bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and characterization of a cDNA clone from Catharanthus roseus encoding NADPH:cytochrome P‐450 reductase, an enzyme essential for reactions catalysed by cytochrome P‐450 mono‐oxygenases in plants
Authors: Meijer Annemarie H. , Cardoso M. Inês Lopes , Voskuilen John Th. , de Waal Anthony , Verpoorte Robert , Hoge J. Harry C.
Published: March 2003

The membrane‐bound flavoprotein NADPH:cytochrome P‐450 (cytochrome c) reductase, that functions in electron transfer to cytochrome P‐450 mono‐oxygenases, was purified from a cell suspension culture of the higher plant Catheranthus roseus. Anti‐serum raised against the purified protein was found to inhibit NADPH:cytochrome c reductase activity as well as the activities of the cytochrome P‐450 enzymes geraniol 10‐hydroxylase and trans‐cinnamate 4‐hydroxylase, which are involved in alkaloid biosynthesis and phenylpropanoid biosynthesis, respectively. Immunoscreening of a C. roseus cDNA expression library resulted in the isolation of a partial NADPH: cytochrome P‐450 reductase cDNA clone, which was identified on the basis of sequence homology with NADPH:cytochrome P‐450 reductases from yeast and animal species. The identity of the cDNA was confirmed by expression in Escherichia coli as a functional protein capable of NADPH‐dependent reduction of cytochrome c and neotetrazolium, two in vitro substrates for the reductase. The N‐terminal sequence of the reductase, which was not present in the cDNA clone, was determined from a genomic NADPH: cytochrome P‐450 reductase clone. It was demonstrated that the reductase probably is encoded by a single copy gene. A sequence comparison of this plant NADPH:cytochrome P‐450 reductase with the corresponding enzymes from yeast and animal species showed that functional domains involved in binding of the cofactors FMN, FAD and NADPH are highly conserved between all kingdoms. In C. roseus cell cultures a rapid increase of the reductase steady state mRNA level was observed after the addition of fungal elicitor preparations that are known to induce cytochrome P‐450‐dependent biosynthetic pathways.
Influence of bacterial strain genotype on transient expression of plasmid DNA in plant protoplasts
Authors: Torres Jorge Tovar , Block Annette , Hahlbrock Klaus , Somssich Imre E.
Published: March 2003

It is demonstrated that transient expression of plasmid DNA in plant protoplasts can be strongly influenced by the bacterial strain used for plasmid propagation. Four different promoter constructs containing two light‐responsive and two fungal elicitor‐responsive parsley promoters translationally fused to the reporter gene, β‐glucuronidase (GUS), were amplified in bacterial strains MC1061, DH5α or GM2163 and tested individually. Marked differences in basal expression levels and in fold inducibilities were observed upon transfection of parsley protoplasts. Low levels of basal expression and strong light or elicitor inducibilities were observed only with plasmid DNA derived from the methylation‐deficient GM2163 strain. In vitro methylation of DNA prior to transformation also drastically increased basal expression levels. The results suggest that DNA methylation may be partly responsible for deregulating promoter activity in the transient expression system.
Inheritance and genetic mapping of fruit sucrose accumulation in Lycopersicon chmielewskii
Authors: Chetelat Roger T. , Klann Ellen , DeVerna Joseph W. , Yelle Serge , Bennett Alan B.
Published: March 2003

Fruit of the domestic tomato (Lycopersicon esculentum Mill.) accumulate soluble sugars primarily in the form of the hexoses, glucose and fructose. In contrast, the predominant sugar in fruit of the wild tomato relative, L. chmielewskii, is sucrose. In the present study, the inheritance and linkage relations of sucrose accumulation were examined in interspecific L. esculentum x L. chmielewskii populations. In backcrosses to either the wild or domestic tomato, segregation for sucrose accumulation permitted qualitative analysis of the trait and indicated monogenic recessive control, although deviations from Mendelian inheritance were observed in some populations. This major gene, designated sucr, was mapped in F2, F3, and BC1F2 populations using a set of 95 informative RFLP and isozyme markers covering the tomato genome. A map location near the centromere of chromosome 3 was established, with tight linkage to the genomic clone TG102. Association of sucrose accumulation with yellow fruit, encoded by an allele of the r gene, permitted alignment with the classical map, thereby confirming the map location of sucr. A linkage map of the region surrounding sucr was obtained by monitoring recombination between flanking markers in the back‐crosses to tomato. A cDNA clone of tomato fruit acid invertase, TIV1, was mapped to TG102 and sucr, with no recombination between the two RFLP markers observed in over 1700 meiotic products. Despite the tight linkage, TG102 and TIV1 hybridize to distinct restriction fragments, hence do not represent the same gene. The genetic data strongly suggest that sucr is an allele of the invertase gene and thus support previous biochemical studies that demonstrated low invertase activity in sucrose‐accumulating fruit. L. hisutum, another low‐invertase, sucrose‐accumulating species, was hybridized with L. chmielewskii and the resulting F1 plants accumulated sucrose, indicating that genetic control of soluble sugar composition is conserved in these two species.
An improved method of plant megabase DNA isolation in agarose microbeads suitable for physical mapping and YAC cloning
Authors: Wing Rod A. , Rastogi Vipin K. , Zhang Hong‐Bin , Paterson Andrew H. , Tanksley Steven D.
Published: March 2003

