bluuebunny/crossref_metadata_2025 huggingface dataset:
Towards improved ground models for slope instability evaluations through better characterization of sediment‐hosted gas‐hydrates
Authors: Gunn D. A. , Nelder L. M. , Rochelle C. A. , Bateman K. , Jackson P. D. , Lovell M. A. , Hobbs P. R. N. , Long D. , Rees J. G. , Schultheiss P. , Roberts J. , Francis T.
Published: March 2003

ABSTRACT Recently developed effective stress‐controlled geophysical property models are used in passive‐margin slope instability analyses including simulated earthquake motion. The pressure–temperature (P–T) history of sediment‐hosted gas hydrate may significantly alter the geophysical property profile of the sediment column (e.g. metastable cement or increased pore pressures). This can result in significant amplification of earthquake ground motion, and thus seabed instability, where hydrates are present. Published studies suggest destabilization of these high‐pressure/low‐temperature sediment‐hosted hydrates could trigger catastrophic slope failures with consequent liberation of ‘greenhouse’ gases and significant effects on global climate. To provide improved ground models for slope instability analyses we are investigating the influence of P–T history on hydrate distribution in sediments through the development of laboratory techniques to enable geophysical quantification of hydrate morphology and fabric on hydrate stability.
Differences in DNA‐methylation are associated with a paramutation phenomenon in transgenic petunia
Authors: Meyer Peter , Heidmann Iris , Niedenhof Ingrid
Published: March 2003

The transgenic petunia line 17‐R contains one copy of the maize A1 gene which mediates brick‐red pelargonidin pigmentation of the flower. A white derivative, 17‐W, was isolated from homozygous progeny of this line in which no pelargonidin pigmentation was observed. In 17‐W the 35S promoter driving the A1 gene was hypermethylated, in contrast to its hypomethylated state in 17‐R. Progeny plants carrying both the 17‐R and 17‐W allele did not show the expected A1 phenotype. Predominantly white progeny and variable plants were observed which showed a continuous change in pattern and intensity of pelargonidin pigmentation. This reduction of A1 activity argues for a semidominant effect of the 17‐W allele which inhibits the activity of its homologue, 17‐R. This system shows striking similarities to some paramutation phenomena in plants which represent a heritable change in gene function of a paramutable allele directed by a paramutagenic homologue. The analysis of the methylation patterns of the A1 alleles suggests that interactions between differentially methylated alleles are responsible for the paramutation‐like effect which is mediated by somatic pairing. The analogy of this system to other phenomena based on homology‐dependent interlocus trans‐inactivation supports the assumption that those may be based on a related mechanism which includes an interaction between ectopic homologues.
Expression of an Arabidopsis sucrose synthase gene indicates a role in metabolization of sucrose both during phloem loading and in sink organs
Authors: Martin Thomas , Frommer Wolf B. , Salanoubat Marcel , Willmitzer Lothar
Published: March 2003

Sucrose synthase, an important enzyme in carbohydrate metabolism, catalyzes the reversible conversion of sucrose and UDP to UDP‐glucose and fructose in vitro. To investigate the in vivo function of sucrose synthase, both the gene (Asus1) and a corresponding cDNA from roots of Arabidopsis were isolated. The Asus1 gene has homologies of 67–72% to sucrose synthase genes from other species. Histochemical GUS analysis of Arabidopsis and tobacco plants transformed with a 1.5 kb Asus1 promoter fragment transcriptionally fused to the β‐glucuronidase reporter gene showed that the Asus1 gene is expressed in the phloem of leaves, and in roots. Induction is found under conditions of limited ATP supply and increased demand for translocation of carbohydrates such as anaerobic or cold treatment. During anaerobiosis the increase in RNA level leads to increased sucrose synthase activity in roots. The expression pattern and regulation of the gene suggest that sucrose synthase is involved in the supply of energy for phloem loading in source tissues, and in metabolization of sucrose in sink tissues after unloading.
A mutation in the Arabidopsis DET3 gene uncouples photoregulated leaf development from gene expression and chloroplast biogenesis
Authors: Cabrera y Poch Héctor L. , Peto Charles A. , Chory Joanne
Published: March 2003

The genetic and phenotypic characterization of a new Arabidopsis mutant, de‐etiolated‐3, (det3), involved in light‐regulated seedling development is described. A recessive mutation in the DET3 gene uncouples light signals from a subset of light‐dependent processes. The det3 mutation causes dark‐grown Arabidopsis thaliana seedlings to have short hypocotyls, expanded cotyledons, and differentiated leaves, traits characteristic of light‐grown seedlings. Despite these morphological changes, however, the det3 mutant does not develop chloroplasts or show elevated expression of nuclear‐ and chloroplast‐encoded light‐regulated mRNAs. The det3 mutation thus uncovers a downstream branch of the light transduction pathways that separates leaf development from chloroplast differentiation and light‐regulated gene expression. In addition, light‐grown det3 plants have reduced stature and apical dominance, suggesting that DET3 functions during growth in normal light conditions as well. The genetic interactions between mutations in det1, det2, and det3 are described. The phenotypes of doubly mutant strains suggest that there are at least two parallel pathways controlling light‐mediated development in Arabidopsis.
A plant gene for photolyase: an enzyme catalyzing the repair of UV‐light‐induced DNA damage
Authors: Batschauer Alfred
Published: March 2003

