bluuebunny/crossref_metadata_2025 huggingface dataset:
Temporal Differences in the Ability of Ethanol to Modulate Endotoxin‐Induced Increases in Inflammatory Cytokines in Muscle Under In Vivo Conditions
Authors: Frost Robert A. , Nystrom Gerald , Burrows Patricia V. , Lang Charles H.
Published: July 2005

Background:

Acute alcohol (EtOH) intoxication may both antagonize and potentiate the ability of monocytes/macrophages to respond to endotoxin (lipopolysaccharide LPS). The suppressive effects of EtOH predominate when the duration between EtOH and LPS administration is relatively short, whereas sensitization is observed under conditions when there is a relatively longer delay between EtOH and LPS exposure. Striated muscle is now recognized to possess components of both the afferent and efferent limbs of the innate immune system. The aim of the present study was to determine whether the interval between EtOH and LPS administration differentially affects the mRNA content for selected elements of the innate immune response in skeletal and cardiac muscle and to compare such changes with those occurring in liver and spleen.

Methods:

The content of mRNA for interleukin (IL)‐6, IL‐1β, tumor necrosis factor (TNF)‐α, and high‐mobility group box (HMGB)‐1, as well as toll‐like receptors (TLRs)‐2 and ‐4, were measured in gastrocnemius, heart, liver and spleen from rats orally gavaged with EtOH and then injected with LPS either two or 24 hr thereafter.

Results:

EtOH intoxication two hr before LPS acutely suppressed the increased IL‐6 mRNA in all tissues and antagonized the increase in plasma and tissue IL‐6 protein concentration. Similarly, EtOH blunted the LPS‐induced increase in tissue mRNA expression of TNF‐α and IL‐1β. In contrast, when LPS was given 24 hr after EtOH, the increased IL‐6 in striated muscle, but not in liver or spleen, was selectively potentiated. An enhanced LPS responsiveness was also observed for the late‐phase cytokine HMGB1 in all tissues; however, the increased tissue expression of TNF‐α and IL‐1β induced by LPS was not augmented. TLR4 mRNA was decreased in both heart and spleen (but unaltered in skeletal muscle and liver) of rats injected with LPS, and this change was prevented by pretreatment with EtOH. In contrast, EtOH alone increased TLR‐2 mRNA content of heart, liver, and spleen but not muscle. LPS also markedly increased TLR2 mRNA in the same three tissues under control conditions, but this increase was attenuated by EtOH administered either two or 24 hr before LPS.

Conclusions:

Under in vivo conditions, the interval between EtOH exposure and LPS differentially affected the synthesis of various cytokines. In this regard, EtOH administered within two hr of LPS generally suppressed IL‐6, IL‐1β, and TNF‐α mRNAs in muscle, heart, liver, and spleen. Delaying the exposure of animals to LPS for 24 hr after EtOH, however, accentuated the increase in IL‐6 and HMGB1, and for IL‐6, this increased sensitivity appeared localized to striated muscle.
Toward Efficient and Comprehensive Measurement of the Alcohol Problems Continuum in College Students: The Brief Young Adult Alcohol Consequences Questionnaire
Authors: Kahler Christopher W. , Strong David R. , Read Jennifer P.
Published: July 2005

Background:

Although a number of measures of alcohol problems in college students have been studied, the psychometric development and validation of these scales have been limited, for the most part, to methods based on classical test theory. In this study, we conducted analyses based on item response theory to select a set of items for measuring the alcohol problem severity continuum in college students that balances comprehensiveness and efficiency and is free from significant gender bias.

Method:

We conducted Rasch model analyses of responses to the 48‐item Young Adult Alcohol Consequences Questionnaire by 164 male and 176 female college students who drank on at least a weekly basis. An iterative process using item fit statistics, item severities, item discrimination parameters, model residuals, and analysis of differential item functioning by gender was used to pare the items down to those that best fit a Rasch model and that were most efficient in discriminating among levels of alcohol problems in the sample.

Results:

The process of iterative Rasch model analyses resulted in a final 24‐item scale with the data fitting the unidimensional Rasch model very well. The scale showed excellent distributional properties, had items adequately matched to the severity of alcohol problems in the sample, covered a full range of problem severity, and appeared highly efficient in retaining all of the meaningful variance captured by the original set of 48 items.

Conclusions:

The use of Rasch model analyses to inform item selection produced a final scale that, in both its comprehensiveness and its efficiency, should be a useful tool for researchers studying alcohol problems in college students. To aid interpretation of raw scores, examples of the types of alcohol problems that are likely to be experienced across a range of selected scores are provided.
Prevention of Ethanol‐Induced Behavioral Stimulation by d‐Penicillamine: A Sequestration Agent for Acetaldehyde
Authors: Font Laura , Miquel Marta , Aragon Carlos M. G.
Published: July 2005

Background:

d‐Penicillamine, a sulfhydryl amino acid derived from penicillin, is a highly selective agent for sequestering in vivo acetaldehyde, the first metabolic product of ethanol. A substantial amount of research supports the idea that brain acetaldehyde, produced by central ethanol metabolism, plays a key role in determining some of the behavioral effects of ethanol administration. This study addressed two questions. First, we tested if d‐penicillamine was able to modify the depressant effects of acetaldehyde on behavior. Second, we studied the effect of d‐penicillamine on ethanol‐induced behavioral stimulation.

