bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel nucleic acid helicase gene identified by promoter trapping in Arabidopsis
Authors: Wei Wenbin , Twell David , Lindsey Keith
Published: March 2003

A gene encoding a predicted nucleic acid helicase was identified in Arabidopsis thaliana by activation of a promoter trap. An in vivo transcriptional fusion between the helicase gene and a promoterless uidA (gusA) gene was generated, which was expressed specifically in the tapetum and vascular tissues. The tagged gene, designated HVT1 (Helicase in Vascular tissue and Tapetum), encodes a native transcript of approximately 4.4 kb, which is of very low abundance. The predicted HVT1 protein, of 1291 amino acid residues, is homologous to the Drosophila MALELESS, human RNA helicase A and bovine nuclear DNA helicase proteins, and represents the first identified member of a new subgroup within the mle group of the DEAH family. Low stringency genomic Southern blot analysis indicates that at least two structurally related genes exist in Arabidopsis. We suggest that the HVT1 protein may play a role in nucleic acid unwinding in restricted cell types during both vegetative and reproductive phases of the Arabidopsis life cycle.
Recognition of bacterial avirulence proteins occurs inside the plant cell: a general phenomenon in resistance to bacterial diseases?
Authors: Bonas Ulla , Van den Ackerveken Guido
Published: March 2003

One of the recent exciting developments in the research area of plant‐microbe interactions is a breakthrough in understanding part of the initial signalling between avirulent Gram‐negative bacteria and resistant plants. For resistance to occur, both interacting organisms need to express matching genes, the plant resistance gene and the bacterial avirulence gene. The biochemical function of bacterial avirulence genes and the nature of the signal molecules recognized by the plant have been a mystery for a long time. Recently, several laboratories have shown that bacterial avirulence proteins function as elicitors that are perceived within the plant cell.
Conditional transgenic expression of the ipt gene indicates a function for cytokinins in paracrine signaling in whole tobacco plants
Authors: Faiss Martin , Zalubìlová Jana , Strnad Miroslav , Schmülling Thomas
Published: March 2003

This study investigated whether an increased production of the plant hormone cytokinin in roots, the main site of its synthesis and putative signaling organ, can influence developmental events, such as growth of axillary shoot meristems or leaf senescence, in the plant shoot. To this end, transgenic tobacco plants (Nicotiana tabacum L.) were generated that conditionally overproduce cytokinins. These plants harbour the ipt gene under the transcriptional control of a modified 35S promoter that is repressed in plants with high titers of tetracycline repressor protein. De‐repression of transcription led to a rapid more than 50‐ fold increase of hormone concentration. The time course of changes in the steady‐state levels of 16 different cytokinin metabolites, as a consequence of IPT enzyme activity, was monitored in different plant tissues. Zeatin riboside was the first and most dramatically increased product; zeatin, dihydrozeatin and glucosides accumulated later. The consequences of enhanced cytokinin synthesis remained mainly restricted to the site of hormone production. For example, de‐repression of ipt gene transcription in lateral buds caused the growth of single buds only at the site of tetracycline application. In reciprocal grafts of transgenic plants with wild‐type plants, no biological cytokinin effects, i.e. growth of lateral shoot meristems or sequential leaf senescence, were observed in the non‐transgenic plant part. Also, the increase in steady‐state levels of cytokinins remained restricted mainly to the transgenic part, despite a specific increase of the zeatin riboside concentration in the transpiration stream. These results question the role of cytokinins as a long‐range root‐to‐shoot signal in correlative control of apical dominance and sequential leaf senescence of tobacco, and support the assumption that this hormone is relevant to paracrine signaling.
Expression and activation of the vacuolar processing enzyme in Saccharomyces cerevisiae
Authors: Hiraiwa Nagako , Nishimura Mikio , Hara‐Nishimura Ikuko
Published: March 2003

Vacuolar processing enzymes (VPEs) are cysteine proteinases responsible for maturation of various vacuolar proteins in plants. A larger precursor to VPE synthesized on rough endoplasmic reticulum is converted to an active enzyme in the vacuoles. In this study, a precursor to castor bean VPE was expressed in a pep4 strain of the yeast Saccharomyces cerevisiae to examine the mechanism of activation of VPE. Two VPE proteins of 59 and 46 kDa were detected in the vacuoles of the transformant. They were glycosylated in the yeast cells, although VPE is not glycosylated in plant cells in spite of the presence of two N‐linked glycosylation sites. During the growth of the transformant, the level of the 59 kDa VPE increased slightly until a rapid decrease occurred after 9 h. By contrast, the 46 kDa VPE appeared simultaneously with the disappearance of the 59 kDa VPE. Vacuolar processing activity increased with the accumulation of the 46 kDa VPE, but not of the 59 kDa VPE. The specific activity of the 46 kDa VPE was at a similar level to that of VPE in plant cells. The 46 kDa VPE instead of proteinase A mediated the conversion of procarboxypeptidase Y to the mature form. This indicates that proteinase A responsible for maturation of yeast vacuolar proteins can be replaced functionally by plant VPE. These findings suggest that an inactive VPE precursor synthesized on the endoplasmic reticulum is transported to the vacuoles in the yeast cells and then processed to make an active VPE by self‐catalytic proteolysis within the vacuoles.
Efficient in vitro transcription of plant nuclear tRNASer genes in a nuclear extract from tobacco cultured cells
Authors: Yukawa Yasushi , Sugita Mamoru , Sugiura Masahiro
Published: March 2003

