bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Role of Pyruvate Dehydrogenase and Acetyl-Coenzyme A Synthetase in Fatty Acid Synthesis in Developing Arabidopsis Seeds
Authors: Ke Jinshan , Behal Robert H. , Back Stephanie L. , Nikolau Basil J. , Wurtele Eve Syrkin , Oliver David J. 
Published: July 2002

Abstract
Acetyl-coenzyme A (acetyl-CoA) formed within the plastid is the precursor for the biosynthesis of fatty acids and, through them, a range of important biomolecules. The source of acetyl-CoA in the plastid is not known, but two enzymes are thought to be involved: acetyl-CoA synthetase and plastidic pyruvate dehydrogenase. To determine the importance of these two enzymes in synthesizing acetyl-CoA during lipid accumulation in developing Arabidopsis seeds, we isolated cDNA clones for acetyl-CoA synthetase and for the ptE1α- and ptE1β-subunits of plastidic pyruvate dehydrogenase. To our knowledge, this is the first reported acetyl-CoA synthetase sequence from a plant source. The Arabidopsis acetyl-CoA synthetase preprotein has a calculated mass of 76,678 D, an apparent plastid targeting sequence, and the mature protein is a monomer of 70 to 72 kD. During silique development, the spatial and temporal patterns of the ptE1β mRNA level are very similar to those of the mRNAs for the plastidic heteromeric acetyl-CoA carboxylase subunits. The pattern of ptE1β mRNA accumulation strongly correlates with the formation of lipid within the developing embryo. In contrast, the level of mRNA for acetyl-CoA synthetase does not correlate in time and space with lipid accumulation. The highest level of accumulation of the mRNA for acetyl-CoA synthetase during silique development is within the funiculus. These mRNA data suggest a predominant role for plastidic pyruvate dehydrogenase in acetyl-CoA formation during lipid synthesis in seeds.
At-ACA8 Encodes a Plasma Membrane-Localized Calcium-ATPase of Arabidopsis with a Calmodulin-Binding Domain at the N Terminus
Authors: Bonza Maria Cristina , Morandini Piero , Luoni Laura , Geisler Markus , Palmgren Michael Gjedde , De Michelis Maria Ida 
Published: July 2002

AbstractA Ca2+-ATPase was purified from plasma membranes (PM) isolated from Arabidopsis cultured cells by calmodulin (CaM)-affinity chromatography. Three tryptic fragments from the protein were microsequenced and the corresponding cDNA was amplified by polymerase chain reaction using primers designed from the microsequences of the tryptic fragments. At-ACA8 (Arabidopsis-autoinhibited Ca2+-ATPase, isoform 8, accession no. AJ249352) encodes a 1,074 amino acid protein with 10 putative transmembrane domains, which contains all of the characteristic motifs of Ca2+-transporting P-type Ca2+-ATPases. The identity of At-ACA8p as the PM Ca2+-ATPase was confirmed by immunodetection with an antiserum raised against a sequence (valine-17 through threonine-31) that is not found in other plant CaM-stimulated Ca2+-ATPases. Confocal fluorescence microscopy of protoplasts immunodecorated with the same antiserum confirmed the PM localization of At-ACA8.At-ACA8 is the first plant PM localized Ca2+-ATPase to be cloned and is clearly distinct from animal PM Ca2+-ATPases due to the localization of its CaM-binding domain. CaM overlay assays localized the CaM-binding domain of At-ACA8p to a region of the N terminus of the enzyme around tryptophan-47, in contrast to a C-terminal localization for its animal counterparts. Comparison between the sequence ofAt-ACA8p and those of endomembrane-localized type IIB Ca2+-ATPases of plants suggests that At-ACA8 is a representative of a new subfamily of plant type IIB Ca2+-ATPases.
A Maize Vacuolar Invertase, IVR2, Is Induced by Water Stress. Organ/Tissue Specificity and Diurnal Modulation of Expression
Authors: Kim Jae-Yean , Mahé Aline , Brangeon Judy , Prioul Jean-Louis 
Published: July 2002

