bluuebunny/crossref_metadata_2025 huggingface dataset:
A new subfamily, two new genera and three new species of Aclerdidae (Hemiptera: Coccoidea) from southern Africa, with a phylogenetic analysis of relationships
Authors: Hodgson Chris J. , Millar Ian M.
Published: March 2003

Abstract A new aclerdid genus, Lecanaclerda Hodgson & Millar, closely related to Aclerda but with fully developed legs and antennae, is introduced for a new new species, L. macropoda Hodgson & Millar, for which the male and female are described. In addition, genus Rhodesaclerda McConnell is revised and all known stages of existing species, R. combreticola McConnell and R. halli McConnell, are redescribed. The crawler, second‐instar female, pupa and the adult male and female of a new species, R. insleyae Hodgson & Millar, from South Africa are also described. Another new genus, Kwazulaclerda Hodgson & Millar, close to Rhodesaclerda, is introduced and the crawler, second‐instar female, second‐instar male, pupa and adult male and female of the new species K. loranthi Hodgson & Millar from Kwazulu‐Natal are described. A phylogenetic analysis based on adult macropterous male characters was undertaken to look at the relationships of these species with other Coccoidea. It is concluded that Lecanaclerda is closely related to Aclerda, Kwazulaclerda is closely related to Rhodesaclerda, Kwazulaclerda and Rhodesaclerda belong to Aclerdidae, and Aclerdidae should be split into two subfamilies, Rhodesaclerdinae Hodgson & Millar (to include Rhodesaclerda and Kwazulaclerda) and Aclerdinae Cockerell (to include Aclerda, Lecanaclerda and Nipponaclerda). These two subfamilies are diagnosed and a key is given for the separation of the genera now included in Aclerdidae. The relationships of Aclerdidae and Coccidae are discussed.
Resolving relationships over a wide taxonomic range in Delphacidae (Homoptera) using the COI gene
Authors: Dijkstra Eddy , Rubio Jose M. , Post Rory J.
Published: March 2003

Abstract. Using a combination of different methods to investigate the suitability of a fragment of the cytochrome c oxidase I gene (COI), we succeeded in partially resolving phylogenetic relationships in Delphacidae from the level of species to subfamily. Spectral analysis applied to the relatively noisy COI data proved to be especially useful. It clearly showed when phylogenetic signals were not completely randomized and it was very helpful for identifying problem areas in the dataset. Relationships among the four sampled subfamilies were completely resolved. In contrast to the tree based on morphological characters, we found evidence that Asiraca and Ugyops are sister groups (supporting monophyly of Asiracinae) and that Stenocraninae are the sister group of Kelisiinae. Contradictory signals were observed within Delphacini, but there are characters that support a close relationship between Conomelus and Megamelus. Other than this, the COI data gave support for the monophyly of Kelisiinae, Delphacinae, Chloriona and Javesella. Although third codon positions may appear to be saturated within the ‘modern’ Delphacidae (Delphacini), they still contain important phylogenetic signals at the deepest taxonomic level. The easiest explanation for this is the difference in amino acid usage between Asiracinae and non‐Asiracinae. Overall, this fragment of the COI gene seems to be useful for a rather wide taxonomic range in Delphacidae, except maybe for resolving generic relationships in the large tribe Delphacini.
Basal lherzolites in the Bay of Islands Ophiolite: origin by detachment‐related telescoping of a ridge‐parallel melting gradient
Authors: Suhr  , Batanova
Published: March 2003

Decompression melting under a spreading centre should generate a residual upper mantle section with a weak vertical gradient in the degree of chemical depletion. Chemical data from the 6 km‐thick ophiolitic mantle section of the Table Mountain massif (Bay of Islands Ophiolite, Newfoundland) reveal, however, a depletion gradient of 1% melting/100 m in the basal mantle section of the massif. Within the framework of a segmented ridge we suggest that during ophiolite detachment intermittent accretion of progressively less depleted peridotites occurred to the (former) base of the ophiolite, amounting to a several hundred metre‐thick lherzolitic sole. In this view, the basal mantle section in Table Mountain extends a structural concept previously established for the metamorphic sole: both metamorphic gradient in the sole and melting gradient in the basal mantle rocks are apparent and of tectonic nature. Like the sole, the basal mantle sequence may thus expose mantle material derived from a scale larger than the preserved ophiolite complex.
Abrupt shifts in the late Cenozoic environment of north‐western China recorded in loess–palaeosol–red clay sequences
Authors: Xiong Shangfa , Ding Zhongli , Yang Shiling
Published: March 2003

Aeolian deposits can provide a sensitive record of climatic change. Two abrupt changes in grain size and accumulation rate have been identified at c. 2.4–2.6 Ma and 1.1–1.2 Ma in the loess–palaeosol–red clay sequences from the Loess Plateau of China. These changes are apparent in two major stepwise increases in grain size and accumulation rate following two abrupt prominent change events of grain size, implying a two‐step reorganization that was probably connected with two abrupt environmental change events. The correlation among different sections suggests that regional rather than local environmental processes are responsible for these changes. These changes likely record abrupt fluctuations in winter monsoon and the provenance of sediments associated with renewed Plio‐Pleistocene uplift of the Tibetan plateau, and its role on climate and detrital sediment yield.
Collisional processes at the junction of the Aleutian–Kamchatka arcs: new evidence from fission track analysis and field observations
Authors: Freitag Ralf , Gaedicke Christoph , Baranov Boris , Tsukanov Nikolay
Published: March 2003