Methods:

Mice were pretreated with 75.00 mg/kg of d‐penicillamine, and 30 min later, they received acetaldehyde at 0, 100, 200, or 300 mg/kg intraperitoneally. Different groups of animals were treated with 0.0, 37.5, 75, 150, or 300 mg/kg of d‐penicillamine simultaneously 30, 90, 150, or 210 min before the intraperitoneal administration of saline or 1.2, 1.8, 2.4, 3.0, or 3.6 g/kg of ethanol, respectively. The specificity of d‐penicillamine effects was addressed using two drugs: cocaine (4 mg/kg) and caffeine (15 mg/kg).

Results:

Our results revealed that behavioral depression caused by acetaldehyde (200 and 300 mg/kg) could be attenuated by d‐penicillamine treatment. In addition, d‐penicillamine was also effective in lowering behavioral locomotion induced by ethanol (1.8 and 2.4 g/kg), without altering spontaneous locomotor activity. This sulfhydryl amino acid specifically modified the effect of ethanol on locomotion because cocaine‐ or caffeine‐induced locomotion was unaffected. In addition, blood ethanol levels were not different between d‐penicillamine‐ and saline‐pretreated mice.

Conclusions:

Behavioral effects produced by acetaldehyde and ethanol are blocked when animals are treated with d‐penicillamine, an effective sequestration agent for acetaldehyde. These results suggest that some of the psychopharmacological effects, classically attributed to ethanol, could be mediated by its first metabolite, acetaldehyde.
RECENT DEVELOPMENTS IN THE GENETICS OF ALCOHOL‐RELATED PHENOTYPES
Authors: Hesselbrock Victor , Higuchi Susumu , Soyka Michael
Published: July 2005

This article presents the proceedings of a symposia held at the International Society for Biomedical Research on Alcoholism Congress in Mannheim, Germany, in October, 2004 and focused on recent developments in alcohol‐related phenotypes from three different research groups. The first presentation focused on the possible contribution of polymorphisms of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase‐2 (ALDH2) as contributors to alcohol‐related organ damage. As polymorphisms of both ADH and ALDH, these genes may explain individual differences in the concentration and elimination of ethanol and acetaldehyde in the blood after heavy drinking; they may also be useful for determining their possible contribution to possible alcohol‐related types of organ damage, including amnestic problems and polyneuropathy. A second presentation examined externalizing behavior phenotypes, including conduct disorder symptoms, aggression, and suicidal behavior, which are all prevalent among individuals with alcohol dependence. As part of the Collaborative Study on the Genetics of Alcoholism, a genome screen was performed in multiplex alcohol‐dependent families to identify chromosomal regions related to these three types of externalizing behaviors. Both the quantitative and qualitative phenotypes were examined, and evidence of linkage was found for several chromosomal regions. An area of chromosome 2 demonstrated linkages for suicidal behavior, conduct problems, and alcohol dependence, suggesting a possible sharing of genes for different externalizing behavior phenotypes. The last presentation focused on serotonin (5‐HT) as being a key neurotransmitter in antisocial alcoholism and related phenotypes. In a study of adult alcoholics, an association was found between a lower frequency of the 5‐HT 1B 861C allele, antisocial personality traits, and conduct disorder in alcohol dependence. Adult antisocial personality was more often found in male subjects. Based on the presented analyses, inconsistent but encouraging results were found to support the role of the 5‐HT 1B G 861 C polymorphism and antisocial behavior in alcohol dependence.
Involvement of Ethylene in Stress-Induced Expression of the TLC1.1 Retrotransposon from Lycopersicon chilense Dun.
Authors: Tapia Gerardo , Verdugo Isabel , Yañez Mónica , Ahumada Iván , Theoduloz Cristina , Cordero Cecilia , Poblete Fernando , González Enrique , Ruiz-Lara Simón
Published: July 2005

The TLC1 family is one of the four families of long terminal repeat (LTR) retrotransposons identified in the genome of Lycopersicon chilense. Here, we show that this family of retroelements is transcriptionally active and its expression is induced in response to diverse stress conditions such as wounding, protoplast preparation, and high salt concentrations. Several stress-associated signaling molecules, including ethylene, methyl jasmonate, salicylic acid, and 2,4-dichlorophenoxyacetic acid, are capable of inducing TLC1 family expression in vivo. A representative of this family, named TLC1.1, was isolated from a genomic library from L. chilense. Transient expression assays in leaf protoplasts and stably transformed tobacco (Nicotiana tabacum) plants demonstrate that the U3 domain of the 5′-LTR region of this element can drive stress-induced transcriptional activation of the β-glucuronidase reporter gene. Two 57-bp tandem repeated sequences are found in this region, including an 8-bp motif, ATTTCAAA, previously identified as an ethylene-responsive element box in the promoter region of ethylene-induced genes. Expression analysis of wild-type LTR and single and double ethylene-responsive element box mutants fused to the β-glucuronidase gene shows that these elements are required for ethylene-responsive gene expression in protoplasts and transgenic plants. We suggest that ethylene-dependent signaling is the main signaling pathway involved in the regulation of the expression of the TLC1.1 element from L. chilense.
A Role for Auxin Redistribution in the Responses of the Root System Architecture to Phosphate Starvation in Arabidopsis
Authors: Nacry Philippe , Canivenc Geneviève , Muller Bertrand , Azmi Abdelkrim , Van Onckelen Harry , Rossignol Michel , Doumas Patrick
Published: July 2005

