bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytogenetics for the model system Arabidopsis thaliana
Authors: Fransz Paul , Armstrong Sue , Alonso‐blanco Carlos , Fischer Thilo C. , Torres‐ruiz Ramón A. , Jones Gareth
Published: March 2003

A detailed karyotype of Arabidopsis thaliana is presented using meiotic pachytene cells in combination with fluorescence in situ hybridization. The lengths of the five pachytene bivalents varied between 50 and 80 μm, which is 20–25 times longer than mitotic metaphase chromosomes. The analysis confirms that the two longest chromosomes (1 and 5) are metacentric and the two shortest chromosomes (2 and 4) are acrocentric and carry NORs subterminally in their short arms, while chromosome 3 is submetacentric and medium sized. Detailed mapping of the centromere position further revealed that the length variation between the pachytene bivalents comes from the short arms. Individual chromosomes were unambiguously identified by their combinations of relative lengths, arm‐ratios, presence of NOR knobs and FISH signals with a 5S rDNA probe and chromosome specific DNA probes. Polymorphisms were found among six ecotypes with respect to the number and map positions of 5S rDNA loci. All ecotypes contain 5S rDNA in the short arms of chromosomes 4 and 5. Three different patterns were observed regarding the presence and position of a 5S rDNA locus on chromosome 3. Repetitive DNA clones enabled us to subdivide the pericentromeric heterochromatin into a central domain, characterized by pAL1 and 106B repeats, which accommodate the functional centromere and two flanking domains, characterized by the 17 A20 repeat sequences. The upper flanking domains of chromosomes 4 and 5, and in some ecotypes also chromosome 3, contain a 5S rDNA locus. The detection of unique cosmids and YAC sequences demonstrates that detailed physical mapping of Arabidopsis chromosomes by cytogenetic techniques is feasible. Together with the presented karyotype this makes Arabidopsis a model system for detailed cytogenetic mapping.
Recurrent onset of epigenetic silencing in rice harboring a multi‐copy transgene
Authors: Kumpatla Siva P. , Hall Timothy C.
Published: March 2003

Extensive methylation of the Ubi1 promoter has been shown to be associated with transcriptional silencing and bialaphos herbicide sensitivity in several R1 progeny derived from a transgenic rice line, JKA 52, containing multiple copies of the introduced genes (Kumpatla et al. 1997;Plant Physiol. 115, 361–373). All of the progeny from silenced R1 lines were silenced, and a proportion (24%) of the R2 seedlings from JKA 52–6, a non‐silenced homozygous R1 line, were found to be silenced. Several non‐silenced (bialaphos‐resistant) R2 progeny derived from the selfed seed of JKA 52–6 were tested at various stages throughout the growth period and found to be resistant to bialaphos, indicating the absence of silencing. Five of these were investigated further to determine whether silencing could recur in subsequent generations. R3 seedlings were raised from selfed R2 seed and herbicide sensitivity was detected in 5–40% of the plants in three of the five lines. This silencing was accompanied by methylation of the Ubi1 promoter and the absence of steady‐state bar mRNA. This recurrent onset of silencing and instability of expression associated with a transgene locus containing several repeated sequences is undesirable. The data strongly suggest that multi‐generation molecular analysis should be undertaken for transgenic lines to be used for agricultural purposes and that lines containing methylated inserts should be carefully evaluated before being included in further breeding strategies.
Microinjection of pollen‐specific actin‐depolymerizing factor, ZmADF1, reorientates F‐actin strands in Tradescantia stamen hair cells
Authors: Hussey Patrick J. , Yuan Ming , Calder Grant , Khan Safina , Lloyd Clive W.
Published: March 2003

Maize actin‐depolymerizing factor (ADF) binds both monomeric and filamentous actin and increases actin dynamics in vitro. To test its effects in vivo, recombinant pollen ADF1 was expressed in bacteria and microinjected into Tradescantia stamen hair cells. Initially, all cytoplasmic streaming ceased and the central, longitudinal transvacuolar strands were disrupted. After 20–45 min, streaming resumed but in the form of conspicuous transverse pathways of movement in the cortex. Staining the actin filaments by a second injection of fluorescein‐conjugated phalloidin showed that the longitudinal actin cables seen in controls had been replaced by a thickening of the transverse cortical arrays, whose orientation matched the new pattern of streaming. Microinjection of rhodamine–tubulin confirmed that the microtubules also formed a transverse cortical array and it is suggested that the spatial cues for re‐modelling the actin after ADF1 injection may be provided by the microtubular system.
Efficient foreign gene expression in Chlamydomonas reinhardtii mediated by an endogenous intron
Authors: Lumbreras Victoria , Stevens David R. , Purton Saul
Published: March 2003

Heterologous genes introduced into the nuclear genome of Chlamydomonas reinhardtii are often poorly expressed. To understand the molecular mechanisms underlying this effect, we examined the influence of various factors on the expression of a chimeric transgene that confers resistance to zeomycin. This marker comprises the bacterial ble gene flanked by 5′ and 3′ sequences from the Chlamydomonas RBCS2 gene. We found that the frequency with which transformants are recovered is significantly increased when ble is fused to shorter versions of the RBCS2 promoter and when Chlamydomonas introns are introduced into the coding region of ble. The latter effect is particularly evident in the case of the first intron of RBCS2, which dramatically stimulates the transformation frequency and the level of ble expression. We found that this improvement is mediated in part by an enhancer element within the intron sequence, and that this element acts in an orientation‐independent manner and is effective when placed either upstream or downstream of the promoter. Our results demonstrate that stable high‐level expression of a foreign gene in Chlamydomonas is possible, and highlight a potential role of introns as modulators of gene expression in this alga.
Four differently chromatin‐associated maize HMG domain proteins modulate DNA structure and act as architectural elements in nucleoprotein complexes
Authors: Ritt Christoph , Grimm Rudi , Fernández Silvia , Alonso Juan C. , Grasser Klaus D.
Published: March 2003

