bluuebunny/crossref_metadata_2025_split huggingface dataset:
An Arabidopsis mutant deficient in sterol biosynthesis: heterologous complementation by ERG 3 encoding a Δ7‐sterol‐C‐5‐desaturase from yeast
Authors: Gachotte Daniel , Meens Roger , Benveniste Pierre 
Published: March 2003

The mutant STE 1 was isolated by screening an ethylmethane sulfonate (EMS)‐mutagenized population of Arabidopsis thaliana which consisted of 22 000 M2 plants divided into 1100 pools of 20 plants by gas chromatography of sterols extracted from small leaf samples.STE 1 was characterized by the accumulation of three Δ7‐sterols concomitantly with the decrease of the three corresponding Δ5‐sterols which are the end products of the sterol pathway in wild‐type leaves. The structure of these Δ7‐sterols was determined after two steps of purification on HPLC, by gas chromatography coupled with mass spectrometry (GC‐MS) and proton nuclear magnetic resonance spectrometry (1H‐NMR). The accumulation of Δ7‐sterols suggested that the mutant is deficient in the activity of the Δ7‐sterol‐C‐5‐desaturase.Genetic analysis showed that the accumulation of Δ7‐sterols was due to a single recessive nuclear mutation. The mutant line STE 1 was backcrossed four times to the wild‐type. The resulting STE 1 plants had wild‐type morphology and set seeds normally, suggesting that the Δ7‐sterols in STE 1 are good surrogates of physiologically active Δ5‐sterols to sustain normal development.STE 1 roots were transformed with the Saccharomyces cerevisiae ERG 3 gene encoding the Δ7‐sterol‐C‐5‐desaturase under the control of the CaMV 35S promoter. Seven transgenic STE 1 root‐derived calli showed an increase in Δ5‐sterols and a concomitant decrease in Δ7‐sterols in comparison with STE 1 untransformed root‐derived calli. Northern blot analysis using the ERG 3 probe showed a strong expression of ERG 3 in three of the seven transgenic calli. These results suggest that the accumulation of Δ7‐sterols in the STE 1 mutant is due to a deficiency of the Δ7‐sterol‐C‐5‐desaturation step in the plant sterol biosynthesis pathway.
One of three nuclear localization signals of maize Activator (Ac) transposase overlaps the DNA‐binding domain
Authors: Boehm Ulrich , Heinlein Manfred , Behrens Ute , Kunze Reinhard 
Published: March 2003

SummaryThe nuclear localization sequences (NLSs) of the Ac transposase (TPase) protein have been characterized by indirect immunofluorescence detection of TPase deletion derivatives and TPase/β‐glucuronidase (GUS) fusion proteins in transiently transfected Petunia cells. The TPase contains three NLSs near its amino‐terminal end, NLS(44–62), NLS(159–178) and NLS(174–206), each of which is sufficient to redirect GUS to the nucleus. Deletion of the N‐terminal 102 TPase residues including NLS(44–62) results in strongly reduced nuclear import of the truncated TPase. NLS(44–62) and NLS(159–178) are bipartite NLSs, whereas the structure of NLS(174–206) does not allow a classification into one of the three major NLS categories. NLS(174–206) overlaps with the basic DNA‐binding domain of TPase. A substitution of two amino acids in this segment (HiS191→Arg and Arg193→His) results in a total loss of DNA‐binding activity, but retains reduced NLS activity. Accordingly, the two functions can be separated. In addition, we show that a NLS‐deficient 71 kDa TPase derivative is co‐imported into the nucleus in the presence of wildtype TPase.
Characterization and partial purification of two pre‐tRNA 5′‐processing activities from Daucus carrota (carrot) suspension cells
Authors: Franklin Scott E. , Zwick Michael G. , Johnson Jerry D. 
Published: March 2003

