bluuebunny/crossref_metadata_2025 huggingface dataset:
Possible involvement of differential splicing in regulation of the activity of Arabidopsis ANP1 that is related to mitogen‐activated protein kinase kinase kinases (MAPKKKs)
Authors: Nishihama Ryuichi , Banno Hiroharu , Kawahara Emi , Irie Kenji , Machida Yasunori
Published: March 2003

Three types of Arabidopsis cDNA (cANP1, cANP2 and cANP3) have been isolated that encode putative protein kinases, designated ANP1, ANP2 and ANP3. These kinases exhibit a high degree of hornology to NPK1, a tobacco protein that is a member of the family of mitogen‐activated protein kinase kinase kinases (MAPKKKs), which appears to function in the proliferation of tobacco cells. The predicted amino acid sequences of the kinase domains in the amino‐terminal halves of the ANPs were more than 80% identical to that of NPK1, while the kinase‐unrelated regions in the carboxy‐terminal halves exhibited relatively low homology. Two species of cANP1 were identified, ANP1L cDNA (cANPIL) and ANP1S cDNA (cANP1S), which were derived from a single ANP1 gene: the former had an intron‐like sequence in the coding region for the kinase‐unrelated region, while the latter did not include such an intron‐like sequence. cANP1L encoded a putative protein with both kinase and kinase‐unrelated domains, resembling NPK1, whereas cANPIS encoded only the amino‐terminal kinase domain because the intron‐like sequence was absent, with resulting elimination of most of the kinase‐unrelated region. Genetic analysis with mutant yeast cells showed that over‐expression of cANPIL or of cANPIS activated the mating pheromone‐responsive signal pathway which is mediated by a MAP kinase cascade. Moreover, the extent of such activation by cANPIS was greater than that by cANPIL. These results predict that differential splicing of the intron‐like sequence in the ANP1 transcript might be at least one of the molecular mechanisms involved in the generation of active ANP1 protein kinase.
Molecular characterization of a phloem‐specific gene encoding the filament protein, Phloem Protein 1 (PP1), from Cucurbita maxima
Authors: Clark Anna M. , Jacobsen Karin R. , Bostwick Dwight E. , Dannenhoffer Joanne M. , Skaggs Megan I. , Thompson Gary A.
Published: March 2003

Sieve elements in the phloem of most angiosperms contain proteinaceous filaments and aggregates called P‐protein. In the genus Cucurbita, these filaments are composed of two major proteins: PP1, the phloem filament protein, and PP2, the phloem lectin. The gene encoding the phloem filament protein in pumpkin (Cucurbita maxima Duch.) has been isolated and characterized. Nucleotide sequence analysis of the reconstructed gene gPP1 revealed a continuous 2430 bp protein coding sequence, with no introns, encoding an 809 amino acid polypeptide. The deduced polypeptide had characteristics of PP1 and contained a 15 amino acid sequence determined by N‐terminal peptide sequence analysis of PP1. The sequence of PP1 was highly repetitive with four 200 amino acid sequence domains containing structural motifs in common with cysteine proteinase inhibitors. Expression of the PP1 gene was detected in roots, hypocotyls, cotyledons, stems, and leaves of pumpkin plants. PP1 and its mRNA accumulated in pumpkin hypocotyls during the period of rapid hypocotyl elongation after which mRNA levels declined, while protein levels remained elevated. PP1 was immunolocalized in slime plugs and P‐protein bodies in sieve elements of the phloem. Occasionally, PP1 was detected in companion cells. PP1 mRNA was localized by in situ hybridization in companion cells at early stages of vascular differentiation. The developmental accumulation and localization of PP1 and its mRNA paralleled the phloem lectin, further suggesting an interaction between these phloem‐specific proteins.
Very‐long‐chain fatty acid biosynthesis is controlled through the expression and specificity of the condensing enzyme
Authors: Millar Anthony A. , Kunst Ljerka
Published: March 2003

The Arabidopsis FATTY ACID ELONGATION1 (FAE1) gene encodes a putative seed‐specific condensing enzyme. It is the first of four enzyme activities that comprise the microsomal fatty acid elongase (FAE) involved in the biosynthesis of very‐long‐chain fatty acids (VLCFAs). FAE1 has been expressed in yeast and in tissues of Arabidopsis and tobacco, where significant quantities of VLCFAs are not found. The introduction of FAE1 alone in these systems is sufficient for the production of VLCFAs, for wherever FAE1 was expressed, VLCFAs accumulated. These results indicate that FAE1 is the rate‐limiting enzyme for VLCFA biosynthesis in Arabidopsis seed, because introduction of extra copies of FAE1 resulted in higher levels of the VLCFAs. Furthermore, the condensing enzyme is the activity of the elongase that determines the acyl chain length of the VLCFAs produced. In contrast, it appears that the other three enzyme activities of the elongase are found ubiquitously throughout the plant, are not rate‐limiting and play no role in the control of VLCFA synthesis. The ability of yeast containing FAE1 to synthesize VLCFAs suggests that the expression and the acyl chain length specificity of the condensing enzyme, along with the apparent broad specificities of the other three FAE activities, may be a universal eukaryotic mechanism for regulating the amounts and acyl chain length of VLCFAs synthesized.
A programmed cell death pathway activated in carrot cells cultured at low cell density
Authors: McCabe Paul F. , Levine Alex , Meijer Per‐Johan , Tapon Nicolas A. , Pennell Roger I.
Published: March 2003

