bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification and Ds‐tagged isolation of a new gene at the Cf‐4 locus of tomato involved in disease resistance to Cladosporium fulvum race 5
Authors: Takken Frank L. W. , Schipper Danny , Nijkamp H. John J. , Hille Jacques 
Published: March 2003

SummaryLeaf mould disease in tomato is caused by the biotrophic fungus Cladosporium fulvum. An Ac/Ds targeted transposon tagging strategy was used to isolate the gene conferring resistance to race 5 of C. fulvum, a strain expressing the avirulence gene Avr4. An infection assay of 2‐week‐old seedlings yielded five susceptible mutants, of which two had a Ds element integrated in the same gene at different positions. This gene, member of a gene family, showed high sequence homology to the C. fulvum resistance genes Cf‐9 and Cf‐2. The gene is predicted to encode an extracellular transmembrane protein containing a divided domain of 25 leucine‐rich repeats. Three mutants exhibited a genomic deletion covering most of the Lycopersicon hirsutum introgressed segment, including the Cf‐4 locus. Southern blot analysis revealed that this deletion includes the tagged gene and five homologous sequences. To test whether the tagged gene confers resistance to C. fulvum via Avr4 recognition, the Avr4 gene was expressed in planta. Surprisingly, expression of the Avr4 gene still triggered a specific necrotic response in the transposon‐tagged plants, indicating that the tagged resistance gene is not, or is not the only gene, involved in Avr4 recognition. Mutants harbouring the genomic deletion did not show this Avr4‐specific response. The deleted segment apparently contains, in addition to the tagged gene, one or more other genes, which play a role in the Avr4 responses. The tagged gene is present at the Cf‐4 locus, but it does not necessarily recognize Avr4 and is therefore designated Cf‐4A.
Physical strain‐mediated microtubule reorientation in the epidermis of gravitropically or phototropically stimulated maize coleoptiles
Authors: Fischer Karin , Schopfer Peter 
Published: March 2003

SummaryDuring gravitropic and phototropic curvature of the maize coleoptile, the cortical microtubules (MTs) adjacent to the outer epidermal cell wall assume opposite orientations at the two sides of the organ. Starting from a uniformly random pattern during straight growth in darkness, the MTs reorientate perpendicularly to the organ axis at the outer (faster growing) side and parallel to the organ axis at the inner (slower growing) side. As similar reorientations can be induced during straight growth by increasing or decreasing the effective auxin concentration, it has been proposed that these reorientations may be used as a diagnostic test for assessing the auxin status of the epidermal cells during tropic curvature. This idea was tested by determining the MT orientations in the coleoptile of intact maize seedlings in which the gravitropic or phototropic curvature was prevented or inversed by an appropriate mechanical counterforce. Forces that just prevented the coleoptile from curving in a gravity or light field prevented reorientations of the MTs. Forces strong enough to overcompensate the tropic stimuli by enforcing curvature in the opposite direction induced reorientations of the MTs opposite to those produced by tropic stimulation. These results show that the MTs at the outer surface of the coleoptile respond to changes in mechanical tissue strain rather than to gravitropic or phototropic stimuli and associated changes at the level of auxin or any other element in the signal transduction chain between perception of tropic stimuli and asymmetric growth response. It is proposed that cortical MTs can act as strain gauges in a positive feed‐back regulatory circle utilized for amplification and stabilization of environmentally induced changes in the direction of elongation growth.
Expression studies of Nrt2:1Np, a putative high‐affinity nitrate transporter: evidence for its role in nitrate uptake
Authors: Krapp Anne , Fraisier Vincent , Scheible Wolf‐Rüdiger , Quesada Alberto , Gojon Alain , Stitt Mark , Caboche Michel , Daniel‐Vedele Françoise 
Published: March 2003

SummaryExpression of the gene Nrt2Np, which encodes a putative high‐affinity nitrate transporter of Nicotiana plumbaginifolia was studied under variable physiological conditions. Nrt2Np is rapidly induced by very low nitrate concentrations and repressed by reduced nitrogen metabolites. Furthermore, Nrt2Np is expressed in coordination with other genes involved in nitrate assimilation (Nia, Nii). A deficiency in nitrate reductase activity, which is accompanied by high internal nitrate concentration and low levels of nitrogen metabolites, e.g. glutamine, leads to an overexpression of Nrt2Np, showing that high nitrate concentration per se does not repress Nrt2Np expression. By investigating plants with altered nitrate uptake properties, we showed a correlation between Nrt2 mRNA accumulation and 15N nitrate influx rates, providing the first evidence that the expression of Nrt2 correlates with the rate of nitrate uptake. In situ hybridization revealed a tissue‐specific expression pattern. Nrt2Np mRNA accumulation is localized throughout all layers of the root tip, being highest in epidermal and endodermal cells. However, in mature root tissue, Nrt2 expression was detected mainly in the lateral root primordia and in the epidermis.
Alfalfa malate dehydrogenase (MDH): molecular cloning and characterization of five different forms reveals a unique nodule‐enhanced MDH
Authors: Miller Susan S. , Driscoll Brian T. , Gregerson Robert G. , Gantt J. Stephen , Vance Carroll P. 
Published: March 2003

