bluuebunny/crossref_metadata_2025 huggingface dataset:
Contrasting patterns in the spread of two seed‐borne viruses in pea embryos
Authors: Wang Daowen , Maule Andrew J.
Published: March 2003

The temporal and spatial patterns of pea seed‐borne mosaic potyvirus (PSbMV) and pea early browning tobra‐virus (PEBV) accumulation in pea embryos were analyzed using in‐situ hybridization and immunohistochemistry. For PSbMV, which infects embryos after fertilization, the distribution changed as the embryo developed and some tissues remained free of virus infection. In contrast, embryos were uniformly infected with PEBV from the earliest stages of embryo development, and PEBV was detected in the egg cell and pollen grains, indicative of gametic transmission into the embryo. These observations suggest that gametically transmitted viruses may be appropriate as potential vectors for the ectopic and uniform expression of novel genes in embryonic tissues. Functional complementarity in the two processes of embryo invasion was tested following co‐inoculation with PSbMV and PEBV. Instead of complementation, interference in PSbMV seed transmission by PEBV was observed; PEBV seed transmission remained unaffected by PSbMV.
Roscovitine, a novel cyclin‐dependent kinase inhibitor, characterizes restriction point and G2/M transition in tobacco BY‐2 cell suspension
Authors: Planchais Séverine , Glab Nathalie , Tréhin Christophe , Perennes Claudette , Bureau Jean‐Marc , Meijer Laurent , Bergounioux Catherine
Published: March 2003

Although the developmental programs of plants and animals differ, key regulatory components of their cell cycle have been conserved. Particular attention has been paid to the role of the complexes between highly conserved cyclin and cyclin‐dependent kinases in regulating progression through the cell cycle. The recent demonstration that roscovitine is a potent and selective inhibitor of the animal cyclin‐dependent kinases cdc2 (CDK1), CDK2 and CDK5 prompted an investigation into its effects on progression through the plant cell cycle. Roscovitine induced arrests both in late G1 and late G2 phase in BY‐2 tobacco cell suspensions. Both blocks were fully reversible when roscovitine was used at concentrations similar to those used in the animal system. Stationary‐phase cells subcultured in the presence of roscovitine were arrested at a 2C DNA content. This arrest was more efficient without exogenous addition of plant growth regulator. Roscovitine induced a block in G1 earlier than that induced by aphidicolin. S‐phase synchronized cells treated with roscovitine were arrested at a 4C DNA content at the G2/ M transition. The expression analysis of a mitotic cyclin (NTCYC1) indicated that the roscovitine‐induced G2 block probably occurs in late G2. Finally, cells in metaphase were insensitive to roscovitine. The purified CDK/cyclin kinase activities of late G1 and early M arrested cells were inhibited in vitro by roscovitine. The implications of these experimental observations for the requirement for CDK activity during progression through the plant cell cycle are discussed.
Transgenic potato tubers accumulate high levels of 1‐kestose and nystose: functional identification of a sucrose sucrose 1‐fructosyltransferase of artichoke (Cynara scolymus) blossom discs
Authors: Hellwege Elke M. , Gritscher Dominique , Willmitzer Lothar , Heyer Arnd G.
Published: March 2003

By screening a cDNA library of artichoke (Cynara scolymus) blosssom discs for fructosyltransferases, we isolated a clone designated Cy21. The deduced amino acid sequence shows homology to acid β‐fructosyl hydrolases and to the sucrose‐fructan 6‐fructosyltransferase (6‐SFT) of barley. Transiently expressed in Nicotiana tabacum protoplasts, the Cy21 gene‐product synthesized 1‐kestose, indicating that Cy21 codes for a sucrose sucrose 1‐fructosyltransferase (1‐SST). The enzyme worked at physiologically relevant sucrose concentrations (25 mM sucrose). In the protoplast system, 1‐kestose seemed to be the only fructan product of the 1‐SST.

The enzyme activity was not affected by pyridoxal‐HCl, an inhibitor of both the β‐fructosyl hydrolase and the fructosyltransferase activity of invertases. The fructosyltransferase activity of the Cy21 gene‐product, however, could be inhibited by Zn2+, Ag+ and Cu2+ ions. In artichoke plants the Cy21 transcript was highly abundant in primary roots and blossom discs. Transgenic potato tubers expressing Cy21 contain high levels of 1‐kestose along with nystose and traces of fructosyl‐nystose, supporting the conclusion that the Cy21 clone encodes a sucrose sucrose 1‐fructosyltransferase.
Developmental and light‐dependent changes of the cytosolic chaperonin containing TCP‐1 (CCT) subunits in maize seedlings, and the localization in coleoptiles
Authors: Himmelspach Regina , Nick Peter , Schäfer Eberhard , Ehmann Bruno
Published: March 2003

The cytosolic chaperonin containing TCP‐1 (CCT) is known to keep fold cytoskeletal proteins and is involved in the proper organization of the cytoskeleton. These studies are based on the assumption that growth responses linked to structural rearrangement of the plant cytoskeleton include the action of CCT and the need for newly synthesized tubulin. The presence of the α‐ and ɛ‐ subunits of CCT was investigated in soluble fractions of protein extracts from maize mesocotyls and coleoptiles at distinct growth stages. The CCT‐subunits, tubulins and actin decreased in the coleoptile in response to far‐red light. In addition, independent from light treatment, the amount of CCTɛ abundance declined with age in coleoptiles and mesocotyls between 2 and 4.5 days after sowing. In contrast to CCTɛ, no significant light regulation of CCTα was found in the mesocotyl. In two day old, light‐grown rapidly elongating coleoptiles part of the CCTα subunit and the bulk of actin and tubulin was found shifted into fractions of high molecular weight complexes when compared to slowly elongating, dark grown coleoptiles. In 4.5 day old, etiolated and elongating coleoptiles, part of both CCT‐subunits and cytoskeleton proteins were found in fractions of high molecular weight. A complete disappearance of these polypeptides was observed in old far‐red irradiated growth‐arrested coleoptiles. CCTɛ was found to be co‐localized to microtubular structures and to the nucleus. We conclude from our data that abundance of CCT‐subunits in soluble extracts is dependent on age and light treatment, but independent from the growth stage of mesocotyl and coleoptile.
Ectopic expression of oat phytochrome A in hybrid aspen changes critical daylength for growth and prevents cold acclimatization
Authors: Olsen Jorunn E. , Junttila Olavi , Nilsen Jarle , Eriksson Maria E. , Martinussen Inger , Olsson Olof , Sandberg Göran , Moritz Thomas
Published: March 2003

