bluuebunny/crossref_metadata_2025_split huggingface dataset:
Double mutation in Eleusine indica α‐tubulin increases the resistance of transgenic maize calli to dinitroaniline and phosphorothioamidate herbicides
Authors: Anthony Richard G. , Hussey Patrick J. 
Published: March 2003

SummaryThe repeated use of dinitroaniline herbicides on the cotton and soybean fields of the southern United States has resulted in the appearance of resistant biotypes of one of the world's worst weeds, Eleusine indica. Two biotypes have been characterized, a highly resistant (R) biotype and an intermediate resistant (I) biotype. In both cases the resistance has been attributed to a mutation in α‐tubulin, a component of the α/β tubulin dimer that is the major constituent of microtubules. We show here that the I‐biotype mutation, like the R‐biotype mutation shown in earlier work, can confer dinitroaniline resistance on transgenic maize calli. The level of resistance obtained is the same as that for E. indica I‐ or R‐biotype seedlings. The combined I‐ and R‐biotype mutations increase the herbicide tolerance of transgenic maize calli by a value close to the summation of the maximum herbicide tolerances of calli harbouring the single mutations. These data, taken together with the position of the two different mutations within the atomic structure of the α/β tubulin dimer, imply that each mutation is likely to exert its effect by a different mechanism. These mechanisms may involve increasing the stability of microtubules against the depolymerizing effects of the herbicide or changing the conformation of the α/β dimer so that herbicide binding is less effective, or a combination of both possibilities.
Footprinting in vivo reveals changing profiles of multiple factor interactions with the β‐phaseolin promoter during embryogenesis
Authors: Li Guofu , Hall Timothy C. 
Published: March 2003

SummaryWhereas in vitro techniques are essentially limited to the analysis of interactions with a single or limited number of cis‐elements, in vivo footprinting techniques can be used to assess the total profile of factor interactions with a promoter. By probing with dimethylsulphate and using sensitive ligation‐mediated PCR analytical techniques, the in vivo status of the phas promoter was determined in transcriptionally active (embryo) and inactive (leaf) tissues. Changes in factor occupancy were detected during embryogenesis, and the greatest complexity seen (at mid‐maturation) was in accordance with the many potential binding sites predicted on the basis of sequence comparison. Evidence was obtained that several cis‐elements not previously shown to be used for factor binding in plant promoters are occupied. The great complexity of footprints may represent the need for multiple factor interaction to achieve high levels of transcription. Alternatively, it is possible that the differential levels of expression in individual regions of the embryo evident from histochemical analysis of the GUS reporter result from the interaction of relatively few factors, with the overall footprinting pattern representing a summation of patterns from various tissues.
5′ to 3′ exoribonucleolytic activity is a normal component of chloroplast mRNA decay pathways
Authors: Drager Robert G. , Higgs David C. , Kindle Karen L. , Stern David B. 
Published: March 2003

SummaryMolecular genetic studies have shown that determinants of chloroplast mRNA stability lie in both the 5′ and 3′ untranslated regions. While it is well‐known that chloroplast mRNAs are unstable in the absence of certain nucleus‐encoded factors, little is known of the decay mechanisms for chloroplast mRNA in wild‐type cells. Here we used a poly(G)18 sequence, which impedes both 5′→3′ and 3′→5′ exoribonucleolytic RNA decay in vivo, to study the degradation pathway of petD mRNA in wild‐type and mcd1 mutant chloroplasts of Chlamydomonas; the mcd1 mutant lacks a nucleus‐encoded factor required for petD mRNA accumulation. Upon inserting poly(G) at positions –20, +25, +165 or +25/+165 relative to the mature petD 5′ end, mRNAs accumulate with 5′ ends corresponding to the poly(G) sequence, in addition to the normal RNA with its 5′ end at +1. We interpret these results as evidence for continuous degradation of petD mRNA in wild‐type cells by a 5′→3′ exoribonucleolytic activity. In the case of the –20 insertion, the accumulating RNA can be interpreted as a processing intermediate, suggesting that 5′ end maturation may also involve this activity. When examined in the mcd1 mutant background, petD mRNAs with the poly(G) 5′ ends, but not normal +1 ends, accumulated. However, no expression of SUIV, the petD gene product, was detected. Insertion of poly(G) at +165 in wild‐type cells did not demonstrably affect SUIV accumulation, suggesting that ribosomal scanning does not occur upstream of this position. However, since neither poly(G) –20 nor +165 RNA could be translated in mcd1 cells, this raises the possibility that the MCD1 product is essential for translation.
Sequences throughout the basic β‐1,3‐glucanase mRNA coding region are targets for homology dependent post‐transcriptional gene silencing
Authors: Jacobs John J. M. R. , Sanders Matthew , Bots Marc , Andriessen Marco , van Eldik Gerben J. , Litière Katia , Van Montagu Marc , Cornelissen Marc 
Published: March 2003