The Aleutian island arc collides with the Kuril–Kamchatka arc in the area of the Cape Kamchatka peninsula. Field studies of neotectonic structures and apatite fission track analysis provide evidence for crustal plate shortening onshore the Cape Kamchatka peninsula. Tectonic blocks show differential mean exhumation rates varying from 0.18 ± 0.04 mm yr−1 in the north up to 1.2 ± 0.18 mm yr−1 in the south of the peninsula. A few of the fission track length data point to an unsteady exhumation rate. The blocks are separated by major dextral fault zones splaying off from Aleutian island arc fault zones. Across the western segment of the North American–Pacific Plate boundary the strain is partitioned along the fault zones and increases from north to south. Results from this study suggest that indentation and accretion of island arc fragments has recently occurred in the southeastern part of the Cape Kamchatka peninsula.
Hydrate phase equilibria in porous media: effect of pore size and salinity
Authors: Østergaard Kasper K. , Anderson Ross , Llamedo Maria , Tohidi Bahman
Published: March 2003

ABSTRACT Considering the ever‐increasing importance of marine gas hydrates, it is crucial to gain a better understanding of clathrate formation and decomposition in porous media. It is well established that, due to capillary effects, small‐diameter pores – similar to those found in natural sediments – act to inhibit hydrate stability. However, accurate data constraining these effects are still lacking. Here, we present experimental methane clathrate dissociation data for 3.5 mass% methanol aqueous solutions in confined silica glass pores of narrow distribution (30.6, 15.8, and 9.2 nm mean diameters). These data have been used to validate a thermodynamic model for clathrate stability porous media. Experimental data show a marked improvement on literature data – which we attribute to the experimental and interpretative methods used – and are in good agreement with the model predictions. Results suggest that mass transfer of inhibitors (methanol) and dissolved gas during clathrate formation/dissociation within the porous network plays an important role in controlling gas hydrate equilibria.
The TACPyAT repeats in the chalcone synthase promoter of Petunia hybrida act as a dominant negative cis‐acting module in the control of organ‐specific expression
Authors: van der Meer Ingrid M. , Brouwer Margreet , Spelt Cornelis E. , Mol Joseph‐N. M. , Stuitje Antoine R.
Published: March 2003

Analysis of the expression of the GUS reporter gene driven by various region of the Petunia hybrida chalcone synthase (chsA) promoter revealed that the developmental and organ‐specific expression of the chsA gene is conferred by a TATA proximal module located between −67 and −53, previously designated as the TACPyAT repeats.

Histochemical analysis of GUS reporter gene expression revealed that the organ‐specific 67 bp promoter fragment directs the same cell‐type specificity as a 530 bp promoter, whereas additional enhancer sequences are present within the more TATA distal region. Moreover, the region between −800 and −530 is also involved in extending the cell‐type specificity to the trichomes of flower organs and of young seedlings.

The mechanism by which the TACPyAT repeats modulate expression during plant development was studied by analysing the expression of the GUS gene driven by chimeric promoters consisting of the CaMV 35S enhancer(domain B, −750 to −90) fused to various chsA5′ upstream sequences. Detailed enzymatic and histochemical analysis revealed that in the presence of the TACPyAT module the CaMV 35S region only enhance GUS activity in those organs in which the chs A promoter is normally active. Furthermore, this analysis shows that enhancement in the presence of the CaMV 35S domain B is accomplished by increasing the number of cell types expressing the GUS gene within the organ, rather than enhancement of the chsA cell‐type‐specific expression within these organs. Deletion of the TACPyAT sequences in the chimeric promoter construct completely restores the well‐documented CaMV 35S domain B cell‐type specificity, showing that the TACPyAT module acts as a dominant negative cis‐acting element which controls both organ and developmental regulation of the chsA promoter activity.
A functional promoter shift of a chloroplast gene: a transcriptional fusion between a novel psbA gene copy and the trnK(UUU) gene in Pinus contorta
Authors: Lidholm Jonas , Gustafsson Petter
Published: March 2003

A comparative transcription analysis of the chloroplast trnK‐psbA‐trnH region of the two pine species Pinus contorta and Pinus sylvestris is reported. The chloroplast genome of P. contorta has previously been shown to contain a duplicated psbA gene copy integrated closely upstream of the split trnK gene. This rearrangement has resulted in the gene order psbAI‐trnK‐psbAII‐trnH, where psbAII is the ancestral psbA gene copy. In P. sylvestris, a species which lacks the psbA duplication, transcription of the trnK gene originates from a position 291 bp upstream of the trnK 5′ exon, adjacent to a canonical promoter structure. In P. contorta, the corresponding promoter structure has been separated from the trnK gene by the insertion of psbAI, and has, in addition, been partially deleted. Analysis of the transcriptional organization of the trnK‐psbA‐trnH region of the two pine species revealed that the trnK gene in P. contorta is transcriptionally fused to the inserted psbAI gene copy. As a result, trnK is under the control of the psbA promoter in this species and has therefore acquired psbA‐like expression characteristics. In P. sylvestris, accumulation of trnK transcripts is not significantly higher in light‐grown than in dark‐grown seedlings. In contrast, the level of trnK transcripts in P. contorta is approximately 12‐fold higher in the light than in the dark. When light‐grown seedlings of the two pine species were compared, an approximately 20‐fold higher level of trnK RNAs was found in P. contorta. In both pine species, evidence was obtained for trnK‐psbA and psbA‐trnH co‐transcription.
Temporal and spatial expression of a thiolprotease gene during pea ovary senescence, and its regulation by gibberellin
Authors: Granell A. , Harris N. , Pisabarro A. G. , Carbonell J.
Published: March 2003