The isolation of high quality megabase DNA from plant cells that is susceptible to a variety of molecular reagents is a critical first step in the physical analysis of complex genomes. A method for the isolation of such DNA by encapsulating plant protoplasts in agarose microbeads is presented. In comparison with the conventional agarose plug method, microbeads provide a dramatic increase in the surface area yielding megabase DNA that can be treated essentially as an aqueous DNA solution. Examples of the utility of DNA prepared by this technique for physical mapping, partial restriction enzyme digestion and cloning of large inserts as YACs are presented.
Promoter tagging with a promoterless ipt gene leads to cytokinin‐induced phenotypic variability in transgenic tobacco plants: implications of gene dosage effects
Authors: Hewelt Andreas , Prinsen Els , Schell Jeff , Van Onckelen Harry , Schmülling Thomas
Published: March 2003

Tobacco plants have been transformed with a T‐DNA construct harboring a promoterless cytokinin‐synthesizing ipt gene close to the right T‐DNA border. Eighteen out of 85 transgenic clones displayed phenotypic alterations typical for an enhanced cytokinin production. Northern blot analysis confirmed the transcriptional activation of the introduced gene by tagged plant promoters. The concentration of cytokinins, expressed as zeatinriboside equivalents, was increased up to sevenfold in transgenic tissues. These increases in cytokinin levels resulted in major developmental changes. Transgenic clones exhibited to different levels traits of a general cytokinin‐syndrome, i.e. reduced root growth, reduced apical dominance, reduced leaf surface, reduced growth of the stem and retarded leaf senescence or displayed localized and developmentally specific cytokinin‐induced alterations in otherwise normally developing plants. These traits were in particular a simultaneous break of dormancy in all axillary buds before or at the onset of flowering or the reorientation of the developmental pathway of secondary meristems or terminally differentiated cells. This indicates that endogenously produced cytokinins not only influence different growth parameters but have the potential to alter differentiation pattern. The results show that stably inherited developmental alterations due to a general or localized cytokinin overproduction can be obtained by the promoter‐tagging approach. The investigation of gene dosage effects in homozygote plants readresses the question of threshold levels for cytokinin effects on the developmental program of plants.
The effects of allelic variation at the Mla resistance locus in barley on the early development of Erysiphe graminis f.sp. hordei and host responses
Authors: Boyd Lesley A. , Smith Philip H. , Foster Elaine M. , Brown James K.M.
Published: March 2003

The development of Erysiphe graminis f.sp. hordei isolate CC1 was followed on the susceptible barley variety Pallas, and the near‐isogenic resistant varieties, P‐01 (Mla1), P‐02 (Mla3), P‐03 (Mla6) and P‐04B (Mla7). The development of CC1 was examined in relation to the host’s cellular resistance responses, papilla formation and hypersensitive cell death, and to the transcription of five barley defence‐related genes. The infection type exhibited by each variety was positively correlated with the time of CC1 developmental arrest and with the timing of the hypersensitive response, indicating a positive role for hypersensitive cell death in Mla resistance. On P‐01 and P‐03, a rapid hypersensitive response in the epidermis was associated with a higher percentage of germlings arrested in their development at the haustorial stage. On P‐02 and P‐04B, a slower hypersensitive response was associated with haustoria, and more elongating secondary hyphae were formed. There was a later, and more extensive combined epidermal and mesophyll hypersensitive response associated with elongating secondary hyphae on these two varieties. A significant papilla response by P‐03 corresponded to a larger number of germlings being arrested at the secondary lobed stage on this variety. In general, defence gene transcript levels were lower in Pallas than in the four resistant varieties. Levels of defence gene transcripts were often greater in tissues where a larger number of cells were undergoing hypersensitive cell death, suggesting an association between defence gene expression and the hypersensitive response.
Expression of recombinant Renilla luciferase in transgenic plants results in high levels of light emission
Authors: Mayerhofer R. , Langridge W.H.R. , Cormier M.J. , Szalay A.A.
Published: March 2003

A 970 bp DNA fragment which encodes the luciferase enzyme of the marine soft coral Renilla reniformis was fused to the cauliflower mosaic virus (CaMV) promoter. The construct pPCV702‐ruc was transferred into alfalfa protoplasts by Ca‐PEG‐mediated transformation and into tobacco, tomato and potato plants by Agrobacterium‐mediated transformation. The light emission from homogenates of alfalfa protoplasts transformed with pPCV702‐ruc was 16‐fold higher than that of protoplasts transformed with the same vector carrying the bacterial luxF gene. Application of a 3 µM aequous solution of 2‐benzyl luciferin (luciferin) on to calli, leaves, roots and slices of tomato fruits and potato tubers of transformed plants resulted in strong light emission within seconds which could be easily visualized by a photon counting camera. Light emissions obtained from tissue homogenates of tobacco plants containing a single copy of the pPCV702‐ruc construct were around 20‐fold higher than those from plants carrying multiple copies of the firefly luciferase gene and around 360‐fold higher than those from plants transformed with the bacterial luciferase gene. Owing to its high efficiency the Renilla luciferase may become a useful and novel tool for gene expression studies in plants and other systems.
Isolation of a flax (Linum usitatissimum) gene induced during susceptible infection by flax rust (Melampsora lini)
Authors: Roberts J.K. , Pryor A.
Published: March 2003

