bluuebunny/crossref_metadata_2025_split huggingface dataset:
Developmental and environmental regulation of tissue‐ and cell‐specific expression for a pea protein farnesyltransferase gene in transgenic plants
Authors: Zhou Dafeng , Qian Daqi , Cramer Carole L. , Yang Zhenbiao 
Published: March 2003

SummaryFarnesylation mediates membrane targeting and in vivo activities of several key regulatory proteins such as Ras and Ras‐related GTPases and protein kinases in yeast and mammals, and is implicated in cell cycle control and abscisic acid (ABA) signaling in plants. In this study, the developmental expression of a pea protein farnesyl‐transferase (FTase) gene was examined using transgenic expression of the β‐glucuronidase (GUS) gene fused to a 3.2 kb 5′ upstream sequence of the gene encoding the pea FTase β subunit. Coordinate expression of the GUS transgene and endogenous tobacco FTase β subunit gene in tobacco cell lines suggests that the 3.2 kb region contains the key FTase promoter elements. In transgenic tobacco plants, GUS expression is most prominent in meristematic tissues such as root tips, lateral root primordia and the shoot apex, supporting a role for FTase in the control of the cell cycle in plants. GUS activity was also detected in mature embryos and imbibed embryos, in accordance with a role for FTase in ABA signaling that modulates seed dormancy and germination. In addition, GUS activity was detected in regions that border two organs, e.g. junctions between stems and leaf petioles, cotyledons and hypocotyls, roots and hypocotyls, and primary and secondary roots. GUS is expressed in phloem complexes that are adjacent to actively growing tissues such as young leaves, roots of light‐grown seedlings, and hypocotyls of dark‐grown seedlings. Both light and sugar (e.g. sucrose) treatments repressed GUS expression in dark‐grown seedlings. These expression patterns suggest a potential involvement of FTase in the regulation of nutrient allocation into actively growing tissues.
Analysis of SCARECROW expression using a rapid system for assessing transgene expression in Arabidopsis roots
Authors: Malamy Jocelyn E. , Benfey Philip N. 
Published: March 2003

SummaryAlthough the introduction of foreign genes into Arabidopsis has become routine, the production of transgenic Arabidopsis plants still requires several months. A transgene expression system (TES) has been developed that allows characterization of gene expression patterns and the effects of foreign genes in the Arabidopsis root in 2–4 weeks. The method is based on regeneration of stably transformed roots directly from callus tissue. TES has been used to study the expression of the SCARECROW gene, which is involved in establishing radial patterning in the root. The 2.5 kb region directly upstream of the SCARECROW coding region was found to be sufficient to confer cell‐type specific expression. Furthermore, this promoter is active in the scr mutant background, indicating that factors essential for cell‐type specific expression are present even in the absence of correct radial patterning. Finally, TES was used to demonstrate that the SCARECROW gene under control of this promoter complements the root organization defect of the scr mutant. These experiments demonstrate the utility of the TES system for studying gene expression in roots in wild‐type and mutant backgrounds and for molecular complementation of root mutant phenotypes. It is possible that the method will also be applicable to other organs.
Promoter elements required for phloem‐specific gene expression from the RTBV promoter in rice
Authors: Yin Yanhai , Chen Lili , Beachy Roger 
Published: March 2003

SummaryPrevious studies indicated that a DNA fragment comprising nucleotides (nt) −164 to +45 of the RTBV promoter is sufficient to drive phloem‐specific expression of a reporter gene in transgenic rice plants. In addition, two potential cis elements, Box I (nt −3 to +5) and Box II (nt −53 to −39) were identified by DNA‐protein interaction assays. In this study, the results of further in vivo studies involving mutagenesis of selected DNA sequences and analysis of expression of a reporter gene in transgenic rice plants revealed that, in addition to Box I and Box II, other elements are required for phloem‐specific gene expression, among which are a direct repeat (ASL Box, nt −98 to −79) and a GATA motif (nt −143 to −135). All the these elements bind rice nuclear factors specifically, and mutations of the elements were identified that resulted in loss‐of‐competition in electrophoretic mobility shift assays. A DNA fragment comprising nt −164 to −32, which contains Box II, the ASL Box and the GATA motif, conferred phloem‐specific reporter gene expression independent of Box I when fused to a heterologous CaMV 35S minimal promoter and introduced to transgenic rice plants. Studies that introduced point mutations suggested that in the context of the −103 to +45 promoter fragment, Box II and the ASL Box act synergistically to confer tissue‐specific gene expression. Similar studies in the −164 to +45 promoter fragment indicated that the −164 to −103 region, which includes the GATA motif, contains sequences that are functionally redundant with those in the −103 to −32 region, including the ASL Box and Box II. It is concluded that these regions act additively to direct phloem‐specific gene expression.
Expression of a dominant negative form of cpSRP54 inhibits chloroplast biogenesis in Arabidopsis
Authors: Pilgrim Marsha L. , Va Klaas‐Jan , Wijk  , Parry Devin H. , Sy Donna A. C. , Hoffman Neil E. 
Published: March 2003

