bluuebunny/crossref_metadata_2025 huggingface dataset:
Radial clastic dykes formed by a salt diapir in the Dead Sea Rift, Israel
Authors: Marco Shmuel , Weinberger Ram , Agnon Amotz
Published: March 2003

ABSTRACT Fanning structures radiating from a central perturbation are known in various geological environments, where different processes have produced similar geometry. The present contribution describes and analyses fanning clastic dykes in the Dead Sea Rift, a new example of diapir‐related deformation. The dykes are opening‐mode fractures exposed in lacustrine varved marl of the Lisan Formation, deposited 70–15 ka. They are arranged mainly in a radial and tangential geometry. The radial traces converge at the ‘Black Hill’ structural dome. The geometry of the fractures is consistent with stresses exerted by the rise of a salt diapir located underneath the Black Hill. The estimated extension of the radial fractures is in good agreement with the present topographic elevation of the hill. The absence of fractures in the overlying Holocene alluvium probably indicates that either the rise of the Black Hill salt diapir paused or is associated now with a different style of deformation.
Mud volcano gas hydrates in the Gulf of Cadiz
Authors: Mazurenko L. L. , Soloviev V. A. , Belenkaya I. , Ivanov M. K. , Pinheiro L. M.
Published: March 2003

ABSTRACT This paper presents new geochemical data on gas‐hydrate‐bearing mud volcanoes discovered for the first time in the Gulf of Cadiz during cruises TTR9 and TTR10 of the R/V Professor Logachev in 1999–2000. The estimated gas hydrate content is 3–16% of sediment volume and 5–31% of pore space volume. Estimated values of the water isotopic composition for the Ginsburg mud volcano are very heavy for δ18O (up to +53‰) and light for δD (up to − 210‰). Gas released from the hydrates contains 81% of C1 and 19% of C2+. The inferred source of the gas in the hydrates is enriched in C2–C6 (≤ 5%), indicating that the gas has a thermogenic origin. Gas hydrate of cubic structure II should be formed from a gas of such composition. It is interpreted that the composition of the mud volcano fluid corresponds to deep oil basins below the Gulf of Cadiz.
Magnetostratigraphic and cyclostratigraphic calibration of a prospective Palaeocene/Eocene stratotype at Zumaia (Basque Basin, northern Spain)
Authors: Dinarès‐Turell Jaume , Baceta Juan Ignacio , Pujalte Victoriano , Orue‐Etxebarria Xabier , Bernaola Gilen
Published: March 2003

ABSTRACT Detailed magnetostratigraphic work at the Palaeocene/Eocene boundary interval in the Zumaia section has led to a precise delineation of the position of polarity Chron C25n from −35.0 m (or −35.7 m) to −25.2 m below the base of the Carbon Isotopic Excursion that marks the Palaeocene/Eocene boundary. This chronostratigraphic constraint suggests that the lithological carbonate–marl couplets making up most of the studied section are the expression of orbital precession cycles, and confirms the expanded and continuous nature of the section, with mean sediment accumulation rates of ∼ 2.5 cm kyr−1. The new data render Zumaia a unique land‐based marine section with available magnetostratigraphic data coupled to cyclostratigraphy. The revised chronostratigraphic framework presented supports previous cyclostratigraphic calibration of the latest Palaeocene geomagnetic timescale; it should facilitate better assessment of the different chemobiostratigraphic events that span this critical interval as a consequence of past climate and oceanographic change. Consequently, the Zumaia section is proposed as a candidate Global Stratotype Section and Point (GSSP) for the Palaeocene/Eocene boundary.
Benthic foraminiferal distribution and sedimentary structures suggest tectonic erosion at the Costa Rica convergent plate margin
Authors: Meschede Martin , Weiss Regine , Schmiedl Gerhard , Hemleben Christoph
Published: March 2003

ABSTRACT Distribution patterns of benthic foraminiferal faunas from ODP Leg 170 Sites 1041 and 1042 show that the Costa Rican convergent margin subsided from coastal to abyssal depth from Middle Miocene to Present. This favours the model of a margin undergoing active subduction erosion. We propose that subduction erosion leads to the removal of material from the base of the forearc wedge and, as a consequence, to progressive subsidence of the forearc. A mean subsidence rate is estimated to be approximately 0.4 mm yr−1.
Re‐interpretation of the existence of a primitive plume under Australia based on neon isotope fractionation during step heating
Authors: Gautheron Cécile , Moreira Manuel
Published: March 2003

ABSTRACT The origin of the volcanism in south‐eastern Australia is poorly understood. Matsumoto and collaborators found near solar neon isotope values in measurements obtained by step heating apatites in ultramafic xenoliths from Bullenmerri (SE Australia), and proposed that these values result from the presence of a primitive plume. However, the results of simple diffusion models indicate that the neon isotopic signature observed by Matsumoto et al. can be generated by mass fractionation during step heating. If such mass fractionation does occur during step heating, then neither neon nor helium isotopes require the presence of primitive mantle, and the rare gas observations are consistent with the involvement of MORB‐source mantle in the generation of south‐eastern Australian volcanics. These results show that both the acquisition and the interpretation of step heating data should be carefully controlled and that a mineral crushing technique may yield more reliable analyses.
Cloning and characterization of the gene encoding the endo polygalacturonase‐inhibiting protein (PGIP) of Phaseolus vulgaris L.
Authors: Toubart Patrick , Desiderio Angiola , Salvi Giovanni , Cervone Felice , Daroda Lorenza , De Lorenzo Giulia , Bergmann Carl , Darvill Alan G. , Albersheim Peter
Published: March 2003

