bluuebunny/crossref_metadata_2025 huggingface dataset:
Cyclitol production in transgenic tobacco
Authors: Vernon Daniel M. , Tarczynski Mitchell C. , Jensen Richard G. , Bohnert Hans J.
Published: March 2003

High levels of cyclic sugar alcohols (cyclitols) correlate with tolerance to osmotic stress in a number of plant species. A gene encoding a cyclitol biosynthesis enzyme from a halophyte, Mesembryanthemum crystallinum has been introduced into tobacco. The gene, lmt1, encodes a myo‐inositol O‐methyl transferase that, in M. crystallinum, catalyzes the first step in the stress‐induced accumulation of the cyclitol pinitol. Tobacco transformed with the lmt1 cDNA under the control of the CaMV 35S promoter appeared phenotypically normal and exhibited IMT1 enzyme activity. Transformants accumulated a carbohydrate product not detectable in non‐transformed control plants. This product was identified by HPLC and NMR as ononitol (1‐d‐4‐O‐methyl myo‐inositol). Ononitol was a major carbohydrate constituent in leaf tissue of plants expressing the lmt1 gene, accumulating to up to 25% the level of sucrose in transformant seedlings. The identification of ononitol as the IMT1 product and the specific accumulation of this compound in transformed tobacco support a role for ononitol as a stable intermediate in pinitol biosynthesis and indicate that an epimerization activity lacking in tobacco is responsible for the conversion of ononitol to pinitol in M. crystallinum. The production of ononitol in tobacco indicates that plant carbohydrate metabolism is flexible and can accommodate the synthesis and accumulation of non‐endogenous metabolites. The transgenic system described here will serve as a useful model to test the ability of cyclitols such as ononitol to confer tolerance to environmental stress in a normally glycophytic plant.
A basic‐type PR‐1 promoter directs ethylene responsiveness, vascular and abscission zone‐specific expression
Authors: Eyal Yoram , Meller Yael , Lev‐Yadun Simcha , Fluhr Robert
Published: March 2003

Pathogenesis‐related (PR) proteins form a heterogeneous group of host‐encoded, low‐molecular‐mass proteins that are secreted through the exocytic pathway. They are synthesized by the plant in response to various stimuli, including pathogen attack or exposure to certain chemicals. The PRB‐1b gene of Nicotiana tabacum codes for a basic‐type PR‐1 protein whose transcription is regulated by ethylene. A minimal ethylene‐responsive promoter element was defined by deletion analysis in transgenic tobacco plants. Promoter sequences containing 213 bp or more were sufficient to enhance a 20‐fold increase of β‐glucuronidase reporter gene expression in transgenic tobacco leaves exposed to 20 µl l−1 of ethylene, while 67 bp were not sufficient to trigger ethylene responsiveness. All the constructs that retained ethylene inducibility exhibited phloem‐specific activity, which was constitutive in petiole and pedicel abscission zones. This functional study was correlated to an in vitro screening of the major nuclear proteins’ binding sites present on the promoter. Gel‐shift analysis using nuclear extracts from ethylene‐treated and non‐treated plants revealed five sequence‐specific protein‐DNA complexes on promoter sequences spanning −863 to −142 bp. Constitutive expression of the basic‐type PR‐1 genes at the leaf and petiole or flower and pedicel interfaces may represent pre‐emption of plant defenses against potential pathogens, suggesting a functional similarity to pathogen‐induced expression in the leaf.
RNA editing in maize chloroplasts is a processing step independent of splicing and cleavage to monocistronic mRNAs
Authors: Freyer Regina , Hoch Brigitte , Neckermann Kai , Maier Rainer M. , Kössel Hans
Published: March 2003

The psbB operon contained in the plastomes of higher plants consists of the genes psbB, psbH, petB and petD. The primary transcript of this operon is subject to a series of processing steps which include cleavages resulting in four monocistronic mRNAs and splicing of the petB and petD transcripts. A search for editing sites within the two latter transcripts from maize led us to the detection of one editing site within the petB coding region which is conserved at the DNA level in other graminean species and in tobacco. This shows that editing must be considered as an additional processing step of the psbB operon encoded primary transcript. As is evident from cDNA sequences derived from the dicistronic and/or unspliced petB/D transcripts which are completely edited, editing is an early step of mRNA processing which precedes both splicing and cleavage to the monocistronic mRNAs and which must, therefore, be independent of the latter two steps. This conclusion is confirmed by a similar observation with the editing site of the rpl2 transcript which is contained in the polycistronic transcript of the rpoA operon, although here only partial editing is observed for the unspliced dicistronic rpl23/rpl2 transcript.
DIP: a member of the MIP family of membrane proteins that is expressed in mature seeds and dark‐grown seedlings of Antirrhinum majus
Authors: Culianez‐Macia Francisco A. , Martin Cathie
Published: March 2003

DiP, a gene from Antirrhinum majus, which encodes a protein with striking homology to other integral membrane proteins, was cloned. The gene was specifically expressed in mature seeds and during seedling germination, particularly in cotyledons of seedlings grown in the dark. The deduced product, called DiP, for dark intrinsic protein, shows strong homology with the MIP family of channel transporters which include; the bovine major intrinsic protein (MIP), the Escherichia coli glycerol facilitator (GlpF), the peribacteroid nodulin‐26 (Nod26), and the tonoplast protein from kidney bean (TIP). DiP is most similar to other plant members of this family, and in particular to the tobacco protein TobRB7 which is expressed specifically in roots. However, the expression pattern of diP suggests that its product is functionally more similar to the tonoplast intrinsic protein from kidney bean since it is most highly expressed in the cotyledons of germinating seedlings, before the cells undergo expansion growth and become photosynthetic.
Lipo‐oligosaccharides of Rhizobium induce infection‐related early nodulin gene expression in pea root hairs
Authors: Horvath Beatrix , Heidstra Renze , Lados Miklos , Moerman Marja , Spaink Herman P. , Promé Jean‐Claude , van Kammen Albert , Bisseling Ton
Published: March 2003

