-INSENSITIVE 1 (BRI1) encodes a putative Leucine-rich repeat receptor kinase in Arabidopsis that has been shown by genetic and molecular analysis to be a critical component of brassinosteroid signal transduction. In this study we examined some of the biochemical properties of the BRI1 kinase domain (BRI1-KD) in vitro, which might be important predictors of in vivo function. Recombinant BRI1-KD autophosphorylated on serine (Ser) and threonine (Thr) residues with p-Ser predominating. Matrix-assisted laser desorption/ionization mass spectrometry identified a minimum of 12 sites of autophosphorylation in the cytoplasmic domain of BRI1, including five in the juxtamembrane region (N-terminal to the catalytic KD), five in the KD (one each in sub-domains I and VIa and three in sub-domain VIII), and two in the carboxy terminal region. Five of the sites were uniquely identified (Ser-838, Thr-842, Thr-846, Ser-858, and Thr-872), whereas seven were localized on short peptides but remain ambiguous due to multiple Ser and/or Thr residues within these peptides. The inability of an active BRI1-KD to transphosphorylate an inactive mutant KD suggests that the mechanism of autophosphorylation is intramolecular. It is interesting that recombinant BRI1-KD was also found to phosphorylate certain synthetic peptides in vitro. To identify possible structural elements required for substrate recognition by BRI1-KD, a series of synthetic peptides were evaluated, indicating that optimum phosphorylation of the peptide required R or K residues atP − 3, P − 4, andP + 5 (relative to the phosphorylated Ser atP = 0).
Multiple Signaling Pathways in Gene Expression during Sugar Starvation. Pharmacological Analysis of din Gene Expression in Suspension-Cultured Cells of Arabidopsis
Authors: Fujiki Yuki , Ito Masaki , Nishida Ikuo , Watanabe Akira
Published: July 2002

We have identified many dark-inducible (din) genes that are expressed in Arabidopsis leaves kept in the dark. In the present study we addressed the question of how plant cells sense the depletion of sugars, and how sugar starvation triggersdin gene expression in suspension-cultured cells of Arabidopsis. Depletion of sucrose in the medium triggered marked accumulation of din transcripts. Suppression ofdin gene expression by 2-deoxy-Glc, and a non-suppressive effect exerted by 3-O-methyl-Glc, suggested that sugar-repressible expression of din genes is mediated through the phosphorylation of hexose by hexokinase, as exemplified in the repression of photosynthetic genes by sugars. We have further shown that the signaling triggered by sugar starvation involves protein phosphorylation and dephosphorylation events, and have provided the first evidence that multiple pathways of protein dephosphorylation exist in sugar starvation-induced gene expression. An inhibitor of serine/threonine protein kinase, K-252a, inhibiteddin gene expression in sugar-depleted cells. Okadaic acid, which may preferentially inhibit type 2A protein phosphatases over type 1, enhanced the transcript levels of all dingenes, except din6 and din10, under sugar starvation. Conversely, a more potent inhibitor of type 1 and 2A protein phosphatases, calyculin A, increased transcripts fromdin2 and din9, but decreased those from other din genes, in sugar-depleted cells. On the other hand, calyculin A, but not okadaic acid, completely inhibited the gene expression of chlorophyll a/b-binding protein under sugar starvation. These results indicate that multiple signaling pathways, mediated by different types of protein phosphatases, regulate gene expression during sugar starvation.
Cloning and Functional Analysis of Sucrose:Sucrose 1-Fructosyltransferase from Tall Fescue
Authors: Lüscher Marcel , Hochstrasser Urs , Vogel Guido , Aeschbacher Roger , Galati Virginie , Nelson Curtis J. , Boller Thomas , Wiemken Andres
Published: July 2002

Enzymes of grasses involved in fructan synthesis are of interest since they play a major role in assimilate partitioning and allocation, for instance in the leaf growth zone. Several fructosyltransferases from tall fescue (Festuca arundinacea) have previously been purified (Lüscher and Nelson, 1995). It is surprising that all of these enzyme preparations appeared to act both as sucrose (Suc):Suc 1-fructosyl transferases (1-SST) and as fructan:fructan 6G-fructosyl transferases. Here we report the cloning of a cDNA corresponding to the predominant protein in one of the fructosyl transferase preparations, its transient expression in tobacco protoplasts, and its functional analysis in the methylotrophic yeast,Pichia pastoris. When the cDNA was transiently expressed in tobacco protoplasts, the corresponding enzyme preparations produced 1-kestose from Suc, showing that the cDNA encodes a 1-SST. When the cDNA was expressed in P. pastoris, the recombinant protein had all the properties of known 1-SSTs, namely 1-kestose production, moderate nystose production, lack of 6-kestose production, and fructan exohydrolase activity with 1-kestose as the substrate. The physical properties were similar to those of the previously purified enzyme, except for its apparent lack of fructan:fructan  6G-fructosyl transferase activity. The expression pattern of the corresponding mRNA was studied in different zones of the growing leaves, and it was shown that transcript levels matched the 1-SST activity and fructan content.
A Nuclear Casein Kinase 2 Activity Is Involved in Early Events of Transcriptional Activation Induced by Salicylic Acid in Tobacco
Authors: Hidalgo Perla , Garretón Virginia , Berrı́os Carmen Gloria , Ojeda Héctor , Jordana Xavier , Holuigue Loreto
Published: July 2002

