bluuebunny/crossref_metadata_2025_split huggingface dataset:
A novel composite locus of Arabidopsis encoding two polypeptides with metabolically related but distinct functions in lysine catabolism
Authors: Tang Guiliang , Zhu Xiaohong , Tang Xiaohu , Galili Gad 
Published: March 2003

SummaryBoth plants and animals catabolise lysine via saccharopine by two consecutive enzymes, lysine‐ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH), which are linked on a single polypeptide. We recently demonstrated that Arabidopsis plants possess not only a bifunctional LKR/SDH but in addition a monofunctional SDH enzyme. We also speculated that these two enzymes may be controlled by a single gene (G. Tang et al. Plant Cell, 1997, 9, 1305–1316). By expressing several epitope‐tagged and GUS reporter constructs, we demonstrate in the present study that the Arabidopsis monofunctional SDH is encoded by a distinct gene, which is, however, nested entirely within the coding and 3′ non‐coding regions of the larger bifunctional LKR/SDH gene. The entire open reading frame of the monofunctional SDH gene, as well as some components of its promoter, are also parts of the translated coding sequence of the bifunctional LKR/SDH gene. These special structural characteristics, combined with the fact that the two genes encode simultaneously two metabolically related but distinct enzymes, render the LKR/SDH locus a novel type of a composite locus. Not all plant species possess an active monofunctional SDH gene and the production of this enzyme is correlated with an increased flux of lysine catabolism. Taken together, our results suggest that the composite LKR/SDH locus serves to control an efficient, highly regulated flux of lysine catabolism
Abscisic aldehyde oxidase in leaves of Arabidopsis thaliana
Authors: Seo Mitsunori , Koiwai Hanae , Akaba Shuichi , Komano Teruya , Oritani Takayuki , Kamiya Yuji , Koshiba Tomokazu 
Published: March 2003

SummaryAbscisic acid (ABA) is a plant hormone involved in seed development and responses to various environmental stresses. Oxidation of abscisic aldehyde is the last step of ABA biosynthesis and is catalysed by aldehyde oxidase (EC 1.2.3.1). We have reported the occurrence of three isoforms of aldehyde oxidase, AOα, AOβ and AOγ, in Arabidopsis thaliana seedlings, but none oxidized abscisic aldehyde. Here we report a new isoform, AOδ, found in rosette leaf extracts, which efficiently oxidizes abscisic aldehyde. AOδ was specifically recognized by antibodies raised against a recombinant peptide encoded by AAO3, one of four Arabidopsis aldehyde oxidase genes (AAO1, AAO2, AAO3 and AAO4). Functionally expressed AAO3 protein in the yeast Pichia pastoris showed a substrate preference very similar to that of rosette AOδ. These results indicate that AOδ is encoded by AAO3. AOδ produced in P. pastoris exhibited a very low Km value for abscisic aldehyde (0.51 μm), and the oxidation product was determined by gas chromatography–mass spectrometry to be ABA. Northern analysis showed that AAO3 mRNA is highly expressed in rosette leaves. When the rosette leaves were detached and exposed to dehydration, AAO3 mRNA expression increased rapidly within 3 h of the treatment. These results suggest that AOδ, the AAO3 gene product, acts as an abscisic aldehyde oxidase in Arabidopsis rosette leaves.
A dominant negative mutant of Sar1 GTPase inhibits protein transport from the endoplasmic reticulum to the Golgi apparatus in tobacco and Arabidopsis cultured cells
Authors: Takeuchi Masaki , Ueda Takashi , Sato Ken , Abe Hiroshi , Nagata Toshiyuki , Nakano Akihiko 
Published: March 2003