In a susceptible infection of flax (Linum usitatissimum), the obligate rust pathogen (Melampsora lini) can grow in the leaf without triggering the hypersensitive resistance response. The rust establishes specialized structures (haustoria) in plant mesophyll cells and induces changes in plant subcellular organization. Subtraction hybridization methods were used to isolate cDNA clones of mRNAs that have altered expression in infected leaves. Most of the cDNAs recovered were of fungal origin, but one clone, pFIS1 (flax inducible sequence No. 1), recovered from several independent experiments, was a plant‐specified mRNA that showed a 10‐fold increase in steady‐state levels during susceptible growth. The increase in fis1 mRNA levels was not seen in the resistant reaction (hypersensitive reaction) and the predicted protein sequence (551 amino acids with a predicted molecular weight of 61 kDa) has no similarity to known pathogenesis‐related proteins. Searches of sequence data bases showed that fis1 encodes a protein which contains amino acid sequence motifs that are conserved in all previously characterized aldehyde dehydrogenases.
Mutations throughout an Arabidopsis blue‐light photoreceptor impair blue‐light‐responsive anthocyanin accumulation and inhibition of hypocotyl elongation
Authors: Ahmad Margaret , Lin Chentao , Cashmore Anthony R.
Published: March 2003

This paper reports the characterization of novel mutations within the Arabidopsis thaliana HY4 gene, which has previously been shown to encode a protein (CRY1) with characteristics of a blue‐light photoreceptor. Several point mutations were identified within the amino‐terminal domain of CRY1—this region of CRY1 has high homology to photolyase and is likely to be involved in blue‐light‐mediated electron transfer. Mutations were found within the region of homology to the known chromophore binding domains of photolyase. Point mutations within the 200 amino acid carboxy‐terminal extension distinguishing CRY1 from photolyase, likewise disrupt function of the protein. CRY1 was originally defined as the photoreceptor responsible for blue‐light‐mediated inhibition of hypocotyl elongation and we now report that anthocyanin accumulation in germinating seedlings is an additional phenotype under the control of this photoreceptor—this is shown to be mediated in part by modulation of mRNA levels of chalcone synthase, one of the anthocyanin biosynthetic enzymes. The effect of the novel mutations on both inhibition of hypocotyl elongation and anthocyanin biosynthesis have been evaluated, and it is demonstrated that mutations with less severe effects on hypocotyl elongation show a similarly reduced effect on anthocyanin biosynthesis. These results are consistent with the cryptochrome photoreceptor mediating multiple regulatory pathways by the same primary mode of action.
Arabidopsis late‐flowering fve mutants are affected in both vegetative and reproductive development
Authors: Martínez‐Zapater José M. , Jarillo José A. , Cruz‐Alvarez Marilyn , Roldán Marta , Salinas Julio
Published: March 2003

The development of wild‐type Arabidopsis thaliana (L.) Heyhn and two late‐flowering fve mutants has been analysed under different environmental conditions. In wild‐type plants, short‐day photoperiods delay the floral transition as a consequence of lengthening all the developmental phases of the plant. Moreover, short days also alter the inflorescence structure by reducing the internode elongation and delaying the establishment of the floral developmental programme in the lateral meristems of the inflorescence and co‐florescences. Mutations at the FVE locus cause a delay in flowering time, and a change in the inflorescence structure, similar to the effect of short photoperiods on wild‐type plants. However, the effect of the fve mutations is additive to the effect of short days, and all the aspects of the Fve phenotype are corrected by vernalization. These results seem to indicate that FVE is not simply involved in timing the transition from vegetative to reproductive growth, but that it could play a role during all stages of plant development.
Immunolocalization of freezing‐tolerance‐associated proteins in the cytoplasm and nucleoplasm of wheat crown tissues
Authors: Houde Mario , Daniel Claude , Lachapelle Mario , Allard France , Laliberté Sylvie , Sarhan Fathey
Published: March 2003

A protein family associated with the development of freezing tolerance in wheat has been identified. This protein family is Gramineae‐specific and coordinately regulated by low temperature. Antibodies directed against the 50 kDa (WCS120) protein recognize at least 5 members of this family. Using these antibodies, the cellular content and location of this protein family was determined in cold‐acclimated wheat seedlings. Western analyses of subcellular fractions indicated the presence of all members of the family in the cytosolic and purified nuclear fractions. These proteins accumulated to 0.9% of soluble proteins after 21 days of cold acclimation in winter wheat. This represents a cellular concentration of 1.34 µM. Immunohistochemical localization showed that these proteins are highly expressed in the vascular transition zone. No detectable expression was found in mature xylem, in the shoot apical meristem or lateral root primordia. This differential tissue expression suggests that the sensitive cells near the regions where water tends to freeze first require a higher amount of these proteins. This observation is consistent with the fact that regrowth after freezing stress is highly dependent on the viability of this region of the crown. Electron microscopy analysis using immunogold labelling showed that these proteins are present in the cytoplasm and in the nucleoplasm. They are not found in cell walls or other organelles. In vitro cryoprotective assays indicated that the WCS120 protein (PD50 of 10 µg ml−1 or 0.2 µM) are as effective as BSA and sucrose (at 250 mM) against freezing denaturation of lactate dehydrogenase. These results suggest that this protein family may be involved in a general mechanism of protection in the soluble fraction of the cell. Their presence in the nucleoplasm may also suggest a possible protective function of the transcriptional machinery. The high hydrophilicity, the abundance and stability of these proteins to boiling suggest that they may provide a particular micro‐environment needed for cell survival in the sensitive vascular transition zone during freezing stress.
Ethylene regulates the timing of leaf senescence in Arabidopsis
Authors: Grbić Vojislava , Bleecker Anthony B.
Published: March 2003