SummaryThe chloroplast homolog of the 54 kDa subunit of signal recognition particle is required for the in vitro targeting of chlorophyll a/b binding proteins (LHCP) to the thylakoid membrane. To explore the function of cpSRP54 in vivo, plants that are mutated in cpSRP54 function were generated. Dominant negative forms of cpSRP54 altered in single amino acids within the conserved guanine nucleotide binding domain were expressed in Arabidopsis. Transformed plants contained less than 30% of the wild‐type level of cpSRP54 protein. As a consequence of the reduced cpSRP54 protein content, the first emerging leaves were yellow and contained immature chloroplasts. Although the chlorophyll (chl) content of the leaves was reduced by 75%, the chl a/b ratio was unaffected, indicating a role of cpSRP54 in the biogenesis of proteins besides LHCP. Many chloroplast proteins were less abundant in the first emerging leaves, including non‐pigmented proteins, thylakoid proteins known to be targeted by alternative pathways, and soluble proteins. These observations indicate that the cpSRP54 mutation also has a pleiotropic effect on chloroplast biogenesis. Whereas the level of cpSRP54 remained low as the plants aged, leaves emerging subsequently had a wild‐type appearance, suggesting that the adult plants compensated for the reduction in cpSRP54 protein.
Transcription of Arabidopsis and wheat Cab genes in single tobacco transgenic seedlings exhibits independent rhythms in a developmentally regulated fashion
Authors: Kolar Claudia , Fejes Erzsébet , Ádám Éva , Schäfer Eberhard , Kay Steven , Nagy Ferenc 
Published: March 2003

SummaryTranscription of Cab genes has been previously shown to be regulated by a circadian oscillator coupled to the red light‐absorbing plant photoreceptor phytochrome in various plant species. In addition, it has recently been suggested that rhythmic expression of the Cab genes could also be affected by a phytochrome‐independent circadian oscillator in a developmentally regulated fashion. This study has shown that a red light‐insensitive oscillator and a phytochrome‐coupled circadian clock indeed co‐regulate the oscillating expression of individual Cab genes at the level of transcription at an early developmental stage. The study involved analysing the expression patterns of transgenes, containing short fragments of the Arabidopsis thaliana Cab2 or the wheat Cab‐1 promoter fused to the firefly luciferase reporter gene, by a video‐imaging system in single, etiolated tobacco seedlings. Germination and red/far‐red light treatments applied between 12 and 36 h after sowing lead to the appearance of two independent circadian rhythms. These rhythms co‐exist, both exhibiting period lengths close to 25 h but phased differently. However, repeated red‐light treatments given 60 h or later after sowing synchronize these free‐running rhythms and induce a single new circadian oscillation. These data indicate that both oscillators regulate the expression of the Cab genes studied at the level of transcription and that the cis‐acting element(s) of the wheat Cab‐1 and A. thaliana Cab2 genes mediating these responses are located on short, 250 bp promoter regions. Furthermore, these red‐light induced rhythms are also inducible by far‐red light treatments alone. Therefore, in tobacco, the phytochrome‐coupled oscillator is regulated, at least partially, by the very low fluence response of phytochrome A.
Mode of phytochrome B action in the photoregulation of seed germination in Arabidopsis thaliana
Authors: Shinomura Tomoko , Hanzawa Hiroko , Schäfer Eberhard , Furuya Masaki 
Published: March 2003