Abstract
The expression of invertases was analyzed in vegetative organs of well-watered and water-stressed maize (Zea mays) plants. Early changes in sucrose metabolism and in acid soluble invertase expression were observed in vegetative sink and source organs under mild water stress. The organ-specific induction of acid invertase activity was correlated with an increase in theIvr2 gene transcripts and in the vacuolar invertase proteins. In addition diurnal changes in activity andIvr2 transcripts for vacuolar invertase were noted in shoots. Hexoses (glucose and fructose) accumulated in all organs examined from water-stressed plants. In situ localization studies showed that glucose accumulation, vacuolar invertase activity, invertase protein, and the Ivr2 transcripts colocalized specifically in bundle sheath and vascular tissue cells of mature stressed leaf; in primary roots the stress-induced increase ofIvr2 transcripts was detected only in root tips. Based on these results different regulatory roles are proposed in sink and source organs for the stress induced Ivr2 vacuolar invertase.
Strategies for the Allocation of Resources under Sulfur Limitation in the Green Alga Dunaliella salina
Authors: Giordano Mario , Pezzoni Valerio , Hell Rüdiger 
Published: July 2002

Abstract
The effect of sulfur limitation on the partitioning of carbon, nitrogen, and sulfur was investigated in Dunaliella salina. D. salina was able to adapt to 6 μm sulfate; under these conditions, the cells showed reduced growth and photosynthetic rates. Whereas intracellular sulfate was depleted, phosphate, nitrate, and ammonium increased. Amino acids showed a general increase, and alanine became the most abundant amino acid. The activities of four key enzymes of carbon, sulfur, and nitrogen metabolism were differentially regulated: Adenosine 5′ triphosphate sulfurylase activity increased 4-fold, nitrate reductase and phosphoenolpyruvate (PEP) carboxylase activities decreased 4- and 11-fold, respectively, whereas carbonic anhydrase activity remained unchanged. Sulfur limitation elicited specific increase or decrease of the abundance of several proteins, such us Rubisco, PEP carboxylase, and a light harvesting complex protein. The accumulation of potentially toxic ammonium indicates an insufficient availability of carbon skeletons. Sulfur deficiency thus induces an imbalance between carbon and nitrogen. The dramatic reduction in PEP carboxylase activity suggests that carbon was diverted away from anaplerosis and possibly channeled into C3 metabolism. These results indicate that it is the coordination of key steps and components of carbon, nitrogen, and sulfur metabolism that allows D. salina to adapt to prolonged sulfur limitation.
Expression of an Expansin Is Associated with Endosperm Weakening during Tomato Seed Germination
Authors: Chen Feng , Bradford Kent J. 
Published: July 2002

AbstractExpansins are extracellular proteins that facilitate cell wall extension, possibly by disrupting hydrogen bonding between hemicellulosic wall components and cellulose microfibrils. In addition, some expansins are expressed in non-growing tissues such as ripening fruits, where they may contribute to cell wall disassembly associated with tissue softening. We have identified at least three expansin genes that are expressed in tomato (Lycopersicon esculentumMill.) seeds during germination. Among these, LeEXP4mRNA is specifically localized to the micropylar endosperm cap region, suggesting that the protein might contribute to tissue weakening that is required for radicle emergence. In gibberellin (GA)-deficient (gib-1) mutant seeds, which germinate only in the presence of exogenous GA, GA induces the expression ofLeEXP4 within 12 hours of imbibition. Whengib-1 seeds were imbibed in GA solution combined with 100 μm abscisic acid, the expression ofLeEXP4 was not reduced, although radicle emergence was inhibited. In wild-type seeds, LeEXP4 mRNA accumulation was blocked by far-red light and decreased by low water potential but was not affected by abscisic acid. The presence ofLeEXP4 mRNA during seed germination parallels endosperm cap weakening determined by puncture force analysis. We hypothesize that LeEXP4 is involved in the regulation of seed germination by contributing to cell wall disassembly associated with endosperm cap weakening.
Specific Binding of vf14-3-3a Isoform to the Plasma Membrane H+-ATPase in Response to Blue Light and Fusicoccin in Guard Cells of Broad Bean
Authors: Emi Takashi , Kinoshita Toshinori , Shimazaki Ken-ichiro 
Published: July 2002