SummaryIn the transgenic tobacco line T17, plants homozygous for the gn1 transgene display developmentally regulated post‐transcriptional silencing of basic β‐1,3‐glucanase genes. Previously, it has been shown that silencing involves a markedly increased turnover of silencing‐target glucanase mRNAs. Using a two‐component viral reporter system facilitated a comparison, in a quantitat‐ ive manner, of the relative silencing efficiencies of various sequences derived from the gn1 transgene. The results show that target sites for the silencing mechanism are present throughout the coding region of the gn1 mRNA. Similar‐sized coding region sequences along the entire gn1 mRNA display a similar susceptibility to the silencing mechanism. The susceptibility to silencing increases as the coding region elements increase in size. Relative to internal sequences, the 5′ and 3′ terminal regions of the gn1 mRNA are inefficient targets for the silencing machinery. Importantly, sequences of the gn1 transgene that are not part of the mature gn1 mRNA are not recognized by the silencing machinery when expressed in chimeric viral RNAs. These results show that the glucanase silencing mechanism in T17 plants is primarily directed against gn1 mRNA‐internal sequences and that terminal sequences of the gn1 mRNA are relatively unaffected by the silencing mechanism.
S‐protein mutants indicate a functional role for SBP in the self‐incompatibility reaction of Papaver rhoeas
Authors: Jordan Nicholas D. , Kakeda Katsuyuki , Conner Alex , Ride Jon P. , Franklin‐Tong Vernonica E. , Franklin F. Christopher H. 
Published: March 2003

SummaryThe self‐incompatibility response involves S‐allele specific recognition between stigmatic S proteins and incompatible pollen, resulting in S‐specific pollen inhibition. In Papaver rhoeas, the pollen S gene product is predicted to be a receptor that interacts with the stigmatic S protein in an S specific manner. We recently identified an S protein binding protein (SBP) in pollen that binds stigmatic S proteins, although apparently not in an S‐allele‐specific manner. In order to investigate the functional significance of the interaction between S proteins and SBP, we constructed mutant derivatives of the S1 protein and tested their SBP‐binding activity and their biological activity. Here we present an evaluation of nine mutant derivatives of the S1 protein. Western ligand blotting was used to show that mutations to amino acid residues in predicted loops 2 and 6 of the S1 protein cause significant reductions in their SBP‐binding activity. These same mutants show a concomitant reduction in their ability to inhibit incompatible pollen. This establishes a direct link between SBP binding and inhibition of incompatible pollen and implicates SBP as a pollen component playing a key role in the self‐incompatibility reaction. We discuss the possible nature of the contribution of SBP in the S‐specific rejection of incompatible pollen.
Retrotransposon BARE‐1: expression of encoded proteins and formation of virus‐like particles in barley cells
Authors: Jääskeläinen Marko , Mykkänen Anna‐Helena , Arna Tiina , Vicient Carlos M. , Suoniemi Annu , Kalendar Ruslan , Savilahti Harri , Schulman Alan H. 
Published: March 2003

SummaryRetrotransposons are ubiquitous and major components of plant genomes, and are characteristically retroviral‐like in their genomic structure and in the major proteins encoded. Nevertheless, few have been directly demonstrated to be transcribed or reverse transcribed. The BARE‐1 retrotransposon family of barley (Hordeum vulgare) is highly prevalent, actively transcribed, and contains well conserved functional regions. Insertion sites for BARE‐1 are highly polymorphic in the barley genome. Here we show that BARE‐1 is translated and the capsid protein (GAG) and integrase (IN) components of the predicted polyprotein are processed into polypeptides of expected size. Some of the GAG sediments as virus‐like particles together with IN and with BARE‐1 cDNA. Reverse transcriptase activity is also present in gradient fractions containing BARE‐1 translation products. Virus‐like particles have also been visualized in fractions containing BARE‐1 components. Thus BARE‐1 components necessary for carrying out the life cycle of an active retrotransposon appear to be present in vivo, and to assemble. This would suggest that post‐translational mechanisms may be at work to prevent rapid genome inflation through unrestricted integration.
Display and isolation of transposon‐flanking sequences starting from genomic DNA or RNA
Authors: Yephremov Alexander , Saedler Heinz 
Published: March 2003

