bluuebunny/crossref_metadata_2025 huggingface dataset:
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

We 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

Pectin 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

Transgenic 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

Red 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

Through 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

Ethylene‐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

Modifications 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

The 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

Upon 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

The 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

Two 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

‘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

We 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

The 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.
Novel auxin transport inhibitors phenocopy the auxin influx carrier mutation aux1
Authors: Parry Geraint , Delbarre Alain , Marchant Alan , Swarup Ranjan , Napier Richard , Perrot‐Rechenmann Catherine , Bennett Malcolm J.
Published: March 2003

The hormone auxin is transported in plants through the combined actions of diffusion and specific auxin influx and efflux carriers. In contrast to auxin efflux, for which there are well documented inhibitors, understanding the developmental roles of carrier‐mediated auxin influx has been hampered by the absence of specific competitive inhibitors. However, several molecules that inhibit auxin influx in cultured cells have been described recently. The physiological effects of two of these novel influx carrier inhibitors, 1‐naphthoxyacetic acid (1‐NOA) and 3‐chloro‐4‐hydroxyphenylacetic acid (CHPAA), have been investigated in intact seedlings and tissue segments using classical and new auxin transport bioassays. Both molecules do disrupt root gravitropism, which is a developmental process requiring rapid auxin redistribution. Furthermore, the auxin‐insensitive and agravitropic root‐growth characteristics of aux1 plants were phenocopied by 1‐NOA and CHPAA. Similarly, the agravitropic phenotype of inhibitor‐treated seedlings was rescued by the auxin 1‐naphthaleneacetic acid, but not by 2,4‐dichlorophenoxyacetic acid, again resembling the relative abilities of these two auxins to rescue the phenotype of aux1. Further investigations have shown that none of these compounds block polar auxin transport, and that CHPAA exhibits some auxin‐like activity at high concentrations. Whilst results indicate that 1‐NOA and CHPAA represent useful tools for physiological studies addressing the role of auxin influx in planta, 1‐NOA is likely to prove the more useful of the two compounds.
Quinone oxidoreductase message levels are differentially regulated in parasitic and non‐parasitic plants exposed to allelopathic quinones
Authors: Matvienko Marta , Wojtowicz Angela , Wrobel Russell , Jamison Denneal , Goldwasser Yaakov , Yoder John I.
Published: March 2003

Allelopathic chemicals released by plants into the rhizosphere have effects on neighboring plants ranging from phytoxicity to inducing organogenesis. The allelopathic activity of naturally occurring quinones and phenols is primarily a function of reactive radicals generated during redox cycling between quinone and hydroquinone states. We isolated cDNAs encoding two distinct quinone oxidoreductases from roots of the parasitic plant Triphysaria treated with the allelopathic quinone 2,6‐dimethoxybenzoquinone (DMBQ). TvQR1 is a member of the ζ‐crystallin quinone oxidoreductase family that catalyzes one‐electron quinone reductions, generating free radical semiquinones. TvQR2 belongs to a family of detoxifying quinone oxidoreductases that catalyze bivalent redox reactions which avoid the radical intermediate. TvQR1 and TvQR2 message levels are rapidly upregulated in Triphysaria roots as a primary response to treatment with various allelopathic quinones. Inhibition of quinone oxidoreductase enzymatic activity with dicumarol prior to quinone treatment resulted in increased transcript levels. While TvQR2 homologs were upregulated by DMBQ in roots of all plants examined, TvQR1 homologs were upregulated only in roots of parasitic plants. Phylogenetic trees constructed of TvQR1 and TvQR2 protein homologs in Archea, Eubacteria and Eukaryotes indicated that both gene families are ancient, yet the families have dissimilar evolutionary histories in angiosperms. We hypothesize that TvQR2‐like proteins function to detoxify allelopathic quinones in the rhizosphere, while TvQR1 has specific functions associated with haustorium development in parasitic plants.
Evidence for the plant‐specific intercellular transport of the Arabidopsis copper chaperone CCH
Authors: Mira Helena , Martínez‐García Fernando , Peñarrubia Lola
Published: March 2003

