bluuebunny/crossref_metadata_2025 huggingface dataset:
Epidermal Growth Factor Prevents Acetaldehyde‐Induced Paracellular Permeability in Caco‐2 Cell Monolayer
Authors: Sheth P. , Seth A. , Thangavel M. , Basuroy S. , Rao R. K.
Published: May 2004

Abstract: Background: Intestinal permeability and endotoxemia play a crucial role in the pathogenesis of alcoholic liver disease. Previous studies showed that acetaldehyde disrupts intestinal epithelial barrier function and increases paracellular permeability by a tyrosine kinase‐dependent mechanism. In the present study, the role of epidermal growth factor (EGF) in protection of epithelial barrier function from acetaldehyde was evaluated in Caco‐2 intestinal epithelial cell monolayer.

Methods: Caco‐2 cells on Transwell inserts were exposed to acetaldehyde in the absence or presence of EGF, and the paracellular permeability was evaluated by measuring transepithelial electrical resistance and unidirectional flux of inulin. Integrity of epithelial tight junctions and adherens junctions was analyzed by confocal immunofluorescence microscopy and immunoblot analysis of occludin, zonula occludens (ZO)‐1, E‐cadherin, and β‐catenin in the actin cytoskeleton. Reorganization of actin cytoskeletal architecture was examined by confocal microscopy.

Results: Acetaldehyde increased paracellular permeability to inulin and lipopolysaccharide, and EGF significantly reduced these effects of acetaldehyde in a time‐ and dose‐dependent manner. EGF prevented acetaldehyde‐induced reorganization of occludin, ZO‐1, E‐cadherin, and β‐catenin from the cellular junctions to the intracellular compartments. Acetaldehyde treatment induced a reorganization of actin cytoskeletal network and reduced the levels of occludin, ZO‐1, E‐cadherin, and β‐catenin associated with the actin cytoskeleton. EGF effectively prevented acetaldehyde‐induced reorganization of actin cytoskeleton and the interaction of occludin, ZO‐1, E‐cadherin, and β‐catenin with the actin cytoskeleton.

Conclusion: These results indicate that EGF attenuates acetaldehyde‐induced disruption of tight junctions and adherens junctions and prevents acetaldehyde‐induced reorganization of actin cytoskeleton and its interaction with occludin, ZO‐1, E‐cadherin, and β‐catenin.
Legionella drancourtii sp. nov., a strictly intracellular amoebal pathogen
Authors: Scola Bernard La , Birtles Richard J. , Greub Gilbert , Harrison Timothy J. , Ratcliff Rodney M. , Raoult Didier
Published: May 2004

A Legionella-like amoebal pathogen (LLAP), formerly named LLAP12T, was characterized on the basis of microscopic appearance, staining characteristics, growth in Acanthamoeba polyphaga at different temperatures, DNA G+C content, serological cross-reactivity and 16S rRNA and macrophage infectivity potentiator (mip) gene sequence analysis. LLAP12T was found to be a motile, Gram-negative bacterium that grew within cytoplasmic vacuoles in infected amoebae. The infecting bacteria induced lysis of their amoebal hosts and time taken to do so was dependent on incubation temperature. Recovery of LLAP12T from amoebae onto axenic media could not be achieved. Phylogenetic analysis of LLAP12T, based on 16S rRNA and mip gene sequence analysis, indicated that it lay within the radiation of the Legionellaceae and that it clustered specifically with Legionella lytica and Legionella rowbothamii. The divergence observed between LLAP12T and these two species was of a degree equal to, or greater than, that observed between other members of the family. In support of this delineation, LLAP12T was found not to cross-react serologically with any other Legionella species. The mip and 16S rRNA gene sequence-based analyses also indicated that LLAP12T was related very closely to two other previously identified LLAP isolates, LLAP4 and LLAP11. Taken together, these results support the proposal of LLAP12T as the type strain of Legionella drancourtii sp. nov.
Catellibacterium nectariphilum gen. nov., sp. nov., which requires a diffusible compound from a strain related to the genus Sphingomonas for vigorous growth
Authors: Tanaka Yasuhiro , Hanada Satoshi , Manome Akira , Tsuchida Takayasu , Kurane Ryuichiro , Nakamura Kazunori , Kamagata Yoichi
Published: May 2004

A bacterial strain, designated AST4T, was isolated from activated sludge. The bacterium did not show significant growth on nutrient broth, but growth was clearly stimulated by addition of supernatant from other bacterial cultures. Culture filtrate of a strain related to the genus Sphingomonas in particular increased the cell yield and growth rate of strain AST4T. Phylogenetic analysis based on the 16S rRNA gene sequences showed that strain AST4T is located within the ‘Rhodobacter group’ in the α-3 subclass of Proteobacteria, but is clearly distant from related genera in this group such as Paracoccus, Rhodobacter and Rhodovulum. Strain AST4T is a Gram-negative, non-motile, rod-shaped (0·6–0·8×1·3–2·0 μm) and aerobic bacterium. It was not able to reduce nitrate to nitrite or N2. No phototrophic growth was observed. Optimal growth occurred at 30 °C and pH 6·5–7·5. The dominant cellular fatty acid in the isolate was C18 : 1
cis11. Ubiquinone-10 was the major respiratory quinone. The G+C content was 64·5 mol% (by HPLC). Based on the phylogenetic and phenotypic traits, the name Catellibacterium nectariphilum gen. nov., sp. nov. is proposed for this isolate; the type strain is AST4T (=NBRC 100046T=JCM 11959T=DSM 15620T).
Jeotgalicoccus pinnipedialis sp. nov., from a southern elephant seal (Mirounga leonina)
Authors: Hoyles Lesley , Collins Matthew D. , Foster Geoffrey , Falsen Enevold , Schumann Peter
Published: May 2004

