bluuebunny/crossref_metadata_2025_split huggingface dataset:
An anion current at the plasma membrane of tobacco protoplasts shows ATP‐dependent voltage regulation and is modulated by auxin
Authors: Zimmermann Sabine , Thomine Sébastien , Guern Jean , Barbier‐Brygoo Hélène 
Published: March 2003

SummaryPlasma membrane ion channels of protoplasts from tobacco cell suspensions were characterized by patch‐clamp experiments. In the whole‐cell configuration, a voltage‐dependent current with a current maximum around −90 mV was observed that displayed a reversal potential close to the Nernst potential for chloride. This whole‐cell current was identified as an anion current by replacing the internal Cl− with glutamate. The tobacco suspension anion channel (TSAC) was characterized by fast activation/deactivation and slow inactivation kinetics with voltage‐dependent time constants in the range of milliseconds and seconds, respectively. Among the plant channels, TSAC exhibits original properties in terms of phosphorylation‐dependent voltage regulation while sharing similarities with the fast anion channel from stomatal guard cells (GCAC1). The voltage dependence of the whole‐cell current reflecting the fast deactivation of the current at potentials of less than −100 mV was observed only in the presence of internal ATP or when ATP was replaced by the protein phosphatase inhibitor okadaic acid, and was suppressed by staurosporine. This suggests that protein phosphorylation may be involved in regulating the activity of the anion channel. As observed on GCAC1 the active auxin 1‐NAA caused a time‐ and concentration‐dependent shift of the activation potential of TSAC. In addition, TSAC reacted to the auxin agonist antibody D16 providing evidence for the recognition of the auxin signal at the outer face of the plasma membrane.
The forever young gene encodes an oxidoreductase required for proper development of the Arabidopsis vegetative shoot apex
Authors: Callos Joseph D. , DiRado Marion , Xu Bibo , Behringer Friedrich J. , Link Bruce M. , Medford June I. 
Published: March 2003

SummaryIn plant development, leaf primordia are formed on the flanks of the shoot apical meristem in a highly predictable pattern. The cells that give rise to a primordium are sequestered from the apical meristem. Maintenance of the meristem requires that these cells be replaced by the addition of new cells. Despite the central role of these activities in development, the mechanism controlling and coordinating them is poorly understood. These processes have been characterized in the Arabidopsis mutant forever young (fey). The fey mutation results in a disruption of leaf positioning and meristem maintenance. The predicted FEY protein shares significant homology to a nodulin and limited homology to various reductases. It is proposed that FEY plays a role in communication in the shoot apex through the modification of a factor regulating meristem development.
Morphogenesis in pinoid mutants of Arabidopsis thaliana
Authors: Bennett Sally R.M. , Alvarez John , Bossinger Gerd , Smyth David R. 
Published: March 2003

SummaryA series of mutants of Arabidopsis thaliana was selected in which the inflorescence stem elongates but loses the ability to produce flower primordia on its flanks. Mutants fell into two classes, further occurrences of pin‐formed mutants and mutations at a new locus named pinoid. As well as causing inflorescence defects, pinoid mutations result in pleiotropic defects in the development of floral organs, cotyledons and leaves. Most changes involve the number of organs produced rather than their differentiation suggesting that PINOID controls an early general step in meristem development. pinoid mutant defects are similar to those seen in pin‐formed mutants for inflorescences and flowers, but different for cotyledons and leaves indicating that the two genes have separate but overlapping functions. A defect in polar auxin transport is implicated in the pin‐formed mutant phenotype, but in young inflorescence stems of even the strongest pinoid mutants it occurs at close to wild‐type levels. It is markedly reduced only after stems have ceased elongating. Thus, it is likely that polar auxin transport is secondarily affected in pinoid mutants rather than being directly controlled by the PINOID gene product. Even so, double mutant studies indicate that the process controlled by PINOID overlaps with that specified by the AUXIN RESISTANT1 gene, suggesting that PINOID plays some role in an auxin‐related process.
Calcium and cGMP target distinct phytochrome‐responsive elements
Authors: Wu Yan , Hiratsuka Kazuyuki , Neuhaus Gunther , Chua Nam‐Hai 
Published: March 2003