The changes in root system architecture (RSA) triggered by phosphate (P) deprivation were studied in Arabidopsis (Arabidopsis thaliana) plants grown for 14 d on 1 mm or 3 μm P. Two different temporal phases were observed in the response of RSA to low P. First, lateral root (LR) development was promoted between days 7 and 11 after germination, but, after day 11, all root growth parameters were negatively affected, leading to a general reduction of primary root (PR) and LR lengths and of LR density. Low P availability had contrasting effects on various stages of LR development, with a marked inhibition of primordia initiation but a strong stimulation of activation of the initiated primordia. The involvement of auxin signaling in these morphological changes was investigated in wild-type plants treated with indole-3-acetic acid or 2,3,5-triiodobenzoic acid and in axr4-1, aux1-7, and eir1-1 mutants. Most effects of low P on RSA were dramatically modified in the mutants or hormone-treated wild-type plants. This shows that auxin plays a major role in the P starvation-induced changes of root development. From these data, we hypothesize that several aspects of the RSA response to low P are triggered by local modifications of auxin concentration. A model is proposed that postulates that P starvation results in (1) an overaccumulation of auxin in the apex of the PR and in young LRs, (2) an overaccumulation of auxin or a change in sensitivity to auxin in the lateral primordia, and (3) a decrease in auxin concentration in the lateral primordia initiation zone of the PR and in old laterals. Measurements of local changes in auxin concentrations induced by low P, either by direct quantification or by biosensor expression pattern (DR5::β-glucuronidase reporter gene), are in line with these hypotheses. Furthermore, the observation that low P availability mimicked the action of auxin in promoting LR development in the alf3 mutant confirmed that P starvation stimulates primordia emergence through increased accumulation of auxin or change in sensitivity to auxin in the primordia. Both the strong effect of 2,3,5-triiodobenzoic acid and the phenotype of the auxin-transport mutants (aux1, eir1) suggest that low P availability modifies local auxin concentrations within the root system through changes in auxin transport rather than auxin synthesis.
Autophagic Nutrient Recycling in Arabidopsis Directed by the ATG8 and ATG12 Conjugation Pathways
Authors: Thompson Allison R. , Doelling Jed H. , Suttangkakul Anongpat , Vierstra Richard D.
Published: July 2005

Autophagy is an important mechanism for nonselective intracellular breakdown whereby cytosol and organelles are encapsulated in vesicles, which are then engulfed and digested by lytic vacuoles/lysosomes. In yeast, this encapsulation employs a set of autophagy (ATG) proteins that direct the conjugation of two ubiquitin-like protein tags, ATG8 and ATG12, to phosphatidylethanolamine and the ATG5 protein, respectively. Using an Arabidopsis (Arabidopsis thaliana) atg7 mutant unable to ligate either tag, we previously showed that the ATG8/12 conjugation system is important for survival under nitrogen-limiting growth conditions. By reverse-genetic analyses of the single Arabidopsis gene encoding ATG5, we show here that the subpathway that forms the ATG12-ATG5 conjugate also has an essential role in plant nutrient recycling. Similar to plants missing ATG7, those missing ATG5 display early senescence and are hypersensitive to either nitrogen or carbon starvation, which is accompanied by a more rapid loss of organellar and cytoplasmic proteins. Multiple ATG8 isoforms could be detected immunologically in seedling extracts. Their abundance was substantially elevated in both the atg5 and atg7 mutants, caused in part by an increase in abundance of several ATG8 mRNAs. Using a green fluorescent protein-ATG8a fusion in combination with concanamycin A, we also detected the accumulation of autophagic bodies inside the vacuole. This accumulation was substantially enhanced by starvation but blocked in the atg7 background. The use of this fusion in conjunction with atg mutants now provides an important marker to track autophagic vesicles in planta.
Functional Redundancy of AtFtsH Metalloproteases in Thylakoid Membrane Complexes
Authors: Yu Fei , Park Sungsoon , Rodermel Steven R.
Published: July 2005

FtsH is an ATP-dependent metalloprotease found in bacteria, mitochondria, and plastids. Arabidopsis (Arabidopsis thaliana) contains 12 AtFtsH proteins, three in the mitochondrion and nine in the chloroplast. Four of the chloroplast FtsH proteins are encoded by paired members of closely related genes (AtFtsH1 and 5, and AtFtsH2 and 8). We have previously reported that AtFtsH2 and 8 are interchangeable components of AtFtsH complexes in the thylakoid membrane. In this article, we show that the var1 variegation mutant, which is defective in AtFtsH5, has a coordinate reduction in the AtFtsH2 and 8 pair, and that the levels of both pairs are restored to normal in var1 plants that overexpress AtFtsH1. Overexpression of AtFtsH1, but not AtFtsH2/VAR2, normalizes the pattern of var1 variegation, restoring a nonvariegated phenotype. We conclude that AtFtsH proteins within a pair, but not between pairs, are interchangeable and functionally redundant, at least in part. We further propose that the abundance of each pair is matched with that of the other pair, with excess subunits being turned over. The variegation phenotype of var1 (as well as var2, which is defective in AtFtsH2) suggests that a threshold concentration of subunits is required for normal chloroplast function. AtFtsH1, 2, 5, and 8 do not show evidence of tissue or developmental specific expression. Phylogenetic analyses revealed that rice (Oryza sativa) and Arabidopsis share a conserved core of seven FtsH subunit genes, including the AtFtsH1 and 5 and AtFtsH2 and 8 pairs, and that the structure of the present-day gene families can be explained by duplication events in each species following the monocot/dicot divergence.
Arabidopsis Peroxin 16 Coexists at Steady State in Peroxisomes and Endoplasmic Reticulum
Authors: Karnik Sheetal K. , Trelease Richard N.
Published: July 2005

