bluuebunny/crossref_metadata_2025 huggingface dataset:
A high-performance cryogenic amplifier based on a radio-frequency single electron transistor
Authors: Segall K. , Lehnert K. W. , Stevenson T. R. , Schoelkopf R. J. , Wahlgren P. , Aassime A. , Delsing P.
Published: December 2002

We demonstrate a high-performance cryogenic amplifier based on a radio-frequency single-electron- transistor (rf-SET). The high charge sensitivity and large bandwidth of the rf-SET, along with low power dissipation, low capacitance and on-chip integrability, make it a good candidate for a general-purpose cryogenic amplifier for high impedance sources. We measure a large-gate rf-SET with an open-loop voltage noise of 30 nV/(Hz), among the lowest reported voltage noise figures for a SET. Using a closed-loop transimpedance configuration, the amplifier shows almost 2 orders of magnitude increase in dynamic range, a 3 dB bandwidth of 30 kHz, and a transimpedance gain of 50 V/μA for a cryogenic 1 MΩ load resistor. The performance of this amplifier is already sufficient for use as an integrated readout with some types of high-performance cryogenic detectors for astrophysics.
Activity of single ion channel proteins detected with a planar microstructure
Authors: Fertig Niels , Klau Michèle , George Michael , Blick Robert H. , Behrends Jan C.
Published: December 2002

We present recordings of currents mediated by single ion channel proteins obtained using planar, microstructured glass chips. In these chips, pores with diameters of 1–2 μm are produced by ion track etching and are used for patch clamping instead of using the classical micropipette. Our results represent success in using such devices to record from single channels in cell membranes. The planar chip greatly enhances the accessibility of the ion channel containing membrane and can serve as a workbench for experiments on single ion channels using combinations of patch clamp current recording with other single molecule techniques.
Reversed truncated cone composition distribution of In0.8Ga0.2As quantum dots overgrown by an In0.1Ga0.9As layer in a GaAs matrix
Authors: Lenz A. , Timm R. , Eisele H. , Hennig Ch. , Becker S. K. , Sellin R. L. , Pohl U. W. , Bimberg D. , Dähne M.
Published: December 2002

We present cross-sectional scanning tunneling microscopy results of self-organized In0.8Ga0.2As quantum dots covered by an In0.1Ga0.9As film inside a GaAs matrix prepared by metalorganic chemical vapor deposition. From images of quantum dots with atomic resolution, we determine a spatial distribution of the In composition within the dots with a shape of a reversed truncated cone. The wetting layer and the overgrown In0.1Ga0.9As layer show vertical intermixing.
Cell Death Processes during Expression of Hybrid Lethality in Interspecific F1 Hybrid between Nicotiana gossei Domin and Nicotiana tabacum
Authors: Mino Masanobu , Maekawa Kenji , Ogawa Ken'ichi , Yamagishi Hiroshi , Inoue Masayoshi
Published: December 2002

Hybrid lethality, a type of reproductive isolation, is a genetically controlled event appearing at the seedling stage in interspecific hybrids. We characterized the lethality of F1hybrid seedlings from Nicotiana  gosseiDomin and Nicotiana  tabacum cv Bright-Yellow 4 using a number of traits including growth rate, microscopic features of tissues and cells, ion leakage, DNA degradation, reactive oxygen intermediates including superoxide radical (O2  −) and hydrogen peroxide (H2O2), and expression of stress response marker genes. Lethal symptoms appeared at 4 d after germination in the basal hypocotyl and extended toward both the hypocotyl and root of the plants grown at 26°C. Microscopic analysis revealed a prompt lysis of cell components during cell death. Membrane disruption and DNA degradation were found in the advanced stage of the lethality. The death of mesophyll cells in the cotyledon was initiated by the vascular bundle, suggesting that a putative factor inducing cell death diffused into surrounding cells from the vascular tissue. In contrast, these symptoms were not observed in the plants grown at 37°C. Seedlings grown at 26°C generated larger amounts of reactive oxygen intermediate in the hypocotyl than those grown at 37°C. A number of stress response marker genes were expressed at 26°C but not at 37°C. We proposed that a putative death factor moving systemically through the vascular system induced a prompt and successive lysis of the cytoplasm of cells and that massive cell death eventually led to the loss of the hybrid plant.
Starch Biosynthesis and Intermediary Metabolism in Maize Kernels. Quantitative Analysis of Metabolite Flux by Nuclear Magnetic Resonance
Authors: Glawischnig Erich , Gierl Alfons , Tomas Adriana , Bacher Adelbert , Eisenreich Wolfgang
Published: December 2002

The seeds of cereals represent an important sink for metabolites during the accumulation of storage products, and seeds are an essential component of human and animal nutrition. Understanding the metabolic interconversions (networks) underpinning storage product formation could provide the foundation for effective metabolic engineering of these primary nutritional sources. In this paper, we describe the use of retrobiosynthetic nuclear magnetic resonance analysis to establish the metabolic history of the glucose (Glc) units of starch in maize (Zea mays) kernels. Maize kernel cultures were grown with [U-13C6]Glc, [U-13C12]sucrose, or [1,2-13C2]acetate as supplements. After 19 d, starch was hydrolyzed, and the isotopomer composition of the resulting Glc was determined by quantitative nuclear magnetic resonance analysis. [1,2-13C2]Acetate was not incorporated into starch. [U-13C6]Glc or [U-13C12]sucrose gave similar labeling patterns of polysaccharide Glc units, which were dominated by [1,2,3-13C3]- and [4,5,6-13C3]-isotopomers, whereas the [U-13C6]-, [3,4,5,6-13C4]-, [1,2-13C2]-, [5,6-13C2], [3-13C1], and [4-13C1]-isotopomers were present at lower levels. These isotopomer compositions indicate that there is extensive recycling of Glc before its incorporation into starch, via the enzymes of glycolytic, glucogenic, and pentose phosphate pathways. The relatively high abundance of the [5,6-13C2]-isotopomer can be explained by the joint operation of glycolysis/glucogenesis and the pentose phosphate pathway.
Starch Biosynthesis during Pollen Maturation Is Associated with Altered Patterns of Gene Expression in Maize
Authors: Datta Rupali , Chamusco Karen C. , Chourey Prem S.
Published: December 2002