SummaryPrevious work using microinjection into single cells of the tomato aurea mutant demonstrated that phytochrome A‐dependent activation of rbcS and chs genes was mediated by calcium and cGMP, respectively. This work sought to identify promoter cis‐elements that respond to these two small molecules. Box II and Unit I, derived from rbcS‐3A and chs promoters, respectively, were previously shown to function as light‐responsive cis‐elements. Eleven copies of Box II and four copies of Unit I were linked 5′ to the −90 and −46 35 S promoters, respectively, and, both constructs were fused to the β‐glucuronidase (GUS) reporter gene. GUS activities were obtained upon co‐injection of either Box II/‐90GUS or Unit I/‐46GUS with oat phytochrome A (phyA) and GTPγS, an activator of heterotrimeric G proteins. The activation of Box II/‐90GUS by phyA was insensitive to the cGMP antagonist, Rp‐cGMPS, although anthocyanin accumulation, but not chloroplast development, was totally blocked in the injected cells. Consistent with this result, calcium, but not cGMP, induced Box II/‐90GUS activity. In contrast to Box II/‐90GUS, phyA‐dependent activation of Unit I/‐46GUS activity was blocked by Rp‐cGMPS. Moreover, cGMP, not calcium, induced Unit I/‐46GUS activity. Control experiments showed that −90 GUS and −46 GUS were inactive in the presence of calcium and cGMP, respectively. These results provide evidence that Box II and Unit I are targets of the calcium and cGMP pathways, respectively. Interestingly, calcium activation of Box II/‐90GUS was repressed by a high concentration of cGMP and cGMP induction of Unit I/‐46GUS was blocked by a high concentration of calcium/CaM. Thus, these two small cis‐elements can also serve as targets of the reciprocal control mechanisms that operate to regulate the activities of the two phyA signaling branches.
PRK1, a receptor‐like kinase of Petunia inflata, is essential for postmeiotic development of pollen
Authors: Lee Hyun‐Sook , Karunanandaa Balasulojini , McCubbin Andrew , Gilroy Simon , Kao Teh‐hui 
Published: March 2003

SummaryA pollen‐expressed gene of Petunia inflata that encodes a receptor‐like kinase named PRK1 was previously identified. The extracellular domain of PRK1 contains leucine‐rich repeats which have been implicated in protein—protein interactions, and the cytoplasmic domain was found to autophosphorylate on serine and possibly tyrosine. To investigate the function of PRK1 in pollen development, P. inflata plants were transformed with a construct containing the promoter of a pollen‐expressed gene of tomato, LAT52, fused to an antisense PRK1 cDNA corresponding to part of the extracellular domain of PRK1. Three transgenic plants were found to each produce approximately equal amounts of normal and aborted pollen. Analysis of the inheritance of the transgene inserts in two of the transgenic plants, ASRK‐13 and ASRK‐20, to their progeny revealed that certain transgene inserts co‐segregated with the pollen abortion phenotype. Microscopic examination of the aborted pollen grains showed that their outer wall, the exine, was essentially normal, but that their cytoplasm contained only starch‐like granules. Staining of the nuclei of the microspores at different stages of anther development revealed that the microspores of the transgenic plants developed normally until the uninucleate stage. However, in subsequent stages half of the microspores completed mitosis and developed into normal binucleate pollen, but the other half initially remained uninucleate and subsequently lost their nuclei. Analysis of the amounts of PRK1 mRNA and the antisense PRK1 transcript suggested that the pollen abortion phenotype most likely resulted from downregulation of the PRK1 gene by the antisense PRK1 transgene. These results suggest that PRK1 plays an essential role in a signal transduction pathway that mediates postmeiotic development of microspores.
Spatial expression dynamics of Men‐9 delineate the third floral whorl in male and female flowers of dioecious Silene latifolia
Authors: Robertson Shona E. , Li Yi , Scutt Charles P. , Willis Malcolm E. , Gilmartin Philip M. 
Published: March 2003

