bluuebunny/crossref_metadata_2025 huggingface dataset:
Three 1-Aminocyclopropane-1-Carboxylate Synthase Genes Regulated by Primary and Secondary Pollination Signals in Orchid Flowers1
Authors: Bui Anhthu Q. , Neill Sharman D. O'
Published: July 2002

The temporal and spatial expression patterns of three 1-aminocyclopropane-1-carboxylate (ACC) synthase genes were investigated in pollinated orchid (Phalaenopsis spp.) flowers. Pollination signals initiate a cascade of development events in multiple floral organs, including the induction of ethylene biosynthesis, which coordinates several postpollination developmental responses. The initiation and propagation of ethylene biosynthesis is regulated by the coordinated expression of three distinct ACC synthase genes in orchid flowers. One ACC synthase gene (Phal-ACS1) is regulated by ethylene and participates in amplification and interorgan transmission of the pollination signal, as we have previously described in a related orchid genus. Two additional ACC synthase genes (Phal-ACS2 andPhal-ACS3) are expressed primarily in the stigma and ovary of pollinated orchid flowers. Phal-ACS2 mRNA accumulated in the stigma within 1 h after pollination, whereasPhal-ACS1 mRNA was not detected until 6 h after pollination. Similar to the expression of Phal-ACS2, thePhal-ACS3 gene was expressed within 2 h after pollination in the ovary. Exogenous application of auxin, but not ACC, mimicked pollination by stimulating a rapid increase in ACC synthase activity in the stigma and ovary and inducing Phal-ACS2and Phal-ACS3 mRNA accumulation in the stigma and ovary, respectively. These results provide the basis for an expanded model of interorgan regulation of three ACC synthase genes that respond to both primary (Phal-ACS2 and Phal-ACS3) and secondary (Phal-ACS1) pollination signals.
Characterization of Recombinant Rhamnogalacturonan α-l-Rhamnopyranosyl-(1,4)-α-d-Galactopyranosyluronide Lyase from Aspergillus aculeatus1
Authors: Mutter Margien , Colquhoun Ian J. , Beldman Gerrit , Schols Henk A. , Bakx Edwin J. , Voragen Alphons G.J.
Published: July 2002

The four major oligomeric reaction products from saponified modified hairy regions (MHR-S) from apple, produced by recombinant rhamnogalacturonan (RG) α-l-rhamnopyranosyl-(1,4)-α-d-galactopyranosyluronide lyase (rRG-lyase) from Aspergillus aculeatus, were isolated and characterized by 1H-nuclear magnetic resonance spectroscopy. They contain an alternating RG backbone with a degree of polymerization of 4, 6, 8, and 10 and with an α-Δ-(4,5)-unsaturated d-galactopyranosyluronic acid at the nonreducing end and an l-rhamnopyranose at the reducing end. l-Rhamnopyranose units are substituted at C-4 with β-galactose. The maximum reaction rate of rRG-lyase toward MHR-S at pH 6.0 and 31°C was 28 units mg−1. rRG-lyase and RG-hydrolase cleave the same alternating RG I subunit in MHR. Both of these enzymes fragment MHR by a multiple attack mechanism. The catalytic efficiency of rRG-lyase for MHR increases with decreasing degree of acetylation. Removal of arabinose side chains improves the action of rRG-lyase toward MHR-S. In contrast, removal of galactose side chains decreased the catalytic efficiency of rRG-lyase. Native RG-lyase was purified from A. aculeatus, characterized, and found to be similar to the rRG-lyase expressed inAspergillus oryzae.
Family of MADS-Box Genes Expressed Early in Male and Female Reproductive Structures of Monterey Pine
Authors: Mouradov Aidyn , Glassick Tina V. , Hamdorf Britt A. , Murphy Lawrence C. , Marla Soma S. , Yang Yumin , Teasdale Robert D.
Published: July 2002

Three MADS-box genes isolated from Monterey pine (Pinus radiata), PrMADS1, PrMADS2, and PrMADS3, are orthologs to members of the AGL2 and AGL6 gene subfamilies in Arabidopsis. These genes were expressed during early stages of pine shoot development in differentiating seed- and pollen-cone buds. Their transcripts were found within a group of cells that formed ovuliferous scale and microsporophyll primordia. Expression ofPrMADS3 was also detected in a group of cells giving rise to needle primordia within differentiated vegetative buds, and in needle primordia.
(+)-Germacrene A Biosynthesis
Authors: de Kraker Jan-Willem , Franssen Maurice C.R. , de Groot Aede , König Wilfried A. , Bouwmeester Harro J.
Published: July 2002

The leaves and especially the roots of chicory (Cichorium intybus L.) contain high concentrations of bitter sesquiterpene lactones such as the guianolides lactupicrin, lactucin, and 8-deoxylactucin. Eudesmanolides and germacranolides are present in smaller amounts. Their postulated biosynthesis through the mevalonate-farnesyl diphosphate-germacradiene pathway has now been confirmed by the isolation of a (+)-germacrene A synthase from chicory roots. This sesquiterpene cyclase was purified 200-fold using a combination of anion-exchange and dye-ligand chromatography. It has a Km value of 6.6 μm, an estimated molecular mass of 54 kD, and a (broad) pH optimum around 6.7. Germacrene A, the enzymatic product, proved to be much more stable than reported in literature. Its heat-induced Cope rearrangement into (−)-β-elemene was utilized to determine its absolute configuration on an enantioselective gas chromatography column. To our knowledge, until now in sesquiterpene biosynthesis, germacrene A has only been reported as an (postulated) enzyme-bound intermediate, which, instead of being released, is subjected to additional cyclization(s) by the same enzyme that generated it from farnesyl diphosphate. However, in chicory germacrene A is released from the sesquiterpene cyclase. Apparently, subsequent oxidations and/or glucosylation of the germacrane skeleton, together with a germacrene cyclase, determine whether guaiane- or eudesmane-type sesquiterpene lactones are produced.
Light and Excess Manganese
Authors: González Alonso , Steffen Kenneth L. , Lynch Jonathan P.
Published: July 2002

