bluuebunny/crossref_metadata_2025 huggingface dataset:
Late embryogenesis‐abundant genes encoding proteins with different numbers of hydrophilic repeats are regulated differentially by abscisic acid and osmotic stress
Authors: Espelund Mari , Sæbøe‐Larssen Stein , Hughes D. Wayne , Galau Glenn A. , Larsen Frank , Jakobsen1 Kjetill S.
Published: March 2003

The late embryogenesis‐abundant (Lea) genes, which are suggested to act as desiccation protectants during seed desiccation and in water‐stressed seedlings, can be induced by abscisic acid (ABA) and various kinds of water‐related stress. Using cotton Lea cDNAs as probes it was found that several of the Lea genes are conserved at the mRNA level in dicots and monocots. By screening a barley cDNA library with a cotton Lea D19 cDNA a family of three members was isolated. The putative B19 proteins have strong similarities to the Em protein in wheat and to LEA proteins from several dicots. However, the middle part of the B19 proteins consists of a 20‐amino acid motif repeated three and four times in B19.3 and B19.4, respectively, but only once in B19.1. The gene products are strongly hydrophilic, the internal 20‐amino acid motif being the most hydrophilic part. This motif is found once in cotton Lea D19 but is repeated twice in cotton Lea D132, indicating that the repeats are universal among monocot and dicot B19‐like genes. The B19 genes are regulated similarly during embryo development, but to very different levels. In contrast, they are differentially regulated by ABA and various types of osmotic stress. In immature embryos all three genes are responsive to ABA and mannitol. However, B19.1 is also responsive to salt. Cold stress does not induce B19 mRNAs; only a stabilization of the transcript levels is seen. These results suggest that the responses to salt stress and exogenous ABA operate through different pathways.
Correlation between arrested secondary plasmodesmal development and onset of accelerated leaf senescence in yeast acid invertase transgenic tobacco plants
Authors: Ding Biao , Haudenshield James S. , Willmitzer Lothar , Lucas William J.
Published: March 2003

Mature leaves of a transgenic tobacco plant (Nicotiana tabacum L var. Samsun, line A41‐10) that constitutively express a yeast‐derived acid invertase gene develop symptoms which are characterized by the presence of greenish‐yellow and green sectors in the same leaf, and onset of early, leaf senescence. Previous studies indicated that invertase activity was two‐ to threefold higher in the greenish‐yellow sectors than in the green sectors. Our structural analyses revealed that development of secondary plasmodesmata, via modification of existing primary plasmodesmata, between mesophyll cells was inhibited severely in the greenish‐yellow sectors, but only marginally in the green sectors. In contrast, the structure and function of primary plasmodesmata in the same symptomatic sectors remained unaltered as determined by structural and dye coupling studies. It is hypothesized that secondary plasmodesmata differ from primary plasmodesmata in having special abilities to traffic information molecules to coordinate leaf development and physiological function(s). Arrest of secondary plasmodesmal development by high invertase activity in the transgenic tobacco leaf may have prevented this type of trafficking and hence resulted in early leaf senescence. The results also indicate that the yeast acid invertase‐expressing tobacco may provide an effective experimental system for the molecular characterization of cellular mechanisms that regulate the development, function, and possible turnover of secondary plasmodesmata.
Molecular characterization of the gene for carrot cell wall β‐fructosidase
Authors: Ramloch‐Lorenz Kathrin , Knudsen Soeren , Sturm Arnd
Published: March 2003

Carrot cell wall β‐fructosidase, previously purified and cloned, is encoded by a single, wound‐ and pathogen‐inducible gene. The developmental regulation of the gene was studied by determining the steady‐state mRNA levels in different organs during carrot development: cell wall β‐fructosidase mRNA was detected in roots and leaves of young plants but not during tap root development. A genomic clone was isolated and characterized. The transcription start site was determined by primer extension analysis. Inspection of the promoter sequence (1488 bp) revealed the presence of sequences with high homology to cis‐acting elements for the regulation of plant genes by wounding and infection. The 5′‐regulatory sequence was fused to the reporter gene β‐glucuronidase (GUS) and tested in a transient expression assay with carrot suspension cells and wounded carrot root tissue (aged disks of carrot roots). The expression of the GUS gene in the transfected cells proved that the isolated promoter was functional. In transgenic tobacco plants containing the cell wall β‐fructosidase promoter fused to GUS, the reporter gene was predominantly expressed in the shoot and root meristems of young seedlings. No GUS expression was detected in mature tobacco plants, showing that the development‐specific regulation of the cell wall β‐fructosidase promoter seen in carrot was maintained in tobacco plants. In contrast, expression of the GUS reporter gene in transgenic tobacco was not wound inducible. To analyze the functional organization of the cell wall β‐fructosidase promoter, a 5′‐deletion series was generated and tested in a transient expression assay in protoplasts of Nicotiana plumbaginifolia. Two regions containing putative silencer elements were identified. A comparison of these regions with known silencer elements identified in both regions one copy of the negative dominant cis‐acting element found in a chalcone synthase promoter of petunia.
Abortion of infection during the Rhizobium meliloti—alfalfa symbiotic interaction is accompanied by a hypersensitive reaction
Authors: Vasse Jacques , de Billy Françoise , Truchet Georges
Published: March 2003