In contrast to other eukaryotes which usually express two closely related HMG1‐like proteins, plant cells have multiple relatively variable proteins of this type. A systematic analysis of the DNA‐binding properties of four chromosomal HMG domain proteins from maize revealed that they bind linear DNA with similar affinity. HMGa, HMGc1/2 and HMGd specifically recognise diverse DNA structures such as DNA mini‐circles and supercoiled DNA. They induce DNA‐bending, and constrain negative superhelical turns in DNA. In the presence of DNA, the HMG domain proteins can self‐associate, whereas they are monomeric in solution. The maize HMG1‐like proteins have the ability to facilitate the formation of nucleoprotein structures to different extents, since they can efficiently replace a bacterial chromatin‐associated protein required for the site‐specific β‐mediated recombination. A variable function of the HMG1‐like proteins is indicated by their differential association with maize chromatin, as judged by their ‘extractability’ from chromatin with spermine and ethidium bromide. Collectively, these findings suggest that the various plant chromosomal HMG domain proteins could be adapted to act in different nucleoprotein structures in vivo.
Immunolocalisation of the cytoskeleton to plasmodesmata of Chara corallina
Authors: Blackman Leila M. , Overall Robyn L.
Published: March 2003

The macromolecular structure of plasmodesmata in the giant celled freshwater alga, Chara corallina, was examined using antibodies against cytoskeletal elements. The large internodal cells of Chara are separated by a nodal complex of smaller cells which are interconnected by plasmodesmata. Putative plasmodesmata‐associated proteins can be identified by a comparison of proteins extracted from preparations of clean walls of nodal complexes and those extracted from the external walls of internodal cells which have no plasmodesmata. Actin and tubulin were identified in the protein extracts of nodal walls and the cytoplasm of nodes and internodes but not in the extracts of internodal external walls. Immunogold labelling confirmed the localisation of actin and myosin to plasmodesmata of Chara.
The GCN4 motif in a rice glutelin gene is essential for endosperm‐specific gene expression and is activated by Opaque‐2 in transgenic rice plants
Authors: Wu Chuan‐Yin , Suzuki Akihiro , Washida Haruhiko , Takaiwa Fumio
Published: March 2003

The GCN4 motif is conserved in a number of seed storage protein genes, and promoter fragments containing this motif have been shown to be involved in controlling seed‐specific expression of the genes studied. All genes encoding the rice seed storage protein glutelin contain the GCN4 motif at similar sites in their 5′ flanking regions. Using a stable homologous transgenic system, we have analysed the promoter of the rice glutelin gene GluB‐1 and demonstrated that the GCN4 motif functions as an essential cis‐element for endosperm‐specific gene expression. Moreover, a 21 bp GluB‐1 promoter fragment spanning the GCN4 motif, as a multimer, directed GUS gene expression in endosperm of transgenic rice plants, when fused directly to the core promoter (–46) of CaMV 35S. In transiently transfected rice protoplasts, over a hundred‐fold transactivation was observed from the 21 bp sequence by the bZIP type transcriptional activator Opaque‐2 (O2) co‐expressed under a CaMV 35S promoter. The transactivation was also evident in transgenic plants containing both O2 and the 21 bp sequence/GUS fusion. The O2‐mediated activation requires binding of O2 to an intact GCN4 motif. Our results suggest that a bZIP protein functionally similar to O2 may exist in rice and participate in controlling the endosperm‐specific expression of GluB‐1 through the GCN4 motif.
A xyloglucan oligosaccharide‐active, transglycosylating‐D‐glucosidase from the cotyledons of nasturtium (Tropaeolum majus L) seedlings – purification, properties and characterization of a cDNA clone
Authors: Crombie Hazel J. , Chengappa Sumant , Hellyer Amanda , Reid J. S. Grant
Published: March 2003