Homologs of peroxin 16 genes (PEX16) have been identified only in Yarrowia lipolytica, humans (Homo sapiens), and Arabidopsis (Arabidopsis thaliana). The Arabidopsis gene (AtPEX16), previously reported as the SSE1 gene, codes for a predicted 42-kD membrane peroxin protein (AtPex16p). Lin et al. (Y. Lin, J.E. Cluette-Brown, H.M. Goodman [2004] Plant Physiol 135: 814–827) reported that SSE1/AtPEX16 was essential for endoplasmic reticulum (ER)-dependent oil and protein body biogenesis in peroxisome-deficient maturing seeds and likely also was involved in peroxisomal biogenesis based on localization of stably expressed green fluorescent protein::AtPex16p in peroxisomes of Arabidopsis plants. In this study with Arabidopsis suspension-cultured cells, combined in vivo and in vitro experiments revealed a novel dual organelle localization and corresponding membrane association/topology of endogenous AtPex16p. Immunofluorescence microscopy with antigen affinity-purified IgGs showed an unambiguous, steady-state coexistence of AtPex16p in suspension cell peroxisomes and ER. AtPex16p also was observed in peroxisomes and ER of root and leaf cells. Cell fractionation experiments surprisingly revealed two immunorelated polypeptides, 42 kD (expected) and 52 kD (unexpected), in homogenates and microsome membrane pellets derived from roots, inflorescence, and suspension cells. Suc-gradient purifications confirmed the presence of both 42-kD and 52-kD polypeptides in isolated peroxisomes (isopycnic separation) and in rough ER vesicles (Mg2+ shifted). They were found peripherally associated with peroxisome and ER membranes but not as covalently bound subunits of AtPex16p. Both were mostly on the matrix side of peroxisomal membranes and unexpectedly mostly on the cytosolic side of ER membranes. In summary, AtPex16p is the only authentic plant peroxin homolog known to coexist at steady state within peroxisomes and ER; these data provide new insights in support of its ER-related, multifunctional roles in organelle biogenesis.
Arabidopsiscyp51Mutant Shows Postembryonic Seedling Lethality Associated with Lack of Membrane Integrity
Authors: Kim Ho Bang , Schaller Hubert , Goh Chang-Hyo , Kwon Mi , Choe Sunghwa , An Chung Sun , Durst Francis , Feldmann Kenneth A. , Feyereisen René
Published: July 2005

CYP51 exists in all organisms that synthesize sterols de novo. Plant CYP51 encodes an obtusifoliol 14α-demethylase involved in the postsqualene sterol biosynthetic pathway. According to the current gene annotation, the Arabidopsis (Arabidopsis thaliana) genome contains two putative CYP51 genes, CYP51A1 and CYP51A2. Our studies revealed that CYP51A1 should be considered an expressed pseudogene. To study the functional importance of the CYP51A2 gene in plant growth and development, we isolated T-DNA knockout alleles for CYP51A2. Loss-of-function mutants for CYP51A2 showed multiple defects, such as stunted hypocotyls, short roots, reduced cell elongation, and seedling lethality. In contrast to other sterol mutants, such as fk/hydra2 and hydra1, the cyp51A2 mutant has only minor defects in early embryogenesis. Measurements of endogenous sterol levels in the cyp51A2 mutant revealed that it accumulates obtusifoliol, the substrate of CYP51, and a high proportion of 14α-methyl-Δ8-sterols, at the expense of campesterol and sitosterol. The cyp51A2 mutants have defects in membrane integrity and hypocotyl elongation. The defect in hypocotyl elongation was not rescued by the exogenous application of brassinolide, although the brassinosteroid-signaling cascade is apparently not affected in the mutants. Developmental defects in the cyp51A2 mutant were completely rescued by the ectopic expression of CYP51A2. Taken together, our results demonstrate that the Arabidopsis CYP51A2 gene encodes a functional obtusifoliol 14α-demethylase enzyme and plays an essential role in controlling plant growth and development by a sterol-specific pathway.
Possible Involvement of Phototropins in Leaf Movement of Kidney Bean in Response to Blue Light
Authors: Inoue Shin-ichiro , Kinoshita Toshinori , Shimazaki Ken-ichiro
Published: July 2005

The leaf of kidney bean (Phaseolus vulgaris) moves in response to blue light. The movement is induced by a decrease in the turgor pressure of pulvinar motor cells on the irradiated side. In this study, we investigated the initial event of the movement with respect to function of phototropin and the plasma membrane H+-ATPase in the motor cells. The results indicated that, in dark conditions, phototropin existed in a dephosphorylated state and the H+-ATPase existed in a phosphorylated state. A pulse of blue light (30 s) induced the phosphorylation of phototropin and the dephosphorylation of the H+-ATPase as determined by the binding behavior of 14-3-3 protein. Phototropin phosphorylation occurred rapidly, followed by the transient gradual dephosphorylation of the H+-ATPase. When the specific flavoprotein inhibitor diphenyleneiodonium and the protein kinase inhibitors K-252a and staurosporine were administered to pulvinar cells, both phototropin phosphorylation and H+-ATPase dephosphorylation were inhibited. The phosphorylation and dephosphorylation exhibited similar fluence rate dependencies to blue light. These results indicated that phototropin may function upstream of the plasma membrane H+-ATPase and decrease the activity of H+-ATPase by dephosphorylation. We provide evidence for the existence of three kinds of phototropins in pulvinar motor cells.
SEC8, a Subunit of the Putative Arabidopsis Exocyst Complex, Facilitates Pollen Germination and Competitive Pollen Tube Growth
Authors: Cole Rex A. , Synek Lukás , Zarsky Viktor , Fowler John E.
Published: July 2005