Nodulation, the organogenetic process resulting from the symbiotic interaction between Rhizobium and legumes, is under the feedback control of the plant. However, the autoregulatory mechanisms controlling root nodule formation are poorly understood. In this paper it is shown that alfalfa can react to infection by its symbiont Rhizobium meliloti by eliciting a defence mechanism similar to the hypersensitive reaction (HR) observed in incompatible plant‐pathogen interactions. After the first nodule primordia have been induced, an increasing proportion of infection threads abort in a single or a few root cortical cells in which both symbionts simultaneously undergo necrosis. Autofluorescent, cytochemical and immunolocalization assays revealed that phenolic compounds and proteins associated with defence mechanisms in plants have accumulated in the necrotic cells. These results lead to the proposition that the elicitation of a HR is part of the mechanism by which the plant controls infection and, therefore, regulates nodulation.
Plasmodesmata between sieve element and companion cell of extrafascicular stem phloem of Cucurbita maxima permit passage of 3 kDa fluorescent probes
Authors: Kempers R. , Prior D.A.M. , Van Bel A.J.E. , Oparka K.J.
Published: March 2003

Fluorescent probes of differing Mr were introduced into the sieve element—companion cell (SE‐CC) complexes of the extrafascicular stem phloem of Cucurbita maxima by either immersing the cut ends of internode segments in aldehyde‐fixable probes, or directly by micro‐injection. In both cases probes of up to Mr 3000 (3 kDa) were able to move freely along the extrafascicular SE and, more significantly, between SE and CC. No movement occurred out of the SE‐CC complex. In the case of the fascicular phloem, occlusion of the sieve plates was ineffectual at impeding the longitudinal movement of low molecular mass probes such as Lucifer Yellow CH (LYCH), but was effective at preventing the movement of probes of Mr 3000 or greater. The results show that the molecular exclusion limit of plasmodesmata can be considerably in excess of Mr 800 and provide direct evidence for the long‐held suspicion that the plasmodesmata which connect the CC with the enucleate SE have a special permeability to allow the intercellular passage of macromolecules essential for the maintenance of the SE.
Interconversion of the salicylic acid signal and its glucoside in tobacco
Authors: Hennig Jacek , Malamy Jocelyn , Grynkiewicz Grzegorz , Indulski Janusz , Klessig Daniel F.
Published: March 2003

Salicylic acid (SA) has been proposed to play a role in the induction of pathogenesis‐related (PR) proteins and systemic acquired resistance (SAR) in tobacco. Since SA is rapidly converted to salicylic acid β‐glucoside (SAG) in tobacco, we have attempted to assess the role of SAG in pathogenesis by application of chemically synthesized SAG to tobacco leaves. SAG was as active as SA in induction of PR‐1 gene expression. This induction was preceded by a transient release of SA, which occurred in the extracellular spaces. The existence of a mechanism that releases SA from SAG suggests a possible role for SAG in SAR.
Constitutive, light‐responsive and circadian clock‐responsive factors compete for the different I box elements in plant light‐regulated promoters
Authors: Borello Ugo , Ceccarelli Elena , Giuliano Giovanni
Published: March 2003

The I box is a conserved regulatory motif which is found upstream of plant genes (rbcS, cab and nia) whose transcription is regulated by light and the circadian clock. Gel retardation and UV cross‐linking assays were used to resolve two different groups of I box binding factors (IBFs) in tomato nuclear extracts.

Active components of the first group (IBF‐1) recognize the I box of the light‐responsive rbcS promoter; one factor within this group, IBF‐1a, also recognizes the adjacent G box, which has been shown previously to bind a different class of plant transcription factors, the G box binding factors (GBFs). To the limit of experimental resolution, IBF‐1a and GBF compete for the same nucleotides on the G box. Nevertheless, these two activities are biochemically and immunologically distinct. The relative abundance of IBF‐1a shows a vast decrease in dark‐adapted plants.

Factors in the second group (IBF‐2), recognize the I box of the nia promoter, which is regulated both by light and the circadian clock; one factor within this group, IBF‐2a, also binds the I box of a second promoter showing similar regulation, the cab promoter. The IBF‐2a binding sites on the cab and nia promoters show extensive homology to a circadian clock‐responsive promoter element from wheat. The abundance of IBF‐2a is diurnally regulated and shows a dramatic induction around the onset of the light period. Transfer of the plants in continuous darkness demonstrates that this induction is under the control of a circadian clock. These data suggest that I box binding factors may be involved in regulation of transcription by light and the circadian clock.
Vesicle transport and processing of the precursor to 2S albumin in pumpkin
Authors: Hara‐Hishimura Ikuko , Takeuchi Yuka , Inoue Kaori , Nishimura Mikio
Published: March 2003

Cell fractionation of pulse‐chase‐labeled developing pumpkin cotyledons demonstrated that proprotein precursor to 2S albumin is transported from the endoplasmic reticulum to dense vesicles and then to the vacuoles, in which pro2S albumin is processed to the mature 2S albumin. Immunocytochemical analysis showed that dense vesicles of about 300 nm in diameter mediate the transport of pro2S albumin to the vacuoles.