The effect of light intensity on antioxidants, antioxidant enzymes, and chlorophyll content was studied in common bean (Phaseolus vulgaris L.) exposed to excess Mn. Leaves of bean genotypes contrasting in Mn tolerance were exposed to two different light intensities and to excess Mn; light was controlled by shading a leaflet with filter paper. After 5 d of Mn treatment ascorbate was depleted by 45% in leaves of the Mn-sensitive genotype ZPV-292 and by 20% in the Mn-tolerant genotype CALIMA. Nonprotein sulfhydryl groups and glutathione reductase were not affected by Mn or light treatment. Ten days of Mn-toxicity stress increased leaf ascorbate peroxidase activity of cv ZPV-292 by 78% in low light and by 235% in high light, and superoxide dismutase activity followed a similar trend. Increases of ascorbate peroxidase and superoxide dismutase activity observed in cv CALIMA were lower than those observed in the susceptible cv ZPV-292. The cv CALIMA had less ascorbate oxidation under excess Mn-toxicity stress. Depletion of ascorbate occurred before the onset of chlorosis in Mn-stressed plants, especially in cv ZPV-292. Lipid peroxidation was not detected in floating leaf discs of mature leaves exposed to excess Mn. Our results suggest that Mn toxicity may be mediated by oxidative stress, and that the tolerant genotype may maintain higher ascorbate levels under stress than the sensitive genotype.
Two Genetically Separable Phases of Growth Inhibition Induced by Blue Light in Arabidopsis Seedlings
Authors: Parks Brian M. , Cho Myeon H. , Spalding Edgar P.
Published: July 2002

High fluence-rate blue light (BL) rapidly inhibits hypocotyl growth in Arabidopsis, as in other species, after a lag time of 30 s. This growth inhibition is always preceded by the activation of anion channels. The membrane depolarization that results from the activation of anion channels by BL was only 30% of the wild-type magnitude inhy4, a mutant lacking the HY4 BL receptor. High-resolution measurements of growth made with a computer-linked displacement transducer or digitized images revealed that BL caused a rapid inhibition of growth in wild-type and hy4seedlings. This inhibition persisted in wild-type seedlings during more than 40 h of continuous BL. By contrast, hy4escaped from the initial inhibition after approximately 1 h of BL and grew faster than wild type for approximately 30 h. Wild-type seedlings treated with 5-nitro-2-(3-phenylpropylamino)-benzoic acid, a potent blocker of the BL-activated anion channel, displayed rapid growth inhibition, but, similar to hy4, these seedlings escaped from inhibition after approximately 1 h of BL and phenocopied the mutant for at least 2.5 h. The effects of 5-nitro-2-(3-phenylpropylamino)-benzoic acid and the HY4mutation were not additive. Taken together, the results indicate that BL acts through HY4 to activate anion channels at the plasma membrane, causing growth inhibition that begins after approximately 1 h. Neither HY4 nor anion channels appear to participate greatly in the initial phase of inhibition.
petit1, a Conditional Growth Mutant of Arabidopsis Defective in Sucrose-Dependent Elongation Growth
Authors: Kurata Tetsuya , Yamamoto Kotaro T.
Published: July 2002

The hypocotyl of Arabidopsis is well suited for the analysis of cell elongation because it elongates without cell division. We have isolated a new class of recessive mutants,petit1 (pet1), which are defective in aspects of hypocotyl elongation. The short-hypocotyl phenotype ofpet1 is caused by shortened cells. The cells of the elongation zone of the hypocotyl are often deformed.pet1 also shows defects in elongation of the roots, flower stalk, leaves, petals, pedicels, and siliques, and these defects cannot be repaired by the application of auxin, gibberellin, brassinolide, or an inhibitor of ethylene biosynthesis. The short-hypocotyl phenotype of pet1 is pronounced only in growth medium supplemented with sucrose, which has promotive effects on hypocotyl elongation. In pet1 this effect is much reduced, causing the sucrose-dependent short-hypocotyl phenotype ofpet1. pet1 accumulates more soluble sugars than the wild type and also shows more intensive iodo-starch staining in the cotyledon and hypocotyl. These results indicate that PETIT1 is involved in a sugar-dependent elongation process that may include correct assembly of expanding cell wall architecture.
Altered Zn Compartmentation in the Root Symplasm and Stimulated Zn Absorption into the Leaf as Mechanisms Involved in Zn Hyperaccumulation in Thlaspi caerulescens
Authors: Lasat Mitch M. , Baker Alan J.M. , Kochian Leon V.
Published: July 2002

We investigated Zn compartmentation in the root, Zn transport into the xylem, and Zn absorption into leaf cells in Thlaspi caerulescens, a Zn-hyperaccumulator species, and compared them with those of a related nonaccumulator species, Thlaspi arvense. 65Zn-compartmental analysis conducted with roots of the two species indicated that a significant fraction of symplasmic Zn was stored in the root vacuole ofT. arvense, and presumably became unavailable for loading into the xylem and subsequent translocation to the shoot. InT. caerulescens, however, a smaller fraction of the absorbed Zn was stored in the root vacuole and was readily transported back into the cytoplasm. We conclude that in T. caerulescens, Zn absorbed by roots is readily available for loading into the xylem. This is supported by analysis of xylem exudate collected from detopped Thlaspi species seedlings. When seedlings of the two species were grown on either low (1 μm) or high (50 μm) Zn, xylem sap ofT. caerulescens contained approximately 5-fold more Zn than that of T. arvense. This increase was not correlated with a stimulated production of any particular organic or amino acid. The capacity of Thlaspi species cells to absorb 65Zn was studied in leaf sections and leaf protoplasts. At low external Zn levels (10 and 100 μm), there was no difference in leaf Zn uptake between the twoThlaspi species. However, at 1 mmZn2+, 2.2-fold more Zn accumulated in leaf sections ofT. caerulescens. These findings indicate that altered tonoplast Zn transport in root cells and stimulated Zn uptake in leaf cells play a role in the dramatic Zn hyperaccumulation expressed inT. caerulescens.
Differential Responses of Abaxial and Adaxial Guard Cells of Broad Bean to Abscisic Acid and Calcium
Authors: Wang Xi-Qing , Wu Wei-Hua , Assmann Sarah M.
Published: July 2002