Polygalacturonase‐inhibiting protein (PGIP) is a cell wall protein purified from hypocotyls of true bean (Phaseolus vulgaris L.). PGIP inhibits fungal endopolygalacturonases and is considered to be an important factor for plant resistance to phytopathogenic fungi (Albersheim and Anderson, 1971; Cervone et al., 1987). The amino acid sequences of the N‐terminus and one internal tryptic peptide of the PGIP purified from P. vulgaris cv. Pinto were used to design redundant oligonucleotides that were successfully utilized as primers in a polymerase chain reaction (PCR) with total DNA of P. vulgaris as a template. A DNA band of 758 bp (a specific PCR amplification product of part of the gene coding for PGIP) was isolated and cloned. By using the 758‐bp DNA as a hybridization probe, a lambda clone containing the PGIP gene was isolated from a genomic library of P. vulgaris cv. Saxa. The coding and immediate flanking regions of the PGIP gene, contained on a subcloned 3.3 kb Sa/I–Sa/I DNA fragment, were sequenced. A single, continuous ORF of 1026 nt (342 amino acids) was present in the genomic clone. The nucleotide and deduced amino acid sequences of the PGIP gene showed no significant similarity with any known databank sequence. Northern blotting analysis of poly(A)+ RNAs, isolated from various tissues of bean seedlings or from suspension‐cultured bean cells, were also performed using the cloned PCR‐generated DNA as a probe. A 1.2 kb transcript was detected in suspension‐cultured cells and, to a lesser extent, in leaves, hypocotyls, and flowers. A cDNA clone containing part of the coding region for the PGIP (from nt 345 to the poly(A) tail) was isolated from a λgt11 cDNA library prepared from poly(A)+ RNA isolated from suspension‐cultured cells of P. vulgaris cv. Pinto. The nucleotide and deduced amino acid sequences of the PGIP genes from the two different cultivars shared 96.4% and 97.4% identity, respectively.
Constitutive and cell‐division‐inducible protein—DNA interactions in two maize histone gene promoters
Authors: Brignon Pierre , Chaubet Nicole
Published: March 2003

Protein—DNA interactions in the promoter regions of two maize histone genes have been analyzed by DNase I and DMS in vivo footprinting combined with LMPCR amplification. Both promoters present a bimodular structure characterized by a proximal cell division‐specific set of interactions and a distal region which displays constitutive footprints but enhancement of these footprints upon cell proliferation. The inducible region contains two cis‐elements common to all replication‐dependent plant histone genes, one of them having previously been shown to be a target for the wheat nuclear protein HBP‐2. In the constitutive region, the first demonstration for the existence of a transcription factor binding to the highly conserved plant histone‐specific octamer CGCGGATC is provided. Exchange of cell‐type‐specific factors is postulated to occur at that site. Additional immediate upstream constitutive elements binding regulatory proteins include a degenerate octameric sequence, a CCAAT‐box, a CACCC sequence and composite ACGTCA/ACGTGG hexameric sequences binding HBP‐1‐related trans‐acting factors. The close proximity of these elements within the constitutive region and the redundancy of some of them suggest complex cooperation and competition mechanisms contributing to achieve the final expression level and likely also to mediate the interplay between constitutive and inducible factors.
Methyl jasmonate represses translation initiation of a specific set of mRNAs in barley
Authors: Reinbothe Steffen , Reinbothe Christiane , Parthier Benno
Published: March 2003

Jasmonic acid methyl ester (methyl jasmonate, JaMe) causes accumulation of novel abundant proteins in excised leaf segments of barley, and concomitantly represses synthesis of most pre‐existing (‘control’) proteins. The changes in control protein synthesis do not correspond to equivalent alterations at the in vitro translatable mRNA level, suggesting a post‐transcriptional mode of regulation. Methyl jasmonate did not interfere with the in vitro translation of either control or JaMe‐induced mRNAs. Polysome runoff translation, in combination with two‐dimensional separations of the products formed, however, revealed a reduced synthesis of control proteins in JaMe‐exposed leaf tissues, in contrast to a continued translation of these polypeptides in water‐treated leaf segments. In vitro translation of polysomal RNAs demonstrated the preferential association of control mRNAs with polysomes of water‐treated leaf tissues but not with polysomes of JaMe‐treated tissues. Polysomes isolated from the latter leaves contained primarily JaMe‐induced mRNAs, as shown by in vitro translation and Northern hybridization with gene‐specific probes. Treatment of JaMe‐incubated leaf tissues with cycloheximide prior to harvesting caused an increase of in vitro translatable control mRNAs recovered from polysomes, thus highlighting an impairment of control protein synthesis by JaMe at the level of translation initiation.
Phenotypic effects of overexpression of Agrobacterium rhizogenes T‐DNA ORF13 in transgenic tobacco plants are mediated by diffusible factor(s)
Authors: Hansen Geneviève , Vaubert Danièle , Héron Jean Noël , Clérot Danielle , Tempe Jacques , Brevet Jean
Published: March 2003

Nicotiana tabacum cv. Xanthi transgenic plants expressing ORF13 of Agrobacterium rhizogenes 8196 T‐DNA under the 35S RNA promoter from the cauliflower mosaic virus displayed developmental abnormalities. They were small, with short and variable internodal lengths, their root systems were poorly developed; leaves were small, asymmetric, rounded, wrinkled and dark green; flowers were short, and irregularly shaped. They exhibited reduced apical dominance and regularly produced offshoots at the base of the plant. This phenotype was also exhibited by offshoots of normal N. tabacum cv. Xanthi stock grafted with a transgenic scion indicating that expression of ORF13 influences plant development via diffusible factor(s).
Phenotypic characterization and molecular mapping of the Arabidopsis thaliana locus RPP5, determining disease resistance to Peronospora parasitica
Authors: Parker Jane E. , Szabó Véronique , Staskawicz Brian J. , Lister Clare , Dean Caroline , Daniels Michael J. , Jones Jonathan D.G.
Published: March 2003

