bluuebunny/crossref_metadata_2025_split huggingface dataset:
Trehalose‐6‐phosphate synthase 1, which catalyses the first step in trehalose synthesis, is essential for Arabidopsis embryo maturation
Authors: Eastmond Peter J. , Van Dijken Anja J. H. , Spielman Melissa , Kerr Aimie , Tissier Alain F. , Dickinson Hugh G. , Jones Jonathan D. G. , Smeekens Sjef C. , Graham Ian A. 
Published: March 2003

SummaryDespite the recent discovery that trehalose synthesis is widespread in higher plants very little is known about its physiological significance. Here we report on an Arabidopsis mutant (tps1), disrupted in a gene encoding the first enzyme of trehalose biosynthesis (trehalose‐6‐phosphate synthase). The tps1 mutant is a recessive embryo lethal. Embryo morphogenesis is normal but development is retarded and stalls early in the phase of cell expansion and storage reserve accumulation. TPS1 is transiently up‐regulated at this same developmental stage and is required for the full expression of seed maturation marker genes (2S2 and OLEOSN2). Sucrose levels also increase rapidly in seeds during the onset of cell expansion. In Saccharomyces cerevisiae trehalose‐6‐phosphate (T‐6‐P) is required to regulate sugar influx into glycolysis via the inhibition of hexokinase and a deficiency in TPS1 prevents growth on sugars (Thevelein and Hohmann, 1995). The growth of Arabidopsis tps1–1 embryos can be partially rescued in vitro by reducing the sucrose level. However, T‐6‐P is not an inhibitor of AtHXK1 or AtHXK2. Nor does reducing hexokinase activity rescue tps1–1 embryo growth. Our data establish for the first time that an enzyme of trehalose metabolism is essential in plants and is implicated in the regulation of sugar metabolism/embryo development via a different mechanism to that reported in S. cerevisiae.
Geminivirus‐based vectors for gene silencing in Arabidopsis
Authors: Turnage Michael A. , Muangsan Nooduan , Peele Charles G. , Robertson Dominique 
Published: March 2003

SummaryGene silencing, or RNA interference, is a powerful tool for elucidating gene function in Caenorhabditis elegans and Drosophila melanogaster. The vast genetic, developmental and sequence information available for Arabidopsis thaliana makes this an attractive organism in which to develop reliable gene‐silencing tools for the plant world. We have developed a system based on the bipartite geminivirus cabbage leaf curl virus (CbLCV) that allows silencing of endogenous genes singly or in combinations in Arabidopsis. Two vectors were tested: a gene‐replacement vector derived from the A component; and an insertion vector derived from the B component. Extensive silencing was produced in new growth from the A component vectors, while only minimal silencing and symptoms were seen in the B component vector. Two endogenous genes were silenced simultaneously from the A component vector and silencing of the genes was maintained throughout new growth. Because the CbLCV vectors are DNA vectors they can be inoculated directly from plasmid DNA. Introduction of these vectors into intact plants bypasses transformation and extends the kinds of silencing studies that can be carried out in Arabidopsis.
Extended leaf longevity in the ore4‐1 mutant of Arabidopsis with a reduced expression of a plastid ribosomal protein gene
Authors: Woo Hye Ryun , Goh Chang‐Hyo , Park Joon‐Hyun , de la Serve Bernard Teyssendier , Kim Jin‐Hee , Park Youn‐Il , Nam Hong Gil 
Published: March 2003

SummaryThe longevity of plant leaf organs is genetically determined. However, the molecular mechanisms underlying the control of longevity are still largely unknown. Here, we describe a T‐DNA‐insertional mutation of Arabidopsis thaliana that confers extended leaf longevity. The mutation, termed ore4‐1, delays a broad spectrum of age‐dependent leaf senescence, but has little effect on leaf senescence artificially induced by darkness, abscisic acid (ABA), methyl jasmonate (MeJA), or ethylene. The T‐DNA was inserted within the promoter region of the plastid ribosomal small subunit protein 17 (PRPS17) gene, and this insertion dramatically reduced PRPS17 mRNA expression. In the ore4‐1 mutant, the leaf growth rate is decreased, while the maturation timing is similar to that of wild‐type. In addition, the activity of the photosystem I (PSI) is significantly reduced in the ore4‐1 mutant, as compared to wild‐type. Thus, the ore4‐1 mutation results in a deficiency in various chloroplast functions, including photosynthesis, which may decrease leaf growth. Our results suggest a possible link between reduced metabolism and extended longevity of the leaf organs in the ore4‐1 mutation.
pGD vectors: versatile tools for the expression of green and red fluorescent protein fusions in agroinfiltrated plant leaves
Authors: Goodin Michael M. , Dietzgen Ralf G. , Schichnes Denise , Ruzin Steven , Jackson Andrew O. 
Published: March 2003

