bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional analysis of HD2 histone deacetylase homologues in Arabidopsis thaliana
Authors: Wu Keqiang , Tian Lining , Malik Kamal , Brown Daniel , Miki Brian 
Published: March 2003

SummaryPost‐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

SummaryReversible 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

SummaryThe 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

SummaryA 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

SummaryWe 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

SummaryThe 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

SummaryThe 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

SummaryIn 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

SummaryDuring 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

SummaryThe 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

SummaryThree 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

Summary 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

SummaryNuclear 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

SummaryAn 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.