bluuebunny/crossref_metadata_2025 huggingface dataset:
Some thaumatin‐like proteins hydrolyse polymeric β‐1,3‐glucans
Authors: Grenier Jean , Potvin Claude , Trudel Jean , Asselin Alain
Published: March 2003

Thaumatin and 12 purified thaumatin‐like (TL) proteins were surveyed for their capacity to hydrolyse β‐1,3‐glucans by using an in‐gel glucanase assay. Six TL proteins identified by N‐terminal amino acid microsequencing were found to be active on carboxymethyl(CM)‐pachyman: a barley leaf stress‐related permatin, two tomato fruit osmotins, a cherry fruit and two tobacco stigma proteins. TL enzymes ranged in specific activity from 0.07 to 89 nkat mg–1 with CM‐pachyman as substrate. Hydrolytic activities were not restricted to TL proteins strongly binding to water‐insoluble β‐1,3‐glucans since the two osmotins were active without tight binding to pachyman. Some TL proteins hydrolysed crude fungal walls and one barley TL enzyme even lysed fungal spores. No activity was observed on laminarin in the in‐gel hydrolase assay. Thin‐layer chromatography revealed that the six enzymes acted as endo‐β‐1,3‐glucanases leading to the formation of various oligoglucosides. Thus far, the TL enzymes (EC 3.2.1.x) appeared different from the well‐known β‐1,3‐glucanases (EC 3.2.1.39). No activity was found with thaumatin, zeamatin, tobacco leaf PR‐R protein and four stress‐related TL proteins from barley and pea. This is the first demonstration that diverse TL proteins are enzymatically active. The functions of some TL proteins must be reassessed because they display endo‐β‐1,3‐glucanase activity on polymeric β‐1,3‐glucans.
Molecular analysis of rice plants harboring an Ac/Ds transposable element‐mediated gene trapping system
Authors: Chin Hang Gyeong , Choe Mi Sook , Lee Sung‐Ho , Park Sung Han , Park Su Hyun , Koo Ja Choon , Kim No Youl , Lee Jeung Joo , Oh Byeong Geun , Yi Gi Hwan , Kim Soon Chul , Choi Hae Chune , Cho Moo Je , Han Chang‐deok
Published: March 2003

In rice, limited efforts have been made to identify genes by the use of insertional mutagens, especially hetero‐ logous transposons such as the maize Ac/Ds. We constructed Ac and gene trap Ds vectors and introduced them into the rice genome by Agrobacterium‐mediated transformation. In this report, rice plants that contained single and simple insertions of T‐DNA were analysed in order to evaluate the gene‐tagging efficiency. The 3′ end of Ds was examined for putative splicing donor sites. As observed in maize, three splice donor sites were identified at the 3′ end of the Ds in rice. Nearly 80% of Ds elements were excised from the original T‐DNA sites, when Ac cDNA was expressed under a CaMV 35S promoter. Repetitive ratoon culturing was performed to induce new transpositions of Ds in new plants derived from cuttings. About 30% of the plants carried at least one Ds which underwent secondary transposition in the later cultures. Eight per cent of transposed Ds elements expressed GUS in various tissues of rice panicles. With cloned DNA adjacent to Ds, the genomic complexities of the insertion sites were examined by Southern hybridization. Half of the Ds insertion sites showed simple hybridization patterns which could be easily utilized to locate the Ds. Our data demonstrate that the Ac/Ds‐mediated gene trap system could prove an excellent tool for the analysis of functions of genes in rice. We discuss genetic strategies that could be employed in a large scale mutagenesis using a hetero‐ logous Ac/Ds family in rice.
Millisecond UV‐B irradiation evokes prolonged elevation of cytosolic‐free Ca2+ and stimulates gene expression in transgenic parsley cell cultures
Authors: Frohnmeyer Hanns , Loyall Linda , Blatt Michael R. , Grabov Alexander
Published: March 2003

Chalcone synthase (CHS) is a key enzyme leading to the generation of protective flavonoids in plants under environmental stress. Expression of the CHS gene is strongly upregulated by exposures to UV light, a response also observed in heterotrophic parsley cell cultures. Although there are hints that the stimulus for CHS expression may be coupled to UV‐B irradiation through a rise in cytosolic‐free Ca2+ ([Ca2+]i), the temporal relationship of these events has never been investigated critically. To explore this question, we have used a CHS promoter/luciferase (CHS/LUC) reporter gene fusion and recorded its expression and [Ca2+]i elevation in a transgenic parsley cell culture following millisecond light pulses. Luciferase expression was enhanced maximally seven‐ (± 2) fold by 30 10 ms flashes of UV‐B light. The response was specific to wavelengths of 300–330 nm and could be inhibited in the presence of the Ca2+ channel blocker nifedipine. In parallel measurements, using Fura‐2 fluorescence ratio microphotometry, we found that 10 ms UV‐B flashes also evoked a gradual and prolonged rise of [Ca2+]i in the parsley cells which was irreversible within the timescale of these experiments, but could be prevented by prior treatment with nifedipine. These, and additional results, indicate a remarkably high temporal sensitivity to, and specificity for, UV‐B light in CHS gene expression independent of UV‐mediated DNA damage by thymine dimerization. The ability of transient UV‐B stimulation to evoke prolonged elevations of [Ca2+]i suggests a functional coupling between the initial light stimulus and subsequent gene expression that takes place many tens of minutes later.
The UNUSUAL FLORAL ORGANS gene of Arabidopsis thaliana is an F‐box protein required for normal patterning and growth in the floral meristem
Authors: Samach Alon , Klenz Jennifer E. , Kohalmi Susanne E. , Risseeuw Eddy , Haughn George W. , Crosby William L.
Published: March 2003