This paper shows that lipo‐oligosaccharides (Nod factors) synthesized by Rhizobium bacteria elicit the induction of infection‐related early nodulin genes (PsENOD5 and PsENOD12) in pea root hairs. R. leguminosarum bv. viciae secretes a mixture of Nod factors containing a C18 fatty acid chain with 4 (C18:4) or 1 double bond (C18:1). Purified Nod factors harbouring either a C18:4 or a C18:1 acyl moiety induce the expression of the pea early nodulin genes, PsENOD5 and PsENOD12, but the kinetics of induction are different. The expression of both early nodulin genes is induced in a transient manner by the purified Nod factors while a mixture of the Nod factors extends the period during which these genes are expressed. In spite of the host‐specific nature of the infection process, heterologous Nod factors of R. meliloti also induce the expression of PsENOD5 and PsENOD12 genes, though with a marked delay compared with the homologous compounds.
The anther‐specific protein encoded by the Brassica napus and Arabidopsis thaliana A6 gene displays similarity to β‐1,3‐glucanases
Authors: Hird Diane L. , Worrall Dawn , Hodge Rachel , Smartt Sarah , Paul Wyatt , Scott Rod
Published: March 2003

An anther‐specific Brassica napus cDNA, A6, and two corresponding Arabidopsis thaliana genes have been isolated. Sequence analyses of A6 revealed similarity to β‐1,3‐glucanases. The deduced A6 protein differs from other β‐1,3‐glucanases in the possession of a long C‐terminus. Immunoblotting using an antibody raised to the A6 protein detects a temporal 60 kDa protein in B. napus buds, suggesting that the long C‐terminal region is present in the mature protein. A6 promoter—GUS and RNase fusions demonstrate that the A6 gene is tapetum‐specific and temporally expressed with a peak in activity when the plant normally expresses callase (a complex of endo‐ and exo‐β‐1,3‐glucanase activities). The sequence similarity of A6 to other β‐1,3‐glucanases, coupled with the temporal and spatial expression data, suggests that A6 may be part of the callase enzyme complex.
An inventory of 1152 expressed sequence tags obtained by partial sequencing of cDNAs from Arabidopsis thaliana†
Authors: Höfte Herman , Desprez Thierry , Amselem Joëlle , Chiapello Hélène , Caboche Michel , Moisan Annick , Jourjon Marie‐Françoise , Charpenteau Jean‐Louis , Berthomieu Pierre , Guerrier Danièle , Giraudat Jérome , Quigley Françoise , Thomas Frank , Yu De‐Yao , Mache Régis , Raynal Monique , Cooke Richard , Grellet Françoise , Delseny Michel , Parmentier Yves , de Marcillac Guy , Gigot Claude , Fleck Jacqueline , Philipps Gabriel , Axelos Michèle , Bardet Claude , Tremousaygue Dominique , Lescure Bernard
Published: March 2003

As part of the goal to generate a detailed transcript map for Arabidopsis thaliana, 1152 single run sequences (expressed sequence tags or ESTs) have been determined from cDNA clones taken at random in libraries prepared from different sources of plant material: developing siliques, etiolated seedlings, flower buds, and cultured cells. Eight hundred and ninety‐five different genes could be identified, 32% of which showed significant similarity to existing sequences in Arabidopsis and an array of other organisms. These sequences in combination with their positioning on the Arabidopsis genetic map will not only constitute a new set of molecular markers for genome analysis in Arabidopsis but also provide a direct route for the in vivo analysis of their gene products. The sequences have been made available to the public databases.
Repression of the high‐methionine zein gene in the maize inbred line Mo17
Authors: Schickler Hedva , Benner Michael S. , Messing Joachim
Published: March 2003

The methionine content in maize kernels is largely determined by the level of a methionine‐rich 10 kDa protein, encoded by the gene Zps10/(22). In the maize inbred BSSS53 this gene is over‐expressed compared with its normal expression in inbred lines like W64A and W22. This is in part due to the trans‐acting factor Zpr10/(22). However, compared with the normal level of expression, this gene is under‐expressed in the inbred Mo17 and the high‐methionine zein accumulates to very low levels. Therefore, differential expression of this gene between BSSS53 and Mo17 is even larger than in other inbred lines of maize. Repression of 10 kDa zein expression in Mo17 is not due to the translatability of 10 kDa zein mRNA or a cis‐acting mutation in the mRNA and neither is it due to a change in the transcription rate per se. However, steady state levels of mRNA appear to be controlled in trans by narrowing the developmental time frame of mRNA accumulation.
Cloning and in vivo expression of functional triose phosphate/phosphate translocators from C3‐ and C4‐plants: evidence for the putative participation of specific amino acid residues in the recognition of phosphoenolpyruvate
Authors: Fischer Karsten , Arbinger Bettina , Kammerer Birgit , Busch Christine , Brink Susanne , Wallmeier Holger , Sauer Norbert , Eckerskorn Christoph , Flügge Ulf‐Ingo
Published: March 2003

