bluuebunny/crossref_metadata_2025_split huggingface dataset:
Over‐expression of a single Ca2+‐dependent protein kinase confers both cold and salt/drought tolerance on rice plants
Authors: Saijo Yusuke , Hata Shingo , Kyozuka Junko , Shimamoto Ko , Izui Katsura 
Published: March 2003

SummaryA rice gene encoding a calcium‐dependent protein kinase (CDPK), OsCDPK7, was induced by cold and salt stresses. To elucidate the physiological function of OsCDPK7, we generated transgenic rice plants with altered levels of the protein. The extent of tolerance to cold and salt/drought stresses of these plants correlated well with the level of OsCDPK7 expression. Therefore, OsCDPK7 was shown to be a positive regulator commonly involved in the tolerance to both stresses in rice. Over‐expression of OsCDPK7 enhanced induction of some stress‐responsive genes in response to salinity/drought, but not to cold. Thus, it was suggested that the downstream pathways leading to the cold and salt/drought tolerance are different from each other. It seems likely that at least two distinct pathways commonly use a single CDPK, maintaining the signalling specificity through unknown post‐translational regulation mechanisms. These results demonstrate that simple manipulation of CDPK activity has great potential with regard to plant improvement.
Developmental and light‐dependent regulation of a phloem‐localised K+ channel of Arabidopsis thaliana
Authors: Deeken Rosalia , Sanders Christoph , Ache Peter , Hedrich Rainer 
Published: March 2003

SummaryK+ channels in plants can currently be classified into six families with individual members being involved in nutrient uptake, loading of the xylem and the physiology of stomatal movement. In this study we have focused on akt2/3. This K+ channel, as shown by GUS‐expression analysis, is expressed in the phloem and xylem of the aerial parts of Arabidopsis thaliana. Northern blot analyses revealed the highest akt2/3‐concentrations in the flower stalk, followed by the leaf, flower and stem. During the light period (8 am to 4 pm), transcripts reached a peak around noon (11 am), decayed to almost 50% in the afternoon and reached a low background level in the following dark period. In continuous darkness, however, the K+ channel mRNA content had already decreased beyond the background level by noon. In leaves and flower stalk, the light‐induced transcription of akt2/3 was suppressed by CO2‐free air, indicating that gene activity is under the control of photosynthates. Additionally, when rosette leaves were illuminated and flower stalks shaded, akt2/3‐mRNA transcription was still inhibited in the shaded region. This indicates that channel gene activation is sensitive to photosynthesis‐derived factors from neighboring cells rather than factors mobile in the phloem. We propose that the coupling between sugar production and allocation involves the photosynthate‐ and light‐dependent phloem K+ channel AKT2/3.
Fungal cell wall phosphomannans facilitate the toxic activity of a plant PR‐5 protein
Authors: Ibeas José I. , Lee Hyeseung , Damsz Barbara , Prasad Doddananjappa T. , Pardo José M. , Hasegawa Paul M. , Bressan Ray A. , Narasimhan Meena L. 
Published: March 2003

SummaryOsmotin is a plant PR‐5 protein. It has a broad spectrum of antifungal activity, yet also exhibits specificity for certain fungal targets. The structural bases for this specificity remain unknown. We show here that full sensitivity of Saccharomyces cerevisiae cells to the PR‐5 protein osmotin is dependent on the function of MNN2, MNN4 and MNN6. MNN2 is an α‐1,2‐mannosyltransferase catalyzing the addition of the first mannose to the branches on the poly l,6‐mannose backbone of the outer chain of cell wall N‐linked mannans. MNN4 and MNN6 are required for the transfer of mannosylphosphate to cell wall mannans. Null mnn2, mnn4 or mnn6 mutants lack phosphomannans and are defective in binding osmotin to the fungal cell wall. Both antimannoprotein antibody and the cationic dye alcian blue protect cells against osmotin cytotoxicity. MNN1 is an α‐1,3‐mannosyltransferase that adds the terminal mannose to the outer chain branches of N‐linked mannan, masking mannosylphosphate. Null mnn1 cells exhibit enhanced osmotin binding and sensitivity. Several cell wall mannoproteins can bind to immobilized osmotin, suggesting that their polysaccharide constituent determines osmotin binding. Our results demonstrating a causal relationship between cell surface phosphomannan and the susceptibility of a yeast strain to osmotin suggest that cell surface polysaccharides of invading pathogens control target specificity of plant PR‐5 proteins.
Functional identification of an Arabidopsis Snf4 ortholog by screening for heterologous multicopy suppressors of snf4 deficiency in yeast
Authors: Kleinow Tatjana , Bhalerao Rishikesh , Breuer Frank , Umeda Masaaki , Salchert Klaus , Koncz Csaba 
Published: March 2003