Summary Malate dehydrogenase (MDH) catalyzes the readily reversible reaction of oxaloacetate ; malate using either NADH or NADPH as a reductant. In plants, the enzyme is important in providing malate for C
4
metabolism, pH balance, stomatal and pulvinal movement, respiration, β‐oxidation of fatty acids, and legume root nodule functioning. Due to its diverse roles the enzyme occurs as numerous isozymes in various organelles. While antibodies have been produced and cDNAs characterized for plant mitochondrial, glyoxysomal, and chloroplast forms of MDH, little is known of other forms. Here we report the cloning and characterization of cDNAs encoding five different forms of alfalfa MDH, including a plant cytosolic MDH (cMDH) and a unique novel nodule‐enhanced MDH (neMDH). Phylogenetic analyses show that neMDH is related to mitochondrial and glyoxysomal MDHs, but diverge from these forms early in land plant evolution. Four of the five forms could effectively complement an

E. coliMdh– mutant. RNA and protein blots show that neMDH is most highly expressed in effective root nodules. Immunoprecipitation experiments show that antibodies produced to cMDH and neMDH are immunologically distinct and that the neMDH form comprises the major form of total MDH activity and protein in root nodules. Kinetic analysis showed that neMDH has a turnover rate and specificity constant that can account for the extraordinarily high synthesis of malate in nodules.
Amplicons of maize zein genes are conserved within genic but expanded and constricted in intergenic regions
Authors: Llaca Victor , Messing Joachim 
Published: March 2003

SummaryThe 78 101 base pair long sequence of a cluster of 22‐kDa alpha zein genes in the maize inbred BSSS53 was determined. Each zein gene is contained within a repeat unit that varies in length. If such a repeat, or amplicon, is aligned along the entire sequence, a 10.5‐fold sequence amplification is delineated. Because of insertions and deletions in intergenic regions, many of the zein genes are spaced over different distances. Only three out of 10 zein‐related sequences have an intact open reading frame, indicating an unusual large number of genes unable to contribute to the accumulation of normal‐size 22‐kDa zein proteins. It is proposed that the seven remaining zein‐related sequences be considered gene reserves because of their potential to be restored by gene conversion. Intergenic insertions in the cluster range from 1098 to 14 896 base pairs. Although they are composed of transposable element sequences, they also contain additional open reading frames, two of them showing homology to rice cDNA sequences. The average amplicon is 4423 base pairs long, with the sequence surrounding each zein gene more than 90 % conserved. Coincidently, the size of the amplicon is equivalent to the average gene density (one gene within 4640 bp) in the Arabidopsis thaliana genome, one of the smallest in plants. Successive steps of amplification and insertion of DNA might explain to a certain degree how genome size variation has been generated in plants.
Gibberellin‐photoaffinity labelling of two polypeptides in plant plasma membranes
Authors: Lovegrove Alison , Barratt D. H. Paul , Beale Michael H. , Hooley Richard 
Published: March 2003

Summary Two polypeptides of

M

r 68 kDa and 18 kDa were gibberellin (GA)‐photoaffinity labelled

in vitro

in plasma membrane preparations from oat (

Avena sativa

L.) aleurone and from leaves and stems of wild‐type and GA‐sensitivity mutants of different species. Labelling of these polypeptides could be competed by biologically active, but not by inactive, GAs, indicating the likely biological significance of these interactions. On 2‐dimensional gels the radiolabelled polypeptides were each resolved as one intensely labelled low abundance spot with a slightly lower pI form adjacent to it. There was a strong pH dependency for both labelling events, which correlated well with pH values at which GA are known to be most biologically active. A semi‐dwarf GA‐sensitivity mutant of sweet pea (

Lathyrus odoratus

L.),

lb

, showed reduced photoaffinity labelling of both polypeptides compared with the wild type,

