bluuebunny/crossref_metadata_2025 huggingface dataset:
Drinking to Cope in Socially Anxious Individuals: A Controlled Study
Authors: Thomas Suzanne E. , Randall Carrie L. , Carrigan Maureen H.
Published: December 2003

Background: Several hypotheses exist to account for the higher than normal rate of alcoholism in individuals with high trait anxiety (or anxiety disorders). Most of these suggest that the practice of drinking alcohol to reduce anxiety leads to an increased risk of alcoholism in vulnerable individuals. The first assumption of the hypothesis is that anxious individuals use alcohol to cope with their anxiety. Few studies have examined this issue systematically, and none have used a nonanxious matched control group.

Methods: Twenty‐three individuals with high social anxiety and 23 nonsocially anxious matched controls were included in the study. Groups were similar on demographic variables and alcohol use. All participants were queried regarding the use of alcohol to cope, the practice of avoiding social situations if alcohol was not available, and the degree of relief attained by alcohol. Participants also were asked about using alcohol in 11 specific situations.

Results: The socially anxious group was significantly more likely than controls to report using alcohol to feel more comfortable in social situations and to avoid social situations if alcohol was unavailable. They also reported a greater degree of relief of anxiety from alcohol. Exploratory analyses revealed that socially anxious individuals reported using alcohol more to cope with social interactions than with social performance situations.

Conclusions: Individuals high in social anxiety deliberately drink alcohol to cope with their social fears. They report that alcohol is moderately effective at reducing their anxiety, which is seemingly sufficient to allow them to endure social situations. The data support the first assumption of the self‐medication hypothesis—that alcohol is used to reduce social discomfort in socially anxious individuals; however, the study was not designed to address the veracity of the self‐medication hypothesis as a whole. Results can help guide future studies that examine the relationship between social anxiety and alcohol.
Complexity Changes of the EEG Induced by Alcohol Cue Exposure in Alcoholics and Social Drinkers
Authors: Kim Dai‐Jin , Jeong Jaeseung , Kim Kwang‐Soo , Chae Jeong‐Ho , Jin Seung‐Hyun , Ahn Kook Jin , Myrick Hugh , Yoon Su‐Jung , Kim Hyung‐Rae , Kim Soo Yong
Published: December 2003

Background: An understanding of the neurophysiological mechanisms underlying alcohol craving is important in the effective treatment of alcohol dependence. The aim of this study was to examine the utility of the electroencephalogram (EEG) to measure the changes in electrical brain activity of alcoholics when exposed to alcohol‐specific cues.

Methods: Fifteen adult alcoholic subjects (four women) with a mean age of 35 (SD = 4.5) and 10 healthy social drinking controls (three women) with a mean age of 34 (SD = 5.6) were recruited. Subjects were serially rated for alcohol craving after presentations of pictures of control nonalcoholic and alcohol beverages. After the picture presentation, the EEG was recorded (16,384 data points for each epoch) with eyes closed. The dimensional complexity (D2) was estimated as a measure of complexity of the EEG.

Results: Alcoholic subjects exhibited a significant increase in the D2 values of the EEG in frontal (F3, F4), right posterior temporal (T6), and occipital (O1, O2) regions after viewing alcohol cues compared with viewing other beverage cues. These results indicate that more complex (or higher) cortical activity is induced over specific brain regions of alcoholic subjects by alcohol‐specific cues. Changes in subscale of alcohol craving between nonalcoholic and alcohol pictures were correlated with changes in D2 values in the left frontal (F3) region in alcoholic subjects.

Conclusions: These findings suggest that, when subjects are exposed to alcohol cues, changes in the EEG complexity are induced in frontal, right posterior temporal, and occipital areas, which may be key brain structures for alcohol craving. In addition, nonlinear measures like the D2 are useful in evaluating alcohol cue‐induced brain activity from the EEG.
1-Mb Resolution Array-Based Comparative Genomic Hybridization Using a BAC Clone Set Optimized for Cancer Gene Analysis
Authors: Greshock Joel , Naylor Tara L. , Margolin Adam , Diskin Sharon , Cleaver Stephen H. , Futreal P. Andrew , deJong Pieter J. , Zhao Shaying , Liebman Michael , Weber Barbara L.
Published: December 2003

Array-based comparative genomic hybridization (aCGH) is a recently developed tool for genome-wide determination of DNA copy number alterations. This technology has tremendous potential for disease-gene discovery in cancer and developmental disorders as well as numerous other applications. However, widespread utilization of a CGH has been limited by the lack of well characterized, high-resolution clone sets optimized for consistent performance in aCGH assays and specifically designed analytic software. We have assembled a set of ∼4100 publicly available human bacterial artificial chromosome (BAC) clones evenly spaced at ∼1-Mb resolution across the genome, which includes direct coverage of ∼400 known cancer genes. This aCGH-optimized clone set was compiled from five existing sets, experimentally refined, and supplemented for higher resolution and enhancing mapping capabilities. This clone set is associated with a public online resource containing detailed clone mapping data, protocols for the construction and use of arrays, and a suite of analytical software tools designed specifically for aCGH analysis. These resources should greatly facilitate the use of aCGH in gene discovery.
Auxin Responsiveness of a Novel Cytochrome P450 in Rice Coleoptiles
Authors: Chaban Christina , Waller Frank , Furuya Masaki , Nick Peter
Published: December 2003