Survival of temperate‐zone tree species under the normal summer‐winter cycle is dependent on proper timing of apical growth cessation and cold acclimatization. This timing is primarily based on the perception of daylength, and through evolution many tree species have developed photoperiodic ecotypes which are closely adapted to the local light conditions. The longest photoperiod inducing growth cessation, the critical photoperiod, is inherited as a quantitative character. The phytochrome pigment family is the probable receptor of daylength, but the exact role of phytochrome and the physiological basis for the different responses between photoperiodic ecotypes are not known. This report shows for the first time that over‐expression of the oat phytochrome A gene (PHYA) in a tree significantly changes the critical daylength and effectively prevents cold acclimatization. While the critical daylength for elongation growth in the wild‐type of hybrid aspen (Populus tremula × tremuloides) was approximately 15 h, transgenic lines with a strong expression of the oat PHYA gene did not stop growing even under a photoperiod of 6 h. Quantitative analysis of gibberellins (GA) as well as indole‐3‐acetic acid (IAA) revealed that levels of these were not down‐regulated under short days in the transgenic plants expressing high levels of oat PHYA, as in the wild‐type. These results indicate that photoperiodic responses in trees might be regulated by the amount of PHYA gene expressed in the plants, and that the amount of phytochrome A (phyA) affects the metabolism of GAs and IAA.
In vivo evidence for 5′→3′ exoribonuclease degradation of an unstable chloroplast mRNA
Authors: Drager Robert G. , Girard‐bascou Jacqueline , Choquet Yves , Kindle Karen L. , Stern David B.
Published: March 2003

The acetate‐requiring Chlamydomonas reinhardtii nuclear mutant F16 harbors the mutation mcd1–1 and fails to accumulate the cytochrome b6/f complex. The primary defect of mcd1–1 was determined to be the instability of petD mRNA, which encodes subunit IV of the complex. Chimeric reporter genes introduced by chloroplast transformation demonstrated that the determinant of petD mRNA instability in the mcd1–1 background is located in the 5′ untranslated region (UTR). However, when this 5′ UTR was present downstream of other sequences in dicistronic or chimeric transcripts, the RNAs were no longer destabilized in the mcd1–1 background. Together, these results suggest that the 5′ end of the petD 5′ UTR interacts with the MCD1 product. The insertion of a polyguanosine sequence into the petD 5′ UTR fused to a reporter gene allowed accumulation of the reporter gene transcript in the mutant background. Since polyguanosine forms a structure that is known to impede exonucleases, these data provide in vivo evidence that petD mRNA can be degraded by 5′→3′ exoribonuclease activity. Furthermore, the data support a model in which protein binding to the petD 5′ UTR protects the mRNA from 5′→3′ degradation in wild‐type cells.
The role of ion fluxes in Nod factor signalling in Medicago sativa
Authors: Felle Hubert H. , Kondorosi Éva , Kondorosi Ádám , Schultze Michael
Published: March 2003

Using ion‐selective microelectrodes, the basis of Nod factor‐induced changes in the plasma membrane potential was analysed by measuring the extracellular free concentrations of Ca2+, K+, H+ and Cl– in the root hair zone of alfalfa. After addition of the Rhizobium meliloti Nod factor NodRm‐IV(C16:2,S) at a concentration of 0.1 μM, a decrease in [Ca2+] was observed first, which was followed after a few seconds by an increase of [Cl–], by an alkalinization, and then by a delayed increase of [K+], all of which were transient changes. Simultaneously with the appearance of Cl– ions in the root hair zone, a decrease in cytosolic [Cl–] was measured. It was concluded that the depolarization was caused by temporary short‐circuiting of the proton pump through the rapid release of Cl– ions along their steep electrochemical gradient. Since under resting conditions the driving force for K+ ions was inwardly directed, their release was delayed until their driving force was inverted. This indicates that K+ serves as a charge balance that eventually stops depolarization and initiates repolarization. Since the decrease in [Ca2+] was observed seconds before the increase in [Cl–] and the depolarization, it is argued that Ca2+ entering into the cell does not cause the depolarization directly, but might initiate it by triggering the activation of an anion channel that then releases the chloride ions. The observations that the Ca2+ ionophore A23187 mimicks the Nod factor response, and that the Ca2+ channel antagonist nifedipine inhibits this response, support the idea that Ca2+ plays a primary role in the transduction of the Nod signal in alfalfa.
Solubilization and characterization of a galacturonosyltransferase that synthesizes the pectic polysaccharide homogalacturonan
Authors: Doong Ron Lou , Mohnen Debra
Published: March 2003