Photolyases are thought to be critical components of the defense of plants against damage to DNA by solar ultraviolet light, but nothing is known about their molecular or enzymatic nature. The molecular cloning of a photolyase from mustard (Sinapis alba) described here is intended to increase the knowledge about this important repair mechanism in plant species at a molecular level. The gene encodes a polypeptide of 501 amino acids with a predicted molecular mass of 57 kDa. There is a strong sequence similarity to bacterial and yeast photolyases, with a close relationship to enzymes with a deazaflavin chromophor. The plant photolyase is shown to be functional in Escherichia coli which also indicates conservation of photolyases during evolution. It is demonstrated that photolyase expression in plants is light induced, thus providing good evidence for the adaptation of plants to their environment in order to diminish the harmful effects of sunlight.
A repeating 11‐mer amino acid motif and plant desiccation
Authors: Dure Leon
Published: March 2003

Among the proteins that accumulate as plant seeds desiccate are several protein families that are composed principally of a tandemly repeated 11‐mer amino acid motif. Proteins containing the same motif accumulate in the desiccating leaves of a desiccation‐tolerant plant species.

This motif is characterized by apolar residues in positions 1, 2, 5 and 9, and charged or amide residues in positions 3, 6, 7, 8 and 11. An α helical arrangement of the 11‐mer repeating unit gives an amphiphilic helix whose hydrophobic stripe twists in a right‐handed fashion around the helix. Should these proteins dimerize via binding of their hydrophobic faces, a right‐handed coiled coil would be formed. Such a structure has not previously been observed. A conceivable function for these proteins in ion sequestration in the desiccated state is proposed.
Mechanosensory calcium‐selective cation channels in epidermal cells
Authors: Ding Jiu Ping , Pickard Barbara G.
Published: March 2003

This paper explores the properties and likely functions of an epidermal Ca2+‐selective cation channel complex activated by tension. As many as eight or nine linked or linkable equivalent conductance units or co‐channels can open together. Open time for co‐channel quadruplets and quintuplets tends to be relatively long with millimolar Mg2+ (but not millimolar Ca2+) at the cytosolic face of excised plasma membrane. Sensitivity to tension is regulated by transmembrane voltage and temperature. Under some circumstances channel activity is synchronized in rhythmic pulses. Certain lanthanides and a cytoskeleton‐disturbing herbicide that inhibit gravitropic reception act on the channel system at low concentrations. Specifically, ethyl‐N‐phenylcarbamate promotes tension‐dependent activity at micromolar levels. With moderate suction, Gd3+ provided at about 0.5 μM at the extracellular face of the membrane promotes for several seconds but may then become inhibitory. Provision at 1–2 μM promotes and subsequently inhibits more vigorously (often abruptly and totally), and at high levels inhibits immediately. La3+, a poor gravitropic inhibitor, acts similarly but much more gradually and only at much higher concentrations. These properties, particularly these susceptibilities to modulation, indicate that in vivo the mechanosensitive channel must be mechano‐sensory and mechanoregulatory. It could serve to transduce the shear forces generated in the integrated wall‐membrane‐cytoskeleton system during turgor changes and cell expansion as well as transducing the stresses induced by gravity, touch and flexure. In so far as such transduction is modulated by voltage and temperature, the channels would also be sensors for these modalities as long as the wall‐membrane‐cytoskeleton system experiences mechanical stress.
Increased cell cycle‐dependent staining of plant cells by the antibody MPM‐2 correlates with preprophase band formation
Authors: Young T. , Hyams J.S. , Lloyd C.W.
Published: March 2003

Cytoplasmic microtubules of animal cells catastrophically depolymerize upon entry into mitosis but in higher plants there is a longer transition during which cortical microtubules form an increasingly narrow preprophase band, and the chromatin gradually condenses. Progression towards mitosis in onion root tip cells was analysed using a CCD camera and image processing to quantify fluorescence staining by the monoclonal antibody MPM‐2, which recognizes mitotic phosphoproteins in a range of eukaryotic cells. MPM‐2 fluorescence, which was predominantly nuclear, was categorized relative to the stage of the DNA cycle (using DAPI), and to the microtubule cycle (using anti‐tubulin) in individual cells. Cells with the characteristic interphase cortical microtubule arrays had a bimodal distribution of DAPI fluorescence, indicating that some were in G1 (2C DNA) whilst the double value suggested the others to be in G2 (4C). There was no difference in MPM‐2 fluorescence between 2C and 4C cells possessing the cortical array in which microtubules were evenly distributed. However, in 4C cells possessing a preprophase band MPM‐2 values doubled; this relationship applied not only to tight PPBs but to early, broad PPBs in which the individual microtubules could still be distinguished. Since alkaline phosphatase abolished MPM‐2 reactivity it is concluded that mitotic phosphoproteins do not necessarily begin to accumulate in G2 per se, but during that part of G2 when the preprophase band first becomes recognizable as a distinct entity.
A (1→3,1→4)‐β‐glucan‐specific monoclonal antibody and its use in the quantitation and immunocytochemical location of (1→3,1→4)‐β‐glucans
Authors: Meikle Peter J. , Hoogenraad Nicholas J. , Bonig Ingrid , Clarke Adrienne E. , Stone Bruce A.
Published: March 2003

Monoclonal antibodies were raised against a (1→3,1→4)‐β‐glucan‐bovine serum albumin (BSA) conjugate. One antibody (BG1) selected for further characterization, was specific for (1→3,1→4)‐β‐glucan, displaying no binding activity against a (1→3)‐β‐glucan‐BSA conjugate and minimal binding against a cellopentaose‐BSA conjugate. A range of oligosaccharides was prepared by enzymatic digestion of (1→3,1→4)‐β‐glucan, purified by size exclusion chromatography and characterized by 1H‐NMR and anion exchange chromatography.

