bluuebunny/crossref_metadata_2025_split huggingface dataset:
Female Reproductive Tissues Are the Primary Target ofAgrobacterium-Mediated Transformation by the Arabidopsis Floral-Dip Method
Authors: Desfeux Christine , Clough Steven J. , Bent Andrew F. 
Published: July 2002

Abstract
The floral-dip method for Agrobacterium-mediated transformation of Arabidopsis allows efficient plant transformation without need for tissue culture. To facilitate use with other plant species, we investigated the mechanisms that underlie this method. In manual outcrossing experiments, application of Agrobacterium tumefaciens to pollen donor plants did not produce any transformed progeny, whereas application ofAgrobacterium to pollen recipient plants yielded transformants at a rate of 0.48%. Agrobacterium strains with T-DNA carrying gusA (encoding β-glucuronidase [GUS]) under the control of 35S, LAT52, or ACT11 promoters revealed delivery of GUS activity to developing ovules, whereas no GUS staining of pollen or pollen tubes was observed. Transformants derived from the same seed pod contained independent T-DNA integration events. In Arabidopsis flowers, the gynoecium develops as an open, vase-like structure that fuses to form closed locules roughly 3 d prior to anthesis. In correlation with this fact, we found that the timing ofAgrobacterium infection was critical. Transformants were obtained and GUS staining of ovules and embryo sacs was observed only if the Agrobacterium were applied 5 d or more prior to anthesis. A 6-fold higher rate of transformation was obtained with a CRABS-CLAW mutant that maintains an open gynoecium. Our results suggest that ovules are the site of productive transformation in the floral-dip method, and further suggest that Agrobacterium must be delivered to the interior of the developing gynoecium prior to locule closure if efficient transformation is to be achieved.
Involvement of Polyamines in the Chilling Tolerance of Cucumber Cultivars
Authors: Shen Wenyun , Nada Kazuyoshi , Tachibana Shoji 
Published: July 2002

Abstract
The possible involvement of polyamines (PAs) in the chilling tolerance of cucumber (Cucumis sativus L. cv Jinchun No. 3 and cv Suyo) was investigated. Plants with the first expanded leaves were exposed to 3°C or 15°C in the dark for 24 h (chilling), and then transferred to 28°C/22°C under a 12-h photoperiod for another 24 h (rewarming). Chilling-tolerant cv Jinchun No. 3 showed a marked increase of free spermidine (Spd) in leaves, once during chilling and again during rewarming. Putrescine increased significantly during rewarming, but the increase of spermine was slight. Any of these PAs did not increase in chilling-sensitive cv Suyo during either period. PA-biosynthetic enzyme activities appear to mediate these differences between cultivars. Pretreatment of Spd to cv Suyo prevented chill-induced increases in the contents of hydrogen peroxide in leaves and activities of NADPH oxidases and NADPH-dependent superoxide generation in microsomes and alleviated chilling injury. Pretreatment of methylglyoxal-bis-(guanylhydrazone), a PA biosynthesis inhibitor, to chilled cv Jinchun No. 3 prevented Spd increase and enhanced microsomal NADPH oxidase activity and chilling injury. The results suggest that Spd plays important roles in chilling tolerance of cucumber, probably through prevention of chill-induced activation of NADPH oxidases in microsomes.
Gene Induction of Stilbene Biosynthesis in Scots Pine in Response to Ozone Treatment, Wounding, and Fungal Infection
Authors: Chiron Hélène , Drouet Alain , Lieutier François , Payer Hans-Dieter , Ernst Dieter , Sandermann Heinrich 
Published: July 2002

Abstract
TheS-adenosyl-l-methionine:pinosylvin-O-methyltransferase (PMT)2 gene was sequenced from Scots pine (Pinus sylvestris). The open reading frame is arranged in two exons spaced by one 102-bp intron. Promoter regulatory elements such as two “CAAT” boxes and one “TATA” box were identified. Several cis-regulatory elements were recognized: stress-responsive elements (Myb-responsive elements) as well as G, H, and GC boxes. Moreover, elicitor-responsive elements (W boxes) and a sequence resembling the simian virus 40 enhancer core were found. In phloem and needles of control trees, the transcripts of stilbene synthase (STS) and PMT were hardly detectable. Increased ozone fumigation up to 0.3 μL L−1 enhanced the transcript level of STSand PMT in needles but not in healthy phloem. Wounding, e.g. mock inoculation, of stem-phloem was characterized by a transient increase in STS and PMT transcripts, which was more pronounced in the case of fungal inoculation. Combination of fungal-challenge or mock treatment with ozone resulted in a positive interaction at 0.3 μL L−1. Scots pine stilbene formation appeared to be induced via STS andPMT gene expression upon ozone and fungal stress as well as wounding. The broad stress-responsiveness is in agreement with the range of various cis-acting elements detected in the STSand PMT promoters.
Efficient Screening of Arabidopsis T-DNA Insertion Lines Using Degenerate Primers
Authors: Young Jeffery C. , Krysan Patrick J. , Sussman Michael R. 
Published: July 2002