Abstract
The plasma membrane H+-ATPase is activated by blue light with concomitant binding of the 14-3-3 protein to the C terminus in guard cells. Because several isoforms of the 14-3-3 protein are expressed in plants, we determined which isoform(s) bound to the H+-ATPase in vivo. Four cDNA clones (vf14-3-3a, vf14-3-3b,vf14-3-3c, and vf14-3-3d) encoding 14-3-3 proteins were isolated from broad bean (Vicia faba) guard cells. Northern analysis revealed that mRNAs encoding vf14-3-3a and vf14-3-3b proteins were expressed predominantly in guard cells. The 14-3-3 protein that bound to the H+-ATPase in guard cells had the same molecular mass as the recombinant vf14-3-3a protein. The H+-ATPase immunoprecipitated from mesophyll cell protoplasts, which had been stimulated by fusicoccin, coprecipitated with the 32.5-kD 14-3-3 protein, although three 14-3-3 isoproteins were found in mesophyll cell protoplasts. Digestions of the bound 14-3-3 protein and recombinant vf14-3-3a with cyanogen bromide gave the identical migration profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but that of vf14-3-3b gave a different profile. Mass profiling of trypsin-digested 14-3-3 protein bound to the H+-ATPase gave the predicted peptide masses of vf14-3-3a. Far western analysis revealed that the H+-ATPase had a higher affinity for vf14-3-3a than for vf14-3-3b. These results suggest that the 14-3-3 protein that bound to the plasma membrane H+-ATPase in vivo is vf14-3-3a and that it may play a key role in the activation of H+-ATPase in guard cells.
Mapping Maize Sequences to Chromosomes Using Oat-Maize Chromosome Addition Materials,
Authors: Okagaki Ron J. , Kynast Ralf G. , Livingston Suzanne M. , Russell Charles D. , Rines Howard W. , Phillips Ronald L. 
Published: July 2002

Abstract
Oat- (Avena sativa) maize (Zea mays) chromosome additions are produced by crossing maize and oat. During early embryo development maize chromosomes are preferentially eliminated, and oat plants are often recovered that retain a single maize chromosome. Each of the 10 maize chromosomes recently has been isolated as a separate oat-maize addition. We describe here the mapping of 400 maize sequences to chromosomes using polymerase chain reaction and DNA from the oat-maize addition material. Fifty of the sequences were from cloned markers that had been previously mapped by linkage analysis, and our results were consistent with those obtained using Southern-blot analysis. Previously unmapped expressed sequence tags and sequence tagged sites (350) were mapped to chromosomes. Maize gene sequences and expression data are rapidly being accumulated. Coupling this information with positional information from high throughput mapping programs provides plant biologists powerful tools for identifying candidate genes of interest.
Xylem Cavitation in the Leaf of Prunus laurocerasusand Its Impact on Leaf Hydraulics
Authors: Nardini Andrea , Tyree Melvin T. , Salleo Sebastiano 
Published: July 2002

