bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cloning and characterization ofMS5fromArabidopsis: a gene critical in male meiosis
Authors: Glover Julie , Grelon Mathilde , Craig Stuart , Chaudhury Abdul , Dennis Elizabeth 
Published: March 2003

Summary In this paper, we describe the cloning of the

MS5

gene, a gene essential for male fertility in

Arabidopsis

. We previously defined the

MS5

locus by characterizing an EMS‐induced allele,

ms5–1

. We identified a new allele of

MS5

(

ms5–2

) that was T‐DNA‐generated and used the T‐DNA tag to clone the gene. Sequencing of mutant and wild‐type alleles together with complementation of the

ms5–1

mutant phenotype with a wild‐type genomic clone confirmed the identity of the gene. Differences between the phenotypes of the two mutant alleles could be attributed to differences in mutant gene structure. The semi‐dominant and dominant negative phenotypes of the

ms5–2

mutant probably result from production of a truncated polypeptide. An unknown locus in Landsberg erecta can counteract the dominant negative phenotype of

ms5–2

. Mutations in

MS5

cause the formation of ‘polyads’– tetrads with more than four pools of chromosomes after male meiosis. Similarities between the MS5 sequence and that of a number of proteins were found; two that may be significant were with a synaptonemal complex protein and with a regulatory subunit of a cyclin‐dependent kinase. The

MS5

gene is a member of a small gene family highly conserved amongst plant species.
Two different genes encode ferrochelatase in Arabidopsis: mapping, expression and subcellular targeting of the precursor proteins
Authors: Chow Keng See , Singh Davinder Pal , Walker Amanda R. , Smith Alison G. 
Published: March 2003

Summary Ferrochelatase is the last enzyme of haem biosynthesis. We have isolated 27 independent ferrochelatase cDNAs from

Arabidopsis thaliana

by functional complementation of a yeast mutant. Twenty‐two of these cDNAs were similar to a previously isolated clone, AF3, and although they varied in length at the 5′ and 3′ ends, their nucleotide sequences were identical, indicating that they were derived from the same gene (ferrochelatase‐I). The remaining five cDNAs all encoded a separate ferrochelatase isoform (ferrochelatase‐II), which was 69% identical at the amino acid level to ferrochelatase‐I. Using RFLP analysis in recombinant inbred lines, the ferrochelatase‐I gene was mapped to chromosome V and that for ferrochelatase‐II to chromosome II. Northern analysis showed that both ferrochelatase genes are expressed in leaves, stems and flowers, and expression in the leaves is higher in the light than in the dark. However, in roots only ferrochelatase‐I transcripts were detected. High levels of sucrose stimulated expression of ferrochelatase‐I, but had no effect, or repressed slightly, the expression of the ferrochelatase‐II isoform. Import experiments into isolated chloroplasts and mitochondria showed that the ferrochelatase‐II gene encodes a precursor which is imported solely into the chloroplast, in contrast to ferrochelatase‐I which is targeted to both organelles. The significance of these results for haem biosynthesis and the production of haemoproteins, both within the plant cell and in different plant tissues, is discussed.
Characterization of a tobacco epoxide hydrolase gene induced during the resistance response to TMV
Authors: Guo Ailan , Durner Jörg , Klessig Daniel F. 
Published: March 2003

SummaryA clone encoding a putative soluble epoxide hydrolase (EH‐1), an enzyme which converts epoxides to diols, was isolated by differential screening of a cDNA library prepared from tobacco mosaic virus (TMV)‐infected tobacco leaves. To confirm that EH‐1 encodes an epoxide hydrolase, the recombinant EH‐1 protein produced in bacteria was shown to have high epoxide hydrolase activity in vitro. Infection of resistant but not susceptible tobacco cultivars induced the accumulation of EH‐1 transcripts in both the inoculated and uninoculated, systemic leaves. EH‐1 expression was also induced in the inoculated and systemic tissues of TMV‐infected NahG plants, which are unable to accumulate salicylic acid (SA). However, EH‐1 expression in the inoculated leaves of NahG plants was delayed, whilst in the systemic leaves the induction was both later and weaker, compared to that observed in wild‐type plants. Furthermore, exogenously applied SA or its functional analog 2,6‐dichloroisonicotinic acid (INA) caused a rapid and transient accumulation of EH‐1 transcripts, whereas an inactive SA analog did not. Thus, the induction of EH‐1 gene expression appears to be regulated by both SA‐independent and SA‐dependent pathways. Since EH‐1 was expressed only in TMV‐resistant tobacco after infection, and the encoded enzyme is thought to help metabolize toxic compounds, we propose that EH‐1 may play a role in protection from oxidative damage associated with defense responses. It may also play a role in generating signals for activation of certain defense responses.
The glutathione‐deficient, cadmium‐sensitive mutant,cad2–1, ofArabidopsis thalianais deficient in γ‐glutamylcysteine synthetase
Authors: Cobbett Christopher S. , May Mike J. , Howden Ross , Rolls Barbara 
Published: March 2003

