bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of a simple transient assay for Ac/Ds activity in cells of intact barley tissue
Authors: McElroy David , Louwerse Jeanine D. , McElroy Suzin M. , Lemaux Peggy G.
Published: March 2003

The development of a barley (Hordeurn vulgare L.) transformation system made it possible to consider the use of maize Activator/Dissociation (Ac/Ds) transposable elements for gene tagging in transgenic barley plants. However, barley transformation is time‐consuming, and therefore a simple transient assay for Ac/Ds activity in intact barley tissues was developed to test the components of a proposed gene tagging system, prior to their stable introduction into plants. In this assay, barley scutellar tissue is co‐transformed with constructs containing the maize Ac transposase gene and an Escherichia coli uidA reporter gene (Gus), the expression of which is interrupted by a maize Ds element. In transformed barley scutellar cells, Ac transposase‐mediated excision of the Ds element generates a functional Gus gene, leading to histochemically detectable GUS activity. Characterization of the excision products showed that they had a pattern of nucleotide deletions and/or transversions similar to that found in maize and other heterologous plant systems. In addition, although contrary to the situation observed in heterologous dicot systems, efficient Ds excision in barley, a heterologous monocot system, appears to be inversely associated with Ac copy number, a finding similar to the Ac dosage effects observed in maize. The transient assay was used to demonstrate functional transposase activity in barley callus lines stably transformed with an Ac transposase gene.
Subcellular localization of H2O2 in plants. H2O2 accumulation in papillae and hypersensitive response during the barley—powdery mildew interaction
Authors: Thordal‐Christensen Hans , Zhang Ziguo , Wei Yangdou , Collinge David B.
Published: March 2003

Active oxygen species (AOS) are believed to have important roles in plants in general and in plant—pathogen interactions in particular. They are believed to be involved in signal transduction, cell wall reinforcement, hypersensitive response (HR) and phytoalexin production, and to have direct antimicrobial effects. Since current methods are inadequate for localizing AOS in intact plant tissue, most studies have been conducted using cell suspension culture/elicitors systems. 3,3‐diaminobenzidine (DAB) polymerizes instantly and locally as soon as it comes into contact with H2O2 in the presence of peroxidase, and it was found that, by allowing the leaf to take up this substrate, in‐vivo and in‐situ detection of H2O2 can be made at subcellular levels. This method was successfully used to detect H2O2 in developing papillae and surrounding haloes (cell wall appositions) and whole cells of barley leaves interacting with the powdery mildew fungus. Thus, H2O2 can be detected in the epidermal cell wall subjacent to the primary germ tube from 6 h after inoculation, and subjacent to the appressorium from 15 h. The earliest time point for observation of H2O2 in relation to epidermal cells undergoing HR is 15 h after inoculation, first appearing in the zones of attachment to the mesophyll cells underneath, and eventually in the entire epidermal cell. Furthermore, it was observed that proteins in papillae and HR cells are cross‐linked, a process believed to be fuelled by H2O2. This cross‐linking reinforces the apposition, presumably assisting the arrest of the pathogen.
Regulatory elements in vivo in the promoter of the abscisic acid responsive gene rab17 from maize
Authors: Busk Peter Kamp , Jensen Anders Bøgh , Pagès Montserrat
Published: March 2003

The rab17 gene from maize is transcribed in late embryonic development and is responsive to abscisic acid and water stress in embryo and vegetative tissues. In vivo footprinting and transient transformation of rab17 were performed in embryos and vegetative tissues to characterize the cis‐elements involved in regulation of the gene. By in vivo footprinting, protein binding was observed to nine elements in the promoter, which correspond to five putative ABREs (abscisic acid responsive elements) and four other sequences. The footprints indicated that distinct proteins interact with these elements in the two developmental stages. In transient transformation, six of the elements were important for high level expression of the rab17 promoter in embryos, whereas only three elements were important in leaves. The cis‐acting sequences can be divided in embryo‐specific, ABA‐specific and leaf‐specific elements on the basis of protein binding and the ability to confer expression of rab17. We found one positive, new element, called GRA, with the sequence CACTGGCCGCCC. This element was important for transcription in leaves but not in embryos. Two other non‐ABRE elements that stimulated transcription from the rab17 promoter resemble previously described abscisic acid and drought‐inducible elements. There were differences in protein binding and function of the five ABREs in the rab17 promoter. The possible reasons for these differences are discussed. The in vivo data obtained suggest that an embryo‐specific pathway regulates transcription of the rab genes during development, whereas another pathway is responsible for induction in response to ABA and drought in vegetative tissues.
Microinjection of fluorescent tubulin into plant cells provides a representative picture of the cortical microtubule array
Authors: Wymer Carol L. , Shaw Peter J. , Warn Richard M. , Lloyd Clive W.
Published: March 2003