SummaryProtein secretion plays an important role in plant cells as it does in animal and yeast cells, but the tools to study molecular events of plant secretion are very limited. We have focused on the Sar1 GTPase, which is essential for the vesicle formation from the endoplasmic reticulum (ER) in yeast, and have previously shown that tobacco and Arabidopsis SAR1 complement yeast sar1 mutants. In this study, we have established a transient expression system of GFP‐fusion proteins in tobacco and Arabidopsis cultured cells. By utilizing confocal laser scanning microscopy, we demonstrate that a dominant negative mutant of Arabidopsis Sar1 inhibits the ER‐to‐Golgi transport of Golgi membrane proteins, AtErd2 and AtRer1B, and locates them to the ER. The same mutant Sar1 also blocks the exit from the ER of a vacuolar storage protein, sporamin. These results not only provide the first evidence that the Sar1 GTPase functions in the ER‐to‐Golgi transport in plant cells, but also prove that conditional expression of dominant mutants of secretory machinery can be a useful tool in manipulating vesicular trafficking.
Functional analysis of the two Arabidopsis homologues of Toc34, a component of the chloroplast protein import apparatus†
Authors: Gutensohn Michael , Schulz Burkhard , Nicolay Peter , Flügge Ulf‐Ingo 
Published: March 2003

SummaryTwo Arabidopsis Toc34 homologues, atToc34 and atToc33, components of the chloroplast protein import machinery located in the outer envelope membrane, were recently isolated. Both proteins insert into the outer envelope, are supposed to bind GTP and to interact with Toc75 as demonstrated by in vitro import assays. We studied the expression of the two genes by RNA gel blot analysis, promoter‐GUS plants and in situ hybridisations as well as immunoblot analysis. The atToc34 and atToc33 genes are expressed in green as well as non‐green tissues and are developmentally regulated. Despite these similarities, however, the two Arabidopsis Toc34 homologues are differentially expressed in various plant organs. To gain more insight into the in vivo function of both proteins, antisense plants were created. While antisense plants of atToc33 are characterized by a pale yellowish phenotype, antisense plants of atToc34 show a weaker phenotype. Protein interaction studies using an in vitro translated precursor protein and heterologously expressed atToc34 and atToc33 proteins showed a direct GTP‐dependent interaction, but demonstrated different affinities of the two atToc proteins towards the precursor protein. Thus, our results indicate a more specialized function for both atToc34 and atToc33, suggesting specificity for certain imported precursor proteins.
NO way back: nitric oxide and programmed cell death in Arabidopsis thaliana suspension cultures
Authors: Clarke Andrew , Desikan Radhika , Hurst Roger D. , Hancock John T. , Neill Steven J. 
Published: March 2003

SummaryRecent research has implicated nitric oxide (NO) in the induction of the hypersensitive response (HR) during plant–pathogen interactions. Here we demonstrate that Arabidopsis suspension cultures generate elevated levels of NO in response to challenge by avirulent bacteria, and, using NO donors, show that these elevated levels of NO are sufficient to induce cell death in Arabidopsis cells independently of reactive oxygen species (ROS). We also provide evidence that NO‐induced cell death is a form of programmed cell death (PCD), requiring gene expression, and has a number of characteristics of PCD of mammalian cells: NO induced chromatin condensation and caspase‐like activity in Arabidopsis cells, while the caspase‐1 inhibitor, Ac‐YVAD‐CMK, blocked NO‐induced cell death. A well‐established second messenger mediating NO responses in mammalian cells is cGMP, produced by the enzyme guanylate cyclase. A specific inhibitor of guanylate cyclase blocked NO‐induced cell death in Arabidopsis cells, and this inhibition was reversed by the cell‐permeable cGMP analogue, 8Br‐cGMP, although 8Br‐cGMP alone did not induce cell death or potentiate NO‐induced cell death. This suggests that cGMP synthesis is required but not sufficient for NO‐induced cell death in Arabidopsis. In‐gel protein kinase assays showed that NO activates a potential mitogen‐activated protein kinase (MAPK), although a specific inhibitor of mammalian MAPK activation, PD98059, which blocked H2O2‐induced cell death, did not inhibit the effects of NO.
Multiple pathways for Cre/lox‐mediated recombination in plastids
Authors: Hajdukiewicz Peter T. J. , Gilbertson Larry , Staub Jeffrey M. 
Published: March 2003

