bluuebunny/crossref_metadata_2025_split huggingface dataset:
Antisense inhibition of the GDP‐mannose pyrophosphorylase reduces the ascorbate content in transgenic plants leading to developmental changes during senescence
Authors: Keller Ruth , Renz Franziska Springer andreas , Kossmann Jens 
Published: March 2003

SummaryGDP‐mannose pyrophosphorylase (GMPase, EC 2.7.7.22) catalyses the synthesis of GDP‐D‐mannose and represents the first committed step in the formation of all guanosin‐containing sugar nucleotides found in plants which are precursors for cell wall biosynthesis and, probably more important, the synthesis of ascorbate. A full‐length cDNA encoding GMPase from S. tuberosum was isolated. Transgenic potato plants were generated in which the GMPase cDNA was introduced in antisense orientation to the 35S promoter. Transformants with reduced GMPase activity were selected. Transgenic plants were indistinguishable from the wild‐type when held under tissue culture conditions, however, a major change was seen 10 weeks after transfer into soil. Transgenic plants showed dark spots on leaf veins and stems with this phenotype developing from the bottom to the top of the plant. In case of the line with the strongest reduction, all aerial parts finally dried out after 3 months in soil, in contrast to the wild‐type plants which did not start to senesce at this time. This coincides with a reduction of ascorbate contents in the transgenic plants, which is in agreement with the recently proposed pathway of ascorbate biosynthesis. Furthermore, leaf cell walls of the transgenic potato plants had mannose contents that were reduced to 30–50% of the wild‐type levels, whereas the composition of tuber cell walls was unchanged. The glycosylation pattern of proteins was unaffected by GMPase inhibition, as studied by affinoblot analysis.
Isolation and characterization of a flavonoid 3′‐hydroxylase cDNA clone corresponding to the Ht1 locus of Petunia hybrida
Authors: Brugliera Filippa , Barri‐Rewell Gina , Holton Timothy A. , Mason John G. 
Published: March 2003

SummaryWe have isolated a cDNA clone that corresponds to the Ht1 locus of petunia which controls the hydroxylation of dihydrokaempferol to dihydroquercetin and of naringenin to eriodictyol by the action of flavonoid 3′‐hydroxylase (F3′H). The cDNA encodes a 512 amino acid polypeptide with regions of similarity to petunia flavonoid 3′,5′‐hydroxylases (F3′5′H). Both F3′H and F3′5′H are cytochromes P450 and are key enzymes in the flavonoid pathway leading to the production of the coloured anthocyanins. The F3 ′H transcript is most abundant in petals from flowers at an early stage of development and levels decline as the flower matures. Transcripts are also detected in the ovaries, sepals, peduncles, stems and anthers of the petunia plant. No or very reduced levels of transcripts are detected in ht1/ht1 lines. This is the first report of isolation of a F3 ′H cDNA clone from any species.
In vitro synthesis and properties of pectin/Acetobacter xylinus cellulose composites
Authors: Chanliaud Elisabeth , Gidley Michael J. 
Published: March 2003

SummaryPectin and cellulose are major components of most primary cell walls, yet little is known about the way in which they interact either during assembly or in subsequent functional performance of the wall. As a mimic of cell wall assembly, we studied the formation of molecular composites formed by deposition of cellulose from Acetobacter xylinus into pectin/calcium systems, and the molecular, architectural and mechanical properties of the composites obtained. The formation of interpenetrating cellulose/pectin composite networks (as envisaged in current models for primary cell walls) required a pre‐existing, but not too strong, pectin network. For pectin either in solution or strongly networked, phase separation from cellulose occurred, providing two physical models for the formation of middle lamellae. Composite networks showed no evidence of direct molecular interaction between the components, but pectin networks became more aggregated following deposition of cellulose into them. The shear strength under small deformation conditions for cellulose/pectin composites was very similar to that of cellulose alone. In contrast, under uniaxial tension, extensibility was greatly increased and stiffness decreased. These major changes were due to the effect of pectin on cellulose network architecture at deposition, as they were maintained upon removal of the pectin component. These results show that the presence and physical state of pectin at the time of cellulose deposition in muro may be a significant determinant of subsequent extensibility without compromising strength.
Polyhydroxyalkanoate synthesis in transgenic plants as a new tool to study carbon flow through β‐oxidation
Authors: Mittendorf Volker , Bongcam Vanessa , Allenbach Laure , Coullerez Géraldine , Martini Nobert , Poirier Yves 
Published: March 2003