The primary structure of the precursor (16 578 Da) to pumpkin 2S albumin has been deduced from the nucleotide sequence of an isolated cDNA insert. The presence of a hydrophobic signal peptide at the N‐terminus indicates that the precursor is a preproprotein that is converted into pro2S albumin after cleavage of the signal peptide. N‐terminal sequencing of the pro2S albumin in the isolated vesicles revealed that the signal peptide is cleaved off co‐translationally on the C‐terminal side of alanine residue 22 of prepro2S albumin. By contrast, post‐translational cleavages occur on the C‐terminal sides of asparagine residues 35 and 74, which are conserved among precursors to 2S albumin from different plants. Hydropathy analysis revealed that the two asparagine residues are located in the hydrophilic regions of pro2S albumin. These findings suggest that a vacuolar processing enzyme can recognize exposed asparagine residues on the molecular surface of pro2S albumin and cleave the peptide bond on the C‐terminal side of each asparagine residue to produce mature 2S albumin in the vacuoles.
PCR amplification of microdissected wheat chromosome arms in a simple ‘single tube’ reaction
Authors: Albani Diego , Côté Marie‐José , Armstrong Ken C. , Chen Qianfa , Segal Allan , Robert Laurian S.
Published: March 2003

A novel method for the adaptor‐mediated PCR amplification of microdissected chromosome arms is described. This simple and versatile protocol eliminates the need for enzymatic micromanipulation in nanoliter volumes and permits the efficient amplification of as little as two wheat chromosome arms in a ‘single tube’ reaction.
Tissue‐specific expression of sunflower heat shock proteins in response to water stress
Authors: Almoguera Concepción , Coca María A. , Jordano Juan
Published: March 2003

Accumulation of mRNA and synthesis of low‐molecular‐weight heat shock proteins (LMW HSPs) was investigated in water‐stressed sunflower, under experimental conditions resulting in little or no thermal stress. Using probes and antibodies derived from developmentally expressed LMW HSPs, it was shown that homologous mRNAs and proteins accumulate in the stem and root of water‐stressed plants. This expression is quantitatively comparable with the response to heat shock: protein and mRNA accumulate to similar, high, levels and persist for comparable times during recovery from either environmental stress. However, it is shown that LMW HSPs with different molecular weights and isoelectric points are expressed in response to heat shock or water stress. Furthermore in situ localizations show a differential tissue‐specificity for the water‐stress‐ and heat‐shock‐induced LMW HSPs. Whereas the latter are localized mostly around the xylem vessels in the stem, the water‐stress‐induced proteins accumulate in the fascicular and interfascicular cambium. The possible functional implications for this specific expression are discussed.
Molecular characterization and expression of alfalfa isoliquiritigenin 2′‐O‐methyltransferase, an enzyme specifically involved in the biosynthesis of an inducer of Rhizobium meliloti nodulation genes
Authors: Maxwell Carl A. , Harrison Maria J. , Dixon Richard A.
Published: March 2003

A cDNA clone encoding an O‐methyltransferase (OMT) from alfalfa has been isolated, which methylates the 2′‐hydroxyl of isoliquiritigenin (2′,4,4′‐trihydroxychalcone) to form 4,4′‐dihydroxy‐2′‐methoxychalcone, the most potent of the nod‐gene‐inducing flavonoid derivatives released from alfalfa roots. The cDNA clone was identified on the basis of N‐terminal sequence identity to purified S‐adenosyl‐l‐methionine: isoliquiritigenin 2′‐O‐methyltransferase (chalcone OMT) and expression of enzymatically active chalcone OMT protein in Escherichia coli. The deduced amino acid sequence showed significant similarities to other OMTs. Chalcone OMT is encoded by a small gene family in alfalfa and related sequences are present in other legumes. The chalcone OMT gene is expressed primarily in alfalfa roots; transcript levels were highest during the first 2 weeks of development. The OMT transcript was also detected, to a much lesser extent, in root nodules. In contrast, chalcone isomerase (CHI), although expressed at high levels in roots, was found in all plant organs and had a somewhat different developmental expression pattern. Chalcone OMT transcripts were localized primarily to epidermal and cortical cells starting 1.5–2.0 mm behind the root tip, whereas CHI transcripts were present at aproximately equal levels in epidermal, cortical and vascular tissues, both at the root tip and throughout the root. Chalcone OMT transcripts were elicitor‐inducible in alfalfa cell suspension cultures, although only low levels of methoxychalcone accumulated. The implications of these results for plant‐microorganism interactions are discussed.
Multiple functions of promoter sequences involved in organ‐specific expression and ammonia regulation of a cytosolic soybean glutamine synthetase gene in transgenic Lotus corniculatus
Authors: Marsolier Marie‐Claude , Carrayol Elisa , Hirel Bertrand
Published: March 2003

A 3.5 kbp promoter fragment fused to the reporter β‐glucuronidase (GUS) gene had previously been shown to contain the regulatory elements controlling the ammonia‐stimulated transcription of GS15, a soybean gene encoding root and root nodule cytosolic glutamine synthetase (GS). Further studies using an RNase protection assay allowed detection of large amounts of GS15 transcripts in flowers and pulvinus of soybean plants, whereas in other vegetative parts of the plant, the corresponding mRNAs were expressed at low levels. A comparable GUS expression pattern was obtained in transgenic Lotus corniculatus containing the GS15‐GUS construct. Histochemical localization of GUS activity revealed that the cytosolic GS gene is expressed in anther theca and pollen at a late stage of flower development. Strong GUS staining was also visible in transgenic Lotus pulvini. Progressive 5′ deletion of GS15 promoter showed that regulatory elements necessary for ammonia stimulation both in roots and nodules are located between −3.5 and −1.3 kbp relative to the transcription start site. Elements involved in pulvinus‐specific expression are localized between −1.3 and −0.7 kbp, whereas 240 bp are sufficient to control the expression of the GS15 gene in the anthers. It therefore appears that multiple and separate promoter sequences control the organ‐specific and ammonia‐regulated expression of the cytosolic GS15 gene.
Purification, characterization, and cell wall localization of an α‐fucosidase that inactivates a xyloglucan oligosaccharin
Authors: Augur Christopher , Benhamou Nicole , Darvill Alan , Albersheim Peter
Published: March 2003