Peronospora parasitica causes downy mildew on crucifers. An isolate of P. parasitica (denoted NoCO2) was identified that infected Arabidopsis plants of the land race Columbia (Col‐0) but not plants of land race Landsberg erecta (La‐er). Segregation analysis of F2 plants derived from a La‐er x Col‐0 cross established that the resistance was inherited as a single locus, denoted RPP5. Macroscopic and microscopic examinations of inoculated La‐er and Col‐0 cotyledons showed that restriction of fungal growth in La‐er was accompanied by massive callose accumulation and death of plant cells in direct contact with points of attempted fungal penetration. La‐er x Col‐0 F1 plants exhibited an intermediate resistance response in all aspects of fungal development, indicating that RPP5 is semi‐dominant in its action. F8 recombinant inbred lines generated between La‐er and Col‐0 were used to map RPP5 to a narrow interval (<1.1 cM) on chromosome 4, utilizing existing restriction fragment length polymorphic (RFLP) markers and newly generated random amplified polymorphic DNA (RAPD) markers. The data provide a basis for the isolation of the RPP5 locus by positional cloning as a first step towards understanding recognitional specificity in plant‐pathogen interactions at a molecular level.
Developmental and pathogen‐induced expression of three barley genes encoding lipid transfer proteins
Authors: Molina Antonio , García‐Olmedo Francisco
Published: March 2003

Clones for three barley non‐specific lipid transfer proteins (LTP2, LTP3, and LTP4; formerly Cw18, Cw20 and Cw21, respectively) which had been previously shown to inhibit growth of plant pathogens, were selected and characterized from a cDNA library derived from young etiolated leaves. Genes Ltp2 and Ltp4 were located in chromosome 3H and gene Ltp3 was assigned to chromosome 7H by Southern blot analysis of wheat—barley disomic addition lines, using gene‐specific probes (3′‐ends of cDNAs). These assignments were confirmed by the polymerase chain reaction, using specific primers. The three genes were expressed in stem, shoot apex, leaves and roots (at low levels) throughout development. Genes Ltp3 and Ltp4 were expressed at high levels, and Lpt2 at low levels, in the spike (rachis, lemma plus palea and grain coats). Neither of the mRNAs was detected in endosperm. The proteins were localized by tissue‐printing with polyclonal antibodies in the outer cell layer of the exposed surfaces of the plant, throughout the embryo, and in vascular tissues. Expression levels in leaves were moderately increased by 0.34 M NaCl and by 0.1 mM abscisic acid and were not affected by cold, drought, salicylate, 2,6‐dichloro‐isonicotinic acid, ethylene or ethephon. Methyl Jasmonate (10 µM) switched off all three genes. Inoculation with Av6 or vir6 isolates of the fungal pathogen Erysiphe graminis increased the three mRNAs, especially that of LTP4, which reached a maximum nine‐fold increase 12–16 h after infection.
Manipulation of lignin quality by downregulation of cinnamyl alcohol dehydrogenase
Authors: Halpin Claire , Knight Mary E. , Foxon Geoffrey A. , Campbell Malcolm M. , Boudet Alain M. , Boon Jaap J. , Chabbert Brigitte , Tollier Marie‐Thérèse , Schuch Wolfgang
Published: March 2003

The composition of lignin in tobacco stems has been altered by genetic engineering. Antisense expression of sequences encoding cinnamyl alcohol dehydrogenase (CAD), the enzyme catalysing the final step in lignin precursor synthesis, leads to the production of a modified lignin in otherwise normal plants. Although Klason and acetyl bromide lignin determinations show little quantitative change in lignin deposition in CAD antisense plants, a number of qualitative changes have been identified. The lignin is altered in both composition and structure and is more susceptible to chemical extraction. Consistent with a block in CAD activity, antisense plants incorporate less cinnamyl alcohol monomers and more cinnamyl aidehyde monomers into lignin than corresponding control plants. Antisense plants with very low levels of CAD activity also show a novel phenotype with the appearance of a red‐brown colour in xylem tissues. A similar phenotype is correlated with altered lignification and improved digestibility in brownmidrib mutants of maize and sorghum. The improved chemical extractability of lignin in CAD antisense plants supports a role for this technology in improving the pulp and paper‐making value of forest trees while the similarity with brown‐midrib mutants suggests a route to more digestible forage crops.
Isolation and characterization of a cDNA clone corresponding to the Rt locus of Petunia hybrida
Authors: Brugliera Filippa , Holton Timothy A. , Stevenson Trevor W. , Farcy Eliane , Lu Chin‐Yi , Cornish Edwina C.
Published: March 2003

