bluuebunny/crossref_metadata_2025 huggingface dataset:
Assessment of temporal changes in coastal sand dune environments using the log‐hyperbolic grain‐size method
Authors: Knight Jasper , Orford Julian D. , Wilson Peter , Braley Sharon M.
Published: March 2003

Abstract Three cores (<9 m long) recovered from late Holocene coastal sand dunes in Northumberland, north‐eastern England, were examined stratigraphically and dated by the infrared stimulated luminescence (IRSL) method to the time period between the Medieval Warm Period (MWP; ≈ 900–1200 AD) and Little Ice Age (LIA; ≈ 1550–1800 AD). Grain‐size variability and CaCO3 content were determined at 10‐mm intervals throughout parts of the cores. The sahara computer program was used to plot the log‐hyperbolic distribution (LHD) of grain‐size spectra from different stratigraphic levels and estimate the LHD's statistical components. Core samples plot variously in the depositional and erosional domains of the hyperbolic chi (χ) vs. xi (ξ) shape triangle, which describes components of sediment sorting. Depositional and erosional phases, discriminated within the LHD's shape triangle, cannot readily be resolved from core stratigraphies or correlated between cores. IRSL dates from single cores show a clustering about the period 1430–1540. Between the dated horizons, dunes accreted at ≈ 0·02–0·05 m year−1, enabling the timing of excursions in χ and ξ to be estimated. These excursions, which can be resolved to the subannual level between the dated horizons, may be related to deposition by episodic storms or storm surges in the North Sea region, which is supported by historical documentary evidence. This innovative use of the LHD method and its derived statistics can be used successfully to describe temporal trends in coastal depositional environments and identify likely storm events in dune sediment records.
A new genus of Paramacronychiinae (Diptera: Sarcophagidae), argued from a genus‐level cladistic analysis
Authors: PAPE THOMAS
Published: March 2003

Primorya gen.n. is described as a new genus of the flesh fly subfamily Paramacronychiinae, including the type species and sole member Primorya ussuriensis sp.n. from the Russian Far East. Arguments for erecting a new genus are obtained from a phylogenetic analysis of all paramacronychiine genera. Goniophyto suenagai (Kurahashi), comb.n. is transferred from Wohlfahrtiodes Villeneuve and included in a broadened concept of Goniophyto Townsend, and Agria cicadina (Kato) , comb.n. is transferred from Angiometopa Brauer & Bergenstamm. A key to all paramacronychiine genera is provided.
The genus Pnyxiopalpus gen.n. (Diptera: Sciaridae)
Authors: Vilkamaa Pekka , Hippa Heikki
Published: March 2003

Pnyxiopalpus gen.n. (type‐species P. raptor sp.n.), Oriental in distribution, includes the following species: P. acanthipes sp.n. (Malay Peninsula), P. aculeatus sp.n. (Borneo), P. adebratti sp.n. (Borneo), P. aphrodite sp.n. (Sumatra), P. dentaneus sp.n. (Sulawesi), P. fossor sp.n. (Borneo), P. fuscinellus sp.n. (Borneo), P. hamatus sp.n. (Borneo), P. latifalx sp.n. (Borneo), P. macrocellus sp.n. (Malay Peninsula), P. microdon sp.n. (Sumatra), P. nepenthophilus sp.n. (Malay Peninsula), P. noona sp.n. (Palawan), P. raptor sp.n. (Borneo), P. reticulatus sp.n. (Malay Peninsula) and P. simplex sp.n. (Borneo). Males and females are keyed and described. Pnyxiopalpus has a number of characters previously unknown in Sciaridae. In contrast to other Sciaridae, interspecific morphological variation is greater for the females compared to the males. Based on a parsimony analysis of sixty‐four morphological characters, Pnyxiopalpus is monophyletic, and its sister group appears to be Spathobdella Frey + {[Peyerimhoffia Kieffer + (Faratsiho Paulian + Pnyxia Johannsen)] + [Hyperlasion Schmitz + (Hermapterosciara Mohrig & Mamaev + Parapnyxia Mohrig & Mamaev)]}. According to the most parsimonious solution, the monophyly of Hyperlasion, Lycoriella Frey and Plastosciara Berg in their current sense is questioned. Aptery and one‐segmented maxillary palp, usually regarded as important in sciarid classification, show a lot of homoplasy.
In situ stress data from the Jura Mountains — new results and interpretation
Authors: Becker
Published: March 2003

Recently completed in situ stress measurements using the borehole slotter at 33 new test sites within the Swiss and French Jura Mountains are presented. When combined with previously published stress data, these new measurements allow a detailed description of the contemporary state of stress in this fold‐and‐thrust belt and the adjacent foreland. The data now allow the recognition of five stress provinces, within which two general and different orientations of maximum horizontal stress SH can be distinguished: the Central and the Southwest Provinces, with a NNW–SSE to NNE–SSW orientation, and the East, Northwest and Southeast Provinces, orientated E–W to NW–SE. Each province has its own characteristic orientation. Measurements in three study areas are presented for comparison with already published acoustic anisotropy measurements, borehole breakout data, overcoring tests and triangulation measurements. The results show a generally good agreement with the new borehole slotter measurements. The tectonic implications of these stress data are discussed in terms of a large‐scale stress reorientation of the NW–SE orientated contemporary Central European stress field in the foreland of the western Alpine arc.
Mantle upwelling within the Pannonian Basin: evidence from xenolith lithology and mineral chemistry
Authors: Falus György , Szabó Csaba , Vaselli Orlando
Published: March 2003

