bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

Summary 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

SummaryTo 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

SummaryIn 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

SummaryImportant 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.
A novel parsley 4CL1 cis‐element is required for developmentally regulated expression and protein‐DNA complex formation
Authors: Neustaedter David A. , Lee Stephen P. , Douglas Carl J. 
Published: March 2003

SummaryThe phenylpropanoid enzyme 4‐coumarate:coenzyme A ligase (4CL) participates in the biosynthesis of a wide range of secondary products with specialized function and tissue distribution in plants. The parsley 4CL1 promoter directs a complex tissue‐ and cell‐specific pattern of reporter gene expression in transgenic tobacco, consistent with the distribution of phenylpropanoid products and sites of 4CL expression in tobacco vegetative and floral organs. We generated mutants in a 4CL1 promoter element previously implicated as a site for protein–DNA complex formation to analyze its role in vivo4CL1 promoter surrounding the FP56 element is the site for formation of two large protein–DNA complexes, and that an intact FP56 element is required for formation of these complexes. Finally, the detergent deoxycholate was used to investigate the role of protein–protein interactions in FP56 complex formation. Our data suggest that the FP56 cis‐element plays a central role in transcriptional activation from the 4CL1 promoter, and that its role may be to nucleate formation of a large protein complex on the promoter.
Construction of tomato genomic DNA libraries in a binary‐BAC (BIBAC) vector
Authors: Hamilton Carol M.. , Frary Anne , Xu Yimin , Tanksley Steven D.. , Zhang Hong‐Bin 
Published: March 2003

SummaryThis is the first report of large insert genomic DNA libraries constructed in a binary‐BAC (BIBAC) vector. Genomic DNA libraries containing approximately 4.6 haploid nuclear genomic equivalents were constructed for Lycopersicon esculentum (cv. Mogeor) and Lycopersicon pennellii (LA716) in the BIBAC2 vector. The L. esculentum library has an average insert size of 125 kb and is comprised of 42 272 individual colonies stored as frozen cultures in a 384‐well format (108 plates). The L. pennellii library has an average insert size of 90 kb and is comprised of 53 760 individual clones (140 384‐well plates). In each of the libraries, it is estimated that 90% of the colonies contain genomic DNA inserts. The composition of the L. esculentum and L. pennellii libraries was determined by analyzing a series of randomly selected clones. The L. esculentum library was surveyed for clones containing chloroplast DNA (1.4%), mitochondrial DNA (0.012%) and repetitive DNA motifs. BIBAC clones that may contain a gene of interest can be identified from these libraries by colony hybridization with homologous or heterologous probes or by PCR pooling techniques. Once identified, BIBAC genomic DNA library clones are immediately suitable for Agrobacterium tumefaciens‐mediated plant transformation.
In vitro activities of four xyloglucan endotransglycosylases from Arabidopsis
Authors: Campbell Paul , Braam Janet 
Published: March 2003

SummaryXyloglucan endotransglycosylases (XETs) are encoded by a gene family in Arabidopsis thaliana. These enzymes modify a major structural component of the plant cell wall, xyloglucan, and therefore may influence plant growth and development. We have produced four Arabidopsis XETs (TCH4, Meri‐5, EXGT and XTR9) using the baculovirus/insect cell system and compared their biochemical activities. TCH4, as previously demon‐ strated, and the other three proteins are capable of carrying out transglycosylation of xyloglucans. The Km for XLLGol acceptor oligosaccharide is in the range of 20–40 μm for all the XETs except XTR9, which has a Km of 5 μm and is significantly inhibited by high levels of XLLGol. All four enzymes are most active between pH 6.0 and 6.5. TCH4 and XTR9 have temperature optima of 18°C, whereas Meri‐5 and EXGT are most active at 28 and 37°C, respectively. Although the activity levels of three of the XETs are not influenced by the presence of fucose on the xyloglucan polymer, XTR9 has a clear preference for non‐fucosylated xyloglucan polymer. The four XETs show a marked preference for XLLGol over either XXFGol or XXXGol as acceptor oligosaccharide. All four XETs are glycosylated; however, only the activities of TCH4 and Meri‐5 are affected by the removal of the N‐glycan with PNGase F. These four enzymes most likely function solely as transglycosylases because xyloglucan endoglucanase activity was not apparent. Subtle differences in biochemical activities may influence the physiological functions of the distinct XETs in vivo.
Aquaporin Nt‐TIPa can account for the high permeability of tobacco cell vacuolar membrane to small neutral solutes
Authors: Gerbeau Patricia , Güçlü Josette , Ripoche Pierre , Maurel Christophe 
Published: March 2003