The plant hormone ethylene influences many aspects of plant growth and development, including some specialized forms of programmed senescence such as fruit ripening and flower petal senescence. To study the relationship between ethylene and leaf senescence, etr1‐1, an ethylene‐insensitive mutant in Arabidopsis, was used. Comparative analysis of rosette leaf senescence between etr1‐1 and wild‐type plants revealed that etr1‐1 leaves live approximately 30% longer than the wild‐type leaves. Delayed leaf senescence in etr1‐1 coincided with delayed induction of senescence‐associated genes (SAGs) and higher expression levels of photosynthesis‐associated genes (PAGs). In wild‐type plants, exogenous ethylene was able to further accelerate induction of SAGs and decrease expression of PAGs. The extended period of leaf longevity in etr1‐1 was associated with low levels of photosynthetic activity. Therefore, the leaves in etr1‐1 functionally senesced even though the apparent life span of the leaf was prolonged.
Derepression of the activity of genetically engineered heat shock factor causes constitutive synthesis of heat shock proteins and increased thermotolerance in transgenic Arabidopsis
Authors: Lee Jeong Hee , Hübel Anja , Schöffl Fritz
Published: March 2003

ATHSF1 is a heat shock transcription factor (HSF) of Arabidopsis that is constitutively expressed but its activity for DNA binding, trimer formation and transcriptional activation of heat shock (hs) genes is repressed at normal temperatures. In this study the functional properties of chimeric HSF‐glucuronidase (GUS) fusion proteins were tested. Ectopic expression of HSF—GUS or GUS—HSF in transgenic Arabidopsis plants resulted in a derepression of HSF functions as shown by trimer formation, specific DNA binding, and the constitutive expression of heat shock proteins (HSPs) at normal temperature. A novel GUS activity‐staining protocol was used to show the specific binding of trimeric HSF fusion proteins to DNA and following hs, an interaction between chimeric HSF—GUS and authentic HSF proteins. The chimeric HSFs were insensitive to the negative regulation that counteracts activation of the authentic HSF at normal temperature. Heterotrimer complexes were reconstituted in vitro from recombinant ATHSF1 and HSF—GUS proteins expressed in Escherichia coli and using this protocol, the temperature‐dependent activation of wt HSF was monitored in vivo and in vitro. Transgenic plants expressing constitutively active HSF—GUS fusion proteins are also constitutive for HSPs. Approximately 20% of the maximum heat‐inducible levels of HSP18 were already present at normal temperature. The level of basic thermotolerance was significantly enhanced in these plants. The results indicate that genetic engineering using protein fusion is a very effective means to derepress the activity of an important regulatory protein in plants, that consequently activates a constitutive hs response in the absence of heat stress and eventually alters the thermotolerance phenotype.
Expression of Escherichia coli branching enzyme in tubers of amylose‐free transgenic potato leads to an increased branching degree of the amylopectin
Authors: Kortstee Anne J. , Vermeesch Angela M.S. , De Vries Beja J. , Jacobsen Evert , Visser Richard G.F.
Published: March 2003

In order to increase the branching degree of potato tuber starch, the gene encoding branching enzyme (glgB) of Escherichia coli was expressed in the amylose‐free potato mutant. The E. coli glgB was cloned in the binary vector pBIN19 under the transcriptional control of the potato Granule Bound Starch Synthase (GBSS) promoter and transitpeptide sequence. The E. coli glgB was cloned behind the two N‐terminal amino acids of the GBSS mature protein, creating a chimeric protein. Transgenic plants were obtained which expressed the E. coli branching enzyme as was shown by the presence of mRNA and protein in the tubers. Correctly processed protein was found both in the soluble and starch granule bound protein fraction. Analysis of the starch showed an increase in the branching degree (DE) of up to 25% more branchpoints. The increase in the number of branchpoints was due to the presence of more short chains, with a degree of polymerization (DP) of 16 glucose‐residues or less in the amylopectin. Changes in other characteristics of the starch, such as average chain length (CL) and λmax, indicated a more branched structure for starch of transformed plants as well.
Evidence that actin filaments are involved in controlling the permeability of plasmodesmata in tobacco mesophyll
Authors: Ding Biao , Kwon Myoung‐Ok , Warnberg Leif
Published: March 2003

The role of actin filaments in regulating plasmodesmal transport has been studied by microinjection experiments in mesophyll cells of tobacco (Nicotiana tabacum L. cv. Samsun). When fluorescent dextrans of various molecular sizes were each co‐injected with specific actin filament perturbants cytochalasin D (CD) or profilin into these cells, dextrans up to 20 kilodalton (kDa) moved from the injected cell into surrounding cells within 3–5 min. In contrast, when such dextrans were injected alone or co‐injected with phalloidin into the mesophyll cells, they remained in the injected cells. Phalloidin co‐injection slowed down or even inhibited CD‐ or profilin‐elicited dextran cell‐to‐cell movement. Dextrans of 40 kDa or larger were unable to move out of the injected cell in the presence of CD or profilin. These data suggest that actin filaments may participate in the regulation of plasmodesmal transport by controlling the permeability of plasmodesmata.
The Arabidopsis homeobox gene ATHB‐7 is induced by water deficit and by abscisic acid
Authors: Söderman Eva , Mattsson Jim , Engström Peter
Published: March 2003