A nuclear extract derived from tobacco cultured BY‐2 cells supports RNA polymerase III‐dependent transcription of Arabidopsis tRNASer genes. Primer extension analysis indicated that the transcription starts at 6 bp upstream from the 5′ end of tRNA coding region. Procedures for nuclear extraction and in vitro reaction conditions have been optimized for tRNA transcription, which allows direct detection of de novo synthesized tRNA by gel electrophoresis. This improved in vitro system yields a mature‐sized tRNA of 85 nucleotides from the Arabidopsis tRNASer gene, indicating that efficient processing of the pre‐tRNA also occurs in the tobacco nuclear extract.
Light‐induced transcriptional repression of the pea AS1 gene: identification of cis‐elements and transfactors
Authors: Ngai Nora , Tsai Fong‐Ying , Coruzzi Gloria
Published: March 2003

Here, we examine the cis‐elements and trans‐factors affecting the expression of asparagine synthetase (AS) genes whose transcription is negatively regulated by light. The promoters for the AS1 and AS2 genes of pea were isolated, sequenced, and functionally dissected for their ability to confer regulated expression to the GUS reporter gene in transgenic tobacco. Histochemical analysis of transgenic plants demonstrated that the AS1 and AS2 promoters show identical patterns of cell‐specific expression. The more highly active AS1 promoter was further demonstrated to confer negative light‐regulation to the GUS gene in transgenic tobacco. Deletion analysis and gain‐of‐function experiments showed that 124 bp of the AS1 promoter was sufficient to confer light‐activated repression to a heterologous promoter. Potential conserved transcription regulatory elements, Box B, Box C, and Box C′ within this region were shown to bind to nuclear proteins by gel shift analysis. A light‐specific DNA:protein interaction was detected with Box B. The nuclear factors that bind to Box C and C′ elements of AS1 are competed by a putative repressor element ‘RE1’ defined previously in the oat phytochrome gene whose transcription is also repressed by light. The Box B and C/ C′‐Box/REl‐binding factors were found in nuclear extracts of tobacco, pea, and Arabidopsis and may therefore be universal factors involved in light‐activated transcriptional repression.
Nicotiana plumbaginifolia hlg mutants have a mutation in a PHYB‐type phytochrome gene: they have elongated hypocotyls in red light, but are not elongated as adult plants
Authors: Hudson Matthew , Robson Paul R.H. , Kraepiel Yvan , Caboche Michel , Smith Harry
Published: March 2003

Two new allelic mutants of Nicotiana plumbaginifolia have been isolated which display a hypocotyl which is long (hlg) when seedlings are grown in continuous white light (W). This can be accounted for by the decreased response to red light (R) of the hypocotyl elongation rate in these mutants. Responses to other wavelengths are unaffected in the mutants. When grown in white light, mature hlg mutants are not elongated with respect to the wild‐type; they also bolt and flower later. The shade‐avoidance responses to red/far red ratio (R:FR) are intact in these mutants. Both mutants are deficient in a phyB‐like polypeptide that is immunodetectable in the wild‐type; both have wild‐type levels of a phyA‐like polypeptide. These alleles are inherited in a partially dominant manner, and correspond to single‐base missense mutations in a gene highly homologous to N. tabacum PHYB, which codes for a phytochrome B‐type photoreceptor. One allele, hlg‐1, has an introduced amino acid substitution; this may define a residue essential for phytochrome protein stability. The other allele, hlg‐2, has a stop codon introduced C‐terminal to the chromophore binding domain. As these phyB mutants are unaffected in shade‐avoidance responses, but deficient in perception of R, it is concluded that the phyB absent in these mutants is responsible for R perception in the N. plumbaginifolia seedling, but is not a R:FR sensor in light‐grown plants.
Seven zinc‐finger transcription factors are expressed sequentially during the development of anthers in petunia
Authors: Kobayashi Akira , Sakamoto Ayako , Kubo Kenichi , Rybka Zbigniew , Kanno Yoshiaki , Takatsuji Hiroshi
Published: March 2003

The development of anthers and pollen involves several stages. In an attempt to identify the transcription factors that are involved in the regulation of gene expression associated with the development of anthers, seven genes for new zinc‐finger proteins that are expressed in anthers were detected using RT‐PCR. Subsequent cloning and sequence analysis of the full‐length cDNA clones revealed that they all encoded zinc‐finger proteins of the EPF type. However, the encoded proteins were very different from each other in terms of size, number of zinc fingers (two, three and four) and the spacings between the fingers. Northern blot analysis revealed that the genes were expressed preferentially in anthers. Moreover, it was found that the seven genes were expressed transiently for short periods that corresponded to different developmental stages. It appeared that the genes were activated sequentially during the development of the anther. Considering these observations, a possible mechanism that could account for the autonomous progression of the development of the anther via a regulatory cascade of these transcription factors is discussed.
Pathogen‐derived resistance targeted against the negative‐strand RNA of tobacco mosaic virus: RNA strand‐specific gene silencing?
Authors: Marano María Rosa , Baulcombe David
Published: March 2003

Tobacco plants transformed with the open‐reading frame (ORF) of tobacco mosaic virus strain U1 (TMV‐U1) encoding a 54 kDa (54K) region of the viral replicase are resistant against TMV strain U1. These plants are not resistant against the crucifer strain of TMV or the heterologous virus, potato virus X (PVX). However, they are resistant against derivatives of PVX containing fragments of the 54K ORF inserted either in the sense or anti‐sense orientation. The smallest fragment that was a target of the resistance mechanism was a 383 nucleotide region from the central part of the 54K ORF. A transient gene expression assay revealed that this central region was also the target of a post‐transcriptional gene silencing mechanism. However, unlike other examples of gene silencing associated with virus resistance, the silencing was specific for the anti‐sense rather than the coding strand of the target RNA. Based on these data the authors propose that the TMV resistance is due, at least in part, to a type of transgene silencing.
Accumulation of small heat shock proteins, including mitochondrial HSP22, induced by oxidative stress and adaptive response in tomato cells
Authors: Banzet Nathalie , Richaud Christiane , Deveaux Yves , Kazmaier Michaël , Gagnon Jean , Triantaphylidès Christian
Published: March 2003