Regulation by abscisic acid (ABA) and Ca2+ of broad bean (Vicia faba) abaxial and adaxial guard cell movements and inward K+ currents were compared. One millimolar Ca2+ in the bathing medium inhibited abaxial stomatal opening by 60% but only inhibited adaxial stomatal opening by 15%. The addition of 1 μm ABA in the bathing medium resulted in 80% inhibition of abaxial but only 45% inhibition of adaxial stomatal opening. Similarly, ABA and Ca2+ each stimulated greater abaxial stomatal closure than adaxial stomatal closure. Whole-cell patch-clamp results showed that the inward K+ currents of abaxial guard cells were inhibited by 60% (−180 mV) in the presence of 1.5 μmCa2+ in the cytoplasm, whereas the inward K+currents of adaxial guard cells were not affected at all by the same treatment. Although 1 μm ABA in the cytoplasm inhibited the inward K+ currents to a similar extent for both abaxial and adaxial guard cells, the former were more sensitive to ABA applied externally. These results suggest that the abaxial stomata are more sensitive to Ca2+ and ABA than adaxial stomata in regard to stomatal opening and closing processes and that the regulation of the inward K+ currents by ABA may not proceed via a Ca2+-signaling pathway in adaxial guard cells. Therefore, there may be different pathways for ABA- and Ca2+-mediated signal transduction in abaxial and adaxial guard cells.
Red Bell Pepper Chromoplasts Exhibit in Vitro Import Competency and Membrane Targeting of Passenger Proteins from the Thylakoidal Sec and ΔpH Pathways but Not the Chloroplast Signal Recognition Particle Pathway1
Authors: Summer Elizabeth J. , Cline Kenneth
Published: July 2002

Chloroplast to chromoplast development involves new synthesis and plastid localization of nuclear-encoded proteins, as well as changes in the organization of internal plastid membrane compartments. We have demonstrated that isolated red bell pepper (Capsicum annuum) chromoplasts contain the 75-kD component of the chloroplast outer envelope translocon (Toc75) and are capable of importing chloroplast precursors in an ATP-dependent fashion, indicating a functional general import apparatus. The isolated chromoplasts were able to further localize the 33- and 17-kD subunits of the photosystem II O2-evolution complex (OE33 and OE17, respectively), lumen-targeted precursors that utilize the thylakoidal Sec and ΔpH pathways, respectively, to the lumen of an internal membrane compartment. Chromoplasts contained the thylakoid Sec component protein, cpSecA, at levels comparable to chloroplasts. Routing of OE17 to the lumen was abolished by ionophores, suggesting that routing is dependent on a transmembrane ΔpH. The chloroplast signal recognition particle pathway precursor major photosystem II light-harvesting chlorophyll a/b protein failed to associate with chromoplast membranes and instead accumulated in the stroma following import. The Pftf (plastidfusion/translocation factor), a chromoplast protein, integrated into the internal membranes of chromoplasts during in vitro assays, and immunoblot analysis indicated that endogenous plastid fusion/translocation factor was also an integral membrane protein of chromoplasts. These data demonstrate that the internal membranes of chromoplasts are functional with respect to protein translocation on the thylakoid Sec and ΔpH pathways.
Further Studies of the Role of Cyclic β-Glucans in Symbiosis. An ndvC Mutant of Bradyrhizobium japonicumSynthesizes Cyclodecakis-(1→3)-β-Glucosyl1
Authors: Bhagwat Arvind A. , Mithöfer Axel , Pfeffer Philip E. , Kraus Christine , Spickers Nicole , Hotchkiss Arland , Ebel Jürgen , Keister Donald L.
Published: July 2002

The cyclic β-(1→3),β-(1→6)-d-glucan synthesis locus ofBradyrhizobium japonicum is composed of at least two genes, ndvB and ndvC. Mutation in either gene affects glucan synthesis, as well as the ability of the bacterium to establish a successful symbiotic interaction with the legume host soybean (Glycine max). B. japonicumstrain AB-14 (ndvB::Tn5) does not synthesize β-glucans, and strain AB-1 (ndvC::Tn5) synthesizes a cyclic β-glucan lacking β-(1→6)-glycosidic bonds. We determined that the structure of the glucan synthesized by strain AB-1 is cyclodecakis-(1→3)-β-d-glucosyl, a cyclic β-(1→3)-linked decasaccharide in which one of the residues is substituted in the 6 position with β-laminaribiose. Cyclodecakis-(1→3)-β-d-glucosyl did not suppress the fungal β-glucan-induced plant defense response in soybean cotyledons and had much lower affinity for the putative membrane receptor protein than cyclic β-(1→3),β-(1→6)-glucans produced by wild-typeB. japonicum. This is consistent with the hypothesis presented previously that the wild-type cyclic β-glucans may function as suppressors of a host defense response.
Characterization of Two Novel Type I Ribosome-Inactivating Proteins from the Storage Roots of the Andean CropMirabilis expansa1
Authors: Vivanco Jorge M. , Savary Brett J. , Flores Hector E.
Published: July 2002

