bluuebunny/crossref_metadata_2025_split huggingface dataset:
A deletion mutation at the ep locus causes low seed coat peroxidase activity in soybean
Authors: Gijzen Mark 
Published: March 2003

SummaryThe Ep locus severely affects the amount of peroxidase enzyme in soybean seed coats. Plants containing the dominant Ep allele accumulate large amounts of peroxidase in the hourglass cells of the sub‐epidermis. Homozygous recessive epep genotypes do not accumulate peroxidase in the hourglass cells and are much reduced in total seed coat peroxidase activity. To isolate the gene encoding the seed coat peroxidase and to determine whether it corresponds to the Ep locus, a cDNA library was constructed from developing seed coats and an abundant 1.3 kb peroxidase transcript was cloned. The corresponding structural gene was also isolated from a genomic library. Sequence analysis shows that the seed coat peroxidase is translated as a 352 amino acid precursor protein of 38 kDa. Processing of a putative 26 amino acid signal sequence results in a mature protein of 326 residues with a calculated mass of 35 kDa and a pl of 4.4. Using probes derived from the cDNA, genomic DNA blot hybridization and polymerase chain reaction analysis detected polymorphisms that distinguished EpEp and epep genotypes. Co‐segregation of the polymorphisms in an F2 population from a cross of EpEp and epep plants shows that the Ep locus encodes the seed coat peroxidase protein. Comparison of Ep and ep alleles indicates that the recessive gene lacks 87 bp of sequence encompassing the translation start codon. Analysis by RNA blot hybridization shows that epep plants have drastically reduced amounts of peroxidase transcript compared with EpEp plants. The peroxidase mRNA is abundant in seed coat tissues of EpEp plants during the late stages of seed maturation, and could also be detected in root tissues, but not in the flower, embryo, pod or leaf. The results indicate that the lack of peroxidase accumulation in seed coats of homozygous recessive epep plants is due to a mutation of the structural gene that reduces transcript abundance.
A moderate decrease of plastid aldolase activity inhibits photosynthesis, alters the levels of sugars and starch, and inhibits growth of potato plants
Authors: Haake Volker , Zrenner Rita , Sonnewald Uwe , Stitt Mark 
Published: March 2003

SummaryAntisense expression of a full length cDNA encoding plastid aldolase led to decreased expression of aldolase at the transcript and protein level in several ‘antisense’ potato transformants. To quantify the inhibition, activity was compared in corresponding leaves down a plant and in plants of different ages. Aldolase activity was decreased by 32–43%, 56–71%, 79–83% and 91–97% in A‐70, A‐3, A‐51 and A‐2. Separation on a Q‐Sepharose–FF column showed the decrease was due to inhibition of plastid aldolase. The transformants showed a small increase of Rubisco activity, a small decrease of phosphoribulokinase activity, and larger but subproportional decreases of sedoheptulose‐1,7‐biphosphatase and plastid fructose‐1,6‐bisphosphatase activity. Ambient photosynthesis was inhibited by 10%, 40%, 66% and 85% in A‐70, A‐3, A‐51 and A‐2. The transformants contained increased triose phosphates, and very low ribulose‐1,5‐bisphosphate and glycerate‐3‐phosphate. Chlorophyll fluorescence indicated that photosystem II was more reduced and thylakoid energization was increased. Starch synthesis was decreased by 16% and 36% in A‐70 and A‐3, whereas sucrose synthesis was less strongly inhibited. Plant growth was not significantly altered in A‐70, was decreased by 41% in A‐3, and was severely inhibited in plants with under 20% of wild‐type aldolase activity. Although plastid aldolase catalyses a readily reversible reaction, possesses no known regulatory properties, and would appear irrelevant for the control of metabolism and growth, small changes in its activity have marked consequences for photosynthesis, carbon partitioning and growth.
Effects of mevinolin on cell cycle progression and viability of tobacco BY‐2 cells
Authors: Hemmerlin Andréa , Bach Thomas J. 
Published: March 2003