These (1→3,1→4)‐β‐oligoglucosides, together with (1→3)‐β‐ and (1→4)‐β‐oligoglucosides were used to characterize the binding site of the monoclonal antibody (BG1) by competitive inhibition. The monoclonal antibody showed maximal binding to a heptasaccharide with the structure Glc(1→3) Glc(1→4) Glc(1→4) Glc(1→3) Glc(1→4) Glc(1→4) Glc and was determined to have an affinity constant of 3.8 × 104 M−1 for this oligoglucoside.

The monoclonal antibody (BG1) has been used to develop a sensitive sandwich ELISA for the specific quantitation of (1→3,1→4)‐β‐glucans. The assay operates in the range 1–10 ng ml−1 and shows no significant cross‐reaction with tamarind xyloglucan, wheat endosperm arabinoxylan or carboxymethyl‐pachyman ((1→3)‐β‐glucan). When used with a second‐stage, rabbit anti‐mouse gold conjugate and viewed under the electron microscope, the monoclonal antibody probe was found to bind strongly to the walls of the aleurone in thin sections of immature wheat (Triticum aestivum) cv. Millewa grains but not to the middle lamella region. A previously described specific anti‐(1→3)‐β‐glucan antibody (Meikle et al., 1991) bound to discrete patches on the aleurone walls, believed to be plasmodesmata.
Molecular analysis of protein domain function encoded by the myb‐homologous maize genes C1, Zm 1 and Zm 38
Authors: Franken Philipp , Schrell Susanne , Peterson Peter A. , Saedler Heinz , Wienand Udo
Published: March 2003

Two maize genes, Zm 1 and Zm 38, related to the regulatory anthocyanin gene C1 were analyzed molecularly and used for fusion constructs in transient domain swapping experiments with the C1 wild‐type gene. It was shown that both genes (Zm 1 and Zm 38) influence the expression of the A1 locus, a target gene for C1. Zm 1 activates the A1 promoter, however it does not turn on the whole anthocyanin pathway. The Zm 38 gene product shows functions similar to C1‐I, a dominant inhibitor of the C1 wild‐type gene. Concerning the trans‐inhibition by C1‐I two effects seem to be involved, competition for binding and formation of heterodimers. Further analysis of C1 function was carried out by a fine structure analysis of C1 mutants induced by the insertion and excision of transposable elements. These experiments indicate that for the activating domain of the protein, the formation of an alpha helix seems to be more important than a high negative charge.
Isolation and characterization of two cDNA clones encoding ATP‐sulfurylases from potato by complementation of a yeast mutant
Authors: Klonus Dirk , Höfgen Rainer , Willmitzer Lothar , Riesmeier Jörg W.
Published: March 2003

Sulfur plays an important role in plants, being used for the biosynthesis of amino acids, sulfolipids and secondary metabolites. After uptake sulfate is activated and subsequently reduced to sulfide or serves as donor for sulfurylation reactions. The first step in the activation of sulfate in all cases studied so far is catalyzed by the enzyme ATP‐sulfurylase (E.C. 2.7.7.4.) which catalyzes the formation of adenosine‐5′‐phosphosulfate (APS). Two cDNA clones from potato encoding ATP‐sulfurylases were identified following transformation of a Saccharomyces cerevisiae mutant deficient in ATP‐sulfurylase activity with a cDNA library from potato source leaf poly(A)+ RNA cloned in a yeast expression vector. Several transformants were able to grow on a medium with sulfate as the only sulfur source, this ability being strictly linked to the presence of two classes of cDNAs. The clones StMet3‐1 and StMet3‐2 were further analyzed. DNA analysis revealed an open reading frame encoding a protein with a molecular mass of 48 kDa in the case of StMet3‐1 and 52 kDa for StMet3‐2. The deduced polypeptides are 88% identical at the amino acid level. The clone StMet3‐2 has a 48 amino acid N‐terminal extension which shows common features of a chloroplast transit peptide. Sequence comparison of the ATP‐sulfurylase Met3 from Saccharomyces cerevisiae with the cDNA StMet3‐1 (StMet3‐2) reveals 31% (30%) identity at the amino acid level. Protein extracts from the yeast mutant transformed with the clone StMet3‐1 displayed ATP‐sulfurylase activity. RNA blot analysis demonstrated the expression of both genes in potato leaves, root and stem, but not in tubers. To the best of the authors' knowledge this is the first cloning and identification of genes involved in the reductive sulfate assimilation pathway from higher plants.
Rhizobium meliloti Nod factors elicit cell‐specific transcription of the ENOD12 gene in transgenic alfalfa
Authors: Journet E.P. , Pichon M. , Dedieu A. , De Billy F. , Truchet G. , Barker D.G.
Published: March 2003