Five spinel lherzolite xenoliths hosted in Neogene alkali basalts from the marginal parts of the Pannonian Basin (Styrian Basin in Austria and Persani Mts. in the Eastern Transylvanian Basin, Romania) contain orthopyroxene–clinopyroxene–spinel clusters, which are believed to represent former garnet in lherzolitic mantle material. ‘Palaeo’ equilibrium pressure of this former garnet lherzolite was estimated to be equivalent to depths of 90–120 km using calculated garnet compositions and measured orthopyroxene compositions from the clusters. ‘Neo’ equilibrium pressures of the xenoliths indicate depths of 55–65 km, suggesting c. 50–60 km uprise of the mantle section represented by these xenoliths. This petrological result confirms the observations from previous geophysical studies that significant mantle uplift has occurred beneath the Pannonian Basin.
Formation of nonintersecting nested calderas: insights from analogue models
Authors: Acocella V. , Cifelli F. , Funiciello R.
Published: March 2003

Approximately 56% of 60 known nested calderas are pairs of nonintersecting subcircular structures with similar eccentricities. The consistent configuration suggests an interdependence in the formation of the two annular structures. This possibility has been investigated using 11 analogue models with sand (crust analogue) overlying silicone (magma analogue). Silicone withdrawal induces collapse within the sand, forming two concentric depressions: the first‐formed inner depression, bordered by outward‐dipping reverse ring faults (mean dip of 75°), induces the collapse of the outer depression, bordered by inward dipping, normal ring faults (mean dip of 84°). An original linear relationship is determined among the ratios between the diameters of the two depressions and the depth of the ring faults. Selected pairs of natural nested calderas show similar proportions, highlighting a consistent architecture. This suggests that some pairs of nonintersecting nested calderas result from a single collapse event.
Aeromagnetic map constrains cratonization of the Arabian Shield
Authors: Nehlig Pierre , Asfirane Fawsia , Genna Antonin , Guerrot Catherine , Nicol Nadège , Salpeteur Ignace , Shanti Mohammed , Thiéblemont Denis
Published: March 2003

A new aeromagnetic map together with new geological and geochronological data has led to a reinterpretation of the geological history of the Arabian Shield.

The magnetic anomalies outline an orogenic complex containing a network of mostly left‐lateral strike‐slip faults, including the Nabitah Belt and several peripheral mountain ranges. Oblique accretion resulted in obliteration of early volcanic‐arc magnetic fabrics, which were almost completely replaced by a NW–SE magnetic fabric in the northern Shield; the southern Shield, however, reveals extensive E–W anomalies related to post‐accretion magmatic intrusions. This complex web of orogenic zones is intimately associated with synchronous molasse basins that formed 680–610 Ma.

The distribution and chronology of orogenic zones, related to the closing of East and West Gondwana, brings into question several earlier assumptions, such as high continental growth rates, palaeogeodynamic reconstructions, the definitions of the Nabitah and Najd faults, and the significance of molasse basins.
A Brassica napus transcript encoding a protein related to the Kunitz protease inhibitor family accumulates upon water stress in leaves, not in seeds.
Authors: Downing WL , Mauxion F , Fauvarque MO , Reviron MP , De Vienne D , Vartanian N , Giraudat J
Published: March 2003

A cDNA clone encoding a Brassica napus drought‐induced 22 kDa (BnD22) protein has been isolated and characterized. The BnD22 transcript accumulated in response to drought reversibly, and to other conditions of leaf water deficit such as rapid water stress or salt acclimation, but not to cold acclimation or heat shock. Exogenously applied abscisic acid induced both changes in leaf morphology similar to the drought‐ adaptive response and a pronounced accumulation of the BnD22 mRNA. In control and drought‐adapted plants, the BnD22 transcript was expressed in an organ‐specific manner: the mRNA level was highest in leaves, low in hypocotyls and undetectable in roots. Sequence analysis indicates that the BnD22 protein is related to the Kunitz family of protease inhibitors. In contrast to most members of this family, and also to most polypeptides expressed in vegetative tissues upon drought, the BnD22 mRNA was absent in seeds, before or during the seed desiccation phase. The BnD22 gene represents a new class of genes which are strictly induced in vegetative tissues upon environmental stress, and its pattern of expression shows that the responses to water deficit differ, at least partially, in seeds and in leaves.
Pattern of somatic transposition in a high copy Ac tomato line
Authors: Belzile Francois , Yoder John I.
Published: March 2003

A transgenic tomato line containing between eight and ten copies per genome of an exceptionally active maize transposable element Ac has previously been described. Southern analyses indicated that these elements are somatically active in these plants. In order to characterize further the pattern of somatic transposition in this line, 24 independent Ac insertion events from a single plant were cloned. In 21 cases, Ac inserted into single copy genomic DMA while in three cases Ac inserted into sequences present at two to four copies per genome; none of the insertions occurred into more highly repetitive DMA. The chromosomal locations of 20 insertion sites were determined by RFLP mapping and a pattern of small dispersed clusters emerged. Thirteen of the 20 insertion sites were linked to at least one other insertion site but these were distributed over nine of the 12 tomato chromosomes. Only one Ac insertion was linked to the T‐DNA locus. The structural integrity of these Ac elements was examined and no evidence of deletions or other rearrangements suggestive of Ds elements was found. The implications of these findings with respect to the use of Ac as a transposon tag in heterologous species are discussed.
The pea plastocyanin promoter directs cell‐specific but not full light‐regulated expression in transgenic tobacco plants
Authors: Pwee Keng‐Hock , Gray John C.
Published: March 2003