SummaryPlastid transformation technology involves the insertion by homologous recombination and subsequent amplification of plastid transgenes to ≈10 000 genome copies per leaf cell. Selection of transformed genomes is achieved using a selectable antibiotic resistance marker that has no subsequent role in the transformed line. We report here a feasibility study in the model plant tobacco, to test the heterologous Cre/lox recombination system for antibiotic marker gene removal from plastids. To study its efficiency, a green fluorescent protein reporter gene activation assay was utilized that allowed visual observation of marker excision after delivery of Cre to plastids. Using a combination of in vivo fluorescence activation and molecular assays, we show that transgene excision occurs completely from all plastid genomes early in plant development. Selectable marker‐free transplastomic plants are obtained in the first seed generation, indicating a potential application of the Cre/lox system in plastid transformation technology. In addition to the predicted transgene excision event, two alternative pathways of Cre‐mediated recombination were also observed. In one alternative pathway, the presence of Cre in plastids stimulated homologous recombination between a 117 bp transgene expression element and its cognate sequence in the plastid genome. The other alternative pathway uncovered a plastid genome ‘hot spot’ of recombination composed of multiple direct repeats of a 5 bp sequence motif, which recombined with lox independent of sequence homology. Both recombination pathways result in plastid genome deletions. However, the resultant plastid mutations are silent, and their study provides the first insights into tRNA accumulation and trans‐splicing events in higher plant plastids.
NtSET1, a member of a newly identified subgroup of plant SET‐domain‐containing proteins, is chromatin‐associated and its ectopic overexpression inhibits tobacco plant growth
Authors: Shen Wen‐Hui 
Published: March 2003

SummaryThe SET‐ and chromo‐domains are recognized as signature motifs for proteins that contribute to epigenetic control of gene expression through effects on the regional organization of chromatin structure. This paper reports the identification of a novel subgroup of SET‐domain‐containing proteins in tobacco and Arabidopsis, which show highest homologies with the Drosophila position‐effect‐variegation repressor protein SU(VAR)3–9 and the yeast centromer silencing protein CLR4. The tobacco SET‐domain‐containing protein (NtSET1) was fused to the green fluorescence protein (GFP) that serves as a visual marker for localization of the recombinant protein in living cells. Whereas control GFP protein alone was uniformly dispersed within the nucleus and cytoplasm, the NtSET1‐GFP fusion protein showed a non‐uniform localization to multiple nuclear regions in interphase tobacco TBY2 cells. During mitosis, the NtSET1‐GFP associated with condensed chromosomes with a non‐random distribution. The NtSET1 thus appears to have distinct target regions in the plant chromatin. Overexpression of the NtSET1‐GFP in transgenic tobacco inhibited plant growth, implicating the possible involvement of the NtSET1 in transcriptional repression of growth control genes through the formation of higher‐order chromatin domains.
Functional importance of conserved domains in the flowering‐time gene CONSTANS demonstrated by analysis of mutant alleles and transgenic plants
Authors: Robson Frances , Costa M. Manuela R , Hepworth Shelley R , Vizir Igor , Pin˜eiro Manuel , Reeves Paul H , Putterill Joanna , Coupland George 
Published: March 2003

Summary CONSTANS promotes flowering of Arabidopsis in response to long‐day conditions. We show that CONSTANS is a member of an Arabidopsis gene family that comprises 16 other members. The CO‐Like proteins encoded by these genes contain two segments of homology: a zinc finger containing region near their amino terminus and a CCT (CO, CO‐Like, TOC1) domain near their carboxy terminus. Analysis of seven classical co mutant alleles demonstrated that the mutations all occur within either the zinc finger region or the CCT domain, confirming that the two regions of homology are important for CO function. The zinc fingers are most similar to those of B‐boxes, which act as protein–protein interaction domains in several transcription factors described in animals. Segments of CO protein containing the CCT domain localize GFP to the nucleus, but one mutation that affects the CCT domain delays flowering without affecting the nuclear localization function, suggesting that this domain has additional functions. All eight co alleles, including one recovered by pollen irradiation in which DNA encoding both B‐boxes is deleted, are shown to be semidominant. This dominance appears to be largely due to a reduction in CO dosage in the heterozygous plants. However, some alleles may also actively delay flowering, because overexpression from the CaMV 35S promoter of the co‐3 allele, that has a mutation in the second B‐box, delayed flowering of wild‐type plants. The significance of these observations for the role of CO in the control of flowering time is discussed.
RNA splicing in higher plant mitochondria: determination of functional elements in group II intron from a chimeric cox II gene in electroporated wheat mitochondria
Authors: Farré Jean‐Claude , Araya Alejandro 
Published: March 2003