Polygalacturonate 4‐α‐galacturonosyltransferase (PGA‐GalAT), the glycosyltransferase that synthesizes the plant cell wall pectic polysaccharide homogalacturonan, has previously been identified and partially characterized in tobacco membranes. Membrane bound PGA‐GalAT catalyzes the transfer of galacturonic acid from UDP‐galacturonic acid (UDP‐GalA) onto an endogenous acceptor to produce polymeric homogalacturonan ( Doong et al. (1995) Plant Physiol. 109, 141
–152). It is shown here that a galacturonosyltransferase is solubilized from tobacco membranes with a HEPES buffer, pH 6.8, containing 40 mM CHAPS and 2 mM EDTA. The solubilized galacturonosyltransferase was identified as putative PGA‐GalAT because it transfered [14C]GalA from UDP‐[14C]GalA onto exogenous homogalacturonan acceptors with degrees of polymerization (DP) of ≥ 10. Maximal solubilized PGA‐GalAT activity in the presence of 0.9 μM UDP‐[14C]GalA required approximately 125 μM exogenous homogalacturonan acceptor [e.g. oligogalacturonide (OGA) of DP 15]. Solubilized PGA‐GalAT was active over a broad pH range of 6.3–7.8, and had an apparent Km for UDP‐GalA of 37 μM and a Vmax of 290 pmol min–1 mg–1 protein. Approximately 44% of the PGA‐GalAT activity in detergent‐dispersed membranes, corresponding to 21% of the PGA‐GalAT activity in intact membranes, was solubilized. PGA‐GalAT solubilized with 40 mM CHAPS was shown, by exopolygalacturonase treatment in combination with size exclusion and high performance anion exchange chromatographies, to add a single α‐1,4‐linked galacturonic acid residue onto an OGA exogenous acceptor of DP 15 to yield an OGA product of DP 16. The significance of the apparent lack of processivity of the solubilized PGA‐GalAT is discussed.
Inhibition of plant viral systemic infection by non‐toxic concentrations of cadmium
Authors: Ghoshroy Soumitra , Freedman Karen , Lartey Robert , Citovsky Vitaly
Published: March 2003

Heavy metals, such as cadmium, have a significant impact on plant physiology. However, their potential effect on plant–pathogen interaction, an important biological process, has not been examined. This study shows that exposure of tobacco plants to non‐toxic concentrations of cadmium completely blocked viral disease caused by turnip vein clearing virus. Cadmium‐mediated viral protection was due to inhibition of the systemic movement of the virus, i.e. its spread from the inoculated into uninoculated leaves. Exposure of plants to cadmium had no effect on viral replication, assembly and local movement within the inoculated leaf. Analysis of the viral presence in different tissues suggested that cadmium treatment inhibited virus exit from the vascular tissue into uninoculated leaves rather than its entry into the host plant vasculature. Higher, toxic levels of cadmium did not produce this inhibitory effect on viral movement, allowing the systemic spread of the virus and development of the viral disease. These observations suggest that cadmium‐induced viral protection requires a relatively healthy, unpoisoned plant in which non‐toxic levels of cadmium may trigger the production of cellular factors which interfere with the viral systemic movement.
Induction of systemic resistance to Pythium damping‐off in cucumber plants by benzothiadiazole: ultrastructure and cytochemistry of the host response
Authors: Benhamou Nicole , Bélanger Richard R.
Published: March 2003

Benzo (1,2,3) thiadiazole‐7‐carbothioic acid S‐methyl ester (BTH, CGA 245704), a non‐toxic, synthetic chemical, was applied as a foliar spray to cucumber plants and evaluated for its potential to induce defense mechanisms in root tissues infected by the soilborne pathogen, Pythium ultimum Trow. In non‐treated cucumber plants, fungal colonization was intense and paralleled marked host tissue damage, whereas in BTH‐treated plants, pathogen ingress towards the vascular stele was apparently halted by the massive deposition of a phenolic‐enriched material which occluded a large number of cortical and vascular parenchyma cells. This considerable increase in the accumulation of phenolics was accompanied by cytological disorders of the invading pathogen at a time when the wall‐bound cellulose component was preserved. In addition to phenolic compounds, the occluding material contained large amounts of β‐glucoside residues. These residues gradually decreased in the areas neighboring fungal cells whereas phenolic deposition appeared to be more uniformly distributed throughout the occluded host cells. Pathogen penetration in non‐occluded cucumber root cells coincided with other changes, mainly characterized by both the deposition onto the inner surface of the cell walls of some heterogeneous wall appositions and the coating of some intercellular spaces with an electron‐opaque material. Evidence is provided in this study that BTH has the ability to induce SAR in cucumber. Exogenous, foliar applications of the chemical sensitize susceptible cucumber plants to react more rapidly and more efficiently to P. ultimum attack, mainly through the massive accumulation of phenolic compounds at sites of attempted pathogen penetration.
Identification and characterization of a locus (RTM1) that restricts long‐distance movement of tobacco etch virus in Arabidopsis thaliana
Authors: Mahajan Sunita K. , Chisholm Stephen T. , Whitham Steven A. , Carrington James C.
Published: March 2003