Homeodomain‐leucine zipper (HD‐Zip) proteins are putative transcription factors encoded by a class of recently discovered homeobox genes as yet found only in plants. This paper reports on the characterization of one of these genes, ATHB‐7, in Arabidopsis thaliana. ATHB‐7 transcripts were present in all organs of the plant at low levels, but expression was induced several‐fold by water deficit, osmotic stress as well as by exogenous treatment with abscisic acid (ABA), a response being detectable at 10−8 M and reaching a maximum at 10−6 M ABA. The ATHB‐7 transcript was detected within 30 min after treatment with ABA and the transcript level was rapidly reduced after removal of the hormone. The induction of ATHB‐7 was shown to be mediated strictly via ABA, since no induction of ATHB‐7 was detectable in the ABA‐deficient mutant aba‐3 subjected to drought treatment. Induction levels in two ABA‐insensitive mutants abi2 and abi3 were similar to the wild‐type response. In the abi1 mutant, however, induction was impaired as 100‐fold higher concentrations of ABA were required for a maximum induction as compared with wild‐type. In this mutant the ATHB‐7 response was reduced also after drought and osmotic stress treatments. These results indicate that ATHB‐7 is transcriptionally regulated in an ABA‐dependent manner and may act in a signal transduction pathway which mediates a drought response and also includes ABI1.
rbohA, a rice homologue of the mammalian gp91phox respiratory burst oxidase gene
Authors: Groom Quentin J. , Torres Miguel Angel , Fordham‐Skelton Anthony P. , Hammond‐Kosack Kim E. , Robinson Nigel J. , Jones Jonathan D.G.
Published: March 2003

It has been hypothesized that plants contain respiratory burst oxidases which, upon activation, oxidize NADPH and generate extracellular superoxide, O2°−. These proteins are proposed to play a central role in defence against pathogens. However, plant DNA sequences that encode proteins with similarity to components of respiratory burst oxidases have not previously been reported. This paper describes the complete cDNA and genomic DNA sequence of the rice rbohA (for respiratory burst oxidase homologue) gene. The predicted RbohA product is most similar to the main catalytic subunit, gp91phox, of the respiratory burst oxidase of neutrophils. Reverse transcriptase PCR detects rbohA transcripts in both roots and shoots of healthy rice plants.
Arabidopsis mutants showing an altered response to vernalization
Authors: Chandler John , Wilson Allison , Dean Caroline
Published: March 2003

Flowering in many plant species is accelerated by a long period of cold temperature, known as a vernalization period. This research investigates how this cold temperature signal is perceived by plant cells and the mechanism by which it influences the transition to flowering. Mutagenesis of the late‐flowering, vernalization‐responsive, Arabidopsis mutant, fca, has yielded five independent mutations (termed vrn mutations) conferring an altered vernalization response. Allelism tests showed that these mutations fall into at least three complementation groups defining three loci named VRN 1, 2 and 3. The vrn1 and vrn2 mutations did not affect the acclimation response as judged by expression of cold‐induced transcripts and freezing tolerance assays. vrn1‐1 affected the short‐day vernalization response of Landsberg erecta and reduced the vernalization response of other late‐flowering Arabidopsis mutants. The acceleration of flowering by GA3 was not affected by vrn1‐1. The VRN 1 locus was mapped to chromosome 3.
Analysis of fast neutron‐generated mutants at the Arabidopsis thaliana HY4 locus
Authors: Bruggemann Edward , Handwerger Korie , Essex Carrie , Storz Gisela
Published: March 2003

Ionizing radiation is expected to produce mutants with deletions or other chromosomal rearrangements. These mutants are useful for a variety of purposes, such as creating null alleles and cloning genes whose existence is known only from their mutant phenotype; however, only a few mutations generated by ionizing radiation have been characterized at the molecular level in Arabidopsis thaliana. Twenty fast neutron‐generated alleles of the Arabidopsis HY4 locus, which encodes a blue light receptor, CRY1, were isolated and characterized. Nine of the mutant alleles displayed normal genetic behavior. The other 11 mutant alleles were poorly transmitted through the male gametophyte and were lethal in homozygous plants. Southern blot analysis demonstrated that alleles of the first group generally contain small or moderate‐sized deletions at HY4, while alleles of the second group contain large deletions at this locus. These results demonstrate that fast neutrons can produce a range of deletions at a single locus in Arabidopsis. Many of these deletions would be suitable for cloning by genomic subtraction or representational difference analysis. The results also suggest the presence of an essential locus adjacent to HY4.
Stereospecificity of NADH‐ferricyanide reductase is a convenient marker for the endoplasmic reticulum of plant cells
Authors: Fredlund Kenneth M. , Widell Susanne , Møller Ian M.
Published: March 2003