Two novel type I ribosome-inactivating proteins (RIPs) were found in the storage roots of Mirabilis expansa, an underutilized Andean root crop. The two RIPs, named ME1 and ME2, were purified to homogeneity by ammonium sulfate precipitation, cation-exchange perfusion chromatography, and C4 reverse-phase chromatography. The two proteins were found to be similar in size (27 and 27.5 kD) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their isoelectric points were determined to be greater than pH 10.0. Amino acid N-terminal sequencing revealed that both ME1 and ME2 had conserved residues characteristic of RIPs. Amino acid composition and western-blot analysis further suggested a structural similarity between ME1 and ME2. ME2 showed high similarity to the Mirabilis jalapa antiviral protein, a type I RIP. Depurination of yeast 26S rRNA by ME1 and ME2 demonstrated their ribosome-inactivating activity. Because these two proteins were isolated from roots, their antimicrobial activity was tested against root-rot microorganisms, among others. ME1 and ME2 were active against several fungi, includingPythium irregulare, Fusarium oxysporumsolani, Alternaria solani,Trichoderma reesei, and Trichoderma harzianum, and an additive antifungal effect of ME1 and ME2 was observed. Antibacterial activity of both ME1 and ME2 was observed against Pseudomonas syringae, Agrobacterium tumefaciens, Agrobacterium radiobacter, and others.
Oxidative Turnover of Soybean Root Glutamine Synthetase. In Vitro and in Vivo Studies1
Authors: Ortega Jose Luis , Roche Dominique , Sengupta-Gopalan Champa
Published: July 2002

Glutamine synthetase (GS) is the key enzyme in ammonia assimilation and catalyzes the ATP-dependent condensation of NH3 with glutamate to produce glutamine. GS in plants is an octameric enzyme. Recent work from our laboratory suggests that GS activity in plants may be regulated at the level of protein turnover (S.J. Temple, T.J. Knight, P.J. Unkefer, C. Sengupta-Gopalan [1993] Mol Gen Genet 236: 315–325; S.J. Temple, S. Kunjibettu, D. Roche, C. Sengupta-Gopalan [1996] Plant Physiol 112: 1723–1733; S.J. Temple, C. Sengupta-Gopalan [1997] In C.H. Foyer, W.P. Quick, eds, A Molecular Approach to Primary Metabolism in Higher Plants. Taylor & Francis, London, pp 155–177). Oxidative modification of GS has been implicated as the first step in the turnover of GS in bacteria. By incubating soybean (Glycine max) root extract enriched in GS in a metal-catalyzed oxidation system to produce the ·OH radical, we have shown that GS is oxidized and that oxidized GS is inactive and more susceptible to degradation than nonoxidized GS. Histidine and cysteine protect GS from metal-catalyzed inactivation, indicating that oxidation modifies the GS active site and that cysteine and histidine residues are the site of modification. Similarly, ATP and particularly ATP/glutamate give the enzyme the greatest protection against oxidative inactivation. The roots of plants fed ammonium nitrate showed a 3-fold increase in the level of GS polypeptides and activity compared with plants not fed ammonium nitrate but without a corresponding increase in the GS transcript level. This would suggest either translational or posttranslational control of GS levels.
Heavy-Metal Regulation of Thioredoxin Gene Expression inChlamydomonas reinhardtii
Authors: Lemaire Stéphane , Keryer Eliane , Stein Mariana , Schepens Isabelle , Issakidis-Bourguet Emmanuelle , Gérard-Hirne Catherine , Miginiac-Maslow Myroslawa , Jacquot Jean-Pierre
Published: July 2002

Heavy metals are highly toxic compounds for cells. In this report we demonstrate that the expression of Chlamydomonas reinhardtii thioredoxins (TRX)m and h is induced by heavy metals. Upon exposure of the cells to Cd and Hg, a strong accumulation of both messengers was observed. Western-blot experiments revealed that among these two TRXs, only TRX h polypeptides accumulated in response to the toxic cations. A biochemical analysis indicated that heavy metals inhibit TRX activity, presumably by binding at the level of their active site. Sequence analysis of the C. reinhardtii TRX h promoter revealed the presence of cis-acting elements related to cadmium induction. The origins and purposes of this regulation are discussed. Our data suggest, for the first time to our knowledge, a possible implication of TRXs in defense mechanisms against heavy metals.
Rapid and Systemic Accumulation of Chloroplast mRNA-Binding Protein Transcripts after Flame Stimulus in Tomato
Authors: Vian Alain , Henry-Vian Chantal , Davies Eric
Published: July 2002

It has been shown that tomato (Lycopersicon esculentum) plants respond to flame wounding and electrical stimulation by a rapid (15 min) and systemic up-regulation of proteinase inhibitor (pin) genes. To find other genes having a similar expression pattern, we used subtractive cDNA screening between flamed and control plants to select clones up-regulated by flame wounding. We report the characterization of one of them, a chloroplast mRNA-binding protein encoded by a single gene and expressed preferentially in the leaves. Systemic gene expression in response to flaming in the youngest terminal leaf exhibited three distinct phases: a rapid and transient increase (5–15 min) in transcript accumulation, a decline to basal levels (15–45 min), and then a second, more prolonged increase (60–90 min). In contrast, after a mechanical wound the rapid, transient increase (5 min) was followed by a rapid decline to basal levels but no later, prolonged accumulation. In the petiole, the initial flame-wound-evoked transient increase (15 min) was followed by a continuous decline for 3 h. The nature of the wound signal(s) causing such rapid changes in transcript abundance is discussed in relation to electrical signaling, which has recently been implicated in plant responses to wounding.
Redox Control of psbA Gene Expression in the Cyanobacterium Synechocystis PCC 6803. Involvement of the Cytochrome b6/fComplex
Authors: Alfonso Miguel , Perewoska Irène , Kirilovsky Diana
Published: July 2002