Salicylic acid (SA) activates immediate early transcription of genes controlled by a family of DNA promoter elements namedas-1-like elements. These elements are functional in the promoter of glutathione S-transferase genes. We have previously shown that SA increases the binding of tobacco (Nicotiana tabacum cv Xanthi nc) nuclear factors to theas-1 sequence in a process mediated by protein phosphorylation. In this study we give evidence for the participation of a nuclear protein kinase CK2 (casein kinase 2) in the pathway activated by SA in tobacco. The first line of evidence comes from the evaluation of the CK2 activity in nuclear extracts prepared from tobacco plants treated with SA or water as a control. Results from these experiments indicate that SA increases the nuclear CK2 activity. The second line of evidence derives from the evaluation of the in vivo effect of 5,6-dichloro-1-(β-d-ribofuranosyl) benzimidazole (DRB), a cell-permeable CK2 inhibitor, on the responsiveness of the as-1 sequence to SA. Results from these experiments indicate that DRB impairs the activating effect of SA on the transcription of both, the GUS reporter gene controlled by a tetramer of the as-1 element, and the endogenous gnt35 gene encoding a glutathioneS-transferase, in transgenic tobacco plants. DRB also impaired the increasing effect of SA on the binding of nuclear factors to the as-1 element. Furthermore, transcription of theas-1/GUS reporter gene activated by the synthetic auxin 2,4-dichlorophenoxyacetic acid and by methyl jasmonate was also inhibited by DRB. To our knowledge, this is the first report in which activation of a CK2 enzyme by a plant hormone is reported.
Characterization of a Tobacco Bright Yellow 2 Cell Line Expressing the Tetracycline Repressor at a High Level for Strict Regulation of Transgene Expression
Authors: David Karine M. , Perrot-Rechenmann Catherine
Published: July 2002

Manipulating the expression of a transgene in transient and stable transformed cells is a requirement for many functional analyses. We have investigated the use of the tetracycline-dependent gene expression system developed by Gatz et al. (1992) in tobacco (Nicotiana tabacum L. cv Bright Yellow 2 [BY2]) cells, the most widely used plant cell culture. We have selected a BY2 cell line, named BY2-tetracycline repressor (tetR) 17, which expresses the tetR at a high level, and have evaluated the capacity of this cell line to suppress the expression of a green fluorescent protein reporter gene under the control of the “Triple-Op” promoter in the absence of tetracycline in a large number of independent transformants. The ability to induce the expression of green fluorescent protein after treatment by anhydrotetracycline in the same transformants was also analyzed. BY2-tetR17 cells were demonstrated to be excellent recipient cells for recovery of clonal cell lines with a highly controlled regulation of the introduced transgene.
ADP-Glucose Pyrophosphorylase Is Located in the Plastid in Developing Tomato Fruit
Authors: Beckles Diane M. , Craig Josephine , Smith Alison M.
Published: July 2002

The subcellular location of activity and protein of ADP-glucose pyrophosphorylase (AGPase) in developing tomato (Lycopersicon esculentum) fruit was determined following a report that the enzyme might be present inside and outside the plastids in this organ. Plastids prepared from crude homogenates of columella and pericarp, the starch-accumulating tissues of developing fruit, contained 8% to 18% of the total activity of enzymes known to be confined to plastids, and 0.2% to 0.5% of the total activity of enzymes known to be confined to the cytosol. The proportion of the total activity of AGPase in the plastids was the same as that of the enzymes known to be confined to the plastid. When samples of plastid and total homogenate fractions were subjected to immunoblotting with an antiserum raised to AGPase, most or all of the protein detected was plastidial. Taken as a whole, these data provide strong evidence that AGPase is confined to the plastids in developing tomato fruit.
Induction of Lipid Metabolic Enzymes during the Endoplasmic Reticulum Stress Response in Plants
Authors: Shank Karin J. , Su Pei , Brglez Irena , Boss Wendy F. , Dewey Ralph E. , Boston Rebecca S.
Published: July 2002

