bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of calcium in blue‐light‐dependent chloroplast movement in Lemna trisulca L.
Authors: Tlałka Monika , Fricker Mark
Published: March 2003

Chloroplast 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

Conditions 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

To 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

Since 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

The 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

The 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

The 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

Ferulate 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

Photomorphogenesis 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

A 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.
Antisense inhibition of plastidial phosphoglucomutase provides compelling evidence that potato tuber amyloplasts import carbon from the cytosol in the form of glucose‐6‐phosphate
Authors: Tauberger Eva , Fernie Alisdair R. , Emmermann Michael , Renz Andreas , Kossmann Jens , Willmitzer Lothar , Trethewey Richard N.
Published: March 2003

The aim of this work was to establish whether plastidial phosphoglucomutase is involved in the starch biosynthetic pathway of potato tubers and thereby to determine the form in which carbon is imported into the potato amyloplast. For this purpose, we cloned the plastidial isoform of potato PGM (StpPGM), and using an antisense approach generated transgenic potato plants that exhibited decreased expression of the StpPGM gene and contained significantly reduced total phosphoglucomutase activity. We confirmed that this loss in activity was due specifically to a reduction in plastidial PGM activity. Potato lines with decreased activities of plastidial PGM exhibited no major changes in either whole‐plant or tuber morphology. However, tubers from these lines exhibited a dramatic (up to 40%) decrease in the accumulation of starch, and significant increases in the levels of sucrose and hexose phosphates. As tubers from these lines exhibited no changes in the maximal catalytic activities of other key enzymes of carbohydrate metabolism, we conclude that plastidial PGM forms part of the starch biosynthetic pathway of the potato tuber, and that glucose‐6‐phosphate is the major precursor taken up by amyloplasts in order to support starch synthesis.
Ectopic expression of a tomato 9‐cis‐epoxycarotenoid dioxygenase gene causes over‐production of abscisic acid
Authors: Thompson Andrew J. , Jackson Alison C. , Symonds Rachael C. , Mulholland Barry J. , Dadswell A. Ruth , Blake Patrick S. , Burbidge Alan , Taylor Ian B.
Published: March 2003

The tomato mutant notabilis has a wilty phenotype as a result of abscisic acid (ABA) deficiency. The wild‐type allele of notabilis, LeNCED1, encodes a putative 9‐cis‐epoxycarotenoid dioxygenase (NCED) with a potential regulatory role in ABA biosynthesis. We have created transgenic tobacco plants in which expression of the LeNCED1 coding region is under tetracycline‐inducible control. When leaf explants from these plants were treated with tetracycline, NCED mRNA was induced and bulk leaf ABA content increased by up to 10‐fold. Transgenic tomato plants were also produced containing the LeNCED1 coding region under the control of one of two strong constitutive promoters, either the doubly enhanced CaMV 35S promoter or the chimaeric ‘Super‐Promoter’. Many of these plants were wilty, suggesting co‐suppression of endogenous gene activity; however three transformants displayed a common, heritable phenotype that could be due to enhanced ABA biosynthesis, showing increased guttation and seed dormancy. Progeny from two of these transformants were further characterized, and it was shown that they also exhibited reduced stomatal conductance, increased NCED mRNA and elevated seed ABA content. Progeny of one transformant had significantly higher bulk leaf ABA content compared to the wild type. The increased seed dormancy was reversed by addition of the carotenoid biosynthesis inhibitor norflurazon. These data provide strong evidence that NCED is indeed a key regulatory enzyme in ABA biosynthesis in leaves, and demonstrate for the first time that plant ABA content can be increased through manipulating NCED.
Development of an efficient maintenance and screening system for large‐insert genomic DNA libraries of hexaploid wheat in a transformation‐competent artificial chromosome (TAC) vector
Authors: Liu Yao‐Guang , Nagaki Kiyotaka , Fujita Masako , Kawaura Kanako , Uozumi Masahiko , Ogihara Yasunari
Published: March 2003