Extracellular lipo‐oligosaccharides of Rhizobium, known as Nod factors, play a key role in the molecular signal exchange which leads to the specific nitrogen‐fixing symbiotic association between the soil microbe and Its host legume. The biological activity of Nod factors and their perception by the host plant during the earliest stages of the Rhizobium/legume interaction have been studied using transgenic alfalfa carrying a fusion between the promoter of the early nodulin gene MtENOD12 and the β‐glucuronidase (GUS) reporter gene. Histochemical staining has shown that GUS accumulates specifically in the differentiating root epidermis, prior to and during root hair emergence, within 2–3 h following the addition of purified Rhizobium meliloti Nod factors. This precocious transcriptional activation of the MtENOD12 gene, reminiscent of that observed after inoculation with intact Rhizobium, implies that the Nod factor signal can be perceived at a developmental stage preceding root hair formation. GUS activity can be detected following treatment with a wide range of R. meliloti Nod factor concentrations down to 10−13 M, and furthermore, this rapid response to the bacterial elicitor appears to be non‐systemic. Significantly, MtENOD12—GUS expression is not observed after inoculation with a R. meliloti nodH mutant which synthesizes exclusively non‐sulphated Nod factors. Indeed purified Nod factors which lack the sulphate substituent are approximately 1000‐fold less active than their sulphated counterparts. Thus, the triggering of ENOD12 transcription in the alfalfa root epidermis is a rapid molecular response which is subject to the same host‐specificity determinant (Nod factor sulphation) that governs the interaction between alfalfa and its bacterial symbiont.
Jellyfish green fluorescent protein as a reporter for virus infections
Authors: Baulcombe David C. , Chapman Sean , Santa Cruz Simon
Published: March 2003

The gene encoding green fluorescent protein (GFP) of Aequorea victoria was introduced into the expression cassette of a virus vector based on potato virus X (PVX). Host plants of PVX inoculated with PVX.GFP became systemically infected. Production of GFP in these plants was detected initially between 1 and 2 days postinoculation by the presence of regions on the inoculated leaf that fluoresced bright green under UV light. Subsequently, this green fluorescence was evident in systemically infected tissue. The fluorescence could be detected by several methods. The simplest of these was by looking at the UV‐illuminated plants in a darkened room. The PVX.GFP‐infected tissue has been analysed either by epifluorescence or confocal laser scanning microscopy. These microscopical methods allow the presence of the virus to be localized to individual infected cells. It was also possible to detect the green fluorescence by spectroscopy or by electrophoresis of extracts from infected plants. To illustrate the potential application of this reporter gene in virological studies a derivative of PVX.GFP was constructed in which the coat protein gene of PVX was replaced by GFP. Confocal laser scanning microscopy of the inoculated tissue showed that the virus was restricted to the inoculated cells thereby confirming earlier speculation that the PVX coat protein is essential for cell‐to‐cell movement. It is likely that GFP will be useful as a reporter gene in transgenic plants as well as in virus‐infected tissue.
Functional dissection of the promoter of the pollen‐specific gene NTP303 reveals a novel pollen‐specific, and conserved cis‐regulatory element
Authors: Weterings Koen , Schrauwen Jan , Wullems George , Twell David
Published: March 2003

Regulatory elements within the promoter of the pollen‐specific NTP303 gene from tobacco were analysed by transient and stable expression analyses. Analysis of precisely targeted mutations showed that the NTP303 promoter is not regulated by any of the previously described pollen‐specific cis‐regulatory elements. However, two adjacent regions from −103 to −86 bp and from −86 to −59 bp were shown to contain sequences which positively regulated the NTP303 promoter. Both of these regions were capable of driving pollen‐specific expression from a heterologous promoter, independent of orientation and in an additive manner. The boundaries of the minimal, functional NTP303 promoter were determined to lie within the region −86 to −51 bp. The sequence AAATGA localized from −94 to −89 bp was identified as a novel cis‐acting element, of which the TGA triplet was shown to comprise an active part. This element was shown to be completely conserved in the similarly regulated promoter of the Bp10 gene from Brassica napus encoding a homologue of the NTP303 gene.
Is hydrogen peroxide a second messenger of salicylic acid in systemic acquired resistance?
Authors: Neuenschwander Urs , Vernooij Bernard , Friedrich Leslie , Uknes Scott , Kessmann Helmut , Ryals John
Published: March 2003

Elevated levels of salicylic acid (SA) are required for the induction of systemic acquired resistance (SAR) in plants. Recently, a salicylic acid‐binding protein (SABP) isolated from tobacco was shown to have catalase activity. Based on this finding elevated levels of hydrogen peroxide (H2O2) were postulated to act as a second messenger of SA in the SAR signal transduction pathway. A series of experiments have been carried out to clarify the role of H2O2 in SAR‐signaling. No increase of H2O2 was found during the onset of SAR. Induction of the SAR gene, PR‐1, by H2O2 and H2O2‐inducing chemicals is strongly suppressed in transgenic tobacco plants that express the bacterial salicylate hydroxylase gene, indicating that H2O2 induction of SAR genes is dependent on SA accumulation. Following treatment of plants with increasing concentrations of H2O2, a dose‐dependent accumulation of total SA species was found, suggesting that H2O2 may induce PR‐1 gene expression through SA accumulation. While the results do not support a role for H2O2 in SAR signaling, it is suggested that SA inhibition of catalase activity may be important in tissues undergoing a hypersensitive response.
Hydrogen peroxide does not function downstream of salicylic acid in the induction of PR protein expression
Authors: Bi Yong‐Mei , Kenton Paul , Mur Luis , Darby Robert , Draper John
Published: March 2003