An early auxin-induced gene was isolated from rice (Oryza sativa L. subsp. japonica cv Nihonmasari) coleoptiles by a fluorescent-labeled differential display screen. The full-length gene contains conserved domains characteristic for the cytochrome P450 superfamily. This gene, designated as CYP87A3, was weakly expressed in dark-grown coleoptiles but was up-regulated rapidly and transiently when coleoptile segments were incubated in 5 μm indole-3-acetic acid. This induction by auxin could not be suppressed by cycloheximide. Depletion of segments from endogenous auxin reduced the amount of CYP87A3 transcripts. The CYP87A3 transcript level was rapidly, although transiently, up-regulated in response to light as well. The observed pattern of gene regulation might indicate a role in the suppression of auxin-induced coleoptile growth. The role of CYP87A3 is discussed with respect to auxin signaling in the regulation of coleoptile growth.
Glycosylphosphatidylinositol Lipid Anchoring of Plant Proteins. Sensitive Prediction from Sequence- and Genome-Wide Studies for Arabidopsis and Rice
Authors: Eisenhaber Birgit , Wildpaner Michael , Schultz Carolyn J. , Borner Georg H.H. , Dupree Paul , Eisenhaber Frank
Published: December 2003

Posttranslational glycosylphosphatidylinositol (GPI) lipid anchoring is common not only for animal and fungal but also for plant proteins. The attachment of the GPI moiety to the carboxyl-terminus after proteolytic cleavage of a C-terminal propeptide is performed by the transamidase complex. Its four known subunits also have obvious full-length orthologs in the Arabidopsis and rice (Oryza sativa) genomes; thus, the mechanism of substrate protein processing appears similar for all eukaryotes. A learning set of plant proteins (substrates for the transamidase complex) has been collected both from the literature and plant sequence databases. We find that the plant GPI lipid anchor motif differs in minor aspects from the animal signal (e.g. the plant hydrophobic tail region can contain a higher fraction of aromatic residues). We have developed the “big-Π plant” program for prediction of compatibility of query protein C-termini with the plant GPI lipid anchor motif requirements. Validation tests show that the sensitivity for transamidase targets is approximately 94%, and the rate of false positive prediction is about 0.1%. Thus, the big-Π predictor can be applied as unsupervised genome annotation and target selection tool. The program is also suited for the design of modified protein constructs to test their GPI lipid anchoring capacity. The big-Π plant predictor Web server and lists of potential plant precursor proteins in Swiss-Prot, SPTrEMBL, Arabidopsis, and rice proteomes are available at http://mendel.imp.univie.ac.at/gpi/plants/gpi_plants.html. Arabidopsis and rice protein hits have been functionally classified. Several GPI lipid-anchored arabinogalactan-related proteins have been identified in rice.
Primary Inhibition of Hypocotyl Growth and Phototropism Depend Differently on Phototropin-Mediated Increases in Cytoplasmic Calcium Induced by Blue Light
Authors: Folta Kevin M. , Lieg Erin J. , Durham Tessa , Spalding Edgar P.
Published: December 2003

The phototropin photoreceptors transduce blue-light signals into several physiological and developmental responses in plants. A transient rise in cytoplasmic calcium (Ca2+) that begins within seconds of phototropin 1 (phot1) excitation is believed to be an important element in the transduction pathways leading to one or more of the phot1-dependent responses. The goal of the present work was to determine whether the Ca2+ response was necessary for (a) the inhibition of hypocotyl elongation that develops within minutes of the irradiation, and (b) hypocotyl phototropism (curved growth of the stem in response to asymmetric illumination). After determining that pulses of light delivering photon fluences of between 1 and 1,000 μmol m-2 induced growth inhibition mediated by phot1 without significant interference from other photosensory pathways, the effect of blocking the Ca2+ rise was assessed. Treatment of seedlings with a Ca2+ chelator prevented the rise in cytoplasmic Ca2+ and prevented phot1-mediated growth inhibition. However, the same chelator treatment did not impair phot1-mediated phototropism. Thus, it appears that the early, transient rise in cytoplasmic Ca2+ is an important intermediary process in at least one but not all phot1-signaling pathways.
HY5, Circadian Clock-Associated 1, and a cis-Element, DET1 Dark Response Element, Mediate DET1 Regulation ofChlorophyll a/b-Binding Protein 2Expression
Authors: Maxwell Bridey B. , Andersson Carol R. , Poole Daniel S. , Kay Steve A. , Chory Joanne
Published: December 2003

DET1 is a pleiotropic regulator of Arabidopsis development and controls the expression of many light-regulated genes. To gain a better understanding of the mechanism by which DET1 controls transcription from light-regulated promoters, we identified elements in the chlorophyll a/b-binding protein 2 (CAB2) promoter that are required for DET1-mediated expression. Using a series of reporter constructs in which the luciferase gene is controlled by CAB2 promoter fragments, we defined two DET1-responsive elements in the CAB2 promoter that are essential for proper CAB2 transcription. A 40-bp DET1 dark-response element (DtRE) is required for both dark and root-specific repression of CAB2, whereas the known CAB upstream factor-1 element is required for DET1 activation-associated effects in the light and repression in the roots. HY5, a factor that binds CAB upstream factor-1, is also required for DET1 effects in the light. DtRE binds two distinct activities in Arabidopsis seedling extracts: a novel activity with binding site CAAAACGC that we have named CAB2 DET1-associated factor 1 plus an activity that is likely to be the myb transcription factor Circadian Clock-Associated 1. Both activities are altered in dark-grown det1 extracts as compared with wild type, correlating a change in extractable DNA binding activity with a major change in CAB2 expression. We conclude that DET1 represses the CAB2 promoter in the dark by regulating the binding of two factors, CAB2 DET1-associated factor 1 and Circadian Clock-Associated 1, to the DtRE.
Monitoring Expression Profiles of Rice Genes under Cold, Drought, and High-Salinity Stresses and Abscisic Acid Application Using cDNA Microarray and RNA Gel-Blot Analyses
Authors: Rabbani M. Ashiq , Maruyama Kyonoshin , Abe Hiroshi , Khan M. Ayub , Katsura Koji , Ito Yusuke , Yoshiwara Kyoko , Seki Motoaki , Shinozaki Kazuo , Yamaguchi-Shinozaki Kazuko
Published: December 2003