A previously unknown Gram-positive, catalase-positive, facultatively anaerobic, non-spore-forming, coccus-shaped bacterium (A/G14/99/10T), originating from the mouth of a female southern elephant seal, was subjected to a taxonomic analysis. Comparative 16S rRNA gene-sequencing showed that the organism formed a hitherto unknown subline within the catalase-positive, low-G+C, Gram-positive cocci, exhibiting a specific association with species of the genus Jeotgalicoccus. Sequence divergence values of approximately 7 %, together with phenotypic differences, showed the unknown bacterium to be distinct from the two described species of this genus, Jeotgalicoccus halotolerans and Jeotgalicoccus psychrophilus. Based on phenotypic and phylogenetic considerations, it is proposed that strain A/G14/99/10T=CCUG 42722T=CIP 107946T from the mouth of a seal be classified as the type strain of a novel species of the genus Jeotgalicoccus, Jeotgalicoccus pinnipedialis sp. nov.
Phylogeny of the ring-forming bacterium Arcicella aquatica gen. nov., sp. nov. (ex Nikitin et al. 1994), from a freshwater neuston biofilm
Authors: Nikitin Denis I. , Strömpl Carsten , Oranskaya M. S. , Abraham Wolf-Rainer
Published: May 2004

Arcicella aquatica NO-502T, obtained from a neuston film on a freshwater lake and belonging to the phylum Bacteroidetes, is characterized by ring-forming cells. The bacterium is a strict aerobe, with optimal growth between 28 and 30 °C. Carbohydrates, but no organic acids or amino acids, are used as substrates. The G+C content of strain NO-502T is 34·5 mol%; its genome size is 2·9×109 Da. The genus Arcicella and its type species Arcicella aquatica (type strain NO-502T=LMG 21963T=CIP 107990T) are proposed, and descriptions of this genus and species are given.
Corynebacterium halotolerans sp. nov., isolated from saline soil in the west of China
Authors: Chen Hua-Hong , Li Wen-Jun , Tang Shu-Kun , Kroppenstedt Reiner M. , Stackebrandt Erko , Xu Li-Hua , Jiang Cheng-Lin
Published: May 2004

A halotolerant, non-spore-forming actinobacterium was isolated from a soil sample from the west of China. The strain, designated YIM 70093T (=CCTCC AA 001024T=DSM 44683T), comprised Gram-positive, non-motile, diphtheroid and irregular rods. It grew in 0–25 % KCl (KCl could be substituted by NaCl or MgCl2.6H2O), with optimum growth at 10 % KCl, and its optimal pH and cultivation temperature were 7·2 and 28 °C, respectively. On the basis of its morphological, physiological and phylogenetic characteristics, strain YIM 70093T should be classified in the genus Corynebacterium. However, it is sufficiently different from hitherto described Corynebacterium species to be considered as a novel species, for which the name Corynebacterium halotolerans sp. nov. is proposed.
Collimonas fungivorans gen. nov., sp. nov., a chitinolytic soil bacterium with the ability to grow on living fungal hyphae
Authors: de Boer Wietse , Leveau Johan H. J. , Kowalchuk George A. , Gunnewiek Paulien J. A. Klein , Abeln Edwin C. A. , Figge Marian J. , Sjollema Klaas , Janse Jaap D. , van Veen Johannes A.
Published: May 2004

A polyphasic approach was used to describe the phylogenetic position of 22 chitinolytic bacterial isolates that were able to grow at the expense of intact, living hyphae of several soil fungi. These isolates, which were found in slightly acidic dune soils in the Netherlands, were strictly aerobic, Gram-negative rods. Cells grown in liquid cultures were flagellated and possessed pili. A wide range of sugars, alcohols, organic acids and amino acids could be metabolized, whereas several di- and trisaccharides could not be used as substrates. The major cellular fatty acids were C16 : 0, C16 : 1
ω7c and C18 : 1
ω7c. DNA G+C contents were 57–62 mol%. Analysis of nearly full-length 16S rDNA sequences showed that the isolates were related closely to each other (>98·6 % sequence similarity) and could be assigned to the β-Proteobacteria, family ‘Oxalobacteraceae’, order ‘Burkholderiales’. The most closely related species belonged to the genera Herbaspirillum and Janthinobacterium, exhibiting 95·9–96·7 % (Herbaspirillum species) and 94·3–95·6 % (Janthinobacterium species) 16S rDNA sequence similarity to the isolates. Several physiological and biochemical properties indicated that the isolates could be distinguished clearly from both of these genera. Therefore, it is proposed that the isolates described in this study are representatives of a novel genus, Collimonas gen. nov. Genomic fingerprinting (BOX-PCR), detailed analysis of 16S rDNA patterns and physiological characterization (Biolog) of the isolates revealed the existence of four subclusters. The name Collimonas fungivorans gen. nov., sp. nov. has been given to one subcluster (four isolates) that appears to be in the centre of the novel genus; isolates in the other subclusters have been tentatively named Collimonas sp. The type strain of Collimonas fungivorans gen. nov., sp. nov. is Ter6T (=NCCB 100033T=LMG 21973T).
Pseudomonas lutea sp. nov., a novel phosphate-solubilizing bacterium isolated from the rhizosphere of grasses
Authors: Peix Alvaro , Rivas Raúl , Santa-Regina Ignacio , Mateos Pedro F. , Martínez-Molina Eustoquio , Rodríguez-Barrueco Claudino , Velázquez Encarna
Published: May 2004