SummaryMevinolin, an inhibitor of 3‐hydroxy‐3‐methylglutaryl‐CoA reductase, was used to study the importance of mevalonic acid (MVA) for cell cycle progression of tobacco (Nicotiana tabacum L.) BY‐2 cells. After treatment with 5 μM mevinolin, the cell cycle progression was completely blocked and two cell populations accumulated (80% in phase G0/G1 and 20% in G2/M). The arrest could be released by subsequent addition of MVA. Effects were compared to those caused by aphidicolin, an inhibitor of α‐like DNA polymerases that blocks cell cycle at the entry of the S phase. The 80% proportion of mevinolin‐treated TBY‐2 cells was clearly arrested before the aphidicolin‐inducible block. By the aid of a double‐blocking technique, it was shown that the mevinolin‐induced cell arrest of highly synchronized cells was due to interaction with a control point located at the mitotic telophase/entry G1 phase. Depending on the developmental stage, mevinolin induced rapid cell death in a considerable percentage of cells. Mevinolin treatment led to a partial synchronization, as shown by the increase in mitotic index. The following decrease was correlated with the above‐mentioned induction of cell death.
Systemic and local induction of an Arabidopsis thionin gene by wounding and pathogens
Authors: Vignutelli Alberto , Wasternack Claus , Apel Klaus , Bohlmann Holger 
Published: March 2003

SummaryThe Arabidopsis Thi2.1 thionin gene was cloned and sequenced. The promoter was fused to the uidA gene and stably transformed into Arabidopsis to study its regulation. GUS expression levels correlated with the steady‐state levels of Thi2.1 mRNA, thus demonstrating that the promoter is sufficient for the regulation of the Thi2.1 gene. The sensitivity of the Thi2.1 gene to methyl jasmonate was found to be developmentally determined. Systemic and local expression could be induced by wounding and inoculation with Fusarium oxysporum f sp. matthiolae. A deletion analysis of the promoter identified a fragment of 325 bp upstream of the start codon, which appears to contain all the elements necessary for the regulation of the Thi2.1 gene. These results support the view that thionins are defence proteins, and indicate the possibility that resistance of Arabidopsis plants to necrotrophic fungal pathogens is mediated through the octadecanoid pathway.
Translation initiation factors are differentially regulated in cereals during development and following heat shock
Authors: Gallie Daniel R. , Le Hanh , Tanguay Robert L. , Browning Karen S. 
Published: March 2003

SummaryThe level of protein synthetic activity varies greatly in plants during development or following many environmental stresses. In order to determine whether these global changes in protein synthesis involve changes in the expression of the translational machinery itself, the expression patterns of the initiation factors (eIFs) during cereal seed development, germination, and in leaves following a heat shock were investigated. The expression patterns of initiation factors during seed development fell into four classes: those whose levels were high during early to mid‐development but decreased during late seed development (eIF4 A, eIFiso4E, eIFiso4G, and most eIF3 subunits); those whose levels increased from early to mid‐development followed by a decrease during late seed development (eIF4B, eIF4E, eIF2α, eIF2β, and PABP); those whose levels steadily increased throughout seed development (eIF4G); and those whose levels remained constant (the p34 subunit of eIF3). In contrast to these observations, the expression patterns of the factors appeared to be coordinately regulated during early germination although differences were observed at later stages. Following a heat shock, the changes in initiation factor expression in wheat leaves fell into two classes, those whose level increased (eIF4G, eIFiso4G, eIF3, and PABP) and those whose level remained unchanged (eIF4 A, eIF4B, eIF4E, eIFiso4E). These observations reveal the complexity of the expression patterns of the initiation factors during seed development, germination, and following thermal stress and may have mechanistic importance for the selective translation of certain mRNAs under these conditions.
KAS IV: a 3‐ketoacyl‐ACP synthase fromCuphea sp. is a medium chain specific condensing enzyme
Authors: Dehesh Katayoon , Edwards Patricia , Fillatti JoAnne , Slabaugh Mary , Byrne James 
Published: March 2003

Summary cDNA clones encoding a novel 3‐ketoacyl‐ACP synthase (KAS) have been isolated from

Cuphea

. The amino acid sequence of this enzyme is different from the previously characterized classes of KASs, designated KAS I and III, and similar to those designated as KAS II. To define the acyl chain specificity of this enzyme, we generated transgenic

Brassica

plants over‐expressing the cDNA encoded protein in a seed specific manner. Expression of this enzyme in transgenic

Brassica

seeds which normally do not produce medium chain fatty acids does not result in any detectable modification of the fatty acid profile. However, co‐expression of the

Cuphea

KAS with medium chain specific thioesterases, capable of production of either 12:0 or 8:0/10:0 fatty acids in seed oil, strongly enhances the levels of these medium chain fatty acids as compared with seed oil of plants expressing the thioesterases alone. By contrast, co‐expression of the

Cuphea

KAS along with an 18:0/18:1‐ACP thioesterase does not result in any detectable modification of the fatty acids. These data indicate that the