Genetic and molecular studies have suggested that the UNUSUAL FLORAL ORGANS (UFO) gene, from Arabidopsis thaliana, is expressed in all shoot apical meristems, and is involved in the regulation of a complex set of developmental events during floral development, including floral meristem and floral organ identity. Results from in situ hybridization using genes expressed early in floral development as probes indicate that UFO controls growth of young floral primordia. Transgenic constructs were used to provide evidence that UFO regulates floral organ identity by activating or maintaining transcription of the class B organ‐identity gene APETALA 3, but not PISTILLATA. In an attempt to understand the biochemical mode of action of the UFO gene product, we show here that UFO is an F‐box protein that interacts with Arabidopsis SKP1‐like proteins, both in the yeast two‐hybrid system and in vitro. In yeast and other organisms both F‐box proteins and SKP1 homologues are subunits of specific ubiquitin E3 enzyme complexes that target specific proteins for degradation. The protein selected for degradation by the complex is specified by the F‐box proteins. It is therefore possible that the role of UFO is to target for degradation specific proteins controlling normal growth patterns in the floral primordia, as well as proteins that negatively regulate APETALA 3 transcription.
Interactions of the developmental regulator ABI3 with proteins identified from developing Arabidopsis seeds
Authors: Kurup Smita , Jones Huw D. , Holdsworth Michael John
Published: March 2003

The ABI3 locus is a major regulator of embryo development in Arabidopsis and is essential for the simultaneous activation of the maturation pathway, as well as repression of germination and seedling development. We used a two‐hybrid screen in yeast in order to identify proteins that interact with ABI3. Four ABI3‐interacting proteins (AIPs) were identified which showed specific in vivo and in vitro interactions with the C‐terminal region of ABI3 that contains the B2 and B3 domains, previously shown to have DNA binding activity. The expression characteristics of the genes encoding the AIPs have also been analysed in wild‐type and abi3, lec1 and fus3 embryo mutants. This analysis demonstrated differential expression of these genes during normal embryo development and in the mutant lines. All the AIPs show homology to existing transcription factors and therefore they may function with ABI3 within the network of transcriptional regulators that control embryo development in Arabidopsis.
Functional analysis of HD2 histone deacetylase homologues in Arabidopsis thaliana
Authors: Wu Keqiang , Tian Lining , Malik Kamal , Brown Daniel , Miki Brian
Published: March 2003

Post‐translational modification of histones, in particular acetylation, is an important mechanism in the regulation of eukaryotic gene expression. Histone deacetylases are enzymes that remove acetyl groups from the core histones and play a key role in the repression of transcription. HD2 is a maize histone deacetylase, which shows no sequence homology to the histone deacetylases identified from other eukaryotes. We have identified two putative HD2‐like histone deacetylase cDNA clones, AtHD2A and AtHD2B, from Arabidopsis thaliana by screening the expressed sequence tag database. AtHD2A and AtHD2B encode putative proteins of 246 and 305 amino acids, and share 44% and 46% amino acid identity to the maize HD2, respectively. Northern blot analysis indicated that AtHD2A was highly expressed in flowers and young siliques of Arabidopsis plants, whereas AtHD2B was widely expressed in stems, leaves, flowers and young siliques. AtHD2A repressed transcription when directed to a promoter containing GAL4‐binding sites as a GAL4 fusion protein. Deletion of the extended acidic domain or the domain containing predicted catalytic residues of AtHD2A resulted in the loss of gene repression activity, revealing the importance of both domains to AtHD2A function. Arabidopsis plants were transformed with a gene construct comprising an AtHD2A cDNA in the antisense orientation driven by a strong constitutive promoter, −394tCUP. Silencing of AtHD2A expression resulted in aborted seed development in transgenic Arabidopsis plants, suggesting that the AtHD2A gene product was important in the reproductive development of Arabidopsis thaliana.
Inhibition of serine/threonine‐specific protein phosphatases causes premature activation of cdc2MsF kinase at G2/M transition and early mitotic microtubule organisation in alfalfa
Authors: Ayaydin Ferhan , Vissi Emese , Mészáros Tamás , Miskolczi Pál , Kovács Izabella , Fehér Attila , Dombrádi Viktor , Erdödi Ferenc , Gergely Pál , Dudits Dénes
Published: March 2003

Reversible phosphorylation of serine/threonine residues of cell cycle‐regulatory proteins is one of the key molecular mechanisms controlling eukaryotic cell division. In plants, the protein kinase partners (i.e. p34cdc2/CDC28‐related kinases) have been extensively studied, while the role of counter‐acting protein phosphatases is less well understood. We used endothall (ET) as a cell‐permeable inhibitor of serine/threonine‐specific protein phosphatases to alter cytological and biochemical characteristics of cell division in cultured alfalfa cells. A high concentration of ET (10 and 50 μm) inhibited both protein phosphatases 1 and 2 (PP1 and PP2A), while a low concentration (1 μm) of ET‐treatment primarily reduced the PP2A activity. High concentrations of the inhibitor increased the frequency of hypercondensed early and late prophase chromosomes that could not enter metaphase. In contrast, a low concentration of ET did not interfere with chromosomal events but caused significant alterations in the organisation of microtubules. Exposure of cells to 1 μm ET resulted in disturbance of preprophase band formation, increase in the number of nuclei with prophase microtubule assembly, premature polarisation of the spindle, and abnormal phragmoplast maturation. Under the same conditions, the ET‐treated cells exhibited an early increase in cdc2MsF kinase activity. These results suggest that PP2A contributes to the control of mitotic kinase activities and microtubule organisation. Normal chromosome condensation and mitotic progression are dependent on both PP1 and PP2A activities. The presented data support the functional role of protein phosphatases in the co‐ordination of chromosomal and microtubule events in dividing plant cells.
DNA‐interactions and nuclear localisation of the chromosomal HMG domain protein SSRP1 from maize
Authors: Röttgers Karin , Krohn Nicholas M. , Lichota Jacek , Stemmer Christian , Merkle Thomas , Grasser Klaus D.
Published: March 2003