Summary This paper reports that the glutathione (GSH)‐deficient mutant,

cad2–1

, of Arabidopsis is deficient in the first enzyme in the pathway of GSH biosynthesis, γ‐glutamylcysteine synthetase (GCS). The mutant accumulates a substrate of GCS, cysteine, and is deficient in the product, γ‐glutamylcysteine.

In vitro

enzyme assays showed that the

cad2–1

mutant has 40% of wild‐type levels of GCS activity but is unchanged in the activity of the second enzyme in the pathway, GSH synthetase. The

CAD2

locus maps to chromosome 4 and is tightly linked to a gene,

GSHA

, identified by a previously isolated cDNA. A genomic clone of

GSHA

complements both the phenotypic and biochemical deficiencies of the

cad2–1

mutant. The nucleotide sequence of the gene has been determined and, in the mutant, this gene contains a 6 bp deletion within an exon. These data demonstrate that the

CAD2

gene encodes GCS. The

cad2–1

mutation is close to the conserved cysteine which is believed to bind the substrate glutamate and the specific inhibitor L‐buthionine‐[S,R] sulfoximine (BSO). Both root growth and GCS activity of the

cad2–1

mutant was less sensitive than the wild‐type to inhibition by BSO, indicating that the mutation may alter the affinity of the inhibitor binding site.
A flax transposon identified in two spontaneous mutant alleles of theL6rust resistance gene
Authors: Luck Joanne E. , Lawrence Greg J. , Finnegan E. Jean , Jones David A. , Ellis Jeff G. 
Published: March 2003

Summary Two spontaneous mutant alleles of the

L6

flax rust resistance gene,

l6‐X3A

and

l6‐X117

, contain the same transposable element designated

dLute

(defective

Linum usitatissimum

transposable element). The element is 314 bp long, 70% AT‐rich and, because it contains no extended open reading frame, is probably non‐autonomous. It has 14 bp imperfect terminal inverted repeats related to those in the

Ac

family of plant transposons and, like

Ac

, causes 8 bp target site duplications upon insertion. Multiple copies of

dLute

‐related sequences exist in the flax genome. Rust resistant revertants were recovered amongst the progeny of both mutants and reversion was associated with excision of

dLute

. Excision either restored the wild‐type

L6

sequence or was imprecise, leaving sequence alterations (‘footprints’) resulting in one to three amino acid alterations in the L6 protein. No phenotypic differences were discerned between plants containing the standard and revertant

L6

alleles.
Molecular characterization of the mutableflakedallele for flower variegation in the common morning glory
Authors: Habu Yoshiki , Hisatomi Yasuyo , Iida Shigeru 
Published: March 2003

Summary The mutable

flaked

(or

af

) lines of the common morning glory bear white flowers with colored flakes and sectors. The

af

allele shows incomplete dominance. Plants in the heterozygous state

A/af

bear lightly colored flowers with intensely colored flakes and occasionally with white sectors. We showed that the mutable

af

allele is caused by insertion of a new transposable element,

Tip100

, into the

CHS‐D

gene intron.

Tip100

is 3.9 kb long and belongs to the

Ac/Ds

family. Although the timing and frequency of the flower variegation vary in different lines, they carry an identical mutable allele. We also noticed that a

flaked

subline, bearing variegated flowers, carries a

Tip100

derivative,

Tip100–1

. The structure of

Tip100–1

contains an additional 48 bp terminal sequence as tandem repeats and its integration site is identical to that of

Tip100

. Another line, with stable white flowers, is a double mutant carrying two copies of

Tip100

in the

CHS‐D

gene. These results are discussed with regard to the variegated phenotypes of flowers in various mutable lines.
Diversity and similarity among recognition sequences of Dof transcription factors
Authors: Yanagisawa Shuichi , Schmidt Robert J. 
Published: March 2003