Arabidopsis copper chaperone (CCH) belongs to a family of eukaryotic proteins that participates in intracellular copper homeostasis by delivering this metal to the secretory pathway. In this work we show that the CCH protein is mainly located along the vascular bundles of senescing leaves and petioles, as shown by tissue prints and immunohistochemical detection. CCH protein also accumulates in stem sieve elements and is collected in phloem exudates. Accordingly, Arabidopsis CCH is the only member of the metallochaperone family described to function intercellularly to date. Moreover, the CCH protein remains stable when plants are subjected to excess copper that causes a rapid and specific decrease in its mRNA. These facts point to a role for CCH in copper mobilization from decaying organs towards reproductive structures, as a result of metalloprotein breakdown.
A potato tuber‐expressed mRNA with homology to steroid dehydrogenases affects gibberellin levels and plant development
Authors: Bachem Christian W.B. , Horvath Beatrix , Trindade Luisa , Claassens Margo , Davelaar Evert , Jordi Wilco , Visser Richard G. F.
Published: March 2003

Using cDNA‐AFLP RNA fingerprinting throughout potato tuber development, we have isolated a transcript‐derived fragment (TDF511) with strong homology to plant steroid dehydrogenases. During in vitro tuberization, the abundance profile of the TDF shows close correlation to the process of tuber formation. However, when tuberization is inhibited by the addition of gibberellins (GAs) to the growth medium, the appearance of TDF511 in the fingerprint is delayed, then steadily increases in intensity during later stages of development. TDF511 was used to isolate the corresponding cDNA (CB12). The DNA and deduced amino‐acid sequences of the cDNA show high homology to a fruit‐ripening gene from tomato, a series of steroid dehydrogenases, and the maize Ts2 gene. A section of the cDNA was cloned in antisense orientation behind a 35S CaMV promoter and transformed into potato. Transgenic plants expressing the antisense gene showed significantly earlier emergence, an increase in height, and longer tuber shape. In vitro tuberization experiments reveal extended stolon lengths in comparison to the controls. The analysis of endogenous GA levels showed that the transgenic antisense plants have elevated levels of biologically active GAs and their respective precursors. We propose that this gene plays a role in the metabolism of plant‐growth substances important for tuber life cycle and plant development.
Collateral gene expression changes induced by distinct plant viruses during the hypersensitive resistance reaction in Chenopodium amaranticolor
Authors: Cooper Bret
Published: March 2003

Hypersensitive reactions to plant diseases are typically mediated by R genes. Many R genes that have been cloned only confer resistance to a particular pathogen. However, Chenopodium spp. have multivirus hypersensitive resistance, thus making the understanding of this broad‐spectrum resistance mechanism attractive. Using tobacco mosaic virus (TMV) tagged with green fluorescent protein to follow infection over time, cDNA‐AFLP to find genes up‐regulated during virus infection in C. amaranticolor and quantitative RT–PCR to accurately measure gene expression at different time points, the first dissection of this significant defense response pathway is presented. The detected disease‐expressed sequences in C. amaranticolor (DESCA) are similar to those that encode p450 monooxegenases, hypersensitivity‐related genes, cellulases, ABC transporters, receptor‐like kinases, serine/threonine kinases, phosphoribosylanthranilate transferases and hypothetical R genes, many of which are associated with pathogen defense in other plants. The expressions of these DESCA genes are also induced by infection with the taxonomically distinct tobacco rattle virus (TRV) in C. amaranticolor. In particular, DESCA1, one of the gene fragments from C. amaranticolor that lacks similarity to any other sequence in the GenBank database, is induced at least 200 fold 4 d after infection (dai) by both TMV and TRV. The potential role of DESCA genes in a C. amaranticolor multivirus defense response with regard to their levels and time of gene expression is discussed.
A deficiency of coproporphyrinogen III oxidase causes lesion formation in Arabidopsis
Authors: Ishikawa Atsushi , Okamoto Hiroki , Iwasaki Yukimoto , Asahi Tadashi
Published: March 2003