Lb

. In the GA‐insensitive

Arabidopsis thaliana

mutant,

gai

, the level of labelling was the same as in wild type,

GAI

. This is the first report of GA‐binding proteins in plant plasma membranes. Some preliminary sequence data are given for one of the labelled polypeptides. We discuss these mutants and consider their possible roles in GA perception or action.
Specific accumulation of GFP in a non‐acidic vacuolar compartment via a C‐terminal propeptide‐mediated sorting pathway
Authors: Di Sansebastiano Gian‐Pietro , Paris Nadine , Marc‐Martin Sophie , Neuhaus Jean‐Marc 
Published: March 2003

Summary The green fluorescent protein (GFP) from

Aequorea victoria

can be detected in living plant cells after transient transformation of protoplasts. Expression of the GFP can be used to monitor protein trafficking in a mixed cell population and also to study the different function and importance of organelles in different cell types. We developed a vacuolar form of GFP that was obtained by replacing the C‐terminal endoplasmic reticulum (ER)‐retention motif of mGFP5‐ER by the vacuolar targeting peptide of tobacco chitinase A. The vacuolar GFP was transported and accumulated in the vacuole as expected. However, we found two patterns of GFP accumulation after prolonged incubation (18–24 h) depending on the cell type. Most chloroplast‐rich protoplasts had a fluorescent large central vacuole. In contrast, most chloroplast‐poor protoplasts accumulated the GFP in one smaller vacuole but not in the large central vacuole, which was visible under a light microscope in the same cell. This differential accumulation reflected the existence of two different vacuolar compartments as described recently by immunolocalization of several vacuolar markers. We were able to characterize the vacuolar compartment to which GFP is specifically targeted as non‐acidic, since it did not accumulate neutral red while acidic vacuoles did not accumulate GFP.
A cytokinesis‐defective mutant ofArabidopsis(cyt1) characterized by embryonic lethality, incomplete cell walls, and excessive callose accumulation
Authors: Nickle Todd C. , Meinke David W. 
Published: March 2003

Summary The genetic control of cell division in eukaryotes has been addressed in part through the analysis of cytokinesis‐defective mutants. Two allelic mutants of

Arabidopsis

(

cyt1–1

and

cyt1–2

) altered in cytokinesis and cell‐wall architecture during embryogenesis are described in this report. Mutant embryos appear slightly abnormal at the heart stage and then expand to form a somewhat disorganized mass of enlarged cells with occasional incomplete walls. In contrast to the

keule and knolle

mutants of

Arabidopsis and

the

cyd

mutant of pea, which also exhibit defects in cytokinesis during embryogenesis,

cyt1

embryos cannot be rescued in culture, are desiccation‐intolerant at maturity, and produce cell walls with excessive callose as revealed through staining with the aniline blue fluorochrome, Sirofluor. Some

cyt1

defects can be partially phenocopied by treatment with the herbicide dichlobenil, which is thought to interfere with cellulose biosynthesis. The distribution of unesterified pectins in

cyt1

cell walls is also disrupted as revealed through immunocytochemical localization of JIM 5 antibodies. These features indicate that

CYT1

plays an essential and unique role in plant growth and development and the establishment of normal cell‐wall architecture.
A 45 kDa protein isolated from the nodal walls ofChara corallinais localised to plasmodesmata
Authors: Blackman Leila M. , Gunning Brian E. S. , Overall Robyn L. 
Published: March 2003

Summary The cellular anatomy of the green alga,

Chara corallina,

was exploited to isolate putative plasmodesmataassociated proteins. In

C. corallina

, large internodal cells are symplastically connected via intervening nodal complexes of smaller cells which have plasmodesmata in their cell walls. Comparison of proteins extracted from walls with plasmodesmata (nodal complexes) with those from walls without plasmodesmata (external internodal walls) identified four putative plasmodesmata‐associated proteins. These putative plasmodesmata‐associated proteins were approximately 95, 45, 44 and 33 kDa. A monoclonal antibody (MAB45/22) was raised against the 45 kDa putative plasmodesmata‐associated protein (CPAP45). Using immunofluorescence, this antibody co‐localised with aniline blue induced fluorescence of callose in the source cell walls. MAB45/22 was localised to the plasmodesmata of

C. corallina

and, in particular, to the central cavity using immunogold cytochemistry. In contrast, a monoclonal antibody to callose specifically labelled the mouth of

C. corallina

plasmodesmata. MAB45/22 also labelled higher plant plasmodesmata.
Anions permeate and gate GCAC1, a voltage‐dependent guard cell anion channel
Authors: Dietrich Petra , Hedrich Rainer 
Published: March 2003