Abstract
The sequencing of the Arabidopsis plant genome is providing a fuller understanding of the number and types of plant genes. However, in most cases we do not know which genes are responsible for specific metabolic and signal transduction pathways. Analysis of gene function is also often confounded by the presence of multiple isoforms of the gene of interest. Recent advances in PCR-based reverse genetic techniques have allowed the search for plants carrying T-DNA insertions in any gene of interest. Here we report preliminary screening results from an ordered population of nearly 60,470 independently derived T-DNA lines. Degenerate PCR primers were used on large DNA pools (n = 2,025 T-DNA lines) to screen for more than one gene family member at a time. Methods are presented that facilitated the identification and isolation of isoform-specific mutants in almost all members of the Arabidopsis H+-proton ATPase gene family. Multiple mutant alleles were found for several isoforms.
The Arabidopsis ISR1 Locus Controlling Rhizobacteria-Mediated Induced Systemic Resistance Is Involved in Ethylene Signaling
Authors: Ton Jurriaan , Davison Sylke , Van Wees Saskia C.M. , Van Loon L.C. , Pieterse Corné M.J. 
Published: July 2002

Abstract
In Arabidopsis, the rhizobacterial strain Pseudomonas fluorescens WCS417r triggers an induced systemic resistance (ISR) response that is effective against different types of pathogens. The ISR signaling pathway functions independent of salicylic acid, but requires responsiveness to jasmonate (JA) and ethylene. Using the genetic variability of ISR inducibility between Arabidopsis accessions, we recently identified a locus (ISR1) on chromosome III that is involved in ISR signaling. Accessions RLD and Wassilewskija (Ws) are recessive at the ISR1 locus and are, therefore, unable to develop ISR. Here we investigated whether theISR1 locus is involved in JA or ethylene signaling. Compared with the ISR-inducible accession Columbia (Col), accessions RLD and Ws were not affected in JA-induced inhibition of root growth and expression of the JA-responsive gene Atvsp, suggesting that the ISR1 locus is not involved in JA signaling. However, RLD and Ws showed an affected expression of the triple response and a reduced expression of the ethylene responsive genes Hel and Pdf1.2 after exogenous application of the ethylene precursor 1-aminocyclopropane-1-carboxylate. Moreover, in contrast to Col, RLD and Ws did not develop resistance against P. syringaepv. tomato DC3000 after treatment of the leaves with 1-aminocyclopropane-1-carboxylate. Analysis of the F2 and F3 progeny of a cross between Col (ISR1/ISR1) and RLD (isr1/isr1) revealed that reduced sensitivity to ethylene cosegregates with the recessive alleles of theISR1 locus. These results suggest that theISR1 locus encodes a component of the ethylene response, which is required for the expression of rhizobacteria-mediated ISR.
Cabbage Cryoprotectin Is a Member of the Nonspecific Plant Lipid Transfer Protein Gene Family
Authors: Hincha Dirk K. , Neukamm Birgit , Sror Hany A.M. , Sieg Frank , Weckwarth Wolfgang , Rückels Markus , Lullien-Pellerin Valérie , Schröder Werner , Schmitt Jürgen M. 
Published: July 2002