Screens of Arabidopsis thaliana for susceptibility to tobacco etch virus (TEV) revealed that each of 10 ecotypes were able to support genome replication and cell‐to‐cell movement in inoculated leaves. However, only four ecotypes, including C24 and La‐er, supported complete infections in which TEV was able to replicate and move from cell to cell and long distances through the vasculature. The rates of cell‐to‐cell movement of a reporter‐tagged TEV strain (TEV–GUS) in inoculated leaves of C24 and Columbia (Col‐3) were similar, and infection foci continued to expand in both ecotypes through 10 days post‐inoculation. No visible or microscopic hypersensitive or cell death responses were evident in inoculated leaves of Col‐3 plants. Infection of neither C24 nor Col‐3 plants with TEV–GUS resulted in induction of PR‐1a gene expression, which is normally associated with active defence responses and systemic acquired resistance. The genetic basis for the restriction of long‐distance movement of TEV–GUS in Columbia was investigated using C24 × Col‐3 crosses and backcrosses and using La‐er× Col‐0 recombinant inbred lines. A dominant locus conditioning the restricted TEV infection phenotype was identified on chromosome 1 between markers ATEAT1 and NCC1 at approximately 14 cM in both genetic analyses. This locus was designated RTM1 (restricted TEV movement 1). It is proposed that RTM1 mediates a restriction of long‐distance movement through a mechanism that differs substantially from those conditioned by the dominant resistance genes normally associated with gene‐for‐gene interactions.
dCAPS, a simple technique for the genetic analysis of single nucleotide polymorphisms: experimental applications in Arabidopsis thaliana genetics
Authors: Neff Michael M. , Neff Joseph D. , Chory Joanne , Pepper Alan E.
Published: March 2003

PCR‐based detection of single nucleotide polymorphisms is a powerful tool for the plant geneticist. Cleaved amplified polymorphic sequence analysis is the most widely used approach for the detection of single nucleotide polymorphisms. However, this technique is limited to mutations which create or disrupt a restriction enzyme recognition site. This paper presents a modification of this technique where mismatches in a PCR primer are used to create a polymorphism based on the target mutation. This technique is useful for following known mutations in segregating populations and genetic mapping of isolated DNAs used for positional based cloning of new genes. In addition, a computer program has been developed that facilitates the design of these PCR primers.
Magnesium ions promote assembly of channel‐like structures from beticolin 0, a non‐peptide fungal toxin purified from Cercospora beticola
Authors: Goudet Cyril , Véry Anne‐Aliénor , Milat Marie‐Louise , Ildefonse Michèle , Thibaud Jean‐Baptiste , Sentenac Hervé , Blein Jean‐Pierre
Published: March 2003

Beticolins are toxins produced by the fungus Cercospora beticola. Using beticolin 0 (B0), we have produced a strong and Mg2+‐dependent increase in the membrane conductance of Arabidopsis protoplasts and Xenopus oocytes. In protein‐free artificial bilayers, discrete deflexions of current were observed (12 pS unitary conductance in symmetrical 100 mM KCl) in the presence of B0 (approximately 10 μM) and in the presence of nominal Mg2+. Addition of 50 μM Mg2+ induced a macroscopic current which could be reversed to single channel current by chelating Mg2+ with EDTA. Both unitary and macroscopic currents were ohmic. The increase in conductance of biological membranes triggered by B0 is therefore likely to originate from the ability of this toxin to organize itself into transmembrane pores in the presence of Mg2+. The pore is poorly selective, displaying permeability ratios PCl/PK, PNa/PK and PCa/PK close to 0.3, 0.65 and 0.4, respectively. Such channel‐like activity could be involved in the deleterious biological activity of the toxin, by causing the collapse of ionic and electrical gradients through biological membranes together with Ca2+ influx and scrambling of cellular signals.
Six Arabidopsis thaliana homologues of the human respiratory burst oxidase (gp91phox)
Authors: Torres Miguel Angel , Onouchi Hitoshi , Hamada Susuma , Machida Chiyoko , Hammond‐Kosack Kim E. , Jones Jonathan D. G.
Published: March 2003

An NADPH oxidase analogous to that in mammalian phagocytes has been hypothesized to produce reactive oxygen species (ROS) in the plant defence response. A. thaliana contains at least six gp91phox homologues, designated AtrbohA‐F (A. thaliana Respiratory Burst Oxidase Homologues), which map to different positions. Transcripts of three of these genes can be detected in healthy plants by RNA gel blot analyses. The Atrboh gene products are closely related to gp91phox and the intron locations suggest a common evolutionary origin. A putative EF‐hand Ca2+‐binding motif in the extended N‐terminal region of the Atrboh proteins suggests a direct regulatory effect of Ca2+ on the activity of the NADPH oxidase in plants.
Rhizobial lipochitooligosaccharide nodulation factors activate expression of the legume early nodulin gene ENOD12 in rice
Authors: Reddy Pallavolu M. , Ladha Jagdish K. , Ramos Marilou C. , Maillet Fabienne , Hernandez Rowena J. , Torrizo Lina B. , Oliva Norman P. , Datta Swapan K. , Datta Karabi
Published: March 2003

Lipochitooligosaccharide nodulation factors (Nod factors) produced by rhizobia are a major host range determinant. These factors play a pivotal role in the molecular signal exchange, infection and induction of symbiotic developmental responses in legumes leading to the formation of a nodule in which rhizobia carry out N2 fixation. Determining whether rice (

Oryza sativa) can respond to Nod factors could lead to strategies that would make rice amenable to develop a nitrogen‐fixing endosymbiotic association with rhizobia. We introduced into rice the promoter of the infection‐related gene

MtENOD12
(from

Medicago truncatula) fused to the β‐glucuronidase (GUS) reporter gene to serve as a molecular marker to aid in the detection of Nod factor signal perception by rice cells. Treatment of the transgenic rice roots with Nod factors (10–6–10–9m) under nitrogen‐limiting conditions induced MtENOD12‐GUS expression in cortical parenchyma, endodermis and pericycle. In contrast, chitooligosaccharide backbone alone failed to elicit such a response in the root tissues. These findings demonstrate that rice roots perceive Nod factors and that these lipochitooligosaccharides, but not simple chitin oligomers, act as signal molecules in activatingMtENOD12
in cortical parenchyma as in legumes. Exogenous application ofN‐naphthaleneacetic acid mimicked the Nod factor‐elicited tissue‐specific expression ofMtENOD12
in roots while cytokinins inhibited it, thus evidencing that Nod factors, auxin and cytokinins probably act on similar signaling elements responsible for the regulation ofMtENOD12
activation in rice. Taken together, these results suggest that at least a portion of the signal transduction machinery important for legume nodulation is likely to exist in rice.
Spatial specificity of H2O2‐generating oxalate oxidase gene expression during wheat embryo germination
Authors: Caliskan Mahmut , Cuming Andrew C.
Published: March 2003