Changes in gene expression, by application of H2O2, O2°–generating agents (methyl viologen, digitonin) and gamma irradiation to tomato suspension cultures, were investigated and compared to the well‐described heat shock response. Two‐dimensional gel protein mapping analyses gave the first indication that at least small heat shock proteins (smHSP) accumulated in response to application of H2O2 and gamma irradiation, but not to O2°– generating agents. While some proteins seemed to be induced specifically by each treatment, only part of the heat shock response was observed. On the basis of Northern hybridization experiments performed with four heterologous cDNA, corresponding to classes I–IV of pea smHSP, it could be concluded that significant amounts of class I and II smHSP mRNA are induced by H2O2 and by irradiation. Taken together, these results demonstrate that in plants some HSP genes are inducible by oxidative stresses, as in micro‐organisms and other eukaryotic cells. HSP22, the main stress protein that accumulates following H2O2 action or gamma irradiation, was also purified. Sequence homology of amino terminal and internal sequences, and immunoreactivity with Chenopodium rubrum mitochondrial smHSP antibody, indicated that the protein belongs to the recently discovered class of plant mitochondrial smHSP. Heat shock or a mild H2O2 pretreatment was also shown to lead to plant cell protection against oxidative injury. Therefore, the synthesis of these stress proteins can be considered as an adaptive mechanism in which mitochondrial protection could be essential.
Regulation of Rubisco activation in antisense plants of tobacco containing reduced levels of Rubisco activase
Authors: Hammond Edward T. , Andrews T. John , Mott Keith A. , Woodrow Ian E.
Published: March 2003

Following an increase in photon flux density (PFD), ribulose bisphosphate carboxylase/oxygenase (Rubisco) undergoes a slow activation which substantially limits the rate of photosynthesis. This activation process is mediated in part by Rubisco activase. Antisense DNA plants of tobacco were used to quantify the degree to which activase limits Rubisco activation. Reductions in leaf activase content caused proportional reductions in the rate of Rubisco activation following a PFD increase from 110 to 1200 μmol m–2 sec–1. This was the case for activase levels up to and slightly beyond normal wild‐type activase levels. Activase therefore has a flux control coefficient of unity with respect to the Rubisco activation flux. Such a high control coefficient has rarely been measured for any metabolic system, and this is the highest control coefficient measured for an important photosynthetic flux. In contrast, the rate of Rubisco inactivation in leaves following a drop in PFD of 1200 to 110 μmol m–2 sec–1 was unchanged by a 60% reduction in activase levels. Despite the high degree of control that activase exerts over the rate of activation, and thus non‐steady‐state photosynthesis, it was shown that steady‐state photosynthesis was largely unaffected by activase concentration until it was reduced below approximately 15% of the wild‐type level. The significance of these results and their implications for published models of Rubisco activation are discussed.
Genetic evidence that the endodermis is essential for shoot gravitropism in Arabidopsis thaliana
Authors: Fukaki Hidehiro , Wysocka‐Diller Joanna , Kato Takehide , Fujisawa Hisao , Benfey Philip N. , Tasaka Masao
Published: March 2003

Shoots of higher plants exhibit negative gravitropism. However, little is known about the mechanism or site of gravity perception in shoots. We have identified two loci that are essential for normal shoot gravitropism in

Arabidopsis thaliana. Genetic analysis demonstrated that the shoot gravitropism mutants

sgr1and

sgr7are allelic to the radial pattern mutants,

scrand

shr, respectively. Characterization of the aerial phenotype of these mutants revealed that the primary defect is the absence of a normal endodermis in hypocotyls and inflorescence stems. This indicates that the endodermis is essential for shoot gravitropism and strongly suggests that this cell layer functions as the gravity‐sensing cell layer in dicotyledonous plant shoots. These results also demonstrate that, in addition to their previously characterized role in root radial patterning,

SCRand

SHRregulate the radial organization of the shoot axial organs in

Arabidopsis.
Molecular cloning of starch synthase I from maize (W64) endosperm and expression in Escherichia coli
Authors: Knight Mary E. , Harn Chee , Lilley Caroline E. R. , Guan Hanping , Singletary George W. , Mu‐Forster Chen , Wasserman Bruce P. , Keeling Peter L.
Published: March 2003

A full length cDNA clone encoding a starch synthase (zSS) from maize endosperm (inbred line W64) was isolated and characterized. The cDNA clone (Ss1) is 2907 bp in length and contains an open reading frame of 1866 bp corresponding to a polypeptide of 622 amino acid residues including a transit peptide of 39 amino acids. The Ss1 cDNA clone was identified as zSSI by its direct alignment with sequences to: (i) the N‐terminus obtained from the granule‐associated form of the zSSI polypeptide, (ii) four internal peptide fragments obtained from the granule‐associated form of the zSSI protein, and (iii) one internal fragment from the soluble form of the zSSI protein. The deduced amino acid sequence of Ss1 shares 75.7 % sequence identity with rice soluble Ss and contains the highly conserved KSGGLGDV putative ADP–Glc binding site. Moreover, Ss1 exhibited significant activity when expressed in E. coli and the expressed protein is recognized by the antibody raised against the granule associated zSSI protein. Ss1 transcripts were detected in endosperm beginning at 15 days after pollination, but were not found in embryo, leaf or root. Maize contains a single copy of the Ss1 gene, which maps close to the Waxy locus of chromosome 9.
Phytepsin, a barley vacuolar aspartic proteinase, is highly expressed during autolysis of developing tracheary elements and sieve cells
Authors: Runeberg‐Roos Pia , Saarma Mart
Published: March 2003