A ‐D‐glucosidase has been purified to apparent homogeneity from the cotyledons of germinated nasturtium (Tropaeolum majus L.) seedlings during the mobilization of the xyloglucan stored in the cotyledonary cell walls. The purified protein (Mr 76 000; a glycoprotein; pI > 9.5; apparent pH optimum 4.5; temperature optimum 30°C) catalysed the hydrolysis of p‐nitrophenyl‐‐D‐glucopyranoside, cello‐oligosaccharides, ‐linked glucose disaccharides, and certain xyloglucan oligosaccharides. Glucose disaccharides with different linkages were hydrolysed at different rates [(1ν3) > (1ν4) > (1ν2) > (1ν6)] with significant transglycosylation occurring in the early stages of the reaction. Cello‐oligosaccharide hydrolysis was also accompanied by extensive transglycosylation to give transitory accumulations of higher oligosaccharides. At least some of the glycosyl linkages formed during transglycosylation were (1ν6)‐. Xyloglucan oligosaccharides xylose‐substituted at the non‐reducing terminal glucose residue (XXXG, XXLG, XLXG and XLLG, where G is an unsubstituted glucose residue, X is a xylose‐substituted glucose residue, and L is a galactosylxylose‐substituted glucose residue) were not hydrolysed. Some xyloglucan oligosaccharides with an unsubstituted non‐reducing terminal glucose residue (GXXG, GXLG and GXG) were hydrolysed, but others (GLXG and GLLG) were not. This indicated steric hindrance by L but not X substitution at the glucose residue next to the one at the non‐reducing end of the oligosaccharide. Hydrolysis of xyloglucan oligosaccharides was not accompanied by transglycosylation. Natural xyloglucan subunit oligosaccharides (XXXG, XXLG, XLXG, XLLG) were totally degraded to their monosaccharide components when treated with nasturtium ‐D‐galactosidase (
Edwards et al. (1988
) J. Biol. Chem. 263, 4333–4337), followed by alternations of nasturtium xyloglucan‐specific α‐xylosidase (
Fanutti et al. (1991
) Planta 184, 137–147) and this enzyme. Several extensively overlapping cDNA clones were obtained by RT–PCR and by screening cDNA libraries. A composite, full‐length DNA had an open reading frame of 1962 bp, encoding a polypeptide of 654 amino acids, including all N‐terminal and internal sequences obtained from the purified ‐glucosidase protein, and a motif resembling plant signal sequences thought to direct proteins to the cell wall. Database searches revealed homology with ‐glucosidases from several sources (plant, bacteria, yeast), notably with glycosylhydrolases of ‘Family 3’, according to the classification of Henrissat (
Henrissat (1991) Biochem. J. 280, 309–316). There was strong sequence homology with a ‐glucan exo‐hydrolase from barley (
Hrmova et al. (1996
) J. Biol. Chem. 271, 5277–5286). The nasturtium ‐glucosidase is ascribed a role in xyloglucan mobilization, and its interaction with the α‐xylosidase and the ‐galactosidase is modelled.
Phytobilin biosynthesis: cloning and expression of a gene encoding soluble ferredoxin‐dependent heme oxygenase from Synechocystis sp. PCC 6803
Authors: Cornejo Juan , Willows Robert D. , Beale Samuel I.
Published: March 2003

The phytobilin chromophores of phycobiliproteins and phytochromes are biosynthesized from heme in a pathway that begins with the opening of the tetrapyrrole macrocycle of protoheme to form biliverdin IXα, in a reaction catalyzed by heme oxygenase. A gene containing an open reading frame with a predicted polypeptide that has a sequence similar to that of a conserved region of animal microsomal heme oxygenases was identified in the published genomic sequence of Synechocystis sp. PCC 6803. This gene, named ho1, was cloned and expressed in Escherichia coli under the control of the lacZ promoter. Cells expressing the gene became green colored due to the accumulation of biliverdin IXα. The size of the expressed protein was equal to the predicted size of the Synechocystis gene product, named HO1. Heme oxygenase activity was assayed in incubations containing extract of transformed E. coli cells. Incubations containing extract of induced cells, but not those containing extract of uninduced cells, had ferredoxin‐dependent heme oxygenase activity. With mesoheme as the substrate, the reaction product was identified as mesobiliverdin IXα by spectrophotometry and reverse‐phase HPLC. Heme oxygenase activity was not sedimented by centrifugation at 100 000 g. Expression of HO1 increased several‐fold during incubation of the cells for 72 h in iron‐deficient medium.
The ArabidopsisERECTAgene is expressed in the shoot apical meristem and organ primordia
Authors: Yokoyama Ryusuke , Takahashi Taku , Kato Atsushi , Torii Keiko U. , Komeda Yoshibumi
Published: March 2003

In

Arabidopsis thaliana

(L.) Heynh, the mutation in

ERECTA

is known to confer a compact inflorescence by a reduction in the lengths of internodes and pedicels. We analyzed the expression pattern of this gene during plant development.

In situ

hybridization and histochemical analysis using transgenic plants carrying chimeric gene fusions, with the

ERECTA

promoter fused to the β‐glucuronidase (GUS) gene, showed that

ERECTA

was predominantly expressed in the shoot apical meristems and organ primordia.

ERECTA

expression in the shoot apical meristem was weak early in plant development but increased with the transition from the vegetative to the reproductive growth phase.

ERECTA

was also strongly expressed in organ primordia and immature organs but weakly in mature organs. Thus,

ERECTA

was expressed in a cell‐specific and developmentally regulated manner. In order to identify the regulatory mechanism responsible for the expression pattern of

ERECTA

, the

cis

‐acting regions in the

ERECTA

promoter were defined by study of the expression of the chimeric genes that consist of the 5′‐ or internal deleted promoter and a GUS reporter gene in transgenic plants. The results showed that the essential

cis

‐regulatory elements governing the spatially and temporally specific expression of

ERECTA

are located between positions –462 and – 228 bp and between positions –228 and –153 bp with respect to the transcriptional initiation site.
Over‐expression of a tobacco homeobox gene,NTH15, decreases the expression of a gibberellin biosynthetic gene encoding GA 20‐oxidase
Authors: Tanaka‐Ueguchi Miyako , Itoh Hironori , Oyama Naomi , Koshioka Masaji , Matsuoka Makoto
Published: March 2003

Ectopic expression of the homeobox gene,

NTH15

(

Nicotiana tabacum

homeobox 15) in transgenic tobacco leads to abnormal leaf and flower morphology, accompanied by a decrease in the content of the active gibberellin, GA1. Quantitative analysis of intermediates in the GA biosynthetic pathway revealed that the step from GA19 to GA20 was blocked in transgenic tobacco plants overexpressing

NTH15

. To investigate the relationship between the expression of

NTH15

and genes involved in GA biosynthesis, we isolated three cDNA clones from tobacco encoding two types of GA 20‐oxidase and a 3β‐hydroxylase. RNA gel blot analysis revealed that the expression of one gene (

Ntc12

, encoding GA 20‐oxidase), which in wild‐type tobacco plants was abundantly expressed in leaves, was strongly suppressed in the transformants. The expression level of

Ntc12

decreased with increasing severity of phenotype of transgenic tobacco leaves. The abnormal leaf morphology was largely overcome by treatment with GA20 or GA1 but not by GA19. These data strongly suggest that overexpression of

NTH15

inhibits the expression of

Ntc12

, resulting in reduced levels of active GA and abnormal leaf morphology in transgenic tobacco plants.