A phosphate-solubilizing bacterial strain designated OK2T was isolated from rhizospheric soil of grasses growing spontaneously in a soil from Spain. Cells of the strain were Gram-negative, strictly aerobic, rod-shaped and motile. Phylogenetic analysis of the 16S rRNA gene indicated that this bacterium belongs to the γ-subclass of Proteobacteria within the genus Pseudomonas and that the closest related species is Pseudomonas graminis. The strain produced catalase but not oxidase. Cellulose, casein, starch, gelatin and urea were not hydrolysed. Aesculin was hydrolysed. Growth was observed with many carbohydrates as carbon sources. The main non-polar fatty acids detected were hexadecenoic acid (16 : 1), hexadecanoic acid (16 : 0) and octadecenoic acid (18 : 1). The hydroxy fatty acids detected were 3-hydroxydecanoic acid (3-OH 10 : 0), 3-hydroxydodecanoic acid (3-OH 12 : 0) and 2-hydroxydodecanoic acid (2-OH 12 : 0). The G+C DNA content determined was 59·3 mol%. DNA–DNA hybridization showed 48·7 % relatedness between strain OK2T and P. graminis DSM 11363T and 26·2 % with respect to Pseudomonas rhizosphaerae LMG 21640T. Therefore, these results indicate that strain OK2T (=LMG 21974T=CECT 5822T) belongs to a novel species of the genus Pseudomonas, and the name Pseudomonas lutea sp. nov. is proposed.
Auriculibuller fuscus gen. nov., sp. nov. and Bullera japonica sp. nov., novel taxa in the Tremellales
Authors: Sampaio José Paulo , Inácio João , Fonseca Álvaro , Gadanho Mário , Spencer-Martins Isabel , Scorzetti Gloria , Fell Jack W.
Published: May 2004

Seven phylloplane yeast strains that were collected in the Arrábida Natural Park, Portugal, and identified preliminarily asBullera alba, the anamorphic stage ofBulleromyces albus, were investigated. In contrast toBulleromyces albus, these isolates produced a brownish pigment when grown on potato dextrose agar. The pigment caused darkening of the cultures and diffused into the culture medium. Mating studies revealed that the Arrábida isolates did not react with the different mating types ofBulleromyces albus, but were sexually compatible with them and produced mycelium with clamp connections, haustoria and transversally septate basidia that ejected the basidiospores. Various taxonomic criteria that were evaluated during the present study and comparison with other sexual taxa of the Tremellales indicated that this teleomorph should be classified in a novel genus. Therefore,Auriculibuller fuscusgen. nov., sp. nov. (type strain, PYCC 5690T=CBS 9648T) is proposed. In addition, during the course of this investigation, a member of a novelBulleraspecies,Bullera japonicasp. nov. (type strain, PYCC 4534T=CBS 2013T), was found among collection isolates that were identified formerly asBullera alba. In molecular phylogenetic analysis of the D1/D2 domains of the 26S rDNA and the internal transcribed spacer region, the two taxa were found to be closely related, but distinct at the species level.
Stress Tolerance and Glucose Insensitive Phenotypes in Arabidopsis Overexpressing theCpMYB10Transcription Factor Gene
Authors: Villalobos Miguel Angel , Bartels Dorothea , Iturriaga Gabriel
Published: May 2004

The resurrection plant Craterostigma plantagineum has the ability to survive complete dehydration. In an attempt to further understand desiccation tolerance in this plant, the CpMYB10 transcription factor gene was functionally characterized. CpMYB10 is rapidly induced by dehydration and abscisic acid (ABA) treatments in leaves and roots, but no expression was detected in fully hydrated tissues. Electrophoretic mobility shift assay experiments showed binding of rCpMYB10 to specific mybRE elements within the LEA Cp11-24 and CpMYB10 promoters. Localization of CpMYB10 transcript by in situ reverse transcription-PCR reactions showed expression in vascular tissues, parenchyma, and epidermis both in leaves and roots in response to ABA. Transgenic Arabidopsis plants transformed with CpMYB10 promoter fused to GUS gene showed reporter expression under ABA and stress conditions in several organs. Overexpression of CpMYB10 cDNA in Arabidopsis led to desiccation and salt tolerance of transgenics lines. Interestingly, it was found that plants overexpressing CpMYB10 exhibited Glc-insensitive and ABA hypersensitive phenotypes. Therefore, our results indicate that CpMYB10 in Arabidopsis is mediating stress tolerance and altering ABA and Glc signaling responses.
Interactions between Auxin Transport and the Actin Cytoskeleton in Developmental Polarity ofFucus distichusEmbryos in Response to Light and Gravity
Authors: Sun Haiguo , Basu Swati , Brady Shari R. , Luciano Randy L. , Muday Gloria K.
Published: May 2004