Vacuolarisation, formation of autophagocytotic vacuoles and tonoplast disruption have been reported in plant cells undergoing developmentally regulated programmed cell death (PCD), but little is known about the vacuolar proteins involved. In HeLa cells, cathepsin D, a lysosomal aspartic proteinase has been shown to mediate PCD. Based on immunohistochemical staining of barley roots, we show here that the previously well characterised barley vacuolar aspartic proteinase (phytepsin), a plant homologue to cathepsin D, is highly expressed both during formation of tracheary elements and during partial autolysis of sieve cells. In serial transverse sections of the vascular cylinder, starting from the root tip, phytepsin is expressed in root cap cells, in the tracheary elements of early and late metaxylem, and in the sieve cells of the protophloem and metaphloem. Aleurain, a barley vacuolar cysteine proteinase, is expressed similarly in root cap cells but differently in the tracheary elements of protoxylem and early metaxylem. This is the first evidence that a vacuolar aspartic proteinase, in analogy to cathepsin D in animals, may play a role in the active autolysis of plant cells.
A gene encoding phosphatidylinositol‐4‐phosphate 5‐kinase is induced by water stress and abscisic acid inArabidopsis thaliana
Authors: Mikami Koji , Katagiri Takeshi , Iuchi Satoshi , Yamaguchi‐Shinozaki Kazuko , Shinozaki Kazuo
Published: March 2003

Phosphatidylinositol‐4‐phosphate 5‐kinase (PIP5K) phosphorylates phosphatidylinositol‐4‐phosphate to produce phosphatidylinositol‐4,5‐bisphosphate as a precursor of two second messengers, inositol‐1,4,5‐triphosphate and diacylglycerol, and as a regulator of many cellular proteins involved in signal transduction and cytoskeletal organization. Despite PIP5K playing such an essential role in a number of physiological processes, much still remains to be made clear about its association with plants. Searching the

Arabidopsis

expression sequence tag database against already known yeast and mammalian PIP5K cDNAs, we identified two clones which partly encode the same

Arabidopsis

PIP5K and isolated a corresponding full‐length cDNA encoding a protein that we designated AtPIP5K1. Recombinant AtPIP5K1 expressed in

Escherichia coli

possessed a PIP5K activity

in vitro

. Due to some structural and biochemical differences, AtPIP5K1 was not categorized as either a type I or type II PIP5K. The expression of the AtPIP5K1 mRNA was induced rapidly by treating

Arabidopsis

plants with drought, salt and abscisic acid, which suggests that AtPIP5K1 is involved in water‐stress signal transduction. These data give evidence for a close link between phosphoinositide signaling cascades and water‐stress responses in plants.
Endomycorrhizae and rhizobial Nod factors both require SYM8 to induce the expression of the early nodulin genesPsENOD5 and PsENOD12A
Authors: Albrecht Catherine , Geurts René , Lapeyrie Frederic , Bisseling Ton
Published: March 2003

We report here that the pea early nodulin genesPsENOD5andPsENOD12Aare induced during the interaction of pea roots and the endomycorrhizal fungusGigaspora margarita. Using the pea nodulation mutant Sparkle‐R25, which is mutated inSYM8, it is shown that SYM8 is essential for the induction ofPsENOD5andPsENOD12Ain pea roots interacting either withRhizobiumor the endomycorrhizal fungusGigaspora margarita. Our results suggest that mycorrhizal signals activate a signal transduction cascade sharing at least one common step with the Nod factor‐activated signal transduction cascade.
Developmental abnormalities associated with deoxyadenosine methylation in transgenic tobacco
Authors: Van Blokland Rik , Ross Simon , Corrado Giandomenico , Scollan Claire , Meyer Peter
Published: March 2003

As in other higher eukaryotes, DNA methylation in plants is predominantly found at deoxycytosine residues, while deoxyadenosine residues are not methylated at significant levels. 6mdA methylation has been successfully introduced into yeast and

Drosophila

via expression of a heterologous methyltransferase, but similar attempts in tobacco had, up until now, proved unsuccessful despite the correct expression of a methyltransferase construct. It was unclear whether this result reflected the failure of heterologous methyltransferases to enter the nucleus, or whether 6mdA methylation, which has been shown to interfere with promoter activity, was toxic for plants. Here we show that 6mdA methylation can be successfully introduced into transgenic tobacco plants via expression of the bacterial

dam

enzyme. The efficiency of 6mdA methylation was directly proportional to expression levels of the

dam

construct, and methylation of all GATC sites was observed in a highly expressing line. Increasing expression levels of the enzyme in different plants correlated with increasingly abnormal phenotypes affecting leaf pigmentation, apical dominance, and leaf and floral structure. Whilst introduction of

dam

‐specific methylation does not cause any developmental abnormalities in yeast or

Drosophila

, our data suggest that methylation of deoxyadenine residues in plants interferes with the expression of genes involved in leaf and floral development.
Ozone‐induced oxidative burst in the ozone biomonitor plant, tobacco Bel W3
Authors: Schraudner Martina , Moeder Wolfgang , Wiese Cosima , Camp Wim Van , Inzé Dirk , Langebartels Christian , Sandermann Heinrich
Published: March 2003

