bluuebunny/crossref_metadata_2025_split huggingface dataset:
Self‐processing 2A‐polyproteins – a system for co‐ordinate expression of multiple proteins in transgenic plants
Authors: Halpin Claire , Cooke Susan E. , Barakate Abdellah , Amrani Abdel El , Ryan Martin D. 
Published: March 2003

Summary Achieving co‐ordinate, high‐level and stable expression of multiple transgenes in plants is currently difficult. Expression levels are notoriously variable and influenced by factors that act independently on transgenes at different genetic loci. Instability of expression due to loss, re‐arrangement or silencing of transgenes may occur, and is exacerbated by increasing numbers of transgenic loci and repeated use of homologous sequences. Even linking two or more genes within a T‐DNA does not necessarily result in co‐ordinate expression. Linking proteins in a single open reading frame – a polyprotein – is a strategy for co‐ordinate expression used by many viruses. After translation, polyproteins are processed into constituent polypeptides, usually by proteinases encoded within the polyprotein itself. However, in foot‐and‐mouth disease virus (FMDV), a sequence (2A) of just 16–20 amino acids appears to have the unique capability to mediate cleavage at its own C‐terminus by an apparently enzyme‐independent, novel type of reaction. This sequence can also mediate cleavage in a heterologous protein context in a range of eukaryotic expression systems. We have constructed a plasmid in which the 2A sequence is inserted between the reporter genes chloramphenicol acetyltransferase (CAT) and β‐glucuronidase (GUS), maintaining a single open reading frame. Here we report that expression of this construct in wheatgerm lysate and transgenic plants results in efficient cleavage of the polyprotein and co‐ordinate expression of active CAT and GUS. Self‐processing polyproteins using the FMDV 2A sequence could therefore provide a system for ensuring coordinated, stable expression of multiple introduced proteins in plant cells.
Phosphorylation‐dependent interactions between enzymes of plant metabolism and 14‐3‐3 proteins
Authors: Moorhead Greg , Douglas Pauline , Cotelle Valérie , Harthill Jean , Morrice Nick , Meek Sarah , Deiting Uta , Stitt Mark , Scarabel Marie , Aitken Alastair , MacKintosh Carol 
Published: March 2003

SummaryFar‐Western overlays of soluble extracts of cauliflower revealed many proteins that bound to digoxygenin (DIG)‐labelled 14‐3‐3 proteins. Binding to DIG‐14‐3‐3s was prevented by prior dephosphorylation of the extract proteins or by competition with 14‐3‐3‐binding phosphopeptides, indicating that the 14‐3‐3 proteins bind to phosphorylated sites. The proteins that bound to the DIG‐14‐3‐3s were also immunoprecipitated from extracts with anti‐14‐3‐3 antibodies, demonstrating that they were bound to endogenous plant 14‐3‐3 proteins. 14‐3‐3‐binding proteins were purified from cauliflower extracts, in sufficient quantity for amino acid sequence analysis, by affinity chromatography on immobilised 14‐3‐3 proteins and specific elution with a 14‐3‐3‐binding phosphopeptide. Purified 14‐3‐3‐binding proteins included sucrose–phosphate synthase, trehalose‐6‐phosphate synthase, glutamine synthetases, a protein (LIM17) that has been implicated in early floral development, an approximately 20 kDa protein whose mRNA is induced by NaCl, and a calcium‐dependent protein kinase that was capable of phosphorylating and rendering nitrate reductase (NR) sensitive to inhibition by 14‐3‐3 proteins. In contrast to the phosphorylated NR‐14‐3‐3 complex which is activated by dissociation with 14‐3‐3‐binding phosphopeptides, the total sugar–phosphate synthase activity in plant extracts was inhibited by up to 40% by a 14‐3‐3‐binding phosphopeptide and the phosphopeptide‐inhibited activity was reactivated by adding excess 14‐3‐3 proteins. Thus, 14‐3‐3 proteins are implicated in regulating several aspects of primary N and C metabolism. The procedures described here will be valuable for determining how the phosphorylation and 14‐3‐3‐binding status of defined target proteins change in response to extracellular stimuli.
Glycine and serine catabolism in non‐photosynthetic higher plant cells: their role in C1 metabolism
Authors: Mouillon Jean‐Marie , Aubert Serge , Bourguignon Jacques , Gout Elisabeth , Douce Roland , Rébeillé Fabrice 
Published: March 2003

