bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular cloning and functional expression in yeast of CYP76B1, a xenobiotic‐inducible 7‐ethoxycoumarin O‐de‐ethylase from Helianthus tuberosus
Authors: Batard Yannick , LeRet Monique , Schalk Michel , Robineau Tiburce , Durst Francis , Werck‐Reichhart Danièle 
Published: March 2003

SummaryIn order to obtain plant markers of chemical stress and possible tools for the bio‐monitoring of pollution, a protein purification/PCR approach was used to isolate cDNAs of xenobiotic‐inducible P450 oxygenases. O‐dealkylation of 7‐ethoxycoumarin is catalysed in Helianthus tuberosus by cytochromes P450 strongly inducible by a wide range of xenobiotics. Therefore, a 7‐ethoxycoumarin O‐de‐ethylase (ECOD) was purified from induced tuber tissues (Batard et al. 1995). A primer designed from an internal peptide sequence, but also corresponding to a conserved P450 haem‐binding region, led to the generation of a gene‐specific probe corresponding to a P450 strongly inducible by aminopyrine. Two partial and 98% identical coding sequences were isolated from a cDNA library prepared from aminopyrine‐induced tuber. A full‐length cDNA was reconstituted by 5′‐RACE elongation. The protein deduced from this full‐length sequence, with 41.1% amino acid identity to CYP76A1 and high phylogenetic relationship to other CYP76s, was termed CYP76B1. CYP76B1 was expressed in yeast. Microsomes from the transformed yeast catalysed the NADPH‐dependent O‐deakylation of 7‐ethoxycoumarin. However, protein sequence as well as enzymological data indicated that CYP76B1 does not correspond to the purified ECOD protein. These results confirm previous data and demonstrate that several P450s in H. tuberosus are capable of actively catalysing the O‐de‐ethylation of ethoxycoumarin. Determination of the steady‐state level of CYP76B1 transcripts after slicing tuber tissues and ageing them in water, alone or in the presence of various chemicals, showed that the expression of this P450 was not responsive to mechanical stress, but was strongly induced by chemical treatments. CYP76B1 thus appears to be a good potential marker of chemical stress and of environmental pollution.
Sensing trehalose biosynthesis in plants
Authors: Goddijn Oscar , Smeekens Sjef 
Published: March 2003

SummaryA most unexpected finding in research on plant carbohydrate metabolism is the recent discovery that angiosperms encode genes whose products are involved in trehalose metabolism. The presence and functionality of such genes has been elegantly shown by expressing Arabidopsis‐derived trehalose phosphate synthase and trehalose phosphate phosphatase genes in yeast mutants lacking these enzymatic activities. Homologue sequences have now been cloned from a number of different plant species suggesting that the capacity to synthesise trehalose is ubiquitous in angiosperms. Except for Myrothamnus flabellifolius, trehalose biosynthesis has never been observed in tissues of higher plants, probably due to the presence of high levels of trehalase activity. The function of trehalose metabolism in plants is still a mystery. One of the postulated functions of trehalose metabolism in yeast is in the control of glucose repression and a similar function in sugar sensing can be proposed for plants as well.
Co‐operation between cytosolic and plastidic oxidative pentose phosphate pathways revealed by 6‐phosphogluconate dehydrogenase‐deficient genotypes of maize
Authors: Averill Rachel H. , Bailey‐Serres Julia , Kruger Nicholas J. 
Published: March 2003