We investigated the role of the redox state of the photosynthetic and respiratory electron transport chains on the regulation ofpsbA expression in Synechocystis PCC 6803. Different means to modify the redox state of the electron carriers were used: (a) dark to oxidize the whole electron transport chain; (b) a shift from dark to light to induce its reduction; (c) the chemical interruption of the electron flow at different points to change the redox state of specific electron carriers; and (d) the presence of glucose to maintain a high reducing power in darkness. We show that changes in the redox state of the intersystem electron transport chain induce modifications of psbA transcript production and psbA mRNA stability. Reduction of the intersystem electron carriers activates psbAtranscription and destabilizes the mRNA, while their oxidation induces a decrease in transcription and a stabilization of the transcript. Furthermore, our data suggest that the redox state of one of the electron carriers between the plastoquinone pool and photosystem I influences not only the expression of the psbA gene, but also that of other two photosynthetic genes, psaE andcpcBA. As a working hypothesis, we propose that the occupancy of the Q0 site in the cytochromeb  6/f complex may be involved in this regulation.
Expression of Tryptophan Decarboxylase and Tyrosine Decarboxylase Genes in Tobacco Results in Altered Biochemical and Physiological Phenotypes
Authors: Guillet Gabriel , Poupart Julie , Basurco Juan , De Luca Vincenzo
Published: July 2002

The substrate specificity of tryptophan (Trp) decarboxylase (TDC) for Trp and tyrosine (Tyr) decarboxylase (TYDC) for Tyr was used to modify the in vivo pools of these amino acids in transgenic tobacco. Expression of TDC and TYDC was shown to deplete the levels of Trp and Tyr, respectively, during seedling development. The creation of artificial metabolic sinks for Trp and Tyr also drastically affected the levels of phenylalanine, as well as those of the non-aromatic amino acids methionine, valine, and leucine. Transgenic seedlings also displayed a root-curling phenotype that directly correlated with the depletion of the Trp pool. Non-transformed control seedlings could be induced to display this phenotype after treatment with inhibitors of auxin translocation such as 2,3,5-triiodobenzoic acid or N-1-naphthylphthalamic acid. The depletion of aromatic amino acids was also correlated with increases in the activities of the shikimate and phenylpropanoid pathways in older, light-treated transgenic seedlings expressing TDC, TYDC, or both. These results provide in vivo confirmation that aromatic amino acids exert regulatory feedback control over carbon flux through the shikimate pathway, as well as affecting pathways outside of aromatic amino acid biosynthesis.
Phytoene Desaturase Is Localized Exclusively in the Chloroplast and Up-Regulated at the mRNA Level during Accumulation of Secondary Carotenoids in Haematococcus pluvialis (Volvocales, Chlorophyceae),
Authors: Grünewald Kay , Eckert Manfred , Hirschberg Joseph , Hagen Christoph
Published: July 2002

The unicellular green alga Haematococcus pluvialis Flotow is known for its massive accumulation of ketocarotenoids under various stress conditions. Therefore, this microalga is one of the favored organisms for biotechnological production of these antioxidative compounds. Astaxanthin makes up the main part of the secondary carotenoids and is accumulated mostly in an esterified form in extraplastidic lipid vesicles. We have studied phytoene desaturase, an early enzyme of the carotenoid biosynthetic pathway. The increase in the phytoene desaturase protein levels that occurs following induction is accompanied by a corresponding increase of its mRNA during the accumulation period, indicating that phytoene desaturase is regulated at the mRNA level. We also investigated the localization of the enzyme by western-blot analysis of cell fractions and by immunogold labeling of ultrathin sections for electron microscopy. In spite of the fact that secondary carotenoids accumulate outside the chloroplast, no extra pathway specific for secondary carotenoid biosynthesis in H. pluvialis was found, at least at this early stage in the biosynthesis. A transport process of carotenoids from the site of biosynthesis (chloroplast) to the site of accumulation (cytoplasmatic located lipid vesicles) is implicated.
Selenium Assimilation and Volatilization from Dimethylselenoniopropionate by Indian Mustard
Authors: de Souza Mark P. , Lytle C. Mel , Mulholland Maria M. , Otte Marinus L. , Terry Norman
Published: July 2002

Earlier work from our laboratory on Indian mustard (Brassica juncea L.) identified the following rate-limiting steps for the assimilation and volatilization of selenate to dimethyl selenide (DMSe): (a) uptake of selenate, (b) activation of selenate by ATP sulfurylase, and (b) conversion of selenomethionine (SeMet) to DMSe. The present study showed that shoots of selenate-treated plants accumulated very low concentrations of dimethylselenoniopropionate (DMSeP). Selenonium compounds such as DMSeP are the most likely precursors of DMSe. DMSeP-supplied plants volatilized Se at a rate 113 times higher than that measured from plants supplied with selenate, 38 times higher than from selenite, and six times higher than from SeMet. The conversion of SeMet to selenonium compounds such as DMSeP is likely to be rate-limiting for DMSe production, but not the formation of DMSe from DMSeP because DMSeP was the rate of Se volatilization from faster than from SeMet and SeMet (but no DMSeP) accumulated in selenite- or SeMet-supplied wild-type plants and in selenate-supplied ATP-sulfurylase transgenic plants. DMSeP-supplied plants absorbed the most Se from the external medium compared with plants supplied with SeMet, selenate, or selenite; they also accumulated more Se in shoots than in roots as an unknown organic compound resembling a mixture of DMSeP and selenocysteine.
Metabolism of Methanol in Plant Cells. Carbon-13 Nuclear Magnetic Resonance Studies
Authors: Gout Elizabeth , Aubert Serge , Bligny Richard , Rébeillé Fabrice , Nonomura Arthur R. , Benson Andrew A. , Douce Roland
Published: July 2002