Abstract
This paper reports how water stress correlates with changes in hydraulic conductivity of stems, leaf midrib, and whole leaves ofPrunus laurocerasus. Water stress caused cavitation-induced dysfunction in vessels of P. laurocerasus. Cavitation was detected acoustically by counts of ultrasonic acoustic emissions and by the loss of hydraulic conductivity measured by a vacuum chamber method. Stems and midribs were approximately equally vulnerable to cavitations. Although midribs suffered a 70% loss of hydraulic conductance at leaf water potentials of −1.5 MPa, there was less than a 10% loss of hydraulic conductance in whole leaves. Cutting and sealing the midrib 20 mm from the leaf base caused only a 30% loss of conduction of the whole leaf. A high-pressure flow meter was used to measure conductance of whole leaves and as the leaf was progressively cut back from tip to base. These data were fitted to a model of hydraulic conductance of leaves that explained the above results, i.e. redundancy in hydraulic pathways whereby water can flow around embolized regions in the leaf, makes whole leaves relatively insensitive to significant changes in conductance of the midrib. The onset of cavitation events in P. laurocerasus leaves correlated with the onset of stomatal closure as found recently in studies of other species in our laboratory.
Brassicaceae Express Multiple Isoforms of Biotin Carboxyl Carrier Protein in a Tissue-Specific Manner
Authors: Thelen Jay J. , Mekhedov Sergei , Ohlrogge John B. 
Published: July 2002

Abstract
Plastidial acetyl-coenzyme A carboxylase from most plants is a multi-enzyme complex comprised of four different subunits. One of these subunits, the biotin carboxyl carrier protein (BCCP), was previously proposed to be encoded by a single gene in Arabidopsis. We report and characterize here a second Arabidopsis BCCP (AtBCCP2) cDNA with 42% amino acid identity to AtBCCP1 and 75% identity to a class of oilseed rape (Brassica napus) BCCPs. Both Arabidopsis BCCP isoforms were expressed in Escherichia coli and found to be biotinylated and supported carboxylation activity when reconstituted with purified, recombinant Arabidopsis biotin carboxylase. In vitro translated AtBCCP2 was competent for import into pea (Pisum sativum) chloroplasts and processed to a 25-kD polypeptide. Extracts of Arabidopsis seeds contained biotinylated polypeptides of 35 and 25 kD, in agreement with the masses of recombinant AtBCCP1 and 2, respectively. AtBCCP1 protein was present in developing tissues from roots, leaves, flowers, siliques, and seeds, whereas AtBCCP2 protein was primarily expressed in 7 to 10 d-after-flowering seeds at levels approximately 2-fold less abundant than AtBCCP1. AtBCCP1 transcript reflected these protein expression profiles present in all developing organs and highest in 14-d leaves and siliques, whereas AtBCCP2 transcript was present in flowers and siliques. In protein blots, four different BCCP isoforms were detected in developing seeds from oilseed rape. Of these, a 35-kD BCCP was detected in immature leaves and developing seeds, whereas developing seeds also contained 22-, 25-, and 37-kD isoforms highly expressed 21 d after flowering. These data indicate that oilseed plants in the familyBrassicaceae contain at least one to three seed-up-regulated BCCP isoforms, depending upon genome complexity.
Differential Expression of 1-Aminocyclopropane-1-Carboxylate Synthase Genes during Orchid Flower Senescence Induced by the Protein Phosphatase Inhibitor Okadaic Acid
Authors: Wang Ning Ning , Yang Shang Fa , Charng Yee-yung 
Published: July 2002