Localized cell death is a common feature of ozone phytotoxicity and is generally thought to be initiated by the strong oxidant ozone itself as well as by ozone‐derived reactive oxygen intermediates (ROIs). Here we report that ozone (150 nl l–1, 5 h) elicits cellular ROI production in the ozone‐sensitive tobacco cv. Bel W3, but not in the tolerant cv. Bel B. Both cultivars exhibited a transient first maximum of apoplastic ROI accumulation followed by a comparable induction of glutathione peroxidase transcript levels. During postcultivation in pollutant‐free air, a second and sustained peak of apoplastic ROI accumulation was detected only in cv. Bel W3. Histochemical staining revealed a spot‐like accumulation of H2O2 and, to a lesser extent, of superoxide anion radicals in this cultivar. The H2O2 spots (‘burst initiation sites’) occurred mainly in the vicinity of leaf veins and correlated in number and distribution with discrete sites of local cell death and with visible symptoms that evolved between 15 and 72 h. The results indicate that ozone effects are amplified in the sensitive tobacco cv. Bel W3 by an oxidative burst which participates in the generation of hypersensitive cell death‐like lesions.
A GFP‐mouse talin fusion protein labels plant actin filamentsin vivoand visualizes the actin cytoskeleton in growing pollen tubes
Authors: Kost Benedikt , Spielhofer Pius , Chua Nam‐Hai
Published: March 2003

The C‐terminus of mouse talin (amino acids 2345–2541) is responsible for all of the protein’s f‐actin binding capacity. Unlike full‐length talin, the C‐terminal f‐actin binding domain is unable to nucleate actin polymerization. We have found that transient and stable expression of the talin actin‐binding domain fused to the C‐terminus of the green fluorescent protein (GFP‐mTn) can visualize the actin cytoskeleton in different types of living plant cells without affecting cell morphology or function. Transiently expressed GFP‐mTn co‐localized with rhodamine‐phalloidin in permeabilized tobacco BY‐2 suspension cells, showing that the fusion protein can specifically label the plant actin cytoskeleton. Constitutive expression of GFP‐mTn in transgenic

Arabidopsis thaliana

plants visualized actin filaments in all examined tissues with no apparent effects on plant morphology or development at any stage during the life cycle. This demonstrates that in a number of different cell types GFP‐mTn can serve as a non‐invasive marker for the actin cytoskeleton. Confocal imaging of GFP‐mTn labeled actin filaments was employed to reveal novel information on the

in vivo

organization of the actin cytoskeleton in transiently transformed, normally elongating tobacco pollen tubes.
An oligo selection procedure for identification of sequence‐specific DNA‐binding activities associated with the plant defence response
Authors: Wang Zeping , Yang Peizhen , Fan Baofang , Chen Zhixiang
Published: March 2003

Sequence‐specific DNA‐binding (SSDB) factors play central roles in transcription, DNA replication, recombination and repair. This report describes a simple procedure for high‐throughput identification of SSDB activities without prior knowledge of their target genes or binding sequences. The procedure starts with a population of completely random oligo(nucleotide) sequences and selects for those oligo molecules that specifically bind to cellular SSDB factors by use of common gel‐retardation assays and PCR. Amplification and subsequent cloning of these selected oligo molecules result in the establishment of oligo DNA libraries enriched in DNA molecules containing specific sequences recognized by SSDB factors. These oligo libraries can be rapidly screened to identify a large number of SSDB activities, including those that are differentially regulated by developmental and environmental signals. With identified oligo DNA as probes, the corresponding SSDB factors can be isolated and analysed with respect to their structures, regulation and functions. Using this procedure, we have identified approximately 100 SSDB activities from tobacco leaves, including seven that are differentially regulated during the tobacco mosaic virus‐induced hypersensitive response in resistant tobacco plants.
Intimate association of microsatellite repeats with retrotransposons and other dispersed repetitive elements in barley
Authors: Ramsay Luke , Macaulay Malcolm , Cardle Linda , Morgante Michele , Ivanissevich Stephania degli , Maestri Elena , Powell Wayne , Waugh Robbie
Published: March 2003

Simple sequence repeat (SSR)‐based genetic markers are being actively developed for the majority of crop plant species. In barley, characterization of 290 dinucleotide repeat‐containing clones from SSR‐enriched libraries has revealed that a high percentage are associated with cereal retrotransposon‐like and other dispersed repetitive elements. Associations found were with BARE‐1, WIS2‐1A, PREM1 and the dispersed repetitive element R173. Additional similarities between different SSR clones, which have no matches in DNA sequence databases, indicate that this phenomenon is probably widespread in the barley genome. Sequence homologies to the non‐coding regions of several cereal genes were also explained by homology to mobile genetic elements. The SSRs found can therefore be classified into two types: (1) those with unique sequences on either flank, and (2) those which are intimately associated with retro‐transposons and other dispersed repetitive elements. As the cereal genome is thought to consist largely of this type of DNA, some random association would be expected. However, the conserved positions of the SSRs, relative to repetitive elements, indicate that they have arisen non‐randomly. Furthermore, this class of SSRs can be classified into three subtypes: (1) those which are positioned 3′ of a transposable element with unique sequence on the other flank, (2) those positioned 5′ of a transposable element, and (3) those which have arisen from an internal sequence and so have transposable element sequence on both flanks. The first appear to be analogous to the class of SSRs in mammalian systems which are associated with Alu elements and SINEs (short interspersed elements) and which have been postulated to arise following integration of an extended and polyadenylated retro‐transcript into the host genome, followed by mutation of the poly(A) tract and expansion into an SSR. For the second, we postulate that a proto‐SSR (A‐rich sequence) has acted as a ‘landing pad' for transposable element insertion (rather than being the result of insertion), while the third includes those which have evolved as a component of an active transposable element which has spread throughout the genome during bursts of transposition activity. The implications of these associations for genome and SSR evolution in barley are discussed.
Re‐evaluation of phytohormone‐independent division of tobacco protoplast‐derived cells
Authors: Schell Jeff , Bisseling Ton , Dülz Marion , Franssen Henk , Fritze Klaus , John Michael , Kleinow Tatjana , Leßnick Angela , Miklashevichs Edvins , Pawlowski Katharina , Röhrig Horst , De Sande Karin van , Schmidt Jürgen , Steinbiß Hans‐Henning , Stoll Marion
Published: March 2003