Germin, a molecular marker of wheat embryo germination, is a protease‐resistant, apoplastic, homopentameric glycoprotein with peroxide‐generating oxalate oxidase activity. The spatial specificity of germin‐like oxalate oxidase (gl‐OXO) gene expression has been determined in tissues of germinating wheat embryos by a combination of histochemical, immunocytochemical and in situ hybridization techniques. The synthesis and accumulation of gl‐OXO mRNA and protein is localised within the enveloping tissues of the embryonic axis (particularly the coleorhiza) during the first 24 h of imbibition. By 48 h germination, gl‐OXO accumulation is detected throughout the root, with the exception of the postmitotic zone of cell elongation, where accumulation of its transcript is restricted to outer cell layers. At this time in the elongating shoot, gl‐OXO is restricted to the coleoptile where it is detected only in the epidermal cell layer, the vascular bundles and bundle sheath cells. In older seedlings (approximately 9 days post‐imbibition) gl‐OXO activity is detected in leaves, but only within the vascular bundles. These patterns of expression are consistent with the hypothesis that the biological function of gl‐OXO is to restrict cell growth by participating in cell‐wall restructuring through the local provision of hydrogen peroxide for cross‐linking of wall components.
Expression cloning of a fungal proline‐rich glycoprotein specific to the biotrophic interface formed in the Colletotrichum–bean interaction
Authors: Perfect Sarah E. , O’Connell Richard J. , Green Elaine F. , Doering‐Saad Christiane , Green Jonathan R.
Published: March 2003

The monoclonal antibody, UB25, recognises a glycoprotein specifically located at the biotrophic interface formed in the Colletotrichum lindemuthianum–bean interaction. The antibody labels the walls of intracellular hyphae and the interfacial matrix which separates them from the invaginated host plasma membrane. In Western blots, UB25 recognises a ladder of bands which are multiples of Mr 40.5 kDa. A full length cDNA encoding the glycoprotein recognised by UB25 has been isolated by expression cloning and designated CIH1 (Colletotrichum Intracellular Hypha 1). In vitro transcription/translation of CIH1, and transfection of mammalian COS cells, showed that UB25 recognised the expressed product in both procedures confirming that the clones isolated were true positives. Southern analysis of bean and C. lindemuthianum genomic DNA indicated that the CIH1 glycoprotein is fungally encoded and Northern analysis showed that it is only expressed in planta. Analysis of the deduced amino acid sequence of CIH1 indicates the presence of an N‐terminal signal sequence and two possible sites for N‐glycosylation. The N‐terminal domain of the mature protein is rich in proline and contains several short repetitive motifs. CIH1 is thus a fungal proline‐rich glycoprotein which appears to form a cross‐linked structure in planta and, as such, resembles plant cell wall proline‐ and hydroxyproline‐rich proteins. Possible functions for the CIH1 protein in the establishment and maintenance of biotrophy are discussed.
Co‐ and/or post‐translational modifications are critical for TCH4 XET activity
Authors: Campbell Paul , Braam Janet
Published: March 2003

TCH4

encodes a xyloglucan endotransglycosylase (XET) of

Arabidopsis thaliana

. XETs endolytically cleave and religate xyloglucan polymers; xyloglucan is one of the primary structural components of the plant cell wall. Therefore, XET function may affect cell shape and plant morphogenesis. To gain insight into the biochemical function of TCH4, we defined structural requirements for optimal XET activity. Recombinant baculoviruses were designed to produce distinct forms of TCH4. TCH4 protein engineered to be synthesized in the cytosol and thus lack normal co‐ and post‐translational modifications is virtually inactive. TCH4 proteins, with and without a polyhistidine tag, that harbor an intact N‐terminus are directed to the secretory pathway. Thus, as predicted, the N‐terminal region of TCH4 functions as a signal peptide. TCH4 is shown to have at least one disulfide bond as monitored by a mobility shift in SDS‐PAGE in the presence of dithiothreitol (DTT). This disulfide bond(s) is essential for full XET activity. TCH4 is glycosylated

in vivo

; glycosidases that remove N‐linked glycosylation eliminated 98% of the XET activity. Thus, co‐ and/or post‐translational modifications are critical for optimal TCH4 XET activity. Furthermore, using site‐specific mutagenesis, we demonstrated that the first glutamate residue of the conserved DEIDFEFL motif (E97) is essential for activity. A change to glutamine at this position resulted in an inactive protein; a change to aspartic acid caused protein mislocalization. These data support the hypothesis that, in analogy to

Bacillus

β‐glucanases, this region may be the active site of XET enzymes.
Immediate early induction of mRNAs for ethylene‐responsive transcription factors in tobacco leaf strips after cutting
Authors: Suzuki Kaoru , Suzuki Nao , Ohme‐Takagi Masaru , Shinshi Hideaki
Published: March 2003