SummaryThe aim of this work was to examine the extent to which the oxidative steps of the pentose phosphate pathway in the cytosol contribute to the provision of reductant for biosynthetic reactions. Maize (Zea mays L.) contains at least two loci (pgd1 and pgd2) that encode 6‐phosphogluconate dehydrogenase. Ten genotypic combinations of wild‐type (Pgd1+3.8;Pgd2+5) and null alleles of pgd1 and pgd2 were constructed in the B73 background. The maximum catalytic activity of 6‐phosphogluconate dehydrogenase in the roots of seedlings of these lines correlated with the number of functional pgd1 and pgd2 alleles. Enzyme activity in the double‐null homozygote (pgd1‐null;pgd2‐null) was 32% of that in B73 wild‐type suggesting the presence of at least one other isozyme of 6‐phosphogluconate dehydrogenase in maize. Subcellular fractionation studies and latency measurements confirmed that the products of pgd1 and pgd2 are responsible for the vast majority, if not all, of the cytosolic 6‐phosphogluconate dehydrogenase activity in maize roots. Essentially, all of the residual activity in the double‐null homozygote was confined to the plastids. Low concentrations (0.1–0.5 mM) of sodium nitrite stimulated 14CO2 production by detached root tips of both wild‐type and 6‐phosphogluconate dehydrogenase‐deficient maize seedlings fed [U‐14C]glucose. Analysis of the ratio of 14CO2 released from [1–14C]glucose relative to [6–14C]glucose (C1/C6 ratio) showed that stimulation of the oxidative pentose phosphate pathway by nitrite correlated with the dosage of wild‐type alleles of pgd1 and pgd2. The failure of 6‐phosphogluconate dehydrogenase‐deficient lines to respond to nitrite indicates that perturbation of the cytosolic oxidative pentose phosphate pathway can influence the provision of reductant in the plastid. We conclude that the plastidic and cytosolic oxidative pentose phosphate pathways are able to co‐operate in the provision of NADPH for biosynthesis.
Post‐transcriptional silencing of pectin methylesterase gene in transgenic tomato fruits results from impaired pre‐mRNA processing
Authors: Mishra Krishna K. , Handa Avtar K. 
Published: March 2003

SummaryTransgenic tomato fruit expressing a truncated fruit pectin methylesterase (PME) sense transgene under the control of CaMV 35S promoter show developmentally regulated co‐silencing of the endogenous fruit PME and sense transgene. This co‐silencing is associated with the onset of fruit PME gene expression. Detectable levels of PME protein and mRNA were not present in ripening transgenic fruits. However, several smaller transgene RNA species hybridizing to the 3′ end of the transgene are present in transgenic fruits, indicating degradation of sense transgene transcript. RT–PCR analyses of nuclear RNA from wild‐type and transgenic fruits, primed with oligo(dT), show the presence of spliced and unspliced transcripts for the fruit PME and the transgene, indicating a post‐transcriptional regulation of gene silencing. However, an accumulation of unspliced fruit PME transcript was obtained in the nuclei of transgenic fruit, using the randomly primed cDNAs in PCR amplification. Taken together, these results suggest that the ectopic expression of the fruit PME sense transgene interferes with processing of the endogenous PME pre‐mRNA.
Transcription of the Arabidopsis CPD gene, encoding a steroidogenic cytochrome P450, is negatively controlled by brassinosteroids
Authors: Mathur Jaideep , Molnár Gergely , Fujioka Shozo , Takatsuto Suguru , Sakurai Akira , Yokota Takao , Adam Günter , Voigt Brunhilde , Nagy Ferenc , Maas Christoph , Schell Jeff , Koncz Csaba , Szekeres Miklós 
Published: March 2003

SummaryThe Arabidopsis CPD gene encodes a cytochrome P450 steroid side‐chain hydroxylase (CYP90) that plays an essential role in the biosynthesis of the plant hormone brassinolide. Expression of the CPD gene is confined to cotyledons and leaf primordia in etiolated seedlings and detectable in the adaxial parenchyma of expanding leaves in light‐grown plants. Transcription of the CPD gene is not affected by the plant growth factors auxin, ethylene, gibberellin, cytokinin, jasmonic acid and salicylic acid, but is specifically down‐regulated by brassinolide in both dark and light. Steady‐state mRNA levels of a CPD promoter‐driven uidA reporter gene correlate with the expression of resident CPD gene in transgenic plants. Intermediates of the early and late C‐6 oxidation pathways of brassinolide, carrying C‐22 and C‐23 side‐chain hydroxyls, efficiently inhibit the activity of the CPD promoter. Repression of CPD transcription by brassinosteroids is sensitive to the protein synthesis inhibitor cycloheximide, indicating a requirement for de novo synthesis of a regulatory factor.
Localization of a myosin‐like protein to plasmodesmata
Authors: Radford Janine E. , White Rosemary G. 
Published: March 2003

SummaryMyosin has been localized to plasmodesmata in root tissues of Allium cepa, Zea mays and Hordeum vulgare using a polyclonal antibody to animal myosin in both fluorescence and electron microscopy. Labelling was also observed throughout the cytoplasm, mainly associated with the endoplasmic reticulum and plasma membrane. On Western blots, bands of 180 and 110 kDa were consistently labelled in all three species. These bands were also labelled when the blot was incubated in actin prior to staining with antibodies to actin, raising the possibility that either of these proteins (180 kDa or 110 kDa) may be present in plasmodesmata. Pre‐treatment of the tissue with 2,3‐butanedione monoxime (BDM), an inhibitor of actin–myosin motility, resulted in a strong constriction of the neck region of plasmodesmata. These results indicate that a myosin‐like protein may be present in plasmodesmata and may also play a role in the regulation of transport at the neck region.
Sur2 mutations of Arabidopsis thaliana define a new locus involved in the control of auxin homeostasis
Authors: Delarue Marianne , Prinsen Els , Va Harry , Onckelen  , Caboche Michel , Bellini Catherine 
Published: March 2003