SummaryGlycine and serine are two interconvertible amino acids that play an important role in C1 metabolism. Using 13C NMR and various 13C‐labelled substrates, we studied the catabolism of each of these amino acids in non‐photosynthetic sycamore cambial cells. On one hand, we observed a rapid glycine catabolism that involved glycine oxidation by the mitochondrial glycine decarboxylase (GDC) system. The methylenetetra‐ hydrofolate (CH2‐THF) produced during this reaction did not equilibrate with the overall CH2‐THF pool, but was almost totally recycled by the mitochondrial serine hydroxymethyltransferase (SHMT) for the synthesis of one serine from a second molecule of glycine. Glycine, in contrast to serine, was a poor source of C1 units for the synthesis of methionine. On the other hand, catabolism of serine was about three times lower than catabolism of glycine. Part of this catabolism presumably involved the glycolytic pathway. However, the largest part (about two‐thirds) involved serine‐to‐glycine conversion by cytosolic SHMT, then glycine oxidation by GDC. The availability of cytosolic THF for the initial SHMT reaction is possibly the limiting factor of this catabolic pathway. These data support the view that serine catabolism in plants is essentially connected to C1 metabolism. The glycine formed during this process is rapidly oxidized by the mitochondrial GDC–SHMT enzymatic system, which is therefore required in all plant tissues.
The role of calcium in blue‐light‐dependent chloroplast movement in Lemna trisulca L.
Authors: Tlałka Monika , Fricker Mark 
Published: March 2003

SummaryChloroplast movements are a normal physiological response to changes in light intensity and provide a good model system to analyse the signal transduction pathways following light perception. Blue‐light‐dependent chloroplast movements were observed in Lemna trisulca using confocal optical sectioning and 3‐D reconstruction or photometric measurements of leaf transmission. Chloroplasts moved away from strong blue light (SBL) towards the anticlinal walls (profile position), and towards the periclinal walls (face position) under weak blue light (WBL) over about 20–40 min. Cytoplasmic calcium ([Ca2 + ]cyt) forms part of the signalling system in response to SBL as movements were associated with small increases in [Ca2 + ]cyt and were blocked by antagonists of calcium homeostasis, including EGTA, nifedipine, verapamil, caffeine, thapsigargin, TFP (trifluoperazine), W7 and compound 48/80. Treatments predicted to affect internal Ca2 +  stores gave the most rapid and pronounced effects. In addition, artificially increasing [Ca2 + ]cyt in darkness using the Ca2 +  ionophore A23187 and high external Ca2 +  (or Sr2 + ), triggered partial movement of chloroplasts to profile position analogous to a SBL response. These data are all consistent with [Ca2 + ]cyt acting as a signal in SBL responses; however, the situation is more complex given that both WBL and SBL responses were inhibited to a similar extent by all the Ca2 + ‐signalling antagonists used. As the direction of chloroplast movement in WBL is exactly opposite to that in SBL, we conclude that, whilst proper regulation of [Ca2 + ]cyt homeostasis is critical for both SBL and WBL responses, additional factors may be required to specify the direction of chloroplast movement.
Sucrose availability on the aerial part of the plant promotes morphogenesis and flowering of Arabidopsis in the dark
Authors: Roldán Marta , Gómez‐Mena Concepción , Ruiz‐García Leonor , Salinas Julio , Martínez‐Zapater José M. 
Published: March 2003

SummaryConditions to promote dark morphogenesis and flower‐ing in Arabidopsis have previously been limited to liquid cultures and to a few laboratory ecotypes. We have obtained development and flowering of Arabidopsis plants under complete darkness by growing them on vertical Petri dishes containing solid agar medium with sucrose. Under these conditions, all the ecotypes tested were able to develop, giving rise to etiolated plants that flowered after producing a certain number of leaves. Dark‐grown plants showed similarities with phytochrome‐deficient mutants and were different from de‐etiolated or constitutive photomorphogenesis mutants such as det and cop. Late‐ and early‐flowering ecotypes, showing large differences in flowering time and leaf number under long days, flowered with a similar number of leaves when grown in the dark. Rapid dark flowering of late‐flowering ecotypes was not an effect of darkness but the result of the interaction between dark and sucrose availability at the aerial part of the plant, since sucrose also had an effect when plants were grown in the light. Gibberellin‐deficient and insensitive mutants were delayed in the initiation of flowers in the dark, indicating a role for these hormones in flowering promotion in the dark. The late‐flowering phenotype of mutants at different loci of the autonomous and long‐day‐dependent flowering induction pathways was rescued in dark growth conditions. However, the late‐flowering phenotype of ft and fwa mutants was not rescued by sucrose either in the dark or in the light, suggesting a different role for these genes in flowering induction.
Differential regulation of three functional ammonium transporter genes by nitrogen in root hairs and by light in leaves of tomato
Authors: Von Wirén Nicolaus , Lauter Frank‐Roman , Ninnemann Olaf , Gillissen Bernd , Walch‐Liu Pia , Engels Christof , Jost Wolfgang , Frommer Wolf B. 
Published: March 2003