Starch biosynthesis during pollen maturation is not well understood in terms of genes/proteins and intracellular controls that regulate it in developing pollen. We have studied two specific developmental stages: “early,” characterized by the lack of starch, before or during pollen mitosis I; and “late,” an actively starch-filling post-pollen mitosis I phase in S-type cytoplasmic male-sterile (S-CMS) and two related male-fertile genotypes. The male-fertile starch-positive, but not the CMS starch-deficient, genotypes showed changes in the expression patterns of a large number of genes during this metabolic transition. In addition to a battery of housekeeping genes of carbohydrate metabolism, we observed changes in hexose transporter, plasma membrane H+-ATPase, ZmMADS1, and 14-3-3 proteins. Reduction or deficiency in 14-3-3 protein levels in all three major cellular sites (amyloplasts [starch], mitochondria, and cytosol) in male-sterile relative to male-fertile genotypes are of potential interest because of interorganellar communication in this CMS system. Further, the levels of hexose sugars were significantly reduced in male-sterile as compared with male-fertile tissues, not only at “early” and “late” stages but also at an earlier point during meiosis. Collectively, these data suggest that combined effects of both reduced sugars and their reduced flux in starch biosynthesis along with a strong possibility for altered redox passage may lead to the observed temporal changes in gene expressions, and ultimately pollen sterility.
Humic Acids Isolated from Earthworm Compost Enhance Root Elongation, Lateral Root Emergence, and Plasma Membrane H+-ATPase Activity in Maize Roots
Authors: Canellas Luciano Pasqualoto , Olivares Fabio Lopes , Okorokova-Façanha Anna L. , Façanha Arnoldo Rocha
Published: December 2002

Earthworms (Eisenia foetida) produce humic substances that can influence plant growth by mechanisms that are not yet clear. In this work, we investigated the effects of humic acids (HAs) isolated from cattle manure earthworm compost on the earliest stages of lateral root development and on the plasma membrane H+-ATPase activity. These HAs enhance the root growth of maize (Zea mays) seedlings in conjunction with a marked proliferation of sites of lateral root emergence. They also stimulate the plasma membrane H+-ATPase activity, apparently associated with an ability to promote expression of this enzyme. In addition, structural analysis reveals the presence of exchangeable auxin groups in the macrostructure of the earthworm compost HA. These results may shed light on the hormonal activity that has been postulated for these humic substances.
Molecular Characterization of a Novel Gene Family Encoding ACT Domain Repeat Proteins in Arabidopsis
Authors: Hsieh Ming-Hsiun , Goodman Howard M.
Published: December 2002

In bacteria, the regulatory ACT domains serve as amino acid-binding sites in some feedback-regulated amino acid metabolic enzymes. We have identified a novel type of ACT domain-containing protein family in Arabidopsis whose members contain ACT domain repeats (the “ACR” protein family). There are at least eight ACR genes located on each of the five chromosomes in the Arabidopsis genome. Gene structure comparisons indicate that the ACR gene family may have arisen by gene duplications. Northern-blot analysis indicates that each member of the ACR gene family has a distinct expression pattern in various organs from 6-week-old Arabidopsis. Moreover, analyses of an ACR3 promoter-β-glucuronidase (GUS) fusion in transgenic Arabidopsis revealed that the GUS activity formed a gradient in the developing leaves and sepals, whereas low or no GUS activity was detected in the basal regions. In 2-week-old Arabidopsis seedlings grown in tissue culture, the expression of the ACR gene family is differentially regulated by plant hormones, salt stress, cold stress, and light/dark treatment. The steady-state levels of ACR8 mRNA are dramatically increased by treatment with abscisic acid or salt. Levels of ACR3 and ACR4 mRNA are increased by treatment with benzyladenine. The amino acid sequences of Arabidopsis ACR proteins are most similar in the ACT domains to the bacterial sensor protein GlnD. The ACR proteins may function as novel regulatory or sensor proteins in plants.
Independent Control of Gibberellin Biosynthesis and Flowering Time by the Circadian Clock in Arabidopsis
Authors: Blázquez Miguel A. , Trénor Marta , Weigel Detlef
Published: December 2002

Flowering of the facultative long-day plant Arabidopsis is controlled by several endogenous and environmental factors, among them gibberellins (GAs) and day length. The promotion of flowering by long days involves an endogenous clock that interacts with light cues provided by the environment. Light, and specifically photoperiod, is also known to regulate the biosynthesis of GAs, but the effects of GAs and photoperiod on flowering are at least partially separable. Here, we have used a short-period mutant,toc1, to investigate the role of the circadian clock in the control of flowering time by GAs and photoperiod. We show thattoc1 affects expression of several floral regulators and a GA biosynthetic gene, but that these effects are independent.
Forms of Zinc Accumulated in the HyperaccumulatorArabidopsis halleri
Authors: Sarret Géraldine , Saumitou-Laprade Pierre , Bert Valérie , Proux Olivier , Hazemann Jean-Louis , Traverse Agnès , Marcus Matthew A. , Manceau Alain
Published: December 2002