An α‐fucosidase that releases fucosyl residues from oligosaccharide fragments of xyloglucan, a plant cell wall hemicellulosic polysaccharide, was purified to homogeneity from pea (Pisum sativum) epicotyls using a combination of cation exchange chromatography and isoelectric focusing. The α‐fucosidase has a molecular mass of 20 kDa according to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The α‐fucosidase has an isoelectric point of 5.5. The substrate specificity of the α‐fucosidase was determined by high performance anion exchange chromatographic analysis of oligosaccharide substrates and products. The enzyme hydrolyzes the terminal α‐1,2‐fucosidic linkage of oligosaccharides and does not cleave p‐nitrophenyl‐α‐L‐fucoside. The enzyme does not release measurable amounts of fucosyl residues from large polysaccharides. The subcellular localization of α‐fucosidase in pea stems and leaves has been studied by immunogold cytochemistry. The α‐fucosidase accumulates in primary cell walls and is not detectable in the middle lamella or in the cytoplasm of 8‐day‐old stem tissue and 14‐day‐old leaf tissue. α‐Fucosidase activity was readily detected in extracts of 8‐day‐old stem tissue. No significant α‐fucosidase acitivity or immunogold labeling of the α‐fucosidase was detected in 2‐ and 4‐day‐old stem tissue indicating that production of α‐fucosidase is developmentally regulated.
A phloem‐specific sucrose‐H+ symporter from Plantago major L. supports the model of apoplastic phloem loading
Authors: Gahrtz Manfred , Stolz Jürgen , Sauer Norbert
Published: March 2003

In this paper the cloning of a full‐length cDNA clone encoding the PmSUC2 sucrose‐H+ symporter from Plantago major is described. This plant allows the simple preparation of vascular bundles from the basal regions of fully developed source leaves and thus a separation of vascular and non‐vascular tissue. A cDNA library was constructed from poly(A)+ RNA isolated from vascular bundles and used for the subsequent cloning of cDNAs. The respective mRNA is specifically expressed in the vascular bundles as shown on Northern blots of total RNA from vascular and non‐vascular tissues. The PmSUC2 protein has 12 putative transmembrane helices and is highly homologous to other plant sucrose transporters. Substrate specificity and energy dependence of the transporter encoded by this cDNA were determined by expression in baker's yeast Saccharomyces cerevisiae. The PmSUC2 protein catalyses the transport of sucrose into transgenic yeast cells. Invertase null mutants of yeast expressing PmSUC2 accumulate sucrose more than 200‐fold. This transport was sensitive to uncouplers or SH‐group inhibitors. Plasma membranes from yeast cells expressing the PmSUC2 protein were purified and fused to proteoliposomes containing cytochrome‐c‐oxidase. In this system sucrose is accumulated only when proton motive force is generated, indicating that PmSUC2 is a sucrose‐H+ symporter. The apparent molecular weight of the PmSUC2 protein is 35 kDa on 10% SDS‐polyacrylamide gels. The presented data strongly support the theory of phloem loading from the apoplastic space by a sucrose‐H+ symporter.
The promoter of the barley aleurone‐specific gene encoding a putative 7 kDa lipid transfer protein confers aleurone cell‐specific expression in transgenic rice
Authors: Kalla Roger , Shimamoto Ko , Potter Robert , Nielsen Peter Stein , Linnestad Casper , Olsen Odd‐Arne
Published: March 2003

This paper describes the aleurone‐specific gene Ltp2 from barley, which encodes a putative 7 kDa non‐specific lipid transfer protein. As shown by Northern and in situ hybridization analyses, the Ltp2 transcript is present in barley aleurone cells shortly after the initiation of aleurone cell differentiation. The expression of Ltp2 increases until grain mid‐maturity, but the mRNA is absent from mature grains. The Ltp2 transcript is undetectable in the embryo and vegetative tissues, confirming the aleurone specificity of the Ltp2 gene. The ability of the isolated 801 bp Ltp2 promoter to direct aleurone‐specific expression in immature barley grains is demonstrated by particle bombardment experiments. In these experiments, the activity of the Ltp2 promoter is 5% of the activity of the strong constitutive Actin1 promoter from rice, as quantified by GUS activity measurements. In stably transformed rice plants containing the Ltp2 promoter—Gus construct, the specificity of the Ltp2 promoter is confirmed in vivo by the presence of GUS activity exclusively in the aleurone layer. This study demonstrates the conserved nature of the regulatory signals involved in aleurone‐specific gene transcription in cereal grains.
Production of fertile transgenic maize plants by silicon carbide whisker‐mediated transformation
Authors: Frame Bronwyn R. , Drayton Paul R. , Bagnall Susan V. , Lewnau Carol J. , Bullock W. Paul , Wilson H. Martin , Dunwell James M. , Thompson John A. , Wang Kan
Published: March 2003

A simple and inexpensive system for the generation of fertile, transgenic maize plants has been developed. Cells from embryogenic maize suspension cultures were transformed using silicon carbide whiskers to deliver plasmid DNA carrying the bacterial bar and uidA (gus) genes. Transformed cells were selected on medium containing the herbicide bialaphos. Integration of the bar gene and activity of the enzyme phosphinothricin acetyl transferase (PAT) were confirmed in all bialaphos‐resistant callus lines analysed. Fertile transgenic maize plants were regenerated. Herbicide spraying of progeny plants revealed that the bar gene was transmitted in a Mendelian fashion.
Two classes of cis sequences contribute to tissue‐specific expression of a PAL2 promoter in transgenic tobacco
Authors: Hatton Diane , Sablowski Robert , Yung Mei‐Hing , Smith Caroline , Schuch Wolfgang , Bevan Michael
Published: March 2003