The primary sequences of the chloroplast triose phosphate/phosphate translocator precursor proteins from C4‐plants (maize mesophyll cells and Flaveria trinervia) and from the C3‐type Flaveria pringlei were determined. The mature parts of these translocators possess 83–94% identical amino acid residues. The C4‐translocator protein can be correctly targeted to C3‐type chloroplasts and inserted into the envelope membrane. Expression of the mature parts of these chloroplast translocators (cTPT) in transformed yeast cells and subsequent reconstitution of the functional proteins reveals the difference between the recombinant translocator proteins from the two cell types with respect to the transport of phosphoenolpyruvate. Comparison of the cTPT sequences from F. pringlei and F. trinervia in combination with computer‐aided molecular modelling of the substrate translocation pore leads to the suggestion, that only minor exchanges of amino acid residues between the C3‐ and C4‐translocator proteins are sufficient to extend their substrate specificities to recognize also phosphoenolpyruvate.
Evidence for a role of β‐1,3‐glucanase in dicot seed germination
Authors: Vögeli‐Lange Regina , Fründt Corinne , Hart Craig M. , Beffa Roland , Nagy Ferenc , Meins Frederick
Published: March 2003

Class I β‐1,3‐glucanases are antifungal vacuolar proteins implicated in plant defense that show developmental, hormonal, and pathogenesis‐related regulation. The expression was studied in germinating tobacco seeds of a chimeric β‐glucuronidase (GUS) reporter gene fused to 1.6 kb of the 5′ flanking sequence of the tobacco class I β‐1,3‐glucanase B (GLB) promoter. Histological staining for GUS activity showed that expression of the GLB promoter is highly localized in a specific zone of the endosperm in germinating seeds. The temporal and spatial patterns of GUS and β‐1,3‐glucanase activity found, suggest a novel function for class I β‐1,3‐glucanases during seed germination in a dicotyledonous plant.
A cotyledon regulatory region is responsible for the different spatial expression patterns of Arabidopsis 2S albumin genes
Authors: Da Silva Conceição Alexandre , Krebbers Enno
Published: March 2003

The 2S albumin genes of Arabidopsis thaliana are a model system to study gene expression during late embryogenesis. The at2S1 gene has previously been shown to be expressed essentially in the embryo axis, unlike at2S2, which is expressed throughout the embryo. Hybrid promoter constructs between at2S1 and at2S2 were introduced into Arabidopsis and used to identify a cotyledon regulatory region necessary for 2S albumin expression in palisade parenchyma and specific epidermal cells. Other promoter sequences flanking this tissue‐specific promoter element were shown to control mRNA expression levels independently of the mRNA distribution throughout the embryos. Certain hybrid promoters resulted in the alteration of the time course of expression in cotyledons. Differential expression of 2S albumin genes is discussed in terms of layered cellular organization and mitotic activity throughout the embryo.
The maize chromosomal HMGa protein recognizes structural features of DNA and increases DNA flexibility
Authors: Grasser Klaus D. , Krech Anja B. , Feix Günter
Published: March 2003

The abundant maize high‐mobility group protein HMGa belongs to the chromosomal, non‐histone proteins and consists of a basic region containing the HMG‐box DNA‐binding domain and a highly acidic carboxy‐terminal tail. The full‐length HMGa protein and a truncated version lacking the acidic tail were synthesized in Escherichia coli and tested for their ability to induce DNA‐bending in a ligase mediated circularization assay with short DNA fragments. It is shown that the recombinant HMGa protein as well as its truncated form efficiently cause circularization of the tested DNA fragments without an obvious requirement for stable DNA‐binding. They bind furthermore preferentially to A/T‐rich linear DNA or bent DNA structures such as four‐way junctions and DNA minicircles. The DNA‐binding properties and the ability to increase DNA flexibility suggest a general role of the HMGa protein in assisting the formation of nucleoprotein complexes, possibly by facilitating interactions of proteins bound to adjacent DNA sites.
Promoter and leader sequences of the spinach PsaD and PsaF genes direct an opposite light response in tobacco cotyledons: PsaD sequences downstream of the ATG codon are required for a positive light response
Authors: Flieger K. , Wicke A. , Herrmann R.G. , Oelmüller R.
Published: March 2003

Subunits II and III of the photosystem I reaction centre are encoded by the nuclear genes PsaD and PsaF, respectively. In spinach, the expression of both genes is highly synchronized with regard to time, space and in response to stimulators such as light. Nevertheless, promoter sequences as well as the design and location of regulatory elements are strikingly different. Promoter and leader of PsaF, when fused to the GUS reporter gene, direct a positive light response in the cotyledons of transgenic tobacco seedlings. In contrast, the equivalent PsaD regions confer a negative‐light regulation to the GUS gene. If a 6‐kb fragment that contains 1802 bp of the promoter, the transcription unit as well as additional 2.5 kb downstream of the PsaD gene is introduced into tobacco, the transcript level from the PsaD transgene is positively light‐regulated in tobacco cotyledons. Thus, regulatory elements of the spinach PsaD and PsaF promoters are arranged in a very different way and essential cis‐determinants for the positive light response of the PsaD gene can be located within the coding region and/or even further downstream.
Altered body morphology is caused by increased stearate levels in a mutant of Arabidopsis
Authors: Lightner Jonathan , James Douglas W. , Dooner Hugo K. , Browse John
Published: March 2003