The chemical forms of zinc (Zn) in the Zn-tolerant and hyperaccumulator Arabidopsis halleri and in the non-tolerant and nonaccumulator Arabidopsis  lyrata subsp. petraea were determined at the molecular level by combining chemical analyses, extended x-ray absorption spectroscopy (EXAFS), synchrotron-based x-ray microfluorescence, and μEXAFS. Plants were grown in hydroponics with various Zn concentrations, and A. halleri specimens growing naturally in a contaminated site were also collected. Zn speciation in A. halleri was independent of the origin of the plants (contaminated or non-contaminated) and Zn exposure. In aerial parts, Zn was predominantly octahedrally coordinated and complexed to malate. A secondary organic species was identified in the bases of the trichomes, which contained elevated Zn concentrations, and in which Zn was tetrahedrally coordinated and complexed to carboxyl and/or hydroxyl functional groups. This species was detected thanks to the good resolution and sensitivity of synchrotron-based x-ray microfluorescence and μEXAFS. In the roots of A. halleri grown in hydroponics, Zn phosphate was the only species detected, and is believed to result from chemical precipitation on the root surface. In the roots of A. halleri grown on the contaminated soil, Zn was distributed in Zn malate, Zn citrate, and Zn phosphate. Zn phosphate was present in both the roots and aerial part of A. lyrata subsp. petraea. This study illustrates the complementarity of bulk and spatially resolved techniques, allowing the identification of: (a) the predominant chemical forms of the metal, and (b) the minor forms present in particular cells, both types of information being essential for a better understanding of the bioaccumulation processes.
Cloning, Functional Expression, and Subcellular Localization of Multiple NADPH-Cytochrome P450 Reductases from Hybrid Poplar
Authors: Ro Dae-Kyun , Ehlting Jürgen , Douglas Carl J.
Published: December 2002

NADPH:cytochrome P450 reductase (CPR) provides reducing equivalents to diverse cytochrome P450 monooxygenases. We isolated cDNAs for three CPR genes (CPR1,CPR2, and CPR3) from hybrid poplar (Populus trichocarpa × Populus deltoides). Deduced CPR2 and CPR3 amino acid sequences were 91% identical, but encoded isoforms divergent from CPR1 (72% identity). CPR1 and CPR2 were co-expressed together with the P450 enzyme cinnamate-4-hydroxylase (C4H) in yeast (Saccharomyces cerevisiae). Microsomes isolated from strains expressing CPR1/C4H or CPR2/C4H enhanced C4H activities approximately 10-fold relative to the C4H-only control strain, and catalyzed NADPH-dependent cytochrome c reduction. The divergent CPR isoforms (CPR1 and CPR2/3) contained entirely different N-terminal sequences, which are conserved in other plant CPRs and are diagnostic for two distinct classes of CPRs within the angiosperms. C-terminal green fluorescent protein fusions to CPR1 and CPR2 were constructed and expressed in both yeast and Arabidopsis. The fusion proteins expressed in yeast retained the ability to support C4H activity and, thus, were catalytically active. Both CPR::green fluorescent protein fusion proteins were strictly localized to the endoplasmic reticulum in transgenic Arabidopsis. The lack of localization of either isoform to chloroplasts, where P450s are known to be present, suggests that an alternative P450 reduction system may be operative in this organelle. Transcripts for the three poplar CPR genes were present ubiquitously in all tissues examined, but CPR2 showed highest expression in young leaf tissue.
Expression and Biochemical Properties of a Ferredoxin-Dependent Heme Oxygenase Required for Phytochrome Chromophore Synthesis
Authors: Muramoto Takuya , Tsurui Noriyuki , Terry Matthew J. , Yokota Akiho , Kohchi Takayuki
Published: December 2002

The HY1 gene of Arabidopsis encodes a plastid heme oxygenase (AtHO1) required for the synthesis of the chromophore of the phytochrome family of plant photoreceptors. To determine the enzymatic properties of plant heme oxygenases, we have expressed theHY1 gene (without the plastid transit peptide) inEscherichia coli to produce an amino terminal fusion protein between AtHO1 and glutathione S-transferase. The fusion protein was soluble and expressed at high levels. Purified recombinant AtHO1, after glutathione S-transferase cleavage, is a hemoprotein that forms a 1:1 complex with heme. In the presence of reduced ferredoxin, AtHO1 catalyzed the formation of biliverdin IXα from heme with the concomitant production of carbon monoxide. Heme oxygenase activity could also be reconstituted using photoreduced ferredoxin generated through light irradiation of isolated thylakoid membranes, suggesting that ferredoxin may be the electron donor in vivo. In addition, AtHO1 required an iron chelator and second reductant, such as ascorbate, for full activity. These results show that the basic mechanism of heme cleavage has been conserved between plants and other organisms even though the function, subcellular localization, and cofactor requirements of heme oxygenases differ substantially.
Temperature Response of Mesophyll Conductance. Implications for the Determination of Rubisco Enzyme Kinetics and for Limitations to Photosynthesis in Vivo
Authors: Bernacchi Carl J. , Portis Archie R. , Nakano Hiromi , von Caemmerer Susanne , Long Stephen P.
Published: December 2002