SummaryTransgenic plants producing peroxisomal polyhydroxy‐ alkanoate (PHA) from intermediates of fatty acid degradation were used to study carbon flow through the β‐oxidation cycle. Growth of transgenic plants in media containing fatty acids conjugated to Tween detergents resulted in an increased accumulation of PHA and incorporation into the polyester of monomers derived from the β‐oxidation of these fatty acids. Tween–laurate was a stronger inducer of β‐oxidation, as measured by acyl‐CoA oxidase activity, and a more potent modulator of PHA quantity and monomer composition than Tween–oleate. Plants co‐expressing a peroxisomal PHA synthase with a capryl‐acyl carrier protein thioesterase from Cuphea lanceolata produced eightfold more PHA compared to plants expressing only the PHA synthase. PHA produced in double transgenic plants contained mainly saturated monomers ranging from 6 to 10 carbons, indicating an enhanced flow of capric acid towards β‐oxidation. Together, these results support the hypothesis that plant cells have mechanisms which sense levels of free or esterified unusual fatty acids, resulting in changes in the activity of the β‐oxidation cycle as well as removal and degradation of these unusual fatty acids through β‐oxidation. Such enhanced flow of fatty acids through β‐oxidation can be utilized to modulate the amount and composition of PHA produced in transgenic plants. Furthermore, synthesis of PHAs in plants can be used as a new tool to study the quality and relative quantity of the carbon flow through β‐oxidation as well as to analyse the degradation pathway of unusual fatty acids.
Molecular cloning, functional expression and characterisation of RCC reductase involved in chlorophyll catabolism
Authors: Wüthrich Karin L. , Bovet Lucien , Hunziker Peter E. , Donnison Iain S. , Hörtensteiner Stefan 
Published: March 2003

SummaryRed chlorophyll catabolite (RCC) reductase (RCCR) and pheophorbide (Pheide) a oxygenase (PaO) catalyse the key reaction of chlorophyll catabolism, porphyrin macrocycle cleavage of Pheide a to a primary fluorescent catabolite (pFCC). RCCR was purified from barley and a partial gene sequence was cloned (pHvRCCR). The gene was expressed at all stages of leaf development and in roots. By comparison with different databases, genomic sequences and expressed sequence tags similar to RCCR were found in phylogenetically diverse species, and activity of RCCR was demonstrated in two of them, Arabidopsis thaliana and Marchantia polymorpha. The gene of A. thaliana (AtRCCR) was employed for molecular cloning, heterologous expression and the production of polyclonal antibodies. With recombinant RCCR, the major product of RCC reduction was pFCC‐1, but small quantities of its C1 epimer, pFCC‐2, also accumulated. The reaction required reduced ferredoxin and was sensitive to oxygen. AtRCCR encoded a 35 kDa protein which was used for chloroplast import experiments. Upon transport, it was processed to a mature form of 31 kDa. The significance of cloning of RCCR is discussed in respect to the evolution of chlorophyll catabolism and to the cloning of PaO.
Activation of a diverse set of genes during the tobacco resistance response to TMV is independent of salicylic acid; induction of a subset is also ethylene independent
Authors: Guo Ailan , Salih Gaza , Klessig Daniel F. 
Published: March 2003

SummaryThrough differential screening of a cDNA library, we cloned six groups of genes that are expressed relatively early in the inoculated leaves of tobacco resisting infection by tobacco mosaic virus (TMV). Induction of all these genes was subsequently detected in the uninoculated leaves; thus, their expression is associated with the development of both local and systemic acquired resistance. Exogenously applied salicylic acid (SA) was observed to induce these genes transiently. However, analyses with transgenic NahG plants, which are unable to accumulate SA, demonstrated that expression of these genes in TMV‐inoculated leaves is mediated via an SA‐independent pathway. Because the expression kinetics of these genes differ from those associated with the well‐characterized pathogenesis‐related protein (PR‐1) and phenylalanine ammonia‐lyase (PAL) genes, we propose that they belong to a group which we designate SIS, for SA‐independent, systemically induced genes. Interestingly, the expression of several SIS genes in the uninoculated leaves of TMV‐infected NahG plants was delayed and/or reduced, raising the possibility that SA is involved in activating some of these genes in systemic tissue. Most of the SIS genes were induced by exogenous ethylene. However, analyses of infected NahG plants treated with ethylene action and/or synthesis inhibitors indicated that the TMV‐induced expression of several SIS genes is independent of ethylene as well as SA.
Three ethylene‐responsive transcription factors in tobacco with distinct transactivation functions
Authors: Ohta Masaru , Ohme‐Takagi Masaru , Shinshi Hideaki 
Published: March 2003