We isolated an Arabidopsis lesion initiation 2 (lin2) mutant, which develops lesion formation on leaves and siliques in a developmentally regulated and light‐dependent manner. The phenotype of the lin2 plants resulted from a single nuclear recessive mutation, and LIN2 was isolated by a T‐DNA tagging approach. LIN2 encodes coproporphyrinogen III oxidase, a key enzyme in the biosynthetic pathway of chlorophyll and heme, a tetrapyrrole pathway, in Arabidopsis. The lin2 plants express cytological and molecular markers associated with the defense responses, usually activated by pathogen infection. These results demonstrate that a porphyrin pathway impairment is responsible for the lesion initiation phenotype, which leads to the activation of defense responses, in Arabidopsis. Lesion formation was not suppressed, and was even enhanced when accumulation of salicylic acid (SA) was prevented in lin2 plants by the expression of an SA‐degrading salicylate hydroxylase (nahG) gene. This suggests that the lesion formation triggered in lin2 plants is determined prior to or independently of the accumulation of SA but that the accumulation is required to limit the spread of lesions in lin2 plants.
The mobility of the tobacco Tnt1 retrotransposon correlates with its transcriptional activation by fungal factors
Authors: Melayah Delphine , Bonnivard Eric , Chalhoub Boulos , Audeon Colette , Grandbastien Marie‐Angèle
Published: March 2003

We have analyzed the stress‐induced amplification of the tobacco Tnt1 element, one of the rare active plant retrotransposons. Tnt1 mobility was monitored using the retrotransposon‐anchored SSAP strategy that allows the screening of multiple insertion sites of high copy number elements. We have screened for Tnt1 insertion polymorphisms in plants regenerated from mesophyll leaf cells, either via explant culture or via protoplast isolation. The second procedure includes an overnight exposure to fungal extracts known to induce high levels of Tnt1 transcription. Newly transposed Tnt1 copies were detected in nearly 25% of the plants regenerated via protoplast isolation, and in less than 3% of the plants derived from explant culture. These results show that Tnt1 transcription is followed by transposition, and that fungal extracts efficiently activate Tnt1 mobility. Transcription appears to be the key step to controlling Tnt1 amplification, as newly transposed Tnt1 copies show high sequence similarities to the subpopulations of transcribed Tnt1 elements. Our results provide direct evidence that factors of microbial origin are able to induce retrotransposon amplification in plants, and strengthen the hypothesis that stress modulation of transposable elements might play a role in generating host genetic plasticity in response to environmental stresses.
Evidence for structurally specific negative feedback in the Nod factor signal transduction pathway
Authors: Oldroyd Giles E. D. , Mitra Raka M. , Wais Rebecca J. , Long Sharon R.
Published: March 2003

Nod factor is a critical signalling molecule in the establishment of the legume/rhizobial symbiosis. The Nod factor of Sinorhizobium meliloti carries O‐sulphate, O‐acetate and C16:2N‐acyl attachments that define its activity and host specificity. Here we assess the relative importance of these modifications for the induction of calcium spiking in Medicago truncatula. We find that Nod factor structures lacking the O‐sulphate, structures lacking the O‐acetate and N‐acyl groups, and structures lacking the O‐acetate combined with a C18:1N‐acyl group all show calcium spiking when applied at high concentrations. These calcium responses are blocked in dmi1 and dmi2 mutants, suggesting that they function through the Nod factor signal transduction pathway. The dmi3 mutant, which is proposed to function in the Nod factor signal transduction pathway downstream of calcium spiking, shows increased sensitivity to Nod factor. This increased sensitivity is only active with wild‐type Nod factor and was not present when the plants were treated with mutant Nod factor structures. We propose that the Nod factor signal transduction pathway is under negative feedback regulation that is activated at or downstream of DMI3 and requires structural components of the Nod factor molecule for activity.
In situ analysis of lignins in transgenic tobacco reveals a differential impact of individual transformations on the spatial patterns of lignin deposition at the cellular and subcellular levels
Authors: Chabannes Matthieu , Ruel Katia , Yoshinaga Arata , Chabbert Brigitte , Jauneau Alain , Joseleau Jean‐Paul , Boudet Alain‐Michel
Published: March 2003