SummaryMembers of the major intrinsic protein (MIP) family, described in plants as water‐selective channels (aquaporins), can also transport small neutral solutes in other organisms. In the present work, we characterize the permeability of plant vacuolar membrane (tonoplast; TP) and plasma membrane (PM) to non‐electrolytes and evaluate the contribution of MIP homologues to such transport. PM and TP vesicles were purified from tobacco suspension cells by free‐flow electrophoresis, and membrane permeabilities for a wide range of neutral solutes including urea, polyols of different molecular size, and amino acids were investigated by stopped‐flow spectrofluorimetry. For all solutes tested, TP vesicles were found to be more permeable than their PM counterparts, with for instance urea permeabilities from influx experiments of 74.9 ± 9.6 × 10–6 and 1.0 ± 0.3 × 10–6 cm sec–1, respectively. Glycerol and urea transport in TP vesicles exhibited features of a facilitated diffusion process. This and the high channel‐mediated permeability of the same TP vesicles to water suggested a common role for MIP proteins in water and solute transport. A cDNA encoding a novel tonoplast intrinsic protein (TIP) homologue named Nicotiana tabacum TIPa (Nt‐TIPa) was isolated from tobacco cells. Immunodetection of Nt‐TIPa in purified membrane fractions confirmed that the protein is localized in the TP. Functional expression of Nt‐TIPa in Xenopus oocytes showed this protein to be permeable to water and solutes such as urea and glycerol. These features could account for the transport selectivity profile determined in purified TP vesicles. These results support the idea that plant aquaporins have a dual function in water and solute transport. Because Nt‐TIPa diverges in sequence from solute permeable aquaporins characterized in other organisms, its identification also provides a novel tool for investigating the molecular determinants of aquaporin transport selectivity.
Cloning and expression of a cDNA encoding betanidin 5‐O‐glucosyltransferase, a betanidin‐ and flavonoid‐specific enzyme with high homology to inducible glucosyltransferases from the Solanaceae
Authors: Vogt Thomas , Grimm Rudi , Strack Dieter 
Published: March 2003

SummaryBased on protein sequence data and RT–PCR, a full length cDNA encoding betanidin 5‐O‐glucosyltransferase (5‐GT) was obtained from a cDNA library of Dorotheanthus bellidiformis (Burm.f.) N.E.Br. (Aizoaceae). 5‐GT catalyses the transfer of glucose from UDP‐glucose to the 5‐hydroxyl group of the chromogenic betanidin. Betanidin and its conjugates, referred to as betacyanins, are characteristic fruit and flower pigments in most members of the Caryophyllales, which fail to synthesise anthocyanins. The 5‐GT cDNA displayed homology to previously published glucosyltransferase sequences and exhibited high identity to sequences of several inducible glucosyltransferases of tobacco and tomato (Solanaceae). The open reading frame encodes a polypeptide of 489 amino acids with a calculated molecular mass of 55.24 kDa. The corresponding cDNA was expressed in Escherichia coli. The recombinant protein displayed identical substrate specificity compared to the native enzyme purified from D. bellidiformis cell suspension cultures. In addition to the natural substrate betanidin, ortho‐dihydroxylated flavonols and flavones were glycosylated preferentially at the B‐ring 4′‐hydroxyl group. 5‐GT is the first enzyme of betalain biosynthesis in plants, of which the corresponding cDNA has been cloned and expressed. The results are discussed in relation to molecular evolution of plant glucosyl‐ transferases.
Inter‐organellar crosstalk in higher plants: impaired chloroplast development affects mitochondrial gene and transcript levels
Authors: Hedtke Boris , Wagner Ina , Börner Thomas , Hess Wolfgang R. 
Published: March 2003