Genes encoding phenylalanine ammonia‐lyase (PAL) are expressed in a complex pattern during plant development and in response to light, pathogen ingress, mechanical damage and other stresses. Analysis of the promoter of the bean PAL2 gene in transgenic tobacco has shown that some regions responsible for developmental expression are functionally compensatory. The minimum sequence containing all cis sequences necessary for developmental patterns of expression is within −254 bp of the transcription start site. Footprinting and electrophoretic mobility shift assay studies of this region revealed potential cis sequences which coincided with the functional domains defined by small deletions and promoter fusions. Mutations in these potential cis sequences in the context of the minimal −254 bp promoter altered tissue‐specific expression patterns, confirming the importance of these sequences for expression in vivo. A functional model for the promoter is presented which predicts that three AC‐elements, which are possible Myb protein binding sites, together with a G‐box, interact to direct the complex patterns of tissue‐specific expression observed.
Twin autonomous bipartite nuclear localization signals direct nuclear import of GT‐2
Authors: Dehesh Katayoon , Smith Laurie G. , Tepperman James M. , Quail Peter H.
Published: March 2003

GT‐2 is a DNA‐binding protein with high target‐sequence specificity toward functionally defined, positively acting cis elements in the rice phytochrome A gene promoter. Using immunocytochemical procedures, it is shown here that GT‐2 is localized to the nucleus, consistent with a function in transcriptional regulation. Immunoblot and immunocytochemical analyses show that rice shoots contain higher levels of GT‐2 protein than roots, and that no photo‐induced changes in GT‐2 abundance or spatial distribution are detectable in these tissues, a result consistent with the proposed constitutive activity of GT‐2. In both shoots and roots, GT‐2 protein is undetectable in meristematic tissue but becomes expressed at later stages of cellular development, consistent with a role in contributing to the pattern of phytochrome A gene expression. By transfecting protoplasts with a series of constructs containing deletion derivatives of GT‐2 fused to β‐glucuronidase (GUS), followed by in situ localization of GUS activity, two independent, functionally active nuclear localization sequences (NLSs) have been identified in GT‐2. One NLS resides within each of a pair of previously identified, spatially separate, trihelix motifs in the protein. Sequence inversion and alanine‐scanning mutagenesis has identified residues within these NLSs necessary for nuclear localization. Each NLS contains two basic domains separated by 10 amino acids, conforming to the bipartite class of NLS involved in the targeting of numerous other nuclear localized proteins.
A mutant of Arabidopsis thaliana displaying altered patterns of cellulose deposition
Authors: Potikha Tamara , Delmer Deborah P.
Published: March 2003

A homozygous recessive mutant of Arabidopsis thaliana has been selected which displays altered patterns of cellulose deposition. The mutant was selected because leaf and stem trichomes lacked the strong birefringence under polarized light which is characteristic of plant cells which contain highly ordered cellulose in their secondary cell walls. Compared with wild‐type A. thaliana, this mutant (designated tbr for trichome birefringence) also displays reduced birefringence in the xylem of the leaf. Direct chemical analyses of root, stem, and leaf tissues, including isolated leaf trichomes, support the conclusion that tbr is impaired in its ability to deposit secondary wall cellulose in specific cell types, most notably in trichomes where the secondary wall appears to be totally absent. Altered patterns of wound‐induced callose deposition in trichomes and surrounding cells is another trait which also co‐segregates with the tbr mutation.
The amylose content in rice endosperm is related to the post‐transcriptional regulation of the waxy gene
Authors: Wang Zong‐Yang , Zheng Fei‐Qin , Shen Ge‐Zhi , Gao Ji‐Ping , Snustad D. Peter , Li Min‐Gang , Zhang Jing‐Liu , Hong Meng‐Min
Published: March 2003

The waxy (Wx) gene of rice encodes a granule‐bound starch synthase (GBSS = waxy protein) required for the synthesis of amylose in endosperm. An analysis of Wx transcripts, Wx protein, and amylose content of 31 rice cultivars revealed that endosperm amylose and Wx protein contents are correlated with the ability of the cultivar to excise intron I from the leader sequence of the Wx transcript. Cultivars with high endosperm amylose content (group I) contain high levels of amylose, Wx protein, and the mature 2.3 kb Wx mRNA. Cultivars with intermediate amylose content (group II) produce substantial amounts of a large 3.3 kb Wx pre‐mRNA, with intron I still present, in addition to the mature Wx mRNA, and intermediate levels of Wx protein. Glutinous rice (group III cultivars) contains no amylose, no Wx protein, and no mature Wx mRNA; only the incompletely spliced 3.3 kb Wx pre‐mRNA is present in group III cultivars. Based on these results, it is hypothesized that the amylose content of rice endosperm is regulated at the level of Wx transcript processing, and, more specifically, at the stage of intron I excision from the Wx pre‐mRNA.
Further progress towards a catalogue of all Arabidopsis genes: analysis of a set of 5000 non‐redundant ESTs
Authors: Cooke Richard , Raynal Monique , Laudié Michele , Grellet Françoise , Delseny Michel , Morris Peter‐Christian , Guerrier Danièle , Giraudat Jérôme , Quigley Françoise , Clabault Gérard , Li You‐Fang , Mache Régis , Krivitzky Micheline , Gy Isabelle Jean‐Jacques , Kreis Martin , Lecharny Alain , Parmentier Yves , Marbach Jacqueline , Fleck Jacqueline , Clément Bernadette , Philipps Gabriel , Hervé Christine , Bardet Claude , Tremousaygue Dominique , Lescure Bernard , Lacomme Christophe , Roby Dominique , Jourjon Marie‐Françoise , Chabrier Patrick , Charpenteau Jean‐Louis , Desprez Thierry , Amselem Joelle , Chiapello Helen , Höfte Herman
Published: March 2003