We have used a [3H] thymidine incorporation assay and microscopic observation in order to reassess recently published data dealing with the response of tobacco protoplasts to phytohormones, lipochitooligosaccharides and peptides (

Harling
et al
. 1997

;

Hayashi
et al
. 1992

;

Miklashevichs
et al
. 1996

;

Miklashevichs
et al
. 1997

;

Röhrig
et al
. 1995

;

Röhrig
et al
. 1996

;

van de Sande
et al
. 1996

;

Walden
et al
. 1994

). These proliferation assays reveal that, in contrast to published data, isolated cells of the investigated mutant plant lines axi159 (

Hayashi
et al
. 1992

;

Walden
et al
. 1994

), axi4/1 (

Harling
et al
. 1997

) and cyi1 (

Miklashevichs
et al
. 1997

), which were generated by activation T‐DNA tagging, were unable to grow in the absence of auxin or cytokinin. Furthermore, lipochitooligosaccharides which play a key role in the induction of nodules on roots of legumes were unable to promote auxin‐ or cytokinin‐independent cell division in tobacco protoplasts as claimed by

Röhrig
et al
. (1995

,

1996

). The finding of

van de Sande
et al
. (1996

) that
ENOD40
confers tolerance of high auxin concentration to wild‐type tobacco protoplasts was also reinvestigated. The results of our investigations show that we were unable to reproduce the proliferation data presented in this study, which were obtained by counting tobacco protoplast‐derived cells undergoing division. In total, none of the published data on phytohormone‐independent division of tobacco cells could be reproduced.
Biological functions of proline in morphogenesis and osmotolerance revealed in antisense transgenic Arabidopsis thaliana
Authors: Nanjo Tokihiko , Kobayashi Masatomo , Yoshiba Yoshu , Sanada Yukika , Wada Keishiro , Tsukaya Hirokazu , Kakubari Yoshitaka , Yamaguchi‐Shinozaki Kazuko , Shinozaki Kazuo
Published: March 2003

Many organisms, including higher plants, accumulate free proline (Pro) in response to osmotic stress. Although various studies have focused on the ability of Pro as a compatible osmolyte involved in osmotolerance, its specific role throughout plant growth is still unclear. It has been reported that Pro is synthesized from Glu catalyzed by a key enzyme, Δ1‐pyrroline‐5‐carboxylate synthetase (P5CS), in plants. To elucidate essential roles of Pro, we generated antisense transgenic Arabidopsis plants with a P5CS cDNA. Several transgenics accumulated Pro at a significantly lower level than wild‐type plants, providing direct evidence for a key role of P5CS in Pro production in Arabidopsis. These antisense transgenics showed morphological alterations in leaves and a defect in elongation of inflorescences. Furthermore, transgenic leaves were hypersensitive to osmotic stress. Microscopic analysis of transgenic leaves, in which the mutated phenotype clearly occurred, showed morphological abnormalities of epidermal and parenchymatous cells and retardation of differentiation of vascular systems. These phenotypes were suppressed by exogenous L‐Pro but not by D‐Pro or other Pro analogues. In addition, Pro deficiency did not broadly affect all proteins but specifically affected structural proteins of cell walls in the antisense transgenic plants. These results indicate that Pro is not just an osmoregulator in stressed plants but has a unique function involved in osmotolerance as well as in morphogenesis as a major constituent of cell wall structural proteins in plants.
Isolation of a gene encoding Arabidopsis membrane‐associated acyl‐CoA binding protein and immunolocalization of its gene product
Authors: Chye Mee‐Len , Huang Bing‐Quan , Zee Sze Yong
Published: March 2003