The endoplasmic reticulum (ER) stress response is a signal transduction pathway activated by the perturbation of normal ER metabolism. We used the maize (Zea mays)floury-2 (fl2) mutant and soybean (Glycine max) suspension cultures treated with tunicamycin (Tm) to investigate the ER stress response as it relates to phospholipid metabolism in plants. Four key phospholipid biosynthetic enzymes, including DG kinase and phosphatidylinositol (PI) 4-phosphate 5-kinase were up-regulated in the fl2 mutant, specifically in protein body fractions where the mutation has its greatest effect. The third up-regulated enzyme, choline-phosphate cytidylyltransferase, was regulated by fl2 gene dosage and developmental signals. Elevated accumulation of the fourth enzyme, PI 4-kinase, was observed in the fl2 endosperm and soybean cells treated with Tm. The activation of these phospholipid biosynthetic enzymes was accompanied by alterations in membrane lipid synthesis and accumulation. The fl2 mutant exhibited increased PI content in protein body membranes at 18 d after pollination and more than 3-fold higher triacylglycerol accumulation in the endosperm by 36 d after pollination. Incorporation of radiolabeled acetate into phospholipids in soybean culture cells increased by about 30% with Tm treatment. The coordinated regulation of ER stress related proteins and multiple components of phospholipid biosynthesis is consistent with signaling through a common pathway. We postulate that the plant ER stress response has an important role in general plant metabolism, and more specifically in integrating the synthesis of protein and lipid reserves to allow proper seed formation.
Wound-Inducible Proteinase Inhibitors in Pepper. Differential Regulation upon Wounding, Systemin, and Methyl Jasmonate
Authors: Moura Daniel S. , Ryan Clarence A.
Published: July 2002

Seven small (approximately 6,000 D) wound-inducible proteinase inhibitor proteins were isolated from leaves of pepper (Capsicum annuum) plants that are members of the potato inhibitor II family. N-terminal sequences obtained indicated that the pepper leaf proteinase inhibitors (PLPIs) exhibit homology to two GenBank accessions that code for preproteins containing three isoinhibitors domains each that, when post-translationally processed, can account for the mixture of isoinhibitors that are reported herein from pepper leaves. A constitutive level of PLPI proteins was found in pepper leaves, and these levels increased up to 2.6-fold upon wounding of the lower leaves. Exposing intact plants to methyl jasmonate vapors induced the accumulation of PLPIs. Supplying excised young pepper plants with water through the cut stems induced PLPI proteins to levels higher than those found in intact plants, but with high variability. Supplying the excised plants with systemin did not result in an increase of PLPI levels that were statistically higher than levels found in excised plants. Gel-blot analyses of PLPI induction revealed the presence of two mRNA bands, having slightly different mobilities in agarose gels. Only the low M  r mRNA is present in untreated control plants, and it appears to be responsible for the constitutive levels of PLPI found in leaves. Both mRNA species are wound- and methyl jasmonate-inducible. Only the low- M  rspecies is weakly induced by systemin, indicating a differential expression of the two PLPI species.
Possible Involvement of Protein Phosphorylation in Aluminum-Responsive Malate Efflux from Wheat Root Apex
Authors: Osawa Hiroki , Matsumoto Hideaki
Published: July 2002

In many plants, efflux of organic anions from roots has been proposed as one of the major Al resistance mechanisms. However it remains unknown how plants regulate efflux of organic anions in response to Al. In this study, the regulatory mechanisms of Al-responsive malate efflux in wheat (Triticum aestivum) were characterized focusing on the role of protein phosphorylation. Al-resistant wheat (cv Atlas) initiated malate efflux at 5 min after addition of Al, and this response was sensitive to temperature. K-252a, a broad range inhibitor of protein kinases, effectively blocked the Al-induced malate efflux accompanied with an increased accumulation of Al and intensified Al-induced root growth inhibition. A transient activation of a 48-kD protein kinase and an irreversible repression of a 42-kD protein kinase were observed preceding the initiation of malate efflux, and these changes were canceled by K-252a. Malate efflux was accompanied with a rapid decrease in the contents of organic anions in the root apex, such as citrate, succinate, and malate but with no change in the contents of inorganic anions such as chloride, nitrate, and phosphate. These results suggest that protein phosphorylation is involved in the Al-responsive malate efflux in the wheat root apex and that the organic anion-specific channel might be a terminal target that responds to Al signaling mediated by phosphorylation.