SummaryYeast Snf4 is a prototype of activating γ‐subunits of conserved Snf1/AMPK‐related protein kinases (SnRKs) controlling glucose and stress signaling in eukaryotes. The catalytic subunits of Arabidopsis SnRKs, AKIN10 and AKIN11, interact with Snf4 and suppress the snf1 and snf4 mutations in yeast. By expression of an Arabidopsis cDNA library in yeast, heterologous multicopy snf4 suppressors were isolated. In addition to AKIN10 and AKIN11, the deficiency of yeast snf4 mutant to grown on non‐fermentable carbon source was suppressed by Arabidopsis Myb30, CAAT‐binding factor Hap3b, casein kinase I, zinc‐finger factors AZF2 and ZAT10, as well as orthologs of hexose/UDP‐hexose transporters, calmodulin, SMC1‐cohesin and Snf4. Here we describe the characterization of AtSNF4, a functional Arabidopsis Snf4 ortholog, that interacts with yeast Snf1 and specifically binds to the C‐terminal regulatory domain of Arabidopsis SnRKs AKIN10 and AKIN11.
A gain‐of‐function phenotype conferred by over‐expression of functional subunits of the COP9 signalosome in Arabidopsis
Authors: Kang Dingming , Wang Xiping , Cao Kaiming , Sun Chongrong , Deng Xing‐Wang , Wei Ning 
Published: March 2003

SummaryThe COP9 signalosome is a conserved cellular regulator present in diverse organisms. To understand the structural and functional relationship of the COP9 signalosome with its subunits, we expressed in wild‐type and mutant Arabidopsis backgrounds two orthologues of subunit 1, rice FUS6 (rFUS6) and human GPS1, and Arabidopsis subunit 8 (COP9). In Arabidopsis, rFUS6 can functionally replace Arabidopsis endogenous FUS6 to form the COP9 signalosome complex and rescue the null fus6‐1 mutant phenotype. Moreover, light‐grown rFUS6 over‐expression seedlings displayed longer hypocotyls and reduced anthocyanin accumulation in comparison to wild‐type seedlings, which is opposite to the fus6/cop11 mutant phenotype. The long‐hypocotyl phenotype was also observed in transgenic seedlings over‐expressing Arabidopsis COP9. This finding indicates that over‐expression of a functional subunit 1 or subunit 8 of the COP9 signalosome confers a gain‐of‐function phenotype relative to the complex. Human GPS1, when expressed in the fus6‐1 null mutant of Arabidopsis, can assemble into a chimeric COP9 signalosome at low efficiency, demonstrating the structural conservation of the complexes between human and Arabidopsis. This low‐abundancy chimeric complex is insufficient to fully rescue the mutant but is able to attenuate the mutant severity.
Biochemical characterization of the Arabidopsis K+ channels KAT1 and AKT1 expressed or co‐expressed in insect cells
Authors: Urbach Serge , Chérel Isabelle , Sentenac Hervé , Gaymard Frédéric 
Published: March 2003