SummaryTo elucidate the role of transporters in N nutrition of tomato, two new transporter genes were isolated from cDNA libraries of root hairs or leaves of tomato. While LeAMT1;2 is closely related to LeAMT1;1 (75.6% amino acid identity), LeAMT1;3 is more distantly related (62.8% identity) and possesses two short upstream open reading frames in the 5′ end of the mRNA and a particularly short N‐terminus of the protein as unique features. When expressed in yeast mutants defective in uptake, all three genes complemented uptake. In roots of hydroponically grown plants, transcript levels of LeAMT1;2 increased after

or NO3– supply, while LeAMT1;1 was induced by N deficiency coinciding with low glutamine concentrations, and LeAMT1;3 was not detected. In aeroponic culture, expression of LeAMT1;1 and LeAMT1;2 was higher in root hairs than in the remaining root fraction. Growth of plants at elevated CO2 slightly decreased expression of LeAMT1;2 and LeAMT1;3 in leaves, but strongly repressed transcript levels of chloroplast glutamine synthetase and photorespiratory serine hydroxymethyl‐transferase. Expression of LeAMT1;2 and LeAMT1;3 showed a reciprocal diurnal regulation with highest transcript levels of LeAMT1;3 in darkness and highest levels of LeAMT1;2 after onset of light. These results indicate that in tomato at least two high‐affinity

transporters, LeAMT1;1 and LeAMT1;2, are differentially regulated by N and contribute to root hair‐mediated

acquisition from the rhizosphere. In leaves, the reciprocally expressed transporters LeAMT1;2 and LeAMT1;3 are supposed to play different roles in N metabolism,

uptake and/or NH3 retrieval during photorespiration.
c‐MYB oncogene‐like genes encoding three MYB repeats occur in all major plant lineages
Authors: Kranz Harald , Scholz Kai , Weisshaar Bernd 
Published: March 2003

SummarySince the identification of the first plant MYB‐like protein, the Zea mays factor C1, the number of MYB‐related genes described has greatly increased. All of the more than 150 plant MYB‐like proteins known so far contain either two or only one sequence‐related helix‐turn‐helix motif in their DNA‐binding domain. Animal c‐MYB genes contain three such helix‐turn‐helix motif‐encoding repeats (R1R2R3 class genes). It has therefore been concluded that R2R3‐MYB genes are the plant equivalents of c‐MYB and that there are significant differences in the basic structure of MYB genes of plants and animals. Here, we describe expressed R1R2R3‐MYB genes from Physcomitrella patens and Arabidopsis thaliana, designated PpMYB3R‐1 and AtMYB3R‐1. The amino acid sequences of their DNA‐binding domains show high similarity to those of animal MYB factors, and less similarity to R2R3‐MYB proteins from plants. In addition, R1R2R3‐MYB genes were identified in different plant evolutionary lineages including mosses, ferns and monocots. Our data show that a DNA‐binding domain consisting of three MYB repeats existed before the divergence of the animal and plant lineages. R1R2R3‐MYB genes may have a conserved function in eukaryotes, and R2R3‐MYB genes may predominantly regulate plant‐specific processes which evolved during plant speciation.
Gene regulation during late embryogenesis: the RY motif of maturation‐specific gene promoters is a direct target of the FUS3 gene product
Authors: Reidt Wim , Wohlfarth Thomas , Ellerström Mats , Czihal Andreas , Tewes Annegret , Ezcurra Ines , Rask Lars , Bäumlein Helmut 
Published: March 2003