Using tobacco transgenic lines altered in the monolignol biosynthetic pathway and which differ in their lignin profiles we have evaluated lignin deposition at the cellular and subcellular levels using several microanalytical techniques. Surprisingly, whereas a Cinnamoyl CoA reductase (CCR) down‐regulated line with a strong decrease in lignin content exhibited an overall reduction in lignin deposition in the walls of the different xylem cell types, this reduction was selectively targeted to the fibers in a double transformant (down‐regulated for both CCR and Cinnamyl alcohol dehydrogenase (CAD)) displaying a similar degree of global lignin content decrease. Fiber and vessel secondary walls of the transgenic tobacco line homozygous for the ccr antisense gene (CCR.H) down‐regulated plants were dramatically destructured, particularly in the S2 sublayer, whereas the deposition of lignins in the S1 sublayer was not significantly modified. In contrast, cell wall organization was slightly altered in xylem cells of the double transformant. The relative distribution of non‐condensed and condensed units in lignin, evaluated microscopically with specific antibodies, was differentially affected in the transgenics studied and, in a general way, a drop in non‐condensed lignin units (β− 0–4 interunit linkages) was associated with a loss of cohesion and extensive disorganization of the secondary wall. These results demonstrate that lignification is tightly and independently regulated in individual cell types and cell wall sublayers. They also show that down‐regulation of specific genes may induce targeted changes in lignin structure and in spatial deposition patterns of the polymer.
Two glucosyltransferases are involved in detoxification of benzoxazinoids in maize
Authors: Von Rad Uta , Hüttl Regina , Lottspeich Friedrich , Gierl Alfons , Frey Monika
Published: March 2003

Benzoxazinoids are major compounds involved in chemical defence in grasses. These toxins are stored in the vacuole as glucosides. Two glucosyltransferases, BX8 and BX9, that catalyse this last step of benzoxazinoid biosynthesis have been isolated via functional cloning. No close relative of these maize genes was found among the known glucosyltransferases. The enzymes display a very high degree of substrate specificity. DIMBOA, the major benzoxazinoid in young maize, is the preferred substrate. Both genes are highly expressed in young maize seedlings, the developmental stage with the highest activity of benzoxazinoid biosynthesis. Bx8 is included in the cluster of DIMBOA biosynthesis genes located on the short arm of chromosome 4. Hence, the gene cluster comprises three different enzymatic functions and a complete set of genes for the biosynthesis of DIBOA glucoside. Bx9 mapped to chromosome 1. Expression of Bx8 and Bx9 in Arabidopsis corroborated the potency of the enzymes in detoxification of their substrates. This capacity might have implications for allelopathic interactions.
The participation of AtXPB1, the XPB/RAD25 homologue gene from Arabidopsis thaliana, in DNA repair and plant development
Authors: Costa Renata M. A. , Morgante Patrícia G. , Berra Carolina M. , Nakabashi Myna , Bruneau Dominique , Bouchez David , Sweder Kevin S. , Van Sluys Marie‐Anne , Menck Carlos F. M.
Published: March 2003