Programmed cell death (PCD) occurs in plants during development and defense, but the processes and mechanisms are not yet defined. Culture of carrot single cells at a cell density of <104 cells ml−1 activates a cell death process involving condensation and shrinkage of the cytoplasm and nucleus and fragmentation of the DNA. Modest abiotic stress treatments also cause cell condensation and shrinkage and the formation of DNA fragments, but the same abiotic stresses at high levels cause rapid necrosis with cell swelling and lysis. The common morphological features of cells dying at low cell density and following modest abiotic stress treatments suggest that these features reveal a PCD pathway in carrot. The addition of a cell‐conditioned growth medium allows cells at low cell density to remain alive, demonstrating that cell‐derived signal molecules suppress a pathway that is otherwise induced by default. Differences in the morphology of the dead cells suggest that proteolysis during PCD differs in detail in plants and animals; however, these findings show that plants, like animals, can control PCD by social signaling, and imply that the mechanism of PCD in plants and animals may be similar. Consistent with this, manipulation of signal pathway intermediates that regulate PCD in animals shows that Ca2+ and protein phosphorylation events are PCD pathway intermediates in carrot.
Plant male germ line transformation
Authors: Touraev A. , Stöger E. , Voronin V. , Heberle‐Bors E.
Published: March 2003

A new method to produce transgenic plants ‐ male germ line transformation (MAGELITR) is reported. Unicellular tobacco microspores were isolated from excised anthers, and DNA carrying two marker genes was transferred biolistically. The bombarded microspores were matured in vitro for 6 days, and the mature pollen was used for in vivo pollination. Seeds were recovered and putative transformants were selected on the basis of their antibiotic resistance. Five kanamycin‐resistant plants were chosen for further analysis, four contained the first construct, one the second. Parallel experiments with bicellular immature pollen did not produce any transgenic plants. A detailed DNA blot and expression analysis confirmed the transgenic nature of the five plants, and a genetic analysis showed that the transgenes are transmitted to subsequent generations. MAGELITR is a fast, regeneration‐independent method, not prone to chimerism and somaclonal variation which should be genotype‐independent and may be applicable in a wide range of species once in vitro maturation of pollen is established.
The male determinant of self‐incompatibility in Brassica oleracea is located in the pollen coating
Authors: Stephenson Andrew G. , Doughty James , Dixon Suzanne , Elleman Carole , Hiscock Simon , Dickinson Hugh G.
Published: March 2003

An in vitro bioassay has been developed to explore the role of the pollen coating in the pollen/stigma interaction in Brassica oleracea. In the assay, coating is removed from pollen grains, supplemented with protein fractions isolated from coatings of different S (self incompatibility) haplotypes, and then—using micromanipulation—interposed between individual pollen grains and the stigmatic surface. Normally, the coating used is of the same haplotype as the pollen in the experiment—thus constituting an ‘extension’ of its own coat—but carrying the supplemented protein fractions. Initial experiments confirmed preliminary data that the pollen coating contained the male determinant of self incompatibility (SI); not only did the addition of ‘self’ coating (i.e. that with the same S‐haplotype as the stigma) prevent the success of a compatible cross pollination, but a ‘cross’ coating (i.e. that with a different S‐haplotype from the stigma) could induce the germination and growth of self pollen. Protein supplementation experiments demonstrated that the pollen‐held determinant is contained within the water soluble component of the pollen coat, while further analysis revealed that the active molecular species possesses an Mr10 kDa. More extensive fractionation by gel filtration and reverse phase HPLC was used to isolate a family of basic, cysteine‐rich proteins (PCP‐A: Pollen Coat Proteins‐class A)—one of which is known to bind to stigmatically‐expressed components of the S‐locus in Brassica. Introduction of the PCP‐A protein fraction into the bioassay confirmed the male determinant of SI as a protein, and probably a member of the PCP‐A protein family.
N‐glycans harboring the Lewis a epitope are expressed at the surface of plant cells
Authors: Fitchette‐Lainé Anne‐Catherine , Gomord Véronique , Cabanes Marion , Michalski Jean‐Claude , Saint Macary Mireille , Foucher Bernard , Cavelier Brigitte , Hawes Chris , Lerouge Patrice , Faye Loïc
Published: March 2003

In plants, N‐linked glycans are processed in the Golgi apparatus to complex‐type N‐glycans of limited size containing a β(1,2)‐xylose and/or an α(1,3)‐fucose residue. Larger mono‐ and bi‐antennary N‐linked complex glycans have not often been described. This study has re‐examined the structure of such plant N‐linked glycans, and, through both immunological and structural data, it is shown that the antennae are composed of Lewis a (Lea) antigens, comprising the carbohydrate sequence Galβ1‐3[Fucα1‐4]GlcNAc. Furthermore, a fucosyltransferase activity involved in the biosynthesis of this antigen was detected in sycamore cells. This is the first characterization in plants of a Lewis antigen that is usually found on cell‐surface glycoconjugates in mammals and involved in recognition and adhesion processes. Lea‐containing N‐linked glycans are widely distributed in plants and highly expressed at the cell surface, which may suggest a putative function in cell/cell communication.
Genetic transformation of dinoflagellates (Amphidinium and Symbiodinium): expression of GUS in microalgae using heterologous promoter constructs
Authors: Te Michael R. , Lohuis  , Miller David J.
Published: March 2003