Three large‐insert genomic DNA libraries of common wheat, Triticum aestivum cv. Chinese Spring, were constructed in a newly developed transformation‐competent artificial chromosome (TAC) vector, pYLTAC17, which accepts and maintains large genomic DNA fragments stably in both Escherichia coli and Agrobacterium tumefaciens. The vector contains the cis sequence required for Agrobacterium‐mediated gene transfer into grasses. The average insert sizes of the three genomic libraries were approximately 46, 65 and 120 kbp, covering three haploid genome equivalents. Genomic libraries were stored as frozen cultures in a 96‐well format, each well containing approximately 300–600 colonies (12 plates for small library, four for medium‐size library and four for large library). In each of the libraries, approximately 80% of the colonies harbored genomic DNA inserts of >50 kbp. TAC clones containing gene(s) of interest were identified by the pooled PCR technique. Once the target TAC clones were isolated, they could be immediately transferred into grass genomes with the Agrobacterium system. Five clones containing the thionin type I genes (single copy per genome), corresponding to each of the three genomes (A, B and D), were successfully selected by the pooled PCR method, in addition to an STS marker (aWG464; single copy per genome) and CAB (a multigene family). TAC libraries constructed as described here can be used to isolate genomic clones containing target genes, and to carry out genome walking for positional cloning.
Elevated levels of intrachromosomal homologous recombination in Arabidopsis overexpressing the MIM gene
Authors: Hanin Moez , Mengiste Tesfaye , Bogucki Augustyn , Paszkowski Jerzy
Published: March 2003

The Arabidopsis MIM gene encodes a protein belonging to the SMC family (structure maintenance of chromosomes) which is required for intrachromosomal homologous recombination (ICR). Both ICR and MIM gene expression are enhanced by DNA‐damaging treatments, suggesting that MIM is a factor limiting DNA repair by homologous recombination (HR) under genotoxic stress. We tested this hypothesis by measuring the levels of recombination in the mim mutant under genotoxic stress, using methyl methanesulfonate. Although the mutant clearly showed diminished basal and induced levels of ICR, enhancement of ICR by DNA‐damaging treatments was similar to that observed in the wild type. This suggests that the MIM gene product is required for DNA repair by HR, but is not critical for HR induction. To determine whether enhanced availability of MIM would increase basal HR levels in Arabidopsis, we examined ICR frequencies in transgenic Arabidopsis strains overexpressing the MIM gene after ectopic insertion of additional MIM copies. Two independent lines showed a twofold increase in ICR frequency relative to the wild type. Thus MIM is required for efficient ICR in plants, and its manipulation can be used to change homologous recombination frequencies. Since MIM is one of the components responsible for chromatin dynamics, our results suggest that the chromatin environment determines the frequency of homologous recombination.
An estrogen receptor‐based transactivator XVE mediates highly inducible gene expression in transgenic plants
Authors: Zuo Jianru , Niu Qi‐Wen , Chua Nam‐Hai
Published: March 2003

We have developed an estrogen receptor‐based chemical‐inducible system for use in transgenic plants. A chimeric transcription activator, XVE, was assembled by fusion of the DNA‐binding domain of the bacterial repressor LexA (X), the acidic transactivating domain of VP16 (V) and the regulatory region of the human estrogen receptor (E; ER). The transactivating activity of the chimeric XVE factor, whose expression was controlled by the strong constitutive promoter G10‐90, was strictly regulated by estrogens. In transgenic Arabidopsis and tobacco plants, estradiol‐activated XVE can stimulate expression of a GFP reporter gene controlled by the target promoter, which consists of eight copies of the LexA operator fused upstream of the −46 35S minimal promoter. Upon induction by estradiol, GFP expression levels can be eightfold higher than that transcribed from a 35S promoter, whereas the uninduced controls have no detectable GFP transcripts, as monitored by Northern blot analysis. Neither toxic nor adverse physiological effects of the XVE system have been observed in transgenic Arabidopsis plants under all the conditions tested. The XVE system thus appears to be a reliable and efficient chemical‐inducible system for regulating transgene expression in plants.
The S‐methylmethionine cycle in angiosperms: ubiquity, antiquity and activity
Authors: Ranocha Philippe , McNeil Scott D. , Ziemak Michael J. , Li Changjiang , Tarczynski Mitchell C. , Hanson Andrew D.
Published: March 2003