We have isolated, via differential screening of a Petunia hybrida petal cDNA library, a cDNA clone that corresponds to the Rt locus which controls the conversion of anthocyanidin‐3‐glucosides to anthocyanidin‐3‐rutinosides by the UDP rhamnose: anthocyanidin‐3‐glucoside rhamnosyltransferase (3RT). The cDNA encodes a 469 amino acid long polypeptide with regions of similarity to the UDP glucose: flavonoid glucosyltransferases (3GT) from barley and maize. Some sequence similarity was also observed with non‐plant glycosyltransferases. Two aberrant transcripts are present in most of the rt/rt petunia lines examined. Excision of a transposon from an unstable Rt locus of one petunia line (Tr38) is associated with a change in transcript size back to wild‐type. The Rt transcript is most abundant in petals from flowers at an early stage of development and levels decline as the flower matures. Transcripts are also detected in the style and anthers but not in leaf, stem, root, petiole, ovary or sepals. Incubation of leaves in glucose under high light conditions induces the expression of the Rt gene as well as other flavonoid pathway genes. In situ hybridization revealed that the Rt transcript predominantly accumulates in the epidermal cells of the petal, the site of anthocyanin accumulation.
Transposition of a Ds element from a plasmid into the plant genome in Nicotiana plumbaginifolia protoplast‐derived cells
Authors: Houba‐Hérin Nicole , Domin Monique , Pédron Jacques
Published: March 2003

Nicotiana plumbaginifolia haploid protoplasts were co‐transformed with two plasmids, one with a NPT‐II/Ds element and one with a gene encoding an amino‐terminal truncated Ac transposase. It is shown that Ds can efficiently transpose from extrachromosomal DNA to N. plumbaginifolia chromosomes when the Ac transposase gene is present in trans. Ds has been shown to have transposed into the plant genome in a limited number of copies (1.9 copies per genome), for 21/32 transgenic lines tested. The flanking sequences present in the original plasmid are missing in these 21 plants. In only two of 21 plants was part of the transposase construct integrated. By segregation analysis of transgenic progeny, Ds was shown to be present in the heterozygous state in 10 lines even though haploid protoplasts had been originally transformed. This observation could indicate that integration occurred after or during DNA replication that leads to protoplast diploidization.
Molecular characterization of Arabidopsis thaliana cDNAs encoding three purine biosynthetic enzymes
Authors: Schnorr Kirk M. , Nygaard Per , Laloue Michel
Published: March 2003

Glycinamide ribonucleotide (GAR) synthetase, GAR transformylase and aminoimidazole ribonucleotide (AIR) synthetase are the second, third and fifth enzymes in the 10‐step de novo purine biosynthetic pathway. From a cDNA library of Arabidopsis thaliana, cDNAs encoding the above three enzymes were cloned by functional complementation of corresponding Escherichia coli mutants. Each of the cDNAs encode peptides comprising the complete enzymatic domain of either GAR synthetase, GAR transformylase or AIR synthetase. Comparisons of the three Arabidopsis purine biosynthetic enzymes with corresponding enzymes/polypeptide‐fragments from procaryotic and eucaryotic sources indicate a high degree of conserved homology at the amino acid level, in particular with procaryotic enzymes. Assays from extracts of E. coli expressing the complementing clones verified the specific enzymatic activity of Arabidopsis GAR synthetase and GAR transformylase. Sequence analysis, as well as Northern blot analysis indicate that Arabidopsis has single and monofunctional enzymes. In this respect the organization of these three plant purine biosynthesis genes is fundamentally different from the multifunctional purine biosynthesis enzymes characteristic of other eucaryotes and instead resembles the one gene, one enzyme relationship found in procaryotes.
Brij 58, a polyoxyethylene acyl ether, creates membrane vesicles of uniform sidedness. A new tool to obtain inside‐out (cytoplasmic side‐out) plasma membrane vesicles
Authors: Johansson Fredrik , Olbe Malin , Sommarin Marianne , Larsson Christer
Published: March 2003

Most of the plasma membrane vesicles formed upon homogenization of plant tissue have a right‐side‐out (cytoplasmic side‐in) orientation. Subsequent purification of plasma membrane vesicles using aqueous two‐phase partitioning leads to a further enrichment in right‐side‐out vesicles resulting in preparations with 80–90% of the vesicles in this orientation. Thus, to be able to assay, e.g. the ion‐pumping activities of the H+‐ATPase and the Ca2+‐ATPase, which expose their active sites towards the cytoplasm, the vesicles have to be inverted. This is very efficiently achieved by including 0.05% of the detergent Brij 58 (C16E20) in the assay medium, which produces 100% sealed, inside‐out (cytoplasmic side‐out) vesicles from preparations of 80–90% right‐side‐out vesicles. This was shown by assaying ATP‐dependent H+ pumping using the ΔpH probe acridine orange and dissipating the H+ gradient with nigericin, and by assaying ATP‐dependent Ca2+ transport using 45CA2+ and dissipating the Ca2+ gradient with the ionophore A23187. The presence of intact vesicles was confirmed by electronmicroscopy. The detergent Brij 58 is a polyoxyethylene acyl ether and a survey among some other members of this series revealed that those with a head group of relatively large size (E20–23) showed this ‘non‐detergent behavior’, whereas those with smaller head groups (E8–10) behaved as normal detergents and permeabilized the membranes. Thus, a very convenient system for studies on ion‐pumping activities and other vectorial properties of the plasma membrane is obtained by simply including the detergent Brij 58 in the assay medium.
Flavanone 3‐hydroxylase transcripts and flavonol accumulation are temporally coordinate in maize anthers
Authors: Deboo Gulshan B. , Albertsen Marc C. , Taylor Loverine P.
Published: March 2003