Land plants orient their growth relative to light and gravity through complex mechanisms that require auxin redistribution. Embryos of brown algae use similar environmental stimuli to orient their developmental polarity. These studies of the brown algae Fucus distichus examined whether auxin and auxin transport are also required during polarization in early embryos and to orient growth in already developed tissues. These embryos polarize with the gravity vector in the absence of a light cue. The auxin, indole-3-acetic acid (IAA), and auxin efflux inhibitors, such as naphthylphthalamic acid (NPA), reduced environmental polarization in response to gravity and light vectors. Young rhizoids are negatively phototropic, and NPA also inhibits rhizoid phototropism. The effect of IAA and NPA on gravity and photopolarization is maximal within 2.5 to 4.5 h after fertilization (AF). Over the first 6 h AF, auxin transport is relatively constant, suggesting that developmentally controlled sensitivity to auxin determines the narrow window during which NPA and IAA reduce environmental polarization. Actin patches were formed during the first hour AF and began to photolocalize within 3 h, coinciding with the time of NPA and IAA action. Treatment with NPA reduced the polar localization of actin patches but not patch formation. Latrunculin B prevented environmental polarization in a time frame that overlaps the formation of actin patches and IAA and NPA action. Latrunculin B also altered auxin transport. Together, these results indicate a role for auxin in the orientation of developmental polarity and suggest interactions between the actin cytoskeleton and auxin transport in F. distichus embryos.
High-Frequency Transformation of Undeveloped Plastids in Tobacco Suspension Cells
Authors: Langbecker Camri L. , Ye Guang-Ning , Broyles Debra L. , Duggan Lisa L. , Xu Charles W. , Hajdukiewicz Peter T.J. , Armstrong Charles L. , Staub Jeffrey M.
Published: May 2004

Although leaf chloroplast transformation technology was developed more than a decade ago, no reports exist of stable transformation of undeveloped plastids or other specialized plastid types, such as proplastids, etioplasts, or amyloplasts. In this work we report development of a dark-grown tobacco suspension cell model system to investigate the transformation potential of undeveloped plastids. Electron microscope analysis confirmed that the suspension cells carry plastids that are significantly smaller (approximately 50-fold less in volume) and have a very different subcellular localization and developmental state than leaf cell chloroplasts. Using antibiotic selection in the light, we demonstrated that both plastid and nuclear transformation of these cell suspensions is efficient and reproducible, with plastid transformation frequency at least equal to that of leaf chloroplast transformation. Homoplasmic plastid transformants are readily obtained in cell colonies, or in regenerated plants, providing a more consistent and versatile model than the leaf transformation system. Because of the uniformity of the cell suspension model, we could further show that growth rate, selection scheme, particle size, and DNA amount influence the frequency of transformation. Our results indicate that the rate-limiting steps for nuclear and plastid transformation are different, and each must be optimized separately. The suspension cell system will be useful as a model for understanding transformation in those plant species that utilize dark-grown embryogenic cultures and for characterizing the steps that lead to homoplasmic plastid transformation.
Capsicum annuumTobacco Mosaic Virus-Induced Clone 1 Expression Perturbation Alters the Plant's Response to Ethylene and Interferes with the Redox Homeostasis
Authors: Shin Ryoung , An Jong-Min , Park Chang-Jin , Kim Young Jin , Joo Sunjoo , Kim Woo Taek , Paek Kyung-Hee
Published: May 2004

Capsicum annuum tobacco mosaic virus (TMV)-induced clone 1 (CaTin1) gene was expressed early during incompatible interaction of hot pepper (Caspsicum annuum) plants with TMV and Xanthomonas campestris. RNA-blot analysis showed that CaTin1 gene was expressed only in roots in untreated plants and induced mainly in leaf in response to ethylene, NaCl, and methyl viologen but not by salicylic acid and methyl jasmonate. The ethylene dependence of CaTin1 induction upon TMV inoculation was demonstrated by the decrease of CaTin1 expression in response to several inhibitors of ethylene biosynthesis or its action. Transgenic tobacco (Nicotiana tabacum) plants expressing CaTin1 gene in sense- or antisense-orientation showed interesting characteristics such as the accelerated growth and the enhanced resistance to biotic as well as abiotic stresses. Such characteristics appear to be caused by the elevated level of ethylene and H2O2. Moreover, in transgenic plants expressing antisense CaTin1 gene, the expression of some pathogenesis-related genes was enhanced constitutively, which may be mainly due to the increased ethylene level. The promoter of CaTin1 has four GCC-boxes, two AT-rich regions, and an elicitor-inducible W-box. The induction of the promoter activity by ethylene depends on GCC-boxes and by TMV on W-box. Taken together, we propose that the CaTin1 up-regulation or down-regulation interferes with the redox balance of plants leading to the altered response to ethylene and biotic as well as abiotic stresses.
Light Induces Phosphorylation of Glucan Water Dikinase, Which Precedes Starch Degradation in Turions of the Duckweed Spirodela polyrhiza
Authors: Reimann Rezarta , Hippler Michael , Machelett Bernd , Appenroth Klaus-J.
Published: May 2004

Degradation of storage starch in turions, survival organs of Spirodela polyrhiza, is induced by light. Starch granules isolated from irradiated (24 h red light) or dark-stored turions were used as an in vitro test system to study initial events of starch degradation. The starch-associated pool of glucan water dikinase (GWD) was investigated by two-dimensional gel electrophoresis and by western blotting using antibodies raised against GWD. Application of this technique allowed us to detect spots of GWD, which are light induced and absent on immunoblots prepared from dark-adapted plants. These spots, showing increased signal intensity following incubation of the starch granules with ATP, became labeled by randomized [βγ-33P]ATP but not by [γ-33P]ATP and were removed by acid phosphatase treatment. This strongly suggests that they represent a phosphorylated form(s) of GWD. The same light signal that induces starch degradation was thus demonstrated for the first time to induce autophosphorylation of starch-associated GWD. The in vitro assay system has been used to study further effects of the light signal that induces autophosphorylation of GWD and starch degradation. In comparison with starch granules from dark-adapted plants, those from irradiated plants showed increase in (1) binding capacity of GWD by ATP treatment decreased after phosphatase treatment; (2) incorporation of the β-phosphate group of ATP into starch granules; and (3) rate of degradation of isolated granules by starch-associated proteins, further enhanced by phosphorylation of starch. The presented results provide evidence that autophosphorylation of GWD precedes the initiation of starch degradation under physiological conditions.
CINCINNATA Controls Both Cell Differentiation and Growth in Petal Lobes and Leaves of Antirrhinum
Authors: Crawford Brian C.W. , Nath Utpal , Carpenter Rosemary , Coen Enrico S.
Published: May 2004