Genetic transformation of two dinoflagellates (Amphidinium sp., Symbiodinium microadriaticum) was achieved using plasmid constructs containing the neomycin phosphotransferase gene (nptII) fused to the Agrobacterium nos promoter, or the hygromycin B phosphotransferase gene (hpt) fused to the bidirectional Agrobacterium p1′2′ promoter. Gene transfer into intact (walled) dinoflagellate cells was achieved by agitation in the presence of silicon carbide (SiCa) whiskers. Transformation rates of 5–24 transformants per 107 cells were obtained. Southern hybridization of transformants revealed stable integration of multiple copies of the constructs. Activity of integrated copies of the β‐glucoronidase (GUS) reporter gene coupled to the cauliflower mosaic virus 35S promoter or the p1′2′ promoter was confirmed both histochemically and fluorometrically. This is the first report of successful application of heterologous and widely used promoter and reporter genes in microalgae, and is the first demonstration of transformation of a dinoflagellate. There appear to be no substantial barriers to transformation of Amphidinium and Symbiodinium, which must now be considered as the first of the dinoflagellate genera accessible to genetic manipulation.
Plasma membrane depolarization‐activated calcium channels, stimulated by microtubule‐depolymerizing drugs in wild‐type Arabidopsis thaliana protoplasts, display constitutively large activities and a longer half‐life in ton 2 mutant cells affected in the organization of cortical microtubules
Authors: Thion Laurence , Mazars Christian , Nacry Philippe , Bouchez David , Moreau Marc , Ranjeva Raoul , Thuleau Patrice
Published: March 2003

Depolarization‐activated plasma membrane calcium channels have been suggested to play prominent roles in signal perception and transduction processes during growth and development of higher plants. The existence of such channels has recently been established in higher plant cells. However, patch–clamp experiments have shown that their activity is very low and decreases very rapidly after the establishment of the whole‐cell configuration, due most probably to protein–protein interactions involving microtubules. The present study takes advantage of the existence of Arabidopsis thaliana mutants referred to as ton 2 mutants reported to be affected in their microtubule organization, to address the physiological relevance of such a hypothesis based on a pharmacological approach. Patch–clamp studies showed that depolarization‐activated calcium channel activities in ton 2 protoplasts were 10‐fold higher and their relative half‐life three‐times longer than in wild‐type protoplasts. In addition, oryzalin and colchicine, which disrupt the microtubule organization, stimulated and stabilized calcium channel activities in wild‐type but remained ineffective on ton 2 protoplasts. However, although the microtubules appeared important in the regulation of calcium channels in A. thaliana, immunocytological staining of tubulin demonstrated that there was no visible difference in the general organization of microtubule networks or in the amount of microtubules bound to the plasma membrane in ton 2 and wild‐type protoplasts. It is suggested that the down‐regulation of calcium channels implicating microtubules involves additional component(s) corresponding probably to gene product(s) defective in ton 2 mutant cells.
Molecular cloning and functional expression in yeast of CYP76B1, a xenobiotic‐inducible 7‐ethoxycoumarin O‐de‐ethylase from Helianthus tuberosus
Authors: Batard Yannick , LeRet Monique , Schalk Michel , Robineau Tiburce , Durst Francis , Werck‐Reichhart Danièle
Published: March 2003

In order to obtain plant markers of chemical stress and possible tools for the bio‐monitoring of pollution, a protein purification/PCR approach was used to isolate cDNAs of xenobiotic‐inducible P450 oxygenases. O‐dealkylation of 7‐ethoxycoumarin is catalysed in Helianthus tuberosus by cytochromes P450 strongly inducible by a wide range of xenobiotics. Therefore, a 7‐ethoxycoumarin O‐de‐ethylase (ECOD) was purified from induced tuber tissues (Batard et al. 1995). A primer designed from an internal peptide sequence, but also corresponding to a conserved P450 haem‐binding region, led to the generation of a gene‐specific probe corresponding to a P450 strongly inducible by aminopyrine. Two partial and 98% identical coding sequences were isolated from a cDNA library prepared from aminopyrine‐induced tuber. A full‐length cDNA was reconstituted by 5′‐RACE elongation. The protein deduced from this full‐length sequence, with 41.1% amino acid identity to CYP76A1 and high phylogenetic relationship to other CYP76s, was termed CYP76B1. CYP76B1 was expressed in yeast. Microsomes from the transformed yeast catalysed the NADPH‐dependent O‐deakylation of 7‐ethoxycoumarin. However, protein sequence as well as enzymological data indicated that CYP76B1 does not correspond to the purified ECOD protein. These results confirm previous data and demonstrate that several P450s in H. tuberosus are capable of actively catalysing the O‐de‐ethylation of ethoxycoumarin.

 Determination of the steady‐state level of CYP76B1 transcripts after slicing tuber tissues and ageing them in water, alone or in the presence of various chemicals, showed that the expression of this P450 was not responsive to mechanical stress, but was strongly induced by chemical treatments. CYP76B1 thus appears to be a good potential marker of chemical stress and of environmental pollution.
Sensing trehalose biosynthesis in plants
Authors: Goddijn Oscar , Smeekens Sjef
Published: March 2003

A most unexpected finding in research on plant carbohydrate metabolism is the recent discovery that angiosperms encode genes whose products are involved in trehalose metabolism. The presence and functionality of such genes has been elegantly shown by expressing Arabidopsis‐derived trehalose phosphate synthase and trehalose phosphate phosphatase genes in yeast mutants lacking these enzymatic activities. Homologue sequences have now been cloned from a number of different plant species suggesting that the capacity to synthesise trehalose is ubiquitous in angiosperms. Except for Myrothamnus flabellifolius, trehalose biosynthesis has never been observed in tissues of higher plants, probably due to the presence of high levels of trehalase activity. The function of trehalose metabolism in plants is still a mystery. One of the postulated functions of trehalose metabolism in yeast is in the control of glucose repression and a similar function in sugar sensing can be proposed for plants as well.
Co‐operation between cytosolic and plastidic oxidative pentose phosphate pathways revealed by 6‐phosphogluconate dehydrogenase‐deficient genotypes of maize
Authors: Averill Rachel H. , Bailey‐Serres Julia , Kruger Nicholas J.
Published: March 2003