In situ

hybridization in wild‐type tobacco revealed that expression of

Ntc12

occurred mainly in the rib meristem, cells surrounding the procambium and in leaf primordia. Expression was not seen in the tunica, corpus and procambium, tissues in which

NTH15

was predominantly expressed. The contrasting expression patterns of these genes may reflect their antagonistic functions in the formation of lateral organs from the shoot apical meristem.
Gain of function assays identify non‐rolgenes fromAgrobacterium rhizogenesTL‐DNA that alter plant morphogenesis or hormone sensitivity
Authors: Lemcke Kai , Schmülling Thomas
Published: March 2003

This study tested the morphogenetic potential of 15 open reading frames of the TL‐DNA of

Agrobacterium rhizogenes

strain HRI. These open reading frames were expressed individually under the control of the 35S RNA promoter in transgenic tobacco plants (

Nicotiana tabacum

L.). Expression of three T‐DNA loci, ORF3n, ORF8 and ORF13, alters plant morphogenesis or the response of transgenic tissues to plant hormones. ORF3n transgenic plants are characterized by retarded flowering, altered internode elongation, altered leaf shape and, in particular, leaf tip necrosis. ORF3n and ORF8 expression reduces the sensitivity to auxin and cytokinin in combination or auxin alone. Tetracycline‐dependent expression of ORF13 overcomes a selection of low levels of expression during plant regeneration and reveals a strong inhibitory effect of the ORF13 gene product on cell division and cell elongation. We conclude that the

A. rhizogenes

TL‐DNA harbors genetic information that is important for pathogenicity apart from the well studied

rol

genes. We propose that these genes play mainly a negative regulatory role during pathogenesis. Moreover, these loci might be relevant to successful infections in specific host plants.
Disruption of chloroplast biogenesis and plant development upon down‐regulation of a chloroplast processing enzyme involved in the import pathway
Authors: Wan Jiangxin , Bringloe David , Lamppa Gayle K.
Published: March 2003

Typically, nuclear‐encoded chloroplast proteins are synthesized as precursors and require proteolytic processing upon import before their assembly into functional complexes within the organelle. A cDNA encoding a chloroplast processing enzyme (CPE), which was originally identified as a protease that cleaves the precursor for the major light‐harvesting chlorophyll binding protein (preLHCP), was introduced into the tobacco genome in an antisense orientation to investigate the role of the enzyme

in vivo

. The presence of the antisense‐CPE gene resulted in chlorotic leaves, and retarded shoot and root growth. The introduction of the antisense‐CPE gene disrupted the normal pattern of plastid division. Chloroplast numbers in cotyledon and first leaf cells were reduced 25% compared to the control plants. Chloroplasts contained fewer thylakoids and large starch grains, the latter an indication of a change in carbon flux. CPE levels and activity were significantly lower in stromal extracts in the transgenic plants. Interestingly,

in vitro

import of precursor proteins was defective. Most of the preLHCP remained on the exterior of the organelle, and only a small fraction of preRBCA was imported, suggesting that a change in CPE levels can influence translocation across the envelope. Our

in vivo

results support the conclusion that CPE plays a critical role during chloroplast biogenesis, and that the pleiotropic effects of CPE down‐regulation reflect its function as a general stromal processing peptidase as part of the import machinery. Furthermore, these findings indicate the importance of regulating the expression of components of the import machinery for normal plant development.
ICK1, a cyclin‐dependent protein kinase inhibitor fromArabidopsis thalianainteracts with both Cdc2a and CycD3, and its expression is induced by abscisic acid
Authors: Wang Hong , Qi Qungang , Schorr Peter , Cutler Adrian J. , Crosby William L. , Fowke Larry C.
Published: March 2003

Cyclin‐dependent kinase (CDK) inhibitor genes encode low molecular weight proteins which have important functions in cell cycle regulation, development and perhaps also in tumorigenesis. The first plant CDK inhibitor gene

ICK1

was recently identified from

Arabidopsis thaliana

. Although the C‐terminal domain of ICK1 contained an important consensus sequence with the mammalian CDK inhibitor p27Kip1, the remainder of the deduced ICK1 sequence showed little similarity to any known CDK inhibitors.

In vitro

assays showed that recombinant ICK1 exhibited unique kinase inhibitory properties. In the present study we characterized ICK1 in terms of its gene structure, its interaction with both

A. thaliana

Cdc2a and CycD3, and its induction by the plant growth regulator, abscisic acid (ABA).

ICK1

was expressed at a relatively low level in the tissues surveyed. However,

ICK1

was induced by ABA, and along with

ICK1

induction there was a decrease in Cdc2‐like histone H1 kinase activity. These results suggest a molecular mechanism by which plant cell division might be inhibited by ABA.