To understand how differentiation and growth may be coordinated during development, we have studied the action of the CINCINNATA (CIN) gene of Antirrhinum. We show that in addition to affecting leaf lamina growth, CIN affects epidermal cell differentiation and growth of petal lobes. Strong alleles of cin give smaller petal lobes with flat instead of conical cells, correlating with lobe-specific expression of CIN in the wild type. Moreover, conical cells at the leaf margins are replaced by flatter cells, indicating that CIN has a role in cell differentiation of leaves as well as petals. A weak semidominant cin allele affects cell types mainly in the petal but does not affect leaf development, indicating these two effects can be separated. Expression of CIN correlates with expression of cell division markers, suggesting that CIN may influence petal growth, directly or indirectly, through effects on cell proliferation. For both leaves and petals, CIN affects growth and differentiation of the more distal and broadly extended domains (leaf lamina and petal lobe). However, while CIN promotes growth in petals, it promotes growth arrest in leaves, possibly because of different patterns of growth control in these systems.
Phosphatase Under-Producer Mutants Have Altered Phosphorus Relations
Authors: Tomscha Jennifer L. , Trull Melanie C. , Deikman Jill , Lynch Jonathan P. , Guiltinan Mark J.
Published: May 2004

Phosphorus (P) acquisition and partitioning are essential for plant homeostasis. P is available for plant uptake when in its inorganic form (H2PO4  −, or Pi), but Pi is often limiting in soils. Plants secrete acid phosphatases (APases) into the apoplastic space, which may be important for obtaining Pi from organic P sources; however, the relative importance of these enzymes for plant P nutrition has yet to be determined. We demonstrate that the root-associated APase pool is increased in Arabidopsis when Pi is limiting and document five APase isoforms secreted from Arabidopsis roots. Previously, we presented the identification of the phosphatase under-producer (pup) mutants, which have decreased in vivo root APase staining when grown under low P conditions. Here, we present the characterization of one of these, pup3, and further studies with pup1. pup3 has 49%, 38%, and 37% less specific APase activity in exudates, roots, and shoots, respectively. Root-associated APase activity is decreased by 16% in pup1 and 25% in pup3, regardless of P treatment. Two APase activity isoforms are reduced in pup3 exudates, and root and shoot isoforms are also affected. One of the two exudate isoforms is recognized by a polyclonal antibody raised to an Arabidopsis purple APase recombinant protein (AtPAP12); however, AtPAP12 transcript levels are unaffected in the mutant. The pup3 mutation was mapped to 68.4 ± 6.0 centimorgans on chromosome 5. Although P concentrations were not altered in pup1 and pup3 tissues when grown in nutrient solution in which Pi was the sole source of P, the mutants had 10% (pup1) and 17% (pup3) lower shoot P concentrations when grown in a peat-vermiculite mix in which the majority of the total P was present as organic P. Therefore, the pup defects, which include secreted APases, are functionally important for plant P nutrition.
Evidence for Apoplasmic Phloem Unloading in Developing Apple Fruit
Authors: Zhang Ling-Yun , Peng Yi-Ben , Pelleschi-Travier Sandrine , Fan Ying , Lu Yan-Fen , Lu Ying-Min , Gao Xiu-Ping , Shen Yuan-Yue , Delrot Serge , Zhang Da-Peng
Published: May 2004

The phloem unloading pathway remains unclear in fleshy fruits accumulating a high level of soluble sugars. A structural investigation in apple fruit (Malus domestica Borkh. cv Golden Delicious) showed that the sieve element-companion cell complex of the sepal bundles feeding the fruit flesh is symplasmically isolated over fruit development. 14C-autoradiography indicated that the phloem of the sepal bundles was functional for unloading. Confocal laser scanning microscopy imaging of carboxyfluorescein unloading showed that the dye remained confined to the phloem strands of the sepal bundles from the basal to the apical region of the fruit. A 52-kD putative monosaccharide transporter was immunolocalized predominantly in the plasma membrane of both the sieve elements and parenchyma cells and its amount increased during fruit development. A 90-kD plasma membrane H+-ATPase was also localized in the plasma membrane of the sieve element-companion cell complex. Studies of [14C]sorbitol unloading suggested that an energy-driven monosaccharide transporter may be functional in phloem unloading. These data provide clear evidence for an apoplasmic phloem unloading pathway in apple fruit and give information on the structural and molecular features involved in this process.
Both Subunits of ADP-Glucose Pyrophosphorylase Are Regulatory
Authors: Cross Joanna M. , Clancy Maureen , Shaw Janine R. , Greene Thomas W. , Schmidt Robert R. , Okita Thomas W. , Hannah L. Curtis
Published: May 2004