SummaryThe Arabidopsis mutants fus3 and abi3 show pleiotropic effects during embryogenesis including reduced levels of transcripts encoding embryo‐specific seed proteins. To investigate the interaction between the B3‐domain‐containing transcription factors FUS3 and ABI3 with the RY cis‐motif, conserved in many seed‐specific promoters, a promoter analysis as well as band‐shift experiments were performed. The analysis of promoter mutants revealed the structural requirements for the function of the RY cis‐element. It is shown that both the nucleotide sequence and the alternation of purin and pyrimidin nucleotides (RY character) are essential for the activity of the motif. Further, it was shown that FUS3 and ABI3 can act independently of each other in controlling promoter activity and that the RY cis‐motif is a target for both transcription factors. For FUS3, which is so far the smallest known member of the B3‐domain family, a physical interaction with the RY motif was established. The functional and biochemical data demonstrate that the regulators FUS3 and ABI3 are essential components of a regulatory network acting in concert through the RY‐promoter element to control gene expression during late embryogenesis and seed development.
Conserved expression of Arabidopsis thaliana poly (A) binding protein 2 (PAB2) in distinct vegetative and reproductive tissues
Authors: Palanivelu Ravishankar , Belostotsky Dmitry A. , Meagher Richard B. 
Published: March 2003

SummaryThe poly(A) tails of eukaryotic mRNAs are complexed with poly(A) binding protein (PABP). The poly(A)–PABP complex is central to the efficient translation initiation and control of poly (A) tail length and is required in some pathways of mRNA decay. A large gene family encodes PABPs in Arabidopsis thaliana. In striking contrast to the floral and root specific expression of three previously reported Arabidopsis PABPs, we demonstrate that RNA and protein for one highly diverse member of this family, PAB2, are expressed in roots, stems, leaves, flowers, pollen and siliques of Arabidopsis. However, cell‐type specific analysis of a PAB2 reporter gene fusion revealed that PAB2 is spatially and temporally regulated in each organ. For example, strong expression was detected only in the stele and meristem region of roots and a dramatic decrease in expression was observed upon fertilization of ovules. Furthermore, the PAB2‐reporter construct gave a nearly identical expression pattern in transgenic tobacco, demonstrating that PAB2 expression is under strong selective constraint. The PAB2‐reporter was also strongly expressed in the transmittal tissues of both Arabidopsis and tobacco, raising the possibility of its involvement in the pollination‐dependent poly(A) tail shortening of transmittal tissue specific mRNAs previously reported in tobacco (
Wang et al. 1996
, Plant J. 9, 715–727). In view of its potential role in poly(A) tail shortening, we demonstrated the strong and distinct presence of PAB2 protein in transmittal tissues of Arabidopsis. The evolutionary and functional implications of the expression pattern of PAB2 and its possible functional roles in post‐transcriptional regulation in transmittal tissues are discussed.
Arabidopsis thaliana poly (A) binding protein 2 (PAB2) functions in yeast translational and mRNA decay processes
Authors: Palanivelu Ravishankar , Belostotsky Dmitry A. , Meagher Richard B. 
Published: March 2003

SummaryThe single yeast gene (PAB1) encoding poly (A) binding protein (PABP) has several roles in post‐transcriptional processes, including translation initiation and mRNA decay. PABP is encoded by a large gene family in plants. Within Arabidopsis thaliana, the several characterized PABP genes exhibit an extreme degree of sequence divergence and are differentially expressed. Arabidopsis PAB2 is expressed in distinct tissues or during defined developmental windows in most plant organs. In this study we demonstrate that PAB2 restores viability to a yeast pab1 mutant strain. Yeast strains containing wild‐type, null (PAB2s) and temperature sensitive (PAB2ts) alleles of PAB2 were used to explore the molecular functions of the plant protein. PAB2 can participate in poly (A) tail shortening, thus demonstrating that it interacts with the yeast poly(A) nuclease complex. PAB2 is required for translation, helping to maintain intact polysome structures. Consistent with its role in translation initiation, poly (A) was found to enhance PAB2 binding to Arabidopsis eIF‐iso4G in vitro. In addition, PAB2 can partially restore the linkage between deadenylation, decapping and mRNA decay in yeast. Taken together, our results suggest that Arabidopsis PAB2 participates in many of the same complex post‐transcriptional processes identified for yeast PAB1, and is functionally distinct from other characterized Arabidopsis PABPs.
Modified lignin in tobacco and poplar plants over‐expressing the Arabidopsis gene encoding ferulate 5‐hydroxylase
Authors: Franke Rochus , McMichael Colleen M. , Meyer Knut , Shirley Amber M. , Cusumano Joanne C. , Chapple Clint 
Published: March 2003