ICK1

clones were also identified from independent yeast two‐hybrid screens using the CycD3 construct. The implication that ICK1 protein could interact with both Cdc2a and CycD3 was confirmed by

in vitro

binding assays. Furthermore, deletion analysis indicated that different regions of ICK1 are required for the interactions with Cdc2a and CycD3. These results provide a mechanistic basis for understanding the role of CDK inhibitors in cell cycle regulation in plant cells.
An Arabidopsis immunophilin, AtFKBP12, binds to AtFIP37 (FKBP interacting protein) in an interaction that is disrupted by FK506
Authors: Faure Jean‐Denis , Gingerich Derek , Howell Stephen H.
Published: March 2003

AtFKBP12 is an Arabidopsis cDNA that encodes a protein similar to the mammalian immunophilin, FKBP12. AtFKBP12 was used as ‘bait’ in a yeast 2‐hybrid system to screen for cDNAs in Arabidopsis encoding proteins that bind to FKBP12. Two partial cDNAs were recovered encoding the C‐terminus of a protein we have called

Arabidopsis thaliana

FKBP12 interacting protein 37 (AtFIP37). AtFIP37 is similar to a mammalian protein, FAP48, that also binds to FKBP12. The interaction between AtFKBP12 and AtFIP37 in the 2‐hybrid system, as assessed by histidine auxotrophy and β‐galactosidase activity, was disrupted by FK506, but not by cyclosporin A, a drug that binds to cyclophilin A. AtFIP37 was also shown to bind

in vitro

to AtFKBP12 in GST‐fusion protein binding assays. The binding was abolished by prior incubation of AtFKBP12 with FK506. These findings indicate that an Arabidopsis FKBP12 ortholog encodes a protein that binds FK506 and that the interaction between AtFKBP12 and AtFIP37 may involve the FK506 binding site of AtFKBP12. The interaction provides interesting new opportunities for controlling protein:protein interactions

in vivo

in plants.
Distribution of TMV movement protein in single living protoplasts immobilized in agarose
Authors: Más Paloma , Beachy Roger N.
Published: March 2003

Recent studies of the tobacco mosaic virus (TMV) P30 movement protein (MP) fused with green fluorescent protein (GFP) during TMV infection described the involvement of elements of the cytoskeleton and components of the endoplasmic reticulum (ER) in the intracellular trafficking of MP:GFP from the sites of synthesis in the cytoplasm to plasmodesmata. To examine in real‐time the pattern of synthesis, accumulation and degradation of MP:GFP, we developed a method to immobilize protoplasts in agarose such that they are maintained alive for extended periods of time. The pattern of MP:GFP accumulation in single living protoplasts visualized by confocal laser scanning microscopy (CLSM) was parallel to that previously described in a population of protoplasts harvested at different times post‐infection. Additionally, a network of weakly fluorescent filaments, which are apparently different from microtubules, was observed to surround the nucleus and these filaments were associated with fluorescent bodies (previously identified as ER‐derived structures). Later in infection, the fluorescent bodies increased in size and coalesced to form larger structures that accumulated near the periphery of the cells while highly fluorescent non‐cortical filaments were observed distributed in the cytoplasm. The putative involvement of these filaments in targeting the fluorescent bodies to the periphery of the cell is discussed. Studies of single, embedded protoplasts make it possible to observe changes in amount and subcellular localization of viral and other proteins.
Activation of MAPK homologues by elicitors in tobacco cells
Authors: Lebrun‐Garcia Angela , Ouaked Fatma , Chiltz Annick , Pugin Alain
Published: March 2003

Elicitors of plant defence reactions (such as cryptogein, an elicitin produced by

Phytophthora cryptogea

, or oligogalacturonides (OGs)), induced in tobacco cell suspensions (

Nicotiana tabacum

var Xanthi) a rapid and transient activation of two protein kinases (PKs) with apparent molecular masses of 50 and 46 kDa, respectively. These PKs activated and phosphorylated at tyrosine residues, phosphorylated myelin basic protein (MBP) at serine/threonine residues. Both are recognized by anti‐MAPK antibodies. The two MBP kinases possessed the same kinetics of activation, and their activation depended, to the same extent, on different exogenously applied compounds (staurosporine, lanthanum, EGTA). We demonstrate here that the activation of the MBP kinases is calcium dependent and sensitive to staurosporine, a protein kinase inhibitor which annihilates all known responses of tobacco cells to cryptogein. The activation of MBP kinases appeared to be independent of the production of active oxygen species (AOS) and insensitive to calyculin A, a protein phosphatase type 1 and 2A inhibitor. The activation of MAPKs is discussed in relation to the early responses induced by cryptogein.
Suppression of endogenous α and β tubulin synthesis in transgenic maize calli overexpressing α and β tubulins
Authors: Anthony Richard G. , Hussey Patrick J.
Published: March 2003

Maize Black Mexican Sweetcorn cells have been transformed with constructs containing α and β tubulin coding sequences either singly or together. It is shown that recovery of stable maize transformants is dependent on the co‐expression of transfected α and β tubulin in the same lines, indicating that plant cells cannot tolerate an imbalance in the ratio of α tubulin to β tubulin within the cytoplasm. The co‐expression of transfected α and β tubulin in maize cells results in an increase in the overall tubulin content (approximately threefold). The transfected α and β tubulins are incorporated into cortical, spindle and phragmoplast microtubule arrays indicating that they are functional. Furthermore, the co‐expression of the transfected α and β tubulins results in the suppression of endogenous α and β tubulin synthesis. This suppression increases both with the strength of the promoter in the constructs and with the number of copies of the transgenes inserted into the maize genome. The implications for the post‐transcriptional and post‐translational regulation of tubulin synthesis in plant cells are discussed.
Molecular characterization of plastidic phosphoserine aminotransferase in serine biosynthesis fromArabidopsis
Authors: Ho Chai‐Ling , Noji Masaaki , Saito Maiko , Yamazaki Mami , Saito Kazuki
Published: March 2003