Cuphea

KAS reported here has a different acyl‐chain specificity to the previously characterized KAS I, II and III. Therefore, we designate this enzyme KAS IV, a medium chain specific condensing enzyme.
Two dehydration‐inducible transcripts from the resurrection plantCraterostigma plantagineumencode interacting homeodomain‐leucine zipper proteins
Authors: Frank Wolfgang , Phillips Jonathan , Salamini Francesco , Bartels Dorothea 
Published: March 2003

Summary The molecular dissection of desiccation tolerance in the resurrection plant

Craterostigma plantagineum

led to the isolation of two dehydration‐stress inducible homeodomain‐leucine zipper genes (

CPHB‐1

and

‐2

). When the coding region of

CPHB‐1

was used as bait in the yeast two‐hybrid system, the ability of CPHB‐1 to form homodimers was demonstrated. The two‐hybrid system was also used to isolate CPHB‐2, which heterodimerises with CPHB‐1. Both transcripts are inducible by dehydration in leaves and roots, but steady state levels vary in response to exogenously applied ABA. Although expression of

CPHB‐1

is not inducible by ABA, the transcript level of

CPHB‐2

increases during ABA‐treatment. Both genes are expressed at very early stages of dehydration and thus may be involved in the regulation of gene expression during dehydration.

CPHB‐1

and ‐

2

differential expression in response to ABA suggests that they act in different branches of the dehydration‐induced signalling network.

In vitro

binding studies revealed that CPHB‐1 specifically binds to the pseudopalindromic sequence CAAT(C/G)ATTG. Using this element for

in vitro

binding studies with nuclear proteins from dehydrated leaves, an inducible DNA‐protein complex was identified.
Characterization of the TAC box, acis‐element within an elicitor‐inducible sesquiterpene cyclase promoter
Authors: Newman Jeffrey D. , Yin Shaohui , Chappell Joseph 
Published: March 2003

Summary The first unique step in the synthesis of the tobacco phytoalexin capsidiol is cyclization of farnesyl pyrophosphate catalyzed by 5‐epi‐aristolochene synthase (EAS), a sesquiterpene cyclase. Earlier work demonstrated that the elicitor‐inducibility of this enzyme activity corresponded to the transcriptional activation of at least one gene,

EAS4

, of a rather complex gene family consisting of > 10 members. To investigate the mechanism(s) controlling expression of this gene, fragments of the

EAS4

promoter were examined for binding by proteins in nuclear and whole‐cell extracts. A strong protein binding site (TAC box; ACTCTACAGTACTC) was identified between –245 and –232 by the electrophoretic mobility shift assay, and DNAase I and methylation interference footprinting. Several distinctly migrating bands representing protein–TAC box complexes were also observed in the mobility shift assays, and the relative abundance of these bands varied in extracts from cells at different stages of EAS induction. The TAC box binding factor (TacBBF) was purified > 450‐fold from crude whole‐cell extracts by a combination of DNA affinity and cation exchange chromatography. The purified fractions were enriched for polypeptides of 17 and 19 kDa and the DNA binding properties of these preparations were characterized. Mutation of 2 bp in the TAC box prevented protein binding

in vitro

and increased both basal and elicitor‐inducible gene expression 2.5‐fold in transgenic tobacco plants harboring promoter–GUS fusions, consistent with the notion that this

cis

‐element functions as a silencer or repressor of EAS gene expression.
Functional analysis of regulatory sequences controllingPR‐1 gene expression in Arabidopsis
Authors: Lebel Edouard , Heifetz Peter , Thorne Leigh , Uknes Scott , Ryals John , Ward Eric 
Published: March 2003

Summary The Arabidopsis PR‐1 gene is one of a suite of genes induced co‐ordinately during the onset of systemic acquired resistance (SAR), a plant defense pathway triggered by pathogen infection or exogenous application of chemicals such as salicylic acid (SA) and 2,6‐dichloroisonicotinic acid (INA). We have characterized

cis

‐acting regulatory elements in the PR‐1 promoter involved in INA induction using deletion analysis, linker‐scanning mutagenesis, and