Summary GCAC1 is a strongly voltage‐dependent anion channel in the guard‐cell plasma membrane of

Vicia faba

. In patch–clamp experiments, we have investigated the permeation and gating properties of GCAC1 with respect to its anion dependence in the whole‐cell and excised‐patch configuration. The relative permeability followed the order SCN– > NO3– > Br– > Cl–, while the single‐channel conductances in symmetrical anionic solutions exhibited a nearly inverse sequence. The Cl– dependence of inward currents (Cl– release) is characterized by a maximum single‐channel conductance of 89 pS half‐saturating at 87 mM cytoplasmic chloride. In addition to this substrate saturation, anion release was also dependent on the external Cl– activity (

K

m = 16 mM). In the presence of SCN– and Cl–, the single‐channel conductance exhibited an anomalous mole‐fraction dependence, identifying GCAC1 as a multi‐ion single‐file pore. Using anions with increasing ionic size, a minimum pore diameter of 0.5 nm was assumed from their relative permeabilities. In line with an anion‐selective channel, a tenfold increase in the extracellular anion activity shifted the reversal potential by –59.8 mV. Simultaneously, the half‐activation potential shifted negatively by about 23 mV. A further analysis of the anion dependence revealed that extracellular rather than cytosolic anions affect the gating process of GCAC1. From anion substitution experiments, we conclude that anion concentration and species determines both permeation and gating of the plant anion channel GCAC1.
Molecular characterization of a plant FKBP12 that does not mediate action of FK506 and rapamycin
Authors: Xu Qiang , Liang Shuping , Kudla Jörg , Luan Sheng 
Published: March 2003

Summary Immuonosuppressive drugs FK506 and rapamycin block a number of signal transduction pathways in eukaryotic systems. The 12 kDa FK506 binding protein (FKBP12) mediates the action of both FK506 and rapamycin against their functional targets. In this report, we cloned, sequenced and characterized a gene encoding FKBP12 in

Vicia faba

(

Vf

FKBP12). While

Vf

FKBP12 is highly homologous to animal and yeast FKBP12, it does not mediate the action of FK506 and rapamycin. There are unique features in plant FKBP12 sequences that cause the variation in their function. One lies in the domain that is critical for interaction with calcineurin (CaN), the mammalian and yeast target of FKBP12‐FK506 complex. Protein–protein interaction assays revealed a low‐affinity and unstable

Vf

FKBP12‐FK506‐CaN ternary complex. In the genetic assay,

Vf

FKBP12 did not restore the sensitivity of yeast FKBP12 mutant to rapamycin or FK506, supporting that plant FKBP12‐ligand complexes are unable to block the function of the drug target. Also unique to plant FKBP12 proteins, a pair of cysteines is spatially adjacent to potentially form disulfide linkage. Treatment of

Vf

FKBP12 with reductant dithiothreitol (DTT) abolished the formation of

Vf

FKBP12‐FK506‐CaN ternary complex. Site‐directed mutagenesis to substitute one of the cysteines, Cys26, with Ser produced a similar effect as DTT treatment. These results indicate that an intramolecular disulfide bond is a novel structural feature required for the low–affinity interaction between plant FKBP12 and CaN. In conclusion, plant FKBP12 proteins have evolved structural changes that modify their protein‐protein interacting domains and cause loss of function against the drug targets.
Co‐ordinated expression of multiple enzymes in different subcellular compartments in plants
Authors: Dasgupta Santanu , Collins Glenn B. , Hunt Arthur G. 
Published: March 2003

Summary A gene expression system designed for coordinated expression of multiple genes in plants and their targeting to specified subcellular locations was tested. A series of genes encoding polyproteins containing the tobacco vein mottling virus (TVMV) NIa proteinase along with two other reporter genes (those encoding the

Escherichia coli

acetate kinase (ACK) and Tn9 chloramphenicol acetyl transferase (CAT) enzymes) were assembled. The respective coding sequences of these genes were separated by a TVMV NIa proteinase recognition sequence. In addition, in some instances, chloroplast targeting information (a transit peptide (TP) from a pea

rbcS

gene) was incorporated into the polyprotein. We found that the NIa proteinase can be used to express, as individual polypeptides, the ACK and CAT proteins, and that these proteins retain enzymatic activity. Polyproteins with the structure TP‐NIa‐ACK‐CAT or TP‐ACK‐CAT‐NIa failed to yield chloroplast‐localized ACK and CAT proteins, although the latter did give rise to a chloroplast‐localized ACK‐CAT polyprotein. These results indicate that the NIa proteinase acts