SummaryCo‐ordination of gene expression between the three genomes present in plastids, mitochondria and nucleus is of crucial importance for plant cells. Previous studies revealed that in white leaves of the albostrians (Hordeum vulgare cv. Haisa) mutant, photosynthesis‐related plastid and nuclear genes are expressed only at an extremely low level. The plastids of this mutant lack ribosomes, photosynthetic activity and have only rudimentary membrane systems. Here we report on the expression of mitochondrial genes in albostrians barley. Steady‐state RNA levels of the mitochondrial genes encoding cytochrome oxidase or ATPase subunits, coxII, coxIII, atpA, atp6, atp9 and cob, were observed to be consistently elevated in the white leaves but not in roots. Investigation of mitochondrial DNA revealed an about three‐fold enhanced mitochondrial gene copy number in white compared to green leaf cells, but no differential amplification of mitochondrial genes. Analysis of plants in which the white albostrians plastids were combined with a new nuclear background showed that the enhanced transcript levels were a consequence of the impaired plastids and not of the nuclear albostrians allele. Furthermore, plants bleached by the carotenoid biosynthesis inhibitor norflurazon also showed an enhanced mitochondrial transcript level. These findings allow the conclusion that lack of chloroplast activity in an otherwise fully differentiated leaf leads to an increase in mitochondrial gene copy number and an elevated level of mitochondrial transcripts. Our results indicate an influence of plastids on the genetic apparatus of mitochondria in leaves but not in roots.
A glucocorticoid‐inducible transcription system causes severe growth defects in Arabidopsis and induces defense‐related genes
Authors: Kang Hong‐Gu , Fang Yiwen , Singh Karam B. 
Published: March 2003

SummaryA glucocorticoid‐inducible transcription system was employed to control the expression of AtEBP, an Arabidopsis transcription factor. A number of the transgenic AtEBP lines had developmental and growth defects when grown on dexamethasone (DEX), a strong synthetic glucocorticoid. However, these growth defects were not confined to the AtEBP lines but were observed with other transgenic lines that were generated using the same system, including empty vector lines. In about 25% of the AtEBP or empty vector transgenic lines, these growth defects were severe and in some cases led to death. As AtEBP has been linked to the plant defense response, the expression of specific defense‐related genes, including a number of pathogenesis‐related (PR) genes was also examined. PDF1.2, a plant defensin gene, was strongly induced in all transgenic lines examined following treatment with DEX, including empty vector lines that did not show any observable DEX‐induced growth defect. PR‐5 was induced to a lesser extent in all the lines, while the expression of PR‐1, PR‐2 and phenylalanine ammonia‐lyase 3 (PAL3) did not change significantly. While the induction of the AtEBP transgene and PDF1.2 had similar DEX concentration requirements, the kinetics of induction differed significantly, with the AtEBP transgene being induced within 1 h and PDF1.2 only being induced between 24 and 48 h. Although the molecular mechanisms underlying the growth defects and changes in gene expression remain to be determined, these changes appear to result from the glucocorticoid‐inducible system itself, and may therefore limit the usefulness of this system for controlling gene expression in Arabidopsis.
Homologues of yeast and bacterial rotenone‐insensitive NADH dehydrogenases in higher eukaryotes: two enzymes are present in potato mitochondria
Authors: Rasmusson Allan G. , Svensson Å. Staffan , Knoop Volker , Grohmann Lutz , Brennicke Axel 
Published: March 2003