The exocyst, a complex of eight proteins, contributes to the morphogenesis of polarized cells in a broad range of eukaryotes. In these organisms, the exocyst appears to facilitate vesicle docking at the plasma membrane during exocytosis. Although we had identified orthologs for each of the eight exocyst components in Arabidopsis (Arabidopsis thaliana), no function has been demonstrated for any of them in plants. The gene encoding one exocyst component ortholog, AtSEC8, is expressed in pollen and vegetative tissues of Arabidopsis. Genetic studies utilizing an allelic series of six independent T-DNA mutations reveal a role for SEC8 in male gametophyte function. Three T-DNA insertions in SEC8 cause an absolute, male-specific transmission defect that can be complemented by expression of SEC8 from the LAT52 pollen promoter. Microscopic analysis shows no obvious abnormalities in the microgametogenesis of the SEC8 mutants, and the mutant pollen grains appear to respond to the signals that initiate germination. However, in vivo assays indicate that these mutant pollen grains are unable to germinate a pollen tube. The other three T-DNA insertions are associated with a partial transmission defect, such that the mutant allele is transmitted through the pollen at a reduced frequency. The partial transmission defect is only evident when mutant gametophytes must compete with wild-type gametophytes, and arises in part from a reduced pollen tube growth rate. These data support the hypothesis that one function of the putative plant exocyst is to facilitate the initiation and maintenance of the polarized growth of pollen tubes.
Adaptations Required for Mitochondrial Import following Mitochondrial to Nucleus Gene Transfer of Ribosomal Protein S10
Authors: Murcha Monika W. , Rudhe Charlotta , Elhafez Dina , Adams Keith L. , Daley Daniel O. , Whelan James
Published: July 2005

The minimal requirements to support protein import into mitochondria were investigated in the context of the phenomenon of ongoing gene transfer from the mitochondrion to the nucleus in plants. Ribosomal protein 10 of the small subunit is encoded in the mitochondrion in soybean and many other angiosperms, whereas in several other species it is nuclear encoded and thus must be imported into the mitochondrial matrix to function. When encoded by the nuclear genome, it has adopted different strategies for mitochondrial targeting and import. In lettuce (Lactuca sativa) and carrot (Daucus carota), Rps10 independently gained different N-terminal extensions from other genes, following transfer to the nucleus. (The designation of Rps10 follows the following convention. The gene is indicated in italics. If encoded in the mitochondrion, it is rps10; if encoded in the nucleus, it is Rps10.) Here, we show that the N-terminal extensions of Rps10 in lettuce and carrot are both essential for mitochondrial import. In maize (Zea mays), Rps10 has not acquired an extension upon transfer but can be readily imported into mitochondria. Deletion analysis located the mitochondrial targeting region to the first 20 amino acids. Using site directed mutagenesis, we changed residues in the first 20 amino acids of the mitochondrial encoded soybean (Glycine max) rps10 to the corresponding amino acids in the nuclear encoded maize Rps10 until import was achieved. Changes were required that altered charge, hydrophobicity, predicted ability to form an amphiphatic α-helix, and generation of a binding motif for the outer mitochondrial membrane receptor, translocase of the outer membrane 20. In addition to defining the changes required to achieve mitochondrial localization, the results demonstrate that even proteins that do not present barriers to import can require substantial changes to acquire a mitochondrial targeting signal.
Virus-Induced Gene Silencing-Based Functional Characterization of Genes Associated with Powdery Mildew Resistance in Barley
Authors: Hein Ingo , Barciszewska-Pacak Maria , Hrubikova Katarina , Williamson Sandie , Dinesen Malene , Soenderby Ida E. , Sundar Suresh , Jarmolowski Artur , Shirasu Ken , Lacomme Christophe
Published: July 2005

We successfully implemented virus-induced gene silencing (VIGS) in barley (Hordeum vulgare) for the functional characterization of genes required for Mla13-mediated resistance toward the biotrophic barley pathogen Blumeria graminis f. sp. hordei. Initially, barley cultivars were screened for their ability to host the barley stripe mosaic virus (BSMV)-VIGS vector by allowing its replication and systemic movement without causing excessive symptoms. Phytoene desaturase silencing leading to photobleaching was used as a phenotypic marker alongside reverse transcription-PCR data to characterize the silencing response at the molecular level. Barley cultivar Clansman, harboring the Mla13 resistance gene, was chosen as the most suitable host for BSMV-VIGS-based functional characterization of Rar1, Sgt1, and Hsp90 in the Mla-mediated resistance toward powdery mildew. BSMV-induced gene silencing of these candidate genes, which are associated in many but not all race-specific pathways, proved to be robust and could be detected at both mRNA and protein levels for up to 21 d postinoculation. Systemic silencing was observed not only in the newly developed leaves from the main stem but also in axillary shoots. By examining fungal development from an incompatible mildew strain carrying the cognate Avr13 gene on plants BSMV silenced for Rar1, Sgt1, and Hsp90, a resistance-breaking phenotype was observed, while plants infected with BSMV control constructs remained resistant. We demonstrate that Hsp90 is a required component for Mla13-mediated race-specific resistance and that BSMV-induced VIGS is a powerful tool to characterize genes involved in pathogen resistance in barley.
Structure-Based in Vitro Engineering of the Anthranilate Synthase, a Metabolic Key Enzyme in the Plant Tryptophan Pathway
Authors: Kanno Takuya , Komatsu Akira , Kasai Koji , Dubouzet Joseph G. , Sakurai Minako , Ikejiri-Kanno Yasuko , Wakasa Kyo , Tozawa Yuzuru
Published: July 2005