SummaryFerulate 5‐hydroxylase (F5H) is a cytochrome P450‐dependent monooxygenase that catalyses the hydroxylation of ferulic acid, coniferaldehyde and coniferyl alcohol in the pathways leading to sinapic acid and syringyl lignin biosynthesis. Earlier studies in Arabidopsis have demonstrated that F5H over‐expression increases lignin syringyl monomer content and abolishes the tissue‐specificity of its deposition. To determine whether this enzyme has a similar regulatory role in plants that undergo secondary growth, we over‐expressed the F5H gene in tobacco and poplar. In tobacco, over‐expression of F5H under the control of the cauliflower mosaic virus 35S promoter increased lignin syringyl monomer content in petioles, but had no detectable effect on lignification in stems. By contrast, when the cinnamate 4‐hydroxylase (C4H) promoter was used to drive F5H expression, there was a significant increase in stem lignin syringyl monomer content. Yields of thioglycolic acid and Klason lignin in C4H–F5H lines were lower than in the wild‐type, suggesting that F5H over‐expression leads to a reduced deposition or an altered extractability of lignin in the transgenic plants. Histochemical analysis suggested that the novel lignin in C4H–F5H transgenic lines was altered in its content of hydroxycinnamyl aldehydes. Transgenic poplar trees carrying the C4H–F5H transgene also displayed enhanced lignin syringyl monomer content. Taken together, these data show that hydroxylation of guaiacyl‐substituted lignin precursors controls lignin monomer composition in woody plants, and that F5H over‐expression is a viable metabolic engineering strategy for modifying lignin biosynthesis in forest species.
Photocontrol of subcellular partitioning of phytochrome‐B:GFP fusion protein in tobacco seedlings
Authors: Gil Patricia , Kircher Stefan , Adam Eva , Bury Erik , Kozma‐Bognar Laszlo , Schäfer Eberhard , Nagy Ferenc 
Published: March 2003

SummaryPhotomorphogenesis of higher plants is regulated by photoreceptors including the red/far‐red light‐absorbing phytochromes, blue‐UV/A sensing cryptochromes and as yet uncharacterized UV/B receptors. Specific phototransduction pathways that are controlled by either individual or interacting photoreceptors mediate regulation. Phytochrome B (phyB) is the major red light‐sensing photoreceptor. Phototransduction mediated by this light sensor has been shown to include light‐dependent nuclear import and interaction of phyB with transcription factor‐like proteins in the nucleus. Here we report that nuclear import of phyB and physiological responses regulated by this photoreceptor exhibit very similar wavelength‐ and fluence rate‐dependence. Nuclear import of phyB is insensitive to single red, blue and far‐red light pulses. It is induced by continuous red light and to a lesser extent by continuous blue light, whereas far‐red light is completely ineffective. The data presented indicate that light‐dependent partitioning of phyB exhibits features characteristic of blue light responsiveness amplification, a phenomenon that is thought to be mediated by interaction of phyB with CRY1.
Arabidopsis seed coat development: morphological differentiation of the outer integument
Authors: Windsor J. Brian , Symonds V. Vaughan , Mendenhall John , Lloyd Alan M. 
Published: March 2003

SummaryA morphological description of the differentiation of the outer integument of the Arabidopsis thaliana seed is presented. The period covered starts at about the octant embryo stage, extends to the mature seed, and concludes beyond that at the initial stages of seed imbibition. During this period the two‐cell‐layered outer integument goes through a dramatic differentiation process. The outer cell layer secretes mucilage in a ring between the plasma membrane and the outer cell wall at the corners of the cell. This secretion forces the cytoplasm into a columnar shape in the center of the cell. Before and during this process, starch granules are produced, initially at the center of the outer wall and later within the column. Late in differentiation, the starch granules are degraded as the cell produces a highly reinforced wall surrounding the columnar protoplast and at the radial walls between adjacent cells. This results in a cell containing large amounts of mucilage surrounding and completely outside of a highly reinforced columella. The mucilage and outer wall then dehydrate to leave the columella and radial walls visible as the epidermal plateau and reticulations visible on the mature seed. The inner cell layer of the outer integument also produces and degrades starch granules concomitantly with the outer layer but produces no mucilage. In the mature dry seed the collapsed outer wall remains connected to the top of the columella and the radial walls, but these connections are rapidly broken as the mucilage fully hydrates.