The roles of salicylic acid (SA) and H2O2 in the induction of PR proteins in tobacco have been examined. Studies were conducted on wild‐type tobacco and plants engineered to express a bacterial salicylate hydroxylase capable of metabolizing SA to catechol (SH‐L plants). Wild‐type and PR‐1a—GUS‐transformed plants express PR‐1a following challenge with Pseudomonas syringae pathovar syringae, SA or 2,6‐dichloro‐isonicotinic acid (INA). In contrast, SH‐L plants failed to respond to SA but did express PR‐1a following INA treatment. H2O2 and the irreversible catalase inhibitor 3‐amino‐1,2,4‐triazole (3‐AT) were found to be weak inducers of PR‐1a expression (relative to SA) in wild‐type tobacco but were unable to induce PR‐1a in SH‐L plants, suggesting that the action of these compounds depends upon the accumulation of SA. A model has been proposed suggesting that SA binds to and inhibits a catalase inducing an increase in H2O2 leading to PR protein expression. Catalase activity has been measured in tobacco and no significant changes in activity following infection with P. syringae pv. syringae were detected. Furthermore, inhibition of catalase activity in vitro in plant extracts requires pre‐incubation and only occurs at SA concentrations above 250 µM. Leaf disks pre‐incubated with 1 mM SA do accumulate SA to these levels and PR‐1a is efficiently induced but there is no apparent inhibition of catalase activity. It is also shown that a SA‐responsive gene, PR‐1a, and a H2O2‐sensitive gene, AoPR‐1, are both relatively insensitive to 3‐AT suggesting that induction of these genes is unlikely to be due entirely to inhibition of an endogenous catalase.
Characterization of a plasma membrane‐associated phosphoinositide‐specific phospholipase C from soybean
Authors: Shi Jinrui , Gonzales Robert A. , Bhattacharyya Madan K.
Published: March 2003

Phosphoinositide‐specific phospholipase C (PI‐PLC) is a key signal transducing enzyme which generates the second messengers inositol trisphosphate and diacylglycerol in mammalian cells. A cDNA clone (PI‐PLC1) encoding a phosphoinositide‐specific phospholipase C was isolated from soybean by screening a cDNA expression library using an anti‐(plasma membrane) serum. Genomic DNA gel blot analysis suggested that the corresponding gene is a member of a multigene family. The deduced amino acid sequence of the soybean PI‐PLC1 isozyme contains the conserved X and Y regions, found in other PI‐PLCs. It is closely related to mammalian δ‐type PI‐PLCs, Dictyostelium discoideum PI‐PLC and yeast PI‐PLC1 in terms of the arrangement of the conserved region. Unlike mammalian δ‐type PI‐PLCs and yeast PI‐PLC1, the putative Ca2+‐binding site of the soybean PI‐PLC1 is located in the region spanning the X and Y domains, and the N‐terminal region is truncated. FLAG epitope‐tagged PI‐PLC1 fusion protein purified from transgenic tobacco plants showed phosphoinositide‐specific phospholipase C activity. Heterologous expression of the soybean PI‐PLC1 cDNA in a yeast PI‐PLC1 deletion mutant complemented the lethality phenotype of haploid PI‐PLC1 disruptants. Immunoblot analysis of the cell fractions prepared from transgenic tobacco plants over‐expressing the FLAG epitope‐tagged PI‐PLC1 fusion protein indicated that the protein encoded by the PI‐PLC1 cDNA was localized in the cytosol and plasma membrane.
Green‐fluorescent protein as a new vital marker in plant cells
Authors: Sheen Jen , Hwang Seongbin , Niwa Yasuo , Kobayashi Hirokazu , Galbraith David W.
Published: March 2003

The green‐fluorescent protein (GFP) from jellyfish Aequorea victoria has been used as a convenient new vital marker in various heterologous systems. However, it has been problematic to express GFP in higher eukaryotes, especially in higher plants. This paper reports that either a strong constitutive or a heat‐shock promoter can direct the expression of GFP which is easily detectable in maize mesophyll protoplasts. In this single‐cell system, bright green fluorescence emitted from GFP is visible when excited with UV or blue light even in the presence of blue fluorescence from the vacuole or the red chlorophyll autofluorescence from chloroplasts using a fluorescence microscope. No exogenous substrate, co‐factor, or other gene product is required. GFP is very stable in plant cells and shows little photobleaching. Viable cells can be obtained after fluorescence‐activated cell sorting based on GFP. The paper further reports that GFP can be detected in intact tissues after delivering the constructs into Arabidopsis leaf and root by microprojectile bombardment. The successful detection of GFP in plant cells relies on the use of a universal transcription enhancer from maize or the translation enhancer from tobacco mosaic virus (TMV) to boost the expression. This new reporter could be used to monitor gene expression, signal transduction, co‐transfection, transformation, protein trafficking and localization, protein‐protein interaction, cell separation and purification, and cell lineage in higher plants.
Chromosomal integration is required for spatial regulation of expression from the β‐phaseolin promoter
Authors: Frisch David A. , Van Der Geest Apolonia H.M. , Dias Kalyani , Hall Timothy C.
Published: March 2003