Using 13C-NMR, we demonstrate that [13C]methanol readily entered sycamore (Acer pseudoplatanus L.) cells to be slowly metabolized to [3-13C]serine, [13CH3]methionine, and [13CH3]phosphatidylcholine. We conclude that the assimilation of [13C]methanol occurs through the formation of 13CH3H4Pte-glutamate (Glu)n and S-adenosyl-methionine, because feeding plant cells with [3-13CH3]serine, the direct precursor of13CH2H4Pte-Glun, can perfectly mimic [13CH3]methanol for folate-mediated single-carbon metabolism. On the other hand, the metabolism of [13C]methanol in plant cells revealed assimilation of label into a new cellular product that was identified as [13CH3]methyl-β-d-glucopyranoside. The de novo synthesis of methyl-β-d-glucopyranoside induced by methanol did not require the formation of13CH3H4Pte-Glun and was very likely catalyzed by a “transglycosylation” process.
Regulation of High-Affinity Nitrate Transporter Genes and High-Affinity Nitrate Influx by Nitrogen Pools in Roots of Barley
Authors: Vidmar Joseph John , Zhuo Degen , Siddiqi M. Yaeesh , Schjoerring Jan K. , Touraine Bruno , Glass Anthony D.M.
Published: July 2002

To investigate the regulation of HvNRT2, genes that encode high-affinity NO3  − transporters in barley (Hordeum vulgare) roots, seedlings were treated with 10 mm NO3  − in the presence or absence of amino acids (aspartate, asparagine, glutamate [Glu], and glutamine [Gln]), NH4  +, and/or inhibitors of N assimilation. Although all amino acids decreased high-affinity13NO3  − influx andHvNRT2 transcript abundance, there was substantial interconversion of administered amino acids, making it impossible to determine which amino acid(s) were responsible for the observed effects. To clarify the role of individual amino acids, plants were separately treated with tungstate, methionine sulfoximine, or azaserine (inhibitors of nitrate reductase, Gln synthetase, and Glu synthase, respectively). Tungstate increased the HvNRT2 transcript by 20% to 30% and decreased NO3  − influx by 50%, indicating that NO3  − itself does not regulate transcript abundance, but may exert post-transcriptional effects. Experiments with methionine sulfoximine suggested that NH4  + may down-regulate HvNRT2gene expression and high-affinity NO3  − influx by effects operating at the transcriptional and post-transcriptional levels. Azaserine decreased HvNRT2 transcript levels and NO3  − influx by 97% and 95%, respectively, while decreasing Glu and increasing Gln levels. This suggests that Gln (and not Glu) is responsible for down-regulating HvNRT2expression, although it does not preclude a contributory effect of other amino acids.
Molecular Characterization of CYP73A9 andCYP82A1 P450 Genes Involved in Plant Defense in Pea
Authors: Whitbred Joy M. , Schuler Mary A.
Published: July 2002

Cytochrome P450 monooxygenases (P450s) mediate a wide range of oxidative reactions involved in the biosynthesis of phenylpropanoids, terpenes, and alkaloids. Two pea (Pisum sativum) P450 cDNAs (CYP73A9v1, encoding trans-cinnamic acid hydroxylase [t-CAH] in the core phenylpropanoid pathway, and CYP82A1v1, possibly encoding an activity in a late branch of the phenylpropanoid pathway) have previously been described. Of three CYP73A9 genes now isolated, theCYP73A9v1 gene is full-length with two introns at positions conserved in other t-CAH genes, and theCYP73A9v2 and CYP73A9v3 gene fragments are 5′-truncated and lack introns. The full-lengthCYP82A1v2 gene contains a single intron at an alternate position. Nucleotide searches of the CYP73A9v1 andCYP82A1v2 promoters have indicated that the regulatory sequences for these early and late phenylpropanoid transcripts are substantially different. The P-, L-, and H-boxes identified in white light-, ultraviolet light-, and elicitor-induced footprints in early phenylpropanoid promoters (phenylalanine ammonia lyase [PAL], 4-coumarate coenzyme A:ligase [4-CL], and chalcone synthase [CHS]) are conserved in the t-CAH promoter and are absent from the CYP82A1v2 promoter. Both promoters contain TCA motifs identified in stress-responsive promoters, box IV elements identified in elicitor-responsive PAL andCHS promoters, and spatially conserved wound-response elements potentially coordinating regulation of these wound-responsive promoters.
Overexpression of Auxin-Binding Protein Enhances the Sensitivity of Guard Cells to Auxin
Authors: Bauly James M. , Sealy Ian M. , Macdonald Heather , Brearley Jane , Dröge Swenja , Hillmer Stefan , Robinson David G. , Venis Michael A. , Blatt Michael R. , Lazarus Colin M. , Napier Richard M.
Published: July 2002

To explore the role of auxin-binding protein (ABP1) in planta, a number of transgenic tobacco (Nicotiana tabacum) lines were generated. The wild-type KDEL endoplasmic reticulum targeting signal was mutated to HDEL, another common retention sequence in plants, and to KEQL or KDELGL to compromise its activity. The auxin-binding kinetics of these forms of ABP1 were found to be similar to those of ABP1 purified from maize (Zea mays). To test for a physiological response mediated by auxin, intact guard cells of the transgenic plants were impaled with double-barreled microelectrodes, and auxin-dependent changes in K+ currents were recorded under voltage clamp. Exogenous auxin affected inwardly and outwardly rectifying K+ currents in a dose-dependent manner. Auxin sensitivity was markedly enhanced in all plants overexpressing ABP1, irrespective of the form present. Immunogold electron microscopy was used to investigate the localization of ABP1 in the transgenic plants. All forms were detected in the endoplasmic reticulum and the KEQL and KDELGL forms passed further across the Golgi stacks than KDEL and HDEL forms. However, neither electron microscopy nor silver-enhanced immunogold epipolarization microscopy revealed differences in cell surface ABP1 abundance for any of the plants, including control plants, which indicated that overexpression of ABP1 alone was sufficient to confer increased sensitivity to added auxin. Jones et al. ([1998] Science 282: 1114–1117) found increased cell expansion in transgenic plants overexpressing wild-type ABP1. Single cell recordings extend this observation, with the demonstration that the auxin sensitivity of guard cell K+ currents is mediated, at least in part, by ABP1.
Trienoic Fatty Acids Are Required to Maintain Chloroplast Function at Low Temperatures
Authors: Routaboul Jean-Marc , Fischer Steven F. , Browse John
Published: July 2002