The structure‐specific recognition protein 1 (SSRP1) is a member of the protein family containing a high mobility group (HMG) domain DNA‐binding motif. We have functionally characterised the 71.4 kDaZm‐SSRP1 protein from maize. The chromatin‐associatedZm‐SSRP1 is detected by immunoblot analysis in maize leaves, kernels and suspension culture cells, but not in roots. Mediated by its HMG domain, recombinantZm‐SSRP1 interacts structure‐specifically with supercoiled DNA and DNA minicircles when compared with linear DNA. In linear duplex DNA, the protein does not recognise a specific sequence, but it binds preferentially to sequences containing the deformable dinucleotide TG, as demonstrated by a random oligonucleotide selection experiment.Zm‐SSRP1 modulates DNA structure by bending the target sequence, since it promotes the circularisation of short DNA fragments in the presence of DNA ligase. Moreover,Zm‐SSRP1 facilitates the formation of nucleoprotein structures, as measured using the bacterial site‐specific β‐mediated recombination reaction. Analysis of the subcellular localisation of various SSRP1‐GFP fusions revealed that, in contrast to HMG domain transcription factors, the nuclear localisation sequence ofZm‐SSRP1 is situated within a 20‐amino acid residue region adjacent to the HMG domain rather than within the DNA‐binding domain. The results are discussed in the context of the likely function of SSRP1 proteins in transcription and replication.
An increase in phosphoinositide‐specific phospholipase C activity precedes induction of C4 phosphoenolpyruvate carboxylase phosphorylation in illuminated and NH4Cl‐treated protoplasts from Digitaria sanguinalis
Authors: Coursol Sylvie , Giglioli‐Guivarc'h Nathalie , Vidal Jean , Pierre Jean‐Noël
Published: March 2003

A Ca2+‐dependent phosphoinositide‐specific phospholipase C (PI‐PLC) activity has been characterized in the microsomal fraction of Digitaria sanguinalis mesophyll cell protoplasts. Microsomal PI‐PLC was found to be inhibited in vitro by a mammalian anti‐PLC‐δ1 antibody and by the aminosteroide U‐73122, an inhibitor of PI‐PLC activity in animal cells. In Western blot experiments, the antibody recognized an 85 kDa protein in both microsomal protein extracts from mesophyll protoplasts and rat brain protein extracts containing the authentic enzyme. The involvement of the microsomal PI‐PLC in the light‐dependent transduction pathway leading to the phosphorylation of C4 phosphoenolpyruvate carboxylase (PEPC) was investigated in D. sanguinalis protoplasts. A transient increase in the PI‐PLC reaction product inositol‐1,4,5‐trisphosphate (Ins(1,4,5)P3) was observed in situ during early induction of the C4 PEPC phosphorylation cascade. U‐73122, but not the inactive analogue U‐73343, efficiently blocked the transient accumulation of Ins(1,4,5)P3, and both the increase in C4 PEPC kinase activity and C4 PEPC phosphorylation in illuminated and weak base‐treated protoplasts. Taken together, these data suggest that PI‐PLC‐based signalling is a committed step in the cascade controlling the regulation of C4 PEPC phosphorylation in C4 leaves.
Identification of transposon‐tagged genes by the random sequencing of Mutator‐tagged DNA fragments from Zea mays
Authors: Hanley Steven , Edwards David , Stevenson David , Haines Stephen , Hegarty Matthew , Schuch Wolfgang , Edwards Keith J.
Published: March 2003

We have used a universal adaptor amplification procedure to isolate random Mutator‐tagged fragments from Mutator‐active maize plants. Direct sequence characterization of 761 Mutator‐tagged fragments indicated that a significant number were homologous to sequences within the public databases. The ability of Mutator‐tagged fragments to detect homology was not related to the length of the sequence within the range 100–400 bp. However, fragments above this size did show an increased chance of detecting homology to either expressed sequence tags or genes. Characterization of the insertion sites of the Mutator elements suggested that while it does target transcribed regions, Mutator does not appear to have any site preference within the transcription unit. Hybridization of previously unidentified Mutator‐tagged fragments to arrayed cDNA libraries confirmed that many of these also showed homology to transcribed regions of the genome. Examination of back‐crossed progeny confirmed that all the insertions examined were germinal; however, in all but one case, selfing five individual Mutator‐tagged lines failed to reveal an obvious phenotype. This study suggests that the random sequencing of Mutator‐tagged fragments is capable of producing both a significant number of interesting transposon tagged genes and mutant plant lines, all of which could be extremely valuable in future gene discovery and functional genomics programmes.
Studies on the topology of the protein import channel in relation to the plant mitochondrial processing peptidase integrated into the cytochrome bc1 complex
Authors: Dessi Patrick , Rudhe Charlotta , Glaser Elzbieta
Published: March 2003

