bluuebunny/crossref_metadata_2025_split huggingface dataset:
Higher plant phosphoenolpyruvate carboxylase kinase is regulated at the level of translatable mRNA in response to light or a circadian rhythm
Authors: Hartwell James , Smith Lucy H. , Wilkins Malcolm B. , Jenkins Gareth I. , Nimmo Hugh G. 
Published: March 2003

SummaryPhosphoenolpyruvate carboxylase is regulated by reversible phosphorylation in response to light in C3 and C4 plants and to a circadian oscillator in CAM plants. Increases in phosphoenolpyruvate carboxylase kinase activity require protein synthesis. This requirement has been analysed by quantifying translatable mRNA for this protein kinase using in vitro translation of isolated RNA followed by direct assay of kinase activity. In leaves of the CAM plant Bryophyllum (Kalanchoë) fedtschenkoi, in normal diurnal conditions, kinase mRNA was 20‐fold more abundant at night than in the day. In constant environmental conditions (continuous darkness, CO2‐free air, 15°C) kinase mRNA exhibited circadian oscillations. The circadian disappearance of kinase mRNA and kinase activity was delayed by lowering the temperature to 4°C and accelerated by raising the temperature to 30°C. The appearance of kinase mRNA and activity was blocked by cordycepin and puromycin. In maize and barley, kinase mRNA increased in response to light. For all three plants, the phosphoenolpyruvate carboxylase kinase activity generated during in vitro translation was Ca2+‐independent. These results demonstrate that phosphoenolpyruvate carboxylase kinase activity is regulated at the level of translatable mRNA in C3, C4 and CAM plants.
A transcriptional activation domain of ATMYB2, a drought‐inducible Arabidopsis Myb‐related protein
Authors: Urao Takeshi , Noji Masa‐aki , Yamaguchi‐Shinozaki Kazuko , Shinozaki Kazuo 
Published: March 2003

Summary Trans‐activation activity of ATMYB2, a drought‐inducible Myb‐related protein in Arabidopsis thaliana, was analyzed using a transient assay of Arabidopsis leaf protoplasts. ATMYB2 activated the transcription of a reporter gene from the MYB‐binding site in a sequence‐specific manner. Deletion of the C‐terminal region of ATMYB2 reduced the trans‐activation of the reporter gene, indicating that the acidic region at the C‐terminus of ATMYB2 is required for transcriptional activation. The domain exchange analysis with the yeast GAL4 revealed that the C‐terminal acidic region of ATMYB2 contains a sufficient domain for trans‐activation. These results indicate that ATMYB2 acts as a transcriptional activator and that the C‐terminal acidic region of ATMYB2 can function as a transcriptional activation domain.
A plant in vitro system for the nuclear import of proteins
Authors: Merkle Thomas , Leclerc Denis , Marshallsay Christopher , Nagy Ferenc 
Published: March 2003

SummaryThis paper reports the development of an in vitro system that allows the direct assay of protein import into plant nuclei. In this assay the import of fluorescently labelled karyophilic protein substrates into nuclei isolated from evacuolated tobacco BY‐2 suspension cells is monitored. It is demonstrated that import of the fluorescently labelled peptide conjugates is rapid, saturable and nuclear localization signal (NLS)‐dependent. Exclusion of high molecular weight (70 kDa) dextran and substrates carrying mutated NLS sequences further underline the specificity of this system. Nuclear translocation of karyophilic import substrates in tobacco, similar to mammalian systems, is inhibited by the non‐hydrolysable GTP analogue GTP‐γ‐S. In contrast, protein uptake is not blocked by wheat germ agglutinin, N‐ethyl‐maleinimide and iodoacetic acid. Furthermore, it is shown that nuclear import of proteins is only partially inhibited by low temperature (0–4°C). The in vitro nuclear import assay does not depend on exogenously added ATP or cytosolic factors. However, a block of nuclear import with GTP‐γ‐S could be overcome by the addition of cytosolic extract, suggesting the dependence on cytosolic factors or proteins. These data indicate that the characteristics of nuclear protein import in plant and mammalian cells are similar, but may be, at least in some respects, also different from each other.
The blue‐light receptor cryptochrome 1 shows functional dependence on phytochrome A or phytochrome B in Arabidopsis thaliana
Authors: Ahmad Margaret , Cashmore Anthony R. 
Published: March 2003