By microinjecting rhodamine‐labelled tubulin into living plant cells, it is possible to observe microtubules (MTs) directly and to see how the cortical array reorganizes itself. The validity of the conclusions drawn from such observations depends upon the assumption that most, if not all, of the native MTs are dynamic and incorporate labelled tubulin. However, if arrays also contain MTs that are not exchanging tubulin subunits, such MTs will remain unlabelled, and the labelled MT population will be under‐representative of the whole array. To address this potential problem, we microinjected pea epidermal cells with rhodamine‐labelled tubulin, then fixed the cells and used fluorescein‐conjugated antibodies against tubulin to detect the entire MT array. The two fluorescent patterns corresponded well, confirming that the MTs labelled with exogenous tubulin were evenly distributed throughout the entire array. Also, by comparing the MT image before and after aldehyde fixation, we observed that, although some of the MTs were lost in the procedure, the fixation was able to preserve the arrangement of MTs seen in the living cell. We conclude that fluorescence analogue cytochemistry provides a valid representation of the entire cortical MT array.
Fine‐scale molecular genetic (RFLP) and physical mapping of a 8.9 cM region on the top arm of Arabidopsis chromosome 5 encompassing the male sterility gene, ms1
Authors: Thorlby Glenn J. , Shlumukov Leonid , Vizir Igor Y. , Yang Cai‐Yun , Mulligan Bernard J. , Wilson Zoe A.
Published: March 2003

Fine‐scale molecular mapping has been conducted using 183 recombinants between the markers lutescens (lu; 17.6 cM) and transparent testa glabra (ttg; 35.5 cM) on the top arm of Arabidopsis thaliana chromosome 5. This region contains a number of genes involved in floral development including Ms1, a gene required for the post‐meiotic development of pollen. In homozygous ms1 mutant plants, pollen development is aborted soon after microspore release, regardless of environmental conditions. The ms1 mutation is located at 29.8 ± 0.8 cM on chromosome 5. Markers have been identified which co‐segregate with ms1 and should lie within 39 kb of the gene. The fine‐scale map of the lu‐ms1‐ttg region that has been generated is significantly different from the published integrated map and provides substantially more accurate and higher marker density than the current recombinant inbred map for this region. Using clones derived from four yeast artificial chromosome libraries, a contig has been established between the RFLP markers 4111 and 4556, which encompasses the ms1 gene. This covers a genetic distance of 8.9 cM which corresponds to a physical distance of approximately 1.44 Mb, representing about 1.5–2.0% of the Arabidopsis genome. In this region, 1 cM represents a physical distance of approximately 160 kb.
The enzyme involved in sulfation of the turgorin, gallic acid 4‐O‐(β‐d‐glucopyranosyl‐6′‐sulfate) is pulvini‐localized in Mimosa pudica
Authors: Varin Luc , Chamberland Héléne , Lafontaine Jean Guy , Richard Martine
Published: March 2003

A sulfotransferase (ST) which catalyzes the transfer of sulfate from 3′‐phosphoadenosine 5′‐phosphosulfate (PAPS) to gallic acid glucoside was characterized from microsomal preparations of Mimosa pudica. The product of the reaction was found to co‐elute on HPLC with the periodic leaf movement factor 1 (PLMF‐1) (gallic acid β‐d‐gluco‐pyranosyl‐6′‐sulfate). The distribution of the enzyme activity was restricted to plasma membrane preparations from primary, secondary and tertiary pulvini. The M. pudica ST activity was inhibited in a dose‐dependent manner in the presence of an antibody raised against the flavonol 3‐sulfotransferase of Flaveria chloraefolia, suggesting structural similarities between the two proteins. Western blot analysis of M. pudica protein extracts using these antibodies indicated the presence of a cross‐reactive polypeptide with an apparent molecular mass of 42 000 Da whose distribution correlates with the presence of the gallic acid glucoside ST activity. Indirect immunogold labeling of resin‐embedded sections from tertiary pulvini showed a specific localization of gold particles on the sieve‐tube plasma membranes. The label distribution was uniform and other cellular organelles and membrane systems displayed little or no labeling. The results of the Western blot and immunocytochemical studies are consistent with the detection of the gallic acid glucoside ST activity in plasma membrane preparations of M. pudica pulvini cells. The specific tissue distribution of the ST in motor organ phloem cells suggests that this is the site of synthesis and/or accumulation of PLMF‐1 and supports the proposed hypothesis that PLMF‐1 may be acting as a chemical signal during the seismonastic response of M. pudica.
Post‐transcriptional gene silencing of ACC synthase in tomato results from cytoplasmic RNA degradation
Authors: Lee Kathleen Y. , Baden Catherine , Howie William J. , Bedbrook John , Dunsmuir Pamela
Published: March 2003

Transgenic tomato plants with the coding region of a predominantly fruit specific ACC synthase gene (LE‐ACS2) driven by the CAMV35S promoter, fall into two phenotypic classes: (i) epinastic plants and (ii) plants which appear vegetatively normal with ripening impaired fruit. In leaf tissue from epinastic plants the transgene RNA pool is high, and in the vegetatively normal plants the transgene transcript level is very low. While the epinastic phenotype results from the expected high level expression of the ACC synthase transgene throughout the plant, the vegetatively normal, ripening impaired phenotype is a consequence of the silencing of both the transgene and the corresponding endogenous gene. As in several other cases of gene silencing, the mechanism is post‐transcriptional. Transgene RNA pools are similar in nuclei of epinastic and vegetatively normal tissue. However, the transgene mRNA pool is dramatically reduced in the cytoplasm of vegetatively normal plants, and furthermore, this reduction results from degradation from the 3′ end of the transcript. This degradation probably occurs on the polysomes where the transgene transcripts are localized.
Blocking polyadenylation of mRNA in the chloroplast inhibits its degradation
Authors: Lisitsky Irena , Klaff Petra , Schuster Gadi
Published: March 2003