SummaryInsertion mutagenesis using transposons has become a powerful tool for the isolation of genes involved in any given biochemical or developmental pathway. We describe here ligation‐mediated PCR techniques for the isolation of sequences flanking the transposable elements En/Spm, Mu1 and Cin4 in Zea mays and Arabidopsis thaliana. Two versions of this transposon insertion display (TID) method use biotinylated linkers or biotinylated primers to rapidly isolate transposon‐flanking sequences starting from digested genomic DNA. TID protocols have been employed to clone several genes from En/Spm insertion mutants of Arabidopsis. A novel procedure, expression TID (ETID), is also introduced, which provides a direct approach for the isolation of transposon insertions that tag transcribed portions of genes. ETID uses RNA as a starting material and exploits 5′ RACE PCR to identify transposon copies that form parts of gene transcripts. The detection of several En/Spm insertion mutations in Arabidopsis illustrates the power of this method. ETID offers important advantages for the isolation of mutant alleles of novel genes that are expressed in specific tissues in plants and animals.
Post‐translational regulation of cytosolic glutamine synthetase by reversible phosphorylation and 14‐3‐3 protein interaction
Authors: Finnemann Jørgen , Schjoerring Jan K. 
Published: March 2003

SummaryRegulation of the cytosolic isozyme of glutamine synthetase (GS1; EC 6.3.1.2) was studied in leaves of Brassica napus L. Expression and immunodetection studies showed that GS1 was the only active GS isozyme in senescing leaves. By use of [γ‐32P]ATP followed by immunodetection, it was shown that GS1 is a phospho‐protein. GS1 is regulated post‐translationally by reversible phosphorylation catalysed by protein kinases and microcystin‐sensitive serine/threonine protein phosphatases. Dephosphorylated GS1 is much more susceptible to degradation than the phosphorylated form. The phosphorylation status of GS1 changes during light/dark transitions and depends in vitro on the ATP/AMP ratio. Phosphorylated GS1 interacts with 14‐3‐3 proteins as verified by two different methods: a His‐tag 14‐3‐3 protein column affinity method combined with immunodetection, and a far‐Western method with overlay of 14‐3‐3–GFP. The degree of interaction with 14‐3‐3‐proteins could be modified in vitro by decreasing or increasing the phosphorylation status of GS1. Thus, the results demonstrate that 14‐3‐3 protein is an activator molecule of cytosolic GS and provide the first evidence of a protein involved in the activation of plant cytosolic GS. The role of post‐translational regulation of cytosolic GS and interactions between phosphorylated cytosolic GS and 14‐3‐3 proteins in senescing leaves is discussed in relation to nitrogen remobilization.
Gibberellin response mutants identified by luciferase imaging
Authors: Meier Carsten , Bouquin Thomas , Nielsen Mads Eggert , Raventos Dora , Mattsson Ole , Rocher Anne , Schomburg Fritz , Amasino Richard M. , Mundy John 
Published: March 2003

SummaryThe gibberellin (GA) 20‐oxidase encoded by Arabidopsis GA5 catalyzes the synthesis of active GAs. GA5 is a regulatory step in GA biosynthesis as GA5 mRNA levels are negatively regulated by its bioactive GA products. A fusion between the GA5 promoter and the firefly luciferase reporter (GA5‐LUC) was shown to be similarly regulated, indicating GA feedback of GA5 occurs at the transcriptional level. The fidelity of the GA5‐LUC reporter permitted a fusion genetic screen to identify mutants altered in transgene expression. This bioimaging screen identified two types of recessive mutants with increased LUC activity and apparent GA‐related growth phenotypes, a dwarf (lue1) and two late flowering mutants (fpa1‐3 and fpa1‐4). Mutant progeny exhibited altered levels of LUC and of endogenous GA5 and other GA‐regulated mRNAs. SSLP‐based mapping localized lue1 to chromosome I near the ga2 locus, although complementation analyzes showed that lue1 is not allelic to ga2. Mapping and complementation analyzes showed that the late flowering mutants are allelic to fpa1. This provides genetic evidence for crosstalk between the autonomous and gibberellin‐dependent flowering pathways.
Restructuring of wall‐bound xyloglucan by transglycosylation in living plant cells
Authors: Thompson James E. , Fry Stephen C. 
Published: March 2003