Summary Arabidopsis thaliana seeds imbibed for a short duration show phytochrome B (PhyB)‐specific photo‐induction of germination. Using this system, the relationship was determined between the amount of PhyB in seeds and photon energy required for PhyB‐specific germination in two transgenic Arabidopsis lines transformed with either the Arabidopsis PhyB cDNA (ABO) or the rice PhyB cDNA (RBO). Immunochemical detection of PhyB apoprotein (PHYB) showed that the expression level of PHYB in ABO seeds was at least two times higher than that in the wild‐type seeds, but in RBO seeds the PHYB level was indistinguishable from that in wild‐type seeds. The photon fluence required for induction and photoreversible inhibition of germination was examined using the Okazaki large spectrograph. At the wavelengths of 400–710 nm, the ABO seeds required significantly less photon fluence than wild‐type seeds for induction of germination, whereas the RBO seeds required similar fluence to wild‐type seeds. A critical threshold wavelength for either induction or inhibition of germination of ABO seeds shifted towards the longer wavelengths relative to wild‐type seeds. By assuming that PhyA and PhyB are similar in their photochemical parameters, amounts of Pfr at each wavelength were calculated. The photon fluence required for 50% germination was equivalent to the fluence generating a Pfr/Ptot ratio of 0.21–0.43 in wild‐type seeds, and of 0.035–0.056 in ABO seeds. These results indicate that PhyB‐specific seed germination is not strictly a function of the Pfr/Ptot ratio, but is probably a function of the absolute Pfr concentration.
Combined expression of glucokinase and invertase in potato tubers leads to a dramatic reduction in starch accumulation and a stimulation of glycolysis
Authors: Trethewey Richard N. , Geigenberger Peter , Riedel Kerstin , Hajirezaei Mohammad‐Reza , Sonnewald Uwe , Stitt Mark , Riesmeier Jörg W. , Willmitzer Lothar 
Published: March 2003

SummaryThe original aim of this work was to increase starch accumulation in potato tubers by enhancing their capacity to metabolise sucrose. We previously reported that specific expression of a yeast invertase in the cytosol of tubers led to a 95% reduction in sucrose content, but that this was accompanied by a larger accumulation of glucose and a reduction in starch. In the present paper we introduced a bacterial glucokinase from Zymomonas mobilis into an invertase‐expressing transgenic line, with the intention of bringing the glucose into metabolism. Transgenic lines were obtained with up to threefold more glucokinase activity than in the parent invertase line and which did not accumulate glucose. Unexpectedly, there was a further dramatic reduction in starch content, down to 35% of wild‐type levels. Biochemical analysis of growing tuber tissue revealed large increases in the metabolic intermediates of glycolysis, organic acids and amino acids, two‐ to threefold increases in the maximum catalytic activities of key enzymes in the respiratory pathways, and three‐ to fivefold increases in carbon dioxide production. These changes occur in the lines expressing invertase, and are accentuated following introduction of the second transgene, glucokinase. We conclude that the expression of invertase in potato tubers leads to an increased flux through the glycolytic pathway at the expense of starch synthesis and that heterologous overexpression of glucokinase enhances this change in partitioning.
Plant chitinase/lysozyme isoforms show distinct substrate specificity and cleavage site preference towards lipochitooligosaccharide Nod signals
Authors: Schultze Michael , Staehelin Christian , Brunner Frédéric , Genetet Isabelle , Legrand Michel , Fritig Bernard , Kondorosi Éva , Kondorosi Ádám 
Published: March 2003

Summary The ability of 16 chitinases from seven different plant species to hydrolyze a collection of several structurally related lipochitooligosaccharides (LCOs) of

Rhizobium

was analyzed. It was found that the enzymes differed to a large extent in their activity on different LCOs. Differences were attributed to (i) the relative activity on different LCOs as substrate (e.g. sulfated versus non‐sulfated LCOs); (ii) the relative cleavage site preference on a given LCO molecule (hydrolysis of either the second, third or fourth glycosidic bond from the non‐reducing end of the molecule); and (iii) the stereochemistry of the reaction (retention or inversion of the anomeric configuration). A graphic representation of the different substrate specificities resulted in a ‘fingerprint’ that is characteristic for a given enzyme or a family of related enzymes. By comparing the LCO‐fingerprint of unknown enzymes with those obtained for already characterized proteins, it is possible to identify new glycosyl hydrolases. The high diversity of substrate specificity found among plant chitinases may reflect variations in the natural substrates of the enzymes, such as substitutions on the chitin moiety of fungal cell walls or, in plants, the presence of putative endogenous substrates related to LCOs.
Mutational analyses of a type 2 peroxisomal targeting signal that is capable of directing oligomeric protein import into tobacco BY‐2 glyoxysomes
Authors: Flynn C. Robb , Mullen Robert T. , Trelease Richard N. 
Published: March 2003