To identify cold-, drought-, high-salinity-, and/or abscisic acid (ABA)-inducible genes in rice (Oryza sativa), we prepared a rice cDNA microarray including about 1,700 independent cDNAs derived from cDNA libraries prepared from drought-, cold-, and high-salinity-treated rice plants. We confirmed stress-inducible expression of the candidate genes selected by microarray analysis using RNA gel-blot analysis and finally identified a total of 73 genes as stress inducible including 58 novel unreported genes in rice. Among them, 36, 62, 57, and 43 genes were induced by cold, drought, high salinity, and ABA, respectively. We observed a strong association in the expression of stress-responsive genes and found 15 genes that responded to all four treatments. Venn diagram analysis revealed greater cross talk between signaling pathways for drought, ABA, and high-salinity stresses than between signaling pathways for cold and ABA stresses or cold and high-salinity stresses in rice. The rice genome database search enabled us not only to identify possible known cis-acting elements in the promoter regions of several stress-inducible genes but also to expect the existence of novel cis-acting elements involved in stress-responsive gene expression in rice stress-inducible promoters. Comparative analysis of Arabidopsis and rice showed that among the 73 stress-inducible rice genes, 51 already have been reported in Arabidopsis with similar function or gene name. Transcriptome analysis revealed novel stress-inducible genes, suggesting some differences between Arabidopsis and rice in their response to stress.
Functional Analysis and Intracellular Localization of Rice Cryptochromes
Authors: Matsumoto Nanako , Hirano Tomoharu , Iwasaki Toshisuke , Yamamoto Naoki
Published: December 2003

Blue-light-receptor cryptochrome (CRY), which mediates cotyledon expansion, increased accumulation of anthocyanin, and inhibition of hypocotyl elongation, was first identified in Arabidopsis. Two Arabidopsis cryptochromes (AtCRY1 and AtCRY2) have been reported to be localized to the nucleus. However, there is no information on the cryptochromes in monocotyledons. In this study, we isolated two cryptochrome cDNAs, OsCRY1 and OsCRY2, from rice (Oryza sativa) plants. The deduced amino acid sequences of OsCRY1 and OsCRY2 have a photolyase-like domain in their N termini and are homologous to AtCRY1. To investigate the function of OsCRY1, we overexpressed a green fluorescence protein-OsCRY1 fusion gene in Arabidopsis and assessed the phenotypes of the resulting transgenic plants. When the seedlings were germinated in the dark, no discernible effect was observed. However, light-germinated seedlings showed pronounced inhibition of hypocotyl elongation and increased accumulation of anthocyanin. These phenotypes were induced in a blue-light-dependent manner, indicating that OsCRY1 functions as a blue-light-receptor cryptochrome. We also examined the intracellular localization of green fluorescence protein-OsCRY1 in the transgenic plants. It was localized to both the nucleus and the cytoplasm. We identified two nuclear localization domains in the primary structure of OsCRY1. We discuss the relationship between the function and intracellular localization of rice cryptochromes by using additional data obtained with OsCRY2.
Ethylene Stimulates Endoreduplication But Inhibits Cytokinesis in Cucumber Hypocotyl Epidermis
Authors: Dan Haruka , Imaseki Hidemasa , Wasteneys Geoffrey O. , Kazama Haruko
Published: December 2003

The effects of ethylene on cell division are generally considered inhibitory. In this study, we demonstrate that transient ethylene exposure, while suppressing cytokinesis, stimulates DNA synthesis. We monitored DNA synthesis and cytokinesis in the epidermis of cucumber (Cucumis sativus) hypocotyls, an organ whose post-germination development involves strictly limited cell division. During exposure to ethylene, DNA synthesis, assessed by the incorporation of the thymidine homolog 5-bromo-2′-deoxyuridine, was detected in 20% of the epidermal cells, whereas DNA synthesis was nearly undetectable in normal air. Cytofluorometric analysis of nuclei in affected cells showed an up to 8-fold increase in DNA content. During this time, new cell plate formation was not detected. However, shortly after ethylene was removed, DNA content was rapidly restored to 2C (diploid) levels in all cells, and new cell plate formation dramatically increased. These results demonstrate that ethylene promotes DNA synthesis and its endoreduplication but inhibits cytokinesis, thereby maintaining some cells in G2 phase.
Decreased Content of Leaf Ferredoxin Changes Electron Distribution and Limits Photosynthesis in Transgenic Potato Plants
Authors: Holtgrefe Simone , Bader Klaus P. , Horton Peter , Scheibe Renate , von Schaewen Antje , Backhausen Jan E.
Published: December 2003