Flavanone 3‐hydroxylase (F3H) activity is necessary for the production of both flavonols and anthocyanins. Flavonols are required for functional pollen in maize whereas anthocyanins are non‐essential pigments. A cDNA for F3H was isolated from Zea mays using a heterologous sequence from Antirrhinum majus. Comparison of the deduced amino acid sequence of maize F3H with other F3H sequences confirmed that the protein is highly conserved among widely divergent plant species. The F3H gene is present in a single copy located at the tip of chromosome 2S. High levels of F3H gene expression were detected in pigmented husk and 26‐day postpollination kernels; lower levels in 18‐day postpollination kernels and in coleoptiles of germinating seedlings. Slot blot analysis showed that F3H transcript levels in young seedlings are increased by high fluence‐rate white light treatment in the presence of the anthocyanin regulatory gene ‐r. HPLC analysis of extracts from developmentally staged anthers showed that flavonol accumulation begins at the uninucleate microspore stage, continues until maturity, and is not controlled by ‐r. When the expression pattern of several flavonoid biosynthetic genes was analyzed during microsporogenesis, only F3H transcript accumulation was coordinate with the appearance of flavonols in anthers.
A mutation affecting etiolation and cell elongation in Nicotiana plumbaginifolia causes abnormal division plane alignment and pattern formation in the root meristem†
Authors: Traas Jan , Laufs Patrick , Jullien Marc , Caboche Michel
Published: March 2003

A mutant of Nicotiana plumbaginifolia, hyp2, which is abnormal in the regulation of post‐embryonic cell elongation is described. All cells in the plant are affected by the mutation, and, in general, cells are shorter and thicker than in wild‐type seedlings. Not only is the mutant perturbed in cell expansion, but it also shares a number of characteristics with some of the de‐etiolated mutants of Arabidopsis. In the dark it has spread cotyledons and it expresses the photosynthetic gene RBCS. In the light it shows accumulation of anthocyanins and cells in the upper part of the root develop chloroplasts. An important characteristic of the mutant is that not only cell shape but also pattern formation in the root meristem is perturbed. First, the mutant has an extra cortical cell layer because the cells that usually give rise to the endodermis go through an additional round of longitudinal, periclinal cell divisions. In addition, the amount of cells per cell layer is increased when compared with the wild‐type, showing that the mutation causes extra anticlinal cell divisions in all cell types. Experiments using gibberellic acid (GA) show that the gibberellin GA3 can partially compensate for abnormal HYP2 function. In the presence of this growth factor the mutant shows a hypocotyl which is even longer than in the untreated wild‐type, and root meristem structure is normal, combined with a reduced anthocyanin content. However, a detailed analysis of meristem structure using the hormone and the inhibitor paclobutrazol shows that both HYP2 and gibberellic acid independently control cell elongation and pattern formation in the root meristem. The results also show that gibberellins have a more direct effect on the decision of a cell to divide in a particular plane than is usually proposed in the literature.
An aldose reductase homologous gene from barley: regulation and function
Authors: Roncarati Renza , Salamini Francesco , Bartels Dorothea
Published: March 2003

The expression of a barley gene homologous to aldose reductase and aldehyde reductase is restricted to the embryo and temporally correlated with its acquisition of desiccation tolerance. In the work presented, two aspects of this barley gene were investigated: its transcriptional regulation and the initial characterization of the enzymatic function. The transcriptional regulation of the gene was studied in transgenic tobacco by analysing the expression of chimeric genes containing 5′ sequences of the barley gene transcriptionally fused to the GUS reporter gene. This functional analysis of the promoter revealed that a 1364 bp 5′ fragment confers the appropriate pattern of expression to the reporter gene in tobacco and that a short promoter fragment (−114 to +75) containing the sequence TACGTGGC, homologous to plant G‐box elements, is sufficient for developmental expression during embryogenesis.

To investigate the enzymatic properties of the gene product the wild‐type protein and a mutant carrying a lysine 259 to methionine substitution were overexpressed in a procaryotic system and purified to homogeneity. The wild‐type protein exhibits aldose reductase activity in the reduction of dl‐glyceraldehyde and d‐erythrose specifically using NADPH as co‐factor whereas the mutant shows markedly reduced activity. However, the barley protein possesses some properties different to those of animal aldose and aldehyde reductases and its biological target still needs to be identified.
Effects of ABA on 86Rb+ fluxes at plasmalemma and tonoplast of stomatal guard cells
Authors: MacRobbie E.A.C.
Published: March 2003

Abscisic acid (ABA) induces a transient stimulation of 86Rb+ from isolated guard cells of Commelina communis L. When ABA is added after 30–50 min of wash‐out in the absence of ABA, when tracer is almost entirely vacuolar, its effects on vacuolar release are measured. When ABA is added early in the wash‐out (at 2–4 min), when both cytoplasm and vacuole are labelled, the resulting efflux includes both vacuolar and cytoplasmic contributions. Detailed comparison of rates of efflux in the absence of ABA, and in the presence of ABA added early and late in the wash‐out, allows the effects of ABA on plasmalemma and tonoplast fluxes to be assessed. Three effects of ABA can be distinguished: these are stimulation of the 86Rb+ flux from vacuole to cytoplasm (by twofold to 6.7‐fold); stimulation of the plasmalemma efflux, by up to twofold, a smaller factor than that of the tonoplast effect and variable between experiments; and a doubling of the half‐time for cytoplasmic exchange in ABA, taken to reflect an increase in cytoplasmic ion content as ions flood out of the vacuole. Concentrations of ABA of 0.1–0.2 µM and 1–10 µM are equally effective in the stimulation of plasmalemma efflux, but the effects on tonoplast fluxes are both delayed and reduced at low external concentrations of ABA. It is argued that the delay reflects the need for a threshold internal ABA to be reached before the initiation of vacuolar release, and the reduction reflects the sensitivity of the extent of activation of tonoplast ion channels to concentration of internal ABA. It is likely that the plasmalemma change is mediated by external ABA, and could be the result of the modulation of the stretch‐activated channel suggested previously.
Homology‐dependent resistance: transgenic virus resistance in plants related to homology‐dependent gene silencing
Authors: Mueller Elisabeth , Gilbert Julie , Davenport Guy , Brigneti Gianinna , Baulcombe David C.
Published: March 2003