Rice (Oryza sativa) anthranilate synthase α-subunit, OASA2, was modified by in vitro mutagenesis based on structural information from bacterial homologs. Twenty-four amino acid residues, predicted as putative tryptophan binding sites or their proximal regions in the OASA2 sequence, were selected and 36 mutant OASA2 genes were constructed by PCR-based site-directed mutagenesis. Corresponding mutant proteins were synthesized in a combination of two in vitro systems, transcription with a bacteriophage SP6 RNA polymerase and translation with a wheat-embryo cell-free system. Enzymatic functions of the mutant proteins were simultaneously examined, and we found six mutants with elevated catalytic activity and five mutants with enhanced tolerance to feedback inhibition by tryptophan. Moreover, we observed that some sets of specific combinations of the novel mutations additively conferred both characteristics to the mutant enzymes. The functions of the mutant enzymes were confirmed in vivo. The free tryptophan content of mutant rice calli expressing OASA2 enzyme with a double mutation was 30-fold of that of untransformed calli. Thus, our in vitro approach utilizing structural information of bacterial homologs is a potent technique to generate designer enzymes with predefined functions.
Expression of Arabidopsis MIRNA Genes
Authors: Xie Zhixin , Allen Edwards , Fahlgren Noah , Calamar Adam , Givan Scott A. , Carrington James C.
Published: July 2005

MicroRNAs (miRNAs) are approximately 21-nucleotide noncoding RNAs that regulate target transcripts in plants and animals. In addition to miRNAs, plants contain several classes of endogenous small interfering RNAs (siRNAs) involved in target gene regulation and epigenetic silencing. Small RNA libraries were constructed from wild-type Arabidopsis (Arabidopsis thaliana) and mutant plants (rdr2 and dcl3) that were genetically enriched for miRNAs, and a computational procedure was developed to identify candidate miRNAs. Thirty-eight distinct miRNAs corresponding to 22 families were represented in the libraries. Using a 5′ rapid amplification of cDNA ends procedure, the transcription start sites for 63 miRNA primary transcripts from 52 MIRNA loci (99 loci tested) were mapped, revealing features consistent with an RNA polymerase II mechanism of transcription. Ten loci (19%) yielded transcripts from multiple start sites. A canonical TATA box motif was identified upstream of the major start site at 45 (86%) of the mapped MIRNA loci. The 5′-mapping data were combined with miRNA cloning and 3′-PCR data to definitively validate expression of at least 73 MIRNA genes. These data provide a molecular basis to explore regulatory mechanisms of miRNA expression in plants.
Analysis of the Rice Mutant dwarf and gladius leaf 1. Aberrant Katanin-Mediated Microtubule Organization Causes Up-Regulation of Gibberellin Biosynthetic Genes Independently of Gibberellin Signaling
Authors: Komorisono Masahiko , Ueguchi-Tanaka Miyako , Aichi Ikuko , Hasegawa Yasuko , Ashikari Motoyuki , Kitano Hidemi , Matsuoka Makoto , Sazuka Takashi
Published: July 2005

Molecular genetic studies of plant dwarf mutants have indicated that gibberellin (GA) and brassinosteroid (BR) are two major factors that determine plant height; dwarf mutants that are caused by other defects are relatively rare, especially in monocot species. Here, we report a rice (Oryza sativa) dwarf mutant, dwarf and gladius leaf 1 (dgl1), which exhibits only minimal response to GA and BR. In addition to the dwarf phenotype, dgl1 produces leaves with abnormally rounded tip regions. Positional cloning of DGL1 revealed that it encodes a 60-kD microtubule-severing katanin-like protein. The protein was found to be important in cell elongation and division, based on the observed cell phenotypes. GA biosynthetic genes are up-regulated in dgl1, but the expression of BR biosynthetic genes is not enhanced. The enhanced expression of GA biosynthetic genes in dgl1 is not caused by inappropriate GA signaling because the expression of these genes was repressed by GA3 treatment, and degradation of the rice DELLA protein SLR1 was triggered by GA3 in this mutant. Instead, aberrant microtubule organization caused by the loss of the microtubule-severing function of DGL1 may result in enhanced expression of GA biosynthetic genes in that enhanced expression was also observed in a BR-deficient mutant with aberrant microtubule organization. These results suggest that the function of DGL1 is important for cell and organ elongation in rice, and aberrant DGL1-mediated microtubule organization causes up-regulation of gibberellin biosynthetic genes independently of gibberellin signaling.
An RNA-Dependent RNA Polymerase Prevents Meristem Invasion by Potato Virus X and Is Required for the Activity But Not the Production of a Systemic Silencing Signal
Authors: Schwach Frank , Vaistij Fabian E. , Jones Louise , Baulcombe David C.
Published: July 2005