SummaryHigher plant mitochondria mainly contain group II introns presenting a secondary structure with six helical domains linked to a central hub. Experimental evidence of functional elements in higher plant mitochondria introns is limited since they are unable to undergo self‐splicing and the definition of functional domains is based on data obtained from yeast autocatalytic introns. Here we study the role of putative functional elements required for the splicing reaction. The exon‐binding and intron‐binding sites (EBS and IBS, respectively), and the domain 6, which is involved in lariat formation, were analysed by site‐directed mutagenesis and transient expression in electroporated mitochondria. The data presented here demonstrate the role of EBS1‐IBS1 and EBS2‐IBS2 interactions and reveal a new secondary‐structure interaction. The role of the C to U editing conversion in the IBS1 motif is discussed.
Vacuolar membrane localization of the Arabidopsis‘two‐pore’ K+ channel KCO1
Authors: Czempinski Katrin , Frachisse Jean‐Marie , Maurel Christophe , Barbier‐Brygoo Helene , Mueller‐Roeber Bernd 
Published: March 2003

SummaryPotassium (K+) channels play multiple roles in higher plants, and have been characterized electrophysiologically in various subcellular membranes. The K+ channel AtKCO1 from Arabidopsis thaliana is the prototype of a new family of plant K+ channels. In a previous study the protein has been functionally characterized after heterologous expression in Baculovirus‐infected insect cells. In order to obtain further information on the physiological function of AtKCO1, the gene expression pattern and subcellular localization of the protein in plants were investigated. The regulatory function of the 5′ region of the AtKCO1 gene was examined in transgenic A. thaliana plants carrying β‐glucuronidase (GUS) fusion constructs. Our analysis demonstrates that the AtKCO1 promoter is active in various tissues and cell types, and the highest GUS activity could be detected in mitotically active tissues of the plant. Promoter activity was strongly dependent on the presence of a 5′ leader intron. The same overall structure was identified in two genes encoding AtKCO1‐like K+ channels from Solanum tuberosum (StKCO1α and StKCO1β). To investigate the subcellular localization of AtKCO1, the channel protein, as well as a fusion protein of AtKCO1 with green fluorescence protein (GFP), were expressed in transgenic tobacco BY2 cells. In sucrose density gradients, both proteins co‐fractionate with tonoplast markers (Nt‐TIPa, vATPase). In fluorescence images from transgenic AtKCO1–GFP BY2 cells fluorescence was exclusively detected in the tonoplast. Thus AtKCO1 is the first cloned K+ channel demonstrated to be a vacuolar K+ channel.
Loading of nitrate into the xylem: apoplastic nitrate controls the voltage dependence of X‐QUAC, the main anion conductance in xylem‐parenchyma cells of barley roots
Authors: Köhler Barbara , Wegner Lars H. , Osipov Viktor , Raschke Klaus 
Published: March 2003

SummaryWe report here that NO3– in the xylem exerts positive feedback on its loading into the xylem through a change in the voltage dependence of the Quickly Activating Anion Conductance, X‐QUAC. Properties of this conductance were investigated on xylem‐parenchyma protoplasts prepared from roots of Hordeum vulgare by applying the patch‐clamp technique. Chord conductances were minimal around − 40 mV and increased with plasma membrane depolarisation as well as with hyperpolarisation. Two gates with opposite voltage dependences were postulated. When 30 mm Cl– in the bath was replaced by NO3–, a shift in the midpoint potential of the depolarisation‐activated gate by about − 60 mV from 43 to − 16 mV occurred (Km = 3.4 mm). No such effect was seen when chloride was replaced by malate. Addition of 10 mm NO3– to the pipette solution and reduction of [Cl–] from 124 to 4 mm (to simulate cytoplasmic concentrations) did not interfere with the voltage dependence of X‐QUAC activation, nor was it affected by changes in external [K+]. If only the NO3– effect on gating was considered, an increase of the NO3– concentration in the xylem sap to 5 mm would result in an enhancement of NO3– efflux by about 30%. Although the driving force for NO3– efflux would be reduced simultaneously, NO3– efflux into the xylem through X‐QUAC would be maintained with high NO3– concentrations in the xylem sap; a situation which occurs for instance during the night.
cDNA microarray analysis of gene expression during Fe‐deficiency stress in barley suggests that polar transport of vesicles is implicated in phytosiderophore secretion in Fe‐deficient barley roots
Authors: Negishi Takashi , Nakanishi Hiromi , Yazaki Junshi , Kishimoto Naoki , Fujii Fumiko , Shimbo Kanako , Yamamoto Kimiko , Sakata Katsumi , Sasaki Takuji , Kikuchi Shoshi , Mori Satoshi , Nishizawa Naoko K. 
Published: March 2003