The aim of this work was to examine the extent to which the oxidative steps of the pentose phosphate pathway in the cytosol contribute to the provision of reductant for biosynthetic reactions. Maize (Zea mays L.) contains at least two loci (pgd1 and pgd2) that encode 6‐phosphogluconate dehydrogenase. Ten genotypic combinations of wild‐type (Pgd1+3.8;Pgd2+5) and null alleles of pgd1 and pgd2 were constructed in the B73 background. The maximum catalytic activity of 6‐phosphogluconate dehydrogenase in the roots of seedlings of these lines correlated with the number of functional pgd1 and pgd2 alleles. Enzyme activity in the double‐null homozygote (pgd1‐null;pgd2‐null) was 32% of that in B73 wild‐type suggesting the presence of at least one other isozyme of 6‐phosphogluconate dehydrogenase in maize. Subcellular fractionation studies and latency measurements confirmed that the products of pgd1 and pgd2 are responsible for the vast majority, if not all, of the cytosolic 6‐phosphogluconate dehydrogenase activity in maize roots. Essentially, all of the residual activity in the double‐null homozygote was confined to the plastids. Low concentrations (0.1–0.5 mM) of sodium nitrite stimulated 14CO2 production by detached root tips of both wild‐type and 6‐phosphogluconate dehydrogenase‐deficient maize seedlings fed [U‐14C]glucose. Analysis of the ratio of 14CO2 released from [1–14C]glucose relative to [6–14C]glucose (C1/C6 ratio) showed that stimulation of the oxidative pentose phosphate pathway by nitrite correlated with the dosage of wild‐type alleles of pgd1 and pgd2. The failure of 6‐phosphogluconate dehydrogenase‐deficient lines to respond to nitrite indicates that perturbation of the cytosolic oxidative pentose phosphate pathway can influence the provision of reductant in the plastid. We conclude that the plastidic and cytosolic oxidative pentose phosphate pathways are able to co‐operate in the provision of NADPH for biosynthesis.
Post‐transcriptional silencing of pectin methylesterase gene in transgenic tomato fruits results from impaired pre‐mRNA processing
Authors: Mishra Krishna K. , Handa Avtar K.
Published: March 2003

Transgenic tomato fruit expressing a truncated fruit pectin methylesterase (PME) sense transgene under the control of CaMV 35S promoter show developmentally regulated co‐silencing of the endogenous fruit PME and sense transgene. This co‐silencing is associated with the onset of fruit PME gene expression. Detectable levels of PME protein and mRNA were not present in ripening transgenic fruits. However, several smaller transgene RNA species hybridizing to the 3′ end of the transgene are present in transgenic fruits, indicating degradation of sense transgene transcript. RT–PCR analyses of nuclear RNA from wild‐type and transgenic fruits, primed with oligo(dT), show the presence of spliced and unspliced transcripts for the fruit PME and the transgene, indicating a post‐transcriptional regulation of gene silencing. However, an accumulation of unspliced fruit PME transcript was obtained in the nuclei of transgenic fruit, using the randomly primed cDNAs in PCR amplification. Taken together, these results suggest that the ectopic expression of the fruit PME sense transgene interferes with processing of the endogenous PME pre‐mRNA.
Transcription of the Arabidopsis CPD gene, encoding a steroidogenic cytochrome P450, is negatively controlled by brassinosteroids
Authors: Mathur Jaideep , Molnár Gergely , Fujioka Shozo , Takatsuto Suguru , Sakurai Akira , Yokota Takao , Adam Günter , Voigt Brunhilde , Nagy Ferenc , Maas Christoph , Schell Jeff , Koncz Csaba , Szekeres Miklós
Published: March 2003

The Arabidopsis CPD gene encodes a cytochrome P450 steroid side‐chain hydroxylase (CYP90) that plays an essential role in the biosynthesis of the plant hormone brassinolide. Expression of the CPD gene is confined to cotyledons and leaf primordia in etiolated seedlings and detectable in the adaxial parenchyma of expanding leaves in light‐grown plants. Transcription of the CPD gene is not affected by the plant growth factors auxin, ethylene, gibberellin, cytokinin, jasmonic acid and salicylic acid, but is specifically down‐regulated by brassinolide in both dark and light. Steady‐state mRNA levels of a CPD promoter‐driven uidA reporter gene correlate with the expression of resident CPD gene in transgenic plants. Intermediates of the early and late C‐6 oxidation pathways of brassinolide, carrying C‐22 and C‐23 side‐chain hydroxyls, efficiently inhibit the activity of the CPD promoter. Repression of CPD transcription by brassinosteroids is sensitive to the protein synthesis inhibitor cycloheximide, indicating a requirement for de novo synthesis of a regulatory factor.
Localization of a myosin‐like protein to plasmodesmata
Authors: Radford Janine E. , White Rosemary G.
Published: March 2003