Nearly 7000 Arabidopsis thaliana‐expressed sequence tags (ESTs) from 10 cDNA libraries have been sequenced, of which almost 5000 non‐redundant tags have been submitted to the EMBL data bank. The quality of the cDNA libraries used is analysed. Similarity searches in international protein data banks have allowed the detection of significant similarities to a wide range of proteins from many organisms. Alignment with ESTs from the rice systematic sequencing project has allowed the detection of amino acid motifs which are conserved between the two organisms, thus identifying tags to genes encoding highly conserved proteins. These genes are candidates for a common framework in genome mapping projects in different plants.
Molecular cloning and characterization of the CER2 gene of Arabidopsis thaliana
Authors: Negruk Valentin , Yang Ping , Subramanian Mohan , McNevin John P. , Lemieux Bertrand
Published: March 2003

Plants with defects in the synthesis of their epicuticular wax layer, eceriferum mutants (cer), are readily detected by the naked eye as bright green glossy plants when compared with the more glaucous normal plants. In a previous report, evidence was presented for the isolation of three lines from the Arabidopsis thaliana transformant collection (BRL5, BRL7 and BRL9) which failed to complement the cer2 mutant isolated previously. The analysis of the chemical composition of the epicuticular wax of these mutants suggests that the cer2 mutant of Arabidopsis is defective in very long chain fatty acid elongation. This paper reports the molecular cloning of the CER2 gene of Arabidopsis through the isolation of plant DNA flanking the site of T‐DNA insertion as well as the characterization of the two independent T‐DNA insertion mutant alleles, BRL5 and BRL9, of this gene. In the mutant line BRL5, T‐DNA was found to be inserted in the second exon of the CER2 gene whereas in BRL9, T‐DNA is inserted in the only intron of this gene. Nucleotide sequence analysis suggests that the ORF encodes a 47.3 kDa polypeptide. High levels of CER2 transcripts were detected in stems and flowers. The predicted amino acid sequence of the CER2 gene product reveals little homology with known protein sequences. In accordance with structural characterization of the T‐DNA insertion mutants, no evidence of transcripts derived from the CER2 gene was found in either BRL5 or BRL9.
New insights into the composition, molecular mass and stoichiometry of the protein complexes of plant mitochondria
Authors: Jänsch Lothar , Kruft Volker , Schmitz Udo K. , Braun Hans‐Peter
Published: March 2003

Recently a powerful electrophoresis method for the native preparation and characterization of the respiratory protein complexes of mitochondria from fungi and mammals has been developed, which employs Coomassie dyes to introduce charge shifts on proteins (Schägger and von Jagow (1991) Anal. Biochem. 199, 223–231). The procedure, which is called ‘blue native‐polyacrylamide gel electrophoresis’ (BN‐PAGE), was modified and introduced for the analysis of mitochondria from higher plants. BN‐PAGE of mitochondrial protein from potato allows the separation of nine distinct protein complexes between 100 and 1000 kDa and reveals novel results for their composition, molecular mass and stoichiometry. For the first time soluble mitochondrial protein complexes, like the HSP60 complex (750 kDa) and a complex of 200 kDa, which includes a formate dehydrogenase, are analysed by BN‐PAGE. Complex I from potato (1000 kDa) is about 100 kDa larger than the corresponding enzyme from beef and can be resolved into more than 30 different subunits on a second gel dimension. The F1F0 ATP synthase (580 kDa) and the cytochrome c oxidase (160 kDa) from potato seem to contain more subunits than hitherto reported. Direct sequencing of subunits revealed that the F1 part of the F1F0 ATP synthase lacks the oligomycin sensitivity conferring protein (OSCP), which was reported to be present in F1 parts of dicotyledonous plants, but contains the ATPase inhibitory protein. N‐terminal sequences of 16 mitochondrial proteins were obtained, several of which are presented for the first time from a plant source. BN‐PAGE allows the preparation of mitochondrial protein complexes from gram amounts of plant tissue, as the procedure only requires milligram amounts of organelles. This potential of BN‐PAGE is demonstrated by the separation and characterization of the mitochondrial enzyme complexes from Arabidopsis thaliana. Further analysis of organellar protein complexes by BN‐PAGE will allow the generation of ‘protein maps’ from different tissues and developmental stages or from mutant plants.
Salicylic acid potentiates defence gene expression in tissue exhibiting acquired resistance to pathogen attack
Authors: Mur Luis A.J. , Naylor Grant , Warner Simon A.J. , Sugars Jane M. , White Ray F. , Draper John
Published: March 2003