One of the functions of RNA silencing in plants is antiviral defense. A hallmark of RNA silencing is spreading of the silenced state through the plant. Little is known about the nature of the systemic silencing signal and the proteins required for its production, transport, and reception in plant tissues. Here, we show that the RNA-dependent RNA polymerase RDR6 in Nicotiana benthamiana is involved in defense against potato virus X at the level of systemic spreading and in exclusion of the virus from the apical growing point. It has no effect on primary replication and cell-to-cell movement of the virus and does not contribute significantly to the formation of virus-derived small interfering (si) RNA in a fully established potato virus X infection. In grafting experiments, the RDR6 homolog was required for the ability of a cell to respond to, but not to produce or translocate, the systemic silencing signal. Taking these findings together, we suggest a model of virus defense in which RDR6 uses incoming silencing signal to generate double-stranded RNA precursors of secondary siRNA. According to this idea, the secondary siRNAs mediate RNA silencing as an immediate response that slows down the systemic spreading of the virus into the growing point and newly emerging leaves.
Fractionation of the Three Stable Oxygen Isotopes by Oxygen-Producing and Oxygen-Consuming Reactions in Photosynthetic Organisms
Authors: Helman Yael , Barkan Eugeni , Eisenstadt Doron , Luz Boaz , Kaplan Aaron
Published: July 2005

The triple isotope composition (δ 17O and δ 18O) of dissolved O2 in the ocean and in ice cores was recently used to assess the primary productivity over broad spatial and temporal scales. However, assessment of the productivity with the aid of this method must rely on accurate measurements of the 17O/16O versus 18O/16O relationship in each of the main oxygen-producing and -consuming reactions. Data obtained here showed that cleavage of water in photosystem II did not fractionate oxygen isotopes; the δ 18O and δ 17O of the O2 evolved were essentially identical to those of the substrate water. The fractionation slopes for the oxygenase reaction of Rubisco and respiration were identical (0.518 ± 0.001) and that of glycolate oxidation was 0.503 ± 0.002. There was a considerable difference in the slopes of O2 photoreduction (the Mehler reaction) in the cyanobacterium Synechocystis sp. strain PCC 6803 (0.497 ± 0.004) and that of pea (Pisum sativum) thylakoids (0.526 ± 0.001). These values provided clear and independent evidence that the mechanism of O2 photoreduction differs between higher plants and cyanobacteria. We used our method to assess the magnitude of O2 photoreduction in cyanobacterial cells maintained under conditions where photorespiration was negligible. It was found that electron flow to O2 can be as high as 40% that leaving photosystem II, whereas respiratory activity in the light is only 6%. The implications of our findings to the evaluation of specific O2-producing or -consuming reactions, in vivo, are discussed.
Sites and Regulation of Polyamine Catabolism in the Tobacco Plant. Correlations with Cell Division/Expansion, Cell Cycle Progression, and Vascular Development
Authors: Paschalidis Konstantinos A. , Roubelakis-Angelakis Kalliopi A.
Published: July 2005

We previously gave a picture of the homeostatic characteristics of polyamine (PA) biosynthesis and conjugation in tobacco (Nicotiana tabacum) plant organs during development. In this work, we present the sites and regulation of PA catabolism related to cell division/expansion, cell cycle progression, and vascular development in the tobacco plant. Diamine oxidase (DAO), PA oxidase (PAO), peroxidases (POXs), and putrescine N-methyltransferase expressions follow temporally and spatially discrete patterns in shoot apical cells, leaves (apical, peripheral, and central regions), acropetal and basipetal petiole regions, internodes, and young and old roots in developing plants. DAO and PAO produce hydrogen peroxide, a plant signal molecule and substrate for POXs. Gene expression and immunohistochemistry analyses reveal that amine oxidases in developing tobacco tissues precede and overlap with nascent nuclear DNA and also with POXs and lignification. In mature and old tissues, flow cytometry indicates that amine oxidase and POX activities, as well as pao gene and PAO protein levels, coincide with G2 nuclear phase and endoreduplication. In young versus the older roots, amine oxidases and POX expression decrease with parallel inhibition of G2 advance and endoreduplication, whereas putrescine N-methyltransferase dramatically increases. In both hypergeous and hypogeous tissues, DAO and PAO expression occurs in cells destined to undergo lignification, suggesting a different in situ localization. DNA synthesis early in development and the advance in cell cycle/endocycle are temporally and spatially related to PA catabolism and vascular development.
Characteristics of Electrical Signals in Poplar and Responses in Photosynthesis
Authors: Lautner Silke , Grams Thorsten Erhard Edgar , Matyssek Rainer , Fromm Jörg
Published: July 2005

To gain an understanding of the role of electrical signaling in trees, poplar (Populus trichocarpa, Populus tremula × P. tremuloides) shoots were stimulated by chilling as well as flaming. Two kinds of signal propagation were detected by microelectrode measurements (aphid technique) in the phloem of leaf veins: (1) basipetal, short-distance signaling that led to rapid membrane hyperpolarization caused by K+-efflux within the leaf lamina; and (2) acropetal, long-distance signaling that triggered depolarization of the membrane potential in the leaf phloem. In the latter, the depolarizing signals travel across the stem from the manipulated leaves to adjacent leaves where the net CO2 uptake rate is temporarily depressed toward compensation. With regard to photosystem II, both heat-induced long-distance and short-distance signaling were investigated using two-dimensional “imaging” analysis of chlorophyll fluorescence. Both types of signaling significantly reduced the quantum yield of electron transport through photosystem II. Imaging analysis revealed that the signal that causes yield reduction spreads through the leaf lamina. Coldblocking of the stem proved that the electrical signal transmission via the phloem becomes disrupted, causing the leaf gas exchange to remain unaffected. Calcium-deficient trees showed a marked contrast inasmuch as the amplitude of the electrical signal was distinctly reduced, concomitant with the absence of a significant response in leaf gas exchange upon flame wounding. In summary, the above results led us to conclude that calcium as well as potassium is involved in the propagation of phloem-transmitted electrical signals that evoke specific responses in the photosynthesis of leaves.
Molecular Identification and Characterization of the Arabidopsis Δ3,5,Δ2,4-Dienoyl-Coenzyme A Isomerase, a Peroxisomal Enzyme Participating in the β-Oxidation Cycle of Unsaturated Fatty Acids
Authors: Goepfert Simon , Vidoudez Charles , Rezzonico Enea , Hiltunen J. Kalervo , Poirier Yves
Published: July 2005