Myosin has been localized to plasmodesmata in root tissues of Allium cepa, Zea mays and Hordeum vulgare using a polyclonal antibody to animal myosin in both fluorescence and electron microscopy. Labelling was also observed throughout the cytoplasm, mainly associated with the endoplasmic reticulum and plasma membrane. On Western blots, bands of 180 and 110 kDa were consistently labelled in all three species. These bands were also labelled when the blot was incubated in actin prior to staining with antibodies to actin, raising the possibility that either of these proteins (180 kDa or 110 kDa) may be present in plasmodesmata. Pre‐treatment of the tissue with 2,3‐butanedione monoxime (BDM), an inhibitor of actin–myosin motility, resulted in a strong constriction of the neck region of plasmodesmata. These results indicate that a myosin‐like protein may be present in plasmodesmata and may also play a role in the regulation of transport at the neck region.
Sur2 mutations of Arabidopsis thaliana define a new locus involved in the control of auxin homeostasis
Authors: Delarue Marianne , Prinsen Els , Va Harry , Onckelen  , Caboche Michel , Bellini Catherine
Published: March 2003

A new auxin homeostasis gene in Arabidopsis called SUR2 has been identified. This gene, mapped to the bottom of chromosome 4, is defined by two recessive nuclear mutants designated superroot2 (sur2), which display several abnormalities reminiscent of auxin effects. A number of these characteristics are similar to the phenotype of the previously described auxin‐overproducing mutant superroot1 (sur1); however, several lines of evidences reveal that the SUR2 gene defines a new key point in the regulation of endogenous auxin concentrations. The phenotype of the sur1 sur2 double mutant is additive. Analysis by gas chromatography coupled to mass spectrometry indicated increased levels of free indole‐3‐acetic acid correlated with a decreased level of bound auxin in the sur2 mutant. These results suggest that SUR2 may be involved in the control of auxin conjugation.
Promoters of nuclear‐encoded respiratory chain Complex I genes from Arabidopsis thaliana contain a region essential for anther/pollen‐specific expression
Authors: Zabaleta  , Heiser  , Grohmann  , Brennicke
Published: March 2003

Regulatory promoter regions responsible for the enhanced expression in anthers and pollen are defined in detail for three nuclear encoded mitochondrial Complex I (nCI) genes from Arabidopsis thaliana. Specific regulatory elements were found conserved in the 5′ upstream regions between three different genes encoding the 22 kDa (PSST), 55 kDa NADH binding (55 kDa) and 28 kDa (TYKY) subunits, respectively. Northern blot analysis and transgenic Arabidopsis plants carrying progressive deletions of the promoters fused to the β‐glucuronidase (GUS) reporter gene by histochemical and fluorimetric methods showed that all three promoters drive enhanced expression of GUS specifically in anther tissues and in pollen grains. In at least two of these promoters the –200/–100 regions actively convey the pollen/anther‐specific expression in gain of function experiments using CaMV 35S as a minimal promoter. These nCI promoters thus contain a specific regulatory region responding to the physiological demands on mitochondrial function during pollen maturation. Pollen‐specific motifs located in these regions appear to consist of as little as seven nucleotides in the respective promoter context.
Expression of snowdrop lectin (GNA) in transgenic rice plants confers resistance to rice brown planthopper
Authors: Rao K. V. , Rathore Keerti S. , Hodges Thomas K. , Fu X. , Stoger Eva , Sudhakar D. , Williams Sarah , Christou Paul , Bharathi M. , Bown David P. , Powell Kevin S. , Spence Jacqui , Gatehouse Angharad M. R. , Gatehouse John A.
Published: March 2003

Snowdrop lectin (

Galanthus nivalis

agglutinin; GNA) has been shown previously to be toxic towards rice brown planthopper (

Nilaparvata lugens

; BPH) when administered in artificial diet. BPH feeds by phloem abstraction, and causes ‘hopper burn’, as well as being an important virus vector. To evaluate the potential of the

gna

gene to confer resistance towards BPH, transgenic rice (

Oryza sativa

L.) plants were produced, containing the

gna

gene in constructs where its expression was driven by a phloem‐specific promoter (from the rice sucrose synthase

RSs1

gene) and by a constitutive promoter (from the maize ubiquitin

ubi1

gene). PCR and Southern analyses on DNA from these plants confirmed their transgenic status, and that the transgenes were transmitted to progeny after self‐fertilization. Western blot analyses revealed expression of GNA at levels of up to 2.0% of total protein in some of the transgenic plants. GNA expression driven by the

RSs1

promoter was tissue‐specific, as shown by immunohistochemical localization of the protein in the non‐lignified vascular tissue of transgenic plants. Insect bioassays and feeding studies showed that GNA expressed in the transgenic rice plants decreased survival and overall fecundity (production of offspring) of the insects, retarded insect development, and had a deterrent effect on BPH feeding.

gna

is the first transgene to exhibit insecticidal activity towards sap‐sucking insects in an important cereal crop plant.
Polyunsaturated membranes are required for photosynthetic competence in a mutant ofArabidopsis
Authors: McConn Michele , Browse John
Published: March 2003

High levels of polyunsaturation are characteristic of all the membranes of plant and animal cells. For example, the chloroplasts of leaf cells contain about 75–80% polyunsaturated fatty acids. For the extra‐chloroplast membranes in leaf cells and the membranes of non‐photosynthetic tissues, values of 60–65% are typical. We report here the production of

Arabidopsis

double mutants that contain negligible levels of polyunsaturated fatty acids. The mutants were not capable of autotrophic growth and produced extremely chlorotic cotyledons and leaves. However, on sucrose media, the double mutants were robust plants showing strong leaf and root development. These observations indicate that the vast majority of receptor‐mediated and transport‐related membrane functions required to sustain the organism and induce proper development are adequately supported in the absence of polyunsaturated lipids. By contrast, photosynthesis is one process that does require high levels of membrane polyunsaturation.
A transgene with repeated DNA causes high frequency, post‐transcriptional suppression of ACC‐oxidase gene expression in tomato
Authors: Hamilton Andrew J. , Brown Stephen , Yuanhai Han , Ishizuka Masakatsu , Lowe Alex , Solis Angel‐Gabriel Alpuche , Grierson Don
Published: March 2003