To investigate the functional relationship between the expression of genes for ethylene‐responsive transcription factors (ERFs) and the expression of ethylene‐responsive genes, we examined the expression of genes for ERFs and the expression of a reporter gene in transgenic tobacco that carried a gene for β‐glucuronidase (GUS) under the control of the ethylene‐responsive element, which includes four copies of the 11‐bp consensus sequence (designated the GCC‐box, TAAGAGCCGCC). In strips of leaves of transgenic tobacco, the GCC‐box‐mediated expression of the reporter gene was induced in response to treatment with ethylene. We also observed the ethylene‐independent immediate early induction of the synthesis of mRNAs for ERFs in wounded leaves and the enhancement of this induction by cycloheximide (CHX). Since CHX suppressed the induction of mRNAs for chitinase and GUS by ethylene, protein synthesis de novo was required for induction of the ethylene‐dependent GCC‐box‐mediated transcription of genes. In contrast, the enhancement by CHX of the wound‐induced expression of ERFs suggested that no synthesis of new proteins was required for the wounding signal transduction leading to rapid expression of ERFs. Methyl jasmonate did not stimulate the wounding‐responsive accumulation of ERF mRNAs, but it reduced such accumulation of mRNAs for ERF1, ERF2, ERF4 and the ethylene‐dependent GCC‐box‐mediated transcription of the reporter gene. Thus, the immediate early induction of the expression of genes for ERFs in strips of tobacco leaves appears to be a novel type of wound‐responsive activation of transcription. These results suggested that the expression of ERFs was not sufficient for activation of the GCC‐box‐mediated transcription but the expression of ERF1, ERF2 and ERF4, and that conversion of these ERFs by ethylene to their active form might be crucial for the GCC‐box‐mediated activation of the transcription of defense genes.
Genetic ablation of flowers in transgenicArabidopsis
Authors: Nilsson Ove , Wu Eric , Wolfe Diana S. , Weigel Detlef
Published: March 2003

We have created transgenic

Arabidopsis

plants in which a gene encoding the cell‐autonomous diphtheria toxin A chain (DT‐A) was expressed under the control of the

LEAFY (LFY)

promoter. This promoter is active both in emerging leaf primordia and young flowers, with the highest activity in flowers. The majority of

LFY::DT‐A

plants had normal vegetative development but lacked flowers, demonstrating that relatively widespread activity of a promoter does not exclude its possible use for ablating selected tissues, as long as differences in activity levels between different tissues are significant. We also found that flowers were replaced by empty bracts in

LFY::DT‐A

plants, suggesting that flower‐derived signals normally suppress bract development in

Arabidopsis

.
Divalent cation gating of an ammonium permeable channel in the symbiotic membrane from soybean nodules
Authors: Whitehead Lynne F. , Day David A. , Tyerman Stephen D.
Published: March 2003

The symbiotic membrane between N2‐fixing bacteroids and plant cytoplasm in nodules of soybean contains a sub‐picoSiemen cation channel permeable to NH4+. With millimolar concentrations of Ca2+ or Mg2+ on the cytoplasmic side, the channel rectifies current in the direction of cation influx to the cytoplasm. When Ca2+ is present on the bacteroid side of the membrane the current is rectified in the opposite direction. With submicromollar concentrations of divalent on both sides, the channel no longer rectifies. The channel is inhibited by verapamil on the bacteroid side of the membrane with a Kd of 2.6 μM. In the presence of millimolar concentrations of divalents on the cytoplasmic side, the conductance as a function of voltage is fitted by a simple Boltzmann equation with an effective gating charge equal to one. The voltage at which the conductance reaches 50% of maximum is dependent on external NH4+, shifting negative at lower concentrations. The time‐course of activation upon hyperpolarisation can be described by the Hodgkin‐Huxley equation with Ca2+present on the cytoplasmic side. With Mg2+ the channel activates with single exponential kinetics. The time constant for activation is weakly voltage dependent. Upon depolarisation of the membrane the channel deactivates with double exponential kinetics, the time constants being slightly voltage dependent. We propose a model of the channel in which divalent block is relieved when the blocking ion is dislodged by univalent cation flux into the pore. Mg2+ on the cytoplasmic side may function

in vivo

as the gating particle of the channel.
A novel NADPH‐dependent aldehyde reductase gene fromVigna radiataconfers resistance to the grapevine fungal toxin eutypine
Authors: Guillén Pedro , Guis Monique , Martínez‐Reina Gracia , Colrat Ségolène , Dalmayrac Sylvie , Deswarte Corine , Bouzayen Mondher , Roustan Jean‐Paul , Fallot Jean , Pech Jean‐Claude , Latché Alain
Published: March 2003

Eutypine, 4‐hydroxy‐3‐(3‐methyl‐3‐butene‐1‐ynyl) benzyl aldehyde, is a toxin produced by

Eutypa lata,

the causal agent of eutypa dieback of grapevines. It has previously been demonstrated that tolerance of some cultivars to this disease was correlated with their capacity to convert eutypine to the corresponding alcohol, eutypinol, which lacks phytotoxicity. We have thus purified to homogeneity a protein from

Vigna radiata

that exhibited eutypine‐reducing activity and have isolated the corresponding cDNA. This encodes an NADPH‐dependent reductase of 36 kDa that we have named

Vigna radiata

eutypine‐reducing enzyme (VR‐ERE), based on the capacity of a recombinant form of the protein to reduce eutypine into eutypinol. The strongest homologies (86.8%) of VR‐ERE at the amino acid level were found with CPRD14, a drought‐inducible gene of unknown function, isolated from

Vigna unguiculata

and with an aromatic alcohol dehydrogenase (71.7%) from

Eucalyptus gunnii

. Biochemical characterization of VR‐ERE revealed that a variety of compounds containing an aldehyde group can act as substrates. However, the highest affinity was observed with 3‐substituted benzaldehydes. Expression of a