SummarySex determination in Silene latifolia is controlled by heteromorphic sex chromosomes. Female flowers have five fused carpels and ten arrested stamen primordia. The male‐determining Y chromosome overrides female development to suppress carpel formation and promote stamen development. The isolation and characterization of two S. latifoliaMale enhanced cDNAs, Men‐9a and Men‐9b, which probably represent different alleles of a novel gene are reported here. Men‐9a and Men‐9b share 91.8% coding sequence nucleotide identity, yet only 85.4% amino acid identity. The Men‐9 cDNAs are related to the previously reported MROS3 cDNA from S. latifolia. However, MROS3 is not present in the S. latifolia population used in these studies and the expression dynamics of Men‐9a and Men‐9b contrast dramatically with those reported for MROS3. Men‐9 cDNAs are expressed primarily in anthers of young male flowers, with highest expression in 1–2 mm buds. Men‐9 expression is also observed at a low level in female flowers. In situ hybridization analysis reveals two phases of Men‐9 expression. The first phase is during a common stage of early stamen development in male and female flowers prior to stamen arrest in female flowers. The second phase of Men‐9 expression is maximal in the epidermis and endothecium of Y chromosome‐ and Ustilago violacea‐induced stamens; expression in male and female flowers extends to the epidermis of the staminal nectaries with strict boundaries at the second and fourth whorls. Men‐9 gene expression therefore delineates the boundaries of the third floral whorl in S. latifolia flowers.
A nuclear gene, erd1, encoding a chloroplast‐targeted Clp protease regulatory subunit homolog is not only induced by water stress but also developmentally up‐regulated during senescence in Arabidopsis thaliana
Authors: Nakashima Kazuo , Kiyosue Tomohiro , Yamaguchi‐Shinozaki Kazuko , Shinozaki Kazuo 
Published: March 2003

SummaryA cDNA, ERD1, isolated from one‐hour‐dehydrated plants of Arabidopsis thaliana L. encodes a putative protein that is similar to the regulatory ATPase subunit (ClpA) of the Clp protease and contains a putative chloroplast‐targeting transit‐peptide at the N‐terminus. A chimeric gene with the putative plastid‐targeting sequence of the erd1 gene fused to the synthetic green‐fluorescent protein (sGFP) gene was constructed and introduced into Arabidopsis protoplasts. The N‐terminal region of the ERD1 protein directed the sGFP protein into the plastids of the protoplasts, and functioned as a transit peptide. Northern blot analysis indicated that expression of the erd1 gene was induced not only by water stress, such as dehydration and high salinity, but also by natural senescence and dark‐induced etiolation. The erd1 gene was not strongly induced by exogenous abscisic acid. A chimeric gene with the 0.9 kb promoter region of the erd1 gene fused to the β‐glucuronidase (GUS) reporter gene was constructed, and tobacco plants transformed with the construct. The GUS reporter gene driven by the erd1 promoter was induced by dehydration and high salt stress at significant levels in the transgenic plants. The GUS gene was strongly expressed in older leaves without dehydration, and was induced by dark‐induced etiolation. Furthermore, GUS activity was reduced by cytokinin treatment during dark‐induced etiolation. These results indicate that expression of the erd1 gene is developmentally up‐regulated by senescence as well as by water stress.
Mg‐chelatase of tobacco: identification of a Chl D cDNA sequence encoding a third subunit, analysis of the interaction of the three subunits with the yeast two‐hybrid system, and reconstitution of the enzyme activity by co‐expression of recombinant CHL D, CHL H and CHL I
Authors: Papenbrock Jutta , Gräfe Susanna , Kruse Elisabeth , Hänel Frank , Grimm Bernhard 
Published: March 2003