Nucleotide excision repair in Arabidopsis thaliana differs from other eukaryotes as it contains two paralogous copies of the corresponding XPB/RAD25 gene. In this work, the functional characterization of one copy, AtXPB1, is presented. The plant gene was able to partially complement the UV sensitivity of a yeast rad25 mutant strain, thus confirming its involvement in nucleotide excision repair. The biological role of AtXPB1 protein in A. thaliana was further ascertained by obtaining a homozygous mutant plant containing the AtXPB1 genomic sequence interrupted by a T‐DNA insertion. The 3′ end of the mutant gene is disrupted, generating the expression of a truncated mRNA molecule. Despite the normal morphology, the mutant plants presented developmental delay, lower seed viability and a loss of germination synchrony. These plants also manifested increased sensitivity to continuous exposure to the alkylating agent MMS, thus suggesting inefficient DNA damage removal. These results indicate that, although the duplication seems to be recent, the features described for the mutant plant imply some functional or timing expression divergence between the paralogous AtXPB genes. The AtXPB1 protein function in nucleotide excision repair is probably required for the removal of lesions during seed storage, germination and early plant development.
Determination of the elemental composition of mature wheat grain using a modified secondary ion mass spectrometer (SIMS)
Authors: Heard Peter J. , Feeney Kevin A. , Allen Geoffrey C. , Shewry Peter R.
Published: March 2003

An imaging secondary ion mass spectrometry system has been developed that allows the distribution of elements or ions to be superimposed on an image of the plant cell or tissue generated by ion‐induced secondary electrons. This system has been evaluated by analysing the aleurone and sub‐aleurone cells of mature wheat grain, showing high spatial resolution (100–200 nm) images of O–, PO2–, Mg+, Ca+, Na+ and K+ within the phytate granules of the aleurone, with CN– being diagnostic for proteins and C2– being diagnostic for starch in the starchy endosperm cells. This system should provide improved localization of elements in a range of other plant systems.
Over‐expression of TGA5, which encodes a bZIP transcription factor that interacts with NIM1/NPR1, confers SAR‐independent resistance in Arabidopsis thaliana to Peronospora parasitica
Authors: Kim Han Suk , Delaney Terrence P.
Published: March 2003

The Arabidopsis thaliana NIM1/NPR1 gene product is required for induction of systemic acquired resistance (SAR) by pathogens, salicylic acid (SA) or synthetic SA analogs. We identified, in a yeast two‐hybrid screen, two NIM1/NPR1 interacting proteins, TGA2 and TGA5, which belong to the basic region, leucine zipper (bZIP) family of transcription factors. Both TGA2 and TGA5 strongly interact with NIM1/NPR1 in yeast and in vitro, and recognize the as‐1 cis element found within the promoter of several pathogenesis‐related genes, such as PR‐1. To determine the role TGA2 and TGA5 may play in NIM1/NPR1‐mediated disease resistance, we introduced sense and antisense versions of both genes into transgenic Arabidopsis plants. Characterization of TGA2 transgenic plants revealed that inhibition or overexpression of TGA2 does not significantly affect PR‐1 expression or induction of SAR after pathogen infection or INA treatment. Surprisingly, all TGA5‐antisense transgenic plants produced showed increased accumulation of TGA5 transcripts compared with untransformed control plants, while the TGA5‐sense lines showed no significant increase in TGA5 mRNA levels. Interestingly, the high level of TGA5 mRNA in the antisense lines was accompanied by significant resistance to a highly virulent isolate of the oomycete pathogen Peronospora parasitica. Further, resistance was not coupled to accumulation of products from the SAR‐linked PR‐1 gene following inoculation with P. parasitica or treatment with INA, indicating that these plants express a robust, PR‐1‐independent resistance mechanism. Resistance was retained when a TGA5‐accumulating line was combined genetically with a nim1‐1 mutation or nahG (salicylate hydroxylase) transgene, indicating that resistance in these plants is due to an SA and SAR‐independent mechanism.
Impaired sucrose‐induction mutants reveal the modulation of sugar‐induced starch biosynthetic gene expression by abscisic acid signalling
Authors: Rook Fred , Corke Fiona , Card Roderick , Munz Georg , Smith Caroline , Bevan Michael W.
Published: March 2003