in vivo

footprinting. Compared to promoter fragments of 815 bp or longer (which show greater than 10‐fold inducibility after INA treatment), induction of a 698 bp long promoter fragment is reduced by half and promoter fragments of 621 bp or shorter have lost all inducibility. Additionally, two 10‐bp linker‐scanning mutations centered at 640 bp and 610 bp upstream from the transcription initiation site are each sufficient to abolish chemical inducibility of a GUS reporter fusion. The –640 linker‐scanning mutation encompasses a region highly homologous to recognition sites for transcription factors of the basic leucine zipper class, while the –610 linker‐scanning mutation contains a sequence similar to a consensus recognition site for the transcription factor NF‐κB. Furthermore, several inducible

in vivo

footprints located at or nearby these motifs demonstrate significant and highly reproducible changes in DNA accessibility following SAR induction. This

in vivo

signature of protein–DNA interactions after INA induction is tightly correlated with the functionally important regions of the promoter identified by mutation analysis.
Use of the IGF BAC library for physical mapping of theArabidopsis thalianagenome
Authors: Mozo Teresa , Fischer Sabine , Meier‐Ewert Sebastian , Lehrach Hans , Altmann Thomas 
Published: March 2003

Summary In order to generate a physical map of the

Arabidopsis thaliana

genome based on bacterial artificial chromosome clones (BACs), an iterative high throughput hybridisation strategy was applied and its efficiency was evaluated. Thus, probes generated from both ends of 500 BAC clones selected from the

Arabidopsis

–IGF–BAC library were hybridised to the entire library gridded on high density filters. The 1000 hybridisation reactions identified 4496 clones (41.8% of the complete library, or 50.3% if organellar, centromeric, and ribosomal DNA carrying clones are excluded) which were assembled into a minimum of 220 contigs. These results demonstrate the viability of the applied ‘double‐end clone‐limited/sampling without replacement’ hybridisation strategy for the generation of a high resolution physical map, and provide a highly useful resource for map‐based gene cloning approaches and further genome analysis.
Regulation of gibberellin biosynthesis genes during flower and early fruit development of tomato
Authors: Rebers Mariken , Kaneta Tsuyoshi , Kawaide Hiroshi , Yamaguchi Shinjiro , Yang Young‐Yell , Imai Ryozo , Sekimoto Hiroyuki , Kamiya Yuji 
Published: March 2003

Summary Gibberellins (GAs) are essential for the development of fertile flowers in tomato, and may also be required immediately after fertilization. In the GA‐biosynthetic pathway, the reactions catalyzed by GA 20‐oxidases have been implicated as site of regulation. To study the regulation of GA biosynthesis in flower and early fruit development, we isolated three tomato GA 20‐oxidase cDNA clones,

Le20ox‐1, ‐2 and ‐3

. The three genes showed different organ‐specific patterns of mRNA accumulation. Analysis of the transcript levels of the three

GA 20‐oxidase

genes, as well as those of

copalyl diphosphate synthase

(

LeCPS

) and

GA 3β‐hydroxylase (Le3OH‐2)

during flower bud and early fruit development, revealed temporally distinct patterns of mRNA accumulation. Up until anthesis, transcripts were observed for

LeCPS, Le20ox‐1, ‐2 and Le3OH‐2

, with an accumulation of

Le20ox‐1

mRNA. In contrast to the high level of

Le3OH‐2

transcripts in the fully open flower, mRNA levels of

Le20ox‐1, ‐2 and LeCPS

were reduced at this stage. After anthesis,

LeCPS

and

Le20ox‐1

transcripts increased again. In addition,

Le20ox‐3

transcripts increased whereas the transcripts of

Le3OH‐2

decreased to an undetectable level.

In situ

hybridization results demonstrated that during early stages of bud development,

Le20ox‐2

transcripts were localized in the tapetum and placenta. The presented results supply novel data about localization of GA biosynthesis gene transcripts, and indicate that transcript levels of GA biosynthesis genes are all highly regulated during flower bud development.
Regional insertional mutagenesis of genes onArabidopsis thalianachromosome V using theAc/Dstransposon in combination with a cDNA scanning method
Authors: Ito Takuya , Seki Motoaki , Hayashida Nobuaki , Shibata Daisuke , Shinozaki Kazuo 
Published: March 2003

Summary For regional insertional mutagenesis of

Arabidopsis thaliana

genes, we combined a cDNA scanning method (Hayashida

et al

.