SummaryBlue‐light responses in higher plants are mediated by specific photoreceptors, which are thought to be flavoproteins; one such flavin‐type blue‐light receptor, CRY1 (for cryptochrome), which mediates inhibition of hypocotyl elongation and anthocyanin biosynthesis, has recently been characterized. Prompted by classical photobiological studies suggesting possible co‐action of the red/far‐red absorbing photoreceptor phytochrome with blue‐light photoreceptors in certain plant species, the role of phytochrome in CRY1 action in Arabidopsis was investigated. The activity of the CRY1 photoreceptor can be substantially altered by manipulating the levels of active phytochrome (Pfr) with red or far‐red light pulses subsequent to blue‐light treatments. Furthermore, analysis of severely phytochrome‐deficient mutants showed that CRY1‐mediated blue‐light responses were considerably reduced, even though Western blots confirmed that levels of CRY1 photoreceptor are unaffected in these phytochrome‐deficient mutant backgrounds. It was concluded that CRY1‐mediated inhibition of hypocotyl elongation and anthocyanin production requires active phytochrome for full expression, and that this requirement can be supplied by low levels of either phyA or phyB.
The LS locus of pea encodes the gibberellin biosynthesis enzyme ent‐kaurene synthase A
Authors: Ait‐Ali Tahar , Swain Stephen M. , Reid James B. , Sun Tai‐ping , Kamiya Yuji 
Published: March 2003

SummaryGibberellins (GAs) are hormones required for several aspects of plant development, including internode elongation and seed development in pea (Pisum sativum L.). The first committed step in the GA biosynthesis pathway is the conversion of geranylgeranyl diphosphate (GGDP) to ent‐kaurene via copalyl diphosphate (CDP). These two reactions are catalyzed by the cyclases ent‐kaurene synthase A (KSA) and ent‐kaurene synthase B (KSB), respectively. Previous genetic and biochemical analysis of the GA‐responsive ls‐1 mutant of pea suggested that GA levels are reduced in a developmental‐ and organ‐specific manner due to reduced GA biosynthesis. Analysis of cell‐free enzyme preparations from WT and ls‐1 embryos at contact point reveals that ls‐1 reduces the activity of KSA but not KSB. To characterize the ls‐1 mutation in more detail, a cDNA coding for a pea KSA was cloned and shown to be encoded by the LS locus. The ls‐1 mutation results from an intronic G to A substitution that causes impaired RNA splicing. To determine the activity of the KSAs encoded by the LS and ls‐1 alleles, a new in vitro assay for combined KSA and KSB activity has been developed using the KSB gene of pumpkin. Using recombinant WT KSA and KSB fusion proteins, GGDP is converted to ent‐kaurene in vitro. Based on the sequence of RT‐PCR products, three different truncated KSA proteins are predicted to exist in ls‐1 plants. The most abundant mutant KSA protein does not possess detectable activity in vitro. Nevertheless, the ls‐1 allele is not null and is able to encode at least a partially functional KSA since a more severe ls allele has been identified. The ls‐1 mutation has played a key role in identifying a role for GAs in pea seed development in the first few days after fertilization, but not in older seeds. KSA expression in seeds is developmentally regulated and parallels overall GA biosynthesis, suggesting that KSA expression may play an important role in the regulation of GA biosynthesis and seed development.
Green‐fluorescent protein fusions for efficient characterization of nuclear targeting
Authors: Grebenok Robert J. , Pierson Elizabeth , Lambert Georgina M. , Gong Fang‐Cheng , Afonso Claudio L. , Haldeman‐Cahill Ruth , Carrington James C. , Galbraith David W. 
Published: March 2003