A complete ferredoxin (Fd) cDNA clone was isolated from potato (Solanum tuberosum L. cv Desiree) leaves. By molecular and immunoblot analysis, the gene was identified as the leaf-specific Fd isoform I. Transgenic potato plants were constructed by introducing the homologous potato fed 1 cDNA clone as an antisense construct under the control of the constitutive cauliflower mosaic virus 35S promoter. Stable antisense lines with Fd contents between 40% and 80% of the wild-type level were selected by northern- and western-blot analysis. In short-term experiments, the distribution of electrons toward their stromal acceptors was altered in the mutant plants. Cyclic electron transport, as determined by the quantum yields of photosystems I and II, was enhanced. The CO2 assimilation rate was decreased, but depending on the remaining Fd content, some lines showed photoinhibition. The leaf protein content remained largely constant, but the antisense plants had a lower total chlorophyll content per unit leaf area and an increased chlorophyll a/b ratio. In the antisense plants, the redox state of the quinone acceptor A in photosystem II (QA) was more reduced than that of the wild-type plants under all experimental conditions. Because the plants with lower Fd amounts reacted as if they were grown under a higher light intensity, the possibility that the altered chloroplast redox state affects light acclimation is discussed.
Activation of Anthocyanin Biosynthesis inGerbera hybrida(Asteraceae) Suggests Conserved Protein-Protein and Protein-Promoter Interactions between the Anciently Diverged Monocots and Eudicots
Authors: Elomaa Paula , Uimari Anne , Mehto Merja , Albert Victor A. , Laitinen Roosa A.E. , Teeri Teemu H.
Published: December 2003

We have identified an R2R3-type MYB factor, GMYB10, from Gerbera hybrida (Asteraceae) that shares high sequence homology to and is phylogenetically grouped together with the previously characterized regulators of anthocyanin pigmentation in petunia (Petunia hybrida) and Arabidopsis. GMYB10 is able to induce anthocyanin pigmentation in transgenic tobacco (Nicotiana tabacum), especially in vegetative parts and anthers. In G. hybrida, GMYB10 is involved in activation of anthocyanin biosynthesis in leaves, floral stems, and flowers. In flowers, its expression is restricted to petal epidermal cell layers in correlation with the anthocyanin accumulation pattern. We have shown, using yeast (Saccharomyces cerevisiae) two-hybrid assay, that GMYB10 interacts with the previously isolated bHLH factor GMYC1. Particle bombardment analysis was used to show that GMYB10 is required for activation of a late anthocyanin biosynthetic gene promoter, PGDFR2. cis-Analysis of the target PGDFR2 revealed a sequence element with a key role in activation by GMYB10/GMYC1. This element shares high homology with the anthocyanin regulatory elements characterized in maize (Zea mays) anthocyanin promoters, suggesting that the regulatory mechanisms involved in activation of anthocyanin biosynthesis have been conserved for over 125 million years not only at the level of transcriptional regulators but also at the level of the biosynthetic gene promoters.
Overexpression of SOD2 Increases Salt Tolerance of Arabidopsis
Authors: Gao Xiuhua , Ren Zhonghai , Zhao Yanxiu , Zhang Hui
Published: December 2003

The yeast (Schizosaccharomyces pombe) SOD2 (Sodium2) gene was introduced into Arabidopsis under the control of the cauliflower mosaic virus 35S promoter. Transformants were selected for their ability to grow on medium containing kanamycin. Southern- and northern-blot analyses confirmed that SOD2 was transferred into the Arabidopsis genome. There were no obvious morphological or developmental differences between the transgenic and wild-type (wt) plants. Several transgenic homozygous lines and wt plants (control) were evaluated for salt tolerance and gene expression. Overexpression of SOD2 in Arabidopsis improved seed germination and seedling salt tolerance. Analysis of Na+ and K+ contents of the symplast and apoplast in the parenchyma cells of the root cortex and mesophyll cells in the spongy tissue of the leaf showed that transgenic lines accumulated less Na+ and more K+ in the symplast than the wt plants did. The photosynthetic rate and the fresh weight of the transgenic lines were distinctly higher than that of wt plants after NaCl treatment. Results from different tests indicated that the expression of the SOD2 gene promoted a higher level of salt tolerance in vivo in transgenic Arabidopsis plants.
Methylated DNA-Binding Proteins from Arabidopsis
Authors: Ito Mikako , Koike Akiko , Koizumi Nozomu , Sano Hiroshi
Published: December 2003

The 5-methylcytosines (m5C) play a critical role in epigenetic control, often being recognized by proteins containing a methyl-CpG-binding domain (MBD). Database screening has identified at least 12 putative methyl-CpG-binding proteins from Arabidopsis; we have isolated corresponding cDNAs for seven of them. Despite variation in size and amino acid sequence, all seven proteins exclusively migrate into the nucleus as revealed by green fluorescent protein fusion protein assay, suggesting a relationship with chromatin structure. However, DNA-binding assays using bacterially expressed proteins and synthetic oligonucleotides containing m5C in CpGs showed only one to specifically bind, designated AtMBD5. Further analysis showed that AtMBD5 efficiently binds to m5C in CpNpN (N is A, T, or C) but not in CpNpG sequences, both frequently found in plant DNA. The other six proteins showed either nonspecific DNA binding or no ability to recognize m5C. RNA-blot hybridization and immunoblot analysis indicated AtMBD5 to be present essentially in all tissues. Using green fluorescent protein driven by the authentic promoter, AtMBD5 was found to be actively expressed in pistils and root tips. Because m5Cs in CpG and CpNpN are considered to function in gene expression and gene silencing, respectively, the present results suggest that AtMBD5 may have distinct functions in regulation and/or self defense of genes in actively proliferating cells.
Photosynthetic Acclimation Is Reflected in Specific Patterns of Gene Expression in Drought-Stressed Loblolly Pine
Authors: Watkinson Jonathan I. , Sioson Allan A. , Vasquez-Robinet Cecilia , Shukla Maulik , Kumar Deept , Ellis Margaret , Heath Lenwood S. , Ramakrishnan Naren , Chevone Boris , Watson Layne T. , van Zyl Leonel , Egertsdotter Ulrika , Sederoff Ronald R. , Grene Ruth
Published: December 2003