SummaryEthylene‐responsive factors (ERFs) have conserved DNA‐binding domains and interact directly with the GCC box in the ethylene‐responsive element that is necessary and sufficient for the regulation of transcription by ethylene. ERFs were shown to be localized to nucleus in transient transfection experiments. Transient expression assays using tobacco protoplasts and a heterologous system in yeast were used to examine the transactivation functions of ERFs. ERF2 and ERF4 enhanced the GCC box‐mediated transcription of a reporter gene in tobacco protoplasts. When fused to the DNA‐binding domain of yeast GAL4, a carboxy‐terminal region of ERF2, as well as both amino‐terminal and carboxy‐terminal regions of ERF4, functioned as a transactivation domain in tobacco protoplasts. The amino‐terminal regions of ERF2 and ERF4 functioned as transactivation domains in yeast. In contrast to ERF2 and ERF4, ERF3 reduced the transcription of the reporter gene in tobacco protoplasts, indicating that ERF3 functions as a repressor. Thus, it appears that ERFs exert their regulatory functions in different ways, with ERF2 and ERF4 being activators and ERF3 being a repressor of transcription.
Temporal and spatial regulation of pectic (1→4)‐β‐D‐galactan in cell walls of developing pea cotyledons: implications for mechanical properties
Authors: McCartney Lesley , Ormerod Andrew P. , Gidley Michael J. , Knox J. Paul 
Published: March 2003

SummaryModifications in cell wall pectic polysaccharides are thought to influence cell–cell adhesion and the mechanical properties of plant tissues. Monoclonal antibodies to epitopes occurring in homo‐ galacturonan and side chains of rhamnogalacturonan I have been used in an immunolocalization study of cell wall architecture of developing pea cotyledons. Pectic (1→4)‐β‐ d‐galactan appears in cotyledon cell walls at a defined stage late in development (approximately 26–30 days after anthesis), whereas homogalacturonan and pectic (1→5)‐α‐
l‐arabinan are present in cotyledon cell walls throughout development. (1→4)‐β‐galactan was restricted to a distinct thin layer at the plasma membrane face of the cell wall. Anion exchange and immunoaffinity chromatography indicated that the (1→4)‐β‐galactan was associated with acidic pectic components. Mechanical compressive testing of pea cotyledons, before and after (1→4)‐β‐galactan appearance, indicated that the cotyledons with the galactan‐rich cell wall layer were twice as firm as those with no detectable (1→4)‐β‐galactan.
Decreased and increased expression of the subunit CHL I diminishes Mg chelatase activity and reduces chlorophyll synthesis in transgenic tobacco plants
Authors: Papenbrock Jutta , Pfündel Erhard , Mock Hans‐Peter , Grimm Bernhard 
Published: March 2003

SummaryThe chelation of Fe2+ and Mg2+ ions forms protoheme IX and Mg‐protoporphyrin IX, respectively, and the latter is an intermediate in chlorophyll synthesis. Active magnesium protoporphyrin IX chelatase (Mg‐chelatase) is an enzyme complex consisting of three different subunits. To investigate the function of the CHL I subunit of Mg‐chelatase and the effects of modified Mg‐chelatase activity on the tetrapyrrole biosynthetic pathway, we characterized N. tabacum transformants carrying gene constructs with the Chl I cDNA sequence in antisense and sense orientation under the control of the CaMV 35S promoter. Both elevated and diminished levels of Chl I mRNA and Chl I protein led to reduced Mg‐chelatase activities, reflecting a perturbation of the assembly of the enzyme complex. The transformed plants did not accumulate the substrate of Mg‐chelatase, protoporphyrin IX, but the leaves contained less chlorophyll and possessed increased chlorophyll a/b ratios, as well as a deficiency of light‐harvesting chlorophyll binding proteins of photosystems I and II. The expression and activity of several tetrapyrrolic enzymes were reduced in parallel to lower the Mg‐chelatase activity. Consistent with the lower chlorophyll contents, the rate‐limiting synthesis of 5‐aminolevulinate was also decreased in the transgenic lines analyzed. The consequence of reduced Mg‐chelatase on early and late steps of chlorophyll synthesis, and on the organization of light harvesting complexes is discussed.
A pollen tube growth‐promoting arabinogalactan protein from Nicotiana alata is similar to the tobacco TTS protein
Authors: Wu Hen‐ming , Wong Eric , Ogdahl Justyne , Cheung Alice Y. 
Published: March 2003