A mutant of Arabidopsis thaliana, fab2, in which a profound developmental phenotype, miniature growth, is caused by an increase in the level of the membrane fatty acid, stearate has been characterized. Miniature growth in fab2 results from changes in cell expansion and maturation processes. Leaf anatomy of fab2 is characterized by cell‐specific changes in expansion growth, by a lack of differentiation in the cells of the leaf blade, and by the absence of air spaces. Leaves of fab2 lack the basipetal gradient in cellular development which is ubiquitous in higher plants. These cellular changes occur without distorting the chronology of development, or destroying the biological integrity of the organism. A second site suppressor mutation, shs, substantially restores both normal fatty acid composition and normal body size, causally linking the two phenotypes. Growth of the fab2 mutant at high temperature substantially corrects the miniature phenotype without altering the fatty acid composition, suggesting a role for membrane structure in the production of the aberrant morphology.
Physical mapping of the mitochondrial genome of Arabidopsis thaliana by cosmid and YAC clones
Authors: Klein Mathieu , Eckert‐Ossenkopp Ute , Schmiedeberg Ina , Brandt Petra , Unseld Michael , Brennicke Axel , Schuster Wolfgang
Published: March 2003

As part of the worldwide efforts at molecular analysis of Arabidopsis thaliana as a model plant the complete structure of the mitochondrial genome has been determined. The mitochondrial DNA molecules were mapped by restriction fragment analysis of more than 300 cosmid clones and purified mitochondrial DNA. The entire genome of 372 kb is contained in three different configurations of circular molecules and is split into two additional subgenomic molecules of 234 kb and 138 kb, respectively. These arrangements result from recombinations of the two sets of repeats present in combinations of inverted and/or direct orientation. Alignment of YAC clones confirms the in vivo presence of continuous DNA molecules of more than 300 kb in A. thaliana mitochondria. The presence of this comparatively large mitochondrial genome in a plant with one of the smallest nuclear genomes shows that different size constraints act upon the different genomes in plant cells.
Mutation analysis of the C‐terminal vacuolar targeting peptide of tobacco chitinase: low specificity of the sorting system, and gradual transition between intracellular retention and secretion into the extracellular space
Authors: Neuhaus Jean‐Marc , Pietrzak Maciej , Boller Thomas
Published: March 2003

The C‐terminal propeptide of tobacco (Nicotiana tabacum) chitinase A has been shown to be necessary and sufficient for targeting of chitinases to the plant vacuole. The sequence specificity of this vacuolar targeting peptide (VTP) has now been analysed using transient expression of chitinases in Nicotiana plumbaginifolia protoplasts. An extracellular cucumber chitinase, previously used as a secreted reporter protein in transgenic tobacco, was also secreted into the incubation medium by the transiently transformed protoplasts. Addition of six to seven amino acids at the C‐terminus to generate the VTP of tobacco chitinase A were sufficient to cause retention of most of the cucumber chitinase within the protoplasts. The chitinase A itself, as well as a mutant lacking the N‐terminal chitin‐binding domain, were retained to 80% in the protoplasts when low concentrations of the plasmid were used in the transient expression system. At high concentrations of plasmid, causing high levels of transiently expressed chitinase, retention was reduced, indicating saturation of the sorting system. Deletion of the C‐terminal methionine did not affect the intracellular location, but deletion of even a single internal amino acid of the VTP caused predominantly secretion of tobacco chitinase A. In contrast, exchanges of amino acids in the VTP as well as substitution of the VTP with random sequences had intermediary effects that covered the whole range from retention to secretion. The results suggest that the sorting system responsible for the diversion of secretory proteins to the vacuole has a low specificity for the sequence of C‐terminal targeting peptides, and that sequence changes in the VTP allow a gradual transition from vacuolar retention to secretion.
Binding of BiP to an assembly‐defective protein in plant cells
Authors: Pedrazzini Emanuela , Giovinazzo Giovanna , Bollini Roberto , Ceriotti Aldo , Vitale Alessandro
Published: March 2003

The binding protein (BiP) has been implicated as a mediator of protein folding and assembly in the endoplasmic reticulum of mammalian cells and has often been found in stable association with structurally defective proteins. To acquire information on the activity of BiP in plant cells, we have expressed in tobacco protoplasts the wild type form and an assembly‐defective form of bean phaseolin. Phaseolin (PHSL) is a soluble, trimeric, storage glycoprotein co‐translationally inserted into the lumen of the endoplasmic reticulum and then transported along the secretory pathway to the protein storage vacuoles. We have previously shown that a PHSL mutant in which the last 59 amino acids have been deleted (Δ363PHSL) is unable to form trimers and is retained in a pre‐Golgi compartment when synthesized in Xenopus oocytes. When transiently expressed in tobacco leaf protoplasts, wild‐type PHSL is correctly glycosylated and assembles efficiently and rapidly into trimers. Δ363PHSL is also correctly glycosylated but does not trimerize. Tobacco BiP and Δ363PHSL are co‐immunoselected using either anti‐PHSL or anti‐BiP antibodies. Under the same conditions, co‐immunoselection of BiP with wild‐type PHSL is not detectable. The BiP bound to Δ363PHSL can be released by treatment of the complex with ATP, indicating that the binding is related to the proposed function of BiP in protein folding and assembly in the endoplasmic reticulum. These data indicate that BiP stably binds structurally defective proteins in plant cells.
A NaCl‐regulated plant gene encoding a brain protein homolog that activates ADP ribosyltransferase and inhibits protein kinase C
Authors: Chen Zutang , Fu Haian , Liu Dong , Chang Pi‐Fang Linda , Narasimhan Meena , Ferl Robert , Hasegawa Paul M. , Bressan Ray A.
Published: March 2003