SummaryThe green‐fluorescent protein (GFP) from Aequorea victoria has been shown to be a convenient and flexible reporter molecule within a variety of eukaryotic systems, including higher plants. It is particularly suited for applications in vivo, since the mechanism of fluorophore formation involves an intramolecular autoxidation and does not require exogenous co‐factors. Unlike standard histochemical procedures of fixation and staining required for analysis of the cellular or tissue‐specific expression of other popular reporter molecules, such as the β‐glucuronidase (GUS) marker, analysis of GFP can be done in living cells with no specific pretreatments. This implies that GFP might also be particularly suited for studies of intracellular protein targeting. In this paper, the use of GUS is compared with that of GFP for the analysis of nuclear targeting in tobacco. A novel oligopeptide motif from a tobacco protein is described which confers nuclear localization of GUS. The use of this oligopeptide and two from potyviral proteins to target GFP to the nucleus is examined. An essential modification of GFP is described, which specifically increases its molecular weight to eliminate its passive penetration into the nucleus. Three examples of the targeting of these enlarged GFP molecules to the nucleus are illustrated. GFP, in combination with confocal microscopy, offers significant advantages over traditional methods of studying nuclear targeting.
The green fluorescent protein as a marker to visualize plant mitochondria in vivo
Authors: Köhler Rainer H. , Zipfel Warren R. , Webb Watt W. , Hanson Maureen R. 
Published: March 2003

SummaryTo determine how to utilize the green fluorescent protein (GFP) as a marker for subcellular localization and as a label for plant mitochondria in vivo, transgenic suspension cells and tobacco plants expressing GFP with and without a mitochondrial localization signal were generated. The first GFP form used, GFP1, is easily observable in cells with low autofluorescence, such as suspension cells or trichomes, but masked in green tissue. For the visualization of GFP in cells and tissues with high autofluorescence, such as leaf, the use of a very strong promoter (35S35SAMV), a highly expressed modified mGFP4 coding region and a brighter mutant form of GFP (S65T) was necessary. Confocal or two‐photon laser scanning microscopy reveal a distinct subcellular localization of the fluorescence in cells expressing GFP or coxIVGFP. In cells expressing untargeted GFP, fluorescence accumulates in the nucleoplasm but is also distributed throughout the cytoplasm. It is excluded from vacuoles, nucleoli and from round bodies that are likely to be leucoplasts. In contrast, fluorescence is localized specifically to mitochondria in cells expressing coxIVGFP fusion protein as shown by co‐localization with a mitochondrial‐specific dye. This permits the direct observation of mitochondria and mitochondrial movements in living plant cells and tissues throughout plant development. Three‐dimensional reconstruction of individual cells can give additional information about the distribution and numbers of mitochondria.
Characterization of three loci controlling resistance of Arabidopsis thaliana accession Ms‐0 to two powdery mildew diseases
Authors: Xiao Shunyuan , Ellwood Simon , Findlay Kim , Oliver Richard P. , Turner John G 
Published: March 2003