Until recently, only cytosolic acyl‐CoA binding proteins (ACBPs) have been characterized. The isolation of an Arabidopsis thaliana cDNA encoding a novel membrane‐associated ACBP that accumulates in developing seeds, designated ACBP1, has provided evidence for the existence of membrane‐associated forms of ACBPs (
Chye 1998, Plant Mol. Biol. 38, 827–838). We now report on the isolation of its corresponding gene from an A. thaliana Columbia genomic library using the ACBP1 cDNA as a hybridization probe. Nucleotide sequence analysis of Arabidopsis ACBP1 showed that its promoter lacks a TATA box, resembling the promoters of rat, Drosophila and human genes encoding cytosolic ACBP and suggesting that it is a housekeeping gene. We show by Western blot analysis that ACBP1 expression in developing seeds coincides with lipid deposition and that homologues of membrane‐associated ACBP1 exist in other plants. Using light microscopy, we show that ACBP1 is strongly expressed in the embryo at the cotyledons, hypocotyl, procambium of the axis and in most peripheral cells of the cotyledons and hypocotyl. Immunogold labelling localized ACBP1 to vesicles, to the plasma membrane especially at epidermal cells of heart, torpedo and cotyledonary stage embryos, and to the cell wall of the outer integument cells at the seed coat. Our results suggest that ACBP1 is involved in intermembrane lipid transport from the ER via vesicles to the plasma membrane where it could maintain a membrane‐associated acyl pool; its immunolocalization to the cell wall of outer integument cells at the seed coat suggests a role in cuticle and cutin formation.
A G‐box motif (GCCACGTGCC) tetramer confers high‐level constitutive expression in dicot and monocot plants
Authors: Ishige Fumiharu , Takaichi Miyuki , Foster Randy , Chua Nam‐Hai , Oeda Kenji
Published: March 2003

GUS reporter expression from 11 basal promoters (CaMV –90) with G‐box cores (CACGTG) was analysed to evaluate the regulatory roles of G‐box flanking sequences. While most G‐box motifs exhibited some tissue preference of gene expression, a distinct tissue‐specific expression was not apparent. However, one of 11 G‐box sequences, the G‐box 10 (GCCACGTGCC) tetramer, conferred a high‐level constitutive expression in seed, root, leaf, axillary bud, almost all parts of flower buds and pollen of transgenic tobacco plants. Furthermore, the G‐box 10 tetramer promoter exhibited high‐level expression in transgenic dicot carrot and monocot rice. This is apparently the first report of a G‐box motif conferring a high‐level constitutive expression in a non‐tissue‐specific manner.
The Nicotiana tabacum plasma membrane aquaporin NtAQP1 is mercury‐insensitive and permeable for glycerol
Authors: Biela Alexander , Grote Karsten , Otto Beate , Hoth Stefan , Hedrich Rainer , Kaldenhoff Ralf
Published: March 2003

A new aquaporin from Nicotiana tabacum (cv. Samsun) was characterized. It shares sequence homology to the Arabidopsis thaliana PIP1 protein family. By two‐phase partitioning and immunoblot analysis, plasma membrane localization could be demonstrated. The corresponding mRNA is highly abundant in roots and flowers, while it is rarely expressed in leaves and stems. Functional expression in Xenopus oocytes revealed that NtAQP1 can mediate glycerol transport in addition to water flow. However, NtAQP1 is impermeable for Na

+

, K

+

and Cl

–

ions. The water permeability and selectivity could not be modulated by addition of mercurials or the activity of cAMP‐dependent protein kinases.
Regulation of phytochrome B signaling by phytochrome A and FHY1 in Arabidopsis thaliana
Authors: Cerdán Pablo D. , Yanovsky Marcelo J. , Reymundo F. Carolina , Nagatani Akira , Staneloni Roberto J. , Whitelam Garry C. , Casal Jorge J.
Published: March 2003

Phytochrome A (phyA) and phytochrome B (phyB) share the control of many processes but little is known about mutual signaling regulation. Here, we report on the interactions between phyA and phyB in the control of the activity of an Lhcb1*2 gene fused to a reporter, hypocotyl growth and cotyledon unfolding in etiolated Arabidopsis thaliana. The very‐low fluence responses (VLFR) induced by pulsed far‐red light and the high‐irradiance responses (HIR) observed under continuous far‐red light were absent in the phyA and phyA phyB mutants, normal in the phyB mutant, and reduced in the fhy1 mutant that is defective in phyA signaling. VLFR were also impaired in Columbia compared to Landsberg erecta. The low‐fluence responses (LFR) induced by red‐light pulses and reversed by subsequent far‐red light pulses were small in the wild type, absent in phyB and phyA phyB mutants but strong in the phyA and fhy1 mutants. This indicates a negative effect of phyA and FHY1 on phyB‐mediated responses. However, a pre‐treatment with continuous far‐red light enhanced the LFR induced by a subsequent red‐light pulse. This enhancement was absent in phyA
, phyB
, or phyA phyB and partial in fhy1
. The levels of phyB were not affected by the phyA or fhy1 mutations or by far‐red light pre‐treatments. We conclude that phyA acting in the VLFR mode (i.e. under light pulses) is antagonistic to phyB signaling whereas phyA acting in the HIR mode (i.e. under continuous far‐red light) operates synergistically with phyB signaling, and that both types of interaction require FHY1.
Stable chloroplast transformation in potato: use of green fluorescent protein as a plastid marker
Authors: Sidorov Vladimir A. , Kasten Daniel , Pang Sheng‐Zhi , Hajdukiewicz Peter T. J. , Staub Jeffrey M. , Nehra Narender S.
Published: March 2003

We describe here the development of a reproducible plastid transformation system for potato and regeneration of plants with uniformly transformed plastids. Two distinct tobacco‐specific plastid vectors, pZS197 (Prrn/aadA/TpsbA) and pMON30125 (Prrn/GFP/Trps16::PpsbA/aadA/TpsbA), designed for integration into the large single copy and inverted repeat regions of the plastid genome, respectively, were bombarded into leaf explants of potato line FL1607. A total of three transgenic lines were selected out of 46 plates bombarded with pZS197 and three transgenic lines out of 104 plates were obtained with pMON30125. Development of a high frequency leaf‐based regenera‐ tion system, a stringent selection scheme and optimization of biolistic transformation protocol were critical for recovery of plastid transformants. Plastid‐expressed green fluorescent protein was used as a visual marker for identification of plastid transformants at the early stage of selection and shoot regeneration. The establishment of a plastid transformation system in potato, which has several advantages over routinely used nuclear transformation, offers new possibilities for genetic improvement of this crop.
PR‐1 protein inhibits the differentiation of rust infection hyphae in leaves of acquired resistant broad bean
Authors: Rauscher Martina , Ádám Attila L. , Wirtz Sabine , Guggenheim Richard , Mendgen Kurt , Deising Holger B.
Published: March 2003