CO2 transfer conductance from the intercellular airspaces of the leaf into the chloroplast, defined as mesophyll conductance (g  m), is finite. Therefore, it will limit photosynthesis when CO2 is not saturating, as in C3 leaves in the present atmosphere. Little is known about the processes that determine the magnitude ofg  m. The process dominatingg  m is uncertain, though carbonic anhydrase, aquaporins, and the diffusivity of CO2 in water have all been suggested. The response ofg  m to temperature (10°C–40°C) in mature leaves of tobacco (Nicotiana tabacum L. cv W38) was determined using measurements of leaf carbon dioxide and water vapor exchange, coupled with modulated chlorophyll fluorescence. These measurements revealed a temperature coefficient (Q10) of approximately 2.2 for g  m, suggesting control by a protein-facilitated process because the Q10for diffusion of CO2 in water is about 1.25. Further,g  m values are maximal at 35°C to 37.5°C, again suggesting a protein-facilitated process, but with a lower energy of deactivation than Rubisco. Using the temperature response of g  m to calculate CO2 at Rubisco, the kinetic parameters of Rubisco were calculated in vivo from 10°C to 40°C. Using these parameters, we determined the limitation imposed on photosynthesis byg  m. Despite an exponential rise with temperature, g  m does not keep pace with increased capacity for CO2 uptake at the site of Rubisco. The fraction of the total limitations to CO2uptake within the leaf attributable tog  m rose from 0.10 at 10°C to 0.22 at 40°C. This shows that transfer of CO2 from the intercellular air space to Rubisco is a very substantial limitation on photosynthesis, especially at high temperature.
Transcriptome Changes for Arabidopsis in Response to Salt, Osmotic, and Cold Stress,
Authors: Kreps Joel A. , Wu Yajun , Chang Hur-Song , Zhu Tong , Wang Xun , Harper Jeff F.
Published: December 2002

To identify genes of potential importance to cold, salt, and drought tolerance, global expression profiling was performed on Arabidopsis plants subjected to stress treatments of 4°C, 100 mm NaCl, or 200 mm mannitol, respectively. RNA samples were collected separately from leaves and roots after 3- and 27-h stress treatments. Profiling was conducted with a GeneChip microarray with probe sets for approximately 8,100 genes. Combined results from all three stresses identified 2,409 genes with a greater than 2-fold change over control. This suggests that about 30% of the transcriptome is sensitive to regulation by common stress conditions. The majority of changes were stimulus specific. At the 3-h time point, less than 5% (118 genes) of the changes were observed as shared by all three stress responses. By 27 h, the number of shared responses was reduced more than 10-fold (< 0.5%), consistent with a progression toward more stimulus-specific responses. Roots and leaves displayed very different changes. For example, less than 14% of the cold-specific changes were shared between root and leaves at both 3 and 27 h. The gene with the largest induction under all three stress treatments was At5g52310 (LTI/COR78), with induction levels in roots greater than 250-fold for cold, 40-fold for mannitol, and 57-fold for NaCl. A stress response was observed for 306 (68%) of the known circadian controlled genes, supporting the hypothesis that an important function of the circadian clock is to “anticipate” predictable stresses such as cold nights. Although these results identify hundreds of potentially important transcriptome changes, the biochemical functions of many stress-regulated genes remain unknown.
Comparative Molecular and Functional Analyses of the Tobacco Cyclin-Dependent Kinase Inhibitor NtKIS1a and Its Spliced Variant NtKIS1b
Authors: Jasinski Sophie , Perennes Claudette , Bergounioux Catherine , Glab Nathalie
Published: December 2002

In all eukaryotes, cell cycle progression is controlled by cyclin-dependent kinases (CDKs) whose activity is regulated at several levels including inhibition by CDK inhibitors. Here, we report a comparative molecular and functional analysis of the tobacco (Nicotiana tomentosiformis) CDK inhibitor, NtKIS1a, and its spliced variant, NtKIS1b. The C-terminal end of NtKIS1a shares strong sequence similarity with mammalian CIP/KIP inhibitors, which is not the case for NtKIS1b. Consistent with this, NtKIS1a but not NtKIS1b inhibits in vitro the kinase activity of CDK/cyclin complexes, and tobacco (Nicotiana tabacum) D-type cyclins and an A-type CDK are NtKIS1a, but not NtKIS1b, interacting partners. Although both NtKIS1a and NtKIS1b transcripts are mainly found in flowers and more precisely in stamens, NtKIS1b transcript levels are cell cycle regulated, whereas those of NtKIS1a remain constant during the cell cycle. NtKIS1a and NtKIS1b fused to fluorescent proteins are localized in the nucleus when transiently expressed in onion epidermal cells. Furthermore, there is no competition for their nuclear localization when they are simultaneously overexpressed. In vitro competition toward CDK kinase activity suggests that NtKIS1b is a strong competitor of NtKIS1a. Arabidopsis plants overexpressing NtKIS1a-green fluorescent protein (GFP) or NtKIS1b-GFP fusion proteins were obtained. In these plants, the fusion proteins are still localized in the nucleus. Interestingly, NtKIS1a-GFP-overexpressing plants display strong morphological modifications and a reduced CDK kinase activity, whereas NtKIS1b-GFP-overexpressing plants display a wild-type phenotype including a wild-type CDK kinase activity. Our results strongly suggest that the inhibition of the kinase activity is responsible for the phenotypic modifications.
Differential Regulation of RNA Levels of Gibberellin Dioxygenases by Photoperiod in Spinach
Authors: Lee Dong Ju , Zeevaart Jan A.D.
Published: December 2002

Previous work with spinach (Spinacia oleracea) has shown that the level of gibberellin (GA) 20-oxidase is strongly up-regulated by long days (LD). In the present work, the effect of photoperiod on expression of other GA dioxygenases was investigated and compared with that of GA 20-oxidase. Two GA 2-oxidases and one GA 3-oxidase were isolated from spinach by reverse transcription-polymerase chain reaction with degenerate primers and by 5′- and 3′-rapid amplification of cDNA ends. As determined by high-performance liquid chromatography with on-line radioactivity detection, the SoGA3ox1 gene product catalyzed 3β-hydroxylation of GA9 to GA4 and GA20 to GA1. The SoGA2ox1 and the SoGA2ox2 gene products catalyzed 2β-hydroxylation of GA9 to GA51 and GA20 to GA29. The product of GA20 metabolism by SoGA3ox1 was identified as GA1 by gas chromatography-mass spectrometry, whereas the products of GA1 and GA20 metabolism by SoGA2ox1 and SoGA2ox2 were identified as GA8 and GA29, respectively. SoGA2ox1 also metabolized GA53 to GA97. The levels ofSoGA20ox1 transcripts were greatly increased in all organs tested in LD conditions, but the levels ofSoGA3ox1 transcripts were only slightly increased in blades and petioles. A decrease in the levels of theSoGA2ox1 transcripts in young leaves and tips in LD conditions is opposite to the expression pattern of theSoGA20ox1. Expression of SoGA20ox1 in petioles and young leaves was strongly up-regulated by a supplementary 16 h of light, but the levels of SoGA3ox1 andSoGA2ox1 transcripts did not change. It is concluded that regulation and maintenance of GA1 concentration in spinach are primarily attributable to changes in expression ofSoGA20ox1.
Characterization of Arabidopsis AtAMT2, a High-Affinity Ammonium Transporter of the Plasma Membrane
Authors: Sohlenkamp Christian , Wood Craig C. , Roeb Gerhard W. , Udvardi Michael K.
Published: December 2002