Because the product of a single gene can influence many aspects of plant growth and development, it is necessary to understand how gene products act in concert and upon each other to effect adaptive changes to stressful conditions. We conducted experiments to improve our understanding of the responses of loblolly pine (Pinus taeda) to drought stress. Water was withheld from rooted plantlets of to a measured water potential of -1 MPa for mild stress and -1.5 MPa for severe stress. Net photosynthesis was measured for each level of stress. RNA was isolated from needles and used in hybridizations against a microarray consisting of 2,173 cDNA clones from five pine expressed sequence tag libraries. Gene expression was estimated using a two-stage mixed linear model. Subsequently, data mining via inductive logic programming identified rules (relationships) among gene expression, treatments, and functional categories. Changes in RNA transcript profiles of loblolly pine due to drought stress were correlated with physiological data reflecting photosynthetic acclimation to mild stress or photosynthetic failure during severe stress. Analysis of transcript profiles indicated that there are distinct patterns of expression related to the two levels of stress. Genes encoding heat shock proteins, late embryogenic-abundant proteins, enzymes from the aromatic acid and flavonoid biosynthetic pathways, and from carbon metabolism showed distinctive responses associated with acclimation. Five genes shown to have different transcript levels in response to either mild or severe stress were chosen for further analysis using real-time polymerase chain reaction. The real-time polymerase chain reaction results were in good agreement with those obtained on microarrays.
The Arabidopsis Locus RCB Mediates Upstream Regulation of Mitotic Gene Expression
Authors: Himanen Kristiina , Reuzeau Christophe , Beeckman Tom , Melzer Siegbert , Grandjean Olivier , Corben Liz , Inzé Dirk
Published: December 2003

Transcriptional regulation of cell cycle regulatory genes, such as B-type cyclins, is tightly linked with the mitotic activity in the meristems. To study the regulation of a B-type cyclin gene, a targeted genetic approach was undertaken. An Arabidopsis line containing a fusion construct between the CYCB1;1 promoter and a bacterial β-glucuronidase marker gene (uidA) was used in ethyl methanesulfonate mutagenesis. The mutants were screened for altered CYCB1;1::uidA expression patterns. In a reduced CYCB1;1 expression mutant (rcb), the CYCB1;1::uidA expression was severely affected, being excluded from the shoot and root apical meristems and leaf primordia and shifted to cells associated with root cap and stomata. In addition to the overall reduction of the endogenous CYCB1;1 transcript levels, other G2-to-M phase-specific genes were also down-regulated by the mutation. In the mutant plants, the inflorescence stem growth was reduced, indicating low meristem activity. Based on the altered CYCB1;1::uidA expression patterns in rcb root meristem, a model is proposed for RCB that mediates the tissue specificity of CYCB1;1 promoter activity.
Expression of Arabidopsis γ-Tubulin in Fission Yeast Reveals Conserved and Novel Functions of γ-Tubulin
Authors: Horio Tetsuya , Oakley Berl R.
Published: December 2003

γ-Tubulin localizes to microtubule-organizing centers in animal and fungal cells where it is important for microtubule nucleation. Plant cells do not have morphologically defined microtubule organizing centers, however, and γ-tubulin is distributed in small, discrete structures along microtubules. The great difference in distribution has prompted speculation that plant γ-tubulins function differently from animal and fungal γ-tubulins. We tested this possibility by expressing Arabidopsis γ-tubulin in the fission yeast Schizosaccharomyces pombe. At high temperatures, the plant γ-tubulin was able to bind to microtubule-organizing centers, nucleate microtubule assembly, and support the growth and replication of S. pombe cells lacking endogenous γ-tubulin. However, the distribution of microtubules was abnormal as was cell morphology, and at low temperatures, cells were arrested in mitosis. These results reveal that Arabidopsis γ-tubulin can carry out essential functions in S. pombe and is, thus, functionally conserved. The morphological abnormalities reveal that it cannot carry out some nonessential functions, however, and they underscore the importance of γ-tubulin in morphogenesis of fission yeast cells and in maintaining normal interphase microtubule arrays.
Impaired Induction of the Jasmonate Pathway in the Rice Mutant hebiba
Authors: Riemann Michael , Müller Axel , Korte Arthur , Furuya Masaki , Weiler Elmar W. , Nick Peter
Published: December 2003