Gene silencing with sense genes is an important method for down‐regulating the expression of endogenous plant genes, but the frequency of silencing is unpredictable. Fifteen per cent of tomato plants transformed with a 35S‐ACC‐oxidase (

ACO

1) sense gene had reduced ACC‐oxidase activity. However, 96% of plants transformed with an ACC‐oxidase sense gene, containing two additional upstream inverted copies of its 5′ untranslated region, exhibited reduced ACC‐oxidase activity compared to wild‐type plants. In the three plants chosen for analysis, there were substantially reduced amounts of both endogenous and transgenic ACO RNA, indicating that this was an example of co‐suppression. Ribonuclease protection assays using probes spanning intron‐exon borders showed that the reduced accumulation of endogenous ACO mRNA occurred post‐transcriptionally since the abundance of unprocessed transcripts was not affected. The

ACO1

transgene with the repeated 5′UTR also strongly inhibited the accumulation of RNA from the related

ACO

2 gene in flowers, although there is little homology between the 5′UTRs of

ACO

1 and

ACO

2. These results indicate that although repeated DNA in a transgene greatly enhances the probability of gene silencing of an endogenous gene, it also involves generation of a

trans

‐acting silencing signal produced, at least partly, from sequences external to the repeat.
FUSCA3encodes a protein with a conserved VP1/ABI3‐like B3 domain which is of functional importance for the regulation of seed maturation inArabidopsis thaliana
Authors: Luerßen Hartmut , Kirik Viktor , Herrmann Petra , Miséra Simon
Published: March 2003

Conditionally lethal mutant alleles of the

FUSCA3

(

FUS3

) gene of

Arabidopsis thaliana

are specifically defective in the gene expression program responsible for seed maturation.

FUS3

was isolated by map‐based cloning and expression of the

FUS3

cDNA resulted in complementation of the Fus3– phenotype. In the predicted FUS3 gene product, a continuous stretch of more than 100 amino acids shows significant sequence similarity to the B3 domains of the polypeptides encoded by

ABI3

(

Arabidopsis

) and

VP1

(maize).

FUS3

transcription was detected mainly in siliques and was found to be developmentally regulated during embryogenesis. Transcripts of abnormal sizes were observed in

fus3

mutants due to aberrant splicing caused by point mutations at intron termini. Sequence analysis of mutant and wild‐type

FUS3

alleles, as well as sequencing of

fus3

cDNAs, revealed small in‐frame deletions at two different sites of the coding region. While a deletion between B3 and the C‐terminus of the predicted polypeptide was found in conjunction with normal FUS3 function, another deletion located within the conserved B3 domain (as well as truncations therein) were associated with the Fus3– phenotype. It is apparent, therefore, that an intact B3 domain is essential for the regulation of seed maturation by FUS3.
An endosperm‐specific DOF protein from barley, highly conserved in wheat, binds to and activates transcription from the prolamin‐box of a native B‐hordein promoter in barley endosperm
Authors: Mena Montaña , Vicente‐Carbajosa Jesus , Schmidt Robert J. , Carbonero Pilar
Published: March 2003

A cDNA encoding a DNA‐binding protein of the DOF class of transcription factors was isolated from a barley endosperm library. The deduced amino acid sequence for the corresponding protein is 94% identical through the DOF domain to the prolamin‐box (P‐box) binding factor PBF from maize. The gene encoding the barley PBF (BPBF) maps to chromosome 7H, and its expression is restricted to the endosperm where it precedes that of the hordein genes. The BPBF expressed in bacteria as a GST‐fusion binds a P‐box 5′‐TGTAAAG‐3′ containing oligonucleotide derived from the promoter region of an

Hor2

gene. Binding was prevented when the P‐box motif was mutated to 5′‐TGTAgAc‐3′. A P‐box binding activity, present in barley and wheat endosperm nuclei, interacted similarly to BPBF with this synthetic oligonucleotide, and the binding was abolished by 1,10‐phenanthroline. Transient expression experiments in developing barley endosperms demonstrate that BPBF transactivates transcription from the P‐box element of a native

Hor2

promoter and that direct binding of BPBF to its target site is essential for transactivation since mutations in the DOF DNA‐binding domain or in the P‐box motif of this promoter abolished both binding and transactivation. Evidence was also obtained for the presence in wheat of a

Pbf

homologue having similar DNA‐binding properties to that of BPBF. These results strongly implicate this endosperm‐specific DOF protein from barley as an important activator of hordein gene expression and suggest the evolutionary conservation of the

Pbf

gene function among small grain cereals.
Expression of a yeast‐derived invertase in developing cotyledons ofVicia narbonensisalters the carbohydrate state and affects storage functions
Authors: Weber Hans , Heim Ute , Golombek Sabine , Borisjuk Ljudmilla , Manteuffel Renate , Wobus Ulrich
Published: March 2003

In plants the carbohydrate state provides signals to adjust metabolism to specific physiological conditions. Storage‐active sink organs like seeds often contain high levels of sucrose. In order to change the sugar status during seed development a yeast‐derived invertase gene was expressed in