AtAMT2 is an ammonium transporter that is only distantly related to the five members of the AtAMT1 family of high-affinity ammonium transporters in Arabidopsis. The short-lived radioactive ion13NH4  + was used to show that AtAMT2, expressed in yeast (Saccharomyces cerevisiae), is a high-affinity transporter with a K  m for ammonium of about 20 μm. Changes in external pH between 5.0 and 7.5 had little effect on the K  m for ammonium, indicating that NH4  +, not NH3, is the substrate for AtAMT2. TheAtAMT2 gene was expressed in all organs of Arabidopsis and was subject to nitrogen (N) regulation, at least in roots where expression was partially repressed by high concentrations of ammonium nitrate and derepressed in the absence of external N. Although expression of AtAMT2 in shoots responded little to changes in root N status, transcript levels in leaves declined under high CO2 conditions. Transient expression of an AtAMT2-green fluorescent protein fusion protein in Arabidopsis leaf epidermal cells indicated a plasma membrane location for the AtAMT2 protein. Thus, AtAMT2 is likely to play a significant role in moving ammonium between the apoplast and symplast of cells throughout the plant. However, a dramatic reduction in the level ofAtAMT2 transcript brought about by dsRNA interference with gene expression had no obvious effect on plant growth or development, under the conditions tested.
Nitric Oxide Improves Internal Iron Availability in Plants
Authors: Graziano Magdalena , Beligni Marı́a Verónica , Lamattina Lorenzo
Published: December 2002

Iron deficiency impairs chlorophyll biosynthesis and chloroplast development. In leaves, most of the iron must cross several biological membranes to reach the chloroplast. The components involved in the complex internal iron transport are largely unknown. Nitric oxide (NO), a bioactive free radical, can react with transition metals to form metal-nitrosyl complexes. Sodium nitroprusside, an NO donor, completely prevented leaf interveinal chlorosis in maize (Zea mays) plants growing with an iron concentration as low as 10 μmFe-EDTA in the nutrient solution.S-Nitroso-N-acetylpenicillamine, another NO donor, as well as gaseous NO supply in a translucent chamber were also able to revert the iron deficiency symptoms. A specific NO scavenger, 2-(4-carboxy-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, blocked the effect of the NO donors. The effect of NO treatment on the photosynthetic apparatus of iron-deficient plants was also studied. Electron micrographs of mesophyll cells from iron-deficient maize plants revealed plastids with few photosynthetic lamellae and rudimentary grana. In contrast, in NO-treated maize plants, mesophyll chloroplast appeared completely developed. NO treatment did not increase iron content in plant organs, when expressed in a fresh matter basis, suggesting that root iron uptake was not enhanced. NO scavengers 2-(4-carboxy-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and methylene blue promoted interveinal chlorosis in iron-replete maize plants (growing in 250 μm Fe-EDTA). Even though results support a role for endogenous NO in iron nutrition, experiments did not establish an essential role. NO was also able to revert the chlorotic phenotype of the iron-inefficient maize mutants yellow stripe1 and yellow stripe3, both impaired in the iron uptake mechanisms. All together, these results support a biological action of NO on the availability and/or delivery of metabolically active iron within the plant.
The Elicitor Cryptogein Blocks Glucose Transport in Tobacco Cells
Authors: Bourque Stéphane , Lemoine Rémi , Sequeira-Legrand Anabelle , Fayolle Léon , Delrot Serge , Pugin Alain
Published: December 2002

Cryptogein is a 10-kD protein secreted by the oomycetePhytophthora cryptogea that induces a hypersensitive response on tobacco (Nicotiana tabacum var. Xanthi) plants and a systemic acquired resistance against various pathogens. The mode of action of this elicitor has been studied using tobacco cell suspensions. Our previous data indicated that within minutes, cryptogein signaling involves various events including changes in ion fluxes, protein phosphorylation, sugar metabolism, and, eventually, cell death. These results suggested that transport of sugars could be affected and, thus, involved in the complex relationships between plant and microorganisms via elicitors. This led us to investigate the effects of cryptogein on glucose (Glc) uptake and mitochondrial activity in tobacco cells. Cryptogein induces an immediate inhibition of Glc uptake, which is not attributable to plasma membrane (PM) depolarization. Conversely, cryptogein-induced valine uptake is because of PM depolarization. Inhibition of the PM Glc transporter(s) was shown to be mediated by a calcium-dependent phosphorylation process, and is independent of active oxygen species production. This inhibition was associated with a strong decrease in O2 uptake rate by cells and a large mitochondrial membrane depolarization. Thus, inhibition of Glc uptake accompanied by inhibition of phosphorylative oxidation may participate in hypersensitive cell death. These results are discussed in the context of competition between plants and microorganisms for apoplastic sugars.
Germanium Does Not Substitute for Boron in Cross-Linking of Rhamnogalacturonan II in Pumpkin Cell Walls
Authors: Ishii Tadashi , Matsunaga Toshiro , Iwai Hiroaki , Satoh Shinobu , Taoshita Junji
Published: December 2002