A cDNA clone pCZ1, with a 1.1 kb insert, was isolated from a NaCI‐adapted tobacco cell cDNA library that encodes an apparently full‐length 29 kDa protein (251 amino acids) with a calculated pl of 5.7. The encoded peptide had a high amino acid sequence identity with bovine 14‐3‐3 protein which was originally found as an abundant protein in the animal central nervous system. Recently, proteins with sequence identity to 14‐3‐3 protein have also been found in plants, insects and yeast, and appear to have diverse physiological functions. Similar to the bovine brain 14‐3‐3 protein, the recombinant pCZ1 protein stimulated ADP‐ribosylation of protein substrate by ADP‐ribosyl‐transferase from the plant and animal pathogenic bacterium Pseudomonas aeruginosa. This recombinant protein also inhibited protein kinase C activity in vitro. Southern blot analyses indicated that most likely five genes encoding 14‐3‐3‐like proteins are present in tobacco. The pCZ1 cDNA insert hybridized to a single mRNA of 1.1 kb from cultured tobacco cells. The level of this mRNA transcript in tobacco cells was downregulated upon adaptation to NaCl but was unaffected by short‐term treatment with NaCl, ABA or ethylene. In tobacco plants, expression of transcript that hybridized to pCZ1 was tissue specific, and was most abundant in roots and flower parts. Monoclonal antibody raised against GF14 protein, a maize protein with substantial sequence identity with 14‐3‐3 protein detected two bands on SDS‐PAGE of total proteins from unadapted tobacco cells and only a single band from cells adapted to NaCl. The GF14 antibody was also used to illustrate that the G‐box element of a salt‐induced gene is associated with a 14‐3‐3‐type protein.
Molecular characterization of two Brassica napus genes related to oleosins which are highly expressed in the tapetum
Authors: Robert Laurian S. , Gerster Jean , Allard Sharon , Cass Leslie , Simmonds John
Published: March 2003

Two highly homologous Brassica napus flower cDNA clones, Sta 41‐2 and Sta 41‐9, were isolated and characterized. These clones were shown to correspond to genes expressed in the tapetum from the early uninucleate microspore stage to the dinucleate stage. The predicted Sta 41‐2 and Sta 41‐9 proteins possessed characteristics similar to oleosins such as a polar N‐terminal domain, a large relatively conserved hydrophobic domain and a long C‐terminal domain which consisted of four different groups of repeats. In addition, like oleosins, the Sta 41‐2 and Sta 41‐9 proteins have a basic pl, lack a signal peptide and are found in a tissue which accumulates lipids in small lipid bodies.
Genetic transformation of the C4 plant, Flaveria bidentis
Authors: Chitty Julie A. , Furbank Robert T. , Marshall Jerry S. , Chen Zhenghua , Taylor William C.
Published: March 2003

An efficient and rapid transformation system for the dicotyledonous C4 plant, Flaveria bidentis has been developed. The method involves Agrobacterium tumefaciens infection of explants followed by regeneration of shoots from kanamycin‐resistant callus. Shoots first appear on callus 3–4 weeks after Agrobacterium infection, with plants in soil after another 12–15 weeks. Transformation has been verified by measuring the activity of the gusA and npt‐II transgenes and by genomic Southern blots to show the stable integration of T‐DNA. Most regenerated plants show normal morphology, are self‐fertile and transgenes show expected Mendelian segregations in the next generation. This system allows one to test a number of gene constructions rapidly and should accelerate progress in studies of the cell‐specific expression of genes coding for C4 enzymes and the mechanisms regulating the activities of C4 enzymes.
A 20 bp cis‐acting element is both necessary and sufficient to mediate elicitor response of a maize PRms gene
Authors: Raventós Dora , Jensen Anders B. , Rask Maj‐Britt , Casacuberta Josep M. , Mundy John , San Segundo Blanca
Published: March 2003

Transient gene expression assays in barley aleurone protoplasts were used to identify a cis‐regulatory element involved in the elicitor‐responsive expression of the maize PRms gene. Analysis of transcriptional fusions between PRms 5′ upstream sequences and a chloramphenicol acetyltransferase reporter gene, as well as chimeric promoters containing PRms promoter fragments or repeated oligonucleotides fused to a minimal promoter, delineated a 20 bp sequence which functioned as an elicitor—response element (ERE). This sequence contains a motif (−246 AATTGACC) similar to sequences found in promoters of other pathogen‐responsive genes. The analysis also indicated that an enhancing sequence(s) between −397 and −296 is required for full PRms activation by elicitors. The protein kinase inhibitor staurosporine was found to completely block the transcriptional activation induced by elicitors. These data indicate that protein phosphorylation is involved in the signal transduction pathway leading to PRms expression.
Wild‐type ovule development in Arabidopsis thaliana: a light microscope study of cleared whole‐mount tissue
Authors: Schneitz Kay , Hülskamp Martin , Pruitt Robert E.
Published: March 2003

A detailed morphological description of wild‐type ovule development in Arabidopsis thaliana is presented. The entire process from the formation of the ovule protrusion until the eight‐nuclear endosperm stage is described. The study is based on a light‐microscopical analysis of stained and subsequently optically cleared whole‐mount ovules. It is supplemented by confocal laser scanning microscopy of propidium iodide‐stained whole‐mount ovules. It has been shown that the combination of both techniques eliminates the need for sections to a large extent, and hence allows the rapid morphological inspection of a large number of ovules in Arabidopsis. The ovule constitutes a relatively simple organ. During development, three discrete major pattern elements are laid down along the proximal‐distal axis: the nucellus at the distal end (harbors the megaspore/gametophyte lineage), the chalaza (flanked by the integuments) and the funiculus (includes the vascular strand) at the proximal end. These three pattern elements already appear at a very early stage, when the initially formed protrusion, consisting of files of uniform cells, is transformed into a patterned primordium. Subsequent morphogenesis results in the manifestation of the morphological characters of each pattern element. It was possible to dissect this developmental process into distinct, morphologically discernible steps at a high resolution. A classification scheme of ovule developmental stages is proposed, which is based on ovule‐specific, discrete, and easy‐to‐score markers.
A novel Ti‐plasmid‐convertible λ phage vector system suitable for gene isolation by genetic complementation of Arabidopsis thaliana mutants
Authors: Fuse Takuichi , Kodama Hiroaki , Hayashida Nobuaki , Shinozaki Kazuo , Nishimura Mitsuo , Iba Koh
Published: March 2003