A series of 5′ deletions of the pea plastocyanin gene (petE) promoter fused to the β‐glucuronidase (GUS) reporter gene has been examined for expression in transgenic tobacco plants. Strong positive and negative cis‐elements which modulate quantitative expression of the transgene in the light and the dark have been detected within the petE promoter. Disruption of a negative regulatory element at −784 bp produced the strongest photosynthesis‐gene promoter so far described. Histochemical analysis demonstrated that all petE‐GUS constructs directed expression in chloroplast‐containing cells, and that a region from −176 bp to +4 bp from the translation start site was sufficient for such cell‐specific expression. The petE‐promoter fusions were expressed at high levels in etiolated transgenic tobacco seedlings but there was no marked induction of GUS activity in the light. The endogenous tobacco plastocyanin genes and the complete pea plastocyanin gene in transgenic tobacco plants were also expressed in the dark, but showed a three‐ to sevenfold increase in the light. This indicates a requirement for sequences 3′ to the promoter for the full light response of the petE gene.
Rescue of a maize mitochondrial cytochrome oxidase mutant by tissue culture†
Authors: Gu Jianying , Dempsey Sandra , Newton Kathleen J.
Published: March 2003

The maize NCS6 mitochondrial mutation is a partial deletion of the cytochrome oxidase subunit 2 gene (cox2) that survives heteroplasmically in the plant. Mutant mitochondria segregate from normal mitochondria during somatic development giving rise to defective sectors on the plants, including areas of kernel abortion on the ears. Embryos from NCS6 kernels can be rescued by tissue culture. Slowly growing Type II callus derived from one of these embryos has been shown by PCR analysis to be homoplasmic for the mutation, carrying only the defective mitochondrial cox2 gene. Most of the rescued embryos were heteroplasmic for normal and mutant genes and heteroplasmy was maintained in the callus cultures. However, when suspension cultures were initiated from heteroplasmic calli, normal cells were shown to have a selective advantage. When the homoplasmic cox2 mutant callus cultures were placed on regeneration medium, plantlets did not regenerate. Heteroplasmic calli were capable of regeneration under the same conditions. These studies suggest that the functioning of mitochondrial cytochrome oxidase is not essential for growth as callus, but is required for the differentiation and development of plants.
Rice phytochrome A controls apical hook opening after a single light pulse in transgenic tobacco seedlings
Authors: Schäfer Eberhard , Emmler Karlheinz , Chua Nam‐Hai
Published: March 2003

Apical hook opening in tobacco seedlings can be induced by a single red light pulse and this induction can be reverted by a subsequent far‐red light pulse. The slow hook opening kinetics and the reversibility of an inductive light pulse even after 8 h of darkness indicate the involvement of stable phytochrome. Compared with wild‐type, transgenic BN1 seedlings which overexpress rice phytochrome A exhibit a higher sensitivity to low irradiance red light pulses. Moreover, in BN1 seedlings an inductive red light pulse is only partially reversible even after 30 min, whereas wild‐type tobacco seedlings show complete reversibility during the entire hook opening process. The data found show that rice phytochrome A is active in transgenic tobacco seedlings in controlling hook opening and that the introduced rice phytochrome A and the endogenous stable phytochrome behave differently in this response.
DNA rearrangement associated with the integration of T‐DNA in tobacco: an example for multiple duplications of DNA around the integration target
Authors: Ohba Toshiharu , Yoshioka Yasushi , Machida Chiyoko , Machida Yasunori
Published: March 2003

Transferred DNA (T‐DNA) of the tumor‐inducing (Ti) plasmid is transferred from Agrobacterium tumefaciens to plant cells and is stably integrated into the plant nuclear genome. By the inverse polymerase chain reaction DNA fragments were amplified that contained the T‐DNA/plant DNA junctions from the total DNA of a transgenic tobacco plant that had a single copy of the T‐DNA in a repetitive region of its genome. A DNA fragment containing the target site was amplified from the total DNA of non‐transformed tobacco by the polymerase chain reaction using high‐stringency conditions. Comparison of the nucleotide sequence of the target site with those of the T‐DNA/plant DNA junctions revealed that various duplications of short stretches of nucleotide sequences around the target and in the incoming T‐DNA had accompanied the integration of the T‐DNA. A deletion of 16 bp at the target site was also found and the target site was similar, in terms of nucleotide sequence, to regions around the breakpoints of the T‐DNA. This finding provides a clear example of the occurrence of complex rearrangements during the integration of T‐DNA.
Circadian regulation of the Cat3 catalase gene in maize (Zea mays L.): entrainment of the circadian rhythm of Cat3 by different light treatments
Authors: Boldt Ralf , Scandalios John G.
Published: March 2003