Summary Arabidopsis thaliana accession La‐er was susceptible, and accession Ms‐0 was resistant, to powdery mildew diseases caused by Erysiphe cruciferarum UEA1 and E. cichoracearum UCSC1. The resistance reaction phenotype of A. thaliana Ms‐0 to both pathogens was characterized, and the resistance loci were genetically mapped. Growth of E. cruciferarum UEA1 on Ms‐0 leaves was arrested after formation of the first appressorium: the underlying host epidermal cell collapsed, and occasionally there was necrosis of one or two host mesophyll cells. Growth of E. cichoracearum UCSC1 on Ms‐0 leaves was arrested after emergence of several germ tubes from the conidium, and there was necrosis of host mesophyll cells at the sites of infection. Examination of F2 progeny of a cross La‐er x Ms‐0 indicated that two independently‐segregating dominant loci were required for resistance to E. cruciferarum UEA1. One locus, named RPW6, was genetically mapped to chromosome 5, in a 5.6 cM interval flanked by pCITf16 and PI. The other locus, named RPW7, mapped to chromosome 3 in a 8.5 cM interval flanked by CDC2A and AFC1. Independent effects of RPW6 and RPW7 on E. cruciferarum UEA1 could be detected by quantitative measurements of growth of mycelium and production of conidia. Resistance to E. cichoracearum UCSC1 mapped to a single locus, named RPW8, at a location on chromosome 3 which we could not distinguish from RPW7. Evidently, RPW7 and RPW8 define either a complex resistance locus, or a common resistance gene with dual specificity.
Interaction of Arabidopsis kinesin‐like calmodulin‐binding protein with tubulin subunits: modulation by Ca2+‐calmodulin
Authors: Narasimhulu Soma B. , Kao Yu‐Lin , Reddy A.S.N. 
Published: March 2003

Summary Kinesin‐like calmodulin‐binding protein (KCBP) is a recently identified novel kinesin‐like protein that appears to be unique to and ubiquitous in plants. KCBP is distinct from all other known KLPs in having a calmodulin‐binding domain adjacent to its motor domain. We have used different regions of KCBP to study its interaction with tubulin subunits and the regulation of this interaction by Ca2+‐calmodulin. The results show that the carboxy‐terminal part of the KCBP, with or without calmodulin‐binding domain, binds to tubulin subunits and this binding is sensitive to nucleotides. In the presence of Ca2+‐calmodulin the motor with calmodulin‐binding domain does not bind to tubulin. This Ca2+‐calmodulin modulation is abolished in the presence of antibodies specific to the calmodulin‐binding domain of KCBP. Similar binding studies with the carboxy‐terminal part of KCBP lacking the calmodulinbinding domain show no effect of Ca2+‐calmodulin. These results indicate that Ca2+‐calmodulin modulates the interaction of KCBP with tubulin subunits and this modulation is due to the calmodulin‐binding domain in the KCBP. Calcium‐dependent calmodulin modulation of KCBP interaction with tubulin suggests regulation of KCBP function by calcium, the first such regulation of a kinesin heavy chain among all the known kinesin‐like proteins.
A Cupheaβ‐ketoacyl‐ACP synthase shifts the synthesis of fatty acids towards shorter chains in Arabidopsis seeds expressing Cuphea FatB thioesterases
Authors: Leonard Jeffrey M. , Knapp Steven J. , Slabaugh Mary B. 
Published: March 2003

SummaryAcyl–acyl carrier protein (ACP) thioesterases with specificities on medium chain substrates (C8–C14) are requisite enzymes in plants that produce 8:0, 10:0, 12:0 and 14:0 seed oils, but they may not be the sole enzymatic determinants of chain length. The contribution to chain length regulation of a β‐ketoacyl‐ACP synthase, Cw KAS A1, derived from Cuphea wrightii, a species that accumulates 30% 10:0 and 54% 12:0 in seed oils, was investigated. Expression of Cw KAS A1 in Arabidopsis seeds reduced 16:0 from 8.2 to 6.2 mol%, suggesting a KAS II‐type activity. In the presence of the KAS I inhibitor cerulenin, however, transgenic seed extracts extended 6:0‐ and 8:0‐ACP at a rate four‐ to fivefold greater than extracts from untransformed plants, whereas no difference was observed in extension of 14:0‐ and 16:0‐ACP. The effect of KAS A1 on seed oils was tested by combining it with the C. wrightii medium chain‐specific thioesterases, Cw FatB1 and Cw FatB2, in crosses of transformed plants. Fatty acid synthesis shifted towards shorter chains in progeny expressing both classes of enzymes. KasA1/FatB1 homozygotes produced threefold more 12:0 than the FatB1 parent while 14:0 and 16:0 were reduced by one‐third and one‐half, respectively. F2 progeny expressing KasA1 and FatB2 produced twofold more 10:0 and 1.4‐fold more 12:0 than the FatB2 parent, and the double‐transgenic progeny produced one‐quarter less 14:0 and one‐half less 16:0 than the FatB2 parent. It is hypothesized that the shift towards production of shorter chains resulted from increased pools of medium chain acyl‐ACP resulting from KAS A1 activity. The combined activities of KAS A1 and FatB thioesterases appear to determine the C. wrightii phenotype.
Auxin transport inhibition precedes root nodule formation in white clover roots and is regulated by flavonoids and derivatives of chitin oligosaccharides
Authors: Mathesius Ulrike , Schlaman Helmi R. M. , Spaink Herman P. , Of Sautter Christ , Rolfe Barry G. , Djordjevic Michael A. 
Published: March 2003