The stereospecificity of NADH‐ferricyanide reductase and NADH‐cytochrome c reductase in the endoplasmic reticulum (ER) for the α‐hydrogen on the nicotinamide ring is presented as a very sensitive and convenient assay to detect ER contamination in preparations of membranes lacking α‐specific NADH‐acceptor reductase, such as the plasma membrane and the tonoplast. The experimental details of the assay are given and the limitations explored (time‐course, amount of protein, possible side reactions, speed, reproducibility, etc.). The NADH‐ferricyanide reductase activity of plasma membranes from spinach and sugarbeet leaf was completely β‐specific and always showed a latency (increase upon addition of Triton X‐100), whereas the α‐specificity in the ER was non‐latent. This is consistent with the presence of mainly right‐side‐out vesicles in preparations of plasma membranes with the binding site for NADH and ferricyanide on the inner, cytoplasmic surface. In contrast, right‐side‐out ER vesicles have the binding site on the outer, cytoplasmic surface. The addition of as little as 1% of the α‐specific ER (on an NADH‐ferricyanide activity basis) to the spinach leaf plasma membrane could be detected with the stereospecificity assay. Wheat root plasma membrane showed some α‐specificity (in addition to β‐specificity) which was probably due to ER contamination since the activity was non‐latent. The stereospecificity assay is also shown to be useful in monitoring the separation of tonoplast vesicles from ER vesicles by countercurrent distribution of a light microsomal fraction. It follows that the NADH‐acceptor reductase activities in preparations of plasma membrane and tonoplast are due to distinct enzymes characteristic for those membranes.
The flow karyotype of Arabidopsis thaliana interphase chromosomes
Authors: Samoylova Tatiana I. , Meister Armin , Miséra Simon
Published: March 2003

The DNA contents of various aneuploid lines of Arabidopsis thaliana were measured by flow cytometry of 4′, 6‐diamidino‐2‐phenylindole‐stained interphase nuclei in suspensions and compared with each other as well as with the wild‐type. The fluorescence intensities for all lines were highly reproducible as were the deviations from the wild‐type. The results allowed the estimation of the relative DNA contents of each Arabidopsis chromosome and of chromosome arms. The sum of the surplus values for all trisomics was close to the value expected for the haploid (2C) DNA content. Only the line with the smallest telotrisome (Tr 3A) did not significantly differ in DNA content from that of the wild‐type. It is concluded that approximately 3% of the genome represents the limit for resolution of differences in DNA content in this system. Thus, the approach allows a fast and reliable screening for duplications and deficiencies extending to 3% of the Arabidopsis genome. Regarding chromosome sizes a comparison of the flow karyotype with existing karyotypes revealed differences which are discussed.
fhy1 defines a branch point in phytochrome A signal transduction pathways for gene expression
Authors: Barnes Simon A. , Quaggio Ronaldo B. , Whitelam Garry C. , Chua Nam‐Hai
Published: March 2003

Physiological analysis of the fhy1 mutant of Arabidopsis has led to the proposal that the mutant is deficient in a downstream component of the phytochrome A signal transduction pathway. To define this lesion at the molecular level, we have examined the expression of a range of phytochrome‐regulated genes in fhy1. In far‐red light, the regulation of genes such as CHS and CHI is blocked in fhy1, whereas the induction of CAB and NR genes is affected minimally. In contrast, the induction of all genes tested is blocked in a phytochrome A‐deficient mutant, confirming that gene expression in far‐red light is regulated solely by phytochrome A. Thus, fhy1 defines a branch point in phytochrome A signal transduction pathways for gene expression. Contrary to the general opinion that responses to continuous red light are mediated by phytochrome B and other photostable phytochromes, we have shown also that red light‐induction of CHS is mediated almost entirely by phytochrome A. Furthermore, phytochrome A‐mediated induction of CHS by red light is blocked in fhy1. The induction of CHS by blue light, however, is normal in fhy1, suggesting that although FHY1 is a component of the phytochrome A signaling pathway, it is not a component of the blue‐light signaling pathway for CHS expression.
The ubiquitin‐activating enzyme (E1) gene family in Arabidopsis thaliana
Authors: Hatfield Peggy M. , Gosink Mark M. , Carpenter Tami B. , Vierstra Richard D.
Published: March 2003

Conjugation of multiple ubiquitins serves as a committed step in the degradation of a variety of intracellular eukaryotic proteins by the 26S proteasome. Conjugates are formed via a three‐enzyme cascade; the initial step requires ubiquitin‐activating enzyme (E1), which couples ubiquitin activation to ATP hydrolysis. Previously, we showed that many higher plants contain multiple E1 proteins and described several E1 genes from wheat. To facilitate understanding of the roles of the different plant E1s, we characterized the E1 gene and protein family from Arabidopsis thaliana. Arabidopsis E1s are encoded by two genes (AtUBA1 and AtUBA2) that synthesize approximately 123‐kDa proteins with 81% amino acid sequence identity to each other and 44–75% sequence identity with confirmed E1s from other organisms. Like other E1 proteins, AtUBA1 and 2 contain a cysteine residue in the putative active site for forming the ubiquitin thiol‐ester intermediate. Enzymatic analysis of the corresponding proteins expressed in Escherichia coli demonstrated that both proteins activate ubiquitin in an ATP‐dependent reaction and transfer the activated ubiquitin to a variety of Arabidopsis E2s with near equal specificity. Expression studies by quantitative RT‐PCR and histochemistry with transgenic plants containing AtUBA promoter‐β‐glucuronidase‐coding region fusions showed that the AtUBA1 and 2 genes are co‐expressed in most, if not all, Arabidopsis tissues and cells. Collectively, the data indicate that E1 proteins, and presumably the rest of the ubiquitin pathway, are present throughout Arabidopsis. They also show that the AtUBA1 and 2 genes are not differentially expressed nor do they encode E1s with dramatically distinct enzymatic properties.
Identification and functional significance of a new class of non‐consensus‐type plastid promoters
Authors: Kapoor Sanjay , Suzuki Jon Y. , Sugiura Masahiro
Published: March 2003