The Cat3 maize catalase gene is a non‐photosynthetic, non‐chloroplast‐associated gene that exhibits a transcriptionally regulated circadian rhythm. Cat3 transcript accumulation was examined in leaves of maize seedlings grown under different light regimes. Plants grown in a 12 h light/12 h dark photoperiod (LD) show a distinct diurnal pattern with maximal Cat3 transcript accumulation late in the light period (CT 10, circadian time) and low to undetectable levels late in the dark and early light period (CT 22‐02). The Cat3 gene is expressed at high and constant levels without any diurnal fluctuations in leaves of maize seedlings grown post‐imbibition in constant dark (DD) or constant light (LL). The circadian rhythm of Cat3 is inducible by transferring DD‐grown plants and/ or LL‐grown plants to a 12 h (light/dark LD) or 12 h (dark/light DL) cycle. Entrainment of the rhythm requires a cyclic light signal consisting of one DL‐transition (‘light on’‐signal) followed by one LD transition (‘light off’‐signal). When etiolated maize seedlings were treated with single light pulses of white light and different monochromatic light and transferred to constant darkness (DD) a similar induction pattern of the Cat3 circadian rhythm was observed. Cat3 mRNA accumulates to peak levels 4 h after the light pulse; the next peak appeared 24–28 h later. Induction by far red‐light (FR) exhibits characteristics of a very low fluence phytochrome response. Also blue light and ultraviolet light pulses cause induction of the Cat3 circadian rhythm, but do not influence the periodicity of Cat3 expression. In order to demonstrate differences of the Cat3 circadian regulation from photosynthetic genes, the maize cab gene expression was used for comparison.
Characterization of a gene transcribed in the L2 and L3 layers of the tobacco shoot apical meristem
Authors: Kelly Alan J. , Meeks‐Wagner D. Ry
Published: March 2003

Recent genetic and molecular studies have been initiated to uncover the fundamental cellular processes unique to the shoot apical meristem. Our previous work resulted in the isolation of a cDNA clone derived from tobacco apex RNA, A3, that appeared to be transcriptionally restricted to the shoot apex regardless of developmental stage. Here the DNA sequence and in situ RNA analysis of A3 is presented. The A3 gene potentially encodes a small hydrophilic polypeptide, the sequence of which is unique to current data bases. It has been found that transcripts of the A3 gene are confined to the subepidermal and internal cell layers of the tobacco shoot apical meristem throughout development, become localized to undifferentiated floral organ primordia, and diminish as the developmental potential of the meristematic tissue becomes restricted.
Determinants of the high‐methionine trait in wild and exotic germplasm may have escaped selection during early cultivation of maize
Authors: Swarup Sanjay , Timmermans Marja C.P. , Chaudhuri Sumita , Messing Joachim
Published: March 2003

The 18 kDa high‐methionine δ‐class zein gene from maize has been cloned, and its regulation, structure, and map position studied. These studies have shown that (i) zein genes may also contain tryptophan and lysine codons, (ii) the 18 kDa and the related 10 kDa zein gene are coordinately regulated, but their products accumulate to different levels in a genotype‐dependent manner, (iii) the duplication of δ‐zein genes probably involved unequal crossing over, (iv) no copy correction in either direction has occurred from teosinte to modern corn, and (v) the duplication of of the 18 kDa zein gene probably occurred before the tetraploidization of a progenitor chromosome. The work shows that important nutritional quality determinants like the high‐methionine seed proteins are abundant in several exotic and wild corn varieties and low in most of the inbreds screened. The lack of a selectable phenotype for such quality traits during initial domestication and breeding of corn would have eliminated cis and trans regulatory determinants from the germplasm used in modern corn breeding. Examples of the high‐methionine δ‐class zeins shown here may be generally applicable in explaining the low nutritional quality of most present‐day corn grown.
Metabolism of cyclic carotenoids: a model for the alteration of this biosynthetic pathway in Capsicum annuum chromoplasts
Authors: Hugueney Philippe , Badillo Alfredo , Chen Hsu‐Ching , Klein Annelyse , Hirschberg Joseph , Camara Bilal , Kuntz Marcel
Published: March 2003

The biosynthetic pathway of cyclic carotenoid is known to be quantitatively and qualitatively different in the non‐green plastids of Capsicum annuum fruits compared with chloroplasts. Here, the cloning is described of a novel cDNA from this organism, which encodes an enzyme catalyzing the cyclization of lycopene to β‐carotene when expressed in Escherichia coli. The corresponding gene is constitutively expressed during fruit development. Significant amino acid sequence identity was observed between this enzyme and capsanthin/capsorubin synthase which is involved in the synthesis of the species‐specific red carotenoids of C. annuum fruits. The latter enzyme was found also to possess a lycopene β‐cyclase activity when expressed in E. coli. A model is proposed for the origin of the capsanthin/capsorubin synthase gene and the role of this enzyme, together with the newly cloned lycopene cyclase, in the specific re‐channeling of linear carotenoids into β‐cyclic carotenoids in C. annuum ripening fruits.
Characterization of a binding site for chemically synthesized lipo‐oligosaccharidic NodRm factors in particulate fractions prepared from roots
Authors: Bono Jean‐Jacques , Riond Joëlle , Nicolaou K.C. , Bockovich Nicholas J. , Estevez Virginia A. , Cullimore Julie V. , Ranjeva Raoul
Published: March 2003

This paper describes the characteristics of a binding site for the major, lipo‐oligosaccharide Nod factor of Rhizobium meliloti in roots of the symbiotic host plant, Medicago truncatula. Chemically synthesized NodRm‐IV(Ac, S, C16:2) was labelled by tritiation to a specific activity of 56 Ci mmol−1 and this ligand was shown to be biologically active in the root hair deformation assay at 10−11 M. Binding of the ligand to a particulate fraction from roots of M. truncatula was found to be saturable and reversible with an affinity (Kd) of 86 nM and the binding characteristics were consistent with a single class of binding sites. Competition with modified Nod factors showed that the binding was independent of both the O‐acetyl and the sulphyl group and did not depend on the unsaturation of the fatty acid. However, both moieties of the lipo‐oligosaccharide are required for high‐affinity binding since tetra‐N‐acetyl‐chitotetraose and palmitate were found to be poor competitors of ligand binding. A binding site with analogous characteristics was also found in a similarly prepared particulate fraction of tomato roots. This binding site for Nod factors, termed NFBS1, which is present in both a leguminous and a non‐leguminous plant, may have a more general role than symbiosis.
T‐DNA tagging of genes influencing polyamine metabolism: isolation of mutant plant lines and rescue of DNA promoting growth in the presence of a polyamine biosynthetic inhibitor
Authors: Fritze Klaus , Czaja Inge , Walden Richard
Published: March 2003