The mitochondrial processing peptidase (MPP) specifically cleaves N‐terminal targeting signals from hundreds of nuclear‐encoded, matrix‐targeted precursor proteins. In contrast to yeast and mammals, the plant MPP is an integral component of the respiratory cytochrome bc1 complex. The topology of the protein import channel in relation to MPP/bc1 in plants was studied using chimeric precursors containing truncated cytochrome b2 (cyt b2) proteins of 55–167 residues in length, fused to dihydrofolate reductase (DHFR). The DHFR domain could be tightly folded by methotrexate (MTX), generating translocation intermediates trapped in the import channel with only the cyt b2 pre‐sequence/mature domain protruding into the matrix. Spinach and soybean mitochondria imported and processed unfolded precursors. MTX‐folded intermediates were not processed in spinach but the longest (1–167) MTX‐folded cyt b2–DHFR construct was processed in soybean, while yeast mitochondria successfully processed even shorter MTX‐folded constructs. The MTX‐folded precursors were cleaved with high efficiency by purified spinach MPP/bc1 complex. We interpret these results as indicating that the protein import channel is located distantly from the MPP/bc1 complex in plants, and that there is no link between protein translocation and protein processing.
Localization of single‐copy T‐DNA insertion in transgenic shallots (Allium cepa) by using ultra‐sensitive FISH with tyramide signal amplification
Authors: Khrustaleva Ludmila I. , Kik Chris
Published: March 2003

The sensitivity of fluorescence in situ hybridization (FISH) for mapping plant chromosomes of single‐copy DNA sequences is limited. We have adapted for plant cytogenetics a new signal‐amplification method termed tyramide‐FISH (Tyr‐FISH). Until present this technique has only been applied to human chromosomes. The method is based on enzymatic deposition of fluorochrome‐conjugated tyramide. With Tyr‐FISH it was possible to detect target T‐DNA sequences on plant metaphase chromosomes as small as 710 bp without using a cooled CCD camera. Short detection time and high sensitivity, in combination with a low background, make the Tyr‐FISH method very suitable for routine application in plant cytogenetic research. With Tyr‐FISH we analysed the position of T‐DNA inserts in transgenic shallots. We found that the inserts were preferentially located in the distal region of metaphase chromosomes. Sequential fluorescence in situ hybridization with a 375 bp satellite sequence suggested that a specific T‐DNA insert was located within the satellite sequence hybridization region on a metaphase chromosome. Analysis of less‐condensed prophase and interphase chromosomes revealed that the T‐DNA was integrated outside the satellite DNA‐hybridization region in a more proximal euchromatin region.
Analysis of a contiguous 211 kb sequence in diploid wheat (Triticum monococcum L.) reveals multiple mechanisms of genome evolution
Authors: Wicker Thomas , Stein Nils , Albar Laurence , Feuillet Catherine , Schlagenhauf Edith , Keller Beat
Published: March 2003

In plant species with large genomes such as wheat or barley, genome organization at the level of DNA sequence is largely unknown. The largest sequences that are publicly accessible so far from Triticeae genomes are two 60 kb and 66 kb intervals from barley. Here, we report on the analysis of a 211 kb contiguous DNA sequence from diploid wheat (Triticum monococcum L.). Five putative genes were identified, two of which show similarity to disease resistance genes. Three of the five genes are clustered in a 31 kb gene‐enriched island while the two others are separated from the cluster and from each other by large stretches of repetitive DNA. About 70% of the contig is comprised of several classes of transposable elements. Ten different types of retrotransposons were identified, most of them forming a pattern of nested insertions similar to those found in maize and barley. Evidence was found for major deletion, insertion and duplication events within the analysed region, suggesting multiple mechanisms of genome evolution in addition to retrotransposon amplification. Seven types of foldback transposons, an element class previously not described for wheat genomes, were characterized. One such element was found to be closely associated with genes in several Triticeae species and may therefore be of use for the identification of gene‐rich regions in these species.
The Arabidopsis compact inflorescence genes: phase‐specific growth regulation and the determination of inflorescence architecture
Authors: Goosey Lynn , Sharrock Robert
Published: March 2003

During their life cycle, higher plants pass through a series of growth phases that are characterized by the production of morphologically distinct vegetative and reproductive organs and by different growth patterns. Three major phases have been described in Arabidopsis: juvenile vegetative, adult vegetative, and reproductive. In this report we describe a novel, phase‐specific mutant in Arabidopsis, compact inflorescence (cif). The most apparent aspect of the cif phenotype is a strong reduction in the elongation of internodes in the inflorescence, resulting in the formation of a floral cluster at the apical end of all reproductive shoots. Elongation and expansion of adult vegetative rosette leaves are also compromised in mutant plants. The onset of the cif trait correlates closely with morphological changes marking the phase transition from juvenile to adult, and mutant plants produce normal flowers and are fully fertile. Hence the cif phenotype appears to be adult vegetative phase‐specific. Histological sections of mutant inflorescence internodes indicate normal tissue specification, but reduced cell elongation compared to wild‐type. compact inflorescence is inherited as a two‐gene trait involving the action of a recessive and a dominant locus. These two cif genes appear to be key components of a growth regulatory pathway that is closely linked to phase change, and specifies critical aspects of plant growth and architecture including inflorescence internode length.
Biolistic transformation of Arabidopsis root hairs: a novel technique to facilitate map‐based cloning
Authors: Kemp Alison , Parker Jill , Grierson Claire
Published: March 2003