SummaryThe expression of the auxin responsive reporter construct, GH3:gusA, was examined in transgenic white clover plants to assess changes in the auxin balance during the earliest stages of root nodule formation. Reporter gene expression was monitored at marked locations after the application of bacteria or signal molecules using two precise inoculation techniques: spot‐inoculation and a novel method for ballistic microtargeting. Changes in GH3:gusA expression were monitored after the inoculation of Rhizobium leguminosarum biovar trifolii, non‐host rhizobia, lipo‐chitin oligosaccharides (LCOs), chitin oligosaccharides, a synthetic auxin transport inhibitor (naphthylphthalamic acid; NPA), auxin, the ENOD40–1 peptide or different flavonoids. The results show that clover‐nodulating rhizobia induce a rapid, transient and local downregulation of GH3:gusA expression during nodule initiation followed by an upregulation of reporter gene expression at the site of nodule initiation. Microtargeting of auxin caused a local and acropetal upregulation of GH3:gusA expression, whereas NPA caused local and acropetal downregulation of expression. Both spot‐inoculation and microtargeting of R. l. bv. trifolii LCOs or flavonoid aglycones induced similar changes to GH3:gusA expression as NPA. O‐acetylated chitin oligosaccharides caused similar changes to GH3:gusA expression as R. l. bv. trifolii spot‐inoculation, but only after delivery by microtargeting. Non‐O‐acetylated chitin oligosaccharides, flavonoid glucosides or the ENOD40–1 peptide failed to induce any detectable changes in GH3:gusA expression. GH3:gusA expression patterns during the later stages of nodule and lateral root development were similar. These results support the hypothesis that LCOs and chitin oligosaccharides act by perturbing the auxin flow in the root during the earliest stages of nodule formation, and that endogenous flavonoids could mediate this response.
Immunolocalization of a novel annexin‐like protein encoded by a stress and abscisic acid responsive gene in alfalfa
Authors: Kovács Izabella , Ayaydin Ferhan , Oberschall Attila , Ipacs Ildikó , Bottka Sándor , Pongor Sándor , Dudits Dénes , Tóth Éva Csordás 
Published: March 2003

SummaryWe report here on the isolation and characterization of a full‐length cDNA clone from alfalfa termed AnnMs2 encoding a 333 amino acid long polypeptide that shows 32–37% sequence identity with both mammalian and plant annexins, and has four tandem repeats. While other plant annexins exhibit a high level of sequence similarity to each other (up to 77% identity at amino acid level), AnnMs2 appears to be a distinct type of plant annexins. All the four endonexin folds contain the conserved eukaryotic motif within this alfalfa protein, but this element is considerably different in the second repeat. The AnnMs2 gene is expressed in various tissues of alfalfa with elevated mRNA accumulation in root and flower. This gene is activated in cells or tissues exposed to osmotic stress, abscisic acid (ABA) or water deficiency. The recombinant AnnMs2 protein is able to bind to phospholipid in the presence of Ca2+. Indirect immunofluorescence studies using affinity purified rabbit anti‐AnnMs2 peptide antibody show mainly nucleolar localization, but the protein sequence lacks the usual nuclear localization signal. The potential role of this novel annexin‐like protein in the basic and stress‐induced cellular functions is discussed.
Differential regulation of the tomato ETR gene family throughout plant development
Authors: Lashbrook Coralie C. , Tieman Denise M. , Klee Harry J. 
Published: March 2003