Tobacco plants transformed with the RNA polymerase (RdRp) gene of potato virus X (PVX) that are extremely resistant to infection by potato virus X have previously been described. The PVX‐resistant plants accumulated the RdRp protein at a lower level than fully susceptible plants transformed with the same RdRp construct. In this paper the difference between the PVX‐resistant and susceptible transformed plants is investigated and it is demonstrated that there are three associated phenotypes of the RdRp transgene that vary in parallel between transformed lines. These phenotypes are: accumulation of the transgenic RdRp RNA at a low level; strain‐specific resistance to PVX; and the ability of the transgene to trans‐inactivate homologous transgenes. This gene‐silencing potential of the transgenes conferring PVX resistance was illustrated by analysis of progeny from a cross between a transformant that was extremely resistant to PVX and a second PVX‐susceptible transformant. In other transformants, in which the resistance was less extreme, the same three phenotypes were associated but in a transgene dosage‐dependent manner. The same association of strain‐specific resistance and low‐level accumulation of the transgenic RdRp RNA was observed with plants that were transformed with mutant or wild‐type versions of the RdRp gene. Strain‐specific resistance was also produced in plants transformed with untranslatable versions of the RdRp transgene. Based on these data it is proposed that homology‐dependent gene silencing and transgenic resistance to PVX may be due to the same RNA‐based mechanism. An undefined genomic feature is proposed to account for the variation in the resistance and trans‐inactivation phenotypes of different transformants. It is further proposed that this genome feature influences a cytoplasmic mechanism that degrades RNA with sequence homology to the silencing transgene.
MYB.Ph3 transcription factor from Petunia hybrida induces similar DNA‐bending/distortions on its two types of binding site
Authors: Solano Roberto , Nieto Concepción , Paz‐Ares Javier
Published: March 2003

MYB.Ph3 from Petunia is a member of the MYB transcription factor family that recognizes two types of binding site, resembling direct and inverted repeats of the motif GTTA, respectively, and flanked on their 5′ side by an Arich sequence. In this study, it is shown that MYB.Ph3 induces similar conformational changes in both of its DNA‐binding sites. Circular permutation assays indicate that the center of the distortions induced by MYB.Ph3 maps near the center of either MYB.Ph3‐binding site. The degree of the distortion induced by MYB.Ph3 appears to be greatly affected by regions of the protein other than the DNA‐binding domain, and differs from the distortion caused by animal c‐MYB. Phasing analysis reveals that part of the distortion induced by MYB.Ph3 is DNA bending, oriented towards the minor groove, as is the case of its animal counterpart, c‐MYB. DNA‐binding by both MYB.Ph3 and animal c‐MYB is more efficient in the presence of Ba2+, a divalent cation known to promote/stabilize DNA bending, than in the presence of other cations which do not favor this distortion, such as Na+ and Mg2+. In addition, both MYB proteins show higher affinity to selectively nicked DNA, which has increased DNA flexibility, strongly suggesting that DNA binding by these MYB proteins and DNA structural properties are mutually influenced.
Expression of a cloned plant K+ channel in Xenopus oocytes: analysis of macroscopic currents
Authors: Véry Anne‐Aliénor , Gaymard Frédéric , Bosseux Corinne , Sentenac Hervé , Thibaud Jean‐Baptiste
Published: March 2003

The open reading frame from the Arabidopsis thaliana KAT1 cDNA was cloned in a transcription plasmid between the 3′ and 5′ untranslated regions of a β‐globin cDNA from Xenopus oocyte. The polyadenylated transcripts resulting from in vitro transcription gave rise to high levels of expression of KAT1 channel when injected in Xenopus oocytes. Upon hyperpolarization, a slow activating current could be recorded, inwardly‐ or outwardly‐directed, depending on K+ external concentration. Predictions of the voltage‐gated channel theory were shown to fit the data well. The equivalent gating charge and the half‐activation potential ranged around 2 and −145 mV, respectively. KAT1 gating characteristics did not depend on K+ external concentration nor on external pH in the 5.0–7.5 range. KAT1 conductance was, however, increased (40%) when external pH was decreased from 6.5 to 5.0. The apparent affinity constant of KAT1 for K+ lay in the range 15–30 mM, at external pH 7.4. As for many K+ channels of animal cells, external caesium caused a voltage‐dependent blockage of inward (but not outward) KAT1 current, whereas tetraethylammonium caused a voltage‐independent block of both inward and outward KAT1 currents. In conclusion, high levels of expression made it possible to carry out the first quantitative analysis of KAT1 macroscopic currents. KAT1 channel was shown to display features similar to those of as yet uncloned inward‐rectifying voltage‐gated channels described in both plant cells (namely guard cells) and animal cells.
Generation of a high‐quality P1 library of Arabidopsis suitable for chromosome walking
Authors: Liu Yao‐Guang , Mitsukawa Norihiro , Vazquez‐Tello Alejandro , Whittier Robert F.
Published: March 2003