SummaryTwo distinct RNase P‐like activities which cleave leader sequences from pre‐tRNA molecules to give mature 5′ ends have been identified in carrot suspension‐culture cells. An Escherichia coli pre‐tRNAPhe and a tobacco pre‐tRNATyr were transcribed in vitro then used as substrates for processing reactions in a cell‐free extract. The pre‐tRNATyr transcript was used to establish optimal salt and divalent cation requirements for processing. Kinetic experiments were then carried out on both substrates to determine if 5′ and 3′ processing were ordered. Primer extension analysis of processing intermediates and stable products verified that an ammonium sulfate fraction of the extract was indeed capable of accurately processing the 5′ ends of both pre‐tRNAs. Subsequent fractionation of the 5′ end‐processing activity by chromatography on phosphocellulose revealed two distinct activities, eluting at 0.1 and 0.5 M KCl, when assayed with the tobacco pre‐tRNATyr substrate. When the same fractions were assayed with the E. coli pre‐tRNAPhe, only the 0.1 M KCl fraction exhibited activity. Both of the active fractions display sensitivity to micrococcal nuclease (MN) and proteinase K indicating each is a ribonucleoprotein, a result not seen with other plant RNase Ps. Subsequent FPLC fractionation of the two activities using Mono Q and Mono S columns demonstrated that the two activities could be further distinguished on the basis of their chromatographic behavior.
The AXR1 and AUX1 genes of Arabidopsis function in separate auxin‐response pathways
Authors: Timpte Candace , Lincoln Cynthia , Pickett F. Bryan , Turner Jocelyn , Estelle Mark 
Published: March 2003

SummaryThe recessive mutations aux1 and axr1 of Arabidopsis confer resistance to the plant hormone auxin. The axr1 mutants display a variety of morphological defects. In contrast, the only morphological defect observed in aux1 mutants is a loss of root gravitropism. To learn more about the function of these genes in auxin response, the expression of the auxin‐regulated gene SAUR‐AC1 in mutant and wild‐type plants has been examined. It has been found that axr1 plants display a pronounced deficiency in auxin‐induced accumulation of SAUR‐AC1 mRNA in seedlings as well as rosette leaves and mature roots. In contrast, the aux1 mutation has a modest effect on auxin induction of SAUR‐AC1. To determine if the AUX1 and AXR1 genes interact to facilitate auxin response, plants which are homozygous for both aux1 and axr1 mutations have been constructed and characterized. The two mutations are additive in their effects on auxin response, suggesting that each mutation confers resistance by a different mechanism. However, the morphology of double mutant plants indicates that there is an interaction between the AXR1 and AUX1 genes. In mature plants, the aux1‐7 mutation acts to partially suppress the morphological defects conferred by the axr1‐12 mutation. This suppression is not accompanied by an increase in auxin response, as measured by SAUR‐AC1 expression, suggesting that the interaction between the AUX1 and AXR1 genes is indirect.
Alteration of anion channel kinetics in wild‐type and abi1‐1 transgenic Nicotiana benthamiana guard cells by abscisic acid
Authors: Grabov Alexander , Leung Jeffrey , Giraudat Jerome , Blatt Michael R. 
Published: March 2003

SummaryThe influence of the plant water‐stress hormone abscisic acid (ABA) on anion channel activity and its interaction with protein kinase and phosphatase antagonists was examined in stomatal guard cells of wild‐type Nicotiana benthamiana L. and of transgenic plants expressing the dominant‐negative (mutant) Arabidopsis abi1‐1 protein phosphatase. Intact guard cells were impaled with double‐barrelled micro‐electrodes and membrane current was recorded under voltage clamp in the presence of 15 mM CsCI and 15 mM tetraethylammonium chloride (TEA‐CI) to eliminate K+ channel currents. Under these conditions, the free‐running voltage was situated close to 0 mV (+9 ± 6 mV, n = 18) and the membrane under voltage clamp was dominated by anion channel current (ICl) as indicated from tail current reversal near the expected chloride equilibrium potential, current sensitivity to the anion channel blockers 9‐anthracene carboxylic acid and niflumic acid, and by its voltage‐dependent kinetics. Pronounced activation of ICl was recorded on stepping from a conditioning voltage of −250 mV to voltages between −30 and +50 mV, and the current deactivated with a voltage‐dependent halftime at more negative voltages (τ⋍ 0.3 sec at −150 mV). Challenge with 20 µM ABA increased the steady‐state current conductance, gCl, near 0 mV by 1.2‐ to 2.8‐fold and at −150 mV by 4.5‐ to sixfold with a time constant of 40 ± 4 sec, and it slowed ICl deactivation as much as fourfold at voltages near −50 mV, introducing two additional voltage‐sensitive kinetic components to these current relaxations. Neither the steady‐state and kinetic characteristics of ICl, nor its sensitivity to ABA were influenced by H7 or staurosporine, both broad‐range protein kinase antagonists. However, the protein phosphatase 1/2A antagonist calyculin A mimicked the effects of ABA on gCl and current relaxations on its own and exhibited a synergistic interaction with ABA, enhancing ICl sensitivity to ABA three‐ to four‐fold. Quantitatively similar current characteristics were recorded from guard cells of abi1‐1 transgenic N. bentamiana, indicating that the abi1‐1 protein phosphatase does not influence the ànion current or its response to ABA directly. These results demonstrate that ABA stimulates ICl and modulates its voltage sensitivity. Furthermore, they show that ABA promotes ICl, either by introducing additional long‐lived states of the channel or by activating a second anion channel with similar permeation characteristics but with a very long dwell time in the open state. Overall, the data are broadly consistent with the view that ABA action engenders coordinate control of ICl together with guard cell K+ channels to effect solute loss and stomatal closure.
Genetics of homology‐dependent gene silencing in Arabidopsis; a role for methylation
Authors: Davies Gareth J. , Sheikh Mazhar A. , Ratcliffe Oliver J. , Coupland George , Furner Ian J. 
Published: March 2003