SummaryA new auxin homeostasis gene in Arabidopsis called SUR2 has been identified. This gene, mapped to the bottom of chromosome 4, is defined by two recessive nuclear mutants designated superroot2 (sur2), which display several abnormalities reminiscent of auxin effects. A number of these characteristics are similar to the phenotype of the previously described auxin‐overproducing mutant superroot1 (sur1); however, several lines of evidences reveal that the SUR2 gene defines a new key point in the regulation of endogenous auxin concentrations. The phenotype of the sur1 sur2 double mutant is additive. Analysis by gas chromatography coupled to mass spectrometry indicated increased levels of free indole‐3‐acetic acid correlated with a decreased level of bound auxin in the sur2 mutant. These results suggest that SUR2 may be involved in the control of auxin conjugation.
Promoters of nuclear‐encoded respiratory chain Complex I genes from Arabidopsis thaliana contain a region essential for anther/pollen‐specific expression
Authors: Zabaleta  , Heiser  , Grohmann  , Brennicke  
Published: March 2003

SummaryRegulatory promoter regions responsible for the enhanced expression in anthers and pollen are defined in detail for three nuclear encoded mitochondrial Complex I (nCI) genes from Arabidopsis thaliana. Specific regulatory elements were found conserved in the 5′ upstream regions between three different genes encoding the 22 kDa (PSST), 55 kDa NADH binding (55 kDa) and 28 kDa (TYKY) subunits, respectively. Northern blot analysis and transgenic Arabidopsis plants carrying progressive deletions of the promoters fused to the β‐glucuronidase (GUS) reporter gene by histochemical and fluorimetric methods showed that all three promoters drive enhanced expression of GUS specifically in anther tissues and in pollen grains. In at least two of these promoters the –200/–100 regions actively convey the pollen/anther‐specific expression in gain of function experiments using CaMV 35S as a minimal promoter. These nCI promoters thus contain a specific regulatory region responding to the physiological demands on mitochondrial function during pollen maturation. Pollen‐specific motifs located in these regions appear to consist of as little as seven nucleotides in the respective promoter context.
Expression of snowdrop lectin (GNA) in transgenic rice plants confers resistance to rice brown planthopper
Authors: Rao K. V. , Rathore Keerti S. , Hodges Thomas K. , Fu X. , Stoger Eva , Sudhakar D. , Williams Sarah , Christou Paul , Bharathi M. , Bown David P. , Powell Kevin S. , Spence Jacqui , Gatehouse Angharad M. R. , Gatehouse John A. 
Published: March 2003

Summary Snowdrop lectin (

Galanthus nivalis

agglutinin; GNA) has been shown previously to be toxic towards rice brown planthopper (

Nilaparvata lugens

; BPH) when administered in artificial diet. BPH feeds by phloem abstraction, and causes ‘hopper burn’, as well as being an important virus vector. To evaluate the potential of the

gna

gene to confer resistance towards BPH, transgenic rice (

Oryza sativa

L.) plants were produced, containing the

gna

gene in constructs where its expression was driven by a phloem‐specific promoter (from the rice sucrose synthase

RSs1

gene) and by a constitutive promoter (from the maize ubiquitin

ubi1

gene). PCR and Southern analyses on DNA from these plants confirmed their transgenic status, and that the transgenes were transmitted to progeny after self‐fertilization. Western blot analyses revealed expression of GNA at levels of up to 2.0% of total protein in some of the transgenic plants. GNA expression driven by the

RSs1

promoter was tissue‐specific, as shown by immunohistochemical localization of the protein in the non‐lignified vascular tissue of transgenic plants. Insect bioassays and feeding studies showed that GNA expressed in the transgenic rice plants decreased survival and overall fecundity (production of offspring) of the insects, retarded insect development, and had a deterrent effect on BPH feeding.

gna

is the first transgene to exhibit insecticidal activity towards sap‐sucking insects in an important cereal crop plant.
Polyunsaturated membranes are required for photosynthetic competence in a mutant ofArabidopsis
Authors: McConn Michele , Browse John 
Published: March 2003