Using improved techniques, a representative P1 library of Arabidopsis was constructed and characterized. Megabase genomic DNA was prepared from nuclei and partially digested with Sau3AI. DNA fragments of 75–100 kb were selected by size fractionation in low melting agarose, concentrated by a spot‐evaporation/dialysis method, and cloned in the pAd10sacBII P1 vector. The library contains 10 080 clones individually stored in microtiter plates. With an average insert size of about 80 kb, the library represents about eight haploid genomic equivalents of this plant. This library can be screened rapidly by dot hybridization of plate and well position pools. Characterization of the library by restriction analysis, screening with RFLP probes, RFLP mapping of insert end sequences, and chromosome walking shows that the library is of high quality with respect to insert site, completeness, and absence of chimeric artifacts. With this library a contig of about 600 kb has been constructed in the cer9 locus region. This P1 library is expected to be useful for genome mapping and gene cloning in Arabidopsis research programs.
Three families of thiol peptides are induced by cadmium in maize
Authors: Meuwly Philippe , Thibault Pierre , Schwan Adrian L. , Rauser Wilfried E.
Published: March 2003

Phytochelatins ((γGluCys)nGly) are synthesized from glutathione by plants exposed to metals like Cd2+, Cu2+ and Zn2+. An intracellular complex formed by phytochelatins with Cd2+ and sulfide is thought to detoxify the metal possibly by sequestration in the vacuole. It was found that maize seedlings exposed to Cd2+ produced phytochelatins and two additional families of cysteina‐containing peptides, (γGluCys)n and (γGluCys)nGlu. All thiol peptides with n = 2 and 3 were purified and their structure characterized by tandem mass spectrometry. For maize plants exposed to Cd2+ for 7 days, phytochelatins were synthesized preferentially in the first 24 h whereas the amounts of (γGluCys)n and (γGluCys)nGlu were the highest thereafter. This was probably due to an initial large pool of glutathione available in control plants compared with a dearth of γGluCys and no detectable γGluCysGlu. The (γGluCys)nGlu peptides were induced exclusively by Cd2+ as they were below the detection limit in control seedlings that contained low amounts of phytochelatins and (γGluCys)n. Since the Cys moiety of the peptides is essential for binding Cd2+, a role for accumulated (γGluCys)n and (γGluCys)nGlu in detoxifying Cd2+ in plants must be considered.
Effects of cytosolic calcium and limited, possible dual, effects of G protein modulators on guard cell inward potassium channels
Authors: Kelly Walter B. , Esser John E. , Schroeder Julian I.
Published: March 2003

The cellular mechanisms that regulate potassium (K+) channels in guard cells have been the subject of recent research, as K+ channel modulation has been suggested to contribute to stomatal movements. Patch clamp studies have been pursued on guard cell protoplasts of Vicia faba to analyze the effects of physiological cytosolic free Ca2+ concentrations, Ca2+ buffers and GTP‐binding protein modulators on inward‐rectifying K+ channels. Ca2+ inhibition of inward‐rectifying K+ currents depended strongly on the concentration and effectiveness of the Ca2+ buffer used, indicating a large Ca2+ buffering capacity and pH increases in guard calls. When the cytosolic Ca2+ concentration was buffered to micromolar levels using BAPTA, inward‐rectifying K+ channels were strongly inhibited. However, when EGTA was used as the Ca2+ buffer, much less inhibition was observed, even when pipette solutions contained 1 µM free Ca2+. Under the imposed conditions, GTPγS did not significantly inhibit inward‐rectifying K+ channel currents when cytosolic Ca2+ was buffered to low levels or when using EGTA as the Ca2+ buffer. Furthermore, GDPβS reduced inward K+ currents at low cytosolic Ca2+, indicating a novel mode of inward K+ channel regulation by G‐protein modulators, which is opposite in effect to that from previous reports. On the other hand, when Ca2+ was effectively elevated in the cytosol to 1 µM using BAPTA, GTPγS produced an additional inhibition of the inward‐rectifying K+ channel currents in a population of cells, indicating possible Ca2+‐dependent action of GTP‐binding protein modulators in K+ channel inhibition. Assays of stomatal opening show that 90% inhibition of inward K+ currents does not prohibit, but slows, stomatal opening and reduces stomatal apertures by only 34% after 2 h light exposure. These data suggest that limited K+ channel down‐regulation alone may not be rate‐limiting, and it is proposed that the concerted action of proton‐pump inhibition and additional anion channel activation is likely required for inhibition of stomatal opening. Furthermore, G‐protein modulators regulate inward K+ channels in a more complex and limited, possibly Ca2+‐dependent, manner than previously proposed.
The pea chloroplast membrane‐associated protein, IEP96, is a subunit of acetyl‐CoA carboxylase
Authors: Shorrosh Basil S. , Savage Linda J. , Soll Jürgen , Ohlrogge John B.
Published: March 2003