Summary In this study of the type 2 peroxisomal targeting signal (PTS2) pathway, we examined the apparent discontinuity and conservation of residues within the PTS2 nonapeptide and demonstrated that this topogenic signal is capable of directing heteromultimeric protein import in plant cells. Based on cumulative data showing that at least 26 unique, putative PTS2 nonapeptides occur within 12 diverse peroxisomal‐destined proteins, the current (‐R/K‐L/V/I‐X5‐H/Q‐L/A‐) as well as the original (‐R‐L‐X5‐H/Q‐L‐) PTS2 motif appear to be oversimplified. To assess the functionality of residues within the motif, rat liver thiolase (rthio) and various chimeric chloramphenicol acetyltransferase (CAT) proteins were expressed transiently in suspension‐cultured tobacco (

Nicotiana tabacum

L.) cv Bright Yellow cells (BY‐2), and their subcellular location was determined by immunofluorescence microscopy. Hemagglutinin (HA)‐epitope‐tagged‐CAT subunits, lacking a PTS2 (CAT‐HA), were ‘piggybacked’ into glyoxysomes by PTS2‐bearing CAT subunits (rthio‐CAT), whereas signal‐depleted CAT‐HA subunits that were modified to prevent oligomerization did not import into glyoxysomes. These results provided direct evidence that signal‐depleted subunits imported into peroxisomes were targeted to the organelle as oligomers (heteromers) by a PTS2. Mutational analysis of residues within PTS2 nonapeptides revealed that a number of amino acid substitutions were capable of maintaining targeting function. Furthermore, functionality of residues within the PTS2 nonapeptide did not appear to require a context‐specific environment conferred by adjacent residues. These results collectively suggest that the functional PTS2 is not solely defined as a sequence‐specific motif, i.e. ‐R/K‐X6‐H/Q‐A/L/F‐, but defined also by its structural motif that is dependent upon the physiochemical properties of residues within the nonapeptide.
Construction of two ordered cDNA libraries enriched in genes encoding plasmalemma and tonoplast proteins from a high‐efficiency expression library
Authors: Galaud Jean‐Philippe , Carrière Marguerite , Pauly Nicolas , Canut Hervé , Chalon Pascale , Caput Daniel , Pont‐Lezica Rafael F. 
Published: March 2003

Summary We constructed a high‐efficiency expression library from

Arabidopsis

cDNA clones by introducing a poly (dC) stretch at the 5′ end of the clones. This library enables the synthesis of proteins from all the cDNA clones present. We have screened the high‐efficiency expression library with antibodies raised against total proteins from

Arabidopsis

plasmalemma and tonoplast. With the positive clones, we have constructed two cDNA ordered libraries enriched in genes encoding plasmalemma (522 clones) and tonoplast proteins (594 clones). Partial sequencing of both libraries shows that a high proportion (47%) of the clones encoded putative membrane proteins, or membrane‐associated proteins. When sequenced, 55% of the cDNAs were new EST sequences for

Arabidopsis

, 26% were similar to genes present in other plants or organisms, and 29% were not referenced in any databank. Immunoscreening of the two cDNA ordered libraries with antibodies raised against proteins from

Arabidopsis

cells submitted to osmotic stress allows the selection of genes over‐ and under‐expressed in stress conditions.
A bacterial haloalkane dehalogenase gene as a negative selectable marker in Arabidopsis
Authors: Næsted Henrik , Fennema Marko , Hao Lin , Andersen Mathias , Janssen Dick B. , Mundy John 
Published: March 2003