The final stage of map‐based gene isolation is complementation of the mutant phenotype with wild‐type DNA to determine the exact location of the gene of interest. This usually involves Agrobacterium tumefaciens‐mediated transformation, which is reliable and produces stable transformants. However, the process of Agrobacterium transformation may take up to three months to complete. If the mutant phenotype can be seen in a single cell, and the wild‐type copy of the gene can act cell autonomously, then complementation of the whole plant is not strictly necessary. We have developed a technique for the biolistic transformation of Arabidopsis thaliana root hairs, and used this to test large insert clones for complementation of two recessive mutant phenotypes, a procedure that takes less than a day. Our results show that biolistic transformation can be used with transient assays to conduct rapid tests for complementation by large insert clones.
Contrasting modes of evolution acting on the complex N locus for rust resistance in flax
Authors: Dodds Peter N. , Lawrence Gregory J. , Ellis Jeffrey G.
Published: March 2003

Three rust resistance specificities, N, N1 and N2, map to the complex N locus of flax. We used a degenerate PCR approach, with primers directed to the nucleotide binding site (NBS) domain characteristic of many plant resistance genes, to isolate resistance gene analogs (RGAs) from flax. One RGA clone detected RFLPs co‐segregating with alleles of the N locus. With this probe we isolated four related genes that occur within a 30kbp region and encode proteins with NBS and leucine‐rich repeat (LRR) domains and N‐terminal Toll/Interleukin‐1 Receptor homology (TIR) domains. One of these four genes was identified as the N resistance gene by sequence analysis of three mutant alleles and by transgenic expression. We isolated homologous genes from two flax lines containing the N1 or N2 specificities and from flax lines carrying no N locus resistance specificities. Analysis of shared polymorphisms among this set of 18 N locus sequences revealed three groups of genes with independent lineages. Sequence exchanges have only occurred between genes within each group, but not between groups. Two of the groups contain only one sequence from each haplotype and probably represent orthologous genes. However, the third group contains two genes from each haplotype. We suggest that the re‐assortment of variation by recombination/gene conversion at this locus is limited by the degree of sequence identity between genes.
Characterization of an Arabidopsis–Phytophthora Pathosystem: resistance requires a functional PAD2 gene and is independent of salicylic acid, ethylene and jasmonic acid signalling
Authors: Roetschi Alexandra , Si‐Ammour Azeddine , Belbahri Lassaâd , Mauch Felix , Mauch‐Mani Brigitte
Published: March 2003

Arabidopsis accessions were screened with isolates of Phytophthora porri originally isolated from other crucifer species. The described Arabidopsis–Phytophthora pathosystem shows the characteristics of a facultative biotrophic interaction similar to that seen in agronomically important diseases caused by Phytophthora species. In susceptible accessions, extensive colonization of the host tissue occurred and sexual and asexual spores were formed. In incompatible combinations, the plants reacted with a hypersensitive response (HR) and the formation of papillae at the sites of attempted penetration. Defence pathway mutants such as jar1 (jasmonic acid‐insensitive), etr1 (ethylene receptor mutant) and ein2 (ethylene‐insensitive) remained resistant towards P. porri. However, pad2, a mutant with reduced production of the phytoalexin camalexin, was hyper‐susceptible. The accumulation of salicylic acid (SA) and PR1 protein was strongly reduced in pad2. Surprisingly, this lack of SA accumulation does not appear to be the cause of the hyper‐susceptibility because interference with SA signalling in nahG plants or sid2 or npr1 mutants had only a minor effect on resistance. In addition, the functional SA analogue benzothiadiazol (BTH) did not induce resistance in susceptible plants including pad2. Similarly, the complete blockage of camalexin biosynthesis in pad3 did not cause susceptibility. Resistance of Arabidopsis against P. porri appears to depend on unknown defence mechanisms that are under the control of PAD2.
A tobacco nuclear extract supporting transcription, processing, splicing and modification of plant intron‐containing tRNA precursors
Authors: Yukawa Yasushi , Fan Hao , Akama Kazuhito , Beier Hildburg , Gross Hans J. , Sugiura Masahiro
Published: March 2003

Nuclear tRNA genes are transcribed by RNA polymerase III (Pol III) and pre‐tRNAs are processed into mature tRNAs via complex processes in the nucleus. We have developed an in vitro Pol III‐dependent transcription system derived from tobacco cultured cells, which supports efficiently not only transcription of a variety of plant tRNA genes but also 5′‐and 3′‐end processing, nucleotide modification and splicing of intron‐containing pre‐tRNAs. The structures of in vitro transcripts have been confirmed by primer extension analysis and by RNase T1 fingerprinting. The optimal Mg2+ concentration differed for each step so that each reaction can be controlled by adjusting the Mg2+ concentration. At 1 mm Mg2+, only transcription occurs so that pre‐tRNAs accumulate. The splicing reaction can be initiated by raising Mg2+ ions (> 5 mm) and enhanced by adding 1 mm hexamminecobalt chloride. Using the optimized system for the Nicotiana intron‐containing tRNATyr gene, the precise initiation and termination sites of transcription and the splice sites were determined. The presence of 1 mm NAD+ in the reaction mixture leads to the removal of the 2′‐phosphate at the splice junction of tRNATyr, demonstrating the activity of a 2′‐phosphotransferase in the tobacco nuclear extract. Many modified nucleosides such as m2G, m22G, m1A, Ψ27 and Ψ35 are introduced in either of the studied transcripts. As shown in other systems, the conversion of U35 to Ψ requires an intron‐containing substrate.
The K+ channel SKT1 is co‐expressed with KST1 in potato guard cells – both channels can co‐assemble via their conserved KT domains
Authors: Zimmermann Sabine , Hartje Stefanie , Ehrhardt Thomas , Plesch Gunnar , Mueller‐Roeber Bernd
Published: March 2003