Clones encoding a thiolprotease (tpp) have been isolated from a cDNA library of unpollinated, senescent pea ovaries and its pattern of expression during both ovary senescence and parthenocarpic development have been studied. The sequence of the tpp cDNA displays a high similarity with other plant and animal thiolproteases of the papain group. The homology is highest around the Cys‐His of the active centre; a 109 amino acid sequence at the carboxy terminus was found to be homologous only to thiolproteases of plant origin; this part of the mRNA is also present in another pea mRNA that exhibits similar patterns of induction. tpp mRNA shows a temporal pattern of accumulation that precedes that observed for proteolytic activity. Such accumulation did not occur when ovaries were induced to grow parthenocarpically by gibberellic acid (GA) treatment; furthermore the initial low level of expression present in ovaries decreased after GA treatment, indicating that the gene is down‐regulated by gibberellins. Spatially, tpp mRNA is localized mainly within the ovule and ovary vascular elements, and transiently within the endocarp of senescent ovaries. This pattern of expression precedes the development of the cytopathogenic effects observed as unpollinated ovaries undergo senescence.
Combinatorial interactions between positive and negative cis‐acting elements control spatial patterns of 4CL‐1 expression in transgenic tobacco
Authors: Hauffe Karl D. , Lee Stephen P. , Subramaniam Rajagopal , Douglas Carl J.
Published: March 2003

The phenylpropanoid enzyme 4‐coumarate:coenzyme A ligase (4CL) plays a key role in linking general phenylpropanoid metabolism to end‐product specific biosynthetic pathways. During vascular system and floral organ differentiation, the parsley 4CL‐1 gene is expressed in a restricted set of tissues and cell types where 4CL activity is required to supply precursors for the synthesis of diverse phenylpropanoid‐derived products such as lignin and flavonoids. In order to localize cis‐acting elements which specify complex patterns of 4CL‐1 expression, we analyzed the expression of internally deleted promoter fragment—GUS fusions in tobacco plants and parsley protoplasts. Elements located between −244 and −78 were required for most aspects of developmentally regulated expression. Within this region, three separate promoter domains containing partially redundant cis‐elements directed vascular‐specific expression when combined with a TATA‐proximal domain. A negative cis‐acting element which represses phloem expression was revealed in one of the domains and appears to be responsible for restricting vascular expression to the xylem. Distinct but overlapping promoter domain combinations were required for expression in floral organs, suggesting that different combinations of cis‐acting elements may direct expression in different organs. Gel retardation assays were used to demonstrate the formation of DNA‐protein complexes between factors present in nuclear extracts of parsley tissue culture cells and various tobacco organs and a 4CL‐1 promoter fragment. Competition experiments showed that complex formation required the presence of a 42 bp promoter domain shown to be critical for 4CL‐1 expression in vascular and floral tissues. The results are discussed in light of the coordinate expression of 4CL and other phenylpropanoid genes.
Phytochrome regulates GTP‐binding protein activity in the envelope of pea nuclei
Authors: Clark Gregory B. , Memon Abdul R. , Tong Chii‐Gong , Thompson Guy A. , Roux Stanley J.
Published: March 2003

Three GTP‐binding proteins with apparent molecular masses of 27, 28 and 30 kDa have been detected in isolated nuclei of etiolated pea plumules. After LDS‐PAGE and transfer to nitrocellulose these proteins bind [32p]GTP in the presence of excess ATP, suggesting that they are monomeric G proteins. When nuclei are disrupted, three proteins co‐purify with the nuclear envelope fraction and are highly enriched in this fraction. The level of [32P]GTP‐binding for all three protein bands is significantly increased when harvested pea plumules are irradiated by red light, and this effect is reversed by far‐red light. The results indicate that GTP‐binding activity associated with the nuclear envelope of plant cells is photoreversibly regulated by the pigment phytochrome.
Myb genes from Hordeum vulgare: tissue‐specific expression of chimeric Myb promoter/Gus genes in transgenic tobacco
Authors: Wissenbach Margit , Überlacker Bärbel , Vogt Friederike , Becker Dieter , Salamini Francesco , Rohde Wolfgang
Published: March 2003

The structures of the three Myb‐related genes Hv1, Hv5 and Hv33 from barley were determined. They contain a single intron located in the second repeat unit of the Myb‐related domain. By analogy to the animal MYB oncoproteins this conserved region of the gene product was shown by filter‐binding experiments to exhibit nucleic acid‐binding activity. Tobacco plants transgenic for chimeric Myb promoter/Gus genes express the enzyme in a developmentally controlled and tissue‐specific manner. During germination and early stages of plant growth, GUS activity is seen in the root cap and adjacent meristematic tissue. At later stages of plant development, GUS activity is predominantly observed in the shoot apical meristem, the roots and the nodal regions of the stem. Within the stem at stages of secondary growth, Myb promoters are active in defined cell types. In the internode low GUS activity is displayed by the innermost cell layer of the cortex, the starch sheath, that surrounds the vascular cylinder of secondary xylem and phloem tissue, as well as in pith rays originating from vascular cambium initials. In the nodal region Myb promoter‐controlled Gus expression is mainly confined to the abaxial starch sheath of the leaf trace, to the branch traces and to internal strands of primary phloem. It is suggested that in addition to their activity in meristematically active plant tissues Myb genes are expressed in conductive tissues that are closely associated with vascular bundles.
Detection of antisense transcripts in transgenic plants by RT—PCR
Authors: Brown John W.S. , Simpson Gordon G. , Clark Gillian , Lyon Jackie , Kumar Amar , Simpson Craig
Published: March 2003