The promoter regions of most plastid transcription units have been reported to consist of prokaryotic −10 and −35‐like consensus sequences. However, a few promoters with no homology to the consensus elements have also been characterized. A novel class of non‐consensus plastid promoters ‐ designated as non consensus type II (NC‐II) promoters ‐ that effect low‐level constitutive expression of respective genes in photosynthetic as well as non‐photosynthetic plastids is described in this paper. The abundance of NC‐II promoter‐derived transcripts remains unaltered even when light‐grown seedlings are shifted to the dark. In contrast, transcripts from −10 and −35‐like elements containing consensus type (CT) promoters accumulate to high levels in chloroplasts as compared with non‐photosynthetic plastids of roots and cultured cells. Moreover, accumulation of these transcripts is greatly affected by light. The inhibition of plastid protein synthesis has no apparent effect on the abundance of the NC‐II transcripts whereas levels of CT transcripts are greatly reduced. In vivo tagetitoxin (a plastid transcription inhibitor) treatment also reduces the levels of CT transcripts with no apparent inhibitory effect on the accumulation of NC‐II transcripts. The accumulation of transcripts from both classes of promoters, however, is reduced when cytoplasmic protein synthesis is inhibited by in vivo treatment with cycloheximide. The results are suggestive of the possible existence of at least two distinctive systems for the synthesis and/or maintenance of plastid transcripts which differentiate between two classes of transcripts in a promoter‐type specific manner.
Fructan of the inulin neoseries is synthesized in transgenic chicory plants (Cichorium intybus L.) harbouring onion (Allium cepa L.) fructan:fructan 6G‐fructosyltransferase
Authors: Vijn I. , Van Dijken A. , Sprenger N. , Van Dun K. , Weisbeek P. , Wiemken A. , Smeekens S.
Published: March 2003

Fructan (polyfructosylsucrose) is an important storage carbohydrate in many plant families. fructan:fructan 6G‐fructosyltransferase (6G‐FFT) is a key enzyme in the formation of the inulin neoseries, a type of fructan accumulated by members of the Liliales. We have cloned the 6G‐FFT from onion by screening a cDNA library using barley sucrose:fructan 6‐fructosyltransferase (6‐SFT) as a probe. The deduced amino acid sequence showed a high homology with plant invertases and 6‐SFT. Incubation of protein extracts from transgenic tobacco plants with the trisaccharide 1‐kestose and sucrose resulted in the formation of neokestose and fructans of the inulin neoseries with a degree of polymerization up to six. Introduction of the onion 6G‐FFT into chicory resulted in the synthesis of fructan of the inulin neoseries, in addition to the synthesis of linear inulin.
Introns act post‐transcriptionally to increase expression of the Arabidopsis thaliana tryptophan pathway gene PAT1
Authors: Rose Alan B. , Last Robert L.
Published: March 2003

The expression of the Arabidopsis thaliana PAT1 gene, which encodes the tryptophan biosynthetic enzyme phosphoribosylanthranilate transferase, was investigated using translational fusions of the PAT1 promoter to the GUS reporter gene. Independent stably transformed A. thaliana lines containing a single copy of a fusion that includes the entire plastid transit peptide and the first two introns of PAT1 had on average 30 times more GUS enzyme activity than plants transformed with a construct in which GUS was fused a short distance downstream of the PAT1 start codon. Plants containing the construct without introns or leader peptide accumulated undetectable amounts of PAT1‐GUS fusion protein and mRNA, even though the transcriptional rate of both fusion constructs was comparable. Fusions containing the entire transit peptide and either of the first two introns yield as much GUS activity as constructs containing both introns, but constructs containing the transit peptide but no introns give rise to much lower levels. Therefore, introns greatly enhance the expression of PAT1‐GUS fusions, and they act post‐transcriptionally to increase the steady‐state level of mRNA.
Inducible overexpression of oat arginine decarboxylase in transgenic tobacco plants
Authors: Masgrau Carles , Altabella Teresa , Farrás Rosa , Flores Dante , Thompson Andrew J. , Besford Robert T. , Tiburcio Antonio F.
Published: March 2003

To test the possible interaction of polyamines in plant growth responses, transgenic tobacco plants containing the Avena sativa L. (oat) arginine decarboxylase (ADC) gene under the control of a tetracycline‐inducible promoter were generated. Inducible overexpression of oat ADC in transgenic tobacco led to an accumulation of ADC mRNA, increased ADC activity and changes in polyamine levels. Transgenic lines, induced during vegetative stage, displayed different degrees of an altered phenotype, the severity of which was correlated with putrescine content. These phenotypic changes were characterized by short internodes, thin stems and leaves, leaf chlorosis and necrosis, as well as reduced root growth. This is the first report to show altered phenotypes as a consequence of polyamine changes under tetracycline‐induction in in vivo conditions. Interestingly, overexpression of oat ADC in tobacco resulted in similar detrimental effects to those observed by ADC activation induced by osmotic stress in the homologous oat leaf system. In the context of the role of specific polyamines in plant growth and development, the present results indicate that activation of the ADC pathway leading to high levels of endogenous putrescine (or its catabolytes) is toxic for the vegetative growth of the plant. In contrast, no visible phenotypic effects were observed in flowering plants following tetracycline induction. Further characterization of the different transgenic lines may shed light on the action of specific polyamines in different plant developmental processes.
Stilbene synthase gene expression causes changes in flower colour and male sterility in tobacco
Authors: Fischer Regina , Budde Ina , Hain Rüdiger
Published: March 2003