Treatment of broad bean leaves with salicylic acid (SA) or 2,6‐dichloro‐isonicotinic acid (DCINA) induces resistance against the rust fungus Uromyces fabae resulting in reduced rust pustule density. Light‐microscopy studies showed that in induced resistant plants the rust fungus is inhibited immediately after penetration through the stomatal pore. The differentia‐ tion of infection structures growing within the intercellular space of the leaf, i.e. infection hyphae and haustorial mother cells, is inhibited. Furthermore, low‐temperature scanning electron microscopy studies of freeze fractures revealed protrusions at the tips of infection hyphae growing in induced resistant broad bean leaves. Treatment of in vitro‐differentiating rust infection structures with intercellular fluids (IFs) from induced resistant plants confirmed that the fungus is sensitive towards an apoplastic anti‐fungal activity only after having formed appressoria. Other legume rusts such as U. vignae and U. appendiculatus were likewise inhibited in the presence of IF from SA‐treated broad bean leaves. Heterologous antibodies were used to study changes in the extracellular pathogenesis‐related (PR) protein pattern after resistance induction. Western blots indicated that chitinases and β‐1,3‐glucanases were present in both induced and control plants. In contrast, PR‐1 proteins were newly synthesized in response to SA or DCINA application. The major induced PR‐1 protein was purified and exhibited strong differentiation‐inhibiting activity towards U. fabae infection structures. We conclude that the inhibition of rust infection hyphae in acquired resistant broad bean plants is mainly due to the anti‐fungal activity of this induced basic PR‐1 protein.
Long‐chain acyl‐CoA oxidases of Arabidopsis
Authors: Hooks Mark A. , Kellas Fiona , Graham Ian A.
Published: March 2003

Full‐length cDNAs coding for two distinct acyl‐CoA oxidases were isolated by screening an Arabidopsis cDNA library. The genes for the two acyl‐CoA oxidases have been termed AtACX1 and AtACX2. AtACX1 encodes a peptide of 664 amino acids possessing a molecular mass of 74.3 kDa. AtACX2 encodes a peptide of 691 amino acids in length with a molecular mass of 77.5 kDa. Peroxisomal targeting signals were identified in the primary sequences. AtACX1 has a putative PTS1, whereas AtACX2 has a characteristic PTS2. Expression of AtACX1 and AtACX2 in Escherichia coli gave active enzymes for enzymatic and biochemical analysis. AtACX1 was active with both medium‐and long‐chain saturated fatty acyl‐CoAs and showed maximal activity with C14‐CoA. Activity with mono‐unsaturated acyl‐CoAs was slightly higher than with the corresponding saturated acyl‐CoA. AtACX2 was active with long‐chain acyl‐CoAs and showed maximal activity with C18‐CoA. AtACX2 activity with mono‐unsaturated acyl‐CoAs was approximately twice as high as with the corresponding saturated acyl‐CoA. Both enzymes have an apparent Km of approximately 5 μm with the preferred substrate. Northern analysis was conducted to determine the expression patterns of AtACX1 and AtACX2 during germination and in various tissues of a mature plant. The two genes showed generally similar expression profiles and steady‐state mRNA levels in seedlings and mature tissues, but subtle differences were observed. Enzymatic analyses of plant extracts revealed that AtACX1 and AtACX2 are members of a family that includes acyl‐CoA oxidases specific for shorter‐chain acyl‐CoAs. Through expression of antisense constructs of the individual genes, we were able to decrease long‐chain oxidase activity only in antisense AtACX1 plants. Seedlings with long‐chain oxidase activity reduced down to 30% of wild‐type levels germinated and established normally; however, reduced root growth appeared to be a general feature of antisense AtACX1 plants.
Seed‐specific overexpression of phytoene synthase: increase in carotenoids and other metabolic effects
Authors: Shewmaker Christine K. , Sheehy Julie A. , Daley Maureen , Colburn Susan , Ke Dang Yang
Published: March 2003

A bacterial phytoene synthase (crtB) gene was overexpressed in a seed‐specific manner and the protein product targeted to the plastid in Brassica napus (canola). The resultant embryos from these transgenic plants were visibly orange and the mature seed contained up to a 50‐fold increase in carotenoids. The predominant carotenoids accumulating in the seeds of the transgenic plants were alpha and beta‐carotene. Other precursors such as phytoene were also detected. Lutein, the predominant carotenoid in control seeds, was not substantially increased in the transgenics. The total amount of carotenoids in these seeds is now equivalent to or greater than those seen in the mesocarp of oil palm. Other metabolites in the isoprenoid pathway were examined in these seeds. Sterol levels remained essentially the same, while tocopherol levels decreased significantly as compared to non‐transgenic controls. Chlorophyll levels were also reduced in developing transgenic seed. Additionally, the fatty acyl composition was altered with the transgenic seeds having a relatively higher percentage of the 18 : 1 (oleic acid) component and a decreased percentage of the 18 : 2 (linoleic acid) and 18 : 3 (linolenic acid) components. This dramatic increase in flux through the carotenoid pathway and the other metabolic effects are discussed.