SummaryKAT1 and AKT1 belong to the multigenic family of the inwardly rectifying Shaker‐like plant K+ channels. They were biochemically characterized after expression in insect cells using recombinant baculoviruses. The channels were solubilized from microsomal fractions prepared from infected cells (among eight different detergents only one,
l‐α‐lysophosphatidylcholine, was efficient for solubilization), and purified to homogeneity using immunoaffinity (KAT1) or ion‐exchange and size exclusion (AKT1) techniques. The following results were obtained with the purified polypeptides: (i) neither KAT1 nor AKT1 was found to be glycosylated; (ii) both polypeptides were mainly present as homotetrameric structures, supporting the hypothesis of a tetrameric structure for the functional channels; (iii) no heteromeric KAT1/AKT1 assembly was detected when the two polypeptides were co‐expressed in insect cells. The use of the two‐hybrid system in yeast also failed to detect any interaction between KAT1 and AKT1 polypeptides. Because of these negative results, the hypothesis that plant K+‐channel subunits are able to co‐assemble without any discrimination, previously put forward based on co‐expression in Xenopus oocytes of various K+‐channel subunits (including KAT1 and AKT1), has still to be supported by independent approaches. Co‐localization of channel subunits within the same plant tissue/cell does not allow us to conclude that the subunits form heteromultimeric channels.
In vivo imaging of an elicitor‐induced nitric oxide burst in tobacco
Authors: Foissner Ilse , Wendehenne David , Langebartels Christian , Durner Jörg 
Published: March 2003

SummaryA growing body of evidence suggests that nitric oxide (NO), an important signalling and defence molecule in mammals, plays a key role in activating disease resistance in plants, acting as signalling molecule and possibly as direct anti‐microbial agent. Recently, a novel fluorophore (diaminofluorescein diacetate, DAF‐2 DA) has been developed which allows bio‐imaging of NO in vivo. Here we use the cell‐permeable DAF‐2 DA, in conjunction with confocal laser scanning microscopy, for real‐time imaging of NO in living plant cells. Epidermal tobacco cells treated with cryptogein, a fungal elicitor from Phytophthora cryptogea, respond to the elicitor with a strong increase of intracellular NO. NO‐induced fluorescence was found in several cellular compartments, and could be inhibited by a NO scavenger and an inhibitor of nitric oxide synthase. The NO burst was triggered within minutes, reminiscent of the oxidative burst during hypersensitive response reactions. These results reveal additional similarities between plant and animal host responses to infection.
Characterization of cis‐elements required for vascular expression of the Cinnamoyl CoA Reductase gene and for protein–DNA complex formation
Authors: Lacombe Eric , Van Doorsselaere Jan , Boerjan Wout , Boudet Alain M. , Grima‐Pettenati Jacqueline 
Published: March 2003

SummaryCinnamoyl‐CoA reductase (CCR) catalyses the first specific step in the biosynthesis of monolignols, the monomeric units of lignins. We examined the developmental regulation of the Eucalyptus gunnii EgCCR promoter by analysing the expression of EgCCR–GUS fusions in tobacco. EgCCR promoter activity was strongest in lignified organs (stems and roots) consistent with the EgCCR mRNA level in these organs. Histochemical analysis showed expression in vascular tissues (cambium, young differentiating xylem, ray cells, internal and external phloem) of stems and roots in agreement with in situ hybridization data. Promoter deletion analysis and gain‐of‐function experiments identified the sequences between positions −119 and −77 as necessary and sufficient for expression in vascular tissues of stems. Electrophoretic mobility‐shift assays showed that this region is specifically recognized by nuclear proteins present in tobacco stems, giving rise to two retarded complexes, LMC1 and LMC2. Using overlapping EgCCR fragments and mutated oligonucleotides as competitors in gel‐shift assays, two DNA–protein interaction sites were mapped. Finally, the role of protein–protein interactions in the formation of the LMC1 and LMC2 complexes was investigated using the detergent sodium deoxycholate, and protein fractionation onto a heparin Sepharose column.
Identification of plant actin‐binding proteins by F‐actin affinity chromatography
Authors: Hu Shiquan , Brady Shari R. , Kovar David R. , Staiger Christopher J. , Clark Greg B. , Roux Stanley J. , Muday Gloria K. 
Published: March 2003

SummaryProteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin‐binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin‐binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G‐actin) or actin filaments (filamentous actin, F‐actin) have been used to identify actin‐binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G‐actin, F‐actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane‐associated actin‐binding proteins. Extracts of soluble actin‐binding proteins yield distinct patterns when eluted from the G‐actin and F‐actin columns, respectively, leading to the identification of a putative F‐actin‐binding protein of approximately 40 kDa. When plasma membrane‐associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F‐actin column and cross‐reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F‐actin column. These experiments provide a new approach that may help to identify novel actin‐binding proteins from plants.
Plastidic metabolite transporters and their physiological functions in the inducible crassulacean acid metabolism plant Mesembryanthemum crystallinum
Authors: Häusler Rainer E. , Baur Bernhard , Scharte Judith , Teichmann Thomas , Eicks Michael , Fischer Katrin L. , Flügge Ulf‐Ingo , Schubert Sabine , Weber Andreas , Fischer Karsten 
Published: March 2003