in cis

more rapidly than transport of proteins into the chloroplast, but that chloroplast localization can take place before complete processing of the polyprotein. Polyproteins with the structures ACK‐NIa‐TP‐CAT and TP‐ACK‐NIa‐TP‐CAT yielded appropriately processed and targeted ACK and CAT. Our results show that subcellular localization signals can be effectively recognized in the context of a polyprotein, and they suggest an appropriate strategy for simultaneous engineering of multiple subcellular compartments in plants.
Mutations inArabidopsis thalianagenes involved in the tryptophan biosynthesis pathway affect root waving on tilted agar surfaces
Authors: Rutherford Robert , Gallois Patrick , Masson Patrick H. 
Published: March 2003

Summary
Arabidopsis thaliana

roots grow in a wavy pattern upon a slanted surface. A novel mutation in the

anthranilate synthase α1

(

ASA1

) gene, named

trp5–2wvc1

, and mutations in the

tryptophan synthase α and β1

genes (

trp3–1

and

trp2–1

, respectively) confer a compressed root wave phenotype on tilted agar surfaces. When

trp5–2wvc1

seedlings are grown on media supplemented with anthranilate metabolites, their roots wave like wild type. Genetic and pharmacological experiments argue that the compressed root wave phenotypes of

trp5–2wvc1

,

trp2–1

and

trp3–1

seedlings are not due to reduced IAA biosynthetic potential, but rather to a deficiency in L‐tryptophan (L‐Trp), or in a L‐Trp derivative. Although the roots of 7‐day‐old seedlings possess higher concentrations of free L‐Trp than the shoot as a whole,

trp5–2wvc1

mutants show no detectable alteration in L‐Trp levels in either tissue type, suggesting that a very localized shortage of L‐Trp, or of a L‐Trp‐derived compound, is responsible for the observed phenotype.
Enhancement of the tolerance ofArabidopsisto high temperatures by genetic engineering of the synthesis of glycinebetaine
Authors: Alia  , Hayashi Hidenori , Sakamoto Atsushi , Murata Norio 
Published: March 2003

Summary
Arabidopsis thaliana

was transformed with the

codA

gene for choline oxidase from

Arthrobacter globiformis

under control of the 35S RNA promoter of cauliflower mosaic virus. As a result, high levels of glycinebetaine accumulated in the seeds of transformed plants. Transformation with the

codA

gene significantly enhanced the tolerance to high temperatures during the imbibition and germination of seeds, as well as during growth of young seedlings. The extent of enhancement of the tolerance to high temperature was correlated with levels of choline oxidase expressed and of glycinebetine accumulated in the transformed plants. The induction of homologues of heat shock protein 70 at high temperature was less conspicuous in the transformed plants than in the wild‐type plants, suggesting that the transformation alleviated the high‐temperature stress.
In situmonitoring of DNA: the plant nuclear envelope allows passage of short DNA fragments
Authors: Gisel Andreas , Rothen Barbara , Iglesias Victor A. , Potrykus Ingo , Of Sautter Christ 
Published: March 2003

Summary We monitored the cellular localization of fluorescently labeled foreign DNA in living plant cells. After physical delivery of labeled DNA fragments to the cytoplasm, short fragments up to 1.5 kb in length were found equally distributed between the cytoplasm and nucleus after 60 min. In contrast, 2.5 kb DNA fragments did not appear inside the nucleus. Thus, foreign DNA can enter plant nuclei through the intact nuclear envelope, but the efficiency of this process declines with increasing size of fragment.
Identification of a dual‐specificity protein phosphatase that inactivates a MAP kinase fromArabidopsis
Authors: Gupta Rajeev , Huang Yafan , Kieber Joe , Luan Sheng 
Published: March 2003

Summary Mitogen‐activated protein kinases (MAPKs) play a key role in plant responses to stress and pathogens. Activation and inactivation of MAPKs involve phosphorylation and dephosphorylation on both threonine and tyrosine residues in the kinase domain. Here we report the identification of an

Arabidopsis

gene encoding a dual‐specificity protein phosphatase capable of hydrolysing both phosphoserine/threonine and phosphotyrosine in protein substrates. This enzyme, designated AtDsPTP1 (

Arabidopsis thaliana

dual‐specificity protein tyrosine phosphatase), dephosphorylated and inactivated AtMPK4, a MAPK member from the same plant. Replacement of a highly conserved cysteine by serine abolished phosphatase activity of AtDsPTP1, indicating a conserved catalytic mechanism of dual‐specificity protein phosphatases from all eukaryotes.