Serine biosynthesis in plants proceeds by two pathways; a photorespiratory pathway which is associated with photorespiration and a pathway from phosphoglycerate. A cDNA encoding plastidic phosphoserine aminotransferase (PSAT) which catalyzes the formation of phosphoserine from phosphohydroxypyruvate has been isolated from

Arabidopsis thaliana

. Genomic DNA blot analysis indicated that this enzyme is most probably encoded by a single gene and is mapped on the lower arm of chromosome 4. The deduced protein contains an N‐terminal extension exhibiting the general features of a plastidic transit peptide, which was confirmed by subcellular organelle localization using GFP (green flourescence protein). Northern analysis indicated preferential expression of PSAT in roots of light‐grown plants, supporting the idea that the phosphorylated pathway may play an important role in supplying the serine requirement of plants in non‐green tissues.

In situ

hybridization analysis of PSAT revealed that the gene is generally expressed in all types of cells with a significantly higher amount in the meristem tissue of root tips.
A relic S‐RNase is expressed in the styles of self‐compatibleNicotiana sylvestris
Authors: Golz John F. , Clarke Adrienne E. , Newbigin Ed , Anderson Marilyn
Published: March 2003

We surveyed ribonuclease activity in the styles of

Nicotiana

spp. and found little or no activity in self‐compatible species and in a self‐compatible accession of a self‐incompatible species. All self‐incompatible species had high levels of ribonuclease activity in their style. Interestingly, one self‐compatible species,

N. sylvestris

, had a level of stylar ribonuclease activity comparable to that of some self‐incompatible

Nicotiana

species. A ribonuclease with biochemical properties similar to those of the self‐incompatibility (S‐)RNases of

N. alata

was purified from

N. sylvestris

styles. The N‐terminal sequence of this protein was used to confirm the identity of a cDNA corresponding to the stylar RNase. The amino acid sequence deduced from the cDNA was related to those of the S‐RNases and included the five conserved regions characteristic of these proteins. It appears that the

N. sylvestris

RNase may have evolved from the S‐RNases and is an example of a ‘relic S‐RNase’. A number of features distinguish the

N. sylvestris

RNase from the S‐RNases, and the role these may have played in the presumed loss of the self‐incompatibility response during the evolution of this species are discussed.
Retrotransposon‐based insertion polymorphisms (RBIP) for high throughput marker analysis
Authors: Flavell Andrew J. , Knox Maggie R. , Pearce Stephen R. , Ellis T. H. Noel
Published: March 2003

Two assays based upon PCR detection of a polymorphic PDR1 retrotransposon insertion in

Pisum sativum

have been developed. Both methods involve PCR with primers derived from the transposon and flanking DNA. The first method uses a dot assay for PCR product detection which could be fully automated for handling thousands of samples. The second method, which is designed to handle lower numbers, requires a single PCR and gel lane per sample. Both methods yield co‐dominant markers, with presence and absence of the transposon insertion independently scorable, and both could in principle be applied to any transposable element in any plant species.
Interaction of maize actin‐depolymerising factor with actin and phosphoinositides and its inhibition of plant phospholipase C.
Authors: Gungabissoon Ravine A. , Jiang Chang‐Jie , Drøbak Bjørn K. , Maciver Sutherland K. , Hussey Patrick J.
Published: March 2003

We have previously reported the isolation of three

Zea mays

genes that encode actin‐depolymerising factors/cofilins, a family of low molecular weight actin regulating proteins. In the present study, we have characterised one of these proteins, ZmADF3. We report that ZmADF3 binds G‐actin with a 1:1 stoichiometry, and that the interaction with F‐actin is pH‐sensitive. ZmADF3 co‐sediments mainly with F‐actin at pH 6.0 and mainly with G‐actin at pH 9.0. This response is more similar to that of vertebrate cofilin and ADF than to that of

Acanthamoeba

actophorin which, although more similar in primary sequence to ZmADF3, is not pH sensitive. However, ZmADF3 requires a more basic environment to depolymerise actin relative to either vertebrate ADF or cofilin. Filaments decorated with ZmADF3 at low pH are very rapidly depolymerised upon raising the pH, which is consistent with a severing mechanism for the disruption of actin filaments. Also, we demonstrate that ZmADF3 binds specific polyphosphatidylinositol lipids, especially phosphatidylinositol 4,5‐bisphosphate (PIP2), and we show that this binding inhibits the actin‐depolymerising function of ZmADF3. Moreover, we show that a consequence of ZmADF3 binding PIP2 is the inhibition of the activity of polyphosphatidylinositol specific plant phospholipase C, indicating the possibility of reciprocal modulation of this major signalling pathway and the actin cytoskeleton.
Molecular and cytogenetic characterization of a transgene locus that induces silencing and methylation of homologous promoters intrans
Authors: Jakowitsch Johannes , Papp István , Moscone Eduardo A. , Van Der Winden Johannes , Matzke Marjori , Matzke Antonius J. M.
Published: March 2003

One type of homology‐dependent gene silencing in transgenic plants involves a silencing locus that is able to transcriptionally inactivate and methylate an unlinked target locus with which it shares sequence identity in promoter regions. In a manner resembling paramutation of endogenous genes, the target locus reactivates and loses methylation progressively over several generations after segregating away from the silencing locus, which autonomously acquires stable methylation. To investigate the origins of

trans

‐silencing ability and susceptibility, we have analyzed the structures, flanking DNA sequences and chromosomal locations of a nopaline synthase promoter silencing locus,