Plants both produce and utilize carbohydrates and have developed mechanisms to regulate their sugar status and co‐ordinate carbohydrate partitioning. High sugar levels result in a feedback inhibition of photosynthesis and an induction of storage processes. We used a genetic approach to isolate components of the signalling pathway regulating the induction of starch biosynthesis. The regulatory sequences of the sugar inducible ADP‐glucose pyrophosphorylase subunit ApL3 were fused to a negative selection marker. Of the four impaired sucrose induction (isi) mutants described here, two (isi1 and isi2) were specific to this screen. The other two mutants (isi3 and isi4) showed additional phenotypes associated with sugar‐sensing screens that select for seedling establishment on high‐sugar media. The isi3 and isi4 mutants were found to be involved in the abscisic acid signalling pathway. isi3 is allelic to abscisic acid insensitive4 (abi4), a gene encoding an Apetala2‐type transcription factor; isi4 was found to be allelic to glucose insensitive1 (gin1) previously reported to reveal cross‐talk between ethylene and glucose signalling. Here we present an alternative interpretation of gin1 as an allele of the ABA‐deficient mutant aba2. Expression analysis showed that ABA is unable to induce ApL3 gene expression by itself, but greatly enhances ApL3 induction by sugar. Our data suggest a major role for ABA in relation to sugar‐signalling pathways, in that it enhances the ability of tissues to respond to subsequent sugar signals.
Evidence of mitochondrial involvement in the transduction of signals required for the induction of genes associated with pathogen attack and senescence
Authors: Maxwell Denis P. , Nickels Roxy , McIntosh Lee
Published: March 2003

Using the mRNA differential display technique, seven cDNAs have been isolated that are rapidly induced when cultured tobacco (Nicotiana tabacum) cells are treated with the mitochondrial electron transport inhibitor antimycin A (AA). Interestingly, six of the cDNAs show distinct similarity to genes known to be induced by processes that involve programmed cell death (PCD), such as senescence and pathogen attack. All of the cDNAs as well as Aox1, a gene encoding the alternative oxidase, were found to also be strongly induced by H2O2 and salicylic acid (SA). AA, H2O2 and SA treatment of tobacco cells caused a rapid rise in intracellular ROS accumulation that, when prevented by antioxidant treatment, resulted in inhibition of gene induction. Besides AA, both H2O2 and SA were found to disrupt normal mitochondrial function resulting in decreased rates of electron transport and a lowering of cellular ATP levels. Furthermore, the pre‐treatment of tobacco cells with bongkrekic acid, a known inhibitor of the mitochondrial permeability transition pore in animal cells, was found to completely block gene induction when AA, H2O2 or SA were subsequently added. These findings suggest that the mitochondrion may serve an important role in conveying intracellular stress signals to the nucleus, leading to alterations in gene expression.
Local and systemic wound‐induction of RNase and nuclease activities in Arabidopsis: RNS1 as a marker for a JA‐independent systemic signaling pathway
Authors: LeBrasseur Nicole D. , MacIntosh Gustavo C. , Pérez‐Amador Miguel A. , Saitoh Maki , Green Pamela J.
Published: March 2003

Induction of defense‐related genes is one way in which plants respond to mechanical injury. We investigated whether RNases are involved in the wound response in Arabidopsis thaliana. As in other plant systems, several activities are induced with various timings in damaged leaves, stems and seedlings in Arabidopsis, including at least three bifunctional nucleases, capable of degrading both RNA and DNA, as well as RNS1, a member of the ubiquitous RNase T2 family of RNases. The strong induction of RNS1 is particularly interesting because it occurs both locally and systemically following wounding. The systemic induction of this RNase indicates that members of this family may be involved in defense mechanisms in addition to their previously hypothesized functions in nutrient recycling and remobilization. Additionally, the systemic induction appears to be controlled independently of jasmonic acid, and the local induction of RNS1 and the nuclease activities are independent of both JA and oligosaccharide elicitors. Consequently, a novel systemic pathway, likely involving a third signal, appears to exist in Arabidopsis.