SummaryMg‐protoporphyrin IX chelatase catalyzes insertion of the magnesium ion into protoporphyrin IX, the last common intermediate precursor in chlorophyll and heme biosynthesis, to form Mg‐protoporphyrin IX. In Rhodobacter sphaeroides, and Synechocystis, the three open reading frames bchD/chlD, bchH/chlH and bchl/chll encode proteins which are required for in vitro Mg‐chelatase activity. In higher plants also, three proteins are necessary for the Mg chelation, and genes homologous to bchH and bchl have been isolated previously. In this study, a novel tobacco cDNA sequence homologous to bchD is isolated and initially characterized. Together with the tobacco clones encoding the other two subunits, full‐length cDNAs are now available for the first time for all three subunits of one plant species. The CHL D polypeptide deduced from the open reading frame encodes a protein of 758 aa (82.9 kDa) with an amino terminal extension that resembles a plastid transit peptide. Sequence comparison of tobacco CHL D revealed similarities to the D subunit of Rhodobacter and Synechocystis of 44% and 75%. The amino terminal half of CHL D shows significant similarity (46%) to the entire CHL I peptide sequence, indicating a gene duplication from an ancestral gene. The carboxy terminal half seemed to be unique. Both parts of CHL D are linked with a glutamine/asparagine/proline‐rich region flanked by a highly acid‐rich segment. Protein‐protein interaction among the three subunits CHL D, H and I was studied using the yeast two‐hybrid system. Physical interaction was demonstrated between CHL D and CHL I indicating that CHL D is part of the Mg‐chelatase. Heterodimer formation of CHL H with CHL I or CHL D could not be demonstrated by transactivation of the lacZ reporter gene. Homodimerization of the CHL D subunit was indicated in the more sensitive assay on X‐Gal‐containing agar plates. In vitro Mg2+ insertion into protoporphyrin IX was demonstrated in protein extracts of yeast strains expressing the three subunits of tobacco Mg‐chelatase. The reconstitution of the recombinant enzyme activity required additional ATP.
Compromising early salicylic acid accumulation delays the hypersensitive response and increases viral dispersal during lesion establishment in TMV‐infected tobacco
Authors: Mur Luis A.J. , Bi Yong‐Mei , Darby Robert M. , Firek Simon , Draper John 
Published: March 2003

SummaryTo investigate the role of salicylic acid (SA) in the hypersensitive response (HR) its accumulation was compromised during different phases of lesion development by differential expression of a salicylate hydroxylase gene (SH‐L). Constitutive suppression of SA accumulation was achieved by expression of a gene fusion between the CaMV35S promoter (35S) and SH‐L. Using the H2O2‐responsive AoPR1 promoter to drive SH‐L SA accumulation could be compromised at an early stage, on lesion formation and possibly prior to visible necrosis, whilst use of the salicylate‐responsive PR1a promoter reduced SA accumulation at a later stage as lesions expand. TMV infection of 35S‐SH‐L and AoPR1‐SH‐L, but not PR1a‐SH‐L, tobacco resulted in significantly greater rates of lesion growth than in wild‐type tobacco. TMV was detected in asymptomatic tissue surrounding lesions only in 35S‐SH‐L and AoPR1‐SH‐L lines; subsequently these transgenic lines exhibited a ‘spreading‐necrosis’ originating from the lesion which entered the stem and eventually other leaves, a phenotype which could be correlated with the presence of TMV particles. Analysis of TMV‐infected and ‘temperature‐shifted’ tobacco indicated that both 35S‐SH‐L and AoPR1‐SH‐L, but not PR1a‐SH‐L, transgenics exhibited delayed cell‐death compared to wild‐type infections. We propose that the SH‐L phenotypes indicate that early SA accumulation is a major factor in preventing viral escape, via mechanism(s) which may include influencing the rate of host‐cell death and, possibly, an effect on viral function.
Overexpressed phytochrome C has similar photosensory specificity to phytochrome B but a distinctive capacity to enhance primary leaf expansion
Authors: Qin Minmin , Kuhn Robert , Moran Sharon , Quail Peter H. 
Published: March 2003

SummaryPhytochrome C (phyC) is a low‐abundance member of the five‐membered phytochrome family of photoreceptors in Arabidopsis. Towards developing an understanding of the photosensory and physiological functions of phyC, transgenic Arabidopsis plants were generated that overexpress cDNA‐encoded phyC and seedling responses to continuous white, red, or far‐red light (Wc, Rc or FRc, respectively) were examined. Transgenic seedlings overexpressing phyC displayed enhanced inhibition of hypocotyl elongation in Rc, but were unchanged in responsiveness to FRc relative to wild‐type. These data indicate that phyC has photosensory specificity that is similar to that of phyB and thus distinct from that of phyA. phyC overexpressors with levels only 3 to 4 times the level of endogenous phyC exhibited enhanced primary leaf expansion in Wc. This is in contrast to phyA or phyB overexpressors which respectively have levels that are 500‐and 100‐fold that of overexpressed phyC but showed no enhancement of primary leaf expansion. Therefore, phyC may have some physiological roles that are different to those of phyA and phyB in the control of seedling responses to light signals.
In vitro and in vivo evidence for actin association of the naphthylphthalamic acid‐binding protein from zucchini hypocotyls
Authors: Butler John H. , Hu Shiquan , Brady Shari R. , Dixon Michael W. , Muday Gloria K. 
Published: March 2003