H2

, and a sensitive target locus,

K81

. A partially resistant target locus,

Kα

, has been characterized molecularly. The complex and scrambled

H2

locus comprises six copies of the nopaline synthase promoter, two of which are collinear with prokaryotic non‐T‐DNA sequences, and is integrated close to a region of intercalary heterochromatin. These features probably contribute collectively to the silencing ability because

H2

subclones reintroduced into random locations in the K81 genome did not frequently induce silencing. Both the

K81

and

Kα

loci have simple structures, although the former contains non‐T‐DNA prokaryotic sequences that are also present at

H2

, and they are flanked by low copy plant DNA.

H2

and

K81

might interact effectively because they are present on morphologically similar chromosomes from the T subgenome of allotetraploid tobacco.
Localisation of glutathione and homoglutathione inMedicago truncatulais correlated to a differential expression of genes involved in their synthesis
Authors: Frendo Pierre , Gallesi Daniela , Turnbull Ruth , Van de Sype Ghislaine , Hérouart Didier , Puppo Alain
Published: March 2003

Glutathione (GSH) and homoglutathione (hGSH) were quantified in

Medicago truncatula

during plant development. hGSH was detectable only 48 h after seed germination whereas GSH was present in the dry seeds, indicating that only GSH is used for sulphur storage in seeds. The hGSH was detectable only in the underground part of mature plants whereas GSH was present in all the organs. γ‐EC synthetase (γ‐ECS) and GSH synthetase (GSHS) activities were found in roots and leaves whereas hGSH synthetase (hGSHS) was found only in roots. Full‐length cDNA encoding γ‐ECS and two partial cDNAs (

gshs1

and

gshs2

) showing high identity with GSHS were isolated in

M. truncatula

. High γ‐ECS activity was detected in protein extracts of a γ‐ECS‐deficient

E. coli

strain expressing the

M. truncatula

γ‐ECS. Northern blot analysis showed that the γ‐ECS gene was similarly expressed in all the mature plant organs tested, whereas

gshs1

had a higher expression in leaves and flowers and

gshs2

was preferentially expressed in roots and nodules. We hypothesise that

gshs1

and

gshs2

encode a GSHS and an hGSHS, respectively.
Expression of cysteine proteinase during developmental events associated with programmed cell death in brinjal
Authors: Xu Fang‐Xiu , Chye Mee‐Len
Published: March 2003

Here we show that the expression of a cysteine proteinase coincides with several developmental events associated with programmed cell death (PCD) in

Solanum melongena

(brinjal), i.e. during leaf senescence, fruit senescence, xylogenesis, nucellar cell degeneration and anther senescence. We have isolated a cDNA encoding brinjal cysteine proteinase (SmCP) that shares high (90–92%) amino acid identity to cysteine proteinases of tobacco (CYP‐8) and tomato (LCYP‐2) that have not been previously reported to be senescence‐associated. In contrast, SmCP shows lower (39–41%) amino acid identity to other senescence‐related cysteine proteinases and, unlike most of them, it is not preferentially expressed in certain organs or cell types. Northern analysis of leaves, fruits and flowers at different stages of development showed that

SmCP

expression increased significantly at senescence in leaf and fruit, but was highly expressed throughout flower development.

In situ

hybridization studies on flower sections using an antisense RNA probe localized the

SmCP

mRNA to the xylem, the epidermis and the endothecium of the anther and the nucellar cells, suggesting its involvement in PCD during xylogenesis, anther senescence and ovule development, respectively. Its expression during nucellar cell degeneration suggests that protein reserves of the nucellus are released to the developing embryo. Polarity in its pattern of expression in the nucellus of the developing seed (40DAP) further implies a directional flow of these nutrients.
Ozone‐induced cell death occurs via two distinct mechanisms in Arabidopsis: the role of salicylic acid
Authors: Rao Mulpuri V. , Davis Keith R.
Published: March 2003

Previous studies suggest that salicylic acid (SA) plays an important role in influencing plant resistance to ozone (O3). To further define the role of SA in O3‐induced responses, we compared the responses of two
Arabidopsis
genotypes that accumulate different amounts of SA in response to O3 and a SA‐deficient transgenic Col‐0 line expressing salicylate hydroxylase (NahG). The differences observed in O3‐induced changes in SA levels, the accumulation of active oxygen species, defense gene expression, and the kinetics and severity of lesion formation indicate that SA influences O3 tolerance via two distinct mechanisms. Detailed analyses indicated that features associated with a hypersensitive response (HR) were significantly greater in O3‐exposed Cvi‐0 than in Col‐0, and that NahG plants failed to exhibit these HR‐like responses. Furthermore, O3‐induced antioxidant defenses, including the redox state of glutathione, were greatly reduced in NahG plants compared to Col‐0 and Cvi‐0. This suggests that O3‐induced cell death in NahG plants is due to the loss of SA‐mediated potentiation of antioxidant defenses, while O3‐induced cell death in Cvi‐0 is due to activation of a HR. This hypothesis is supported by the observation that inhibition of NADPH‐oxidases reduced O3‐induced H2O2 levels and the O3‐induced cell death in Cvi‐0, while no major changes were observed in NahG plants. We conclude that although SA is required to maintain the cellular redox state and potentiate defense responses in O3 exposed plants, high levels of SA also potentiate activation of an oxidative burst and a cell death pathway that results in apparent O3 sensitivity.
The interaction between plastocyanin and photosystem I is inefficient in transgenic Arabidopsis plants lacking the PSI‐N subunit of photosystem
Authors: Haldrup Anna , Naver , Helle , Scheller Henrik Vibe
Published: March 2003