The allosteric enzyme ADP-Glc pyrophosphorylase (AGPase) catalyzes the synthesis of ADP-Glc, a rate-limiting step in starch synthesis. Plant AGPases are heterotetramers, most of which are activated by 3-phosphoglyceric acid (3-PGA) and inhibited by phosphate. The objectives of these studies were to test a hypothesis concerning the relative roles of the two subunits and to identify regions in the subunits important in allosteric regulation. We exploited an Escherichia coli expression system and mosaic AGPases composed of potato (Solanum tuberosum) tuber and maize (Zea mays) endosperm subunit fragments to pursue this objective. Whereas potato and maize subunits have long been separated by speciation and evolution, they are sufficiently similar to form active mosaic enzymes. Potato tuber and maize endosperm AGPases exhibit radically different allosteric properties. Hence, comparing the kinetic properties of the mosaics to those of the maize endosperm and potato tuber AGPases has enabled us to identify regions important in regulation. The data herein conclusively show that both subunits are involved in the allosteric regulation of AGPase. Alterations in the small subunit condition drastically different allosteric properties. In addition, extent of 3-PGA activation and extent of 3-PGA affinity were found to be separate entities, mapping to different regions in both subunits.
The Dominant MutantWavy auricle in blade1Disrupts Patterning in a Lateral Domain of the Maize Leaf
Authors: Hay Angela , Hake Sarah
Published: May 2004

Mature maize leaves have defined cell types along the proximal distal and medial lateral axes. The patterning events that establish these axes take place early in leaf initiation. We have identified a new dominant mutation, Wavy auricle in blade1 (Wab1), which affects patterning in both axes in a dose-dependent manner. Wab1 leaves are narrower than normal leaves and displace proximal tissues distally. We show that the proximal distal patterning defects are not due to misexpression of knox genes. Genetic analyses suggest that the action of dominant Wab1 alleles is localized to a lateral domain of the leaf, located between the midvein and the marginal domain that is determined by narrow sheath function. Thus, Wab1 defines a knox-independent pathway that affects specification of the proximal distal axis of the maize leaf. We suggest that failure to elaborate a normal lateral domain in the Wab1 leaf is responsible for disrupting patterning of the proximal distal axis.
Expression of Phosphoenolpyruvate Carboxylase and Phosphoenolpyruvate Carboxylase Kinase Genes. Implications for Genotypic Capacity and Phenotypic Plasticity in the Expression of Crassulacean Acid Metabolism
Authors: Taybi Tahar , Nimmo Hugh G. , Borland Anne M.
Published: May 2004

In plants with crassulacean acid metabolism (CAM), dark CO2 uptake is mediated by phosphoenolpyruvate carboxylase (PEPC), an enzyme that can be regulated at transcriptional and posttranslational levels. Reversible phosphorylation of PEPC is catalyzed by a dedicated PEPC kinase, which in turn is regulated at the transcriptional level over the 24-h cycle in CAM plants. PEPC kinase controls the day/night regulation of PEPC during the CAM cycle, thus facilitating plasticity for optimizing CO2 uptake under different environmental conditions. To understand the importance of PEPC kinase in relation to its target PEPC in terms of CAM performance, the expression of the genes encoding the two enzymes was investigated in four species of Clusia that have photosynthetic patterns ranging from C3 photosynthesis to constitutive CAM. By linking changes in the expression of PEPC and PEPC kinase to day/night patterns of leaf gas exchange, organic acid, and soluble sugar contents under different environmental conditions, the genetic and metabolic limitations to CAM plasticity were assessed. The results indicate that PEPC expression is a major factor underpinning the genotypic capacity for CAM and that PEPC kinase expression does not appear to limit CAM. The day/night regulation of Ppck transcript abundance was found to be a consequence of CAM and the day/night cycling of associated metabolites, rather than the primary controlling factor for the temporal separation of carboxylation processes.
CHLOROPLAST BIOGENESIS Genes Act Cell and Noncell Autonomously in Early Chloroplast Development
Authors: Gutiérrez-Nava María de la Luz , Gillmor C. Stewart , Jiménez Luis F. , Guevara-García Arturo , León Patricia
Published: May 2004

In order to identify nuclear genes required for early chloroplast development, a collection of photosynthetic pigment mutants of Arabidopsis was assembled and screened for lines with extremely low levels of chlorophyll. Nine chloroplast biogenesis (clb) mutants that affect proplastid growth and thylakoid membrane formation and result in an albino seedling phenotype were identified. These mutations identify six new genes as well as a novel allele of cla1. clb mutants have less than 2% of wild-type chlorophyll levels, and little or no expression of nuclear and plastid-encoded genes required for chloroplast development and function. In all but one mutant, proplastids do not differentiate enough to form elongated stroma thylakoid membranes. Analysis of mutants during embryogenesis allows differentiation between CLB genes that act noncell autonomously, where partial maternal complementation of chloroplast development is observed in embryos, and those that act cell autonomously, where complementation during embryogenesis is not observed. Molecular characterization of the noncell autonomous clb4 mutant established that the CLB4 gene encodes for hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (HDS), the next to the last enzyme of the methylerythritol 4-phosphate (MEP) pathway for the synthesis of plastidic isoprenoids. The noncell autonomous nature of the clb4 mutant suggests that products of the MEP pathway can travel between tissues, and provides in vivo evidence that some movement of MEP intermediates exists from the cytoplasm to the plastid. The isolation and characterization of clb mutants represents the first systematic study of genes required for early chloroplast development in Arabidopsis.
Lysine Metabolism Is Concurrently Regulated by Synthesis and Catabolism in Both Reproductive and Vegetative Tissues
Authors: Zhu Xiaohong , Galili Gad
Published: May 2004