Summary High levels of polyunsaturation are characteristic of all the membranes of plant and animal cells. For example, the chloroplasts of leaf cells contain about 75–80% polyunsaturated fatty acids. For the extra‐chloroplast membranes in leaf cells and the membranes of non‐photosynthetic tissues, values of 60–65% are typical. We report here the production of

Arabidopsis

double mutants that contain negligible levels of polyunsaturated fatty acids. The mutants were not capable of autotrophic growth and produced extremely chlorotic cotyledons and leaves. However, on sucrose media, the double mutants were robust plants showing strong leaf and root development. These observations indicate that the vast majority of receptor‐mediated and transport‐related membrane functions required to sustain the organism and induce proper development are adequately supported in the absence of polyunsaturated lipids. By contrast, photosynthesis is one process that does require high levels of membrane polyunsaturation.
A transgene with repeated DNA causes high frequency, post‐transcriptional suppression of ACC‐oxidase gene expression in tomato
Authors: Hamilton Andrew J. , Brown Stephen , Yuanhai Han , Ishizuka Masakatsu , Lowe Alex , Solis Angel‐Gabriel Alpuche , Grierson Don 
Published: March 2003

Summary Gene silencing with sense genes is an important method for down‐regulating the expression of endogenous plant genes, but the frequency of silencing is unpredictable. Fifteen per cent of tomato plants transformed with a 35S‐ACC‐oxidase (

ACO

1) sense gene had reduced ACC‐oxidase activity. However, 96% of plants transformed with an ACC‐oxidase sense gene, containing two additional upstream inverted copies of its 5′ untranslated region, exhibited reduced ACC‐oxidase activity compared to wild‐type plants. In the three plants chosen for analysis, there were substantially reduced amounts of both endogenous and transgenic ACO RNA, indicating that this was an example of co‐suppression. Ribonuclease protection assays using probes spanning intron‐exon borders showed that the reduced accumulation of endogenous ACO mRNA occurred post‐transcriptionally since the abundance of unprocessed transcripts was not affected. The

ACO1

transgene with the repeated 5′UTR also strongly inhibited the accumulation of RNA from the related

ACO

2 gene in flowers, although there is little homology between the 5′UTRs of

ACO

1 and

ACO

2. These results indicate that although repeated DNA in a transgene greatly enhances the probability of gene silencing of an endogenous gene, it also involves generation of a

trans

‐acting silencing signal produced, at least partly, from sequences external to the repeat.
FUSCA3encodes a protein with a conserved VP1/ABI3‐like B3 domain which is of functional importance for the regulation of seed maturation inArabidopsis thaliana
Authors: Luerßen Hartmut , Kirik Viktor , Herrmann Petra , Miséra Simon 
Published: March 2003

Summary Conditionally lethal mutant alleles of the

FUSCA3

(

FUS3

) gene of

Arabidopsis thaliana

are specifically defective in the gene expression program responsible for seed maturation.

FUS3

was isolated by map‐based cloning and expression of the

FUS3

cDNA resulted in complementation of the Fus3– phenotype. In the predicted FUS3 gene product, a continuous stretch of more than 100 amino acids shows significant sequence similarity to the B3 domains of the polypeptides encoded by

ABI3

(

Arabidopsis

) and

VP1

(maize).

FUS3

transcription was detected mainly in siliques and was found to be developmentally regulated during embryogenesis. Transcripts of abnormal sizes were observed in

fus3

mutants due to aberrant splicing caused by point mutations at intron termini. Sequence analysis of mutant and wild‐type

FUS3

alleles, as well as sequencing of

fus3

cDNAs, revealed small in‐frame deletions at two different sites of the coding region. While a deletion between B3 and the C‐terminus of the predicted polypeptide was found in conjunction with normal FUS3 function, another deletion located within the conserved B3 domain (as well as truncations therein) were associated with the Fus3– phenotype. It is apparent, therefore, that an intact B3 domain is essential for the regulation of seed maturation by FUS3.
An endosperm‐specific DOF protein from barley, highly conserved in wheat, binds to and activates transcription from the prolamin‐box of a native B‐hordein promoter in barley endosperm
Authors: Mena Montaña , Vicente‐Carbajosa Jesus , Schmidt Robert J. , Carbonero Pilar 
Published: March 2003