SummaryNinety‐eight independent transformed (T1) Arabidopsis plants were generated, containing additional copies of the chalcone synthase (CHS) gene. Three T2 generation families (A, B and C) were found that showed reduced anthocyanin biosynthesis, consistent with homology‐ dependent gene silencing of CHS. Clonal sectors of tissue showing CHS silencing were seen in the early generations. Affected individuals in family A showed only slight silencing, in family C such plants were almost completely silenced, and in family B affected individuals were intermediate. Plants homozygous for a single silencing insert were isolated from each family. Plants homozygous or hemizygous for insert A showed variable penetrance and expressivity of silencing. Self‐fertilization of plants hemizygous for the B and C‐inserts suggested that these CHS‐silencing inserts each behave as single Mendelian dominant traits. The CHS mRNA of the C‐insert homozygotes was reduced to undetectable levels.Outcrosses of B‐ and C‐insert homozygotes to wild‐type plants resulted in F1 plants that were variegated. This variegation appears to be due to expression of the CHS allele from the wild‐type parent, since use of a CHS mutant, tt4, as untransformed parent resulted in uniform green F1 plants. Southern blots revealed a correlation between DNA methylation and CHS silencing. In addition, derivative plants were generated from C‐insert homozygotes that had lost the silencing inserts, and these showed a partial reversion towards wild‐type phenotype and methylation of the cellular CHS gene at the TT4 locus. This result suggests that the TT4 copy of CHS became methylated during the C‐insert‐induced silencing and retained methylation and partial silencing after the silencing T‐DNA was lost.
Re‐evaluation of the role of a transmitting tract‐specific glycoprotein on pollen tube growth
Authors: Sommer‐Knudsen Jens , Lush W. Mary , Bacic Antony , Clarke Adrienne E. 
Published: March 2003

SummaryIt has been proposed that a stylar glycoprotein, the transmitting tissue‐specific (TTS) protein isolated from Nicotiana tabacum, serves both as a growth stimulant, by providing a source of nutrients, and as an attractant for pollen tubes during their growth through the style. Working with a galactose‐rich style glycoprotein (GaRSGP) that is the N. alata homologue (97% homology) of the TTS protein, a series of experiments, similar to those done with the TTS protein, was performed. Evidence was found for inhibition of pollen tube growth at high concentrations, but no evidence was found for stimulation of growth at concentrations up to 2 mg ml–1. No effect as a pollen tube attractant was detected. The discrepancies in the features and functionality between these homologous glycoproteins in the closely related Nicotiana species warrants further investigation before a general function is assigned to these molecules.
Early stages of the apoptotic pathway in plant cells are reversible
Authors: O’brien Iona E. W. , Baguley Bruce C. , Murray Brian G. , Morris Bret A. M. , Ferguson Ian B. 
Published: March 2003

SummaryChromatin condensation and nDNA fragmentation, indicators of apoptosis in mammalian cells, occur in plant cells during senescence and following induction by chemical agents. In Nicotiana plumbaginifolia cells, camptothecin, okadaic acid, salicylic acid, hydrogen peroxide, and the calcium ionophore A23187 induced chromatin condensation and nDNA fragmentation. Exposure of cells to low concentrations or removal of the chemical agent resulted in an initial phase of chromatin condensation, followed by its reversal. A further feature of apoptosis in mammalian cells, annexin V binding, indicative of phosphotidylserine exposure, was also confirmed in relation to the other events in the apoptotic pathway. With respect to flow cytometric characteristics, apoptosis triggered by a variety of chemicals occurs in plant cells in a manner closely related to that in mammalian cells. However, the extent of chromatin condensation is substantially greater, and in the early stages is reversible.
Hybrids from pea chloroplast thioredoxins f and m: physicochemical and kinetic characteristics
Authors: Jaramillo Javier López , Chueca Ana , Sahrawy Mariam , Gorgé Julio López 
Published: March 2003