The functional role of Lys catabolism in balancing Lys levels in plants has only been directly demonstrated in developing seeds. Seed-specific expression of a bacterial feedback-insensitive dihydrodipicolinate synthase (DHPS) in an Arabidopsis knockout mutant of the AtLKR/SDH gene that regulates Lys catabolism synergistically boosted Lys accumulation in mature seeds, but it also severely reduced the growth of seedlings derived from them. Here we further tested whether the inhibition of seedling growth was due to a negative physiological effect of excess Lys on seed maturation or to defective postgermination catabolism of Lys, which accumulated in the mature seeds. To address these questions, we coexpressed a bacterial DHPS gene with an RNAi construct of AtLKR/SDH, both under control of the same seed-specific promoter, to restrict Lys synthesis and catabolism to the developing seeds. Coexpression of these genes boosted seed Lys content and caused a significant, metabolically unanticipated increase in Met content, similarly to our previous report using plants expressing the bacterial DHPS on an AtLKR/SDH knockout background. However, postgermination seedling growth was significantly improved when the reduction of Lys catabolism was restricted to seed development, suggesting that defective postgermination Lys catabolism was responsible for inhibition of seedling growth in the AtLKR/SDH knockout plants expressing the bacterial DHPS gene in a seed-specific manner. Constitutive expression of the bacterial DHPS in the AtLKR/SDH knockout mutant boosted Lys levels in vegetative tissues in a similar manner to that observed in seeds, further demonstrating that Lys catabolism plays an important regulatory role in balancing Lys levels.
Identification of a New Chloroplast Carbonic Anhydrase inChlamydomonas reinhardtii
Authors: Mitra Mautusi , Lato Scott M. , Ynalvez Ruby A. , Xiao Ying , Moroney James V.
Published: May 2004

Carbonic anhydrases (CA) are zinc-containing metalloenzymes that catalyze the reversible hydration of CO2. The three evolutionarily unrelated families of CAs are designated α-, β-, and γ-CA. Aquatic photosynthetic organisms have evolved different forms of CO2 concentrating mechanisms (CCMs) to aid Rubisco in capturing CO2 from the surrounding environment. One aspect of all CCMs is the critical roles played by various specially localized extracellular and intracellular CAs. Five CAs have previously been identified in Chlamydomonas reinhardtii, a green alga with a well-studied CCM. Here we identify a sixth gene encoding a β-type CA. This new β-CA, designated Cah6, is distinct from the two mitochondrial β-CAs in C. reinhardtii. Nucleotide sequence data show that the Cah6 cDNA contains an open reading frame encoding a polypeptide of 264 amino acids with a leader sequence likely targeting the protein to the chloroplast stroma. We have fused the Cah6 open reading frame to the coding sequence of maltose-binding protein in a pMal expression vector. The purified recombinant fusion protein is active and was used to partially characterize the Cah6 protein. The purified recombinant fusion protein was cleaved with protease Factor Xa to separate Cah6 from the maltose-binding protein and the purified Cah6 protein was used to raise an antibody. Western blots, immunolocalization studies, and northern blots collectively indicated that Cah6 is constitutively expressed in the stroma of chloroplasts. A possible role for Cah6 in the CCM of C. reinhardtii is proposed.
Contribution of Different Carbon Sources to Isoprene Biosynthesis in Poplar Leaves
Authors: Schnitzler Jörg-Peter , Graus Martin , Kreuzwieser Jürgen , Heizmann Ulrike , Rennenberg Heinz , Wisthaler Armin , Hansel Armin
Published: May 2004

This study was performed to test if alternative carbon sources besides recently photosynthetically fixed CO2 are used for isoprene formation in the leaves of young poplar (Populus × canescens) trees. In a 13CO2 atmosphere under steady state conditions, only about 75% of isoprene became 13C labeled within minutes. A considerable part of the unlabeled carbon may be derived from xylem transported carbohydrates, as may be shown by feeding leaves with [U-13C]Glc. As a consequence of this treatment approximately 8% to 10% of the carbon emitted as isoprene was 13C labeled. In order to identify further carbon sources, poplar leaves were depleted of leaf internal carbon pools and the carbon pools were refilled with 13C labeled carbon by exposure to 13CO2. Results from this treatment showed that about 30% of isoprene carbon became 13C labeled, clearly suggesting that, in addition to xylem transported carbon and CO2, leaf internal carbon pools, e.g. starch, are used for isoprene formation. This use was even increased when net assimilation was reduced, for example by abscisic acid application. The data provide clear evidence of a dynamic exchange of carbon between different cellular precursors for isoprene biosynthesis, and an increasing importance of these alternative carbon pools under conditions of limited photosynthesis. Feeding [1,2-13C]Glc and [3-13C]Glc to leaves via the xylem suggested that alternative carbon sources are probably derived from cytosolic pyruvate/phosphoenolpyruvate equivalents and incorporated into isoprene according to the predicted cleavage of the 3-C position of pyruvate during the initial step of the plastidic deoxyxylulose-5-phosphate pathway.
Copper Transport Across Pea Thylakoid Membranes
Authors: Shingles Richard , Wimmers Larry E. , McCarty Richard E.
Published: May 2004

The initial rate of Cu2+ movement across the thylakoid membrane of pea (Pisum sativum) chloroplasts was directly measured by stopped-flow spectrofluorometry using membranes loaded with the Cu2+-sensitive fluorophore Phen Green SK. Cu2+ transport was rapid, reaching completion within 0.5 s. The initial rate of uptake was dependent upon Cu2+ concentration and saturated at about 0.6 μ  m total Cu2+. Cu2+ uptake was maximal at a thylakoid lumen pH of 7.0. Cu2+ transport was inhibited by Zn2+ but was largely unaffected by Mn2+ and Cu+. Zn2+ inhibited Cu2+ transport to a maximum of 60%, indicating that there may be more than one transporter for copper in pea thylakoid membranes.
Structure and Expression Profile of the Arabidopsis PHO1 Gene Family Indicates a Broad Role in Inorganic Phosphate Homeostasis
Authors: Wang Yong , Ribot Cécile , Rezzonico Enea , Poirier Yves
Published: May 2004