A new λ phage vector system, λTI, has been constructed to facilitate genetic complementation of higher plant mutations. The λTI vectors are stable, and by using the Cre—lox site‐specific recombination, are automatically convertible into Ti‐plasmid binary vectors which are capable of expressing genes in higher plants. Two λTI vectors were constructed: (i) λTI1, which can generate a Ti‐plasmid that contains the cauliflower mosaic virus (CaMV) 35S promoter and is suitable for the expression of cDNA in transformed plants and (ii) λTI2, which can generate a Ti‐plasmid with the multicloning site (MCS). cDNA and genomic libraries, which were constructed from the cruciferous plant Arabidopsis thaliana in these λTI vectors, can be probed by large DNA fragments of more than 100 kb, such as yeast artificial chromosomes (YACs), enabling the direct screening of the clones in the chromosome region containing a specified genetic locus. These libraries will certainly become powerful tools for the genetic complementation of Arabidopsis mutant phenotypes by quickly providing transformation‐competent clones.
Jasmonate‐induced lipoxygenase forms are localized in chloroplasts of barley leaves (Hordeum vulgare cv. Salome)
Authors: Feussner Ivo , Hause Bettina , Vörös Kirsten , Parthier Benno , Wasternack Claus
Published: March 2003

Barley leaves respond to application of (−)‐jasmonic acid (JA), or its methylester (JM) with the synthesis of abundant proteins, so‐called jasmonate induced proteins (JIPs). Here Western blot analysis is used to show a remarkable increase upon JM treatment of a 100 kDa lipoxygenase (LOX), and the appearance of two new LOX forms of 98 and 92 kDa. The temporal increase of LOX‐100 protein upon JM treatment was clearly distinguishable from the additionally detectable LOX forms. JM‐induced LOX forms in barley leaves were compared with those of Arabidopsis and soybean leaves. Both dicot species showed a similar increase of one LOX upon JM induction, whereas, leaves from soybean responded with additional synthesis of a newly formed LOX of 94 kDa.

Using immunofluorescence analysis and isolation of intact chloroplasts, it is demonstrated that JM‐induced LOX forms of barley leaves are exclusively located in the chloroplasts of all chloroplast‐containing cells. Analogous experiments carried out with Arabidopsis and soybean revealed a similar plastidic location of JM‐induced LOX forms in Arabidopsis but a different situation for soybean. In untreated soybean leaves the LOX protein was mainly restricted to vacuoles of paraveinal mesophyll cells. Additionally, LOX forms could be detected in cytoplasm and nuclei of bundle sheath cells. Upon JM treatment cytosolic LOX was detectable in spongy mesophyll cells, too. The intracellular location of JM‐induced LOX is discussed in terms of stress‐related phenomena mediated by JM.
Enhanced quantitative resistance against fungal disease by combinatorial expression of different barley antifungal proteins in transgenic tobacco
Authors: Jach Guido , Görnhardt Birgit , Mundy John , Logemann Jürgen , Pinsdorf Elke , Leah Robert , Schell Jeff , Maas Christoph
Published: March 2003

cDNAs encoding three proteins from barley (Hordeum vulgare), a class‐II chitinase (CHI), a class‐II β‐1,3‐glucanase (GLU) and a Type‐I ribosome‐inactivating protein (RIP) were expressed in tobacco plants under the control of the CaMV 35S‐promoter. High‐level expression of the transferred genes was detected in the transgenic plants by Northern and Western blot analysis. The leader peptides in CHI and GLU led to accumulation of these proteins in the intercellular space of tobacco leaves. RIP, which is naturally deposited in the cytosol of barley endosperm cells, was expressed either in its original cytosolic form or fused to a plant secretion peptide (spRIP). Fungal infection assays revealed that expression of the individual genes in each case resulted in an increased protection against the soilborne fungal pathogen Rhizoctonia solani, which infects a range of plant species including tobacco. To create a situation similar to ‘multi‐gene’ tolerance, which traditional breeding experience has shown to provide crops with a longer‐lasting protection, several of these antifungal genes were combined and protection against fungal attack resulting from their co‐expression in planta was evaluated. Transgenic tobacco lines were generated with tandemly arranged genes coding for RIP and CHI as well as GLU and CHI. The performance of tobacco plants co‐expressing the barley transgenes GLU/ CHI or CHI/RIP in a Rhizoctonia solani infection assay revealed significantly enhanced protection against fungal attack when compared with the protection levels obtained with corresponding isogenic lines expressing a single barley transgene to a similar level. The data indicate synergistic protective interaction of the co‐expressed anti‐fungal proteins in vivo.
Thioredoxin activity in the C terminus of Phalaris S protein
Authors: Li Xinmin , Nield Jan , Hayman David , Langridge Peter
Published: March 2003