Activation T‐DNA tagging has been used to generate cell lines characterized by their ability to grow and form callus in vitro under selective concentrations of the polyamine biosynthetic inhibitor methylglyoxal‐bis(guanylhydrazone) (MGBG). These have been regenerated into plants of which the majority have been shown by Southern analysis to contain single T‐DNA inserts. In the mutant lines the T‐DNA is contained on differing restriction fragments of genomic DNA, suggesting that they represent different insertion events. Regenerated plants display characteristic phenotypic changes when compared with untransformed plants including leaf and floral malformation as well as parthenocarpy. In two of the plant lines studied in detail, the activity of S‐adenosylmethionine decarboxylase, the target of MGBG, was increased in callus tissue and in one line this is coupled with elevated levels of putrescine and spermidine. Plant genomic DNA flanking the T‐DNA insert has been rescued from one mutant plant line and Northern blot analysis indicates that transcripts corresponding to this DNA accumulate to higher levels in the T‐DNA tagged line compared with untransformed tobacco. Rescued genomic DNA flanking the T‐DNA, once re‐introduced into untransformed tobacco protoplasts by PEG‐mediated uptake, confers the ability to grow under selective levels of MGBG. This confirms that sequences responsible for the selected phenotype have been isolated. It is anticipated that the analysis of such mutants and the further characterization of the tagged ganomic sequences may shed light on the action of polyamines in plant growth and development.
Post‐transcriptional regulation of gene expression in oxygen‐deprived roots of maize
Authors: Fennoy Sheila L. , Bailey‐Serres Julia
Published: March 2003

Maize (Zea mays L.) seedling roots undergo transient changes in gene expression in response to O2 deprivation (hypoxia or anoxia). Previous research has demonstrated the induction of transcription of alcohol dehydrogenase‐1 (adh1) and implicated post‐transcriptional and translational processes in the regulation of gene expression. In this study, the effects of O2 deprivation on expression of mRNA encoding ADH1 and an aerobically synthesized protein, the mitochondrial adenine nucleotide translocator (ANT) were compared. The transcription of adh1 in isolated nuclei and steady‐state level of poly(A)+ mRNA encoding ADH1 increased in response to O2 deprivation. In contrast, transcription of ant was not significantly affected, whereas the steady‐state level of ANT mRNA steadily decreased under O2 deprivation. To further understand the contrasting effect of low‐O2 on the stability of ADH1 and ANT mRNAs, post‐transcriptional processes of gene expression were examined. RNA blot analysis of sucrose density gradient fractions of ribosomes revealed that ADH1 and ANT mRNAs are associated with polyribosomes under aerobic and O2‐deprived growth conditions. However, the number of ribosomes loaded per ANT mRNA was significantly reduced during O2 deprivation. Immunoprecipitation analysis showed that synthesis of ANT was impaired relative to accumulation of ANT mRNA in O2‐deprived roots. The differences in synthesis of ADH1 and ANT in O2‐deprived roots appears to be due not only to increased transcription of adh1, but to competitive differences between transcripts for translation initiation factors and differences in ability to elongate during translation.
Two‐dimensional RFLP analyses reveal megabase‐sized clusters of rRNA gene variants in Arabidopsis thaliana, suggesting local spreading of variants as the mode for gene homogenization during concerted evolution
Authors: Copenhaver Gregory P. , Pikaard Craig S.
Published: March 2003

Eukaryotic genes encoding the precursor of 18S, 5.8S and 25S ribosomal RNA (rRNA genes or rDNA) are virtually identical within a species, yet they evolve rapidly between species, a phenomenon known as concerted evolution. The mechanisms by which sequence homogenization and fixation of new rRNA gene variants occurs within a genome are not clear. In diploid Arabidopsis thaliana, approximately 1500 rRNA genes are tandemly arrayed at two nucleolus organizer regions, one on chromosome 2 (NOR2), the other on chromosome 4 (NOR4). This paper shows that NOR2 and NOR4 are similar in size, each spanning approximately 3.5–4.0 Mbp. Using two‐dimensional mapping techniques involving a combination of pulsed‐field and conventional gel electrophoresis, the distributions of four distinct rRNA gene variants at NOR2 and NOR4 have been determined. rRNA genes at NOR4 are homogeneous with respect to a HindIII site occurring once per gene. In contrast, fewer than 10% of the rRNA genes at NOR2 are HindIII‐bearing variants. A single intergenic spacer length is found among rRNA genes at NOR2 but three classes of spacer length variants are present at NOR4. The NOR4 variants are not intermingled with one another; instead, they are highly clustered over distances as large as 1.5 Mbp. These data suggest that in the concerted evolution of rRNA genes, homogenization is a consequence of local spreading of new rRNA gene variants.
Regulation of expression of the tobacco Tnt1 retrotransposon in heterologous species following pathogen‐related stresses
Authors: Moreau‐Mhiri Corinne , Morel Jean‐Benoît , Audeon Colette , Ferault Madina , Grandbastien Marie‐Angéle , Lucas Hélène
Published: March 2003