Abstract
We have recently purified a protein (cryoprotectin) from the leaves of cold-acclimated cabbage (Brassica oleracea) to electrophoretic homogeneity, which protects thylakoids isolated from the leaves of nonacclimated spinach (Spinacia oleracea) from freeze-thaw damage. Sequencing of cryoprotectin showed the presence of at least three isoforms of WAX9 proteins, which belong to the class of nonspecific lipid transfer proteins. Antibodies raised against two synthetic peptides derived from the WAX9 proteins recognized a band of approximately 10 kD in western blots of crude cryoprotectin preparations. This protein and the cryoprotective activity could be precipitated from solution by the antiserum. We show further that cryoprotectin is structurally and functionally different from WAX9 isolated from the surface wax of cabbage leaves. WAX9 has lipid transfer activity for phosphatidylcholine, but no cryoprotective activity. Cryoprotectin, on the other hand, has cryoprotective, but no lipid transfer activity. The cryoprotective activity of cryoprotectin was strictly dependent on Ca2+ and Mn2+ and could be inhibited by chelating agents, whereas the lipid transfer activity of WAX9 was higher in the presence of ethylenediaminetetraacetate than in the presence of Ca2+and Mn2+.
Amyloplasts That Sediment in Protonemata of the MossCeratodon purpureus Are Nonrandomly Distributed in Microgravity
Authors: Kern Volker D. , Smith Jeffrey D. , Schwuchow Jochen M. , Sack Fred D. 
Published: July 2002

Abstract
Little is known about whether or how plant cells regulate the position of heavy organelles that sediment toward gravity. Dark-grown protonemata of the moss Ceratodon purpureus displays a complex plastid zonation in that only some amyloplasts sediment along the length of the tip cell. If gravity is the major force determining the position of amyloplasts that sediment, then these plastids should be randomly distributed in space. Instead, amyloplasts were clustered in the subapical region in microgravity. Cells rotated on a clinostat on earth had a roughly similar non-random plastid distribution. Subapical clusters were also found in ground controls that were inverted and kept stationary, but the distribution profile differed considerably due to amyloplast sedimentation. These findings indicate the existence of as yet unknown endogenous forces and mechanisms that influence amyloplast position and that are normally masked in stationary cells grown on earth. It is hypothesized that a microtubule-based mechanism normally compensates forg-induced drag while still allowing for regulated amyloplast sedimentation.
Enzymes That Scavenge Reactive Oxygen Species Are Down-Regulated Prior to Gibberellic Acid-Induced Programmed Cell Death in Barley Aleurone
Authors: Fath Angelika , Bethke Paul C. , Jones Russell L. 
Published: July 2002

AbstractGibberellins (GAs) initiate a series of events that culminate in programmed cell death, whereas abscisic acid (ABA) prevents this process. Reactive oxygen species (ROS) are key elements in aleurone programmed cell death. Incubation of barley (Hordeum vulgare) aleurone layers in H2O2 causes rapid death of all cells in GA- but not ABA-treated layers. Sensitivity to H2O2 in GA-treated aleurone cells results from a decreased ability to metabolize ROS. The amounts and activities of ROS scavenging enzymes, including catalase (CAT), ascorbate peroxidase, and superoxide dismutase are strongly down-regulated in aleurone layers treated with GA. CAT activity, protein, andCat2 mRNA decline rapidly following exposure of aleurone layers to GA. In ABA-treated layers, on the other hand, the amount and activity of CAT and Cat2 mRNA increases. Incubation in ABA maintains high amounts of ascorbate peroxidase and superoxide dismutase, whereas GA brings about a rapid reduction in the amounts of these enzymes. These data imply that GA-treated cells loose their ability to scavenge ROS and that this loss ultimately results in oxidative damage and cell death. ABA-treated cells, on the other hand, maintain their ability to scavenge ROS and remain viable.
Genetic Control of Natural Variation in Arabidopsis Glucosinolate Accumulation
Authors: Kliebenstein Daniel J. , Kroymann Juergen , Brown Paul , Figuth Antje , Pedersen Deana , Gershenzon Jonathan , Mitchell-Olds Thomas 
Published: July 2002

Abstract
Glucosinolates are biologically active secondary metabolites of the Brassicaceae and related plant families that influence plant/insect interactions. Specific glucosinolates can act as feeding deterrents or stimulants, depending upon the insect species. Hence, natural selection might favor the presence of diverse glucosinolate profiles within a given species. We determined quantitative and qualitative variation in glucosinolates in the leaves and seeds of 39 Arabidopsis ecotypes. We identified 34 different glucosinolates, of which the majority are chain-elongated compounds derived from methionine. Polymorphism at only five loci was sufficient to generate 14 qualitatitvely different leaf glucosinolate profiles. Thus, there appears to be a modular genetic system regulating glucosinolate profiles in Arabidopsis. This system allows the rapid generation of new glucosinolate combinations in response to changing herbivory or other selective pressures. In addition to the qualitative variation in glucosinolate profiles, we found a nearly 20-fold difference in the quantity of total aliphatic glucosinolates and were able to identify a single locus that controls nearly three-quarters of this variation.
Class I β-1,3-Glucanase and Chitinase Are Expressed in the Micropylar Endosperm of Tomato Seeds Prior to Radicle Emergence
Authors: Wu Chun-Ta , Leubner-Metzger Gerhard , Meins Frederick , Bradford Kent J. 
Published: July 2002