Summary Dof proteins are a family of transcription factors that share a unique DNA‐binding domain. Dof proteins were found recently in association with diverse promoters of plant‐specific genes, suggesting various roles of Dof proteins in plants. Through binding site selection experiments using randomly synthesized DNA, the recognition sequences of four maize Dof proteins were systematically analyzed. All selected oligonucleotides contained an AAAG sequence, suggesting that this sequence is the recognition core of Dof proteins. In fact, a single mutation in this sequence abolished binding of all four Dof proteins. Furthermore, the preference of each Dof protein for the sequence flanking the core motif was also analyzed using oligonucleotides containing a fixed AAAG and random flanking sequences. Similar, as well as distinct, flanking sequences were observed among the optimal binding sites. Changes in the flanking sequences did affect DNA‐binding of Dof proteins.
Modification of gibberellin production and plant development in Arabidopsis by sense and antisense expression of gibberellin 20‐oxidase genes
Authors: P Jeremy , Phillips . Coles andrew L. , Croker Stephen J. , García‐Lepe Raol , Lewis Mervyn J. , Hedden Peter 
Published: March 2003

Summary Gibberellin (GA) 20‐oxidase catalyses consecutive steps late in GA biosynthesis in plants. In
Arabidopsis
, the enzyme is encoded by a gene family of at least three members (
AtGA20ox1
,
AtGA20ox2
and
AtGA20ox3
) with differential patterns of expression. The genes are regulated by feedback from bioactive GAs, suggesting that the enzymes may be involved in regulating GA biosynthesis. To investigate this, we produced transgenic
Arabidopsis
expressing sense or antisense copies of each of the GA 20‐oxidase cDNAs. Over‐expression of any of the cDNAs gave rise to seedlings with elongated hypocotyls; the plants flowered earlier than controls in both long and short days and were 25% taller at maturity. GA analysis of the vegetative rosettes showed a two‐ to threefold increase in the level of GA4, indicating that GA 20‐oxidase normally limits bioactive GA levels. Plants expressing antisense copies of
AtGA20ox1
had short hypocotyls and reduced rates of stem elongation. This was reflected in reduced levels of GA4 in both rosettes and shoot tips. In short days, flowering was delayed and the reduction in the rate of stem elongation was greater. Antisense expression of A
tGA20ox2
had no apparent effects in long days, but stem growth in one transgenic line grown in short days was reduced by 20%. Expression of antisense copies of
AtGA20ox3
had no visible effect, except for one transgenic line that had short hypocotyls. These results demonstrate that GA levels and, hence, plant growth and development can be modified by manipulation of GA 20‐oxidase expression in transgenic plants.
A chromosomal paracentric inversion associated with T‐DNA integration in Arabidopsis
Authors: Laufs Patrick , Autran Daphné , Traas Jan 
Published: March 2003

SummaryT‐DNA integration in the nuclear plant genome may lead to rearrangements of the plant target site. Here we present evidence for a chromosomal inversion of 26 cM bordered by two T‐DNAs in direct orientation, which is linked to the mgoun2 mutation. The integration sites of the T‐DNAs map at positions 80 and 106 of chromosome I and we show that each T‐DNA is bordered by plant sequences from positions 80 and 106, respectively. Although the T‐DNAs are physically distant, they are genetically closely linked. In addition, three markers located on the chromosome segment between the two T‐DNA integration sites show no recombination with the mgo2 mutation. We show that the inversion cannot be a consequence of a recombination event between the two T‐DNAs, but that the integration of the T‐DNAs and the inversion were two temporally linked events. T‐DNA integration mechanisms that could have led to this inversion are discussed.
Gravity‐induced reorientation of cortical microtubules observed in vivo
Authors: Himmelspach Regina , Wymer Carol L. , Lloyd Clive W. , Nick Peter 
Published: March 2003

SummaryCortical microtubules play an important role during morphogenesis by determining the direction of cellulose deposition. Although many triggers are known that can induce the reorientation of cortical plant microtubules, the reorientation mechanism has remained obscure. In our approach, we used gravitropic stimulation which is a strong trigger for microtubule reorientation in epidermal cells of maize coleoptiles. To visualize the gravitropically induced microtubule reorientation in living cells, we injected rhodamine‐conjugated tubulin into epidermal cells of intact maize coleoptiles that were exposed to gravitropic stimulation. From these in vivo observations, we propose a reorientation mechanism consisting of four different stages: (1) a transitional stage with randomly organized microtubules; (2) emergence of a few microtubules in a slightly oblique orientation; (3) co‐alignment: neighbouring microtubules adopt the oblique orientation resulting in parallel organized microtubules; and (4) the angle of these parallel, organized micro‐ tubules increases gradually. Thus, the overall reorienta‐ tion process could include selective stabilization/disassembly of microtubules (stage 2) as well as move‐ ment of individual microtubules (stages 3 and 4).
Targeted disruption of the plastid RNA polymerase genes rpoA, B and C1: molecular biology, biochemistry and ultrastructure
Authors: De Santis‐Maciossek Giovanna , Kofer Waltraud , Bock Armin , Schoch Siegrid , Maier Rainer M. , Wanner Gerhard , Rüdiger Wolfhart , Koop Hans‐Ulrich , Herrmann Reinhold G. 
Published: March 2003