Transcriptional regulation of the tobacco retrotransposon Tnt1 was monitored in Arabidopsis thaliana and Lycopersicon esculentum. In these two heterologous host species, Tnt1 expression is developmentally regulated, and its tissue‐specific pattern of transcription is similar to that observed in tobacco, with the exception that Tnt1 is expressed in their flowers. Induction of Tnt1 by microbial elicitors, previously found in tobacco, is maintained in Arabidopsis and tomato. Tnt1 activation is not strictly dependent upon a Hypersensitive Response‐like necrotic response, but also occurs in a plant‐pathogen compatible interaction triggered by viral or bacterial infections. The putative role of Tnt1 in generating genetic variability in its host plant is discussed.
Isolation of pl 4.6 extensin peroxidase from tomato cell suspension cultures and identification of Val—Tyr—Lys as putative intermolecular cross‐link site
Authors: Schnabelrauch Linda S. , Kieliszewski Marcia , Upham Brad L. , Alizedeh Hasan , Lamport Derek. T.A.
Published: March 2003

Extensins and kindred hydroxyproline‐rich glycoproteins occur in dicot cell walls mainly as insoluble integral components that may form an intermolecularly cross‐linked network interpenetrated by other polymers. Extensins also occur in muro as a small pool of soluble monomeric precursors to network extensin. These precursors were prepared in milligram quantities by salt elution from the surface of intact cells grown as tomato suspension cultures. Based on an FPLC (Superose‐6) gel filtration assay of cross‐linked extensin oligomers, a pl 4.6 extensin cross‐linking peroxidase isozyme was partially purified from the culture growth medium. Purification involved: volume reduction, ultracentrifugation to remove pectin and co‐adsorbed cationic peroxidase, followed by chromatography of anionic extensin peroxidase (pl 4.6) on DEAE—Trisacryl and TSK‐gel DEAE‐5PW columns. With tomato P1 extensin as substrate and 60 µM H2O2 as co‐substrate, at 23° pl 4.6 extensin peroxidase gave a K,m of 0.22 mM P1 and a Vmax of 70 µmol P1 cross‐linked min−1 mg−1 enzyme, at the optimum pH 5.5. Assayed with 12 different extensins from representative monocots, dicots, and gymnosperms, the pl 4.6 isozyme cross‐linked highly selectively, indicating two natural groups: cross‐linking or CL‐extensins and non‐cross‐linking or NCL‐extensins. CL‐extensins contained the X—Hyp—Val—Tyr—Lys motif and were also highly glycosylated. However, the simplest motif common to CL‐extensins but absent from NCL‐extensins was Val—Tyr—Lys. Thus, peroxidative coupling of extensin monomers and resistance of the resultant oligomers to depolymerization by anhydrous HF suggests that the intermolecular cross‐link involves tyrosine or lysine.
The Petunia tra1 gene controls cell elongation and plant development, and mediates responses to cytokinins
Authors: Dubois F. , Dang Ha D. Bui , Sangwan R.S. , Durand J.
Published: March 2003

The molecular control of cell elongation, one of the basic processes of plant morphogenesis, is still largely not understood. This paper describes a Petunia hybrida mutant of dumpy phenotype, trapu, which identifies tra1, a gene required for cell elongation and mediating responses to applied cytokinin. This mutant displayed an extreme reduction in length, due to a single recessive mutation which was expressed in every part of the plant and during the entire life of the plant, including the mature embryo. The mutant was unable to flower. The mutant roots, as well as the leafy organs, were short and thick, and the root elongation zone, hypocotyl and petioles were absent. The mutant plantlets responded neither to applied auxin nor to gibberellin, indicating that this phenotype was not caused by a deprivation of these phytohormones. However, unlike the wildtype, the mutant growth was stimulated by applied cytokinin, even though its morphology remained abnormal. A histological study revealed the presence of all tissue types in normal positions, including root hairs and vascular bundles. The mutant's cells were rounder in every tissue. Both shoot and root meristems were disorganized, without consistent cell shape and size. The regular cell files, which are typical of a normal root apex organization, were totally absent in the mutant root apex. Indirect immunofluorescence of α‐tubulin on root apices showed the cortical microtubules in the mutant cells to be unable to form the parallel arrays in elongating cells and the preprophase band in dividing cells. This default resulted in the prevention of unidirectional cell elongation and formation of regular cell files, thus causing the trapu phenotype. This paper discusses the similarities and differences of trapu to the Arabidopsis mutants, fass and ton, trapu confirming that the establishment of plant body pattern and differentiation can be dissociated from cell elongation.
Purification of the Chlorella HUP1 hexose—proton symporter to homogeneity and its reconstitution in vitro
Authors: Caspari Thomas , Robl Ingrid , Stolz Jürgen , Tanner Widmar
Published: March 2003