SummaryXyloglucan endotransglycosylases (XETs) cleave and then re‐join xyloglucan chains and may thus contribute to both wall‐assembly and wall‐loosening. The present experiments demonstrate the simultaneous occurrence in vivo of two types of interpolymeric transglycosylation: ‘integrational’ (in which a newly secreted xyloglucan reacts with a previously wall‐bound one) and ‘restructuring’ (in which one previously wall‐bound xyloglucan reacts with another). Xyloglucans synthesised by cultured rose (Rosa sp.) cells in ‘heavy’ or ‘light’ media (with [13C,2H]glucose or [12C,1H]glucose, respectively) had buoyant densities of 1.643 and 1.585 g ml−1, respectively, estimated by isopycnic centrifugation in caesium trifluoroacetate. To detect transglycosylation, we shifted heavy rose cells into light medium, then supplied a 2‐h pulse of l‐[1−3H]arabinose. Light [3H]xyloglucans were thus secreted into heavy, non‐radioactive walls and chased by light, non‐radioactive xyloglucans. At 2 h after the start of radiolabelling, the (neutral) [3H]xyloglucans were on average 29% heavy, indicating molecular grafting during integrational transglycosylation. The [3H]xyloglucans then gradually increased in density until, by 11 h, they were 38% heavy. This density increase suggests that restructuring transglycosylation reactions occurred between the now wall‐bound [3H]xyloglucan and other (mainly older, i.e. heavy) wall‐bound non‐radioactive xyloglucans. Brefeldin A (BFA), which blocked xyloglucan secretion, did not prevent the increase in density of wall‐bound [3H]xyloglucan (2−11 h). This confirms that restructuring transglycosylation occurred between pairs of previously wall‐bound xyloglucans. After 7 d in BFA, the 3H was in hybrid xyloglucans in which on average 55% of the molecule was heavy. Exogenous xyloglucan oligosaccharides (competing acceptor substrates for XETs) did not affect integrational transglycosylation whereas they inhibited restructuring transglycosylation. Possible reasons for this difference are discussed. This is the first experimental evidence for restructuring transglycosylation in vivo. We argue that both integrational and restructuring transglycosylation can contribute to both wall‐assembly and ‐loosening.
Coregulation of light‐harvesting complex II phosphorylation and lhcb mRNA accumulation in winter rye
Authors: Pursiheimo Saijaliisa , Mulo Paula , Rintamäki Eevi , Aro Eva‐Mari 
Published: March 2003

SummaryWinter rye plants grown under contrasting environmental conditions or just transiently shifted to varying light and temperature conditions, were studied to elucidate the chloroplast signal involved in regulation of photosynthesis genes in the nucleus. Photosystem II excitation pressure, reflecting the plastoquinone redox state, and the phosphorylation level of thylakoid light‐harvesting proteins (LHCII and CP29) were monitored together with changes occurring in the accumulation of lhcb, rbcS and psbA mRNAs. Short‐term shifts of plants to changed conditions, from 1 h to 2 d, were postulated to reveal signals crucial for the initiation of the acclimation process. Comparison of these results with those obtained from plants acclimated during several weeks' growth at contrasting temperature and in different light regimes, allow us to make the following conclusions: (1) LHCII protein phosphoylation is a sensitive tool to monitor redox changes in chloroplasts; (2) LHCII protein phosphorylation and lhcb mRNA accumulation occur under similar conditions and are possibly coregulated via an activation state of the same kinase (the LHCII kinase); (3) Maximal accumulation of lhcb mRNA during the diurnal light phase seems to require an active LHCII kinase whereas inactivation of the kinase is accompanied by dampening of the circadian oscillation in the amount of lhcb mRNA; (4) Excitation pressure of photosystem II (reduction state of the plastoquinone pool) is not directly involved in the regulation of lhcb mRNA accumulation. Instead (5) the redox status of the electron acceptors of photosystem I in the stromal compartment seems to be highly regulated and crucial for the regulation of lhcb gene expression in the nucleus.
Molecular cloning and functional identification of (+)‐δ‐cadinene‐8‐hydroxylase, a cytochrome P450 mono‐oxygenase (CYP706B1) of cotton sesquiterpene biosynthesis
Authors: Luo Ping , Wang Yan‐Hong , Wang Guo‐Dong , Essenberg Margaret , Chen Xiao‐Ya 
Published: March 2003