The addition of poly(A)‐rich sequences to endonuclease cleavage products of chloroplast mRNA has recently been suggested to target the polyadenylated RNA for rapid exonucleolytic degradation. This study analyzed whether the addition of a poly(A)‐rich tail to RNA molecules is required for degradation by chloroplast exonuclease(s). In lyzed chloroplasts from spinach, addition of the polyadenylation inhibitor, cordycepin triphosphate (3′‐dATP), inhibited the degradation of psbA and rbcL mRNAs. Furthermore, degradation intermediates generated by endonucleolytic cleavages accumulated. Similar results were obtained when yeast tRNA was added to the mRNA degradation system as a non‐specific exoribonuclease inhibitor. Nevertheless, the stabilization mechanisms differ: while tRNA directly affects the exonuclease activity, 3′dATP has an indirect effect by inhibiting polyadenylation. The results indicate that the addition of poly(A)‐rich sequences to endonucleolytic cleavage products of chloroplast mRNA is required to target these RNAs for rapid exonucleolytic degradation. Together with previous work, the data reported here support a model for mRNA degradation in the chloroplast in which endonucleolytic cleavages are followed by the addition of poly(A)‐rich sequences to the proximal cleavage products, targeting these RNAs for rapid exonucleolytic decay.
Oxidative cross‐linking of plasma membrane arabinogalactan proteins
Authors: Kjellbom Per , Snogerup Lars , Stöhr Christine , Reuzeau Christophe , McCabe Paul F. , Pennell Roger I.
Published: March 2003

Monoclonal antibodies which recognize carbohydrate in arabinogalactan proteins (AGPs) have revealed that certain carbohydrate epitopes at the outer plasma membrane surface are demonstratively regulated. Some epitomes are expressed according to cell position, and AGES are thought to play a role in cell—cell interaction during development. This study demonstrates that sugar beet plasma membranes contain two subagencies of AGES, with apparent molecular masses of 82 and 97 kDa, and that each subfamily consists of a small number of acidic AGP isoforms. Excision of leaves generates three additional AGP complexes with apparent molecular masses of 120, 170 and 210 kDa, with the 170 kDa complex being the major form induced by excision. The addition of millimolar concentrations of H2O2 to a partially purified fraction of the 82 and 97 kDa AGPs also generates AGP complexes, with the 170 kDa complex as the major form. These results indicate that the plasma membrane AGPs are a target for endogenous H2O2.
The influence of small changes in transgene transcription on homology‐dependent virus resistance and gene silencing
Authors: English James J. , Baulcombe David C.
Published: March 2003

A tissue culture/regeneration scheme was employed to introduce epigenetic variation into tobacco lines that either do (T4) or do not (T19) exhibit post‐transcriptional silencing of β‐glucuronidase (GUS) transgenes. Homology‐dependent resistance to a potato virus X derivative carrying GUS (PVX‐GUS), an indicator of post‐transcriptional gene silencing, was used as a screen for epigenetic changes. Regenerant families derived from the high‐expressing line T19 and nontransformed tobacco remained completely susceptible to PVX‐GUS, like the progenitors. Twenty out of twenty T4 regenerant families were also susceptible to PVX‐GUS and were therefore very distinct from the low‐expressing, PVX‐GUS‐resistant T4 progenitor line. This change to PVX‐GUS susceptibility was not associated with DNA structural changes, but was associated with slight increases in GUS mRNA accumulation, increased 35S promoter methylation and correspondingly decreased transcription of GUS. These results suggest that homology‐dependent virus resistance and gene silencing are very sensitive to small changes in transgene transcription.
In transformed tobacco cells the apoplasmic invertase inhibitor operates as a regulatory switch of cell wall invertase
Authors: Krausgrill Silke , Greiner Steffen , Köster Ulrike , Vogel Rolf , Rausch Thomas
Published: March 2003

Agrobacterium tumefaciens‐transformed tobacco suspension‐cultured cells (TSCC) exhibit no significant quantitative changes of cell wall invertase protein (CWI) during a culture period of 40 days, whereas CWI activity decreases strongly between 10 and 30 days after cell transfer to fresh medium. Western blot analysis revealed that the apoplasmic invertase inhibitor (INH) is equally expressed throughout the entire culture period. When apoplasmic protein fractions from 4 and 28 days old cell cultures are chromatographed on Concanavalin A(ConA)‐Sepharose, the non‐glycosylated INH always coelutes with the ConA‐bound fraction, suggesting that (i) INH and the glycosylated CWI form a complex in the apoplasmic space, and (ii) INH binding is not sufficient for CWI inhibition. The high specificity of INH binding to CWI was confirmed by native cathodic polyacrylamide gel electrophoresis. Expression analysis of CWI and INH indicates that, at least during certain stages of plant development (seedlings, roots of adult plants), CWI activity may be modulated by INH, the latter operating as a regulatory switch.
cDNA cloning and characterisation of an α‐glucosidase gene from potato (Solanum tuberosum L.)
Authors: Taylor Mark A. , George Lesley A. , Ross Heather A. , Davies Howard V.
Published: March 2003