Salicylic acid (SA) is absolutely required for establishment of acquired resistance in non‐infected tissues following localized challenge of other leaves with a necrotizing pathogen. Although not directly responsive to SA, or induced systemically following pathogen challenge, the expression of defence gene promoter fusions AoPR1—GUS and PAL‐3—GUS after wounding or pathogen challenge could be enhanced by pre‐treating tobacco plants hydroponically with SA, a phenomenon designated ‘potentiation’. Potentiation of AoPR1—GUS wound‐responsiveness was also demonstrated locally, but not systemically, in tobacco tissue exhibiting acquired resistance following infection with either viral or bacterial pathogens. Potentiation of wound‐responsive expression by prior wounding could not be demonstrated. In contrast, potentiation of pathogen‐responsive AoPR1—GUS expression was exhibited both locally and systemically in non‐infected tissue. The spatial and temporal exhibition of defence gene potentiation correlated directly with the acquisition of resistance in non‐infected tissue. Pathogen‐responsive potentiation was obtained at about 10‐fold lower levels of salicylic acid than wounding‐responsive potentiation in AoPR1—GUS tobacco plants prefed with salicylate. These results may explain the failure to observe systemic potentiation of the wound‐responsive defence gene expression. The data suggest a dual role for SA in terms of gene induction in acquired immunity: a direct one by induction of genes such as pathogenesis‐related proteins, and an indirect one by potentiation of expression of other local defence genes (such as PAL and AoPR1) which do not respond directly to SA but become induced on pathogen attack or wounding.
Identification of a peptide methionine sulphoxide reductase gene in an oleosin promoter from Brassica napus‡
Authors: Sadanandom Arivananthan , Piffanelli Pietro , Knott Tracy , Robinson Colin , Sharpe Andrew , Lydiate Derek , Murphy Denis , Fairbairn David J.
Published: March 2003

A bidirectional promoter can be defined operationally as a short segment of DNA that regulates divergent transcription. In an attempt to investigate whether the intergenic region between the oleosin and a second open reading frame (ORFII) in Brassica napus (L.) is a divergent promoter, and also to characterize the ORFII, cDNA clones homologous to ORFII were isolated from a leaf cDNA library. A representative cDNA (clone D) of one of the two classes identified was identical, in DNA sequence, to the genomic ORFII. The second representative cDNA (clone O) was 97% identical at the nucleotide level to the genomic ORFII. The predicted amino acid sequence of the cDNA clones each exhibit homology with the peptide methionine sulphoxide reductase (PMSR) of Escherichia coli. The gene structure of ORFII was elucidated and the relative positions of the oleosin, ORFII, and the intergenic promoter region were determined. This confirms that the B. napus oleosin‐ORFII intergenic region has divergent promoter activity. Consequently this is the first such plant nuclear divergent promoter identified. RFLP‐mapping results showed that all four ORFII genes are linked to four of the six copies of the oleosin genes. This suggested that the bidirectional promoter locus is conserved within the B. napus genome. The ORFII gene product is targeted to the chloroplast, which is consistent with previous data indicating the presence of PMSR activity in the chloroplast. The over‐expressed recombinant fusion protein (minus the transit‐peptide) showed the capability to reduce peptide methionine sulphoxide residues in vitro, indicating PMSR activity. This study demonstrates that ORFII is transcribed and encodes a plant PMSR, and is the first example of the isolation of a eukaryotic PMSR gene.
Developmentally regulated silencing and reactivation of tobacco chitinase transgene expression
Authors: Kunz Christian , Schöb Hanspeter , Stam Maike , Kooter Jan M. , Meins Frederick
Published: March 2003

The tobacco class I chitinase gene CHN48 driven by CaMV 35S RNA expression signals introduced into Nicotiana sylvestris can inactivate its own expression as well as expression of homologous host genes at the mRNA level. Comparison of the silencing of chitinase and β‐1,3‐glucanase transgenes with the same expression signals and breeding experiments with transgenes inserted in different chromosomes showed that chitinase‐gene silencing depends on transcribed sequence hornology and trans‐gene dose. The CHN48 transgene silenced host class I but not class II chitinases suggesting that greater than about 60% identity of sequences encoding the catalytic domain is required for silencing. Although steady‐state mRNA contents were greatly reduced in silent plants, the levels of nascent chitinase transcripts detected in nuclear run‐on experiments were the same in nuclei from high‐expressing and silent plants. This strongly suggests that chitinase‐gene silencing is a post‐transcriptional phenomenon. Silencing and resetting to high‐level expression of chitinase genes is developmentally regulated. Silencing occurs stochastically in 25–100% of seedlings at the 6–10 leaf stage of development and is preceded by a marked, transient increase in transgene‐encoded chitinase. Resetting of silent genes to the high‐expressing state is a non‐stochastic event that occurs in developing seeds 8–11 days after pollination. Experiments with lateral buds and plants regenerated from cultured leaf disks show that once established, competence for silencing can persist in dormant, actively growing, and de novo established shoot meristems. Some plants show a variegated pattern of silencing. Analyses of these patterns indicate that chitinase‐gene silencing is not a clonal event. Thus, it is likely that communication between cells rather than the lineage of the cells is important in stabilizing the silent state. Possible mechanisms for developmentally regulated post‐transcriptional silencing are discussed.
Introduction and expression of foreign DNA in isolated spinach chloroplasts by electroporation
Authors: To Kin‐Ying , Cheng Ming‐Chih , Chen Long‐Fang Oliver , Chen Shu‐Chen Grace
Published: March 2003