Summary The dhlA gene of Xanthobacter autotrophicus GJ10 encodes a dehalogenase which hydrolyzes dihalo‐ alkanes, such as 1,2‐dichloroethane (DCE), to a halo‐ genated alcohol and an inorganic halide (
Janssen et al. 1994
, Annu. Rev. Microbiol. 48, 163–191). In Xanthobacter, these alcohols are further catabolized by alcohol and aldehyde dehydrogenase activities, and by the product of the dhlB gene to a second halide and a hydroxyacid. The intermediate halogenated alcohols and, in particular, the aldehydes are more toxic than the haloalkane substrates or the pathway products. We show here that plants, including Arabidopsis, tobacco, oil seed rape and rice, do not express detectable haloalkane dehalogenase activities, and that wild‐type Arabidopsis grows in the presence of DCE. In contrast, DCE applied as a volatile can be used to select on plates or in soil transgenic Arabidopsis which express dhlA. The dhlA marker therefore provides haloalkane dehalogenase reporter activity and substrate dependent negative selection in transgenic plants.
The late pollen‐specific actins in angiosperms
Authors: Kandasamy Muthugapatti K. , McKinney Elizabeth C. , Meagher Richard B. 
Published: March 2003

SummaryThe actin gene family of Arabidopsis has eight functional genes that are grouped into two ancient classes, vegetative and reproductive, and into five subclasses based on their phylogeny and mRNA expression patterns. Progress in deciphering the functional significance of this diversity is hindered by the lack of tools that can distinguish the highly conserved subclasses of actin proteins at the biochemical and cellular level. In order to address the functional diversity of actin isovariants, we have used Arabidopsis recombinant actins as immunogens and produced several new anti‐actin monoclonal antibodies. One of them, MAb45a, specifically recognizes two closely related reproductive subclasses of actins. On immunoblots, MAb45a reacts strongly with actins expressed in mature pollen but not with actins in other Arabidopsis tissues. Moreover, immunocytochemical studies show that this antibody can distinguish actin filaments in pollen tubes from those in most vegetative tissues. Peptide competition analyses demonstrate that asparagine at position 79 (Asn79) within an otherwise conserved sequence is essential for MAb45a specificity. Actins with the Asn79 epitope are also expressed in the mature pollen from diverse angiosperms and Ephedra but not from lower gymnosperms, suggesting that this epitope arose in an ancestor common to angiosperms and advanced gymnosperms more than 220 million years ago. During late pollen development in angio‐ sperms there is a switch in expression of actins from vegetative to predominantly reproductive subclasses, perhaps to fulfil the unique functions of pollen in fertilization.
Plant interstitial telomere motifs participate in the control of gene expression in root meristems
Authors: Tremousaygue Dominique , Manevski Alexandra , Bardet Claude , Lescure Nicole , Lescure Bernard 
Published: March 2003

SummaryThe promoters of Arabidopsis eEF1A genes contain a telomere motif, the telo‐box, associated with an activating sequence, the tef‐box. Database searches indicated the presence of telo‐boxes in the 5′ region of numerous genes encoding components of the translational apparatus. By using several promoter constructs we demonstrate that the telo‐box is required for the expression of a β‐glucoronidase gene in root primordia of transgenic Arabidopsis. This effect was observed when a telo‐box was inserted upstream or downstream from the transcription initiation site, and occurred in synergy with the tef‐box. These results clearly indicate that interstitial telomere motifs in plants are involved in control of gene expression. South‐western screening of a λZAP library with a double‐stranded Arabidopsis telomere motif resulted in characterization of a protein related to the conserved animal protein Purα. The possibility of a regulation process similar to that achieved by the Rap1p in Saccharomyces cerevisiae is discussed.
Identification of a novel peptide motif that mediates cross‐linking of proteins to cell walls
Authors: Domingo Concha , Saurí Asunción , Mansilla Elena , Conejero Vicente , Vera Pablo 
Published: March 2003

SummaryA cDNA clone representing a member of a novel class of cell wall proteins was isolated from tobacco plants. We have designated this protein NtTLRP for tyrosine‐ and lysine‐rich protein. It is structurally related to the previously identified TLRP from tomato plants, sharing a high amino‐acid sequence similarity at the C‐terminal region. This region contains what appears to be a novel peptide motif which we call CD for cysteine‐rich domain, and which is common to several other cell‐wall proteins. By using a functional test in transgenic plants, we demonstrate that the presence of the CD domain is per se sufficient to cross‐link previously soluble proteins to the cell wall. We present evidence that NtTLRP is cross‐linked and specifically localizes to the cell wall of lignified cells. The highly localized deposition of NtTLRP in these cells indicates that this class of cell‐wall proteins may have a specialized function in the formation of xylem tissue.