A reverse transcriptase—polymerase chain reaction (RT—PCR) where one oligonucleotide primer is end‐labelled has been used to analyse expression in transgenic plants carrying antisense gene constructs. Specific detection of both sense and antisense RNA transcripts of the spliceosomal protein gene, U2B″, was achieved using the same pair of oligonucleotide primers. To maintain specificity, a reaction step in which reverse transcriptase was inactivated and RNA digested was found to be essential.
Detection of point mutations in the chloroplast genome by single‐stranded conformation polymorphism analysis
Authors: To Kin‐Ying , Liu Chiu‐I. , Liu Shih‐Tung , Chang Yu‐Sun
Published: March 2003

Single‐stranded conformation polymorphism (SSCP) analysis was used to examine the mutations of the chloroplast 16S rRNA locus of streptomycin‐resistant mutants in Nicotiana plumbaginifolia. DNA fragments of 121, 517, 968 and 1578 bp, each possessing a known point mutation, were generated by polymerase chain reaction (PCR). The resulting fragments were denatured and separated by nondenaturing polyacrylamide gel electrophoresis. Compared to the patterns of the wild‐type DNA fragments, the bands of the single‐stranded DNA fragments of 121 and 517 bp with base changes were shifted. However, no pattern variations were detected from the DNA fragments of 968 and 1578 bp generated from both wild‐type and mutants.
Molecular characterization of a 70 kDa heat‐shock protein of bean mitochondria
Authors: Vidal Vincent , Ranty Benoit , Dillenschneider Marietta , Charpenteau Martine , Ranjeva Raoul
Published: March 2003

A bean cDNA clone that specifies a 70 kDa heat‐shock protein (hsp70) has been isolated and sequenced. The nucleotide sequence analysis shows that the cDNA could encode a 72 kDa protein that is highly related to prokaryotic and mitochondrial members of the hsp70 family. The predicted protein was found to contain an amino‐terminal extension typical of transit sequences. The in vitro transcription/translation product of the cDNA behaved as a 72 kDa polypeptide as predicted from the longest open reading frame. This polypeptide could be imported into isolated mitochondria and recovered as a 68 kDa product. The imported protein is identical in size to a mitochondrial protein that cross‐reacts with hsp70‐specific antibodies. The import data and Western blot analysis suggest that the cDNA clone encodes a mitochondrial member of the hsp70 family. Electrophoretic and immunoblot analysis reveal that the protein is loosely associated to the mitochondrial envelope and also exists as discrete soluble protein aggregates of about 270 and 420 kDa. Hsp70 of bean mitochondria can be in vitro phosphorylated on threonine residues in a calcium‐dependent manner, and the modified protein was detected as an oligomer of about 160 kDa only. The data are discussed with respect to the chaperone function of hsp70 in mitochondria.
Fruit‐specific expression of the A. tumefaciens isopentenyl transferase gene in tomato: effects on fruit ripening and defense‐related gene expression in leaves
Authors: Martineau Belinda , Houck Catherine M. , Sheehy Raymond E. , Hiatt William R.
Published: March 2003

This paper describes the analysis of tomato plants transformed with a chimeric gene consisting of the promoter region of a fruit specifically expressed tomato gene linked to the ipt gene coding sequences from the Ti plasmid of Agrobacterium tumefaciens. The pattern of expression of this chimeric gene was found to be consistent with the expression of the endogenous fruit‐specific gene and consequently, plants expressing the chimeric gene were phenotypically normal until fruit maturation and ripening. A dramatically altered fruit phenotype, islands of green pericarp tissue remaining on otherwise deep red ripe fruit, was then evident in many of the transformed plants. Cytokinin levels in transformed plant fruit tissues were 10 to 100‐fold higher than in control fruit. In the leaves of a fruit‐bearing transformant, despite a lack of detectable ipt mRNA accumulation, approximately fourfold higher than control leaf levels of cytokinin were detected. It is suggested that cytokinin produced in fruit is being transported to the leaves since accumulation in leaves of PR‐1 and chitinase mRNAs, which encode defense‐related proteins known to be induced by cytokinin, occurred only when the transformant was reproductively active. Effects of elevated cytokinin levels on tomato fruit gene expression and cellular differentiation processes are also described.
A cis‐acting element and a trans‐acting factor involved in the wound‐induced expression of a horseradish peroxidase gene
Authors: Kawaoka Akiyoshi , Kawamoto Tomohiro , Sekine Masami , Yoshida Kazuya , Takano Mitsuo , Shinmyo Atsuhiko
Published: March 2003