Angiosperms synthesize S‐methylmethionine (SMM) from methionine (Met) and S‐adenosylmethionine (AdoMet) in a unique reaction catalyzed by Met S‐methyltransferase (MMT). SMM serves as methyl donor for Met synthesis from homocysteine, catalyzed by homocysteine S‐methyltransferase (HMT). MMT and HMT together have been proposed to constitute a futile SMM cycle that stops the free Met pool from being depleted by an overshoot in AdoMet synthesis. Arabidopsis and maize have one MMT gene, and at least three HMT genes that belong to two anciently diverged classes and encode enzymes with distinct properties and expression patterns. SMM, and presumably its cycle, must therefore have originated before dicot and monocot lineages separated. Arabidopsis leaves, roots and developing seeds all express MMT and HMTs, and can metabolize [35S]Met to [35S]SMM and vice versa. The SMM cycle therefore operates throughout the plant. This appears to be a general feature of angiosperms, as digital gene expression profiles show that MMT and HMT are co‐expressed in leaves, roots and reproductive tissues of maize and other species. An in silico model of the SMM cycle in mature Arabidopsis leaves was developed from radiotracer kinetic measurements and pool size data. This model indicates that the SMM cycle consumes half the AdoMet produced, and suggests that the cycle serves to stop accumulation of AdoMet, rather than to prevent depletion of free Met. Because plants lack the negative feedback loops that regulate AdoMet pool size in other eukaryotes, the SMM cycle may be the main mechanism whereby plants achieve short‐term control of AdoMet level.
Form I Rubiscos from non‐green algae are expressed abundantly but not assembled in tobacco chloroplasts
Authors: Whitney Spencer M. , Baldet Pierre , Hudson Graham S. , Andrews T. John
Published: March 2003

Non‐green algae have Rubiscos that are phylogenetically distinct from their counterparts in green algae and higher plants. Some non‐green‐algal Rubiscos are more specific for CO2, relative to O2, than higher‐plant Rubiscos, sometimes coupled with lower Michaelis constants for CO2. If these Rubiscos could be substituted for the higher‐plant enzyme, and if they functioned successfully in the higher‐plant chloroplast and were regulated appropriately, they would improve the CO2 use and quantum efficiency of higher‐plant photosynthesis. To assess the feasibility of expressing non‐green algal Rubiscos in higher‐plant chloroplasts, we inserted the rbcLS operons from the rhodophyte Galdieria sulphuraria and the diatom Phaeodactylum tricornutum into the inverted repeats of the plastid genome of tobacco, leaving the tobacco rbcL gene unaltered. Homoplasmic transformants were selected. The transgenes directed the synthesis of abundant amounts of transcripts and both subunits of the foreign Rubiscos. In some circumstances, leaves of the transformants with the P. tricornutum Rubisco contained as much foreign Rubisco protein as endogenous tobacco Rubisco (>30% of the soluble leaf protein). However, the subunits of the foreign Rubiscos were not properly folded and/or assembled. All the foreign large subunits and most of the foreign small subunits were recovered in the insoluble fractions of leaf extracts. Edman sequencing yielded the expected N‐terminal sequences for the foreign small subunits but the N‐termini of the foreign large subunits were blocked. Accumulation of large amounts of denatured foreign Rubisco in the leaves, particularly of the P. tricornutum transformants, caused a reduction in the amount of tobacco Rubisco present, with concomitant reductions in leaf CO2 assimilation and plant growth.
Efficient elimination of selectable marker genes from the plastid genome by the CRE‐lox site‐specific recombination system
Authors: Corneille Sylvie , Lutz Kerry , Svab Zora , Maliga Pal
Published: March 2003