Using an Arabidopsis thaliana expressed sequence tag with sequence similarity to human lysosomal α‐glucosidase as a probe, a potato cDNA was isolated. The cDNA encodes a polypeptide with an Mr value of 105,400 and the most significant matches of the deduced amino acid sequence are with members of family 31 of glucosyl transferase. The potato cDNA was expressed in a strain of Saccharomyces cerevisiae that is deficient in maltase activity and unable to grow using maltose as a carbon source (ABYSMAL81). Expression of the potato cDNA in the mutant yeast strain restores its ability to use maltose as a carbon source for growth. Additionally, α‐glucosidase activity could be measured in extracts of the yeast cells following complementation. A range of maltodextrins were substrates for this activity. The steady‐state expression level of the potato α‐glucosidase gene was low in most tissues examined, the highest levels occurring in sprouting tubers and source leaves.
The characterization ofgio, a new pea mutant, shows the role of indoleacetic acid in the control of fruit development by the apical shoot
Authors: Rodrigo Ma Jesús , López‐Díaz Isabel , García‐Martínez José L.
Published: March 2003

Fruit‐set and fruit growth in pea (Pisum sativumL.) depend on gibberellins (GAs). The authors have isolated a new pea mutant,gio, which appeared spontaneously within the population of the cultivar Alaska, characterized by unpollinated ovaries much less sensitive to applied GAs. The mutant also has elongated peduncles, and is taller than the wild‐type (WT) because the upper plant internodes are longer. Contrary to WT, thegioovaries respond very little to benzylaminopurine (BAP) and 2,4‐dichlorophenoxyacetic acid, but become fully sensitive to GA3when this hormone is applied together with BAP. Thegiophenotype is determined by a mutation at a single mendelian locus. The mutation is recesive, shows incomplete penetrance, and its expression depends on environmental culture conditions. The sensitivity of the ovaries to GA3can be recovered by removing the apical shoot (plant decapitation) and by blocking the transport of indoleacetic acid (IAA) from the apical shoot with 2,3,5‐triiodobenzoic acid. The content of IAA in methanolic extracts and phloematic exudates of the apical shoot ofgiois about double that in the WT. The rate of transport of [3H]IAA applied to the apex of the mutant is also twice that in the WT. This indicates that the insensitivity of thegioovaries to GAs is due to the inhibitory effect of the higher basipetal IAA transport from the shoot. The interaction between the fruit and the apical shoot mediated by IAA probably also involves cytokinins transported from the basal part of the plant.
Signs of translational regulation within the transcript leader of a plant plasma membrane H+‐ATPase gene
Authors: Lukaszewicz Marcin , Jérouville Bénédicte , Boutry Marc
Published: March 2003

Transcripts of most plant plasma membrane H+‐ATPase genes possess a leader (5′ untranslated region) that is unusually long and that contains a short upstream open reading frame (uORF), two features which suggest post‐transcriptional regulation. To investigate the putative role of the transcript leader, we have placed the leader of pma3, one of the Nicotiana plumbaginifolia H+‐ATPase genes, between the CaMV 35S promoter and the sequence coding for the β‐glucuronidase (GUS) reporter gene. Transient expression of this chimeric gene and of derived mutants was analysed in electroporated tobacco protoplasts. The whole leader had a positive effect on translation, since deletion of most of its sequence reduced GUS activity. Suppression of the uORF by point mutation of its initiating AUG increased GUS activity by about 55%. Analysis of various deletions and mutations suggested that the uORF is translated by at least two‐thirds of scanning ribosomes, half of which subsequently reinitiate downstream translation under our experimental conditions. Reinitiation did not depend on the nucleotide sequence of the uORF, nor on that separating the uORF and the main open reading frame. We conclude that the pma3 transcript possesses features of translational regulation, whose mode of functioning has yet to be discovered.
C4‐type gene expression is not directly dependent on Kranz anatomy in an amphibious sedge Eleocharis vivipara Link
Authors: Uchino Akira , Sentoku Naoki , Nemoto Keisuke , Ishii Ryuichi , Samejima Muneaki , Matsuoka Makoto
Published: March 2003

Eleocharis vivipara Link alters its photosynthetic mode depending on the growth environment. It utilizes C4 photosynthesis when grown under terrestrial conditions (terrestrial form) and C3 photosynthesis when grown under submerged conditions (submerged form). The photosynthetic organ (the mature internodal region of the culm) of the terrestrial form shows typical Kranz anatomy with well‐developed bundle sheath cells, while the bundle sheath cells of the submerged form are not developed. In the mature internodal region of the terrestrial form, expression of the genes encoding two carboxylases, the small subunit of ribulose 1,5‐bisphosphate carboxylase (RbcS) and phosphoenolpyruvate carboxylase (Ppc), occurred mainly in bundle sheath cells and in mesophyll cells, respectively, as seen in a typical C4 leaf. In the submerged form, RbcS was expressed in both bundle sheath cells and mesophyll cells, and no expression of Ppc was observed. In the immature internodal region with undeveloped bundle sheath cells, both life forms showed the same expression pattern as in C3 plants: RbcS expression was localized in mesophyll cells and no Ppc expression was observed. The C4‐type expression pattern was established concomitantly with the development of bundle sheath cells during tissue maturation in the terrestrial internode. In contrast to the terrestrial form, the submerged form maintains C3‐type gene expression during tissue maturation. When the terrestrial culm was submerged, a region of transition from the terrestrial form to the submerged form was established in newly sprouting culms. In this transitional region, C4‐type expression of the two carboxylase genes was still maintained even though the development of bundle sheath cells was repressed. This observation suggests that the C4‐type cell‐specific gene expression pattern does not depend on the formation of Kranz anatomy.
NADPH is a specific inhibitor of protein import into glyoxysomes
Authors: Pool Martin R. , López‐Huertas Eduardo , Horng Jim‐Tong , Baker Alison
Published: March 2003