A prokaryotic biotin acceptor domain was fused to the carboxy terminal end of the Chlorella hexose—proton sym‐ porter. The plant symporter is biotinylated in vivo when expressed in Schizosaccharomyces pombe. The extended biotinylated transport protein is fully active, catalyzes accumulation of d‐glucose analogs and restores growth of a glucose‐uptake‐deficient yeast strain. Crude membranes were solubilized with octyl‐β‐d‐glucoside in the presence of Escherichia colil‐α‐phosphatidylethanolamine. Biotinylated symporter was purified to homogeneity by biotinavidin affinity chromatography. The symporter protein was reconstituted together with cytochrome‐c oxidase prepared from beef heart mitochondria into proteo‐liposomes. Cytochrome‐c oxidase is a redox‐driven H+‐pump generating a proton motive force (inside negative and alkaline) while transferring electrons from cytochrome‐c to oxygen; this energy is used by the symporter to accumulate d‐glucose at least 30‐fold. In the absence of the driving force the transport protein facilitates diffusion of d‐glucose until the concentration equilibrium is reached. It was shown that maximal transport activity depends highly on the amount of co‐reconstituted cytochrome‐c oxidase and that the symporter possesses 10% of its in vivo turnover number under optimized in vitro transport conditions.
Gene identification in a complex chromosomal continuum by local genomic cross‐referencing
Authors: Avramova Zoya , Tikhonov Alexander , SanMiguel Phillip , Jin Young‐Kwan , Liu Changnong , Woo Sung‐Sick , Wing Rod A. , Bennetzen Jeffrey L.
Published: March 2003

Most higher plants have complex genomes containing large quantities of repetitive DNA interspersed with low‐copy‐number sequences. Many of these repetitive DNAs are mobile and have homology to RNAs in various cell types. This can make it difficult to identify the genes in a long chromosomal continuum. It was decided to use genic sequence conservation and grass genome co‐linearity as tools for gene identification. A bacterial artificial chromosome (BAC) clone containing sorghum genomic DNA was selected using a maize Adh1 probe. The 165 kb sorghum BAC was tested for hybridization to a set of clones representing the contiguous 280 kb of DNA flanking maize Adh1. None of the repetitive maize DNAs hybridized, but most of the low‐copy‐number sequences did. A low‐copy‐number sequence that did cross‐hybridize was found to be a gene, while one that did not was found to be a low‐copy‐number retrotransposon that was named Reina. Regions of cross‐hybridization were co‐linear between the two genomes, but closer together in the smaller sorghum genome. These results indicate that local genomic cross‐referencing by hybridization of orthologous clones can be an efficient and rapid technique for gene identification and studies of genome organization.
Immunofluorescent quantitation of chloroplast proteins
Authors: Leech Rachel M. , Marrison Joanne L.
Published: March 2003

Using scanning light microscopy software to detect and measure immunofluorescence in leaf sections Rubisco concentration in situ in chloroplasts has been accurately determined throughout development. The fluorescence measurements were calibrated by comparison with values for Rubisco accumulation obtained from rocket immuno‐ electrophoresis profiles of soluble protein from isolated cells and from chloroplasts using a purified sample of Rubisco as the standard. It has been shown that in situ immunofluorescence can be used for cytoquantitation of proteins within individual chloroplasts to a sensitivity of 1fg and also for the comparison of the protein levels in adjacent chloroplasts and cells. Several important applica‐ tions of this new technique are discussed.
mCCG methylation in angiosperms
Authors: Jeddeloh Jeffrey A. , Richards Eric J.
Published: March 2003

Two different methods were used to investigate the abundance of cytosine methylation at the outer (5′) position in 5′‐CCG‐3′ trinucleotides in angiosperm genomes. Mspl is unable to cut its target site if the outer cytosine is methylated (5′‐mCCGG‐3′). Using Mspl restriction analysis, it was shown that 5′‐mCCG‐3′ is present in all angiosperm genomes examined, and that the amount of cytosine methylation at this site varies between species. Subsequently, direct measurements were made of the amount of methylation at both cytosines in a subset of 5′‐CCG‐3′ trinucleotides in the Arabidopsis thaliana genome. Based upon these analyses, it was estimated that approximately 20–30% of 5′‐CCG‐3′ trinucleotides in A. thaliana are methylated at the outer cytosine. Approximately 20% of the 5′‐CCG‐3′ trinucleotides contain 5‐methyl‐cytosine at the inner cytosine position, which corresponds to a previous determination of 5′‐mCG‐3′ methylation in A. thaliana. The implications of 5′‐mCCG‐3′ methylation are discussed.
Pollination induces mRNA poly(A) tail‐shortening and cell deterioration in flower transmitting tissue
Authors: Wang Hong , Wu Hen‐ming , Cheung Alice Y.
Published: March 2003

Pollination induces many physiological responses in the flower, including deterioration and death in specific pistil cell types. It is shown here that within the style of tobacco, pollination‐induced cell deterioration was restricted to the transmitting tissue while the surrounding cortical tissue was not affected. It was distinct from general senescence since exogenously applying the senescence‐inducing hormone ethylene, or its precursor aminocyclopropane‐1‐carboxylic acid (ACC), to the flower or the pistil induced overall deterioration in the entire flower. Furthermore, both pollen tube growth and ethylene action were needed for the entire spectrum of cellular changes associated with this pollination‐induced transmitting tissue deterioration process. It is also shown that pollination‐induced mRNA poly(A) tail‐shortening for at least three major classes of transmitting tissue‐specific mRNAs. As is commonly observed for poly(A) tail‐shortened mRNAs, the levels of two of these three mRNA classes decline after pollination. On the other hand, the third class of mRNAs, transmitting tissue‐specific (TTs) mRNAs, was maintained at a very high level subsequent to pollination, even after substantial poly(A)‐tail shortening. TTS mRNAs encode a pollen tube growth‐promoting and ‐attracting protein needed for optimal in vivo pollen tube growth. The specific preservation of TTS mRNAs in the deteriorating transmitting tissue cells suggests that these cells can distinguish molecules needed in the pollinated styles from those that are dispensable, and protect them from degradation. It is suggested that the pollination‐induced mRNA poly(A) tail‐shortening and cell death are programmed processes suited to the post‐pollination transmitting tissue environment. Results showing that ACC is a candidate signal molecule for the pollination‐induced mRNA‐shortening which is accentuated by ethylene and mediated via a protein phosphorylation‐dependent signal transduction pathway are also presented.
Genetic transformation of the actinorhizal tree Allocasuarina verticillata by Agrobacterium tumefaciens
Authors: Franche C. , Diouf D. , Le Q.V. , Bogusz D. , N'Diaye A. , Gherbi H. , Gobé C. , Duhoux E.
Published: March 2003