PHO1 has been recently identified as a protein involved in the loading of inorganic phosphate into the xylem of roots in Arabidopsis. The genome of Arabidopsis contains 11 members of the PHO1 gene family. The cDNAs of all PHO1 homologs have been cloned and sequenced. All proteins have the same topology and harbor a SPX tripartite domain in the N-terminal hydrophilic portion and an EXS domain in the C-terminal hydrophobic portion. The SPX and EXS domains have been identified in yeast (Saccharomyces cerevisiae) proteins involved in either phosphate transport or sensing or in sorting proteins to endomembranes. The Arabidopsis genome contains additional proteins of unknown function containing either a SPX or an EXS domain. Phylogenetic analysis indicated that the PHO1 family is subdivided into at least three clusters. Reverse transcription-PCR revealed a broad pattern of expression in leaves, roots, stems, and flowers for most genes, although two genes are expressed exclusively in flowers. Analysis of the activity of the promoter of all PHO1 homologs using promoter-β-glucuronidase fusions revealed a predominant expression in the vascular tissues of roots, leaves, stems, or flowers. β-Glucuronidase expression is also detected for several promoters in nonvascular tissue, including hydathodes, trichomes, root tip, root cortical/epidermal cells, and pollen grains. The expression pattern of PHO1 homologs indicates a likely role of the PHO1 proteins not only in the transfer of phosphate to the vascular cylinder of various tissues but also in the acquisition of phosphate into cells, such as pollen or root epidermal/cortical cells.
Biosynthesis of the Nitrile Glucosides Rhodiocyanoside A and D and the Cyanogenic Glucosides Lotaustralin and Linamarin in Lotus japonicus
Authors: Forslund Karin , Morant Marc , Jørgensen Bodil , Olsen Carl Erik , Asamizu Erika , Sato Shusei , Tabata Satoshi , Bak Søren
Published: May 2004

Lotus japonicus was shown to contain the two nitrile glucosides rhodiocyanoside A and rhodiocyanoside D as well as the cyanogenic glucosides linamarin and lotaustralin. The content of cyanogenic and nitrile glucosides in L. japonicus depends on plant developmental stage and tissue. The cyanide potential is highest in young seedlings and in apical leaves of mature plants. Roots and seeds are acyanogenic. Biosynthetic studies using radioisotopes demonstrated that lotaustralin, rhodiocyanoside A, and rhodiocyanoside D are derived from the amino acid l-Ile, whereas linamarin is derived from Val. In silico homology searches identified two cytochromes P450 designated CYP79D3 and CYP79D4 in L. japonicus. The two cytochromes P450 are 94% identical at the amino acid level and both catalyze the conversion of Val and Ile to the corresponding aldoximes in biosynthesis of cyanogenic glucosides and nitrile glucosides in L. japonicus. CYP79D3 and CYP79D4 are differentially expressed. CYP79D3 is exclusively expressed in aerial parts and CYP79D4 in roots. Recombinantly expressed CYP79D3 and CYP79D4 in yeast cells showed higher catalytic efficiency with l-Ile as substrate than with l-Val, in agreement with lotaustralin and rhodiocyanoside A and D being the major cyanogenic and nitrile glucosides in L. japonicus. Ectopic expression of CYP79D2 from cassava (Manihot esculenta Crantz.) in L. japonicus resulted in a 5- to 20-fold increase of linamarin content, whereas the relative amounts of lotaustralin and rhodiocyanoside A/D were unaltered.
Production of Coumaroylserotonin and Feruloylserotonin in Transgenic Rice Expressing Pepper Hydroxycinnamoyl-Coenzyme A:Serotonin N-(Hydroxycinnamoyl)transferase
Authors: Jang Sun-Mi , Ishihara Atsushi , Back Kyoungwhan
Published: May 2004

Transgenic rice (Oryza sativa) plants were engineered to express a N-(hydroxycinnamoyl)transferase from pepper (Capsicum annuum), which has been shown to have hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase activity, a key enzyme in the synthesis of hydroxycinnamic acid amides, under the control of constitutive maize (Zea mays) ubiquitin promoter. The transgenic rice plants require foliar application of amines to support synthesis of hydroxycinnamic acid amides, suggestive of limiting amine substrates in rice shoots. In addition, when T2 homozygous transgenic rice plants were grown in the presence of amines or phenolic acids, two novel compounds were exclusively identified in the leaves of the transgenic plants. These compounds eluted earlier than p-coumaroyltyramine and feruloyltyramine during HPLC chromatography and were identified as p-coumaroylserotonin and feruloylserotonin by liquid chromatography/mass spectrometry and other methods. To test whether the unpredicted production of serotonin derivatives is associated with the pepper N-(hydroxycinnamoyl)transferase, the substrate specificity of the pepper enzyme was analyzed again. Purified recombinant pepper N-(hydroxycinnamoyl)transferase exhibited a serotonin N-hydroxycinnamoyltransferase (SHT) activity, synthesized p-coumaroylserotonin and feruloylserotonin in vitro, and demonstrated a low K  m for serotonin. SHT activity was inhibited by 10 to 50 mm tyramine. In addition, SHT activity was predominantly found in the root tissues of wild-type rice in parallel with the synthesis of serotonin derivatives, suggesting that serotonin derivatives are synthesized in the root of rice. This is the first report of SHT activity and the first demonstration, to our knowledge, that serotonin derivatives can be overproduced in vivo in transgenic rice plants that express serotonin N-(hydroxycinnamoyl)transferase.