Boron (B)-deficient pumpkin (Cucurbita  moschata Duchesne) plants exhibit reduced growth, and their tissues are brittle. The leaf cell walls of these plants contain less than one-half the amount of borate cross-linked rhamnogalacturonan II (RG-II) dimer than normal plants. Supplying germanium (Ge), which has been reported to substitute for B, to B-deficient plants does not restore growth or reduce tissue brittleness. Nevertheless, the leaf cell walls of the Ge-treated plants accumulated considerable amounts of Ge. Dimeric RG-II (dRG-II) accounted for between 20% and 35% of the total RG-II in the cell walls of the second to fourth leaves from Ge-treated plants, but only 2% to 7% of the RG-II was cross-linked by germanate (dRG-II-Ge). The ability of RG-II to form a dimer is not reduced by Ge treatment because approximately 95% of the monomeric RG-II generated from the walls of Ge-treated plants is converted to dRG-II-Ge in vitro in the presence of germanium oxide and lead acetate. However, dRG-II-Ge is unstable and is converted to monomeric RG-II when the Ge is removed. Therefore, the content of dRG-II-Ge and dRG-II-B described above may not reflect the actual ratio of these in muro.10B-Enriched boric acid and Ge are incorporated into the cell wall within 10 min after their foliar application to B-deficient plants. Foliar application of 10B but not Ge results in an increase in the proportion of dRG-II in the leaf cell wall. Taken together, our results suggest that Ge does not restore the growth of B-deficient plants.
Biosynthesis of UDP-Xylose. Cloning and Characterization of a Novel Arabidopsis Gene Family, UXS, Encoding Soluble and Putative Membrane-Bound UDP-Glucuronic Acid Decarboxylase Isoforms
Authors: Harper April D. , Bar-Peled Maor
Published: December 2002

UDP-xylose (Xyl) is an important sugar donor for the synthesis of glycoproteins, polysaccharides, various metabolites, and oligosaccharides in animals, plants, fungi, and bacteria. UDP-Xyl also feedback inhibits upstream enzymes (UDP-glucose [Glc] dehydrogenase, UDP-Glc pyrophosphorylase, and UDP-GlcA decarboxylase) and is involved in its own synthesis and the synthesis of UDP-arabinose. In plants, biosynthesis of UDP-Xyl is catalyzed by different membrane-bound and soluble UDP-GlcA decarboxylase (UDP-GlcA-DC) isozymes, all of which convert UDP-GlcA to UDP-Xyl. Because synthesis of UDP-Xyl occurs both in the cytosol and in membranes, it is not known which source of UDP-Xyl the different Golgi-localized xylosyltransferases are utilizing. Here, we describe the identification of several distinct Arabidopsis genes (named AtUXS for UDP-Xyl synthase) that encode functional UDP-GlcA-DC isoforms. The Arabidopsis genome contains fiveUXS genes and their protein products can be subdivided into three isozyme classes (A–C), one soluble and two distinct putative membrane bound. AtUxs from each class, when expressed inEscherichia coli, generate active UDP-GlcA-DC that converts UDP-GlcA to UDP-Xyl. Members of this gene family have a large conserved C-terminal catalytic domain (approximately 300 amino acids long) and an N-terminal variable domain differing in sequence and size (30–120 amino acids long). Isoforms of class A and B appear to encode putative type II membrane proteins with their catalytic domains facing the lumen (like Golgi-glycosyltransferases) and their N-terminal variable domain facing the cytosol. Uxs class C is likely a cytosolic isoform. The characteristics of the plant Uxs support the hypothesis that unique UDP-GlcA-DCs with distinct subcellular localizations are required for specific xylosylation events.
Ethylene Synthesis Regulated by Biphasic Induction of 1-Aminocyclopropane-1-Carboxylic Acid Synthase and 1-Aminocyclopropane-1-Carboxylic Acid Oxidase Genes Is Required for Hydrogen Peroxide Accumulation and Cell Death in Ozone-Exposed Tomato
Authors: Moeder Wolfgang , Barry Cornelius S. , Tauriainen Airi A. , Betz Christian , Tuomainen Jaana , Utriainen Merja , Grierson Donald , Sandermann Heinrich , Langebartels Christian , Kangasjärvi Jaakko
Published: December 2002

We show that above a certain threshold concentration, ozone leads to leaf injury in tomato (Lycopersicon esculentum). Ozone-induced leaf damage was preceded by a rapid increase in 1-aminocyclopropane-1-carboxylic acid (ACC) synthase activity, ACC content, and ethylene emission. Changes in mRNA levels of specific ACC synthase, ACC oxidase, and ethylene receptor genes occurred within 1 to 5 h. Expression of the genes encoding components of ethylene biosynthesis and perception, and biochemistry of ethylene synthesis suggested that ozone-induced ethylene synthesis in tomato is under biphasic control. In transgenic plants containing anLE-ACO1 promoter-β-glucuronidase fusion construct, β-glucuronidase activity increased rapidly at the beginning of the O3 exposure and had a spatial distribution resembling the pattern of extracellular H2O2 production at 7 h, which coincided with the cell death pattern after 24 h. Ethylene synthesis and perception were required for active H2O2 production and cell death resulting in visible tissue damage. The results demonstrate a selective ozone response of ethylene biosynthetic genes and suggest a role for ethylene, in combination with the burst of H2O2production, in regulating the spread of cell death.
Expressed Sequence Tag-Based Gene Expression Analysis under Aluminum Stress in Rye,
Authors: Milla Miguel A. Rodriguez , Butler Ed , Huete Alicia Rodriguez , Wilson Cindy F. , Anderson Olin , Gustafson J. Perry
Published: December 2002