Incorporation of a selectable marker gene during transformation is essential to obtain transformed plastids. However, once transformation is accomplished, having the marker gene becomes undesirable. Here we report on adapting the P1 bacteriophage CRE‐lox site‐specific recombination system for the elimination of marker genes from the plastid genome. The system was tested by the elimination of a negative selectable marker, codA, which is flanked by two directly oriented lox sites (>codA>). Highly efficient elimination of >codA> was triggered by introduction of a nuclear‐encoded plastid‐targeted CRE by Agrobacterium transformation or via pollen. Excision of >codA> in tissue culture cells was frequently accompanied by a large deletion of a plastid genome segment which includes the tRNA‐ValUAC gene. However, the large deletions were absent when cre was introduced by pollination. Thus pollination is our preferred protocol for the introduction of cre. Removal of the >codA> coding region occurred at a dramatic speed, in striking contrast to the slow and gradual build‐up of transgenic copies during plastid transformation. The nuclear cre gene could subsequently be removed by segregation in the seed progeny. The modified CRE‐lox system described here will be a highly efficient tool to obtain marker‐free transplastomic plants.
Single ribosomal transcription units are linear, compacted Christmas trees in plant nucleoli
Authors: González‐Melendi Pablo , Wells Brian , Beven Alison F. , Shaw Peter J.
Published: March 2003

The rDNA transcription units are enormous macromolecular structures located in the nucleolus and containing 50–100 RNA polymerases together with the nascent pre‐rRNA attached to the rDNA. It has not previously been possible to visualize nucleolar transcription units directly in intact nucleoli, although highly spread preparations in the electron microscope have been imaged as ‘Christmas trees’ 2–3 µm long. Here we determine the relative conformation of individual transcription units in Pisum sativum plant nucleoli using a novel labelling technique. Nascent transcripts were detected by a highly sensitive silver‐enhanced 1 nm gold procedure, followed by 3D electron microscopy of entire nucleoli. Individual transcription units are seen as conical, elongated clusters approximately 300 nm in length and 130 nm in width at the thickest end. We further show that there were approximately 300 active ribosomal genes in the nucleoli examined. The underlying chromatin structure of the transcribing rDNA was directly visualized by applying a novel limited extraction procedure to fixed specimens in order to wash out the proteins and RNA, thus specifically revealing DNA strands after uranyl acetate staining. Using this technique, followed by post‐embedding in situ hybridization, we observed that the nucleolar rDNA fibres are not extended but show a coiled, thread‐like appearance. Our results show for the first time that native rDNA transcription units are linear, compacted Christmas trees.
Assessment of the food safety issues related to genetically modified foods
Authors: Kuiper Harry A. , Kleter Gijs A. , Noteborn Hub P. J. M. , Kok Esther J.
Published: March 2003

International consensus has been reached on the principles regarding evaluation of the food safety of genetically modified plants. The concept of substantial equivalence has been developed as part of a safety evaluation framework, based on the idea that existing foods can serve as a basis for comparing the properties of genetically modified foods with the appropriate counterpart. Application of the concept is not a safety assessment per se, but helps to identify similarities and differences between the existing food and the new product, which are then subject to further toxicological investigation. Substantial equivalence is a starting point in the safety evaluation, rather than an endpoint of the assessment. Consensus on practical application of the principle should be further elaborated. Experiences with the safety testing of newly inserted proteins and of whole genetically modified foods are reviewed, and limitations of current test methodologies are discussed. The development and validation of new profiling methods such as DNA microarray technology, proteomics, and metabolomics for the identification and characterization of unintended effects, which may occur as a result of the genetic modification, is recommended. The assessment of the allergenicity of newly inserted proteins and of marker genes is discussed. An issue that will gain importance in the near future is that of post‐marketing surveillance of the foods derived from genetically modified crops. It is concluded, among others that, that application of the principle of substantial equivalence has proven adequate, and that no alternative adequate safety assessment strategies are available.
Impaired expression of the plastidic ferrochelatase by antisense RNA synthesis leads to a necrotic phenotype of transformed tobacco plants
Authors: Papenbrock Jutta , Mishra Sanjay , Mock Hans‐Peter , Kruse Elisabeth , Schmidt Eva‐Kathrin , Petersmann Astrid , Braun Hans‐Peter , Grimm Bernhard
Published: March 2003