Two forms of acetyl‐CoA carboxylase (ACCase) have been characterized in pea (Pisum sativum L.) leaves; a heteromeric chloroplast enzyme and a homomeric, presumably cytosolic enzyme. The biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and β‐carboxyltransferase (CT) subunits of the plastidial‐ACCase have recently been characterized and cloned. To further characterize the carboxyl‐transferase, an improved assay for CT was developed and used to follow its partial purification. CT activity co‐purifies with ACCase activity during gel permeation chromatography. However, upon anion‐exchange chromatography or native PAGE, CT separates from the BC and BCCP subunits of plastidiaI‐ACCase and ACCase activity is lost. In addition, it is demonstrated that a previously sequenced pea chloroplast cDNA of unknown function (IEP96) with a predicted molecular weight of 91 kDa encodes the α‐CT subunit of the MS‐ACCase. Antibodies raised against the first 404 amino acids of IEP96 protein detected a polypeptide with molecular weight of 91 kDa that co‐eluted during gel permeation chromatography with plastidial CT and ACCase activities. These antibodies also immunoprecipitated the activities of both ACCase and CT with the concomitant precipitation of the β‐CT subunit. Furthermore, antibodies against β‐CT immunoprecipitated the IEP96 protein. Two‐dimensional PAGE and DEAE purification of ACCase protein demonstrated that the β‐CT forms a tight association with the IEP96 protein. Pea leaf was fractionated into soluble and membrane fractions and the α‐CT subunit was primarily associated with the membrane fraction. Together, these data demonstrate that IEP96 is the α‐CT subunit of pea chloroplast ACCase.
Arabidopsis profilins are functionally similar to yeast profilins: identification of a vascular bundle‐specific profilin and a pollen‐specific profilin
Authors: Christensen Hans E.M. , Ramachandran Srinivasan , Tan Chio‐Tee , Surana Uttam , Dong Chun‐Hai , Chua Nam‐Hai
Published: March 2003

Four members of the Arabidopsis profilin (pfn) multigene family have been cloned, sequenced and analyzed. By RNA gel blot analysis it has been shown that these four genes fall into two groups: one group (pfn1 and pfn2) is expressed in all organs of the plant and the other group (pfn3 and pfn4) in floral tissues only. Based on amino acid sequence alignment Arabidopsis profilins can be divided into the same two groups: PFN1 and PFN2 are 89% identical and PFN3 and PFN4 are 91% identical. Between these two groups they are 71–75% identical. The Arabidopsis profilins bind poly‐l‐proline and can complement both the Saccharomyces cerevisiae profilin deletion mutant and the Schizosaccharomyces pombe cdc3‐124/profilin mutation, showing that the plant profilins are functionally similar to yeast profilins despite the low amino acid sequence homology. Analysis of pfn promoter‐GUS fusion genes in transgenic Arabidopsis shows that pfn2 is specifically expressed in the vascular bundles of roots, hypocotyls, cotyledons, leaves, sepals, petals, stamen filaments and stalks of developing seeds, whereas expression of pfn4 is restricted to mature and germinating pollen grains.
Rapid alkalinization in alfalfa root hairs in response to rhizobial lipochitooligosaccharide signals
Authors: Felle Hubert H. , Kondorosi Éva , Kondorosi Adám , Schultze Michael
Published: March 2003

Rhizobial lipochitooligosaccharides (Nod factors) function as symbiotic signals that trigger root hair deformations and cortical cell divisions on the roots of leguminous plants in a host‐specific manner. By using pH‐sensitive microelectrodes, it is shown that alfalfa root hair cells respond to Rhizobium meliloti Nod factors with a rapid intracellular alkalinization of 0.2–0.3 pH units. This alkalinization remained as long as the Nod factor was present, but slowly reversed after removal of the signal. The response was most sensitive to the sulfated tetrameric Nod factor, NodRm‐IV(C16:2,S), which is morphogenic on the host plant alfalfa, suggesting a role in a signal transduction cascade. Non‐sulfated Nod factor as well as chitooligosaccharides elicited a pHc change only at elevated concentrations. The increase of PHc in response to sulfated Nod factor was concomitant with a depolarization of the plasma membrane potential whereas the PHc change in response to non‐sulfated Nod factor occurred in the absence of membrane depolarization. In addition, whereas a first dose of sulfated Nod factor inhibited the subsequent response to a second dose of the same molecule, it did not significantly repress the activity of non‐sulfated Nod factor. These results indicate that sulfated and non‐sulfated Nod factors act independently and suggest the existence of two Nod signal perception systems, one transmitting the host‐specific signal, the other representing an ancient reception system for a generic Nod factor structure.
Identification of cis‐elements regulating the expression of an Arabidopsis histone H4 gene
Authors: Chaubet Nicole , Flénet Martine , Clément Bernadette , Brignon Pierre , Gigot Claude
Published: March 2003

Protein—DNA interactions in the proximal region of an Arabidopsis H4 histone gene promoter were analyzed by DMS in vivo footprinting combined with LMPCR amplification. Interactions were identified over six particular sequence motifs, five of which were previously shown to bind proteins in maize histone H3 and H4 promoters and are commonly found in the corresponding regions of other plant histone gene promoters. These motifs are located within a 126 bp fragment which was previously shown to confer preferential expression in meristems of transgenic plants. The contribution of each cis‐element to the overall expression level and specificity was investigated by testing individual or combined mutations in transgenic Arabidopsis plants. All five motifs behaved as positive cis‐elements of unequal strength. The GCCAAT‐like sequence GCCACT behaved as a strong positive cis‐element but had no influence on the specificity. In contrast, the nonamer AGATCGACG and to a lesser extent the closely linked hexamer CCGTCG proved to be essential for meristem‐specific expression. Involvement of the highly conserved histone‐specific octamer CGCGGATC in specific expression was revealed at some stages of meristem development. Importance of these three cis‐elements, nonamer, hexamer, and octamer, was further confirmed by the fact that combining mutations of two of them either abolished the promoter activity or completely modified the promoter specificity. Mutation of the fifth cis‐element, a degenerate copy of the octamer, little perturbed the promoter function. However disruption of both octamers had a dramatic negative effect, thus suggesting that the two copies cooperate to achieve maximal function in the wild‐type promoter, possibly by mobilizing the proliferation‐specific factors binding to the nonamer and CCGTCG cis‐elements.