Summary A cDNA encoding a DNA‐binding protein of the DOF class of transcription factors was isolated from a barley endosperm library. The deduced amino acid sequence for the corresponding protein is 94% identical through the DOF domain to the prolamin‐box (P‐box) binding factor PBF from maize. The gene encoding the barley PBF (BPBF) maps to chromosome 7H, and its expression is restricted to the endosperm where it precedes that of the hordein genes. The BPBF expressed in bacteria as a GST‐fusion binds a P‐box 5′‐TGTAAAG‐3′ containing oligonucleotide derived from the promoter region of an

Hor2

gene. Binding was prevented when the P‐box motif was mutated to 5′‐TGTAgAc‐3′. A P‐box binding activity, present in barley and wheat endosperm nuclei, interacted similarly to BPBF with this synthetic oligonucleotide, and the binding was abolished by 1,10‐phenanthroline. Transient expression experiments in developing barley endosperms demonstrate that BPBF transactivates transcription from the P‐box element of a native

Hor2

promoter and that direct binding of BPBF to its target site is essential for transactivation since mutations in the DOF DNA‐binding domain or in the P‐box motif of this promoter abolished both binding and transactivation. Evidence was also obtained for the presence in wheat of a

Pbf

homologue having similar DNA‐binding properties to that of BPBF. These results strongly implicate this endosperm‐specific DOF protein from barley as an important activator of hordein gene expression and suggest the evolutionary conservation of the

Pbf

gene function among small grain cereals.
Expression of a yeast‐derived invertase in developing cotyledons ofVicia narbonensisalters the carbohydrate state and affects storage functions
Authors: Weber Hans , Heim Ute , Golombek Sabine , Borisjuk Ljudmilla , Manteuffel Renate , Wobus Ulrich 
Published: March 2003

Summary In plants the carbohydrate state provides signals to adjust metabolism to specific physiological conditions. Storage‐active sink organs like seeds often contain high levels of sucrose. In order to change the sugar status during seed development a yeast‐derived invertase gene was expressed in

Vicia narbonensis

under control of the LeguminB4 promoter. A signal sequence targeted the invertase to the apoplast in maturing embryos. In the cotyledons, sucrose was decreased whereas hexoses strongly accumulated. There was a major reduction of starch whereas proteins were less affected. Vacuoles of cotyledon cells were enlarged and dry seeds wrinkled. Transcripts and enzyme activity of sucrose synthase, the small and large subunit of ADP‐glucose pyrophosphorylase as well as vicilin were downregulated. Sucrose phosphate synthase and legumin‐mRNAs were not affected. Analysing single seeds with different sucrose levels revealed a positive correlation of sucrose concentration to mRNA levels of sucrose synthase and most pronounced to ADP‐glucose pyrophosphorylase‐mRNA levels as well as to starch content. Glucose on the other hand did not show any correlation. After feeding 14C‐sucrose

in vitro

, the invertase‐expressing cotyledons partitioned less carbon into starch compared to the wild‐type. In the transgenic cotyledons, a relatively higher amount was directed into proteins compared to starch. We conclude that starch accumulation in developing cotyledons could be a function of sucrose concentration. Our results are consistent with a possible sucrose‐mediated induction of storage‐associated differentiation indicated by upregulation of specific genes of the starch synthesis pathway.
Changes in the cytoskeleton accompanying infection‐induced nuclear movements and the hypersensitive response in plant cells invaded by rust fungi
Authors: Skalamera Dubravka , Heath Michèle C. 
Published: March 2003

Summary During the infection of cowpea

(Vigna unguiculata)

by the cowpea rust fungus

(Uromyces vignae,

race 1

)

the plant nucleus moves towards and away from the invading hypha and eventually moves close to the fungus in the susceptible cultivar while it remains away in two cultivars which subsequently respond by resistance gene‐dependent plant cell death (the hypersensitive response, HR). The role of plant cytoskeleton in these responses was investigated by fluorescent microscopy and treatments with anticytoskeletal drugs. Observations of microtubule organization prior to cell death revealed that the sequence of events leading to protoplast collapse differed between the two resistant cultivars, suggesting a possibility of multiple pathways for cellular degradation during the HR. Different fixations produced two different microfilament patterns: a filament network and cables. Microfilament network remained visible even at later stages of cell death. Oryzalin and taxol reduced the incidence of autofluorescence that develops late in the death process, indicating a role of microtubules in the deposition of phenolics by adjacent living cells. Cell death and nuclear movements were not affected by oryzalin and taxol but were inhibited by cytochalasin E, suggesting that the microfilaments are required for the HR.