The chloroplast membranes of all higher plants contain very high proportions of trienoic fatty acids. To investigate how these lipid structures are important in photosynthesis, we have generated a triple mutant line of Arabidopsis that contains negligible levels of trienoic fatty acids. For mutant plants grown at 22°C, photosynthetic fluorescence parameters were indistinguishable from wild type at 25°C. Lowering the measurement temperature led to a small decrease in photosynthetic quantum yield, ΦII, in the mutant relative to wild-type controls. These and other results indicate that low temperature has only a small effect on photosynthesis in the short term. However, long-term growth of plants at 4°C resulted in decreases in fluorescence parameters, chlorophyll content, and thylakoid membrane content in triple-mutant plants relative to wild type. Comparisons among different mutant lines indicated that these detrimental effects of growth at 4°C are strongly correlated with trienoic fatty acid content with levels of 16:3 + 18:3, approximately one-third of wild type being sufficient to sustain normal photosynthetic function. In total, our results indicate that trienoic fatty acids are important to ensure the correct biogenesis and maintenance of chloroplasts during growth of plants at low temperatures.
Study of the Role of Antimicrobial Glucosinolate-Derived Isothiocyanates in Resistance of Arabidopsis to Microbial Pathogens
Authors: Tierens Koenraad F.M.-J. , Thomma Bart P.H.J. , Brouwer Margreet , Schmidt Jürgen , Kistner Katherine , Porzel Andrea , Mauch-Mani Brigitte , Cammue Bruno P.A. , Broekaert Willem F.
Published: July 2002

Crude aqueous extracts from Arabidopsis leaves were subjected to chromatographic separations, after which the different fractions were monitored for antimicrobial activity using the fungus Neurospora crassa as a test organism. Two major fractions were obtained that appeared to have the same abundance in leaves from untreated plants versus leaves from plants challenge inoculated with the fungusAlternaria brassicicola. One of both major antimicrobial fractions was purified to homogeneity and identified by 1H nuclear magnetic resonance, gas chromatography/electron impact mass spectrometry, and gas chromatography/chemical ionization mass spectrometry as 4-methylsulphinylbutyl isothiocyanate (ITC). This compound has previously been described as a product of myrosinase-mediated breakdown of glucoraphanin, the predominant glucosinolate in Arabidopsis leaves. 4-Methylsulphinylbutyl ITC was found to be inhibitory to a wide range of fungi and bacteria, producing 50% growth inhibition in vitro at concentrations of 28 μm for the most sensitive organism tested (Pseudomonas syringae). A previously identified glucosinolate biosynthesis mutant, gsm1-1, was found to be largely deficient in either of the two major antimicrobial compounds, including 4-methylsulphinylbutyl ITC. The resistance ofgsm1-1 was compared with that of wild-type plants after challenge with the fungi A. brassicicola,Plectosphaerella cucumerina, Botrytis cinerea, Fusarium oxysporum, orPeronospora parasitica, or the bacteria Erwinia carotovora or P. syringae. Of the tested pathogens, only F. oxysporum was found to be significantly more aggressive on gsm1-1 than on wild-type plants. Taken together, our data suggest that glucosinolate-derived antimicrobial ITCs can play a role in the protection of Arabidopsis against particular pathogens.
The Maize MADS Box Gene ZmMADS3 Affects Node Number and Spikelet Development and Is Co-Expressed withZmMADS1 during Flower Development, in Egg Cells, and Early Embryogenesis
Authors: Heuer Sigrid , Hansen Susanne , Bantin Jörg , Brettschneider Reinhold , Kranz Erhard , Lörz Horst , Dresselhaus Thomas
Published: July 2002

MADS box genes represent a large gene family of transcription factors with essential functions during flower development and organ differentiation processes in plants. Addressing the question of whether MADS box genes are involved in the regulation of the fertilization process and early embryo development, we have isolated two novel MADS box cDNAs, ZmMADS1 and ZmMADS3, from cDNA libraries of maize (Zea mays) pollen and egg cells, respectively. The latter gene is allelic to ZAP1. Transcripts of both genes are detectable in egg cells and in in vivo zygotes of maize. ZmMADS1 is additionally expressed in synergids and in central and antipodal cells. During early somatic embryogenesis, ZmMADS1 expression is restricted to cells with the capacity to form somatic embryos, and to globular embryos at later stages. ZmMADS3 is detectable only by more sensitive reverse transcriptase-PCR analyses, but is likewise expressed in embryogenic cultures. Both genes are not expressed in nonembryogenic suspension cultures and in isolated immature and mature zygotic embryos. During flower development, ZmMADS1 andZmMADS3 are co-expressed in all ear spikelet organ primordia at intermediate stages. Among vegetative tissues,ZmMADS3 is expressed in stem nodes and displays a gradient with highest expression in the uppermost node. Transgenic maize plants ectopically expressing ZmMADS3are reduced in height due to a reduced number of nodes. Reduction of seed set and male sterility were observed in the plants. The latter was due to absence of anthers. Putative functions of the genes during reproductive and vegetative developmental processes are discussed.
Morphology of Ni-base superalloys via very small-angle neutron scattering technique
Authors: Aizawa K. , Tomimitsu H. , Tamaki H. , Yoshinari A.
Published: July 2002