SummaryUpon germination on the stigma, pollen tubes elongate in the stylar transmitting tract, aided by female factors, with speed and directionality not mimicked in in vitro pollen tube growth cultures. We have shown that a stylar transmitting tissue arabinogalactan protein (AGP) from Nicotiana tabacum (tobacco), TTS protein, stimulates pollen tube growth in vivo and in vitro and attracts pollen tubes grown in a semi‐in vivo culture system. It has been reported that the self‐incompatible Nicotiana alata produced a stylar glycoprotein, GaRSGP, which had a backbone polypeptide that shared 97% identity with those of TTS proteins but some of its properties were different from those described for TTS proteins. We report here the characterization of a family of stylar transmitting tissue glycoproteins from N. alata that is virtually identical to tobacco TTS proteins and which we refer to as NaTTS proteins. Like their tobacco counterparts, NaTTS proteins are recognized by the traditional AGP‐diagnostic reagent β‐glucosyl Yariv reagent, and they are also recognized by JIM13, a monoclonal antibody against AGP. NaTTS proteins also stimulate pollen tube elongation in vitro and attract pollen tubes in a semi‐in vivo pollen tube culture system. Biochemical and immunological characterization of NaTTS proteins revealed that they have extraordinary variability in the extent of sugar modifications of their polypeptide backbones. The extent of sugar modifications on NaTTS proteins significantly affects their biochemical properties, influences how they interact with the transmitting tissue extracellular matrix, and affects their solubility from this matrix. Our results suggest that the strategy used to purify GaRSGP only recovered a less glycosylated, more tightly extracellular matrix‐bound sub‐population of the entire spectrum of N. alata TTS proteins.
Complex structure of a maize Myb gene promoter: functional analysis in transgenic plants
Authors: Sidorenko Lyudmila V. , Li Xianggan , Cocciolone Suzy M. , Chopra Surinder , Tagliani Laura , Bowen Ben , Daniels Michael , Peterson Thomas 
Published: March 2003

SummaryThe maize P gene encodes a Myb‐like transcription factor that regulates synthesis of red flavonoid pigments in floral organs. To study the transcriptional regulation of the P gene, candidate regulatory sequences of the P1‐rr gene promoter were identified by Ac insertional mutagenesis and subjected to functional testing in transgenic maize plants. The results indicate that a 561 bp fragment (Pb) encompassing the transcription start site (−235 to +326) supports weak expression of a GUS reporter gene in floral organs, including husk, silk, kernel pericarp, cob and male inflorescence. Two other fragments, located approximately 1 and 5 kb 5′ of the transcription start site, increased the levels of GUS activity in floral tissues and thus appear to contain enhancer elements. All of the tested constructs gave similar patterns of GUS expression, suggesting that the 561 bp Pb fragment that is common among the transgene constructs contains regulatory elements that promote activation in floral organs. The basal promoter and proximal enhancer fragments contain putative binding sites for bZip regulatory factors, and a complex arrangement of palindromes including a large inverted repeat of two tRNA‐like genes. Possibly, interconversions between linear and cruciform conformations of the palindromes may affect protein/DNA interactions and thereby modulate P1‐rr expression.
Transformation of Medicago truncatula via infiltration of seedlings or flowering plants with Agrobacterium
Authors: Trieu Anthony T. , Burleigh Stephen H. , Kardailsky Igor V. , Maldonado‐Mendoza Ignacio E. , Versaw Wayne K. , Blaylock Laura A. , Shin Heungsop , Chiou Tzyy‐Jen , Katagi Hiroaki , Dewbre Gary R. , Weigel Detlef , Harrison Maria J. 
Published: March 2003

SummaryTwo rapid and simple in planta transformation methods have been developed for the model legume Medicago truncatula. The first approach is based on a method developed for transformation of Arabidopsis thaliana and involves infiltration of flowering plants with a suspension of Agrobacterium. The second method involves infiltration of young seedlings with Agrobacterium. In both cases a proportion of the progeny of the infiltrated plants is transformed. The transformation frequency ranges from 4.7 to 76% for the flower infiltration method, and from 2.9 to 27.6% for the seedling infiltration method. Both procedures resulted in a mixture of independent transformants and sibling transformants. The transformants were genetically stable, and analysis of the T2 generation indicates that the transgenes are inherited in a Mendelian fashion. These transformation systems will increase the utility of M. truncatula as a model system and enable large‐scale insertional mutagenesis. T‐DNA tagging and the many adaptations of this approach provide a wide range of opportunities for the analysis of the unique aspects of legumes.
Sense transformation reveals a novel role for class I β‐1,3‐glucanase in tobacco seed germination
Authors: Leubner‐Metzger Gerhard , Meins Frederick 
Published: March 2003