An appreciable number of potassium channels mediating K+ uptake have been identified in higher plants. Promoter‐β‐glucuronidase reporter gene studies were used here to demonstrate that SKT1, encoding a potato K+ inwardly rectifying channel, is expressed in guard cells in addition to KST1 previously reported. However, whereas KST1 was found to be expressed in essentially all mature guard cells, SKT1 expression was almost exclusively restricted to guard cells of the abaxial leaf epidermis. This suggests that different types of K+ channel subunits contribute to channel formation in potato guard cells and therefore differential regulation of stomatal movements in the two leaf surfaces. The overlapping expression pattern of SKT1 and KST1 in abaxial guard cells indicates that K+in channels of different sub‐families contribute to ionic currents in this cell type, thus explaining the different properties of channels expressed solely in heterologous systems and those endogenous to guard cells. Interaction studies had previously suggested that plant K+ inward rectifiers form clusters via their conserved C‐terminal domain, KT/HA. K+ channels co‐expressed in one cell type may therefore form heteromers, which increase functional variability of K+ currents, a phenomenon well described for animal voltage‐gated K+ channels. Co‐expression of KST1 and SKT1 in Xenopus oocytes resulted in currents with an intermediate sensitivity towards Cs+, suggesting the presence of heteromers, and a sensitivity towards external Ca2+, which reflected the property of the endogenous K+in current in guard cells. Modulation of KST1 currents in oocytes by co‐expressing KST1 with a SKT1 pore‐mutant, which by itself was not able to confer activating K+ currents, demonstrated the possibility that KST1 and SKT1 co‐assemble to hetero‐oligomers. Furthermore, various C‐terminal deletions of the mutated SKT1 channel restored KST1 currents, showing that the C‐terminal KT motif is essential for heteromeric channel formation.
Mechanism of cell death and disease resistance induction by transgenic expression of bacterio‐opsin
Authors: Pontier Dominique , Mittler Ron , Lam Eric
Published: March 2003

One of the earliest signal transduction events that trigger the hypersensitive response (HR) of plants against pathogen attack is thought to be an alteration of proton flux across the plasma membrane (PM). However, no direct genetic evidence for the involvement of PM‐localised proton channels or pumps in the induction of this response has been reported. We previously showed that expression of the bacterial proton pump bacterio‐opsin (bO) in transgenic plants resulted in the spontaneous activation of the HR. Here we show that the bO protein is likely localised to the PM in transgenic tobacco plants. Furthermore, mutational analysis shows that induction of the HR by bO expression is dependent upon the capability of bO to translocate protons. Although bO functions as a light‐driven proton pump in Halobacteria when assembled with retinal, we also show by mutational analysis that this chromophore binding is unnecessary for its in planta activity. Taken together, our results suggest that expression of bO in plants leads to the insertion of a passive proton channel into the PM. The activity of this channel in the PM results in spontaneous activation of cell death and HR‐associated phenotypes including enhanced resistance to a broad spectrum of plant pathogens. Our work provides direct molecular evidence to support a working model in which alterations in ionic homeostasis at the level of the PM may work as one of the critical steps in the signalling pathway for the activation of the HR.
Decreased Rubisco activity leads to dramatic changes of nitrate metabolism, amino acid metabolism and the levels of phenylpropanoids and nicotine in tobacco antisense RBCS transformants
Authors: Matt Petra , Krapp Anne , Haake Volker , Mock Hans‐Peter , Stitt Mark
Published: March 2003

Tobacco transformants that express an antisense RBCS construct were used to investigate the consequences of a lesion in photosynthetic carbon metabolism for nitrogen metabolism and secondary metabolism. The results show that an inhibition of photosynthesis and decrease in sugar levels leads to a general inhibition of nitrogen metabolism, and dramatic changes in the levels of secondary metabolites. The response was particularly clear in plants that received excess nitrogen. In these conditions, a decrease of Rubisco activity led to an inhibition of nitrate reductase activity, accumulation of nitrate, a decrease of amino acid levels that was larger than the decrease of sugars, and a large decrease of chlorogenic acid and of nicotine, which are the major carbon‐ and nitrogen‐rich secondary metabolites in tobacco leaves, respectively. Similar changes were seen when nitrogen‐replete wild‐type tobacco was grown in low light. The inhibition of nitrogen metabolism was partly masked when wild‐type plants and antisense RBCS transformants were compared in marginal or in limiting nitrogen, because the lower growth rate of the transformants alleviated the nitrogen deficiency, leading to an increase of amino acids. In these conditions, chlorogenic acid always decreased but the decrease of nicotine was ameliorated or reversed. When the changes in internal pools are compared across all the genotypes and growth conditions, two conclusions emerge. First, decreased levels of primary metabolites lead to a dramatic decrease in the levels of secondary metabolites. Second, changes of the amino acid : sugar ratio are accompanied by changes of the nicotine:chlorogenic acid ratio.
Maize DRE‐binding proteins DBF1 and DBF2 are involved in rab17 regulation through the drought‐responsive element in an ABA‐dependent pathway
Authors: Kizis Dimosthenis , Pagès Montserrat
Published: March 2003