SummaryWe have constructed a matched set of binary vectors designated pGD, pGDG and pGDR for the expression and co‐localization of native proteins and GFP or DsRed fusions in large numbers of plant cells. The utility of these vectors following agroinfiltration into leaves has been demonstrated with four genes from Sonchus yellow net virus, a plant nucleorhabdovirus, and with a nucleolar marker protein. Of the three SYNV proteins tested, sc4 gave identical localization patterns at the cell wall and nucleus when fused to GFP or DsRed. However, some differences in expression patterns were observed depending on whether DsRed or GFP was the fusion partner. In this regard, the DsRed:P fusion showed a similar pattern of localization to GFP:P, but localized foci appeared in the nucleus and near the periphery of the nucleus. Nevertheless, the viral nucleocapsid protein, expressed as a GFP:N fusion, co‐localized with DsRed:P in a subnuclear locale in agreement with our previous observations (Goodin et al., 2001). This locale appears to be distinct from the nucleolus as indicated by co‐expression of the N protein, DsRed:P and a nucleolar marker AtFib1 fused to GFP. The SYNV M protein, which is believed to be particularly prone to oligomerization, was detectable only as a GFP fusion. Our results indicate that agroinfiltration with bacteria containing the pGD vectors is extremely useful for transient expression of several proteins in a high proportion of the cells of Nicotiana benthamiana leaves. The GFP and DsRed elements incorporated into the pGD system should greatly increase the ease of visualizing co‐localization and interactions of proteins in a variety of experimental dicotyledonous hosts.
Mutations at CRE1 impair cytokinin‐induced repression of phosphate starvation responses in Arabidopsis
Authors: Franco‐Zorrilla José M. , Martin Ana C. , Solano Roberto , Rubio Vicente , Leyva Antonio , Paz‐Ares Javier 
Published: March 2003

SummaryPlants display a number of responses to low phosphate availability, involving biochemical and developmental changes. Recently we have shown that many of these responses can be repressed in roots by exogenous addition of cytokinins. In order to understand the genetic basis to this effect of cytokinins, and its relation with the better known roles of cytokinins in the control of cell‐cycle and differentiation, we have undertaken mutant screening and characterization using a transgenic line of Arabidopsis thaliana harbouring a reporter gene specifically responsive to Pi starvation (AtIPS1::GUS). One type of mutant identified displayed reduced sensitivity of AtIPS1::GUS to cytokinin repression. Several other Pi starvation response genes showed reduced cytokinin sensitivity in these lines. These mutants also showed reduced cytokinin repression of the anthocyanin accumulation induced by Pi starvation in the aerial part of the plants. Mapping and molecular characterization of these mutants showed that they were allelic of CRE1/WOL, a locus known to encode a cytokinin receptor. CRE1 is downregulated by Pi starvation and induced by cytokinins, both in the wild‐type and in the cre1 mutants, in which cre1 mRNA levels are higher. These results reveal the existence of a positive feed‐back loop, in addition to the already established negative feedback loop, in cytokinin signalling and indicate that the negative regulation of Pi starvation responses by cytokinins involves a two‐component signalling circuitry, as it is the case of other types of cytokinin response.
Heavy metal toxicity: cadmium permeates through calcium channels and disturbs the plant water status
Authors: Perfus‐Barbeoch Laetitia , Leonhardt Nathalie , Vavasseur Alain , Forestier Cyrille 
Published: March 2003