Self‐incompatibility in the grass Phalaris coerulescens is controlled by two genes S and Z. Isolation and sequencing of two S alleles showed that they encode proteins that are highly conserved at the C terminus with significant homology to thioredoxin H proteins. In particular, the residues in and around the active site of thioredoxin, Trp‐Cys‐Gly‐Pro‐Cys, are perfectly conserved. The C terminus of the S protein has been expressed in Eschericia coli and purified to homogeneity on Ni‐NTA resin. Functional assays showed that the protein has thioredoxin activity; it can act as a substrate for E. coli thioredoxin reductase and also catalyse the reduction of insulin by dithiothreitol. The possible role of thioredoxin‐like activity of the S protein in mediating the incompatibility reaction in Phalaris is discussed.
Non‐destructive detection of firefly luciferase (LUC) activity in single plant cells using a cooled, slow‐scan CCD camera and an optimized assay
Authors: Kost Benedikt , Schnorf Martin , Potrykus Ingo , Neuhaus Gunther
Published: March 2003

A flexible, comparatively inexpensive system based on a liquid nitrogen‐cooled slow‐scan CCD (charge coupled device) camera is presented, which can be employed for quantitative low‐light (bioluminescence, chemiluminescence or fluorescence) imaging. Using this camera system and the firefly luciferase (LUC) as a screenable marker, transgenic tobacco lines have been produced by direct gene transfer. Bioluminescence emitted from single tobacco cells transiently expressing the firefly luciferase gene (Luc) as well as from stably transformed calli, regenerated shoots, plantlets and T1 seedlings could be monitored in vivo with no effect on the viability of the material analysed. The patterns of light emission from sections through Luc‐expressing leaves and bioluminescent single protoplasts isolated from such leaves were also imaged microscopically. The assay used to detect in vivo LUC activity was optimized by quantifying bioluminescence emitted from Luc‐expressing tobacco protoplasts and leaves incubated in different substrate solutions and determining the kinetics of light emission during incubation in the substrate solution.
In vitro synthesis of a microfibrillar (1→3)‐β‐glucan by a ryegrass (Lolium multiflorum) endosperm (1→3)‐β‐glucan synthase enriched by product entrapment
Authors: Bulone Vincent , Fincher Geoffrey B. , Stone Bruce A.
Published: March 2003

A simple, rapid procedure has been developed to purify (1→3)‐β‐glucan synthase (UDP‐Glc:(1→3)‐β‐glucan 3‐β‐d‐glucosyl transferase (EC 2.4.1.34)) over 400‐fold from membrane preparations of Italian ryegrass (Lolium multiflorum) and 60‐fold from CHAPS‐extracted membranes. When a CHAPS‐extract of the membranes is treated with 8 mM CaCl2, proteinaceous material is precipitated. Although less than 10% of CHAPS‐solubilized protein is removed in this step, the total activity recovered in the supernatant increases fourfold. Thus, CaCl2 precipitation appears to be important in removing inhibitors of the (1→3)‐β‐glucan synthase. In the presence of 1 mM UDP‐glucose, the supernatant after CaCl2 treatment produces a high molecular weight, insoluble product that entraps a (1→3)‐β‐glucan synthase of high specific activity. The product‐entrapped enzyme preparation contains six major polypeptides, and comparison of the SDS—PAGE pattern of this fraction with the polypeptide profile of an immunoprecipitated (1→3)‐β‐glucan synthase preparation suggests that polypeptides at 30–31 and 55–58 kDa are the most likely candidates for participation in (1→3)‐β‐glucan synthesis. When the reaction is performed on a larger scale, milligram quantities of product can be seen precipitating from the reaction mixture within 1 h of substrate addition. This product has been characterized by methylation analysis, 1H‐ and 13C‐nmr spectroscopy, X‐ray diffraction, electron microscopy, size exclusion chromatography, UV‐induced fluorescence in the presence of the (1→3)‐β‐glucan‐specific fluorochrome from aniline blue, and enzymic hydrolysis with a specific (1→3)‐β‐glucanase. These physical, chemical and enzymic analyses clearly demonstrate that the product is a microfibrillar (1→3)‐β‐glucan with a degree of polymerization of about 1500.
Molecular cloning of cDNAs encoding the protein backbones of arabinogalactan‐proteins from the filtrate of suspension‐cultured cells of Pyrus communis and Nicotiana alata
Authors: Mau Shaio‐Lim , Chen Chao‐Guang , Pu Zhao‐Yan , Moritz Robert L. , Simpson Richard J. , Bacic Antony , Clarke Adrienne E.
Published: March 2003

This paper reports the isolation of cDNAs encoding the protein backbone of two arabinogalactan‐proteins (AGPs), one from pear cell suspension cultures (AGPPc2) and the other from suspension cultures of Nicotiana alata (AGPNa2). The proteins encoded by these cDNAs are quite different from the ‘classical’ AGP backbones described previously for AGPs isolated from pear suspension cultures and extracts of N. alata styles. The cDNA for AGPPc2 encodes a 294 amino acid protein, of which a relatively short stretch (35 amino acids) is Hyp/Pro rich; this stretch is flanked by sequences which are dominated by Asn residues. Asn residues are not a feature of the ‘classical’ AGP backbones in which Hyp/Pro, Ser, Ala and Thr account for most of the amino acids. The cDNA for AGPNa2 encodes a 437 amino acid protein, which contains two distinct domains: one rich in Hyp/Pro, Ser, Ala, Thr and the other rich in Asn, Tyr and Ser. The composition and sequence of the Pro‐rich domain resembles that of the ‘classical’ AGP backbone. The Asn‐rich domains of the two cDNAs described have no sequence similarity; in both cases they are predicted to be processed to give a mature backbone with a composition similar to that of the ‘classical’ AGPs. The study shows that different AGPs can differ in the amino acid sequence in the protein backbone, as well as the composition and sequence of the arabinogalactan side‐chains. It also shows that differential expression of genes encoding AGP protein backbones, as well as differential glycosylation, can contribute to the tissue specificity of AGPs.
The minimal ribosomal RNA gene promoter of Arabidopsis thaliana includes a critical element at the transcription initiation site
Authors: Doelling Jed H. , Pikaard Craig S.
Published: March 2003