Protoporphyrin IX is the last common intermediate of tetrapyrrole biosynthesis. The chelation of a Mg2+ ion by magnesium chelatase and of a ferrous ion by ferrochelatase directs protoporphyrin IX towards the formation of chlorophyll and heme, respectively. A full length cDNA clone encoding a ferrochelatase was identified from a Nicotiana tabacum cDNA library. The encoded protein consists of 497 amino acid residues with a molecular weight of 55.4 kDa. In vitro import of the protein into chloroplasts and its location in stroma and thylakoids confirm its close relationship to the previously described Arabidopsis thaliana plastid‐located ferrochelatase (FeChII). A 1700‐bp tobacco FeCh cDNA sequence was expressed in Nicotiana tabacum cv. Samsun NN under the control of the CaMV 35S promoter in antisense orientation allowing investigation into the consequences of selective reduction of the plastidic ferrochelatase activity for protoporphyrin IX channeling in chloroplasts and for interactions between plastidic and mitochondrial heme synthesis. Leaves of several transformants showed a reduced chlorophyll content and, during development, a light intensity‐dependent formation of necrotic leaf lesions. In comparison with wild‐type plants the total ferrochelatase activity was decreased in transgenic lines leading to an accumulation of photosensitizing protoporphyrin IX. Ferrochelatase activity was reduced only in plastids but not in mitochondria of transgenic plants. By means of the specifically diminished ferrochelatase activity consequences of the selective inhibition of protoheme formation for the intracellular supply of heme can be investigated in the future.
Constitutive expression of a celery mannitol dehydrogenase in tobacco enhances resistance to the mannitol‐secreting fungal pathogen Alternaria alternata
Authors: Jennings Dianne B. , Daub Margaret E. , Pharr D. Mason , Williamson John D.
Published: March 2003

Our previous observation that host plant extracts induce production and secretion of mannitol in the tobacco pathogen Alternaria alternata suggested that, like their animal counterparts, plant pathogenic fungi might produce the reactive oxygen quencher mannitol as a means of suppressing reactive oxygen‐mediated plant defenses. The concurrent discovery that pathogen attack induced mannitol dehydrogenase (MTD) expression in the non‐mannitol‐containing host tobacco suggested that plants, unlike animals, might be able to counter this fungal suppressive mechanism by catabolizing mannitol of fungal origin. To test this hypothesis, transgenic tobacco plants constitutively expressing a celery Mtd cDNA were produced and evaluated for potential changes in resistance to both mannitol‐ and non‐mannitol‐secreting pathogens. Constitutive expression of the MTD transgene was found to confer significantly enhanced resistance to A. alternata, but not to the non‐mannitol‐secreting fungal pathogen Cercospora nicotianae. These results are consistent with the hypothesis that MTD plays a role in resistance to mannitol‐secreting fungal plant pathogens.
Nitrate and phosphate availability and distribution have different effects on root system architecture of Arabidopsis
Authors: Linkohr Birgit I. , Williamson Lisa C. , Fitter Alastair H. , Leyser H.M. Ottoline
Published: March 2003