The mechanisms that control the wound‐induced expression of the prxC2 gene for horseradish peroxidase (HRP) have been investigated. Analysis of the regulatory properties of 5′‐deleted promoters showed that a positive element involved in the response to wounding was located between −307 and −99 bp from the site of initiation of translation. In in vitro binding assays of tobacco nuclear proteins and DNA fragments of prxC2 promoter, the binding site was the Box 1 from −296 to −283 containing the CACGTG motif. To identify the functional role of Box 1, the prxC2 promoter that has been digested from the 5′ end to −289 with a disrupted Box 1 was fused to a reporter gene for β‐glucuronidase (GUS). No induction of GUS activity was observed in transgenic tobacco plants with the prxC2(−289)/GUS construct. These data indicated that the expression of prxC2 in response to wounding required the Box 1 sequence from −296 to −283. Furthermore, a tobacco cDNA expression library was screened and a cDNA clone for a protein, designated TFHP‐1, that bound specifically to the Box 1 sequence was identified. The putative TFHP‐1 protein contains a basic region and leucine zipper (bZip) motif and a helix—loop—helix (HLH) motif. The mRNA for TFHP‐1 was abundant in roots and stems, and it was not induced by wounding in leaves. In tobacco protoplasts, antisense TFHP‐1 suppressed the expression of prxC2 (−529)/GUS.
Functional expression of Saccharomyces cerevisiae CYP51A1 encoding lanosterol‐14‐demethylase in tobacco results in bypass of endogenous sterol biosynthetic pathway and resistance to an obtusifoliol‐14‐demethylase herbicide inhibitor
Authors: Grausem Bernard , Chaubet Nicole , Gigot Claude , Loper John C. , Benveniste Pierre
Published: March 2003

Nicotiana tabacum protoplasts have been transformed by Agrobacterium tumefaciens containing a T‐DNA in which the gene CYP51A1 encoding lanosterol‐14‐demethylase (LAN14DM) from Saccharomyces cerevisiae is under the control of a cauliflower mosaic virus (CaMV) 35S promoter. Two transformants strongly expressed the LAN14DM as shown by Northern and Western experiments. These transgenic calli were killed by LAB 170250F (LAB) (a phytotoxic fungicide inhibiting both plant obtusifoliol‐14‐demethylase (OBT14DM) and LAN14DM) but were resistant to γ‐ketotriazole (γ‐kt), a herbicide which has been shown to inhibit OBT14DM but not LAN14DM at a concentration that was lethal to control calli. However, these transgenic calli were killed by mixtures of γ‐kt plus fungicide inhibitors of LAN14DM such as ketoconazole, itraconazole or flusilazole which alone were not effective.

Further analysis of the transgenic calli grown in the presence of γ‐kt showed that their Δ5‐sterol content was close to that of untreated control calli obtained from protoplasts transformed with control plasmid; this is in agreement with evidence that the LAN14DM expressed from the transgene could bypass the blocked OBT14DM by using the plant substrate obtusifoliol. In contrast, control calli when treated with γ‐kt, displayed a sterol content strongly enriched in 14α‐methyl sterols and depressed in physiological Δ5‐sterols. When the transgenic calli were cultured in mixtures of γ‐kt and LAN14DM inhibitors sterol compositions enriched in 14α‐methyl sterols were obtained, reflecting a strong inhibition of both ‘endogenous’ OBT14DM and ‘exogenous’ LAN14DM.

Taken together these results show that in tobacco calli transformed with CYP51A1, resistance to a triazole herbicide arises from expression of a functional LAN14DM enzyme; its activity in transgenic tissues creates a bypass of the sterol biosynthetic pathway at the 14‐demethylase level when this latter is blocked by an OBT14DM herbicide inhibitor.
Construction of a trans‐dominant inhibitor for members of the TGA family of transcription factors conserved in higher plants
Authors: Miao Zhong‐He , Lam Eric
Published: March 2003

Activating sequence factor 1 (ASF‐1) is a conserved DNA‐binding activity that interacts with sequence elements containing TGACG motifs, some of which have been demonstrated to respond to exogenous application of auxin and salicylic acid. Genes encoding transcription factors with similar DNA‐binding specificity to ASF‐1 have been cloned from diverse plant species and these factors all contain a distinct basic‐leucine‐zipper (bZIP) motif. Members of this family of DNA‐binding proteins, designated as TGA factors, have been shown to interact with similar DNA sequences and at least seven distinct TGA genes are present in the Arabidopsis genome. To study the roles that this family of factors may play in plant development, a trans‐dominant inhibitor of ASF‐1 was constructed by deleting the basic portion of the bZIP domain in the tobacco factor TGA1a. In vitro co‐expression studies demonstrated that this deletion mutant, named TGA1a‐D, suppresses the DNA‐binding activity of wild‐type tobacco TGA1a, and three different members of the Arabidopsis TGA family. In contrast, co‐expression of TGA1a‐D with another class of bZIP proteins, the G‐Box Binding Factor family, showed no suppression of DNA‐binding activity. Over‐expression of TGA1a‐D in transgenic tobacco significantly decreased nuclear ASF‐1 relative to several other known factors, indicating that the proteins comprising ASF‐1 activity in vivo are likely TGA family members. Thus, TGA1a‐D may be a family‐specific inhibitor for the TGA family and should facilitate the study of ASF‐1 function in vivo.
The N‐terminal extension of the ADP/ATP translocator is not involved in targeting to plant mitochondria in vivo
Authors: Mozo Teresa , Fischer Karsten , Flügge Ulf Ingo , Schmitz Udo Klaus
Published: March 2003