Abstract
Applying 10 pmol of okadaic acid (OA), a specific inhibitor of type 1 or type 2A serine/threonine protein phosphatases, to the orchid (Phalaenopsis species) stigma induced a dramatic increase in ethylene production and an accelerated senescence of the whole flower. Aminoethoxyvinylglycine or silver thiosulfate, inhibitors of ethylene biosynthesis or action, respectively, effectively inhibited the OA-induced ethylene production and retarded flower senescence, suggesting that the protein phosphatase inhibitor induced orchid flower senescence through an ethylene-mediated signaling pathway. OA treatment induced a differential expression pattern for the 1-aminocyclopropane-1-carboxylic acid synthase multigene family. Accumulation of Phal-ACS1 transcript in the stigma, labelum, and ovary induced by OA were higher than those induced by pollination as determined by “semiquantitative” reverse transcriptase-polymerase chain reaction. In contrast, the transcript levels of Phal-ACS2 and Phal-ACS3 induced by OA were much lower than those induced by pollination. Staurosporine, a protein kinase inhibitor, on the other hand, inhibited the OA-inducedPhal-ACS1 expression in the stigma and delayed flower senescence. Our results suggest that a hyper-phosphorylation status of an unidentified protein(s) is involved in up-regulating the expression of Phal-ACS1 gene resulting in increased ethylene production and accelerated the senescence process of orchid flower.
Rapid Accumulation of Phosphatidylinositol 4,5-Bisphosphate and Inositol 1,4,5-Trisphosphate Correlates with Calcium Mobilization in Salt-Stressed Arabidopsis
Authors: DeWald Daryll B. , Torabinejad Javad , Jones Christopher A. , Shope Joseph C. , Cangelosi Amanda R. , Thompson James E. , Prestwich Glenn D. , Hama Hiroko 
Published: July 2002

Abstract
The phosphoinositide phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is a key signaling molecule in animal cells. It can be hydrolyzed to release 1,2-diacyglycerol and inositol 1,4,5-trisphosphate (IP3), which in animal cells lead to protein kinase C activation and cellular calcium mobilization, respectively. In addition to its critical roles in constitutive and regulated secretion of proteins, PtdIns(4,5)P2 binds to proteins that modify cytoskeletal architecture and phospholipid constituents. Herein, we report that Arabidopsis plants grown in liquid media rapidly increase PtdIns(4,5)P2 synthesis in response to treatment with sodium chloride, potassium chloride, and sorbitol. These results demonstrate that when challenged with salinity and osmotic stress, terrestrial plants respond differently than algae, yeasts, and animal cells that accumulate different species of phosphoinositides. We also show data demonstrating that whole-plant IP3 levels increase significantly within 1 min of stress initiation, and that IP3 levels continue to increase for more than 30 min during stress application. Furthermore, using the calcium indicators Fura-2 and Fluo-3 we show that root intracellular calcium concentrations increase in response to stress treatments. Taken together, these results suggest that in response to salt and osmotic stress, Arabidopsis uses a signaling pathway in which a small but significant portion of PtdIns(4,5)P2 is hydrolyzed to IP3. The accumulation of IP3 occurs during a time frame similar to that observed for stress-induced calcium mobilization. These data also suggest that the majority of the PtdIns(4,5)P2 synthesized in response to salt and osmotic stress may be utilized for cellular signaling events distinct from the canonical IP3 signaling pathway.
Testing the role of chain connectivity on the stability and structure of dihydrofolate reductase fromE. coli: Fragment complementation and circular permutation reveal stable, alternatively folded forms
Authors: Smith Virginia F. , Matthews C. Robert 
Published: July 2002

AbstractThe effects of chain cleavage and circular permutation on the structure, stability, and activity of dihydrofolate reductase (DHFR) fromEscherichia coliwere investigated by various spectroscopic and biochemical methods. Cleavage of the backbone after position 86 resulted in two fragments, {1–86} and {87–159}, each of which are poorly structured and enzymatically inactive. When combined in a 1 : 1 molar ratio, however, the fragments formed a high‐affinity (Ka= 2.6 × 107M−1) complex that displays a weakly cooperative urea‐induced unfolding transition at micromolar concentrations. The retention of about 15% of the enzymatic activity of full‐length DHFR is surprising, considering that the secondary structure in the complex is substantially reduced from its wild‐type counterpart. In contrast, a circularly permuted form with its N‐terminus at position 86 has similar overall stability to full‐length DHFR, about 50% of its activity, substantial secondary structure, altered side‐chain packing in the adenosine binding domain, and unfolds via an equilibrium intermediate not observed in the wild‐type protein. After addition of ligand or the tight‐binding inhibitor methotrexate, both the fragment complex and the circular permutant adopt more native‐like secondary and tertiary structures. These results show that changes in the backbone connectivity can produce alternatively folded forms and highlight the importance of protein‐ligand interactions in stabilizing the active site architecture of DHFR.
Disulfide bond effects on protein stability: Designed variants of Cucurbita maxima trypsin inhibitor‐V
Authors: Zavodszky Maria , Chen Chao‐Wei , Huang Jenq‐Kuen , Zolkiewski Michal , Wen Lisa , Krishnamoorthi Ramaswamy 
Published: July 2002