Very small-angle neutron scattering (VSANS) was used to develop a nondestructive inspecting technique for predicting the residual life time of turbine blades made from single crystal Ni-base superalloy. The VSANS curves, obtained from single crystalline CMSX-4 samples with the various degrees of damage, clearly show the first order peak of the lamellar structure created by a sequence of γ and γ' phases in this material. The peak shifts in the direction of lower scattering vector magnitudes when the creep damage increases, which reflects a corresponding increase of the period of the lamellar structure. According to the VSANS data, the interphase distance in the samples increases gradually (but does not grow rapidly) with increasing creep damage when the latter is < 54% of the creep leading to rupture. Thus, the interphase distance is a sensitive parameter which can be used to describe the morphological changes in Ni-base superalloys related to creep damage. The results are consistent with those obtained from scanning electron microscopy.
Double-crystal diffraction imaging with a small effective divergence source: application to the magneto-acoustic vibrations in FeBO3
Authors: Matsouli Ioanna , Kvardakov Vladimir V. , Baruchel José
Published: July 2002

Ultrasonic standing waves, excited in FeBO3(111) crystal plates through magneto-elastic coupling, were visualized using monochromatic Bragg diffraction imaging (topography) with synchrotron radiation. The images depend strongly on whether diffraction by the sample occurs in the same plane as in the double-crystal monochromator, or in the perpendicular plane. The observations are explained by taking into account (a) the strong spatial dispersion which prevails because of the small effective divergence (angular size of the source as seen from a point in the specimen), which is less than one microradian in this experiment, and (b) the sample vibration and curvature.
Structural studies of the 30S subunit of ribosomes Thermus thermophilus by small-angle neutron and X-ray scattering
Authors: Fan L. , Svergun D. I. , Volkov V. V. , Aksenov V. L. , Algalarov C. Ch. , Selivanova O. M. , Shcherbakova I. V. , Koch M. , Gilles R. , Wiedenmann A. , May R. , Serdyuk I. N.
Published: July 2002

Three different small-angle neutron scattering facilities, the "YUMO" camera at JINR (Dubna, Russia), the V4 camera at HMI (Berlin, Germany) and D11 camera at ILL (Grenoble, France), were used in the investigation of 30S ribosomal subunit from Thermus thermophilus. Parallel X-ray measurements on X33 camera at DESY (Hamburg, Germany) were used to control the identity of deuterated particles. The neutron and x-ray contrast variation data were interpreted by direct method using spherical harmonics. Structural models of the 30S ribosomal subunit from Thermus thermophilus and its RNA -rich core with the resolution 3.5 nm were constructed. A comparison between the 30S Thermus thermophilus and 30S E.coli models shows that primary difference between them consists in the special distributions of the protein component whereas the special distributions of the RNA component coincide well in both models.
The new SANS instrument at the Swiss spallation source SINQ
Authors: Kohlbrecher J. , Wagner W.
Published: July 2002

With the start-up of the neutron spallation source SINQ at PSI an instrument for small angle neutron scattering became operational and is open for the user community. The instrument is equipped with state-of-the-art components and compares well with the world's largest and most powerful facilities of this kind. Great emphasis was put on providing a flexible, universal multi-user facility which guaranties a comfortable and reliable operation. In the present paper, the layout of the instrument is presented, the main components are described, and the performance is illustrated by selected examples
Development of spin-echo small-angle neutron scattering
Authors: Bouwman W. G. , van Oossanen M. , Uca O. , Kraan W.H. , Rekveldt M.T.
Published: July 2002

A polarised neutron spin echo technique is used to build a novel kind of small angle neutron scattering (SANS) instrument. The basis of this instrument is a symmetric set-up with a spin flipper in the centre, which creates a spin echo, even with a divergent beam. The precession regions on either side of the spin flipper are shaped such as to produce a very sensitive relation between the vertical angle of the neutron path and the total precession angle. Any SANS of a sample placed in the instrument reduces the symmetry of the neutron path and therefore decreases the echo. Magnetised foils define the precession regions by rotating the neutron spin from being parallel to the magnetic field to perpendicular to the field, to start the precession. These foils and the flipper were built and tested. A spin echo SANS signal is measured with the complete set-up . It should be possible with this technique to measure within minutes a full correlation function in samples over distances from 5 to 1000 nm.
Microstructure analysis using temperature-induced contrast variation in small-angle scattering
Authors: Barnes J.D. , Kolb R. , Barnes K. , Nakatani A.I. , Hammouda B.
Published: July 2002

We propose to introduce Temperature-Induced Contrast Variation (TICV) as a technique for improving the specificity of microstructure analyses obtained using small-angle scattering methods. TICV exploits the fact that, at temperatures well removed from melting or annealing regimes, the scattering contrast in many multicomponent systems exhibits a significant temperature dependence while the form factors for the scattering processes change only slightly. Difference scattering patterns formed by subtracting the pattern measured at a suitable reference temperature from the pattern obtained at another temperature emphasize those components of the microstructure that vary most strongly with temperature over the chosen range. This can facilitate an effective separation of the observed scattering patterns into separate components for differing scattering mechanisms. Examples of distinct scattering mechanisms commonly found in polymers include liquid-like scattering from amorphous components, contributions from the intramolecular amorphous halo, Guinier scattering from inclusions, and the scattering from ordered stacks of crystalline lamellae. The experimental data presented here provide qualitative demonstrations of general features of the method. Further work is needed to obtain improved descriptors to characterize microstructure in these systems.