Vicia narbonensis

under control of the LeguminB4 promoter. A signal sequence targeted the invertase to the apoplast in maturing embryos. In the cotyledons, sucrose was decreased whereas hexoses strongly accumulated. There was a major reduction of starch whereas proteins were less affected. Vacuoles of cotyledon cells were enlarged and dry seeds wrinkled. Transcripts and enzyme activity of sucrose synthase, the small and large subunit of ADP‐glucose pyrophosphorylase as well as vicilin were downregulated. Sucrose phosphate synthase and legumin‐mRNAs were not affected. Analysing single seeds with different sucrose levels revealed a positive correlation of sucrose concentration to mRNA levels of sucrose synthase and most pronounced to ADP‐glucose pyrophosphorylase‐mRNA levels as well as to starch content. Glucose on the other hand did not show any correlation. After feeding 14C‐sucrose

in vitro

, the invertase‐expressing cotyledons partitioned less carbon into starch compared to the wild‐type. In the transgenic cotyledons, a relatively higher amount was directed into proteins compared to starch. We conclude that starch accumulation in developing cotyledons could be a function of sucrose concentration. Our results are consistent with a possible sucrose‐mediated induction of storage‐associated differentiation indicated by upregulation of specific genes of the starch synthesis pathway.
Changes in the cytoskeleton accompanying infection‐induced nuclear movements and the hypersensitive response in plant cells invaded by rust fungi
Authors: Skalamera Dubravka , Heath Michèle C.
Published: March 2003

During the infection of cowpea

(Vigna unguiculata)

by the cowpea rust fungus

(Uromyces vignae,

race 1

)

the plant nucleus moves towards and away from the invading hypha and eventually moves close to the fungus in the susceptible cultivar while it remains away in two cultivars which subsequently respond by resistance gene‐dependent plant cell death (the hypersensitive response, HR). The role of plant cytoskeleton in these responses was investigated by fluorescent microscopy and treatments with anticytoskeletal drugs. Observations of microtubule organization prior to cell death revealed that the sequence of events leading to protoplast collapse differed between the two resistant cultivars, suggesting a possibility of multiple pathways for cellular degradation during the HR. Different fixations produced two different microfilament patterns: a filament network and cables. Microfilament network remained visible even at later stages of cell death. Oryzalin and taxol reduced the incidence of autofluorescence that develops late in the death process, indicating a role of microtubules in the deposition of phenolics by adjacent living cells. Cell death and nuclear movements were not affected by oryzalin and taxol but were inhibited by cytochalasin E, suggesting that the microfilaments are required for the HR.
Molecular characterization of CDSP 34, a chloroplastic protein induced by water deficit inSolanum tuberosumL. plants, and regulation ofCDSP 34expression by ABA and high illumination
Authors: Gillet Benjamin , Beyly Audrey , Peltier Gilles , Rey Pascal
Published: March 2003

We previously described the accumulation of a 34 kDa thylakoid protein, named CDSP 34 for chloroplastic drought‐induced stress protein, in

Solanum tuberosum

plants subjected to water deficit. A full‐length CDSP 34 cDNA has been isolated and we report here that mature CDSP 34 is highly similar to two chromoplastic proteins, fibrillin from

Capsicum annuum and

CHRC (for chromoplast protein C) from

Cucumis sativus

, components of carotenoid‐accumulating structures. Northern and Western analyses showed that both CDSP 34 transcript and protein accumulated from early stages of water deficit. In water‐stressed tomato plants, similar increases in the CDSP 34‐related transcript amount were noticed in wild‐type and ABA‐deficient

flacca

mutant, but protein accumulation was observed only in wild‐type, suggesting a post‐transcriptional role of ABA in CDSP 34 synthesis regulation. Substantial increases in CDSP 34 transcript and protein abundances were also observed in potato plants subjected to high illumination. The CDSP 34 protein is proposed to play a structural role in stabilizing stromal lamellae thylakoids upon osmotic or oxidative stress.
Auxin and ethylene promote root hair elongation inArabidopsis
Authors: Pitts R. Jason , Cernac Alex , Estelle Mark
Published: March 2003

Genetic and physiological studies implicate the phytohormones auxin and ethylene in root hair development. To learn more about the role of these compounds, we have examined the root hair phenotype of a number of auxin‐ and ethylene‐related mutants. In a previous study,

Masucci & Schiefelbein (1996

) showed that neither the auxin response mutations

aux1 and axr1

nor the ethylene response mutations

etr1 and ein2

have a significant effect on root hair initiation. In this study, we found that mutants deficient in either auxin or ethylene response have a pronounced effect on root hair length. Treatment of wild‐type,

axr1

and

etr1

seedlings with the synthetic auxin, 2,4‐D, or the ethylene precursor ACC, led to the development of longer root hairs than untreated seedlings. Furthermore,

axr1

seedlings grown in the presence of ACC produce ectopic root hairs and an unusual pattern of long root hairs followed by regions that completely lack root hairs. These studies indicate that both auxin and ethylene are required for normal root hair elongation.
A combined reduction in activity of starch synthases II and III of potato has novel effects on the starch of tubers
Authors: Edwards Anne , Fulton Daniel C.. , Hylton Christopher M.. , Jobling Stephen A.. , Gidley Michael , Rössner Ute , Martin Cathie , Smith Alison M..
Published: March 2003

A chimeric antisense construct has been used to generate transgenic potatoes (

Solanum tuberosum

L.) in which activities of both of the main starch synthases responsible for amylopectin synthesis in the tuber (SSII and SSIII) are reduced. The properties of starch from tubers of these plants have been compared with those of starches from transgenic plants in which activity of either SSII or SSIII has been reduced. Starches from the three types of transgenic plant are qualitatively different from each other and from the starch of control plants with unaltered starch synthase activities, with respect to granule morphology, the branch lengths of amylopectin, and the gelatinisation behaviour analysed by viscometry. The effects of reducing SSII and SSIII together cannot be predicted from consideration of the effects of reducing these two isoforms individually. These results indicate that different isoforms of starch synthase make distinct contributions to the synthesis of amylopectin, and that they act in a synergistic manner, rather than independently, during amylopectin synthesis.
Site‐directed mutagenesis of serine 158 demonstrates its role in spinach leaf sucrose‐phosphate synthase modulation
Authors: Toroser Dikran , McMichael Robert , Krause Klause‐Peter , Kurreck Jens , Sonnewald Uwe , Stitt Mark , Huber Steven C.
Published: March 2003