An electroporation‐mediated method for the study of foreign gene expression within chloroplasts has been developed. The chloroplast expression vector pHD203‐GUS, which consists of coding regions for β‐glucuronidase (GUS) and chloramphenicol acetyltransferase (CAT) separated by a double psbA promoter fragment from pea (in opposite orientation) was electroporated into spinach chloroplasts and the transient gene expression was examined. Conditions for the expression of the reporter genes have been optimized. Both CAT and GUS activities were detected in chloroplasts electroporated with pHD203‐GUS, but not with nuclear expression vector pBI221 or negative control pUC18. No GUS activity was detected when pHD203‐GUS was electroporated into spinach protoplasts. Dot immunoblot analysis using anti‐GUS antibody confirmed the existence of GUS protein in chloroplasts electroporated with chloroplast‐specific vector but not the negative controls, excluding the possibilities of endogenous GUS or bacterial contamination. The expression of GUS protein in treated chloroplasts was further confirmed by Western blot analysis.
The shoot controls zeatin riboside export from pea roots. Evidence from the branching mutant rms4
Authors: Beveridge Christine A. , Murfet Ian C. , Kerhoas Lucien , Sotta Bruno , Miginiac Emile , Rameau Catherine
Published: March 2003

The rms4 mutant of pea (Pisum sativum L.) was used in grafting studies and cytokinin analyses of the root xylem sap to provide evidence that, at least for pea, the shoot can modify the import of cytokinins from the root. The rms4 mutation, which confers a phenotype with increased branching in the shoot, causes a very substantial decrease (down to 40‐fold less) in the concentration of zeatin riboside (ZR) in the xylem sap of the roots. Results from grafts between wild‐type (WT) and rms4 plants indicate that the concentration of cytokinins in the xylem sap of the roots is determined almost entirely by the genotype of the shoot. WT scions normalize the cytokinin concentration in the sap of rms4 mutant roots, whereas mutant scions cause WT roots to behave like those of self‐grafted mutant plants. The mechanism whereby rms4 shoots of pea cause a down‐regulation in the export of cytokinins from the roots is unknown at this time. However, our data provide evidence that the shoot transmits a signal to the roots and thereby controls processes involved in the regulation of cytokinin biosynthesis in the root.
Identification of a methyl jasmonate‐responsive region in the promoter of a lipoxygenase 1 gene expressed in barley grain
Authors: Rouster Jacques , Leah Robert , Mundy John , Cameron‐Mills Verena
Published: March 2003

A genomic DNA fragment was isolated containing 5′ upstream sequences and part of the open reading frame corresponding to the lipoxygenase 1 cDNA (LoxA) expressed in barley grains during development and germination. Lox1 transcription was shown to be methyl jasmonate (MeJA)‐ and wound‐inducible in leaves, but Lox1 transcripts were not detected in mildew‐infected leaves, although this is a commonly observed response to pathogenic attack in various plants. Transient gene expression assays were used to identify a promoter region involved in MeJA‐responsive expression. Analysis of 5′ and 3′ promoter deletions indicated that sequences between −363 and −294 conferred MeJA‐responsive expression. Deletions/replacements covering this part of the promoter further defined a MeJA‐responsive region between −331 and −291. Insertion of the region −328 to −293 into the constitutive CaMV 35S promoter conferred MeJA‐responsive expression. The 36 bp fragment contains the motif TGACG as inverted repeats, which has been previously identified as a binding site for bZIP transactivating factors. Site‐directed mutagenesis on these TGACG motifs abolished MeJA‐responsive expression, clearly identifying them as MeJA‐responsive elements. Sequence comparisons found no similar motif in other characterized promoters of MeJA‐inducible genes, but suggested a common spatial structure which may serve as a binding site for transacting factors involved in the MeJA signal transduction pathway.
Intra‐ and extracellular lipid composition and associated gene expression patterns during pollen development in Brassica napus
Authors: Piffanelli Pietro , Ross Joanne H.E. , Murphy Denis J.
Published: March 2003

Pollen development in angiosperms is regulated by the interaction of products contributed by both the gametophytic (haploid) and sporophytic (diploid) genomes. In entomophilous species, lipids are major products of both sporophytic and gametophytic metabolism during pollen development. Mature pollen grains of Brassica napus are shown to contain three major acyl lipid pools as follows: (i) the extracellular tryphine mainly consisting of medium‐chain neutral esters; (ii) the intracellular membranes, particularly endoplasmic reticulum, mainly containing phospholipids; and (iii) the intracellular storage lipids, which are mostly triacylglycerols. This paper reports on the kinetics of accumulation of these lipid classes during pollen maturation and the expression patterns of several lipid biosynthetic genes and their protein products that are differentially regulated in developing microspores/ pollen grains (gametophyte) and tapetal cells (sporophyte) of B. napus. Detailed analysis of three members of the stearoyl‐ACP desaturase (sad) gene family by Northern blotting, in situ hybridization and RT‐PCR showed that the same individual genes were expressed both in gametophytic and sporophytic tissues, although under different temporal regulation. In the tapetum, maximal expression of two marker genes for lipid biosynthesis (sad and ear) occurred at a bud length of 2–3 mm, and the corresponding gene products SAD and EAR were detected by Western blotting in 3–4 mm buds, coinciding with the maximal rates of tapetal lipid accumulation. These lipids are released following tapetal cell disintegration and are relocated to form the major structural component of the extracellular tryphine layer that coats the mature pollen grain. In contrast, in developing microspores/pollen grains, maximal expression of the lipid marker genes sad, ear, acp and cyb5 was at the 3–5 mm bud stages, with the SAD and EAR gene products detected in 4–7 mm buds. This pattern of expression coincided with accumulation of the intracellular storage and membrane lipid components of pollen. These results suggest that, although the same genes may be expressed in the sporophytic tapetal cells and in gametophytic tissues, they are regulated differentially leading to the production of the various contrasting lipidic structures that are assembled together to give rise to a viable, fertile pollen grain.