SummaryBecause plant wilting has been described as a consequence of cadmium (Cd2+) toxicity, we investigate Cd2+ effects on plant water losses, gas exchanges and stomatal behaviour in Arabidopsis thaliana L. Effects of 1‐week Cd2+ application in hydroponic condition (CdCl2 10–100 µm) were analyzed. A 10‐µm Cd2+ concentration had no significant effect on the plant–water relationship and carbon assimilation. At higher Cd2+ concentrations, a Cd2+ ‐dependent decrease in leaf conductance and CO2 uptake was observed despite the photosynthetic apparatus appeared not to be affected as probed by fluorescence measurements. In epidermal strip bioassays, nanomolar Cd2+ concentrations reduced stomatal opening under light in A. thaliana, Vicia faba and Commelina communis. Application of 5 µm ABA limited the root‐to‐shoot translocation of cadmium. However, the Cd2+‐induced stomatal closure was likely ABA‐independent, since a 5‐day treatment with 50 µm Cd2+ did not affect the plant relative water content. Additionally, a similar Cd2+‐induced stomatal closure was observed in the ABA insensitive mutant abi1‐1. Interestingly, this mutant displayed a higher transpiration rate than the wild type but did not accumulate more Cd2+, arguing that Cd2+ uptake is not dependent only on the transpiration flow. Application of putative calcium channels inhibitors suppressed the inhibitory effect of Cd2+ in epidermal strip experiments, suggesting that Cd2+ could enter the guard cell through calcium channels. Patch‐clamp studies with V. faba guard cell protoplasts showed that plasma membrane K+ channels were insensitive to external Cd2+ application whereas Ca2+ channels were found permeable to Cd2+. In conclusion, we propose that Cd2+ affects guard cell regulation in an ABA‐independent manner by entering the cytosol via Ca2+ channels.
The stability of cortical microtubules depends on their orientation
Authors: Wiesler Bodo , Wang Qi‐Yan , Nick Peter 
Published: March 2003

SummaryAuxin controls the orientation of cortical microtubules in maize coleoptile segments. We used tyrosinylated α‐tubulin as a marker to assess auxin‐dependent changes in microtubule turnover. Auxin‐induced tyrosinylated α‐tubulin, correlated with an elevated sensitivity of growth to antimicrotubular compounds such as ethyl‐N‐phenylcarbamate (EPC). We determined the affinity of α‐tubulin to EPC and found that it was dramatically increased when the tubulin was de‐tyrosinylated. By proteolytic cleavage of the carboxy terminal tyrosine, such an increased affinity could be induced in vitro. Thus, the auxin‐induced sensitivity of growth to EPC is not caused by an increased affinity for this inhibitor, but caused by a reduced microtubule turnover. Double visualization assays revealed that the transverse microtubules induced by auxin consist predominantly of tyrosinylated α‐tubulin, whereas the longitudinal microtubules induced by auxin depletion contain de‐tyrosinylated α‐tubulin. The results are discussed in terms of direction‐dependent differences in the lifetime of microtubules.
A role for HvGAMYB in anther development
Authors: Murray Fiona , Kalla Roger , Jacobsen John , Gubler Frank 
Published: March 2003

SummaryHvGAMYB is a transcription factor that was first identified in barley aleurone cells and shown to be upregulated by gibberellin (GA). Using RNA and immunoblot analysis we have shown HvGAMYB is also strongly expressed in barley anthers. Transgenic barley expressing a HvGAMYB:GFP fusion gene have been created and GAMYB expression in anthers analysed. GFP expression was clearly visible during early anther development in the nuclei of the epidermis, endothecium, middle layer and tapetum. Expression in the epidermis and endothecium persists until just prior to anther dehiscence, expression in the other two cell layers is visible until they are compressed and broken down as the microspores develop. Further evidence of a role for HvGAMYB in anther development was provided by the creation of transgenic barley over‐expressing the HvGAMYB gene. Associated with the increase in HvGAMYB levels was a progressive decrease in anther size, particularly a decrease in anther length. Anthers also became increasingly lighter in colour. Anthers with fourfold more HvGAMYB protein than non‐transgenic controls failed to dehisce and were male sterile, anthers with approximately three to fourfold endogenous GAMYB protein levels were smaller and paler but still shed normally. To investigate the hormonal regulation of HvGAMYB expression in anthers, HvGAMYB and SLN1 protein levels in anthers were analysed following application of GA3. As in cereal aleurone, HvGAMYB levels were found to increase and SLN1 levels decrease following GA3 application suggesting a similar GA‐signalling pathway to that in aleurone exists in anthers.
OsDREB genes in rice, Oryza sativa L., encode transcription activators that function in drought‐, high‐salt‐ and cold‐responsive gene expression
Authors: Dubouzet Joseph G. , Sakuma Yoh , Ito Yusuke , Kasuga Mie , Dubouzet Emilyn G. , Miura Setsuko , Seki Motoaki , Shinozaki Kazuo , Yamaguchi‐Shinozaki Kazuko 
Published: March 2003