The mitochondrial ADP/ATP translocator, also called adenine nucleotide translocase (ANT), is synthesized in plants with an N‐terminal extension which is cleaved upon import into mitochondria. In contrast, the homologous proteins of mammals or fungi do not contain such a transient amino terminal presequence. To investigate whether the N‐terminal extension is needed for correct intracellular sorting in vivo, translational fusions were constructed of the translocator cDNA—with and without presequence—with the β‐glucuronidase (gus) reporter gene. The distribution of reporter enzymatic activity in the subcellular compartments of transgenic plants and transformed yeast cells was subsequently analysed. The results show that: (i) the plant translocator presequence is not necessary for the correct localization of the ANT to the mitochondria; (ii) the mitochondrial targeting information contained in the mature part of the protein is sufficient to overcome, to some extent, the presence of plastid transit peptides; and (iii) the presequence alone is not able to target a passenger protein to mitochondria in vivo.
Evidence for K+ channel control in Vicia guard cells coupled by G‐proteins to a 7TMS receptor mimetic
Authors: Armstrong Fiona , Blatt Michael R.
Published: March 2003

To explore the involvement of a class of seven‐trans‐membrane‐span (7TMS) receptors in cellular signalling, a synthetic analogue (mas7) of the amphipathic tetradecapeptide mastoparan was used to mimic hormonal stimulus in guard cells of Vicia faba. The ability for mas7 to substitute for an activated receptor complex was assayed by the effect on guard cell ion channel activities in the absence of any hormonal stimulus. Currents carried by inward‐(IK,in) and outward‐(IK,out) rectifying potassium channels were determined under voltage clamp conditions before, during, and after exposure to mas7. The dominant effect of mas7 was to inactivate IK,in within 30 sec of application. By contrast, IK,out was largely unaffected under these conditions. The effect of mas7 on IK,in was both concentration‐ and voltage‐dependent. At any one clamp voltage, mas7 inactivation showed Michaelian behaviour, with a mean Ki of 0.05 ± 0.02 µM at −240 mV. Increasing mas7 concentration also shifted the voltage for half‐maximal activation of the current negative, with 0.5 µM mas7 effecting a −13 ± 2 mV displacement and lengthening the halftime for activation of the current by up to threefold. By contrast, the non‐amphipathic analogue of mas7, masCP, had no appreciable effect on the steady‐state current or its activation kinetics; nor was the poly‐cation polylysine able to substitute for mas7 in its action on the K+ channels. Application of the non‐hydrolysable analogue of GDP, GDP‐β‐S, either by iontophoresis or by diffusion from the microelectrode, effectively blocked mas7‐induced inactivation of IK,in. These, and additional results provide in vivo evidence for the involvement of G‐protein‐linked 7TMS receptors in the regulation of membrane transport in a higher plant cell.
Multiple phosphoinositide‐specific phospholipases C in oat roots: characterization and partial purification
Authors: Huang Chiung‐Hua , Tate Bonnie F. , Crain Richard C. , Coté Gary G.
Published: March 2003

Phosphoinositide‐specific phospholipases C are critical enzymes in the transduction of hormonal and environmental signals into animal cells. Several different isozymes of phospholipase C are known in mammalian systems which differ in their expression and regulation. Elucidation of the regulation of these phospholipases C has greatly advanced our understanding of the control of mammalian cell growth and development. Plant cells, too, contain phospholipases C specific for phosphoinositides, and there is evidence that they may be involved in plant cell responses to environmental stimuli. This paper reports that there are at least four variants of phosphoinositide‐specific phospholipase C in the roots of oat seedlings, two cytosolic and two plasma membrane‐associated. The two cytosolic and two plasma membrane variants can be separated on the basis of their affinity for binding to heparin. Both the cytosolic and the plasma membrane heparin‐binding forms have apparent molecular weights of about 50–70 kDa by size exclusion chromatography. The two heparin‐binding forms have been partially purified. The partially purified enzymes are activated by micromolar calcium and are specific for phosphorylated phosphoinositides; in their substrate specificities they resemble mammalian phospholipases C ɛ.
A system for insertional mutagenesis and chromosomal rearrangement using the Ds transposon and Cre—lox
Authors: Osborne Brian I. , Wirtz Uwe , Baker Barbara
Published: March 2003

A system for insertional mutagenesis and chromosomal rearrangement in Arabidopsis has been developed. The T‐DNA vectors are based on the maize transposon Ds, lox sites from the Cre—lox site‐specific recombination system, and transcriptional fusions expressing Ac transposase or Cre recombinase. The engineered transposon is termed Dslox. Transposed Dslox insertions were created by crossing plants bearing Dslox with plants expressing Ac transposase, then simultaneously selecting for excision and re‐insertion in F2 seedlings using the herbicides chlorsulfuron and phosphonothricin, respectively. F2 plants bearing stable Dslox insertions were identified by scoring for the absence of the Ac transposase T‐DNA, using a novel, visual marker in that T‐DNA. Two independent Dslox insertions were characterized and placed 5.6 and 16.5 cM from their T‐DNAlox, which mapped close to m506 on chromosome 4. Plants bearing either of the two different transposed Dsloxs and T‐DNAlox were crossed to plants expressing Cre recombinase, which catalyzed recombination between the lox site in transposed Dslox and the lox site in T‐DNAlox. Lox—lox recombinants were identified selectively amongst progeny of these crosses. Molecular and genetic analysis of the lox—lox rearrangements indicated that both were inversions. The smaller inversion was germinally transmitted from generation to generation as a simple trait, whereas the larger inversion was not transmitted to progeny of plants bearing the rearrangement.
Lectin genes are expressed throughout root nodule development and during nitrogen‐fixation in the Rhizobium—Medicago symbiosis
Authors: Bauchrowitz Martina A. , Barker David G. , Truchet Georges
Published: March 2003