SummaryTwo hybrid thioredoxins (Trx) have been constructed from cDNA clones coding for pea chloroplast Trxs m and f. The splitting point was the AvaII site situated between the two cysteines of the regulatory cluster. One hybrid, Trx m/f, was purified from Escherichia coli‐expressed cell lysates as a high yielding 12 kDa protein. Western blot analysis showed a positive reaction with antibodies against pea Trxs m and f and, like the parenteral pea Trx m, displayed an acidic pI (5.0) and a high thermal stability. In contrast, the opposite hybrid Trx f/m appeared in E. coli lysates as inclusion bodies, where it was detected by Western blot against pea Trx f antibodies as a 40 kDa protein. Trx f/m was very unstable, sensitive to heat denaturation, and could not be purified. Trx m/f showed a higher affinity for pea chloroplast fructose‐1,6‐bisphosphatase (FBPase) and a smaller Trx/FBPase saturation ratio than both parenterals; however, the FBPase catalytic rate was lower than that with Trxs m and f. Surprisingly, the hybrid Trx m/f appeared to be incompetent in the activation of pea NADP‐malate dehydrogenase. Computer‐assisted models of pea Trxs m and f, and of the chimeric Trx m/f, showed a change in the orientation of the α4‐helix in the hybrid, which could explain the kinetic modifications with respect to Trxs m and f. We conclude that the stability of Trxs lies on the N‐side of the regulatory cluster, and is associated with the acidic character of this fragment and, as a consequence, with the acidic pI of the whole molecule. In contrast, the ability of FBPase binding and enzyme catalysis depends on the structure on the C‐side of the regulatory cysteines.
Identification of a novel D6‐acyl‐group desaturase by targeted gene disruption in Physcomitrella patens
Authors: Girke Thomas , Schmidt Hermann , Zähringer Ulrich , Reski Ralf , Heinz Ernst 
Published: March 2003

SummaryThe moss Physcomitrella patens contains high levels of arachidonic acid. For its synthesis from linoleic acid by desaturation and elongation, novel D5‐ and D6‐ desaturases are required. To isolate one of these, PCR‐based cloning was used, and resulted in the isolation of a full‐length cDNA coding for a putatively new desaturase. The deduced amino acid sequence has three domains: a N‐terminal segment of about 100 amino acids, with no similarity to any sequence in the data banks, followed by a cytochrome b5‐related region and a C‐terminal sequence with low similarity (27% identity) to acyl‐lipid desaturases. To elucidate the function of this protein, we disrupted its gene by transforming P. patens with the corresponding linear genomic sequence, into which a positive selection marker had been inserted. The molecular analysis of five transformed lines showed that the selection cartridge had been inserted into the corresponding genomic locus of all five lines. The gene disruption resulted in a dramatic alteration of the fatty acid pattern in the knockout plants. The large increase in linoleic acid and the concomitant disappearance of γ‐linolenic and arachidonic acid in all knockout lines suggested that the new cDNA coded for a D6‐desaturase. This was confirmed by expression of the cDNA in yeast and analysis of the resultant fatty acids by GC–MS. Only the transformed yeast cells were able to introduce a further double bond into the D6‐position of unsaturated fatty acids. To our knowledge, this is the first report of a successful gene disruption in a multicellular plant resulting in a specific biochemical phenotype.
Low‐oxygen stress and water deficit induce cytosolic pyruvate orthophosphate dikinase (PPDK) expression in roots of rice, a C3 plant
Authors: Moons Ann , Valcke Roland , Van Marc , Montagu  
Published: March 2003

SummaryPyruvate orthophosphate dikinase (PPDK) is known for its role in C4 photosynthesis but has no established function in C3 plants. Abscisic acid, PEG and submergence were found to markedly induce a protein of about 97 kDa, identified by microsequencing as PPDK, in rice roots (C3). The rice genome was found to contain two ppdk loci, osppdka and osppdkb. We isolated osppdka cDNA, which encodes a cytosolic rice PPDK isoform of 96.6 kDa, that corresponded to the ABA‐induced protein from roots. Western blot analysis showed a PPDK induction in roots of rice seedlings during gradual drying, cold, high salt and mannitol treatment, indicating a water deficit response. PPDK was also induced in the roots and sheath of submerged rice seedlings, and in etiolated rice seedlings exposed to an oxygen‐free N2 atmosphere, which indicated a low‐oxygen stress response. None of the stress treatments induced PPDK protein accumulation in the lamina of green rice seedlings. Ppdk transcripts were found to accumulate in roots of submerged seedlings, concomitant with the induction of alcohol dehydrogenase 1. Low‐oxygen stress triggered an increase in PPDK activity in roots and etiolated rice seedlings, accompanied by increases in phosphoenolpyruvate carboxylase and malate dehydrogenase activities. The results indicate that cytosolic PPDK is involved in a metabolic response to water deficit and low‐oxygen stress in rice, an anoxia‐tolerant species.
Post‐transcriptional silencing of chalcone synthase in petunia using a geminivirus‐based episomal vector
Authors: Atkinson Ross G. , Gleave Lara R. F. Bieleski andrew P. , Janssen Bart‐Jan , Morris Bret A. M. 
Published: March 2003