VR‐ERE

transgene in

Vitis vinifera

cells cultured

in vitro

conferred resistance to the toxin. This discovery opens up new biotechnological approaches for the generation of grapevines resistant to eutypa dieback.
Molecular characterization of higher plant NAD‐dependent isocitrate dehydrogenase: evidence for a heteromeric structure by the complementation of yeast mutants
Authors: Lancien Muriel , Gadal Pierre , Hodges Michael
Published: March 2003

NAD‐dependent isocitrate dehydrogenase (IDH) is a key enzyme controlling the activity of the citric acid cycle. Despite more than 30 years of work, the plant enzyme remains poorly characterized. In this paper, a molecular characterization of the plant IDH is presented. Starting from probes defined according to sequence comparisons, three full‐length cDNAs named

Ntidha

,

Ntidhb

and

Ntidhc

encoding different IDH subunits have been isolated from a

Nicotiana tabacum

cell suspension library. Sequence comparisons of the tobacco IDH subunits with the

E. coli

NADP‐dependent enzyme, and the yeast IDH1 and IDH2 subunits suggested that only IDHa had the capacity to be catalytic as IDHb and IDHc were lacking certain residues implied in catalysis. The ability of antibodies raised against the recombinant IDHa protein to preferentially cross‐react with IDH2 indicated that IDHa was more closely related to IDH2 than to IDH1. Complementation of yeast single IDH mutants showed that IDHb and IDHc could replace the function of the yeast regulatory IDH1 subunit. Although IDHa was unable to complement the IDH2 mutant, its catalytic function was revealed by the ability of two heteromeric enzymes, composed of either IDHa with IDHb or IDHa with IDHc, to replace IDH function in a yeast double mutant lacking both subunits. Expression studies at the protein and mRNA levels show that each subunit is present in both root and leaf tissues and that the three IDH genes respond in the same way to nitrate addition. Taken together, such observations suggest that the physiologically active enzyme is composed of the three different subunits. These results show for the first time that the plant IDH is heteromeric and that IDH subunit composition appears to be conserved between plant and animal kingdoms.
Genetic identification of FIN2, a far red light‐specific signaling component ofArabidopsis thaliana
Authors: Soh Moon Soo , Hong Sung Hyun , Hanzawa Hiroko , Furuya Masaki , Nam Hong Gil
Published: March 2003

Phytochrome A (PhyA) mediates most, if not all various plant responses to far‐red (FR) light. Here, we report a novel genetic mutation that impairs a variety of responses in the PhyA‐signaling pathway of

Arabidopsis thaliana

. The mutation was isolated by screening seedlings that show reduced sensitivity to continuous far‐red (FRc) light irradiation, but not to continuous red (Rc) light irradiation. The mutation named

fin2–1

is not allelic to a

PHYA

mutation. Furthermore, immunoblot analysis indicated that the amount of the phytochrome A apoprotein in the

fin2–1

mutant was comparable to that in wild type. Seedling of the

fin2–1

mutant showed defects in hypocotyl growth inhibition and apical hook and cotyledon opening in FRc light but not in Rc light. The results showed that the mutation occurred in a downstream signaling component potentially specific to PhyA. Other PhyAmediated responses such as FR‐preconditioned blocking of greening, anthocyanin accumulation, reduction of gravitropic response, and expression of the

CAB

and

CHS

genes were impaired by the

fin2–1

mutation: the degree of the mutant effect on the responses was variable. However, FR light‐mediated seed germination and photoperiodic flowering responses were not affected significantly in the mutant. These results showed that FIN2 defines an upstream branch point in the PhyA signaling pathway.
Direct repeats of T‐DNA integrated in tobacco chromosome: characterization of junction regions
Authors: Krizkova Lucie , Hrouda Milan
Published: March 2003

Plant transformation via

Agrobacterium

frequently results in formation of multiple copy T‐DNA arrays at one target site of the chromosome. The T‐DNA copies are arranged in repeats, direct or inverted around one of the T‐DNA borders. A Ti plasmid‐derived transformation vector has been constructed enabling direct selection of transformants carrying at least two linked copies of T‐DNA in the same orientation. The selection is based on expression of a promoterless neomycin phosphotransferase gene on one T‐DNA copy from a promoter located on the other T‐DNA copy. After co‐cultivation of tobacco protoplasts with

Agrobacterium

, as many as 30% of regenerated transformed plants carried directly repeated T‐DNA copies. The junction regions between two T‐DNAs were amplified and 13 amplified fragments were cloned and sequenced. The involvement of T‐DNA left and right border sequences in direct repeat junctions was determined. In some junctions, additional filler DNA was detected. The length of filler DNA varied from a few up to almost 300 bp. The longer filler DNAs from two clones were found to be T‐DNA fragments in direct or reverse orientation. We discuss the recently suggested models for T‐DNA integration and propose that the formation of direct repeats in genomes does not necessarily result from ligation of intermediates (i.e. T‐strands), but more likely from the co‐integration of several intermediates into one target site.
Treatment ofVicia fabaroot tip cells with specific inhibitors to cyclin‐dependent kinases leads to abnormal spindle formation
Authors: Binarová Pavla , Doležel Jaroslav , Draber Pavel , Heberle‐Bors Erwin , Strnad Miroslav , Bögre László
Published: March 2003

Many events during cell division are triggered by an evolutionary conserved regulator, the cyclin‐dependent kinase (Cdk). Here we used two novel drugs, the purine analogues bohemine and roscovitine, to study the role of Cdks in cell cycle progression and microtubule organisation in