In order to study the spatio‐temporal patterns of lectin gene expression throughout the various developmental stages of the Rhizobium—legume interaction, promoter‐gusA fusions for each of three lectin genes from Medicago truncatula (Mtlec1, 2 and 3) have been introduced into transgenic Medicago varia plants. Histochemical staining for β‐glucuronidase activity has shown that Mtlec1 and Mtlec3 are expressed independently of symbiosis in inner cortical and vascular tissues of the root, respectively. Transcription of Mtlec3 in the root stele is markedly enhanced following inoculation with Rhizobium meliloti. Furthermore, both Mtlec1 and Mtlec3 are transcribed in dividing cells of the root nodule primordium, whether elicited by R. meliloti or by purified nodulation (Nod) factors. These findings suggest that these two lectins are involved in developmental processes early during nodule ontogeny. In contrast, Mtlec2 is not expressed in uninoculated roots or during early stages of the symbiotic interaction. At later stages, all three lectin promoters are functional in mature nodules. In this context, lectin gene expression is restricted to non‐infected cells of both central and certain peripheral tissues and appears to be correlated with the nitrogen‐fixing capacity of the nodule.
Mutation of putative branchpoint consensus sequences in plant introns reduces splicing efficiency
Authors: Simpson Craig G. , Clark Gillian , Davidson Diane , Smith Philip , Brown John W.S.
Published: March 2003

Intron lariat formation between the 5′ end of an intron and a branchpoint adenosine is a fundamental aspect of the first step in animal and yeast nuclear pre‐mRNA splicing. Despite similarities in intron sequence requirements and the components of splicing, differences exist between the splicing of plant and vertebrate introns. The identification of AU‐rich sequences as major functional elements in plant introns and the demonstration that a branchpoint consensus sequence was not required for splicing have led to the suggestion that the transition from AU‐rich intron to GC‐rich exon is a major potential signal by which plant pre‐mRNA splice sites are recognized. The role of putative branchpoint sequences as an internal signal in plant intron recognition/definition has been re‐examined. Single nucleotide mutations in putative branchpoint adenosines contained within CUNAN sequences in four different plant introns all significantly reduced splicing efficiency. These results provide the most direct evidence to date for preferred branchpoint sequences being required for the efficient splicing of at least some plant introns in addition to the important role played by AU sequences in dicot intron recognition. The observed patterns of 3′ splice site selection in the introns studied are consistent with the scanning model described for animal intron 3′ splice site selection. It is suggested that, despite the clear importance of AU sequences for plant intron splicing, the fundamental processes of splice site selection and splicing in plants are similar to those in animals.
Chemically induced expression of the rolC‐encoded β‐glucosidase in transgenic tobacco plants and analysis of cytokinin metabolism: rolC does not hydrolyze endogenous cytokinin glucosides in planta
Authors: Faiss Martin , Strnad Miroslav , Redig Pascale , Doležal Karel , Hanuš Jan , Van Onckelen Harry , Schmülling Thomas
Published: March 2003

The rolC gene of Agrobacterium rhizogenes T‐DNA plays an essential role in the establishment of hairy root disease and its overexpression in transgenic plants causes pleiotropic developmental alterations. This study investigated whether the biological activity of the rolC β‐glucosidase is due to an alteration of the cytokinin balance in planta. HPLC radiocounting assays of [3H]‐labeled cytokinin glucosides fed exogenously to tobacco leaf disks, to rolC expressing Escherichia coli cells or cell‐free extracts showed that cytokinin N3‐ and O‐glucosides are the preferred substrate of the rolC protein. Hydrolysis of N7‐ and N9‐glucosides was not detected at substrate concentrations close to physiological levels. Furthermore, these conjugates were also not active as cytokinins in biotests when fed to rolC‐expressing tissues. For analysis of the rolC activity on endogenous cytokinin conjugates the gene was expressed under the transcriptional control of a modified tetracycline‐inducible 35S promoter. This was done to avoid possible interference with secondary effects or plant homeostatic mechanisms which could mask primary in planta events when transgenes are expressed constitutively. No changes in the endogenous pool of different cytokinin glucosides, as determined by a newly developed electrospray tandem mass spectroscopy directly coupled to high performance liquid chromatography, were found following chemical induction of the rolC gene. Also the levels of free cytokinins remained unchanged after gene induction. Hybrid tobacco plants expressing the cytokinin‐synthezising ipt gene and the rolC gene showed added phenotypes indicating that the rolC phenotype is mediated on a signalling pathway different from those of cytokinins. RolC/ipt hybrids also accumulated high levels of cytokinin O‐glucosides. It is concluded that the phenotypic alterations caused by the rolC gene product are not due to a release of free cytokinins from inactive conjugates, most likely because of subcellular compartmentation of the putative substrate.
Isolation and characterization of cDNAs encoding transcription factor IIB from Arabidopsis and soybean
Authors: Baldwin Don A. , Gurley William B.
Published: March 2003