The PSI‐N subunit of photosystem I (PSI) is restricted to higher plants and is the only subunit located entirely in the thylakoid lumen. The role of the PSI‐N subunit in the PSI complex was investigated in transgenic
Arabidopsis
plants which were generated using antisense and co‐suppression strategies. Several lines without detectable levels of PSI‐N were identified. The plants lacking PSI‐N assembled a functional PSI complex and were capable of photoautotrophic growth. When grown on agar media for several weeks the plants became chlorotic and developed significantly more slowly. However, under optimal growth conditions, the plants without PSI‐N were visually indistinguishable from the wild‐type although several photosynthetic parameters were affected. In the transformants, the second‐order rate constant for electron transfer from plastocyanin to P700+, the oxidized reaction centre of PSI, was only 55% of the wild‐type value, and steady‐state NADP+ reduction was decreased to a similar extent. Quantum yield of oxygen evolution and PSII photochemistry were about 10% lower than in the wild‐type at leaf level. Photochemical fluorescence quenching was lowered to a similar extent. Thus, the 40–50% lower activity of PSI at the molecular level was much less significant at the whole‐plant level. This was partly explained by a 17% increase in PSI content in the plants lacking PSI‐N.
A signaling pathway, independent of the oxidative burst, that leads to hypersensitive cell death in cultured tobacco cells includes a serine protease
Authors: Yano Akira , Suzuki Kaoru , Shinshi Hideaki
Published: March 2003

To examine the role of reactive oxygen species (ROS) in the signal transduction that leads to hypersensitive cell death, we used a previously established system in which a xylanase from Trichoderma viride (TvX) induces an oxidative burst and cell death in a culture of tobacco cells. Diphenylene iodonium and N‐Acetyl‐L‐cysteine known as an inhibitor of NADPH oxidase and a scavenger of superoxides, respectively, and catalase inhibited the oxidative burst but did not inhibit the induction of cell death. We also found that inhibitors of serine proteases inhibited TvX‐induced cell death. These results suggest that there is a signaling pathway in which a serine protease might be responsible for the signal transduction, which is independent of the oxidative burst, that leads to the hypersensitive cell death of tobacco cells.
Characterisation of a novel gene family of putative cyclic nucleotide‐ and calmodulin‐regulated ion channels in Arabidopsis thaliana
Authors: Köhler Claudia , Merkle Thomas , Neuhaus Gunther
Published: March 2003

In plants, cyclic GMP is involved in signal transduction in response to light and gibberellic acid. For cyclic AMP, a potential role during the plant cell cycle was recently reported. However, cellular targets for cyclic nucleotides in plants are largely unknown. Here we report on the identification and characterisation of a new gene family in Arabidopsis, which share features with cyclic nucleotide‐gated channels from animals and inward‐rectifying K+ channels from plants. The identified gene family comprises six members (Arabidopsis thaliana cyclic nucleotide‐gated channels, AtCNGC1–6) with significant homology among the deduced proteins. Hydrophobicity analysis predicted six membrane‐spanning domains flanked by hydrophilic amino and carboxy termini. A putative cyclic nucleotide binding domain (CNBD) which contains several residues that are invariant in other CNBDs was located in the carboxy terminus. This domain overlaps with a predicted calmodulin (CaM) binding site, suggesting interaction between cyclic nucleotide and CaM regulation. We demonstrated interaction of the carboxy termini of AtCNGC1 and AtCNGC2 with CaM in yeast, indicating that the CaM binding sites are functional. Furthermore, it was shown that both AtCNGC1 and AtCNGC2 can partly complement the K+‐uptake‐deficient yeast mutant CY162. Therefore, we propose that the identified genes constitute a family of plant cyclic nucleotide‐ and CaM‐regulated ion channels.
Induction of RAB18 gene expression and activation of K+ outward rectifying channels depend on an extracellular perception of ABA in Arabidopsis thaliana suspension cells
Authors: Jeannette Emmanuelle , Rona Jean‐Pierre , Bardat Françoise , Cornel Daniel , Sotta Bruno , Miginiac Emile
Published: March 2003

Important progress has been made regarding the characterization of the ABA signalling components using genetic and molecular approaches ( Leung & Giraudat 1998). However, we do not yet know the mechanism of ABA perception. Conflicting results concerning the site of ABA perception have been published. The prevailing view is that since ABA controls many responses, different sites of perception for ABA might exist. In order to establish the cellular localisation of the ABA receptors in Arabidopsis thaliana suspension cells, we developed two physiological tests based upon the capacity of impermeant ABA‐BSA conjugate to mimic permeant free ABA effects. We show that purified ABA‐BSA conjugate is able to trigger RAB18 gene expression and that this response is strictly due to the natural (+)‐ABA enantiomer. The rate of RAB18 gene expression was independent of the level of ABA uptake by the cells. Using the voltage‐clamp technique we show that ABA‐BSA, similarly to ABA, evokes a membrane depolarization and activates time‐and voltage‐dependent outward rectifying currents (ORC). We demonstrate that these ORC are due to a K+ efflux as assessed by tail currents and specific inhibition by both tetraethylammonium (TEA) and Ba2+. These observations provide evidence in favour of an extracellular site for ABA perception.