To understand the mechanisms responsible for aluminum (Al) toxicity and tolerance in plants, an expressed sequence tag (EST) approach was used to analyze changes in gene expression in roots of rye (Secale cereale L. cv Blanco) under Al stress. Two cDNA libraries were constructed (Al stressed and unstressed), and a total of 1,194 and 774 ESTs were generated, respectively. The putative proteins encoded by these cDNAs were uncovered by Basic Local Alignment Search Tool searches, and those ESTs showing similarity to proteins of known function were classified according to 13 different functional categories. A total of 671 known function genes were used to analyze the gene expression patterns in rye cv Blanco root tips under Al stress. Many of the previously identified Al-responsive genes showed expression differences between the libraries within 6 h of Al stress. Certain genes were selected, and their expression profiles were studied during a 48-h period using northern analysis. A total of 13 novel genes involved in cell elongation and division (tonoplast aquaporin and ubiquitin-like protein SMT3), oxidative stress (glutathione peroxidase, glucose-6-phosphate-dehydrogenase, and ascorbate peroxidase), iron metabolism (iron deficiency-specific proteins IDS3a, IDS3b, and IDS1; S-adenosyl methionine synthase; and methionine synthase), and other cellular mechanisms (pathogenesis-related protein 1.2, heme oxygenase, and epoxide hydrolase) were demonstrated to be regulated by Al stress. These genes provide new insights about the response of Al-tolerant plants to toxic levels of Al.
A Rice Mutant Defective in Si Uptake
Authors: Ma Jian Feng , Tamai Kazunori , Ichii Masahiko , Wu Guo Feng
Published: December 2002

Rice (Oryza sativa) accumulates silicon (Si) in the tops to levels up to 10.0% of shoot dry weight, but the mechanism responsible for high Si uptake by rice roots is not understood. We isolated a rice mutant (GR1) that is defective in active Si uptake by screening M2 seeds (64,000) of rice cv Oochikara that were treated with 10−3  m sodium azide for 6 h at 25oC. There were no phenotypic differences between wild type (WT) and GR1 except that the leaf blade of GR1 remained droopy when Si was supplied. Uptake experiments showed that Si uptake by GR1 was significantly lower than that by WT at both low and high Si concentrations. However, there was no difference in the uptake of other nutrients such as phosphorus and potassium. Si concentration in the xylem sap of WT was 33-fold that of the external solution, but that of GR1 was 3-fold higher than the external solution at 0.15 mm Si. Si uptake by WT was inhibited by metabolic inhibitors including NaCN and 2,4-dinitrophenol and by low temperature, whereas Si uptake by GR1 was not inhibited by these agents. These results suggest that an active transport system for Si uptake is disrupted in GR1. Analysis of F2 populations between GR1 and WT showed that roots with high Si uptake and roots with low Si uptake segregated at a 3:1 ratio, suggesting that GR1 is a recessive mutant of Si uptake.
cDNA Cloning, Heterologous Expressions, and Functional Characterization of Malonyl-Coenzyme A:Anthocyanidin 3-O-Glucoside-6"-O-Malonyltransferase from Dahlia Flowers
Authors: Suzuki Hirokazu , Nakayama Toru , Yonekura-Sakakibara Keiko , Fukui Yuko , Nakamura Noriko , Yamaguchi Masa-atsu , Tanaka Yoshikazu , Kusumi Takaaki , Nishino Tokuzo
Published: December 2002

In the flowers of important ornamental Compositae plants, anthocyanins generally carry malonyl group(s) at their 3-glucosyl moiety. In this study, for the first time to our knowledge, we have identified a cDNA coding for this 3-glucoside-specific malonyltransferase for anthocyanins, i.e. malonyl-coenzyme A:anthocyanidin 3-O-glucoside-6"-O-malonyltransferase, from dahlia (Dahlia variabilis) flowers. We isolated a full-length cDNA (Dv3MaT) on the basis of amino acid sequences specifically conserved among anthocyanin acyltransferases of the versatile plant acyltransferase family. Dv3MaT coded for a protein of 460 amino acids. Quantitative real-time PCR analyses of Dv3MaT showed that the transcript was present in accordance with the distribution of 3MaT activities and the anthocyanin accumulation pattern in the dahlia plant. The Dv3MaTcDNA was expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity and characterized. The recombinant Dv3MaT catalyzed the regiospecific transfer of the malonyl group from malonyl-coenzyme A (K  m, 18.8 μm) to pelargonidin 3-O-glucoside (K  m, 46.7 μm) to produce pelargonidin 3-O-6"-O-malonylglucoside with a k  cat value of 7.3 s−1. The other enzymatic profiles of the recombinant Dv3MaT were closely related to those of native anthocyanin malonyltransferase activity in the extracts of dahlia flowers. Dv3MaT cDNA was introduced into petunia (Petunia hybrida) plants whose red floral color is exclusively provided by cyanidin 3-O-glucoside and 3,5-O-diglucoside. Thirteen transgenic lines of petunia were found to produce malonylated products of these anthocyanins (11–63 mol % of total anthocyanins in the flower). The spectral stability of cyanidin 3-O-6"-O-malonylglucoside at the pHs of intracellular milieus of flowers was significantly higher than that of cyanidin 3-O-glucoside. Moreover, 6"-O-malonylation of cyanidin 3-O-glucoside effectively prevented the anthocyanin from attack of β-glucosidase. These results suggest that malonylation should serve as a strategy for pigment stabilization in the flowers.
Auxin and Ethylene Response Interactions during Arabidopsis Root Hair Development Dissected by Auxin Influx Modulators
Authors: Rahman Abidur , Hosokawa Satoko , Oono Yutaka , Amakawa Taisaku , Goto Nobuharu , Tsurumi Seiji
Published: December 2002