Plant root systems can respond to nutrient availability and distribution by changing the three‐dimensional deployment of their roots: their root system architecture (RSA). We have compared RSA in homogeneous and heterogeneous nitrate and phosphate supply in Arabidopsis. Changes in nitrate and phosphate availability were found to have contrasting effects on primary root length and lateral root density, but similar effects on lateral root length. Relative to shoot dry weight (DW), primary root length decreased with increasing nitrate availability, while it increased with increasing phosphate supply. Lateral root density remained constant across a range of nitrate supplies, but decreased with increasing phosphate supply. In contrast, lateral root elongation was suppressed both by high nitrate and high phosphate supplies. Local supplies of high nitrate or phosphate in a patch also had different effects. Primary root growth was not affected by a high nitrate patch, but growth through a high phosphate patch reduced primary root growth after the root left the patch. A high nitrate patch induced an increase in lateral root density in the patch, whereas lateral root density was unaffected by a high phosphate patch. However, both phosphate‐ and nitrate‐rich patches induced lateral root elongation in the patch and suppressed it outside the patch. This co‐ordinated response of lateral roots also occurs in soil‐grown plants exposed to a nutrient‐rich patch. The auxin‐resistant mutants axr1, axr4 and aux1 all showed the wild‐type lateral root elongation responses to a nitrate‐rich patch, suggesting that auxin is not required for this response.
Single‐step transformation for generating marker‐free transgenic rice using the ipt‐type MAT vector system
Authors: Endo Saori , Sugita Koichi , Sakai Miho , Tanaka Hiroshi , Ebinuma Hiroyasu
Published: March 2003

The ipt‐type MAT vector uses the ipt gene for regeneration of marker‐free transgenic plants. However, it was pointed out that this system was not suitable for most economically important crops that regenerated through auxin‐dependent embryogenesis. We report a single‐step transformation system of rice using MAT vector. When we transformed scutellum tissues of 5 days pre‐cultured rice seeds, marker‐free transgenic rice plants directly regenerated from 25.5% infected scutellum tissues without forming ipt‐intermediates within 4 weeks after an infection. Excision of the ipt gene caused the regeneration of marker‐free transgenic rice plants through embryogenic tissues. Therefore, this system needs no selective agent and no sexual crossing for identification of transgenic plants not containing a selectable marker gene. This system is highly effective for generation of marker‐free transgenic plants in economically important crops.
The ethanolic fermentation pathway supports respiration and lipid biosynthesis in tobacco pollen
Authors: Mellema Stefan , Eichenberger Waldemar , Rawyler André , Suter Marianne , Tadege Million , Kuhlemeier Cris
Published: March 2003

Rapid pollen tube growth requires a high rate of sugar metabolism to meet energetic and biosynthetic demands. Previous work on pollen sugar metabolism showed that tobacco pollen carry out efficient ethanolic fermentation concomitantly with a high rate of respiration (Bucher et al., 1995). Here we show that the products of fermentation, acetaldehyde and ethanol, are further metabolised in a pathway that bypasses mitochondrial PDH. The enzymes involved in this pathway are pyruvate decarboxylase, aldehyde dehydrogenase and acetyl‐CoA synthetase. Radiolabelling experiments show that during tobacco pollen tube growth label of 14C‐ethanol is incorporated into CO2 as well as into lipids and other higher molecular weight compounds. A role for the glyoxylate cycle appears unlikely since activity of malate synthase, a key enzyme of the glyoxylate cycle, could not be detected.
A WD40‐repeat containing protein, similar to a fungal co‐repressor, is required for transcriptional gene silencing in Chlamydomonas
Authors: Zhang Chaomei , Wu‐Scharf Dancia , Jeong Byeong‐ryool , Cerutti Heriberto
Published: March 2003

In higher plants, mammals, and filamentous fungi, transcriptional gene silencing is frequently associated with DNA methylation. However, recent evidence suggests that certain transgenes can be inactivated by a methylation independent mechanism. In the unicellular green alga Chlamydomonas reinhardtii, single‐copy transgenes are transcriptionally silenced without discernible cytosine methylation of the introduced DNA. We have isolated a Chlamydomonas gene, Mut11, which is required for the transcriptional repression of single‐copy transgenes. Mut11 appears to have a global role in gene regulation since it also affects transposon mobilization, cellular growth, and sensitivity to DNA damaging agents. In transient expression assays, a fusion protein between the predicted Mut11 gene product (Mut11p) and E. coli β‐glucuronidase localizes predominantly to the nucleus. Mut11p, a polypeptide of 370 amino acids containing seven WD40 repeats, is highly homologous to proteins of unknown function that are widely distributed among eukaryotes. Mut11p also shows similarity to the C‐terminal domain of TUP1, a global transcriptional co‐repressor in fungi. Based on these findings we speculate that, in Chlamydomonas, the silencing of certain single‐copy transgenes and dispersed transposons integrated into euchromatic regions may occur by a mechanism(s) similar to those involving global transcriptional repressors. Our results also support the existence, in methylation‐competent organisms, of a mechanism(s) of transcriptional (trans)gene silencing that is independent of DNA methylation.
Starch granule initiation and growth are altered in barley mutants that lack isoamylase activity
Authors: Burton Rachel A. , Jenner Helen , Carrangis Luke , Fahy Brendan , Fincher Geoffrey B. , Hylton Chris , Laurie David A. , Parker Mary , Waite Darren , Van Wegen Sonja , Verhoeven Tamara , Denyer Kay
Published: March 2003