Abstract
β-1,3-Glucanase (EC 3.2.1.39) and chitinase (EC 3.2.1.14) mRNAs, proteins, and enzyme activities were expressed specifically in the micropylar tissues of imbibed tomato (Lycopersicon esculentum Mill.) seeds prior to radicle emergence. RNA hybridization and immunoblotting demonstrated that both enzymes were class I basic isoforms. β-1,3-Glucanase was expressed exclusively in the endosperm cap tissue, whereas chitinase localized to both endosperm cap and radicle tip tissues. β-1,3-Glucanase and chitinase appeared in the micropylar tissues of gibberellin-deficient gib-1tomato seeds only when supplied with gibberellin. Accumulation of β-1,3-glucanase mRNA, protein and enzyme activity was reduced by 100 μM abscisic acid, which delayed or prevented radicle emergence but not endosperm cap weakening. In contrast, expression of chitinase mRNA, protein, and enzyme activity was not affected by abscisic acid. Neither of these enzymes significantly hydrolyzed isolated tomato endosperm cap cell walls. Although both β-1,3-glucanase and chitinase were expressed in tomato endosperm cap tissue prior to radicle emergence, we found no evidence that they were directly involved in cell wall modification or tissue weakening. Possible functions of these hydrolases during tomato seed germination are discussed.
Ozone Quenching Properties of Isoprene and Its Antioxidant Role in Leaves
Authors: Loreto Francesco , Mannozzi Michela , Maris Christophe , Nascetti Pamela , Ferranti Francesco , Pasqualini Stefania 
Published: July 2002

Abstract
Isoprene is formed in and emitted by plants and the reason for this apparent carbon waste is still unclear. It has been proposed that isoprene stabilizes cell and particularly chloroplast thylakoid membranes. We tested if membrane stabilization or isoprene reactivity with ozone induces protection against acute ozone exposures. The reduction of visible, physiological, anatomical, and ultrastructural (chloroplast) damage shows that clones of plants sensitive to ozone and unable to emit isoprene become resistant to acute and short exposure to ozone if they are fumigated with exogenous isoprene, and that isoprene-emitting plants that are sensitive to ozone do not suffer damage when exposed to ozone. Isoprene-induced ozone resistance is associated with the maintenance of photochemical efficiency and with a low energy dissipation, as indicated by fluorescence quenching. This suggests that isoprene effectively stabilizes thylakoid membranes. However, when isoprene reacts with ozone within the leaves or in a humid atmosphere, it quenches the ozone concentration to levels that are less or non-toxic for plants. Thus, protection from ozone in plants fumigated with isoprene may be due to a direct ozone quenching rather than to an induced resistance at membrane level. Irrespective of the mechanism, isoprene is one of the most effective antioxidants in plants.
Functional Conservation of Plant Secondary Metabolic Enzymes Revealed by Complementation of Arabidopsis Flavonoid Mutants with Maize Genes
Authors: Dong Xiaoyun , Braun Edward L. , Grotewold Erich 
Published: July 2002

Abstract
Mutations in the transparent testa(tt) loci abolish pigment production in Arabidopsis seed coats. The TT4, TT5, andTT3 loci encode chalcone synthase, chalcone isomerase, and dihydroflavonol 4-reductase, respectively, which are essential for anthocyanin accumulation and may form a macromolecular complex. Here, we show that the products of the maize (Zea mays)C2, CHI1, and A1 genes complement Arabidopsis tt4, tt5, andtt3 mutants, restoring the ability of these mutants to accumulate pigments in seed coats and seedlings. Overexpression of the maize genes in wild-type Arabidopsis seedlings does not result in increased anthocyanin accumulation, suggesting that the steps catalyzed by these enzymes are not rate limiting in the conditions assayed. The expression of the maize A1 gene in the flavonoid 3′ hydroxylase Arabidopsis tt7 mutant resulted in an increased accumulation of pelargonidin. We conclude that enzymes involved in secondary metabolism can be functionally exchangeable between plants separated by large evolutionary distances. This is in sharp contrast to the notion that the more relaxed selective constrains to which secondary metabolic pathways are subjected is responsible for the rapid divergence of the corresponding enzymes.
Structural and dynamic characterization of an unfolded state of poplar apo‐plastocyanin formed under nondenaturing conditions
Authors: Bai Yawen , Chung John , Dyson H. Jane , Wright Peter E. 
Published: July 2002