SummaryIn cotton, gossypol and related sesquiterpene aldehydes are present in the glands of aerial tissues and in epidermal cells of roots. A cytochrome P450 was found to be expressed in aerial tissues of glanded cotton cultivars, but not or at an extremely low level in the aerial tissues of a glandless cultivar. Its cDNA was then isolated from Gossypium arboreum L. After expression in Saccharomyces cerevisiae, the P450 was found to catalyse the hydroxylation of (+)‐δ‐cadinene, forming 8‐hydroxy‐(+)‐δ‐cadinene. This P450 mono‐oxygenase has been classified as CYP706B1, and is the first member of the CYP706 family for which a function has been determined. Sesquiterpene aldehydes and CYP706B1 transcripts were detected in roots of both the glanded and glandless cultivars and in aerial tissues of the glanded cultivar. In suspension cultured cells of G. arboreum, elicitors prepared from the phytopathogenic fungus Verticillium dahliae caused a dramatic induction of CYP706B1 expression. The expression pattern of CYP706B1 and the position at which it hydroxylates (+)‐δ‐cadinene suggest that it catalyses an early step in gossypol biosynthesis. Southern blotting revealed a single copy of CYP706B1 in the genome of G. arboreum. CYP706B1 holds good potential for manipulation of gossypol levels in cottonseed via genetic engineering.
Overexpression of RCN1 and RCN2, rice TERMINAL FLOWER 1/CENTRORADIALIS homologs, confers delay of phase transition and altered panicle morphology in rice
Authors: Nakagawa Mayu , Shimamoto Ko , Kyozuka Junko 
Published: March 2003

Summary TERMINAL FLOWER 1 (TFL1)/CENTRORADIALIS (CEN)‐like genes play important roles in determining plant architecture, mainly by controlling the timing of phase transition. To investigate the possibility of similar mechanisms operating in the control of inflorescence architecture in rice, we analysed the functions of RCN1 and RCN2, rice TFL1/CEN homologs. Constitutive overexpression of RCN1 or RCN2 in Arabidopsis caused a late‐flowering and highly branching phenotype, indicating that they possess conserved biochemical functions as TFL1. In 35S::RCN1 and 35S::RCN2 transgenic rice plants, the delay of transition to the reproductive phase was observed. The transgenic rice plants exhibited a more branched, denser panicle morphology. Detailed observation of the panicle structure revealed that the phase change from the branch shoot to the floral meristem state was also delayed, leading to the generation of higher‐order panicle branches. These results suggest rice has a pathway that can respond to the overexpressed TFL1/CEN‐like functions, and the molecular mechanisms controlling the phase transition of meristems are conserved between grass and dicot species, at least to some extent.
Overexpression of Arabidopsis thaliana farnesyl diphosphate synthase (FPS1S) in transgenic Arabidopsis induces a cell death/senescence‐like response and reduced cytokinin levels
Authors: Masferrer Angela , Arró Montserrat , Manzano David , Schaller Hubert , Fernández‐Busquets Xavier , Moncaleán Paloma , Fernández Belén , Cunillera Núria , Boronat Albert , Ferrer Albert 
Published: March 2003

SummaryTo investigate the contribution of farnesyl diphosphate synthase (FPS) to the overall control of the mevalonic acid pathway in plants, we have generated transgenic Arabidopsis thaliana overexpressing the Arabidopsis FPS1S isoform. Despite high levels of FPS activity in transgenic plants (8‐ to 12‐fold as compared to wild‐type plants), the content of sterols and the levels of 3‐hydroxy‐3‐methylglutaryl‐CoA reductase activity in leaves were similar to those in control plants. Plants overexpressing FPS1S showed a cell death/senescence‐like phenotype and grew less vigorously than wild‐type plants. The onset and the severity of these phenotypes directly correlated with the levels of FPS activity. In leaves of plants with increased FPS activity, the expression of the senescence activated gene SAG12 was prematurely induced. Transgenic plants grown in the presence of either mevalonic acid (MVA) or the cytokinin 2‐isopentenyladenine (2‐iP) recovered the wild‐type phenotype. Quantification of endogenous cytokinins demonstrated that FPS1S overexpression specifically reduces the levels of endogenous zeatin‐type cytokinins in leaves. Altogether these results support the notion that increasing FPS activity without a concomitant increase of MVA production leads to a reduction of IPP and DMAPP available for cytokinin biosynthesis. The reduced cytokinin levels would be, at least in part, responsible for the phenotypic alterations observed in the transgenic plants. The finding that wild‐type and transgenic plants accumulated similar increased amounts of sterols when grown in the presence of exogenous MVA suggests that FPS1S is not limiting for sterol biosynthesis.