The plant hormones auxin and ethylene have been shown to play important roles during root hair development. However, cross talk between auxin and ethylene makes it difficult to understand the independent role of either hormone. To dissect their respective roles, we examined the effects of two compounds, chromosaponin I (CSI) and 1-naphthoxyacetic acid (1-NOA), on the root hair developmental process in wild-type Arabidopsis, ethylene-insensitive mutantein2-1, and auxin influx mutants aux1-7,aux1-22, and double mutant aux1-7 ein2. β-Glucuronidase (GUS) expression analysis in the BA-GUS transgenic line, consisting of auxin-responsive domains ofPS-IAA4/5 promoter and GUS reporter, revealed that 1-NOA and CSI act as auxin uptake inhibitors in Arabidopsis roots. The frequency of root hairs in ein2-1roots was greatly reduced in the presence of CSI or 1-NOA, suggesting that endogenous auxin plays a critical role for the root hair initiation in the absence of an ethylene response. All of these mutants showed a reduction in root hair length, however, the root hair length could be restored with a variable concentration of 1-naphthaleneacetic acid (NAA). NAA (10 nm) restored the root hair length ofaux1 mutants to wild-type level, whereas 100 nm NAA was needed for ein2-1 andaux1-7 ein2 mutants. Our results suggest that insensitivity in ethylene response affects the auxin-driven root hair elongation. CSI exhibited a similar effect to 1-NOA, reducing root hair growth and the number of root hair-bearing cells in wild-type andein2-1 roots, while stimulating these traits inaux1-7and aux1-7ein2 roots, confirming that CSI is a unique modulator of AUX1.
Auxin Regulation of the Gibberellin Pathway in Pea
Authors: O'Neill Damian P. , Ross John J.
Published: December 2002

The auxin indole-3-acetic acid (IAA) has been shown to promote the biosynthesis of the active gibberellin (GA1) in shoots of pea (Pisum sativum). We used northern analysis to investigate the timing of IAA-induced changes in transcript levels ofPsGA3ox1 (Mendel's LE),PsGA2ox1, PsGA2ox2, andPsGA20ox1, key genes for the later stages of GA1 biosynthesis and metabolism in pea. Rapid (2–4 h) changes occurred in the transcript levels of PsGA3ox1,PsGA2ox1, and PsGA2ox2 after treatent with IAA. [14C]GA1 metabolism studies in decapitated shoots indicated that IAA inhibits GA1 deactivation, consistent with the suppression ofPsGA2ox1 (SLN) transcript levels by IAA. Studies with the sln mutant also indicated thatPsGA2ox1 activity is involved in GA1deactivation in decapitated shoots. Culture of excised internode tissue in the presence of auxin clearly demonstrated that internode tissue is a site of GA1 biosynthesis per se. Excised internode tissue cultured in the presence/absence of cycloheximide showed that de novo protein synthesis is required for IAA-induced increases inPsGA3ox1 transcript levels. Auxin dose response studies indicated that IAA concentration is a critical determinant of GA1 biosynthesis over 1 to 2 orders of magnitude, and a range of auxins was shown to affect the GA pathway.
Acclimation of Rice Photosynthesis to Irradiance under Field Conditions
Authors: Murchie Erik H. , Hubbart Stella , Chen Yizhu , Peng Shaobing , Horton Peter
Published: December 2002

Acclimation to irradiance was measured in terms of light-saturated photosynthetic carbon assimilation rates (Pmax), Rubisco, and pigment content in mature field-grown rice (Oryza sativa) plants in tropical conditions. Measurements were made at different positions within the canopy alongside irradiance and daylight spectra. These data were compared with a second experiment in which acclimation to irradiance was assessed in uppermost leaves within whole-plant shading regimes (10% low light [LL], 40% medium light [ML], and 100% high light [HL] of full natural sunlight). Two varieties, japonica (tropical; new plant type [NPT]) and indica (IR72) were compared. Values for Rubisco amount, chlorophyll a/b, and Pmax all declined from the top to the base of the canopy. In the artificial shading experiment, acclimation of Pmax (measured at 350 μL L−1 CO2) occurred between LL and ML for IR72 with no difference observed between ML and HL. The Rubisco amount increased between ML and HL in IR72. A different pattern was seen for NPT with higher Pmax (measured at 350 μL L−1CO2) at LL than IR72 and some acclimation of this parameter between ML and HL. Rubisco levels were higher in NPT than IR72 contrasting with Pmax. Comparison of data from both experiments suggests a leaf aging effect between the uppermost two leaf positions, which was not a result of irradiance acclimation. Results are discussed in terms of: (a) acclimation of photosynthesis and radiation use efficiency at high irradiance in rice, and (b) factors controlling photosynthetic rates of leaves within the canopy.
Enhancer Trap Expression Patterns Provide a Novel Teaching Resource
Authors: Geisler Matt , Jablonska Barbara , Springer Patricia S.
Published: December 2002

A collection of Arabidopsis enhancer trap transposants has been identified for use as a teaching tool. This collection serves to assist students in understanding the patterning and organization of plant tissues and cells, and will be useful in plant anatomy, morphology, and developmental biology courses. Each transposant exhibits reporter gene expression in a specific tissue, cell type, or domain, and these lines collectively offer a glimpse of compartments of gene expression. Some compartments correspond to classical definitions of botanical anatomy and can assist in anatomical identification. Other patterns of reporter gene expression are more complex and do not necessarily correspond to known anatomical features. The sensitivity of the β-glucuronidase histochemical stain provides the student with a colorful and direct way to visualize difficult aspects of plant development and anatomy, and provides the teacher with an invaluable tool for a practical laboratory session.