Summary The plastid encoded RNA polymerase subunit genes rpoA
, B and C1 of tobacco were disrupted individually by PEG‐mediated plastid transformation. The resulting off‐white mutant phenotype is identical for inactivation of the different genes. The mutants pass through a normal ontogenetic cycle including flower formation and production of fertile seeds. Their plastids reveal a poorly developed internal membrane system consisting of large vesicles and, occasionally, flattened membranes, reminiscent of stacked thylakoids. The rpo

–

material is capable of synthesising pigments and lipids, similar in composition but at lower amounts than the wild‐type. Western analysis demonstrates that plastids contain nuclear‐coded stroma and thylakoid polypeptides including terminally processed lumenal components of the Sec but not of the ΔpH thylakoid translocation machineries. Components using the latter route accumulate as intermediates. In striking contrast, polypeptides involved in photosynthesis encoded by plastid genes could not be detected by Western analysis, although transcription of plastid genes, including the rrn operon, by the plastid RNA polymerase of nuclear origin is found as expected. Remarkably, ultrastructural, sedimentation and Northern analyses as well as pulse experiments suggest that rpo

–

plastids contain functional ribosomes. The detection of the plastid‐encoded ribosomal protein Rpl2 is consistent with these results. The findings demonstrate that the consequences of rpo gene disruption, and implicitly the integration of the two plastid polymerase types into the entire cellular context, are considerably more complex than presently assumed.
Profilin in Phaseolus vulgaris is encoded by two genes (only one expressed in root nodules) but multiple isoforms are generated in vivo by phosphorylation on tyrosine residues
Authors: Guillén Gabriel , Valdés‐López Víctor , Noguez Raúl , Olivares Juan , Rodríguez‐Zapata Luis Carlos , Pérez Héctor , Vidali Luis , Villanueva Marco A. , Sánchez Federico 
Published: March 2003

SummaryActin‐binding proteins such as profilins participate in the restructuration of the actin cytoskeleton in plant cells. Profilins are ubiquitous actin‐, polyproline‐, and inositol phospholipid‐binding proteins, which in plants are encoded by multigene families. By 2D‐PAGE and immunoblotting, we detected as much as five profilin isoforms in crude extracts from nodules of Phaseolus vulgaris. However, by immunoprecipitation and gel electrophoresis of in vitro translation products from nodule RNA, only the most basic isoform of those found in nodule extracts, was detected. Furthermore, a bean profilin cDNA probe hybridised to genomic DNA digested with different restriction enzymes, showed either a single or two bands. These data indicate that profilin in P. vulgaris is encoded by only two genes. In root nodules only one gene is expressed, and a single profilin transcript gives rise to multiple profilin isoforms by post‐translational modifications of the protein. By in vivo32P‐labelling and immunoprecipitation with both, antiprofilin and antiphosphotyrosine‐specific antibodies, we found that profilin is phosphorylated on tyrosine residues. Since chemical (TLC) and immunological analyses, as well as plant tyrosine phosphatase (AtPTP1) treatments of profilin indicated that tyrosine residues were phosphorylated, we concluded that tyrosine kinases must exist in plants. This finding will focus research on tyrosine kinases/tyrosine phosphatases that could participate in novel regulatory functions/pathways, involving not only this multifunctional cytoskeletal protein, but other plant proteins.
Molecular characterisation of a membrane‐bound galactosyltransferase of plant cell wall matrix polysaccharide biosynthesis
Authors: Edwards Mary E. , Dickson Cathryn A. , Chengappa Sumant , Sidebottom Christopher , Gidley Michael J. , Reid J. S. Grant 
Published: March 2003