Two mutant lines of barley, Risø 17 and Notch‐2, were found to accumulate phytoglycogen in the grain. Like the sugary mutants of maize and rice, these phytoglycogen‐accumulating mutants of barley lack isoamylase activity in the developing endosperm. The mutants were shown to be allelic, and to have lesions in the isoamylase gene, isa1 that account for the absence of this enzyme. As well as causing a reduction in endosperm starch content, the mutations have a profound effect on the structure, number and timing of initiation of starch granules. There are no normal A‐type or B‐type granules in the mutants. The mutants have a greater number of starch granules per plastid than the wild‐type and, particularly in Risø 17, this leads to the appearance of compound starch granules. These results suggest that, as well as suppressing phytoglycogen synthesis, isoamylase in the wild‐type endosperm plays a role in determining the number, and hence the form, of starch granules.
Regulation of ADL6 activity by its associated molecular network
Authors: Lam Bernard C.‐H. , Sage Tammy L. , Bianchi Fabrizio , Blumwald Eduardo
Published: March 2003

Plant dynamin‐like proteins consist of a group of high molecular weight GTPase with diverse structural arrangements and cellular localizations. In addition, unlike animal dynamins, there was no evidence for the involvement of any plant dynamin‐like protein in clathrin‐mediated vesicle trafficking. In this study we demonstrate that ADL6 (Arabidopsis dynamin‐like protein 6), due to its domain arrangement, behaves similarly to the animal dynamins. The association of ADL6 with clathrin‐coated vesicles was demonstrated by co‐fractionation and immunocytochemical studies. ADL6 also interacted via its C‐terminus with γ‐adaptin, an adaptor protein of clathrin‐coated vesicles. Our results suggest that ADL6 participates in clathrin‐mediated vesicle trafficking originating from the Golgi. In addition, our studies demonstrate that ADL6 intrinsic GTPase activity is regulated by its association with acidic phospholipids and an SH3 (Src homology 3)‐containing protein.
GLK gene pairs regulate chloroplast development in diverse plant species
Authors: Fitter David W. , Martin David J. , Copley Martin J. , Scotland Robert W. , Langdale Jane A.
Published: March 2003

Chloroplast biogenesis is a complex process that requires close co‐ordination between two genomes. Many of the proteins that accumulate in the chloroplast are encoded by the nuclear genome, and the developmental transition from proplastid to chloroplast is regulated by nuclear genes. Here we show that a pair of Golden 2‐like (GLK) genes regulates chloroplast development in Arabidopsis. The GLK proteins are members of the GARP superfamily of transcription factors, and phylogenetic analysis demonstrates that the maize, rice and Arabidopsis GLK gene pairs comprise a distinct group within the GARP superfamily. Further phylogenetic analysis suggests that the gene pairs arose through separate duplication events in the monocot and dicot lineages. As in rice, AtGLK1 and AtGLK2 are expressed in partially overlapping domains in photosynthetic tissue. Insertion mutants demonstrate that this expression pattern reflects a degree of functional redundancy as single mutants display normal phenotypes in most photosynthetic tissues. However, double mutants are pale green in all photosynthetic tissues and chloroplasts exhibit a reduction in granal thylakoids. Products of several genes involved in light harvesting also accumulate at reduced levels in double mutant chloroplasts. GLK genes therefore regulate chloroplast development in diverse plant species.