SummaryTo acquire Fe from soil, graminaceous plants secrete mugineic acid family phytosiderophores (MAs) from their roots. The secretion of MAs increases in response to Fe deficiency, and shows a distinct diurnal rhythm. We used a microarray that included 8987 cDNAs of rice EST clones to examine gene expression profiles in barley roots during Fe‐deficiency stress. Approximately 200 clones were identified as Fe‐deficiency‐inducible genes, of which seven had been identified previously. In order to meet the increased demand for methionine to produce MAs, Fe‐deficiency enhances the expression of genes that participate in methionine synthesis, as well as recycling methionine through the Yang cycle. Of these 200 genes, approximately 50 exhibited different transcription levels in Fe‐deficient roots at noon and at night. Northern blot analysis of time course experiments confirmed that five of these genes exhibited a diurnal change in their level of expression. The diurnal changes in the expression of these genes suggest that polar vesicle transport is involved in the diurnal secretion of MAs.
Nitrate signalling on the nitrate reductase gene promoter depends directly on the activity of the nitrate transport systems in Chlamydomonas
Authors: Llamas Angel , Igeño María Isabel , Galván Aurora , Fernández Emilio 
Published: March 2003

SummaryNitrate signalling on the nitrate reductase (Nia1) gene promoter from Chlamydomonas reinhardtii has been studied by using a construct of the Nia1 promoter transcriptionally fused to the Chlamydomonas arylsulphatase gene as a reporter in strains bearing different sets of nitrate/nitrite transport genes. The high‐affinity nitrate transport (HANT) system I is required for efficient signalling by nitrate, even at submicromolar concentrations of the anion. In addition, the autogenous regulation of nitrate reductase has been found to depend on the presence of system I. The low‐affinity nitrate transport system III promoted signalling optimally on the promoter at millimolar nitrate concentrations. The HANT system IV, which is insensitive to ammonium and active at low CO2, allowed nitrate signalling at micromolar concentrations even in the presence of ammonium, suggesting that the balance of these two effectors controls Nia1 transcription. Our data indicate that nitrate signalling on the Nia1 gene promoter occurs intracellularly and depends on the activity of nitrate transporters.
In muro fragmentation of the rhamnogalacturonan I backbone in potato (Solanum tuberosum L.) results in a reduction and altered location of the galactan and arabinan side‐chains and abnormal periderm development
Authors: Oomen Ronald J. F. J. , Doeswijk‐Voragen Chantal H. L. , Bush Maxwell S. , Vincken Jean‐Paul , Borkhardt Bernhard , Van Den Broek Lambertus A. M. , Corsar Julia , Ulvskov Peter , Voragen Alphons G. J. , McCann Maureen C. , Visser Richard G. F. 
Published: March 2003

SummaryRhamnogalacturonan (RG) I is a branched pectic polysaccharide in plant cell walls. Rhamnogalacturonan lyase (eRGL) from Aspergillus aculeatus is able to cleave the RG I backbone at specific sites. Transgenic potato (Solanum tuberosum L.) plants were made by the introduction of the gene encoding eRGL, under the control of the granule‐bound starch synthase promoter. The eRGL protein was successfully expressed and translated into an active form, demonstrated by eRGL activity in the tuber extracts. The transgenic plants produced tubers with clear morphological alterations, including radial swelling of the periderm cells and development of intercellular spaces in the cortex. Sugar compositional analysis of the isolated cell walls showed a large reduction in galactosyl and arabinosyl residues in transgenic tubers. Immunocytochemical studies using the LM5 (galactan) and LM6 (arabinan) antibodies also showed a large reduction in galactan and arabinan side‐chains of RG I. Most of the remaining LM5 epitopes were located in the expanded middle lamella at cell corners of eRGL tubers, which is in contrast to their normal location in the primary wall of wild type tubers. These data suggest that RG I has an important role in anchoring galactans and arabinans at particular regions in the wall and in normal development of the periderm.