SummaryGalactomannan biosynthesis in vitro is catalysed by membrane preparations from developing fenugreek seed endosperms. Two enzymes interact: a GDP‐mannose dependent (1→4)‐β‐d‐mannan synthase and a UDP‐galactose dependent (1→6)‐α‐d‐galactosyltransferase. The statistical distribution of galactosyl substituents along the mannan backbone, and the degree of galactose substitution of the primary product of galactomannan biosynthesis appear to be regulated by the specificity of the galactosyltransferase. We now report the detergent solubilisation of the fenugreek galactosyltransferase with retention of activity, the identification on gels of a putative 51 kDa galactosyltransferase protein, and the isolation, cloning and sequencing of the corresponding cDNA. The solubilised galactosyltransferase has an absolute requirement for added acceptor substrates. Beta‐(1→4)‐linked d‐manno‐oligosaccharides with chain lengths greater than or equal to 5 acted as acceptors, as did galactomannans of low to medium galactose‐substitution. The putative galactosyltransferase cDNA encodes a 51282 Da protein, with a single transmembrane alpha helix near the N terminus. We have also confirmed the identity of the galactosyltransferase by inserting the cDNA in frame into the genome of the methylotrophic yeast Pichia pastoris under the control of an AOX promoter and the yeast alpha secretion factor and observing the secretion of galactomannan α‐galactosyltransferase activity. Particularly high activities were observed when a truncated sequence, lacking the membrane‐spanning helix, was expressed.
Light regulation of assimilatory sulphate reduction in Arabidopsis thaliana
Authors: Kopriva Stanislav , Muheim Regula , Koprivova Anna , Trachsel Nadine , Catalano Cinzia , Suter Marianne , Brunold Christian 
Published: March 2003

SummaryAdenosine 5′‐phosphosulphate reductase (APR) is considered to be a key enzyme of sulphate assimilation in higher plants. We analysed the diurnal fluctuations of total APR activity and protein accumulation together with the mRNA levels of three APR isoforms of Arabidopsis thaliana. The APR activity reached maximum values 4 h after light onset in both shoots and roots; the minimum activity was detected at the beginning of the night. During prolonged light, the activity remained stable and low in shoots, but followed the normal rhythm in roots. On the other hand, the activity decreased rapidly to undetectable levels within 24 h of prolonged darkness both in shoots and roots. Subsequent re‐illumination restored the activity to 50% in shoots and to 20% in roots within 8 h. The mRNA levels of all three APR isoforms showed a diurnal rhythm, with a maximum at 2 h after light onset. The variation of APR2 mRNA was more prominent compared to APR1 and APR3. 35SO42– feeding experiments showed that the incorporation of 35S into reduced sulphur compounds in vivo was significantly higher in light than in the dark. A strong increase of mRNA and protein accumulation as well as enzyme activity during the last 4 h of the dark period was observed, implying that light was not the only factor involved in APR regulation. Indeed, addition of 0.5% sucrose to the nutrient solution after 38 h of darkness led to a sevenfold increase of root APR activity over 6 h. We therefore conclude that changes in sugar concentrations are also involved in APR regulation.
A tobacco plasma membrane calmodulin‐binding transporter confers Ni2+ tolerance and Pb2+ hypersensitivity in transgenic plants
Authors: Arazi Tzahi , Sunkar Ramanjulu , Kaplan Boaz , Fromm Hillel 
Published: March 2003

SummaryAll organisms require a minimal amount of metal ions to sustain their metabolism, growth and development. At the same time, their intrinsic metal‐uptake systems render them vulnerable to toxic levels of metals in the biosphere. Using radiolabeled recombinant calmodulin as a probe to screen a tobacco cDNA library, we have discovered a protein designated NtCBP4 (Nicotiana tabacum calmodulin‐binding protein) that can modulate plant tolerance to heavy metals. Structurally, NtCBP4 is similar to vertebrate and invertebrate K+ and to non‐selective cation channels, as well as to recently reported proteins from barley and Arabidopsis. Here we report on the subcellular localization of NtCBP4 and the phenotype of transgenic plants overexpressing this protein. The localization of NtCBP4 in the plasma membrane was manifested by fractionating tobacco membranes on sucrose gradients or by aqueous two‐phase partitioning, and subsequently using immunodetection. Several independent transgenic lines expressing NtCBP4 had higher than normal levels of NtCBP4. These transgenic lines were indistinguishable from wild type under normal growth conditions. However, they exhibited improved tolerance to Ni2+ and hypersensitivity to Pb2+, which are associated with reduced Ni2+ accumulation and enhanced Pb2+ accumulation, respectively. To our knowledge this is the first report of a plant protein that modulates plant tolerance or accumulation of Pb2+. We propose that NtCBP4 is involved in metal uptake across the plant plasma membrane. This gene may prove useful for implementing selective ion tolerance in crops and improving phytoremediation strategies.