The abscisic acid‐responsive gene rab17 of maize is expressed during late embryogenesis, and is induced by ABA and desiccation in embryo and vegetative tissues. ABRE and DRE cis‐elements are involved in regulation of the gene by ABA and drought. Using yeast one‐hybrid screening, we isolated two cDNAs encoding two new DRE‐binding proteins, designated DBF1 and DBF2, that are members of the AP2/EREBP transcription factor family. Analysis of mRNA accumulation profiles showed that DBF1 is induced during maize embryogenesis and after desiccation, NaCl and ABA treatments in plant seedlings, whereas the DBF2 mRNA is not induced. DNA‐binding preferences of DBFs were analysed by electrophoretic mobility shift assays, and showed that both DBF1 and DBF2 bound to the wild‐type DRE2 element, but not to the DRE2 mutant or to the DRE1 element which differs only in a single nucleotide. Transactivation activity using particle bombardment showed that DBF1 functioned as activator of DRE2‐dependent transcription of rab17 promoter by ABA, whereas DBF2 overexpression had a repression action downregulating not only the basal promoter activity, but also the ABA effect. These results show that ABA plays a role in the regulation of DBF activity, and suggests the existence of an ABA‐dependent pathway for the regulation of genes through the C‐repeat/DRE element.
Flux control of sulphate assimilation in Arabidopsis thaliana: adenosine 5′‐phosphosulphate reductase is more susceptible than ATP sulphurylase to negative control by thiols
Authors: Vauclare Pierre , Kopriva Stanislav , Fell David , Suter Marianne , Sticher Liliane , Von Ballmoos Peter , Krähenbühl Urs , Den Camp Roel Op , Brunold Christian
Published: March 2003

The effect of externally applied l‐cysteine and glutathione (GSH) on ATP sulphurylase and adenosine 5′‐phosphosulphate reductase (APR), two key enzymes of assimilatory sulphate reduction, was examined in Arabidopsis thaliana root cultures. Addition of increasing l‐cysteine to the nutrient solution increased internal cysteine, γ‐glutamylcysteine and GSH concentrations, and decreased APR mRNA, protein and extractable activity. An effect on APR could already be detected at 0.2 mm l‐cysteine, whereas ATP sulphurylase was significantly affected only at 2 mm l‐cysteine. APR mRNA, protein and activity were also decreased by GSH at 0.2 mm and higher concentrations. In the presence of l‐buthionine‐S, R‐sulphoximine (BSO), an inhibitor of GSH synthesis, 0.2 mm l‐cysteine had no effect on APR activity, indicating that GSH formed from cysteine was the regulating substance. Simultaneous addition of BSO and 0.5 mm GSH to the culture medium decreased APR mRNA, enzyme protein and activity. ATP sulphurylase activity was not affected by this treatment. Tracer experiments using 35SO42– in the presence of 0.5 mm l‐cysteine or GSH showed that both thiols decreased sulphate uptake, APR activity and the flux of label into cysteine, GSH and protein, but had no effect on the activity of all other enzymes of assimilatory sulphate reduction and serine acetyltransferase. These results are consistent with the hypothesis that thiols regulate the flux through sulphate assimilation at the uptake and the APR step. Analysis of radioactive labelling indicates that the flux control coefficient of APR is more than 0.5 for the intracellular pathway of sulphate assimilation. This analysis also shows that the uptake of external sulphate is inhibited by GSH to a greater extent than the flux through the pathway, and that the flux control coefficient of APR for the pathway, including the transport step, is proportionately less, with a significant share of the control exerted by the transport step.
Lipid peroxidation in cotton: Xanthomonas interactions and the role of lipoxygenases during the hypersensitive reaction
Authors: Jalloul A. , Montillet J. L. , Assigbetsé K. , Agnel J.P. , Delannoy E. , Triantaphylidès C. , Daniel J.F. , Marmey P. , Geiger J.P. , Nicole M.
Published: March 2003

Lipid peroxidation, often associated with hypersensitive cell death, may be initiated either by active oxygen species (AOS) or lipoxygenases (LOX). Here we report a detailed analysis of this oxidative process in both incompatible and compatible interactions between the cotton cultivar Reba B50 and Xanthomonas campestris pv. malvacearum (Xcm). The hypersensitive reaction (HR) was characterized by a massive production of polyunsaturated fatty acid (PUFA) hydroperoxides together with typical tissue dehydration. Among these, isomers peroxidized on carbon 9, largely predominant, were chiral, showing an excess in the S enantiomer. The HR process was accompanied by an increase in 9S‐LOX activity and preceded by transcription of a LOX gene (GhKLox1). These results showed that: (i) AOS produced during the oxidative burst were not involved in PUFA peroxidation during HR; and (ii) as previously described in elicited leaves of tobacco, the massive enzymatic lipid peroxidation was closely associated with hypersensitive cell death. During disease development in this cotton cultivar, the 9‐lipoxygenation of PUFAs was late, weak, preceded by a faint accumulation of GhKLox1 transcripts, and associated with chlorosis but not with necrosis. Consequently, the main difference between incompatible and compatible interactions was in the precocity and intensity of the oxidative process, rather than in its nature. These data provide the evidence for a correlation between lipid peroxidation and hypersensitive cell death induced by pathogens.
NAN fusions: a synthetic sialidase reporter gene as a sensitive and versatile partner for GUS
Authors: Kirby J. , Kavanagh T. A.
Published: March 2003