AbstractAttempts to increase protein stability by insertion of novel disulfide bonds have not always been successful. According to the two current models, cross‐links enhance stability mainly through denatured state effects. We have investigated the effects of removal and addition of disulfide cross‐links, protein flexibility in the vicinity of a cross‐link, and disulfide loop size on the stability of Cucurbita maxima trypsin inhibitor‐V (CMTI‐V; 7 kD) by differential scanning calorimetry. CMTI‐V offers the advantage of a large, flexible, and solvent‐exposed loop not involved in extensive intra‐molecular interactions. We have uncovered a negative correlation between retention time in hydrophobic column chromatography, a measure of protein hydrophobicity, and melting temperature (Tm), an indicator of native state stabilization, for CMTI‐V and its variants. In conjunction with the complete set of thermodynamic parameters of denaturation, this has led to the following deductions: (1) In the less stable, disulfide‐removed C3S/C48S (ΔΔGd50°C = −4 kcal/mole; ΔTm = −22°C), the native state is destabilized more than the denatured state; this also applies to the less‐stable CMTI‐V* (ΔΔGd50°C = −3 kcal/mole; ΔTm = −11°C), in which the disulfide‐containing loop is opened by specific hydrolysis of the Lys44‐Asp45 peptide bond; (2) In the less stable, disulfide‐inserted E38C/W54C (ΔΔGd50°C = −1 kcal/mole; ΔTm = +2°C), the denatured state is more stabilized than the native state; and (3) In the more stable, disulfide‐engineered V42C/R52C (ΔΔGd50°C = +1 kcal/mole; ΔTm = +17°C), the native state is more stabilized than the denatured state. These results show that a cross‐link stabilizes both native and denatured states, and differential stabilization of the two states causes either loss or gain in protein stability. Removal of hydrogen bonds in the same flexible region of CMTI‐V resulted in less destabilization despite larger changes in the enthalpy and entropy of denaturation. The effect of a cross‐link on the denatured state of CMTI‐V was estimated directly by means of a four‐state thermodynamic cycle consisting of native and denatured states of CMTI‐V and CMTI‐V*. Overall, the results show that an enthalpy‐entropy compensation accompanies disulfide bond effects and protein stabilization is profoundly modulated by altered hydrophobicity of both native and denatured states, altered flexibility near the cross‐link, and residual structure in the denatured state.
A “loop entropy reduction” phage‐display selection for folded amino acid sequences
Authors: Minard Philippe , Scalley‐Kim Michelle , Watters Alex , Baker David 
Published: July 2002

AbstractAs a step toward selecting folded proteins from libraries of randomized sequences, we have designed a ‘loop entropy reduction’‐based phage‐display method. The basic premise is that insertion of a long disordered sequence into a loop of a host protein will substantially destabilize the host because of the entropic cost of closing a loop in a disordered chain. If the inserted sequence spontaneously folds into a stable structure with the N and C termini close in space, however, this entropic cost is diminished. The host protein function can, therefore, be used to select folded inserted sequences without relying on specific properties of the inserted sequence. This principle is tested using the IgG binding domain of protein L and the lck SH2 domain as host proteins. The results indicate that the loop entropy reduction screen is capable of discriminating folded from unfolded sequences when the proper host protein and insertion point are chosen.