We have developed an efficient transformation system for the tropical actinorhizal tree Allocasuarina verticillata using Agrobacterium tumefaciens mediated gene transfer. Mature zygotic embryos were inoculated with the disarmed strain C58C1 carrying, in the binary vector BIN19, the nptll gene, providing kanamycin resistance as a selectable marker, and the reporter gene β‐glucuronidase containing an intron. The transformed embryos were cultivated on nutrient medium supplemented with 0.5 µM NAA, 2.5 µM BA, 100 mg l−1 kanamycin and 250 mg l−1 cefotaxime. After 2 months, a 21% transformation frequency was obtained. Within 6–9 months, transgenic plants were recovered from 70% of the transformed calli. The presence of the transgenes was demonstrated by PCR analysis and by the expression of the β‐glucuronidase; integration of the T‐DNA was confirmed by Southern hybridization. More than 100 transgenic plants from a total of 23 independent transformation events have been successfully established in soil. The possibility to obtain nitrogen‐fixing nodules after inoculation of transgenic A. verticillata plants by the actinomycetal strain of Frankia Allo2 was established.
Ribosomal transcription units integrated via T‐DNA transformation associate with the nucleolus and do not require upstream repeat sequences for activity in Arabidopsis thaliana
Authors: Wanzenböck Eva‐Maria , Schöfer Christian , Schweizer Dieter , Bachmair Andreas
Published: March 2003

Ribosomal repeat units of Arabidopsis thaliana were introduced into the A. thaliana genome via Agrobacterium‐mediated transformation. Ribosomal transgenes integrated into chromosomal regions outside the nucleolus organizers. Cytological data suggest that the transgenes associate with a nucleolus. To allow detection of transgenic rRNA, a short extension was inserted into the V1 variable region of the 25S ribosomal gene. The RNA transcript from the transgene undergoes a series of maturation steps, including correct processing of the 5′ end of 25S rRNA. Using primer extension analysis, expression of a complete rDNA repeat unit was compared with the activity of a repeat unit lacking a sequence called ‘upstream Sal repeats’. No qualitative or quantitative differences were detected, suggesting that upstream repeat sequences of the rDNA intergenic region do not act as transcriptional enhancers for RNA polymerase I in A. thaliana.
Transgenic tobacco plants expressing antisense RNA for cytosolic ascorbate peroxidase show increased susceptibility to ozone injury
Authors: Örvar Björn Lárus , Ellis Brian E.
Published: March 2003

Ascorbate peroxidase (APX), as a participant in the ascorbate—glutathione cycle, has been suggested to be a particularly important antioxidant enzyme in helping plants survive oxidative stress, but direct evidence for this has not been reported. We demonstrate that expressing antisense RNA comprising 45% of the 3′‐coding region of the tobacco cytosolic APX, can reduce significantly both the endogenous APX mRNA levels and the APX catalytic activity in transgenic tobacco plants. Those transgenic plants showing a reduction in both endogenous APX mRNA levels and extractable APX activity display a significant increase in ozone injury following high‐level ozone exposure. Lower‐level ozone exposure reveals even more drastic differences between the antisense and control plants, suggesting that even a partial loss of APX function in oxidative defence cannot be fully compensated for by other antioxidant measures.
Developmental regulation of a plant encoded inhibitor of eukaryotic initiation factor 2α phosphorylation
Authors: Langland Jeffrey O. , Langland Lisa , Zeman Cameron , Saha Debabrata , Roth Don A.
Published: March 2003

An inhibitor of elF‐2a phosphorylation was identified in various plant species. The plant protein (termed PKI) specifically cross‐reacts with monoclonal antiserum that recognizes the glycosylated, active form of a Mr 67 kD protein analog (p67) from reticulocytes. Northern blot analysis using a probe to the reticulocyte inhibitor cDNA further supports the presence of analogous transcripts in plant tissue. PKI specifically inhibits the phosphorylation of the plant encoded elF‐2α kinase (pPKR) as well as plant and human elF‐2α phosphorylation. The interaction between PKI and pPKR is indicated by their copurification on dsRNA agarose, despite evidence showing that PKI does not bind dsRNA. Further, wheat PKI inhibits human PKR phosphorylation but activity is recovered by immunodepletion of PKI from wheat germ fractions. PKI is temporally regulated during plant growth and development. It is maximally present in extracts from dormant seeds, however, it is not detectable soon after leaf emergence at approximately 48 h post‐imbibition. PKI levels are again detectable at the mid‐milk stage in seed development. Protein levels of pPKR in ribosomal salt wash and cytosolic extracts from healthy plant tissue remain essentially constant throughout the life cycle. In contrast, pPKR activity levels based upon autophosphorylation vary significantly and are inversely correlated with PKI protein levels. Phosphorylation of elF‐2α is a classical mechanism for the downregulation of protein synthesis suggesting that inhibition of pPKR activity by PKI may contribute to the dramatic and rapid increase in protein synthesis observed during seed germination.