The stringency of spatial expression of phaseolin, the major storage protein of bean (Phaseolus vulgaris) seeds, has been rigorously evaluated using stable and transient transformation techniques. Transgenic tobacco plants known to be homozygous for the β‐glucuronidase (gus) reporter sequence under the regulation of various lengths of the β‐phaseolin gene (phas) promoter were shown to express gus only in developing seed tissues. No expression was detected in calli initiated from stems, leaves and immature seeds, showing that expression was not leaky in undifferentiated tissues. Control plants and cultures containing gus fused to the CaMV 35S promoter actively expressed gus under identical conditions. It was not possible to induce expression in phas/gus calli with ABA, GA or jasmonic acid. Treatment of the cultures with 5‐azacytidine did not result in expression, excluding methyletlon as the major factor regulating the phas promoter. However, strong gus expression was detected in seed of plants regenerated from these callus cultures, confirming that neither gene rearrangements nor deletion were responsible for the lack of activity seen in tissues other than the developing seed. In contrast to the above observations, strong transient expression of gus was detected in tobacco, bean and soybean leaves following introduction of the phas/gus fusion constructs via biolistic approaches and in electroporated bean leaf and hypocotyl protoplasts. These experiments show unequi‐vocally that the phas promoter is under rigorous spatial control when integrated into the genome, but lacks spatial control when present as extrachromosomal naked DNA. A putative model explaining these differences is presented.
Maternal‐specific demethylation and expression of specific alleles of zein genes in the endosperm of Zea mays L.
Authors: Lund Gertrud , Ciceri Pietro , Viotti Angelo
Published: March 2003

Zeins constitute 60–70% of maize endosperm protein. Zein genes are specifically transcribed in the endosperm, and a correlation has been established between tissue‐specific expression and demethylation. Three inbred lines and their reciprocal crosses were analysed to assess for allele‐specific differences in methylation, transcription and translation. DNAs from endosperm, embryo and seedling tissues analysed by cleavage with methylation‐sensitive restriction enzymes and Southern blot hybridization with zein cDNA and genomic sequences show that specific demethylation of zein sequences occurs only in endosperm and is restricted to the maternal complements. Steady‐state transcript accumulation of zein mRNA assessed by RNase protection assay reveals qualitative and quantitative differences among endosperm RNAs of the inbreds and of their reciprocal hybrids. Moreover, two‐dimensional gel electrophoresis of zein proteins identified polypeptides that are maternally imprinted in reciprocal crosses. These results indicate that endosperm‐specific expression of specific zein alleles may occur via parental imprinting and disclose a possible role of methylation in regulating the expression of genes differently contributed in the endosperm by the maternal and paternal genomes.
A rolB regulatory factor belongs to a new class of single zinc finger plant proteins
Authors: De Paolis Angelo , Sabatini Sabrina , De Pascalis Luca , Costantino Paolo , Capone Imerio
Published: March 2003

A protein which binds to the regulatory domain B necessary for expression of the plant oncogene rolB in (root) meristems contains a single zinc finger of a novel type conserved in dicots and monocots. Band shift analysis revealed the presence in tobacco nuclei of a protein selectively binding to domain B, a tetramer of which was used to isolate a cDNA (NtBBF1, Nicotiana tabacum rolB domain B,Factor1) from a tobacco expression library. The corresponding genomic clone was also isolated. The protein encoded by NtBBF1 contains a single C2C2 zinc finger, and its target sequence in domain B was identified by means of mutagenized oligonucleotides. The DNA‐binding capability of the zinc finger was assessed by means of a fusion of this latter with the glutathione‐S‐transferase protein, that was shown to bind the same target sequence as NtBBF1. A number of other tobacco cDNAs encoding different proteins with a domain (BBF domain) encompassing the zinc finger identical to NtBBF1 were also isolated. Furthermore, a cDNA encoding a protein with an almost identical single zinc finger was isolated from Arabidopsis. A very closely related zinc finger has very recently been identified in maize transcription factors and termed the Dof domain. It is proposed that the tobacco, Arabidopsis and maize BBF/Dof domain proteins are members of a new broad family of plant transcription factors acting through a single zinc finger widely utilized in the plant kingdom.
Alkali cation selectivity of the wheat root high‐affinity potassium transporter HKT1
Authors: Gassmann Walter , Rubio Francisco , Schroeder Julian I.
Published: March 2003

The wheat root high‐affinity K+ transporter HKT1 functions as a sodium‐coupled potassium co‐uptake transporter. At toxic millimolar levels of sodium (Na+), HKT1 mediates low‐affinity Na+ uptake while potassium (K+) uptake is blocked. In roots, low‐affinity Na+ uptake and inhibition of K+ uptake contribute to Na+ toxicity. In the present study, the selectivity among alkali cations of HKT1 expressed in Xenopus oocytes and yeast was investigated under various ionic conditions at steady state. The data show that HKT1 is highly selective for uptake of the two physiologically significant alkali cations, K+ and Na+ over Rb+, Cs+ and Li+. In addition, Rb+ and Cs+, and an excess of extracellular K+ over Na+, are shown to partially reduce or block HKT1‐mediated K+‐Na+ uptake. Furthermore, K+, Rb+ and Cs+ also effectively reduce outward currents mediated by HKT1, thereby causing depolarizations. In yeast, HKT1 can produce high‐affinity Rb+ uptake at approximately 15‐fold lower rates than for K+. Rb+ influx in yeast can be mediated by the ability of the yeast plasma membrane proton pump to balance the 35‐fold lower HKT1 conductance for Rb+. A model for HKT1 activity is presented involving a high‐affinity K+ binding site and a high‐affinity Na+ binding site, and competitive interactions of K+, Na+ and other alkali cations for binding to these two sites. Possible implications of the presented results for physiological K+ and Na+ uptake in plants are discussed.
Arabidopsis cryptochrome 1 is a soluble protein mediating blue light‐dependent regulation of plant growth and development
Authors: Lin Chentao , Ahmad Margaret , Cashmore Anthony R.
Published: March 2003