Degradation of unsaturated fatty acids through the peroxisomal β-oxidation pathway requires the participation of auxiliary enzymes in addition to the enzymes of the core β-oxidation cycle. The auxiliary enzyme Δ3,5,Δ2,4-dienoyl-coenzyme A (CoA) isomerase has been well studied in yeast (Saccharomyces cerevisiae) and mammals, but no plant homolog had been identified and characterized at the biochemical or molecular level. A candidate gene (At5g43280) was identified in Arabidopsis (Arabidopsis thaliana) encoding a protein showing homology to the rat (Rattus norvegicus) Δ3,5,Δ2,4-dienoyl-CoA isomerase, and possessing an enoyl-CoA hydratase/isomerase fingerprint as well as aspartic and glutamic residues shown to be important for catalytic activity of the mammalian enzyme. The protein, named AtDCI1, contains a peroxisome targeting sequence at the C terminus, and fusion of a fluorescent protein to AtDCI1 directed the chimeric protein to the peroxisome in onion (Allium cepa) cells. AtDCI1 expressed in Escherichia coli was shown to have Δ3,5,Δ2,4-dienoyl-CoA isomerase activity in vitro. Furthermore, using the synthesis of polyhydroxyalkanoate in yeast peroxisomes as an analytical tool to study the β-oxidation cycle, expression of AtDCI1 was shown to complement the yeast mutant deficient in the Δ3,5,Δ2,4-dienoyl-CoA isomerase, thus showing that AtDCI1 is also appropriately targeted to the peroxisome in yeast and has Δ3,5,Δ2,4-dienoyl-CoA isomerase activity in vivo. The AtDCI1 gene is expressed constitutively in several tissues, but expression is particularly induced during seed germination. Proteins showing high homology with AtDCI1 are found in gymnosperms as well as angiosperms belonging to the Monocotyledon or Dicotyledon classes.
Arabidopsis ARP7 Is an Essential Actin-Related Protein Required for Normal Embryogenesis, Plant Architecture, and Floral Organ Abscission
Authors: Kandasamy Muthugapatti K. , McKinney Elizabeth C. , Deal Roger B. , Meagher Richard B.
Published: July 2005

The actin-related proteins (ARPs) that are localized to the nucleus are present as components of various chromatin-modifying complexes involved in chromatin dynamics and transcriptional regulation. Arabidopsis (Arabidopsis thaliana) ARP7 is a constitutively expressed nuclear protein belonging to a novel plant-specific ARP class. In this study, we demonstrate a vital role for ARP7 protein in embryogenesis and plant development. Knocking out the expression of ARP7 in an arp7-1 T-DNA mutant produced morphologically aberrant, homozygous embryos that were arrested at or before the torpedo stage of development. Hence, the arp7-1 null mutation is homozygous lethal. Knocking down the expression levels of ARP7 protein with RNA interference produced viable Arabidopsis lines affected in multiple developmental pathways and induced dosage-dependent, heritable defects in plant architecture. The transgenic plants containing greatly reduced levels of ARP7 in the nucleus were severely dwarfed with small rosette leaves that are defective in cell expansion and trichome morphology. Moreover, the ARP7-deficient RNA interference plants exhibited retarded root growth, altered flower development, delayed perianth abscission, and reduced fertility. These pleiotropic phenotypic changes suggest a critical role for the Arabidopsis ARP7 protein in the regulation of various phases of plant development through chromatin-mediated, global regulation of gene expression.
The Tobacco Mosaic Virus 126-Kilodalton Protein, a Constituent of the Virus Replication Complex, Alone or within the Complex Aligns with and Traffics along Microfilaments
Authors: Liu Jian-Zhong , Blancaflor Elison B. , Nelson Richard S.
Published: July 2005

Virus-induced cytoplasmic inclusion bodies (referred to as virus replication complexes [VRCs]) consisting of virus and host components are observed in plant cells infected with tobacco mosaic virus, but the components that modulate their form and function are not fully understood. Here, we show that the tobacco mosaic virus 126-kD protein fused with green fluorescent protein formed cytoplasmic bodies (126-bodies) in the absence of other viral components. Using mutant 126-kD:green fluorescent fusion proteins and viral constructs expressing the corresponding mutant 126-kD proteins, it was determined that the size of the 126-bodies and the corresponding VRCs changed in synchrony for each 126-kD protein mutation tested. Through colabeling experiments, we observed the coalignment and intracellular trafficking of 126-bodies and, regardless of size, VRCs, along microfilaments (MFs). Disruption of MFs with MF-depolymerizing agents or through virus-induced gene silencing compromised the intracellular trafficking of the 126-bodies and VRCs and virus cell-to-cell movement, but did not decrease virus accumulation to levels that would affect virus movement or prevent VRC formation. Our results indicate that (1) the 126-kD protein modulates VRC size and traffics along MFs in cells; (2) VRCs traffic along MFs in cells, possibly through an interaction with the 126-kD protein, and the negative effect of MF antagonists on 126-body and VRC intracellular movement and virus cell-to-cell movement correlates with the disruption of this association; and (3) virus movement was not correlated with VRC size.