SummaryA vector that produces DNA replicons (multicopy plant episomes) was constructed using elements of the geminivirus tobacco yellow dwarf virus (TYDV). All plant cells contain an integrated chromosomal T‐DNA copy of the TYDV elements that provides a template for the production of episomes in the cell nucleus. Transgenic Petunia hybrida plants containing a CaMV 35S promoter‐driven chalcone synthase A (ChsA) gene cloned into the episomal vector produced flowers with a white‐spotted phenotype at high frequency. The spots were found at random locations in the petals and occurred in corresponding positions in both the upper and lower epidermis, indicating that the spots were non‐clonal. The spotted phenotype was somatically stable and was inherited through meiosis. In white‐spotted flower tissue, steady‐state ChsA mRNA levels were downregulated but rates of RNA transcription were unaffected, suggesting that the phenotype resulted from post‐transcriptional gene silencing of the endogenous and episomal ChsA genes. Increases in both the frequency and extent of gene silencing in flowers correlated with increases in episome copy number in mature flowers, flower buds and young and fully expanded leaves. Relatively small increases in episome copy number (less than threefold) appeared sufficient to trigger the gene‐silenced phenotype.
A starch‐accumulating mutant ofArabidopsis thalianadeficient in a chloroplastic starch‐hydrolysing enzyme
Authors: Zeeman Samuel C. , Northrop Fred , Smith Alison M. , Rees Tom ap 
Published: March 2003

Summary The aim of this work was to identify enzymes that participate in the degradation of transitory starch in

Arabidopsis

. A mutant line was isolated by screening leaves at the end of the night for the presence of starch. The mutant had a higher starch content than the wild‐type throughout the diurnal cycle. This accumulation was due to a reduction in starch breakdown, leading to an imbalance between the rates of synthesis and degradation. No reduction in the activity of endo‐amylase (α‐amylase), β‐amylase, starch phosphorylase, maltase, pullulanase or D‐enzyme could be detected in crude extracts of leaves of the mutant. However, native PAGE in gels containing amylopectin revealed that a starch‐hydrolysing activity, putatively identified as an endo‐amylase and present in wild‐type chloroplasts, was absent or appreciably reduced in the mutant. This is the first time that a specific enzyme required for starch degradation has been identified in leaves.
UV‐B‐induced photomorphogenesis in Arabidopsis thaliana
Authors: Kim Byung Chul , Tennessen Daniel J. , Last Robert L. 
Published: March 2003

SummaryRelatively little is known about the types of photomorphogenic responses and signal transduction pathways that plants employ in response to ultraviolet‐B (UV‐B, 290–320 nm) radiation. In wild‐type Arabidopsis seedlings, hypocotyl growth inhibition and cotyledon expansion were both reproducibly promoted by continuous UV‐B. The fluence rate response of hypocotyl elongation was examined and showed a biphasic response. Whereas photomorphogenic responses were observed at low doses, higher fluences resulted in damage symptoms. In support of our theory that photomorphogenesis, but not damage, occurs at low doses of UV‐B, photomorphogenic responses of UV‐B sensitive mutants were indistinguishable from wild‐type plants at the low dose. This allowed us to examine UV‐B‐induced photomorphogenesis in photoreceptor deficient plants and constitutive photomorphogenic mutants. The cry1 cryptochrome structural gene mutant, and phytochrome deficient hy1, phyA and phyB mutant seedlings resembled wild‐type seedlings, while phyA/phyB double mutants were less sensitive to the photomorphogenic effects of UV‐B. These results suggest that either phyA or phyB is required for UV‐B‐induced photomorphogenesis. The constitutive photomorphogenic mutants cop1 and det1 did not show significant inhibition of hypocotyl growth in response to UV‐B, while det2 was strongly affected by UV‐B irradiation. This suggests that COP1 and DET1 work downstream of the UV‐B signaling pathway.