Cryptochrome 1 (CRY1) is a flavin‐type blue light receptor of Arabidopsis thaliana which mediates inhibition of hypocotyl elongation. In the work described in this report it is demonstrated that CRY1 is a soluble protein expressed in both young seedlings grown either in the dark or under light, and in different organs of adult plants. The functional role of CRY1 was further investigated using transgenic Arabidopsis plants overexpressing CRY1. It is demonstrated that overexpression of CRY1 resulted in hypersensitivity to blue, UV‐A, and green light for the inhibition of hypocotyl elongation response. Transgenic plants overexpressing CRY1 also exhibited a dwarf phenotype with reduced size in almost every organ. This was in keeping with the previous observation of reciprocal alterations found in hy4 mutant plants and is consistent with a hypothesis that CRY1 mediates a light‐dependent process resulting in a general inhibitory effect on plant growth. In addition, transgenic plants overexpressing CRY1 showed increased anthocyanin accumulation in response to blue, UV‐A, and green light in a fluence rate‐dependent manner. This increase in anthocyanin accumulation in transgenic plants was shown to be concomitant with increased blue light‐induction of CHS gene expression. It is concluded that CRY1 is a photoreceptor mediating blue light‐dependent regulation of gene expression in addition to its affect on plant growth.
Differential activation of the primary auxin response genes, PS‐IAA4/5 and PS‐IAA6, during early plant development
Authors: Wong Lu Min , Abel Steffen , Shen Nancy , De La Foata Monique , Mall Yasmeen , Theologis Athanasios
Published: March 2003

The plant growth hormone auxin typified by indoleacetic acid (IAA) transcriptionally activates early genes in pea, PS‐IAA4/5 and PS‐IAA6, that are members of a multigene family encoding short‐lived nuclear proteins. To gain first insight into the biological role of PS‐IAA4/5 and PSIAA6, promoter‐β‐glucuronidase (GUS) gene fusions were constructed and their expression during early development of transgenic tobacco seedlings was examined. The comparative analysis reveals spatial and temporal expression patterns of both genes that correlate with cells, tissues, and developmental processes known to be affected by auxin. GUS activity in seedlings of both transgenic lines is located in the root meristem, sites of lateral root initiation and in hypocotyls undergoing rapid elongation. In addition, mutually exclusive cell‐specific expression is evident. For instance, PS‐IAA4/5—GUS but not PS‐IAA6—GUS is expressed in root vascular tissue and in guard cells, whereas only PS‐IAA6—GUS activity is detectable in glandular trichomes and redistributes to the elongating side of the hypocotyl upon gravitropic stimulation. Expression of PS‐IAA4/5 and PS‐IAA6 in elongating, dividing, and differentiating cell types indicates multiple functions during development. The common and yet distinct activity patterns of both genes suggest a combinatorial code of spatio‐temporal co‐expression of the various PS‐IAA4/ 5‐like gene family members in plant development that may mediate cell‐specific responses to auxin.
Reduction of the cytosolic fructose‐1,6‐bisphosphatase in transgenic potato plants limits photosynthetic sucrose biosynthesis with no impact on plant growth and tuber yield
Authors: Zrenner Rita , Krause Klaus‐Peter , Apel Peter , Sonnewald Uwe
Published: March 2003

Sucrose produced in source leaves is the predominant carbon source for developing sink tissues in most higher plants. Consequently the rate of sucrose synthesis is likely to be important for sink development and final crop yield. Two sucrose biosynthetic enzymes are believed to possess regulatory properties with respect to the rate of sucrose synthesis: (i) cytosolic FBPase and (ii) sucrose phosphate synthase. To study the impact of reduced photosynthetic sucrose biosynthesis on plant growth and crop yield a cDNA clone encoding cytosolic FBPase was isolated from a potato leaf cDNA library and used for antisense experiments in transgenic potato plants. The cDNA clone cy‐F1, containing an open reading frame of 1020 bp highly homologous (85%) to other known sequences of plant cytosolic FBPases, was cloned in reversed orientation between the 35S CaMV promoter and the octopine synthase polyadenylation signal. Out of 75 independent transformants five transgenic lines having 9 to 55% of the wild‐type FBPase activity were chosen for further analysis.

A 45% reduction of the cytosolic FBPase activity did not cause any measurable change in metabolite concentrations, growth behaviour or photosynthetic parameters of the transgenic plants. Inhibition of cytosolic FBPase activity below 20% of the wild‐type activity led to an accumulation of 3‐PGA, triose‐phosphates and fructose‐1,6bisphosphate in source leaves. This resulted in a reduced light‐saturated rate of assimilation measured via gas exchange and a decreased photosynthetic rate under conditions of the leaf disc electrode with saturating light and CO2. Measuring photosynthetic carbon fluxes by labelling leaf discs with 14CO2 revealed a 53–65% reduction of sucrose synthesis whereas starch synthesis decreased only by 18–24%. The flux into the anionic and cationic fraction was not altered. Despite these changes steadystate sucrose concentrations were not effected in source leaves from transgenic plants. Starch accumulated by more than a factor of 3 compared with wild‐type leaves and was degraded during the night. This provides strong evidence for the hypothesis that hexoses and/or hexosephosphates are exported out of the chloroplasts, thereby circumventing the limitation of sucrose biosynthesis caused by the inhibition of cytosolic FBPase in the dark. Accordingly, plant growth and potato tuber yield remained unaltered. From these data it can be concluded that a reduced photosynthetic sucrose biosynthetic capacity can be efficiently compensated without any reduction in crop yield under greenhouse or growth chamber conditions by changing carbon export strategy. Whether the same holds true for field conditions remains to be elucidated.
Genetic evidence for an essential role of brassinosteroids in plant development
Authors: Kauschmann Annette , Jessop Alison , Koncz Csaba , Szekeres Miklos , Willmitzer Lotbar , Altmann Thomas
Published: March 2003