Summary‘Coat‐enhanced’ seed dormancy of many dicotyledonous species, including tobacco, is released during after‐ripening. Rupture of the endosperm, which is the limiting step in tobacco seed germination, is preceded by induction of class I β‐1,3‐glucanase (βGLU I) in the micropylar endosperm where the radicle will penetrate. Treating after‐ripened tobacco seeds with abscisic acid (ABA) delays endosperm rupture and inhibits βGLU I induction. Sense transformation with a chimeric ABA‐inducible βGLU I transgene resulted in over‐expression of βGLU I in seeds and promoted endosperm rupture of mature seeds and of ABA‐treated after‐ripened seeds. Taken together, these results provide direct evidence that βGLU I contributes to endosperm rupture. Over‐expression of βGLU I during germination also replaced the effects of after‐ripening on endosperm rupture. This suggests that regulation of βGLU I by ABA signalling pathways might have a key role in after‐ripening.
A functional cloning strategy, based on a binary PVX‐expression vector, to isolate HR‐inducing cDNAs of plant pathogens
Authors: Takken Frank L.W. , Luderer Rianne , Gabriëls Suzan H.E.J. , Westerink Nienke , Lu Riu , De Wit Pierre J.G.M. , Joosten Matthieu H.A.J. 
Published: March 2003

SummaryWe have devised a novel, high‐throughput functional cloning method to isolate cDNAs from plant pathogens of which the products elicit a hypersensitive response (HR) in plants. Copy DNA, made from RNA isolated from the tomato pathogen Cladosporium fulvum grown under nutrient‐limiting conditions in vitro, was cloned into a binary, potato virus X (PVX)‐based expression vector and transformed to Agrobacterium tumefaciens. 9600 colonies were individually toothpick‐inoculated onto leaflets of tomato plants resistant to C. fulvum. Four cDNAs were identified whose expression induced formation of a necrotic lesion around the inoculation site. One of these clones, specifically inducing HR on tomato plants carrying the Cf‐4 resistance gene, encodes race‐specific elicitor AVR4. The other three cDNAs, inducing a non‐genotype‐specific HR, encode a protein highly homologous to bZIP, basic transcription factors. To determine whether this approach has general applicability, part of the library was also inoculated onto Nicotiana tabacum var. Samsun NN, which is not a host for C. fulvum. Four independent HR‐inducing cDNAs were identified which all encode ECP2, an extracellular protein of C. fulvum known to induce necrosis in certain Nicotiana species. These observations confirm that this functional screening method is a versatile strategy to identify cDNAs of pathogens that encode (race‐specific) elicitors and other HR‐inducing proteins.
Functional characterization of the EMCV IRES in plants
Authors: Urwin Peter , Yi Li , Martin Helen , Atkinson Howard , Gilmartin Philip M. 
Published: March 2003

SummaryThe translation of eukaryotic messenger RNA is typically dependent upon the presence of an m7GpppN cap structure at the 5′ end of the transcript. However, several animal viruses, including the Picorna viruses, have been shown to exhibit cap‐independent translation through the presence of an internal ribosome entry site or IRES. This IRES‐mediated cap‐independent internal translation initiation has been exploited to generate bicistronic transcripts that function in animal cells. Recently IRES elements have also been identified in a small number of vertebrate, insect and yeast cellular messenger RNAs although no such sequences have been identified in endogenous plant genes and there are no reports of animal virus derived IRES activity in plant cells. Here we have constructed a bicistronic gene containing both green fluorescent protein and luciferase open‐reading frames separated by the encephalomyocarditis IRES element under the control of the CaMV 35S promoter. Northern analysis reveals expression of the bicistronic transcript and in vivo imaging of GFP and luciferase activities demonstrates the functional presence of both proteins. Western blot analysis confirms the independent translation of both reporter proteins. These data suggest that insertion of the encephalomyocarditis virus (EMCV) IRES element between two open‐reading frames of a plant bicistronic transcript can mediate translation of the second open‐reading frame. This activity is more apparent in the leaves, than in the roots, of transgenic seedlings carrying the bicistronic reporter gene construct.