SummaryTwo different cDNAs, homologous to genes for rotenone‐insensitive NADH dehydrogenases of bacteria and yeast, were isolated from potato. The encoded proteins, called NDA and NDB, have calculated molecular masses of 55 and 65 kDa, respectively. The N‐terminal parts show similarity to mitochondrial targeting peptides and the polypeptides are in vitro imported into potato mitochondria. Import processing to a smaller polypeptide is seen for the NDA but not the NDB protein. After import, NDA is intramitochondrially sorted to the matrix side of the inner membrane, whereas NDB becomes exposed to the intermembrane space. Imported proteins are associated to membranes upon digitonin permeabilization. On expression in Escherichia coli, NDB is released from the bacterial membrane in the absence of divalent cations whereas detergents are necessary for solubilization of NDA. Both deduced amino‐acid sequences contain the dual motifs for nucleotide binding with the characteristics of the core criteria, similar to the bacterial homologues. Unique among NADH dehydro‐ genases, the NDB amino‐acid sequence contains a non‐conserved insert, which is similar to EF‐hand motifs for calcium binding. Phylogenetic analyses group the rotenone‐insensitive NADH dehydrogenases largely by species, but suggest ancient gene duplications.
UPL1 and 2, two 405 kDa ubiquitin–protein ligases from Arabidopsis thaliana related to the HECT‐domain protein family
Authors: Bates Paul W. , Vierstra Richard D. 
Published: March 2003

SummaryThe ubiquitin/26S proteasome pathway is a major route for degrading abnormal and important short‐lived regulatory proteins in eukaryotes. Covalent attachment of ubiquitin, which triggers entry of target proteins into the pathway, is accomplished by an ATP‐dependent reaction cascade involving the sequential action of three enzymes, E1s, E2s and E3s. Although much of the substrate specificity of the pathway is determined by E3s (or ubiquitin–protein ligases, UPLs), little is known about these enzymes in plants and how they choose appropriate targets for ubiquitination. Here, we describe two 405 kDa E3s (UPL1 and 2) from Arabidopsis thaliana related to the HECT‐E3 family that is essential in yeast and animals. UPL1 and 2 are encoded by 13 kbp genes 26 cm apart on chromosome I, that are over 95% identical within both the introns and exons, suggesting that the two loci arose from a recent gene duplication. The C‐terminal HECT domain of UPL1 is necessary and sufficient to conjugate ubiquitin in vitro in a reaction that requires the positionally conserved cysteine within the HECT domain, E1, and an E2 of the UBC8 family. Given that HECT E3s help define target specificity of the ubiquitin conjugation, a continued characterization of UPL1 and 2 should be instrumental in understanding the functions of ubiquitin‐dependent protein turnover in plants and for identifying pathway substrates.
Stachyose synthesis in seeds of adzuki bean (Vigna angularis): molecular cloning and functional expression of stachyose synthase
Authors: Peterbauer Thomas , Mucha Jan , Mayer Ulrike , Popp Marianne , Glössl Josef , Richter Andreas 
Published: March 2003

SummaryStachyose is the major soluble carbohydrate in seeds of a number of important crop species. It is synthesized from raffinose and galactinol by the action of stachyose synthase (EC 2.4.1.67). We report here on the identification of a cDNA encoding stachyose synthase from seeds of adzuki bean (Vigna angularis Ohwi et Ohashi). Based on internal amino acid sequences of the enzyme purified from adzuki bean, oligonucleotides were designed and used to amplify corresponding sequences from adzuki bean cDNA by RT–PCR, followed by rapid amplification of cDNA ends (RACE–PCR). The complete cDNA sequence comprised 3046 nucleotides and included an open reading frame which encoded a polypeptide of 857 amino acid residues. The entire coding region was amplified by PCR, engineered into the baculovirus expression vector pVL1393 and introduced into Spodoptera frugiperda (Sf21) insect cells for heterologous expression. The recombinant protein was immunologically reactive with polyclonal antibodies raised against stachyose synthase purified from adzuki bean and was shown to be a functional stachyose synthase with the same catalytic properties as its native counterpart. High levels of stachyose synthase mRNA were transiently accumulated midway through seed development, and the enzyme was also present in mature seeds and during germination.