The elongation of rice (Oryza sativa) coleoptiles is inhibited by light, and this photoinhibition was used to screen for mutants with impaired light response. In one of the isolated mutants, hebiba, coleoptile elongation was stimulated in the presence of red light, but inhibited in the dark. Light responses of endogenous indolyl-3-acetic acid and abscisic acid were identical between the wild type and the mutant. In contrast, the wild type showed a dramatic increase of jasmonate heralded by corresponding increases in the content of its precursor o-phytodienoic acid, whereas both compounds were not detectable in the mutant. The jasmonate response to wounding was also blocked in the mutant. The mutant phenotype was rescued by addition of exogenous methyl jasmonate and o-phytodienoic acid. Moreover, the expression of O. sativa 12-oxophytodienoic acid reductase, an early gene of jasmonic acid-synthesis, is induced by red light in the wild type, but not in the mutant. This evidence suggests a novel role for jasmonates in the light response of growth, and we discuss a cross-talk between jasmonate and auxin signaling. In addition, hebiba represents the first rice mutant in which the induction of the jasmonate pathway is impaired providing a valuable tool to study the role of jasmonates in Graminean development.
T-DNA Integration in Arabidopsis Chromosomes. Presence and Origin of Filler DNA Sequences
Authors: Windels Pieter , De Buck Sylvie , Van Bockstaele Erik , De Loose Marc , Depicker Ann
Published: December 2003

To investigate the relationship between T-DNA integration and double-stranded break (DSB) repair in Arabidopsis, we studied 67 T-DNA/plant DNA junctions and 13 T-DNA/T-DNA junctions derived from transgenic plants. Three different types of T-DNA-associated joining could be distinguished. A minority of T-DNA/plant DNA junctions were joined by a simple ligation-like mechanism, resulting in a junction without microhomology or filler DNA insertions. For about one-half of all analyzed junctions, joining of the two ends occurred without insertion of filler sequences. For these junctions, microhomology was strikingly combined with deletions of the T-DNA ends. For the remaining plant DNA/T-DNA junctions, up to 51-bp-long filler sequences were present between plant DNA and T-DNA contiguous sequences. These filler segments are built from several short sequence motifs, identical to sequence blocks that occur in the T-DNA ends and/or the plant DNA close to the integration site. Mutual microhomologies among the sequence motifs that constitute a filler segment were frequently observed. When T-DNA integration and DSB repair were compared, the most conspicuous difference was the frequency and the structural organization of the filler insertions. In Arabidopsis, no filler insertions were found at DSB repair junctions. In maize (Zea mays) and tobacco (Nicotiana tabacum), DSB repair-associated filler was normally composed of simple, uninterrupted sequence blocks. Thus, although DSB repair and T-DNA integration are probably closely related, both mechanisms have some exclusive and specific characteristics.
Characterization of Four Lectin-Like Receptor Kinases Expressed in Roots of Medicago truncatula. Structure, Location, Regulation of Expression, and Potential Role in the Symbiosis with Sinorhizobium meliloti
Authors: Navarro-Gochicoa Maria-Téresa , Camut Sylvie , Timmers Antonius C.J. , Niebel Andreas , Hervé Christine , Boutet Emmanuel , Bono Jean-Jacques , Imberty Anne , Cullimore Julie V.
Published: December 2003

To study the role of LecRK (lectin-like receptor kinase) genes in the legumerhizobia symbiosis, we have characterized the four Medicago truncatula Gaernt. LecRK genes that are most highly expressed in roots. Three of these genes, MtLecRK7;1, MtLecRK7;2, and MtLecRK7;3, encode proteins most closely related to the Class A LecRKs of Arabidopsis, whereas the protein encoded by the fourth gene, MtLecRK1;1, is most similar to a Class B Arabidopsis LecRK. All four genes show a strongly enhanced root expression, and detailed studies on MtLecRK1;1 and MtLecRK7;2 revealed that the levels of their mRNAs are increased by nitrogen starvation and transiently repressed after either rhizobial inoculation or addition of lipochitooligosaccharidic Nod factors. Studies of the MtLecRK1;1 and MtLecRK7;2 proteins, using green fluorescent protein fusions in transgenic M. truncatula roots, revealed that they are located in the plasma membrane and that their central transmembrane-spanning helix is required for correct sorting. Moreover, their lectin-like domains appear to be highly glycosylated. Of the four proteins, only MtLecRK1;1 shows a high conservation of key residues implicated in monosaccharide binding, and molecular modeling revealed that this protein may be capable of interacting with Nod factors. However, no increase in Nod factor binding was found in roots overexpressing a fusion in which the kinase domain of this protein had been replaced with green fluorescent protein. Roots expressing this fusion protein however showed an increase in nodule number, suggesting that expression of MtLecRK1;1 influences nodulation. The potential role of LecRKs in the legume-rhizobia symbiosis is discussed.
Photosynthesis and State Transitions in Mitochondrial Mutants ofChlamydomonas reinhardtiiAffected in Respiration
Authors: Cardol Pierre , Gloire Geoffrey , Havaux Michel , Remacle Claire , Matagne René , Franck Fabrice
Published: December 2003

Photosynthetic activities were analyzed in Chlamydomonas reinhardtii mitochondrial mutants affected in different complexes (I, III, IV, I + III, and I + IV) of the respiratory chain. Oxygen evolution curves showed a positive relationship between the apparent yield of photosynthetic linear electron transport and the number of active proton-pumping sites in mitochondria. Although no significant alterations of the quantitative relationships between major photosynthetic complexes were found in the mutants, 77 K fluorescence spectra showed a preferential excitation of photosystem I (PSI) compared with wild type, which was indicative of a shift toward state 2. This effect was correlated with high levels of phosphorylation of light-harvesting complex II polypeptides, indicating the preferential association of light-harvesting complex II with PSI. The transition to state 1 occurred in untreated wild-type cells exposed to PSI light or in 3-(3,4-dichlorophenyl)-1,1-dimethylureatreated cells exposed to white light. In mutants of the cytochrome pathway and in double mutants, this transition was only observed in white light in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. This suggests higher rates of nonphotochemical plastoquinone reduction through the chlororespiratory pathway, which was confirmed by measurements of the complementary area above the fluorescence induction curve in dark-adapted cells. Photo-acoustic measurements of energy storage by PSI showed a stimulation of PSI-driven cyclic electron flow in the most affected mutants. The present results demonstrate that in C. reinhardtii mutants, permanent defects in the mitochondrial electron transport chain stabilize state 2, which favors cyclic over linear electron transport in the chloroplast.
Import of the Peroxisomal Targeting Signal Type 2 Protein 3-Ketoacyl-Coenzyme A Thiolase into Glyoxysomes
Authors: Johnson Tanya L. , Olsen Laura J.
Published: December 2003