SummaryThe N‐1‐naphthylphthalamic acid (NPA)‐binding protein is part of the auxin efflux carrier, the protein complex that controls polar auxin transport in plant tissues. This study tested the hypothesis that the NPA‐binding protein (NBP) is associated with the actin cytoskeleton in vitro and that an intact actin cytoskeleton is required for polar auxin transport in vivo. Cytoskeletal polymerization was altered in extracts of zucchini hypocotyls with reagents that stabilized either the polymeric or monomeric forms of actin or tubulin. Phalloidin treatment altered actin polymerization, as demonstrated by immunoblot analyses following native and denaturing electrophoresis. Phalloidin increased both filamentous actin (F‐actin) and NPA‐binding activity, while cytochalasin D and Tris decreased both F‐actin and NPA‐binding activity in cytoskeletal pellets. The microtubule stabilizing drug taxol increased pelletable tubulin, but did not alter either the amount of pelletable actin or NPA‐binding activity. Treatment of etiolated zucchini hypocotyls with cytochalasin D decreased the amount of auxin transport and its regulation by NPA. These experimental results are consistent with an in vitro actin cytoskeletal association of the NPA‐binding protein and with the requirement of an intact actin cytoskeleton for maximal polar auxin transport in vivo.
Dual regulation of a heat shock promoter during embryogenesis: stage‐dependent role of heat shock elements
Authors: Almoguera Concepción , Prieto‐Dapena Pilar , Jordano Juan 
Published: March 2003

SummaryTransgenic tobacco expression was analysed of chimeric genes with point mutations in the heat shock element (HSE) arrays of a small heat shock protein (sHSP) gene from sunflower: Ha hsp17.7 G4. The promoter was developmentally regulated during zygotic embryogenesis and responded to heat stress in vegetative tissues. Mutations in the HSE affected nucleotides crucial for human heat shock transcription factor 1 (HSF1) binding. They abolished the heat shock response of Ha hsp17.7 G4 and produced expression changes that demonstrated dual regulation of this promoter during embryogenesis. Thus, whereas activation of the chimeric genes during early maturation stages did not require intact HSE, expression at later desiccation stages was reduced by mutations in both the proximal (–57 to –89) and distal (–99 to –121) HSE. In contrast, two point mutations in the proximal HSE that did not severely affect gene expression during zygotic embryogenesis, eliminated the heat shock response of the same chimeric gene in vegetative organs. Therefore, by site‐directed mutagenesis, it was possible to separate the heat shock response of Ha hsp17.7 G4 from its developmental regulation. The results indicate the co‐existence, in a single promoter, of HSF‐dependent and ‐independent regulation mechanisms that would control sHSP gene expression at different stages during plant embryogenesis.
Domains of the TMV movement protein involved in subcellular localization
Authors: Kahn Theodore W. , Lapidot Moshe , Heinlein Manfred , Reichel Christoph , Cooper Bret , Gafny Ron , Beachy Roger N. 
Published: March 2003

SummaryTo identify and map functionally important regions of the tobacco mosaic virus movement protein, deletions of three amino acids were introduced at intervals of 10 amino acids throughout the protein. Mutations located between amino acids 1 and 160 abolished the capacity of the protein to transport virus from cell to cell, while some of the mutations in the C‐terminal third of the protein permitted function. Despite extensive tests, no examples were found of intermolecular complementation between mutants, suggesting that function requires each movement protein molecule to be fully competent. Many of the mutants were fused to green fluorescent protein, and their subcellular localizations were determined by fluorescence microscopy in infected plants and protoplasts. Most mutants lost the ability to accumulate in one or more of the multiple subcellular sites targeted by wild‐type movement protein, suggesting that specific functional domains were disrupted. The order in which accumulation at subcellular sites occurs during infection does not represent a targeting pathway. Association of the movement protein with microtubules or with plasmodesmata can occur in the absence of other associations. The region of the protein around amino acids 9–11 may be involved in targeting the protein to cortical bodies (probably associated with the endoplasmic reticulum) and to plasmodesmata. The region around residues 49–51 may be involved in co‐alignment of the protein with microtubules. The region around residues 88–101 appears to play a role in targeting to both the cortical bodies and microtubules. Thus, the movement protein contains independently functional domains.