SummaryThe transcription factors DREBs/CBFs specifically interact with the dehydration‐responsive element/C‐repeat (DRE/CRT) cis‐acting element (core motif: G/ACCGAC) and control the expression of many stress‐inducible genes in Arabidopsis. In rice, we isolated five cDNAs for DREB homologs: OsDREB1A, OsDREB1B, OsDREB1C, OsDREB1D, and OsDREB2A. Expression of OsDREB1A and OsDREB1B was induced by cold, whereas expression of OsDREB2A was induced by dehydration and high‐salt stresses. The OsDREB1A and OsDREB2A proteins specifically bound to DRE and activated the transcription of the GUS reporter gene driven by DRE in rice protoplasts. Over‐expression of OsDREB1A in transgenic Arabidopsis induced over‐expression of target stress‐inducible genes of Arabidopsis DREB1A resulting in plants with higher tolerance to drought, high‐salt, and freezing stresses. This indicated that OsDREB1A has functional similarity to DREB1A. However, in microarray and RNA blot analyses, some stress‐inducible target genes of the DREB1A proteins that have only ACCGAC as DRE were not over‐expressed in the OsDREB1A transgenic Arabidopsis. The OsDREB1A protein bound to GCCGAC more preferentially than to ACCGAC whereas the DREB1A proteins bound to both GCCGAC and ACCGAC efficiently. The structures of DREB1‐type ERF/AP2 domains in monocots are closely related to each other as compared with that in the dicots. OsDREB1A is potentially useful for producing transgenic monocots that are tolerant to drought, high‐salt, and/or cold stresses.
A Mak‐like kinase is a repressor of GAMYB in barley aleurone
Authors: Woodger Fiona J. , Gubler Frank , Pogson Barry J. , Jacobsen John V. 
Published: March 2003

SummaryGAMYB is a gibberellin (GA)‐regulated activator of hydrolase gene expression in the aleurone layer of germinating cereal grains. Although it is clear that GAMYB expression is regulated by GA, more remains to be understood about how this transcription factor operates within the GA‐response pathway. In order to isolate new components from the GA‐response pathway, barley aleurone libraries were screened for GAMYB‐binding proteins using a recently developed yeast two‐hybrid system, which is compatible with the use of transcription factors as baits. We isolated a new member of the emerging Mak‐subgroup of cdc2‐ and MAP kinase‐related protein kinases. We have termed this GAMYB‐binding protein KGM (for kinase associated with GAMYB). Transient expression of KGM specifically repressed α‐amylase promoter activity at the level of GAMYB function but a mutation designed to de‐stabilise the activation loop of KGM alleviated this repression. We propose that KGM is a negative regulator of GAMYB function in aleurone that may prevent precocious hydrolase gene expression.
Phytochrome control of flowering is temperature sensitive and correlates with expression of the floral integrator FT
Authors: Halliday Karen J. , Salter Michael G. , Thingnaes Elin , Whitelam Garry C. 
Published: March 2003

SummaryIn Arabidopsis flowering is accelerated by reduced red:far‐red (R:FR) ratio which signals the presence of neighbouring vegetation. Hastened flowering is one component of the shade‐avoidance syndrome of responses, which alter many aspects of development in response to the threat of potential competition. Of the red/far‐red‐absorbing photoreceptors it is phyB that plays the most prominent role in shade‐avoidance, although other related phytochromes act redundantly with phyB. It is well established that the phyB mutant has a constitutively early flowering phenotype. However, we have shown that the early flowering phenotype of phyB is temperature‐dependent. We have established that this temperature‐sensitive flowering response defines a pathway that appears to be independent of the autonomous‐FLC pathway. Furthermore, we have demonstrated that the phytochromes control the expression of the floral promoter FT. We have also shown that other phyB‐controlled responses, including petiole elongation, are not sensitive to the same temperature change. This suggests that discrete pathways control flowering and petiole elongation, components of the shade‐avoidance response. This work provides an insight into the phytochrome and temperature interactions that maintain flowering control.
Retracted: An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus
Authors: Voinnet Olivier , Rivas Susana , Mestre Pere , Baulcombe David 
Published: March 2003