We have studied the import of proteins into glyoxysomes in vitro and show that this process is specifically inhibited by NADPH. NADPH affects both binding and translocation of proteins into glyoxysomes, and inhibition is determined by the ratio of NADP+ to NADPH. The site of action of NADPH is most likely within the glyoxysome because (1) pretreatment of glyoxysomes with NADPH, followed by re‐isolation of the organelles prior to the import assay, resulted in inhibition of import that could be restored by the addition of NADP+; (2) low concentrations of NADPH inhibited binding of proteins to broken glyoxysome membranes. The sensitivity of protein import to inhibition by NADPH declines as glyoxysomes are converted to leaf‐type peroxisomes. A model is proposed that speculates on a possible role for NADPH in regulating protein import into plant peroxisomes.
STF1 is a novel TGACG‐binding factor with a zinc‐finger motif and a bZIP domain which heterodimerizes with GBF proteins
Authors: Cheong Yong Hwa , Yoo Cheol Min , Park Jeong Mee , Ryu Gyeong Ryeol , Goekjian Virginia H. , Nagao Ron T. , Key Joe L. , Cho Moo Je , Hong Jong Chan
Published: March 2003

Two separate nuclear binding activities (B1 and B2) in the soybean apical hypocotyl have been identified that interact with a palindromic C‐box sequence (TGACGTCA) and which are developmentally regulated in an inverse manner. The bZIP factors responsible for these two binding activities, B1 and B2, were isolated from a cDNA library and designated STGA1 and STFs (STF1 and STF2), respectively. Sequence analysis shows that the STFs contain both a zinc‐finger domain and a bZIP domain. The two zinc finger sequences of Cys4–Cys4 are most related to the RING zinc‐finger motif carrying a Cys3‐His‐Cys4. In addition the bZIP domain of STFs is highly homologous to the HY5 protein of Arabidopsis. DNA binding studies revealed that STF1 binding to the TGACGT sequence requires distinct flanking sequences. Furthermore, STF1 binds to the Hex sequence as a heterodimer with G‐box binding factors (GBFs), a feature not observed with STGA1. Since STF1 expression is most prevalent in apical and elongating hypocotyls, it is proposed that STF1 may be a transcription factor involved in the process of hypocotyl elongation.
Conservation of gene structure and activity in the regulation of reproductive organ development of conifers and angiosperms
Authors: Tandre Karolina , Svenson Marie , Svensson Mats E. , Engström Peter
Published: March 2003

The Norway spruce (Picea abies) gene DAL2 shows distinct structural similarities to angiosperm MADS‐box genes which act in the control of the development of the sexual organs of the flower. Transcription of DAL2 is restricted to the reproductive organs, the unisexual cones, of Norway spruce. In this paper we show that DAL2 in the compound female cone is exclusively expressed in the developing ovule‐bearing organ, the ovuliferous scale. When expressed constitutively in transgenic Arabidopsis the gene causes developmental alterations very similar to those observed in plants ectopically expressing the Arabidopsis gene AGAMOUS and the closely related Brassica napus gene BAG1. These alterations include homeotic transformations of floral organs. On the basis of these data and analysis of the phylogeny of the plant MADS‐box gene family, we propose that DAL2 acts to control reproductive organ development in spruce. We also propose that DAL2 shares a common origin with AGAMOUS and related genes from other angiosperms, in an ancestral MADS‐box gene that was active in the control of ontogeny of ovule‐bearing organs in the unknown last common ancestor of conifers and angiosperms.
A mutant deficient in the plastid lipid DGD is defective in protein import into chloroplasts
Authors: Chen Lih‐Jen , Li Hsou‐min
Published: March 2003

Most proteins in chloroplasts are encoded by the nuclear genome and synthesized in the cytosol with N‐terminal extensions called transit peptides. Transit peptides function as the import signal to chloroplasts. The import process requires several protein components in the envelope and stroma and also requires the hydrolysis of ATP. Lipids have been implicated in the import process based on theories or experiments with

in vitro

model systems. We show here that chloroplasts isolated from an

Arabidopsis

mutant deficient in the plastid lipid digalactosyl diacylglycerol (DGD) were normal in importing a chloroplast outer membrane protein, but were defective in importing precursor proteins targeted to the interior of chloroplasts. The impairment includes the binding, or docking, step of the import process that is supported by 100 μM ATP.
The transient nature of the oligogalaturonide‐induced ion fluxes of tobacco cells is not correlated with fragmentation of the oligogalacturonides
Authors: Mathieu Yves , Guern Jean , Spiro Mark D. , O’Neill Malcolm A. , Kates Keith , Darvill Alan G. , Albersheim Peter
Published: March 2003

The metabolism by suspension‐cultured tobacco cells of oligogalacturonides was investigated. Dodecagalacturonic acid‐[3H]galactitol induces a rapid and transient alkalinization of the incubation medium resulting in part from enhanced K+ efflux from tobacco cells. However, a threefold higher concentration of dodecagalacturonic acid‐[3H]galactitol is required to induce a response with the same amplitude and kinetics as that induced by the unreduced tridecagalacturonic acid. Approximately 20% of the dodecagalacturonic acid‐[3H]galactitol added to suspension‐cultured tobacco ionically binds to the cell walls within 1 min; maximum binding (approximately 30% of the oligogalacturonide) occurs in approximately 25 min. The unbound dodecagalacturonic acid‐[3H]galactitol is rapidly (half‐life, 30 min) fragmented to smaller, biologically inactive fragments by a polygalacturonase present in the growth medium. In contrast, the wall‐bound dodecagalacturonic acid‐[3H]galactitol is not degraded for at least 150 min. However, the kinetics, amplitude and duration of oligogalacturonide‐induced ion fluxes are not correlated with the rate at which oligogalacturonides are converted to biologically inactive fragments. We propose that the transient nature of the oligogalacturonide‐induced responses is likely to result from a temporary desensitization of the plant cells to the bioactive oligogalacturonides.
Plants have a sensitive perception system for the most conserved domain of bacterial flagellin
Authors: Felix Georg , Duran Juliana D. , Volko Sigrid , Boller Thomas
Published: March 2003