The genes encoding the precursor of 18S, 5.8S and 25S ribosomal RNAs are transcribed in the nucleolus by RNA polymerase I. Unlike rRNA gene promoters in animals which differ substantially across species boundaries, plant rRNA gene promoters share sequence similarity for several nucleotides upstream and downstream of the transcription start site (+1). The conserved sequence consists of a near‐consensus TATA box, a critical promoter element of most genes transcribed by RNA polymerase II and certain genes transcribed by RNA polymerase III, followed by a cluster of four to six guanosines. Using transient expression of cloned promoter deletions in Arabidopsis thaliana protoplasts, it is shown that the 5′ boundary of the Arabidopsis rRNA gene promoter is located between −55 and −33 and the 3′ promoter boundary is approximately +6. The critical role of the TATA element at the initiation site was demonstrated by altering this region using tandem point mutations within constructs containing essentially complete intergenic spacer sequences from −2590 to +6. The initiation site mutations either abolished transcription or dramatically reduced transcript abundance relative to the wild‐type promoter. In some mutants, transcription initiation was shifted to a new site, suggesting a role for the TATA‐like initiator region in both start site selection and promoter strength. It is suggested that minimal rRNA gene promoters might be similar in design to minimal promoters of protein‐encoding genes, many of which utilize an initiator element, or INR, as an important promoter element located directly at the transcription start site.
A dinucleotide mutation in dihydrodipicolinate synthase of Nicotiana sylvestris leads to lysine overproduction
Authors: Ghislain Marc , Frankard Valerie , Jacobs Michel
Published: March 2003

By applying a mutagenesis/selection procedure to obtain resistance to a lysine analog, S‐(2‐aminoethyl)l‐cysteine (AEC), a lysine overproducing mutant in Nicotiana sylvestris was isolated. Amino acid analyses performed throughout plant development and of different organs of the N. sylvestris RAEC‐1 mutant, revealed a developmental‐dependent accumulation of free lysine. Lysine biosynthesis in the RAEC‐1 mutant was enhanced due to a lysine feedback‐desensitized dihydrodipicolinate synthase (DHDPS). Several molecular approaches were undertaken to identify the nucleotide change in the dhdps‐r1 gene, the mutated gene coding for the lysine‐desensitized enzyme. The enzyme was purified from wild‐type plants for amino end microsequencing and 10 amino acids were identified. Using dicotyledon dhdps probes, a genomic fragment was cloned from an enriched library of DNA from the homozygote RAEC‐1 mutant plant. A dhdps cDNA, putatively full‐length, was isolated from a tobacco cDNA library. Nucleotide sequence analyses confirmed the presence of the previously identified amino end preceded by a chloroplast transit peptide sequence. Nucleotide sequence comparisons, enzymatic and immunological analyses revealed that the tobacco cDNA corresponds to a normal type of DHDPS, lysine feedback‐regulated, and the genomic fragment to the mutated DHDPS, insensitive to lysine inhibition. Functional complementation of a DHDPS‐deficient Escherichia coli strain was used as an expression system. Reconstruction between the cDNA and genomic fragment led to the production of a cDNA producing an insensitive form of DHDPS. Amino acid sequence comparisons pointed out, at position 104 from the first amino acid of the mature protein, the substitution of Asn to lleu which corresponds to a dinucleotide mutation. This change is unique to the dhdps‐r1 gene when compared with the wild‐type sequence. The identification of the nucleotide and amino acid change of the lysine‐desensitized DHDPS from RAEC‐1 plant opens new perspectives for the improvement of the nutritional value of crops and possibly to develop a new plant selectable marker.
Genetic regulation of gibberellin deactivation in Pisum
Authors: Ross John J. , Reid James B. , Swain Stephen M. , Hasan Omar , Poole Andrew T. , Hedden Peter , Willis Christine L.
Published: March 2003

The regulation of gibberellin (GA) deactivation was examined using the sin (slender) mutation in the garden pea (Pisum sativum L.). This mutation blocks the deactivation of GA20, the precursor of the bioactive GA1. Firstly, crosses were made to combine sin with the GA biosynthesis mutations na, lhi and le‐3. The combination sin na produced a novel phenotype, with long (‘slender’) basal internodes and extremely short (‘nana’) upper internodes. In contrast, the double mutant sin lhi was phenotypically dwarf. The mutation sin causes an accumulation of GA20 in maturing seeds, and this was unaffected by na, since the na mutation is not expressed in seeds. In contrast, lhi seeds did not accumulate GA20, since lhi imposes an early block on GA biosynthesis. Secondly, the effects of sin on several steps in GA deactivation were investigated. In maturing seeds, the mutation sin blocks two steps in GA20 metabolism, namely, GA20 to GA29, and GA29 to GA29‐catabolite. In the vegetative plant, on the other hand, sin blocked the step GA20 to GA29, but not GA29 to GA29‐catabolite; the steps GA20 to GA81 and GA20 to GA1 were also not impaired in this mutant. It is clear that the effects of sin, like those of na, are strongly organ‐specific. The presence of separate enzymes for the steps GA20 to GA29 and GA29 to GA29‐catabolite was suggested by the observation that GA8 inhibited the latter step, but not the former, and by the inability of GA20 and GA29 to inhibit each other's metabolism. It is suggested that the Sin gene may be a regulatory gene controlling the expression of two structural genes involved in GA deactivation.