SummaryThe inducible crassulacean acid metabolism (CAM) plant Mesembryanthemum crystallinum accumulates malic acid during the night and converts it to starch during the day via a pathway that, because it is located in different subcellular compartments, depends on specific metabolite transport across membranes. The chloroplast glucose transporter (pGlcT) and three members of the phosphate translocator (PT) family were isolated. After induction of CAM, transcript amounts of the phosphoenolpyruvate (PEP) phosphate translocator (PPT) and the glucose‐6‐phosphate (Glc6P) phosphate translocator (GPT) genes were increased drastically, while triose phosphate (TP) phosphate translocator (TPT) and the pGlcT transcripts remained unchanged. PPT‐ and GPT‐specific transcripts and transporter activities exhibited a pronounced diurnal variation, displaying the highest amplitude in the light. pGlcT transcripts were elevated towards the end of the light period and at the beginning of the dark period. These findings, combined with diurnal variations of enzyme activities and metabolite contents, helped to elucidate the roles of the PPT, GPT, TPT and pGlcT in CAM. The main function of the PPT is the daytime export from the stroma of PEP generated by pyruvate orthophosphate:dikinase (PPDK). The increased transport activity of GPT in the light suggests a higher requirement for Glc6P import for starch synthesis rather than starch mobilization. Most likely, Glc6P rather than 3‐phosphoglycerate or triose phosphates is the main substrate for daytime starch biosynthesis in M. crystallinum plants in which CAM has been induced (CAM‐induced), similar to non‐green plastids. In the dark, starch is mobilized both phosphorylytically and amylolytically and the products are exported by the GPT, TPT and pGlcT. The transport activities of all three phosphate translocators and the transcript amounts of the pGlcT adapt to changing transport requirements in order to maintain high metabolic fluxes during the diurnal CAM cycle.
Technical Advance: Cytometric analysis of an epitope‐tagged transit peptide bound to the chloroplast translocation apparatus
Authors: Subramanian Chitra , Ivey Robert , Bruce Barry D. 
Published: March 2003

SummaryChloroplast transit peptides are necessary and sufficient for the targeting and translocation of precursor proteins across the chloroplast envelope. However, the mechanism by which transit peptides engage the translocation apparatus has not been investigated. To analyse this interaction, we have developed a novel epitope‐tagged transit peptide derived from the precursor of the small subunit of pea Rubisco. The recombinant transit peptide, His‐S‐SStp, contains a removable dual‐epitope tag, His‐S, at its N‐terminus that permits both rapid purification via immobilized metal affinity chromatography and detection by blotting, flow cytometry and laser‐scanning confocal microscopy. Unlike other chimeric precursors, which place the passenger protein C‐terminal to the transit peptide, His‐S‐SStp bound to the translocation apparatus yet did not translocate across the chloroplast envelope. This early translocation intermediate allowed non‐radioactive detection using fluorescent and chemiluminescent reporters. The physiological relevance of this interaction was confirmed by protein import competitions, sensitivity to pre‐ and post‐import thermolysin treatment, photochemical cross‐linking and organelle fractionation. The interaction was specific for the transit peptide since His‐S alone did not engage the chloroplast translocation apparatus. Quantitation of the bound transit peptide was determined by flow cytometry, showing saturation of binding yet only slight ATP‐dependence. The addition of GTP showed inhibition of the binding of His‐S‐SStp to the chloroplasts indicating an involvement of GTP in the formation of this early translocation intermediate. In addition, direct visualization of His‐S‐SStp and Toc75 by confocal microscopy revealed a patch‐like labeling, suggesting a co‐ordinate localization to discrete regions on the chloroplast envelope. These findings represent the first direct visualization of a transit peptide interacting with the chloroplast translocation apparatus. Furthermore, identification of a chloroplast‐binding intermediate may provide a novel tool to dissect interactions between a transit peptide and the chloroplast translocation apparatus.
Living under a ‘dormant’ canopy: a molecular acclimation mechanism of the desert plant Retama raetam
Authors: Mittler Ron , Merquiol Emmanuelle , Hallak‐Herr Elza , Rachmilevitch Shimon , Kaplan Aaron , Cohen Mira 
Published: March 2003