The flagellum is an important virulence factor for bacteria pathogenic to animals and plants. Here we demonstrate that plants have a highly sensitive chemoperception system for eubacterial flagellins, specifically targeted to the most highly conserved domain within its N terminus. Synthetic peptides comprising 15–22 amino acids of this domain acted as elicitors of defence responses at sub‐nanomolar concentrations in cells of tomato and several other plant species. Peptides comprising only the central 8 to 11 amino acids of the active domain had no elicitor activity but acted as specific, competitive inhibitors in tomato cells. These antagonists suppressed the plant’s response to flagellin, crude bacterial extracts and living bacterial cells. Thus, plants have a highly sensitive and selective perception system for the flagellin of motile eubacteria.
M phase‐specific activation of theNicotiana sylvestris Cyclin B1promoter involves multiple regulatory elements
Authors: Tréhin Christophe , Glab Nathalie , Perennes Claudette , Planchais Séverine , Bergounioux Catherine
Published: March 2003

B‐type cyclins are cell cycle regulatory proteins specifically expressed in G2‐M phases. To understand the mechanisms that regulate their expression, it is important to identify the required promoter element(s). By using synchronized BY‐2 cell cultures stably transformed with chimeric genes composed of sequential Nicsy;

CycB1

; 1 promoter deletions fused to the β‐glucuronidase reporter gene (

gus

), we show that at least five distinct promoter regions are required for maximal M‐phase expression. Furthermore, two distinct promoter regions contain sufficient element(s) to induce M‐phase‐specific expression. In one of these regions, a 23 bp promoter element is able to activate reporter gene expression in cells induced to divide in an orientation‐independent manner but without an M‐phase specific expression. Therefore, this 23 bp element, which contains a 5 bp element identical to the MYB binding core, is a good upstream activating sequence (UAS) candidate. Moreover, electrophoretic mobility shift assays show that this putative UAS specifically binds protein complexes that appear to differ whether cells are cycling or not. The constitutive transcriptional activation mediated by this UAS would suggest that the Nicsy;

CycB1

; 1 gene promoter is regulated through an activation/repression mechanism.
Characterization of the ABA‐deficient tomato mutantnotabilisand its relationship with maizeVp14
Authors: Burbidge Alan , Grieve Teresa M. , Jackson Alison , Thompson Andrew , McCarty Donald R. , Taylor Ian B.
Published: March 2003

The

notabilis

(

not

) mutant of tomato has a wilty phenotype due to a deficiency in the levels of the plant hormone abscisic acid (ABA). The mutant appears to have a defect in a key control step in ABA biosynthesis – the oxidative cleavage of a 9‐

cis

xanthophyll precursor to form the C15 intermediate, xanthoxin. A maize mutant,

viviparous 14

(

vp14

) was recently obtained by transposon mutagenesis. This maize genetic lesion also affects the oxidative cleavage step in ABA synthesis. Degenerate primers for PCR, based on the VP14 predicted amino acid sequence, have been used to provide probes for screening a wilt‐related tomato cDNA library. A full‐length cDNA clone was identified which is specific to the

not

gene locus. The ORFs of the tomato cDNA and maize

Vp14

are very similar, apart from parts of their N‐terminal sequences. The

not

mutation has been characterized at the DNA level. A specific A/T base pair deletion of the coding sequence has resulted in a frameshift mutation, indicating that

not

is a null mutant. This observation is discussed in connection with the relatively mild phenotype exhibited by

not

mutant homozygotes.
The accumulation of triacylglycerols within the endoplasmic reticulum of developing seeds ofHelianthus annuus
Authors: Lacey Dominic J. , Beaudoin Frédéric , Dempsey Christopher E. , Shewry Peter R. , Napier Johnathan A.
Published: March 2003

Microsomes isolated from developing seeds of

Helianthus annuus

were prepared in a medium which ensured that endoplasmic reticulum (ER)‐bound polysomes remained attached to the ER during homogenization. The microsomes were then incubated with the substrates necessary to sustain the synthesis of triacylglycerols (TAGs). Microsomes that contained high activities of the enzymes involved in the synthesis of TAGs (the enzymes of the Kennedy pathway) accumulated TAGs synthesized

in vitro

, resulting in a decrease in their buoyant density. These light membrane fractions could therefore be separated on discontinuous sucrose density gradients from microsomes containing low activities of the enzymes of the Kennedy pathway. Analysis of the microsome fractions by 1H‐NMR spectroscopy showed that the TAGs synthesized in the microsomes

in vitro

were tumbling isotropically in an environment similar to that of the TAGs in oil bodies. Western blot analysis revealed that microsomes which synthesized large amounts of TAGs

in vitro

were also substantially enriched in oleosins. In addition, labelling studies indicated that the oleosins newly synthesized

in vitro

by ‘run‐on' translation of ER‐bound polysomes also localized to light membrane fractions. This indicates that oleosins are specifically enriched in regions of the ER involved in the biogenesis of the oil body.
Molecular characterization of transforming plasmid rearrangements in transgenic rice reveals a recombination hotspot in the CaMV 35S promoter and confirms the predominance of microhomology mediated recombination
Authors: Kohli Ajay , Griffiths Simon , Palacios Natalia , Twyman Richard M. , Vain Philippe , Laurie David A. , Christou Paul
Published: March 2003