SummaryTransient gene expression is a fast, flexible and reproducible approach to high‐level expression of useful proteins. In plants, recombinant strains of Agrobacterium tumefaciens can be used for transient expression of genes that have been inserted into the T‐DNA region of the bacterial Ti plasmid. A bacterial culture is vacuum‐infiltrated into leaves, and upon T‐DNA transfer, there is ectopic expression of the gene of interest in the plant cells. However, the utility of the system is limited because the ectopic protein expression ceases after 2–3 days. Here, we show that post‐transcriptional gene silencing (PTGS) is a major cause for this lack of efficiency. We describe a system based on co‐expression of a viral‐encoded suppressor of gene silencing, the p19 protein of tomato bushy stunt virus (TBSV), that prevents the onset of PTGS in the infiltrated tissues and allows high level of transient expression. Expression of a range of proteins was enhanced 50‐folds or more in the presence of p19 so that protein purification could be achieved from as little as 100 mg of infiltrated leaf material. The effect of p19 was not saturated in cells that had received up to four individual T‐DNAs and persisted until leaf senescence. Because of its simplicity and rapidity, we anticipate that the p19‐enhanced expression system will have value in industrial production as well as a research tool for isolation and biochemical characterisation of a broad range of proteins without the need for the time‐consuming regeneration of stably transformed plants.
Arabidopsisgene expression changes during cyst nematode parasitism revealed by statistical analyses of microarray expression profiles
Authors: Puthoff David P. , Nettleton Dan , Rodermel Steven R. , Baum Thomas J. 
Published: March 2003

SummaryWith the availability of microarray technology, the expression profiles of thousands of genes can be monitored simultaneously to help determine the mechanisms of these biological processes. We conducted Affymetrix GeneChip microarray analyses of theArabidopsis–cyst nematode interaction and employed a statistical procedure to analyze the resultant data, which allowed us to identify significant gene expression changes. Quantitative real‐time RT‐PCR assays were used to confirm the microarray analyses. The results of the expression profiling revealed 128 genes with altered steady‐state mRNA levels following infection by the sugar beet cyst nematode (Heterodera schachtii; BCN), in contrast to only 12 genes that had altered expression following infection by the soybean cyst nematode (H. glycines; SCN). The expression of these 12 genes also changed following infection by BCN, i.e. we did not identify any genes regulated exclusively by SCN. The identification of 116 genes whose expression changes during successful cyst nematode parasitism by BCN suggests a potential involvement of these genes in the infection events starting with successful syncytium induction. Further characterization of these genes will permit the formulation of testable hypotheses to explain successful cyst nematode parasitism.
CYP79F1 and CYP79F2 have distinct functions in the biosynthesis of aliphatic glucosinolates in Arabidopsis
Authors: Chen Sixue , Glawischnig Erich , Jørgensen Kirsten , Naur Peter , Jørgensen Bodil , Olsen Carl‐Erik , Hansen Carsten H. , Rasmussen Hasse , Pickett John A. , Halkier Barbara A. 
Published: March 2003

SummaryCytochromes P450 of the CYP79 family catalyze the conversion of amino acids to oximes in the biosynthesis of glucosinolates, a group of natural plant products known to be involved in plant defense and as a source of flavor compounds, cancer‐preventing agents and bioherbicides. We report a detailed biochemical analysis of the substrate specificity and kinetics of CYP79F1 and CYP79F2, two cytochromes P450 involved in the biosynthesis of aliphatic glucosinolates in Arabidopsis thaliana. Using recombinant CYP79F1 and CYP79F2 expressed in Escherichia coli and Saccharomyces cerevisiae, respectively, we show that CYP79F1 metabolizes mono‐ to hexahomomethionine, resulting in both short‐ and long‐chain aliphatic glucosinolates. In contrast, CYP79F2 exclusively metabolizes long‐chain elongated penta‐ and hexahomomethionines. CYP79F1 and CYP79F2 are spatially and developmentally regulated, with different gene expression patterns. CYP79F2 is highly expressed in hypocotyl and roots, whereas CYP79F1 is strongly expressed in cotyledons, rosette leaves, stems, and siliques. A transposon‐tagged CYP79F1 knockout mutant completely lacks short‐chain aliphatic glucosinolates, but has an increased level of long‐chain aliphatic glucosinolates, especially in leaves and seeds. The level of long‐chain aliphatic glucosinolates in a transposon‐tagged CYP79F2 knockout mutant is substantially reduced, whereas the level of short‐chain aliphatic glucosinolates is not affected. Biochemical characterization of CYP79F1 and CYP79F2, and gene expression analysis, combined with glucosinolate profiling of knockout mutants demonstrate the functional role of these enzymes. This provides valuable insights into the metabolic network leading to the biosynthesis of aliphatic glucosinolates, and into metabolic engineering of altered aliphatic glucosinolate profiles to improve nutritional value and pest resistance.