Clones containing the complete coding regions for transcription factor IIB (TFIIB) were isolated from cDNA libraries of Arabidopsis and soybean. The predicted protein sequences for TFIIB from these two species are highly homologous and contain the same structural motifs and organization as seen in other eukaryotes and Archaebacteria. Southern and Northern blots and primer extension for Arabidopsis TFIIB indicate a low copy number gene encodes a transcript of 1400 nt with a leader of 180 nt. Phylogeny analysis places the plant proteins into one group nearly equidistant from metazoans, fungi, and Archaebacteria.
Cell fate in the development of the Arabidopsis flower
Authors: Furner Ian J.
Published: March 2003

The Arabidopsis flower consists of four concentric whorls of organs. The first (outermost) whorl consists of four sepals and the fourth (innermost) whorl is made up of two carpels. Cell fate in the first and fourth whorls was studied using X‐ray‐induced yellow ch‐42 sectors. Sector boundaries were found to be non‐random around the two whorls and four generalizations relating the marked and unmarked tissues were deduced. In the sepal and carpel whorls the smallest sectors of marked and unmarked tissue were found to be one half of a sepal and one half of a carpel, respectively. A detailed frequency‐distance map of the floral primordium was made and found to be a ridge with the fourth whorl carpels at the summit and the first whorl transverse sepal pair at the base. Consideration of: the rate of loss of chimerism in the inflorescence meristem, the frequency‐distance across the flower and the frequency‐distance between successive flowers, was used to produce an abstract model of the inflorescence meristem.
Salt regulation of transcript levels for the c subunit of a leaf vacuolar H+‐ATPase in the halophyte Mesembryanthemum crystallinum
Authors: Tsiantis Miltos S. , Bartholomew Dolores M. , Smith J. Andrew C.
Published: March 2003

The halophyte Mesembryanthemum crystallinum is an inducible crassulacean acid metabolism (CAM) plant native to seasonally arid coastal environments that has been widely used to study plant responses to environmental stress. On exposure of plants to salt, the activities of both the tonoplast (vacuolar) H+‐ATPase (V‐ATPase) and Na+/ H+ antiporter increase in leaf cells, thereby energizing vacuolar salt accumulation. To investigate the molecular basis of this response, a cDNA (Vmac1) encoding the H+‐conducting c subunit (16.6 kDa) of an M. crystallinum V‐ATPase has been cloned. Northern analysis of RNA from leaves of plants treated with NaCl or with isoosmotic mannitol solutions demonstrated (i) that NaCl increased steady‐state transcript levels for the V‐ATPase c subunit, and (ii) that this effect was caused by the ionic rather than the osmotic component of salt stress. Southern analysis of genomic DNA suggested the probable existence of more than one gene for this subunit of the V‐ATPase in M. crystallinum. Expression studies using the 3′‐untranslated region of the Vmac1 cDNA as a probe showed that the corresponding salt‐inducible transcript was preferentially expressed in leaves. Induction by salt was also observed in juvenile plants in addition to adult ones. These findings, as well as the inability of mannitol to upregulate mRNA levels for this gene, clearly differentiate between the induction of transcript for the V‐ATPase c subunit and for genes involved in the CAM pathway in M. crystallinum. Further, the plant growth regulator abscisic acid (ABA) was able to mimic the effect of salt on transcript levels for the V‐ATPase c subunit, suggesting the possible involvement of ABA in a distinct signal‐transduction pathway linked to vacuolar salt accumulation in this highly salt‐tolerant species.
Cloning and expression in Escherichia coli of the obtusifoliol 14α‐demethylase of Sorghum bicolor (L.) Moench, a cytochrome P450 orthologous to the sterol 14α‐demethylases (CYP51) from fungi and mammals
Authors: Bak Søren , Kahn Rachel Alice , Olsen Carl Erik , Halkier Barbara Ann
Published: March 2003

Obtusifoliol 14β‐demethylase from Sorghum bicolor (L.) Moench has been cloned using a gene‐specific probe generated using PCR primers designed from an internal 14 amino acid sequence. The sequence identifies sorghum obtusifoliol 14α‐demethylase as a cytochrome P450 and it is assigned to the CYP51 family together with the sterol 14α‐demethylases from fungi and mammals. The presence of highly conserved regions in the amino acid sequences, analogous substrates and the same metabolic role demonstrate that the sterol 14α‐demethylases are orthologous enzymes. The sterol 14α‐demethylases catalyse an essential step in sterol biosynthesis as evidenced by the absence of a 14α‐methyl group in all known functional sterols. A functional sorghum obtusifoliol 14α‐demethylase was expressed at high levels in Escherichia coli and purified using an efficient method based on temperature‐induced Triton X‐114 phase partitioning. The recombinant purified enzyme produced a type I spectrum with obtusifoliol as substrate. Reconstitution of purified recombinant enzyme with sorghum NADPH—cytochrome P450 reductase in dilaurylphosphatidylcholine micelles confirms that obtusifoliol 14α‐demethylase catalyses the 14α‐demethylation of obtusifoliol to 4α‐methyl‐5α‐ergosta‐8,14,24(28)‐trien‐3β‐ol as evidenced by GC—MS. The isolation of a cDNA clone encoding the plant sterol 14α‐demethylase, combined with the previously isolated cDNA clones for fungal and mammalian sterol 14α‐demethylases, provides an important tool in the rational design of specific inhibitors towards the individual sterol 14α‐demethylases.