The characterization of plasmid‐genomic DNA junctions following plant transformation has established links between DNA double‐strand break repair (DSBR), illegitimate recombination and plasmid DNA integration. The limited information on plasmid–plasmid junctions in plants comes from the dicot species tobacco and Arabidopsis. We analyzed 12 representative transgenic rice lines, carrying a range of transforming plasmid rearrangements, which predominantly reflected microhomology mediated illegitimate recombination involving short complementary patches at the recombining ends. Direct end‐ligation, in the absence of homology between the recombining molecules, occurred only rarely. Filler DNA was found at some of the junctions. Short, purine‐rich tracts were present, either at the junction site or in the immediate flanking regions. Putative DNA topoisomerase I binding sites were clustered around the junctions. Although different regions of the transforming plasmid were involved in plasmid–plasmid recombination, we showed that a 19 bp palindromic sequence, including the TATA box of the CaMV 35S promoter, acted as a recombination hotspot. The purine‐rich half of the palindromic sequence was specifically involved at the recombination junctions. This recombination hotspot is located within the ‘highly recombinogenic’ region of the full‐length CaMV RNA that has been shown to promote viral recombination in dicot plants. Clustering of plasmid recombination events in this highly recombinogenic region, even in the absence of viral enzymes and other cis‐acting elements proves that the plant cellular machinery alone is sufficient to recognize and act on these viral sequences. Our data also show the similarity between mechanisms underlying junction formation in dicot and monocot plants transformed using different procedures.
Matrix attachment regions increase transgene expression levels and stability in transgenic rice plants and their progeny
Authors: Vain Philippe , Worland Barbara , Kohli Ajay , Snape John W. , Christou Paul , Allen George C. , Thompson William F.
Published: March 2003

To investigate the effect of matrix attachment regions (MARs) on transgene expression levels and stability in cereal crops, we generated 83 independent transgenic rice callus lines containing a gusA expression cassette either as a simple expression unit, or flanked with MARs from tobacco (Rb7) or yeast (ARS1). Transgenic rice plants were regenerated from these callus lines and analysed at the structural and expression levels over two generations. In the first generation (T0), both Rb7 and ARS1 MARs significantly increased transgene expression levels. In the populations of plants containing MARs, we observed a significant reduction in the number of non‐expressing lines compared to the population of plants without MARs. However, variation in β‐glucuronidase (GUS) expression levels between independent lines was similar both in the presence and absence of flanking MARs. In the presence of MARs, GUS activity increased in proportion to transgene copy number up to 20 copies, but was generally reduced in lines carrying a higher copy number. In the population of plants without MARs, there was no correlation between expression level and transgene copy number. In the second generation (T1), transgene expression levels were significantly correlated with those of the T0 parents. The Rb7 MARs significantly improved the stability of transgene expression levels over two generations, and therefore appear to offer protection against transgene silencing. Our study shows that the exploitation of MARs may be an important strategy for stabilising transgene expression levels in genetically engineered cereals.
Double mutation in Eleusine indica α‐tubulin increases the resistance of transgenic maize calli to dinitroaniline and phosphorothioamidate herbicides
Authors: Anthony Richard G. , Hussey Patrick J.
Published: March 2003

The repeated use of dinitroaniline herbicides on the cotton and soybean fields of the southern United States has resulted in the appearance of resistant biotypes of one of the world's worst weeds, Eleusine indica. Two biotypes have been characterized, a highly resistant (R) biotype and an intermediate resistant (I) biotype. In both cases the resistance has been attributed to a mutation in α‐tubulin, a component of the α/β tubulin dimer that is the major constituent of microtubules. We show here that the I‐biotype mutation, like the R‐biotype mutation shown in earlier work, can confer dinitroaniline resistance on transgenic maize calli. The level of resistance obtained is the same as that for E. indica I‐ or R‐biotype seedlings. The combined I‐ and R‐biotype mutations increase the herbicide tolerance of transgenic maize calli by a value close to the summation of the maximum herbicide tolerances of calli harbouring the single mutations. These data, taken together with the position of the two different mutations within the atomic structure of the α/β tubulin dimer, imply that each mutation is likely to exert its effect by a different mechanism. These mechanisms may involve increasing the stability of microtubules against the depolymerizing effects of the herbicide or changing the conformation of the α/β dimer so that herbicide binding is less effective, or a combination of both possibilities.
Footprinting in vivo reveals changing profiles of multiple factor interactions with the β‐phaseolin promoter during embryogenesis
Authors: Li Guofu , Hall Timothy C.
Published: March 2003