GUS continues to be the reporter of choice for many gene fusion applications, due to the unparalleled sensitivity of the encoded enzyme and the ease with which it can be quantified in cell‐free extracts and visualized histochemically in cells and tissues. A compatible and functionally equivalent reporter gene would facilitate dual promoter studies and internal standardization of expression analyses in the same plant. A search for a candidate enzyme activity not found in plants, which might form the basis of a novel GUS‐compatible reporter system, led us to investigate nanH, a Clostridium perfringens gene which encodes the so‐called ‘small’ cytoplasmic sialidase. Expression of the native, AT‐rich nanH gene in transgenic plants did not, however, result in detectable sialidase activity. For this reason, a codon‐optimized derivative, NAN, was synthesized which possesses a GC content similar to that found in highly expressed plant genes. NAN enzyme activity was expressed at high levels in both stably and transiently transformed cells, possessed kinetic and stability properties similar to those of GUS, and showed optimal activity in GUS buffer. Moreover, NAN and GUS activity could be visualized simultaneously in polyacrylamide gels using the corresponding methylumbelliferone‐based substrates, and in whole seedlings and tissue sections using the histochemical substrates 5‐bromo‐4‐chloro‐3‐indolyl α‐d‐N‐acetylneuraminic acid (X‐NeuNAc) and 5‐bromo‐6‐chloro‐3‐indolyl β‐d‐glucuronide (X‐GlucM), respectively.
Chemical regulated production of cDNAs from genomic DNA fragments in plants
Authors: Møller Simon Geir , Chua Nam‐Hai
Published: March 2003

We have developed a new chemical inducible genetic system that allows for the isolation of any cDNA molecule from in vitro generated genomic transgenes in transgenic plants. This system, termed regulated in vivo cDNA generation (RIDE), permits both targeted isolation of individual full‐length cDNA molecules and random isolation of any partial or full‐length cDNA from in planta genomic libraries. The RIDE system makes use of the 17‐β estradiol‐inducible promoter system linked to intron donor and acceptor sites in a new binary vector configuration. In transgenic Arabidopsis and tobacco plants, we show that the RIDE system can isolate low‐abundance full‐length cDNAs previously unattainable by conventional means at high efficiencies (75–85%). The ability to randomly isolate individual exons and exons spliced together from genomic libraries in planta suggest that this system can be used for the isolation of any cDNA molecules. The RIDE system thus appears to be an efficient and versatile system for the generation of potentially any cDNA molecule. Moreover, the ORF structural data generated will be of value in both verifying and correcting computational ORF predications in the databases available to the scientific community.
Pasticcino2 is a protein tyrosine phosphatase‐like involved in cell proliferation and differentiation in Arabidopsis
Authors: Bellec Yannick , Harrar Yaël , Butaeye Christelle , Darnet Sylvain , Bellini Catherine , Faure Jean‐Denis
Published: March 2003

The pasticcino2 (pas2) mutant shows impaired embryo and seedling development associated with cell de‐differentiation and proliferation. This process is specifically enhanced in presence of cytokinins leading to callus‐like structure of the apical part of the seedling. Cell proliferation concerns localized and stochastic nodules of dividing cells. In absence of cytokinins, cell proliferation leads to small calli on stems but, most often, cell proliferation is associated with post‐genital organ fusion. The PAS2 gene was identified by positional cloning. PAS2 expression was found in every plant organ and was not regulated by PAS1 and PAS3 genes. PAS2 encodes the Arabidopsis member of the protein tyrosine phosphatase‐like (Ptpl) family, a new PTP family originally described in mice and humans and characterized by a mutated PTP active site. This family of proteins has a yeast homolog that is essential for cell viability. The absence of yeast PAS2 homolog can be functionally replaced by the Arabidopsis PAS2 protein, demonstrating that PAS2 function is conserved between higher and lower eukaryotes.
Spatio‐temporal expression of patatin‐like lipid acyl hydrolases and accumulation of jasmonates in elicitor‐treated tobacco leaves are not affected by endogenous levels of salicylic acid
Authors: Dhondt Sandrine , Gouzerh Guillaume , Müller Axel , Legrand Michel , Heitz Thierry
Published: March 2003

We have previously isolated three tobacco genes (NtPat) encoding patatin‐like proteins, getting rapidly induced during the hypersensitive response (HR) to tobacco mosaic virus, in advance to jasmonate accumulation. NtPAT enzymes are lipid acyl hydrolases that display high phospholipase A2 (PLA2) activity and may mobilize fatty acid precursors of oxylipins. Here, we performed a detailed study of NtPat gene regulation under various biotic and abiotic stresses. PLA2 activity was poorly induced in response to drought, wounding, reactive oxygen intermediates, salicylic acid (SA) or methyl‐jasmonate (MJ) whereas the ethylene (ET) precursor, 1‐aminocyclopropane‐1‐carboxylic acid (ACC), provoked a moderate induction. In contrast, PLA2 activity was strongly induced when ACC was combined with MJ, and in response to the bacterium Erwinia carotovora or to the fungus Botrytis cinerea, as well as to treatment with β‐megaspermin, a cell death‐inducing protein elicitor. A simplified system based on the infiltration of β‐megaspermin into leaves was used to dissect the spatio‐temporal activation of PLA2 activity with regards to the accumulation of jasmonates and to the influence of endogenous SA. NtPat‐encoded PLA2 activity was rapidly induced in the infiltrated zone before the appearance of cell death and with some delay in the surrounding living cells. A massive accumulation of 12‐oxo‐phytodienoic and jasmonic acids occurred in the elicitor‐infiltrated zone, but only low levels were detectable outside this area. A similar picture was found in SA‐deficient plants, showing that in tobacco, accumulation of jasmonates is not affected by the concomitant HR‐induced build‐up of endogenous SA. Finally, ET‐insensitive plants showed a weakened induction of PLA2 activity outside the elicitor‐infiltrated tissue.