Brassinosteroids which show high structural similarity to animal steroid hormones elicit a variety of growth responses when exogeneously applied to plant tissues. Thus far however, the function of endogeneous brassinosteroids in higher plants has been unclear. This paper describes three extremely dwarfed Arabidopsis thaliana mutants, cbb1 (dwf1‐6), cbb2 and cbb3, which are impared in cell elongation controlled by brassinosteroids. While cbb1 (dwf1‐6) and cbb3 can be phenotypically normalized to wild‐type by feeding with brassinosteroids indicating deficiencies of brassinosteroid biosynthesis, cbb2 is brassinosteroid‐insensitive and defines a function required for further metabolic conversion necessary for biological activity or for perception/signal transduction of these growth‐regulating plant steroid hormones. Expression of the meri5 and TCH4 genes is low in all three cbb mutants and can be restored to wild‐type levels by brassinosteroid treatment in the cbb1 (dwf1‐6) and cbb3 mutants but are unaffected in the cbb2 mutant. These data indicate that brassinosteroids are essential for proper plant development and play an important role in the control of cell elongation.
Cysteine synthesis in plants: protein‐protein interactions of serine acetyltransferase from Arabidopsis thaliana
Authors: Bogdanova Natascha , Hell Rüdiger
Published: March 2003

The biosynthesis of cysteine represents the final step of sulfate assimilation in bacteria and plants. It is catalyzed by the sequential action of serine acetyltransferase (SAT) and O‐acetylserine (thiol) lyase (OAS‐TL) which form a cysteine synthase (CS) complex in vitro. SAT and OAS‐TL from Arabidopsis thaliana have previously been cloned, and now the first evidence is presented for the CS complex and SAT self‐interaction in vivo employing the yeast two‐hybrid system. Application of this method proved to be an efficient tool for the analysis of protein‐protein interactions within a plant metabolic protein complex. Mapping of SAT domain structure revealed two new, independent domains with specific functions in protein‐protein interaction. Analysis using truncated proteins proved the C‐terminus of SAT to be sufficient for association with OAS‐TL and to correlate with the putative transferase activity domain. SAT/SAT interaction was localized in the central region of the protein and occured also between SAT isoforms. Both protein interaction domains coincided with distinct α‐helical and β‐sheet clusters and together correlated with the minimal protein structure required for SAT catalysis as shown by functional complementation of an Escherichia coli mutant. The homo‐ and hetero‐oligomerization properties are discussed with respect to the assumed function of the CS complex in metabolic channeling and activation of SAT by interaction with OAS‐TL.
Xyloglucan sidechains modulate binding to cellulose during in vitro binding assays as predicted by conformational dynamics simulations
Authors: Levy Samuel , Maclachlan Gordon , Staehelin L. Andrew
Published: March 2003

Cross‐links between cellulose microfibrils and xyloglucan (XG) molecules play a major role in defining the structural properties of plant cell walls and the regulation of growth and development of dicotyledonous plants. How these cross‐links are established and how they are regulated has yet to be determined. In a previous study, preliminary data were presented which suggested that the different sidechains of XG may play a role in controlling cellulose microfibril‐XG interactions. In this study, this question is addressed directly by analyzing to what extent the different sidechains of pea cell wall XG and nasturtium seed storage XG affect their binding to cellulose microfibrils. Of particular importance to this study are the chemical data indicating that pea XG possesses a trisaccharide sidechain, which is not found in nasturtium XG. To this end, conformational dynamic simulations have been used to predict whether oligosaccharides representative of pea and nasturtium XG can adopt a hypothesized cellulose‐binding conformation and which of these XGs exhibits a preferential ability to bind cellulose. Extensive analysis of the conformational forms populated during 300 K and high‐temperature Monte Carlo simulations established that a planar, sterically accessible, glucan backbone is essential for optimal cellulose‐binding. For the trisaccharide sidechain‐containing oligosaccharide as found in pea XG, sidechain orientation appeared to regulate the gradual acquisition of this hypothesized cellulose binding conformation. Thus, conformational forms were identified that included the twisted backbone (non‐planar) putative solution form of XG, forms in which the trisaccharide sidechain orientation enables increased backbone planarity and steric accessibility, and finally a planar, sterically accessible, backbone. By applying these conformational requirements for cellulose binding, it has been determined that pea XG possesses a two‐ to threefold occurrence of the cellulose binding conformation than nasturtium XG. Based on this finding, it was predicted that pea XG would bind to cellulose at a higher rate than nasturtium XG. In vitro binding assays showed that pea XG‐avicel binding does indeed occur at a twofold higher rate than nasturtium XG‐avicel binding. The enhanced ability of pea cell wall XG over nasturtium seed storage XG to associate with cellulose is consistent with a structural role of the former during epicotyl growth where efficient association with cellulose is a requirement. In contrast, the relatively low ability of nasturtium XG to bind cellulose is consistent with the need to enhance the accessibility of this polymer to glycanases during germination. These findings suggest potential roles for XG sidechain substitution, enabling XG to function in a variety of different biological contexts.