Site‐directed mutagenesis of spinach sucrose‐phosphate synthase (SPS) was performed to investigate the role of Ser158 in the modulation of spinach leaf SPS. Tobacco plants expressing the spinach wild‐type (WT), S158A, S158T and S157F/S158E SPS transgenes were produced. Expression of transgenes appeared not to reduce expression of the tobacco host SPS. SPS activity in the WT and the S158T SPS transgenics showed light/dark modulation, whereas the S158A and S157F/S158E mutants were not similarly light/dark modulated: the S158A mutant enzyme was not inactivated in the dark, and the S157F/S158E was not activated in the light. The inability to modulate the activity of the S158A mutant enzyme by protein phosphorylation was demonstrated

in vitro

. The WT spinach enzyme immunopurified from dark transgenic tobacco leaves had a low initial activation state, and could be activated by PP2A and subsequently inactivated by SPS‐kinase plus ATP. Rapid purification of the S158A mutant enzyme from dark leaves of transgenic plants using spinach‐specific monoclonal antibodies yielded enzyme that had a high initial activation state, and pre‐incubation with leaf PP2A or ATP plus SPS‐kinase (the PKIII enzyme) caused little modulation of activity. The results demonstrate the regulatory significance of Ser158 as the major site responsible for dark inactivation of spinach SPS

in vivo

, and indicate that the significance of phosphorylation is the introduction of a negative charge at the Ser158 position.
Changes in aldolase activity in wild‐type potato plants are important for acclimation to growth irradiance and carbon dioxide concentration, because plastid aldolase exerts control over the ambient rate of photosynthesis across a range of growth conditions
Authors: Haake Volker , Geiger Michael , Walch‐Liu Pia , Of Engels Christ , Zrenner Rita , Stitt Mark
Published: March 2003

Even though plastid aldolase catalyses a reversible reaction, does not possess properties allowing it to contribute to ‘fine’ regulation, and would therefore be considered unimportant for the control of metabolism and growth, antisense transformants with a 50–70% decrease in aldolase activity showed an inhibition of photosynthesis and growth. We now show that acclimation of photosynthesis to growth conditions includes and requires changes in plastid aldolase activity. Wild‐type potato plants and transformants were grown at low irradiance (70 μmol m–2 sec–1), and at high irradiance (390 μmol m–2 sec–1) at 400 or 800 p.p.m. carbon dioxide. (i) Ambient photosynthesis was always inhibited by a 30–40% decrease of aldolase activity, the strongest inhibition being observed when plants were growing in high irradiance and elevated carbon dioxide. (ii) The inhibition was due to a low rate of ribulose‐1,5‐bisphosphate regeneration in low light, exacerbated by an inadequate rate of starch synthesis in high light and elevated carbon dioxide. Decreased expression of aldolase in antisense transformants was also accompanied by a decrease of fructose‐1,6‐bisphosphatase protein and activity, and Rubisco activity. Transcript levels for the plastid fructose‐1,6‐bisphosphatase and the small subunit of Rubisco did not decrease. (iii) In wild‐type plants, increasing the growth irradiance from 70 to 390 μmol m–2 sec–1 led to a 15–95% increase of the activity of eight Calvin cycle enzymes, and increasing the carbon dioxide concentration from 400 to 800 p.p.m. led to a 5–35% decrease of these enzyme activities. The largest changes occurred for aldolase, and for transketolase which also catalyses a reversible reaction and is not subject to ‘fine’ regulation.
A role for glutathione transferases functioning as glutathione peroxidases in resistance to multiple herbicides in black‐grass
Authors: Cummins Ian , Cole David J. , Edwards Robert
Published: March 2003

Black‐grass (Alopecurus myosuroides) is a major weed of wheat in Europe, with several populations having acquired resistance to multiple herbicides of differing modes of action. As compared with herbicide‐susceptible black‐grass, populations showing herbicide cross‐resistance contained greatly elevated levels of a specific type I glutathione transferase (GST), termed AmGST2, but similar levels of a type III GST termed AmGST1. Following cloning and expression of the respective cDNAs, AmGST2 differed from AmGST1 in showing limited activity in detoxifying herbicides but high activities as a glutathione peroxidase (GPOX) capable of reducing organic hydroperoxides. In contrast to AmGST2, other GPOXs were not enhanced in the herbicide‐resistant populations. Treatment with a range of herbicides used to control grass weeds in wheat resulted in increased levels of hydroperoxides in herbicide‐susceptible populations but not in herbicide‐resistant plants, consistent with AmGST2 functioning to prevent oxidative injury caused as a primary or secondary effect of herbicide action. Increased AmGST2 expression in black‐grass was associated with partial tolerance to the peroxidizing herbicide paraquat. The selective enhancement of AmGST2 expression resulted from a constitutively high expression of the respective gene, which was activated in herbicide‐susceptible black‐grass in response to herbicide safeners, dehydration and chemical treatments imposing oxidative stress. Our results provide strong evidence that GSTs can contribute to resistance to multiple herbicides by playing a role in oxidative stress tolerance in addition to detoxifying herbicides by catalysing their conjugation with glutathione.