Most peroxisomal matrix proteins possess a carboxy-terminal tripeptide targeting signal, termed peroxisomal targeting signal type 1 (PTS1), and follow a relatively well-characterized pathway of import into the organelle. The peroxisomal targeting signal type 2 (PTS2) pathway of peroxisomal matrix protein import is less well understood. In this study, we investigated the mechanisms of PTS2 protein binding and import using an optimized in vitro assay to reconstitute the transport events. The import of the PTS2 protein thiolase differed from PTS1 protein import in several ways. Thiolase import was slower than typical PTS1 protein import. Competition experiments with both PTS1 and PTS2 proteins revealed that PTS2 protein import was inhibited by addition of excess PTS2 protein, but it was enhanced by the addition of PTS1 proteins. Mature thiolase alone, lacking the PTS2 signal, was not imported into peroxisomes, confirming that the PTS2 signal is necessary for thiolase import. In competition experiments, mature thiolase did not affect the import of a PTS1 protein, but it did decrease the amount of radiolabeled full-length thiolase that was imported. This is consistent with a mechanism by which the mature protein competes with the full-length thiolase during assembly of an import complex at the surface of the membrane. Finally, the addition of zinc to PTS2 protein imports increased the level of thiolase bound and imported into the organelles.
LAF3, a Novel Factor Required for Normal Phytochrome A Signaling
Authors: Hare Peter D. , Møller Simon G. , Huang Li-Fang , Chua Nam-Hai
Published: December 2003

Phytochrome A (phyA) is the photolabile plant light receptor that mediates broad spectrum very low-fluence responses and high irradiance responses to continuous far-red light (FRc). An Arabidopsis mutant laf3-1 (long after far-red 3) was recovered from a screen for transposon-tagged mutants that exhibit reduced inhibition of hypocotyl elongation in FRc. The laf phenotype correlated well with a strongly attenuated disappearance of XTR7 transcript in FRc. The effects of laf3-1 on phyA-controlled CAB, CHS, and PET H expression were more subtle, and the mutation had no clear effects on PET E and ASN1 transcript levels in FRc. The use of two alternative transcription initiation sites in the LAF3 gene generates two isoforms that differ only at their N termini. Transcripts encoding both isoforms were induced during germination and were present at slightly higher levels in de-etiolated seedlings than in those grown in darkness. No significant differential regulation of the two isoforms was observed upon exposure to either FRc or continuous red light. Transcripts encoding the shorter isoform (LAF3ISF2) always appear to be more abundant than those encoding the longer isoform (LAF3ISF1). However, both isoforms were capable of full complementation of the laf3-1 hypocotyl phenotype in FRc. When fused to a yellow fluorescent protein, both isoforms localize to the perinuclear region, suggesting that LAF3 encodes a product that might regulate nucleo-cytoplasmic trafficking of an intermediate(s) involved in phyA signal transduction.
Tissue-Specific and Developmentally Regulated Expression of a Cluster of Tandemly Arrayed Cell Wall-Associated Kinase-Like Kinase Genes in Arabidopsis
Authors: Verica Joseph A. , Chae Lee , Tong Hongyun , Ingmire Peter , He Zheng-Hui
Published: December 2003

The Arabidopsis cell wall-associated kinase (WAK) and WAK-like kinase (WAKL) family of receptor-like kinase genes encodes transmembrane proteins with a cytoplasmic serine/threonine kinase domain and an extracellular region containing epidermal growth factor-like repeats. Previous studies have suggested that some WAK members are involved in plant defense and heavy metal responses, whereas others are required for cell elongation and plant development. The WAK/WAKL gene family consists of 26 members in Arabidopsis and can be divided into four groups. Here, we describe the characterization of group 2 members that are composed of a cluster of seven tandemly arrayed WAKL genes. The predicted WAKL proteins are highly similar in their cytoplasmic region but are more divergent in their predicted extracellular ligand-binding region. WAKL7 encodes a truncated WAKL isoform that is predicted to be secreted from the cytoplasm. Ratios of nonsynonymous to synonymous substitutions suggest that the extracellular region is subject to diversifying selection. Comparison of the WAKL and WAK gene clusters suggests that they arose independently. Protein gel-blot and immunolocalization analyses suggest that WAKL6 is associated with the cell wall. Histochemical analyses of WAKL promoters fused with the β-glucuronidase reporter gene have shown that the expressions of WAKL members are developmentally regulated and tissue specific. Unlike WAK members whose expressions were found predominately in green tissues, WAKL genes are highly expressed in roots and flowers. The expression of WAKL5 and WAKL7 can be induced by wounding stress and by the salicylic acid analog 2,6-dichloroisonicotinic acid in an nonexpressor of pathogenesis-related gene 1-dependent manner, suggesting that they, like some WAK members, are wound inducible and can be defined as pathogenesis-related genes.
Isolated Durum Wheat and Potato Cell Mitochondria Oxidize Externally Added NADH Mostly via the Malate/Oxaloacetate Shuttle with a Rate That Depends on the Carrier-Mediated Transport
Authors: Pastore Donato , Di Pede Sergio , Passarella Salvatore
Published: December 2003