AbstractPlastocyanin is a predominantly β‐sheet protein containing a type I copper center. The conformational ensemble of a denatured state of apo‐plastocyanin formed in solution under conditions of low salt and neutral pH has been investigated by multidimensional heteronuclear NMR spectroscopy. Chemical shift assignments were obtained by using three‐dimensional triple‐resonance NMR experiments to trace through‐bond heteronuclear connectivities along the backbone and side chains. The 3JHN,Hα coupling constants, 15N‐edited proton–proton nuclear Overhauser effects (NOEs), and 15N relaxation parameters were also measured for the purpose of structural and dynamic characterization. Most of the residues corresponding to β‐strands in the folded protein exhibit small upfield shifts of the 13Cα and 13CO resonances relative to random coil values, suggesting a slight preference for backbone dihedral angles in the β region of (ϕ,ψ) space. This is further supported by the presence of strong sequential dαN(i, i + 1) NOEs throughout the sequence. The few dNN(i, i + 1) proton NOEs that are observed are mostly in regions that form loops in the native plastocyanin structure. No medium or long‐range NOEs were observed. A short sequence, between residues 59 and 63, was found to populate a nonnative helical conformation in the unfolded state, as indicated by the shift of the 13Cα, 13CO, and 1Hα resonances relative to random coil values and by the decreased values of the 3JHN,Hα coupling constants. The 15N relaxation parameters indicate restriction of motions on a nanosecond timescale in this region. Intriguingly, this helical conformation is present in a sequence that is close to but not in the same location as the single short helix in the native folded protein. The results are consistent with earlier NMR studies of peptide fragments of plastocyanin and confirm that the regions of the sequence that form β‐strands in the native protein spontaneously populate the β‐region of (ϕ,ψ) space under folding conditions, even in the absence of stabilizing tertiary interactions. We conclude that the state of apo‐plastocyanin present under nondenaturing conditions is a noncompact unfolded state with some evidence of nativelike and nonnative local structuring that may be initiation sites for folding of the protein.
Crystal structures of a ddATP‐, ddTTP‐, ddCTP, and ddGTP‐ trapped ternary complex of Klentaq1: Insights into nucleotide incorporation and selectivity
Authors: Li Ying , Waksman Gabriel 
Published: July 2002

AbstractThe mechanism by which DNA polymerase I enzymes function has been the subject of extensive biochemical and structural studies. We previously determined the structure of a ternary complex of the large fragment of DNA polymerase I from Thermus aquaticus (Klentaq1) bound to a primer/template DNA and a dideoxycytidine 5′‐triphosphate (ddCTP). In this report, we present the details of the 2.3‐Å resolution crystal structures of three additional ternary complexes of Klentaq1 bound to a primer/template DNA and a dideoxyguanosine 5′‐triphosphate (ddGTP), a dideoxythymidine 5′‐triphosphate (ddTTP), or a dideoxyadenosine 5′‐triphosphate (ddATP). Comparison of the active site of the four ternary complexes reveals that the protein residues around the nascent base pair (that formed between the incoming dideoxynucleoside triphosphate [ddNTP] and the template base) form a snug binding pocket into which only a correct Watson‐Crick base pair can fit. Except in the ternary complex bound to dideoxyguanosine 5′‐triphosphate, there are no sequence specific contacts between the protein side chains and the nascent base pair, suggesting that steric constraints imposed by the protein onto the nascent base pair is the major contributor to nucleotide selectivity at the polymerase active site. The protein around the polymerase active site also shows plasticity, which may be responsible for the substrate diversity of the enzyme. Two conserved side chains, Q754 and R573, form hydrogen bonds with the N3 atom in the purine base and O2 atom in the pyrimidine base at the minor groove side of the base pair formed by the incorporated ddNMP and the corresponding template base in all the four ternary complexes. These hydrogen‐bonding interactions may provide a means of detecting misincorporation at this position.