Whereas in vitro techniques are essentially limited to the analysis of interactions with a single or limited number of cis‐elements, in vivo footprinting techniques can be used to assess the total profile of factor interactions with a promoter. By probing with dimethylsulphate and using sensitive ligation‐mediated PCR analytical techniques, the in vivo status of the phas promoter was determined in transcriptionally active (embryo) and inactive (leaf) tissues. Changes in factor occupancy were detected during embryogenesis, and the greatest complexity seen (at mid‐maturation) was in accordance with the many potential binding sites predicted on the basis of sequence comparison. Evidence was obtained that several cis‐elements not previously shown to be used for factor binding in plant promoters are occupied. The great complexity of footprints may represent the need for multiple factor interaction to achieve high levels of transcription. Alternatively, it is possible that the differential levels of expression in individual regions of the embryo evident from histochemical analysis of the GUS reporter result from the interaction of relatively few factors, with the overall footprinting pattern representing a summation of patterns from various tissues.
5′ to 3′ exoribonucleolytic activity is a normal component of chloroplast mRNA decay pathways
Authors: Drager Robert G. , Higgs David C. , Kindle Karen L. , Stern David B.
Published: March 2003

Molecular genetic studies have shown that determinants of chloroplast mRNA stability lie in both the 5′ and 3′ untranslated regions. While it is well‐known that chloroplast mRNAs are unstable in the absence of certain nucleus‐encoded factors, little is known of the decay mechanisms for chloroplast mRNA in wild‐type cells. Here we used a poly(G)18 sequence, which impedes both 5′→3′ and 3′→5′ exoribonucleolytic RNA decay in vivo, to study the degradation pathway of petD mRNA in wild‐type and mcd1 mutant chloroplasts of Chlamydomonas; the mcd1 mutant lacks a nucleus‐encoded factor required for petD mRNA accumulation. Upon inserting poly(G) at positions –20, +25, +165 or +25/+165 relative to the mature petD 5′ end, mRNAs accumulate with 5′ ends corresponding to the poly(G) sequence, in addition to the normal RNA with its 5′ end at +1. We interpret these results as evidence for continuous degradation of petD mRNA in wild‐type cells by a 5′→3′ exoribonucleolytic activity. In the case of the –20 insertion, the accumulating RNA can be interpreted as a processing intermediate, suggesting that 5′ end maturation may also involve this activity. When examined in the mcd1 mutant background, petD mRNAs with the poly(G) 5′ ends, but not normal +1 ends, accumulated. However, no expression of SUIV, the petD gene product, was detected. Insertion of poly(G) at +165 in wild‐type cells did not demonstrably affect SUIV accumulation, suggesting that ribosomal scanning does not occur upstream of this position. However, since neither poly(G) –20 nor +165 RNA could be translated in mcd1 cells, this raises the possibility that the MCD1 product is essential for translation.
Sequences throughout the basic β‐1,3‐glucanase mRNA coding region are targets for homology dependent post‐transcriptional gene silencing
Authors: Jacobs John J. M. R. , Sanders Matthew , Bots Marc , Andriessen Marco , van Eldik Gerben J. , Litière Katia , Van Montagu Marc , Cornelissen Marc
Published: March 2003

In the transgenic tobacco line T17, plants homozygous for the gn1 transgene display developmentally regulated post‐transcriptional silencing of basic β‐1,3‐glucanase genes. Previously, it has been shown that silencing involves a markedly increased turnover of silencing‐target glucanase mRNAs. Using a two‐component viral reporter system facilitated a comparison, in a quantitat‐ ive manner, of the relative silencing efficiencies of various sequences derived from the gn1 transgene. The results show that target sites for the silencing mechanism are present throughout the coding region of the gn1 mRNA. Similar‐sized coding region sequences along the entire gn1 mRNA display a similar susceptibility to the silencing mechanism. The susceptibility to silencing increases as the coding region elements increase in size. Relative to internal sequences, the 5′ and 3′ terminal regions of the gn1 mRNA are inefficient targets for the silencing machinery. Importantly, sequences of the gn1 transgene that are not part of the mature gn1 mRNA are not recognized by the silencing machinery when expressed in chimeric viral RNAs. These results show that the glucanase silencing mechanism in T17 plants is primarily directed against gn1 mRNA‐internal sequences and that terminal sequences of the gn1 mRNA are relatively unaffected by the silencing mechanism.
S‐protein mutants indicate a functional role for SBP in the self‐incompatibility reaction of Papaver rhoeas
Authors: Jordan Nicholas D. , Kakeda Katsuyuki , Conner Alex , Ride Jon P. , Franklin‐Tong Vernonica E. , Franklin F. Christopher H.
Published: March 2003

The self‐incompatibility response involves S‐allele specific recognition between stigmatic S proteins and incompatible pollen, resulting in S‐specific pollen inhibition. In Papaver rhoeas, the pollen S gene product is predicted to be a receptor that interacts with the stigmatic S protein in an S specific manner. We recently identified an S protein binding protein (SBP) in pollen that binds stigmatic S proteins, although apparently not in an S‐allele‐specific manner. In order to investigate the functional significance of the interaction between S proteins and SBP, we constructed mutant derivatives of the S1 protein and tested their SBP‐binding activity and their biological activity. Here we present an evaluation of nine mutant derivatives of the S1 protein. Western ligand blotting was used to show that mutations to amino acid residues in predicted loops 2 and 6 of the S1 protein cause significant reductions in their SBP‐binding activity. These same mutants show a concomitant reduction in their ability to inhibit incompatible pollen. This establishes a direct link between SBP binding and inhibition of incompatible pollen and implicates SBP as a pollen component playing a key role in the self‐incompatibility reaction. We discuss the possible nature of the contribution of SBP in the S‐specific rejection of incompatible pollen.