SummaryDesert plants are exposed to a combination of environmental stress conditions, including low water availability, extreme temperature fluctuations, high irradiance and nutrient deprivation. Studying desert plants within their natural habitat may therefore reveal novel mechanisms and strategies that enable plants to resist stressful conditions. We studied the acclimation of Retama raetam, an evergreen stem‐assimilating desert plant, to growth within an arid dune ecosystem. Retama raetam contained two different populations of stems: those of the upper canopy, exposed to direct sunlight, and those of the lower canopy, protected from direct sunlight. During the dry season, stems of the upper canopy contained a very low level of a number of essential proteins, including the large and small subunits of rubisco, ascorbate peroxidase and the D1 subunit of the reaction centre of photosystem II. However, RNA encoding these proteins was present; cytosolic transcripts were associated with polysomes, while chloroplastic transcripts were not. Upon water application, as well as following the first rainfall of the season, these ‘photosynthetically suppressed’ stems recovered and accumulated essential proteins within 6–24 h. In contrast, stems of the lower canopy contained the essential proteins throughout the dry season. We suggest that R. raetam uses an acclimation strategy of ‘partial plant dormancy’ in order to survive the dry season. ‘Dormancy’, as evident by the post‐transcriptional suppression of gene expression, as well as the suppression of photosynthesis, was induced specifically in stems of the upper canopy which protect the lower canopy by shading.
Expression of MsLEC1‐ and MsLEC2‐antisense genes in alfalfa plant lines causes severe embryogenic, developmental and reproductive abnormalities
Authors: Brill Laurence M. , Evans Cory J. , Hirsch Ann M. 
Published: March 2003

SummaryAlthough it has been proposed that plant lectins play a number of roles, the function of these proteins in normal plant growth and development has been unclear. To analyze the functions of putative alfalfa lectin genes, lines of transgenic alfalfa plants expressing approximately half of the open reading frame of MsLEC1 or MsLEC2, in the antisense or sense orientation, were established and analyzed. The antisense plants displayed severe abnormalities in embryogenesis, and both vegetative and reproductive development were perturbed. Some differences were observed between MsLEC1‐ and MsLEC2‐antisense plants, and abnormalities were especially severe during the early stages of development in both the primary and secondary transgenic generations. In contrast, vector‐control and sense‐transgene plants exhibited normal growth and development. MsLEC1 and MsLEC2 mRNA accumulation levels were reduced in cognate antisense plants, especially during the later stages of embryogenesis, but also tended to be low in MsLEC1 sense‐transgene plants. However, correlated with the phenotypic abnormalities observed in the MsLEC1‐antisense plants was the specific reduction in the accumulation of a candidate MsLEC1 protein. Our results suggest that the MsLEC1 and MsLEC2 gene products, in addition to being important for embryogenesis, are required throughout alfalfa development.
CKS1At overexpression in Arabidopsis thaliana inhibits growth by reducing meristem size and inhibiting cell‐cycle progression
Authors: De Veylder Lieven , Beemster Gerrit T.S. , Beeckman Tom , Inzé Dirk 
Published: March 2003

SummaryThe SUC1/CKS1 proteins associate with cyclin‐dependent kinases (CDKs) and play an essential role in the regulation of the cell cycle. Recently, an Arabidopsis thaliana SUC1/CKS1 homologous gene, designated CKS1At, has been cloned. Here, overexpression of CKS1At in Arabidopsis is shown to reduce leaf size and root growth rates. Reduced root growth resulted primarily from an increase of the cell‐cycle duration and a shortening of the meristem. Endoreduplication was unaffected. The increased cell‐cycle duration was associated with an equal extension of both the G1 and G2 phases. This inhibition was due to the binding of CDK subunits with CDKs. The reduced growth rates in response to altered cell‐cycle gene expression demonstrates a direct dependence of plant growth rates on cell‐cycle regulation.