Gene

1995; 165:155–161) and an

Ac/Ds

transposon designed for local mutagenesis, and evaluated this approach with two overlapping yeast artificial chromosome (YAC) clones, CIC7E11 and CIC8B11, on

A. thaliana

chromosome 5. We applied a previously developed novel cDNA selection method using DNA latex particles (cDNA scanning method) to the two YAC clones and constructed two sub‐libraries in which cDNAs for genes on each YAC DNA were concentrated. From each sub‐library we isolated cDNAs for genes on each YAC DNA, partially sequenced them, and produced expressed sequence tags (ESTs). In total, 113 non‐redundant groups of cDNAs were obtained. Forty‐four per cent of these EST clones were novel, and 34% had significant homology to functional proteins from various organisms. In parallel, we transposed

Ds

from a donor

Ds–GUS–

T‐DNA line, Ds4391–20, already mapped to the CIC7E11/8B11 region. We obtained

Ds

‐transposed lines and recovered their

Ds

‐flanking genomic DNAs by thermal asymmetric interlaced (TAIL) polymerase chain reaction (PCR). Dot‐blot analysis indicated that 20% of the lines contained transposed

Ds

in the CIC7E11/8B11 region, suggesting that this

Ac/Ds

transposon system is effective for regional insertional mutagenesis. To isolate

Ds

insertion mutants in the genes identified from the CIC7E11/8B11 region, we carried out PCR screening from 800

Ds

‐containing lines using

Ds

‐specific and gene‐specific primers that were designed from the 113 cDNA sequences identified by the cDNA scanning method. We found that 49 lines contain

Ds

insertion mutations, and that five lines contain

Ds

mutations in genes that are mapped to the sequenced CIC7E11/8B11 genomic DNA region. These results indicate that combining the cDNA scanning method and the

Ac/Ds

transposon gives a powerful tool for regional insertional mutagenesis not only in

Arabidopsis

but also in other plants or crops whose genomes are not sequenced.
The dormancy‐related peroxiredoxin anti‐oxidant, PER1, is localized to the nucleus of barley embryo and aleurone cells
Authors: Stacy Robin A. P. , Nordeng Tommy W. , Culiáñez‐Macià Francisco A. , Aalen Reidunn B. 
Published: March 2003

SummaryProtection against desiccation‐induced injury, including damage by reactive oxygen species (ROS), is a necessary component of the genetic programmes active during late seed development. Likewise, protection against ROS respiration by‐products is required during seed imbibition and germination. Late embryogenesis abundant (LEA) proteins are proposed to protect seed tissues against desiccation‐induced damage. Specifically, the atypical Lea gene Per1 in barley (Hordeum vulgare L.) has been proposed to play a protective role in embryo and aleurone cells against free‐radical damage during late seed development and early imbibition. PER1 represents a subgroup of the peroxiredoxin family of thiol‐requiring anti‐oxidants with one conserved cysteine residue (1‐Cys), and displays in vitro anti‐oxidant activity. In this work, we use antiserum generated against PER1 to study protein accumulation patterns as well as localization at the tissue, cellular and subcellular level. While previous studies have shown the Per1 transcript to be dormancy‐related, we show here that the protein level is maintained in imbibed dormant seeds, but not in non‐dormant seeds. Our data identify the location of this seed‐specific peroxiredoxin as the nucleus of immature embryos and aleurone layers. Highest levels of protein are detected in nucleoli. In contrast, in mature imbibed dormant seeds, cytosolic levels are comparable to that of the nucleus. A putative nuclear localization signal (NLS) of bipartite nature was identified in the C‐terminal end of the PER1 sequence. Protective roles for PER1 in seeds are discussed.
Suppression of pea nuclear topoisomerase I enzyme activity by pea PCNA
Authors: Van Hop Duong , Gaikwad Amos , Yadav Badam Singh , Reddy Malireddy Kodandarami , Sopory Sudhir , Mukherjee Sunil Kumar 
Published: March 2003

SummaryProliferating cell nuclear antigen (PCNA), a highly conserved DNA polymerase accessory protein of eukary‐ otic kingdom, has not been studied thoroughly in bio‐ chemical terms in plants. We describe the isolation of the cDNA encoding PCNA from the pea cDNA library using the PCR approach. The cDNA was used for expression of pea PCNA in bacteria as a fusion protein (GST.PCNA) with the GST tag at the amino terminal end. The GST.PCNA stimulated the partially purified pea DNA polymerases approximately 30‐fold. The stimulation was due to the oligomeric form of GST.PCNA. The pea PCNA interacted with the recombinant type I pea topoiso‐ merase as well as the native pea nuclear topoisomerase I and repressed the DNA relaxation activities. However, the DNA binding activity of Topo I remained undisturbed in the presence of high amounts of PCNA, thereby signify‐ ing that the catalysis of Topo I was probably affected by PCNA.