Vicia faba

root tip cells. Both drugs inhibited the activity of immunopurified

Vicia faba

and alfalfa Cdc2‐kinase. The transcript levels of an A‐ and B‐type cyclin, as well as of the

cdc2

genes, declined in treated root tips, while the mRNA level of a D‐type cyclin gene was not affected. An observed transient arrest at the G1/S and G2/M regulatory points indicated that inhibition of the Cdc2‐kinase had an effect on both transitions. In contrast to the regular bipolar spindle in untreated cell, in drug‐treated metaphase cells abnormally short and dense kinetochore microtubule fibres were observed. These microtubules were randomly arranged in the vicinity of the kinetochores and connected the chromosomes. Thus, the chromosomes were not aligned on the metaphase plate but were arranged in a circle, with kinetochores pointing inwards and chromosome arms pointing outwards. γ‐Tubulin, which plays a role in microtubule nucleation, also localised to the centre of the monopolar spindle. The observed abnormalities in mitosis, after inhibition of Cdc2‐kinase by specific drugs, suggest a role for this enzyme in regulating some of the steps leading to a bipolar spindle structure.
KCS1encodes a fatty acid elongase 3‐ketoacyl‐CoA synthase affecting wax biosynthesis inArabidopsis thaliana
Authors: Todd James , Post‐Beittenmiller Dusty , Jaworski Jan G.
Published: March 2003

An Arabidopsis fatty acid elongase gene,

KCS1

, with a high degree of sequence identity to

FAE1

, encodes a 3‐ketoacyl‐CoA synthase which is involved in very long chain fatty acid synthesis in vegetative tissues, and which also plays a role in wax biosynthesis. Sequence analysis of

KCS1

predicted that this synthase was anchored to a membrane by two adjacent N‐terminal, membrane‐spanning domains. Analysis of a T‐DNA tagged

kcs1–1

mutant demonstrated the involvement of the

KCS1

in wax biosynthesis. Phenotypic changes in the

kcs1–1

mutant included thinner stems and less resistance to low humidity stress at a young age. Complete loss of

KCS1

expression resulted in decreases of up to 80% in the levels of C26 to C30 wax alcohols and aldehydes, but much smaller effects were observed on the major wax components, i.e. the C29 alkanes and C29 ketones on leaves, stems and siliques. In no case did the loss of

KCS1

expression result in complete loss of any individual wax component or significantly decrease the total wax load. This indicated that there was redundancy in the elongase KCS activities involved in wax synthesis. Furthermore, since alcohol, aldehyde, alkane and ketone levels were affected to varying degrees, involvement of the

KCS1

synthase in both the decarbonylation and acyl‐reduction wax synthesis pathways was demonstrated.
A partially assembled complex I in NAD4‐deficient mitochondria of maize
Authors: Karpova Olga V. , Newton Kathleen J.
Published: March 2003

The proton‐translocating NADH:ubiquinone oxidoreductase (respiratory complex I) consists of at least 32 subunits in higher plants, nine of which are mitochondrially encoded (NAD 1–7, NAD4L, NAD9). Complex I (CI) has been analyzed from a mitochondrial mutant of maize, NCS2, that carries a deletion for the 3′ end of the
nad4
gene. Mitochondria from highly defective, near‐homoplasmic mutant plants have only trace amounts of the normal complex I. Instead, a reduced amount of a smaller complex, which also exhibits NADH dehydrogenase activity, is detected on ‘blue‐native’ polyacrylamide gels. Subunits of 76 kDa, 40 kDa and 55 kDa, as well as NAD7 and NAD9, have been identified in the subcomplex by their cross‐reactivity with heterologous antisera. The corresponding subunits in
Neurospora
are localized in a ‘peripheral arm’ of CI, which is known to assemble independently of a ‘membrane arm’. The maize NCS2 CI subcomplex is loosely bound to the membrane and is missing several subunits that could be membrane components. Thus, the mutant CI subcomplex may consist of a peripheral arm. A reduction in the steady‐state levels of NAD7 and NAD9 in NCS2 mitochondria occurs despite normal rates of biosynthesis and there is a concomitant decrease of the nuclear encoded 76 kDa subunit. The reduction in CI‐associated NADH dehydrogenase activity in the
nad4
‐deficient NCS2 mutant mitochondria is not associated with a compensatory increase in the activities or amounts of the putative ‘exogenous’ NAD(P)H dehydrogenases that are found in plant mitochondria.
Cell wall alterations and localized accumulation of feruloyl‐3′‐methoxytyramine in onion epidermis at sites of attempted penetration by Botrytis allii are associated with actin polarisation, peroxidase activity and suppression of flavonoid biosynthesis
Authors: McLusky Sarah R. , Bennett Mark H. , Beale Michael H. , Lewis Mervyn J. , Gaskin Paul , Mansfield John W.
Published: March 2003

Granular deposits of reaction material (RM) were formed in onion epidermal cells at sites of attempted penetration by
Botrytis allii
. Both RM and the adjacent cell wall fluoresced blue under UV excitation. The blue autofluorescence was caused primarily by the accumulation of feruloyl‐3′‐methoxytyramine (FMT) and feruloyltyramine (FT) within challenged tissues. Additional phenolics increasing at infection sites were identified as coumaroyl glucose, coumaroyltyramine (CT) and 2‐hydroxy‐2‐(4‐hydroxyphenyl) ethylferulate. The major autofluorescent components of RM, the hydroxycinnamic acid amides FMT and FT, were bound by ether linkage onto the cell wall as well as being present in methanol soluble granules. Formation of RM was associated with early increases in peroxidase activity detected by histochemistry at reaction sites and striking polarisation of actin microfilaments. Quantitative analysis of quercetin and cyanidin glucosides revealed that the localized synthesis and deposition of feruloyltyramine derivatives was associated with suppression of flavonoid and anthocyanin accumulation in a zone of cells around those accumulating RM. No antifungal activity was detected in FMT, FT or CT, nevertheless it is proposed that the phenolics have a key role in resistance by preventing fungal degradation of the cell wall.