SummaryEthylene perception in plants is co‐ordinated by multiple hormone receptor candidates sharing sequence commonalties with prokaryotic environmental sensor proteins known as two‐component regulators. Two tomato homologs of the Arabidopsis ethylene receptor ETR1 were cloned from a root cDNA library. Both cDNAs, termed LeETR1 and LeETR2, were highly homologous to ETR1, exhibiting ∼ 90% deduced amino acid sequence similarity and 80% deduced amino acid sequence identity. LeETR1 and LeETR2 contained all the major structural elements of two‐component regulators, including the response regulator motif absent in LeETR3, the gene encoding tomato NEVER RIPE (NR). Using RNase protection analysis, the mRNAs of LeETR1, LeETR2 and NR were quantified in tissues engaged in key processes of the plant life cycle, including seed germination, shoot elongation, leaf and flower senescence, floral abscission, fruit set and fruit ripening. LeETR1 was expressed constitutively in all plant tissues examined. LeETR2 mRNA was expressed at low levels throughout the plant but was induced in imbibing tomato seeds prior to germination and was down‐regulated in elongating seedlings and senescing leaf petioles. NR expression was developmentally regulated in floral ovaries and ripening fruit. Notably, hormonal regulation of NR was highly tissue‐specific. Ethylene biosynthesis induced NR mRNA accumulation in ripening fruit but not in elongating seedlings or in senescing leaves or flowers. Furthermore, the abundance of mRNAs for all three LeETR genes remained uniform in multiple plant tissues experiencing marked changes in ethylene sensitivity, including the cell separation layer throughout tomato flower abscission.
Localization of components of the oxidative cross‐linking of glycoproteins and of callose synthesis in papillae formed during the interaction between non‐pathogenic strains ofXanthomonas campestris andFrench bean mesophyll cells
Authors: Brown Ian , Trethowan Jonathan , Kerry Maria , Mansfield John , Bolwell G. Paul 
Published: March 2003

Summary Immunogold labelling was used to probe the responses of mesophyll cells in French bean (

Phaseolus vulgaris
L.) to anhrpAmutant ofXanthomonas campestrispv.vesicatoriaand a saprophytic strain ofX.c.The non‐pathogenic strains both caused localized alterations to the plant cell wall and formation of large papillae in adjacent cells. Immunocytochemistry showed the co‐localization, in the cell wall and paramural deposits, of anMr 42 000 proline‐rich glycoprotein with chitin‐binding activity (CBPRP) and the enzyme responsible for its immobilization, anMr 46 000 peroxidase. The CBPRP appeared to lose antigenicity after cross‐linking, and, unlike the peroxidase, was not detected consistently in the extracellular matrix that encapsulated bacteria onto the plant cell wall. The peroxidase may have a dual function in both the generation and utilization of H2O2 for cross‐linking of proteins and phenolics during the construction of papillae. A burst of H2O2 was detected 1–5 h after inoculation at reaction sites by histochemical staining with cerium chloride. Progressive expansion of papillae and cell‐wall alterations was, however, not associated with the maintenance of high levels of H2O2. Co‐localization of callose and anMr 65 000 polypeptide component of callose synthase was also demonstrated. Synthesis of callose appeared so rapid that the enzyme became embedded in the polysaccharide so that both were detected as integral to the developing papilla. Localized alterations to the cell wall and deposition of papillae were found to involve co‐ordinated synthetic and oxidative activities at microsites within responding cells, without activation of the hypersensitive reaction.