We investigated whether and how mitochondria from durum wheat (Triticum durum Desf.) and potato (Solanum tuberosum), isolated from etiolated shoots and a cell suspension culture, respectively, oxidize externally added NADH via the mitochondrial shuttles; in particular, we compared the shuttles and the external NADH dehydrogenase (NADH DHExt) with respect to their capacity to oxidize external NADH. We found that external NADH and NADPH can be oxidized via two separate DHExt, whereas under conditions in which the activities of NAD(P)H DHExt are largely prevented, NADH (but not NADPH) is oxidized in the presence of external malate (MAL) and MAL dehydrogenase, in a manner sensitive to several non-penetrant compounds according to the occurrence of the MAL/oxaloacetate (OAA) shuttle. In durum wheat mitochondria and potato cell mitochondria, the rate of NADH oxidation was limited by the rate of a novel carrier, the MAL/OAA antiporter, which is different from other carriers thought to transport OAA across the mitochondrial membrane. No NAD(P)H oxidation occurred arising from the MAL/Aspartate and the α-glycerophosphate/dihydroxyacetonphosphate shuttles. We determined the kinetic parameters of the enzymes and the antiporter involved in NADH oxidation, and, on the basis of a kinetic analysis, we showed that, at low physiological NADH concentrations, oxidation via the MAL/OAA shuttle occurred with a higher efficiency than that due to the NADH DHExt (about 100- and 10-fold at 1 μm NADH in durum wheat mitochondria and in potato cell mitochondria, respectively). The NADH DHExt contribution to NADH oxidation increased with increasing NADH concentration.
Characterization of a Novel Non-Constitutive Photomorphogeniccop1Allele
Authors: Dieterle Monika , Büche Claudia , Schäfer Eberhard , Kretsch Thomas
Published: December 2003

A specific light program consisting of multiple treatments with alternating red and far-red light pulses was used to isolate mutants in phytochrome A-dependent signal transduction in Arabidopsis seedlings. Because of their phenotype, the mutants were called eid (empfindlicher im dunkelroten Licht, which means hypersensitive in far-red light). One of the isolated mutants, eid6, is a novel recessive allele of the COP1 gene (constitutive photomorphogenic 1) that carries an amino acid transition in a conserved histidine residue of the RING finger domain. Mutant seedlings exhibited an extreme hypersensitivity towards all tested light qualities, but in contrast to known cop1 alleles, no constitutive photomorphogenic phenotype was detectable in darkness. Thus, the novel cop1eid6 allele seems to encode for a protein whose remaining activity is sufficient for the suppression of photomorphogenesis in dark-grown plants. In adult cop1eid6 plants, the development of the Cop1 phenotype is dominated by phytochrome B. Comparison of the phenotype of the novel cop1eid6 and the weak cop1-4 allele under continuous far-red light indicates that the RING finger and coiled-coil domains of COP1 are sufficient for some specific regulatory function in phytochrome A-dependent high irradiance responses.
Finding Unexpected Patterns in Microarray Data
Authors: Perelman Susana , Mazzella María Agustina , Muschietti Jorge , Zhu Tong , Casal Jorge J.
Published: December 2003

We describe the performance of a protocol based on the sequential application of unsupervised and supervised methods to analyze microarray samples defined by a combination of factors. Correspondence analysis is used to visualize the emerging patterns of three set of novel or previously published data: photoreceptor mutants of Arabidopsis grown under different light/dark conditions, Arabidopsis exposed to different types of biotic and abiotic stress, and human acute leukemia. We find, for instance, that light has a dramatic effect on plants despite the absence of the four major photoreceptors, that bacterial-, fungal-, and viral-induced responses converge at later stages of attack, and that sample preparation procedures used in different hospitals have large effects on transcriptome patterns. We use canonical discriminant analysis to identify the genes associated with these patters and hierarchical clustering to find groups of coregulated genes that are easily visualized in a second round of correspondence analysis and ordered tables. The unconventional combination of standard descriptive multivariate methods offers a previously unrecognized tool to uncover unexpected information.
Phytochrome Modulation of Blue Light-Induced Chloroplast Movements in Arabidopsis
Authors: DeBlasio Stacy L. , Mullen Jack L. , Luesse Darron R. , Hangarter Roger P.
Published: December 2003

Photometric analysis of chloroplast movements in various phytochrome (phy) mutants of Arabidopsis showed that phyA, B, and D are not required for chloroplast movements because blue light (BL)-dependent chloroplast migration still occurs in these mutants. However, mutants lacking phyA or phyB showed an enhanced response at fluence rates of BL above 10 μmol m-2 s-1. Overexpression of phyA or phyB resulted in an enhancement of the low-light response. Analysis of chloroplast movements within the range of BL intensities in which the transition between the low- and high-light responses occur (1.5-15 μmol m-2 s-1) revealed a transient increase in light transmittance through leaves, indicative of the high-light response, followed by a decrease in transmittance to a value below that measured before the BL treatment, indicative of the low-light response. A biphasic response was not observed for phyABD leaves exposed to the same fluence rate of BL, suggesting that phys play a role in modulating the transition between the low- and high-light chloroplast movement responses of Arabidopsis.