Stilbene synthase genes (STS) have previously been used to enhance disease resistance in plants. In order to study the effects of modified STS expression patterns in plants, heterologous promoters were fused to an STS gene and the chimeric genes were transferred to tobacco. Very high constitutive expression of STS mediated by a duplicated upstream region of the 35S RNA promoter from CaMV affected secondary biosynthetic pathways. STS over‐expression caused altered flower pigmentation and male sterility, probably due to competition between the introduced STS and the endogenous chalcone synthase for the substrates 4‐coumaroyl CoA and malonyl CoA. Furthermore, tobacco plants with tapetum‐specific STS expression were male‐sterile. Thus, STS genes are promising tools in strategies for engineering altered flower colours and the development of a novel hybrid seed system.
Light modulates the spatial patterns of 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase gene expression in Arabidopsis thaliana
Authors: Learned R. Marc , Connolly Erin L.
Published: March 2003

Although the coordinated biosynthesis of isoprenoid compounds is thought to be essential to the normal processes of plant growth and development, the mechanisms that regulate the mevalonate pathway in plants are not well understood. As the first committed step in the pathway, the conversion of 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG CoA) to mevalonic acid by HMG CoA reductase and the regulation of the genes encoding this enzyme have been implicated in the network that controls isoprenoid biosynthesis in higher plants. Using histochemical staining for β‐glucuronidase, as well as conventional RNA hybridization analysis, the temporal and spatial regulation of HMG1, one of the genes encoding HMG CoA reductase in the crucifer Arabidopsis thaliana, has been characterized. Furthermore, the HMG1 promoter is shown to be differentially responsive to illumination in different organs, and promoter activation by light deprivation is confined primarily to immature leaves. In contrast, expression of the HMG1 gene in roots is confined to the elongation zone and is not responsive to illumination. Light‐mediated regulation of HMG1 expression is shown to be an organ‐autonomous response that depends on direct illumination, and environmental cues regarding light do not appear to be exchanged between different organs in Arabidopsis. These studies reveal several new features of HMG1 regulation, and indicate that the high levels of HMG CoA reductase expression detected in immature leaves may be primarily attributed to the dark‐induced expression of HMG1, and that HMG1 is expressed at low levels throughout the plant in response to light. Thus, environmental cues interact with the developmental program to define the pattern of HMG1 gene expression in Arabidopsis.
A glucocorticoid‐mediated transcriptional induction system in transgenic plants
Authors: Aoyama Takashi , Chua Nam‐Hai
Published: March 2003

A novel chemical induction system for transcription in plants has been developed, taking advantage of the regulatory mechanism of vertebrate steroid hormone receptors. A chimeric transcription factor, designated GVG was constructed, consisting of the DNA‐binding domain of the yeast transcription factor GAL4, the transactivating domain of the herpes viral protein VP16, and the receptor domain of the rat glucocorticoid receptor (GR). The GVG gene was introduced into transgenic tobacco and Arabidopsis together with a luciferase (Luc) gene which was transcribed from a promoter containing six tandem copies of the GAL4 upstream activating sequence. Induction of luciferase activity was observed when the transgenic tobacco plants were grown on an agar medium containing dexamethasone (DEX), a strong synthetic glucocorticoid. Induction levels of the luciferase activity were well correlated with DEX concentrations in the range from 0.1 to 10 µM and the maximum expression level was over 100 times that of the basal level. Analysis of the induction kinetics by Northern blot analysis showed that the Luc mRNA was first detected 1 h after DEX treatment and increased to the maximum level in 4 h. The stationary induction level and the duration of the induction varied with the glucocorticoid derivative used. The GVG gene activity can also be regulated by DEX in transgenic Arabidopsis plants. The results indicate that a stringent chemical control of transcription can be achieved in plants with the GVG system. Advantages and potential uses of this system are also discussed.
Development of necrosis and activation of disease resistance in transgenic tobacco plants with severely reduced catalase levels
Authors: Takahashi Hideki , Chen Zhixiang , Du He , Liu Yidong , Klessig Daniel F.
Published: March 2003

Numerous studies argue that salicylic acid (SA) is an important component of the plant signal transduction pathway(s) leading to disease resistance. The discovery that the SA‐binding protein is a catalase, whose activity is blocked by SA, led to the proposal that one of SA’s modes of action is to inhibit this H2O2‐degrading enzyme and thus elevate H2O2 levels. To test this model, an attempt was made to mimic the action of SA by reducing the synthesis of catalase using antisense RNA technology. Analyses of transgenic tobacco plants that expressed the tobacco catalase 1 (cat1) or catalase 2 (cat2) gene in an antisense orientation indicate that there is no correlation between modest to high levels of reduction in catalase activity and activation of plant defenses such as pathogenesis‐related (PR)‐1 protein synthesis. However, three independent antisense catalase transgenic plants (ASCAT1 Nos 16, 17, and 28), which exhibited the most severe reduction in catalase activity (∼90% or more), developed chlorosis or necrosis on some of their lower leaves. These same leaves accumulated very high levels of PR‐1 proteins and showed enhanced resistance to tobacco mosaic virus. Necrosis and elevated SA, which appear to result from severe depression of catalase levels, may be responsible for the induction of these defense responses.